Safety and Efficacy of Radiofrequency Ablation for Nonfunctioning Benign Thyroid Nodules in Children and Adolescents in 14 Patients over a 10-Year Period.

PURPOSE: To evaluate the efficacy and safety of radiofrequency (RF) ablation for nonfunctioning benign thyroid nodules in children and adolescents. MATERIALS AND METHODS: Fourteen pediatric patients (10 female, 4 male; mean age 15.7 ± 2.3 years, range 12-19 years) with nonfunctioning benign thyroid nodules (mean longest diameter 3.7 ± 1.1 cm, range 2.0-5.6 cm) treated with the use of RF ablation from 2005 to 2015 were evaluated. The inclusion criteria for RF ablation therapy were (i) age <20 years, (ii) benign cytological confirmation by ≥2 separate fine-needle aspiration or core needle biopsies, (iii) pressure symptoms or cosmetic problems caused by thyroid nodules, (iv) absence of any suspicious feature as determined with the use of ultrasound (US), (v) normal serum levels of thyroid hormone and thyrotropin, and (vi) follow-up of >6 months. RF ablation was performed with the use of an RF generator and an 18-gauge internally cooled electrode. RF ablation was performed under local anesthesia without conscious sedation or general anesthesia. Changes in nodules on follow-up US, changes in symptomatic and cosmetic scores, and complications arising during or after RF ablation were evaluated. RESULTS: Mean follow-up period was 36.9 ± 21.7 months (range 6-69 months). At last follow-up visits, mean longest nodule diameter and volume had decreased significantly (3.7 ± 1.1 cm vs 1.4 ± 0.9 cm and 14.6 ± 13.3 mL vs 1.7 ± 4.4 mL; P < 0.001). Both cosmetic and compressive symptoms significantly improved (3.8 ± 0.6 vs 1.4 ± 0.6 and 3.4 ± 1.0 vs 0.1 ± 0.4; P < 0.001). The mean number of ablation sessions was 2.1 ± 1.2 (range 1-5 sessions) and no major complication was encountered during or after RF ablation. CONCLUSIONS: RF ablation might be a safe and effective treatment modality for nonfunctioning benign thyroid nodules in children and adolescents.

Non-Contact Measurement of the Spectral Emissivity through Active/Passive Synergy of CO2 Laser at 10.6 µm and 102F FTIR (Fourier Transform Infrared) Spectrometer.

In the inversion of land surface temperature (LST) from satellite data, obtaining the information on land surface emissivity is most challenging. How to solve both the emissivity and the LST from the underdetermined equations for thermal infrared radiation is a hot research topic related to quantitative thermal infrared remote sensing. The academic research and practical applications based on the temperature-emissivity retrieval algorithms show that directly measuring the emissivity of objects at a fixed thermal infrared waveband is an important way to close the underdetermined equations for thermal infrared radiation. Based on the prior research results of both the authors and others, this paper proposes a new approach of obtaining the spectral emissivity of the object at 8-14 µm with a single-band CO2 laser at 10.6 µm and a 102F FTIR spectrometer. Through experiments, the spectral emissivity of several key samples, including aluminum plate, iron plate, copper plate, marble plate, rubber sheet, and paper board, at 8-14 µm is obtained, and the measured data are basically consistent with the hemispherical emissivity measurement by a Nicolet iS10 FTIR spectrometer for the same objects. For the rough surface of materials, such as marble and rusty iron, the RMSE of emissivity is below 0.05. The differences in the field of view angle and in the measuring direction between the Nicolet FTIR method and the method proposed in the paper, and the heterogeneity in the degree of oxidation, polishing and composition of the samples, are the main reasons for the differences of the emissivities between the two methods.

Toll-like receptor 4 activity protects against hepatocellular tumorigenesis and progression by regulating expression of DNA repair protein Ku70 in mice.

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.

TLR4 activity is required in the resolution of pulmonary inflammation and fibrosis after acute and chronic lung injury.

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.

Blocking IL-17A promotes the resolution of pulmonary inflammation and fibrosis via TGF-beta1-dependent and -independent mechanisms.

