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Epicardial adipose tissue (EAT) dysfunction mediates chronic inflammation by regulating inflammation-related adipokines and cytokines, and it further promotes coronary artery disease (CAD) development. CD40L/CD40 is involved in multiple inflammatory pathways that contribute to various pathophysiological processes. However, the function of CD40L/CD40 in the expression and production of adipokines and cytokines in epicardial adipocytes remains unclear. The purpose of the present study was to explore the role and underlying mechanisms of CD40L/CD40 in adipokine and cytokine expression and production. We isolated adipocytes from EAT tissues of CAD and non-CAD patients. We noticed that CD40 was dramatically increased in EAT tissues of CAD patients. Loss-of-function and gain-of-function studies were performed. The results showed that CD40 silencing reduced recombinant CD40 ligand (rCD40L)-induced upregulation of plasminogen activator inhibitor-1, leptin, interleukin-6, and monocyte chemotactic protein-1 messenger RNA levels and secretion. Overexpression of CD40 displayed the opposite results. In addition, rCD40L triggered mixed lineage leukemia protein-1 (MLL1) expression both in messenger RNA and protein levels. CD40 depletion apparently blocked MLL1 expression, whereas gain of function of CD40 resulted in augmentation of MLL1 levels. Interestingly, chromatin immunoprecipitation-quantitative real-time polymerase chain reaction analysis revealed that CD40 elimination dampened histone H3 lysine 4 trimethylation enrichment at plasminogen activator inhibitor-1, leptin, interleukin-6, and monocyte chemotactic protein-1 promoter regions in the presence of rCD40L. The reverse pattern was observed upon ectopic expression of CD40. Most important, MLL1 silencing effectively reversed the promotive effects of CD40 on adipokine and cytokine secretion. Taken together, our findings suggest that CD40L/CD40 regulates adipokine and cytokine expression by H3 lysine 4 trimethylation modification in adipocytes.

The aim of this work was to examine the ameliorative effect of foliar application of salicylic acid and potassium on antioxidant enzymes activity in wheat under saline conditions. Two wheat genotypes WL-711 (salt tolerant) and Kohistan-97 (salt sensitive) were used with salicylic acid (SA) and potassium (K+) foliar spray which were applied at both vegetative and grain filling stage under saline conditions. Various enzymatic antioxidant activities include catalase (CAT), peroxidase (POD), superoxide dismutase (SOD) and ascorbate peroxidase (APX) was assesed. Result cleared that foliar spray of salicylic acid and potassium decreased the damaged effect of salinity in both wheat genotypes especially Kohistan-97 which was more salt sensitive. Overall, foliarly applied salicylic acid and potassium reduces the adverse effects of reactive oxygen species and antioxidant enzymes on cellular metabolism that was disturbed by salt stress.

Due to the high recurrence and progression rate of non-muscle invasive bladder cancer after transurethral resection of bladder tumor, some new optical imaging technologies have arisen as auxiliary imaging modes for white light cystoscopy to improve the detection rate of small or occult tumor lesions, such as photodynamic diagnosis, narrow-band imaging, and molecular imaging. White light cystoscopy is inadequate and imperfect for bladder cancer detection, and thus residual tumors or coexisting flat malignant lesions, especially carcinoma in situ, would be ignored during conventional resection. The bladder, a hollow organ with high compliance, provides an ideal closed operation darkroom for endoscopic molecular imaging free from interference of external light sources. Also, intravesical instillation of a molecular fluorescent tracer is simple and convenient before surgery through the urethra. Molecular fluorescent tracer has high sensitivity and specificity to tumor cells, and its mediated molecular imaging allows small or occult tumor lesion detection while minimizing false-positive results. Meanwhile, endoscopic molecular imaging provides a real-time and dynamic image during surgery, which helps urologists to perform high-quality and complete tumor resection through accurate judgment of tumor boundaries and depth of invasion. Photoimmunotherapy is a novel molecular targeted therapeutic pattern of photodynamic therapy that kills malignant cells selectively and minimizes the cytotoxicity to normal tissues. The combination of endoscopic molecular imaging and photoimmunotherapy used in initial treatment may avoid the need of repeat transurethral resection in strictly selected patients and improve oncological outcomes such as recurrence-free survival and overall survival after operation., Graphical Abstract

Graves’ disease is the most common cause of hyperthyroidism in children. The surgery treatment for children Graves’ disease with huge goiter is high risk and controversial. A 14-year-old girl suffered Graves’ disease with huge goiter and failed to the antithyroid drug therapy for nearly 4 years was surgically treated with total thyroidectomy. The excised thyroid weighed 449.1 g and heavier than any excised children goiter reported so far. After operation, the patient’s symptoms of Graves’ disease were significantly improved without any complication, including normal basal metabolic rate, relieved exophthalmia and euthyroidism. So, a children Graves’ disease with huge goiter was cured by total thyroidectomy, suggesting that a total/near-total thyroidectomy is a good option for children Graves’ disease with huge goiter.

To evaluate the surgical safety of en bloc resection of bladder tumor (ERBT) and the effectiveness of ERBT combined with near-infrared (NIR) imaging technique in the diagnosis and treatment of non-muscle invasive bladder cancer (NMIBC). From October 2017 to June 2018, 26 patients newly diagnosed with single NMIBC were included in this retrospectively trial. All patients received ERBT with monopolar current. After surgery, the fresh specimen was incubated with anti-CD47-Alexa Fluor 790, and then imaged under NIR imaging technique. Operative details, intraoperative and postoperative complications of ERBT regarded as safety outcomes, the mean fluorescence intensity (MFI) of tumor tissue and adjacent normal background tissue, and 12 months follow-up data were analyzed. Of 26 collected patients, obturator nerve reflex was occurred in six patients during tumor resection, and only one patient was observed with bladder perforation. In NIR gray image, the gray scale of MFI of tumor tissue were 132.31 ± 6.67 and the adjacent normal background tissue were 52.27 ± 12.09. The result showed a significantly higher MFI signals in tumor tissue compared to adjacent normal background tissue (P < 0.001). The recurrence-free survival rate at 12 month was 96.15%. ERBT with monopolar current is a safe and feasible technique to treat patients with NMIBC. A integrated bladder tumor tissue-bound anti-CD47-Alexa Fluor 790 was detected under NIR light, and the NIR image indicates that higher MFI signals in surgical margin is a predictive factor for residual tumor in patients with NMIBC after ERBT.

Enterovirus 71 (EV71) is the causative pathogen of hand, foot, and mouth disease (HFMD). However, no effective antiviral therapy is currently available. Some viruses could escape the host’s innate immunity by upregulating suppressor of cytokine signaling (SOCS) proteins. Until now, whether EV71 evades the host immune system by regulating the expression of SOCS proteins remains unknown. In this study, we found that EV71 infection promoted SOCS expression at both mRNA and protein levels in vitro and in vivo. Consistently, the infectivity of EV71 was decreased significantly in the SOCS3 or SOCS1 knockdown cells, suggesting that SOCS1 and especially SOCS3 are crucial for EV71 infection. Further investigation showed that SOCS3 promoted virus infection by inhibiting interferon-induced STAT3 phosphorylation. SOCS1 and SOCS3 mRNA expressions were independent on virus-induced type I interferon expression but were blocked by the inhibitor of NF-κB. Therefore, EV71 infection stimulates the expression of SOCS proteins in an interferon-independent way and negatively regulates the JAK/STAT signaling pathway, thus escaping host immunity. All these results may add new information to the mechanism of EV71 in fighting against type I interferon responses.

