CD20 is a surface marker protein of B-cell lymphoma, and its extracellular region is the target of specific antibodies and drugs. To obtain a cheap and easily modified specific preparation targeting CD20, we optimized the gene of CD20 extracellular region according to codon degeneracy to facilitate its expression in Escherichia coli. The optimized gene was cloned into pGEX-4T-1 vector, and the recombinant vector was transformed into E. coli BL21(DE3) for expression. The purified protein was identified by SDS-PAGE and Western blotting. Systematic evolution of ligands by exponential enrichment (SELEX) was employed to screen the ssDNA aptamer that specifically binds to the fusion protein, and the affinity of the aptamer to CD20 was detected by flow cytometry. Then, the cytotoxicity test was carried out to examine the inhibitory effect of the aptamer on B lymphoma cells. In this study, we established the prokaryotic expression method of CD20 and obtained the aptamer specifically binding to the extracellular region of CD20, which laid a foundation for the development of therapeutic drugs targeting CD20.
Humans ; Escherichia coli/metabolism* ; SELEX Aptamer Technique/methods* ; Aptamers, Nucleotide/genetics* ; Antigens, CD20/metabolism* ; Ligands

Neuronomodulation refers to the modulation of neural conduction and synaptic transmission (i.e., the conduction process involved in synaptic transmission) of excitable neurons via changes in the membrane potential in response to chemical substances, from spillover neurotransmitters to paracrine or endocrine hormones circulating in the blood. Neuronomodulation can be direct or indirect, depending on the transduction pathways from the ligand binding site to the ion pore, either on the same molecule, i.e. the ion channel, or through an intermediate step on different molecules. The major players in direct neuronomodulation are ligand-gated or voltage-gated ion channels. The key process of direct neuronomodulation is the binding and chemoactivation of ligand-gated or voltage-gated ion channels, either orthosterically or allosterically, by various ligands. Indirect neuronomodulation involves metabotropic receptor-mediated slow potentials, where steroid hormones, cytokines, and chemokines can implement these actions. Elucidating neuronomodulation is of great significance for understanding the physiological mechanisms of brain function, and the occurrence and treatment of diseases.
Ligands ; Neurons/metabolism* ; Synaptic Transmission/physiology* ; Ion Channels/metabolism* ; Hormones/metabolism*

We identified distinct senescence-related molecular subtypes and critical genes among prostate cancer (PCa) patients undergoing radical prostatectomy (RP) or radical radiotherapy (RT). We conducted all analyses using R software and its suitable packages. Twelve genes, namely, secreted frizzled-related protein 4 (SFRP4), DNA topoisomerase II alpha (TOP2A), pleiotrophin (PTN), family with sequence similarity 107 member A (FAM107A), C-X-C motif chemokine ligand 14 (CXCL14), prostate androgen-regulated mucin-like protein 1 (PARM1), leucine zipper protein 2 (LUZP2), cluster of differentiation 38 (CD38), cartilage oligomeric matrix protein (COMP), vestigial-like family member 3 (VGLL3), apolipoprotein E (APOE), and aldehyde dehydrogenase 2 family member (ALDH2), were eventually used to subtype PCa patients from The Cancer Genome Atlas (TCGA) database and GSE116918, and the molecular subtypes showed good correlations with clinical features. In terms of the tumor immune environment (TME) analysis, compared with cluster 1, cancer-associated fibroblasts (CAFs) scored significantly higher, while endothelial cells scored lower in cluster 2 in TCGA database. There was a statistically significant correlation between both CAFs and endothelial cells with biochemical recurrence (BCR)-free survival for PCa patients undergoing RP. For the GSE116918 database, cluster 2 had significantly lower levels of CAFs and tumor purity and higher levels of stromal, immune, and Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression data (ESTIMATE) scores than cluster 1; in addition, patients with high levels of CAFs, stromal scores, immune scores, and ESTIMATE scores and low levels of tumor purity tended to suffer from BCR. Based on the median of differentially expressed checkpoints, high expression of CD96, hepatitis A virus cellular receptor 2 (HAVCR2), and neuropilin 1 (NRP1) in GSE116918 and high expression of CD160 and tumor necrosis factor (ligand) superfamily member 18 (TNFSF18) in TCGA database were associated with a significantly higher risk of BCR than their counterparts. In conclusion, we first constructed distinct molecular subtypes and critical genes for PCa patients undergoing RP or RT from the fresh perspective of senescence.
Male ; Humans ; Endothelial Cells ; Ligands ; Prostatic Neoplasms/pathology* ; Prostate/pathology* ; Prostatectomy ; Aldehyde Dehydrogenase, Mitochondrial ; DNA-Binding Proteins ; Transcription Factors

