Co-Delivery of siNRF2 and Sorafenib by a "Click" Dual Functioned Hyperbranched Nanocarrier for Synergistically Inducing Ferroptosis in Hepatocellular Carcinoma.

In the course of antitumor therapy, the complex tumor microenvironment and drug-mediated changes in cell signaling and biological processes lead to drug resistance. The effect of sorafenib is greatly limited by the specific tumor microenvironment induced by antiangiogenic therapy and ferroptosis resistance induced by the upregulation of nuclear factor erythroid-2 related factor 2 (NRF2). In this study, a pH responsive and amphiphilic hyperbranched polyglycerol, HDP, is synthesized based on a co-graft click chemistry pathway. This nano-scale carrier provides excellent drug-loading capacity, storing stability and pH responsibility, and effectively co-delivery of sorafenib and siRNA. Sorafenib and siNRF2 plays a greatly synergistic effect in inducing reactive oxygen species (ROS), iron overloading, depleting glutathione (GSH), and promoting lipid peroxidation. Importantly, verified in two different animal experiments, HDP-ss (HDP loaded with both siNRF2 and sorafenib) presents a superior anti-tumor effect, by achieving a tumor inhibition rate of ≈94%. Thus, HDP can serve as an excellent targeted delivery nanocarrier with good biocompatibility in antitumor therapy, and combined application of siNRF2 effectively improves the antitumor effect of sorafenib by overcoming NRF2-mediated ferroptosis resistance. Taken together, this study provides a novel therapeutic strategy to combat the drug resistance in antiangiogenic therapy and ferroptosis.

Laparoscopic ultrasound-guided superselective portal vein injection combined with real-time indocyanine green fluorescence imaging and navigation for accurate resection of localized intrahepatic bile duct dilatation: a case report.

BACKGROUND: Primary intrahepatic bile duct dilatation can be very harmful to patients although it belongs to benign biliary disease. It can occur in any part of the liver, intraoperative laparoscopic ultrasound (LUS) guidance combine with real-time indocyanine green (ICG) fluorescence navigation are the means of choice for accurate surgical resection. CASE PRESENTATION: Herein we reported a 43-year-old female patient presented with repeated right upper abdominal pain and distension for 3 years and aggravated for half a year, without fever and jaundice. A diagnosis of localized bile duct dilatation with lithiasis in segment 4 (S4) was made on the basis of preoperative imaging. Correspondingly, we selected to perform a laparoscopic surgery with LUS guided real time ICG fluorescence imaging (ICG-FI) and navigation to make the operation more simply and accurately, as well as to retain normal tissues in a certain extent. Laparoscopic resection of S4b and partial S4a was successfully performed, without any complications. CONCLUSION: Laparoscopic anatomical surgery for intrahepatic bile duct dilatation is a technically challenging operation. The combined use of preoperative three-dimensional computerized tomography (CT) planning, intraoperative LUS guided super-selection, ICG hepatic segment staining and real-time fluorescence navigation could help surgeons accurately complete the segmentectomy or subsegmentectomy with minimized trauma and maximized liver tissue preservation.

Respiratory Syncytial Virus Utilizes a tRNA Fragment to Suppress Antiviral Responses Through a Novel Targeting Mechanism.

Target identification is highly instructive in defining the biological roles of microRNAs. However, little is known about other small noncoding RNAs; for example, tRNA-derived RNA Fragments (tRFs). Some tRFs exhibit a gene-silencing mechanism distinctly different from that of typical microRNAs. We recently demonstrated that a respiratory syncytial virus (RSV)-induced tRF, called tRF5-GluCTC, promotes RSV replication. RSV is the single most important cause of lower respiratory tract infection in children. By using biochemical screening and bioinformatics analyses, we have identified apolipoprotein E receptor 2 (APOER2) as a target of tRF5-GluCTC. The 3'-portion of tRF5-GluCTC recognizes a target site in the 3'-untranslated region of APOER2 and suppresses its expression. We have also discovered that APOER2 is an anti-RSV protein whose suppression by tRF5-GluCTC promotes RSV replication. Our report represents the first identification of a natural target of a tRF and illustrates how a virus utilizes a host tRF to control a host gene to favor its replication.

