An Automated HIV-1 Env-Pseudotyped Virus Production for Global HIV Vaccine Trials

Background

Infections with HIV still represent a major human health problem worldwide and a vaccine is the only long-term option to fight efficiently against this virus. Standardized assessments of HIV-specific immune responses in vaccine trials are essential for prioritizing vaccine candidates in preclinical and clinical stages of development. With respect to neutralizing antibodies, assays with HIV-1 Env-pseudotyped viruses are a high priority. To cover the increasing demands of HIV pseudoviruses, a complete cell culture and transfection automation system has been developed.

Methodology/Principal Findings

The automation system for HIV pseudovirus production comprises a modified Tecan-based Cellerity system. It covers an area of 5×3 meters and includes a robot platform, a cell counting machine, a CO₂ incubator for cell cultivation and a media refrigerator. The processes for cell handling, transfection and pseudovirus production have been implemented according to manual standard operating procedures and are controlled and scheduled autonomously by the system. The system is housed in a biosafety level II cabinet that guarantees protection of personnel, environment and the product. HIV pseudovirus stocks in a scale from 140 ml to 1000 ml have been produced on the automated system. Parallel manual production of HIV pseudoviruses and comparisons (bridging assays) confirmed that the automated produced pseudoviruses were of equivalent quality as those produced manually. In addition, the automated method was fully validated according to Good Clinical Laboratory Practice (GCLP) guidelines, including the validation parameters accuracy, precision, robustness and specificity.

Conclusions

An automated HIV pseudovirus production system has been successfully established. It allows the high quality production of HIV pseudoviruses under GCLP conditions. In its present form, the installed module enables the production of 1000 ml of virus-containing cell culture supernatant per week. Thus, this novel automation facilitates standardized large-scale productions of HIV pseudoviruses for ongoing and upcoming HIV vaccine trials.     

A proteomic investigation of Fusobacterium nucleatum alkaline-induced biofilms

ABSTRACT: BACKGROUND: The Gram negative anaerobe Fusobacterium nucleatum has been implicated in the aetiology of periodontal diseases. Although frequently isolated from healthy dental plaque, its numbers and proportion increase in plaque associated with disease. One of the significant physico-chemical changes in the diseased gingival sulcus is increased environmental pH. When grown under controlled conditions in our laboratory, F. nucleatum subspecies polymorphum formed mono-culture biofilms when cultured at pH 8.2. Biofilm formation is a survival strategy for bacteria, often associated with altered physiology and increased virulence. A proteomic approach was used to understand the phenotypic changes in F. nucleatum cells associated with alkaline induced biofilms. The proteomic based identification of significantly altered proteins was verified where possible using additional methods including quantitative real-time PCR (qRT-PCR), enzyme assay, acidic end-product analysis, intracellular polyglucose assay and Western blotting. RESULTS: Of 421 proteins detected on two-dimensional electrophoresis gels, spot densities of 54 proteins varied significantly (p < 0.05) in F. nucleatum cultured at pH 8.2 compared to growth at pH 7.4. Proteins that were differentially produced in biofilm cells were associated with the functional classes; metabolic enzymes, transport, stress response and hypothetical proteins. Our results suggest that biofilm cells were more metabolically efficient than planktonic cells as changes to amino acid and glucose metabolism generated additional energy needed for survival in a sub-optimal environment. The intracellular concentration of stress response proteins including heat shock protein GroEL and recombinational protein RecA increased markedly in the alkaline environment. A significant finding was the increased abundance of an adhesin, Fusobacterial outer membrane protein A (FomA). This surface protein is known for its capacity to bind to a vast number of bacterial species and human epithelial cells and its increased abundance was associated with biofilm formation. CONCLUSION: This investigation identified a number of proteins that were significantly altered by F. nucleatum in response to alkaline conditions similar to those reported in diseased periodontal pockets. The results provide insight into the adaptive mechanisms used by F. nucleatum biofilms in response to pH increase in the host environment.     

