The mechanism of macrophage-inducible C-type lectin involved in uremic microinflammatory state / 西安交通大学学报(医学版)

【Objective】 To explore the mechanism of macrophage-inducible C-type lectin (Mincle) and microinflammatory state in uremia. 【Methods】 SD rats were randomly divided into uremia group and sham operation group. The morphology and permeability of intestinal tissue, the morphology of intestinal tissue and macrophages were observed by transmission electron microscope, the expression of Mincle was detected in intestinal tissue sections, and the expressions of Toll-like receptor 4 (TLR-4) and NF-kappa B (NF-κB) protein on the surface of macrophages were detected by Western blotting. After the plasma was separated, the levels of endotoxin, C-reactive protein (CRP), interleulin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were detected by Limulus lysate dynamic turbidimetric assay, and enzyme-linked immunosorbent assay (ELISA). The data were analyzed with IBM SPSS19.0 software. 【Results】 The expression of Mincle in the jejunum, ileum, and colon in uremia group was higher than that in sham-operation group (P<0.05). The expressions of TLR4 and NF-κB protein significantly differed in the ileum, jejunum and colon in uremia group (P<0.001). The levels of endotoxin, CRP, IL-6, and TNF-α were significantly increased in uremia group compared with sham-operation group (P<0.05). 【Conclusion】 In uremia, Mincle on the surface of intestinal macrophages increases and further through TLR4/NF-κB pathway mediates the transformation of intestinal macrophages to M1 type, releasing inflammatory products and causing systemic microinflammation.

Research progress on the mechanism of role of podoplanin in sepsis / 中华危重病急救医学

Podoplanin (PDPN) is a small transmembrane mucin-like glycoprotein which is expressed on the surface of lymphatic endothelial cells, glomerular podocytes, type-Ⅰ alveolar epithelial cells and some tumor cells. PDPN plays crucial function in variety of physiological and pathological processes such as embryonic development, immunoreaction, inflammation and cancer. C-type lectin-like receptor 2 (CLEC2) is mainly expressed on the platelet which specific ligand is PDPN. The interaction between PDPN and CLEC2 has received extensive attention. In this review, we summarized recent researches on the role of in sepsis and elaborated the possible mechanisms and some potential therapies for sepsis by targeting PDPN, which may provide theoretical basis for the mechanism and treatment of sepsis.

Development and functional verification of CAR-T cells targeting CLL-1 / 中华血液学杂志

Objective: To explore the development of a CAR-T cells targeting CLL-1 and verify its function. Methods: The expression levels of CLL-1 targets in cell lines and primary cells were detected by flow cytometry. A CLL-1 CAR vector was constructed, and the corresponding lentivirus was prepared. After infection and activation of T cells, CAR-T cells targeting CLL-1 were produced and their function was verified in vitro and in vivo. Results: CLL-1 was expressed in acute myeloid leukemia (AML) cell lines and primary AML cells. The transduction rate of the prepared CAR T cells was 77.82%. In AML cell lines and AML primary cells, CLL-1-targeting CAR-T cells significantly and specifically killed CLL-1-expressing cells. Compared to untransduced T cells, CAR-T cells killed target cells and secreted inflammatory cytokines, such as interleukin-6 and interferon-γ, at significantly higher levels (P<0.001) . In an in vivo human xenograft mouse model of AML, CLL-1 CAR-T cells also exhibited potent antileukemic activity and induced prolonged mouse survival compared with untransduced T cells [not reached vs 22 days (95%CI 19-24 days) , P=0.002]. Conclusion: CAR-T cells targeting CLL-1 have been successfully produced and have excellent functions.

