NS1 proteins of avian influenza A viruses can act as antagonists of the human alpha/beta interferon response

Many viruses, including human influenza A virus, have developed strategies for counteracting the host type I interferon (IFN) response. We have explored whether avian influenza viruses were less capable of combating the type I IFN response in mammalian cells, as this might be a determinant of host range restriction. A panel of avian influenza viruses isolated between 1927 and 1997 was assembled. The selected viruses showed variation in their ability to activate the expression of a reporter gene under the control of the IFN-beta promoter and in the levels of IFN induced in mammalian cells. Surprisingly, the avian NS1 proteins expressed alone or in the genetic background of a human influenza virus controlled IFN-beta induction in a manner similar to the NS1 protein of human strains. There was no direct correlation between the IFN-beta induction and replication of avian influenza viruses in human A549 cells. Nevertheless, human cells deficient in the type I IFN system showed enhanced replication of the avian viruses studied, implying that the human type I IFN response limits avian influenza viruses and can contribute to host range restriction.

Self-assembly of PEGylated peptide conjugates containing a modified amyloid β-peptide fragment

The self-assembly of PEGylated peptides containing a modified sequence from the amyloid beta peptide, FEK LVFF, has been studied in aqueous solution. PEG molar masses PEG1k, PEG2k, and PEG10k were used in the conjugates. It is shown that the three FFK LVFF-PEG hybrids form fibrils comprising a FFKLVFF core and a PEG corona. The beta-sheet secondary structure of the peptide is retained in the FFK LVFF fibril core. At sufficiently high concentrations, FEK LVFF-PEG1k and FEK LVFF-PEG2k form a nema tic phase, while PEG10k-FEK LVFF exhibits a hexagonal columnar phase. Simultaneous small angle neutron scattering/shear flow experiments were performed to study the shear flow alignment of the nematic and hexagonal liquid crystal phases. On drying, PEG crystallization occurs without disruption of the FFK LVFF beta-sheet structure leading to characteristic peaks in the X-ray diffraction pattern and FTIR spectra. The stability of beta-sheet structures was also studied in blends of FFKLVFF-PEG conjugates with poly(acrylic acid) (PAA). While PEG crystallization is only observed up to 25% PAA content in the blends, the FFK LVFF beta-sheet structure is retained up to 75% PAA.

PEGylated amyloid peptide nanocontainer delivery and release system

A micellar nanocontainer delivery and release system is designed on the basis of a peptide-polymer conjugate. The hybrid molecules self-assemble into micelles comprising a modified amyloid peptide core surrounded by a PEG corona. The modified amyloid peptide previously studied in our group forms helical ribbons based on a beta-sheet motif and contains beta-amino acids that are excluded from the beta-sheet structure, thus being potentially useful as fibrillization inhibitors. In the model peptide-PEG hybrid system studied, enzymatic degradation using alpha-chymotrypsin leads to selective cleavage close to the PEG-peptide linkage, break up of the micelles, and release of peptides in unassociated form. The release of monomeric peptide is useful because aggregation of the released peptide into beta-sheet amyloid fibrils is not observed. This concept has considerable potential in the targeted delivery of peptides for therapeutic applications.

Thiolated mucoadhesive and PEGylated nonmucoadhesive organosilica nanoparticles from 3-Mercaptopropyltrimethoxysilane

A novel approach has been developed to synthesize thiolated sub-100 nm organosilica nanoparticles from 3-mercaptopropyltrimethoxysilane (MPTS) through its self-condensation in dimethylsulfoxide in contact with atmospheric oxygen. The formation of MPTS nanoparticles proceeds through the condensation of methoxysilane groups and simultaneous disulfide bridging caused by partial oxidation of thiol groups. These nanoparticles showed excellent colloidal stability in dilute aqueous dispersions but underwent further self-assembly into chains and necklaces at higher concentrations. They exhibited very good ability to adhere to ocular mucosal surfaces, which can find applications in drug delivery. The thiolated nanoparticles could also be easily modified through PEGylation resulting in a loss of their mucoadhesive properties.

