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1.  Expression and distribution of dendritic cells in nasal polyps 
The aim of the present study was to investigate the expression, distribution and function of dendritic cells (DCs) and to study their role in nasal polyps. The study involved 55 participants, 45 of whom had nasal polyps and were the study group and 10 who had normal inferior turbinates and were the control group. Immunohistochemical staining was used to visualize the expression and distribution of the S-100 protein. A double immunostaining method was used to visualize the CD1a and CD40 expression and the images were analyzed with Axioplan 2 microscopy. The expression level of the S-100 protein in the nasal polyps was higher than that in the normal inferior turbinates with a significant difference (P<0.01). The distribution area, number and density of the double stained cells in the nasal polyps were all greater than in the normal inferior turbinates (P<0.01). The S-100 protein and double stained cells were mainly located in the lamina propria below the mucous membrane. The present study demonstrates that DCs are involved in the pathogenesis of nasal polyps and the presence of CD40-positive DCs suggests that this was related to the reciprocal interaction between the DCs and T lymphocytes.
doi:10.3892/etm.2013.986
PMCID: PMC3671826  PMID: 23737902
nasal polyps; dendritic cells; S-100 protein; CD1a; CD40; pathogenesis
2.  Efficacy of anti–tumor necrosis factor therapy for extra-articular manifestations in patients with ankylosing spondylitis: a meta–analysis 
Background
We performed a meta-analysis to evaluate the effect of anti–tumor necrosis factor (TNF) therapy on the frequency of extra–articular manifestations (EAMs) in patients with ankylosing spondylitis (AS).
Methods
We searched with the terms ‘ankylosing spondylitis’, ‘infliximab’, ‘etanercept’, ‘adalimumab’, ‘golimumab’, ‘certolizumab’, ‘TNF inhibitor/blocker/antagonists’ or ‘anti-TNF’ on MEDLINE, EMBASE and Cochrane Library for randomized controlled trials (RCTs) of ≥12 weeks with parallel or crossover design of TNF inhibitor versus placebo to treat uveitis, inflammatory bowel disease (IBD) and/or psoriasis of AS, published before February 2014.
Results
We found 8 RCTs that fit our criteria. Anti–TNF therapy was associated with less uveitis than placebo in patients with AS (OR: 0.35, 95% CI: 0.15–0.81, P = 0.01). Subgroup analysis showed receptor fusion proteins were more efficacious for uveitis than placebo (OR: 0.30, 95% CI: 0.09–0.94, P = 0.04), but monoclonal antibodies were not (OR: 0.43, 95% CI: 0.12–1.49, P = 0.18). Anti–TNF therapy and placebo group did not significantly differ in treating IBD in AS patients (OR: 0.75, 95% CI: 0.25–2.29, P = 0.61). In subgroup analysis, neither monoclonal antibodies (OR: 0.45, 95% CI: 0.10–1.92, P = 0.28) nor receptor fusion proteins (OR: 1.52, 95% CI: 0.25–9.25, P = 0.65) significantly differed from placebo in treating IBD. We found no suitable reports on psoriasis.
Conclusions
Anti–TNF therapy was preventive for flares or new onset of uveitis in AS patients, and might be an alternative for these patients. However, monoclonal anti–TNF antibodies and TNF receptor fusion proteins were not efficacious for IBD in AS patients.
doi:10.1186/s12891-015-0489-2
PMCID: PMC4328050
Ankylosing spondylitis; Anti-TNF therapy; Extra–articular manifestations; Uveitis; Inflammatory bowel disease; Meta–analysis
3.  Compartment-Specific and Sequential Role of MyD88 and CARD9 in Chemokine Induction and Innate Defense during Respiratory Fungal Infection 
PLoS Pathogens  2015;11(2):e1004589.
Aspergillus fumigatus forms ubiquitous airborne conidia that humans inhale on a daily basis. Although respiratory fungal infection activates the adaptor proteins CARD9 and MyD88 via C-type lectin, Toll-like, and interleukin-1 family receptor signals, defining the temporal and spatial pattern of MyD88- and CARD9-coupled signals in immune activation and fungal clearance has been difficult to achieve. Herein, we demonstrate that MyD88 and CARD9 act in two discrete phases and in two cellular compartments to direct chemokine- and neutrophil-dependent host defense. The first phase depends on MyD88 signaling because genetic deletion of MyD88 leads to delayed induction of the neutrophil chemokines CXCL1 and CXCL5, delayed neutrophil lung trafficking, and fatal pulmonary damage at the onset of respiratory fungal infection. MyD88 expression in lung epithelial cells restores rapid chemokine induction and neutrophil recruitment via interleukin-1 receptor signaling. Exogenous CXCL1 administration reverses murine mortality in MyD88-deficient mice. The second phase depends predominately on CARD9 signaling because genetic deletion of CARD9 in radiosensitive hematopoietic cells interrupts CXCL1 and CXCL2 production and lung neutrophil recruitment beyond the initial MyD88-dependent phase. Using a CXCL2 reporter mouse, we show that lung-infiltrating neutrophils represent the major cellular source of CXCL2 during CARD9-dependent recruitment. Although neutrophil-intrinsic MyD88 and CARD9 function are dispensable for neutrophil conidial uptake and killing in the lung, global deletion of both adaptor proteins triggers rapidly progressive invasive disease when mice are challenged with an inoculum that is sub-lethal for single adapter protein knockout mice. Our findings demonstrate that distinct signal transduction pathways in the respiratory epithelium and hematopoietic compartment partially overlap to ensure optimal chemokine induction, neutrophil recruitment, and fungal clearance within the respiratory tract.
