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Supplemental Figure S1. Contribution of NK and T cells to IFN-gamma production. Supplemental Figure S2. MICA expression on monocytes. Supplemental Figure S3. NKG2D Ligand Expression on C1R and C1R-MICA Cell Lines. Supplemental Figure S4. Monocyte MICA expression does not correlate with NK cell IFN-gamma production. Supplemental Figure S5. NKG2D and MICA expression levels prior to and following co-culture.

Natural killer (NK) cells are large granular lymphocytes that promote the antitumor response via communication with other cell types in the tumor microenvironment. Previously, we have shown that NK cells secrete a profile of immune stimulatory factors (e.g., IFNγ, MIP-1α, and TNFα) in response to dual stimulation with the combination of antibody (Ab)-coated tumor cells and cytokines, such as IL12. We now demonstrate that this response is enhanced in the presence of autologous monocytes. Monocyte enhancement of NK cell activity was dependent on cell-to-cell contact as determined by a Transwell assay. It was hypothesized that NK cell effector functions against Ab-coated tumor cells were enhanced via binding of MICA on monocytes to NK cell NKG2D receptors. Strategies to block MICA–NKG2D interactions resulted in reductions in IFNγ production. Depletion of monocytes in vivo resulted in decreased IFNγ production by murine NK cells upon exposure to Ab-coated tumor cells. In mice receiving trastuzumab and IL12 therapy, monocyte depletion resulted in significantly greater tumor growth in comparison to mock-depleted controls (P < 0.05). These data suggest that NK cell–monocyte interactions enhance NK cell antitumor activity in the setting of monoclonal Ab therapy for cancer. Cancer Immunol Res; 5(9); 778–89. ©2017 AACR.

Wallerian degeneration is a complex biological process that occurs after nerve injury, and involves nerve degeneration and regeneration. Schwann cells play a crucial role in the cellular and molecular events of Wallerian degeneration of the peripheral nervous system. However, Wallerian degeneration regulating nerve injury and repair remains largely unknown, especially the early response. We have previously reported some key regulators of Wallerian degeneration after sciatic nerve injury. Baculoviral inhibitor of apoptosis protein repeat-containing protein 3 (BIRC3) is an important factor that regulates apoptosis-inhibiting protein. In this study, we established rat models of right sciatic nerve injury. In vitro Schwann cell models were also established and subjected to gene transfection to inhibit and overexpress BIRC3. The data indicated that BIRC3 expression was significantly up-regulated after sciatic nerve injury. Both BIRC3 upregulation and downregulation affected the migration, proliferation and apoptosis of Schwan cells and affected the expression of related factors through activating c-fos and ERK signal pathway. Inhibition of BIRC3 delayed early Wallerian degeneration through inhibiting the apoptosis of Schwann cells after sciatic nerve injury. These findings suggest that BIRC3 plays an important role in peripheral nerve injury repair and regeneration. The study was approved by the Institutional Animal Care and Use Committee of Nantong University, China (approval No. 2019-nsfc004) on March 1, 2019.

Hundreds of millions of people worldwide suffer from peripheral nerve damage resulting from car accidents, falls, industrial accidents, residential accidents, and wars. The purpose of our study was to further investigate the effects of Wallerian degeneration (WD) after rat sciatic nerve injury and to screen for critical long noncoding RNAs (lncRNAs) in WD. We found H19 to be essential for nerve degeneration and regeneration and to be highly expressed in the sciatic nerves of rats with WD. LncRNA H19 potentially impaired the recovery of sciatic nerve function in rats. H19 was mainly localized in the cytoplasm of Schwann cells (SCs) and promoted their migration. H19 promoted the apoptosis of dorsal root ganglion (DRG) neurons and slowed the growth of DRG axons. The lncRNA H19 may play a role in WD through the Wnt/β-catenin signaling pathway and is coexpressed with a variety of crucial mRNAs during WD. These data provide further insight into the molecular mechanisms of WD. This article is protected by copyright. All rights reserved.

Radiolabeled bombesin (BBN) analogs have long been used for developing gastrin-releasing peptide receptor (GRPR) targeted imaging probes, and tracers with excellent in vivo performance including high tumor uptake, high contrast, and favorable pharmacokinetics are highly desired. In this study, we compared the 68Ga-labeled GRPR agonist (Gln–Trp–Ala–Val–Gly–His–Leu–Met–NH2, BBN7–14) and antagonist (d-Phe–Gln–Trp–Ala–Val–Gly–His–Sta–Leu–NH2, RM26) for the positron emission tomography (PET) imaging of prostate cancer. The in vitro stabilities, receptor binding, cell uptake, internalization, and efflux properties of the probes 68Ga–1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA)–Aca–BBN7–14 and 68Ga–NOTA–poly(ethylene glycol)3 (PEG3)–RM26 were studied in PC-3 cells, and the in vivo GRPR targeting abilities and kinetics were investigated using PC-3 tumor xenografted mice. BBN7–14, PEG3-RM26, NOTA–Aca–BBN7–14, and NOTA–PEG3–RM26 showed similar binding affinity to GRPR. In PC-3 tumor-bearing mice, the tumor uptake of 68Ga–NOTA–PEG3–RM26 remained at around 3.00 percentage of injected dose per gram of tissue within 1 h after injection, in contrast with 68Ga–NOTA–Aca–BBN7–14, which demonstrated rapid elimination and high background signal. Additionally, the majority of the 68Ga–NOTA–PEG3–RM26 remained intact in mouse serum at 5 min after injection, while almost all of the 68Ga–NOTA–Aca–BBN7–14 was degraded under the same conditions, demonstrating more-favorable in vivo pharmacokinetic properties and metabolic stabilities of the antagonist probe relative to its agonist counterpart. Overall, the antagonistic GRPR targeted probe 68Ga–NOTA–PEG3–RM26 is a more-promising candidate than the agonist 68Ga–NOTA–Aca–BBN7–14 for the PET imaging of prostate cancer patients.

