Increased Cell Activity Research Articles

NKT-Like (CD3+CD56+) Cells in Chronic Myeloid Leukemia Patients Treated With Tyrosine Kinase Inhibitors.

Therapy with Tyrosine Kinase Inhibitors (TKI) aiming stable deep molecular response is the gold standard to treat Chronic Myeloid Leukemia (CML). NKT-like cells (CD3+CD56+) combine characteristics of T and NK cells. The physiopathological role of these cells remains unknown although the literature refers their association with inflammation, autoimmune diseases, and cancer. Since the information regarding the role of NKT-like cells in CML is rare, we aimed at the characterization of these cells in CML patients treated with TKIs. Peripheral blood NKT-like cells from 48 CML patients and 40 healthy donors were analyzed by multiparametric flow cytometry. Functional tests consisting of co-culture with leukemic target cells (K562 cell line) were used to measure degranulation and cytokine production. Our results revealed that NKT-like cells are decreased in treated CML patients, although they present increased expression of activation markers (CD69 and HLA-DR), increased degranulation (CD107a) and impaired IFN-γ production. Significantly alterations on the expression of tumor recognition (NCRs and NKp80), and immune regulation receptors (LAG-3, TIM-3, and CD137) by NKT-like cells were observed in CML patients. Second generation TKIs increased cell activation (CD69) and decreased expression of NKp44 and NKp80 by NKT-like cells from CML patients when compared to Imatinib. CML patients that achieved deep molecular response (MR4.5) presented downregulation of NKp44 and LAG-3. Further studies are needed to clarify the role of these cells as biomarkers of therapy response and also to evaluate their value for discrimination of better candidates for sustained treatment-free remission after TKI discontinuation.

MiR-590-3pInhibits Pyroptosis in Diabetic Retinopathy by TargetingNLRP1and Inactivating the NOX4 Signaling Pathway

To elucidate the mechanism whereby miR-590-3p regulates pyroptosis in diabetic retinopathy (DR). Human retinal microvascular endothelial cells (HRMECs) incubated with high glucose (HG) were used to establish cell models, and the expression levels of miR-590-3p, caspase-1, IL-1β, NLRP1, NOX4, TXNIP, NLRP3, and ROS were determined. Additionally, miR-590-3p was altered using a mimic or an inhibitor, and siRNAs targeting NLRP1 and NOX4 were applied to explore the regulatory mechanism of miR-590-3p in DR. The relationships between miR-590-3p and NLRP1/NOX4 also were investigated using a luciferase reporter assay. Furthermore, vitreous tissue samples were collected to confirm pyroptosis in clinical DR. Downregulated miR-590-3p and upregulated NLRP1/NOX4 levels were observed in a cell culture model of DR. Inhibiting miR-590-3p upregulated NLRP1, the NOX4/ROS/TXNIP/NLRP3 pathway, and caspase-1. NLRP1 and NOX4 were confirmed as direct target genes of miR-590-3p. The overexpression of miR-590-3p or knockdown of NLRP1 and NOX4 increased cell activity and suppressed pyroptosis. Intriguingly, the upregulation of IL-1β induced the downregulation of miR-590-3p by lowering the DNA promoter activity of pri-miR-590. HG induced pyroptosis in a cell culture model of DR, and the downregulation of miR-590-3p promoted pyroptotic death by targeting NLRP1 and activating the NOX4/ROS/TXNIP/NLRP3 pathway via an IL-1β-mediated positive feedback loop.

Calcitonin gene-related peptide down-regulates the inflammatory response induced by lipopolysaccharide of Klebsiella pneumoniae

