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High insulin/IGF is a biologic link between diabetes and cancers, but the underlying molecular mechanism remains unclear. Here we report a previously unrecognized tumour-promoting mechanism for stress protein TRB3, which mediates a reciprocal antagonism between autophagic and proteasomal degradation systems and connects insulin/IGF to malignant promotion. We find that several human cancers express higher TRB3 and phosphorylated insulin receptor substrate 1, which correlates negatively with patient's prognosis. TRB3 depletion protects against tumour-promoting actions of insulin/IGF and attenuates tumour initiation, growth and metastasis in mice. TRB3 interacts with autophagic receptor p62 and hinders p62 binding to LC3 and ubiquitinated substrates, which causes p62 deposition and suppresses autophagic/proteasomal degradation. Several tumour-promoting factors accumulate in cancer cells to support tumour metabolism, proliferation, invasion and metastasis. Interrupting TRB3/p62 interaction produces potent antitumour efficacies against tumour growth and metastasis. Our study opens possibility of targeting this interaction as a potential novel strategy against cancers with diabetes.

Background and Aims: Although the minimally invasive endoscopic Stretta procedure is being increasingly used as an alternative strategy to manage gastroesophageal reflux disease (GERD), the benefits of this procedure have to be further evaluated in clinical settings. This prospective observational study assessed the short-term and midterm outcomes associated with laparoscopic Toupet fundoplication (LTF) and the Stretta procedure., Patients and Methods: From January 2011 to January 2012, we allocated 80 patients to LTF and 85 to the Stretta procedure. Primary outcome measures, including symptom scores of heartburn, regurgitation, chest pain, belching, hiccup, cough, and asthma, as well as proton pump inhibitor (PPI) use, were analyzed at midterm follow-up (1-3 years)., Results: Of the 165 patients, 125 patients following LTF (n=65) or the Stretta procedure (n=60) completed the designated 3-year follow-up and were included in the final analysis. At the end of the 3-year follow-up, the symptom scores were all significantly decreased compared with the corresponding values before the two procedures in both groups (P<.05). After LTF and the Stretta procedure, 47/65 (72.3%) and 41/60 (68.3%) patients, respectively, achieved complete PPI therapy independence (72.3% versus 68.3%, P=.627). Comparing with LTF, however, the Stretta procedure had less effect on improving typical symptoms of heartburn, regurgitation, and chest pain and reducing the rate of re-operation (11.8% versus 0%, P=.006)., Conclusions: LTF and the Stretta procedure were equally effective in controlling GERD symptoms and reducing PPI use. However, LTF can achieve more improvement on typical symptoms and has a lower rate of re-operation.

Background and Aim of the Study: Interleukin (IL)-13 is a major inducer of fibrosis in many chronic infectious diseases, yet few studies have reported its role in valvular fibrosis in patients with rheumatic heart disease (RHD). The study aim was to investigate the role of IL-13 in mitral valvular fibrosis in patients with RHD., Methods: Peripheral blood samples were collected from surgical patients with RHD (n = 18) and from healthy controls (n = 9). Serum levels of IL-13 and interferon (IFN)-gamma were analyzed using ELISA. Rheumatic mitral valves removed from surgical patients with RHD, and normal mitral valves, were obtained at autopsy. The expression and distribution of collagen I, collagen III, and IL-13Ralpha1 were examined by immunohistochemical staining, the degree of which was measured using computed imaging analysis., Results: Higher IL-13 levels were observed in RHD patients (15.16 +/- 9.62 pg/ml; p < 0.05) than in healthy controls (7.78 +/- 3.87 pg/ml). RHD patients had high levels of IFN-gamma (9.95 +/- 0.77 pg/ml; p <0.05) compared to healthy controls (5.95 +/- 0.69 pg/ml). Immunohistochemistry showed that, compared to normal valves, rheumatic mitral valves expressed high levels of collagen I (0.01931 +/- 0.00159 versus 0.01183 +/- 0.00207; p < 0.05), collagen III (0.00726 +/- 0.00078 versus 0.00342 +/- 0.00124; p <0.05), and IL-13Rcxl (0.00454 +/- 0.00086 versus 0.00017 +/- 0.00008; p <0.01). Collagens I and III were each expressed in heart interstitial cells, while IL-13Ralpha1 was expressed in the endothelial cells and smooth muscle cells of the blood vessels, and in interstitial cells., Conclusion: Patients with RHD showed increased serum levels of IL-13 compared to healthy controls. IFN-gamma levels were clearly different among RHD patients and healthy controls. The expression of collagens I and III and IL-13Ralpha1 was higher in rheumatic mitral valves compared to normal mitral valves. IL-13 may induce mitral valvular fibrosis in RHD.

Objective: To investigate the effects of paeonol on neuron cell model of Parkinson disease (PD)., Methods: The cell model of Parkinson disease was induced by treatment of 1-Methyl-4-phenylpyridinium (MPP+) in PC12 cells, the PD model cells were treated with 1 μmol/L, 3 μmol/L or 9 μmol/L paeonol for 24h, respectively. Cell viability and LDH leakage were detected by MTT and lactate dehydrogenase (LDH) assay; the apoptosis of PC12 cells was assessed by Hoechst 33258 staining and flow cytometry; reactive oxygen species (ROS) production was detected by DCFH-DA method; and the ratio of Bax/Bcl-2 and activation of caspase-3 were determined by Western blotting., Results: MPP+ treatment significantly reduced cell viability, increased LDH leakage, enhanced the proportion of apoptotic cells and ROS production. In addition, MPP+ treatment dramatically increased the Bax/Bcl-2 ratio, and the activation of caspase-3. Compared to PD model group, paeonol treatment significantly enhanced cell viability, decreased LDH leakage, inhibited the proportion of apoptotic cells and ROS production, reduced the Bax/Bcl-2 ratio and the activated caspase-3 protein., Conclusion: Paeonol can prevent PC12 cells from apoptosis induced by MPP+, and the mechanism may be associated with the down-regulation of ROS production, Bax/Bcl-2 ratio and Caspase-3 activation.

Cancer and diabetes are 2 multifactorial chronic diseases with tremendous impact on health worldwide. Metabolic risk factors play a critical role in fueling a wide range of cancers, but with undefined mechanisms. We recently reported that TRIB3, a stress-induced protein, mediates a reciprocal antagonism between autophagic and proteasomal degradation systems and connects insulin-IGF1 to malignant promotion. We found that several human cancer tissues express higher TRIB3 and phosphorylated IRS1 (insulin receptor substrate 1), which correlates negatively with patient prognosis. Silencing of TRIB3 not only restores insulin-IGF1-suppressed autophagic flux, but also attenuates tumor growth and metastasis. TRIB3 physically interacts with the autophagic receptor SQSTM1, and this interaction hinders the binding of SQSTM1 to LC3 and ubiquitinated proteins, leading to SQSTM1 accumulation and clearance inhibition of ubiquitinated proteins. Interrupting the TRIB3-SQSTM1 interaction with an α-helical peptide derived from SQSTM1 attenuates tumor growth and metastasis through activating autophagic flux. Our findings indicate that TRIB3 links insulin-IGF1 to cancer development and progression through interacting with SQSTM1. Thus, interrupting the TRIB3-SQSTM1 interaction may provide a potential strategy against cancers in patients with diabetes.

