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This phase II clinical trial investigated the efficacy and safety of intramuscular injection of plasmid pUDK-HGF, which encodes the human hepatocyte growth factor gene in patients with critical limb ischemia. Resting pain patients (

Background To investigate the therapeutic effect of human dental pulp stem cells (DPSCs) transfected with adenovirus expressing hepatocyte growth factor (HGF) in a mouse model of collagen-induced arthritis (CIA). Methods DPSCs were modified with Ad-HGF to produce HGF-overexpressing DPSCs, DPSCs-HGF. In experimental mouse CIA model, DPSCs-HGF and DPSCs-Null (modified with Ad-Null) were engrafted via intravenously after disease onset, which was determined by the presence of joint swelling. The therapeutic effects on joints were evaluated at 49 days after collagen injection by histopathological analysis and microcomputed tomography imaging. The inflammatory cytokines were analyzed both in sera and joints via MILLIPLEX kit and immunohistochemical staining, respectively, and the regulatory T cells (Tregs) were analyzed in peripheral blood by using flow cytometry. Furthermore, primary fibroblast-like synoviocytes were isolated, colony formation analysis and FACS were performed to evaluate the effect of HGF on the proliferation and cell cycle of FLSs. Western blot assay was carried out to clarify the signal pathway of HGF-cMet. Results We found that without HGF modification, DPSC transfusion was helpful in controlling autoimmune status, local synovitis, and bone erosion after intravenous administration. However, HGF-modified DPSCs have dual role in rheumatoid arthritis (RA). In the early phase, HGF overexpression inhibited RA progression by its immunosuppressive effects, while in the late phase, HGF promoted synovitis by activating fibroblast-like synoviocytes to produce pathogenic IL-6, accelerating cell proliferation and inducing apoptosis resistance via phosphorylating the c-Met/Akt pathway. The overall effect of HGF modification attenuated the therapeutic effect of DPSCs. Conclusions Our study provides a comprehensive evaluation of the therapeutic effect of DPSCs in the mouse model and a primary answer to the divergence of whether HGF is harmful or helpful in RA.

Identifying targets for chimeric antigen receptor-modulated T lymphocyte (CAR-T) therapy against solid tumors is an urgent problem to solve. In this study, we showed for the first time that the receptor tyrosine kinase, AXL, is overexpressed in various tumor cell lines and patient tumor tissues including triple negative breast cancer (TNBC) cell lines and patient samples, making AXL a potent novel target for cancer therapy, specifically for TNBC treatment. We also engineered T cells with a CAR consisting of a novel single-chain variable fragment against AXL and revealed its antigen-specific cytotoxicity and ability to release cytokines in a TNBC cell line and other AXL-positive tumors in vitro. Furthermore, AXL-CAR-T cells displayed a significant anti-tumor effect and in vivo persistence in a TNBC xenograft model. Taken together, our findings indicate that AXL-CAR-T cells can represent a promising therapeutic strategy against TNBC.

Conditions in space, such as microgravity, may affect the hematopoietic and bone marrow-derived mesenchymal stromal cells (BM-MSCs) of astronauts. However, to date, few detailed phenotype change data about the different type of hematopoietic cells have reported. In this study, C57BL/6 mice were randomly divided into 2 groups: a control group (control) and a hindlimb suspension group (treated). After four weeks of hindlimb suspension, we found that this simulated microgravity (sµg) condition could increase the percentage of monocytes and macrophages and decrease the percentage of B lymphocytes and mature red cells in bone marrow. The percentage of B lymphocytes in the spleen and the RBC (red blood cell) count in peripheral blood also decreased, consistent with the response of bone marrow. The cytoskeleton in the BM-MSCs was disrupted. The expression levels of hematopoietic-related genes, such as fms-like tyrosine kinase-3 ligand (Flt-3 L), granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3), leptin associated with adipocyte differentiation and proliferator-activated receptor γ type 2 (PPARγ2), were upregulated under sµg conditions. These results indicated that simulating microgravity can affect the phenotype of certain types of hematopoietic cells and the morphology and gene expression pattern of BM-MSCs. This article is protected by copyright. All rights reserved.