Objective To investigate the effects of calcitonin gene-related peptide (CGRP) on the inflammatory response induced by lipopolysaccharide (LPS) of Klebsiella pneumoniae. Methods Klebsiella pneumoniae was cultured in vitro to extracted LPS. Different concentrations of LPS were used to stimulate BEAS-2B cells. The activation of human β defensin 2 (hBD-2) in these cells was detected by immunofluorescence assay before and after adding different concentrations of CGRP. MTT assay and flow cytometry were respectively used to detect the proliferation and apoptosis of BEAS-2B cells after LPS stimulation with and without CGRP treatment. Neutrophil granules released in the cells after CGRP treatment were detected using neutrophil gelatinase-related apolipoprotein (NGAL) as the marker. Results Immunofluorescence assay results showed that LPS at different concentrations could significantly increase the relative expression of hBD-2 in BEAS-2B cells, which was significantly inhibit by CGRP intervention. LPS at 50 ng/ml, 100 ng/ml and 200 ng/ml had no significant effect on the activity of BEAS-2B cells, while treatment with 400 ng/ml of LPS for 24 h could significantly reduce the activity and promote the apoptosis of BEAS-2B cells. In addition, remarkedly increased cell activity and suppressed cell apoptosis were induced when BEAS-2B cells were treated with 10 nmol/L of CGRP in combination with LPS. LPS at different concentrations could induce the release of neutrophil-specific granules, while the LPS-induce release could be significantly inhibited by 10 nmol/L of CGRP. Conclusions CGRP could inhibit the expression of hBD-2, promote cell proliferation and reduce the degranulation of neutrophils to down-regulate the inflammatory response induced by LPS of Klebsiella pneumoniae. Key words: Klebsiella pneumoniae; Calcitonin gene-related peptide; βdefensin; Inflammatory response

Transcriptomic Study of Early Responses to the Bud Dormancy-breaking Agent Hydrogen Cyanamide in ‘TropicBeauty’ Peach

To determine how the dormancy-breaking agent hydrogen cyanamide (HC) advances budbreak in peach (Prunus persica), this study compared the transcriptome of buds of low-chill ‘TropicBeauty’ peach trees treated with 1% (v/v) HC and that of nontreated trees at 3 and 7 days after treatment (DAT), respectively, using an RNA sequencing analysis. The peak of total budbreak occurred 6 weeks earlier in the HC-treated trees (at 32 DAT) than the nontreated trees (at 74 DAT). There were 1312 and 1095 differentially expressed genes (DEGs) at 3 and 7 DAT, respectively. At 3 DAT, DEGs related to oxidative stress, including the response to hypoxia, lipid oxidation, and reactive oxygen species (ROS) metabolic process, were upregulated in HC-treated buds. Additionally, DEGs encoding enzymes for ROS scavenging and the pentose phosphate pathway were upregulated at 3 DAT but they were not differently expressed at 7 DAT, indicating a temporary demand for defense mechanisms against HC-triggered oxidative stress. Upregulation of DEGs for cell division and development at 7 DAT, which were downregulated at 3 DAT, suggests that cell activity was initially suppressed but was enhanced within 7 DAT. At 7 DAT, DEGs related to cell wall degradation and modification were upregulated, which was possibly responsible for the burst of buds. The results of this study strongly suggest that HC induces transient oxidative stress shortly after application, leading to the release of bud dormancy and, subsequently, causing an increase in cell activity and cell wall loosening, thereby accelerating budbreak in peach.

Abstract 2271: Development and characterization of SL-115154 (CSF1R-Fc-CD40L) for cancer immunotherapy