Mitochondrial antiviral-signalling protein plays an essential role in host immunity against human metapneumovirus.

Human metapneumovirus (hMPV) is a common cause of respiratory tract infection in the paediatrics population. Recently, we and others have shown that retinoic acid-inducible gene 1 (RIG-I)-like receptors (RLRs) are essential for hMPV-induced cellular antiviral signalling. However, the contribution of those receptors to host immunity against pulmonary hMPV infection is largely unexplored. In this study, mice deficient in mitochondrial antiviral-signalling protein (MAVS), an adaptor of RLRs, were used to investigate the role(s) of these receptors in pulmonary immune responses to hMPV infection. MAVS deletion significantly impaired the induction of antiviral and pro-inflammatory cytokines and the recruitment of immune cells to the bronchoalveolar lavage fluid by hMPV. Compared with WT mice, mice lacking MAVS demonstrated decreased abilities to activate pulmonary dendritic cells (DCs) and abnormal primary T-cell responses to hMPV infection. In addition, mice deficient in MAVS had a higher peak of viral load at day 5 post-infection (p.i.) than WT mice, but were able to clear hMPV by day 7 p.i. similarly to WT mice. Taken together, our data indicate a role of MAVS-mediated pathways in the pulmonary immune responses to hMPV infection and the early control of hMPV replication.

Parent tRNA Modification Status Determines the Induction of Functional tRNA-Derived RNA by Respiratory Syncytial Virus Infection.

tRNA-derived RNA fragments (tRFs) are a recently discovered family of small noncoding RNAs (sncRNAs). We previously reported that respiratory syncytial virus (RSV) infection induces functional tRFs, which are derived from a limited subset of parent tRNAs, in airway epithelial cells. Such induction is also observed in nasopharyngeal wash samples from RSV patients and correlates to RSV genome copies, suggesting a clinical significance of tRFs in RSV infection. This work also investigates whether the modification of parent tRNAs is changed by RSV to induce tRFs, using one of the most inducible tRFs as a model. We discovered that RSV infection changed the methylation modification of adenine at position 57 in tRNA glutamic acid, with a codon of CTC (tRNA-GluCTC), and the change is essential for its cleavage. AlkB homolog 1, a previously reported tRNA demethylase, appears to remove methyladenine from tRNA-GluCTC, prompting the subsequent production of tRFs from the 5'-end of tRNA-GluCTC, a regulator of RSV replication. This study demonstrates for the first time the importance of post-transcriptional modification of tRNAs in tRF biogenesis following RSV infection, providing critical insights for antiviral strategy development.

B7-H1 up-regulated expression in human hepatocellular carcinoma tissue: correlation with tumor interleukin-10 levels.

BACKGROUND/AIMS: Interleukin-10 (IL-10) plays an important role in hepatocellular carcinoma (HCC) immune evasion. However, the precise mechanism responsible for aberrant IL-10 production remains unknown. B7-H1 expression can predominantly stimulate IL-10 production and contributes to the tumor immune evasion. This study was designed to investigate the expression of B7-H1 and IL-10 in normal liver tissues and HCC samples, and further to evaluate the correlation between B7-H1 expression and IL-10 levels. METHODOLOGY: We detected the B7-H1 and IL-10 expression in sixty HCC tissues and ten healthy liver tissues at mRNA and protein level using RT-PCR and immunohistochemical staining Western-blotting, respectively, and further analyzed the correlation between B7-H1 expression and IL-10 levels in HCC samples. RESULTS: The results showed that all HCC samples demonstrated the up-regulated expression of B7-H1 and IL-10 at mRNA and protein level compared with normal liver tissues and there was a significant correlation between B7-H1 expression and IL-10 levels (p<0.01). CONCLUSION: These findings for the first time suggest the potential role of B7-H1 in promoting IL-10 production in HCC tissue, and implicate a new mechanism of HCC escape from immune surveillance.