Background: BPD is a common pulmonary complication seen in preterm infants which has a multifactorial pathogenesis. Current strategies, albeit successful in moderately reducing morbidity and mortality of BPD, failed to draw overall satisfactory conclusion. Meanwhile, a substantial studies have reported the altered expression profile of microRNA and lncRNA in the pathogenesis of BPD, while detailed circRNA profile as well as comprehensive lncRNA-miRNA-mRNA and circRNA-miRNA-mRNA networks are largely unknown. Methods: Male C57BL6/J newborn pups were exposed to 85% O2 to construct the hyperoxia BPD model and CB-MNCs or UC-MSCs Cells were intravenously injected at PN7 thereafter. Growth rate was recorded, lung morphology were determined by HE staining and transmission electron microscopy, pulmonary function alterations were defined by respiratory function test and cardiovascular-pulmonary function assessment, reshaped expression of cytokines were evaluated by qPCR and immunohistochemistry approach. The underlying mechanism based on theory of ceRNA network were elucidated by whole transcriptome sequencing. Findings: Both CB-MNC and UC-MSC are efficient in alleviating BPD, verified by our mice model mimicking moderate and severe BPD. Notably, CB-MNC implantation have more prominent effects preventing alveolar simplification and pulmonary vessel loss, restoring pulmonary respiratory functions and reshaping inflammatory responses. Moreover, we further systematically investigated the expression profiles of mRNA, miRNA, lncRNA and circRNA. Importantly, those unique alterations induced by transplantation of different stem cells were highly enriched in the processes related to regulation of RhoGTPase activity, chromosome regulation and cell cycle control. Finally, we illustrate the comprehensive mRNA-miRNA-lncRNA and mRNA-miRNA-circRNA networks. Funding: This research was jointly granted by National Key R&D Program of China (2017YFC1001701, 2018YFC1002701) and Chinese National Natural Science Foundation Projects (81901518). Conflict of Interest: All authors declared that they had no competing conflicts of interests to disclose. Ethical Approval: Animal procedures were reviewed and approved by the Animal Care and Ethic Committee of seventh medical center of PLAGeneral Hospital (No. 2020-037). All animals were housed, cared for, and used in compliance with the guidelines regarding the humane use and care of laboratory animals for biomedical research published by the National Institutes of Health (No.85-23, revised 1996).

To investigate alterations of mitochondria in irradiated endothelial cells to further elucidate the mechanism underlying radiation-induced heart disease.Experiments were performed using human umbilical vein endothelial cells (HUVECs). HUVECs were irradiated with single gamma ray dose of 0, 5, 10 and 20 Gy, respectively. Apoptosis was assessed by flow cytometry at 24, 48 and 72 h post-irradiation, respectively. The intracellular reactive oxygen species (ROS) was measured with 2',7'-dichlorofluorescein-diacetate (DCFH-DA) at 24 h post-irradiation. Mitochondrial membrane potential (ΔΨm) by JC-1 and the opening of mitochondrial permeability transition pore (mPTP) by a calcein-cobalt quenching method were detected at 24 h post-irradiation in order to measure changes of mitochondria induced by gamma ray irradiation.Gamma ray irradiation increased HUVECs apoptosis in a dose-dependent and time-dependent manner. Irradiation also promoted ROS production in HUVECs in a dose-dependent manner. At 24 h post-irradiation, the results showed that irradiation decreases ΔΨm, however, paradoxically, flow cytometry showed green fluorescence instensity higher in irradiated HUVECs than in control HUVECs in an irradiation dose-dependent manner which indicated gamma ray irradiation inhibited mPTP opening in HUVECs.Gamma ray irradiation induces apoptosis and ROS production of endothelial cells, and decreases ΔΨm meanwhile contradictorily inhibiting the opening of mPTP.

