Binding of Dengue Virus Partitcles and Dengue Proteins onto Solid Surfaces

The interaction between dengue virus particles (DENV), sedimentation hemagglutinin particles (SHA), dengue virus envelope protein (Eprot), and solid surfaces was investigated by means of ellipsometry and atomic force microscopy (AFM). The surfaces chosen are bare Si/SiO(2) wafers and Si/SiO(2) wafers covered with concanavalin A (ConA), jacalin (Jac), polystyrene (PS), or poly(styrene sulfonate) (PSS) films. Adsorption experiments at pH 7.2 and pH 3 onto all surfaces revealed that (i) adsorption of DENV particles took place only onto ConA under pH 7.2, because of specific recognition between glycans on DENV surface and ConA binding site; (ii) DENV particles did not attach to any of the surfaces at pH 3, suggesting the presence of positive charges on DENV surface at this pH, which repel the positively charged lectin surfaces; (iii) SHA particles are positively charged at pH 7.2 and pH 3 because they adhered to negatively charged surfaces at pH 7.2 and repelled positively charged layers at pH 3; and (iv) SHA particles carry polar groups on the surface because they attached to silanol surfaces at pH 3 and avoided hydrophobic PS films at pH 3 and pH 7.2. The adsorption behavior of Eprot at pH 7.2 revealed affinity for ConA > Jac > PSS > PS approximate to bare Si/SiO(2) layers. These findings indicate that selectivity of the Eprot adsorption is higher when it is part of virus structure than when it is free in solution. The correlation between surface energy values determined by means of contact angle measurements and DENV, SHA, or Eprot adsorption behavior was used to understand the intermolecular forces at the interfaces. A direct correlation was not found because the contributions from surface energy were probably surpassed by specific contributions.

Human papillomavirus type 6b virus-like particles are able to activate the Ras-MAP kinase pathway and induce cell proliferation

The initial step in viral infection is the attachment of the virus to the host cell via an interaction with its receptor. We have previously shown that a receptor for human papillomavirus is the alpha6 integrin. The alpha6 integrin is involved in the attachment of epithelial cells with the basement membrane, but recent evidence suggests that ligation of many integrins results in intracellular signaling events that influence cell proliferation. sere we present evidence that exposure of A431 human epithelial cells to human papillomavirus type 6b L1 virus-like particles (VLPs) results in a dose-dependent increase in cell proliferation, as measured by bromodeoxyuridine incorporation. This proliferation is Lost if VLPs are first denatured or incubated with a monoclonal antibody against L1 protein. The MEK1 inhibitor PB98059 inhibits the VLP-mediated increase in fell proliferation, suggesting involvement of the Ras-MAP kinase pathway, Indeed, VLP binding results in rapid phosphorylation of the beta4 integrin upon tyrosine residues and subsequent recruitment of the adapter protein She to beta4, Within 30 min, the activation of Ras, Raf, and Erk2 was observed. Finally, the upregulation of c-myc mRNA was observed at 60 min, These data indicate that human papillomavirus type 6b is able to signal cells via the Ras-MAP kinase pathway to induce cell proliferation. We hypothesize that such a mechanism would allow papillomaviruses to infect hosts more successfully by increasing the potential pool of cells they are able to infect via the initiation of proliferation in resting keratinocyte stem and suprabasal cells.

Development and characterisation of recombinant hepatitis delta virus-like particles

Injection of particulate hepatitis B virus surface antigen (HBsAg) in mice leads to the induction of a HBsAg-specific class-I-restricted cytotoxic T lymphocyte (CTL) response. It is proposed that any protein internal to HBsAg will also be able to elicit a specific CTL response. In this study, several carboxy-terminal truncations of hepatitis C virus (HCV) core protein were fused to varying lengths of amino-terminal truncated large hepatitis delta antigen (L-HDAg). These constructs were analysed for their ability to be expressed and the particles secreted in the presence of HBsAg after transfection into HuH-7 cells. The secretion efficiency of the various HCV core-HDAg chimeric proteins was generally poor. Constructs containing full length HDAg appeared to be more stable than truncated versions and the length of the inserted protein was restricted to around 40 amino acids. Thus, the use of L-HDAg as a chimera to package foreign proteins is limited. Consequently, a polyepitope (polytope) containing a B-cell epitope from human papillomavirus (HPV 16) and multiple T-cell epitopes from the HCV polyprotein was used to create the construct, L-HDAg-polyB. This chimeric protein was shown to be reliant on the co-expression of HBsAg for secretion into the cell culture fluid and was secreted more efficiently than the previous HCV core-HDAg constructs. These L-HDAg-polyB virus-like particles (VLPs) had a buoyant density of similar to 1.2 g/cm(3) in caesium chloride and similar to 1.15 g/cm(3) in sucrose. The VLPs were also immunoprecipitated using an anti-HBs but not an anti-HD antibody. Thus, these recombinant VLPs have similar biophysical properties to L-HDAg VLPs.