Hydrogen-bonding-driven self-assembly of PEGylated organosilica nanoparticles with poly(acrylic acid) in aqueous solutions and in layer-by-layer deposition at solid surfaces

PEGylated organosilica nanoparticles have been synthesized through self-condensation of (3-mercaptopropyl)trimethoxysilane in dimethyl sulfoxide into thiolated nanoparticles with their subsequent reaction with methoxypoly(ethylene glycol) maleimide. The PEGylated nanoparticles showed excellent colloidal stability over a wide range of pH in contrast to the parent thiolated nanoparticles, which have a tendency to aggregate irreversibly under acidic conditions (pH < 3.0). Due to the presence of a poly(ethylene glycol)-based corona, the PEGylated nanoparticles are capable of forming hydrogen-bonded interpolymer complexes with poly(acrylic acid) in aqueous solutions under acidic conditions, resulting in larger aggregates. The use of hydrogen-bonding interactions allows more efficient attachment of the nanoparticles to surfaces. The alternating deposition of PEGylated nanoparticles and poly(acrylic acid) on silicon wafer surfaces in a layer-by-layer fashion leads to multilayered coatings. The self-assembly of PEGylated nanoparticles with poly(acrylic acid) in aqueous solutions and at solid surfaces was compared to the behavior of linear poly(ethylene glycol). The nanoparticle system creates thicker layers than the poly(ethylene glycol), and a thicker layer is obtained on a poly(acrylic acid) surface than on a silica surface, because of the effects of hydrogen bonding. Some implications of these hydrogen-bonding-driven interactions between PEGylated nanoparticles and poly(acrylic acid) for pharmaceutical formulations are discussed.

The role of myeloid receptors on murine plasmacytoid dendritic cells in induction of type I interferon

This study tested the hypothesis that a set of predominantly myeloid restricted receptors (F4/80, CD36, Dectin-1, CD200 receptor and mannan binding lectins) and the broadly expressed CD200 played a role in a key function of plasmacytoid DC (pDC), virally induced type I interferon (IFN) production. The Dectin-1 ligands zymosan, glucan phosphate and the anti-Dectin-1 monoclonal antibody (mAb) 2A11 had no effect on influenza virus induced IFNα/β production by murine splenic pDC. However, mannan, a broad blocking reagent against mannose specific receptors, inhibited IFNα/β production by pDC in response to inactivated influenza virus. Moreover, viral glycoproteins (influenza virus haemagglutinin and HIV-1 gp120) stimulated IFNα/β production by splenocytes in a mannan-inhibitable manner, implicating the function of a lectin in glycoprotein induced IFN production. Lastly, the effect of CD200 on IFN induction was investigated. CD200 knock-out macrophages produced more IFNα than wild-type macrophages in response to polyI:C, a MyD88-independent stimulus, consistent with CD200's known inhibitory effect on myeloid cells. In contrast, blocking CD200 with an anti-CD200 mAb resulted in reduced IFNα production by pDC-containing splenocytes in response to CpG and influenza virus (MyD88-dependent stimuli). This suggests there could be a differential effect of CD200 on MyD88 dependent and independent IFN induction pathways in pDC and macrophages. This study supports the hypothesis that a mannan-inhibitable lectin and CD200 are involved in virally induced type I IFN induction.