Background To evaluate the surgical safety and quality of transurethral en bloc resection with monopolar current for non-muscle invasive bladder cancer (NMIBC) based on the tumour, node, metastasis (TNM) classification system, and report the midterm oncological outcome. Methods From October 2015 to June 2017, en bloc resection of bladder tumor (ERBT) and transurethral resection of bladder tumor (TURBT) were performed in 96 and 87 patients clinically diagnosed with NMIBC in the prospective case-control trial, respectively. Operative details, intraoperative and postoperative complications regarded as safety outcomes were documented. The quality of ERBT was judged by the histopathological examination of tumor specimens from initial resection and second TURBT, random bladder biopsy and follow-up recurrence rate. Results Operative time, obturator nerve reflex, irrigation and catheterization time were similar in the two groups. Bladder perforation was occurred in 2 patients during ERBT and 9 patients during TURBT (2/96 vs. 9/87, P=0.019). Compared with TURBT group, the ratio of detrusor muscle (DM) identified in pathologic T1 tumor specimens was higher (P=0.024), but lower in pathologic Ta tumor specimens in ERBT group (P

A new coronavirus SARS-CoV-2, also called novel coronavirus 2019 (2019-nCoV), started to circulate among humans around December 2019, and it is now widespread as a global pandemic. The disease caused by SARS-CoV-2 virus is called COVID-19, which is highly contagious and has an overall mortality rate of 6.35% as of May 26, 2020. There is no vaccine or antiviral available for SARS-CoV-2. In this study, we report our discovery of inhibitors targeting the SARS-CoV-2 main protease (M

The maintenance of intestinal tissue homeostasis is vital for the resistance against inflammatory bowel diseases (IBDs). Necroptosis is identified as an alternative mode of regulated cell death, which plays a pivotal role in tissue homeostasis. Thus, the roles of RIP3-mediated necroptosis in intestinal inflammation have been extensively studied. However, the biological implications of the mixed lineage kinase-like protein (MLKL), a molecule downstream of RIP3 in gut remain unclear. In this study, the role of MLKL in DSS-induced colitis was examined, and the contribution of gut microbiota was also determined. Compared with non-littermate WT mice, the survival rate, clinical score, intestinal damage and intestinal mucosal barrier integrity of non-littermate MLKL-deficient mice are significantly improved. MLKL deficiency prevents inflammatory cytokines production and MAPK signaling activation. Hence, MLKL deficiency inhibits DSS-induced colitis. Moreover, we proved that DSS susceptibility difference between two genotypes is not driven by intestinal microbiota based on the co-housing of two non-littermate genotypes and qPCR detection of fecal dominant bacterial taxa.

STING (Stimulator of interferon genes) is known as an important adaptor protein or direct sensor in the detection of nucleotide originating from pathogens or the host. The implication of STING during pulmonary microbial infection remains unknown to date. Herein, we showed that STING protected against pulmonary S.aureus infection by suppressing necroptosis. STING deficiency resulted in increased mortality, more bacteria burden in BALF and lungs, severe destruction of lung architecture, and elevated inflammatory cells infiltration and inflammatory cytokines secretion. STING deficiency also had a defect in bacterial clearance, but did not exacerbate pulmonary inflammation during the early stage of infection. Interestingly, TUNEL staining and LDH release assays showed that STING-/- mice had increased cell death than WT mice. We further demonstrated that STING-/- mice had decreased number of macrophages accompanied by increased dead macrophages. Our in vivo and in vitro findings further demonstrated this cell death as necroptosis. The critical role of necroptosis was detected by the fact that MLKL-/- mice exhibited decreased macrophage death and enhanced host defense to S.aureus infection. Importantly, blocking necroptosis activation rescued host defense defect against S.aureus pneumonia in STING-/- mice. Hence, these results reveal an important role of STING in suppressing necroptosis activation to facilitate early pathogen control during pulmonary S.aureus infection.

Gasdermin D (GSDMD), a member of the gasdermin protein family, is a caspase substrate, and its cleavage is required for pyroptosis and IL-1β secretion. To date, the role and regulatory mechanism of GSDMD during cutaneous microbial infection remain unclear. Here, we showed that GSDMD protected against Staphylococcus aureus skin infection by suppressing Cxcl1–Cxcr2 signalling. GSDMD deficiency resulted in larger abscesses, more bacterial colonization, exacerbated skin damage, and increased inflammatory cell infiltration. Although GSDMD deficiency resulted in defective IL-1β production, the critical role of IL-1β was counteracted by the fact that Caspase-1/11 deficiency also resulted in less IL-1β production but did not aggravate disease severity during S. aureus skin infection. Interestingly, GSDMD-deficient mice had increased Cxcl1 secretion accompanied by increased recruitment of neutrophils, whereas Caspase-1/11-deficient mice presented similar levels of Cxcl1 and neutrophils as wild-type mice. Moreover, the absence of GSDMD promoted Cxcl1 secretion in bone marrow-derived macrophages induced by live, dead, or different strains of S. aureus. Corresponding to higher transcription and secretion of Cxcl1, enhanced NF-κB activation was shown in vitro and in vivo in the absence of GSDMD. Importantly, inhibiting the Cxcl1–Cxcr2 axis with a Cxcr2 inhibitor or anti-Cxcl1 blocking antibody rescued host defence defects in the GSDMD-deficient mice. Hence, these results revealed an important role of GSDMD in suppressing the Cxcl1–Cxcr2 axis to facilitate pathogen control and prevent tissue damage during cutaneous S. aureus infection.

ObjectiveTo evaluate the safety and efficacy of overnight continuous saline bladder irrigation (CSBI) for patients who have received thulium laser en bloc resection of bladder tumor (TmLRBT) combined with immediate intravesical chemotherapy previously.MethodsFrom October 2014 to June 2018, 235 patients with newly diagnosed non-muscle invasive bladder cancer (NMIBC) were included in this retrospective study. All patients received intravesical instillation of pirarubicin immediately after TmLRBT. The patients were divided into two groups according to the duration of postoperative bladder irrigation with normal saline. After immediate intravesical chemotherapy, patients in group 1 received overnight CSBI, while patients in group 2 did not receive overnight CSBI. Data on the time of initial tumor recurrence, recurrence-free survival (RFS) and progression-free survival (PFS) rates, and perioperative complications were collected and analyzed.ResultsOf 235 included patients (129 in group 1 and 106 in group 2), the median follow-up periods were 42 and 38 months, respectively. There were no significant differences in patients’ baseline characteristics between the two groups. The RFS rates of patients in group 1 were 90.7, 82.7, and 76.8% at the end of the first, third, and fifth years, while the corresponding RFS rates of patients in group 2 were 87.7, 78.9, and 73.3%, respectively. Four patients in group 1 and five patients in group 2 experienced tumor progression. No significant differences between the two groups were observed in the time of initial tumor recurrence, RFS, and PFS rates. Only Grade I complications occurred in the two groups, and no significant difference was reached between the two groups.ConclusionsFor patients with NMIBC who have previously received TmLRBT combined with immediate intravesical chemotherapy, overnight CSBI may not improve oncological outcomes and reduce perioperative complications.