Background: Toll-like receptor 4 participates in the process of acute heart injury. The underlying mechanisms of its protection are multifactorial, but we hypothesized that toll-like receptor-mediated autophagy control plays a vital role. The purpose of this study was to clarify the effect of autophagy on cardiac fibrosis., Methods: Cardiac fibrosis was induced by subcutaneous isoproterenol (ISO) injection, and rapamycin was simultaneously administered orally for 14 days. Animal echocardiography was then used to evaluate the success of the cardiac fibrosis model, and the mice were killed after the echocardiography examination., Results: Toll-like receptor 4 knockout (TLR4 KO) mice had better heart function than did wild-type (WT) mice (P < .05). Rapamycin treatment reduced the left ventricular ejection fraction to 23.5% (P < .05), and the collagen volume fraction of the ISO and ISO plus rapamycin groups was 5.9% and 25.9%, respectively, in TLR4 KO mice. Compared with the WT mice, Beclin 1 and autophagy were downregulated in TLR4 KO mice (P < .05); however, the ISO plus rapamycin group had higher autophagy activity than did the ISO group in TLR4 KO mice (P < .05)., Conclusions: Our results suggest that TLR4 KO-induced cardioprotection against ISO-induced cardiac fibrosis is associated with reduced autophagy induction. Cardiac fibroblast autophagy participates in its own activation. The moderate inhibition of autophagic activity may be a new strategy for treating cardiac fibrosis., (© The Author(s) 2014.)

Promoting clearance of intracellular excessive tau is a potential therapeutic strategy for treating Alzheimer's disease. In this work, we designed and synthesized a cyclen-hybrid artificial 'hydrolase' I1-Cu(II) to cleave tau in vitro. Furthermore, a cell-permeable 'hydrolase' I2-Cu(II), derived from I1-Cu(II), was also synthesized to cleave intracellular tau proteins.

Study Design: Descriptive comparison analysis., Objective: To evaluate whether five training cases of International Spinal Cord Injury Core Data Set (ISCICDS) are appropriate for testing the facts within the International Standards for Neurological Classification of Spinal Cord Injury (ISNCSCI) and could thus be used for testing its training effectiveness., Methods: The authors reviewed the five training cases from the ISCICDS and determined the sensory level (SL), motor level (ML) and American Spinal Injury Association Impairment Scale (AIS) for the training cases. The key points from the training cases were compared with our interpretation of the key aspects of the ISNCSCI., Results: For determining SL, three principles of ML, sacral sparing, complete injury, classification of AIS A, B, C and D, determining motor incomplete status through sparing of motor function more than three levels below the ML, there are corresponding case scenarios in ISCICDS. However, no case scenario shows classification of AIS E and the use of voluntary anal sphincter contraction for determination of motor incomplete status. Neurological level of injury could be deduced from the SL and ML. Finally, none of the cases include information about zone of partial preservation, sensory score or motor score., Conclusion: Majority of the facts related to SL, ML and AIS are included in the five training cases of ISCICDS. Thus, using these training cases, it is feasible to test the above facts within the ISNCSCI. It is suggested that the missing fact should be included in an update of the training cases.

Background: Gastroesophageal reflux disease (GERD) is the most common digestive disease, affecting one third of the world's population. The minimally invasive endoscopic Stretta procedure is being increasingly used as an alternative strategy to manage refractory GERD. However, long-term benefits of this procedure have to be further evaluated in clinical settings. This prospective observational study was therefore conducted to evaluate the outcome of patients with refractory GERD 5 years after the Stretta procedure., Methods: A total of 152 patients with refractory GERD underwent the Stretta procedure in our department between April 2007 and September 2008. They were followed up for 5 years, during which the primary outcome measures including symptom scores of heartburn, regurgitation, chest pain, cough and asthma and the secondary outcome measures including proton pump inhibitor (PPI) use and patients' satisfaction were analysed at 6, 12, 24, 36, 48 and 60 months respectively., Results: Of the 152 patients, 138 completed the designated 5-year follow-up and were included in the final analysis. At the end of the 5-year follow-up, the symptom scores of heartburn (2.47 ± 1.22 vs. 5.86 ± 1.52), regurgitation (2.23 ± 1.30 vs. 5.56 ± 1.65), chest pain (2.31 ± 0.76 vs. 4.79 ± 1.59), cough (3.14 ± 1.43 vs. 6.62 ± 1.73) and asthma (3.26 ± 1.53 vs. 6.83 ± 1.46) were all significantly decreased as compared with the corresponding values before the procedure (P < 0.001). After the Stretta procedure, 59 (42.8%) patients achieved complete PPI therapy independence and 104 (75.4%) patients were completely or partially satisfied with the GERD symptom control. Moreover, no severe complications were observed except for complaint of abdominal distention in 12 (8.7%) patients after the Stretta procedure., Conclusion: The Stretta procedure may achieve an effective and satisfactory long-term symptom control and considerably reduce the reliance on medication without significant adverse effects in adult patients with refractory GERD, thereby having profound clinical implications.

Aim: The aim of the present study was to prospectively evaluate the Stretta radiofrequency (RF) treatment for gastroesophageal reflux disease (GERD) -related respiratory symptoms over a 5-years follow-up period., Methods: A total of 132 patients underwent the Stretta procedure between April 2007 and February 2009; 122 of the patients (92.4%) completed the 5-year follow-up. Symptom scores and PPI usage were evaluated at baseline, 6 months, 1 year, 3 years and 5 years after treatment., Results: A total of 122 patients (age, 51.7 ± 13.0 years, M:F, 52: 70) were followed up for 5 years and their outcomes were analyzed. At 5 years after treatment, the symptom scores were significantly reduced (heartburn score, from 5.67 ± 1.52 to 2.41 ± 1.13; regurgitation score, from 5.43 ± 1.66 to 2.27 ± 1.33; chest pain score, from 4.45 ± 1.47 to 2.40 ± 0.88; cough score, from 6.62 ± 1.73 to 3.14 ± 1.43; and asthma score, from 6.83 ± 1.46 to 3.26 ± 1.53, P<0.001). Moreover, 56.6% of the patients were completely off PPIs., Conclusion: Stretta RF significantly improves the symptoms and reduces PPI usage at 5 years. Therefore, it is a viable, effective, and minimally invasive endoluminal procedure for patients with GERD-related respiratory symptoms.

Objective: This study aimed to evaluate the efficacy of antireflux treatment on gastroesophageal reflux (GER)-related cough syncope., Methods: The method used was a retrospective review of the outcomes of antireflux treatment with proton pump inhibitor (PPI), Stretta radiofrequency (SRF), or laparoscopic fundoplication (LF) of 8 patients with chronic cough and cough syncope that was clinically evaluated to be GER related over a period of 2 to 5 years., Results: In the 8 selected cases, the typical GER symptoms disappeared in 7 cases and were significantly eased in 1 case. The chronic cough diminished to mild and occasional occurrence in 6 cases and was completely relieved in 2 cases. Meanwhile, the cough syncope disappeared in all cases. Seven of the patients resumed physical and social functions after the antireflux treatments, except for 1 person, who had a stroke due to other causes., Conclusion: For chronic cough and cough syncope of unknown cause, the GER assessment could be valuable. In treating well-selected GER-related chronic cough and cough syncope, PPI, SRF, and LF can be considered. Moreover, satisfactory restoration of physical and social functions could be achieved after effective antireflux therapy., (© The Author(s) 2014.)