Neutrophils activated by BJcuL, a C-type lectin isolated from Bothrops jararacussu venom, decrease the invasion potential of neuroblastoma SK-N-SH cells in vitro

ABSTRACT Background: Neuroblastoma is a pediatric tumor with a mortality rate of 40% in the most aggressive cases. Tumor microenvironment components as immune cells contribute to the tumor progression; thereby, the modulation of immune cells to a pro-inflammatory and antitumoral profile could potentialize the immunotherapy, a suggested approach for high-risk patients. Preview studies showed the antitumoral potential of BJcuL, a C- type lectin isolated from Bothrops jararacussu venom. It was able to induce immunomodulatory responses, promoting the rolling and adhesion of leukocytes and the activation of neutrophils. Methods: SK-N-SH cells were incubated with conditioned media (CM) obtained during the treatment of neutrophils with BJcuL and fMLP, a bacteria-derived peptide highly effective for activating neutrophil functions. Then we evaluated the effect of the same stimulation on the co-cultivation of neutrophils and SK-N-SH cells. Tumor cells were tested for viability, migration, and invasion potential. Results: In the viability assay, only neutrophils treated with BJcuL (24 h) and cultivated with SK-N-SH were cytotoxic. Migration of tumor cells decreased when incubated directly (p 0.001) or indirectly (p 0.005) with untreated neutrophils. When invasion potential was evaluated, neutrophils incubated with BJcuL reduced the total number of colonies of SK-N-SH cells following co-cultivation for 24 h (p 0.005). Treatment with CM resulted in decreased anchorage-free survival following 24 h of treatment (p 0.001). Conclusion: Data demonstrated that SK-N-SH cells maintain their migratory potential in the face of neutrophil modulation by BJcuL, but their invasive capacity was significantly reduced.

Neutrophils activated by BJcuL, a C-type lectin isolated from Bothrops jararacussu venom, decrease the invasion potential of neuroblastoma SK-N-SH cells in vitro

Neuroblastoma is a pediatric tumor with a mortality rate of 40% in the most aggressive cases. Tumor microenvironment components as immune cells contribute to the tumor progression; thereby, the modulation of immune cells to a pro-inflammatory and antitumoral profile could potentialize the immunotherapy, a suggested approach for high-risk patients. Preview studies showed the antitumoral potential of BJcuL, a C- type lectin isolated from Bothrops jararacussu venom. It was able to induce immunomodulatory responses, promoting the rolling and adhesion of leukocytes and the activation of neutrophils. Methods: SK-N-SH cells were incubated with conditioned media (CM) obtained during the treatment of neutrophils with BJcuL and fMLP, a bacteria-derived peptide highly effective for activating neutrophil functions. Then we evaluated the effect of the same stimulation on the co-cultivation of neutrophils and SK-N-SH cells. Tumor cells were tested for viability, migration, and invasion potential. Results: In the viability assay, only neutrophils treated with BJcuL (24 h) and cultivated with SK-N-SH were cytotoxic. Migration of tumor cells decreased when incubated directly (p < 0.001) or indirectly (p < 0.005) with untreated neutrophils. When invasion potential was evaluated, neutrophils incubated with BJcuL reduced the total number of colonies of SK-N-SH cells following co-cultivation for 24 h (p < 0.005). Treatment with CM resulted in decreased anchorage-free survival following 24 h of treatment (p < 0.001). Conclusion: Data demonstrated that SK-N-SH cells maintain their migratory potential in the face of neutrophil modulation by BJcuL, but their invasive capacity was significantly reduced.(AU)

Research Progress of Receptors of Plant Polysaccharides / 中国药学杂志

Receptors of plant polysaccharides and their signaling pathways have become a hotspot of polysaccharide activity research. As immunomodulators, polysaccharides have attracted much attention due to their immunomodulatory effects mediated by cell surface receptors. Polysaccharide receptors mainly include C-type lectin receptor family, mannose receptor family (MR), Toll-like receptor family (TLR), complement receptor 3 (CR3), scavenger receptor and so on, and these receptors play an important role in the recognition between polysaccharides and cells, which is closely related to polysaccharides activities, such as enhancing cellular immune function, regulating the secretion of cytokines, having immunomodulatory and antineoplastic effects. In this paper, polysaccharide receptors types, biological functions and signal transduction pathways are reviewed, it will be helpful to elucidate the pharmacological activity mechanism of plant polysaccharides and provide theoretical reference for the comprehensive development of polysaccharide drugs.