Although transurethral resection of bladder tumor is the golden standard for the treatment of non-muscle invasive bladder cancer, this surgical procedure still has some serious drawbacks. For example, piecemeal resection of tumor tissue results in exfoliated tumor cells dissemination and implantation, and fragmented tumor specimens make it difficult for pathologists to accurately assess the pathological stage and histologic grade. En bloc tumor resection follows the basic principle of oncological surgery and provides an intact tumor specimen containing detrusor muscle for pathologists to make accurate histopathological assessment. However, there is no robust clinical evidence that en bloc tumor resection is superior to conventional resection in terms of oncological outcomes. Considering the high recurrence rate, small or occult tumor lesions may be overlooked and incomplete tumor resection may occur during white light cystoscopy-assisted transurethral resection. Molecular fluorescent tracers have the ability to bind tumor cells with high sensitivity and specificity. Optical molecular imaging mediated by it can detect small or occult malignant lesions while minimizing the occurrence of false-positive results. Meanwhile, optical molecular imaging can provide dynamic and real-time image guidance in the surgical procedure, which helps urologists to accurately determine the boundary and depth of tumor invasion, so as to perform complete and high-quality transurethral tumor resection. Integrating the advantages of these two technologies, optical molecular imaging-assisted en bloc tumor resection shows the potential to improve the positive detection rate of small or occult tumor lesions and the quality of transurethral resection, resulting in high recurrence-free and progression-free survival rates.

The main protease (Mpro) is one of the best-characterized drug targets among coronaviruses. In the current study, we adopted a multiple cross-docking strategy against different crystal structures of SARS-CoV-2 Mpro to perform computer-based high-throughput virtual screening of possible inhibitors from a drug database using Autodock Vina and SeeSAR software, combined with our in-house automatic processing scripts. The KDs between screened candidates and Mpro were determined using Biacore. Seven drugs were found to fit the substrate-binding pocket of Mpro with a stable conformation, showing high KDs that ranged from 6.79E-7 M to 5.20E-5 M. Finally, mutagenesis studies confirmed that these drugs interact with Mpro specifically, suggesting that our method was reliable and convincing. Given the safety of these old drugs, they may serve as promising candidates to treat the infection of SARS-CoV-2. Our results also provide rational explanations for the behaviour of five drugs evaluated in clinical trials.

Klebsiella pneumoniae (K. pneumoniae) spp. are important nosocomial and community-acquired opportunistic pathogens, which cause various infections. We observed that K. pneumoniae strain K7 abruptly mutates to rough-type phage-resistant phenotype upon treatment with phage GH-K3. In the present study, the rough-type phage-resistant mutant named K7RR showed much lower virulence than K7. Liquid chromatography-tandem mass spectrometry (LC-MS-MS) analysis indicated that WcaJ and two undefined glycosyltransferases (GTs)- named GT-1, GT-2- were found to be down-regulated drastically in K7RR as compared to K7 strain. GT-1, GT-2, and wcaJ are all located in the gene cluster of capsular polysaccharide (CPS). Upon deletion, even of single component, of GT-1, GT-2, and wcaJ resulted clearly in significant decline of CPS synthesis with concomitant development of GH-K3 resistance and decline of virulence of K. pneumoniae, indicating that all these three GTs are more likely involved in maintenance of phage sensitivity and bacterial virulence. Additionally, K7RR and GT-deficient strains were found sensitive to endocytosis of macrophages. Mitogen-activated protein kinase (MAPK) signaling pathway of macrophages was significantly activated by K7RR and GT-deficient strains comparing with that of K7. Interestingly, in the presence of macromolecular CPS residues (>250 KD), K7(ΔGT-1) and K7(ΔwcaJ) could still be bounded by GH-K3, though with a modest adsorption efficiency, and showed minor virulence, suggesting that the CPS residues accumulated upon deletion of GT-1 or wcaJ did retain phage binding sites as well maintain mild virulence. In brief, our study defines, for the first time, the potential roles of GT-1, GT-2, and WcaJ in K. pneumoniae in bacterial virulence and generation of rough-type mutation under the pressure of bacteriophage.

Salmonella effectors translocated into epithelial cells contribute to the pathogenesis of infection. They mediate epithelial cell invasion and subsequent intracellular replication. However, their functions in vivo have not been well-identified. In this study, we uncovered a role for Salmonella outer protein B (SopB) in modulating necroptosis to facilitate bacteria escape epithelial cell and spread to systemic sites through a Salmonella-induced colitis model. Mice infected with SopB deleted strain ΔsopB displayed increased severity to colitis, reduced mucin expression and increased bacterial translocation. In vitro study, we found there was an increased goblet cell necroptosis following ΔsopB infection. Consistently, mice infected with ΔsopB had a strong upregulation of mixed lineage kinase domain-like (MLKL) phosphorylation. Deletion of MLKL rescued severity of tissue inflammatory, improved mucin2 expression and abolished the increased bacterial translocation in mice infected with ΔsopB. Intriguingly, the expression of sopB in LS174T cells was downregulated. The temporally regulated SopB expression potentially switched the role from epithelial cell invasion to bacterial transmission. Collectively, these results indicated a role for SopB in modulating the onset of necroptosis to increased bacteria pathogenesis and translocated to systemic sites.

Interferon-inducible protein (IFI204) (p204, the murine homolog of human IFI16) is known as a cytosolic DNA sensor to recognize DNA viruses and intracellular bacteria. However, little is known about its role during extracellular bacterial infection. Here we show that IFI204 is required for host defense against the infection of Staphylococcus aureus, an extracellular bacterial pathogen. IFI204 deficiency results in decreased survival, increased bacterial loads, severe organs damage, and decreased recruitment of neutrophils and macrophages. Production of several inflammatory cytokines/chemokines including IFN-β and KC is markedly decreased, as well as the related STING-IRF3 and NF-κB pathways are impaired. However, exogenous administration of recombinant KC or IFN-β is unable to rescue the susceptibility of IFI204-deficient mice, suggesting that other mechanisms rather than KC and IFN-β account for IFI204-mediated host defense. IFI204 deficiency leads to a defect in extracellular bacterial killing in macrophages and neutrophils, although bacterial engulf, and intracellular killing activity are normal. Moreover, the defect of bactericidal activity is mediated by decreased extracellular trap formation in the absence of IFI204. Adoptively transferred WT bone marrow cells significantly protect WT and IFI204-deficient recipients against Staphylococcus infection compared with transferred IFI204-deficient bone marrow cells. Hence, this study suggests that IFI204 is essential for the host defense against Staphylococcus infection.