Author Summary
Our understanding of how epithelial and hematopoietic cells in the lung coordinate immunity against inhaled fungal conidia (spores) remains limited. The mold Aspergillus fumigatus is a major cause of infectious mortality in immune compromised patients. Host defense against A. fumigatus involves the activation of two host signal transducers, MyD88 and CARD9, leading to neutrophil recruitment to the infection site. In this study, we define how MyD88- and CARD9-coupled signals operate in epithelial and hematopoietic compartments to regulate neutrophil-mediated defense against A. fumigatus. Our studies support a two-stage model in which MyD88 activation in epithelial cells, via the interleukin-1 receptor, supports the rapid induction of neutrophil-recruiting chemokines. This process is essential for the first phase of neutrophil recruitment. Mortality observed in MyD88-deficient mice can be significantly reversed by administration of a chemokine termed CXCL1 to infected airways. The second phase of neutrophil recruitment is initiated by CARD9 signaling in hematopoietic cells. Loss of both phases of chemokine induction and neutrophil recruitment dramatically increases murine susceptibility to tissue-invasive disease. In sum, our study defines a temporal sequence of events, initiated by interleukin-1 receptor/MyD88 signaling in the pulmonary epithelium and propagated by CARD9 signaling in hematopoietic cells, that induces protective immunity against inhaled fungal conidia.
doi:10.1371/journal.ppat.1004589
PMCID: PMC4306481  PMID: 25621893
4.  Tolerance to etoricoxib in children with nonsteroidal anti-inflammatory drug hypersensitivity 
Asia Pacific Allergy  2015;5(1):40-46.
Background
Cyclooxygenase-2 (COX-2) inhibitors have been found to be safe alternatives in adults with cross-intolerant hypersensitivity to nonsteroidal anti-inflammatory drugs (NSAIDs). However they are usually not prescribed in children and there is little information about their tolerance in the pediatric age group.
Objective
This study aims to evaluate the tolerance to etoricoxib in children with hypersensitivity to multiple antipyretics.
Methods
A retrospective case series of children diagnosed with hypersensitivity reactions to NSAIDs and/or paracetamol who underwent a drug provocation test (DPT) with etoricoxib. Information on atopy, family history of allergic diseases, and medication usage was collected. Outcomes of the DPTs and tolerance to etoricoxib were also evaluated.
Results
A total of 24 children, mean age 13.5 years, had a diagnosis of cross-intolerant hypersensitivity to NSAIDs and/or paracetamol. All except one patient successfully tolerated an oral challenge with etoricoxib. Of those who passed the DPT, the majority continued to use etoricoxib with no problems. It was found to be moderately effective in reducing fever and pain.
Conclusion
Etoricoxib can be used as a safe alternative in older children with hypersensitivity to multiple antipyretics.
doi:10.5415/apallergy.2015.5.1.40
PMCID: PMC4313751  PMID: 25653919
Child; Cyclooxygenase-2; Hypersensitivity; Anti-inflammatory agents, Non-steroidal; Acetaminophen
5.  An improvement of the 2ˆ(–delta delta CT) method for quantitative real-time polymerase chain reaction data analysis 
Background
The 2-ΔΔCT method has been extensively used as a relative quantification strategy for quantitative real-time polymerase chain reaction (qPCR) data analysis. This method is a convenient way to calculate relative gene expression levels between different samples in that it directly uses the threshold cycles (CTs) generated by the qPCR system for calculation. However, this approach relies heavily on an invalid assumption of 100% PCR amplification efficiency across all samples. In addition, the 2-ΔΔCT method is applied to data with automatic removal of background fluorescence by the qPCR software. Since the background fluorescence is unknown, subtracting an inaccurate background can lead to distortion of the results. To address these problems, we present an improved method, the individual efficiency corrected calculation.
Results
Our method takes into account the PCR efficiency of each individual sample. In addition, it eliminates the need for background fluorescence estimation or subtraction because the background can be cancelled out using the differencing strategy. The DNA amount for a certain gene and the relative DNA amount among different samples estimated using our method were closer to the true values compared to the results of the 2-ΔΔCT method.
Conclusions
The improved method, the individual efficiency corrected calculation, produces more accurate estimates in relative gene expression than the 2-ΔΔCT method and is thus a better way to calculate relative gene expression.
PMCID: PMC4280562  PMID: 25558171
6.  Silver Nanowire Exposure Results in Internalization and Toxicity to Daphnia Magna 
ACS nano  2013;7(12):10681-10694.
