Construction and self-activation detection of yeast two-hybrid bait plasmid of human programmed cell death ligand 1 immunoglobulin variable region domain gene / 中国生物制品学杂志

@#Objective To construct a yeast two-hybrid recombinant bait plasmid of human programmed cell death ligand 1(PD-L1)immunoglobulin variable region(IgV)domain gene,detect its expression in yeast and detect the cytotoxicity and self-activation of PD-L1 IgV protein as well as the interaction between PD-L1 IgV and human thioredoxin(hTrx).Methods Human PD-L1 was analyzed by bioinformatics method,and primers were designed to amplify PD-L1 IgV domain based on the coding region of PD-L1 gene registered in NCBI GenBank database. PCR amplification was carried out with pENTERPD-L1 plasmid as template,and then cloned into yeast two-hybrid bait vector pGBKT7. The recombinant bait plasmid and pGBKT7 empty vector were transformed into Y2HGold yeast cells respectively,and the PD-L1 IgV gene and its expression were detected by PCR and Western blot;Meanwhile,the protein toxicity and self-activation of PD-L1 IgV were detected,and the interaction between PD-L1 IgV and hTrx was detected by drip plate method.Results The bioinformatics analysis results of PD-L1 were consistent with related reports. The recombinant bait plasmid pGBKT7-PD-L1 IgV was correctly constructed,and Y2HGold positive clone was obtained,in which PD-L1 IgV was stably expressed. The empty vector pGBKT7 and recombinant bait plasmid pGBKT7-PD-L1 IgV grew well on SD/-Trp and SD/-Trp/X-α-Gal plates with the same colony size and number and white colony,but they did not grow on SD/-Trp/X-α-Gal/AbA plates,which indicated that PD-L1 IgV protein had no toxicity and no self-activation effect on yeast. The results of drip plates test showed that all experimental groups grew well on SD/-Trp/-Leu plate,while only positive control group grew on SD/-Trp/-Leu/X-α-Gal/AbA plate and showed blue color,which indicated that bait protein PD-L1 IgV and hTrx did not self-activate,and there was no interaction between them.Conclusion Recombinant human PD-L1 IgV bait plasmid was successfully constructed. PD-L1 IgV protein showed no toxicity and self-activation effect on yeast cells,and there was no interaction between PD-L1 IgV and hTrx. Subsequently,hTrx can be used to construct a peptide aptamer library,from which peptide aptamers that specifically bind to PD-L1 IgV can be screened.

Research progress on invasive cervical resorption / 口腔疾病防治

@#Tooth absorption can be divided into physiological absorption and pathological absorption. Root absorption of mature deciduous teeth is physiological absorption. Pathological absorption includes internal absorption and external absorption. Internal absorption, also known as intramedullary absorption, includes inflammatory absorption and alternative absorption. External tooth absorption originates from the outer surface of the root or the neck of the tooth and can be divided into inflammatory absorption, alternative absorption, pressure resorption and invasive cervical resorption. Invasive cervical resorption (ICR) is pathological damage caused by many factors, which usually begins in the cemento-enamel junction and extends peripherally or horizontally in the dentin. It hardly invades the pulp. Orthodontic devices, trauma, bleaching, systemic diseases, and the use of certain medications can all lead to invasive cervical resorption. The clinical manifestations of ICR are usually asymptomatic or not obvious, and most of which are found in imaging examinations. Because caries and internal absorption are often misdiagnosed through plain apical radiography, cone beam computed tomography (CBCT) can help to better understand the situation of invasive cervical resorption. Because the pathogenesis and etiology of invasive cervical resorption are not fully understood, clinical negligence and inadequate treatment of invasive cervical resorption can even cause unnecessary tooth loss. This article reviews the latest research progress on the histopathologic features, pathogenic mechanism, susceptibility factors, diagnosis and treatment of ICR, with special emphasis on susceptibility factors and their mechanisms.

Metal-organic Framework Immobilized Enzyme and Its Application in Screening of Enzyme Inhibitors of Traditional Chinese Medicine：A Review / 中国实验方剂学杂志

Enzymes are widely used in chemical and pharmaceutical industries because of their advantages of high efficiency and specificity. However， the shortcomings of the free enzymes， such as poor stability and difficulty in recycling， limit their application. Therefore， the immobilization and application of enzymes have become one of the research hotspots. The selection of the immobilization carriers is a critical step in the process of enzyme immobilization. Metal-organic frameworks（MOFs）， a kind of porous materials， are formed by the coordination of metal ions or metal clusters with organic ligands. As an emerging immobilization carrier， its advantages such as high porosity， strong stability， and surface modifiability make it ideal for immobilized enzyme carriers. By immobilizing the free enzyme on MOFs， the above mentioned deficiencies of the free enzymes can be effectively solved， which greatly broaden the applicable condition. Ligand fishing is a method to find receptor-specific ligands from complex components， which has the advantages of high efficiency， simple sample pretreatment and high specificity. The MOF-enzyme complex formed by enzyme immobilization can act as a "fishing rod" for ligand fishing， which can screen out the targets from the complex system of components. The complex chemical composition and various active ingredients of traditional Chinese medicine（TCM） make the ligand fishing technology to play a big role in the screening of enzyme inhibitors from TCM. And the screened enzyme inhibitors are expected to be further developed into the lead compounds with good efficacy and low adverse effects， so the immobilized enzymes of MOFs have a wide application in the screening of active ingredients from TCM. Based on this， this paper summarized the methods of immobilized enzymes of MOFs in recent years， analyzed the characteristics， advantages and disadvantages of each method， and summarized the laws of preparation conditions and mechanisms. Meanwhile， the application and future development of immobilized enzymes of MOFs in the field of enzyme inhibitor screening from TCM were also summarized and prospected， with a view to providing a reference for the development of natural ingredients and the modernization of TCM.