Pulmonary fibrosis is the pathologic basis for a variety of incurable human chronic lung diseases. IL-17A, a glycoprotein secreted from IL-17-producing cells, has recently been shown to be a proinflammatory cytokine involved in chronic inflammation and autoimmune disease. In this study, we report that IL-17A increased the synthesis and secretion of collagen and promoted the epithelial-mesenchymal transition in alveolar epithelial cells in a TGF-ß1-dependent manner. Using in vivo fibrotic models, we found IL-17A expression to be elevated and IL-17A-associated signaling pathways to be activated in fibrotic lung tissues. Neutralization of IL-17A in vivo promoted the resolution of bleomycin-induced acute inflammation, attenuated pulmonary fibrosis, and increased survival. Additionally, IL-17A antagonism inhibited silica-induced chronic inflammation and pulmonary fibrosis. Targeting IL-17A resulted in a shift of the suppressive immune response in fibrotic lung tissue toward a Th1-type immune response, and it effectively induced autophagy, which promoted the autophagic degradation of collagen and autophagy-associated cell death. Moreover, IL-17A was found to attenuate the starvation-induced autophagy, and autophagy modulators regulated collagen degradation in the alveolar epithelial cells in a TGF-ß1-independent manner. Administration of 3-methylamphetamine, an autophagy inhibitor, reversed the therapeutic efficacy of IL-17A antagonism in pulmonary fibrosis. Our studies indicate that IL-17A participates in the development and progression of pulmonary fibrosis in both TGF-ß1-dependent and -independent manners and that the components of the IL-17A signaling pathway are potential therapeutic targets for the treatment of fibroproliferative lung diseases.

A combined strategy improves the solubility of aggregation-prone single-chain variable fragment antibodies.

Recombinant single-chain variable fragment (scFv) antibodies have wide applications in the areas of biotechnology and medicine. However, there is currently no universal expression-purification system for generating different soluble scFvs. In this study, A15 and E34, two genes coding scFvs against human IL-17A, were fused with N-terminal signal peptide sequences pelB or STII, or with highly hydrophilic tags Trx, NusA, or MBP, respectively. These constructs were expressed in Escherichia coli. We found that the scFvs fused with either NusA or MBP showed a higher solubility than fused with signal peptides or Trx. The scFvs were aggregated when the NusA or MBP was removed by thrombin. Interestingly, we observed a reduction of precipitation when the fusion proteins were expressed in Origami B(DE3)pLysS cells but not in BL21(DE3)pLysS. Because cleaving the tags resulted in the aggregation of scFvs, several solubility-enhancing additives were added in the digestion buffer and only L-arginine (Arg) or Tween20 promoted the solubility. After an affinity chromatography, the scFvs were separated from the tags with the purity up to 90%. The final yield of scFvs from the scFv-MBP system was approximately 8.9 mg/L of culture medium and 1.5 mg/g of wet weight cells, which was 1.6-fold higher than the yield from the scFv-NusA system. The obtained scFvs exhibited normal binding affinities and activities after endotoxin removal. In conclusion, we describe a strategy combining the fusion tags, the Escherichia coli with oxidizing bacterial cytoplasm, and the solubility-enhancing additives for expressing and purifying the soluble and functional scFvs.

Combination therapy with BMP-2 and BMSCs enhances bone healing efficacy of PCL scaffold fabricated using the 3D plotting system in a large segmental defect model.

The three-dimensional (3D) plotting system is a rapidly-developing scaffold fabrication method for bone tissue engineering. It yields a highly porous and inter-connective structure without the use of cytotoxic solvents. However, the therapeutic effects of a scaffold fabricated using the 3D plotting system in a large segmental defect model have not yet been demonstrated. We have tested two hypotheses: whether the bone healing efficacy of scaffold fabricated using the 3D plotting system would be enhanced by bone marrow-derived mesenchymal stem cell (BMSC) transplantation; and whether the combination of bone morphogenetic protein-2 (BMP-2) administration and BMSC transplantation onto the scaffold would act synergistically to enhance bone regeneration in a large segmental defect model. The use of the combined therapy did increase bone regeneration further as compared to that with monotherapy in large segmental bone defects.

