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1.
Front Cardiovasc Med ; 11: 1374881, 2024.
Article in English | MEDLINE | ID: mdl-39045008

ABSTRACT

Background: Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) show tremendous promise for cardiac regeneration following myocardial infarction (MI), but their transplantation gives rise to transient ventricular tachycardia (VT) in large-animal MI models, representing a major hurdle to translation. Our group previously reported that these arrhythmias arise from a focal mechanism whereby graft tissue functions as an ectopic pacemaker; therefore, we hypothesized that hPSC-CMs engineered with a dominant negative form of the pacemaker ion channel HCN4 (dnHCN4) would exhibit reduced automaticity and arrhythmogenic risk following transplantation. Methods: We used CRISPR/Cas9-mediated gene-editing to create transgenic dnHCN4 hPSC-CMs, and their electrophysiological behavior was evaluated in vitro by patch-clamp recordings and optical mapping. Next, we transplanted WT and homozygous dnHCN4 hPSC-CMs in a pig MI model and compared post-transplantation outcomes including the incidence of spontaneous arrhythmias and graft structure by immunohistochemistry. Results: In vitro dnHCN4 hPSC-CMs exhibited significantly reduced automaticity and pacemaker funny current (I f ) density relative to wildtype (WT) cardiomyocytes. Following transplantation with either dnHCN4 or WT hPSC-CMs, all recipient hearts showed transmural infarct scar that was partially remuscularized by scattered islands of human myocardium. However, in contrast to our hypothesis, both dnHCN4 and WT hPSC-CM recipients exhibited frequent episodes of ventricular tachycardia (VT). Conclusions: While genetic silencing of the pacemaker ion channel HCN4 suppresses the automaticity of hPSC-CMs in vitro, this intervention is insufficient to reduce VT risk post-transplantation in the pig MI model, implying more complex mechanism(s) are operational in vivo.

2.
Blood Cancer J ; 9(2): 6, 2019 01 15.
Article in English | MEDLINE | ID: mdl-30647406

ABSTRACT

CD123 (IL-3Rα) is frequently expressed by malignant Hodgkin lymphoma (HL) cells. Naked monoclonal antibodies (mAb) against HL lack clinical benefit, partially due to absence of natural killer (NK) cells in the tumor microenvironment. Here we show that the combination of a fully humanized anti-CD123 mAb (CSL362) and high-affinity Fcγ-receptor NK-92 cells (haNK) effectively target and kill HL cells in vitro. First, we confirmed high expression of CD123 in 2 of the 3 HL cell lines (KM-H2 and L-428), and its absence in NK cells. Cytotoxicity of haNK cells against CD123-positive HL cells was significantly higher in the presence of CSL362. This was also shown with IL-15-activated primary NK cells, although haNK cells showed a 10.87-fold lower estimated half-maximal stimulatory effective concentration (EC50). CSL362 facilitated a significant increase in the expression of CD107a, intracellular IFN-γ and TNF-α and enhanced expression of c-JUN, PLD-1, and ARF6 by NK cells. Inhibition of the ARF6-PLD-1 axis (NAV2729), but not of the MAPK pathway (U0126), completely abrogated CSL362-facilitated antibody-dependent cell-mediated cytotoxicity (ADCC) in haNK and activated primary NK cells. Our results support CD123 as an immunotherapeutic target for HL and the combination of NK cells and CSL362 as a treatment strategy for HL.


Subject(s)
ADP-Ribosylation Factors/antagonists & inhibitors , Antibodies, Monoclonal, Humanized/pharmacology , Antibody-Dependent Cell Cytotoxicity/drug effects , Antineoplastic Agents, Immunological/pharmacology , Interleukin-3 Receptor alpha Subunit/antagonists & inhibitors , Killer Cells, Natural/drug effects , Killer Cells, Natural/immunology , ADP-Ribosylation Factor 6 , Animals , Biomarkers , Cell Degranulation , Cell Line , Cytokines/metabolism , Exocytosis , Humans , Interleukin-3 Receptor alpha Subunit/genetics , Interleukin-3 Receptor alpha Subunit/metabolism , Killer Cells, Natural/metabolism , Mice , Proto-Oncogene Proteins c-jun/genetics , Proto-Oncogene Proteins c-jun/metabolism
3.
Cytotherapy ; 20(8): 1001-1012, 2018 08.
Article in English | MEDLINE | ID: mdl-30076069