Mechanism of action of the bile acid receptor TGR5 in obesity

G protein-coupled receptors (GPCRs) are a large family of membrane protein receptors, and Takeda G protein-coupled receptor 5 (TGR5) is a member of this family. As a membrane receptor, TGR5 is widely distributed in different parts of the human body and plays a vital role in regulating metabolism, including the processes of energy consumption, weight loss and blood glucose homeostasis. Recent studies have shown that TGR5 plays an important role in glucose and lipid metabolism disorders such as fatty liver, obesity and diabetes. With the global obesity situation becoming more and more serious, a comprehensive explanation of the mechanism of TGR5 and filling the gaps in knowledge concerning clinical ligand drugs are urgently needed. In this review, we mainly explain the anti-obesity mechanism of TGR5 to promote the further study of this target, and show the electron microscope structure of TGR5 and review recent studies on TGR5 ligands to illustrate the specific binding between TGR5 receptor binding sites and ligands, which can effectively provide new ideas for ligand research and promote drug research.

Rational drug repurposing for alzheimer's treatment using in-silico ligand and structure-based approaches

Abstract Alzheimer's disease is a devastating neurodegenerative disorder characterized by memory loss and cognitive decline. New AD treatments are essential, and drug repositioning is a promising approach. In this study, we combined ligand-based and structure-based approaches to identify potential candidates among FDA-approved drugs for AD treatment. We used the human acetylcholinesterase receptor structure (PDB ID: 4EY7) and applied Rapid Overlay of Chemical Structures and Swiss Similarity for ligand-based screening.Computational shape-based screening revealed 20 out of 760 FDA approved drugs with promising structural similarity to Donepezil, an AD treatment AChE inhibitor and query molecule. The screened hits were further analyzed using docking analysis with Autodock Vina and Schrodinger glide. Predicted binding affinities of hits to AChE receptor guided prioritization of potential drug candidates. Doxazosin, Oxypertine, Cyclopenthiazide, Mestranol, and Terazosin exhibited favorable properties in shape similarity, docking energy, and molecular dynamics stability.Molecular dynamics simulations confirmed the stability of the complexes over 100 ns. Binding free energy analysis using MM-GBSA indicated favourable binding energies for the selected drugs. ADME, formulation studies offered insights into therapeutic applications and predicted toxicity.This comprehensive computational approach identified potential FDA-approved drugs (especially Doxazosin) as candidates for repurposing in AD treatment, warranting further investigation and clinical assessment.

Prognostic and predictive role of soluble programmed death ligand-1 in head and neck cancer

Abstract Objectives The aim of the study was to investigate clinical significance of soluble PD-L1 (sPD-L1) serum level in head and neck cancer and to evaluate its role as a possible prognostic and predictive biomarker. Methods A prospective analysis of sPD-L1 levels in 60 patients diagnosed and treated due to malignant and non-malignant lesions in the region of head and neck was performed in peripheral blood by an ELISA test. Results The range of sPD-L1 in the study group was 0.16-1.63 ng/mL, mean 0.64 ± 0.32. There were no differences in the mean sPD-L1 regarding patients' age, sex, and the localization of the lesion. Statistically significant difference was revealed in the average sPD-L1 level (p = 0.006) depending on the histopathological advancement of the lesions, 0.704 ± 0.349 and 0.512 ± 0.177 respectively in the malignant and benign group. The separate analysis of laryngeal lesions confirmed statistical difference in sPD-L1 (p = 0.002) for the malignant lesions (0.741 ± 0.353) compared with the benign (0.489 ± 0.175). The sPD-L1 level of 0.765 ng/mL or higher, revealed 35% sensitivity and 95.5% specificity for the diagnosis of head and neck malignant lesions (AUC = 0.664, 95% CI 0.529‒0.8, p-value = 0.039). The 1-year DFS was 83.3% in the group of patients with low sPD-L1 levels (< 0.765 ng/mL) and 53.8% in patients with high sPD-L1 (≥0.765 ng/mL). The 2-year OS were 68% and 69.2% respectively in both groups. The log-rank test confirmed statistically significant prognostic value of sPD-L1 level for 1-year DFS (p-value = 0.035). Conclusions sPD-L1 is a promising prognostic and early recurrence predictive biomarker for head and neck cancers, most significantly for laryngeal lesions. Level of evidence 3.

Diferencia en la expresión del ligando de receptor activador para el factor nuclear K B, en tejido tumoral frente a la infección por diferentes patógenos periodontales / Difference in receptor activator of nuclear factor K B expression in tumoral tissue by different periodontal pathogens

La enfermedad periodontal es una de las principales causas de pérdida dentaria. Clínicamente, esta patología, mediada por la desregulación del sistema inmune producto de una disbiosis ocurrida en el surco gingival, inicia con la inflamación de la encía y evoluciona con el daño irreversible de los tejidos que rodean el diente. El hueso alveolar es uno de los tejidos afectados esta patología, esto debido a la activación de osteoclastos por la sobreexpresión de la proteína RANKL en el huésped. El propósito de este trabajo es determinar el nivel de sobreexpresión de RANKL, en un modelo de células tumorales U2OS, frente a la infección con Porphyromonas gingivalis y Prevotella intermedia. Para identificar el nivel de RANKL, se definieron cuatro grupos: Un grupo control, no tratado; Grupo PG, tratado con P. gingivalis; Grupo PI, tratado con P. Intermedia; y un grupo PG+PI, tratado con ambas bacterias. El nivel relativo de la proteína RANKL fue determinado en el sobrenadante y en los extractos celulares de manera independiente, mediante la técnica Western blot. En sobrenadantes, el grupo PG mostró mayores niveles de RANKL comparados con PI (p < 0,05). En extractos celulares los niveles fueron mayores en el grupo PG+PI (p < 0,05). El grupo PI mostró los niveles más bajos de RANKL. La infección polimicrobiana resulta en una mayor expresión de RANKL en células tumorales U2OS, mientras que frente a la infección P. gingivalis, se observó mayor cantidad de RANKL soluble.