Nanowires (NWs), high-aspect-ratio nanomaterials, are increasingly used in technological materials and consumer products and may have toxicological characteristics distinct from nanoparticles. We carried out a comprehensive evaluation of the physico-chemical stability of four silver nanowires (AgNWs) of two sizes and coatings and their toxicity to Daphnia magna. Inorganic aluminum-doped silica coatings were less effective than organic poly(vinyl pyrrolidone) coatings at preventing silver oxidation or Ag+ release and underwent a significant morphological transformation within one-hour following addition to low ionic strength Daphnia growth media. All AgNWs were highly toxic to D. magna but less toxic than ionic silver. Toxicity varied as a function of AgNW dimension, coating and solution chemistry. Ag+ release in the media could not account for observed AgNW toxicity. Single-particle inductively coupled plasma mass spectrometry (spICPMS) distinguished and quantified dissolved and nanoparticulate silver in microliter-scale volumes of Daphnia magna hemolymph with a limit of detection of approximately 10 ppb. The silver levels within the hemolymph of Daphnia exposed to both Ag+ and AgNW met or exceeded the initial concentration in the growth medium, indicating effective accumulation during filter feeding. Silver-rich particles were the predominant form of silver in hemolymph following exposure to both AgNWs and Ag+. Scanning electron microscopy (SEM) imaging of dried hemolymph found both AgNWs and silver precipitates that were not present in the AgNW stock or the growth medium. Both organic and inorganic coatings on the AgNW were transformed during ingestion or absorption. Pathway, gene ontology and clustering analyses of gene expression response indicated effects of AgNWs distinct from ionic silver on Daphnia magna.
doi:10.1021/nn4034103
PMCID: PMC3912856  PMID: 24099093
Silver; nanowire; ecotoxicology; Daphnia magna; acute toxicity; transcriptomics; single-particle inductively coupled plasma mass spectrometry
7.  Autotaxin-Lysophosphatidic Acid Signaling Axis Mediates Tumorigenesis and Development of Acquired Resistance to Sunitinib in Renal Cell Carcinoma 
Purpose
Sunitinib is currently considered as the standard treatment for advanced renal cell carcinoma (RCC). We aimed to better understand the mechanisms of sunitinib action in kidney cancer treatment and in the development of acquired resistance.
Experimental Design
Gene expression profiles of RCC tumor endothelium in sunitinib-treated and -untreated patients were analyzed and verified by quantitative PCR and immunohistochemistry. The functional role of the target gene identified was investigated in RCC cell lines and primary cultures in vitro and in preclinical animal models in vivo.
Results
Altered expression of autotaxin (ATX), an extracellular lysophospholipase D, was detected in sunitinib-treated tumor vasculature of human RCC and in the tumor endothelial cells of RCC xenograft models when adapting to sunitinib. ATX and its catalytic product, lysophosphatidic acid (LPA), regulated the signaling pathways and cell motility of RCC in vitro. However, no marked in vitro effect of ATX-LPA signaling on endothelial cells was observed. Functional blockage of LPA receptor 1 (LPA1) using an LPA1 antagonist, Ki16425, or gene silencing of LPA1 in RCC cells attenuated LPA-mediated intracellular signaling and invasion responses in vitro. Ki16425 treatment also dampened RCC tumorigenesis in vivo. In addition, coadministration of Ki16425 with sunitinib prolonged the sensitivity of RCC to sunitinib in xenograft models, suggesting that ATX-LPA signaling in part mediates the acquired resistance against sunitinib in RCC.
Conclusions
Our results reveal that endothelial ATX acts through LPA signaling to promote renal tumorigenesis and is functionally involved in the acquired resistance of RCC to sunitinib.
doi:10.1158/1078-0432.CCR-13-1284
PMCID: PMC4191899  PMID: 24122794
autotaxin; lysophosphatidic acid; sunitinib; angiogenesis
8.  Uniaxial Cyclic Stretch Promotes Osteogenic Differentiation and Synthesis of BMP2 in the C3H10T1/2 Cells with BMP2 Gene Variant of rs2273073 (T/G) 
PLoS ONE  2014;9(9):e106598.
Ossification of the posterior longitudinal ligament of the cervical spine (OPLL) is characterized by the replacement of ligament tissues with ectopic bone formation, and this result is strongly affected by genetic and local factors. Two single nucleotide polymorphisms (SNPs) of rs2273073 (T/G) and rs235768 (A/T) of bone morphogenetic protein 2 (BMP2) gene which are associated with OPLL have been reported in our previous report. In this study, we confirmed the connection in 18 case samples analysis of BMP2 gene in OPLL patients; additionally, it was also shown from the OPLL patients with ligament tissues that enchondral ossification and expression of BMP2 were significantly higher compared with the non-OPLL patients by histological examination, immunohistochemistry and Western blotting analysis. To investigate the underlying mechanism, we studied the effect of SNPs in cell model. The C3H10T1/2 cells with different BMP2 gene variants were constructed and then subjected to uniaxial cyclic stretch (0.5 Hz, 10% stretch). In the presence of mechanical stress, the expression of BMP2 protein in C3H10T1/2 cells transfected by BMP2 (rs2273073 (T/G)) and BMP2 (rs2273073 (T/G), rs235768 (A/T)) were significantly higher than the corresponding static groups (P<0.05). In conclusion, these results suggested that BMP2 gene variant of rs2273073 (T/G) could not only increase cell susceptibility to bone transformation similar to pre-OPLL change, but also increase the sensibility to mechanical stress which might play an important role during the progression of OPLL.
doi:10.1371/journal.pone.0106598
PMCID: PMC4156358  PMID: 25191703
9.  Methamphetamine Inhibits Toll-Like Receptor 9-Mediated Anti-HIV Activity in Macrophages 
AIDS Research and Human Retroviruses  2013;29(8):1129-1137.