Aim: Laparoscopic Nissen fundoplication (LNF) and Stretta radiofrequency (RF) are used as main alternative strategies to manage medication-refractory GERD. This study was therefore prospectively evaluated outcomes of patients with refractory GERD 5 years after LNF or Stretta RF., Methods: A total of 215 consecutive patients with refractory GERD underwent LNF (N.=102) and Stretta RF (N.=113) in our department between 2007 and 2008. They were followed-up for 5 years, during which the outcome measures including symptom scores of regurgitation, heartburn, chest pain, belching, hiccup, cough and asthma as well as the proton pump inhibitor (PPI) use and complications., Results: Of the 215 patients, 179 patients following LNF (N.=87) or Stretta RF (N.=92) completed the designated 5-year follow-up and were included in the final analysis. At the end of 5-year follow-up, the post-treatment scores were statistically lower as compared with the pre-treatment scores in both groups, while the symptom improvements after Stretta were significantly lower than that after LNF (p < 0.05). Besides, 81 (91%) patients achieved complete PPI therapy independence after LNF, comparing with 47 (51.1%) after Stretta RF (P<0.05). No significant differences in post-treatment complications were observed except for the abdominal distention., Conclusion: Even though laparoscopic Nissen fundoplication and Stretta RF are capable of controlling GERD symptoms effectively and safely in selected patients, LNF could improve more in symptoms and PPI elimination.

Background: Childhood-to-adult persistent asthma is usually considered to be an atopic disease. However gastroesophageal reflux may also play an important role in this phenotype of asthma, especially when it is refractory to pulmonary medicine., Methods: Fifty-seven consecutive GERD patients who had decades of childhood-to-adult persistent asthmatic symptoms refractory to pulmonary medication were enrolled. GERD was assessed by a symptom questionnaire, endoscopy, reflux monitoring, and manometry, and treated by Stretta radiofrequency (SRF) or laparoscopic Nissen fundoplication (LNF). The outcomes were followed up with a questionnaire for an average of 3.3 ± 1.1 years., Results: Upper esophageal sphincter hypotonia, lower esophageal sphincter (LES) hypotonia, shortened LES, and esophageal body dyskinesia were demonstrated by esophagus manometry in 50.9%, 43.9%, 35.1%, and 45.6% of the patients, respectively. The symptom scores for heartburn, regurgitation, coughing, wheezing, and chest tightness significantly decreased from 5.8 ± 2.0, 5.6 ± 2.0, 7.3 ± 1.6, 8.4 ± 1.2, and 8.1 ± 1.5, to 1.2 ± 1.8, 1.1 ± 1.6, 2.8 ± 2.5, 3.8 ± 2.7, and 3.9 ± 2.7, respectively, after anti-reflux treatment (P < 0.001)., Conclusions: Esophagus dysfunction is high in childhood-to-adult persistent asthmatic patients with GERD. SRF and LNF are both effective for esophagus symptoms as well as persistent asthmatic symptoms for these patients. GER may relate with asthmatic symptoms in some patients. Evaluating asthmatic patients for possible treatment of the underlying cause, such as GERD, may improve symptoms and prevent disease persistence.

Aim: Chronic cough is the most common extra-esophageal manifestation of gastroesophageal reflux disease (GERD). This study aimed to retrospectively analyze outcomes in patients with GERD-related cough following laparoscopic Nissen fundoplication (LNF) and Stretta radiofrequency (RF) respectively., Methods: Medical charts of 83 patients with GERD-related cough that underwent LNF or Stretta RF between 2007 and 2012 were retrieved. Symptom scores (heartburn, regurgitation and cough) and proton pump inhibitors (PPIs) usages were evaluated., Results: A total of 83 patients with GERD-related cough underwent LNF (N.=35) and Stretta RF (N.=48), and were followed up 36.78 ± 16.12 months (range 13-55 months). During the follow-up, the post-treatment scores were statistically lower as compared with the pre-treatment scores in both groups, while the cough improvement after Stretta was significantly lower than that after LNF (P<0.001). Besides, 27 (77.1%) patients achieved complete PPI therapy independence after LNF, comparing with 27 (65.1%) after Stretta (P<0.05). No significant differences in post-treatment complications were observed except for the abdominal distention., Conclusion: Even though laparoscopic Nissen fundoplication and Stretta are capable of controlling GERD-related cough effectively and safely in selected patients, laparoscopic Nissen fundoplication could improve more in symptoms and PPI elimination.

Autophagy is an important homeostatic cellular recycling mechanism responsible for degrading injured or dysfunctional cellular organelles and proteins in all living cells. Aging is a universal phenomenon characterized by progressive deterioration of cells and organs due to accumulation of macromolecular and organelle damage. Growing evidences indicate that the rate of autophagosome formation and maturation and the efficiency of autophagosome/lysosome fusion decline with age. Dysfunctional autophagy has also been observed in age-related diseases. Autophagy disruption resulted accumulation of mutated or misfolded proteins is the essential feature of neurodegenerative disorders. However, in cancers, fibroproliferative diseases or cardiovascular diseases, autophagy can play either a protective or destructive role in different types of disease, and even in different stages of the same disease. The review will discuss the cellular and molecular mechanisms of autophagy and its important role in the pathogenesis of aging and age-related diseases, and the ongoing drug discovery strategies for therapeutic intervention.

Epidemiologic studies have shown a strong association between gastroesophageal reflux (GER) and asthma, especially in children. Diagnosing GER can be difficult in some patients when GER presents solely with asthma. The aim of this study was to explore the relationship between GER and asthma with animal model. Sixty rats were randomly divided into six equal groups, GER group, GER-associated-asthma group, allergic asthma group, and their control groups. The cytokine levels and concentration of inflammatory cells in bronchoalveolar lavage (BAL) were determined. The BAL of the rats with allergic asthma contained higher concentration of Interleukin-5 (IL-5) and more eosinophils than those of rats with GER-associated-asthma. This demonstrates that assaying the concentrations of IL-5 and inflammatory cells in BAL may be an effective method of distinguishing GER-associated asthma from allergic asthma., (© 2013 Wiley Periodicals, Inc. and the International Society for Diseases of the Esophagus.)

To investigate the protective effects and possible mechanism of Mycelium of Hirsutella hepiali Chen et Shen (MHCS) on metabolic syndromes, free fatty acid and MHCS-treated hepatocytes were used for detecting autophagy-related LC3, p62 and lipid accumulation. Moreover, high fat diet fed mice were used to establish metabolic syndromes model. 50-weeks age mice were randomly divided into: control group, model group and MHCS group. At 80-weeks age, 15 mice were randomly chosen from each group separately for examining oral glucose tolerance, serum insulin, insulin-like growth factor 1 (IGF-1), hepatic LC3, p62, p-NF-kappaB p65, NF-kappaB p65, IL-6 and CXCL-8. Moreover, insulin resistance index (IRI) was calculated. Hepatic pathological changes, including vacuoles, lipids accumulation and fibrosis were observed. Remaining mice were fed with diet separately to 110 weeks-age for statistics of mortality. MHCS promoted autophagy of free fatty acid treated hepatocytes. Mice fed with high fat plus MHCS diet exhibited improved oral glucose tolerance, insulin resistance, hepatic pathology, inflammation, mortality and activated autophagy. The protective effects of MHCS against metabolic syndroms might be through the activation of hepatic autophagy.