Antigenicity and immunogenicity of novel chimeric hepatitis B surface antigen particles with exposed hepatitis C virus epitopes

The small envelope protein of hepatitis B virus (HBsAg-S) can self-assemble into highly organized virus like particles (VLPs) and induce an effective immune response. In this study, a restriction enzyme site was engineered into the cDNA of HBsAg-S at a position corresponding to the exposed site within the hydrophilic a determinant region (amino acid [aa] 127-128) to create a novel HBsAg vaccine vector allowing surface orientation of the inserted sequence. We inserted sequences of various lengths from hypervariable region 1 (HVR1) of the hepatitis C virus (HCV) E2 protein containing immunodominant epitopes and demonstrated secretion of the recombinant HBsAg VLPs from transfected mammalian cells. A number of different recombinant proteins were synthesized, and HBsAg VLPs containing inserts up to 36 aa were secreted with an efficiency similar to that of wild-type HBsAg. The HVR1 region exposed on the particles retained an antigenic structure similar to that recognized immunologically during natural infection. VLPs containing epitopes from either HCV-1a or -1b strains were produced that induced strain-specific antibody responses in immunized mice. Injection of a combination of these VLPs induced antibodies against both HVR1 epitopes that resulted in higher titers than were achieved by vaccination with the individual VLPs, suggesting a synergistic effect. This may lead to the development of recombinant particles which are able to induce a broad anti-HCV immune response against the HCV quasispecies or other quasispecies-like infectious agents.

A practical method for identifying intermetallic phase particles in aluminium alloys by electron probe microanalysis

Aluminium alloys that contain Si, Mg, Fe, Mn and/or Cu usually contain one or more types of intermetallic phases that are not readily distinguishable in the microstructure by conventional microscopy methods. It has thus been a challenge to develop a method that will unambiguously identify them. A practical approach has been developed that is based on an inherent linear relationship revealed for the overall distribution of any two elements in a precipitate/matrix geometry and the first-order approximation of electron probe microanalysis (EPMA) results. Application of this approach to a direct chill cast 6082 alloy is demonstrated, and its major limitations are discussed.

Marker concentration patterns of labelled leaf and stem particles in the rumen of cattle grazing bermuda grass (Cynodon dactylon) analysed by reference to a raft model

Large (>1600 mum), ingestively masticated particles of bermuda grass (Cynodon dactylon L. Pers.) leaf and stem labelled with Yb-169 and Ce-144 respectively were inserted into the rumen digesta raft of heifers grazing bermuda grass. The concentration of markers in digesta sampled from the raft and ventral rumen were monitored at regular intervals over approximately 144 h. The data from the two sampling sites were simultaneously fitted to two pool (raft and ventral rumen-reticulum) models with either reversible or sequential flow between the two pools. The sequential flow model fitted the data equally as well as the reversible flow model but the reversible flow model was used because of its greater application. The reversible flow model, hereafter called the raft model, had the following features: a relatively slow age-dependent transfer rate from the raft (means for a gamma 2 distributed rate parameter for leaf 0.0740 v. stem 0.0478 h(-1)), a very slow first order reversible flow from the ventral rumen to the raft (mean for leaf and stem 0.010 h(-1)) and a very rapid first order exit from the ventral rumen (mean of leaf and stem 0.44 h(-1)). The raft was calculated to occupy approximately 0.82 total rumen DM of the raft and ventral rumen pools. Fitting a sequential two pool model or a single exponential model individually to values from each of the two sampling sites yielded similar parameter values for both sites and faster rate parameters for leaf as compared with stem, in agreement with the raft model. These results were interpreted as indicating that the raft forms a large relatively inert pool within the rumen. Particles generated within the raft have difficulty escaping but once into the ventral rumen pool they escape quickly with a low probability of return to the raft. It was concluded that the raft model gave a good interpretation of the data and emphasized escape from and movement within the raft as important components of the residence time of leaf and stem particles within the rumen digesta of cattle.

