Rapid generation of CD19 CAR-T cells by minicircle DNA enables anti-tumor activity and prevents fatal CAR-B leukemia.

Manufacturing chimeric antigen receptor (CAR)-T cells using viral vectors is expensive and time-consuming. In addition, during viral transduction, genes encoding CARs are randomly integrated into the genome, which can cause oncogenesis or produce devastating CAR-tumor cells. Here, using a virus-free and non-transgenic minicircle DNA (mcDNA) vector, we enabled the rapid generation of CD19 CAR-T cells within two days. Furthermore, we demonstrated in vitro and in xenograft models that the antitumor effects of CD19 CAR-T cells produced by mcDNA are as effective as those produced by viral vectors. Finally, we showed that our manufacturing process avoids the production of fatal CAR-tumor cells. Taken together, we have provided a fast, effective, and therapeutically safe method for generating CD19 CAR-T cells for the treatment of leukemia.

Apatinib plus chemotherapy versus chemotherapy alone as neoadjuvant therapy in locally advanced gastric carcinoma patients: a prospective, cohort study.

BACKGROUND: Apatinib, a small molecule targeting VEGFR2, is commonly used for advanced gastric cancer treatment. This prospective cohort study further investigated the efficacy and safety of neoadjuvant apatinib plus chemotherapy in locally advanced gastric carcinoma patients. METHODS: Ninety-six locally advanced gastric carcinoma patients were divided into the apatinib plus chemotherapy group (N = 45) and chemotherapy group (N = 51) according to their chosen treatment. Apatinib was administered (375 mg/day), and S-1 plus oxaliplatin (SOX) or oxaliplatin plus capecitabine (CapOx) was given as chemotherapy, for 3 cycles with 3 weeks a cycle before surgery. RESULTS: The objective response rate (62.2% vs. 37.3%, P = 0.015) and pathological response grade (P = 0.011) were better; meanwhile, the tumor-resection rate (95.6% vs. 84.3%, P = 0.143) and pathological complete response rate (23.3% vs. 9.3%, P = 0.080) exhibited increasing trends (without statistical significance) in the apatinib plus chemotherapy group compared with the chemotherapy group. Additionally, the apatinib plus chemotherapy group achieved prolonged disease-free survival (DFS) (P = 0.019) and overall survival (OS) (P = 0.047) compared with the chemotherapy group. After adjusted by multivariate Cox's regression analysis, neoadjuvant apatinib plus chemotherapy was still superior to chemotherapy regarding DFS (hazard ratio (HR): 0.277, P = 0.014) and OS (HR: 0.316, P = 0.038). Notably, the incidences of adverse events between the two groups were not different (P > 0.050). Moreover, the most common adverse events of neoadjuvant apatinib plus chemotherapy were leukopenia (42.2%), fatigue (37.8%), hypertension (37.8%), and anemia (31.1%). CONCLUSION: Neoadjuvant apatinib plus chemotherapy realizes better clinical response, pathological response, survival profile, and non-inferior safety profile compared to chemotherapy in locally advanced gastric carcinoma.

Therapeutic effects of CXCL9-overexpressing human umbilical cord mesenchymal stem cells on liver fibrosis in rats.

Umbilical cord mesenchymal stem cells (UC-MSCs) transplantation has become a promising treatment for liver fibrosis. However, UC-MSCs have limited anti-fibrosis ability, and their homing ability of UC-MSCs to the injured liver seems to be poor. In our study, we aimed to determine if the CXCL9-overexpressing UC-MSCs could have synergistic anti-fibrosis effects and whether it can promote the homing ability of UC-MSCs. Overexpression of CXCL9 in UC-MSCs (CXCL9-UC-MSCs) was attained by transfecting the lenti-CXCL9-mCherry to naive UC-MSCs. The therapeutic effect of transducted CXCL9-UC-MSCs on both repairing of hepatic fibrosis and target homing were evaluated by comparing with the control of UC-MSCs transfected with empty lenti-mCherry vector. The results revealed that the liver function of CXCL9-UC-MSCs treated group was significantly improved when compared with that of control UC-MSCs (P < 0.05), and the histopathology indicated an obvious decrease of the collagen fiber content and significant disappearing of pseudo-lobules with basically normal morphology of hepatic lobules. Furthermore, liver frozen sections confirmed that CXCL9-UC-MSCs have significantly stronger chemotaxis and stable persistence in the injured liver tissues. In summary, overexpression of CXCL9 could improve the efficacy of UC-MSCs therapy for liver fibrosis repairing on account of an enhanced ability of UC-MSCs in homing to and staying in the injured sites of liver fibrosis in rat models.

ARMS TaqMan real-time PCR for genotyping factor V Leiden mutation in Han Chinese.