OBJECTIVES: To study the expression levels of CD4⁺NKG2D⁺ T cells and NKG2D soluble ligands, the soluble MHC class I chain-related molecules A and B (sMICA/sMICB) in the active stage and stable stage of juvenile idiopathic arthritis (JIA) and their role in the disease activity of JIA. METHODS: Nineteen children with systemic JIA and 20 children with articular JIA who were diagnosed in Children's Hospital of Chongqing Medical University from November 2019 to December 2021 were enrolled in this prospective study. Six healthy children were enrolled as the control group. After peripheral blood samples were collected, ELISA was used to measure the levels of sMICA and sMICB, and flow cytometry was used to measure the percentage of CD4⁺NKG2D⁺ T cells. Systemic Juvenile Arthritis Disease Activity Score-27 (sJADAS-27)/Juvenile Arthritis Disease Activity Score-27 (JADAS-27) was used to evaluate the disease activity in children with JIA. The Pearson correlation analysis and the receiver operating characteristic (ROC) curve were used to assess the role of CD4⁺NKG2D⁺ T cells, sMICA and sMICB in the disease activity of JIA. RESULTS: The active systemic JIA and active articular JIA groups had a significant increase in the percentage of CD4⁺NKG2D⁺ T cells compared with the control group and their corresponding inactive JIA group (P<0.05). The JIA groups had significantly higher levels of sMICA and sMICB than the control group (P<0.05), and the active articular JIA group had a significantly higher level of sMICB than the stable articular JIA group (P<0.05). In the children with JIA, the percentage of CD4⁺NKG2D⁺ T cells and the levels of sMICA and sMICB were positively correlated with sJADAS-27/JADAS-27 disease activity scores (P<0.05). The ROC curve analysis showed that sMICB had an area under the curve of 0.755 in evaluating the disease activity of JIA, with a specificity of 0.90 and a sensitivity of 0.64. CONCLUSIONS The percentage of CD4⁺NKG2D⁺ T cells and the levels of sMICA and sMICB increase in children with JIA compared with healthy children and are positively correlated with the disease activity of JIA, suggesting that CD4⁺NKG2D⁺ T cells and NKG2D ligands can be used as potential biomarkers for evaluating the disease activity of JIA.
Child ; Humans ; Arthritis, Juvenile/pathology* ; Ligands ; NK Cell Lectin-Like Receptor Subfamily K ; Prospective Studies ; T-Lymphocytes/pathology*