Cell-penetrating peptides provide a unique platform to create a new generation of cancer therapeutics with enhanced efficacy and diminished toxicity. In our study, enhanced expression of toll-like receptor 2 (TLR2) was observed in acute myeloid leukemia (AML) cells. Screening of a phage display peptide library using Biopanning and Rapid Analysis of Selective Interactive Ligands (BRASIL) identified a TLR2-binding peptide motif, Pep2. We show that the TLR2-binding peptide motif targeted and penetrated into leukemia cells in a TLR2-dependent manner. Moreover, a synthetic, chimeric peptide composed of the TLR2-binding motif linked to a programmed cell death-inducing sequence, D(KLAKLAK)2, induced apoptosis in AML cells with high TLR2 expression (TLR2(high)) but not in chronic myeloid leukemia (CML) cells with low TLR2 expression (TLR2(low)). The antileukemia activity of this chimeric peptide was confirmed in leukemia patient samples and an animal model of myeloid leukemia, as the development of leukemia was significantly delayed in mice with TLR2(high) AML compared to TLR2(low) CML NOD/SCID mice. TUNEL assays on bone marrow tissue slices revealed that the chimerical peptide induced leukemia cell apoptosis in a TLR2-dependent manner. Together, our findings indicate that TLR2 is a potential therapeutic target for the prevention and treatment of AML, and the prototype, Pep2-D(KLAKLAK)2, is a promising drug candidate in this setting., (© 2013 UICC.)

DEDD, a member of a family of death effector domain-containing proteins, plays crucial roles in mediating apoptosis, regulating cell cycle, and inhibiting cell mitosis. Our recent work demonstrates that DEDD is a novel tumor repressor, which impedes metastasis by reversing the epithelial-mesenchymal transition (EMT) process in breast and colon cancers. DEDD expression therefore may represent a prognostic marker and potential therapeutic target for the prevention and treatment of cancer metastasis. To reveal the anti-metastatic roles of DEDD in these cancer cells, a number of experiments, including immunohistochemistry, the establishment of stably overexpressing or silencing cancer cells, chemoinvasion assay, soft agar assay, protein degradation, and protein-protein interaction were used in our in vitro and in vivo studies. This chapter focuses on the details of these experiments to provide references for the researchers to investigate the function of a gene in the regulation of tumor metastasis.

Background: The treatment of portal hypertension caused by an occlusive lesion in the retrohepatic inferior vena cava and terminal portal venules is complicated because both portal and system venous flow are compromised., Methods: A 47-year-old woman presented with this issue, and we reasoned that the only way to achieve successful management was to create a meso-cavo-jugular shunt. This patient was referred to us after undergoing a splenectomy for hypersplenism, which made her ascites intractable. She had a retrohepatic vena caval stenosis and noncirrhotic portal hypertension. Percutaneous transluminal angioplasty of the inferior vena cava stenosis failed. She underwent substernal placement of a 14-mm ringed GoreTex graft (WL Gore and Associates, Flagstaff, AZ) with end-to-side connections to the superior mesenteric vein, internal jugular vein, and vena cava., Result: Her ascites resolved, and at follow-up 8 years later her graft was patent., Conclusion: The meso-cavo-jugular shunt can simultaneously decompress both portal and systemic venous systems and is worth considering in the rare circumstance of suprahepatic vena caval obstruction coupled with occlusion of the portal venules., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Hepatocellular carcinoma (HCC) remains one of the most deadly solid tumor malignancies worldwide. We recently find that the loss of toll-like receptor 2 (TLR2) activities promotes the diethylnitrosamine (DEN) induced hepatocellular carcinogenesis and tumor progression, which associates with an abundant accumulation of reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress. This finding suggests that the ROS/ER stress plays a role in TLR2 modulated carcinogenesis of HCC. To investigate the mechanism of TLR2 activity defending against hepatocarcinogenesis, the TLR2-deficient mice were treated with or without antioxidant N-acetylcysteine (NAC) before DEN administration. We found that pretreatment of these animals with NAC attenuated carcinogenesis and progression of HCC in the TLR2-deficient mice, declined ROS/ER stress, and alleviated the unfold protein response and inflammatory response in TLR2-deficient liver tissue. Moreover, the NAC treatment significantly reduced the enhanced aggregation of p62 and Mallory-Denk bodies in the DEN-induced HCC liver tissue, suggesting that NAC treatment improves the suppressive autophagic flux in the TLR2-deficient liver. These findings indicate that TLR2 activity defends against hepatocarcinogenesis through diminishing the accumulation of ROS and alleviating ER stress and unfold protein response mediated inflammatory response in the liver.

Aim: Toll-like receptor 2 (TLR2) signaling plays a critical role in the initiation of atherosclerosis. The aim of this study was to investigate whether blocking TLR2 activity could produce therapeutic effects on advanced atherosclerosis., Methods: Forty-week old apolipoprotein E-deficient (ApoE(-/-)) mice fed on a normal diet were intravenously injected with a TLR2-neutralizing antibody or with an isotype-matched IgG for 18 weeks. Double-knockout ApoE(-/-)Tlr2(-/-) mice were taken as a positive control. At the end of the treatments, the plasma lipid levels were measured, and the plaque morphology, pro-inflammatory cytokines expression and apoptosis in arteries were analyzed. In the second part of this study, 6-week old ApoE(-/-) and ApoE(-/-)Tlr2(-/-) mice fed on a high-cholesterol diet for 12 to 24 weeks, the expression levels of TLR2 and apoptotic markers in arteries were examined., Results: Blockade of TLR2 activity with TLR2-neutralizing antibody or knockout of Tlr2 gene did not alter the plasma lipid levels in ApoE(-/-) mice. However, the pharmacologic and genetic manipulations significantly reduced the plaque size and vessel stenosis, and increased plaque stability in the brachiocephalic arteries. The protective effects of TLR2 antagonism were associated with the suppressed expression of pro-inflammatory cytokines IL-6 and TNF-α and the inactivation of transcription factors NF-κB and Stat3. In addition, blocking TLR2 activity attenuated ER stress-induced macrophage apoptosis in the brachiocephalic arteries, which could promote the resolution of necrotic cores in advanced atherosclerosis. Moreover, high-cholesterol diet more prominently accelerated atherosclerotic formation and increased the expression of pro-apoptotic protein CHOP and apoptosis in ApoE(-/-) mice than in ApoE(-/-)Tlr2(-/-) mice., Conclusion: The pharmacologic or genetic blockade of TLR2 activity diminishes and stabilizes advanced atherosclerotic lesions in ApoE(-/-) mice. Thus, targeting TLR2 signaling may be a promising therapeutic strategy against advanced atherosclerosis.