Abstract SL-115154 (CSF1R-Fc-CD40L) is a first-in-class biologic derived from the Agonist Redirected Checkpoint (ARC™) platform developed by Shattuck Labs. The ARC platform was developed to solve a fundamental challenge in cancer immunotherapy, which was to develop combination therapeutics that could block immune checkpoints (such as PD-1), while simultaneously activating Tumor Necrosis Factor (TNF) SuperFamily Receptors (such as OX40, 4-1BB, GITR and CD40). Pre-clinical development of SL-115154 has been completed, and demonstrated that the CD40L end of the ARC binds immobilized CD40 at 0.8 nM affinity and was found to saturate physiological levels of soluble CSF1 at molar ratios of approximate 1 CSF1 to 2-3 ARC molecules; levels which are easily achievable in the clinic. CSF1R-Fc-CD40L binds CD40 on plate bound recombinant receptors and on cells, and in NHP tox immunophenotyping studies, was found to deplete specific populations of CD20+ B cells. Sequestrations of CSF1 and depletion of B cells demonstrated on-target activity culminating in the secretion of cytokines specific to this ARC; including IL-7 & IL-15. SL-115154 was also shown to be highly potent in a series of in potency assays including: CD40 dependent NFkB reporter assays, TCR-independent proliferation/cytokine ELISpot assays, and TCR-dependent SEB super antigen cytokine release assays. In addition, the anti-tumor efficacy of the mouse surrogate CSF1R-Fc-CD40L, was superior to monotherapy or combination treatment with anti-CSF1R and anti-CD40 antibodies and lacked any evidence of toxicity observed with the antibody combination. Interestingly, anti-tumor activity and the rejection of primary and ‘re-challenged’ secondary tumors, was significantly enhanced when CSF1R-Fc-CD40L was sequenced with or following anti-CTLA4 or anti-PD1 therapy. A PD1-resistant in vivo model was established in the mouse to examine CSF1R-Fc-CD40L mono- and combo- efficacy in the molecular context to which most available patients will belong during the phase I study. These data demonstrate feasibility and functional activity of a novel chimeric fusion protein platform, providing cytokine sequestration of CSF1 by CSF1R, along with APC co-stimulation via the CD40/L axis. This strategy may uniquely inhibit immunosuppressive signaling / DC differentiation into pro inflammatory phenotypes, and instead promote an M1 phenotype and co-stimulation of CD40 positive cells, increasing cell activation, antigen presentation/processing, and enhanced anti-tumor response. Citation Format: Taylor H. Schreiber, George Fromm, Suresh De Silva, Arpita Patel, Kellsey Johannes, Kyung Jin Yoo, Kaiwen Huang. Development and characterization of SL-115154 (CSF1R-Fc-CD40L) for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2271.

In vitro evaluation of functionalized decellularized muscle scaffold for in situ skeletal muscle regeneration

Current treatment options for repairing volumetric muscle loss injury involve the use of existing host tissue like muscular flaps or grafts. However, host muscle tissue may not be available and donor site morbidity, such as functional loss and volume deficiency, is often present. In this study, we developed a biofunctionalized muscle-derived decellularized extracellular matrix scaffolding system to utilize endogenous stem/progenitor cells for in situ muscle tissue regeneration. We optimized the decellularization process to enhance cellular infiltration and fabricated an insulin-like growth factor-binding protein 3 (IGFBP-3)-conjugated scaffold for controlled delivery of IGF-I. We then tested in vitro characterization including IGF-I release kinetics and cellular infiltration. In addition, we have analyzed the bioactivities of skeletal muscle cells (C2C12) to assess the indirect effect of released IGF-1 from the scaffold. The IGFBP-3 conjugated scaffolds demonstrated showed sustained release of IGF-1 and 1% SDS decellularized scaffold with IGF-1 showed higher cellular infiltration compared to control scaffolds (no conjugation). In indirect bioactivity assay, IGF-1 conjugated scaffold showed 2.1-fold increased cell activity compared to control (fresh media). Our results indicate that IGFBP-3/IGF-I conjugated scaffold has the potential to be used for in situ muscle tissue regeneration.

THE MORPHOLOGY OF THE ACCIDENTAL INVOLUTION OF THE THYMUS AT THE EXPERIMENTAL CANCEROGENESIS

The development of tumors in the body is a powerful factor in the dysfunction of the immune system. The features of involution processes in the thymus in experimental carcinogenesis are due to impaired metabolism in the body as a whole and the production of lymphoid cells with altered functions. The purpose of the study is to summarize data from modern scientific literature and own research on the accidental transformation of the thymus and the reaction of its cellular structures against the background of experimental carcinogenesis with the development of a mammary gland tumor, stress and the combined effect of these factors. Based on the analysis, it was found that dendritic and mast cells of the thymus respond most actively to tumor growth and stress. Morphometric parameters and changes in organ's cytoarchitecture correspond to the phases of the development of accidental involution, which is most pronounced with the combined effect of two factors. A significant role in these processes belongs to biogenic amines and hormones. The introduction of carcinogens leads to a structural reorganization of the gland: the lobules become more elongated, the interlobular spaces increase, the thickness of the cortical and the area of the medulla increase. There is a decrease in the number of S-100+-cells, an increase in the number of mast cells with a predominance of degranulated and completely devastated forms. The level of histamine in the cells of the pre-medullar area decreases in 1,3 times, in the thymocytes of the cortex and medulla in 1,7 times. Along with the change in the level of biogenic amines, there is a decrease in the ratio of their ratio, which indicates an increase in the functional activity of cells. Against the background of stress, as an additional factor that depresses the immune system, there is a pronounced disorganization of the thymus lobes: a decrease in the thickness of the cortical and the area of the medulla, expansion and filling of the interlobular connective tissue septa with adipose tissue. The number of dendritic cells also decreases and the content of degranulated and completely devastated forms of mast cells increases, the level of histamine significantly decreases in all structures. Thus, data from the literature and our own research indicate that the combined effect of carcinogenesis and stress leads to more pronounced changes in the structure of the thymus in the process of accidental involution and the structural specificity of its manifestations.

