Uptake and depuration of gold nanoparticles in Daphnia magna.

This study presents a series of short-term studies (total duration 48 h) of uptake and depuration of engineered nanoparticles (ENP) in neonate Daphnia magna. Gold nanoparticles (Au NP) were used to study the influence of size, stabilizing agent and feeding on uptake and depuration kinetics and animal body burdens. 10 and 30 nm Au NP with different stabilizing agents [citrate (CIT) and mercaptoundecanoic acid (MUDA)] were tested in concentrations around 0.5 mg Au/L. Fast initial uptake was observed for all studied Au NP, with CIT stabilized Au NP showing similar rates independent of size and MUDA showing increased uptake for the smaller Au NP (MUDA 10 nm > CIT 10 nm, 30 nm > MUDA 30 nm). However, upon transfer to clean media no clear trend on depuration rates was found in terms of stabilizing agent or size. Independent of stabilizing agent, 10 nm Au NP resulted in higher residual whole-animal body burdens after 24 h depuration than 30 nm Au NP with residual body burdens about one order of magnitude higher of animals exposed to 10 nm Au NP. The presence of food (P. subcapitata) did not significantly affect the body burden after 24 h of exposure, but depuration was increased. While food addition is not necessary to ensure D. magna survival in the presented short-term test design, the influence of food on uptake and depuration kinetics is essential to consider in long term studies of ENP where food addition is necessary. This study demonstrates the feasibility of a short-term test design to assess the uptake and depuration of ENP in D. magna. The findings underlines that the assumptions behind the traditional way of quantifying bioconcentration are not fulfilled when ENPs are studied.

Use of dimensionless residence time to study variations in breakthrough behaviour in expanded beds formed from varied particle size distributions.

This work demonstrates an experimental method for studying breakthrough behaviour in expanded beds. The behaviour of beds made with differently sized particles were studied at varying flowrates. The use of a dimensionless residence time measurement allowed a more valid comparison of breakthrough characteristics in expanded bed operation by compensating for the changes in bed volume that occur during expansion. We demonstrate that bed breakthrough behaviour can be compared directly even when the beds contain different-sized particles and hence have different expanded volumes. By utilising this concept we demonstrate that, in the case of the Alcohol Dehydrogenase (ADH) / STREAMLINE Phenyl system used here, there was little or no variation in ADH breakthrough behaviour between beds of differently sized particles operating at flowrates above 100 cm/h. This suggests that the higher specific surface area and hence binding capacity of smaller particles is negated in this case due to mass transfer limitations and the increase in system void volume even at normal operating flowrates of 200-300 cm/h.

Hydrophobic interaction ligand selection and scale-up of an expanded bed separation of an intracellular enzyme from Saccharomyces cerevisiae.

A prototype Streamline-Phenyl matrix was evaluated in a hydrophobic interaction mode for the direct recovery of alcohol dehydrogenase (ADH) from yeast cell homogenate. At 5% breakthrough of ADH, a yield of 100% was obtained for a dynamic expanded bed capacity of 240 U(ADH)/ml matrix with a purification factor of 9.2. This compared with a dynamic capacity of 3013 U(ADH)/ml matrix for the packed bed equivalent and a purification factor of 18. In both systems the purification factor was found to increase simultaneously with a decrease in yield as the load of homogenate or breakthrough of ADH was increased. The expanded bed mode of operation conferred considerable robustness with respect to process fouling. No loss in yield was seen over five cycles of repeat loading with an unclarified homogenate. By contrast the packed bed media showed a decrease in yield from 86 to 56% over the same period. Successful scale up of the expanded bed protocol for a 20% breakthrough was demonstrated over a fourfold increase in column diameter. The application of hydrophobic interaction chromatography mediated expanded bed adsorption and its scale-up is discussed in the context of large-scale operations.

The use of rapid on-line monitoring of products and contaminants from within an expanded bed to control separations exhibiting fast breakthrough characteristics and to maximize productivity.