TLR4 signaling induces B7-H1 expression through MAPK pathways in bladder cancer cells.

TLR4 (Toll-like receptor 4) and B7-H1, which were known to be restricted to immune cells in the past, were found to be aberrantly expressed in a majority of tumor cells, facilitating tumor evasion from immune surveillance. Our study demonstrated that activation of TLR4 signaling in bladder cancer cells up-regulated B7-H1 expression. Furthermore, this regulation was significantly attenuated by ERK or JNK inhibitor. Our results elucidated the molecule mechanism of regulation of B7-H1 expression through TLR4 signaling and may suggest new strategies of down-regulating the cancer-associated B7-H1 expression for bladder cancer treatment.

B7-H1 up-regulated expression in human pancreatic carcinoma tissue associates with tumor progression.

PURPOSE: Aberrant tumor cell B7-H1 expression, a member of B7 family that can predominantly stimulate interleukin 10 (IL-10) products, contributed to the tumor immune evasion and tumor progression. This study was designed to investigate the expression of B7-H1 and IL-10 in normal pancreas tissues and pancreatic carcinoma samples, and to evaluate clinical significance of B7-H1 expression in pancreatic carcinoma. METHODS: First, the B7-H1 and IL-10 expression in 40 pancreatic carcinoma samples and 8 healthy pancreas specimens using reverse transcription-PCR (RT-PCR) and western-blotting was detected. Localization of B7-H1 and IL-10 was confirmed by immunohistochemical (IHC) staining. Next, the association between B7-H1 expression and tumor differentiation and tumor stage was analyzed. Finally, the correlation between tumor-associated B7-H1 and IL-10 was evaluated. RESULTS: Pancreatic carcinoma samples demonstrated the up-regulated expression of B7-H1 and IL-10 at mRNA and protein level compared with normal pancreas tissues. IHC staining revealed that B7-H1 and IL-10 was almost localized in tumor cells. Analysis of relationship between B7-H1 and tumor clinicopathological characteristics showed that B7-H1 expression was significantly associated with poor tumor differentiation (P < 0.01) and advanced tumor stage (P < 0.01). Meanwhile, tumor-associated B7-H1 expression was also correlated with IL-10 products (P < 0.01, R (2) = 0.6985, mRNA level; P < 0.01, R (2) = 0.7236, protein level) in tumor cells. CONCLUSIONS: Our findings for the first time demonstrated up-regulated B7-H1 expression in human pancreatic carcinoma tissues, which might play a role in tumor progression and invasiveness. This expression seemed to be related to the ability of B7-H1 to promoting IL-10 secretion.

Involvement of p38 mitogen-activated protein kinase pathway in honokiol-induced apoptosis in a human hepatoma cell line (hepG2).

BACKGROUND: Honokiol has been known to have antitumour activity. This study was conducted to evaluate the antiproliferative potential of honokiol against the hepG2 heptocellular cell line and its mechanism of action. METHODS: hepG2 cells were treated with honokiol of 0-40 microg/ml concentration. The cytotoxic effect of honokiol was determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The apoptosis was evaluated by flow cytometry. Western blots were used to analyse the expression of various proteins (procaspase-9, procaspase-3, cleaved caspase-3, cytochrome c, Bcl-2, Bax, Bad, Bcl-X(L) and p38). RESULTS: Honokiol induced apoptosis with a decreased expression of procaspase-3 and -9 and an increased expression of active caspase-3. Exposure of hepG2 cells to honokiol resulted in the downregulation of Bcl-X(L) and Bcl-2 expression and the release of mitochondrial cytochrome c to the cytosol. In addition, honokiol activated the p38 mitogen-activated protein kinase (MAPK) pathway, and the inhibition of this pathway by SB203580 reduced honokiol-induced apoptosis and activation of caspase-3. CONCLUSION: Honokiol induces apoptosis of hepG2 human hepatocellular carcinoma cells through activation of the p38 MAPK pathway, and, in turn, activation of caspase-3.

Prediction of CD16+ Monocyte in Acute Rejection after Liver Transplantation.