SUMMARY: Periodontal disease is one of the main causes of tooth loss. Clinically, this pathology, mediated by the deregulation of the immune system due to a dysbiosis occurred in the gingival sulcus, begins with the inflammation of the gum and evolves with the irreversible damage of the tissues that surround the tooth. Alveolar bone is one of the most affected tissues by this disease, due to the activation of osteoclasts by the upregulation of RANKL in the host. The aim of this study is to determine the increase of RANKL, in a U2OS tumor cells model, inoculated with Porphyromonas gingivalis and Prevotella intermedia. To identify the level of RANKL, four groups were defined: A control group, not treated; PG group, treated with P.gingivalis; PI group, treated with P. intermedia; and a PG+PI group, treated with both bacteria. The relative level of RANKL was determined in the supernatant and cell extracts independently, using the Western blot technique. In supernatants, the PG group showed higher RANKL levels compared to PI (p < 0.05). In cell extracts the levels were higher in the PG+PI group (p < 0.05.). The PI group showed the lowest levels of RANKL.Polymicrobial infection results in a greater expression of of soluble RANKL was observed.

Molecular docking and cytotoxicity interactions of naringenin and its nano-structured lipid carriers in ER? positive breast cancer

Phytoestrogens are known to have beneficial properties in various carcinomas. They exhibit its efficacy at cellular levels. Naringenin a flavonoidal phytoestrogen is been explored for its antioxidant, cardio protective and cytotoxic function. The low absorbtion and poor bioavailability of naringenin makes it less efficient in targeting tumours at cellular levels. Due to the structural similarity of naringenin with estradiol and considering the affinity of naringenin with estrogen receptor, this study explores the interactions of naringenin on important signaling proteins involved in ER positive breast cancer through molecular docking studies and the prepared naringenin solid lipid nano particles were characterized and studied for its preventive potential against breast cancer cell lines. The lipidoid form of phytoestrogen shows promising cytotoxic potential compared with naringenin.

Effect of Biling Qutong Prescription on NLRP3， P2X7R， FN， and Hepatic Steatosis in Type 2 Diabetes Mellitus Complicated with Gouty Arthritis / 中国实验方剂学杂志

ObjectiveTo investigate the effects of Biling Qutong prescription （BLQT） on serum levels of NOD-like receptor thermal protein domain associated protein 3 （NLRP3）， purinergic ligand-gated ion channel 7 receptor （P2X7R）， fibronectin （FN）， and hepatic steatosis in patients with type 2 diabetes mellitus （T2DM） complicated with gouty arthritis （GA）. MethodSixty-four patients diagnosed with T2DM comorbid with GA and treated at the First Affiliated Hospital of Anhui University of Chinese Medicine from January 2019 to December 2022 were enrolled and randomly divided into a BLQT group （Chinese medicine group， 32 cases） and the ibuprofen group （western medicine group， 32 cases）. Thirty healthy individuals who underwent routine health examinations during the same period were assigned to the control group. The BLQT group and the western medicine group received basic treatment along with BLQT and ibuprofen， respectively. After 8 weeks of continuous treatment， the traditional Chinese medicine syndrome score （TCMSS） of the patients was evaluated before and after treatment. The differences in fasting plasma glucose （FPG）， 2-hour postprandial plasma glucose （2 h PG）， glycated hemoglobin （HbA1c）， fasting insulin （FINS）， homeostasis model assessment of insulin resistance （HOMA-IR）， serum uric acid （SUA）， serum creatinine （SCr）， total cholesterol （TC）， triglyceride （TG）， high-density lipoprotein cholesterol （HDL-C）， low-density lipoprotein cholesterol （LDL-C）， alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， controlled attenuation parameter （CAP）， liver stiffness measurement （LSM）， NLRP3， P2X7R， and FN levels before and after treatment were compared. Adverse drug reactions that occurred during treatment were recorded. ResultThe TCMSS for joint redness， swelling， pain， joint burning， yellow urine， and red tongue with yellow and greasy coating， as well as total score were significantly reduced in both the BLQT group and the western medicine group as compared with those before treatment （P<0.05， P<0.01）. The BLQT group also showed a significant reduction in symptom scores such as dry mouth， polyuria， polydipsia， and slippery and rapid pulse （P<0.01）. Compared with the western medicine group after treatment， the BLQT group exhibited a more significant reduction in all symptom scores and total score （P<0.05， P<0.01）. The BLQT group and the western medicine group showed a decrease in FPG， 2 h PG， HbA1c， SCr， SUA， TG， TC， and LDL-C levels （P<0.05， P<0.01） after treatment， and the BLQT group showed decreased HOMA-IR， ALT， AST， and HDL-C levels （P<0.05， P<0.01） compared with those before treatment. When compared with the western medicine group after treatment， the BLQT group showed a more significant reduction in all laboratory parameters except for HDL-C （P<0.05， P<0.01）. Before treatment， NLRP3， P2X7R， and FN levels in both the BLQT group and the western medicine group were higher than those in the control group （P<0.01）. After treatment， NLRP3 and P2X7R levels in both groups significantly decreased （P<0.01）， and FN levels in the BLQT group also decreased significantly （P<0.01）. When compared with the western medicine group after treatment， the BLQT group showed a more significant reduction in NLRP3， P2X7R， and FN levels （P<0.01）. Before treatment， CAP and LSM levels in both the BLQT group and the western medicine group were higher than those in the control group （P<0.01）. After treatment， CAP and LSM levels in both groups decreased （P<0.05， P<0.01）. Compared with the western medicine group after treatment， the BLQT group showed a more significant reduction in CAP and LSM （P<0.01）. The incidence of adverse reactions was 3.13% （1/32） in the BLQT group and 15.63% （5/32） in the western medicine group， with no significant difference. ConclusionBLQT has good efficacy in patients with T2DM complicated with GA， which can significantly alleviate joint redness， swelling， heat， pain， limited mobility， dry mouth， and polydipsia， reduce blood glucose， uric acid， and lipid levels， suppress the high expression of NLRP3， P2X7R， and FN， and improve hepatic steatosis.