Conventional control of expanded-bed adsorption (EBA), like that of packed-bed chromatography, is based upon off-line measurements of the column eluant. The relatively high-void volumes in EBA systems means that this approach can lead to significant performance losses caused by the inability to achieve tight control of breakthrough. This problem is made worse if the product has a fast breakthrough characteristic or if it is necessary to operate to low levels of product loss. In this article we examine the utility of constant on-line monitoring from within the expanded bed using stopped-flow analysis (SFA) to provide data for the control of the expanded-bed operation. A modified Streamline 50 column with side ports that enable sampling along the expanded axis of the bed was used. Comparisons between off-line and on-line measurements are presented, showing how the advanced monitoring method can lead to better control and to an analysis of breakthrough development within the bed. The expanded bed was used to purify alcohol dehydrogenase from homogenized suspensions of bakers' yeast. Accurate control of breakthrough to 10% of the target enzyme was achieved using a SFA control system with a response time of 40 seconds. On-line data compared well to assays carried out off-line on the outlet stream for both the product enzyme (ADH), total protein, RNA, and cell debris levels (via UV 650 nm). This information was used to generate a series of graphs with which to track the EBA process in real-time. Results showed that bed utilization was not linear along the bed axis so that, for example, 60% of ADH is bound in the bottom 33% of the column during loading.

Experimental measurement of particle size distribution and voidage in an expanded bed adsorption system.

This paper presents an experimental analysis of matrix bead size distribution and voidage variations with axial height in an expanded bed adsorption system. Use of a specially constructed expanded bed with side ports has enabled sampling from within the expanded bed along the vertical axis. Particles removed from within the bed were measured for their size distributions. Residence time distribution studies were used to estimate bed voidage. Measurements of axial and radial particle size distributions and axial voidage distribution have been made at different flow rates. Particle size was found to be radially constant, indicating constant stratification in the column. The particle size was found to decrease with increasing axial height. Voidage increased with axial height from a settled bed value of 0.39 to approaching unity for high liquid velocities and increased at a constant axial position with increased flowrate. This information provides key insight into bed stability and data for the improved modeling of this important unit operation.

Direct visualisation of plasmid DNA in individual chromatography adsorbent particles by confocal scanning laser microscopy.

Confocal microscopy was used for the measurement of plasmid DNA adsorbed to individual adsorbent particles intended for anion-exchange and triple helix affinity chromatography. Plasmid DNA was visualized with the fluorescent dye YOYO-1, that forms a highly fluorescent complex with double stranded DNA. Confocal images were translated into fluorescence intensity profiles and the distribution of plasmid DNA in the particles was measured. The results that adsorption of plasmid DNA mainly takes place in an outer layer of the particles. The described procedure can also be advantageously used to demonstrate triple helix formation between plasmid DNA and immobilized oligonucleotides.

Immobilised metal ion affinity chromatography purification of alcohol dehydrogenase from baker's yeast using an expanded bed adsorption system.

Alcohol dehydrogenase (ADH) from solutions of homogenised packed bakers' yeast has been successfully purified using immobilised metal-ion affinity chromatography in an expanded bed. Method scouting carried out using pure ADH solutions loaded onto 5-ml HiTrap columns charged with Zn2+, Ni2+ and Cu2+ and eluted using 0-50 mM EDTA gradient found that charging with Zn2+ gave the highest recovery and the lowest EDTA concentration required for elution. These results were used to develop a protocol for the expanded bed system and further tested using clarified yeast homogenate loaded onto XK16/20 packed beds (approximately 30 ml) packed with Chelating Sepharose FastFlow matrix in order to determine the optimum elution conditions using EDTA. The ADH was found to elute at 5 mM EDTA and the dynamic and total binding capacities of Streamline chelating for ADH were found to be 235 U/ml and 1075 U/ml matrix, respectively. Expanded bed work based on a step EDTA elution protocol demonstrated that ADH could be successfully eluted from unclarified homogenised bakers' yeast diluted to 10 mg/ml total protein content with a recovery of 80-100% that was maintained over five consecutive runs with a vigorous clean-in-place procedure between each run.