ABSTRACT

BACKGROUND: Mesenchymal stromal cells (MSCs) promote wound healing, including after radiotherapy (RT) and surgery. The use of MSCs in regenerative medicine in the context of malignancy, such as to enhance wound healing post-RT/surgery in patients with soft tissue sarcomas (STSs), requires safety validation. The aim of this study was to determine the effects of human MSCs on STS growth in vitro and local recurrence and metastasis in vivo. METHODS: Human primary STS and HT-1080 fibrosarcoma lines were transduced to express luciferase/eGFP (enhanced green fluorescent protein). Sarcoma cells were co-cultured or co-injected with bone marrow-derived MSCs for growth studies. Xenograft tumor models were established with STS lines in NOD/SCID/γcnull mice. To emulate a clinical scenario, subcutaneous tumors were treated with RT/surgery prior to MSC injection into the tumor bed. Local and distant tumor recurrence was studied using histology and bioluminescence imaging. RESULTS: MSCs did not promote STS proliferation upon co-culture in vitro, which was consistent among MSCs from different donors. Co-injection of MSCs with sarcoma cells in mice exhibited no significant tumor-stimulating effect, compared with control mice injected with sarcoma cells alone. MSC administration after RT/surgery had no effect on local recurrence or metastasis of STS. DISCUSSION: These studies are important for the establishment of a safety profile for MSC administration in patients with STS. Our data suggest that MSCs are safe in STS management after standard of care RT/surgery, which can be further investigated in early-phase clinical trials to also determine the efficacy of MSCs in reducing morbidity and to mitigate wound complications in these patients.


Subject(s)
Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/physiology , Radiotherapy , Sarcoma/pathology , Sarcoma/therapy , Surgical Procedures, Operative , Adult , Animals , Coculture Techniques , Combined Modality Therapy , HEK293 Cells , Heterografts , Humans , Male , Mesenchymal Stem Cell Transplantation/adverse effects , Mice , Mice, Inbred NOD , Mice, SCID , Mice, Transgenic , Neoplasm Recurrence, Local/etiology , Neoplasm Recurrence, Local/pathology , Radiotherapy/adverse effects , Radiotherapy/methods , Surgical Procedures, Operative/adverse effects , Surgical Procedures, Operative/methods , Tumor Cells, Cultured , Wound Healing , Xenograft Model Antitumor Assays
4.
PLoS One ; 11(1): e0147868, 2016.
Article in English | MEDLINE | ID: mdl-26820314

ABSTRACT

INTRODUCTION: Osteoarthritis (OA) is associated with chronic inflammation, and mesenchymal stromal cells (MSCs) have been shown to provide pain relief and reparative effects in clinical investigations. MSCs are often delivered with hyaluronic acid (HA), although the combined mechanism of action is not fully understood; we thus investigated the immunomodulatory effects of combining MSCs with different molecular weights (MW) of HA. METHODS: HAs with MWs of 1.6 MDa (hHA), 150 kDa or 7.5 kDa, were added to MSCs alone or MSC-immune cell co-cultures. Gene expression analyses, flow cytometry and cytokine measurements were assessed to determine the effect of HAs on the MSC interactions with immune cells. RESULTS: MSCs in the presence of HAs, in both normal and lymphocyte-conditioned medium, showed negligible changes in gene expression. While addition of hHA resulted in increased proliferation of activated lymphocytes, both in the presence and absence of MSCs, the overall combined effect was a more regulated, homeostatic one; this was supported by higher ratios of secreted IL10/IFNγ and IL10/IL2, in lymphocyte cultures, than with lower MW HAs or no HA, both in the presence and absence of MSCs. In addition, examination of monocyte-derived macrophages showed an increased M2 macrophage frequency (CD14+CD163+CD206+) in the presence of hHA, both with and without MSCs. CONCLUSIONS: hHA produces a less pro-inflammatory environment than lower MW HAs. Moreover, combining hHA with MSCs has an additive effect on the MSC-mediated immunomodulation, suggestive of a more potent combination treatment modality for OA.