The entrapment of particles by windbreaks

A theory is developed for calculating the entrapment of particles by a windbreak, with four results. (1) The fraction of particles in the oncoming flow which pass through the windbreak, or transmittance of the windbreak for particles (sigma), is related to the optical porosity (tau). The very simple approximation sigma=tau works well for most applications involving the interception of spray droplets by windbreaks. Results from a field experiment agree with the theoretical predictions. (2) A new equation for the bulk drag coefficient of a windbreak is tested against numerical, wind tunnel and field experiments. This enables the bleed velocity for the flow through the windbreak to be predicted in terms of the screen pressure coefficient (k) of the barrier. (3) The relationship between k and tau is different for a vegetative barrier than for a screen across a confined duct, implying a lower Fc for given tau. (4) The total deposition of particles to a windbreak is determined by a trade-off between particle absorption and throughflow, implying an optimum value of tau for maximum total deposition. For particles larger than 30 mum and vegetation elements smaller than 30 mm, this occurs near tau = 0.2. (C) 2001 Elsevier Science Ltd. All rights reserved.

Spectroscopic study of the penetration depth of grafted polystyrene onto poly (tetrafluoroethylene-co-perfluoropropylvinylether) substrates. 1. Effect of grafting conditions

This study concerns the radiation grafting of styrene onto poly(tetrafluoroethylene-co-perfluoropropylvinylether) (PFA) substrates and the penetration depth of the graft. Grafting was obtained by the simultaneous irradiation method, and the spectroscopic analysis was made with the micro-Raman technique. Effects of grafting conditions such as the type of solvent, dose rate, and irradiation dose on the grafting yield were investigated. Of the different solvents used, the most efficient in terms of increasing grafting yield were dichloromethane, benzene, and methanol, respectively. A mixture of methanol and dichloromethane used as a solvent for styrene achieved a higher degree of grafting and concentration of grafted polystyrene onto the surface of PFA substrates than solutions of the monomer in the separate solvents. The degree of grafting increased with increasing radiation dose up to 500 kGy, stabilizing above this dose. However, the grafting yield decreased with an increase in the dose rate. The increase in the overall grafting yield was accompanied by a proportional increase in the penetration depth of the grafts into the substrate. (C) 2002 Wiley Periodicals, Inc.

Immunogenicity of recombinant HBsAg/HCV particles in mice pre-immunised with hepatitis B virus-specific vaccine

Due to their spatial structure virus-like particles (VLPs) generally induce effective immune responses. VLPs derived from the small envelope protein (HBsAg-S) of hepatitis B virus (HBV) comprise the HBV vaccine. Modified HBsAs-S VLPs, carrying the immunodominant hypervariable region (HVR1) of the hepatitis C virus (HCV) envelope protein E2 within the exposed 'a'-determinant region (HBsAg/HVR1-VLPs), elicited HVR1-specific antibodies in mice. A high percentage of the human population is positive for anti-HBsAg antibodies (anti-HBs), either through vaccination or natural infection. We, therefore, determined if pre-existing anti-HBs could influence immunisation with modified VLPs. Mice were immunised with a commercial HBV vaccine, monitored to ensure an anti-HBs response, then immunised with HBsAg/HVR1-VLPs. The resulting anti-HVR1 antibody titre was similar in mice with or without pre-existing anti-HBs. This suggests that HBsAg/HVR1-VLPs induce a primary immune response to HVR1 in anti-HBs positive mice and, hence, they may be used successfully in individuals already immunised with the HBV vaccine. (C) 2003 Elsevier Science Ltd. All rights reserved.