Over-Expression of MicroRNA-22 Protects Cardiomyocytes from Ischemic Injury

Objective: To investigate the protection effect of over-expressed MicroRNA- 22 on ischemic cardiomyocyte. Methods: Cardiomyocytes cultured in hypoxia and ischemia condition were transfected with recombinant adenovirus vector containing microRNA-22 plus a recombinant green fluorescent protein (GFP). Transduction efficiency and expression of microRNA-22 were assessed. The expression of downstream PTEN protein expression was detected. After successful transduction, cell activities including viability, nuclei proliferation and cell apoptosis were assessed. We used CCK-8 assay to assess cell activities, EdU assay to assess nuclei proliferation and Caspase-3/LDH assay to assess cell apoptosis. Results: MicroRNA-22 was transfected successfully using adeno-virus. The expression of microRNA-22 was significantly increased. The transduction efficiency reached more than 95%. Over-expression of microRNA-22 incardiomyocytes down regulated PTEN protein level. Over- expression of microRNA-22 increased cell activities, DNA proliferation and decreased cell apoptosis. Conclusion: Over-expression of microRNA-22 pro- tected cardiomyocytes from ischemic injury.

Changes in Secretory Activity of Adrenal Zona Fasciculata Cells in Pubertal Rats Exposed to Low Doses of DDT in Different Periods of Ontogeny.

We studied secretory activity of adrenal zona fasciculata cells in pubertal rats exposed to low doses of endocrine disrupter DDT during prenatal and postnatal periods and only during postnatal period. In exposed animals, circulatory disturbances leading to degeneration and necrosis of cells as well as regeneration foci were revealed in the outer zona fasciculata. In rats exposed to DDT during the prenatal and postnatal periods, compensatory increase in secretory activity of cells due to increase in mitochondria content was noted in the inner zona fasciculata, which determined elevated serum concentration of corticosterone. In rats exposed to DDT only during the postnatal development, functional activity of zona fasciculata cells was suppressed, which attested to delayed upregulation of secretion.

Effects of Obesity and Gastric Bypass Surgery on Nutrient Sensors, Endocrine Cells, and Mucosal Innervation of the Mouse Colon.

Background: Nutrient-sensing receptors located on enteroendocrine (EEC) cells modulate appetite via detection of luminal contents. Colonic ‘tasting’ of luminal contents may influence changes to appetite observed in obesity and after weight loss induced by bariatric surgery. We assessed the effects of obesity and gastric bypass-induced weight loss on expression of nutrient-sensing G-protein coupled receptors (GPCRs), EEC and enterochromaffin (EC) cells and mucosal innervation. Methods: qPCR and immunohistochemistry were used to study colonic tissue from (a) chow-fed/lean, (b) high-fat fed/obese, (c) Roux-en-Y gastric bypass surgery (RYGB), and (d) calorie restriction-induced weight loss mice. Results: Expression of GPR41, GPR43, GPR40, GPR120, GPR84, GPR119, GPR93 and T1R3 was increased in obese mice. Obesity-induced overexpression of GPR41, 40, 84, and 119 further increased after RYGB whereas GPR120 and T1R3 decreased. RYGB increased TGR5 expression. L-cells, but not EC cells, were increased after RYGB. No differences in mucosal innervation by protein gene product (PGP) 9.5 and GLP-1R-positive nerve fibers were observed. Stimulation of colonic mucosa with GPR41, GPR40, GPR85, GPR119, and TGR5 agonists increased cell activation marker expression. Conclusions: Several nutrient-sensing receptors induced activation of colonic EEC. Profound adaptive changes to the expression of these receptors occur in response to diet and weight loss induced by RYGB or calorie restriction.