Expanded-bed adsorption in industrial bioprocessing: recent developments.

Expanded-bed adsorption allows the capture of proteins from particle-containing feedstocks without prior removal of particulates, thus enabling clarification of a cell suspension or cell homogenate and the concentration of the desired product in a single operation. This usually results in higher product recovery in a shorter time period. Process development and scale-up of an expanded-bed operation is convenient because both the adsorbent and the equipment are similar to those in conventional packed-bed chromatography. This article reviews the recent developments in expanded-bed adsorption technology and the range of applications that are now being achieved.

The effect of Syntex adjuvant formulation (SAF-m) on humoral immunity to the influenza virus in the mouse.

Syntex adjuvant in its microfluidized form (SAF-m) was equal to or superior to Freund's complete adjuvant in stimulating an enhanced hemagglutination inhibition (HI) antibody response in mice to trivalent influenza virus vaccine (TIV). There was an average 16-fold increase in HI titer for the three components of the vaccine with no significant differences among strains. The increased serum antibodies correlated with an increase in protection against infection. The threonyl-MDP (t-MDP) component of the adjuvant played no role in this activity. The vehicle, in contrast, was so effective that it could be diluted 1:202 (in the presence of (t-MDP) and still retain a statistically significant effect. Vaccine and adjuvant could be stored together at 4 degrees C for 2 years without a statistically significant change in potency. Mice were given a priming immunization with TIV, PBS, or adjuvanted TIV (AIV). A year later, the mice were boosted with heterotypic TIV or AIV. The nature of the priming immunization made no difference in the strong antibody response to an AIV boost. However, priming significantly improved the response to TIV with AIV being the best primer. The enhancement in the antibody response to AlShanghai of the unprimed (PBS) elderly mice caused by AIV (14-fold improvement over TIV) was similar to that in young mice. Female mice had antibody titers which overall were 2.6-fold higher than those of males (P < 0.0001) for AIV and TIV.

Factors affecting the yield of cold-adapted influenza virus vaccine.

Various manipulations to production procedures have been investigated in order to discover methods to attain adequate or augmented titers of cold-adapted influenza virus (CAIV) vaccine. The methods modified include those used for reassortant selection and the determination of virus growth parameters. Increased infectivity titers were achieved through selection of high-yielding mutants by isolating multiple plaques during plaque purification of reassortant clones, as well as through optimization of egg incubation times, age, and lot for individual strains. Up to 6-fold increases in virus yield were obtained by selecting high yielding mutants, up to 9-fold increases were achieved by modifying egg incubation times, and a nearly 1 log increase was realized by determining the ideal egg age for individual strains.

Analysis of some operating parameters of novel adsorbents for recovery of proteins in expanded beds.

We have analysed some operational parameters for two novel adsorbents intended for recovery of proteins from particle-containing feedstocks using expanded bed adsorption. The adsorbents tested were STREAMLINE DEAE and STREAMLINE SP, ion exchangers based on an agarose/crystalline quartz composite. Parameters analysed included bed expansion, adsorption efficiency, washing and elution. Bed expansion was considerably lower for STREAMLINE adsorbents compared to conventional agarose based media, higher flow velocities were thus possible during the expanded bed process. Breakthrough capacity was 63 mg ml-1 for lysozyme on STREAMLINE SP and 36 mg ml-1 for bovine serum albumin on STREAMLINE DEAE at a flow velocity of 300 cm h-1. To achieve high breakthrough capacity, the sedimented bed height should be at least 10 cm. Furthermore, breakthrough capacity increased to some extent when temperature was increased from room temperature to 36 degrees C, a phenomenon which can be useful in some processes. The number of living E. coli cells in the effluent was reduced by a factor of 10(5) after washing with 15 sedimented bed volumes. The optimal flow velocity for elution was 100 cm h-1 considering time for elution and volume of the eluted fraction. Flow direction during elution in packed bed mode had little impact on the elution volume, however, elution in expanded bed mode increased the volume by approx. 40%. The data presented on the performance of STREAMLINE adsorbents show that they are very useful for recovery of proteins from particle-containing feedstocks using expanded bed adsorption.

