Dasatinib enhances anti-leukemia efficacy of chimeric antigen receptor T cells by inhibiting cell differentiation and exhaustion

AbstractRelapses of CD19-expressing leukemia in patients who achieved initial remission after CART cell treatment have been reported to correlate with poor CART cells persistence. Sustained tonic signaling or strong activation drives CART cell differentiation and exhaustion, which limit the therapeutic efficacy and persistence of CART cells. Here, we identified dasatinib as the optimal candidate to prevent or reverse both CD28/CART and 4-1BB/CART cell differentiation and exhaustion during ex vivo expansion, which profoundly enhanced the therapeutic efficacy and in vivo persistence. Moreover, strong activation-induced CART cells differentiation, exhaustion and apoptosis driven by CD3/CD28 stimulation or antigen exposure were dramatically prevented or reversed by dasatinib treatment. Mechanistically, dasatinib markedly reduced the phosphorylation of Src and Lck, and downregulated the expression of genes involved in CAR signaling pathways, which resulted in the optimization of cell differentiation, exhaustion and apoptosis-related gene expression. Our study proposes a promising pharmacological approach for optimizing CART cells manufacture, and provides an experimental basis for reinvigorating CART cells in clinical application.

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EZH2 inhibition reduces cartilage loss and functional impairment related to osteoarthritis

Scientific Reports ◽

10.1038/s41598-020-76724-9 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Lyess Allas ◽

Sybille Brochard ◽

Quitterie Rochoux ◽

Jules Ribet ◽

Cleo Dujarrier ◽

...

Keyword(s):

Functional Disability ◽

Ex Vivo ◽

Cartilage Degradation ◽

Motor Functions ◽

Methyl Transferase ◽

Expression Of Genes ◽

Medial Meniscectomy ◽

The Impact

Abstract Histone methyltransferase EZH2 is upregulated during osteoarthritis (OA), which is the most widespread rheumatic disease worldwide, and a leading cause of disability. This study aimed to assess the impact of EZH2 inhibition on cartilage degradation, inflammation and functional disability. In vitro, gain and loss of EZH2 function were performed in human articular OA chondrocytes stimulated with IL-1β. In vivo, the effects of EZH2 inhibition were investigated on medial meniscectomy (MMX) OA mouse model. The tissue alterations were assayed by histology and the functional disabilities of the mice by actimetry and running wheel. In vitro, EZH2 overexpression exacerbated the action of IL-1β in chondrocytes increasing the expression of genes involved in inflammation, pain (NO, PGE2, IL6, NGF) and catabolism (MMPs), whereas EZH2 inhibition by a pharmacological inhibitor, EPZ-6438, reduced IL-1β effects. Ex vivo, EZH2 inhibition decreased IL-1β-induced degradation of cartilage. In vivo, intra-articular injections of the EZH2 inhibitor reduced cartilage degradation and improved motor functions of OA mice. This study demonstrates that the pharmacological inhibition of the histone methyl-transferase EZH2 slows the progression of osteoarthritis and improves motor functions in an experimental OA model, suggesting that EZH2 could be an effective target for the treatment of OA by reducing catabolism, inflammation and pain.

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Nanocrystals of Fusidic Acid for Dual Enhancement of Dermal Delivery and Antibacterial Activity: In Vitro, Ex Vivo and In Vivo Evaluation

Pharmaceutics ◽

10.3390/pharmaceutics12030199 ◽

2020 ◽

Vol 12 (3) ◽

pp. 199 ◽

Cited By ~ 2

Author(s):

Iman S. Ahmed ◽

Osama S. Elnahas ◽

Nouran H. Assar ◽

Amany M. Gad ◽

Rania El Hosary

Keyword(s):