Stereochemistry of lignans in Phaleria macrocarpa (Scheff.) Boerl

Phaleria macrocarpa (Scheff.) Boerl., a member of the Thymelaeaceae, is traditionally used in Indonesia as medicinal plant against cancer. In this context, we isolated the lignans pinoresinol, lariciresinol and matairesinol from different parts of this plant. The enantiomeric composition of these lignans was determined by chiral column analysis. Pinoresinol and lariciresinol were mixtures of both enantiomers with (79 +/- 4)% and (55 +/- 6)% enantiomeric excess for the (-)-enantiomers, respectively, whereas matairesinol was found as pure (+)-enantiomer.     

Oral Presentations OP02: Myelin and Diseases of Myelin. NPP2, A matricellular protein expressed during myelination,actively modulates oligodendrocyte adhesion

NPP2, also known as phosphodiesterase‐I alpha/autotaxin, is a type‐II membrane protein that belongs to the nucleotide pyrophosphatase/phosphodiesterase family (NPP). We have recently demonstrated that NPP2 is expressed and released by differentiating oligodendrocytes during the critical stages of CNS myelination. The structural domains of this secreted macromolecule suggest a functional role in the regulation of oligodendrocyte adhesion. Here, we present data that demonstrates that NPP2 interferes with the ability of oligodendroglial cells to adhere to known CNS adhesion molecules present during the onset of myelination, such as fibronection, vitronectin, and merosin (laminin2). Responses to NPP2 appear to be regulated by a different mechanism depending on the developmental stage of the oligodendrocyte. Although the exact mechanisms for NPP2 mediated counter‐adhesion are unknown, our studies have implicated that an active signalling mechanism involving heterotrimeric G proteins is responsible for adhesion modulation. These studies clearly define a role of NPP2 as a matricellular protein modulating oligodendrocyte adhesion and suggest that NPP2 function may represent the first step of oligodendrocyte remodelling when differentiating oligodendrocytes are actively involved in the formation of the myelin sheath.     

Chlorophyll fluorescence quenching and violaxanthin deepoxidation of FBPase antisense plants at low light intensities and low temperatures

Genetically modified potato (Solanum tuberosum L. cv. Desiree) and tobacco (Nicotiana tabacum cv. Samsun N.N.) plants were used to analyze the effects exerted by the chloroplastic (cp) fructose- 1,6-bisphosphatase (FBPase) on the regulation of light energy discrimination at the level of photosystem II. The cp-FBPase activity was progressively inhibited by an mRNA antisense to this FBPase. The chlorophyll fluorescence quenching parameters of these transgenic plants were compared to those of wild-type and transgenic plants that were acclimated to low temperatures. In particular various lines of the transgenic potato and tobacco plants were exposed to a temperature treatment of 10 and 20°C for 10 days. Light intensities were kept low to reduce photoinhibition so that we could analyze exclusively the effects of a modification in the carbon fixation cycle on the chlorophyll fluorescence quenching parameters. The photon flux densities (PFDs) employed at the level of the middle leaves of all plants were set to two different values of 10 μmol m^?2 s^?1 and 50 μmol m^?2 s^?1. Subsequent to this 10-day acclimation the chlorophyll-fluorescence parameters of all plants were measured. Photoinhibition as expressed by the F_y/F_m ratio was minor in plants subjected to a PFD of 10 μmol m^?2 s^?1. Higher photon fluence rates of 50 μmol m^?2 s^?1 at temperatures of 10°C gave rise to a significant reduction in the F_y/F_m ratios obtained from the transgenic plants which were characterized by a restriction in cp-FBPase capacity to 20% of normal activity. Furthermore, a progressive inhibition of the cp-FBPase activity induced an amplified nonphotochemical quenching of chlorophyll fluorescence with in the genetically manipulated species (except at 10°C and 50 μmol m^?2 s^?1). The increase in nonphotochemical quenching depended upon light and temperature. Photochemical quenching of light quanta within the antisense plants declined relative to that in the wild type. To further characterize the mechanisms producing higher levels of nonphotochemical fluorescence quenching. we analyzed several of the xanthophyll cycle pigments. The deepoxidation state of the xanthophyll cycle pigments in potato plants increased with attenuating FBPase activities under all conditions. For tobacco plants, this elevation of the deepoxidation state was only observed at a PFD of 50 μmol m^?2 s^?1.     

