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S Reuveny

Publications and source records attributed to S Reuveny.

At least 19 recordsLinked to original sources
PubMed2001

Search for correlates of protective immunity conferred by anthrax vaccine.

Vaccination by anthrax protective antigen (PA)-based vaccines requires multiple immunization, underlying the need to develop more efficacious vaccines or alternative vaccination regimens. In spite of the vast use of PA-based vaccines, the definition of a marker for protective immunity is still lacking. Here we describe studies designed to help define such markers. To this end we have immunized guinea pigs by different methods and monitored the immune response and the corresponding extent of protection against a lethal challenge with anthrax spores. Active immunization was performed by a single injection using one of two methods: (i) vaccination with decreasing amounts of PA and (ii) vaccination with constant amounts of PA that had been thermally inactivated for increasing periods. In both studies a direct correlation between survival and neutralizing-antibody titer was found (r(2) = 0.92 and 0.95, respectively). Most significantly, in the two protocols a similar neutralizing-antibody titer range provided 50% protection. Furthermore, in a complementary study involving passive transfer of PA hyperimmune sera to naive animals, a similar correlation between neutralizing-antibody titers and protection was found. In all three immunization studies, neutralization titers of at least 300 were sufficient to confer protection against a dose of 40 50% lethal doses (LD(50)) of virulent anthrax spores of the Vollum strain. Such consistency in the correlation of protective immunity with anti-PA antibody titers was not observed for antibody titers determined by an enzyme-linked immunosorbent assay. Taken together, these results clearly demonstrate that neutralizing antibodies to PA constitute a major component of the protective immunity against anthrax and suggest that this parameter could be used as a surrogate marker for protection.

Animals↗
PubMed1995

Involvement of oligomerization, N-glycosylation and sialylation in the clearance of cholinesterases from the circulation.

The possible role of post-translational modifications such as subunit oligomerization, protein glycosylation and oligosaccharide processing on the circulatory life-time of proteins was studied using recombinant human acetylcholinesterase (rHuAChE). Different preparations of rHuAChE containing various amounts of tetramers, dimers and monomers are cleared at similar rates from the circulation, suggesting that oligomerization does not play an important role in determining the rate of clearance. An engineered rHuAChE mutant containing only one N-glycosylation site was cleared from the circulation more rapidly than the wild-type triglycosylated enzyme. On the other hand, hyperglycosylated mutants containing either four or five occupied N-glycosylation sites, analagous to those present on the slowly cleared fetal bovine serum acetylcholinesterase (FBS-AChE), were also cleared more rapidly from the bloodstream than the wild-type species. Furthermore, the two different tetraglycosylated mutants were cleared at different rates while the pentaglycosylated mutant exhibited the most rapid clearance profile. These results imply that though the number of N-glycosylation sites plays a role in the circulatory life-time of the enzyme, the number of N-glycan units in itself does not determine the rate of clearance. When saturating amounts of asialofetuin were administered together with rHuAChE, the circulatory half-life of the enzyme was dramatically increased (from 80 min to 19 h) and was found to be similar to that displayed by plasma-derived cholinesterases while desialylation of these enzymes caused a sharp decrease in the circulatory half-life to approximately 3-5 min. Determination of the average number of sialic acid residues per enzyme subunit of the five different N-glycosylation species generated, revealed that the rate of clearance is not a function of the absolute number of appended sialic acid moieties but rather of the number of unoccupied sialic acid attachment sites per enzyme molecule. Specifically, we demonstrate an inverse-linear relationship between the number of vacant sialic acid attachment sites and the values of the enzyme residence time within the bloodstream.

Acetylcholinesterase↗
PubMed1993

Effect of temperature and oxygen on cell growth and recombinant protein production in insect cell cultures.

The effect of temperature and O2 saturation on the production of recombinant proteins beta-galactosidase and human glucocerebrosidase by Spodoptera frugiperda cells (Sf9) infected with recombinant Autographa californica nuclear polyhedrosis virus was investigated. The rates of cell growth, glucose consumption, O2 consumption and product expression were measured at temperatures between 22 degrees C and 35 degrees C. The results indicated that possible O2 limitation may be alleviated without compromising the maximum cell yield by lowering the incubation temperature from 27 degrees C to 25 degrees C. The expression level of the recombinant proteins at 27 degrees C was similar to that obtained at 22 degrees C and 25 degrees C; lower protein yields were obtained at 30 degrees C. An increase in temperature from 22 degrees C to 27 degrees C led to earlier production of the proteins and to an increase in the proportion of the product released outside the cells.

