Safety of allogeneic γδT cell immunotherapy for advanced hepatocellular carcinoma and its effect on patients' immune function / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨使用同种异体Vγ9Vδ2 T细胞回输治疗晚期肝细胞癌（HCC）患者的安全性及治疗后患者免疫功能的变化。方法：选择2021年10月至2022年10月珠海市人民医院收治的4例晚期HCC患者，从健康供体获取外周血单个核细胞（PBMC）后经刺激扩增培养获得Vγ9Vδ2 T细胞，经质控放行后予以回输治疗，回输细胞剂量为5×108个/次，每两周一次，回输次数9次以上，治疗后检测患者αβT细胞、B细胞、NK细胞、γδT细胞各亚群比例，转氨酶、肌酐、肌酸激酶等肝、肾、心功能生化标志物，以及血常规三系（白细胞系统、红细胞系统和血小板系统）细胞数量的变化。结果：4例患者在回输治疗后均显示出对异体Vγ9Vδ2 T细胞良好的耐受性；转氨酶、肌酐、肌酸激酶等肝、肾、心功能生化标志物以及血常规三系细胞数量在回输前后均无明显变化；患者的Tfh1、Tc1、CD127+TEM、HLADR+CD8+ T细胞、CD27- B细胞比例有升高趋势，提示特异性免疫功能的增强。结论：同种异体Vγ9Vδ2 T细胞治疗晚期HCC有较好的安全性并可在一定程度上改善患者的免疫功能。

Ex vivo susceptibilities of Plasmodium vivax isolates from the China-Myanmar border to antimalarial drugs and association with polymorphisms in Pvmdr1 and Pvcrt-o genes.

BACKGROUND: Vivax malaria is an important public health problem in the Greater Mekong Subregion (GMS), including the China-Myanmar border. Previous studies have found that Plasmodium vivax has decreased sensitivity to antimalarial drugs in some areas of the GMS, but the sensitivity of P. vivax to antimalarial drugs is unclear in the China-Myanmar border. Here, we investigate the drug sensitivity profile and genetic variations for two drug resistance related genes in P. vivax isolates to provide baseline information for future drug studies in the China-Myanmar border. METHODOLOGY/PRINCIPAL FINDINGS: A total of 64 P. vivax clinical isolates collected from the China-Myanmar border area were assessed for ex vivo susceptibility to eight antimalarial drugs by the schizont maturation assay. The medians of IC50 (half-maximum inhibitory concentrations) for chloroquine, mefloquine, pyronaridine, piperaquine, quinine, artesunate, artemether, dihydroartemisinin were 84.2 nM, 34.9 nM, 4.0 nM, 22.3 nM, 41.4 nM, 2.8 nM, 2.1 nM and 2.0 nM, respectively. Twelve P. vivax clinical isolates were found over the cut-off IC50 value (220 nM) for chloroquine resistance. In addition, sequence polymorphisms in pvmdr1 (P. vivax multidrug resistance-1), pvcrt-o (P. vivax chloroquine resistance transporter-o), and difference in pvmdr1 copy number were studied. Sequencing of the pvmdr1 gene in 52 samples identified 12 amino acid substitutions, among which two (G698S and T958M) were fixed, M908L were present in 98.1% of the isolates, while Y976F and F1076L were present in 3.8% and 78.8% of the isolates, respectively. Amplification of the pvmdr1 gene was only detected in 4.8% of the samples. Sequencing of the pvcrt-o in 59 parasite isolates identified a single lysine insertion at position 10 in 32.2% of the isolates. The pvmdr1 M908L substitutions in pvmdr1 in our samples was associated with reduced sensitivity to chloroquine, mefloquine, pyronaridine, piperaquine, quinine, artesunate and dihydroartemisinin. CONCLUSIONS: Our findings depict a drug sensitivity profile and genetic variations of the P. vivax isolates from the China-Myanmar border area, and suggest possible emergence of chloroquine resistant P. vivax isolates in the region, which demands further efforts for resistance monitoring and mechanism studies.

Rapid Screening and Characterization of Acetylcholinesterase Inhibitors from Yinhuang Oral Liquid Using Ultrafiltration-liquid Chromatography-electrospray Ionization Tandem Mass Spectrometry.