The ecological rehabilitation of potential toxic metal-contaminated soils in sites disturbed by mining has been a great challenge in recent decades. Phytoremediation is one of the most widely promoted renovation methods due to its environmental friendliness and low cost. However, there is a lack of in situ investigation on the influence of vegetation pattern and spontaneous succession on the rehabilitation of potential toxic metal-polluted soil. To clarify how the vegetation pattern in the early stage of restoration and the spontaneous succession influence the remediation of the soil, we investigated a metal mining dump in Sichuan, China, by field investigation and laboratory analysis. We determined the plant growth, soil fertility, and the capacity of potential toxic metals (PTMs) in metal mining soil under different initial vegetation patterns for different years to understand the role of vegetation pattern and spontaneous succession in PTM pollution phytoremediation projects. The results show that: (1) Phytoremediation with a simple initial vegetation pattern (RP rehabilitative plant pattern) which involves two rehabilitation plants, Agave sisalana and Neyraudia reynaudiana, achieves a PTM pollution index that is 9.28% lower than that obtained with the complex vegetation pattern (RP&amp, LP rehabilitation plants mixed with local plants pattern), 21.86% lower in the soil fertility index, and 73.69% lower in the biodiversity index, (2) The phytoremediation with the 10-year RP&amp, LP pattern was associated with a PTM pollution index that was 4.04% higher than that for the 17-year RP&amp, LP pattern, a soil fertility index that was 4.48% lower, and a biodiversity index that was 12.49% lower. During the process of vegetation succession, if accumulator plants face inhibition of growth or retreat, the reclamation rate will decrease. The vegetation patterns influence the effect of phytoremediation. Spontaneous vegetation succession will cause the phytoremediation process to deviate from the intended target. Therefore, according to the goal of vegetation restoration, choosing a suitable vegetation pattern is the main premise to ensure the effect of phytoremediation. The indispensable manipulation of succession is significant during the succession series, and more attention should be paid to the rehabilitative plants to ensure the stable effect of reclamation. The results obtained in this study could provide a guideline for the in situ remediation of PTM-polluted soil in China.

The beneficial effects of antioxidants against oxidative stress have been well described. However, the pharmacological impacts of antioxidants other than inhibiting the production of reactive oxygen species (ROS) remain less understood. This study demonstrated that diphenyleneiodonium (DPI), a canonical NADPH oxidase 2 (NOX2) inhibitor, effectively promoted non-opsonized bacterial phagocytosis. Indeed, DPI abrogated the elevation in the extracellular ATP level of Escherichia coli (E. coli) -infected murine peritoneal macrophages, thereby restoring the association of the purinergic receptor P2X7 with non-muscle myosin heavy chain 9 (MYH9) to upregulate the P2X7 -dependent phagocytosis of E. coli. DPI also suppressed inflammasome activation and reduced necroptosis in E. coli-infected macrophages by decreasing extracellular ATP levels. Mechanistically, DPI upregulated p38 MAPK phosphorylation to suppress the expression and activity of the hemichannel protein connexin 43 (CX43), leading to the inhibition of CX43-mediated ATP efflux in E. coli-infected macrophages. In a murine E. coli infection model, DPI effectively reduced ATP release, decreased bacterial load and inhibited inflammasome activation, thereby improving survival and ameliorating organ injuries in model mice. In summary, our study demonstrates a previously unknown function of DPI in conferring protection against bacterial infection and suggests a putative antimicrobial strategy of modulating CX43 -dependent ATP leakage.

The mechanism regulating the gastrointestinal epithelial barrier remains poorly understood. We herein demonstrate that Absent in melanoma-2 (AIM2) contributes to the maintenance of intestinal barrier integrity and defense against bacterial infection. AIM2-deficient mice displayed an increased susceptibility to mucosal but not systemic infection by Salmonella typhimurium, indicating a protective role for AIM2 in the gastrointestinal tract. In a Salmonella colitis model, compared with wild-type mice, AIM2(-/-) mice exhibited more severe body weight loss, intestinal damage, intestinal inflammation, and disruption of basal and activated epithelial cell turnover. In vivo and in vitro data showed that AIM2 restricted the early epithelial paracellular invasion of Salmonella and decreased epithelial permeability. The decreased epithelial barrier in AIM2(-/-) mice might be attributed to the altered expression of tight junction proteins that contribute to epithelial integrity. AIM2 promoted the expression of tight junction proteins through Akt activation. Together, these results suggest that AIM2 is required for maintaining the integrity of the epithelial barrier.

// Dong Yang 1 , Xinchuan Zheng 1 , Ning Wang 1 , Shijun Fan 1 , Yongjun Yang 1 , Yongling Lu 1 , Qian Chen 1 , Xin Liu 1 and Jiang Zheng 1 1 Medical Research Center, Southwest Hospital, Third Military Medical University, Chongqing, China Correspondence to: Jiang Zheng, email: // Xin Liu, email: // Keywords : lipopolysaccharide, kukoamine B, hepatocytes, asialoglycoprotein receptor, Immunology and Microbiology Section, Immune response, Immunity Received : April 30, 2016 Accepted : August 11, 2016 Published : August 15, 2016 Abstract Free bacterial lipopolysaccharide (LPS) is generally removed from the bloodstream through hepatic uptake via TLR4, the LPS pattern recognition receptor, but mechanisms for internalization and clearance of conjugated LPS are less clear. Kukoamine B (KB) is a novel cationic alkaloid that interferes with LPS binding to TLR4. In this study, KB accelerated blood clearance of LPS. KB also enhanced LPS distribution in the hepatic tissues of C57 BL/6 mice, along with LPS uptake in primary hepatocytes and HepG2 cells. By contrast, KB inhibited LPS internalization in Kupffer and RAW 264.7 cells. Loss of TLR4 did not affect LPS uptake into KB-treated hepatocytes. We also detected selective upregulation of the asialoglycoprotein receptor (ASGPR) upon KB treatment, and ASGPR colocalized with KB in cultured hepatocytes. Molecular docking showed that KB bound to ASGPR in a manner similar to GalNAc, a known ASGPR agonist. GalNAc dose-dependently reduced KB internalization, suggesting it competes with KB for ASGPR binding, and ASGPR knockdown also impaired LPS uptake into hepatocytes. Finally, while KB enhanced LPS uptake, it was protective against LPS-induced inflammation and hepatocyte injury. Our study provides a new mechanism for conjugated LPS hepatic uptake induced by the LPS neutralizer KB and mediated by membrane ASGPR binding.

This study aimed to explore the effects of glutamine on hypermetabolic reactions in burned rats and its underlying mechanism.Fifty-five Sprague-Dawley rats were randomly divided into three groups, namely, the control (C), burned (B), and burned + glutamine (B + G) groups. Rats in the glutamine treatment group were supplemented with 1 g glutamine per kg body weight. Changes in body weight and resting energy expenditure in all groups were observed daily. Blood glucose and glucose tolerance level were measured on days 1, 3, 7, 10 and 14 after burn injury. On days 7 and 14 after injury, the rats were sacrificed, and the weight and protein content of the skeletal muscle were measured. Moreover, the level of glutamine, inflammatory mediator, nicotinamide adenine dinucleotide phosphate (NADPH), glutathione, and the activity of glutamine metabolic enzymes were measured.The hypermetabolic reaction after burn injury was significantly inhibited by glutamine administration, and the range of variations in the resting energy expenditure and body weight indicators was narrowed remarkably (P0.05 or 0.01), whereas the weight and protein content of the skeletal muscle returned to normal (P0.05 or 0.01). Glutamine could increase glutaminase activity in various tissues, promote the utilization of glutamine, and appropriately reduce the degree of organ damage and inflammatory response (P0.05 or 0.01). Furthermore, glutamine could promote the synthesis of the reducing substances NADPH and glutathione (P0.05 or 0.01).Glutamine administration effectively reduces hypermetabolic reactions by promoting NADPH synthesis, inhibiting oxidative stress, and improving glutamine utilization after burn injury.