A similar immune response is implicated in the pathogenesis of pulmonary fibrosis and allergic disorders. We investigated the potential therapeutic efficacy and mechanism of rupatadine, a dual antagonist of histamine and platelet-activation factor (PAF), in bleomycin- (BLM-) and silica-induced pulmonary fibrosis. The indicated dosages of rupatadine were administered in rodents with bleomycin or silica-induced pulmonary fibrosis. The tissue injury, fibrosis, inflammatory cells and cytokines, and lung function were examined to evaluate the therapeutic efficacy of rupatadine. The anti-fibrosis effect of rupatadine was compared with an H1 or PAF receptor antagonist, and efforts were made to reveal rupatadine's anti-fibrotic mechanism. Rupatadine promoted the resolution of pulmonary inflammation and fibrosis in a dose-dependent manner, as indicated by the reductions in inflammation score, collagen deposition and epithelial-mesenchymal transformation, and infiltration or expression of inflammatory cells or cytokines in the fibrotic lung tissue. Thus, rupatadine treatment improved the declined lung function and significantly decreased animal death. Moreover, rupatadine was able not only to attenuate silica-induced silicosis but also to produce a superior therapeutic efficacy compared to pirfenidone, histamine H1 antagonist loratadine, or PAF antagonist CV-3988. The anti-fibrotic action of rupatadine might relate to its attenuation of BLM- or PAF-induced premature senescence because rupatadine treatment protected against the in vivo and in vitro activation of the p53/p21-dependent senescence pathway. Our studies indicate that rupatadine promotes the resolution of pulmonary inflammation and fibrosis by attenuating the PAF-mediated senescence response. Rupatadine holds promise as a novel drug to treat the devastating disease of pulmonary fibrosis.

Background: Bronchiectasis is a progressive and fatal disease despite the available treatment regimens. Gastroesophageal reflux (GER) may play an important role in the progression of bronchiectasis. However, active anti-reflux intervention such as Stretta radiofrequency (SRF) and/or laparoscopic fundoplication (LF) have rarely been used to treat Bronchiectasis., Case Presentation: Seven patients' clinical outcomes for treating GER-related deteriorated bronchiectasis were retrospective reviewed. All patients were treated by SRF and/or LF, and had follow-up periods ranging from one to five years. Typical GER symptoms, respiratory symptoms, medication consumption and general health status were assessed during the follow-ups. At the latest follow-up all patients were alive. The typical GER symptoms disappeared in five people and were significantly improved in the other two. Two had complete remissions of both respiratory symptoms and bronchiectasis exacerbations; four had significantly improved respiratory symptoms to mild/moderate degrees as well as reduced or zero bronchiectasis exacerbations, which allowed them to resume the physical and social functions; one's respiratory symptoms and bronchiectasis exacerbations were not much improved, yet she was in stable condition and satisfied with the results., Conclusions: Potentially, GER plays an important role in some patients with bronchiectasis, and active anti-reflux treatments can be beneficial. Future clinical studies are suggested to clarify GER's role in bronchiectasis and to further determine whether anti-reflux interventions for GER can improve the outcomes of patients with bronchiectasis.

Gastroesophageal reflux disease may produce esophageal syndromes, such as heartburn and regurgitation. It is a common clinical presentation with extraesophageal manifestations, such as asthma, arrhythmia, snoring, and sleep disturbance, which could make identifying it more difficult than the usual esophageal symptoms. The aim of this study is to characterize the extraesophageal manifestations in patients with gastroesophageal reflux disease and investigate the effect of laparoscopic Nissen fundoplication. We describe the case of a 38-year-old male patient with a history of sleep disturbance attributable to gastroesophageal reflux disease, which resolved on successful laparoscopic Nissen fundoplication treatment. The long-standing sleep apnea obviously improved after laparoscopic Nissen fundoplication treatment. To our knowledge, this is a rare case of successful laparoscopic Nissen fundoplication treatment of a patient with extraesophageal manifestations induced by gastroesophageal reflux disease. The results indicate an underlying mechanism for extraesophageal manifestations and the success of laparoscopic Nissen fundoplication treatment.

Hepatocellular carcinoma (HCC) is among the most lethal and prevalent cancers in the human population. The initiation and progression of HCC is closely associated with chronic liver inflammation. Recent research indicates that nonhomologous end joining (NHEJ), one of the DNA repair mechanisms, autophagy and senescence are all involved in the pathogenesis of HCC induced by carcinogens or oxidative stress. DNA repair proteins including XRCC6/KU70 and XRCC5/KU80 are the critical NHEJ factors that play pivotal roles in genome-maintenance issues such as DNA replication and repair, telomere maintenance and chromosomal instability. Our studies indicate that a deficiency of toll-like receptor 4 (TLR4)-mediated immune activities results in a decreased expression of XRCC5 and XRCC6 in response to insult by the carcinogen diethylnitrosamine (DEN). This effect causes a failure in DNA repair, and promotes the transformation of precancerous hepatocytes and HCC development. Ectopic expression of XRCC6 protects against HCC initiation and progression by restoring the cellular senescent response and activation of immune networks, which induces an effective autophagic degradation, removes the accumulated reactive oxygen species (ROS), decreases DNA damage, attenuates proliferation, and promotes programmed cell death in TLR4-deficient livers. Our work indicates that repairing DNA damage by XRCC6 reverses TLR4-deficiency-worsened HCC development via restoring immunity to support senescence and autophagy in liver cells.

TLR2 activity plays an important role in the pathogenesis of autoimmune diseases, tumor carcinogenesis and cardio-cerebrovascular diseases. To establish a TLR2 receptor-based cell screening model, NF-kappaB promoter-driven luciferase reporter plasmids were transfected into human embryonic kidney cells (HEK293) stably expressing human TLR2 and co-receptors CD14, TLR1 and TLR6. Single clones were then isolated and characterized. Using this screening system, a human TLR2-binding peptide C8 was obtained from the Ph.D.-7 Phage Display Peptide Library through biopanning and rapid analysis of selective interactive ligands (BRASIL). The binding characteristic of C8 with human TLR2 was evaluated by ELISA, flow cytometry and immunofluorescence. The NF-kappaB luciferase activity assay showed that C8 could activate the TLR2/TLR1 signaling pathway and induce the production of cytokines TNF-alpha and IL-6. In conclusion, the TLR2 receptor-based cell screening system is successfully established and a new TLR2-binding peptide is identified by using this system.

We recently found that activation of IL17A signaling promotes the development and progression of acute and chronic pulmonary fibrosis, and that the blockade of IL17A activity attenuates pulmonary fibrosis by promoting the resolution of inflammation and the activation of autophagy. Although the induction of autophagy stimulating the collagen degradation in the fibrotic lung tissue has been identified as a mechanism responsible for the antifibrotic role of targeting IL17A, it remains to be clarified how IL17A signaling suppresses autophagy. Here we report that the phosphorylation of B-cell CLL/lymphoma 2 (BCL2), an apoptosis regulatory protein, was inhibited in the presence of IL17A in lung epithelial cells, and this reduction suppressed the ubiquitination degradation of BCL2, which subsequently attenuated autophagy by promoting the interaction of BCL2 and BECN1. We found that IL17A regulated the phosphorylation of BCL2 through activating the phosphoinositide 3-kinase (PI3K)-glycogen synthase kinase 3 β (GSK3B) signaling cascade. In response to IL17A stimulation, PI3K was activated and resulted in phosphorylation of GSK3B at Ser9, which subsequently attenuated the interaction of GSK3B with BCL2. Interrupting the GSK3B and BCL2 interaction precluded the phosphorylation of BCL2 at Ser70, which could trigger the ubiquitination degradation, and restrained the ubiquitination degradation of BCL2. Consequently, a decrease in the BCL2 degradation induced by IL17A resulted in a suppressed autophagy in lung epithelial cells. These findings indicate that the IL17A-PI3K-GSK3B-BCL2 signaling pathway participates in the attenuation of autophagic activity in lung epithelial cells, which is attributed to be primarily responsible for the development and progression of IL17A-induced pulmonary fibrosis.