[Blocking IL-17A protects against lung injury-induced pulmonary fibrosis through promoting the activation of p50NF-kappaB].

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.

Interleukin-17A is involved in development of spontaneous pulmonary emphysema caused by Toll-like receptor 4 mutation.

AIM: To explore the pathogenic role of Th17 cells and interleukin-17A (IL-17A)-associated signaling pathways in spontaneous pulmonary emphysema induced by a Toll-like receptor 4 mutant (TLR4(mut)). METHODS: Lungs were obtained from wild-type (WT) or TLR4mut mice that were treated with or without recombinant mouse IL-17A (1 µg·kg(-1)·d(-1), ip) from the age of 3 weeks to 3 months. Pulmonary emphysema was determined using histology, immunochemistry, and biochemical analysis. T cell polarization was determined with flow cytometry, the levels of cytokines were measured using ELISA, and the levels of IL-17A-associated signaling molecules were detected using Western blot. RESULTS: Compared to WT mice, 3 month-old TLR4(mut) mice were characterized by significantly reduced infiltration of Th17 cells into lungs (2.49%±1.13 % νs 5.26%±1.39%), and significantly reduced expression levels of IL-17A (3.66±0.99 pg/µg νs 10.67±1.65 pg/µg), IL-23 (12.43±1.28 pg/µg νs 28.71±2.57 pg/µg) and IL-6 (51.82±5.45 pg/µg νs 92.73±10.91 pg/µg) in bronchoalveolar lavage fluid. In addition, p38 MAPK phosphorylation and AP-1 expression were decreased to 27%±9% and 51%±8%, respectively, of that in WT mice. Treatment of TLR4(mut) mice with IL-17A increased the infiltration of Th17 cells into lungs and expression levels of IL-17A, IL-6, and IL-23 in bronchoalveolar lavage fluid, attenuated MDA and apoptosis, and improved emphysema accompanied with increased phosphorylation of p38 MAPK and expression of AP-1. CONCLUSION: Th17 cells, in particular the cytokine IL-17A, play a crucial role in the pathogenesis of TLR4(mut)-induced spontaneous pulmonary emphysema. Both of them are potential targets for therapeutic strategies for pulmonary emphysema.

Toll like receptor 2 mediates bleomycin-induced acute lung injury, inflammation and fibrosis in mice.

Anti-cancer drug bleomycin (BLM) can cause acute lung injury (ALI) which often results in pulmonary fibrosis due to a failure of resolving acute inflammatory response. The aim of this study is to investigate whether toll-like receptor (TLR) 2 mediates BLM-induced ALI, inflammation and fibrosis. BLM-induced dendritic cells (DCs) maturation was analyzed by flow cytometry and cytokine secretion was detected by the ELISA method. The expression and activity of p38 and ERK MAPK were determined with Western blotting. The roles of TLR2 in ALI, inflammation and fibrosis were investigated in C57BL/6 mice administered intratracheally with BLM. The results demonstrated that BLM-administered mice had higher expression of TLR2 (P<0.001) and its signaling molecules. Blocking TLR2 significantly inhibited the maturation of DCs and reversed BLM-stimulated secretion of cytokines in DCs, such as IL-6 (P<0.001), IL-17 (P<0.05) and IL-23 (P<0.05). TLR2 inhibition attenuated BLM-induced increase of inflammatory cells in bronchoalveolar lavage fluid (BALF), and reversed the immunosuppressive microenvironment by enhancing TH1 response (P<0.05) and inhibiting TH2 (P<0.001), Treg (P<0.01) and TH17 (P<0.01) responses. Importantly, blocking TLR2 in vivo significantly protected BLM-administered mice from pulmonary injury, inflammation and fibrosis and subsequently increased BLM-induced animal survival (from 50% to 92%). Therefore, TLR2 is a novel potential target for ALI and pulmonary fibrosis.

[Suppressing myocardial fibrosis to promote myocardial regeneration as a therapeutic strategy for chronic cardiovascular diseases].