Animals↗
PubMed1993

Evaluation of anchorage-dependent cell propagation systems for production of human acetylcholinesterase by recombinant 293 cells.

Production of recombinant human acetylcholinesterase (AChE) by a high producer human embryonic kidney cell line (293) was evaluated by three main cell propagation systems; surface propagator, fixed-bed reactor and stirred microcarrier cultures. The recombinant cell line expresses AChE levels as high as 10-20 mg/l/day. System productivities in either the surface propagator (multitray system), or in the fixed-bed reactor (polyurethane macroporous sponges) were 4-8 mg AChE/l/day during a production period of 8 days. Similar productive rates, yet longer production periods (up to 22 days), were obtained in microcarrier (MC) cultures using either polystyrene beads (Biosilon); collagen-coated dextran beads (Cytodex-3); or gelatin macroporous beads (Cultispher-G). Best results were obtained in an aggregate culture using cellulose beads charged with diethylaminoethyl (DEAE) groups, (Servacel), as carriers. In this culture, a system productivity of 6-10 mg/l/day was maintained for 28 days.

Acetylcholinesterase↗
PubMed1992

Differentiation of myoblasts and CNS cells grown either separately or as co-cultures on microcarriers.

Dispersed neuronal and muscular elements from fetal or neonatal origin, can organize and mature in culture when grown on positively charged cylindrical microcarriers (MCS), to a stage which simulate in vivo maturation. Cells arrange themselves on the MCS to form aggregates which remain floating in the nutrient medium. In such a tridimensional organization, the neuronal tissue is capable of regenerating a network of nerve fibers which establish synapse interconnections and undergo myelination. Oligodendrocytes organize on MCS in a tridimensional pattern and produce extensive myelin-like membranes. Myoblasts in MC-cultures fuse into polynucleated myotubes which become striated and contract spontaneously. Creatine kinase and acetylcholine receptor (AChR) are formed during myogenesis in similar quantities in MC-cultures and in monolayers. When both neuronal and muscle tissues are prepared from the same fetus (autologous nerve-muscle co-cultures) and are cultured on MCS, they interconnect to form neuro-muscular junctions. Cells from both tissues, exhibit better differentiation, for longer periods in MC-cultures than they do in monolayers. The floating functional entities are easy to sample and can be harvested for ultrastructural, immunocytochemical and biochemical analysis. In addition, MC-cultures can be used as a good tool for the study of acute and chronic exposures to toxicological agents, as well as for implantation into demyelinated, injured or dystrophic tissues. In this case the MCS in the implanted entities will serve as identifiable markers.

Animals↗
PubMed1990

Selective adherence of neurons and glial cells from dissociated cerebral and spinal cord microcarrier cultures.

In stationary cultures of dissociated brain and spinal cord grown on microcarriers (MCs), the neuronal and ependymal cells attached to the MCs forming floating aggregates in which they grow in a three-dimensional pattern. The glial and meningeal elements on the contrary, tend to dissociate from the aggregates and adhere to the plastic dish where they divide to form a monolayer. This different behavior of CNS components is not observed in rotating cultures in which all CNS cells remain attached to the MCs and develop into mature floating structures. This cell separation in stationary MC-cultures which is documented here by SEM and immunocytochemistry, may be useful for analysis and evaluation of the metabolic biochemical events of each of the cellular components derived from the same culture.

Animals↗
PubMed1987

Effect of feeding rate on monoclonal antibody production in a modified perfusion-fed fermentor.

Previously we described a perfusion system for production of high yields of monoclonal antibodies in a fermentor. This system incorporated a cylindrically shaped, stainless steel filter mounted around the stirring shaft for retention of cells within a 1 liter fermentor. Modification of this filter by increasing the pore size from 5 micron to 10 micron decreased its tendency to clog and allowed continuous operation for about 3 weeks. Fresh culture medium, containing 6.5 mg glucose/ml and 3% horse serum, was supplied continually at two different perfusion rates, 850 and 1100 ml/day. Spent culture medium containing monoclonal antibody was harvested concomitantly. Highest cell density (5 X 10(7)/ml) and best antibody yield (1.7 g/l culture per day) were obtained at the higher feeding rate.

