Analysis of vibrational dynamics in cell-substrate interactions using nanopipette electrochemical sensors.

Cell-substrate interaction plays a critical role in determining the mechanical status of living cell membrane. Changes of substrate surface properties can significantly alter the cell mechanical microenvironment, leading to mechanical changes of cell membrane. However, it is still difficult to accurately quantify the influence of the substrate surface properties on the mechanical status of living cell membrane without damage. This study addresses the challenge by using an electrochemical sensor made from an ultrasmall quartz nanopipette. With the tip diameter less than 100 nm, the nanopipette-based sensor achieves highly sensitive, noninvasive and label-free monitoring of the mechanical status of single living cells by collecting stable cyclic membrane oscillatory signals from continuous current versus time traces. The electrochemical signals collected from PC12 cells cultured on three different substrates (bare ITO (indium tin oxides) glass, hydroxyl modified ITO glass, amino modified ITO glass) indicate that the microenvironment more favorable for cell adhesion can increase the membrane stiffness. This work provides a label-free electrochemical approach to accurately quantify the mechanical status of single living cells in real-time, which may help to better understand the relationship between the cell membrane and the extra cellular matrix.

Amperometric Identification of Single Exosomes and Their Dopamine Contents Secreted by Living Cells.

Dopamine (DA) is an important neurotransmitter, which not only participates in the regulation of neural processes but also plays critical roles in tumor progression and immunity. However, direct identification of DA-containing exosomes, as well as quantification of DA in single vesicles, is still challenging. Here, we report a nanopipette-assisted method to detect single exosomes and their dopamine contents via amperometric measurement. The resistive-pulse current measured can simultaneously provide accurate information of vesicle translocation and DA contents in single exosomes. Accordingly, DA-containing exosomes secreted from HeLa and PC12 cells under different treatment modes successfully detected the DA encapsulation efficiency and the amount of exosome secretion that distinguish between cell types. Furthermore, a custom machine learning model was constructed to classify the exosome signals from different sources, with an accuracy of more than 99%. Our strategy offers a useful tool for investigating single exosomes and their DA contents, which facilitates the analysis of DA-containing exosomes derived from other untreated or stimulated cells and may open up a new insight to the research of DA biology.

A multi-channel responsive AuNP@COF core-shell nanoprobe for simultaneous subcellular profiling of multiple cancer biomarkers.

The abnormal change in the expression profile of multiple cancer biomarkers is closely related to tumor progression and therapeutic effect. Due to their low abundance in living cells and the limitations of existing imaging techniques, simultaneous imaging of multiple cancer biomarkers has remained a significant challenge. Here, we proposed a multi-modal imaging strategy to detect the correlated expression of multiple cancer biomarkers, MUC1, microRNA-21 (miRNA-21) and reactive oxygen (ROS) in living cells, based on a porous covalent organic framework (COF) wrapped gold nanoparticles (AuNPs) core-shell nanoprobe. The nanoprobe is functionalized with Cy5-labeled MUC1 aptamer, a ROS-responsive molecule (2-MHQ), and a miRNA-21-response hairpin DNA tagged by FITC as the reporters for different biomarkers. The target-specific recognition can induce the orthogonal molecular change of these reporters, producing fluorescence and Raman signals for imaging the expression profiles of membrane MUC1 (red fluorescence channel), intracellular miRNA-21 (green fluorescence channel), and intracellular ROS (SERS channel). We further demonstrate the capability of the cooperative expression of these biomarkers, along with the activation of NF-κB pathway. Our research provides a robust platform for imaging multiple cancer biomarkers, with broad potential applications in cancer clinical diagnosis and drug discovery.

Engineered Extracellular Vesicle-Encapsuled Nanoreactors for Effective Targeting and Cascade Killing of Cancer Cells.

Nanomaterials have presented great potential for cancer therapy. However, their therapeutic efficacy is not always satisfied because of inefficient biocompatibility and targeting efficacy. Here, we report engineered extracellular vesicle (EV)-encapsuled nanoreactors for the targeting and killing of cancer cells. EVs are extracted from engineered cancer cells with surface N-glycans cut and intracellular microRNA-21 (miR-21) silenced to generate cancer-targeting membranes for the following coating of gold-polydopamine (PDA) core-shell nanoparticles. The encapsuled nanoparticles are decorated with doxorubicin (Dox), glucose oxidase (GOx), and miR-21-indicative DNA tags. Once endocytosed, the acidic pH, together with the photothermal effect of the PDA shell, can promote the release of Dox and GOx-catalyzed H2O2 generation/glucose consumption, while the DNA tags allow enhanced fluorescence imaging of miR-21 to indicate the targeting effect. The coadministration of EV-assisted delivery and cascade treatment represents a promising strategy for combination therapy.