Rapidly progressive Whipple's disease of the central nervous system.

We describe a patient with rapidly progressive Whipple's disease confined to the central nervous system (CNS). The diagnosis was made pre-mortem following stereotactic and open brain biopsis and confirmed at autopsy. Despite appropriate antibiotic treatment, the disease ran a fulminant course to death after nine weeks.

Pilot scale recovery of recombinant annexin V from unclarified escherichia coli homogenate using expanded bed adsorption.

Expanded bed adsorption is a new downstream processing technique forcapture of proteins directly from unclarified feedstocks. Expanded bed adsorption reduces the number of operations in purification processes by combining clarification, concentration, and capture into one operation. It is based on stable fluidization and uses adsorbent particles with well-defined size and density distributions, together with columns designed to giveeven liquid flow distribution. The bed expands as the adsorbent particles are lifted by an upward liquid flow through the column. The behavior of the expanded bed is similar to a packed chromatography bed due to very little back-mixing of the adsorbent particles. The major benefit of using anexpanded bed is that adsorption can be carried out with unclarified feedstocks; there is no need for centrifugation or filtration to remove cells and debris. When the feedstock is applied, the target protein is captured by the adsorbent while cells and debris pass through the column unhindered. Washing is performed with the bed in an expanded mode, followed by elution of bound protein in a sedimented mode with downward flowDescribed in this article is the use of expanded bed adsorption for pilot scale recovery of recombinant human placental annexin V from an Escherichia coli ho mogenate. The description includes the whole procedure, from small-scale method optimization to pilot scale. The recovery of annexin V was approximately 95% at both lab scale and pilot scale. During the trials, it was discovered that the expanded bed was affected by the biomass content and viscosity of the homogenate. The upper limits for these parameters were therefore investigated further. For the E. coli used in the application described here, homogenates with biomass dry weightup to 5% and viscosities up to 10 mPa s (at a shear rate of 1 s(-1)) worked best. It was, however, feasible to use homogenates with dry weight up to 7-8% and viscosities up to 50 mPa s (1 s(-1)). (c) 1994 John Wiley & Sons, Inc.

Relevance of the guinea-pig necrotic inflammation footpad reaction to safety concerns with MDP-based adjuvants.

The guinea-pig necrotic inflammatory reaction described by Nagao and Tanaka was investigated to determine its possible relevance to tuberculin-positive individuals who may receive MDP-based adjuvants. The reaction was induced by a preparatory footpad injection of Mycobacterium tuberculosis in Freund's incomplete adjuvant (FIA) followed three weeks later by a provocative gluteal injection of muramyl dipeptide (MDP). Increased footpad swelling and necrosis occurred within 24 h. The reaction was also caused to a significantly lesser degree by a gluteal injection of the threonyl-MDP component of Syntex adjuvant SAF-m. Elicitation of the reaction in responsive animals was absolutely dependent on the presence of both FIA and M. tuberculosis at the reaction site. Animals injected in the right footpad with FIA plus M. tuberculosis and in the left footpad with M. tuberculosis alone responded to the provocative injection with necrosis in the right footpad only. The reaction therefore appears to have little potential relevance to the vaccination of tuberculin-positive humans.

Single-step recovery of a secreted recombinant protein by expanded bed adsorption.