// Xiao-Xia Jiang 1, * , Yu Liu 1, 2, * , Hong Li 1, * , Yaping Gao 1 , Rong Mu 2 , Jianping Guo 2 , Jing Zhang 2 , Yan-Mei Yang 1 , Fengjun Xiao 3 , Bing Liu 4 , Changyong Wang 1 , Beifen Shen 1, 6 , Si-Yi Chen 5 , Zhanguo Li 2 , Guang Yang 1, 6 1 Beijing Institute of Basic Medical Sciences, Beijing, China 2 Department of Rheumatology and Immunology, People’s Hospital, Peking University, Beijing, China 3 Institute of Radiation Medicine, Beijing, China 4 307-Ivy Translational Medicine Center, Laboratory of Oncology, Affiliated Hospital of Academy of Military Medical Sciences, Beijing, China 5 Department of Molecular Microbiology and Immunology, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA 6 State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Institute of Pharmacology and Toxicology, Beijing, China * These authors contributed equally to this work Correspondence to: Guang Yang, email: yangg62033@163.com Zhanguo Li, email: zgli99@aliyun.com Si-Yi Chen, email: siyichen@usc.edu Keywords: MYSM1, miR-150, FLT3, B1a, proliferation Received: June 06, 2016 Accepted: August 25, 2016 Published: August 31, 2016 ABSTRACT The aberrant expansion of B1a cells has been observed in several murine autoimmune disease models; however, the mechanism of such proliferation of B1a cells is still limited. Here, we identify that Myb Like, SWIRM And MPN Domains 1 (MYSM1), a histone H2A deubiquitinase, plays an intrinsic role in the proliferation of B1a cells where MYSM1 deficiency results in the increased proliferation of B1a cells in mice. We demonstrate that MYSM1 recruits c-Myc to the promoter of miR-150 and stimulates the transcription of miR-150. Our further investigation shows that miR-150 decreases FMS-like tyrosine kinase 3 (FLT3) in B1a cells. In agreement with our animal studies, the percentage of FLT3 + B1 cells in Systemic Lupus Erythematosus (SLE) patients is significantly higher than healthy control. Thus, this study uncovers a novel pathway MYSM1/miR-150/FLT3 that inhibits proliferation of B1a, which may be involved in the pathogenesis of SLE.

Mesenchymal stem cells (MSCs) possess an immunoregulatory capacity and are a therapeutic target for many inflammation‐related diseases. However, the detailed mechanisms of MSC‐mediated immunosuppression remain unclear. In this study, we provide new information to partly explain the molecular mechanisms of immunoregulation by MSCs. Specifically, we found that A20 expression was induced in MSCs by inflammatory cytokines. Knockdown of A20 in MSCs resulted in increased proliferation and reduced adipogenesis, and partly reversed the suppressive effect of MSCs on T cell proliferation in vitro and inhibited tumour growth in vivo. Mechanistic studies indicated that knockdown of A20 in MSCs inhibited activation of the p38 mitogen‐activated protein kinase (MAPK) pathway, which potently promoted the production of tumour necrosis factor (TNF)‐α and inhibited the production of interleukin (IL)‐10. Collectively, these data reveal a crucial role of A20 in regulating the immunomodulatory activities of MSCs by controlling the expression of TNF‐α and IL‐10 in an inflammatory environment. These findings provide novel insights into the pathogenesis of various inflammatory‐associated diseases, and are a new reference for the future development of treatments for such afflictions.

Previous studies of Jak–STAT inhibitors have shown promise in treating kidney diseases. The activation of Jak–STAT components is important in cell fate determination in many cell types, including bone marrow–derived cells, which are important contributors in renal interstitial fibrosis. In this study, we tested the effect of a new STAT3 inhibitor, BP-1-102, on monocyte-to-fibrocyte transition and the progression of renal interstitial fibrosis. We tested the effect of BP-1-102 in a mouse model of unilateral ureteral obstruction in vivo and IL-33-treated bone marrow–derived monocytes in vitro. BP-1-102 treatment alleviated renal interstitial fibrosis, reduced collagen deposition and extracellular matrix protein production, inhibited inflammatory cell infiltration, suppressed the percentage of CD45+ PDGFRβ+, CD45+ CD34− Col I+ and CD45+ CD11b+ Col I+ cells within the obstructed kidney and reduced the mRNA levels of the proinflammatory and profibrotic cytokines IL-1β, TGF-β, TNF-α, ICAM-1, and CXCL16. In vitro, BP-1-102 inhibited the IL-33–induced phenotypic transition into fibroblast precursors in bone marrow–derived monocytes, marked by reduced CD45+ CD34− Col I+ and CD45+ CD11b+ Col I+ cell percentage. Our results indicate a potential mechanism by which the STAT3 inhibitor BP-1-102 inhibits bone marrow–derived monocyte transition into fibroblast precursors in an IL-33/STAT3–dependent manner and thereby alleviates renal interstitial fibrosis.