The progress of sepsis is increasingly recognized by the transition from early hyperinflammation to long term immunosuppression, which is characterized in innate immune cells by diminished responsiveness termed as lipopolysaccharide (LPS) tolerance. In this study, we investigated the ability of the antimalarial drug artesunate to reverse LPS tolerance and explored the underlying mechanisms. Initially, we detected a dramatic decline in autophagy accompanied by decreased cytokine production and impaired bacterial clearance by LPS tolerant macrophages. Then we demonstrated that artesunate restored cytokine production and enhanced bacterial clearance by inducing autophagy. Moreover, artesunate caused greater suppression of inhibitory phosphorylation than of activating phosphorylation of Unc-51 like autophagy activating kinase 1 (ULK1), a kinase that is essential for initiating autophagy through the inhibition of excessive AMP-activated protein kinase (AMPK) activation. This effect was shown to be achieved by suppression of Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation, resulting in reduction of the inositol 1,4,5-triphate receptor (IP3R) dependent Ca2+ release from the endoplasmic reticulum (ER) and inhibiting the overactive CaMKKβ-AMPK cascade. Administration of artesunate also upregulated autophagy and reversed the tolerant status in LPS tolerant mice. In summary, our findings reveal a novel immunopharmacological action of artesunate to reverse LPS tolerance by restoring autophagy. Our results may also indicate the significance of autophagy induction for treating immunosuppression in sepsis.

Neospora caninum is an intracellular protozoan parasite closely related to Toxoplasma gondii that mainly infects canids as the definitive host and cattle as the intermediate host, resulting in abortion in cattle and leading to financial losses worldwide. Commercial vaccines or drugs are not available for the prevention and treatment of bovine neosporosis. Knowledge about the hallmarks of the immune response to this infection could form the basis of important prevention strategies. The innate immune system first responds to invading parasite and subsequently initiates the appropriate adaptive immune response against this parasite. Upon infection, activation of host pattern-recognition receptors expressed by immune cells triggers the innate immune response. Toll-like receptors, NOD-like receptors, and C-type lectin receptors play key roles in recognizing protozoan parasite. Therefore, we aimed to explore the role of the NLRP3 inflammasome during the acute period of N. caninum infection. In vitro results showed that N. caninum infection of murine bone marrow-derived macrophages activated the NLRP3 inflammasome, accompanied by the release of IL-1β and IL-18, cleavage of caspase-1, and induction of cell death. K+ efflux induced by N. caninum infection participated in the activation of the inflammasome. Infection of mice deficient in NLRP3, ASC, and caspase-1/11 resulted in decreased production of IL-18 and reduced IFN-γ in serum. Elevated numbers of monocytes/macrophages and neutrophils were found at the initial infection site, but they failed to limit N. caninum replication. These findings suggest that the NLRP3 inflammasome is involved in the host response to N. caninum infection at the acute stage and plays an important role in limiting parasite growth, and it may enhance Th1 response by inducing production of IFN-γ. These findings may help devise protocols for controlling neosporosis.

Staphylococcus aureus (S. aureus) is a common and dangerous pathogen that causes various infectious diseases. Skin damage, such as burn wounds, are at high risk of Staphylococcus aureus colonization and infection, which increases morbidity and mortality. The phage lysin LysGH15 exhibits highly efficient lytic activity against methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) strains. Apigenin (api) significantly decreases haemolysis of rabbit erythrocytes caused by S. aureus and shows anti-inflammatory function. LysGH15 and api were added to Aquaphor to form an LysGH15-api-Aquaphor (LAA) ointment. The LAA ointment simultaneously exhibited bactericidal activity against S. aureus and inhibited haemolysis. In an LAA-treated mouse model of an MRSA-infected skin wound, the mean bacterial colony count decreased to approximately 102 CFU/mg at 18 h after treatment (and the bacteria became undetectable at 96 h), whereas the mean count in untreated mice was approximately 105 CFU/mg of tissue. The LAA ointment also reduced the levels of pro-inflammatory cytokines (TNF-α, IL-1β, and IFN-γ) and accelerated wound healing in the mouse model. These data demonstrate the potential efficacy of a combination of LysGH15 and api for use as a topical antimicrobial agent against S. aureus.

The intestinal mucosal barrier is critical for host defense against pathogens infection. Here, we demonstrate that the mixed lineage kinase-like protein (MLKL), a necroptosis effector, promotes intestinal epithelial barrier function by enhancing inflammasome activation. MLKL−/− mice were more susceptible to Salmonella infection compared with wild-type counterparts, with higher mortality rates, increased body weight loss, exacerbated intestinal inflammation, more bacterial colonization, and severe epithelial barrier disruption. MLKL deficiency promoted early epithelial colonization of Salmonella prior to developing apparent intestinal pathology. Active MLKL was predominantly expressed in crypt epithelial cells, and experiments using bone marrow chimeras found that the protective effects of MLKL were dependent on its expression in non-hematopoietic cells. Intestinal mucosa of MLKL−/− mice had impaired caspase-1 and gasdermin D cleavages and decreased interleukin (IL)-18 release. Moreover, administration of exogenous recombinant IL-18 rescued the phenotype of increased bacterial colonization in MLKL−/− mice. Thus, our results uncover the role of MLKL in enhancing inflammasome activation in intestinal epithelial cells to inhibit early bacterial colonization.

The findings from several studies have suggested that circulating miRNAs are imbalanced with the genesis of gastric cancer (GC). Both normal and cancer cells can generate and secrete exosomes, which are nanosized membrane vesicles that can transport microRNAs and proteins. Emerging evidence indicates that the exosomes secreted by cancer cells can be released into the circulatory system. In this study, we investigated whether circulating exosomal miRNAs can be used to discriminate individuals with GC from healthy controls (NCs). Based on the quantitative reverse transcription polymerase chain reaction (qRT-PCR), four miRNAs (miR-19b-3p, miR-17-5p, miR-30a-5p, and miR-106a-5p) related to GC pathogenesis were identified in serum-circulating exosomes from a cohort of 20 healthy controls and 20 individuals with GC in the initial screening phase. The distinguished miRNAs were further validated in the training (90 GC vs. 90 NCs) and blinded phases 20 GC vs. 20 NCs), and the area under receiver operating characteristic (ROC) curves of these miRNAs were analyzed. We found that miR-19b and miR-106a were markedly overexpressed in individuals with GC compared to NCs (P

Infectious agents can reach the placenta either via the maternal blood or by ascending the genito-urinary tract, and then initially colonizing the maternal decidua. Decidual stromal cells (DSCs) are the major cellular component of the decidua. Although DSCs at the maternal–fetal interface contribute to the regulation of immunity in pregnancy in the face of immunological and physiological challenges, the roles of these DSCs during viral infection remain ill defined. Here, we characterized the response of DSCs to a synthetic double-stranded RNA molecule, polyinosinic-polycytidylic acid [poly(I:C)], which is a mimic of viral infection. We demonstrated that both transfection of cells with poly(I:C) and addition of extracellular (non-transfected) poly(I:C) trigger the necroptosis of DSCs and that this response is dependent on RIG-I-like receptor/IPS-1 signaling and the toll-like receptor 3/TIR-domain-containing adapter-inducing interferon-β pathway, respectively. Furthermore, following poly(I:C) challenge, pregnant mixed lineage kinase domain-like protein-deficient mice had fewer necrotic cells in the mesometrial decidual layer, as well as milder pathological changes in the uterine unit, than did wild-type mice. Collectively, our results establish that necroptosis is a contributing factor in poly(I:C)-triggered abnormal pregnancy and thereby indicate a novel therapeutic strategy for reducing the severity of the adverse effects of viral infections in pregnancy.