HIV entry is mediated by the envelope glycoproteins gp120 and gp41. The gp41 subunit contains several functional domains: the N-terminal heptad repeat (NHR) domains fold a triple stranded coiled-coil forming a meta-stable prefusion intermediate. C-terminal heptad repeat (CHR) subsequently folds onto the hydrophobic grooves of the NHR coiled-coil to form a stable 6-helix bundle, which juxtaposes the viral and cellular membranes for fusion. The C34 which has 34 amino acid residues is known as the core structure in CHR. A highly anti-HIV peptide inhibitor derived from C34 was designed. An artificial salt bridge was added in the 6-helical bundle by substitution of lysine for Ile646. With a cholesterol modification at C-terminal, the inhibitor containing I646K mutation represented higher anti-viral activity than C34-cholesterol combination without mutation., (Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.)

DEDD is a member of the death-effector domain protein family. DEDD inhibits the Smad3 mediated transcriptional activity and participates in the regulation of apoptosis. In this study, how the death-effector domain of DEDD participates in the regulation of Smad3 activity and apoptosis has been further investigated. Immunoblotting, immunofluorescence and immunoprecipitation had been used to detect the effects of the full length DEDD and its two truncated mutants, N-DEDD and C-DEDD on Smad3 subcellular distribution, phosphorylation, and interaction between Smad4. The effects of the full length DEDD and its two truncated mutants on cell apoptosis and proliferation had also been explored by flow cytometry and MTT assay. It showed that DEDD and N-DEDD inhibit TGF-beta1 induced Smad3 nuclear translocation and the formation of Smad3-Samd4 complex. DEDD and its two mutants can induce cell apoptosis and inhibit cell proliferation. These results suggested that DEDD inhibits the activity of Smad3 through its death-effector domain. Both the two truncated mutants of DEDD participate in the regulation of apoptosis and cell proliferation.

Unlabelled: Hepatocellular carcinoma (HCC) is a devastating consequence of chronic inflammatory liver diseases. The goal of this study was to investigate whether Toll-like receptor 4 (TLR4) activity contributes to HCC initiation and progression in mice. A mouse model of diethylnitrosamine (DEN)-induced HCC was generated with wild-type and TLR4 mutant mice, and the development and progression of HCC and senescent responses were assessed using morphologic, immunological, and biochemical criteria. We found that genetic or pharmacologic blocking of TLR4 increased susceptibility to DEN-induced HCC carcinogenesis and progression, which was indicated by increases in number of tumor nodules, tumor volume, and animal death. The enhanced HCC was associated with a broad-spectrum reduction of immune response to DEN liver injury, as indicated by decreases in the liver-infiltrating F4/80+ macrophages, the apoptosis signal-regulating kinase 1/p38 mitogen-activated protein kinase/NF-κB and IRF3 signaling activities, and the expression of inflammatory cytokines. Suppressed immune networks resulted in a halt of cellular senescence induction in TLR4 mutant liver tissue, which promoted proliferation and suppressed programmed cell death. Moreover, TLR4 mutation resulted in a suppressed capacity of DNA repair due to a decrease in TLR4-medicated expression of DNA repair proteins Ku70/80 in liver tissue and cells. Isotopic expression of Ku70 in TLR4 mutant mice restored senescence and interrupted the positive feedback loop of DNA damage and oxidative stress, which reversed TLR4 mutation-deteriorated HCC carcinogenesis and progression., Conclusion: TLR4 plays an integrated defense role against HCC carcinogenesis by enhancing the expression and function of DNA repair protein Ku70. Our studies provide novel insight into TLR4 activity in the regulation of HCC tumorigenesis, which may be useful for the prevention of HCC development., (Copyright © 2013 American Association for the Study of Liver Diseases.)

Unlabelled: Hepatocellular carcinoma (HCC) is a complication at the endstage of chronic inflammatory liver diseases with dismal prognosis. Targeting of Toll-like receptor (TLR) 2 attenuates tumor metastases; we hypothesized that blocking TLR2 might also play a crucial role in reducing hepatocarcinogenesis. Surprisingly, we found that the genetic deletion of TLR2 increased susceptibility to diethylnitrosamine (DEN), a genotoxic carcinogen that can induce HCC. Indeed, TLR2-deficient mice showed a significant increase in carcinogenesis and progression of HCC as indicated by increases in tumor nodule size, tumor volume, and animal death. The enhanced susceptibility to DEN-induced HCC was associated with a broad-spectrum reduction in the immune response to DEN-induced liver injury. We found that TLR2 deficiency caused a decrease in the infiltration of macrophages and an attenuation of apoptosis signal regulating kinase 1 (ASK1) / p38 mitogen-activated protein kinase (p38 MAPK) / nuclear factor kappa B (NF-κB) signaling, which led to a decrease in the expression of interferon-gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin (IL)-1α/β, IL-6, and Cxcl-2 as well as suppression of autophagy flux and increases in oxidative stress and p62 aggregation in liver tissue. The defects in immune networks resulted in suppressed p21- and p16/pRb-dependent senescence, which caused an increase in proliferation and a decrease in apoptotic and autophagy-associated cell death in mouse livers. Restoring cellular senescence and autophagy flux by treating TLR2-deficient mice with IFN-γ, a T helper 1 (Th1) cytokine and positive modulator of senescence and autophagy, could attenuate the carcinogenesis and progression of HCC associated with TLR2-deficient animals., Conclusion: The loss of immune networks supporting cellular senescence and autophagy flux is attributed to enhanced susceptibility to DEN-induced hepatocellular carcinogenesis and progression in TLR2-deficient mice. These findings may be used to prevent the development of liver cancer., (Copyright © 2012 American Association for the Study of Liver Diseases.)

To prepare large naive phage antibody library, the host bacteria with high transformation efficiency is used in the Cre-LoxP recombination system. The variable regions of immunoglobulin light and heavy genes were amplified from lymphocytes collected from adult peripheral blood and newborn cord blood. The genes were spliced to form the single-chain variable fragments (scFv) by overlap PCR, cloned into pDAN5a vector and then transformed into XL2-blue MRF' with the Hte gene. Compared with XL1-blue strain, the size of the primary library was increased by 3.9 times. The primary library infected Cre recombinase-expressing bacteria, and the genes between phagemids created many new VH/VL combinations. The library was calculated to have a diversity of 1.7 x 10(11) and validated by the selection of antibodies against six different protein antigens. This library provides the basis for further selection of antibody-based drugs. It is the first time to report that XL2-blue MRF' can be used to improve the diversity of the library in the recombination system.

