Combined therapy of CAR-IL-15/IL-15Rα-T cells and GLIPR1 knockdown in cancer cells enhanced anti-tumor effect against gastric cancer.

BACKGROUND: Chimeric antigen receptor (CAR) T cell therapy has shown remarkable responses in hematological malignancies with several approved products, but not in solid tumors. Patients suffer from limited response and tumor relapse due to low efficacy of CAR-T cells in the complicated and immunosuppressive tumor microenvironment. This clinical challenge has called for better CAR designs and combined strategies to improve CAR-T cell therapy against tumor changes. METHODS: In this study, IL-15/IL-15Rα was inserted into the extracellular region of CAR targeting mesothelin. In-vitro cytotoxicity and cytokine production were detected by bioluminescence-based killing and ELISA respectively. In-vivo xenograft mice model was used to evaluate the anti-tumor effect of CAR-T cells. RNA-sequencing and online database analysis were used to identify new targets in residual gastric cancer cells after cytotoxicity assay. CAR-T cell functions were detected in vitro and in vivo after GLI Pathogenesis Related 1 (GLIPR1) knockdown in gastric cancer cells. Cell proliferation and migration of gastric cancer cells were detected by CCK-8 and scratch assay respectively after GLIPR1 were overexpressed or down-regulated. RESULTS: CAR-T cells constructed with IL-15/IL-15Rα (CAR-ss-T) showed significantly improved CAR-T cell expansion, cytokine production and cytotoxicity, and resulted in superior tumor control compared to conventional CAR-T cells in gastric cancer. GLIPR1 was up-regulated after CAR-T treatment and survival was decreased in gastric cancer patients with high GLIPR1 expression. Overexpression of GLIPR1 inhibited cytotoxicity of conventional CAR-T but not CAR-ss-T cells. CAR-T treatment combined with GLIPR1 knockdown increased anti-tumor efficacy in vitro and in vivo. CONCLUSIONS: Our data demonstrated for the first time that this CAR structure design combined with GLIPR1 knockdown in gastric cancer improved CAR-T cell-mediated anti-tumor response.

Cardiac glycosides from Streblus asper with potential antiviral activity.

Ten undescribed cardiac glycosides, strasperosides A-J, together with twelve known analogues, were isolated from Streblus asper Lour. Their structures were elucidated on the basis of spectroscopic analysis, electronic circular dichroism data, and chemical methods. These cardiac glycosides showed diversity in steroid skeleton and sugar moiety. Strasperosides A and B are a pair of unusual stereoisomers featuring different orientation of the lactone motif. Ten cardiac glycosides demonstrated potent antiviral effects on HSV-1 in vitro with the IC50 values from 0.19 ± 0.08 to 1.03 ± 0.25 µM and the therapeutic indices from 66.61 ± 5.08 to 326.75 ± 11.75.

Notch Signaling Regulates the Function and Phenotype of Dendritic Cells in Helicobacter pylori Infection.

Notch signaling manipulates the function and phenotype of dendritic cells (DCs), as well as the interaction between DCs and CD4+ T cells. However, the role of Notch signaling in Helicobacter pylori (H. pylori) infection remains elusive. Murine bone marrow-derived dendritic cells (BMDCs) were pretreated in the absence or presence of Notch signaling inhibitor DAPT prior to H. pylori stimulation and the levels of Notch components, cytokines and surface markers as well as the differentiation of CD4+ T cells in co-culture were measured using quantitative real-time PCR (qRT-PCR), Western blot, enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Compared with the control, the mRNA expression of all Notch receptors and Notch ligands Dll4 and Jagged1 was up-regulated in H. pylori-stimulated BMDCs. The blockade of Notch signaling by DAPT influenced the production of IL-1ß and IL-10 in H. pylori-pulsed BMDCs, and reduced the expression of Notch1, Notch3, Notch4, Dll1, Dll3 and Jagged2. In addition, DAPT pretreatment decreased the expression of maturation markers CD80, CD83, CD86, and major histocompatibility complex class II (MHC-II) of BMDCs, and further skewed Th17/Treg balance toward Treg. Notch signaling regulates the function and phenotype of DCs, thus mediating the differentiation of CD4+ T cells during H. pylori infection.

Screening the pathogenic causes of congenital cataract via whole exome sequencing technology in three families: Molecular genetics of congenital cataract.

Congenital cataract is the commonest cause of visual impairment and blindness in children worldwide. Among congenital cataract cases, ~25% are caused by genetic defects, while several genetic mutations have been identified in hereditary cataract. In the present study, a patient with cataract underwent clinical ophthalmic examination and pedigree analysis. Whole exome sequencing and Sanger sequencing were performed to identify and verify gene mutations. The frequency, conservation, pathogenicity and hydrophobicity of the mutated amino acids were analyzed by bioinformatics analysis. The clinical examination and investigation verified that the probands of family A and C suffered from nuclear cataracts. In addition, the proband of family B was diagnosed with white punctate opacity. The pattern of inheritance was autosomal dominant. The sequencing analysis results revealed a mutation c.592-c593insG (p.W198Wfs*22) in exon 6 of CRYBA1/A3, a known mutation c.463C > T (p.Q155X) in exon 6 of CRYBB2 and a third mutation c.865­c.866insC (p.T289Tfs*91) in exon 2 of GJA8. Each variant was co­segregated with disease in family And the mutation frequency in the database was <0.01. It has been reported that the mutation sites are highly conserved among different species, thus greatly affecting the sequence and structure of a protein, while exhibiting high pathogenicity in theory. The two crystallin gene mutations could notably enhance the local hydrophobicity of the protein, eventually resulting in its reduced solubility and destruction of lens transparency. The current study identified pathogenic genes in three families with congenital cataract and analyzed the association between mutation sites and different cataract phenotypes. Overall, the results could expand the genotype spectrum of congenital cataract and provide evidence for its clinical diagnosis.