Macrophage antioxidant protection within atherosclerotic plaques

Macrophage cells within inflammatory lesions are exposed to a wide range of degrading and cytotoxic molecules including reactive oxygen species. Unlike neutrophils, macrophages do not normally die in this environment but continue to generate oxidants, phagocytose cellular remains, and release a range of cytoactive agents which modulate the immune response. It is this potential of the macrophage cell to survive in an oxidative environment that allows the growth and complexity of advanced atherosclerotic plaques. This review will examine the oxidants encountered by macrophages within an atherosclerotic plaque and describe some of the potential antioxidant mechanisms which enable macrophages to function within inflammatory lesions. Ascorbate, alpha-tocopherol, and glutathione appear to be central to the protection of macrophages yet additional antioxidant mechanisms appear to be involved. gamma-Interferon causes macrophages to generate 7,8dihydroneopterin/neopterin and 3-hydroxyanthranilic acid both of which have antioxidant properties. Manganese superoxide dismutase is also upregulated in macrophages. The evidence that these antioxidants provide further protection, so allowing the macrophage cells to survive within sites of chronic inflammation such as atherosclerotic plaques, will be described.

Variation in the ability of human influenza A viruses to induce and inhibit the IFN-beta pathway

We investigated the ability of a selection of human influenza A viruses, including recent clinical isolates, to induce IFN-beta production in cultured cell lines. In contrast to the well-characterized laboratory strain A/PR/8/34, several, but not all, recent isolates of H3N2 viruses resulted in moderate IFN-beta stimulation. Through the generation of recombinant viruses, we were able to show that this is not due to a loss of the ability of the NS1 genes to suppress IFN-beta induction; indeed, the NS1 genes behaved similarly with respect to their abilities to block dsRNA signaling. Interestingly, replication of A/Sydney/5/97 virus was less Susceptible to pre-treatment with IFN-alpha than the other viruses. In contrast to the universal effect on dsRNA signaling, we noted differences in the effect of NS1 proteins on expression of interferon stimulated genes and also genes induced by a distinct pathway. The majority of NS1 proteins blocked expression From both IFN-dependent and TNF-dependent promoters by an apparent post-transcriptional mechanism. The NS1 gene of A/PR/8/34 NS1 did not confer these blocks. We noted striking differences in the Cellular localization of different influenza A virus NS1 proteins during infection, which might explain differences in biological activity. (C) 2005 Elsevier Inc. All rights reserved.

Combined affinity labelling and mass spectrometry analysis of differential cell surface protein expression in normal and prostate cancer cells

Differences in the expression of cell surface proteins between a normal prostate epithelial (1542-NP2TX) and a prostate cancer cell line (1542-CP3TX) derived from the same patient were investigated. A combination of affinity chromatographic purification of biotin-tagged surface proteins with mass spectrometry analysis identified 26 integral membrane proteins and 14 peripheral surface proteins. The findings confirm earlier reports of altered expression in prostate cancer for several cell surface proteins, including ALCAM/CD166, the Ephrin type A receptor, EGFR and the prostaglandin F2 receptor regulatory protein. In addition, several novel findings of differential expression were made, including the voltage-dependent anion selective channel proteins Porin 1 and 2, ecto-5'-nucleotidase (CD73) and Scavenger receptor B1. Cell surface protein expression changed both qualitatively and quantitatively when the cells were grown in the presence of either or both interferon INFalpha and INFgamma. Costimulation with type I and II interferons had additive or synergistic effects on the membrane density of several, mainly peripherally attached surface proteins. Concerted upregulation of surface exposed antigens may be of benefit in immuno-adjuvant-based treatment of interferon-responsive prostate cancer. In conclusion, this study demonstrates that differences in the expression of membrane proteins between normal and prostate cancer cells are reproducibly detectable following vectorial labelling with biotin, and that detailed analysis of extracellular-induced surface changes can be achieved by combining surface-specific labelling with high-resolution two-dimensional gel electrophoresis and mass spectrometry.

