A Novel Allogeneic Rituximab-Conjugated Gamma Delta T Cell Therapy for the Treatment of Relapsed/Refractory B-Cell Lymphoma.

Chimeric antigen receptor T cell (CAR-T) therapy has been applied in the treatment of B-cell lymphoma; however, CAR-T manufacturing requires virus- or non-virus-based genetic modification, which causes high manufacturing costs and potential safety concerns. Antibody-cell conjugation (ACC) technology, which originated from bio-orthogonal click chemistry, provides an efficient approach for arming immune cells with cancer-targeting antibodies without genetic modification. Here, we applied ACC technology in Vγ9Vδ2 T (γδ2 T) cells to generate a novel off-the-shelf CD20-targeting cell therapy ACE1831 (rituximab-conjugated γδ2 T cells) against relapsed/refractory B-cell lymphoma. ACE1831 exhibited superior cytotoxicity against B-cell lymphoma cells and rituximab-resistant cells compared to γδ2 T cells without rituximab conjugation. The in vivo xenograft study demonstrated that ACE1831 treatment strongly suppressed the aggressive proliferation of B-cell lymphoma and prolonged the survival of tumor-bearing mice with no observed toxicity. Mass spectrometry analysis indicated that cell activation receptors including the TCR complex, integrins and cytokine receptors were conjugated with rituximab. Intriguingly, the antigen recognition of the ACC-linked antibody/receptor complex stimulated NFAT activation and contributed to ACE1831-mediated cytotoxicity against CD20-expressing cancer cells. This study elucidates the role of the ACC-linked antibody/receptor complex in cytotoxicity and supports the potential of ACE1831 as an off-the-shelf γδ2 cell therapy against relapsed/refractory B-cell lymphoma.

[Application of the theory of equal emphasis on muscle and bone in percutaneous vertebroplasty of lumbar osteoporotic compression fracture].

OBJECTIVE: To explore the clinical efficacy of percutaneous vertebroplasty(PVP) combined with nerve block in the treatment of lumbar osteoporotic vertebral compression fractures under the guidance of traditional chinese medicine "theory of equal emphasis on muscle and bone". METHODS: Total of 115 patients with lumbar osteoporotic vertebral compression fractures were treated by percutaneous vertebroplasty from January 2015 to March 2022, including 51 males and 64 females, aged 25 to 86 (60.5±15.9) years. Among them, 48 cases were treated with PVP operation combined with erector spinae block and joint block of the injured vertebral articular eminence (intervention group), and 67 cases were treated with conventional PVP operation (control group). The visual analogue scale(VAS) and Oswestry disability index(ODI) before operation, 3 days, 1 month and 6 months after operation between two groups were evaluated. The operation time, number of punctures and intraoperative bleeding between two groups were compared. RESULTS: The VAS and ODI scores of both groups improved significantly after operation compared with those before operation(P<0.05). Moreover, the VAS and ODI scores of 3 days and 1 month after operation of the intervention group improved more significantly than that of the control group(P<0.05). The difference of VAS and ODI scores before operation and 6 months after operation between two groups had no statistical significances(P>0.05). There was no statistically significant difference in the number of punctures and intraoperative bleeding between the two groups (P>0.05). CONCLUSION: Based on the theory of "equal emphasis on muscles and bones", PVP combined with nerve block can effectively relieve paravertebral soft tissue spasm and other "muscle injuries", which can significantly improve short-term postoperative low back pain and lumbar spine mobility compared to conventional PVP treatment, and accelerate postoperative recovery, resulting in satisfactory clinical outcomes.

Enhanced reduction of nitrate by TDER packed with surface-modified plastic particles electrodes.