BACKGROUND: CD16+ monocytes have recently shown the ability to inhibit the proliferation of CD4+CD25+Foxp3+ T cells (Tregs). The inhibitory role of Tregs in acute rejection has been described in patients with liver transplantation. However, the role of CD16+ monocytes in the development of allograft rejection after liver transplantation remains unknown. METHODS: Forty-five liver transplant recipients, including 25 acute rejection diagnosed by liver biopsy and clinical symptoms and 20 stable allograft liver function recipients, were collected from January 2007 to September 2015 at our hospitals. To assay the frequencies of CD16+ monocytes and Tregs in blood samples, flow cytometry was performed. RESULTS: Compared with the stable allograft liver function recipients, a significant increase in CD16+ monocytes (19.45±5.25% vs 7.17±1.69%, P<0.001) and decreased Tregs (1.74±0.59% vs 5.53±2.18%, P<0.001) was observed among recipients with acute rejection. CD16+ monocytes were positively correlated with RAI (rejection activity index) (R2=0.84, P<0.001), but negatively correlated with the frequency of Tregs (R2=0.86, P<0.001). CONCLUSIONS: Based on our data, we concluded that CD16+ monocytes might be responsible for the development of acute allograft rejection after liver transplantation, which may be associated with inhibition of Treg cells.

Preoperative neutrophil-to-lymphocyte ratio and tumor-related factors to predict lymph node metastasis in nonfunctioning pancreatic neuroendocrine tumors.

The lymph node (LN) status is very important for the survival in pancreatic neuroendocrine tumors (PNETs). Therefore, the investigation of factors related to LN metastases has a great clinical significance. The aim of this study was to evaluate the predictive value of the preoperative neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR) and possible clinical parameters on the LN metastases in nonfunctional PNETs (NF-PNETs). A retrospective review of 101 NF-PNET patients following curative resection and lymphadenectomy was conducted. The associations between clinicopathological factors and LN metastases and prognosis were determined. Twenty-seven (26.7%) patients had LN metastases. LN metastases was independently associated with disease-free survival (P = 0.009). Ideal cutoff values for predicting LN metastases were 1.80 for NLR, 168.25 for PLR and 2.5 cm for tumor size according to the receiver operating characteristic curve. On multivariable analysis, NLR (P = 0.017), symptomatic diagnosis (P = 0.028) and tumor size (P = 0.020) were associated with LN metastases. These results indicate that preoperative NLR ≥ 1.80, tumor size ≥2.5 cm and symptomatic diagnosis are independently associated with LN metastases for patients undergoing resection of NF-PNETs. It is anticipated that these findings are useful for further planning of lymphadenectomy before surgery.

Effects of ciclamilast, a new PDE 4 PDE4 inhibitor, on airway hyperresponsiveness, PDE4D expression and airway inflammation in a murine model of asthma.

PDE4 (phosphodiesterase-4) plays a critical role in pathogenesis of allergic asthma and chronic obstructive pulmonary disease (COPD). PDE4 inhibitors are presently under clinical development for the treatment of asthma and/or COPD. Ciclamilast, a new PDE4 inhibitor, is a piclamilast (RP 73401) structural analogue, but has a more potent inhibitory effect on PDE4 and inflammation in the airway tissues and less side effects than that of piclamilast. In this study, we elucidate primarily on the roles of compound on PDE4 enzyme in physiological and pathological processes in a mouse model of asthma. The sensitized/challenged mice were reexposed to ovalbumin and airway response to inhaled methacholine was monitored. Orally administration of ciclamilast, in a dose-dependent manner, significantly inhibited changes in lung resistance and lung dynamic compliance, as well as upregulation of cAMP-PDE activity, increase of PDE4D mRNA expression, but not PDE4B from lung tissue in the murine model. In addition, the compound dose-dependently reduced mRNA expression of eotaxin, tumor necrosis factor (TNF)-alpha and interleukin (IL)-4, but slightly increased mRNA expression of interferon (IFN)-gamma from lung tissue. Further, levels of eotaxin, TNF-alpha and IL-4, and eosinophil and neutrophil accumulation in bronchoalveolar lavage fluid were also significantly reduced. Pathological examination, goblet cell hyperplasia and inflammatory cells infiltration in lung tissue were suppressed by treatment with ciclamilast. A significant correlation was observed between the increases in PDE4D mRNA expression and airway hyperresponsiveness. These studies confirm that inhibitory effect of ciclamilast on airway hyperresponsiveness includes its inhibiting PDE4D mRNA expression, down-modulating PDE4 activity, anti-inflammation and anti-mucus hypersecretion, and ciclamilast may have therapeutic potential for the treatment of asthma.