Parthenolide and arsenic trioxide co-trigger autophagy-accompanied apoptosis in hepatocellular carcinoma cells.

Although arsenic trioxide (ATO) shows a strong anti-tumor effect in the treatment of acute promyelocytic leukemia, it does not benefit patients with hepatocellular carcinoma (HCC). Thus, combination therapy is proposed to enhance the efficacy of ATO. Parthenolide (PTL), a natural compound, selectively eradicates cancer cells and cancer stem cells with no toxicity to normal cells. In this study, we chose PTL and ATO in combination and found that nontoxic dosage of PTL and ATO co-treatment can synergistically inhibit the in vitro and in vivo proliferation activity of HCC cells through suppressing stemness and self-renewal ability and inducing mitochondria-dependent apoptosis. More importantly, USP7-HUWE1-p53 pathway is involved in PTL enhancing ATO-induced apoptosis of HCC cell lines. Meanwhile, accompanied by induction of apoptosis, PTL and ATO evoke autophagic activity via inhibiting PI3K/Akt/mTOR pathway, and consciously controlling autophagy can improve the anti-HCC efficacy of a combination of PTL and ATO. In short, our conclusion represents a novel promising approach to the treatment of HCC.

lncRNA HHIP-AS1/HHIP modulates osteogenic differentiation of BM-MSCs by regulating Hedgehog signaling pathway.

BACKGROUND: lncRNA, a type of non-coding RNA, plays an important role in the osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BM-MSCs). In this study, lncRNA and mRNA microarrays were performed to study the change of gene expression during osteogenic differentiation of BM-MSCs. We focused on Hedgehog interacting protein (HHIP), because HHIP mRNA and lncRNA HHIP-AS1 were gradually down-regulated on days 0, 7, and 14 during osteogenic differentiation. In addition, the gene coding lncRNA HHIP-AS1 is located on the anti-sense of Hhip gene, implying the potential interaction between lncRNA HHIP-AS1 and HHIP mRNA. METHODS: BM-MSCs with over-expressed or silenced lncRNA HHIP-AS1 were constructed to explore the biological role of HHIP-AS1 in osteogenic differentiation. BM-MSCs were lysed to determine the alkaline phosphatase activity. Fluorescence in situ hybridization and immunofluorescence were performed to analyze HHIP-AS1, HHIP, RUNX2 and osteocalcin. RESULTS: Overexpression of lncRNA HHIP-AS1 increased HHIP expression, which suppressed Hedgehog signaling pathway, as indicated by the reduction of SMO, Gli1 and Gli2. The suppression of Hedgehog signal was associated with the inhibited osteogenesis. HHIP knockdown abolished the suppression of osteogenesis induced by lncRNA HHIP-AS1 overexpression. Through binding to HHIP mRNA, lncRNA HHIP-AS1 recruited ELAVL1 to HHIP mRNA, whereby increasing the mRNA stability and the protein level. CONCLUSIONS: This study revealed that down-regulation of HHIP due to lncRNA HHIP-AS1 reduction promoted the osteogenic differentiation of BM-MSCs though removing the suppression of Hedgehog signal.

Specially Resolved Single Living Cell Perfusion and Targeted Fluorescence Labeling Based on Nanopipettes.

