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Rift Valley fever virus (RVFV) causes Rift Valley fever (RVF), resulting in morbidity and mortality in humans and ruminants. Evidence of transboundary outbreaks means that RVFV remains a threat to human health and livestock industries in countries that are free from the disease. To enhance surveillance capability, methods for detection of RVFV are required. The generation of reagents suitable for the detection of RVFV antigen in formalin-fixed, paraffin-embedded tissues from infected animals have been developed and are described herein. Recombinant nucleoprotein (rNP) was expressed in Escherichia coli and purified using immobilized metal ion affinity chromatography. Purified rNP was used as an immunogen to produce anti-NP polyclonal antisera in rabbits for use in detection of RVFV NP in experimentally infected animals by immunohistochemistry. Antisera raised in rabbits against rNP were able to recognize viral NP antigen in fixed infected Vero cell pellets and sheep liver. Therefore, the methods and reagents described herein are useful in assays for detection of RVFV infections in animals, for research and surveillance purposes.

Difficulties associated with efficient delivery and targeting of miRNAs to cells is hampering the real world application of miRNA technology. This study utilized an influenza A-based delivery system to express miR-155 in order to knockdown SOCS1 mRNA. Using qPCR and dual luciferase technology we show that miR-155 delivery resulted in a significant increase in cellular miR-155 which facilitated a downregulation of SOCS1 gene expression and a functional increase in IL-6 and IFN-β cytokines.

We used vertical growth phase (VGP) human VMM5 melanoma cells to ask whether the tumor microenvironment could induce matrix metalloproteinase-1 (MMP-1) in vivo, and whether this induction correlated with metastasis. We isolated two clones from parental VMM5 cells: a low MMP-1 producing clone (C4) and high producing clone (C9). When these clones were injected orthotopically (intradermally) into nude mice, both were equally tumorigenic and produced equivalent and abundant amounts of MMP-1. However, the tumors from the C4 clones displayed different growth kinetics and distinct profiles of gene expression from the C9 population. The C4 tumors, which had low MMP-1 levels in vitro, appeared to rely on growth factors and cytokines in the microenvironment to increase MMP-1 expression in vivo, while MMP-1 levels remained constant in the C9 tumors. C9 cells, but not C4 cells, grew as spheres in culture and expressed higher levels of JARID 1B, a marker associated with melanoma initiating cells. We conclude that VMM5 melanoma cells exhibit striking intra-tumor heterogeneity, and that the tumorigenicity of these clones is driven by different molecular pathways. Our data suggest that there are multiple mechanisms for melanoma progression within a tumor, which may require different therapeutic strategies.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated Cl(-) channel expressed in the apical membrane of fluid-transporting epithelia. The apical membrane density of CFTR channels is determined, in part, by endocytosis and the postendocytic sorting of CFTR for lysosomal degradation or recycling to the plasma membrane. Although previous studies suggested that ubiquitination plays a role in the postendocytic sorting of CFTR, the specific ubiquitin ligases are unknown. c-Cbl is a multifunctional molecule with ubiquitin ligase activity and a protein adaptor function. c-Cbl co-immunoprecipitated with CFTR in primary differentiated human bronchial epithelial cells and in cultured human airway cells. Small interfering RNA-mediated silencing of c-Cbl increased CFTR expression in the plasma membrane by inhibiting CFTR endocytosis and increased CFTR-mediated Cl(-) currents. Silencing c-Cbl did not change the expression of the ubiquitinated fraction of plasma membrane CFTR. Moreover, the c-Cbl mutant with impaired ubiquitin ligase activity (FLAG-70Z-Cbl) did not affect the plasma membrane expression or the endocytosis of CFTR. In contrast, the c-Cbl mutant with the truncated C-terminal region (FLAG-Cbl-480), responsible for protein adaptor function, had a dominant interfering effect on the endocytosis and plasma membrane expression of CFTR. Moreover, CFTR and c-Cbl co-localized and co-immunoprecipitated in early endosomes, and silencing c-Cbl reduced the amount of ubiquitinated CFTR in early endosomes. In summary, our data demonstrate that in human airway epithelial cells, c-Cbl regulates CFTR by two mechanisms: first by acting as an adaptor protein and facilitating CFTR endocytosis by a ubiquitin-independent mechanism, and second by ubiquitinating CFTR in early endosomes and thereby facilitating the lysosomal degradation of CFTR.