Preparation of CD73/PD-L1 bispecific antibody and evaluation of its antitumor function in vitro / 中国生物制品学杂志

@#Objective To prepare bispecific antibody targeting cluster of differentiation 73(CD73) and programmed cell death-ligand 1(PD-L1),and evaluate its binding ability and killing ability in vitro.Methods Using genetic engineering method,PD-L1 single-chain fragment variable(scFv) was inserted into the hinge region of CD73 monoclonal antibody to construct anti-CD73/PD-L1 bispecific antibody(BS-21),which was screened by CHO GS expression system to obtain highly expressed cell line.After purified by Protein A and molecular sieve,the purity of antibody was detected by size exclusion chromatography-high performance liquid chromatography(SEC-HPLC),the binding ability of antibody in vitro was detected by flow cytometry,and the killing ability in vitro was detected by using peripheral blood mononuclear cell(PBMC) to kill Calu 1 lung cancer cells in vitro.Results High-yield cell lines were obtained by pressure screening.A bispecific antibody BS-21 with a purity of 99.6% was obtained by purification,which bound to CD73 and PD-L1 molecules simultaneously.Compared with anti CD73 and anti PD-L1 groups,BS-21 group significantly increased the killing rate of immune cells to Calu 1 tumor cells(F=30.36,each P<0.001).Conclusion Bispecific antibody BS-21 reduced the immunosuppressive effect of CD73 and PD-Ll on immune cells simultaneously,and showed good anti-tumor function.

Effects of VALD-3,a derivative of o-vanilla Schiff base ligand,on proliferation,migration and apoptosis of colorectal cancer cells and its mechanism / 中国生物制品学杂志

@#Objective To the effects of VALD-3,a derivative of o-vanilla Schiff base ligand,on proliferation,migration and apoptosis of colorectal cancer cells and evaluate its mechanism.Methods HT-29 and HCT116 cells were cultured in vitro,and the inhibitory effects of VALD-3(5,10,20 and 40 mg/L) on proliferation of the two kinds of cells were detected by MTT assay;The effects of VALD-3(10,20 and 40 mg/L) on the morphological changes of the cells were observed by inverted microscope,while the effects on the migration ability of HT-29 cells were detected by cell scratch test,and the effects on the apoptosis of HT-29 cells were detected by flow cytometry and Hoechst 33258 fluorescence staining;The effects of VALD-3(5,10,20 and 40 mg/L) on the expression of apoptosis-related proteins in HT-29 cells were detected by Western blot.Negative control groups were set up(with no VALD-3).Results Compared with the negative control group,the survival rates of HT-29 and HCT116 cells in 10,20 and 40 mg/L VALD-3 treated groups significantly decreased(t=7.717～2 006.148,each P <0.05);the number of HT-29 and HCT116 cells in 10,20 and 40 mg/L VALD-3groups decreased significantly with the increase of VALD-3 concentration,the cells appeared irregular morphology and gradually became round and smaller,and cell fragments increased;In 10,20 and 40 mg/L VALD-3 treated groups,the migration rate of HT-29 cell scratches decreased significantly(t=7.596～73.780,each P <0.01),the apoptosis rate increased significantly(t=7.092～8.057,each P <0.01),and the number of apoptotic cells increased significantly with strong bright blue fluorescence,chromatin concentration and nuclear fragmentation;The levels of cleaved caspase-3 and Bax protein in HT-29 cells treated with 5,10,20 and 40 mg/L VALD-3 significantly increased(t=2.998～24.901,each P <0.05),the level of Bcl-2 protein in 40 mg/L VALD-3 group decreased significantly(t=10.035,P <0.05),and the levels of cleaved caspase-8 in 20 and 40 mg/L VALD-3 group significantly increased(t=12.630 and 8.064 respectively,each P <0.01).Conclusion VALD-3 inhibited the proliferation and migration of colorectal cancer cells and induced apoptosis by regulating the expression of cleaved caspase-3,cleaved caspase-8,Bax and Bcl-2 proteins.

Effect of siRNA interference with C-C chemokine ligand 5 on biological characteristics of laryngeal carcinoma cells / 中国生物制品学杂志

@#Objective To investigate the expression of C-C chemokine ligand 5(CCL5) in head and neck squamous cell carcinoma(HNSCC),and explore the effect of CCL5 on the biological characteristics of laryngeal carcinoma cells.Methods Gene Expression Profiling Interactive Analysis(GEPIA) database was used to investigate the expression of CCL5 in HNSCC.The laryngeal carcinoma cells TU177 were transfected with siRNA(siRNA group),and the control(NC) group was set up.The cell proliferation,migration,cycle and apoptosis of each group were detected by CCK8 assay,cell scratch test and flow cytometry respectively.RT-PCR and Western blot were used to detect the knock-down efficiency of CCL5 and the mRNA transcription and protein expression of multidrug resistance protein 2(MRP2) and bcl-2-associated x protein(Bax).Results The expression of CCL5 in HNSCC was higher than that in normal tissues(P <0.05).Compared with NC group,siRNA showed higher knock-down efficiency(t=12.898 and 22.656 respectively,each P <0.01);siRNA interference with CCL5 inhibited the proliferation and migration of laryngeal carcinoma cells,and promoted the late apoptosis of laryngeal carcinoma cells and the expression of apoptosis protein Bax(t=2.600～11.667,each P <0.05).Conclusion CCL5 was highly expressed in HNSCC,while siRNA interference with CCL5 inhibited the proliferation,migration and promoted apoptosis of laryngeal carcinoma cells TU177 by up-regulating the expression of Bax,which laid a foundation of the possibility of CCL5 as a new target for the treatment of laryngeal carcinoma.