Targeted delivery and labeling of single living cells in heterogeneous cell populations are of great importance to understand the molecular biology and physiological functions of individual cells. However, it remains challenging to perfuse fluorescence markers into single living cells with high spatial and temporal resolution without interfering neighboring cells. Here, we report a single cell perfusion and fluorescence labeling strategy based on nanoscale glass nanopipettes. With the nanoscale tip hole of 100 nm, the use of nanopipettes allows special perfusion and high-resolution fluorescence labeling of different subcellular regions in single cells of interest. The dynamic of various fluorescent probes has been studied to exemplify the feasibility of nanopipette-dependent targeted delivery. According to experimental results, the cytoplasm labeling of Sulfo-Cyanine5 and fluorescein isothiocyanate is mainly based on the Brownian movement due to the dyes themselves and does not have a targeting ability, while the nucleus labeling of 4',6-diamidino-2-phenylindole (DAPI) is originated from the adsorption between DAPI and DNA in the nucleus. From the finite element simulation, the precise manipulation of intracellular delivery is realized by controlling the electro-osmotic flow inside the nanopipettes, and the different delivery modes between nontargeting dyes and nucleus-targeting dyes were compared, showcasing the valuable ability of nanopipette-based method for the analysis of specially defined subcellular regions and the potential applications for single cell surgery, subcellular manipulation, and gene delivery.

Vacuolating Cytotoxin A Triggers Mitophagy in Helicobacter pylori-Infected Human Gastric Epithelium Cells.

Helicobacter pylori (H. pylori)-derived vacuolating cytotoxin A (VacA) causes damage to various organelles, including mitochondria, and induces autophagy and cell death. However, it is unknown whether VacA-induced mitochondrial damage can develop into mitophagy. In this study, we found that H. pylori, H. pylori culture filtrate (HPCF), and VacA could activate autophagy in a gastric epithelial cell line (GES-1). VacA-caused mitochondrial depolarization retards the import of PINK1 into the damaged mitochondria and evokes mitophagy. And, among mass spectrometry (LC-MS/MS) identified 25 mitochondrial proteins bound with VacA, Tom20, Tom40, and Tom70, TOM complexes responsible for PINK1 import, were further identified as having the ability to bind VacA in vitro using pull-down assay, co-immunoprecipitation, and protein-protein docking. Additionally, we found that the cell membrane protein STOM and the mitochondrial inner membrane protein PGAM5 also interacted with VacA. These findings suggest that VacA captured by STOM forms endosomes to enter cells and target mitochondria. Then, VacA is transported into the mitochondrial membrane space through the TOM complexes, and PGAM5 aids in inserting VacA into the inner mitochondrial membrane to destroy the membrane potential, which promotes PINK1 accumulation and Parkin recruitment to induce mitophagy. This study helps us understand VacA entering mitochondria to induce the mitophagy process.

Sialidase-Conjugated "NanoNiche" for Efficient Immune Checkpoint Blockade Therapy.

Reactivation of T-cell immunity by blocking the PD-1/PD-L1 immune checkpoint has been considered a promising strategy for cancer treatment. However, the recognition of PD-L1 by antibodies is usually suppressed due to the N-linked glycosylation of PD-L1. In this study, we present an effective PD-L1-blocking strategy based on a sialidase-conjugated "NanoNiche" to improve the antitumor effect via T-cell reactivation. Molecularly imprinted by PD-L1 N-glycans, NanoNiche can specifically recognize glycosylated PD-L1 on the tumor cell surface, thereby resulting in more efficient PD-L1 blockade. Moreover, sialidase modified on the surface of NanoNiche can selectively strip sialoglycans from tumor cells, enhancing immune cell infiltration. In vitro studies confirmed that NanoNiche can specifically bind with PD-L1 while also desialylate the tumor cell surface. The proliferation of PD-L1-positive MDA-MB-231 human breast cancer cells under T-cell killing was significantly inhibited after NanoNiche treatment. In vivo experiments in solid tumors show enhanced therapeutic efficacy. Thus, the NanoNiche-sialidase conjugate represents a promising approach for immune checkpoint blockade therapy.

Reinvestigation of 2-acetylthiazole formation pathways in the Maillard reaction.

2-Acetylthiazole possesses a nutty, cereal-like and popcorn-like aroma and a low odor threshold, and this compound has been identified in some processed foods, while the formation pathway of 2-acetylthiazole has not been clearly elucidated. Here, a model reaction of d-glucose and l-cysteine was constructed to investigate the formation pathway of 2-acetylthiazole. l-Cysteine, d-glucose and the corresponding intermediates, namely, dicarbonyl compounds (DCs), were involved in the formation of 2-acetylthiazole and detected by high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS), high-performance ion chromatography (HPIC) and HPLC, respectively. The carbon module labeling (CAMOLA) technique revealed that the C-4 and C-5 of 2-acetylthiazole were derived from the carbons of glucose. The potential of glyoxal, which is degraded by glucose, to form 2-acetylthiazole was revealed for the first time. A novel route to form 2-acetylthiazole by the reaction of glyoxal and methylglyoxal produced by d-glucose with H2S and NH3 produced by l-cysteine was proposed.