We previously reported that Pseudomonas aeruginosa PA14 secretes a protein that can reduce the apical membrane expression of the c ystic f ibrosis t ransmembrane conductance r egulator (CFTR) protein. Here we report that we have used a proteomic approach to identify this secreted protein as PA2394, and we have named the gene cif , for C FTR i nhibitory f actor. We demonstrate that Cif is a secreted protein and is found associated with outer membrane-derived vesicles. Expression of Cif in Escherichia coli and purification of the C-terminal six-His-tagged Cif protein showed that Cif is necessary and sufficient to mediate the reduction in apical membrane expression of CFTR and a concomitant reduction in CFTR-mediated Cl − ion secretion. Cif demonstrates epoxide hydrolase activity in vitro and requires a highly conserved histidine residue identified in α/β hydrolase family enzymes to catalyze this reaction. Mutating this histidine residue also abolishes the ability of Cif to reduce apical membrane CFTR expression. Finally, we demonstrate that the cif gene is expressed in the cystic fibrosis (CF) lung and that nonmucoid isolates of P. aeruginosa show greater expression of the gene than do mucoid isolates. We propose a model in which the Cif-mediated decrease in apical membrane expression of CFTR by environmental isolates of P. aeruginosa facilitates the colonization of the CF lung by this microbe.

Background: The secretory leukocyte protease inhibitor (SLPI) represents a multifunctional protein with mucosa-protective features. Helicobacter pylori and the usage of low-dose aspirin are two independent risk factors for the development of gastrointestinal diseases. Therefore, the effect of low-dose aspirin on gastrointestinal SLPI expression was analyzed in the context of H. pylori infection. Material and Methods: The study included 20 volunteers (H. pylori positive and negative: n = 10) who received 2 × 50 mg aspirin/day for 7 days. H. pylori-positive subjects underwent eradication therapy and repeated the protocol. Gastroduodenoscopy was performed at day 0, 1, 3, and 7, and biopsies were obtained each from antrum, corpus, and duodenal bulb. SLPI expression was determined by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Results: A reduction of antral SLPI levels, ranging between 582 (day 0) and 941 pg/10 µg protein (day 7), was determined in H. pylori-positive compared to H. pylori-negative and -eradicated subjects (1600–2050 pg/10 µg protein, ANOVA: p = .001–.045). No differences concerning aspirin were observed within the groups. SLPI levels in corpus and duodenal mucosa were neither affected by H. pylori nor low-dose aspirin. There was an inverse correlation between SLPI and H. pylori-induced inflammation (activity: r = −0.575, −0.69 to −0.43, p

Highly pathogenic avian influenza virus infection is associated with severe mortality in both humans and poultry. The mechanisms of disease pathogenesis and immunity are poorly understood although recent evidence suggests that cytokine/chemokine dysregulation contributes to disease severity following H5N1 infection. Influenza A virus infection causes a rapid influx of inflammatory cells, resulting in increased reactive oxygen species production, cytokine expression, and acute lung injury. Proinflammatory stimuli are known to induce intracellular reactive oxygen species by activating NADPH oxidase activity. We therefore hypothesized that inhibition of this activity would restore host cytokine homeostasis following avian influenza virus infection. A panel of airway epithelial and immune cells from mammalian and avian species were infected with A/Puerto Rico/8/1934 H1N1 virus, low-pathogenicity avian influenza H5N3 virus (A/duck/Victoria/0305-2/2012), highly pathogenic avian influenza H5N1 virus (A/chicken/Vietnam/0008/2004), or low-pathogenicity avian influenza H7N9 virus (A/Anhui/1/2013). Quantitative real-time reverse transcriptase PCR showed that H5N1 and H7N9 viruses significantly stimulated cytokine (interleukin-6, beta interferon, CXCL10, and CCL5) production. Among the influenza-induced cytokines, CCL5 was identified as a potential marker for overactive immunity. Apocynin, a Nox2 inhibitor, inhibited influenza-induced cytokines and reactive oxygen species production, although viral replication was not significantly altered in vitro . Interestingly, apocynin treatment significantly increased influenza virus-induced mRNA and protein expression of SOCS1 and SOCS3, enhancing negative regulation of cytokine signaling. These findings suggest that apocynin or its derivatives (targeting host responses) could be used in combination with antiviral strategies (targeting viruses) as therapeutic agents to ameliorate disease severity in susceptible species. IMPORTANCE Highly pathogenic avian influenza virus infection causes severe morbidity and mortality in both humans and poultry. Wide-spread antiviral resistance necessitates the need for the development of additional novel therapeutic measures to modulate overactive host immune responses after infection. Disease severity following avian influenza virus infection can be attributed in part to hyperinduction of inflammatory mediators such as cytokines, chemokines, and reactive oxygen species. Our study shows that highly pathogenic avian influenza H5N1 virus and low-pathogenicity avian influenza H7N9 virus (both associated with human fatalities) promote inactivation of FoxO3 and downregulation of the TAM receptor tyrosine kinase, Tyro3, leading to augmentation of the inflammatory cytokine response. Inhibition of influenza-induced reactive oxygen species with apocynin activated FoxO3 and stimulated SOCS1 and SOCS3 proteins, restoring cytokine homeostasis. We conclude that modulation of host immune responses with antioxidant and/or anti-inflammatory agents in combination with antiviral therapy may have important therapeutic benefits.