Calcific tendinitis is an extremely common shoulder disorder which affects 2.7 to 10.3% of the adult population. However, clear details regarding the formation of calcific tendinitis have only been found recently, and its pathology still under debate. In this report, we present a case of calcific tendinitis affect both the infraspinatus and the teres minor tendon with both X-ray and ultrasound findings. Calcific tendinitis at the infraspinatus is rare (around 8.4% in all rotator cuff calcific tendinitis), and no separate case report on calcific tendinitis has been reported in the teres minor region in the preceding several years, therefore, the rarity of reporting this case was considered.4 We assume that additional case reports can help to further improve our understanding regarding infraspinatus and teres minor tendon calcification.

Background Neospora caninum is an intracellular parasite that causes significant economic losses in cattle industry. Understanding the host resistance mechanisms in the innate immune response to neosporosis could facilitate the exploration of approaches for controlling N. caninum infection. The NLR inflammasome is a multiprotein platform in the cell cytoplasm and plays critical roles in the host response against microbes. Methods Neospora caninum-infected wild-type (WT) macrophages and Nlrp3 −/− macrophages, and inhibitory approaches were used to investigate inflammasome activation and its role in N. caninum infection. Inflammasome RT Profiler PCR Arrays were used to identify the primary genes involved in N. caninum infection. The expression of the sensor protein NLRP3, processing of caspase-1, secretion of IL-1β and cell death were detected. Neospora caninum replication in macrophages was also assessed. Results Many NLR molecules participated in the recognition of N. caninum, especially the sensor protein NLRP3, and further study revealed that the NLRP3 distribution became punctate in the cell cytoplasm, which facilitated inflammasome activation. Inflammasome activation-mediated caspase-1 processing and IL-1β cleavage in response to N. caninum infection were observed and were correlated with the time of infection and number of infecting parasites. LDH-related cell death was also observed, and this death was regarded as beneficial for the clearance of N. caninum. Treatment of N. caninum-infected macrophages with caspase-1, pan-caspase and NLRP3 inhibitors led to the impaired release of active IL-1β and a failure to restrict parasite replication. And Neospora caninum infected peritoneal macrophages from Nlrp3-deficient mice displayed greatly decreased release of active IL-1β and the failure of caspase-1 cleavage. Conclusions The NLRP3 inflammasome can be activated in N. caninum-infected macrophages, and plays a protective role in the host response to control N. caninum. Electronic supplementary material The online version of this article (doi:10.1186/s13071-017-2197-2) contains supplementary material, which is available to authorized users.

A practical issue is present in sustaining and rehabilitating the ecologically vulnerable post-mining area in which the environmental condition varies spatially and therefore influenced by multiple factors. This paper attempts to integrate the ecological vulnerability assessment and rehabilitation treatment to assist land managers in revealing vulnerable features along with developing treatments of vulnerability mitigation. Using a post-mining site in a mountainous area in western China as study area, an indicator system and framework for assessing and reducing vulnerability were developed based on a vulnerability analysis. Geo-informatics, such as satellite image processing and spatial analysis, were employed to perform the assessment and planning. It was found that higher exposure and sensitivity are the main causes of increased vulnerability in a seriously disturbed post-mining area. Rehabilitation treatments were arranged spatially and structurally based on the framework of vulnerability mitigation. A pre-evaluation of the effectiveness shows this type of rehabilitation has a convergence effect that clusters and lowers the ecological vulnerability index (EVI). The average value of EVI will be reduced by 15.02% if the minimum standards of rehabilitation can be completed. Altogether, an integration of rehabilitation treatments and the quantification of vulnerability in a spatially explicit manner are critical for planners to gain more insight into ecological vulnerability in post-mining area, which provides guidance to simplify rehabilitation planning with respect to vulnerability mitigation.

The critical roles of sortase A (SrtA) and listeriolysin O (LLO) in Listeria monocytogenes pathogenicity render these two virulence factors as ideal targets for the development of anti-virulence agents against L. monocytogenes infection. Additionally, the structures of SrtA and LLO are highly conserved among the members of sortase enzyme family and cholesterol dependent toxin family. Here, phloretin, a natural polyphenolic compound derived from apples and pears that has little anti-L. monocytogenes activity, was identified to simultaneously inhibit LLO expression and neutralize SrtA catalytic activity. Phloretin neutralized SrtA activity by causing a conformational change in the protein's active pocket, which prevented engagement with its substrate. Treatment with phloretin simultaneously reduced L. monocytogenes invasion into host cells and blocked the escape of vacuole-entrapped L. monocytogenes into cytoplasm. Further, L. monocytogenes-infected mice that received phloretin showed lower mortality, decreased bacterial burden and reduced pathological injury. Our results demonstrate that phloretin is a promising anti-infective therapeutic for infections caused by L. monocytogenes due to its simultaneous targeting of SrtA and LLO, which may result in fewer side effects than those caused by other antibiotics.

The mechanisms underlying the immune defense by trophoblasts against pathogens remain ill defined. We demonstrated that placental cell death was increased upon in vivo exposure to Listeria monocytogenes. The death of infected cells is an important host innate defense mechanism. Meanwhile, double-stranded DNA (dsDNA) derived from intracellular bacteria or dsDNA viruses is emerging as a potent pathogen-associated molecular pattern recognized by host cells. We sought to characterize trophoblast death in response to cytosolic dsDNA challenge. Our results showed that dsDNA induced caspase-dependent and -independent cell death in human trophoblasts. However, necroptosis, a cell death pathway independent of caspase, could not be induced by dsDNA treatment, even in the presence of exogenously expressed RIPK3. L. monocytogenes-derived genomic DNA triggered a similar cell death pattern. Moreover, the cell death in response to dsDNA was IFI16 dependent. These data suggest that cytosolic dsDNA induces nonnecroptotic cell death in trophoblasts via IFI16, and this could contribute to placental barrier against infection.

Cancers are initiated as a result of changes that occur in the genome. Identification of gains and losses in the structure and expression of tumor-suppressor genes and oncogenes lies at the root of the understanding of cancer cell biology. Here, we show that the mitogen–activated protein kinase (MAPK) MKK3 suppresses the growth of breast cancer, in which it varies in copy number. A pervasive loss of MKK3 gene copy number in patients with breast cancer is associated with an impairment of MKK3 expression and protein level in malignant tissues. To assess the functional role of MKK3 in breast cancer, we showed in an animal model that MKK3 activity is required for suppression of tumor growth. Active MKK3 enhanced expression of the cyclin-dependent kinase inhibitors p21Cip1/Waf1 and p27Kip1, leading to increased cell-cycle arrest in G1 phase of the cell cycle. Our results reveal the functional significance of MKK3 as a tumor suppressor and improve understanding of the dynamic role of the MAPK pathway in tumor progression. Cancer Res; 74(1); 162–72. ©2013 AACR.

Objective Salmonella is known to evolve many mechanisms to avoid or delay inflammasome activation which remain largely unknown. In this study, we investigated whether the SopB protein critical to bacteria virulence capacity was an effector that involved in the regulation of inflammasome activation. Methods BMDMs from NLRC4-, NLRP3-, caspase-1/-11-, IFI16- and AIM2-deficient mice were pretreated with LPS, and subsequently stimulated with a series of SopB-related strains of Salmonella , inflammasome induced cell death, IL-1β secretion, cleaved caspase-1 production and ASC speckle formation were detected. Results We found that SopB could inhibit host IL-1β secretion, caspase-1 activation and inflammasome induced cell death using a series of SopB-related strains of Salmonella ; however the reduction of IL-1β secretion was not dependent on sensor that contain PYD domain, such as NLRP3, AIM2 or IFI16, but dependent on NLRC4. Notably, SopB specifically prevented ASC oligomerization and the enzymatic activity of SopB was responsible for the inflammasome inhibition. Furthermore, inhibition of Akt signaling induced enhanced inflammasome activation. Conclusions These results revealed a novel role in inhibition of NLRC4 inflammasome for Salmonella effector SopB.