Development and validation of a risk assessment nomogram for venous thromboembolism associated with hospitalized postoperative Chinese breast cancer patients.

AIM: The purpose of this study was to develop and validate an individualized nomogram to predict venous thromboembolism (VTE) risk in hospitalized postoperative breast cancer patients. DESIGN: A single-central retrospective and non-interventional trial. METHODS: For model development, we used data from 4,755 breast cancer patients between 1 November 2016-30 June 2018 (3,310 patients in the development group and 1,445 in the validation group). Overall, 216 patients developed VTE (150 in development group and 66 in validation group). The model was validated by receiver operating characteristic curves and the calibration plot. The clinical utility of the model was determined through decision curve analysis. RESULTS: The individualized nomogram consisted of six clinical factors: age, body mass index, number of cardiovascular comorbidities, neoadjuvant chemotherapy, surgical treatment, hospital length of stay and two pre-operative biomarkers of Homocysteine and D-dimer. The model at the 3.9% optimal cut-off had the area under the curve of 0.854 (95% CI, 0.824-0.884) and 0.805 (95% CI, 0.740-0.870) in the development and validation groups. A p = 0.570 of the calibration test showed that the model was well-calibrated. The net benefit of the model was better between threshold probabilities of 5%-30% in decision curve analysis. CONCLUSION: The nomogram of VTE risk assessment, is applicable to hospitalized postoperative breast cancer patients. However, multi-central prospective studies are needed to improve and validate the model. Effectiveness and safety of thromboprophylaxis in high-risk patients are needed to demonstrate in interventional trials. IMPACT: This nomogram can be used in clinical to inform practice of physicians and nurses to predict the VTE probability and maybe direct personalized decision making for thromboprophylaxis in hospitalized postoperative breast cancer patients.

A polydopamine-based biomimetic multifunctional nanoplatform for multilayer imaging of cancer biomarkers carried by extracellular vesicles.

Cancer-derived extracellular vesicles (EVs) have attracted considerable attention for clinical diagnosis. However, a limiting factor in current EV assays is the ability to detect various EV cancer biomarkers expressed at different locations. Here, we report a biomimetic multifunctional nanoplatform for multilayer imaging of cancer biomarkers from the EV surface to the interior without complex pretreatment. Constructed from polydopamine-wrapped gold nanoparticles modified with multiple functional molecules, this nanoplatform can capture EVs from complex samples and target different EV cancer biomarkers for imaging analysis at the single-vesicle level. Combined with 96-well plates, this assay can distinguish cancer cell-derived EVs from normal ones in a high-throughput manner. Using serum samples, EVs from hepatocellular carcinoma (HCC) patients can be distinguished from healthy controls. This convenient workflow represents a promising tool for EV-based cancer diagnosis.

MiR-24 Protects Cardiomyocytes Against Hypoxia/Reoxygenation-Induced Injury Through Regulating Mitogen-Activated Protein Kinase 14.

This study aimed to explore the function of miR-24 in hypoxia/reoxygenation (H/R) -induced cardiomyocyte injury.We constructed a cardiomyocyte model of H/R using the primary cardiomyocytes isolated from Sprague-Dawley rats. To explore the role of miR-24, cells were transfected with a miR-24 mimic or miR-24 inhibitor. The RNA expression levels of miR-24 and Mapk14 were determined using qRT-PCR. The proliferation and apoptosis of cells were determined using a CCK8 assay and a flow cytometer. The TargetScan website was used to predict the targets of miR-24. A dual-luciferase reporter gene assay was conducted to verify whether Mapk14 is indeed a target of miR-24. A Western blot was applied for protein detection.H/R exposure decreased the expression of miR-24 in rat cardiomyocytes. Transfection of the miR-24 mimic into cardiomyocytes reduced H/R-induced injury as evidenced by an increase in proliferation and a decrease in the apoptotic rate. By contrast, transfection of the miR-24 inhibitor aggravated H/R-induced injury. The expression of Bcl-2 was increased while the levels of Bax and Active-caspase 3 were reduced in the H/R+miR-24 mimic group compared to those in the H/R group. H/R+miR-24 inhibitor group showed the opposite results. Mapk14 was identified as a target of miR-24. The mRNA level of Mapk14 and its protein (p38 MAPK) level were negatively affected by miR-24. Furthermore, we discovered that depletion of Mapk14 reduced the promoting effect of the miR-24 inhibitor on cell apoptosis.Overall, our results illustrated that miR-24 could attenuate H/R-induced injury partly by regulating Mapk14.