Based on Iron cathodes, nitrate could be selectively decomposed into other lower-valence nitrogen compounds, including ammonia, nitrogen gas, nitrite and nitric oxide, but the removal efficiencies of nitrate and total nitrogen (TN), are affected significantly by the synergistic effects of anodes, chloride electrolyte and conductive plastic particles electrodes. In this work, the base material Titanium (Ti) metal plates and plastic particles which surfaces were mainly coated with Ru-Sn oxidizing compounds, were applied as plates anodes and conductive particles electrodes in Three Dimensional Electrode Reactors (TDER). The Ti/RuSn plate anodes showed excellent performance on degrading nitrate, more nitrogen gas (83.84%) and less ammonia (15.51%) was produced, less TN and Iron ion (0.02 mg/L) was left in the wastewater, and less amount of chemical sludge (0.20 g/L) was produced. Furthermore, the removal efficiencies of nitrate and TN were further increased by the surface-modified plastic particles, which were cheap, reusable, corrosion-resistance, easy to obtain as manufactured materials and light to be suspended in waters. The degradation of nitrate and its intermediates was enhanced possibly by the continuous synergistic reactions initiated by hydrogen radicals, which was generated on the countless surficial active Ru-Sn sites of Ti/RuSn metal plate anodes and plastic particles electrodes, among residual nitrogen intermediates, most of ammonia was selectively converted to gaseous nitrogen by hypochlorite from chloride ion reaction.

DDX3 suppresses hepatocellular carcinoma progression through modulating the secretion and composition of exosome.

Due to the lack of predictive biomarkers and the lack of conspicuous symptoms at the early stage, hepatocellular carcinoma (HCC) remains difficult to diagnose and treat effectively. During cancer development, exosomes secreted from tumor cells carry functional molecules to surrounding recipient cells, thereby participating in the regulation of cancer progression. DDX3, a DEAD-box RNA helicase, performs many important functions in several cellular processes and is therefore implicated as a tumor suppressor in HCC. However, whether DDX3 affects the secretion and cargo sorting of HCC exosomes remains obscure. In this study, our results revealed that reduced DDX3 expression in HCC cells promoted the release of exosomes and enhanced the expression of several exosome biogenesis-associated proteins, such as exosome markers (e.g., TSG101, Alix, and CD63) and Rab proteins (e.g., Rab5, Rab11, and Rab35). By double knockdown of the expression of DDX3 and these exosome biogenesis-related factors, we confirmed that DDX3 participated in the regulation of exosome secretion by modulating the expression of these cellular factors in HCC cells. In addition, exosomes derived from DDX3-knockdown HCC cells enhanced cancer stem cell properties, including self-renewal capability, migration, and drug resistance, in recipient HCC cells. Moreover, up-regulation of the exosome markers TSG101, Alix, and CD63 as well as down-regulation of tumor-suppressive miR-200b and miR-200c were observed in exosomes derived from DDX3-knockdown HCC cells, which may account for the enhanced hepatic cancer stemness of the recipient cells treated with DDX3-knockdown HCC cell-derived exosomes. Taken together, our findings provide a new molecular mechanism supporting the tumor-suppressor role of DDX3 in HCC, which may contribute to the development of new therapeutic strategies against HCC.

Curcumol Alleviates the Inflammation of Nucleus Pulposus Cells via the PI3K/Akt/NF-κB Signaling Pathway and Delays Intervertebral Disk Degeneration.

BACKGROUND: Intervertebral disk degeneration (IVDD) is closely associated with inflammatory environments. Curcumol has been shown to alleviate inflammation in various disease models, but its effects on IVDD remain unclear. In this study, we sought to determine the mechanism of curcumol in tumor necrosis factor (TNF)-α-induced nucleus pulposus cells and a mouse IVDD model. METHODS: Nucleus pulposus cells were pretreated with curcumol and then exposed to TNF-α. Cell viability was analyzed using CCK-8, and the messenger ribonucleic acid and protein levels of inflammatory cytokines and PI3K/Akt/NF-κB-related signaling molecules were detected using real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and western blotting. The mouse IVDD model was established by puncturing the C6/7 level of the caudal spine, and then it was treated with curcumol after surgery. Alcian blue/orange G staining was performed to evaluate the severity of intervertebral disk damage, and immunohistochemistry was performed to detect the expression of TNF-α. Toxicologic effects of curcumol were measured by performing hematoxylin-eosin staining and enzyme-linked immunosorbent assay. RESULTS: Curcumol reduced IL-1ß, IL-6, and TNF-α production in NPCs, and the phosphorylation of proteins in the PI3K/Akt/NF-κB signaling pathway was also decreased. The PI3K/Akt/NF-κB-related signaling molecules decreased when TNF-α-induced NPCs were treated with a PI3K inhibitor; however, curcumol did not reverse these effects. In vivo, curcumol ameliorated the progression of IVDD at the early stage and did not exert toxicologic effects. CONCLUSIONS: These results suggest a potential therapeutic use of curcumol to alleviate inflammation via the PI3K/Akt/NF-κB signaling pathway and delay the progression of IVDD.

