Biomedical subjects

H Katinger

Publications and source records attributed to H Katinger.

118 records · Page 7Linked to original sources

PubMedDate not supplied

Rational design of an improved induction scheme for recombinant Escherichia coli.

In Escherichia coli, strong overexpression of a recombinant protein has been shown to be deleterious due to a heavy metabolic burden on the host cell, which may completely cease cell growth before maximum product accumulation has occurred. Aiming at a reduction of very high product formation rates, we engineered E. coli strains by mutating the Leloir pathway for galactose metabolization, so that galactose can be utilized to induce lac derived promoters. The induction with galactose was effective in every strain and expression construct tested, and it reduced the metabolic burden on a highly overproducing clone so that cell growth and product accumulation could be maintained for several generations.

Escherichia coli↗

PubMedDate not supplied

Efficient selection of high-producing subclones during gene amplification of recombinant Chinese hamster ovary cells by flow cytometry and cell sorting.

The screening procedure for high-producing cell lines is extremely time- and labor-intensive and costly, and is at present guided by an empirical approach based on individual experience. Flow cytometry and cell sorting, with its ability to analyze and separate single cells, an ideal method in the selection of such rare cells. The isolation of recombinant cell lines is especially difficult due to repeated gene amplification, which introduces high mutational variation into the population. We have established and evaluated a modification of a previous method that traps secreted product on the surface of the secreting cell, thus allowing direct analysis of single cell specific production rates. This method was used to select for high-producing subclones of a recombinant Chinese hamster ovary (CHO) cell line producing a human antibody against HIV-1 by repeated rounds of gene amplification and cell sorting. This cell line has been amplified in previous investigations, so that the amount of work and testing required by traditional methods can be compared with the protocol described herein. Forty-five 96-well plates were necessary to obtain a high-producing subclone by limited dilution methods, whereas only five plates were required when cell sorting was used. The specific production rate of the best clone obtained by sorting, however, was five times that of the clone obtained by traditional methods. In contrast to the clones obtained by limited dilution, which consisted of several populations of low- and high-producing cells even at high methotrexate concentrations (6.4 microM), the clones isolated by sorting were already homogeneous at 0.8 microM methotrexate.

Animals↗

PubMedDate not supplied

Pilot scale production of a human monoclonal antibody against human immunodeficiency virus HIV-1.

Human monoclonal antibodies against the transmembrane protein gp41 of HIV-1 were isolated and purified on a pilot scale. A purification scheme was established for the production of human monoclonal antibodies on the gram scale. 50 1 of culture supernatant can be treated in one purification cycle. The hybridomas were mass cultured in an airlift fermenter. The culture broth was clarified by microfiltration and chromatographed on CM-Sepharose fast flow and protein A Superose. Scale up of the high performance affinity chromatography from 1 ml protein A Superose up to 40 ml is described. All desalting steps were performed by gel filtration on Sephadex G-25 coarse. The yield of the whole purification procedure is in the range of 50-60%. The purity is higher than 99.9%. DNA and reverse transcriptase could not be detected. The whole method is designed as a basis for scale up to industrial scale. Results from quality control assays have proven the validity of this approach.

Antibodies, Monoclonal↗

PubMedDate not supplied

Vaccine potential of influenza vectors expressing Mycobacterium tuberculosis ESAT-6 protein.

We generated several attenuated recombinant influenza A vectors expressing the Mycobacterium tuberculosis early secretory antigenic target (ESAT-6) protein. The ESAT-6 protein was recently identified as one of the most promising protective antigens for cell-mediated immunity. The obtained vectors appeared to be capable of inducing ESAT-6 specific Th1 immune response in mice after intranasal immunization. We found that double immunization with two influenza vectors of different subtypes provided a significant level of protection in mice, when applied as prophylactic vaccine, as well as substantial therapeutic effect in mice with pre-established tuberculosis infection. Moreover, we found a strong synergistic effect when vaccination with Flu/ESAT-6 vectors was combined with isoniazid treatment, resulting in a dramatic reduction of bacterial load in the lungs of infected mice.

Administration, Intranasal↗

PubMedDate not supplied

Inhibition of HIV-1 infection in vitro by monoclonal antibodies to the complement receptor type 3 (CR3): an accessory role for CR3 during virus entry?

Adhesion molecules are known to contribute to infectivity of HIV-1. Here we tested whether the complement receptor type 3 (CR3, CD11b), an alpha(m)beta2 integrin, plays an accessory role in the infection process of HIV-1, because ICAM-1, a ligand of CR3, is present on the envelope of HIV-1. In addition, the viral transmembrane protein gp41 shares four regions of homology with the complement component C3, a further CR3 ligand. Infection of PBMCs with HIV-IIIB and primary isolates was partially inhibited by anti-CR3 antibodies. A peptide derived from the complement component C3, covering the CR3-binding site of C3 and sharing strong similarity to the immunosuppressive region of gp41, significantly reduced the HIV-1 titer in infection assays. Recombinant soluble gp41 (rsgp41) and the peptide covering the immunosuppressive domain of gp41 inhibited the rosetting of iC3b-coated sheep erythrocytes with U937 via complement receptors (CRs) with an efficiency comparable to monoclonal anti-CR antibodies. In addition, sub-populations of CD4 + and CD8 + T-cells isolated from HIV-infected individuals were found to upregulate CR3 as determined by FACS analysis and on the mRNA level. Since gp41 has been implicated in viral fusion, an interaction of its C3-homology region in gp41 or an interaction of ICAM on the surface of free virus with CRs might contribute to facilitate viral entry.