Rolling "wool-balls": rapid live-cell mapping of membrane sialic acids via poly-p-benzoquinone/ethylenediamine nanoclusters.

Here, we report a convenient, fast labeling strategy for the imaging of cell surface sialic acids (SAs, nine-carbon monosaccharides located at the terminals of cell surface sugar chains). This strategy is based on the synthesis of sticky, furry and fluorescent "wool-balls", which are wound into nanoclusters from p-benzoquinone/ethylenediamine polymer "wires". With abundant amino groups at the surface, the wool-balls can easily stick to the C-7 aldehyde group generated at the ends of periodate treated SAs in less than 30 min.

miR­152 regulates TGF­ß1­induced epithelial­mesenchymal transition by targeting HPIP in tubular epithelial cells.

Renal fibrosis is a common pathological feature of chronic kidney diseases, and their development and progression are influenced by epigenetic modifications including aberrant microRNA (miRNA or miR) expression. miRNAs have been demonstrated to modulate the aggressiveness of various cancers and have emerged as possible therapeutic agents for the management of renal fibrosis. Transforming growth factor ß1 (TGF­ß1)­induced epithelial­mesenchymal transition (EMT) of tubular epithelial cells serves a role in the initiation and progression of renal fibrosis. Furthermore, recent results indicated that the progression of EMT is reversible. The present study aimed to clarify the role of miR­152 in EMT of the tubular epithelial cell line HK­2, stimulated by TGF­ß1, using in vitro transfection with a miR­152 mimic and to further investigate the underlying mechanism of miR­152 activity. In the present study, miR­152 expression was significantly reduced in TGF­ß1­treated HK­2 cells, accompanied by an increased expression of hematopoietic pre­B­cell leukemia transcription factor (PBX)­interacting protein (HPIP). Additionally, miR­152 overexpression inhibited TGF­ß1­induced EMT and suppressed HPIP expression by directly targeting the 3' untranslated region of HPIP in HK­2 cells. Furthermore, upregulation of HPIP reversed miR­152­mediated inhibitory effects on the EMT. Collectively, the results suggest that downregulation of miR­152 initiates the dedifferentiation of renal tubules and progression of renal fibrosis, which may provide important targets for prevention strategies of renal fibrosis.

[MiR-155 Inhibits the Expression of MMPs in HK2 Cells by Targeting KLF4].

OBJECTIVES: To determine whether miR -155 inhibits the expression of matrix metalloproteinase 2 (MMP-2) and matrix metalloproteinase 9 (MMP-9) in HK2 cells by targeting Kruppel-like factor 4 (KLF4). METHODS: MiR -155-mimic, miR -155-NC empty plasmid, and culture medium were transfected into renal tubular epithelial cells, respectively. Six hours later, the expression of miR -155 was detected by real time-PCR; the expressions of MMP-2, MMP-9 and KLF4 were detected by Western blot; and the activity of MMP-2 and MMP-9 in the cells was detected by gelatin zymography. Because KLF4 was predicted as the target gene of miR -155 by bioinformatics. The miR-155 overexpressed HK2 cells were transfected with KLF4 overexpression plasmid or empty plasmid. Six hours later, the expressions of MMP-2, MMP-9 and KLF4, and the activity of MMP-2 and MMP-9 were measured again. Finally, cells containing luciferase plasmids with KLF4-3!d-UTR wild type (WT) or mutant (MUT) sequence were constructed and transfected with miR -155-mimic or empty plasmid. Luciferase assay was used to confirm whether KLF4 -3!d-UTR was the binding site in targeting miR -155#. RESULTS: Compared with the cells transfected with empty plasmid, the expression of miR -155 was up-regulated and the expressions of MMP-2, MMP-9 and KLF4 were down-regulated in the cells transfected with miR -155-mimic. Compared with the cells transfected with miR -155 mimic or miR -155 mimic+empty plasmid, the expressions of MMP-2, MMP-9 and KLF4 were up-regulated in the KLF4+miR -155 transfected cells. Luciferase assays confirmed that miR -155 binds to KLF4 , and KLF4 -3!d-UTR is the target gene of miR -155. CONCLUSION: MiR-155 inhibits the expressions of MMP-2 and MMP-9 in HK2 cells by targeting KLF4 -3!d-UTR.