The deregulation of HGF/c-Met signaling is implicated in epithelial-mesenchymal transition (EMT) and progress of hepatocellular carcinoma (HCC). However, the epigenetic mechanisms that HGF/c-Met regulates EMT and metastasis of HCC cells are less explored. In this study, we demonstrated that HCC cells express a high level of SUMO/sentrin-specific protease 1 (Senp1) which is induced by HGF/c-Met signals. Lentivirus-mediated small hairpin RNA (shRNA) transduction results in Senp1 silence in HCC cells. Senp1 silence reduces the HGF-induced proliferation and migration of HCC cells. Senp1 inhibition also induces HCC cell apoptosis and growth arrest. Furthermore, Senp1 knockdown inhibits epithelial-to-mesenchymal transition, with increase of E-cadherin and ZO-1 expression, decrease of fibronectin and N-cadherin expression. The EMT-related transcription factor Zeb1 was SUMO-modified and decreased in Senp1-silenced HCC cells. These results delineate that senp1 might play an important role in the regulation of HGF-induced invasion and migration of HCC cells.

Purpose Radiation-induced pulmonary fibrosis results from thoracic radiation therapy and severely limits radiation therapy approaches. CD4 + CD25 + FoxP3 + regulatory T cells (Tregs) as well as epithelium-to-mesenchyme transition (EMT) cells are involved in pulmonary fibrosis induced by multiple factors. However, the mechanisms of Tregs and EMT cells in irradiation-induced pulmonary fibrosis remain unclear. In the present study, we investigated the influence of Tregs on EMT in radiation-induced pulmonary fibrosis. Methods and Materials Mice thoraxes were irradiated (20 Gy), and Tregs were depleted by intraperitoneal injection of a monoclonal anti-CD25 antibody 2 hours after irradiation and every 7 days thereafter. Mice were treated on days 3, 7, and 14 and 1, 3, and 6 months post irradiation. The effectiveness of Treg depletion was assayed via flow cytometry. EMT and β-catenin in lung tissues were detected by immunohistochemistry. Tregs isolated from murine spleens were cultured with mouse lung epithelial (MLE) 12 cells, and short interfering RNA (siRNA) knockdown of β-catenin in MLE 12 cells was used to explore the effects of Tregs on EMT and β-catenin via flow cytometry and Western blotting. Results Anti-CD25 antibody treatment depleted Tregs efficiently, attenuated the process of radiation-induced pulmonary fibrosis, hindered EMT, and reduced β-catenin accumulation in lung epithelial cells in vivo. The coculture of Tregs with irradiated MLE 12 cells showed that Tregs could promote EMT in MLE 12 cells and that the effect of Tregs on EMT was partially abrogated by β-catenin knockdown in vitro. Conclusions Tregs can promote EMT in accelerating radiation-induced pulmonary fibrosis. This process is partially mediated through β-catenin. Our study suggests a new mechanism for EMT, promoted by Tregs, that accelerates radiation-induced pulmonary fibrosis.