Correlation of KRAS gene mutation and programmed death receptor ligand 1 expression with prognosis of first-line concurrent chemoradiotherapy in patients with locally advanced non-small cell lung cancer / 肿瘤研究与临床

Objective:To investigate the relationship between KRAS gene mutation, programmed death receptor ligand 1 (PD-L1) expression and prognosis of first-line concurrent chemoradiotherapy in patients with locally advanced non-small cell lung cancer.Methods:The clinical data of 50 patients with locally advanced non-small cell lung cancer who were admitted to Nanping First Hospital from January 2018 to December 2021 were retrospectively analyzed. All patients were treated with first-line concurrent chemoradiotherapy. Tissue samples of patients were obtained and paraffin embedded before treatment. Real-time fluorescence quantitative polymerase chain reaction was used to detect the type of KRAS gene mutation in tissues before treatment, and the expression of PD-L1 was determined by immunohistochemistry (the percentage of positive cells in tumor cells ≥1% was positive), and the relationship between KRAS gene status, PD-L1 expression and clinical characteristics and short-term efficacy of patients was analyzed. Patients were followed up for 1 year, and progression-free survival (PFS) curves were plotted by Kaplan-Meier method, and log-rank test was used for comparison. Univariate and multivariate Cox proportional hazards models were used to analyze the influencing factors of PFS.Results:Among the 50 patients, 11 (22.00%) were KRAS mutant, and 36 (72.00%) were PD-L1 positive. Among the 11 patients with KRAS mutation, there were 2 cases of codon 13 mutation and 9 cases of codon 12 mutation in exon 2. The objective response rate (ORR) and clinical control rate (DCR) were 76.00% (38/50) and 86.00% (43/50). There were no significant differences in patients' age, pathological type, TNM stage, ORR and DCR between KRAS mutant group and KRAS wild type group (all P > 0.05). The proportions of male patients [72.73% (8/11) vs. 38.46% (15/39)], patients with smoking history [90.91% (10/11) vs. 20.51% (8/39)] and patients with PD-L1 positive expression [100.00% (11/11) vs. 64.10% (25/39)] in KRAS mutant group were higher than those in KRAS wild type group (all P < 0.05). There were no significant differences in patients' age, pathological type, gender, smoking history, TNM stage, ORR and DCR between PD-L1 positive group and PD-L1 negative group (all P > 0.05). The median PFS time of patients in KRAS mutant group and wild type group was 8.75 and 11.32 months, and the difference in PFS between the two groups was statistically significant ( P = 0.039). The median PFS time of patients with PD-L1 positive and negative was 10.19 and 11.16 months, and there was no statistical significance in PFS between the two ( P = 0.116). Multivariate Cox regression analysis showed that KRAS gene mutation was an independent risk factor for PFS in patients with locally advanced NSCLC after first-line concurrent chemoradiotherapy ( HR = 1.449, 95% CI 1.071-1.196, P = 0.017). PD-L1 expression, smoking history and gender were not independent influencing factors for PFS (all P > 0.05). Conclusions:KRAS gene status is closely related to the prognosis of patients with locally advanced non-small cell lung cancer treated with first-line concurrent chemoradiotherapy, while PD-L1 expression is not.

Clinical significance of CD8 positive tumor-infiltrating lymphocytes density and programmed-death receptor ligand 1 expression in rectal cancer after neoadjuvant chemoradiotherapy / 肿瘤研究与临床

Objective:To investigate the correlation of CD8 positive tumor-infiltrating lymphocytes (CD8 + TIL) density and programmed-death receptor ligand 1 (PD-L1) expression in rectal cancer with clinicopathological characteristics and prognosis of patients after neoadjuvant chemoradiotherapy. Methods:The clinicopathological data of 166 patients with locally advanced rectal cancer (LARC) who received neoadjuvant therapy before surgery in the Beijing Chao-Yang Hospital, Capital Medical University from January 2015 to December 2018 were retrospectively analyzed. CD8 + TIL density and PD-L1 expression were detected by using immunohistochemistry. The correlation of CD8 + TIL density and PD-L1 expression with clinicopathological characteristics of patients after neoadjuvant chemoradiotherapy was analyzed. Kaplan-Meier method was used to analyze the disease-free survival (DFS) and Cox regression risk model was used to make univariate and multivariate analysis of the influencing factors for DFS. Results:Among 166 LARC patients, 81 cases (48.8%) had high density of CD8 + TIL, 85 cases (51.2%) had low density of CD8 + TIL; 63 cases (38.0%) had PD-L1 expression, and 103 cases (62.0%) had non-expression of CD8 + TIL. The expression rate of PD-L1 in CD8 + TIL high density group was higher than that in CD8 + TIL low density group [50.6% (41/81) vs. 25.9%(22/85), χ2 = 10.78, P < 0.001]. According to the density of CD8 + TIL and PD-L1 expression, immunophenotype was divided among 4 groups; the 3-year DFS rate of the CD8 + TIL high density /PD-L1 expression group was 87.1%, which was higher than that of the other groups (CD8 + TIL low density /PD-L1 expression group was 72.8%, CD8 + TIL high density /PD-L1 non-expression group was 67.0%, CD8 + TIL low density /PD-L1 non-expression group was 64.3%), and the difference was statistically significant ( P < 0.05). Univariate analysis showed that tumor differentiation degree, TNM stage, CD8 + TIL density, PD-L1 expression and CD8 + TIL density /PD-L1 expression were correlated with the DFS of patients (all P < 0.05). Multivariate analysis results showed that CD8 + TIL high density /PD-L1 expression was an independent protective factor for DFS ( HR = 0.049, 95% CI 0.005-0.497, P = 0.011), while TNM stage 3 was an independent risk factor for DFS ( HR = 2.752,95% CI 1.300-5.825, P = 0.008). Conclusions:In LARC after neoadjuvant therapy, CD8 + TIL density is positively correlated with the expression of PD-L1, and the high density of CD8 + TIL/PD-L1 expression is an independent influencing factor for good prognosis, suggesting that these patients may benefit from the immunotherapy.

Progress of programmed death factor-1 and its ligand 1 inhibitors in immunotherapy of advanced non-small cell lung cancer / 肿瘤研究与临床

Non-small cell lung cancer (NSCLC) is the most common pathological type of lung cancer. In recent years, with the rapid development of immunotherapy, checkpoint inhibitors, especially programmed death factor-1 (PD-1)/programmed death factor ligand-1 (PD-L1) inhibitors, have made breakthrough progress in the treatment of NSCLC, breaking the pattern of low efficiency and extensive resistance to targeted therapy of traditional chemoradiotherapy, bringing survival benefits to patients. This article reviews the clinical research progress of PD-1/PD-L1 inhibitors in advanced NSCLC.