BACKGROUND: Ischemic acute kidney injury (AKI) is a common syndrome associated with considerable mortality and healthcare costs. Up to now, the underlying pathogenesis of ischemic AKI remains incompletely understood, and specific strategies for early diagnosis and treatment of ischemic AKI are still lacking. Here, this study aimed to define the transcriptomic landscape of AKI patients through single-cell RNA sequencing (scRNA-seq) analysis in kidneys. METHODS: In this study, scRNA-seq technology was applied to kidneys from two ischemic AKI patients, and three human public scRNA-seq datasets were collected as controls. Differentially expressed genes (DEGs) and cell clusters of kidneys were determined. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, as well as the ligand-receptor interaction between cells, were performed. We also validated several DEGs expression in kidneys from human ischemic AKI and ischemia/reperfusion (I/R) injury induced AKI mice through immunohistochemistry staining. RESULTS: 15 distinct cell clusters were determined in kidney from subjects of ischemic AKI and control. The injured proximal tubules (PT) displayed a proapoptotic and proinflammatory phenotype. PT cells of ischemic AKI had up-regulation of novel pro-apoptotic genes including USP47 , RASSF4 , EBAG9 , IER3 , SASH1 , SEPTIN7 , and NUB1 , which have not been reported in ischemic AKI previously. Several hub genes were validated in kidneys from human AKI and renal I/R injury mice, respectively. Furthermore, PT highly expressed DEGs enriched in endoplasmic reticulum stress, autophagy, and retinoic acid-inducible gene I (RIG-I) signaling. DEGs overexpressed in other tubular cells were primarily enriched in nucleotide-binding and oligomerization domain (NOD)-like receptor signaling, estrogen signaling, interleukin (IL)-12 signaling, and IL-17 signaling. Overexpressed genes in kidney-resident immune cells including macrophages, natural killer T (NKT) cells, monocytes, and dendritic cells were associated with leukocyte activation, chemotaxis, cell adhesion, and complement activation. In addition, the ligand-receptor interactions analysis revealed prominent communications between macrophages and monocytes with other cells in the process of ischemic AKI. CONCLUSION Together, this study reveals distinct cell-specific transcriptomic atlas of kidney in ischemic AKI patients, altered signaling pathways, and potential cell-cell crosstalk in the development of AKI. These data reveal new insights into the pathogenesis and potential therapeutic strategies in ischemic AKI.
Humans ; Mice ; Animals ; Transcriptome/genetics* ; Ligands ; Kidney/metabolism* ; Acute Kidney Injury/metabolism* ; Ischemia/metabolism* ; Reperfusion Injury/metabolism* ; Sequence Analysis, RNA ; Adaptor Proteins, Signal Transducing/metabolism* ; Tumor Suppressor Proteins/metabolism*

This study was aimed to investigate the effect of hypoxia on lipopolysaccharide (LPS)-induced CXC-chemokine ligand-10 (CXCL10) expression and the underlying mechanism. C57BL/6J mice were randomly divided into control, hypoxia, LPS, and hypoxia combined with LPS groups. The LPS group was intraperitoneally injected with 0.5 mg/kg LPS, and the hypoxia group was placed in a hypobaric hypoxia chamber (simulated altitude of 6 000 m). The serum and hippocampal tissue samples were collected after 6 h of the treatment. The levels of CXCL10 in the serum and hippocampal tissue of mice were detected by ELISA. The microglia cell line BV2 and primary microglia were stimulated with hypoxia (1% O₂) and/or LPS (100 ng/mL) for 6 h. The mRNA expression level of CXCL10 and its content in culture supernatant were detected by real-time quantitative PCR and ELISA, respectively. The phosphorylation levels of nuclear factor κB (NF-κB) signaling pathway-related proteins, p65 and IκBα, were detected by Western blot. Moreover, after NF-κB signaling pathway being blocked with a small molecular compound, PDTC, CXCL10 mRNA expression level was detected in the BV2 cells. The results showed that in the LPS-induced mouse inflammatory model, hypoxia treatment could promote LPS-induced up-regulation of CXCL10 in both serum and hippocampus. Compared with the cells treated with LPS alone, the expression of CXCL10 mRNA and the content of CXCL10 in the culture supernatant of BV2 cells treated with hypoxia combined with LPS were significantly increased. The CXCL10 mRNA level of primary microglial cells treated with hypoxia combined with LPS was significantly up-regulated. Compared with the cells treated with hypoxia or LPS alone, the phosphorylation levels of p65 and IκBα in the BV2 cells treated with hypoxia combined with LPS were significantly increased. PDTC blocked the induction of CXCL10 gene expression by LPS in the BV2 cells. These results suggest that hypoxia promotes LPS-induced expression of CXCL10 in both animal and cell models, and NF-κB signaling pathway plays an important role in this process.
Animals ; Mice ; Chemokines, CXC/pharmacology* ; Hypoxia ; Ligands ; Lipopolysaccharides/pharmacology* ; Mice, Inbred C57BL ; Microglia/metabolism* ; NF-kappa B/metabolism* ; NF-KappaB Inhibitor alpha/pharmacology* ; RNA, Messenger/metabolism*