Irradiation-induced pulmonary fibrosis results from thoracic radiotherapy and severely limits radiotherapy approaches. CD4(+) CD25(+) FoxP3(+) regulatory T cells (Tregs) are involved in experimentally induced murine lung fibrosis. However, the precise contribution of Tregs to irradiation-induced pulmonary fibrosis still remains unclear. We have previously established the mouse model of irradiation-induced pulmonary fibrosis and observed an increased frequency of Tregs during the process. This study aimed to investigate the effects of Treg depletion on irradiation-induced pulmonary fibrosis and on fibrocyte, Th17 cell response and production of multiple cytokines in mice. Treg-depleted mice were generated by intraperitoneal injection with anti-CD25 mAb 2h after 20 Gy (60)CO γ-ray thoracic irradiation and every 7 days thereafter. Pulmonary fibrosis was semi-quantitatively assessed using Masson's trichrome staining. The proportions of Tregs, fibrocyte and Th17 cells were detected by flow cytometry. Th1/Th2 cytokines were assessed by Luminex assays. We found that Treg depletion decelerated the process of irradiation-induced pulmonary fibrosis and hindered fibrocyte recruitment to the lung. In response to Treg depletion, the number of CD4(+) T lymphocytes and Th17 cells increased. Moreover, Th1/Th2 cytokine balance was disturbed into Th1 dominance upon Treg depletion. Our study demonstrates that Tregs are involved in irradiation-induced pulmonary fibrosis by promoting fibrocyte accumulation, attenuating Th17 response and regulating Th1/Th2 cytokine balance in the lung tissues, which suggests that Tregs may be therapeutically manipulated to decelerate the progression of irradiation-induced pulmonary fibrosis.

Four and a half LIM protein 1 (FHL1) has been characterized as a tumor suppressor in various types of tumor. However, the biological function and underlying mechanism of FHL1 in tongue squamous cell carcinoma (TSCC) remain to be elucidated. The present study demonstrated that FHL1 inhibits anchorage‑dependent and ‑independent growth of TSCC cells in vitro and tumor growth in nude mice, as determined by cell proliferation and soft agar assays. Knockdown of FHL1 with FHL1 small interfering RNA (siRNA) promoted tumor growth in nude mice. Mechanistically, flow cytometric analysis showed that knockdown of FHL1 promoted G1/S cell cycle progression. Furthermore, expression of cell cycle‑associated regulators, cyclin D and cyclin E, were detected by western blotting and reverse transcription‑quantitative polymerase chain reaction. Cyclin D and cyclin E were markedly elevated at both the protein and mRNA level in the FHL1 siRNA‑transfected cells. These results suggested that FHL1 has a tumor suppressive role in TSCC and that FHL1 may be a useful target for TSCC gene therapy.

Radiation-induced pulmonary fibrosis is a frequently occurred complication from radiotherapy of thoracic tumors. The transforming growth factor-β (TGF-β) superfamily plays a key regulatory role in pulmonary fibrosis. As TGF-β3 showed the potential anti-fibrotic properties especially in scar-less wound healing as opposed to the fibrotic function of TGF-β1, we sought to explore the role of TGF-β3 in radiation-induced pulmonary fibrosis. A single thoracic irradiation of 20 Gy was applied in mice to establish the model of radiation-induced pulmonary fibrosis and the mice were treated by intraperitoneal injections of recombinant TGF-β3 weekly after irradiation. We found that TGF-β3 decelerated the progress of radiation-induced pulmonary fibrosis and hindered the recruitment of fibrocytes to lung. In addition, Th1 response was suppressed as shown by diminished IFN-γ in bronchoalveolar lavage fluid (BALF) after irradiation, and enhancement of Th2 response was marked by increased IL-4 in BALF. TGF-β3 administration significantly attenuated these effects and increased the percentage of Tregs in lung during the progression of pulmonary fibrosis. Taken together, these data suggest that TGF-β3 might be involved in the regulatory mechanism for attenuation of radiation-induced pulmonary fibrosis.

pUDK-HGF, recombinant plasmid DNA encoding human HGF (hepatocyte growth factor), is a potential agent for gene therapy of ischaemic disease. Production of pUDK-HGF is essential for its clinical application. In the present paper, a large-scale manufacturing process was developed, including fermentation, cell harvest, alkaline lysis, capturing plasmid DNA with Q-Sepharose XL chromatography, size-exclusion chromatography on a Sephacryl S1000 column and refining with Source 15Q anion-exchange chromatography. The quality criteria of pUDK-HGF such as purity, concentration, homogeneity, residual RNA, chromosomal DNA, contaminated protein, endotoxin and HGF expression efficacy all were analysed and met the requirements for pharmaceutical-grade plasmid DNA.