Influence of Bone Marrow Autotransplantation on the Neurotransmitter Structures of appendix vermiformis in Old Rats

The influence of bone marrow autotransplantation on neurotransmitter structures of appendix vermiformis was studied. The study revealed that an increase in the number of neurotransmitter structures (mast and granular luminescent cells), having a high content of catecholamines and serotonin was noted in old rats in 40 min after bone marrow autotransplantation. In the center of vermiformis appendix lymphoid nodes reproduction cellular programmed differentiations having a high content of neuroamines are determined. In 2 hours after bone marrow autotransplantation their number decreases, the content of catecholamines and serotonin reduces as well. In the immunohistochemical reaction increase in proliferative activity of cells both in proper t. mucosa plate crypts and appendix vermiformis lymphoid nodules is observed up to 40 min of experiment. In 2 hours Ki-67 positive cells reduce both in t. mucosa and t. s/mucosa of appendix vermiformis.

Abstract 4742: Role of tumor-associated macrophages in esophageal cancer: PD-L1 expression and prognosis

Abstract Introduction: It has become clear that macrophages are important cells forming cancer microenvironment and tumor-associated macrophages (TAM) promote their proliferation in various cancer types. In particularly, M2-type macrophages have been reported to promote the production of anti-inflammatory factors and the infiltration of regulatory T cells and suppress anti-tumor immunity, and new antitumor therapies that control tumor growth by controlling TAM have been proposed. In addition, "anti-PD-1 antibody" has attracted attention also in esophageal cancer, but the relationship between TAM and PD-L1 expression has not been clarified. Methods: A retrospective study was performed on 301 patients who underwent esophagectomy for esophageal cancer between March 2005 and June 2013. We performed immunohistochemistry and evaluation of macrophage markers (CD163 and CD204) and PD-L1. In addition, we performed in vitro experiments to clarify the cellular malignancy and relationship with PD-L1 expression. Results: Out of 301 cases of esophageal cancer, 156 cases showed CD163 high expression (51.8%) while 146 cases showed CD204 high expression (48.5%). We divided into three groups of TAM status based on CD204 and CD163 expression (CD163 and CD204 both positive: TAM status high (n=100), either one positive: moderate (n=102), both negative: low (n=99)). In TAM high group, there were more advanced cancers and adenocarcinoma cases (P<0.0001, P=0.042, respectively). Interestingly, TAM status correlated with PD-L1 expression (P=0.0005). We found that there was a significant difference in overall survival, cancer specific survival and disease-free survival among these three groups (log rank P = 0.0062, P= 0.0012, P= 0.0006, respectively), and TAM status correlated with poor prognosis. We performed double immunostaining of PD-L1 and CD163, there is a part where PD-L1 is expressed around TAM, suggesting that the expression of PD-L1 and the presence of TAM are correlated. We performed co-culture of esophageal cancer cell lines (TE-8 and KYSE30) and activated macrophage. In real time PCR PD-L1 expression were elevated at co-culture with activated macrophage. Fluorescence activated cell sorting (FACS) revealed PD-L1 expression positive cells were increased at co-culture with activated macrophage. In invasion assay, when co-cultured with activated macrophages, the number of infiltrating cells were significantly increasing (P=0.00021). In timelapse imaging showed increased cell activity by co-culture with activated macrophage. Conclusion: This study suggested that TAM correlated with poor prognosis in patients with esophageal cancer. In addition, TAM correlated with invasive capacity and migratory capacity. Importantly, TAM correlated PD-L1 expression in esophageal cancer cell lines. Citation Format: Taisuke Yagi, Yoshifumi Baba, Yuki Koga, Tomoyuki Uchihara, Yuki Kiyozumi, Hiroshi Sawayama, Yukiharu Hiyoshi, Masaaki Iwatsuki, Yuji Miyamoto, Naoya Yoshida, Hideo Baba. Role of tumor-associated macrophages in esophageal cancer: PD-L1 expression and prognosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4742.