A Novel off-the-Shelf Trastuzumab-Armed NK Cell Therapy (ACE1702) Using Antibody-Cell-Conjugation Technology.

Natural killer (NK) cells harbor efficient cytotoxicity against tumor cells without causing life-threatening cytokine release syndrome (CRS) or graft-versus-host disease (GvHD). When compared to chimeric antigen receptor (CAR) technology, Antibody-Cell Conjugation (ACC) technology has been developed to provide an efficient platform to arm immune cells with cancer-targeting antibodies to recognize and attack cancer cells. Recently, we established an endogenous CD16-expressing oNK cell line (oNK) with a favorable expression pattern of NK activation/inhibitory receptors. In this study, we applied ACC platform to conjugate oNK with trastuzumab and an anti-human epidermal growth factor receptor 2 (HER2) antibody. Trastuzumab-conjugated oNK, ACE-oNK-HER2, executed in vitro and in vivo cytotoxicity against HER2-expressing cancer cells and showed enhanced T cell-recruiting capability and secretion of IFNγ. The irradiated and cryopreserved ACE-oNK-HER2, designated as ACE1702, retained superior HER2-specific in vitro and in vivo potency with no tumorigenic potential. In conclusion, this study provides the evidence to support the potential clinical application of ACE1702 as a novel off-the-shelf NK cell therapy against HER2-expressing solid tumors.

A novel endogenous CD16-Expressing Natural Killer Cell for cancer immunotherapy.

Natural killer (NK) cells, as a potential source for off-the-shelf cell therapy, attack tumor cells with low risk of severe cytokine release syndrome (CRS) or graft-versus-host disease (GvHD). Fcγ receptor IIIA, also known as CD16, further confers NK cells with antibody-dependent cell-mediated cytotoxicity (ADCC), one mechanism of action of antibody-based immunotherapy. Here, we establish a novel human NK cell line, oNK-1, endogenously expressing CD16 along with high levels of NK activation markers and low levels of NK inhibitory markers. The long-term expansion and CD16 expression of oNK-1 cells were demonstrated. Furthermore, oNK-1 cells elicit superior cytotoxicity against cancer cells than primary NK cells. In conclusion, this study suggests that endogenous CD16-expressing oNK-1 has the potential to develop an effective NK-based therapy.

DDX3 localizes to the centrosome and prevents multipolar mitosis by epigenetically and translationally modulating p53 expression.

The DEAD-box RNA helicase DDX3 plays divergent roles in tumorigenesis, however, its function in mitosis is unclear. Immunofluorescence indicated that DDX3 localized to centrosome throughout the cell cycle and colocalized with centrosome-associated p53 during mitosis in HCT116 and U2OS cells. DDX3 depletion promoted chromosome misalignment, segregation defects and multipolar mitosis, eventually leading to G2/M delay and cell death. DDX3 prevented multipolar mitosis by inactivation and coalescence of supernumerary centrosomes. DDX3 silencing suppressed Ser15 phosphorylation of p53 which is required for p53 centrosomal localization. Additionally, knockout of p53 dramatically diminished the association of DDX3 with centrosome, which was rescued by overexpression of the centrosomal targeting-defective p53 S15A mutant, indicating that centrosomal localization of DDX3 is p53 dependent but not through centrosomal location of p53. Furthermore, DDX3 knockdown suppressed p53 transcription through activation of DNA methyltransferases (DNMTs) along with hypermethylation of p53 promoter and promoting the binding of repressive histone marks to p53 promoter. Moreover, DDX3 modulated p53 mRNA translation. Taken together, our study suggests that DDX3 regulates epigenetic transcriptional and translational activation of p53 and colocalizes with p53 at centrosome during mitosis to ensure proper mitotic progression and genome stability, which supports the tumor-suppressive role of DDX3.

RNA helicase DDX3 maintains lipid homeostasis through upregulation of the microsomal triglyceride transfer protein by interacting with HNF4 and SHP.