The angiotensin AT4 receptor was originally defined as the specific, high-affinity binding site for the hexapeptide angiotensin IV (Ang IV). Subsequently, the peptide LVV-hemorphin 7 was also demonstrated to be a bioactive ligand of the AT4 receptor. Central administration of Ang IV, its analogues or LVV-hemorphin 7 markedly enhance learning and memory in normal rodents and reverse memory deficits observed in animal models of amnesia. The AT4 receptor has a broad distribution and is found in a range of tissues, including the adrenal gland, kidney, lung and heart. In the kidney Ang IV increases renal cortical blood flow and decreases Na+ transport in isolated renal proximal tubules. The AT4 receptor has recently been identified as the transmembrane enzyme, insulin-regulated membrane aminopeptidase (IRAP). IRAP is a type II integral membrane spanning protein belonging to the M1 family of aminopeptidases and is predominantly found in GLUT4 vesicles in insulin-responsive cells. Three hypotheses for the memory-potentiating effects of the AT4 receptor/IRAP ligands, Ang IV and LVV-hemorphin 7, are proposed: (i) acting as potent inhibitors of IRAP, they may prolong the action of endogenous promnestic peptides; (ii) they may modulate glucose uptake by modulating trafficking of GLUT4; (iii) IRAP may act as a receptor, transducing the signal initiated by ligand binding to its C-terminal domain to the intracellular domain that interacts with several cytoplasmic proteins.

Toll-like receptor (TLRs) is a type of pattern-recognition receptor that recognizes pathogen-associated molecular patterns, the important mediator of innate immunity. The aim of this study was to examine the anti-inflammatory effect of TLR2 monoclonal antibody (TLR2 mAb) on concanavalin A (ConA) induced-hepatitis. Our in vitro findings indicated that the TLR2 monoclonal antibody developed by us was able to neutralize the activation of peripherial blood mononucleated cells (PBMC) induced by ConA. Furthermore, pretreatment of mice with TLR2 antibody exerted liver protection against ConA. Compared with the isotype IgG group, the TLR2-antibody-treated group demonstrated less mortality with concomitant decreased serum level of Ast and Alt. The detailed mechanisms underlying the protection effect induced by TLR2 antibody was due to inhibition of infiltration of lymphocytes in liver induced by ConA as well as the expression of pro-inflammatory factors, such as TNF-α, IFN-γ, IL-6. Taken together, our findings demonstrated that TLR2 mAb pretreatment protected liver against ConA-induced hepatitis in mice, suggesting that TLR2 mAb is a potential candidate for therapy of acute hepatitis.

Reverse genetics systems allow artificial generation of non-segmented and segmented negative-sense RNA viruses, like influenza viruses, entirely from cloned cDNA. Since the introduction of reverse genetics systems over a decade ago, the ability to generate ‘designer’ influenza viruses in the laboratory has advanced both basic and applied research, providing a powerful tool to investigate and characterise host–pathogen interactions and advance the development of novel therapeutic strategies. The list of applications for reverse genetics has expanded vastly in recent years. In this review, we discuss the development and implications of this technique, including the recent controversy surrounding the generation of a transmissible H5N1 influenza virus. We will focus on research involving the identification of viral protein function, development of live-attenuated influenza virus vaccines, host–pathogen interactions, immunity and the generation of recombinant influenza virus vaccine vectors for the prevention and treatment of infectious diseases and cancer.

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen chronically infecting the lungs of patients with chronic obstructive pulmonary disease (COPD), pneumonia, cystic fibrosis (CF), and bronchiectasis. Cif (PA2934), a bacterial toxin secreted in outer membrane vesicles (OMV) by P. aeruginosa, reduces CFTR-mediated chloride secretion by human airway epithelial cells, a key driving force for mucociliary clearance. The aim of this study was to investigate the mechanism whereby Cif reduces CFTR-mediated chloride secretion. Cif redirected endocytosed CFTR from recycling endosomes to lysosomes by stabilizing an inhibitory effect of G3BP1 on the deubiquitinating enzyme (DUB), USP10, thereby reducing USP10-mediated deubiquitination of CFTR and increasing the degradation of CFTR in lysosomes. This is the first example of a bacterial toxin that regulates the activity of a host DUB. These data suggest that the ability of P. aeruginosa to chronically infect the lungs of patients with COPD, pneumonia, CF, and bronchiectasis is due in part to the secretion of OMV containing Cif, which inhibits CFTR-mediated chloride secretion and thereby reduces the mucociliary clearance of pathogens., National Institutes of Health (U.S.) (NIH grants R01-HL074175), National Institutes of Health (U.S.) (R01-DK/HL-45881), National Institutes of Health (U.S.) (5P20RR018787-08), National Institutes of Health (U.S.) (R01-AI083256), Cystic Fibrosis Foundation (CF RDP), Cystic Fibrosis Foundation (BOMBER08F0)