Clinical significance of infiltration of M2 tumor-associated macrophage in hepatocellular carcinoma / 中华消化杂志

Objective:To investigate the distribution of M2 tumor-associated macrophage (TAM) in hepatocellular carcinoma (HCC) and their correlation with clinicopathological features, and the significance of programmed death-ligand 1 (PD-L1) expression.Methods:From January 1, 2012 to December 31, 2020, a total of 320 HCC patients who underwent surgical resection at the Third People′s Hospital of Nantong were included. The distribution of CD163 labeled and PD-L1 CD163 double-labeled M2 TAM in HCC tissues was detected by immunohistochemistry, and the cell density was calculated. The cell density> the average cell density (112/mm 2) was judged as high-density, the cell density≤ the average cell density was judged as low-density. The correlation between CD163 positive and PD-L1 CD163 double positive M2 TAM density and the clinical pathological characteristics of HCC and its impact on prognosis were analyzed. Chi-square test was used to analyze the correlation between M2 TAM expression and the clinical pathological characteristics of HCC. Kaplan-Meier method was used to draw survival curves, and log-rank test was used for inter group comparison. Univariate and multivariate Cox proportional hazards regression analysis was used to indentify the relevant factors affecting the prognosis of HCC. Results:TAM were mainly distributed in the tumor edge stroma and tumor sinusoids, CD163 positive M2 TAM were the main macrophage subtype. PD-L1 expression was observed in CD163 positive M2 TAM in HCC tissues, and PD-L1 positive M2 TAM were mainly distributed in the tumor edge stroma. The rate of high-density CD163 positive M2 TAM in HCC tissues was 44.4% (142/320). High-density CD163 positive M2 TAM was correlated with histological grade, TNM stage, and PD-L1 expression on tumor infiltrating immune cells in HCC tissues ( χ2=4.65, 6.72 and 42.19, P=0.031, =0.011 and <0.001). High-density PD-L1 and CD163 double positive M2 TAM in HCC tissues was correlated with microvascular invasion and TNM stage ( χ2=11.96 and 8.74, P=0.001 and 0.004). The median disease-free survival (DFS) time and overall survival (OS) time of patients with high-density CD163 positive M2 TAM were 21 and 36 months, respectively, which were lower than those of patients with low-density CD163 positive M2 TAM (50 and 103 months, respectively); the median DFS time and OS time of patients with high-density PD-L1 CD163 double-positive M2 TAM were 12 and 15 months, respectively, which were lower than those of patients with low-density PD-L1 CD163 double-positive M2 TAM (28 and 45 months, respectively), and the differences were statistically significant (all log-rank tests, all P<0.001). The results of multivariate Cox proportional hazards regression analysis showed that high-density CD163 positive M2 TAM, microvascular invasion and high TNM stage were independent risk factors for evaluating DFS and OS of patients with HCC (DFS time: HR=2.408 (95% confidence interval (95% CI) 1.778 to 3.261), 2.603 (95% CI 1.860 to 3.641), 4.032 (95% CI 2.833 to 5.747), all P<0.001. OS time: HR=2.007 (95% CI 1.457 to 2.764), 4.144 (95% CI 2.881 to 5.960), 4.292 (95% CI 2.915 to 6.329), all P<0.001). Conclusions:High-density of CD163 positive M2 TAM in HCC tissues indicates high malignancy and poor prognosis, and it is an independent prognostic risk factor. The expression of PD-L1 in M2 TAM suggests stronger tumor aggressiveness and worse prognosis in HCC.

Correlations of transglutaminase 2 expression in peripheral blood mononuclear cells with relevant inflammatory factors and disease severity in patients with atopic dermatitis / 中华皮肤科杂志

Objective:To determine the expression of transglutaminase 2 (TGM2) in peripheral blood mononuclear cells (PBMCs) from patients with atopic dermatitis (AD), and to analyze its correlation with AD-related inflammatory factors and disease severity.Methods:A total of 29 AD patients and 15 healthy controls were collected from the First Affiliated Hospital of Fujian Medical University from July 2020 to January 2021. Ten milliliters of peripheral blood samples were collected from each subject, so was the clinical information, including age, gender, course of disease, eosinophil counts, basophil counts, total IgE levels, Scoring AD index (SCORAD), etc. PBMCs were isolated by density gradient centrifugation. Fluorescence-based quantitative PCR was performed to determine the mRNA expression of TGM2 and AD-related inflammatory factors (interleukin [IL]-1β, IL-4, IL-6, IL-8, IL-10, IL-13, IL-17, thymic stromal lymphopoietin [TSLP], P2RX7 [purinergic receptor P2X, ligand-gated ion channel, 7], etc.) in PBMCs from 29 AD patients and 15 healthy controls, and flow cytometry to determine TGM2 protein expression on PBMCs. Mann-Whitney U test was used to analyze differences between groups, and Spearman correlation analysis to evaluate the correlation. Results:The relative mRNA expression of TGM2 in PBMCs did not differ between the AD group and control group ( M[ Q1, Q3]: 0.509 [0.325, 0.958] vs. 0.475 [0.328, 1.051], U = 210.50, P = 0.872). Compared with the control group, the AD group showed significantly decreased IL-4 mRNA expression (0.171[0.049, 0.449] vs. 0.824 [0.397, 1.378], P < 0.001), but significantly increased mRNA expression of IL-8 and IL-13 ( P = 0.011, 0.006, respectively). Spearman correlation analysis showed that the mRNA expression level of TGM2 in PBMCs was positively correlated with the mRNA expression levels of IL-4 and P2RX7 in the AD group ( rs = 0.42, 0.40, P = 0.024, 0.034, respectively), while there were no correlations between TGM2 mRNA expression and AD severity-related indicators (all P>0.05), such as age (21[16, 29] years), course of disease (4[1,10] years), eosinophil counts (0.33[0.18, 0.65] × 10 9/L), basophil counts (0.04[0.03, 0.06] × 10 9/L], SCORAD scores (60.5[46.98, 66.13] points), and serum total IgE levels (373 [40, 1 815] IU/ml). The relative protein expression levels of TGM2 on the surface of PBMCs did not differ between the AD group and control group (54.9 [47.6, 62.8] vs. 55.55 [51.5, 60.25], U = 112.00, P = 0.922) ], and no correlations were observed between the protein expression of TGM2 on PBMCs and AD severity-related indicators in the AD group (all P > 0.05) . Conclusion:No significant differences were observed in TGM2 mRNA expression in PBMCs or TGM2 protein expression on the surface of PBMCs between the AD patients and healthy controls, and there were no correlations between the TGM2 mRNA and protein expression and AD severity.