Abstract
Toll-like receptor 9 (TLR9) is one of the key sensors that recognize viral infection/replication in the host cells. Studies have demonstrated that methamphetamine (METH) dysregulated host cell innate immunity and facilitated HIV infection of macrophages. In this study, we present new evidence that METH suppressed TLR9-mediated anti-HIV activity in macrophages. Activation of TLR9 by its agonist CpG-ODN 2216 inhibits HIV replication, which was demonstrated by increased expression of TLR9, interferon (IFN)-α, IFN regulatory factor-7 (IRF-7), myeloid differentiation factor 88 (MyD88), and myxovirus resistance gene A (MxA) in macrophages. However, METH treatment of macrophages greatly compromised the TLR9 signaling-mediated anti-HIV effect and inhibited the expression of TLR9 downstream signaling factors. Dopamine D1 receptor (D1R) antagonists (SCH23390) could block METH-mediated inhibition of anti-HIV activity of TLR9 signaling. Investigation of the underlying mechanisms of the METH action showed that METH treatment selectively down-regulated the expression of TLR9 on macrophages, whereas it had little effect on the expression of other TLRs. Collectively, our results provide further evidence that METH suppresses host cell innate immunity against HIV infection by down-regulating TLR9 expression and its signaling-mediated antiviral effect in macrophages.
doi:10.1089/aid.2012.0264
PMCID: PMC3715810  PMID: 23751096
10.  RGD Modified Apoferritin Nanoparticles for Efficient Drug Delivery to Tumors 
ACS nano  2013;7(6):4830-4837.
Ferritin is a major iron storage protein found in humans and most living organisms. Each ferritin is comprised of 24 subunits, which self-assemble to form a cage-like nanostructure. FRT nanocages can be genetically modified to present a peptide sequence on the surface. Recently, we demonstrated that Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys (RGD4C)-modified ferritin can efficiently home to tumors through RGD integrin αvβ3 interaction. Though promising, studies on evaluating surface modified ferritin nanocages as drug delivery vehicles have seldom been reported. Herein we showed that after being pre-complexed with Cu(II), doxorubicin can be loaded onto RGD modified apoferritin nanocages with high efficiency (up to 73.49wt%). When studied on U87MG subcutaneous tumor models, these doxorubicin-loaded ferritin nanocages showed a longer circulation half-life, higher tumor uptake, better tumor growth inhibition, and less cardiotoxicity than free doxorubicin. Such a technology might be extended to load a broad range of therapeutics and holds great potential in clinical translation.
doi:10.1021/nn305791q
PMCID: PMC3705644  PMID: 23718215
ferritin; drug delivery; integrin αvβ3; nanocarrier; doxorubicin
11.  Urine interleukin-18 in prediction of acute kidney injury: a systemic review and meta-analysis 
Journal of Nephrology  2014;28:7-16.
Background
Interleukin-18 (IL-18) mediates ischemic acute tubular necrosis; it has been proved as a rapid, reliable, and affordable test marker for the early detection of acute kidney injury (AKI), but its predictive accuracy varies greatly.
Methods
MEDLINE and EMBASE, Cochrane Library, Ovid, and Springerlink (from inception to November 15, 2013) were searched for relevant studies (in English) investigating diagnostic accuracy of urine IL-18 to predict AKI in various clinical settings. The text index was increasing or increased urine IL-18 level and the main outcome was the development of AKI, which was primarily based on serum creatinine level [using risk, injury, failure, loss and end-stage renal disease (RIFLE), acute kidney injury network, or modified pediatric RIFLE criteria in pediatric patients]. Pooled estimates of diagnostic odds ratio (OR), sensitivity and specificity were calculated. Summary receiver operating characteristic curves were used to calculate the measures of accuracy and Q point value (Q*). Remarkable heterogeneity was explored further by subgroup analysis based on the different clinical settings.
Results
We analyzed data from 11 studies of 3 countries covering 2,796 patients. These studies were marked by limitations of threshold and non-threshold effect heterogeneity. Across all settings, the diagnostic OR for urine IL-18 level to predict AKI was 5.11 [95 % confidence interval (CI) 3.22–8.12], with sensitivity and specificity respectively at 0.51 and 0.79. The area under the ROC curve of urine IL-18 level to predict AKI was 0.77 (95 % CI 0.71–0.83). Subgroup analysis showed that urine IL-18 level in pediatric patients (<18 years) and early AKI predictive time (<12 h) were more effective in predicting AKI, with diagnostic ORs of 7.51 (2.99–18.88), 8.18 (2.19–30.51), respectively.
Conclusion
Urine IL-18 holds promise as a biomarker in the prediction of AKI but has only moderate diagnostic value.
doi:10.1007/s40620-014-0113-9
PMCID: PMC4322238  PMID: 24899123
Interleukin-18; Acute kidney injury; Predictive value; Systemic review
12.  Prediction of Protein S-Nitrosylation Sites Based on Adapted Normal Distribution Bi-Profile Bayes and Chou’s Pseudo Amino Acid Composition 
Protein S-nitrosylation is a reversible post-translational modification by covalent modification on the thiol group of cysteine residues by nitric oxide. Growing evidence shows that protein S-nitrosylation plays an important role in normal cellular function as well as in various pathophysiologic conditions. Because of the inherent chemical instability of the S-NO bond and the low abundance of endogenous S-nitrosylated proteins, the unambiguous identification of S-nitrosylation sites by commonly used proteomic approaches remains challenging. Therefore, computational prediction of S-nitrosylation sites has been considered as a powerful auxiliary tool. In this work, we mainly adopted an adapted normal distribution bi-profile Bayes (ANBPB) feature extraction model to characterize the distinction of position-specific amino acids in 784 S-nitrosylated and 1568 non-S-nitrosylated peptide sequences. We developed a support vector machine prediction model, iSNO-ANBPB, by incorporating ANBPB with the Chou’s pseudo amino acid composition. In jackknife cross-validation experiments, iSNO-ANBPB yielded an accuracy of 65.39% and a Matthew’s correlation coefficient (MCC) of 0.3014. When tested on an independent dataset, iSNO-ANBPB achieved an accuracy of 63.41% and a MCC of 0.2984, which are much higher than the values achieved by the existing predictors SNOSite, iSNO-PseAAC, the Li et al. algorithm, and iSNO-AAPair. On another training dataset, iSNO-ANBPB also outperformed GPS-SNO and iSNO-PseAAC in the 10-fold crossvalidation test.
