Combination of microwave-assisted extraction with dispersive micro solid-phase extraction as an efficient sample pretreatment method for the extraction of some antiparasitic drugs from cow liver, meat, and kidney samples.

A simple pretreatment method based on the combination of microwave-assisted extraction and dispersive micro solid phase extraction has been developed for the extraction of eprinomectin, doramectin, ivermectin, and abamectin from cow meat, liver, and kidney samples. The extracted drugs were determined using high-performance liquid chromatography equipped with a diode array detector. In this method, the solid samples were mixed with deionized water and organic solvent mixture, and the resulting mixtures were exposed to microwave irradiations to accelerate the analytes' extraction from the samples into the solution. Then, the supernatant was taken and mixed with a mixture of three sorbents optimized by a simplex centroid design. After vortexing and centrifuging, the sorbent particles were isolated and the adsorbed analytes onto the sorbent surface eluted with a natural deep eutectic solvent for more concentration. After centrifuging, the supernatant was taken and injected into the separation system. Acceptable repeatability (relative standard deviations ≤7.0%), high extraction recoveries (72%-86%) and enrichment factors (216-258), and low limits of detection and quantification (0.06-0.10 and 0.19-0.32 ng/g, respectively) were acquired. The method was successfully applied for the assessment of the analytes in the mentioned samples and ivermectin was found in three samples.

CRISPR/Cas9-mediated TGFßRII disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells in vitro.

BACKGROUND: CAR T-cell therapy has been recently unveiled as one of the most promising cancer therapies in hematological malignancies. However, solid tumors mount a profound line of defense to escape immunosurveillance by CAR T-cells. Among them, cytokines with an inhibitory impact on the immune system such as IL-10 and TGFß are of great importance: TGFß is a pleiotropic cytokine, which potently suppresses the immune system and is secreted by a couple of TME resident and tumor cells. METHODS: In this study, we hypothesized that knocking out the TGFß receptor II gene, could improve CAR T-cell functions in vitro and in vivo. Hereby, we used the CRISPR/Cas9 system, to knockout the TGFßRII gene in T-cells and could monitor the efficient gene knock out by genome analysis techniques. Next, Mesothelin or Claudin 6 specific CAR constructs were overexpressed via IVT-RNA electroporation or retroviral transduction and the poly-functionality of these TGFßRII KO CAR T-cells in terms of proliferation, cytokine secretion and cytotoxicity were assessed and compared with parental CAR T-cells. RESULTS: Our experiments demonstrated that TGFßRII KO CAR T-cells fully retained their capabilities in killing tumor antigen positive target cells and more intriguingly, could resist the anti-proliferative effect of exogenous TGFß in vitro outperforming wild type CAR T-cells. Noteworthy, no antigen or growth factor-independent proliferation of these TGFßRII KO CAR T-cells has been recorded. TGFßRII KO CAR T-cells also resisted the suppressive effect of induced regulatory T-cells in vitro to a larger extent. Repetitive antigen stimulation demonstrated that these TGFßRII KO CAR T-cells will experience less activation induced exhaustion in comparison to the WT counterpart. CONCLUSION: The TGFßRII KO approach may become an indispensable tool in immunotherapy of solid tumors, as it may surmount one of the key negative regulatory signaling pathways in T-cells.

Phase 2 study of a high dose of 186Re-HEDP for bone pain palliation in patients with widespread skeletal metastases.

UNLABELLED: (186)Re-1-hydroxyethylidene-1,1-diphosphonate (HEDP) is an attractive radiopharmaceutical for the treatment of bone pain arising from skeletal metastatic lesions. Currently, (186)Re-HEDP is most commonly used in European countries. The aim of this study was to investigate the palliative efficacy and adverse effects of (186)Re-HEDP in patients with different types of cancers and skeletal bone pain. METHODS: Nineteen (8 male, 11 female) patients with various cancers (breast, prostate, renal cell carcinoma, colon, and neuroendocrine tumors) and painful bone metastases were included in the study. A dose of 1,480-3,330 MBq (40-90 mCi) of (186)Re-HEDP was administered intravenously. The patients' level of pain relief was assessed by the Visual Analog Scale for 8 wk after treatment and by a weekly blood cell count to evaluate for hematologic toxicity. RESULTS: The overall response rate was 89.5%, and the mean pain score assessed by the Visual Analog Scale was reduced from 9.1 to 5.3 after 1 wk (P = 0.003). No adverse effects were reported by patients during intravenous administration or for up to 24 h after administration. A flare reaction was seen in 63.2% of patients, mainly during days 1-3, and lasted for 2-4 d. There was no significant correlation between the response to therapy and the flare reactions (P > 0.05). The nadir of platelet reduction occurred at the fourth or fifth week and led to platelet infusion in only 4 patients with a low baseline platelet count and diffuse skeletal metastases. Bone marrow suppression occurred in patients receiving higher doses, but no clinical problems were seen except in 2 patients who required packed cell transfusion similar to their prior transfusions. CONCLUSION: (186)Re-HEDP is an effective radiopharmaceutical for the palliative treatment of metastatic bone pain and has minimal adverse effects.