Expression of C-type lectin domain family 1 member B in hepatocellular carcinoma and its clinical significance / 第二军医大学学报

Objective To explore the expression of C-type lectin domain 1 member B (CLEC1B) in hepatocellular carcinoma (HCC) tissues and its relationship with the clinicopathological characteristics and prognosis of HCC patients. Methods HCC tissue microarray data (GSE49515, GSE115018) were retrieved from Gene Expression Omnibus (GEO) database to analyze the differential expression of genes between cancer tissues and normal control tissues. HCC transcriptome datasets were screened from The Cancer Genome Atlas (TCGA) database to analyze the differential expression of CLEC1B in HCC tissues. Gene Set Enrichment Analysis (GSEA) was used to search for the signaling pathways related to CLEC1B in HCC. The cancer tissues and corresponding paracancerous tissues were collected from 37 HCC patients. The expression of CLEC1B mRNA was detected by quantitative real-time PCR, the expression of CLEC1B protein was detected by Western blotting. χ2 test was performed to analyze the relationship between CLEC1B expression and clinicopathological characteristics of HCC patients. Kaplan-Meier method and log-rank test were performed to analyze the relationship between CLEC1B mRNA expression and prognosis of HCC patients. The blood samples were collected from 37 HCC patients and 37 healthy volunteers. The concentration of CLEC1B in plasma was measured by enzyme-linked immunosorbent assay. The diagnostic value of CLEC1B for HCC was evaluated by receiver operating characteristic (ROC) curve. Results The expression of CLEC1B was low in HCC cancer tissues, and the low expression of CLEC1B in HCC tissues was associated with tumor hemorrhage (P0.01). The concentration of CLEC1B in plasma could be used as a biomarker for the diagnosis of HCC. The best diagnostic efficiency of CLEC1B was obtained by using 62.44 ng/mL as cut-off value (the area under the ROC curve was 0.966, the sensitivity was 92.7%, and the specificity was 91.3%). The HCC patients with high CLEC1B expression had a longer overall survival than those with low CLEC1B expression, and the difference was significant (P0.01). In HCC, CLEC1B gene showed a consistent trend of differential expression with ATM and Rad3-related pathway (ATR pathway), cell cycle pathway, DNA repair pathway and myc signaling pathway. Conclusion The low expression of CLEC1B in HCC is related to tumor hemorrhage, and the prognosis of HCC patients with low expression of CLEC1B is poor.

Effects of C-type lectin receptors on functional regulation of dendritic cells / 中国免疫学杂志

Dendritic cells are the most potent professional antigen presenting cells.The expression of C-type lectin receptors (CLR) contributes to the intake of glycoprotein and identification of microbial antigens,which results in antigen information presented to T and B lymphocytes and the ensuing adaptive immune response.The process plays an important role in antifungal or anti-parasitic infection immunity,allergic reactions and tumor immunity.Therefore,it is of great significance to explore the effect of C-type lectin receptors on the regulation of DC function and the development of diseases.The review focuses on the recent research findings on the effect of CLR members on the functional regualtion of DC.

Progress in platelet-activating C-type lectin-like receptor-2 and hematogenous metastasis of tumor / 肿瘤

The process of tumor metastasis is extremely complicated. The growing evidence indicates that platelets are closely involved in hematogenous metastasis of tumors. Platelets can secret an array of bioactive factors to help cancer cells evading immune surveillance and natural killer cell-mediated cytotoxicity, induce epithelialmesenchymal transition (EMT), proliferation, invasion of tumor cells, and subsequently promote cancer progression. Recent studies have showed that the interaction between platelet-activating C-type lectin-like receptor-2 (CLEC-2) and its ligand podoplanin (PDPN) can activate platelets and affect the proliferation, migration, and metastasis of tumor cells. Therefore, using anti-CLEC-2 antibody and blocking the CLEC-2/PDPN interaction maybe the novel anticancer treatments. This review illuminates the role of CLEC-2 in hematogenous metastasis of tumors and its clinical application prospect.