We have used an expanded bed adsorption procedure for efficient recovery of a recombinant fusion protein, directly from a crude fermentor broth without prior cell removal. The fusion protein was designed to have a relatively low isoelectric point (pI) to allow anionic exchange adsorption at pH 5.5 where most Escherichia coli host proteins are not adsorbed. The gene product was secreted to the culture medium of the E. coli host cells in high yields (550 mg/l). The separation of cells and the concentration and recovery of the fusion protein could therefore be achieved by a single unit operation. The yield after the expanded bed adsorption exceeded 90 percent. Furthermore, the significant volume reduction by the expanded bed adsorption, enabled efficient and straight-forward polishing of the product by a subsequent affinity chromatography step, for removal of contaminating DNA and pyrogenic compounds to levels acceptable for regulatory authorities. An overall yield exceeding 90 percent was maintained after the affinity chromatography polishing step. The procedure outlined here is suitable for large-scale bioprocesses and allows efficient removal of cells, host proteins, contaminating DNA and endotoxins.

Adenovirus-human immunodeficiency virus (HIV) envelope recombinant vaccines elicit high-titered HIV-neutralizing antibodies in the dog model.

Recombinant human adenoviruses (Ads) (types 4, 5, and 7) expressing the HIV-1 envelope membrane glycoprotein (gp160) were tested for immunogenicity in the dog. Administration of recombinant Ad7-env by intratracheal inoculation resulted in a low serum antibody response to gp160, which developed over several weeks. A strong neutralizing antibody response to the Ad7 vector developed within 1 week of infection. A subsequent booster inoculation 12 weeks later with the heterotypic Ad4-env recombinant virus resulted in significantly enhanced humoral responses directed at the envelope antigen, as measured by both ELISA and Western blot analysis as well as high-titer type-specific neutralizing antibodies, with some animals achieving neutralization titers approaching 1000. Recombinant HIV envelope glycoprotein derived from Ad-HIV-infected cell cultures was used as a subunit booster injection for dogs that had previously received sequential immunizations with heterotypic recombinant Ads. Significant immune responses against the envelope developed as measured by ELISA, Western blot analysis, and neutralization assays. These data indicate that live recombinant Ad-HIV vaccines are capable of inducing high-titer type-specific neutralizing antibodies to gp160 in vivo. Recombinant HIV envelope glycoprotein subunit vaccines, prepared from Ad-env-infected cells, are capable of boosting these responses.

A case of neurofibromatosis 2 presenting with a mononeuritis multiplex.

A patient with neurofibromatosis 2 had an asymmetrical peripheral neuropathy. A nerve biopsy specimen revealed neurofibromatous changes, and the neuropathy may have been a direct consequence of neurofibromatosis. An apparent clinical response to immunosuppressive treatment and plasma exchange is also reported.

A new hamster model for adenoviral vaccination.

Both adult and baby hamsters infected intranasally with human adenovirus type 5 exhibited virologic, serologic, and histologic evidence of infection. When 8-day old hamsters were infected with 4 x 10(6) pfu, concentrations of virus up to 2 x 10(6) pfu/animal were detected in the lung, peaking on day 2. The minimum infectious dose was 1 x 10(3) pfu/animal. This model may be useful in studies of conventional and recombinant adenoviral vaccines for humans.

Isolation of bacterial plasmids by density gradient centrifugation in cesium trifluoroacetate (CsTFA) without the use of ethidium bromide.

Plasmids extracted from bacterial cells by alkaline extraction can easily be isolated from linear DNA by isopycnic centrifugation in CsTFA. This is a fast and simple method which circumvents the use of the intercalating dye, ethidium bromide, and consequently the problems associated with its removal. The buoyant densities for covalently closed circular DNA and linear DNA in CsTFA are 1.60 g/ml and 1.65 g/ml, respectively. The isolation is achieved regardless of plasmid size and can be accomplished at temperatures of between 4 and 30 degrees C. Plasmid DNA isolated in gradients of CsTFA are of a high purity and have been found to be intact when cleaved with restriction enzymes and ligated with T4 DNA ligase.