Inflammasomes are multiprotein complexes that control the production of IL-1β and IL-18. NLRP3 inflammasome, the most characterized inflammasome, plays prominent roles in defense against infection, however aberrant activation is deleterious and leads to diseases. Therefore, its tight control offers therapeutic promise. Liver X receptors (LXRs) have significant anti-inflammatory properties. Whether LXRs regulate inflammasome remains unresolved. We thus tested the hypothesis that LXR's anti-inflammatory properties may result from its ability to suppress inflammasome activation. In this study, LXRs agonists inhibited the induction of IL-1β production, caspase-1 cleavage and ASC oligomerization by NLRP3 inflammasome. The agonists also inhibited inflammasome-associated mtROS production. Importantly, the agonists inhibited the priming of inflammasome activation. In vivo data also showed that LXRs agonist prevented NLRP3-dependent peritonitis. In conclusion, LXRs agonists are identified to potently suppress NLRP3 inflammasome and the regulation of LXRs signaling is a potential therapeutic for inflammasome-driven diseases.

Objective RCAN1 (regulator of calcineurin 1) has been shown to be involved in various physiological and pathological processes. However, the biological implications of RCAN1 during gastrointestinal tract infection remain unclear. In this study, we tried to determine the role of RCAN1 in acute Salmonella infectious colitis. Methods Wild type and RCAN1-deficient mice or macrophages were used to characterize the impacts of RCAN1 on intestinal inflammation, inflammatory cytokines production, animal survival, and pathogen clearance following Salmonella challenge. Results Histologic and quantitative assessments showed increased inflammation and elevated proinflammatory cytokines production in RCAN1-deficient mice. The aberrant inflammatory response was recapitulated in primary bone marrow-derived macrophages. In addition, we reveal a novel regulatory role for RCAN1 in the proinflammatory JNK signaling both in vitro and in vivo. Further analysis showed that the increased inflammation in RCAN1-deficient mice contributed to pathogen clearance and host survival. Conclusions The present study demonstrates that RCAN1 deficiency protects against Salmonella intestinal infection by enhancing proinflammatory JNK signaling.

Inflammasomes are cytoplasmic, multiprotein complexes that trigger caspase-1 activation and IL-1β maturation in response to diverse stimuli. Although inflammasomes play important roles in host defense against microbial infection, overactive inflammasomes are deleterious and lead to various autoinflammatory diseases. In the current study, we demonstrated that genipin inhibits the induction of IL-1β production and caspase-1 activation by NLRP3 and NLRC4 inflammasomes. Furthermore, genipin specifically prevented NLRP3-mediated, but not NLRC4-mediated, ASC oligomerization. Notably, genipin inhibited autophagy, leading to NLRP3 and NLRC4 inflammasome inhibition. UCP2-ROS signaling may be involved in inflammasome suppression by genipin. In vivo, we showed that genipin inhibited NLRP3-dependent IL-1β production and neutrophil flux in LPS- and alum-induced murine peritonitis. Additionally, genipin provided protection against flagellin-induced lung inflammation by reducing IL-1β production and neutrophil recruitment. Collectively, our results revealed a novel role in inhibition of inflammatory diseases for genipin that has been used as therapeutics for centuries in herb medicine.

The presence of eosinophils in the lung is often regarded as a defining feature of asthma. On allergen stimulation, numbers of eosinophils and their progenitors are increased in both the bone marrow and lungs. Eosinophil progenitors provide an ongoing supply of mature eosinophils. Here, we report that deficiency in the regulator of calcineurin 1 gene (Rcan1) leads to a near-complete absence of eosinophilia in ovalbumin-induced allergic asthma in mice. In the absence of Rcan1, bone marrow cells produce significantly fewer eosinophils in vivo and in vitro on interleukin-5 stimulation. Importantly, eosinophil progenitor populations are significantly reduced in both naïve and ovalbumin-challenged Rcan1(-/-) mice. Bone marrow cells from Rcan1(-/-) mice are capable of developing into fully mature eosinophils, suggesting that Rcan1 is required for eosinophil progenitor production but may not be necessary for eosinophil maturation. Thus, Rcan1 represents a novel contributor in the development of eosinophilia in allergic asthma through regulation of eosinophil progenitor production.

Pseudomonas aeruginosa is a major opportunistic pathogen. However, host defense mechanisms involved in P. aeruginosa lung infection remain incompletely defined. The transcription factor IFN regulatory factor 3 (IRF3) is primarily associated with host defense against viral infections, and a role of IRF3 in P. aeruginosa infection has not been reported previously. In this study, we showed that IRF3 deficiency led to impaired clearance of P. aeruginosa from the lungs of infected mice. P. aeruginosa infection induced IRF3 translocation to the nucleus, activation of IFN-stimulated response elements (ISRE), and production of IFN-β, suggesting that P. aeruginosa activates the IRF3–ISRE–IFN pathway. In vitro, macrophages from IRF3-deficient mice showed complete inhibition of CCL5 (RANTES) and CXCL10 (IP-10) production, partial inhibition of TNF, but no effect on CXCL2 (MIP-2) or CXCL1 (keratinocyte-derived chemokine) in response to P. aeruginosa stimulation. In vivo, IRF3-deficient mice showed complete inhibition of CCL5 production and partial or no effects on production of other cytokines and chemokines in the bronchoalveolar lavage fluids and lung tissues. Profiling of immune cells in the airways revealed that neutrophil and macrophage recruitment into the airspace was reduced, whereas B cell, T cell, NK cell, and NKT cell infiltration was unaffected in IRF3-deficient mice following P. aeruginosa lung infection. These data suggest that IRF3 regulates a distinct profile of cytokines and chemokines and selectively modulates neutrophil and macrophage recruitment during P. aeruginosa infection. Thus, IRF3 is an integral component in the host defense against P. aeruginosa lung infection.

The gut microbiota and microRNAs play important roles in the defense against infection. However, the role of miR-146a in L. monocytogenes infection and gut microbiota remains unclear. We tried to determine whether miR-146a controlled L. monocytogenes infection by regulating the gut microbiota. Wild-type and miR-146a-deficient mice or macrophages were used to characterize the impact of miR-146a on animal survival, cell death, bacterial clearance, and gut microbiota following L. monocytogenes challenge. We found that L. monocytogenes infection induced miR-146a expression both in vitro and in vivo. When compared to wild-type mice, miR-146a-deficient mice were more resistant to L. monocytogenes infection. MiR-146a deficiency in macrophages resulted in reduced invasion and intracellular survival of L. monocytogenes. High-throughput sequencing of 16S rRNA revealed that the gut microbiota composition differed between miR-146a-deficient and wild-type mice. Relative to wild-type mice, miR-146a-deficient mice had decreased levels of the Proteobacteria phylum, Prevotellaceae family, and Parasutterella genus, and significantly increased short-chain fatty acid producing bacteria, including the genera Alistipes, Blautia, Coprococcus_1, and Ruminococcus_1. Wild-type mice co-housed with miR-146a-deficient mice had increased resistance to L. monocytogenes, indicating that miR-146a deficiency guides the gut microbiota to alleviate infection. Together, these results suggest that miR-146a deficiency protects against L. monocytogenes infection by regulating the gut microbiota.