doi:10.3390/ijms150610410
PMCID: PMC4100159  PMID: 24918295
S-nitrosylation; post-translational modification; bi-profile Bayes; amino acid physicochemical properties
13.  Quorum Sensing Activity in Pandoraea pnomenusa RB38 
Sensors (Basel, Switzerland)  2014;14(6):10177-10186.
Strain RB38 was recovered from a former dumping area in Malaysia. MALDI-TOF mass spectrometry and genomic analysis identified strain RB-38 as Pandoraea pnomenusa. Various biosensors confirmed its quorum sensing properties. High resolution triple quadrupole liquid chromatography–mass spectrometry analysis was subsequently used to characterize the N-acyl homoserine lactone production profile of P. pnomenusa strain RB38, which validated that this isolate produced N-octanoyl homoserine lactone as a quorum sensing molecule. This is the first report of the production of N-octanoyl homoserine lactone by P. pnomenusa strain RB38.
doi:10.3390/s140610177
PMCID: PMC4118335  PMID: 24919016
matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF); mass spectrometry (MS); cell-to cell communication; triple quodruopole liquid chromatography mass spectrometry (LC-MS/MS); N-octanoyl homoserine lactone (C8-HSL)
14.  FOXC1 regulates the functions of human basal-like breast cancer cells by activating NF-κB signaling 
Oncogene  2012;31(45):4798-4802.
Human basal-like breast cancer (BLBC) is an enigmatic and aggressive malignancy with a poor prognosis. There is an urgent need to identify therapeutic targets for BLBC because current treatment modalities are limited and not effective. The forkhead box transcription factor FOXC1 has recently been identified as a critical functional biomarker for BLBC. However, how it orchestrates BLBC cells was not clear. Here we show that FOXC1 activates the transcription factor NF-κB in BLBC cells by increasing p65/RelA protein stability. High NF-κB activity has been associated with estrogen receptor-negative breast cancer, particularly BLBC. The effect of FOXC1 on p65/RelA protein stability is mediated by increased expression of Pin1, a peptidyl-prolyl isomerase. FOXC1 requires NF-κB for its regulation of cell proliferation, migration, and invasion. Notably, FOXC1 overexpression renders breast cancer cells more susceptible to pharmacologic inhibition of NF-κB. These results suggest that BLBC cells may rely on FOXC1-driven NF-κB signaling. Interventions of this pathway may provide modalities for the treatment of BLBC.
doi:10.1038/onc.2011.635
PMCID: PMC3961006  PMID: 22249250
basal-like breast cancer; FOXC1; NF-κB; p65/RelA; Pin1; protein stability
15.  USP18 inhibits NF-κB and NFAT activation during Th17 differentiation by deubiquitinating the TAK1–TAB1 complex 
The Journal of Experimental Medicine  2013;210(8):1575-1590.
Ubiquitin-specific protease 18 inhibits ubiquitination of TAK1–TAB complexes to restrict IL-2 production and promote Th17 differentiation and autoimmune responses.
Reversible ubiquitin modification of cell signaling molecules has emerged as a critical mechanism by which cells respond to extracellular stimuli. Although ubiquitination of TGF-β–activated kinase 1 (TAK1) is critical for NF-κB activation in T cells, the regulation of its deubiquitination is unclear. We show that USP18, which was previously reported to be important in regulating type I interferon signaling in innate immunity, regulates T cell activation and T helper 17 (Th17) cell differentiation by deubiquitinating the TAK1–TAB1 complex. USP18-deficient T cells are defective in Th17 differentiation and Usp18−/− mice are resistant to experimental autoimmune encephalomyelitis (EAE). In response to T cell receptor engagement, USP18-deficient T cells exhibit hyperactivation of NF-κB and NFAT and produce increased levels of IL-2 compared with the wild-type controls. Importantly, USP18 is associated with and deubiquitinates the TAK1–TAB1 complex, thereby restricting expression of IL-2. Our findings thus demonstrate a previously uncharacterized negative regulation of TAK1 activity during Th17 differentiation, suggesting that USP18 may be targeted to treat autoimmune diseases.
doi:10.1084/jem.20122327
PMCID: PMC3727316  PMID: 23825189
16.  Comparison of clinical features between non-smokers with COPD and smokers with COPD: a retrospective observational study 
Background
Smoking is a major risk factor for chronic obstructive pulmonary disease (COPD); however, the similarities and differences in clinical presentation between smokers and nonsmokers are not fully described in patients with COPD. This study was designed to address this issue in a general teaching hospital in the People’s Republic of China.