Inhibition of phosphodiesterase activity, airway inflammation and hyperresponsiveness by PDE4 inhibitor and glucocorticoid in a murine model of allergic asthma.

Phosphodiesterase 4 (PDE4) isozyme plays important roles in inflammatory and immunomodulatory cells. In this study, piclamilast, a selective PDE4 inhibitor, was used to investigate the role of PDE4 in respiratory function and inflammation in a murine asthma model. Sensitized mice were challenged with aerosolized ovalbumin for 7 days, piclamilast (1, 3 and 10 mg/kg) and dexamethasone (2 mg/kg) were orally administered once daily during the period of challenge. Twenty-four hours after the last challenge, airway hyperresponsiveness to methacholine was determined by whole-body plethysmography, airway inflammation and mucus secretion by histomorphometry, pulmonary cAMP-PDE activity by HPLC, cytokine levels in bronchoalveolar lavage fluid and their mRNA expression in lung by ELISA and RT-PCR, respectively. In control mice, significant induction of cAMP-PDE activity was parallel to the increases of hyperresponsiveness, inflammatory cells, cytokine levels, mRNA expression as well as goblet cell hyperplasia. However, piclamilast dose-dependently and significantly improved airway resistance and dynamic compliance, and the maximal effect was similar to that of dexamethasone. Piclamilast treatment dose-dependently and significantly prevented the increase in inflammatory cell number and goblet cell hyperplasia, as well as production of cytokines, including eotaxin, TNFalpha and IL-4. Piclamilast exerted a weaker inhibitory effect than dexamethasone on eosinophils and neutrophils, had no effect on lymphocyte accumulation. Moreover, piclamilast inhibited up-regulation of cAMP-PDE activity and cytokine mRNA expression; the maximal inhibition of cAMP-PDE was greater than that exerted by dexamethasone, and was similar to dexamethasone on cytokine mRNA expression. This study suggests that inhibition of PDE4 by piclamilast robustly improves the pulmonary function, airway inflammation and goblet cell hyperplasia in murine allergenic asthma.

Effects of cryptoporus polysaccharide on rat allergic rhinitis associated with inhibiting eotaxin mRNA expression.

Aqueous extract from the fruiting body of Cryptoporus volvatus has been reported to present anti-tumor, anti-allergy, anti-inflammation and immunomodulatory activities. However, the effect mechanisms of anti-allergy and anti-inflammation are poorly understood. The aim of study is to evaluate whether Cryptoporus polysaccharides (CP) extracted from fruiting body of Cryptoporus volvatus decrease the development of nasal symptoms, airway hyperresponsiveness (AHR) to methacholine (MCh) and the infiltration of eosinophils in nasal mucosa in rat model of allergic rhinitis, and investigate a possible action mechanism of CP by detecting the expression of eotaxin mRNA in nasal mucosa and lung tissues. Rats were immunized with ovalbumin and consecutive topical antigen instillation was performed. Repeated intranasal ovalbumin challenge caused rhinitis symptom, AHR to MCh, eosinophil infiltration and histological alterations into the nasal mucosa and increase of eotaxin mRNA expression in nasal mucosa and lung tissue were examined. Pretreatment with CP 3, 9 and 27 mg kg(-1) (ig) decreased the numbers of sneezing 27.4%, 38.4% and 44.3% and nasal rubbing 27.5%, 34.9% and 47.7% comparison with model group, respectively. CP caused a dose-related inhibition of MCh-induced AHR. CP 27 mg kg(-1) decreased the expression of eotaxin mRNA in the nasal mucosa by 35%. These results suggest CP can relieve the symptom, eosinophil infiltration and injury of tissue in nasal mucosa and lung tissue and AHR of allergic rhinitis in rats. Its action mechanism may be associated with the decrease of eotaxin mRNA expression.