Fusidic Acid ◽

Therapeutic Efficacy ◽

Ex Vivo ◽

Skin Permeation ◽

Fold Increase ◽

Spherical Particles ◽

Resistant Bacteria ◽

Infection Model

With the alarming rise in incidence of antibiotic-resistant bacteria and the scarcity of newly developed antibiotics, it is imperative that we design more effective formulations for already marketed antimicrobial agents. Fusidic acid (FA), one of the most widely used antibiotics in the topical treatment of several skin and eye infections, suffers from poor water-solubility, sub-optimal therapeutic efficacy, and a significant rise in FA-resistant Staphylococcus aureus (FRSA). In this work, the physico-chemical characteristics of FA were modified by nanocrystallization and lyophilization to improve its therapeutic efficacy through the dermal route. FA-nanocrystals (NC) were prepared using a modified nanoprecipitation technique and the influence of several formulation/process variables on the prepared FA-NC characteristics were optimized using full factorial statistical design. The optimized FA-NC formulation was evaluated before and after lyophilization by several in-vitro, ex-vivo, and microbiological tests. Furthermore, the lyophilized FA-NC formulation was incorporated into a cream product and its topical antibacterial efficacy was assessed in vivo using a rat excision wound infection model. Surface morphology of optimized FA-NC showed spherical particles with a mean particle size of 115 nm, span value of 1.6 and zeta potential of −11.6 mV. Differential scanning calorimetry and powder X-ray diffractometry confirmed the crystallinity of FA following nanocrystallization and lyophilization. In-vitro results showed a 10-fold increase in the saturation solubility of FA-NC while ex-vivo skin permeation studies showed a 2-fold increase in FA dermal deposition from FA-NC compared to coarse FA. Microbiological studies revealed a 4-fofd decrease in the MIC against S. aureus and S. epidermidis from FA-NC cream compared to commercial Fucidin cream. In-vivo results showed that FA-NC cream improved FA distribution and enhanced bacterial exposure in the infected wound, resulting in increased therapeutic efficacy when compared to coarse FA marketed as Fucidin cream.

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Activity of MK1775, a selective Wee1 inhibitor, alone or in combination with gemcitabine, in sarcomas.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.10084 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. 10084-10084

Author(s):

Jenny Kreahling ◽

Damon R. Reed ◽

Parastou Foroutan ◽

Gary Martinez ◽

Robert Gillies ◽

...

Keyword(s):

Cell Cycle ◽

Cell Death ◽

Soft Tissue ◽

Soft Tissue Sarcoma ◽

Therapeutic Efficacy ◽

Ex Vivo ◽

Apoptotic Cell ◽

Apoptotic Cell Death ◽

Primary Therapy

10084 Background: Sarcomas consist of more than 50 subtypes of mesenchymal tumors. Doxorubicin alone or in combination has been the primary therapy for treatment of sarcomas; however, the response rates are suboptimal in many of the more common adult subtypes of soft tissue sarcoma. Accordingly, new agents are needed for the treatment of this heterogeneous group of diseases. Wee1 is a critical component of the G2/M cell cycle checkpoint control and mediates cell cycle arrest by regulating the phosphorylation of CDC2. Methods: MK1775 treatment of multiple sarcoma preclinical models at clinically relevant concentrations leads to unscheduled entry into mitosis and initiation of apoptotic cell death. In our current study we have investigated the therapeutic efficacy of MK1775 in sarcoma cell lines, patient-derived tumor explants ex vivo and in vivo in a xenograft model of osteosarcoma both alone and in combination with gemcitabine. Results: In patient-derived bone and soft tissue sarcoma samples ex vivo treatments show MK1775 in combination with gemcitabine causes significant apoptotic cell death suggesting that this treatment may represent a novel approach in the treatment of sarcomas. The cytotoxic effect of Wee1 inhibition on sarcoma cells appears to be independent of p53 mutational status. Furthermore, in a patient-derived osteosarcoma xenograft mouse model we show the therapeutic efficacy of MK1775 in vivo by utilizing magnetic resonance imaging (MRI) and diffusion MRI methods. Our data shows MK1775 in combination with gemcitabine dramatically slows tumor growth, increases apoptotic cell death and increases CDC2 activity. Cell viability, a clinically established prognostic indicator of survival, was lowest with the combination and very low in animals treated with MK1775 alone. This was mainly due to increased mineralization of the tumors. Caspase-3 was increased in MK1775 treated animals by immunohistochemistry as well. Conclusions: These results together with the promising safety profile of MK1775 strongly suggest that this drug can be used as a potential therapeutic agent alone or in combination with gemcitabine in the treatment of both adult as well as pediatric sarcoma patients.