Dietary intake and risk of rheumatoid arthritis-a cross section multicenter study.

Environmental factors play an important role in the development of rheumatoid arthritis (RA). Among these factors, smoking is generally considered to be an established risk factor for RA. Data regarding the impact of diet on risk of RA development is limited. This study assessed the impact of dietary patterns on RA susceptibility in Chinese populations. This was a large scale, case-control study composed of 968 patients with RA and 1037 matched healthy controls. Subjects were recruited from 18 teaching hospitals. Socio-demographic characteristics and dietary intakes 5 years prior to the onset of RA were reported by a self-administered questionnaire. Differences in quantity of consumption between cases and controls were analyzed by Student's t test. Multiple logistic regression analysis was applied to identify independent dietary risk factor(s) responsible for RA susceptibility. Compared to healthy individuals, RA patients had decreased consumption of mushrooms (P = 0.000), beans (P = 0.006), citrus (P = 0.000), poultry (P = 0.000), fish (P = 0.000), edible viscera (P = 0.018), and dairy products (P = 0.005). Multivariate analyses revealed that several dietary items may have protective effects on RA development, such as mushrooms (aOR = 0.669; 95%CI = 0.518-0.864, P = 0.002), citrus fruits (aOR = 0.990; 95%CI = 0.981-0.999, P = 0.04), and dairy products (aOR = 0.921; 95%CI 0.867-0.977, P = 0.006). Several dietary factors had independent effects on RA susceptibility. Dietary interventions may reduce the risk of RA.

[Detection of epidermal growth factor receptor gene mutations in advanced non-small cell lung cancer].

OBJECTIVE: To investigate the mutations of epidermal growth factor receptor (EGFR) in tumor tissue and pleural effusion in advanced non-small cell lung cancer (NSCLC) patients, and to analyze the relationship between EGFR mutations and the clinicopathologic characteristics. METHODS: Two-hundred and forty-one cases of formalin-fixed, paraffin-embedded tumor tissues and 14 paired pleural effusions from advanced NSCLC patients were collected. Twenty-nine different EGFR mutations in exons 18-21 were assessed by scorpions and amplification refractory mutation system (scorpions ARMS) using real time PCR. The relationship between the EGFR mutations and clinical parameters was analyzed using statistical methods. EGFR mutation of 37 cases were detected with direct sequencing, and assessed the sensitivity, the specificity and the accuracy of scorpions ARMS. RESULTS: EGFR somatic mutations were detected in 114 of 234 advanced NSCLC patients, with the mutation rate of 48.7%, including deletions in exon 19 in 65 patients and point mutation of L858R in exon 21 in 39 patients; both accounting for 91.2% (104/114) of all types of EGFR mutations. The test results of 14 paired pleural effusion specimens were entirely the same to the tissues. The concordance rate of 2 different detection methods was 94.6%. Mutation rate was higher in women (55.9%) than in men (42.2%), and there was no difference in mutation rates between smokers and non-smokers; patients in stage IIIB and stage IV; adenocarcinoma and non-adenocarcinoma. CONCLUSIONS: EGFR somatic mutations appear to occur frequently in Chinese. Scorpions ARMS technology is a sensitive method to detect EGFR mutations and is suitable for screening patients who would likely respond to EGFR inhibitors therapy.

[Survey of tumor necrosis factor inhibitors application in patients with rheumatoid arthritis in China].