Effect of genistein and daidzein on platelet aggregation and monocyte and endothelial function

There has been much recent interest in the cardiovascular benefits of dietary isoflavones. The aim of the present in vitro studies was to investigate potential anti-thrombogenic and anti-atherogenic effects of the isoflavones genistein and daidzein in platelets, macrophages and endothelial cells. Pre-treatment with either isoflavone inhibited collagen-induced platelet aggregation in a dose-dependent manner. In a macrophage cell line (RAW 264-7) activated with interferon gamma plus lipopolysaccharide, both isoflavones were found to inhibit NO production and tumour necrosis factor alpha (TNF-alpha) secretion dose-dependently, but they did not affect mRNA levels for inducible nitric oxide synthase and cyclo-oxygenase-2. Both isoflavones also dose-dependently decreased monocyte chemoattractant protein-1 secretion induced by TNF-alpha in human umbilical vein endothelial cells. Compared with daidzein, genistein exerted greater inhibitory effects for all parameters studied. The present data contributes to our knowledge on the molecular mechanisms by which isoflavones may protect against coronary artery disease. Further studies are required to determine whether the effects of isoflavones observed in the current in vitro studies are relevant to the aetiology of coronary artery disease in vivo.

Targeting αvβ3 and α5β1 for gene delivery to proliferating VSMCs: synergistic effect of TGF-β1

TGF-beta1 levels increase after vascular injury and promote vascular smooth muscle cell (VSMC) proliferation. We define a nonviral gene delivery system that targets alphavbeta3 and alpha5beta1 integrins that are expressed on proliferating VSMCs and strongly induced by TGF-beta1. A 15-amino acid RGDNP-containing peptide from American Pit Viper venom was linked to a Lys(16) peptide as vector (molossin vector) and complexed with Lipofectamine or fusogenic peptide for delivery of luciferase or beta-galactosidase reporter genes to primary cultures of human, rabbit, and rat VSMCs. Preincubation of VSMCs with TGF-beta1 for 24 h, but not with PDGF-BB, interferon-gamma, TNF-alpha, nor PMA, increased alphavbeta3 and alpha5beta1 expressions on VSMCs and enhanced gene delivery of molossin vector. Thus beta-galactosidase activity increased from 35 +/- 5% (controls) to 75 +/- 5% after TGF-beta1 treatment, and luciferase activity increased fourfold over control values. Potential use of this system in vessel bypass surgery was examined in an ex vivo rat aortic organ culture model after endothelial damage. Molossin vector system delivered beta-galactosidase to VSMCs in the vessel wall that remained for up to 12 days posttransfection. The molossin vector system, when combined with TGF-beta1, enhances gene delivery to proliferating VSMCs and might have clinical applications for certain vasculoproliferative diseases.

Multiple lyotropic polymorphism of a PEG-peptide diblock copolymer in aqueous solution

Nematic and hexagonal columnar liquid crystal phase formation by a PEG-peptide conjugate is reported. The results are relevant to peptide-polymer Conjugates and bionanomaterial self-assembly (with relevance to PEGylated peptides used in therapeutic applications). The use of modified fragments of the amyloid beta peptide is especially interesting with respect to amyloid fibrillization and its control.

Combined affinity labelling and mass spectrometry analysis of differential cell surface protein expression in normal and prostate cancer cells

Differences in the expression of cell surface proteins between a normal prostate epithelial (1542-NP2TX) and a prostate cancer cell line (1542-CP3TX) derived from the same patient were investigated. A combination of affinity chromatographic purification of biotin-tagged surface proteins with mass spectrometry analysis identified 26 integral membrane proteins and 14 peripheral surface proteins. The findings confirm earlier reports of altered expression in prostate cancer for several cell surface proteins, including ALCAM/CD166, the Ephrin type A receptor, EGFR and the prostaglandin F2 receptor regulatory protein. In addition, several novel findings of differential expression were made, including the voltage-dependent anion selective channel proteins Porin 1 and 2, ecto-5'-nucleotidase (CD73) and Scavenger receptor B1. Cell surface protein expression changed both qualitatively and quantitatively when the cells were grown in the presence of either or both interferon INF alpha and INF gamma. Costimulation with type I and II interferons had additive or synergistic effects on the membrane density of several, mainly peripherally attached surface proteins. Concerted upregulation of surface exposed antigens may be of benefit in immuno-adjuvant-based treatment of interferon-responsive prostate cancer. In conclusion, this study demonstrates that differences in the expression of membrane proteins between normal and prostate cancer cells are reproducibly detectable following vectorial labelling with biotin, and that detailed analysis of extracellular-induced surface changes can be achieved by combining surface-specific labelling with high-resolution two-dimensional gel electrophoresis and mass spectrometry.