Serum soluble CD40 ligand expression in children with Kawasaki disease in acute phase and its clinical significance / 中国综合临床

Objective:To investigate the expression of soluble CD40 ligand (sCD40L) in serum of children with Kawasaki disease at acute stage and its diagnostic value in coronary artery disease (CAL).Methods:This study adopts case-control study method. Select 127 children with Kawasaki disease admitted to Xuzhou Children's Hospital affiliated to Xuzhou Medical University from August 2021 to August 2022. They are divided into CAL group and non-CAL group according to the degree of coronary artery involvement. Select 30 healthy children who have physical examination in this hospital at the same time as the healthy control group, and select another 30 children with acute upper respiratory tract infection and fever admitted to this hospital at the same time as the fever control group.Compare the sex, age and laboratory indicators of children with Kawasaki disease with or without CAL, and compare the difference between the serum sCD40L level of children with Kawasaki disease with or without CAL and the fever control group and the healthy control group, the serum sCD40L level of children with different degrees of coronary artery dilation, and analyze the correlation between the serum sCD40L and various laboratory indicators of children with Kawasaki disease and the influencing factors of children with Kawasaki disease complicated with CAL, To evaluate the screening effect of serum sCD40L for Kawasaki disease complicated with CAL. The measurement data with normal distribution is expressed by xˉ± s, the comparison between the two groups adopts independent sample t-test, the comparison between multiple groups adopts one-way ANOVA, and the comparison between two groups adopts LSD method and Bonferroni correction; The measurement data of non-normal distribution is expressed by M( Q1, Q3), and the comparison between the two groups is conducted by Mann-Whitney U test. Pearson method and Spearman mothod were used for correlation analysis. Logistic regression model was used to analyze the influencing factors of children with Kawasaki disease complicated with CAL. The diagnostic value of serum sCD40L level in Kawasaki disease complicated with CAL was analyzed by drawing the ROC curve. Results:All 127 children with Kawasaki disease were divided into CAL group (45 cases) and non-CAL group (82 cases) according to the presence or absence of CAL. The serum level of sCD40L in CAL group was higher than that in non-CAL group, healthy control group and fever control group ((7.03±0.91) μg/L vs (4.66±1.23), (1.73±0.96), (2.21±1.08) μg/L), the difference was statistically significant (all P<0.001). The serum level of sCD40L in children with coronary artery dilation in CAL group was lower than that in children with small CAA, medium CAA and large CAA ((6.04±0.22) μg/L vs (6.95±0.69), (8.02±0.57), (8.23±0.26) μg/L), the difference was statistically significant (all P<0.001). Serum sCD40L level and platelet count (PLT), C-reactive protein (CRP), N-terminal pro brain natriuretic peptide (NT-proBNP), interleukin-6 (IL-6), IL-8 and tumor necrosis factor (TNF-α) in children with Kawasaki disease All were positively correlated ( r=0.31, P<0.001, r=0.32, P<0.001, r=0.26, P=0.003, r=0.58, P<0.001, r=0.27, P=0.002, r=0.39, P<0.001). Serum sCD40L, IL-6 and NT-proBNP were the risk factors of complicated CAL in children with Kawasaki disease (odds ratio 1.21, 1.06 and 1.01, 95% confidence interval 1.03-1.43, 1.01-1.12, 1.00-1.01, P values were 0.022, 0.011 and 0.039, respectively). The area under the curve of serum sCD40L in diagnosing Kawasaki disease complicated with CAL was 0.928 (95% confidence interval: 0.885-0.971), and the optimal critical value was 5.60 μg/L, the sensitivity was 97.8% and the specificity was 79.3%. Conclusions:The level of serum sCD40L increased in children with Kawasaki disease in acute phase, especially in children with CAL. The level of serum sCD40L increased with the severity of CAL, which is a risk factor for Kawasaki disease complicated with CAL, and has certain diagnostic value for Kawasaki disease complicated with CAL.

Serum levels of myonectin, corticostatin and Delta like ligand 4 in patients with type 2 diabetic retinopathy and their clinical significance / 中国医师杂志