B cell activation and plasma cell differentiation are inhibited by de novo DNA methylation

B cells provide humoral immunity by differentiating into antibody-secreting plasma cells, a process that requires cellular division and is linked to DNA hypomethylation. Conversely, little is known about how de novo deposition of DNA methylation affects B cell fate and function. Here we show that genetic deletion of the de novo DNA methyltransferases Dnmt3a and Dnmt3b (Dnmt3-deficient) in mouse B cells results in normal B cell development and maturation, but increased cell activation and expansion of the germinal center B cell and plasma cell populations upon immunization. Gene expression is mostly unaltered in naive and germinal center B cells, but dysregulated in Dnmt3-deficient plasma cells. Differences in gene expression are proximal to Dnmt3-dependent DNA methylation and chromatin changes, both of which coincide with E2A and PU.1-IRF composite-binding motifs. Thus, de novo DNA methylation limits B cell activation, represses the plasma cell chromatin state, and regulates plasma cell differentiation.

On the learning control effects in the cancer-immune system competition

The interactions between a tumor and the immune system are modeled at cell scale in the framework of thermostatted kinetic theory. Cell activation and learning are reproduced by the increase of cell activity during interactions. The second moment of the activity of the whole system is controlled by a thermostat which reproduces the regulation of the learning process and memory loss through cell death. An algorithm inspired from the direct simulation Monte Carlo (DSMC) method is used to simulate stochastic trajectories for the numbers of cells and to study the sensitivity of the dynamics to various parameters. The nonintuitive role played by the thermostat is pointed out. For inefficient thermalization, the divergence of the number of cancer cells is obtained in spite of favored production of immune system cells. Conversely, when the activity fluctuations are controlled, the development of cancer is contained even for weakened immune defenses. These results may be correlated to unexpected clinical observations in the case of different cancers, such as carcinoma, lymphoma, and melanoma.

A shift from papillary to reticular fibroblasts enables tumour\u2013stroma interaction and invasion

BackgroundTumour stroma consists of a heterogeneous population of fibroblasts and related mesenchymal cells, collectively dubbed cancer-associated fibroblasts (CAFs). These CAFs are key players in cancer invasion of cutaneous squamous cell carcinoma (SCC). As we have shown earlier, papillary and reticular fibroblasts (Pfs and Rfs, respectively) have distinct effects on epidermal and dermal homeostasis, but their role in cancer invasion and epithelial-to-mesenchymal transition (EMT) remains to be determined.MethodsWe used 3D cultures of human skin equivalents (HSEs) to analyse the effects of Pfs and Rfs on the invasive behaviour of SCC and EMT.ResultsWe reveal for the first time the importance of Pfs versus Rfs in SCC invasion and EMT. Cell lines from different stages of SCC showed significantly more extensive invasion into a dermis composed of Rfs than of Pfs. In addition, Rfs-based HSEs showed increased cell activation and stained positive for CAF biomarkers α-SMA and vimentin. Further analysis revealed that invasively growing cancer cells in Rf-HSEs express markers of EMT transition, like SNAIL2, N-cadherin and ZEB1.ConclusionsConversely, our results show that Pfs contain cancer cells more within the epidermis. Rfs are clearly predisposed to differentiate into CAFs upon SCC signals, assisting invasion and EMT.

Targeting CD19-negative relapsed B-acute lymphoblastic leukemia using trivalent CAR T cells.