Snake venom galactoside-binding lectins: a structural and functional overview

Snake venom galactoside-binding lectins (SVgalLs) comprise a class of toxins capable of recognizing and interacting with terminal galactoside residues of glycans. In the past 35 years, since the first report on the purification of thrombolectin from Bothrops atrox snake venom, several SVgalLs from Viperidae and Elapidae snake families have been described, as has progressive improvement in the investigation of structural/functional aspects of these lectins. Moreover, the advances of techniques applied in protein-carbohydrate recognition have provided important approaches in order to screen for possible biological targets. The present review describes the efforts over the past 35 years to elucidate SVgalLs, highlighting their structure and carbohydrate recognition function involved in envenomation pathophysiology and potential biomedical applications.(AU)

Snake venom galactoside-binding lectins: a structural and functional overview

Snake venom galactoside-binding lectins (SVgalLs) comprise a class of toxins capable of recognizing and interacting with terminal galactoside residues of glycans. In the past 35 years, since the first report on the purification of thrombolectin from Bothrops atrox snake venom, several SVgalLs from Viperidae and Elapidae snake families have been described, as has progressive improvement in the investigation of structural/functional aspects of these lectins. Moreover, the advances of techniques applied in protein-carbohydrate recognition have provided important approaches in order to screen for possible biological targets. The present review describes the efforts over the past 35 years to elucidate SVgalLs, highlighting their structure and carbohydrate recognition function involved in envenomation pathophysiology and potential biomedical applications.

La estimulación de TLR, receptores tipo NOD y dectina-1 en neutrófilos humanos induce la producción de citocinas proinflamatorias / Stimulation of TLRs, Nod-like receptors and Dectin-1 in neutrophils induces the production of proinflammatory cytokines

Objetivo: evaluar la expresión y la función de receptores de reconocimiento de patrones como los de tipo Toll y los de tipo NOD, RIG-I/MDA5, la dectina-1 y moléculas adaptadoras, en neutrófilos humanos. Métodos: a partir de sangre periférica de individuos sanos se purificaron y cultivaron neutrófilos en el medio RMPI-1640, en presencia o ausencia de los agonistas específicos de los receptores de interés. La expresión de los receptores de reconocimiento de patrones se determinó por RT-PCR y la secreción de citocinas proinflamatorias, por ELISA. Resultados: los neutrófilos expresan un amplio espectro de receptores de reconocimiento de patrones y de moléculas adaptadoras. La estimulación de TLR4, TLR5, TLR7/8 induce la secreción de IL-1β e IL-6; la activación de la dectina-1 induce una alta producción de TNF-α, pero bajos niveles de IL-1β e IL-6. Conclusión: los neutrófilos expresan un amplio número de receptores de reconocimiento de patrones y su activación lleva a la expresión de diferentes citocinas proinflamatorias.

Objective: To evaluate the expression and function of pattern recognition receptors such as Toll-like receptors, RIG-I/MDA5, NOD-like receptors, Dectin-1 and adaptor proteins, in human neutrophils. Methods: Neutrophils from peripheral blood of healthy individuals were purified and cultured in RPMI-1640, in the presence or absence of specific agonists of the receptor of interest. The expression of pattern recognition receptors was determined by RT-PCR and the secretion of proinflammatory cytokines, by ELISA. Results: We observed that neutrophils express diverse patterns recognition receptors and adaptor molecules. Stimulation of TLR4, TLR5 and TLR7/8 induces the production of IL-1β and IL-6, and activation of Dectin-1 leads to secretion of high levels of TNF-α, but low levels of IL-1β and IL-6. Conclusion: Neutrophils express a large number of pattern recognition receptors and their activation leads to the expression of proinflammatory cytokines.