Objective:To investigate the serum levels of myonectin, corticostatin and Delta like ligand 4 (DLL4) in patients with type 2 diabetes mellitus (T2DM) diabetes retinopathy (DR) and their clinical significance.Methods:A prospective selection of 341 T2DM patients admitted to Beijing Hospital of Traditional Chinese Medicine, Huairou Hospital from May 2020 to March 2022 was conducted. The patients underwent fundus examination and were divided into a non DR group ( n=85 cases) and a DR group ( n=256 cases) based on DR diagnostic criteria. The DR group was divided into non proliferative and proliferative types according to the staging criteria in China′s DR clinical diagnosis and treatment guidelines, with 142 cases and 114 cases, respectively; 190 healthy individuals who underwent physical examinations in our hospital during the same period were selected as the control group. Enzyme linked immunosorbent assay was used to detect the levels of serum sarconectin, corticostatin, and DLL4 in three groups, collect patient data, and detect biochemical indicators. Logistic regression analysis was used to analyze the influencing factors of DR, and Pearson correlation analysis was used to investigate the relationship between serum sarconectin, corticostatin, DLL4, glucose and lipid metabolism, and insulin resistance; Logistic regression analysis was used to analyze the influencing factors of DR, and Pearson correlation analysis was used to investigate the relationship between serum sarconectin, corticostatin, DLL4, glucose and lipid metabolism, and insulin resistance; The receiver operating characteristic (ROC) curve was used to analyze the diagnostic value of serum sarconectin, corticostatin, and DLL4 in DR. Results:The levels of serum sarconectin and DLL4 in the DR group and non DR group were higher than those in the control group, while the levels of corticostatin were lower than those in the control group (all P<0.05); The levels of sarconectin and DLL4 in the DR group were higher than those in the non DR group, while the levels of corticostatin were lower than those in the non DR group (all P<0.05). The serum levels of sarconectin and DLL4 in proliferative DR patients were higher than those in non proliferative DR patients, while the levels of corticostatin were lower than those in non proliferative DR patients (all P<0.05). The duration of T2DM in the DR group was longer than that in the non DR group, with smoking and alcohol consumption, systolic blood pressure, fasting blood glucose, glycated hemoglobin, triglyceride and insulin resistance index (HOMA-IR) were higher than those in non DR group (all P<0.05). Logistic regression analysis showed that the course of T2DM, systolic blood pressure, smoking and alcohol consumption, glycated hemoglobin, triglycerides, myonectin, corticostatin and DLL4 were the influencing factors of DR (all P<0.05). Pearson correlation analysis results showed that serum sarconectin, DLL4, and fasting blood glucose, glycated hemoglobin, fasting insulin and HOMA-IR were positively correlated (all P<0.05), while cortisol was negatively correlated with fasting blood glucose, glycated hemoglobin and HOMA-IR (all P<0.05). The ROC analysis results showed that the area under the curve (AUC) for the diagnosis of DR was 0.691, 0.745, 0.749, and 0.861 for sarconectin, corticostatin, and DLL4 alone and in combination, respectively. The combined application had higher diagnostic value. Conclusions:Patients with T2DM complicated with DR have elevated levels of serum sarconectin and DLL4, while decreased levels of corticostatin, which are closely related to glucose and lipid metabolism and insulin resistance, and are influencing factors for the occurrence of DR. Combined detection of the three can improve the value of predicting DR.

Role of P2X7/NLRP3 inflammasome pathway in cognitive impairment in sleep deprived mice / 中华行为医学与脑科学杂志

Objective:To investigate the possible role and mechanism of purinergic ligand-gated ion channel 7(P2X7)/nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3) inflammasome pathway in cognitive impairment induced by sleep deprivation (SD)mice.Methods:SPF grade male C57BL / 6J mice aged 6-8 weeks were randomly divided into 3 groups according to the random number table method with 6 mice in each group.They were normal control group (CC group), SD group and SD+ P2X7 receptor antagonist brilliant blue G(BBG) group (SD+ BBG group). Modified multiple platform method was used to establish a 5-day SD model in mice.During the SD intervention period, the mice in SD+ BBG group were injected with BBG(50 mg/kg) intraperitoneally once a day, while the mice in CC group and SD group were injected with the same volume of 0.9% sodium chloride solution.Morris water maze was conducted to evaluate the cognitive function of mice.The protein expression levels of P2X7, NLRP3, caspase-1, apoptosis-associated proteins(ASC) and interleukin-1β(IL-1β) in hippocampus were detected by Western blot.RT-qPCR was used to detect the mRNA expression levels of tumor necrosis factor-α(TNF-α), IL-1β, interleukin-18(IL-18) and microglial polarization surface markers CD206 and CD86 in hippocampus.Graph pad Prism 8.0 software and SPSS 25.0 software were used for statistical analysis and mapping.Results:(1) The interaction effect between time and groups of escape latency in three groups of mice was significant ( F=15.76, P<0.001). From the 2nd to 5th day, the escape latencies of mice in SD group were higher than those of CC group, while the escape latencies of mice in SD+ BBG group were lower than those of SD group (all P<0.05). (2)The results of the space exploration experiment showed that there were statistically significant differences in target quadrant residence time and the times of crossing the platform( F=6.65, P=0.009; F=12.39, P<0.001). The target quadrant residence time ((23.42±0.55) s) and times of crossing the platform ((17.67±0.71) times) of the SD group were both lower than those of the CC group ((29.48±1.78) s, (23.33±0.95) times) (both P<0.05), while the target quadrant residence time ((28.62±1.19) s) and the times of crossing the platforms ((21.33±0.76) times) of the SD+ BBG group were both higher than those of the SD group (both P<0.05). (3)There were statistically significant differences in the protein levels of inflammatory related proteins such as P2X7, NLRP3, caspase-1, ASC and IL-1β in the hippocampus of mice among the 3 groups( F=8.23, 8.97, 8.45, 54.42, 8.12, all P<0.05). Compared with CC group, the protein levels of P2X7 ((0.93±0.02), (0.71±0.04)), NLRP3 ((0.97±0.04), (0.62±0.09)), caspase-1 ((1.00±0.03), (0.76±0.07)), ASC ((0.96±0.02), (0.77±0.04)) and IL-1β ((0.85±0.07), (0.54±0.04)) in SD group were all higher (all P<0.05). Compared with SD group, the protein levels of P2X7 (0.74±0.05), NLRP3 (0.78±0.02), caspase-1 (0.74±0.04), ASC (0.67±0.02), IL-1β (0.53±0.07) in SD+ BBG group were all lower (all P<0.05). (4)There were statistically significant differences in the mRNA levels of IL-18, IL-1β, TNF-α, CD86 and CD206 in hippocampus among the three groups ( F=12.80, 12.28, 105.80, 7.06, 30.19, all P<0.05). The mRNA levels of IL-18, IL-1β, TNF-α, CD86 in SD group were all higher than those in CC group(all P<0.05), while the mRNA level of CD206 in SD group was lower than that in CC group( P<0.05). Compared with SD group, the mRNA levels of IL-18, IL-1β, TNF-α, CD86 were lower in SD+ BBG group (all P<0.05), while the CD206 mRNA level of SD+ BBG group was higher than that in SD group( P<0.05). Conclusion:SD intervention can lead to cognitive impairment and increased expression of P2X7 in hippocampus of mice, which may be related to the activation of P2X7/ NLRP3 inflammasome signaling pathway, promoting the polarization of microglia into pro-inflammatory type and up-regulating the expression of pro-inflammatory cytokines.Inhibition of P2X7 can improve the cognitive function of mice.