In this study, the authors cloned a glycosyltransferase gene PpUGT2 from Paris polyphylla var. yunnanensis with the ORF length of 1 773 bp and encoding 590 amino acids. The phylogenetic tree revealed that PpUGT2 belonged to the UGT80A subfamily and was named as UGT80A49 by the UDP-glycosyltransferase(UGT) Nomenclature Committee. The expression vector pET28a-PpUGT2 was constructed, and enzyme catalytic reaction in vitro was conducted via inducing protein expression and extraction. With UDP-glucose as sugar donor and diosgenin and pennogenin as substrates, the protein was found with the ability to catalyze the C-3 hydroxyl β-glycosylation of diosgenin and pennogenin. To further explore its catalytic characteristic, 15 substrates including steroids and triterpenes were selected and PpUGT2 showed its activity towards the C-17 position of sterol testosterone with UDP-glucose as sugar donor. Homology modelling and molecule docking of PpUGT2 with substrates predicted the key residues interacting with ligands. The re-levant residues of PpUGT2-ligand binding model were scanned to calculate the corresponding mutants, and the optimized mutants were obtained according to the changes in binding affinity of the ligand with protein and the surrounding residues within 5.0 Å of ligands, which had reference value for design of the mutants. This study laid a foundation for further exploring the biosynthetic pathway of polyphyllin as well as the structure of sterol glycosyltransferases.
Ligands ; Glycosyltransferases/genetics* ; Sterols ; Phylogeny ; Ascomycota ; Liliaceae/chemistry* ; Melanthiaceae ; Diosgenin ; Sugars ; Glucose ; Uridine Diphosphate

OBJECTIVE: To compare the consistency of programmed cell death 1-ligand 1 (PD-L1, clone E1L3N, 22C3, SP263) in different immunohistochemical staining methods. METHODS: The first step was to select the optimal process: The PD-L1(clone E1L3N) antibody recommended process, self-built process ①, self-built process ② and self-built process ③ were used to perform immunohistochemical staining in 5 cases of tonsil tissue. The quality of all slides was scored by expert pathologists (0-6 points). The process with the highest score was selected. The second step was to compare the consistency between the optimal procedure and the two standard procedures. Thirty-two cases of lung non-small cell carcinoma diagnosed by pathology in Peking University First Hospital in the past two years were randomly selected. The 32 cases were stained in parallel with the SP263 and 22C3 standard procedures, and all stained slides were scored by specialized pathologists for tumor proportion score (TPS). The scoring results were grouped according to < 1%, ≥1% to < 10%, ≥10% to < 50%, and ≥50%. The consistency of PD-L1 detection antibody clone E1L3N and 22C3, E1L3N and SP263 staining results was analyzed. RESULTS: Tonsil stained slides scores (0-6 points) were as follows: The recommended protocol was 5, 5, 5, 5 and 5. The self-built process ① was 5, 6, 6, 5 and 6. The self-built process ② was 4, 4, 4, 4 and 4.The self-built process ③ was 3, 3, 3, 3 and 3. The self-built process ① was the best with the highest score. The TPSs of 32 non small cell lung carcinoma (NSCLC) cases were as follows: Of self-built process ①, 6 cases were lower than 1%, 5 cases were from 1% to 10%, 10 cases were from 10% to 50%, and 11 cases were higher than 50%; of 22C3 standard procedure, 5 cases were lower than 1%, 3 cases were from 1% to 10%, 13 cases were from 10% to 50%, 11 cases were higher than 50%; of SP263 standard procedure, 7 cases were lower than 1%, 4 cases were from 1% to 10%, 11 cases were from 10% to 50%, 10 cases were higher than 50%. The results of the consistency test were as follows: The κ value for self-built process ① and 22C3 standard procedure was 0.736 (P < 0.001), the agreement was good; the κ value for self-built process ① and SP263 standard procedure was 0.914 (P < 0.001), the agreement was very good. CONCLUSION The immunostaining using PD-L1(E1L3N) with validated self-built staining protocol ① by Ventana Benchmark GX platform can obtain high quality of slides, and the TPSs based on these slides are in good agreement with 22C3 and SP263 standard procedures.
Humans ; Carcinoma, Non-Small-Cell Lung ; Lung Neoplasms/pathology* ; Immunohistochemistry ; B7-H1 Antigen/metabolism* ; Ligands ; Antibodies ; Staining and Labeling ; Apoptosis