Eudragit® E 100 (EE100) was used to improve the transfection efficiency of polyethylenimine (PEI).Mobility of PEI-DNA complexes with and without EE100 were visualized by agarose gel electrophoresis and their transfection efficiencies were investigated in KB human oral carcinoma cells by flow cytometry. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to determine the viability of transfected cells.Gel electrophoresis illustrated formation of complete complexes at N/P ratios above 5. PEI had the highest transfection efficiency at an N/P ratio of 15, whereas in combination with EE100, the transfection efficiency was highest at an N/P ratio of 7.5. High concentrations of EE100 in combination with PEI were found to reduce cell viability.The results show a synergistic action of EE100 in transfection of DNA at low N/P ratios compared to PEI alone.

Gefitinib is a promising agent for the treatment of non-small cell lung cancer. The purpose of this study was to develop a novel liposomal formulation for gefitinib (L-GEF) to improve its therapeutic index.Several L-GEF formulations were prepared and characterized for their physical chemical properties and cytotoxicity. The pharmacokinetic parameters of the liposomes were determined in mice. The effect of lipid composition, transmembrane pH gradient, and incorporation of hydroxypropyl-β-cyclodextrin (HPβCD) on drug-loading efficiency, liposomal stability, and the rate of drug release were investigated.The L-GEF formulation composed of hydrogenated soy phosphatidylcholine (HSPC)/cholesterol (Chol)/monomethoxy polyethylene glycol 2000-distearoyl phosphatidyl-ethanolamine (mPEG-DSPE) encapsulating 0.3 M (NH4)2SO4 and 0.1 M HPβCD (L-GEF-HSPC), had a drug-loading efficiency (DLE) of 85.5%. In vitro release studies showed that gefitinib release from L-GEF-HSPC in the presence of human plasma was slow and exhibited non-Fickian kinetics. Pharmacokinetic study in mice after i.v. bolus administration of L-GEF-HSPC showed that the area under the plasma concentration time curve (AUC) for gefitinib was 32.41 μg·h /ml and six times that of free gefitinib. The elimination half life (t(1/2β)) of L-GEF-HSPC was 7.29 h, while that of free gefitinib was 2.26 h.It was shown that gefitinib can be efficiently loaded into L-GEF-HSPC composed of HSPC/Chol/mPEG-DSPE (55/40/5 mol/mol) with 0.3 M (NH(4))(2)SO(4) and 0.1 M HPβCD as trapping agents. Compared with the free drug, L-GEF-HSPC had high drug loading, good stability, and long-circulating properties.

Folate receptor (FR)-targeted liposomes have been investigated as delivery vehicles for anticancer drugs. A novel lipophilic FR ligand, folate-glutathione-polyethyleneglycol-distearoyl phosphatidylethanolamine (F-GSH-PEG-DSPE), was synthesized, incorporated into liposomes and evaluated for FR targeting efficiency. These liposomes were then evaluated as carriers of the chemotherapy agent vincristine (VIN).F-GSH-PEG-DSPE was synthesized and FR-targeted liposomes loaded with either calcein (F-L-Calcein) or VIN (F-L-VIN) were prepared by thin film hydration followed by polycarbonate membrane extrusion and, in the case of VIN, by remote loading. To assess liposome stability, the uptake of F-L-VIN in KB (FR+) cancer cells was measured after storage under 4°C for 3 months. Comparative pharmacokinetic studies were carried out with F-L-VIN and L-VIN (non-targeted control liposomes).F-L-Calcein showed significantly higher cellular uptake in KB cells compared to non-targeted liposomes. In addition, F-L-VIN showed enhanced cytotoxicity in KB cells in vitro compared to control liposomes. Pharmacokinetic parameters indicated that both F-L-VIN and control liposomes had higher area under the curve (AUC), mean residence time (MRT), elimination half life (t1/2-β) and lower total body clearance (CL) than those of free VIN, while there were no significant differences between these liposomal formulations.F-GSH-PEG-DSPE is effective as a novel ligand for the synthesis of FR-targeted liposomes.