Carboxy-fluorescein diacetate, succinimidyl ester labeled papillomavirus virus-like particles fluoresce after internalization and interact with heparan sulfate for binding and entry

Human papillomaviruses (HPVs) infect epithelial cells and are associated with genital carcinoma. Most epithelial cell lines express cell-surface glycosaminoglycans (GAGs) usually found attached to the protein core of proteoglycans. Our aim was to study how GAGs influenced HPV entry. Using a human keratinocyte cell line (HaCaT), preincubation of HPV virus-like particles (VLPs) with GAGs showed a dose-dependent inhibition of binding. The IC50 (50% inhibition) was only 0.5 mug/ml for heparin, 1 mug/ml for dextran sulfate, and 5-10 mug/ml for heparan sulfate from mucosal origin. Mutated chinese hamster ovary (CHO) cell lines lacking heparan sulfate or all GAGs were unable to bind HPV VLPs. Here we also report a method to study internalization by using VLPs labeled with carboxy-fluorescein diacetate, succinimidyl ester, a fluorochrome that is only activated after cell entry. Pretreatment of labeled HPV VLPs with heparin inhibited uptake, suggesting a primary interaction between HPV and cell-surface heparan sulfate. (C) 2003 Elsevier Science (USA). All rights reserved.

Assessment of exposure to airborne ultrafine particles in the urban environment of Lisbon, Portugal

The aim of this study was the assessment of exposure to ultrafine in the urban environment of Lisbon, Portugal, due to automobile traffic, and consisted of the determination of deposited alveolar surface area in an avenue leading to the town center during late spring. This study revealed differentiated patterns for weekdays and weekends, which could be related with the fluxes of automobile traffic. During a typical week, ultrafine particles alveolar deposited surface area varied between 35.0 and 89.2 μm2/cm3, which is comparable with levels reported for other towns such in Germany and the United States. These measurements were also complemented by measuring the electrical mobility diameter (varying from 18.3 to 128.3 nm) and number of particles that showed higher values than those previously reported for Madrid and Brisbane. Also, electron microscopy showed that the collected particles were composed of carbonaceous agglomerates, typical of particles emitted by the exhaustion of diesel vehicles. Implications: The approach of this study considers the measurement of surface deposited alveolar area of particles in the outdoor urban environment of Lisbon, Portugal. This type of measurements has not been done so far. Only particulate matter with aerodynamic diameters <2.5 (PM2.5) and >10 (PM10) μm have been measured in outdoor environments and the levels found cannot be found responsible for all the observed health effects. Therefore, the exposure to nano- and ultrafine particles has not been assessed systematically, and several authors consider this as a real knowledge gap and claim for data such as these that will allow for deriving better and more comprehensive epidemiologic studies. Nanoparticle surface area monitor (NSAM) equipments are recent ones and their use has been limited to indoor atmospheres. However, as this study shows, NSAM is a very powerful tool for outdoor environments also. As most lung diseases are, in fact, related to deposition of the alveolar region of the lung, the metric used in this study is the ideal one.

Comparison of indoor and outdoor fungi and particles in poultry units

A descriptive study was developed in order to compare indoor and outdoor air contamination caused by fungi and particles in seven poultry units. Twenty eight air samples of 25 litters were collected through the impaction method on malt extract agar. Air sampling and particles concentration measurement were done in the interior and also outside premises of the poultries’ pavilions. Regarding the fungal load in the air, indoor concentration of mold was higher than outside air in six poultry units. Twenty eight species / genera of fungi were identified indoor, being Scopulariopsis brevicaulis (40.5%) the most commonly isolated species and Rhizopus sp. (30.0%) the most commonly isolated genus. Concerning outdoor, eighteen species/genera of fungi were isolated, being Scopulariopsis brevicaulis (62.6%) also the most isolated. All the poultry farms analyzed presented indoor fungi different from the ones identified outdoors. Regarding particles’ contamination, PM2.5, PM5.0 and PM10 had a statistically significant difference (Mann-Whitney U test) between the inside and outside of the pavilions, with the inside more contaminated (p=.006; p=.005; p=.005, respectively). The analyzed poultry units are potential reservoirs of substantial amounts of fungi and particles and could therefore free them in the atmospheric air. The developed study showed that indoor air was more contaminated than outdoors, and this can result in emission of potentially pathogenic fungi and particles via aerosols from poultry units to the environment, which may post a considerable risk to public health and contribute to environmental pollution.