OX40 is a costimulatory receptor that is expressed primarily on activated CD4⁺, CD8⁺, and regulatory T cells. The ligation of OX40 to its sole ligand OX40L potentiates T cell expansion, differentiation, and activation and also promotes dendritic cells to mature to enhance their cytokine production. Therefore, the use of agonistic anti-OX40 antibodies for cancer immunotherapy has gained great interest. However, most of the agonistic anti-OX40 antibodies in the clinic are OX40L-competitive and show limited efficacy. Here, we discovered that BGB-A445, a non-ligand-competitive agonistic anti-OX40 antibody currently under clinical investigation, induced optimal T cell activation without impairing dendritic cell function. In addition, BGB-A445 dose-dependently and significantly depleted regulatory T cells in vitro and in vivo via antibody-dependent cellular cytotoxicity. In the MC38 syngeneic model established in humanized OX40 knock-in mice, BGB-A445 demonstrated robust and dose-dependent antitumor efficacy, whereas the ligand-competitive anti-OX40 antibody showed antitumor efficacy characterized by a hook effect. Furthermore, BGB-A445 demonstrated a strong combination antitumor effect with an anti-PD-1 antibody. Taken together, our findings show that BGB-A445, which does not block OX40-OX40L interaction in contrast to clinical-stage anti-OX40 antibodies, shows superior immune-stimulating effects and antitumor efficacy and thus warrants further clinical investigation.
Mice ; Animals ; Receptors, Tumor Necrosis Factor/physiology* ; Receptors, OX40 ; Membrane Glycoproteins ; Ligands ; Antibodies, Monoclonal/pharmacology* ; Antineoplastic Agents/pharmacology*

OBJECTIVE: Aconite is a traditional Chinese herbal medicine that has been found to inhibit the development of liver cancer; however, its exact molecular mechanisms in this process remain unclear. This study explores how aconite aqueous extract (AAE) inhibits hepatocellular carcinoma (HCC). METHODS: An in vivo mouse model of subcutaneous liver cancer was established. After AAE treatment, immunohistochemistry (IHC) was used to determine the effect of AAE on natural killer (NK) cells. Subsequently, C57BL/6 mice were used to establish the subcutaneous tumor model, and a group of these mice were treated with anti-PK163 antibody to remove NK cells, which was verified by flow cytometry and IHC. The effect of AAE on the proliferation of HCC cells in vitro was determined using cell counting kit-8. The effect of AAE on chemokine production in HCC cells was measured using real-time quantitative polymerase chain reaction and an enzyme-linked immunosorbent assay. The effect of AAE on the migration of NK cells was determined using a transwell assay. Finally, the molecular mechanism was investigated using the Western blotting method. RESULTS: We demonstrated that the ability of AAE to induce overexpression of the cytokine C-C motif chemokine ligand 2 (CCL2) in HCC cells is fundamental to the infiltration of NK cells into the tumor bed. Mechanistically, we found that the upregulation of CCL2 was achieved by the activation of c-Jun N-terminal kinase but not extracellular regulated protein kinase or p38. CONCLUSION Our findings suggest that AAE can be used as an effective immune adjuvant to enhance antitumor immunity by increasing NK cell infiltration into tumors, which could help to improve the efficacy of HCC treatments. Please cite this article as: Yang KD, Zhang X, Shao MC, Wang LN. Aconite aqueous extract inhibits the growth of hepatocellular carcinoma through CCL2-dependent enhancement of natural killer cell infiltration. J Integr Med. 2023; 21(6): 575-583.
Animals ; Mice ; Carcinoma, Hepatocellular/drug therapy* ; Liver Neoplasms/drug therapy* ; Aconitum ; Ligands ; Mice, Inbred C57BL ; Killer Cells, Natural/metabolism* ; Chemokines/pharmacology* ; Cell Line, Tumor