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Epiregulin induces leptin secretion and energy expenditure in high-fat diet-fed mice

Journal of Endocrinology ◽

10.1530/joe-18-0289 ◽

2018 ◽

Vol 239 (3) ◽

pp. 377-388 ◽

Cited By ~ 1

Author(s):

Rumana Yasmeen ◽

Qiwen Shen ◽

Aejin Lee ◽

Jacob H Leung ◽

Devan Kowdley ◽

...

Keyword(s):

Energy Expenditure ◽

High Fat Diet ◽

Ex Vivo ◽

Mapk Pathway ◽

Treated Group ◽

Dependent Manner ◽

High Fat ◽

Fat Depots ◽

Expression Of Genes

Adipokine leptin regulates neuroendocrine circuits that control energy expenditure, thermogenesis and weight loss. However, canonic regulators of leptin secretion, such as insulin and malonyl CoA, do not support these processes. We hypothesize that epiregulin (EREG), a growth factor that is secreted from fibroblasts under thermogenic and cachexia conditions, induces leptin secretion associated with energy dissipation. The effects of EREG on leptin secretion were studied ex vivo, in the intra-abdominal white adipose tissue (iAb WAT) explants, as well as in vivo, in WT mice with diet-induced obesity (DIO) and in ob/ob mice. These mice were pair fed a high-fat diet and treated with intraperitoneal injections of EREG. EREG increased leptin production and secretion in a dose-dependent manner in iAb fat explants via the EGFR/MAPK pathway. After 2 weeks, the plasma leptin concentration was increased by 215% in the EREG-treated group compared to the control DIO group. EREG-treated DIO mice had an increased metabolic rate and core temperature during the active dark cycle and displayed cold-induced thermogenesis. EREG treatment reduced iAb fat mass, the major site of leptin protein production and secretion, but did not reduce the mass of the other fat depots. In the iAb fat, expression of genes supporting mitochondrial oxidation and thermogenesis was increased in EREG-treated mice vs control DIO mice. All metabolic and gene regulation effects of EREG treatment were abolished in leptin-deficient ob/ob mice. Our data revealed a new role of EREG in induction of leptin secretion leading to the energy expenditure state. EREG could be a potential target protein to regulate hypo- and hyperleptinemia, underlying metabolic and immune diseases.

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Translational Paradigms in Cerebrovascular Gene Transfer

Journal of Cerebral Blood Flow & Metabolism ◽

10.1097/01.wcb.0000093324.07718.d4 ◽

2003 ◽

Vol 23 (11) ◽

pp. 1251-1262 ◽

Cited By ~ 10

Author(s):

Vini G Khurana ◽

Fredric B Meyer

Keyword(s):

Gene Transfer ◽

Blood Vessels ◽

Ex Vivo ◽

Present Report ◽

Cerebral Arteries ◽

Experimental Studies ◽

Gene Encoding ◽

Expression Of Genes ◽

Genes Encoding

Gene transfer involves the use of an engineered biologic vehicle known as a vector to introduce a gene encoding a protein of interest into a particular tissue. In diseases with known defects at a genetic level, gene transfer offers a potential means of restoring a normal molecular environment via vector-mediated entry (transduction) and expression of genes encoding potentially therapeutic proteins selectively in diseased tissues. The technology of gene transfer therefore underlies the concept of gene therapy and falls under the umbrella of the current genomics revolution. Particularly since 1995, numerous attempts have been made to introduce genes into intracranial blood vessels to demonstrate and characterize viable transduction. More recently, in attempting to translate cerebrovascular gene transfer technology closer to the clinical arena, successful transductions of normal human cerebral arteries ex vivo and diseased animal cerebral arteries in vivo have been reported using vasomodulatory vectors. Considering the emerging importance of gene-based strategies for the treatment of the spectrum of human disease, the goals of the present report are to overview the fundamentals of gene transfer and review experimental studies germane to the clinical translation of a technology that can facilitate genetic modification of cerebral blood vessels.

