Protocol to generate traceable CAR T cells for syngeneic mouse cancer models.

The efficacy of chimeric antigen receptor (CAR) T cell immunotherapy is limited by insufficient infiltration and activation of T cells due to the immunosuppressive tumor microenvironment. Preclinical studies with optimized mouse CAR T cells in immunocompetent mouse cancer models will help define the mechanisms underlying immunotherapy resistance. Here, we present a protocol for preparing mouse T cells and generating CAR T cells. We then detail procedures for testing their therapeutic efficacy and tracking them in a syngeneic mouse glioma model. For complete details on the use and execution of this protocol, please refer to Zhang et al.1.

Effects of water-aging for 6 months on the durability of a novel antimicrobial and protein-repellent dental bonding agent.

Biofilms at the tooth-restoration bonded interface can produce acids and cause recurrent caries. Recurrent caries is a primary reason for restoration failures. The objectives of this study were to synthesize a novel bioactive dental bonding agent containing dimethylaminohexadecyl methacrylate (DMAHDM) and 2-methacryloyloxyethyl phosphorylcholine (MPC) to inhibit biofilm formation at the tooth-restoration margin and to investigate the effects of water-aging for 6 months on the dentin bond strength and protein-repellent and antibacterial durability. A protein-repellent agent (MPC) and antibacterial agent (DMAHDM) were added to a Scotchbond multi-purpose (SBMP) primer and adhesive. Specimens were stored in water at 37 °C for 1, 30, 90, or 180 days (d). At the end of each time period, the dentin bond strength and protein-repellent and antibacterial properties were evaluated. Protein attachment onto resin specimens was measured by the micro-bicinchoninic acid approach. A dental plaque microcosm biofilm model was used to test the biofilm response. The SBMP + MPC + DMAHDM group showed no decline in dentin bond strength after water-aging for 6 months, which was significantly higher than that of the control (P < 0.05). The SBMP + MPC + DMAHDM group had protein adhesion that was only 1/20 of that of the SBMP control (P < 0.05). Incorporation of MPC and DMAHDM into SBMP provided a synergistic effect on biofilm reduction. The antibacterial effect and resistance to protein adsorption exhibited no decrease from 1 to 180 d (P > 0.1). In conclusion, a bonding agent with MPC and DMAHDM achieved a durable dentin bond strength and long-term resistance to proteins and oral bacteria. The novel dental bonding agent is promising for applications in preventive and restorative dentistry to reduce biofilm formation at the tooth-restoration margin.

Effects of Long-Term Water-Aging on Novel Anti-Biofilm and Protein-Repellent Dental Composite.

The aims of this study were to: (1) synthesize an anti-biofilm and protein-repellent dental composite by combining 2-methacryloyloxyethyl phosphorylcholine (MPC) with quaternary ammonium dimethylaminohexadecyl methacrylate (DMAHDM); and (2) evaluate the effects of water-aging for 180 days on protein resistance, bacteria-killing ability, and mechanical properties of MPC-DMAHDM composite. MPC and DMAHDM were added into a resin composite. Specimens were stored in distilled water at 37 °C for 1, 30, 90, and 180 days. Mechanical properties were measured in three-point flexure. Protein attachment onto the composite was evaluated by a micro bicinchoninic acid approach. An oral plaque microcosm biofilm model was employed to evaluate oral biofilm viability vs. water-aging time. Mechanical properties of the MPC-DMAHDM composite after 180-day immersion matched those of the commercial control composite. The composite with 3% MPC + 1.5% DMAHDM had much stronger resistance to protein adhesion than control (p < 0.05). MPC + DMAHDM achieved much stronger biofilm-eradicating effects than MPC or DMAHDM alone (p < 0.05). Biofilm colony-forming units on the 3% MPC + 1.5% DMAHDM composite were three orders of magnitude lower than commercial control. The protein-repellent and antibacterial effects were durable and showed no loss in water-aging from 1 to 180 days. The novel MPC-DMAHDM composite possessed strong and durable resistance to protein adhesion and potent bacteria-eradicating function, while matching the load-bearing ability of a commercial dental composite. The novel MPC-DMAHDM composite represents a promising means of suppressing oral plaque growth, acid production, and secondary caries.