Factor V Leiden (FVLeiden ) is a missense mutation of 1691 position (G1691A) in exon 10 of FV gene, and being a genetic risk for venous thrombosis. Currently, there are several PCR-based methods for detecting FVLeiden mutation; however, these methods have disadvantages such as time-consuming, cumbersome steps and potentially hazardous gels. The aims of present study were to develop a simple, time-saving, accurate, and gel-free method, called amplification refractory mutation system (ARMS) TaqMan real-time PCR, for detecting FVLeiden mutation. We severally designed two specific reverse primers for mutant and wild-type through intentional introduction of mismatched nucleotide at the penultimate 3' position. Although target amplicon amplification efficiency is reduced, but another corresponding amplicon is almost completely inhibited. Then, specific TaqMan-probe was designed to detect target amplicon. Established method was used to detect 500 unselected samples in Han Chinese, the results showed 499 cases of wild-type and one heterozygote. Afterward, 50 randomly picked wild-type cases and one heterozygote were reexamined by bidirectional DNA sequencing, which is considered as "Gold standard method." Exhilaratingly, the results detected by the two methods were completely consistent. At last, allelic frequency of FVLeiden was calculated the in Han Chinese. Given the above results, A FVLeiden heterozygote has been found in 500 random samples in Han Chinese, and the allelic frequency was 0.1%. In conclusion, the ARMS TaqMan real-time PCR is an ideal detecting system for genotyping FVLeiden mutation in clinical application, and FVLeiden mutation exists in Han Chinese despite extremely low prevalence.

Erratum: Author Correction: Immune cell profiling of COVID-19 patients in the recovery stage by single-cell sequencing.

[This corrects the article DOI: 10.1038/s41421-020-0168-9.].

Immune cell profiling of COVID-19 patients in the recovery stage by single-cell sequencing.

COVID-19, caused by SARS-CoV-2, has recently affected over 1,200,000 people and killed more than 60,000. The key immune cell subsets change and their states during the course of COVID-19 remain unclear. We sought to comprehensively characterize the transcriptional changes in peripheral blood mononuclear cells during the recovery stage of COVID-19 by single-cell RNA sequencing technique. It was found that T cells decreased remarkably, whereas monocytes increased in patients in the early recovery stage (ERS) of COVID-19. There was an increased ratio of classical CD14++ monocytes with high inflammatory gene expression as well as a greater abundance of CD14++IL1ß+ monocytes in the ERS. CD4+ T cells and CD8+ T cells decreased significantly and expressed high levels of inflammatory genes in the ERS. Among the B cells, the plasma cells increased remarkably, whereas the naïve B cells decreased. Several novel B cell-receptor (BCR) changes were identified, such as IGHV3-23 and IGHV3-7, and isotypes (IGHV3-15, IGHV3-30, and IGKV3-11) previously used for virus vaccine development were confirmed. The strongest pairing frequencies, IGHV3-23-IGHJ4, indicated a monoclonal state associated with SARS-CoV-2 specificity, which had not been reported yet. Furthermore, integrated analysis predicted that IL-1ß and M-CSF may be novel candidate target genes for inflammatory storm and that TNFSF13, IL-18, IL-2, and IL-4 may be beneficial for the recovery of COVID-19 patients. Our study provides the first evidence of an inflammatory immune signature in the ERS, suggesting COVID-19 patients are still vulnerable after hospital discharge. Identification of novel BCR signaling may lead to the development of vaccines and antibodies for the treatment of COVID-19.

Efficacy of dendritic cell-based immunotherapy produced from cord blood in vitro and in a humanized NSG mouse cancer model.

AIM: To produce dendritic cells (DCs) from CD34+ stem cells from cord blood and explore their prophylactic and curative effect against tumors by vaccinating humanized NSG mice. MATERIALS & METHODS: Separated CD34+ stem cells from cord blood were cultured for 30 days, and the resultant DCs (CD34-DCs) were collected. The basic function of the CD34-DCs and the cytotoxicity of CD34-cytotoxic-T lymphocytes (CTLs) were tested in vitro, and tumor inhibition in a humanized NSG mouse tumor model was observed. RESULTS: The number of CD34-DCs reached approximately 9 log. These cells performed functions similar to those of DCs derived from monocytes from peripheral blood (PBMC-DCs). The CTLs of the CD34-DCs (CD34-CTLs) presented a better antitumor effect in vitro. The obvious prophylactic and therapeutic antitumor effects of the CD34-DC vaccine were observed in the humanized NSG mouse models. CONCLUSION: CD34-DCs from cord blood were sufficient in quantity and quality as a vaccine agent against tumors in vitro and in vivo.