Dietary synbiotics reduce cancer risk factors in polypectomized and colon cancer patients

Background: Animal studies suggest that prebiotics and probiotics exert protective effects against tumor development in the colon, but human data supporting this suggestion are weak. Objective: The objective was to verify whether the prebiotic concept (selective interaction with colonic flora of nondigested carbohydrates) as induced by a synbiotic preparation-oligofructose-enriched inulin (SYN1) + Lactobacillus rhamnosus GG (LGG) and Bifidobacterium lactis Bb12 (BB12)-is able to reduce the risk of colon cancer in humans. Design: The 12-wk randomized, double-blind, placebo-controlled trial of a synbiotic food composed of the prebiotic SYN1 and probiotics LGG and BB12 was conducted in 37 colon cancer patients and 43 polypectomized patients. Fecal and blood samples were obtained before, during, and after the intervention, and colorectal biopsy samples were obtained before and after the intervention. The effect of synbiotic consumption on a battery of intermediate biomarkers for colon cancer was examined. Results: Synbiotic intervention resulted in significant changes in fecal flora: Bifidobacterium and Lactobacillus increased and Clostridium perfringens decreased. The intervention significantly reduced colorectal proliferation and the capacity of fecal water to induce necrosis in colonic cells and improve epithelial barrier function in polypectomized patients. Genotoxicity assays of colonic biopsy samples indicated a decreased exposure to genotoxins in polypectomized patients at the end of the intervention period. Synbiotic consumption prevented an increased secretion of interleukin 2 by peripheral blood mononuclear cells in the polypectomized patients and increased the production of interferon gamma in the cancer patients. Conclusions: Several colorectal cancer biomarkers can be altered favorably by synbiotic intervention.

Relation between the fatty acid composition of peripheral blood mononuclear cells and measures of immune cell function in health free-living subjects aged 25-72 y

Background: There is little information about the relation between the fatty acid composition of human immune cells and the function of those cells over the habitual range of fatty acid intakes. Objective: The objective of the study was to determine the relation between the fatty acid composition of human peripheral blood mononuclear cell (PBMC) phospholipids and the functions of human immune cells. Design: One hundred fifty healthy adult subjects provided a fasting blood sample. The phagocytic and oxidative burst activities of monocytes and neutrophils were measured in whole blood. PBMCs were isolated and used to measure lymphocyte proliferation in response to the T cell mitogen concanavalin A and the production of cytokines in response to concanavalin A or bacterial lipopolysaccharide. The fatty acid composition of plasma and PBMC phospholipids was determined. Results: Wide variations in fatty acid composition of PBMC phospholipids and immune cell functions were identified among the subjects. The proportions of total Polyunsaturated fatty acids (PUFAs), of total n-6 and n-3 PUFAs, and of several individual PUFAs in PBMC phospholipids were positively correlated with phagocytosis by neutrophils and monocytes, neutrophil oxidative burst, lymphocyte proliferation, and interferon gamma production. The ratios of saturated fatty acids to PUFAs and of n-6 to n-3 PUFAs were negatively correlated with these same immune functions. The relation of PBMC fatty acid composition to monocyte oxidative burst was the reverse of its relation to monocyte phagocytosis and neutrophil oxidative burst. Conclusion: Variations in the fatty acid composition of PBMC phospholipids account for some of the variability in immune cell functions among healthy adults.