Methods
The medical records of patients hospitalized with a lung mass for further evaluation at Zhongshan Hospital, Fudan University, from January 2006 to December 2010 were reviewed and the data of interest were collected. The definition of COPD was according to Global Initiative for Chronic Obstructive Lung Disease (GOLD) spirometric criteria. Participants who had a previous exacerbation within 4 weeks of admission, airflow limitation due to abnormalities in the large airways, or with other pulmonary diseases were excluded. Included subjects were divided into nonsmokers with COPD and smokers with COPD by a cutoff of a 5 pack-year smoking history.
Results
A total of 605 subjects were included in the final analysis. The average age was 64.8±8.5 years and 62.0% (375/605) were smokers. Eighty percent of the patients had mild to moderate disease (GOLD grade 1–2). Age and years with COPD were comparable between the two groups. Compared with smokers with COPD, nonsmokers with COPD were more likely to be female, reported less chronic cough and sputum, have less emphysema on radiologic examination, and higher measures of forced expiratory volume in the first second percent predicted (FEV1), forced expiratory volume in one second/forced vital capacity (FEV1/FVC%) percent predicted, maximal voluntary ventilation percent predicted, diffusing capacity of lung (DLCO) percent predicted, and DLCO/alveolar volume percent predicted, with lower levels of residual volume percent predicted and residual volume/total lung capacity percent predicted. There were no significant differences between the two groups with regard to distribution of disease severity, vital capacity percent predicted, total lung capacity percent predicted, PaO2, PaCO2, modified Medical Research Council dyspnea score, wheezing, airway reversibility, and comorbidities. Smoking amount (pack-years) was correlated negatively with FEV1 percent predicted, FEV1/FVC% percent predicted, inspiratory capacity percent predicted, inspiratory capacity/total lung capacity percent predicted, and DLCO percent predicted, and correlated positively with GOLD grade and symptoms.
Conclusion
Non-smokers with COPD had less impairment in airflow limitation and gas exchange, and a lower prevalence of emphysema, chronic cough, and sputum compared with their smoking counterparts. Tobacco cessation is warranted in smokers with COPD.
doi:10.2147/COPD.S52416
PMCID: PMC3890400  PMID: 24426780
chronic obstructive pulmonary disease; smokers; non-smokers; lung function; symptoms; emphysema
18.  Cord factor and peptidoglycan recapitulate the Th17-promoting adjuvant activity of mycobacteria through Mincle/CARD9 signaling and the inflammasome 
Although adjuvants are critical vaccine components, their modes of action are poorly understood. Here, we investigated the mechanisms by which the heat-killed mycobacteria in complete Freund’s adjuvant (CFA) promote T helper 17 (Th17) CD4+ T cell responses. We found that IL-17 secretion by CD4+ T cells following CFA immunization requires MyD88 and IL-1β/IL-1 receptor (IL-1R) signaling. Through measurement of antigen-specific responses after adoptive transfer of OTII cells, we confirmed that MyD88-dependent signaling controls Th17 differentiation rather than simply production of IL-17. Additional experiments showed that CFA-induced Th17 differentiation involves IL-1β processing by the inflammasome, as mice lacking caspase 1, ASC, or NLRP3 exhibit partially defective responses after immunization. Biochemical fractionation studies further revealed that peptidoglycan is the major component of heat-killed mycobacteria responsible for inflammasome activation. By assaying Il1b transcripts in the injection site skin of CFA-immunized mice, we found that signaling through the adaptor molecule CARD9 plays a major role in triggering pro-IL-1β expression. Moreover, we demonstrated that recognition of the mycobacterial glycolipid trehalose dimycolate (cord factor) by the C type lectin receptor mincle partially explains this CARD9 requirement. Importantly, purified peptidoglycan and cord factor administered in mineral oil synergized to recapitulate the Th17-promoting activity of CFA, and, as expected, this response was diminished in caspase 1-and CARD9-deficient mice. Taken together, these findings suggest a general strategy for the rational design of Th17-skewing adjuvants by combining agonists of the CARD9 pathway with inflammasome activators.
doi:10.4049/jimmunol.1203343
PMCID: PMC3719989  PMID: 23630357
19.  Ferritin Nanocages To Encapsulate and Deliver Photosensitizers for Efficient Photodynamic Therapy against Cancer 
ACS nano  2013;7(8):10.1021/nn402199g.
Photodynamic therapy is an emerging treatment modality that is under intensive preclinical and clinical investigations for many types of disease including cancer. Despite the promise, there is a lack of a reliable drug delivery vehicle that can transport photosensitizers (PSs) to tumors in a site-specific manner. Previous efforts have been focused on polymer- or liposome-based nanocarriers, which are usually associated with a suboptimal PS loading rate and a large particle size. We report herein that a RGD4C-modified ferritin (RFRT), a protein-based nanoparticle, can serve as a safe and efficient PS vehicle. Zinc hexadecafluorophthalocyanine (ZnF16Pc), a potent PS with a high 1O2 quantum yield but poor water solubility, can be encapsulated into RFRTs with a loading rate as high as ~60 wt % (i.e., 1.5 mg of ZnF16Pc can be loaded on 1 mg of RFRTs), which far exceeds those reported previously. Despite the high loading, the ZnF16Pc-loaded RFRTs (P-RFRTs) show an overall particle size of 18.6 ± 2.6 nm, which is significantly smaller than other PS–nanocarrier conjugates. When tested on U87MG subcutaneous tumor models, P-RFRTs showed a high tumor accumulation rate (tumor-to-normal tissue ratio of 26.82 ±4.07 at 24 h), a good tumor inhibition rate (83.64% on day 12), as well as minimal toxicity to the skin and other major organs. This technology can be extended to deliver other metal-containing PSs and holds great clinical translation potential.
