Neutrophil extracellular traps induce pyroptosis of pulmonary microvascular endothelial cells by activating the NLRP3 inflammasome.

Excessive formation of neutrophil extracellular traps (NETs) may lead to myositis-related interstitial lung disease (ILD). There is evidence that NETs can directly injure vascular endothelial cells and play a pathogenic role in the inflammatory exudation of ILD. However, the specific mechanism is unclear. This study aimed to investigate the specific mechanism underlying NET-induced injury to human pulmonary microvascular endothelial cells (HPMECs). HPMECs were stimulated with NETs (200 ng/ml) in vitro. Cell death was detected by propidium iodide staining. The morphological changes of the cells were observed by transmission electron microscopy (TEM). Pyroptosis markers were detected by western blot, immunofluorescence, and quantitative real-time polymerase chain reaction, and the related inflammatory factor Interleukin-1ß (IL-1ß) was verified by enzyme-linked immunosorbent assay (ELISA). Compared with the control group, HPMECs mortality increased after NET stimulation, and the number of pyroptosis vacuoles in HPMECs was further observed by TEM. The pulmonary microvascular endothelial cells (PMECs) of the experimental autoimmune myositis mouse model also showed a trend of pyroptosis in vivo. Cell experiment further confirmed the significantly high expression of the NLRP3 inflammasome and pyroptosis-related markers, including GSDMD and inflammatory factor IL-1ß. Pretreated with the NLRP3 inhibitor MCC950, the activation of NLRP3 inflammasome and pyroptosis of HPMECs were effectively inhibited. Our study confirmed that NETs promote pulmonary microvascular endothelial pyroptosis by activating the NLRP3 inflammasome, suggesting that NETs-induced pyroptosis of PMECs may be a potential pathogenic mechanism of inflammatory exudation in ILD.

SRT1720 inhibits bladder cancer cell progression by impairing autophagic flux.

Bladder cancer (BC) is the most common cancer of the urinary tract, with poor survival, high recurrence rates, and lacking of targeted drugs. In this study, we constructed a library to screen compounds inhibiting bladder cancer cells growth. Among them, SRT1720 was identified to inhibit bladder cancer cell proliferation in vitro and in vivo. SRT1720 treatment also suppressed bladder cancer cells migration, invasion and induced apoptosis. Mechanism studies shown that SRT1720 promoted autophagosomes accumulation by inducing early-stage autophagy but disturbed the late-stage of autophagy by blocking fusion of autophagosomes and lysosomes. SRT1720 appears to induce autophagy related proteins expression and alter autophagy-related proteins acetylation to impede the autophagy flux. LAMP2, an important lysosomal associated membrane protein, may mediate SRT1720-inhibited autophagy flux as SRT1720 treatment significantly deacetylated LAMP2 which may influence its activity. Taken together, our results demonstrated that SRT1720 mediated apoptosis and autophagy flux inhibition may be a novel therapeutic strategy for bladder cancer treatment.

Edaravone promotes viability of random skin flaps via activating PI3K/Akt/mTOR signalling pathway-mediated enhancement of autophagy.

Random skin flap transplantation is a commonly used technique. However, ischemia and ischemia-reperfusion injury always impair its therapeutic effectiveness through acclerating oxidative stress, apoptosis and suppressing angiogenesis. To survive, cells rely on mediating autophagy, DNA repair, immunoregulation to resist these cellular injuries. Thus, mediating autophagy may affect the survival of random skin flaps. The edaravone (EDA), a oxygen radicals scavenger, also possesses autophagy mediator potential, we investigated the effects of EDA on skin flap survival and its autophagy-related mechanisms. In vivo, mice were administered EDA or saline intraperitoneally for 7 days postoperatively. We found that EDA ameliorated the viability of random skin flaps, promoted autophagy and angiogenesis, attenuated apoptosis and oxidative stress. In vitro, mouse umbilical vascular endothelial cells (MUVECs) were administered EDA or 3-methyladenine (3-MA, an autophagy inhibitor) or rapacymin (Rapa, an autophagy activator) at the beginning of oxygen glucose deprivation (OGD). We found that EDA promoted cell viability, activated autophagy, enhanced angiogenesis, alleviated apoptosis and oxidative stress. On one hand, 3-MA reversed the effects of EDA on cell viability, oxidative stress and apoptosis via inhibiting autophagy. On the other hand, Rapa had the similar effects of EDA. Furthermore, EDA-induced autophagy was mediated through downregulating PI3K/Akt/mTOR signalling pathway. The findings showed that EDA ameliorated viability of random skin flaps by promoting angiogenesis, suppressing oxidative stress and apoptosis, which may be mediated by autophagic activation through downregulating PI3K/AKT/mTOR signalling pathway.