121 Background: Chimeric antigen receptor (CAR) T cells targeting CD19 have shown remarkable efficacy in treating relapsed B cell acute lymphoblastic leukemia (B-ALL). However, recent reports show that up to 40% of patients who relapse after CD19 CAR T cell therapy have CD19-negative disease, justifying a need to expand CAR T cell therapy for B-ALL to include additional tumor-associated antigens. We hypothesize that targeting CD19, CD20, and CD22 will improve B-ALL therapy outcomes and control disease progression during CD19-negative relapse. Methods: We designed two trivalent CAR T cell products with exodomains derived from single chain variable fragments (ScFv) targeting CD19, CD20, and CD22. Each CAR contains the 4-1BB and T-cell receptor zeta chains. Donor T cells were engineered to express the CARs using a retroviral system. We used primary CD19-negative relapsed bone marrow samples and CRISPR CD19 knockouts of primary ALL to model CD19 escape and standard cytotoxicity and immune assays to evaluate anti-tumor efficacy. Results: Due to the use of viral 2A sequences we detected near equal expression of each CAR by flow cytometry. The first T cell product expresses three CARs individually (TriCAR), and the second expresses a single CAR targeting CD19 and a second bi-specific CAR targeting CD20 and CD22 via a tandem arrangement (SideCAR). Using primary B-ALL cells, we observed that TriCAR and SideCAR T cells killed ALL cells more robustly than CD19 CAR T cells at low E:T ratios. Further, in ImageStream analysis of single cell interactions between CAR T cells and primary B-ALL cells, TriCAR T cells exhibited increased actin polymerization compared to CD19 CAR T cells, suggesting remodeling and increased cell activation. Finally, in multiple models of CD19 escape in primary ALL, we showed that trivalent CAR T cells mitigated CD19 negative relapse, producing IFN-γ/TNF-α and killing CD19-negative primary ALL, while CD19 CAR T cells remained ineffective. Conclusions: Trivalent CAR T cells effectively target primary ALL cells with varying antigen profiles and mitigate CD19-negative relapse. This strategy has the potential for use as an initial CAR therapy in relapsed ALL or a salvage therapy for patients with CD19-negative disease.

Biocompatibility Evaluation of Feldspathic Porcelain with Nano-Sized Silver Ion Particles.

The major failure of dental restorations is caused by dental caries by S. mutans. This study evaluated the effect of nanosized silver ions in feldspathic porcelain on cytotoxicity and antimicrobial activity. The control group was feldspathic porcelain (Noritake EX-3, Kuraray Noritake Dental Inc., Japan) that did not contain Ag (silver). The experimental groups were feldspathic porcelain mixed with colloidal solutions of nanosized silver ions in mixtures of 5% 10%, 20%, 30%. Cell activity assays evaluated with osteoblast hFOB 1.19 cells (human Fetal Osteoblastic cell line, ATCC® CRL- 11372™) and antimicrobial activity test used the streptococcus mutans (S. mutans). According to the observations, the addition of Ag to feldspathic porcelain led to increased cell activity and showed excellent antibacterial performance. Especially, the feldspathic porcelain with 30% nanosized Ag ion appeared to have a significant antimicrobial effect, this allows for the possibility of various clinical applications for such material, including use in dental prosthesis and implants.

LncRNA LINC00311 Promotes the Proliferation and Differentiation of Osteoclasts in Osteoporotic Rats Through the Notch Signaling Pathway by Targeting DLL3

Osteoporosis is a commonly occurring condition marked by a loss of bone density. Previous evidence has highlighted the roles played by microRNAs as potential treatment tools for the disease. At present, the influence of long non-coding RNAs (lncRNAs) on the progression of osteoporosis remains largely unclear. Thus, an investigation was conducted into the target relationship between LINC00311, which has been reported to be highly expressed in osteoporosis, and delta-like 3 (DLL3), which is involved in the Notch signaling pathway, in connection with a series of bioinformatic methods. An osteoporotic rat model was established by means of ovariectomy (OVX) to evaluate the influence exerted by DLL3-binding LINC00311 on osteoclasts through the Notch signaling pathway. Osteoclasts were extracted from osteoporotic rats and transfected with the LINC00311-vector, shRNA-LINC00311, Notch activator, or a combination of the Notch activator and LINC00311-vector. Western blotting and RT-qPCR techniques were applied to determine the expression levels of LINC00311, DLL3, Notch1, Notch2, Jagged1, Hes-1 and TRAP in tissues and cells, while cell activity was detected by MTT assay. The cell cycle as well as the rate of apoptosis was detected by flow cytometry. The successfully established osteoporotic rats were designated into the OVX-siRNA, OVX-LINC00311 and OVX-control groups to observe the effects of LINC00311 on the proliferation and differentiation of osteoclasts. Cells transfected with the LINC00311-vector exhibited increased expression levels of Notch2 and TRPA as well as increased cell activity, while decreased expression levels of DLL3, Notch1, Jagged1 and Hes-1, along with a decreased cell apoptosis rate, were observed. The opposite tendencies of these parameters were observed in the cells treated with shRNA-LINC00311. A key observation was made when the Notch signaling pathway was activated, in that the cell activity was decreased while the rate of apoptosis increased. In comparison with the OVX-control group, the expression levels of LINC00311, Notch2 and TRAP as well as the positive expression rate of TRAP all exhibited reductions, while those of DLL3, Jagged1 and Notch1 were elevated in the OVX-siRNA group. Compared with those in the sham group, in the OVX-control and OVX-LINC00311 groups, LINC00311 and the expression levels of Notch2 and TRAP were increased; however, decreased levels of DLL3, Jagged1 and Notch1 were noted. Taken together, the key findings of the present study suggest that LINC00311 induces proliferation and inhibits apoptosis of osteoclasts via the regulation of the Notch signaling pathway by inhibiting DLL3 expression, ultimately demonstrating that LINC00311 and its target gene DLL3 may serve as independent factors in cases of osteoporosis.

