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*

OBJECTIVES: This study aims to investigate the genome-wide DNA methylation and transcriptome expression profiles of peripheral blood mononuclear cells (PBMCs) in patients with systemic sclerosis (SSc) with interstitial lung disease (ILD), and to analyze the effects of DNA methylation on Wnt/β-catenin and chemokine signaling pathways. METHODS: PBMCs were collected from 19 patients with SSc (SSc group) and 18 healthy persons (control group). Among SSc patients, there were 10 patients with ILD (SSc with ILD subgroup) and 9 patients without ILD (SSc without ILD subgroup). The genome-wide DNA methylation and gene expression level were analyzed by using Illumina 450K methylation chip and Illumina HT-12 v4.0 gene expression profiling chip. The effect of DNA methylation on Wnt/β-catenin and chemokine signal pathways was investigated. RESULTS: Genome-wide DNA methylation analysis identified 71 hypermethylated CpG sites and 98 hypomethylated CpG sites in the SSc with ILD subgroup compared with the SSc without ILD subgroup. Transcriptome analysis distinguished 164 upregulated genes and 191 downregulated genes in the SSc with ILD subgroup as compared with the SSc without ILD subgroup. In PBMCs of the SSc group, 35 genes in Wnt/β-catenin signaling pathway were hypomethylated, while frizzled-1 (FZD1), mitogen-activated protein kinase 9 (MAPK9), mothers against DPP homolog 2 (SMAD2), transcription factor 7-like 2 (TCF7L2), and wingless-type MMTV integration site family, member 5B (WNT5B) mRNA expressions were upregulated as compared with the control group (all P<0.05). Compared with the SSc without ILD subgroup, the mRNA expressions of dickkopf homolog 2 (DKK2), FZD1, MAPK9 were upregulated in the SSc with ILD subgroup, but the differences were not statistically significant (all P>0.05). In PBMCs of the SSc group, 38 genes in chemokine signaling pathway were hypomethylated, while β-arrestin 1 (ARRB1), C-X-C motif chemokine ligand 10 (CXCL10), C-X-C motif chemokine ligand 16 (CXCL16), FGR, and neutrophil cytosolic factor 1C (NCF1C) mRNA expressions were upregulated as compared with the control group (all P<0.05). Compared with the SSc without ILD subgroup, the mRNA expressions of ARRB1, CXCL10, CXCL16 were upregulated in the SSc with ILD subgroup, but the differences were not statistically significant (all P>0.05). CONCLUSIONS There are differences in DNA methylation and transcriptome profiles between SSc with ILD and SSc without ILD. The expression levels of multiple genes in Wnt/β- catenin and chemokine signaling pathways are upregulated, which might be associatea with the pathogenesis of SSc.
Humans ; DNA Methylation ; Transcriptome ; beta Catenin ; Leukocytes, Mononuclear ; Ligands ; DNA ; RNA, Messenger/genetics*

In order to prevent the maternal immune defenses to the semi-allogeneic fetus, the maternal body will present a special adaptive immune system change represented by acute thymic involution(ATI) during pregnancy, which can be quickly regenerated after delivery. The ATI during pregnancy is related to the level of sex hormones, which is mainly caused by progesterone. Pregnancy-induced ATI is manifested as the continuous shrinkage of thymus volume, especially the cortex, and the wrinkle and phagocytosis of the subcapsular cortical thymic epithelial cells(cTECs), while other thymic epithelial cells(TECs) remain unchanged. The postpartum thymus is regenerated by the co-mediation of forkhead box N1(FOXN1) as well as its target genes chemokine(C-C motif) ligand 25(CCL25), chemokine(C-X-C motif) ligand 12(CXCL12), δ-like ligand 4(DLL4), cathepsin L(CTSL), and serine protease 16(PRSS16). Once the postpartum thymus is poorly repaired, immune dysfunction of the maternal body and several puerperal diseases will be induced, seriously endangering the survival of the mother and the newborn. In traditional Chinese medicine(TCM), Qi and blood are the cornerstone of pregnancy, and the thymus plays a key role in regulating Qi and blood. The deficiency of Qi and blood during pregnancy and childbirth is closely related to the abnormal ATI during pregnancy and the poor regeneration of the postpartum thymus. Based on this theory, TCM has profound academic ideas and rich clinical experience in postpartum recuperation. Based on the systematic description of the mechanism of ATI regeneration during pregnancy, as well as data mining and analysis of two classic gynecological works of TCM, Wan's Gynecology and Fu Qing-zhu's Treatise on Gynecology, this study found that the commonly used TCM for postpartum included Angelicae Sinensis Radix, Ginseng Radix et Rhizoma, Glycyrrhizae Radix et Rhizoma, and Chuanxiong Rhizoma. Among them, Ginseng Radix et Rhizoma, Angelicae Sinensis Radix, and Chuanxiong Rhizoma are high-frequency TCMs with positive effects on postpartum recovery.However, the mechanism of these TCMs in promoting postpartum thymus regeneration needs further investigation.
Female ; Infant, Newborn ; Humans ; Pregnancy ; Ligands ; Drugs, Chinese Herbal/pharmacology* ; Medicine, Chinese Traditional ; Prescriptions ; Postpartum Period ; Chemokines