Integrated bioinformatics analysis shows integrin alpha 3 is a prognostic biomarker for pancreatic cancer.

Integrin subunit alpha 3 (ITGA3) expression correlates with the development and prognosis of human cancers. This study aimed to investigate the association of ITGA3 expression with pancreatic cancer (PCa) prognosis. The ITGA3 gene expression data were extracted from The Cancer Genome Atlas (TCGA) pancreatic adenocarcinoma (PAAD) cohort and 14 Gene Expression Omnibus microarray datasets. The differences in ITGA3 expression levels between tumor and non-tumor tissues were compared using the Mann-Whitney U test. Cox regression analysis and meta-analysis were performed to detect the association of ITGA3 expression with PCa prognosis. ITGA3 expression was higher in tumors than in controls. Tumors with advanced grades (3/4) had higher ITGA3 levels compared with early-grade tumors (1/2). The meta-analysis of the TCGA PAAD cohort and seven microarray datasets (GSE28735, GSE62452, GSE79668, GSE71729, GSE57495, GSE78229, and GSE21501) showed that ITGA3 was a prognostic biomarker in PCa (hazard ratio (HR) = 1.38, 95% confidence interval (CI) 1.26-1.51, p < 0.00001). Five ITGA3-related genes, including ITGB1 (HR = 1.6), ITGB5 (HR = 1.6), ITGB6 (HR = 1.6), LAMA3 (HR = 2.1), and CD9 (HR = 2.3), correlated with PCa prognosis significantly (p < 0.05). Functional enrichment analysis showed that ITGA3 was related to "hsa04151: PI3K-Akt signaling pathway" and "hsa04510: Focal adhesion." We concluded that high ITGA3 expression was a potential prognostic biomarker in PCa.

Prolonged oral ingestion of microplastics induced inflammation in the liver tissues of C57BL/6J mice through polarization of macrophages and increased infiltration of natural killer cells.

Microplastics (< 5 mm diameter) are one of most important environmental pollutants and contaminants worldwide. However, how microplastics affect liver immune microenvironment in not well understood. Microplastics (0.5 µm) were administered orally to C57BL/6J mice for 4 consecutive weeks at the rate of 0.5 mg/day. Non-parenchymal cells were isolated from of the mice through fractionation of fresh hepatic tissues. The immune landscape for four cell populations of B cells, T cells, NK cells and macrophages in the liver tissues was then evaluated using flow cytometry. The secretion level of inflammatory cytokines and associated signaling pathway were investigated using quantitative real-time polymerase chain reaction and western blot. Oral ingestion of microplastics increases liver weight, general liver index as well as expression of serum, liver function-related indicators. Microplastics also increased the infiltration of natural killer cells and macrophages to non-parenchymal liver cells, but reduced that of B cells to the same tissues. However, microplastics had no effect on the infiltration of T cell to non-parenchymal liver cells. Ingestion of MPs also up-regulated the expression of IFN-γ, TNF-α, IL-1ß, IL-6 and IL-33 mRNA, but down-regulated that of IL-4, IL-5, IL-10, IL-18 and TGF-ß1. Overall, the aforementioned processes were regulated via the NF-κB pathway in the hepatic non-parenchymal cells. Microplastics disrupts inflammatory process in liver tissues via the NF-κB signaling pathway. These findings provide a strong foundation on immune processes in hepatic tissues following prolonged ingestion of microplastics.