Abnormal cardiovascular tissue remodeling characterized by myocardial hypertrophy, myocardial cell loss and myocardial fibrosis, are the primary pathological changes for multiple cardiovascular diseases. Targeting tissue remodeling using small molecules (e.g., relaxin, KNK437), biological agents (e.g., BCG, anti-TLR2 antibody), or herb medicine complex CFX and so on, can attenuate tissue fibrosis and improve cardiac functions. Therefore, regulating the property of inflammation and reversing tissue fibrosis should promote the formation of optimal tissue environments for stem cells mobilization and proliferation, which enhances the myocardial regeneration and is potential therapeutic strategy against chronic cardiovascular diseases.

[The identification and advances of regulatory B cells].

B cells are typically characterized by their ability to regulate the immune responses through presenting antigens and producing antibodies. However, a novel B cell subset, named regulatory B cells (Bregs), has been identified. As Tregs, the Bregs are capable of performing both pathogenic and regulatory functions by production of suppressive cytokines, such as IL-10 or TGF-beta1, or by interaction with pathogen T cells or other immune cells. Recent studies indicate that the Bregs play a critical role in the development and resolution of multiple chronic diseases, including inflammatory bowel disease, rheumatoid arthritis, and experimental autoimmune encephalomyelitis. The identification and the clarification of action mechanisms of the Bregs will greatly contribute to understanding the mechanisms of immune tolerance comprehensively and deeply, and to develop the rational therapeutic strategies for arthritis, diabetes, multiple sclerosis, infectious diseases and cancer, etc. In this review, we summarized the recent insights of identification, characterizations, development, and regulation mechanisms of Bregs and these cells' contribution to the pathogenesis of inflammatory diseases.

Modulation of beta-catenin by cyclin-dependent kinase 6 in Wnt-stimulated cells.

Beta-catenin is implicated in quite different cellular processes, which require a fine-tuned regulation of its function. Here we demonstrate that cyclin-dependent kinase 6 (CDK6), in association with cyclin D1 (CCND1), directly binds to beta-catenin. We showed that CCND1-CDK6 phosphorylates beta-catenin on serine 45 (S45). This phosphorylation creates a priming site for glycogen synthase kinase 3beta (GSK3beta) and is both necessary and sufficient to initiate the beta-catenin phosphorylation-degradation cascade. Moreover, co-immunoprecipitation assays using Wnt3a-conditioned medium reveals that while Wnt stimulation leads to the dissociation of beta-catenin from axin and casein kinase Ialpha (CKIalpha), Wnt treatment promotes an increase in CCND1 level and the association of beta-catenin with CCND1-CDK6. Furthermore, Wnt3a-stimulated cytosolic beta-catenin levels were higher in CDK6 knockout mouse embryonic fibroblasts (CDK6-/- MEFs) compared to wild-type MEFs. Thus, the CCND1-CDK6 complex is like to negatively regulate Wnt signaling by mediating beta-catenin phosphorylation and its subsequent degradation in Wnt-stimulated cells.

Antagonism of Interleukin-17A ameliorates experimental hepatic fibrosis by restoring the IL-10/STAT3-suppressed autophagy in hepatocytes.

Interleukin-17A has been identified as a driver of hepatic stellate cell activation and plays a critical role in the pathogenesis of hepatic fibrosis. However, the underlining fibrosis-promoting mechanism of IL-17A is far from understood. Here we aimed to define whether hepatocytes directly respond to IL-17A stimulation and are associated with the development of hepatic fibrosis. The functional significance of IL-17A was evaluated in bile duct ligation (BDL) or thioacetamide (TAA) injection-induced mouse models of hepatic fibrosis. Human cirrhosis and control tissues were obtained from the patients with cirrhosis who received an open surgical repair process. Neutralizing IL-17A promoted the resolution of BDL or TAA-induced acute or chronic inflammation and fibrosis, resulted in a shift of the suppressive immune response in fibrotic liver toward a Th1-type immune response, and restored autophagy activity in both cholestatic and hepatotoxic liver injury induced fibrotic liver tissues, which was accompanied by a significant inhibition of STAT3 phosphorylation. Moreover, we found that IL-17A stimulated the concentration-and time-dependent phosphorylation of STAT3 in AML-12 liver cells. Blocking STAT3 with a specific inhibitor STATTIC or STAT3 siRNA protected from the IL-17A-induced autophagy suppression in AML-12 cells, indicating that STAT3 mediates IL-17A-suppressed autophagy. Administration of IL-10, which activated STAT3 and inhibited autophagy, reversed the therapeutic effect of IL-17A antagonism in vivo. Our study suggests that the IL-17A/STAT3 signaling pathway plays a crucial role in the pathogenesis of hepatic fibrosis through suppressing hepatocellular autophagy and that blocking this pathway may provide therapeutic benefits for the treatment of hepatic fibrosis.