doi:10.1021/nn402199g
PMCID: PMC3819164  PMID: 23829542
photodynamic therapy; photosensitizer; targeted delivery; ferritin; nanoparticle
20.  MicroRNA-20b and ERK1/2 pathway independently regulate the expression of tissue factor in hematopoietic and trophoblastic differentiation of human embryonic stem cells 
Introduction
Tissue factor (TF) is expressed in various types of cells. TF expression is essential for many biological processes, such as blood coagulation and embryonic development, while its high expression in stem cells often leads to failure of transplantation. In this study, we used the human embryonic stem cell (hESC) culture system to understand the molecular mechanisms by which TF expression is regulated in hESC-derived hematopoietic and trophoblastic cells.
Methods
hESCs were induced in vitro to differentiate into hematopoietic and trophoblastic cells. TF expression in various types of cells during these differentiation processes was examined by quantitative real-time polymerase chain reaction analysis and western blot analysis. The regulatory mechanisms of TF expression were investigated by miRNA expression analysis, luciferase report assay, TF mRNA and protein analysis, and pathway phosphorylation analysis.
Results
We first found that TF was expressed only in trophoblasts and granulocyte–monocyte (G-M) cells differentiated from hESCs; and then demonstrated that miR-20b downregulated and Erk1/2 signaling pathway upregulated the TF expression in trophoblasts and G-M cells. Finally, we found that miR-20b downregulated the TF expression independently of the Erk1/2 signaling pathway.
Conclusions
The miR-20b and Erk1/2 pathway independently regulate expression of TF in trophoblasts and G-M cells differentiated from hESCs. These findings will open an avenue to further illustrate the functions of TF in various biological processes.
doi:10.1186/scrt332
PMCID: PMC3854777  PMID: 24405935
21.  EARLY STABILIZING ALVEOLAR VENTILATION PREVENTS ARDS- A NOVEL TIMING-BASED VENTILATORY INTERVENTION TO AVERT LUNG INJURY 
Background
Established ARDS is often refractory to treatment. Clinical trials have demonstrated modest treatment effects, and mortality remains high. Ventilator strategies must be developed to prevent ARDS.
Hypothesis
Early ventilatory intervention will block progression to ARDS if the ventilator mode: 1) maintains alveolar stability and 2) reduces pulmonary edema formation.
Methods
Yorkshire Pigs (38–45kg) were anaesthetized and subjected to "2-hit" Ischemia-Reperfusion and Peritoneal Sepsis. Following injury, animals were randomized into two groups: Early Preventative Ventilation (Airway Pressure Release Ventilation- APRV) vs. Non-Preventative Ventilation (NPV) and followed for 48hr. All animals received anesthesia, antibiotics, and fluid/vasopressor therapy per Surviving Sepsis Campaign. Ventilation parameters: 1) NPV Group - Tidal volume (Vt): 10cc/kg + PEEP- 5 cm/H2O volume-cycled mode, 2) APRV Group - Vt: 10–15 cc/kg; Phigh, Plow, Thigh, Tlow were titrated for optimal alveolar stability. Physiologic data and plasma were collected throughout the 48hr study period, followed by BAL and necropsy.
Results
APRV prevented development of ARDS (p<0.001 vs NPV) by PaO2/FiO2 ratio. Quantitative histological scoring showed APRV prevented lung tissue injury (p<0.001 vs. NPV). BALF showed APRV lowered total protein and IL-6, while preserving surfactant proteins A & B (p<0.05 vs. NPV). APRV significantly lowered lung water (p<0.001 vs. NPV). Plasma IL-6 concentrations were similar between groups.
Conclusions
Early preventative mechanical ventilation with APRV blocked ARDS development, preserved surfactant proteins, and reduced pulmonary inflammation and edema, despite systemic inflammation similar to NPV. These data suggest early preventative ventilation strategies stabilizing alveoli and reducing pulmonary edema can attenuate ARDS after ischemia-reperfusion-sepsis.
doi:10.1097/TA.0b013e31825c7a82
PMCID: PMC3521044  PMID: 22846945
Sepsis; Shock; ARDS; ALI; Ventilator Induced Lung Injury; Airway Pressure Release Ventilation
22.  Tracing Conidial Fate and Measuring Host Cell Antifungal Activity Using a Reporter of Microbial Viability in the Lung 
Cell reports  2012;2(6):1762-1773.
Fluorescence can be harnessed to monitor microbial fate and to investigate functional outcomes of individual microbial cell-host cell encounters at portals of entry in native tissue environments. We illustrate this concept by introducing fluorescent Aspergillus reporter (FLARE) conidia that simultaneously report phagocytic uptake and fungal viability during cellular interactions with the murine respiratory innate immune system. Our studies using FLARE conidia reveal stepwise and cell-type-specific requirements for CARD9 and Syk, transducers of C-type lectin receptor and integrin signals, in neutrophil recruitment, conidial uptake, and conidial killing in the lung. By achieving single-event resolution in defined leukocyte populations, the FLARE method enables host cell profiling on the basis of pathogen uptake and killing and may be extended to other pathogens in diverse model host organisms to query molecular, cellular, and pharmacologic mechanisms that shape host-microbe interactions.
doi:10.1016/j.celrep.2012.10.026
PMCID: PMC3712646  PMID: 23200858
23.  Activation of a silent sesquiterpenoid biosynthetic pathway in Streptomyces avermitilis controlling epi-isozizaene and albaflavenone biosynthesis, and isolation of a new oxidized epi-isozizaene metabolite 
Microbial biotechnology  2010;4(2):184-191.