Effects of interleukin-2 concentration and administration method on proliferation and function of cytokine-induced killer cells.

BACKGROUND: Cytokine-induced killer cells (CIKs) adoptive cell transfer (ACT) is a common malignant tumor treatment method. Interleukin-2 (IL-2) is one of the essential cytokines for CIKs cultures. In different phase of CIKs (quiescent and exponential growth), due to different active states and IL-2R expression of the CIKs surface, different doses of IL-2 are required. However, most studies, only addressed the effects of IL-2 concentrations on the function of CIKs, and the differences between varied administration methods of IL-2 have not been explored. METHODS: This study established a novel sequential administration methods for IL-2. Different concentrations of IL-2 were added during different CIKs induction phases. Then, the proliferation ability of CIKs was evaluated using cell proliferation curves. The immune phenotype was analyzed by flow cytometry (FCM), and IFN-γ secretion ability and cytotoxicity were detected using enzyme-linked immunosorbent assay (ELISA) kits and cell counting kit-8, respectively. Multiple comparisons were conducted between each group to compare the function of CIKs in 12 experimental groups. RESULTS: As the IL-2 concentration increased, the number of CIKs continued to increase in each group, but the function of CIKs was not positively related to its number: CD3+ CD56+ subpopulation ratio, INF-γ secretion ability, and cytotoxicity showed irregular changes. During the quiescent and exponential growth phases, adding 300 and 1,000 U/mL IL-2 respectively achieved powerful CIKs (cell numbers of day 16: (384.37±2.05)×106/mL, proliferation: 128.12, CD3+ CD56+ subpopulation ratio: 40.9%, INF-γ secretion ability: 542 pg/mL, cytotoxicity: 40:1, 74.22). CONCLUSIONS: Different concentrations of IL-2 had a greater influence on the biological function of CIKs in different growth phases, and it is better to add IL-2 sequentially during the quiescent and exponential growth phases of CIKs.

Rho GTPase ROP1 Interactome Analysis Reveals Novel ROP1-Associated Pathways for Pollen Tube Polar Growth in Arabidopsis.

ROP (Rho-like GTPases from plants) GTPases are polarly localized key regulators of polar growth in pollen tubes and other cells in plants. However, how ROP GTPases are regulated and how they control polar growth remains to be fully understood. To gain new insights into ROP-dependent mechanisms underlying polar cell growth, we characterized the interactome of ROP1 GTPase that controls Arabidopsis pollen tube (PT) tip growth, an extreme form of polar cell growth. We established an efficient method for culturing Arabidopsis pollen tubes in liquid medium, which was used for immunoprecipitation/mass spectrometry-based identification of ROP1-associated proteins. A total of 654 candidates were isolated from the ROP1 interactome in Arabidopsis pollen tubes, and GO (Gene Ontology) classification and pathway analysis revealed multiple uncharacterized ROP1-dependent processes including translation, cell wall modification, post transcriptional modification, and ion homeostasis, in addition to known ROP1-dependent pathways. The ROP1-interactome data was further supported by the co-expression of the candidate interactors in highly mature pollen with PT germination and growth defects being discovered in 25% (8/32) of the candidate mutant genes. Taken together, our work uncovers valuable information for the identification and functional elucidation of ROP-associated proteins in the regulation of polar growth, and provides a reliable reference to identify critical regulators of polar cell growth in the future.

Quick stabilization of capillary for rapid determination of potassium ions in the blood of epilepsy patients by capillary electrophoresis without sample pretreatment.