P-glycoprotein (Pgp), a member of the adenosine triphosphate-binding cassette (ABC) transporter superfamily, is a major drug efflux pump expressed in normal tissues, and is overexpressed in many human cancers. Overexpression of Pgp results in reduced intracellular drug concentration and cytotoxicity of chemotherapeutic drugs and is thought to contribute to multidrug resistance of cancer cells. The involvement of Pgp in clinical drug resistance has led to a search for molecules that block Pgp transporter activity to improve the efficacy and pharmacokinetics of therapeutic agents. We have recently identified and characterized a secreted toxin from Pseudomonas aeruginosa, designated cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory factor (Cif). Cif reduces the apical membrane abundance of CFTR, also an ABC transporter, and inhibits the CFTR-mediated chloride ion secretion by human airway and kidney epithelial cells. We report presently that Cif also inhibits the apical membrane abundance of Pgp in kidney, airway, and intestinal epithelial cells but has no effect on plasma membrane abundance of multidrug resistance protein 1 or 2. Cif increased the drug sensitivity to doxorubicin in kidney cells expressing Pgp by 10-fold and increased the cellular accumulation of daunorubicin by 2-fold. Thus our studies show that Cif increases the sensitivity of Pgp-overexpressing cells to doxorubicin, consistent with the hypothesis that Cif affects Pgp functional expression. These results suggest that Cif may be useful to develop a new class of specific inhibitors of Pgp aimed at increasing the sensitivity of tumors to chemotherapeutic drugs, and at improving the bioavailability of Pgp transport substrates.

Enhanced antibiotic resistance of Pseudomonas aeruginosa in the cystic fibrosis (CF) lung is thought to be due to the formation of biofilms. However, there is no information on the antibiotic resistance of P. aeruginosa biofilms grown on human airway epithelial cells or on the effects of airway cells on biofilm formation by P. aeruginosa. Thus we developed a coculture model and report that airway cells increase the resistance of P. aeruginosa to tobramycin (Tb) by >25-fold compared with P. aeruginosa grown on abiotic surfaces. Therefore, the concentration of Tb required to kill P. aeruginosa biofilms on airway cells is 10-fold higher than the concentration achievable in the lungs of CF patients. In addition, CF airway cells expressing ΔF508-CFTR significantly enhanced P. aeruginosa biofilm formation, and ΔF508 rescue with wild-type CFTR reduced biofilm formation. Iron (Fe) content of the airway in CF is elevated, and Fe is known to enhance P. aeruginosa growth. Thus we investigated whether enhanced biofilm formation on ΔF508-CFTR cells was due to increased Fe release by airway cells. We found that airway cells expressing ΔF508-CFTR released more Fe than cells rescued with WT-CFTR. Moreover, Fe chelation reduced biofilm formation on airway cells, whereas Fe supplementation enhanced biofilm formation on airway cells expressing WT-CFTR. These data demonstrate that human airway epithelial cells promote the formation of P. aeruginosa biofilms with a dramatically increased antibiotic resistance. The ΔF508-CFTR mutation enhances biofilm formation, in part, by increasing Fe release into the apical medium.

Although insulin-regulated aminopeptidase (IRAP) was first described as a marker protein for specialized vesicles containing the insulin-responsive glucose transporter, GLUT4, the protein has subsequently been shown to be identical to oxytocinase or the AT4 receptor. In insulin responsive tissues, IRAP is almost exclusively co-localized with GLUT4, being retained in intracellular compartments in the basal state or translocating with GLUT4 to the plasma membrane under insulin stimulation. The function of IRAP in these tissues has not been elucidated — the protein is thought to be involved in the tethering of GLUT4 vesicles to intracellular compartments. In the placenta, IRAP was isolated as the enzyme that degrades oxytocin and is therefore thought to play a role in the prevention of premature labour and maintenance of an adequate blood flow to the uterus. Potentially the most exciting physiological role attributed to IRAP is the involvement of the enzyme in memory processing. AT4 ligands, upon binding to the catalytic site of IRAP, enhance spatial learning, facilitate memory retention and retrieval and reverse amnesia. We postulate that the AT4 ligands act via either one of these mechanisms: (1) The peptides bind to the catalytic site of IRAP and inhibit its enzymatic activity thereby prolonging the half-life of its neuropeptide substrates with memory-enhancing properties or (2) Upon binding to IRAP, the AT4 ligands regulate the level of GLUT4 expressed at the cell surface resulting in an increase in glucose uptake into neurones.