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Functional Antibodies Targeting IsaA ofStaphylococcus aureusAugment Host Immune Response and Open New Perspectives for Antibacterial Therapy

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01144-10 ◽

2010 ◽

Vol 55 (1) ◽

pp. 165-173 ◽

Cited By ~ 40

Author(s):

Udo Lorenz ◽

Birgit Lorenz ◽

Tim Schmitter ◽

Karin Streker ◽

Christian Erck ◽

...

Keyword(s):

Therapeutic Efficacy ◽

Ex Vivo ◽

Venous Catheter ◽

Staphylococcal Infection ◽

Adjunctive Treatment ◽

Infection Model ◽

Reactive Oxygen Metabolites ◽

Dependent Manner ◽

Bacterial Killing

ABSTRACTStaphylococcus aureusis the most common cause of nosocomial infections. Multiple antibiotic resistance and severe clinical outcomes provide a strong rationale for development of immunoglobulin-based strategies. Traditionally, novel immunological approaches against bacterial pathogens involve antibodies directed against cell surface-exposed virulence-associated epitopes or toxins. In this study, we generated a monoclonal antibody targeting the housekeeping protein IsaA, a suggested soluble lytic transglycosylase ofS. aureus, and tested its therapeutic efficacy in two experimental mouse infection models. A murine anti-IsaA antibody of the IgG1 subclass (UK-66P) showed the highest binding affinity in Biacore analysis. This antibody recognized allS. aureusstrains tested, including hospital-acquired and community-acquired methicillin-resistantS. aureusstrains. Therapeutic efficacyin vivoin mice was analyzed using a central venous catheter-related infection model and a sepsis survival model. In both models, anti-IsaA IgG1 conferred protection against staphylococcal infection.Ex vivo, UK-66P activates professional phagocytes and induces highly microbicidal reactive oxygen metabolites in a dose-dependent manner, resulting in bacterial killing. The study provides proof of concept that monoclonal IgG1 antibodies with high affinity to the ubiquitously expressed, single-epitope-targeting IsaA are effective in the treatment of staphylococcal infection in different mouse models. Anti-IsaA antibodies might be a useful component in an antibody-based therapeutic for prophylaxis or adjunctive treatment of human cases ofS. aureusinfections.

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Investigating fibrosis and inflammation in an ex vivo NASH murine model

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00209.2019 ◽

2020 ◽

Vol 318 (2) ◽

pp. G336-G351

Author(s):

Emilia Gore ◽

Emilia Bigaeva ◽

Anouk Oldenburger ◽

Yvette J. M. Jansen ◽

Detlef Schuppan ◽

...

Keyword(s):

Gene Expression ◽

Lipid Metabolism ◽

Liver Disease ◽

Nonalcoholic Steatohepatitis ◽

Ex Vivo ◽

Ex Vivo Model ◽

Nonalcoholic Fatty Liver ◽

Liver Slices ◽

Expression Of Genes

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease, characterized by excess fat accumulation (steatosis). Nonalcoholic steatohepatitis (NASH) develops in 15–20% of NAFLD patients and frequently progresses to liver fibrosis and cirrhosis. We aimed to develop an ex vivo model of inflammation and fibrosis in steatotic murine precision-cut liver slices (PCLS). NASH was induced in C57Bl/6 mice on an amylin and choline-deficient l-amino acid-defined (CDAA) diet. PCLS were prepared from steatohepatitic (sPCLS) and control (cPCLS) livers and cultured for 48 h with LPS, TGFβ1, or elafibranor. Additionally, C57Bl/6 mice were placed on CDAA diet for 12 wk to receive elafibranor or vehicle from weeks 7 to 12. Effects were assessed by transcriptome analysis and procollagen Iα1 protein production. The diets induced features of human NASH. Upon culture, all PCLS showed an increased gene expression of fibrosis- and inflammation-related markers but decreased lipid metabolism markers. LPS and TGFβ1 affected sPCLS more pronouncedly than cPCLS. TGFβ1 increased procollagen Iα1 solely in cPCLS. Elafibranor ameliorated fibrosis and inflammation in vivo but not ex vivo, where it only increased the expression of genes modulated by PPARα. sPCLS culture induced inflammation-, fibrosis-, and lipid metabolism-related transcripts, explained by spontaneous activation. sPCLS remained responsive to proinflammatory and profibrotic stimuli on gene expression. We consider that PCLS represent a useful tool to reproducibly study NASH progression. sPCLS can be used to evaluate potential treatments for NASH, as demonstrated in our elafibranor study, and serves as a model to bridge results from rodent studies to the human system. NEW & NOTEWORTHY This study showed that nonalcoholic steatohepatitis can be studied ex vivo in precision-cut liver slices obtained from murine diet-induced fatty livers. Liver slices develop a spontaneous inflammatory and fibrogenic response during culture that can be augmented with specific modulators. Additionally, the model can be used to test the efficacy of pharmaceutical compounds (as shown in this investigation with elafibranor) and could be a tool for preclinical assessment of potential therapies.