Interleukin 17A inhibits autophagy through activation of PIK3CA to interrupt the GSK3B-mediated degradation of BCL2 in lung epithelial cells.

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 ß (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.

Toll-like receptor (TLR) 2 and TLR4 differentially regulate doxorubicin induced cardiomyopathy in mice.

Recent evidence indicates that toll-like receptor (TLR) 2 and 4 are involved in the pathogenesis of dilated cardiomyopathy (DCM), but the exact mechanisms of their actions have not been elucidated. We explored the therapeutic potential of blocking TLRs in mice with established cardiomyopathy. Cardiomyopathy was generated by a single intraperitoneal injection of doxorubicin (10 mg/kg). Two weeks later, the mice were treated with TLR2 or TLR4 neutralizing antibody. Blocking TLR2, but not TLR4, activity not only reduced mortality, but also attenuated doxorubicin-induced cardiac dysfunction by 20% and inhibited myocardial fibrosis. To determine the differential effects of blocking TLR2 and TLR4 in chronic cardiomyopathy, mice were injected with doxorubicin (3.5 mg/kg) once a week for 8 weeks, followed by treatment with TLR2 or TLR4 neutralizing antibody for 40 days. Blocking TLR2 activity blunted cardiac dysfunction by 13% and inhibited cardiac fibrosis, which was associated with a significant suppression of myocardial inflammation. The underlying mechanism involved interrupting the interaction of TLR2 with its endogenous ligands, resulting in attenuation of inflammation and fibrosis. In contrast, blocking TLR4 exacerbated cardiac dysfunction and fibrosis by amplifying inflammation and suppressing autophagy. Our studies demonstrate that TLR2 and TLR4 play distinct roles in the progression of doxorubicin-induced DCM. TLR4 activity is crucial for the resolution of inflammation and cardiac fibrosis, while blocking TLR2 activity has therapeutic potential for the treatment of DCM.

Timing is critical for an effective anti-metastatic immunotherapy: the decisive role of IFNγ/STAT1-mediated activation of autophagy.

BACKGROUND: Immunotherapy is often recommended as an adjuvant treatment to reduce the chance of cancer recurrence or metastasis. Interestingly, timing is very important for a successful immunotherapy against metastasis, although the precise mechanism is still unknown. METHODS AND FINDINGS: Using a mouse model of melanoma metastasis induced by intravenous injection of B16-F10 cells, we investigated the mechanism responsible for the diverse efficacy of the prophylactic or therapeutic TLR4 and TLR9 agonist complex against metastasis. We found that the activation of TLR4 and TLR9 prevented, but did not reverse, metastasis because the potency of this combination was neither sufficient to overcome the tumor cell-educated immune tolerance nor to induce efficacious autophagy in tumor cells. The prophylactic application of the complex promoted antimetastatic immunity, leading to the autophagy-associated death of melanoma cells via IFNγ/STAT1 activation and attenuated tumor metastasis. IFNγ neutralization reversed the prophylactic benefit induced by the complex by suppressing STAT1 activation and attenuating autophagy in mice. However, the therapeutic application of the complex did not suppress metastasis because the complex could not reverse tumor cell-induced STAT3 activation and neither activate IFNγ/STAT1 signaling and autophagy. Suppressing STAT3 activation with the JAK/STAT antagonist AG490 restored the antimetastatic effect of the TLR4/9 agonist complex. Activation of autophagy after tumor inoculation by using rapamycin, with or without the TLR4/9 agonist complex, could suppress metastasis. CONCLUSION AND SIGNIFICANCE: Our studies suggest that activation of IFNγ/STAT1 signaling and induction of autophagy are critical for an efficacious anti-metastatic immunotherapy and that autophagy activators may overcome the timing barrier for immunotherapy against metastasis.