An attempt to estimate the predatory pressure exerted by the lesser weever, Trachinus vipera Cuvier, in the southern North Sea

In order to evaluate the impact of the lesser weever on the ecosystem of the southern North Sea, geographical distribution, density, growth, production and food requirements have been estimated. High densities were found on and around the Brown Ridge, an area with high tidal current velocities, medium grain-size of the sediment and a poor benthic fauna. Growth is restricted to the months of June October. During the winter cessation of growth a considerable loss of weight (about 20%) takes place. Mortality has been estimated by using the average size frequency distribution of all catches made from 1972 to 1984. The resulting convex type of survival curve indicates a high survival rate of the II to IV-group fishes. The production (estimated with Allen's graphical method) of a population of 100 individuals including all age groups (0-VI) amounts to 123.7 g AFDW-year'. In areas with highest densities, consequently, production amounts to 0.018–0.078 g AFDW-m² -year^-1. With an assumed transfer efficiency of 10% through the year, food requirements amounts to 0.18–0.78 g AFDW-m ² -year ^-1. Since the lesser weever feeds mainly on fish (85.6%), almost exclusively on gobies (Pomatoschistus sp.), and with an assumed transfer efficiency of approximately 10%, the indirect predatory pressure exerted by it may amount to 1.6 6.7g AFDW-m ².year ^-1. A possible feeding by gobies on pelagic organisms (calanoids, mysids) is discussed.     

A comparison of the uptake of [75Se]selenite, [75Se]selenomethionine and [35S]methionine by tissues of ewes and lambs.

The fate of selenium, given as Na2(75)SeO3, or [75Se]selenomethionine, and of [35S]methionine administered intravenously to ewes and lambs, has been examined. The main intention was to follow the incorporation of selenium into protein in a number of tissues, including liver and kidney, and to measure the extent of that incorporation of selenoamino acid, particularly with respect to the administration of selenite. The ewes chosen were lactating ewes with lambs at foot, and the lambs were animals which had been weaned on to fodder low in selenium and were recovering from white muscle disease with selenium therapy. These two experimental situations were chosen as they offered conditions under which selenium incorporation might be considered to be maximal. Entry of isotope into milk was rapid and was greater when 75Se was given as the selenoamino acid than as selenite. In both ewes and lambs greater amounts of activity, derived from selenite, were bound to plasma proteins than to the proteins of milk. This was particularly evident in samples taken some hours after administration. This ability of the plasma to bind selenium was demonstrated by alkaline dialysis. Small, though significant amounts of selenium, derived from Na2(75)SeO3, were incorporated as selenoamino acids into the proteins of liver, kidney and pancreas, as well as into the proteins of milk and plasma. In ewes, both selenomethionine and selenocystine were identified chromatographically in enzyme digests of defatted liver and kidney. Some differences occurred in the distribution of labelled compounds in organs from lactating ewes and recovering lambs. The incorporation of selenium into protein is discussed briefly in relation to the recent findings of an association between selenium and the enzyme glutathione peroxidase.     

Place learning prior to and after telencephalon ablation in bamboo and coral cat sharks (Chiloscyllium griseum and Atelomycterus marmoratus)

This study assessed complex spatial learning and memory in two species of shark, the grey bamboo shark (Chiloscyllium griseum) and the coral cat shark (Atelomycterus marmoratus). It was hypothesized that sharks can learn and apply an allocentric orientation strategy. Eight out of ten sharks successfully completed the initial training phase (by locating a fixed goal position in a diamond maze from two possible start points) within 14.9 ± 7.6 sessions and proceeded to seven sets of transfer tests, in which sharks had to perform under altered environmental conditions. Transfer tests revealed that sharks had oriented and solved the tasks visually, using all of the provided environmental cues. Unintentional cueing did not occur. Results correspond to earlier studies on spatial memory and cognitive mapping in other vertebrates. Future experiments should investigate whether sharks possess a cognitive spatial mapping system as has already been found in several teleosts and stingrays. Following the completion of transfer tests, sharks were subjected to ablation of most of the pallium, which compromised their previously acquired place learning abilities. These results indicate that the telencephalon plays a crucial role in the processing of information on place learning and allocentric orientation strategies.