Mutations in the potassium channel genes may be linked to the development of epilepsy and affect the blood potassium levels. Therefore, accurate determination of potassium in the blood will be critical to diagnose the cause of epilepsy. CE is a competent technique for the fast detection of multiple ions, but complicated matrices of a blood sample may cause significant variation of migration times and the peak shape. In this work, a procedure for rapid stabilization of the capillary inner surface through preflushing of a blood sample was employed. The process takes only 40 min for a capillary and then it can be used for more than 2 weeks. No pretreatment of the blood sample or other surface modification of the capillary is needed for the analysis. The RSDs of the migration time and peak area were reduced to 1.5 and 5.1% from 12.6 and 14.5%, respectively. The proposed method has been successfully applied to the determination of the potassium contents in the blood sample of patients with epilepsy at different stages. The recoveries of potassium ions in these blood samples are in a range from 86.5 to 104.5%.

miR-532 promotes colorectal cancer invasion and metastasis by targeting NKD1.

The aim of this study was to investigate the effect of microRNA-532 (miR-532) on invasion and metastasis of colorectal cancer (CRC) cells, and the underlying mechanism. Human CRC cell line (HCT116) and normal colon (FHC) cells were used for this study. The cells were transfected with naked cuticle homolog 1 (NKD1) overexpression plasmid, miR-532 mimics, miR-532 inhibitor or miR-532 non-homologous sequence using lipofectamine 2000. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to determine the expression of miR-532 in CRC cells, and a combination of scratch and Transwell assays was used to assess the effect of miR-532 on migration and invasion of CRC cells. Western blotting was used to determine the effect of miR-532 on NKD1 expression in CRC cells. Bioinformatics analysis and luciferase reporter gene assay were used to assess the regulatory effect of miR-532 on NKD1. The expression of miR-532 was upregulated in CRC cells relative to normal colon cells (p < 0.05). The HCT116 cells transfected with miR-532 mimics migrated faster than those of miR-532 negative control (miR532-NC) group (p < 0.05). The migration ability of HCT116 cells transfected with miR-532 inhibitor was significantly reduced, when compared with that of miR532-NC group (p < 0.05). The invasive ability of HCT116 cells transfected with miR-532 mimics was significantly higher than that of miR532-NC cells (p < 0.05). However, inhibition of miR-532 expression significantly reduced the invasive ability of HCT116 cells (p < 0.05). Results of bioinformatics showed that miR-532 had specific binding sequence with the 3'UTR region of NKD1. After cloning the sequence into the luciferase reporter plasmid, miR-532 significantly inhibited the expression of NKD1 (p < 0.05). However, miR-532 had no inhibitory effect on mutated NKD1 3'UTR (p > 0.05). Results of Western blotting showed that increased miR-532 expression significantly reduced the expression of NKD1, while decreased miR-532 expression promoted the expression of NKD1 (p < 0.05). Overexpression of NKD1 significantly down-regulated miR-532 overexpression and promoted CRC cell invasion and metastasis (p < 0.05). miR-532 is highly expressed in CRC cells and directly inhibits NKD1 expression, while enhancing invasion and metastasis of CRC cells. It promotes the development of CRC by inhibiting the expression of NKD1.

Clinical effect and safety of dendritic cell-cytokine-induced killer cell immunotherapy for pancreatic cancer: a systematic review and meta-analysis.

BACKGROUND: Although promising results have recently been reported using dendritic cells (DCs) and cytokine-induced killer cells (CIKs) to treat pancreatic cancer (PC), its clinical effect and safety are associated with some controversy, and lack sufficient evidence. Here, we conducted a meta-analysis of 21 clinical trials to better evaluate the efficacy of DC-CIK immunotherapy in clinical practice to treat PC. METHODS: PubMed, Cochrane Library, China National Knowledge Infrastructure (CNKI) and Wanfang Data Knowledge Service Platform (WANFANG Data) were searched to identify clinical trials that used DC-CIK immunotherapy for PC. Meta-analysis was performed using RevMan 5.3 and Stata 12.0. RESULTS: A total of 21 clinical trials involving 1549 patients were included. Compared with traditional treatment, DC-CIK immunotherapy improved and increased the clinical indices such as complete remission, partial remission, overall response rate, disease control rate, overall survival (0.5-y OS, 1-y OS, 1.5-y OS, 2-y OS and 3-y OS), interferon γ and CD3+, CD4+, CD4+/CD8+ and CD3+CD56+ lymphocyte. Additionally, DC-CIK immunotherapy reduced stable disease, progression disease, mortality, CD8+, CD4+CD25+CD127 low lymphocyte and interleukin-4. Furthermore, it showed a low incidence of adverse reactions (22%). CONCLUSION: In contrast to traditional therapy, DC-CIK immunotherapy not only shows improved short-term effect, long-term effect and immunologic function, but also reduces mortality and negative immunoregulatory index, and shows mild adverse reactions. This is the first study to evaluate the clinical effect and safety of DC-CIK immunotherapy for PC, and it indicated that DC-CIK immunotherapy may be suitable for patients with advanced PC or intolerance to radiotherapy and chemotherapy.