Hepatocellular carcinoma (HCC), the most common primary malignant liver tumor, is the third leading cause of cancer deaths. The pathogenesis of HCC is closely associated with chronic liver inflammation fired by a variety of stimulates such as virus infection and metabolic stress. Recent work indicates that autophagy, a homeostatic self-degradation process, which decides cell survival or death upon stress, acts as an effector machinery of immune systems in defending microbial invasion and carcinogenesis. SQSTM1 is a selective target and receptor of autophagy, and the protein content of SQSTM1 reflects the level of autophagic flux in cells. Through degrading SQSTM1, decreasing SQSTM1 aggregates, and therefore interrupting the positive feedback between SQSTM1 aggregates and ROS production, autophagy plays a protective role against hepatocellular carcinoma. Indeed, our studies indicate that toll-like receptor 2 (TLR2)-mediated immune activities in the genotoxic carcinogen diethylnitrosamine (DEN)-injured liver tissue provide essential nutrient stimulates to induce intracellular senescence, which can ensure the activation and maturation of autophagy in liver cells. Loss of TLR2-mediated immune activity and senescence leads to the attenuation of autophagic flux, which cannot eliminate SQSTM1 aggregates, ROS accumulation, and DNA damage, and facilitates the development and progression of HCC.

This study aims to investigate the preventive role and potential mechanisms of blocking extracellular HMGB1 function on doxorubicin induced cardiac injury. Mice were treated with HMGB1 blocker glycyrrhizin 1 h before and one time every day (intraperitoneal, 10 mg per mouse) after doxorubicin injection, and sacrificed on the day 14 after doxorubicin challenge. Cardiac function was evaluated by echocardiography and hemodynamic measurement. Myocardial inflammation and collagen deposition were analyzed by immunohistochemistry and picrosirius red staining. The interaction of HMGB1 and TLR2 was assessed by co-immunoprecipitation and confocal microscopy. The protein contents of HMGB1, MyD88, p65NF-kappaB and phospho-p65NF-kappaB were measured by Immunoblot. Compared with mice treated with saline, doxorubicin treatment led to an upregulation in HMGB1 expression. Blocking HMGB1 activity with glycyrrhizin protected mice against cardiac dysfunction, inflammatory response, and cardiac fibrosis induced by doxorubicin challenge. Glycyrrhizin inhibited the interaction of HMGB1 and TLR2, and blocked the downstream signaling of TLR2. In conclusion, blocking HMGB1 protected against doxorubicin induced cardiac injury by inhibiting TLR2 signaling pathway.

Metastasis is the spread of cancer cells from their primary location to other parts of the body. Metastatic cancer is responsible for most cancer deaths. Increasing evidence indicates that epithelial-mesenchymal transition (EMT), a crucial developmental program, contributes to control cancer invasion and metastasis. We recently reported that death effector domain-containing DNA-binding protein (DEDD), a key effector molecule for cell death signaling receptors, attenuates EMT and acts as an endogenous suppressor of tumor growth and metastasis. We found that DEDD physically interacts with the class III PtdIns 3-kinase complex containing PIK3C3 and BECN1, which controls critical aspects of autophagy; this interaction activates autophagy and induces the autophagy-mediated lysosomal degradation of SNAI/Snail and TWIST, two master inducers of the EMT process. Further study reveals that the DEDD-PIK3C3 interaction can support the stability of PIK3C3 to maintain autophagic activity and promote the degradation of SNAI and TWIST. Our finding indicates that DEDD is a prognostic marker and a potential therapeutic target for the prevention and treatment of cancer metastasis. Moreover, regulation of the DEDD-PIK3C3 interaction may serve as an entry point to translate modifiers of this interaction into clinical endpoints.

With the development of therapeutic antibodies over the past decade, they have become the treatment options for immunity and inflammation diseases. Major limitations of mouse antibodies as therapeutic agents - immunogenicity, lack of effectors' functions and short serum half-life -- were subsequently identified and largely overcome by the advent of humanized and fully human antibody technologies. The therapeutic antibodies for immunity and inflammatory diseases are primarily utilized in the treatment of allograft rejection, autoimmune disease, autoinflammatory syndromes, allergies and other chronic inflammation. The action mechanisms of therapeutic antibody include blocking ligands or receptors, regulating receptor activity, clearing the target cells or activating receptor. Strategies for generating the antibody drugs with high efficacy and low side effects can be realized by modulation of Fc-mediated activities and optimization of antigen-binding domains.

Gastroesophageal reflux disease (GERD) is a commonly encountered condition in children, which at times causes respiratory distress, such as asthmatic symptoms, and results in serious morbidity and even mortality. The complexity is sometimes so obscure, that it can cause paradoxical diagnoses and treatment. Here we present two cases of children with difficult-to-treat asthmatic symptoms, which were eventually found to be related to GERD. The two children were treated with anti-reflux procedures and both became symptom free. Literature was also reviewed to shed a light into this complex disease.

HIV entry is mediated by the envelope glycoproteins gp120 and gp41. The gp41 subunit contains several functional domains: the N-terminal heptad repeat (NHR) domains fold a triple stranded coiled-coil forming a meta-stable prefusion intermediate. The C-terminal heptad repeat (CHR) subsequently folds onto the hydrophobic grooves of the NHR coiled-coil to form a stable 6-helix bundle, which juxtaposes the viral and cellular membranes for fusion. A conserved salt bridge between Lys(574) in NHR and Asp(632) in CHR plays an essential role in the formation of the six-helix bundle. A multi-functional peptide inhibitor for anti-HIV derived from the CHR of gp41 has been designed. It bears a cholesterol group (Chol) at the C-terminal through which the inhibitor can anchor in the cell membrane, and carries an isothiocyanate (NCS) group at the side chain of Asp(632) through which the inhibitor can bind to target covalently at Lys(574) in NHR. The dual functionalized peptide (NCS-C34-Chol) shows high antiviral activity in vitro and in vivo. The inhibitor reacts specifically and rapidly to NHR from gp41. In addition, it exhibits better stability under the digestion of the Proteinase K than C34 and T20.

Epithelial-to-mesenchymal transition (EMT), a crucial developmental program, contributes to cancer invasion and metastasis. In this study, we show that death-effector domain-containing DNA-binding protein (DEDD) attenuates EMT and acts as an endogenous suppressor of tumor growth and metastasis. We found that expression levels of DEDD were conversely correlated with poor prognosis in patients with breast and colon cancer. Both in vitro and in vivo, overexpression of DEDD attenuated the invasive phenotype of highly metastatic cells, whereas silencing of DEDD promoted the invasion of nonmetastatic cells. Via direct interaction with the class III PI-3-kinase (PI3KC3)/Beclin1, DEDD activated autophagy and induced the degradation of Snail and Twist, two master regulators of EMT. The DEDD-PI3KC3 interaction led to stabilization of PI3KC3, which further contributed to autophagy and the degradation of Snail and Twist. Together, our findings highlight a novel mechanism in which the intracellular signaling protein DEDD functions as an endogenous tumor suppressor. DEDD expression therefore may represent a prognostic marker and potential therapeutic target for the prevention and treatment of cancer metastasis., (©2012 AACR.)