Subject(s)
Hyaluronic Acid/pharmacology , Immunologic Factors/pharmacology , Mesenchymal Stem Cells/physiology , Cell Proliferation , Cells, Cultured , Coculture Techniques , Drug Evaluation, Preclinical , Gene Expression/drug effects , Humans , Hyaluronic Acid/chemistry , Immunologic Factors/chemistry , Immunomodulation , Macrophages/physiology , Mesenchymal Stem Cells/drug effects , Molecular Weight , T-Lymphocytes, Helper-Inducer/physiology , T-Lymphocytes, Regulatory/physiology
5.
Proteins ; 82(7): 1542-8, 2014 Jul.
Article in English | MEDLINE | ID: mdl-24470271

ABSTRACT

Pantothenate kinase (PanK) is the rate-limiting enzyme in Coenzyme A biosynthesis, catalyzing the ATP-dependent phosphorylation of pantothenate. We solved the co-crystal structures of PanKs from Staphylococcus aureus (SaPanK) and Klebsiella pneumonia (KpPanK) with N-[2-(1,3-benzodioxol-5-yl)ethyl] pantothenamide (N354-Pan). Two different N354-Pan conformers interact with polar/nonpolar mixed residues in SaPanK and aromatic residues in KpPanK. Additionally, phosphorylated N354-Pan is found at the closed active site of SaPanK but not at the open active site of KpPanK, suggesting an exchange of the phosphorylated product with a new N354-Pan only in KpPanK. Together, pantothenamides conformational flexibility and binding pocket are two key considerations for selective compound design.


Subject(s)
Klebsiella pneumoniae/enzymology , Phosphotransferases (Alcohol Group Acceptor)/chemistry , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Staphylococcus aureus/enzymology , Amides/chemistry , Amides/metabolism , Coenzyme A , Crystallography, X-Ray , Models, Molecular , Protein Conformation
6.
PLoS One ; 7(3): e33943, 2012.
Article in English | MEDLINE | ID: mdl-22470497

ABSTRACT

Kinesin-1 transports various cargos along the axon by interacting with the cargos through its light chain subunit. Kinesin light chains (KLC) utilize its tetratricopeptide repeat (TPR) domain to interact with over 10 different cargos. Despite a high sequence identity between their TPR domains (87%), KLC1 and KLC2 isoforms exhibit differential binding properties towards some cargos. We determined the structures of human KLC1 and KLC2 tetratricopeptide repeat (TPR) domains using X-ray crystallography and investigated the different mechanisms by which KLCs interact with their cargos. Using isothermal titration calorimetry, we attributed the specific interaction between KLC1 and JNK-interacting protein 1 (JIP1) cargo to residue N343 in the fourth TRP repeat. Structurally, the N343 residue is adjacent to other asparagines and lysines, creating a positively charged polar patch within the groove of the TPR domain. Whereas, KLC2 with the corresponding residue S328 did not interact with JIP1. Based on these finding, we propose that N343 of KLC1 can form "a carboxylate clamp" with its neighboring asparagine to interact with JIP1, similar to that of HSP70/HSP90 organizing protein-1's (HOP1) interaction with heat shock proteins. For the binding of cargos shared by KLC1 and KLC2, we propose a different site located within the groove but not involving N343. We further propose a third binding site on KLC1 which involves a stretch of polar residues along the inter-TPR loops that may form a network of hydrogen bonds to JIP3 and JIP4. Together, these results provide structural insights into possible mechanisms of interaction between KLC TPR domains and various cargo proteins.


Subject(s)
Kinesins/chemistry , Models, Molecular , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/metabolism , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Humans , Kinesins/genetics , Kinesins/metabolism , Molecular Sequence Data , Protein Isoforms/chemistry , Protein Isoforms/metabolism , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment
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