The REN4 rheostat dynamically coordinates the apical and lateral domains of Arabidopsis pollen tubes.

The dynamic maintenance of polar domains in the plasma membrane (PM) is critical for many fundamental processes, e.g., polar cell growth and growth guidance but remains poorly characterized. Rapid tip growth of Arabidopsis pollen tubes requires dynamic distribution of active ROP1 GTPase to the apical domain. Here, we show that clathrin-mediated endocytosis (CME) coordinates lateral REN4 with apical ROP1 signaling. REN4 interacted with but antagonized active ROP1. REN4 also interacts and co-localizes with CME components, but exhibits an opposite role to CME, which removes both REN4 and active ROP1 from the PM. Mathematical modeling shows that REN4 restrains the spatial distribution of active ROP1 and is important for the robustness of polarity control. Hence our results indicate that REN4 acts as a spatiotemporal rheostat by interacting with ROP1 to initiate their removal from the PM by CME, thereby coordinating a dynamic demarcation between apical and lateral domains during rapid tip growth.

Identification of two novel mutations in the ATP7B gene that cause Wilson's disease.

BACKGROUND: Wilson's disease is an autosomal recessive disorder characterized by liver disease and/or neurologic deficits due to copper accumulation and is caused by pathogenic mutations in the ATP7B gene. DATA SOURCES: Two unrelated Chinese patients born to nonconsanguineous parents who were diagnosed with earlyonset Wilson's disease. DNA sequencing and bioinformation analysis were conducted. RESULTS: We have identified four mutations in two family trios, of which two were novel, namely, c. 3028A>G (p. K1010E) and c.3992T>G (p.Y1331X), in each patient. CONCLUSIONS: Gene testing is playing an important role in diagnosis of Wilson's disease. The early-onset of Wilson's disease is apparently not associated with P-ATPase domain in the ATP7B protein. Our findings further widen the spectrum of mutations involving the ATP7B gene.

Galectin-1-driven upregulation of SDF-1 in pancreatic stellate cells promotes pancreatic cancer metastasis.

Galectin-1, mainly expressed in activated pancreatic stellate cells (PSCs), is involved in many important cancer-related processes. However, very little is known how Galectin-1 modulates PSCs and subsequently impacts pancreatic cancer cells (PCCs). Our chemokine antibody array and in vitro studies demonstrates that Galectin-1 induces secretion of stromal cell-derived factor-1(SDF-1) in PSCs by activating NF-κB signaling. The secreted SDF-1 increases migration and invasion of PCCs. Knockdown of Galectin-1 and inhibitor-mediated blockade of SDF-1 as well as its ligand CXCR4 and NF-κB verifies the findings. In vivo experiment by knockdown of Galectin-1 in PSCs further demonstrates the conclusion. Collectively, the present studies demonstrate that Galectin-1-driven production of SDF-1 in PSCs through activation of NF-κB promotes metastasis in PDAC, offering a potential target in the treatment of pancreatic cancer.

Modified methods for isolation of pancreatic stellate cells from human and rodent pancreas.