Eukaryotic expression and preparation of polyclonal antibody for osteoclast inhibitory lectin related protein / 中国免疫学杂志

Objective:In order to get recombinant protein OCILRP 2-Fc and anti-OCILRP2 antibody for further study of OCILRP2.Methods:Eukaryotic expression vector pIg-CD5-OCILRP2 which fused with extracellular domain of OCILRP 2 and human IgG1 Fc fragment was constructed.G418 was used for stable expression cell strain after pIg-CD5-OCILRP2 transfected into CHO cells.Recombinant protein OCILRP 2-Fc purified from CHO cell supernatant was used to immunize rabbit and anti-OCILRP2 polyclonal antibody was purified from rabbit serum by using protein G column.Results: ELISA data showed that we got a high-titer anti-serum and anti-OCILRP2 antibody purified from the rabbit serum.Western blot indicated this antibody could specifically bind to OCILRP 2-Fc and OCILRP2 in NIH/3T3 lysate.OCILRP2 expression in murine bone marrow derived dendritic cells ( DC) was detected by this polyclonal antibody ,too.Compared to immature DC ,OCILRP2 expression was elevated in LPS induced-mature DC.Conclusion: This study has offered an available tool and provided a clue for further study of the roles of OCILRP 2 in immune response.

Advances in the study of mincle, a new C-type lectin receptor / 中华实验眼科杂志

Macrophage-inducible C-type lectin (mincle receptor) is a novel unclassical C-type lectin receptor member,which is mainly expressed on the surface of antigen presenting cells.Under the stimulation of fungus,mycobacterium tuberculosis and necrotic cells,mincle receptor plays anatural immune effect in host body by binding to relevant ligand and activate nuclear factor-κB (NF-κB) signaling pathway,which promotes the expression of many inflammatory factors.Up to now,the laboratory and clinical studies on mincle receptor and infected diseases is being a hot topic.However,these study is lack in ophthalmology.Understanding the association of mincle receptor to infected eye diseases will offer a new approach to the immunotherapy.The basic concept,acting mechanism,its relationship to inflammatory eye diseases and outlook in ophthalmology of mincle receptor were reviewed in this paper.

Dectin-1 Stimulation Selectively Reinforces LPS-driven IgG1 Production by Mouse B Cells

Dectin-1, which specifically recognizes beta-glucan of fungal cell walls, is a non-Toll-like receptor (TLR) pattern recognition receptor and a representative of C-type lectin receptors (CLRs). The importance of Dectin-1 in innate immune cells, such as dendritic cells and macrophages, has previously been well studied. However, the function of Dectin-1 in B cells is very poorly understood. To determine the role of Dectin-1 in B cell activation, we first investigated whether mouse B cells express Dectin-1 and then assessed the effect of Dectin-1 stimulation on B cell proliferation and antibody production. Mouse B cells express mRNAs encoding CLRs, including Dectin-1, and surface Dectin-1 was expressed in B cells of C57BL/6 rather than BALB/c strain. Dectin-1 agonists, heat-killed Candida albicans (HKCA) and heat-killed Saccharomyces cerevisiae (HKSC), alone induced B cell proliferation but not antibody production. Interestingly, HKSC, HKCA, and depleted zymosan (a selective Dectin-1 agonist) selectively enhanced LPS-driven IgG1 production. Taken together, these results suggest that, during fungal infection, beta-glucan-stimulated Dectin-1 may cooperate with TLR4 to specifically enhance IgG1 production by mouse B cells.

The Innate Immune Response in House Dust Mite-Induced Allergic Inflammation

Hypersensitivity to house dust mite (HDM; Dermatophagoides sp.) allergens is one of the most common allergic responses, affecting up to 85% of asthmatics. Sensitization to indoor allergens is the strongest independent risk factor associated with asthma. Additionally, >50% of children and adolescents with asthma are sensitized to HDM. Although allergen-specific CD4+ Th2 cells orchestrate the HDM allergic response through induction of IgE directed toward mite allergens, activation of innate immunity also plays a critical role in HDM-induced allergic inflammation. This review highlights the HDM components that lead to activation of the innate immune response. Activation may due to HDM proteases. Proteases may be recognized by protease-activation receptors (PARs), Toll-like receptors (TLRs), or C-type lectin receptors (CTRs), or act as a molecular mimic for PAMP activation signaling pathways. Understanding the role of mite allergen-induced innate immunity will facilitate the development of therapeutic strategies that exploit innate immunity receptors and associated signaling pathways for the treatment of allergic asthma.