The genome-sequenced, Gram-positive bacterium Streptomyces avermitilis harbors an ortholog (SAV_3032) of the previously identified epi-isozizaene synthase (SCO5222) in S. coelicolor A3(2). The sav3032 is translationally coupled with the downstream sav3031 gene encoding the cytochrome P450 CYP170A2 analogous to SCO5223 (CYP170A1) of S. coelicolor A3(2), which exhibits a similar translation coupling. S. avermitilis did not produce epi-isozizaene or any of its oxidized derivatives, albaflavenols and albaflavenone, under in any culture conditions examined. Nonetheless, recombinant SAV_3032 protein expressed in E. coli catalyzed the Mg2+-dependent cyclization of farnesyl diphosphate to epi-isozizaene. To effect the production of epi-isozizaene in S. avermitilis, the sav3032 gene was cloned and placed under control of a copy of the native S. avermilis promoter rpsJp (sav4925). The derived expression construct was introduced by transformation into a large-deletion mutant of S. avermitilis SUKA16. The resulting transformants accumulated epi-isozizaene after 24-hr cultivation. The previously characterized oxidized epi-isozizaene metabolites, (5R)- and (5S)-albaflavenols and albaflavenone, as well as a previously undescribed doubly oxidized epi-isozizaene derivative were isolated from cultures of S. avermitilis SUKA16 transformants in which sav3032 was co-expressed with the P450-encoding sav3031. This new metabolite was identified as 4β,5β-epoxy-7-epi-zizaan-3β-ol which is most likely formed by oxidation of (5S)-albaflavenol.
doi:10.1111/j.1751-7915.2010.00209.x
PMCID: PMC3711710  PMID: 21342464
gene expression/regulation; metabolic design; metabolism; natural products; secondary metabolites
24.  Committed changes in tropical tree cover under the projected 21st century climate change 
Scientific Reports  2013;3:1951.
Warming and drought pose a serious threat to tropical forest. Yet the extent of this threat is uncertain, given the lack of methods to evaluate the forest tree cover changes under future climate predicted by complex dynamic vegetation models. Here we develop an empirical approach based on the observed climate space of tropical trees to estimate the maximum potential tropical tree cover (MPTC) in equilibrium with a given climate. We show that compared to present-day (2000–2009) conditions, MPTC will be reduced by 1 to 15% in the tropical band under equilibrium future (2090–2099) climate conditions predicted by 19 IPCC climate models. Tropical forests are found to regress or disappear mainly in the current transition zones between forest and savanna ecosystems. This climate pressure on tropical forests, added to human-caused land use pressure, poses a grand challenge to the sustainability of the world's largest biomass carbon pool.
doi:10.1038/srep01951
PMCID: PMC3674425  PMID: 23739583
25.  Imaging Tumor-Induced Sentinel Lymph Node Lymphangiogenesis with LyP-1 Peptide 
Amino Acids  2011;42(6):2343-2351.
Lymphangiogenesis in tumor-draining lymph nodes (LNs) starts before the onset of metastasis and is associated with metastasis to distant LNs and organs. In this study, we aimed to visualize tumor induced lymphangiogensis with a tumor lymphatics-specific peptide Lyp-1. The LyP-1 peptide was labeled with a near-infrared fluorophore (Cy5.5) for optical imaging. At days 3, 7, 14 and 21 after subcutaneous 4T1 tumor inoculation, Cy5.5-LyP-1 was administered through the middle phalanges of the upper extremities of the tumor-bearing mice. At 45 min and 24 h postinjection, brachial LN fluorescence imaging was performed. Ex vivo fluorescence images were acquired for quantitative analysis of the fluorescence intensity. Tumor induced lymphangiogenesis was confirmed by LYVE-1 immunostaining and increased size of tumor side brachial LNs. Cy5.5-LyP-1 staining in LNs co-localized with LYVE-1, suggesting lymphatics-specific binding of LyP-1 peptide. The brachial LNs were clearly visualized by optical imaging at both time points. The tumor side LNs showed significantly higher fluorescence intensities than the contralateral brachial LNs at days 7, 14, and 21, but not day 3 after tumor inoculation. At day 21 after tumor inoculation, the average signal of tumor-draining LNs was 78.0 ± 2.44, 24.3 ± 5.43, 25.6 ± 0.25 (×103 photon/cm2/s) using Cy5.5-LyP-1, Cy5.5-LyP-1 with blocking, and Cy5.5 only, respectively. Tumor-draining brachial LNs showed extensive growth of lymphatic sinuses throughout the cortex and medulla. Use of LyP-1 based imaging probes with optical imaging offers a useful tool for the study of tumor-induced lymphangiogenesis. LyP-1 may serve as a marker of lymphangiogenesis useful in detecting “high risk” lymph nodes before tumor metastasis and after micro-metastasis, as well as for screening potential anti-lymphatic therapies.
doi:10.1007/s00726-011-0976-1
PMCID: PMC3257379  PMID: 21769497
Lymphangiogenesis; lymph node; LyP-1 peptide; optical imaging

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