Multifunctional RNA helicase DDX3 participates in HCV infection, one of the major causes of hepatic steatosis. Here, we investigated the role of DDX3 in hepatic lipid metabolism. We found that HCV infection severely reduced DDX3 expression. Analysis of intracellular triglyceride and secreted ApoB indicated that lipid accumulations were increased while ApoB secretion were decreased in DDX3 knockdown HuH7 and HepG2 cell lines. Down-regulation of DDX3 significantly decreased protein and transcript expression of microsomal triglyceride transfer protein (MTP), a key regulator of liver lipid homeostasis. Moreover, DDX3 interacted with hepatocyte nuclear factor 4 (HNF4) and small heterodimer partner (SHP), and synergistically up-regulated HNF4-mediated transactivation of MTP promoter via its ATPase activity. Further investigation revealed that DDX3 interacted with CBP/p300 and increased the promoter binding affinity of HNF4 by enhancing HNF4 acetylation. Additionally, DDX3 partially relieved the SHP-mediated suppression on MTP promoter by competing with SHP for HNF4 binding which disrupted the inactive HNF4/SHP heterodimer while promoted the formation of the active HNF4 homodimer. Collectively, these results imply that DDX3 regulates MTP gene expression and lipid homeostasis through interplay with HNF4 and SHP, which may also reveal a novel mechanism of HCV-induced steatosis.

DDX3 Represses Stemness by Epigenetically Modulating Tumor-suppressive miRNAs in Hepatocellular Carcinoma.

Studies indicate that the presence of cancer stem cells (CSCs) is responsible for poor prognosis of hepatocellular carcinoma (HCC) patients. In this study, the functional role of DDX3 in regulation of hepatic CSCs was investigated. Our results demonstrated that reduced DDX3 expression was not only inversely associated with tumor grade, but also predicted poor prognosis of HCC patients. Knockdown of DDX3 in HCC cell line HepG2 induced stemness gene signature followed by occurrence of self-renewal, chemoreisistance, EMT, migration as well as CSC expansion, and most importantly, DDX3 knockdown promotes tumorigenesis. Moreover, we found positive correlations between DDX3 level and expressions of tumor-suppressive miR-200b, miR-200c, miR-122 and miR-145, but not miR-10b and miR-519a, implying their involvement in DDX3 knockdown-induced CSC phenotypes. In addition, DDX3 reduction promoted up-regulation of DNA methyltransferase 3A (DNMT3A), while neither DNMT3B nor DNMT1 expression was affected. Enriched DNMT3A binding along with hypermethylation on promoters of these tumor-suppressive miRNAs reflected their transcriptional repressions in DDX3-knockdown cells. Furthermore, individual restoration of these tumor-suppressive miRNAs represses DDX3 knockdown-induced CSC phenotypes. In conclusion, our study suggested that DDX3 prevents generation of CSCs through epigenetically regulating a subset of tumor-suppressive miRNAs expressions, which strengthens tumor suppressor role of DDX3 in HCC.

Critical roles of RNA helicase DDX3 and its interactions with eIF4E/PABP1 in stress granule assembly and stress response.

Upon environmental insults, SGs (stress granules) aid cell survival by serving as sites of translational silencing. RNA helicase DDX3 was reported to associate with SGs. However, its role in SG physiology remains undefined. We have demonstrated previously that DDX3 acts as an eIF4E (eukaryotic initiation factor 4E)-inhibitory protein to suppress translation. In the present study, we indentified the SG marker PABP1 [poly(A)-binding protein 1] as another direct interaction partner of DDX3. We established various stimuli as novel stressors that direct DDX3 with eIF4E and PABP1 into SGs, but not to processing bodies. Interestingly, down-regulation of DDX3 interfered with SG assembly, led to nuclear accumulation of PABP1 and reduced cell viability following stress. Conversely, supplementation with a shRNA (short hairpin RNA)-resistant DDX3 restored SG formation, the translocation of PABP1 into SGs and cell survival. Notably, the SG-inducing capacity of DDX3 is independent of its ATPase and helicase activities, but mapped to the eIF4E-binding region. Moreover, the eIF4E-binding-defective mutant DDX3 was impaired in its SG-inducing ability and protective effect on cell survival under adverse conditions. All together, the present study has characterized DDX3 as a pivotal SG-nucleating factor and illustrates co-ordinative roles for DDX3, eIF4E and PABP1 in integrating environmental stress with translational regulation.