Macrophage-inducible C-type lectin wets selectively expressed on macrophages and dendritic cells in synovial fluid from patients with rheumatoid arhritis / 中华风湿病学杂志

Objective To determine the expression pattern of macrophage-inducible c-type lectin (MINCLE)on Macrophage(Mφ),myeloid dendritic cell (mDC)and plasmacytoid DC(pDC)in peripheral blood (PB)and synovial fluid(SF)in patients with rheumatoid arthritis (RA).Methods For mRNA expression of MINCLE,253 RA patients and 71 healthy control subjects were enrolled.The mRNA level of MINCLE was determined by real-time PCR.For protein expression of MINCLE,18 patients with RA,5 patients with osteoarthritis(OA)and 12 healthy control subjects were enrolled.The expression of MINCLE on Mφ,mDC and pDC were detected by flow cytometry.The differences of MINCLE expressions in PB between RA patients,OA patients and healthy controls,or differences between PB and SF in RA patients were analyzed using Mann-Whitney U test or paired-samples t test.Results ①Compared to the healthy controls,RA patients showed elevated mRNA expression level of MINCLE in PBMCs[(1.65±0.36)vs (0.37±0.06),U=6057,P=2.75×10-5].②At protein level,MINCLE was hardly detected in Mφ,mDC and pDC in PB of OA patients and healthy controls.In SF,MINCLE was highiy expressed on mDC in RA patients,compared with that in OA patients[(34.8±4.4)%,U=0,P=2.6×10-3].In RA patients,the expression level of MINCLE was remarkably elevated in Mφ,mDC and pDC in SF compared with that in PB[Mφ(2.01±0.53)%vs(0.273±0.51)%,t=4.879,P=2.23×10-6;mDC(34.8±4.4)%vs(22.7±5.5)%t=2.535.P=0.017].Conclusion MINCLE is selectively expressed on Mφ.mDC and pDC in SF in RA patients.MINCLE may serve as a potential important marker,or even target,for RA and possibly even for inflammation in general.

Toxinas de serpientes con alto potencial terapéutico y su uso en la biomedicina / Animal toxins with high therapeutic potential and their use in biomedicine

Los venenos de animales son mezclas complejas de proteínas, péptidos, enzimas y trazas de elementosno proteicos tales como carbohidratos y sales, cuya finalidad es inmovilizar la presa ycomenzar a digerirla; algunos de estos compuestos han sido aislados y caracterizados o descritoscomo toxinas letales, o se les han atribuido acciones potentes sobre proteínas específicas como, porejemplo, las involucradas en la coagulación sanguínea. Debido al descubrimiento en 1971 del péptidoque dio origen al captopril y al entendimiento de los efectos potenciales de las toxinas, se empezó aconsiderar que los venenos de animales son fuentes ricas en compuestos bioactivos, que no soloproporcionan las herramientas necesarias para descifrar los detalles moleculares de diversos procesosfisiológicos, sino que también sirven como fuente de inspiración para diseñar y desarrollaragentes terapéuticos. Este artículo expone la aplicación de nuevas alternativas terapéuticas y demodelos para el diseño de las mismas basados en algunas moléculas aisladas de venenos de serpientescon alto potencial en campos como la biomedicina y la farmacia.

Animal venoms are complex mixtures of proteins, peptides, enzymes and trace elements such ascarbohydrates and salts, which serve to immobilize preys and to begin their digestion. Some ofthese compounds have been isolated and characterized, or described as lethal toxins, while othershave powerful actions on specific proteins, such as those involved in blood coagulation. Due to thediscovery in 1971 of the peptide that gave rise to captopril and to a better understanding of thepotential effects of toxins, animal venoms started to be considered as rich sources of bioactive compounds, which not only provide the necessary toolsto decipher molecular details of various physiologicalprocesses, but also are a source of inspiration to designand develop a range of new therapeutic agents. Thisreview presents the application of new therapeuticoptions or models to design them based on certainmolecules isolated from snake venoms, with highpotential in fields such as biomedicine and pharmacy.