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Treatment with at Homeopathic Complex Medication Modulates Mononuclear Bone Marrow Cell Differentiation

Evidence-based Complementary and Alternative Medicine ◽

10.1093/ecam/nep119 ◽

2011 ◽

Vol 2011 ◽

pp. 1-10 ◽

Cited By ~ 2

Author(s):

Beatriz Cesar ◽

Ana Paula R. Abud ◽

Carolina C. de Oliveira ◽

Francolino Cardoso ◽

Raffaello Popa Di Bernardi ◽

...

Keyword(s):

Bone Marrow ◽

Cell Differentiation ◽

Mononuclear Cells ◽

Bone Marrow Cells ◽

Ex Vivo ◽

Marrow Cell ◽

Macrophage Colony ◽

Total Bone Marrow

A homeopathic complex medication (HCM), with immunomodulatory properties, is recommended for patients with depressed immune systems. Previous studies demonstrated that the medication induces an increase in leukocyte number. The bone marrow microenvironment is composed of growth factors, stromal cells, an extracellular matrix and progenitor cells that differentiate into mature blood cells. Mice were our biological model used in this research. We now reportin vivoimmunophenotyping of total bone marrow cells andex vivoeffects of the medication on mononuclear cell differentiation at different times. Cells were examined by light microscopy and cytokine levels were measuredin vitro. Afterin vivotreatment with HCM, a pool of cells from the new marrow microenvironment was analyzed by flow cytometry to detect any trend in cell alteration. The results showed decreases, mainly, in CD11b and TER-119 markers compared with controls. Mononuclear cells were used to analyze the effects ofex vivoHCM treatment and the number of cells showing ring nuclei, niche cells and activated macrophages increased in culture, even in the absence of macrophage colony-stimulating factor. Cytokines favoring stromal cell survival and differentiation in culture were inducedin vitro. Thus, we observe that HCM is immunomodulatory, either alone or in association with other products.

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The circadian cryptochrome, CRY1, is a pro-tumorigenic factor that rhythmically modulates DNA repair

Nature Communications ◽

10.1038/s41467-020-20513-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Ayesha A. Shafi ◽

Chris M. McNair ◽

Jennifer J. McCann ◽

Mohammed Alshalalfa ◽

Anton Shostak ◽

...

Keyword(s):

Prostate Cancer ◽

Dna Damage ◽

Dna Repair ◽

Ex Vivo ◽

Functional Studies ◽

Expression Of Genes ◽

Mechanistic Investigation ◽

Transcriptional Coregulator

AbstractMechanisms regulating DNA repair processes remain incompletely defined. Here, the circadian factor CRY1, an evolutionally conserved transcriptional coregulator, is identified as a tumor specific regulator of DNA repair. Key findings demonstrate that CRY1 expression is androgen-responsive and associates with poor outcome in prostate cancer. Functional studies and first-in-field mapping of the CRY1 cistrome and transcriptome reveal that CRY1 regulates DNA repair and the G2/M transition. DNA damage stabilizes CRY1 in cancer (in vitro, in vivo, and human tumors ex vivo), which proves critical for efficient DNA repair. Further mechanistic investigation shows that stabilized CRY1 temporally regulates expression of genes required for homologous recombination. Collectively, these findings reveal that CRY1 is hormone-induced in tumors, is further stabilized by genomic insult, and promotes DNA repair and cell survival through temporal transcriptional regulation. These studies identify the circadian factor CRY1 as pro-tumorigenic and nominate CRY1 as a new therapeutic target.

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