Primary cultures of pancreatic stellate cells (PSCs) remain an important basis for in vitro study. However, effective methods for isolating abundant PSCs are currently lacking. We report on a novel approach to isolating PSCs from normal rat pancreases and human pancreatic ductal adenocarcinoma (PDAC) tissue. After anaesthesia and laparotomy of the rat, a blunt cannula was inserted into the pancreatic duct through the anti-mesentery side of the duodenum, and the pancreas was slowly infused with an enzyme solution until all lobules were fully dispersed. The pancreas was then pre-incubated, finely minced and incubated to procure a cell suspension. PSCs were obtained after the cell suspension was filtered, washed and subject to gradient centrifugation with Nycodenz solution. Fresh human PDAC tissue was finely minced into 1×1×1 mm3 cubes with sharp blades. Tissue blocks were placed at the bottom of a culture plate with fresh plasma (EDTA-anti-coagulated plasma from the same patient, mixed with CaCl2) sprinkled around the sample. After culture for 5-10 days under appropriate conditions, activated PSCs were harvested. An intraductal perfusion of an enzyme solution simplified the procedure of isolation of rat PSCs, as compared with the multiple injections technique, and a modified outgrowth method significantly shortened the outgrowth time of the activated cells. Our modification in PSC isolation methods significantly increased the isolation efficiency and shortened the culture period, thus facilitating future PSC-related research.

Activation of pancreatic stellate cells involves an EMT-like process.

Pancreatic adenocarcinoma (PDAC) and chronic pancreatitis (CP) are characterized by a desmoplastic reaction involving activated pancreatic stellate cells (PSCs). However, the mechanisms of PSC activation remain poorly understood. We examined whether the epithelial-mesenchymal transition (EMT) process might play a role in PSC activation. PSCs were isolated from a rat pancreas and characterized using immunofluorescence and immunocytochemistry. We evaluated changes in cell motility and in the expression levels of a panel of EMT-related genes during the PSC activation process. Activation of PSCs occurred after 48 h of in vitro culture, as indicated by a morphological change to a myofibroblastic shape and a decrease in the number of cytoplasmic lipid droplets. After activation, PSCs showed enhanced cell migration ability compared to quiescent cells. In addition, the expression of epithelial markers (E-cadherin, BMP7 and desmoplakin) decreased, while expression of mesenchymal markers (N-cadherin, vimentin, fibronectin1, collagen1α1 and S100A4) increased in activated PSCs. EMT-related transcription factors (Snail and Slug) were also upregulated after PSC activation. The concurrent increase in cell migration ability and alterations in EMT-related gene expression suggests that the activation of PSCs involves an EMT-like process. The knowledge that PSC activation involves an EMT­like process may help to identify potential new therapeutic targets to alleviate pancreatic fibrosis in diseases like CP and PDAC.

Factors associated with pancreatic infection in patients with severe acute pancreatisis.

OBJECTIVE: To identify potential risk factors associated with pancreatic infection in severe acute pancreatisis (SAP) patients, thus providing evidence for clinical prediction and treatment. METHODS: A total of 42 patients with SAP collected in our hospital from January 2013 to July 2014 were divided into two groups according to the presence or absence of pancreatic infection and retrospectively analyzed. Clinical characteristics and laboratory examine results of the two groups including age, sex, APACHE II score, serum amylase, serum calcium, blood glucose, ALT, AST, hyoxemia, serum albumin, serum creatinine and blood urea nitrogen were investigated for their relevance to pancreatic infection. RESULTS: The overall occurrence of secondary pancreatic infection of the 42 patients was 52.38%. A significantly positive correlation was revealed between the incidence rate of the secondary pancreatic infection and the factors including hyoxemia, blood creatinine and urea nitrogen in SAP patients (P < 0.05 or P < 0.01). Meanwhile, the level of serum albumin was negatively correlated with the rate of secondary infection in SAP patients (P < 0.01). The rest factors showed no significant correlation (P > 0.05). CONCLUSION: Hyoxemia, blood creatinine and urea nitrogen are potential factors leading to pancreatic infection in SAP patients, while an increase of serum albumin may reduce the incidence of infection.