Anti-HIV Agents↗

PubMedDate not supplied

Plant protein hydrolysates: preparation of defined peptide fractions promoting growth and production in animal cells cultures.

A new approach was applied with the aim at producing plant protein hydrolysates less heterogeneous and less contaminated with nonpeptide substances than are the presently available digests. A significant reduction of nonprotein contaminants was achieved by extraction of the plant material, soy flour or wheat flour, with acetone prior to isolation of the protein. Enzymes of nonanimal origin, papain or Pronase, were used for protein hydrolysis. The components of the hydrolysates were resolved by low-pressure liquid chromatography. Separation of peptide fractions and of remaining nonpeptide contaminants was achieved using small-pore size-exclusion chromatography matrices, Sephadex G-15 or Biogel P-2. Individual peptide fractions, both from soy protein and from wheat gluten, varied substantially in their growth-promoting and production-enhancing activities when tested on a mouse hybridoma culture in protein-free medium. The highest enhancement of viable cell density in batch cultures was 180% of control, and the highest enhancement of final immunoglobulin concentration was more than 230% of control. The existence of marked differences in activity of individual peptide fractions leads to a suggestion that the hydrolysates may provide peptides exerting specific positive effects on cultured animal cells.

Animals↗

PubMedDate not supplied

A novel ultrasonic resonance field device for the retention of animal cells.

This article describes two types of flow-through cell retention devices based on the concept of layered piezoelectric resonators. A single-chamber device is compared to a novel optimized steam-sterilizable prototype ultrasonic cell separator with improved acoustic design and an integrated cooling circuit, eliminating the problem of local temperature increase caused by the high amplitudes necessary to achieve the separation of animal cells with low acoustic contrast. This setup yields highly reproducible results and is ideal for studying the long-term effects of ultrasonic sound fields and separation efficiency. The novel two-chamber system has the potential for scaleability due to the reduction in thermal and acoustic flow, increased field stability, and separation efficiency. Finally, the effect of power input on separation and cell viability is reported. Such flow-through cell retention systems could be used as systems to retain biomass within the fermentor or as a substitute for centrifugation, with the major advantage of eliminating high-speed rotational motion.

Animals↗

PubMedDate not supplied

Selective retention of viable cells in ultrasonic resonance field devices.

A double-chamber ultrasonic resonance field device was used for the separation and retention of animal cells. By controlling operational parameters such as flow and power input, the device can retain viable cells more efficiently, allowing for selective removal of nonviable cells and cell debris. A simple model describing the forces acting on spherical particles in a sound field (primary radiation force, Bernoulli force, secondary radiation force) is presented. Field stability increases with decreasing average flow rates and increasing power input. At very high field stability, as achieved with low flow rates and high power input, the selectivity for viable cells is reduced, due to the efficient retention of all types of particles. At high flow rates and resulting low field stability, selectivity is also reduced, due to poor separation efficiency, resulting in equally low retention of viable cells, nonviable cells, and cell debris.

Animals↗

PubMedDate not supplied

Optimization of sorting conditions for the selection of stable, high-producing mammalian cell lines.

The production of Green Fluorescent Protein in recombinant NIH3T3 mouse fibroblast cells was used as a model to determine the optimal conditions for the rapid isolation of high-producing cell lines with a fluorescence-activated cell sorter. "Bulk sorting", that is, sorting of a large number of positive cells, did not result in a stable, high-producing cell line due to overgrowth of high-producing cells by low- or nonproducing cells. The production kinetics and expression of GFP during batch culture was found to differ between NIH3T3 cells and HepG2 hepatoma cells, even though the same plasmid was used for transfection. The kinetics of product formation need therefore to be determined from case to case to select the optimal timepoint for analysis and sorting. Subcloning of sorted cells into microtiter plates only resulted in high-producing subclones when 1 or 2 cells were seeded per well. Higher seeding rates again resulted in overgrowth of low- or nonproducers. By subcloning, two high-producing cells lines could be isolated. They had a 10- and 15-fold higher fluorescent signal compared to the negative control. While one of these subclones started to decrease it's GFP expression after 2 months, the other clone stably expressed GFP for 4 months.

3T3 Cells↗

PubMedDate not supplied

Potent neutralization of primary human immunodeficiency virus clade C isolates with a synergistic combination of human monoclonal antibodies raised against clade B.

OBJECTIVES: We investigated the ability of several human neutralizing monoclonal antibodies (mAbs), originally raised against human immunodeficiency virus (HIV) clade B isolates, to neutralize primary clade C isolates as single agents and in combination. STUDY DESIGN/METHODS: HIV clade C isolates from five different countries were tested for susceptibility to neutralization by anti-clade B mAbs in human peripheral blood mononuclear cells. Monoclonal antibody combinations were evaluated for possible synergy. RESULTS: All 20 primary HIV clade C isolates could be neutralized 97.5% to 100% by a quadruple combination of mAbs IgG1b12, 2G12, 2F5, and 4E10. These mAbs recognized conserved epitopes and were highly synergistic, resulting in strong cross-clade neutralization. CONCLUSIONS: In our previous experiment, a synergistic combination of human neutralizing mAbs protected all macaque neonates against oral challenge with a simian-human immunodeficiency virus encoding HIV env. Together, our data suggest that passive immunization with currently available anti-clade B mAbs could play a role in preventing HIV clade C transmission through breastfeeding.

Antibodies, Monoclonal↗