Aim: To explore the signalling pathways involved in aldosterone-induced inflammation and fibrosis in rat vascular smooth muscle cells (VSMCs)., Methods: Using Western blotting and real-time RT-PCR, we investigated the effects of aldosterone on the expression of cyclooxygenase-2 (Cox-2) and IL-6, two important proinflammatory factors, and TGFβ1, a critical profibrotic factor, in VSMCs., Results: Aldosterone treatment significantly increased the expression of Cox-2 and IL-6 and activation of p38MAPK and NF-κB. The expression of both Cox-2 and IL-6 could be blocked by the mineralocorticoid receptor (MR) antagonist spironolactone and the p38MAPK inhibitor SB203580. Also, the rapid phosphorylation of p38MAPK could be suppressed by SB203580 but not by spironolactone, implicating in nongenomic effects of aldosterone. Similar to SB203580 and spironolactone, the NF-κB inhibitor α-p-tosyl-L-lysine chloromethyl ketone (TLCK) markedly attenuated expression of Cox-2, indicating that MR, p38MAPK and NF-κB are associated with aldosterone-induced inflammatory responses. Furthermore, aldosterone enhanced expression of TGFβ1 in rat VSMCs. This result may be related to activation of the MR/ERK-Sp1 signalling pathway because PD98059, an ERK1/2 inhibitor, significantly blocked the rapid phosphorylation of ERK1/2 and function of Sp1 and led to reduced expression of TGFβ1. Spironolactone was also shown to significantly inhibit TGFβ1 and Sp1 expression but not ERK1/2 phosphorylation., Conclusion: These results suggest that aldosterone-induced inflammatory responses and fibrotic responses may be mediated by the MR/p38MAPK-NF-κB pathways and the MR/ERK-Sp1 pathways in VSMCs, respectively.

This study is to determine the preventive effect and mechanism of targeting IL-17A on pulmonary inflammation and fibrosis after acute lung injury. Mice were treated with anti-IL-17A antibody on the day 7 and sacrificed on the day 14 after bleomycin lung injury. The pulmonary inflammatory status and the deposition of collagen were measured by HE and Sirius stains staining. The contents of hydroxyproline and collagen were measured by using commercial kits. The survival rate of mice was calculated by Kaplan-Meier methods. The inflammatory cytokines in bronchoalveolar lavage fluid were measured by ELISA and the expressions of inflammation-related molecules were detected by Western blotting assay. Targeting of IL-17A could prevent the development of lung inflammation, decrease collagen deposition and the contents of hydroxyproline, and protect against the development of pulmonary fibrosis, which together led to an increase in the animal survival. Moreover, blocking IL-17A decreased the expression ofpro-fibrotic cytokines such as IL-17A, TGF-beta1 and IL-13; increased the expression of anti-fibrotic or anti-inflammatory factors such as IFN-gamma, COX-2, 5-LOX, 15-LOX. Indeed, IL-17A antagonism suppressed the activation of pro-inflammatory p65NF-kappaB but enhanced the activation of pro-resolving p50NF-kappaB. In conclusion, that blockade of IL-17A prevents the development of pulmonary fibrosis from acute lung injury, is because blocking IL-17A may prevent acute inflammation converting to chronic inflammation.

Aim: To develop a rational immunotherapy against tumor metastasis by combining a Toll-like-receptor 2 (TLR2)-neutralizing antibody with a TLR9 agonist CpG ODN, and then investigate the mechanism of action for this combinational regimen., Methods: After mouse melanoma B16-F10 cell inoculation, female C57BL/6 mice were treated with either CpG ODN (0.5 mg/kg) or the anti-TLR2 antibody (200 μg/kg), or with a combination of the two agents. Pulmonary metastases were evaluated by counting metastatic nodes on the lung surface using anatomical microscopy. Flow cytometry was used to evaluate the cytotoxicity of the immune cells in tumor-draining lymph nodes, the cell population in the spleen, and the infiltration of immune cells within the lungs. Cytokine and enzyme expression in the lung tissue was evaluated using ELISA or immunostaining., Results: Anti-metastatic effects were detected in mice treated with either CpG ODN or the anti-TLR2 antibody alone. However, treatment with CpG ODN plus the anti-TLR2 antibody synergistically suppressed the metastasis as compared with treatment with either single agent. The combinational treatment resulted in enhanced infiltration of natural killer cells and cytotoxic T cells, reduced recruitment of type 2 macrophages and Tregs, and decreased expression of immunosuppressive factors including TGF-β1, cyclooxygenase-2 and indoleamine 2,3-dioxygenase, thus stimulated tumor cytotoxicity and suppressed metastasis. The anti-metastatic effect of the combinational regimen was further confirmed in spontaneous metastatic mouse model of Lewis lung carcinoma., Conclusion: Our studies suggest that combining a TLR9 agonist with an anti-TLR2 antibody, which eliminates immunosuppressive factors from the tumor environment, is critical for an effective anti-metastatic immunotherapy.

Pulmonary fibrosis is an inflammation-driven lung disease with a poor prognosis and no cure. Here we report that basal toll-like receptor 4 (TLR4) activity is critical for the resolution of acute and chronic inflammation and pulmonary fibrosis in mouse models of lung injury. We found that genetic or pharmacologic inhibition of TLR4 exacerbates bleomycin-induced pulmonary inflammation, fibrosis, dysfunction, and animal death through promoting formation of an immunosuppressive tissue microenvironment and attenuating autophagy-associated degradation of collagen and cell death in the fibrotic lung tissues. In contrast, pharmacologic activation of TLR4 resulted in a quick resolution of acute inflammation, reversed the established pulmonary fibrosis, improved lung function, and rescued mice from death. Similarly, blocking TLR4 impaired the resolution of silica-induced chronic inflammation and fibrosis. Importantly, altering autophagic activity could reverse the TLR4-regulated lung inflammation, fibrosis, dysfunction, and animal death. Rapamycin, an autophagy activator, reversed the effects of TLR4 antagonism. In contrast, inhibition of autophagy by 3-methyladenine reversed the proresolving and antifibrotic roles of TLR4 agonists and increased animal death. These results not only highlight a pivotal role for TLR4-mediated basal immunity, particularly autophagic activity, in the proresolution of inflammation and fibrosis after chemical-induced lung injury but also provide proof for the concept for activating TLR4 signaling, particularly TLR4-mediated autophagy, as a novel therapeutic strategy against chronic fibroproliferative diseases that are unresponsive to current therapy., (Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Aortic deceleration injury is a common and critical condition following automobile accident with high fatality. The survivors complicated with associated serious injuries are even rare and definitive treatment is required. A 37-year-old male patient had both aortic blunt injury and coronary artery injury after a frontal car collision. After failed coronary artery percutaneous transluminal angioplasty (PTA) and deteriorated aortic lesion, the ruptured aorta was subsequently successfully treated by us with a self-made individualized endograft. The endograft was well in position and the patient functioned well in 11-year followup. With the development of endograft and technique, the endovascular treatment may be an option for patients with complicated aortic blunt injury. Yet careful patient selection and the long-term followup are essential.