BACKGROUND: At present, approximately 17-25 million people in the world suffer from Alzheimer's disease (AD). The most efficacious and acceptable therapeutic drug clinically are the acetylcholinesterase inhibitors (AChEIs). Yinhuang oral liquid is a Chinese medicine preparation which contains AChEIs according to the literatures. However, no strategy has been presented for rapid screening and identification of AChEIs from Yinhuang oral liquid. OBJECTIVE: To develop a method for rapid screening and identification of AChEIs from Yinhuang oral liquid using ultrafiltration-liquid chromatography-electrospray ionization tandem mass spectrometry (UF-LC-ESI-MS/MS). MATERIALS AND METHODS: In this study, UF incubation conditions such as enzyme concentration, incubation time, and incubation temperature were optimized so as to get better screening results. The AChEIs from Yinhuang oral liquid were identified by high-performance liquid chromatography-ESI-MS and the improved Ellman method was used for the AChE inhibitory activity test in vitro. RESULTS: The results showed that Yinhuang oral liquid can inhibit the activity of AChE. We screened and identified seven compounds with potential AChE inhibitory activity from Yinhuang oral liquid, which provided experimental basis for the treatment and prevention of AD. CONCLUSION: The current technique was used to directly screen the active ingredients with acetylcholinesterase inhibition from complex traditional Chinese medicine, which was simple, rapid, accurate, and suitable for high-throughput screening of AChEI from complex systems. SUMMARY: A UF-LC-ESI-MS/MS method for rapid screening and identification of AChEIs from Yinhuang oral liquid was developedSeven compounds were screened and identified with potential AChE inhibitory activity from Yinhuang oral liquidIt provided experimental basis of Yinhuang oral liquid for the treating and preventing AD. Abbreviations used: (AD): Alzheimer's disease; (UF-LC-ESI-MS/MS): ultrafiltration-liquid chromatography-electrospray ionization tandem mass spectrometry; (AChEIs): acetylcholinesterase inhibitors.

[Cloning, expression and identification of gametocyte specific protein Pfgdv1 of Plasmodium falciparum].

OBJECTIVE: To clone a gametocyte specific protein Pfgdv1 of Plasmodium falciparum, express and identify recombinant Pfgdvl protein in vitro. METHODS: PCR was performed to amplify Pfgdv1 from P. falciparum DNA which was got from the patient who was infected with P. falciparum, and the PCR product was inserted into pET28a (+) vector. pET28a-Pfgdv1 recombinant plasmid was constructed and transformed into E. coli host BL21 (DE3+). IPTG was used to induce the recombinant Pfgdv1 protein fused with His tag, and the protein was purified by His-NTA affinity chromatography. The recombinant protein was identified by SDS-PAGE and Western blotting. RESULTS: The PCR product of Pfgdv1 gene was about 1.65 kb, meeting the expectation of predicted fragment size. The recombinant protein was about 67 kDa, which could be recognized by His-Tag monoclonal antibody. CONCLUSION: The Pfgdv1 gene of P. falciparum is successfully cloned, and the recombinant Pfgdv1 protein is expressed, thereby providing an opportunity for further study on transmission blocking vaccine.

[In vitro Effect of Hypericin against Toxoplasma gondii Tachyzoites].

Objective: To investigate the killing effect of hypericin on tachyzoites of Toxoplasma gondii RH strain in vitro. Methods: Normal saline （group A） and different concentrations of hypericin (5 µg/ml, group B; 50 µg/ml, group C; 500 µg/ml, group D） were added to T. gondii tachyzoites in 24-well plate（1×10(6)/well）. The tachyzoites were harvested after 2, 4 and 6 h, and underwent the following treatment: trypan blue staining to calculate the dyeing rate, Giemsa staining to observe the morphological and structural alterations of tachyzoites, and transmission electron microscopy to observe the ultrastructure of tachyzoites. In addition, flow cytometry was performed to calculate the survival rate of YFP-carrying Toxoplasma with the same treatment. Results: The trypan blue dyeing rate at 2 h after treatment in groups B, C and D was（11.0±3.6）%, （25.0±6.3）% and（40.0±2.7）% respectively, with a significant difference of group D versus B and C （P<0.01）, and groups C and D versus group A ［（6.0±3.0）%）］. The dyeing rate at 4 h and 6 h in group D was（97.0±2.0）% and （98.0±1.7）%, respectively, both significantly higher than that of groups C ［（30.0±7.2）%, （42.7±5.5）%ï¼½, B ［（20.0±3.0）%, （34.0±6.6）%ï¼½ and A ［（10.0±1.0）%, （19.3±4.9）%］（P<0.01）. Giemsa staining showed gradual end swelling and necrosis of tachyzoites with increased treatment duration and dosage. Transmission electron microscopy showed swelling of worm body, gap between cell membrane and matrix, increase and enlargement of vacuoles inside worm body, disruption of cell membrane, and dissolving of inner structures, with increased treatment duration. Flow cytometry showed significant difference of tachyzoite survival rate at 2, 4 and 6 h after hypericin treatment with that of the control group（P<0.01）. The survival rate of group C at 2 h after hypericin treatment was（7.9±1.9）%, significantly lower than that of groups B ［（38.1±5.5）%ï¼½ and A ［（81.8±6.0）%ï¼½ （P<0.01）. No tachyzoite was found to survive in group D at 2 h and in group C at 4 h. The survival rate of group B at 4 and 6 h after hypericin treatment was（14.3±7.9）% and （1.4±1.8）%, respectively, both significantly lower than that of group A［（73.8±11.3）% and（64.1±14.4）%, respectivelyï¼½ （P<0.01）. Conclusion: Hypericin has a remarkable killing effect on T. gondii tachyzoites, and the efficacy positively correlates with the dose and treatment duration.