The Baimai Ointment with the effect of relaxing sinew and activating collaterals demonstrates a definite effect on Baimai disease with pain, spasm, stiffness and other symptoms, while the pharmacodynamic characteristics and mechanism of this agent remain unclear. In this study, a rat model of chronic compression of L4 dorsal root ganglion(CCD) was established by lumbar disc herniation, and the efficacy and mechanism of Baimai Ointment in the treatment of CCD were preliminarily explored by behavioral tests, side effect evaluation, network analysis, antagonist and molecular biology verification. The pharmacodynamic experiment indicated that Baimai Ointment significantly improved the pain thresholds(mechanical pain, thermal pain, and cold pain) and gait behavior of CCD model rats without causing tolerance or obvious toxic and side effects. Baimai Ointment inhibited the second-phase nociceptive response of mice in the formalin test, increased the hot plate threshold of normal mice, and down-regulated the expression of inflammatory cytokines in the spinal cord. Network analysis showed that Baimai Ointment had synergistic effect in the treatment of CCD and was related to descending inhibition/facilitation system and neuroinflammation. Furthermore, behavioral tests, Western blot, and immunofluorescence assay revealed that the pain-relieving effect of Baimai Ointment on CCD may be related to the regulation of the interaction between neuroactive ligand and receptors(neuroligands) such as CHRNA7, ADRA2A, and ADRB2, and the down-regulation of the expression of NOS2/pERK/PI3K, the core regulatory element of HIF-1 signaling pathway in spinal microglia. The findings preliminarily reveal the mechanism of relaxing sinew and activating collaterals of Baimai Ointment in the treatment of Baimai disease, providing a reference for the rational drug use and further research of this agent.
Rats ; Mice ; Animals ; Chronic Pain/metabolism* ; Rats, Sprague-Dawley ; Ganglia, Spinal/metabolism* ; Ligands ; Signal Transduction ; Hyperalgesia/metabolism* ; Drugs, Chinese Herbal

In resting platelets, the 17 ^th domain of filamin a (FLNa17) constitutively binds to the platelet membrane glycoprotein Ibα (GPIbα) at its cytoplasmic tail (GPIbα-CT) and inhibits the downstream signal activation, while the binding of ligand and blood shear force can activate platelets. To imitate the pull force transmitted from the extracellular ligand of GPIbα and the lateral tension from platelet cytoskeleton deformation, two pulling modes were applied on the GPIbα-CT/FLNa17 complex, and the molecular dynamics simulation method was used to explore the mechanical regulation on the affinity and mechanical stability of the complex. In this study, at first, nine pairs of key hydrogen bonds on the interface between GPIbα-CT and FLNa17 were identified, which was the basis for maintaining the complex structural stability. Secondly, it was found that these hydrogen bonding networks would be broken down and lead to the dissociation of FLNa17 from GPIbα-CT only under the axial pull force; but, under the lateral tension, the secondary structures at both terminals of FLNa17 would unfold to protect the interface of the GPIbα-CT/FLNa17 complex from mechanical damage. In the range of 0~40 pN, the increase of pull force promoted outward-rotation of the nitrogen atom of the 563 ^rd phenylalanine (PHE ⁵⁶³-N) at GPIbα-CT and the dissociation of the complex. This study for the first time revealed that the extracellular ligand-transmitted axial force could more effectively relieve the inhibition of FLNa17 on the downstream signal of GPIbα than pure mechanical tension at the atomic level, and would be useful for further understanding the platelet intracellular force-regulated signal pathway.
Filamins/metabolism* ; Platelet Glycoprotein GPIb-IX Complex/metabolism* ; Molecular Dynamics Simulation ; Ligands ; Protein Binding ; Blood Platelets/metabolism* ; von Willebrand Factor/metabolism*