Pseudomonas aeruginosa is a common cause of lung infection in immune compromised individuals. Studies in humans and mice have demonstrated that P. aeruginosa lung infection is associated with a predominant Th2 immune response, whereas Th1 responses are accompanied by a better pulmonary outcome. Regulatory T cells (Tregs) are a subpopulation of T cells with unique immunologic characteristics that suppress effector T cell functions. Whether Tregs contribute to P. aeruginosa -induced host responses has not been studied previously. We found that P. aeruginosa lung infection induced an increase in natural Treg cells (CD4 + CD25 + FOXP3 + T cells) in the spleen of mice. To investigate a role of natural CD4 + CD25 + Tregs in the host response to P. aeruginosa lung infection in vivo , anti-CD25 Ab was used to deplete endogenous CD4 + CD25 + Tregs. Anti-CD25 treatment depleted 90% of CD4 + CD25 + FOXP3 + cells. Surprisingly, no differences of P. aeruginosa -induced NF- κ B activation and cytokine/chemokine production (IL-1 β , TNF, IL-6, IL-10, RANTES or MIP-2) were observed between anti-CD25-treated and isotype control Ab-treated animals. Similarly, no differences in lung histology and airway neutrophil infiltration were observed between anti-CD25 and control Ab-treated animals. Furthermore, no difference in survival outcome was found between anti-CD25 and control Ab-treated animals. These data demonstrate that although P. aeruginosa lung infection causes an increase of Tregs, the endogenous natural CD4 + CD25 + Treg cells do not contribute significantly to the host response to this bacterium.

Leptin is a 16-kDa multifunctional protein. Recent reports indicate that leptin is an important molecule during implantation and placentation, implicated in embryonic-maternal cross-talk and cytotrophoblast invasiveness, however, the role of leptin playing in the process of normal blastocyst implantation has not been well characterized. In the present study, the possible mechanisms of leptin playing in mouse blastocyst implantation were investigated. Leptin and receptor isoforms mRNAs were detected in whole mouse uteri during estrous cycle and peri-implantation periods. Immunofluorescent analysis further confirmed Ob-R protein was present in mouse uterus. The differential amounts of leptin and Ob-R isoforms suggested a role for leptin in such endometrial issues as blastocyst implantation. In vitro culture model for studying embryo implantation, leptin promoted mouse blastocyst adhesion and blastocyst outgrowth on fibronectin. Blastocysts treated with 300 ng/ml leptin had the greatest adhesion rate of 76.58+/-6.41% (P=0.046), and blastocysts treated with 30 ng/ml leptin had the greatest outgrowth rate of 78.64+/-8.48% (P=0.005). In isolated endometrial epithelial cells, leptin upregulated amounts of alpha v and beta 3 integrin, and promoted cell adhesion to such extracellular matrix proteins as fibronectin, laminin and type IV collagen, showing a dose- and time-dependent cell-adhesive capacity. Collectively, the information from the present study may partly account for leptin-induced mouse blatocyst implantation.

The expression of tetraspanin CD9 was found on blastocysts in mice and endometrium epithelial cells in human and bovine. However, it remains unknown how CD9 is involved in the precise dialogue between embryo and uterus during early pregnancy. This study was designed to investigate the functional roles of CD9 in the embryo implantation with monoclonal antibody against CD9 protein (anti-CD9 mAb) and antisense oligonucleotide against CD9 gene (AS-CD9). Our results showed that intrauterine injection of anti-CD9 mAb on day 4 of pregnancy significantly increased the number of embryos implanted (7.24±0.39 versus 4.04±0.38). In vitro, anti-CD9 mAb or AS-CD9 significantly enhanced embryo-outgrowth ability on the monolayer of uterus epithelial cells in a dose-dependent manner. However, the attachment of blastocysts to epithelial cells was unaffected. Furthermore, we found that anti-CD9 mAb or AS-CD9 stimulated matrix metalloproteinase 2 (MMP-2) production of blastocysts on Fibronectin. LY294002, a specific inhibitor of phosphoinositide 3-kinase, was able to counteract the effect of anti-CD9 mAb and AS-CD9 on outgrowth ability and production of MMP-2. Our results indicated that CD9 played a role of inhibiting embryo implantation. CD9 was able to impair embryo invasion and the production of MMP-2 through the phosphoinositide 3-kinase signaling pathway.

BACKGROUND: Bacteriophage could be an alternative to conventional antibiotic therapy against multidrug-resistant bacteria. However, the emergence of resistant variants after phage treatment limited its therapeutic application. METHODOLOGY/PRINCIPAL FINDINGS: In this study, an approach, named "Step-by-Step" (SBS), has been established. This method takes advantage of the occurrence of phage-resistant bacteria variants and ensures that phages lytic for wild-type strain and its phage-resistant variants are selected. A phage cocktail lytic for Klebsiella pneumoniae was established by the SBS method. This phage cocktail consisted of three phages (GH-K1, GH-K2 and GH-K3) which have different but overlapping host strains. Several phage-resistant variants of Klebsiella pneumoniae were isolated after different phages treatments. The virulence of these variants was much weaker [minimal lethal doses (MLD)>1.3×10(9) cfu/mouse] than that of wild-type K7 countpart (MLD = 2.5×10(3) cfu/mouse). Compared with any single phage, the phage cocktail significantly reduced the mutation frequency of Klebsiella pneumoniae and effectively rescued Klebsiella pneumoniae bacteremia in a murine K7 strain challenge model. The minimal protective dose (MPD) of the phage cocktail which was sufficient to protect bacteremic mice from lethal K7 infection was only 3.0×10(4) pfu, significantly smaller (p

Mast cells play a central role in allergic inflammation and are activated through cross-linking of FcεRI receptor-bound IgE, initiating a signaling cascade resulting in production of biologically potent mediators. Signaling pathways in the regulation of specific mediators remain incompletely defined. In this study, we examined the role of MAPK kinase 3 (MKK3) in IgE-dependent mast cell activation. In an in vivo model of passive cutaneous anaphylaxis, MKK3-deficient mice showed a deficit in late-phase IgE-dependent inflammation. To characterize the mechanism of this deficiency, we cultured bone marrow-derived mast cells (BMMCs) from wild-type and MKK3-deficient mice. We found that FcεRI-mediated mast cell activation induced rapid MKK3 phosphorylation by 5 min, diminishing slowly after 6 h. In MKK3-deficient BMMCs, phosphorylation of p38 was reduced at early and later time points. Among 40 cytokines tested using a protein array, IL-4 was the only cytokine specifically downregulated in MKK3-deficient BMMCs. Reduced IL-4 expression was seen in the local skin of MKK3-deficient mice following passive cutaneous allergic reaction. Furthermore, early growth response-1 (Egr1) bound to the promoter of IL-4 in FcεRI-activated mast cells, and Egr1 transcription factor activity was diminished in MKK3-deficient BMMCs. Finally, mast cell-deficient mice reconstituted with MKK3-deficient BMMCs displayed a significantly impaired late-phase allergic inflammatory response. Thus, mast cell MKK3 signaling contributes to IgE-dependent allergic inflammation and is a specific regulator of FcεRI-induced IL-4 production.