Tctex1 plays a key role in the α-synuclein autophagy lysosomal degradation pathway

Tctex1 is an important structure of dynein light chain in mammalian cells, clarifying the role of Tctex1 in α-synuclein autophagy lysosomal degradation may offer insights into the formation of Lewy bodies and neuronal death. We constructed dsRED-N1-Tctex1 overexpression, pDsRED2-N1-Tctex1(T94E) mutation and transfected into SH-SY5Y cells. Relative protein expression was measured by Western Blot and mRNA levels were measured by real-time quantitative PCR. Confocal microscopy was used to observe their sublocalizations in cells. We found that: WST assay results show that cell activity decreased after Tctex1 mutation (T94E), while Tctex1 overexpression increased cell activity. In Tctex1 mutation transfected cell lines Tctex1 and dynein protein levels decreased; α-synuclein, LC3-II and LAMP2 protein increased. However, α-synuclein, LC3-II and LAMP2 proteins were reduced in Tctex1 overexpression cell lines, with the same trend was found in mRNA levels. In Tctex1 mutation transfected cell lines Tctex1 fluorescence intensity weakened; α-synuclein, LC3-II and LAMP2 fluorescence was enhanced, while α-synuclein, LC3-II and LAMP2 weakened in Tctex1 overexpressing cells. Our results suggest that Tctex1 mutants interference lead to Tctex1 downregulation and dysfunction. Tctex1 overexpression promoted autophagy lysosome fusion and effectively degraded α-synuclein with increased cell activity. Thus, Tctex1 plays an important role in α-synuclein autophagic degradation.

Glycerol is synthesized and secreted by adipocytes to dispose of excess glucose, via glycerogenesis and increased acyl-glycerol turnover

White adipose tissue (WAT) produces large amounts of lactate and glycerol from glucose. We used mature epididymal adipocytes to analyse the relative importance of glycolytic versus lipogenic glycerol in adipocytes devoid of external stimuli. Cells were incubated (24/48 h) with 7/14 mM glucose; half of the wells contained 14C-glucose. We analysed glucose label fate, medium metabolites, and the expression of key genes coding for proteins controlling glycerol metabolism. The effects of initial glucose levels were small, but time of incubation increased cell activity and modified its metabolic focus. The massive efflux of lactate was uniform with time and unrelated to glucose concentration; however, glycerol-3P synthesis was higher in the second day of incubation, being largely incorporated into the glycerides-glycerol fraction. Glycerophosphatase expression was not affected by incubation. The stimulation of glycerogenic enzymes’ expression was mirrored in lipases. The result was a shift from medium glycolytic to lipolytic glycerol released as a consequence of increased triacylglycerol turnover, in which most fatty acids were recycled. Production of glycerol seems to be an important primary function of adipocytes, maintained both by glycerogenesis and acyl-glycerol turnover. Production of 3C fragments may also contribute to convert excess glucose into smaller, more readily usable, 3C metabolites.