CD226 is an activated receptor on the surface of natural killer (NK) cells. It competes with TIGIT and CD96 to bind to ligands such as CD155 on the surface of tumor cells and mediates the killing function of NK cells. Although TIGIT and CD96 have other binding ligands in the tumor microenvironment, they compete to bind CD115 ligands with higher affinity and inhibit the activity of NK cells, which allows tumor cells to evade killing. Therefore, studying the expression patterns of these three NK cell surface receptors in different tumors and monitoring their binding ability with ligands will help us to explore new tumor treatment strategies. This article reviews the role and mechanism of CD226, TIGIT, CD96 and other NK cell receptor molecules in regulating NK cell function in anti-tumor immune response.
Ligands ; Receptors, Immunologic ; Receptors, Natural Killer Cell ; Killer Cells, Natural ; Antigens, CD

Lectins are proteins responsible for recognizing the signals of sugar molecules in the body. Sialic acid-binding immunoglobulin-like lectins (Siglecs) regulate the innate and adaptive immune responses in the tumor microenvironment by recognizing the glycan structure containing sialic acid and mediating downstream signals through immune receptor tyrosine inhibitory motifs. In recent years, a variety of tumor treatment strategies targeting the sialic acid-Siglecs axis have been introduced, including sialoglycoprotein-mediated drug delivery and antibody mediated inhibition of Siglecs from recognizing tumor surface ligands. In the future, by combining with glycoprotein nanotherapy, antibody therapy and gene therapy, Siglecs can be used to accurately locate tumor targets and release the anti-tumor immunity, so as to achieve the purpose of effective cure of tumors.
Sialic Acid Binding Immunoglobulin-like Lectins/metabolism* ; N-Acetylneuraminic Acid ; Immunoglobulins/metabolism* ; Receptors, Immunologic ; Ligands

OBJECTIVE: To investigate the significance of Tim-3 and Galectin-9 in Th1/Th2 imbalance in patients with multiple myeloma (MM). METHODS: 55 newly diagnosed MM patients and 20 healthy controls were included. Flow cytometry was used to detect the expression of Tim-3 on CD4⁺T cells, the proportion of Th1, Th2, Tim-3⁺Th1 and Tim-3⁺Th2 cells in peripheral blood. ELISA was used to detect the levels of cytokines IFN-γ and IL-4 in serum, and PCR was used to detect the level of Galectin-9 mRNA. Then the correlations between Galectin-9 mRNA expression and Th-cell subsets and related cytokine levels, as well as the relationship between Tim-3⁺Th1/Tim-3⁺Th2 ratio and corresponding clinical features were analyzed. RESULTS: Compared with the control group, the expression of Tim-3 on CD4⁺T cells in peripheral blood of MM patients was significantly increased (P<0.05), the proportions of Tim-3⁺Th1 cells, Tim-3⁺Th2 cells and Tim-3⁺Th1/Tim-3⁺Th2 ratio in MM patients were also increased (P<0.05), while the proportion of Th1 cells and Th1/Th2 ratio in MM patients were significantly decreased (P<0.05). The level of cytokine IFN-γ and IFN-γ/IL-4 ratio in MM patients were significantly decreased (P<0.05), while the level of cytokine IL-4 was increased (P<0.05). The mRNA levels of Galectin-9 in MM patients were significantly increased (P<0.05). The levels of Galectin-9 mRNA were positively correlated with Tim-3⁺CD4⁺T cells (r=0.663), Tim-3⁺Th2 cells (r=0.492) and IL-4 (r=0.470), while negatively correlated with IFN-γ (r=-0.593). The ratios of Tim-3⁺Th1/Tim-3⁺Th2 in MM patients were positively correlated with ISS stage (r=0.511), osteolytic damage (r=0.556) and chromosome abnormality (r=0.632). CONCLUSION These results suggest that Tim-3 and Galectin-9 are involved in Th1/Th2 imbalance in MM patients, and the high ratio of Tim-3⁺Th1/Tim-3⁺Th2 is associated with poor clinical prognosis.
Humans ; Cytokines/metabolism* ; Galectins/metabolism* ; Hepatitis A Virus Cellular Receptor 2/metabolism* ; Interleukin-4/metabolism* ; Ligands ; Multiple Myeloma/metabolism* ; RNA, Messenger/metabolism* ; Th1 Cells/metabolism* ; Th2 Cells/metabolism*

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*