Antibodies, Monoclonal↗
PubMed1987

Microcarriers as a culturing system of insect cells and insect viruses.

There is an increasing interest in culturing of insect cells, which are host for arthropod-born (Arbo) viruses. The potential applications of Arbo viruses are in the following two main fields: 1. Medical applications (e.g. preparation of viral vaccines and viral antigens for diagnostic purposes). 2. As bioinsecticides in pest control in horticulture, agriculture and forestry. One of the potential cell substrates for these applications is an anchorage-dependent-mosquito cell line established from embryonic tissues of Aedes aegypti (AA). The following areas were investigated in the reported research with the AA cell line: The AA cells were successfully propagated in microcarrier (MC)-culturing-systems. Of the tested MC's the cellulose-based microgranular MC (DE-53 of Whatman, having an exchange capacity of 2 meq/g) was found to be the best MC. Cells grew in MCs-cells aggregates in submerged spinner culture. The AA-MC's culture was successfully scaled-up to 8 litre culture volume. "Trypsinization" of the AA cells from the MC surface is successfully done by RDB, a dispersion agent from a plant origin (produced and marketed at the author's Institute). Other known dispersion agents (trypsin, collagenase, pronase) failed to disperse the AA cells from the MC. A serum-free medium was developed for culturing the AA cells on the DE-53 MC's. Bovine serum albumin was the main serum substituant in the developed medium. Arboviruses, from the Toga group, were grown in the AA-MC culture. Sindbis virus (from alpha-group) and West Nile virus (from the Flavi group), chronically infected the AA cells, which continuously produce and liberate these two viruses.(ABSTRACT TRUNCATED AT 250 WORDS)

Aedes↗
PubMed1987

Production of carcinoembryonic antigen from a human colon adenocarcinoma cell line. II. Use of monoclonal antibodies to carcinoembryonic antigen for antigen purification and characterization.

Monoclonal antibodies (McAbs) were prepared against carcinoembryonic antigen (CEA), derived from human metastases to the liver of primary colon adenocarcinoma. A stable clone, secreting monoclonal anti-CEA antibody (Ab), was selected. The hybridoma cells were grown in BALB/C mice, forming ascites fluids containing 2-5 mg/ml of the relevant McAbs. Ascites fluids were purified using a DEAE-Sephacel column. The purified immunoglobulin fraction was characterized as IgG2b, having a dissociation constant of 2 X 10(-9)M. The potential use of these McAbs for CEA diagnosis and purification was evaluated: the IgG2b fraction, when bound to Sepharose 4B, facilitated the purification of CEA from spent media of a human colon adenocarcinoma cell line, resulting in iodination grade CEA; the anti-CEA McAbs demonstrated differential histochemical staining of various sections, both fresh and fixed; the McAbs were also found to be suitable as tagging Abs in a CEA RIA.

Adenocarcinoma↗
PubMed1987

Factors affecting monoclonal antibody production in culture.

Factors that affect production of monoclonal antibodies (McAb) by a murine cell line were investigated. The goal was to estimate the efficiency of large scale production in stirred reactors. It was found that in batch cultures most McAb was produced after the log growth phase; final yield was 100-200 micrograms/ml. Yields of McAb were increased to 290 micrograms/ml by feeding cells glucose and glutamine. Lactic acid, which was produced in culture as a result of glucose metabolism, had no toxic effect on cells, while another waste product, ammonium ion, was probably accumulated at toxic levels during late stages of cell growth. The hybridoma cell line was propagated in four different systems: fed-batch, semi-continuous, two stage and perfusion. These systems were compared to batch cultures for their effect on cell viability and antibody production. Daily addition of fresh medium (fed-batch propagation) increased antibody productivity from 15 (batch culture) to 27 mg/l of culture/day. In the semi-continuous culture productivity was raised to 34 mg/l/day. Further increase in productivity to a level of 62 mg/l/day was achieved by applying a second batch stage to the semi-continuous culture. A perfusion culturing method was the most effective for production of McAb. Average concentrations of 2.2 X 10(7) live cells/ml and 390 micrograms of antibody/ml corresponding to productivity of 660 mg/l/day were achieved. Serum concentration in the medium was reduced to 0.125% resulting in a specific activity of 0.4 mg of McAb per mg of protein in the cell-free culture broth.