OBJECTIVE: To investigate the current status of tumor necrosis factor (TNF) inhibitors application in rheumatoid arthritis (RA) patients in China and to analyze the related factors. METHODS: A retrospective survey was conducted in 21 hospitals from different parts of China. The patients with RA were randomly enrolled. Data of their social backgrounds, clinical conditions, usage and adverse effects of TNF inhibitors were collected. The costs of TNF inhibitors and the indirect costs of the disease were calculated. A multivariate Logistic regression analysis was performed to analyze the factors related to TNF inhibitors application. RESULTS: In the study, 1 095 RA patients from July 2009 to November 2010 were enrolled, of whom 112 had received TNF inhibitors, representing 10.2% of the total patients. The patients who received etanercept and infliximab were 7.4% (86/1 095) of the patients and 2.4% (26/1 095), respectively. There were 0.5% of the patients (5/1 095) who had received both of the TNF inhibitors. The patients who had accepted etanercept and treatment duration for less than 3 months and 3-6 months accounted for 38.5% and 25.0% respectively, while those treated with Infliximab were 38.1%. Their health assessment questionnaire (HAQ) scores were 1.1, 0.5 and 0.1, corresponding to treatment duration of infliximab for less than 3, 3-6 and 6-9 months and those were 1.3, 1.0, 0.3 corresponding to treatment duration of etanercept, respectively. Infliximab costs were RMB 24 525.0, 69 300.0 and 96 800.0 Yuan and etanercept costs were RMB 7 394.8, 9 158.6, 54 910.9 Yuan, respectively. Indirect costs for RA patients who accepted infliximab for less than 3, 3-6 and 6-9 months were RMB 365.6, 0 and 158.9 Yuan and those who accepted etanercept were RMB 2 158.4, 288.5 and 180.1 Yuan, respectively. Allergy and infection were the main side-effects of etanercept and both happened in 3.5% of all the patients. Liver damage happened in 2.3% of all the patients, while allergy and infection happened in 6.5% of all the patients who accepted infliximab. Logistic regression analysis showed that patients with higher education experience increased the odds of entering the TNF inhibitors group (OR: 1.292, 95%CI: 1.132-1.473, P=0.000). CONCLUSION: About one-tenth of RA patients in China have accepted TNF inhibitors. Higher education experience is the key factor for using TNF inhibitors.

[The study of anti-tumor activities of DC vaccine loaded with multi-epotipes of survivin].

AIM: To observe the anti-tumor activity of dendritic cell (DC)vaccine loaded with multi-epitopes of survivin. METHODS: The recombinant plasmid pPIRESneo3.0-survivin (4)/Th which include four survivin HLA-A2-restricted CD8(+); CTL epitopes and a CD4(+);Th epitope, pPIRESneo3.0-survivin (4) which include four survivin CD8(+); CTL epitopes, were transfected into human dendritic cells respectively. There were five groups, which included survivin(4)/Th group, survivin(4)group, empty plasmid group, untransfected group and T lymphocytes group The expression of CD83 and CD86 on the surface of DCs, the expression of CD4 and CD8a on the surface of T lymphocytes, the apoptotic rates of MCF-7 cells after treated by DC vaccine were measured by flow cytometry; IFN-γ levels of all groups were detected by ELISA and the growth inhibition of MCF-7 cells after being treated with DC vaccine was tested by MTT colorimetry. RESULTS: The results of flow cytometry revealed that high levels CD83 and CD86 were expressed on the surface of DCs; high levels CD4 and CD8a were expressed on the surface of T lymphocytes; the IFN-γ levels in survivin(4)/Th group [(66.50±3.34)ng/L]were significantly higher than that in survivin(4)group[(46.10±1.35)ng/L], empty plasmid group[(25.17±0.32)ng/L], untransfected group [(25.47±0.95)ng/L] or T lymphocytes group[(23.73±0.50)ng/L](P<0.05). The inhibition rate of MCF-7 cells in survivin(4)/Th group was significantly higher than that in survivin(4)group, empty plasmid group, untransfected group or T lymphocytes group(P<0.05). The apoptotic rate of MCF-7 cells in survivin(4)/Th group was (10.63±0.29)% after treated by DC vaccine, which was significantly higher than that in in survivin(4)group, empty plasmid group, untransfected group or T lymphocytes group(P<0.05). CONCLUSION: The DCs vaccine loaded with multi- CD8(+); CTL epitopes of survivin has strong anti-tumor effects. CD4(+); Th cells can promote the anti-tumor activity of CD8(+);CTL.