Functional motifs responsible for human metapneumovirus M2-2-mediated innate immune evasion.

Human metapneumovirus (hMPV) is a major cause of lower respiratory infection in young children. Repeated infections occur throughout life, but its immune evasion mechanisms are largely unknown. We recently found that hMPV M2-2 protein elicits immune evasion by targeting mitochondrial antiviral-signaling protein (MAVS), an antiviral signaling molecule. However, the molecular mechanisms underlying such inhibition are not known. Our mutagenesis studies revealed that PDZ-binding motifs, 29-DEMI-32 and 39-KEALSDGI-46, located in an immune inhibitory region of M2-2, are responsible for M2-2-mediated immune evasion. We also found both motifs prevent TRAF5 and TRAF6, the MAVS downstream adaptors, to be recruited to MAVS, while the motif 39-KEALSDGI-46 also blocks TRAF3 migrating to MAVS. In parallel, these TRAFs are important in activating transcription factors NF-kB and/or IRF-3 by hMPV. Our findings collectively demonstrate that M2-2 uses its PDZ motifs to launch the hMPV immune evasion through blocking the interaction of MAVS and its downstream TRAFs.

[Expression of eotaxin mRNA and TNF-alpha mRNA in lung tissues of sensitized mice and modulation by anti-inflammatory drugs].

OBJECTIVE: To establish determination methods of eotaxin mRNA and TNF-alpha mRNA expression in the lung tissue of mice. METHODS: Eotaxin mRNA and TNF-alpha mRNA expressions were determined by semi-quantitative RT-PCR. The functional implications of eotaxin mRNA and TNF-alpha mRNA expression were examined by detecting the numbers of total leucocytes and eosinophils in bronchoalveolar lavage fluid(BALF). RESULT: Eotaxin mRNA and TNF-alpha mRNA expression in lung tissue total numbers of leucocyte and numbers of eosinophil in BALF increased in sensitized mice compared with those in normal mice. Dexamethasone significantly but did not inhibit eotaxin mRNA and TNF-alpha mRNA expressions, and eosinophil infiltration in the lungs of the sensitized mice. A compound preparation of traditional Chinese medicine inhibited eotaxin mRNA and eosinophil infiltration, influenced TNF-alpha mRNA expression. CONCLUSION: Increased eotaxin mRNA expression in lung tissue is associated with eosinophil infiltration in BALF, which indicates that the methods of semi-quantitative RT-PCR may be useful to the study of the mechanism of antiasthmatic medicine.

[Increase of LTB4 level and expression of LTA4-hydrolase mRNA in lung tissue and cerebral cortex in asthmatic rats].

OBJECTIVE: To investigate antigen-induced changes of leukotriene B(4)(LTB(4))content and LTA(4)-hydrolase mRNA expression in lung tissue and cerebral cortex in sensitized rats. METHODS: The contents of LTB(4) in lung tissue and cerebral cortex homogenates and LTA(4)-hydrolase mRNA expression after antigen challenge by aerosol were respectively detected by reverse-phase high performance liquid chromatography(RP-HPLC) and semi-quantitative RT-PCR. RESULT: The LTB(4) levels in lung tissue and cerebral cortex homogenates in asthmatic rats were significantly higher than those in control (P%0.05), and LTA4-hydrolase mRNA expression was also increased in asthmatic group. Dexamethason(DXM, 0.5 mg/kg, i.p.) decreased the LTB(4) content and inhibited the LTA(4)-hydrolase mRNA expression significantly in asthmatic rats(P%0.05). CONCLUSION: LTB(4) content and LTA(4)-hydrolase mRNA expression in cerebral cortex and lung tissue are increased in asthmatic rats, and there may exist neuroimmunological cross-talking between central nervous system and lung tissue in asthma.