Ammonia↗
PubMed1987

Production of carcinoembryonic antigen from a human colon adenocarcinoma cell line. I. Large-scale cultivation of carcinoembryonic antigen-producing cells on cylindric cellulose-based microcarriers.

A continuous cell line from a human colon carcinoma, designated HuCC1-14, was successfully grown on cyclindrical cellulose microcarriers (MC) charged by DEAE. The cells grow on these carriers as large cell-MC aggregates and maintain high cell densities in submerged conditions for extended periods. HuCC1-14 cells secrete into the culture medium significant amounts of carcinoembryonic antigen (CEA). Maximum efficiency in CEA secretion occurred after the cells switched from logarithmic growth to stationary phase. At this state, high CEA levels could be obtained with low-serum medium which greatly facilitates subsequent product purification. The described method provides a system which can be scaled-up and produce this tumor-associated antigen in essentially unlimited amounts and reproducible quality for detection and monitoring of cancer patients.

Adenocarcinoma↗
PubMed1986

Kinetics of monoclonal antibody production in low serum growth medium.

Factors affecting growth and monoclonal antibody production in vitro by a mouse-mouse hybridoma cell line have been investigated in a series of studies. The goal was to maximize antibody yields and demonstrate that antibodies can be produced efficiently on a large-scale in fermentors. This initial report describes (i) development of a radial immunodiffusion assay for accurate determination of antibody levels in culture, (ii) a culture medium formulation that allowed for reduction in the amount of fetal bovine serum required for good cell growth, and (iii) the kinetics of cell growth and monoclonal antibody production in low-serum media. The radial immunodiffusion assay, employing rabbit anti-mouse IgG antibodies in the immobile phase and the monoclonal antibody (an IgG2a to Rhizobium japonicum cells) as the antigen in the mobile phase, was more reproducible and reliable for determining antibody levels in culture broth than was an indirect enzyme-linked immunosorbent assay. Addition of 0.25% Primatone RL and 0.01% Pluronic F-68 to Dulbecco's modified Eagle medium allowed cells to adapt to growth in medium containing as little as 1% fetal bovine serum; without these additives, 5% serum was the lowest level attained. For the kinetic studies, cells were grown in the low-protein medium in 3 liter spinner flasks. Antibody production occurred during the growth phase, however, significant amounts were also produced during later phases when the cells had stopped growing. Final titers were 100-200 micrograms/ml. It was concluded that maintenance of cell viability is more important than growth rate in production of antibody. This conclusion, confirmed in other studies, has developed into the major underlying strategy employed in subsequent investigations to maximize antibody production in stirred reactors.

Animals↗
PubMed1986

Factors affecting cell growth and monoclonal antibody production in stirred reactors.

Environmental and cultural factors that could affect growth and cell viability of mouse-mouse hybridoma cells in culture were investigated. The aim was to determine conditions that could prolong viability and result in increased yields of monoclonal antibodies in stirred reactors. Factors studied included temperature, level of dissolved oxygen, nutrient depletion, and waste product accumulation. Growing cells at temperatures 3-9 degrees lower than optimum (37 degrees C) increased viability but monoclonal antibody production was lowered. A low level of dissolved oxygen (25% air saturation compared to 60% for optimum growth) prolonged cell viability and increased the monoclonal antibody yield by about 50%. Feeding cultures daily to maintain the glucose level above 1 mg/ml and at the same time feeding cells glutamine (150 micrograms/10(6) cells per day) maintained the level of viable cells at 1.7 X 10(6)/ml for at least 9 days and resulted in an antibody yield of 290 micrograms/ml, about a 70% increase over cultures fed either glucose or glutamine alone. Ammonium ion, added to cell populations at levels produced in cultures, stopped cell growth and decreased antibody production. Another waste product, lactic acid, had no toxic effect when added to media at levels found in cultures. These results agree with our suggestion that monoclonal antibody production is enhanced by maintaining cell viability over a prolonged period and provide a base for investigating modes of hybridoma cell propagation in fermentors.

Ammonia↗