Anti-visceral obesity and antioxidant effects of powdered sea buckthorn (Hippophae rhamnoides L.) leaf tea in diet-induced obese mice.

The potential health benefits of tea have long been studied. This study examined the role of powdered sea buckthorn leaf tea (SLT) in high-fat diet-induced obese mice. The mice were fed two different doses of SLT (1% and 5%, wt/wt) for six weeks. SLT suppressed body weight gain in a dose-dependent manner and significantly reduced visceral fat, plasma levels of leptin, triglyceride and total cholesterol and ALT activity compared with the high-fat-fed control mice. SLT also decreased hepatic triglyceride and cholesterol concentrations and lipid accumulation, whereas elevated fecal lipid excretion. High-fat feeding resulted in simultaneously decreasing hepatic FAS and G6PD activities and increasing PAP, ß-oxidation and CPT activities. However, SLT supplementation during high-fat feeding led to a significant decrease in PAP, ß-oxidation and CPT activities with a simultaneous increase in G6PD activity. The hepatic CYP2E1 activity and hepatic and erythrocyte lipid peroxides were significantly lowered with SLT supplements. Hepatic and erythrocyte SOD and CAT activities were also increased with SLT supplements in a dose-dependent manner, whereas GSH-Px activity was increased in erythrocytes only. These results indicate that SLT has potential anti-visceral obesity and antioxidant effects mediated by the regulation of lipid and antioxidant metabolism in high-fat diet-induced obese mice.

Blocking TLR2 activity attenuates pulmonary metastases of tumor.

BACKGROUND: Metastasis is the most pivotal cause of mortality in cancer patients. Immune tolerance plays a crucial role in tumor progression and metastasis. METHODS AND FINDINGS: In this study, we investigated the potential roles and mechanisms of TLR2 signaling on tumor metastasis in a mouse model of intravenously injected B16 melanoma cells. Multiple subtypes of TLRs were expressed on B16 cells and several human cancer cell lines; TLR2 mediated the invasive activity of these cells. High metastatic B16 cells released more heat shock protein 60 than poor metastatic B16-F1 cells. Importantly, heat shock protein 60 released by tumor cells caused a persistent activation of TLR2 and was critical in the constitutive activation of transcription factor Stat3, leading to the release of immunosuppressive cytokines and chemokines. Moreover, targeting TLR2 markedly reduced pulmonary metastases and increased the survival of B16-bearing mice by reversing B16 cells induced immunosuppressive microenvironment and restoring tumor-killing cells such as CD8(+) T cells and M1 macrophages. Combining an anti-TLR2 antibody and a cytotoxic agent, gemcitabine, provided a further improvement in the survival of tumor-bearing mice. CONCLUSIONS AND SIGNIFICANCE: Our results demonstrate that TLR2 is an attractive target against metastasis and that targeting immunosuppressive microenvironment using anti-TLR2 antibody is a novel therapeutic strategy for combating a life-threatening metastasis.

Cyclin-dependent kinase 2 regulates the interaction of Axin with beta-catenin.

Axin, a negative regulator of Wnt, forms a complex with glycogen synthase kinase 3beta, beta-catenin, and adenomatous polyposis coli and promotes GSK3beta-dependent phosphorylation of beta-catenin, thereby stimulating degradation of the beta-catenin. An essential step in that process is the phosphorylation of Axin. Examination of Axin's amino acid sequence revealed it to contain six arginine-X-leucine (RXL) sequences, the cyclin-dependent kinase 2 (CDK2) binding motif, and 10 CDK2 consensus phosphorylation sequences. We also found that cyclin A/CDK2 phosphorylates Axin, thereby enhancing its association with beta-catenin. This suggests that cyclin A/CDK2 is a negative regulator of beta-catenin-mediated signal transduction, which exerts its effects through phosphorylation of Axin.