Biomedical subjects

Hermann Katinger

Publications and source records attributed to Hermann Katinger.

At least 19 recordsLinked to original sources

PubMed2007

Protein-free transfection of CHO host cells with an IgG-fusion protein: selection and characterization of stable high producers and comparison to conventionally transfected clones.

In order to improve the current techniques of cell cultivation in the absence of serum, we have developed a protein-free transfection protocol for CHO cells, based on the Nucleofector technology. After starting with a heterogeneous pool of primary transfectants which express the fusion protein EpoFc, we isolated single clones and compared them with parallel clones generated by lipofection in serum-dependent cultivation. Our intensive characterization program was based on determination of specific productivity (q(p)) and analysis of genetic parameters. In two nucleofection experiments, transfection with 5 microg of DNA resulted in best productivities of the primary cell pools. After subcloning, the q(p) could be raised up to 27 pg x cells(-1) x day(-1). While the serum-dependent transfectants exhibited specific productivities up to 57 pg x cells(-1) x day(-1) in serum-dependent cultivation, a significant decrease that resulted in the range of q(p) of the protein-free transfectants was observed after switching to protein-free conditions. Investigation of genetic parameters revealed higher mRNA levels and gene copy numbers (GCN) for the protein-free adapted serum-dependent transfectants. Therefore, we assume that problems during protein-free adaptation (PFA) lead to a less efficient translation machinery after serum deprivation. We describe the generation of stable-producing recombinant CHO clones by protein-free transfection of a protein-free adapted host cell line, which reduces the risk of adverse clonal changes after PFA. The main advantage of this approach is the earlier predictability of clone behavior, which makes the generation of production clones by protein-free transfection, a viable and highly efficient strategy for recombinant cell line development.

Animals↗

PubMed2007

Mapping of Malus domestica allergens by 2-D electrophoresis and IgE-reactivity.

The importance of apple allergens has been repeatedly emphasized, and their presence has been confirmed both in pollen and in fruits. In the present study, a combination of proteomic tools have been used to build a complete allergen map of apple. The water-soluble fraction of an apple extract was precipitated using a phenol-based procedure and separated by 2-DE. Initially four previously classified allergens, Mal d 1, Mal d 2, Mal d 3 and Mal d 4, could be identified in Western blots with polyclonal rabbit antibodies directed to the four respective allergens, and subsequently matched to the bands recognized by several patient sera. Further, all four known apple allergens were localized on a 2-DE map and they were matched with spots recognized by sera of patients with different allergic patterns. Moreover, a new, putative allergen could be identified using MS. We evaluated the influence of post-translational modifications and the immunoreactivity under different analytical conditions. The comparison of different visualization methods for 2-DE gels and blots revealed that even very low concentrations of the intact epitopes are detectable by IgEs of patients, and therefore might be sufficient to trigger allergic symptoms in sensitized individuals.

Allergens↗

PubMed2006

Aging and the ubiquitinome: traditional and non-traditional functions of ubiquitin in aging cells and tissues.

Ubiquitination of endogenous proteins is one of the key regulatory steps of protein degradation followed by regulation of proteasome activity. During the last years evidence has increased that proteasome activity is decreased during the aging process in various model systems and that these changes might be causally related to aging and aging associated diseases. Since in most instances ubiquitination is the primary event in target selection, the system of ubiquitination and deubiquitination might be of similar importance. Furthermore, ubiquitination and proteasomal degradation are not completely congruent, since ubiquitination also confers functions different from giving "the kiss of death" to proteins. Depending on mono- and polyubiquitination and on how ubiquitin chains are linked together, ubiquitination is involved in transcriptional regulation, receptor internalization, DNA repair, and stabilization of protein complexes. This review is therefore the first to summarize the current knowledge regarding the ubiquitinome and the underlying ubiquitin ligases and deubiquitinating enzymes in replicative senescence, tissue aging as well as in segmental progeroid syndromes and to discuss potential causes and consequences for aging.

Aging↗

PubMed2006

Antibodies to conserved epitopes of the HIV-1 envelope in sera from long-term non-progressors: prevalence and association with neutralizing activity.

OBJECTIVE: Previous studies have shown that broadly neutralizing antibodies (NAb) are more frequent in long-term non-progressors (LTNP) than in other HIV-1 infected patients, but nothing is known about the envelope regions targeted by these broadly NAb. We investigated whether the breadth of neutralizing activity of sera was associated with the presence of specific antibodies (2F5- and/or 4E10-like, b12-like or 2G12-like antibodies) directed against conserved epitopes known to be involved in broad neutralization. METHODS: We assessed the ability of sera from 67 LTNP of the French ANRS cohort (ANRS CO15) to neutralize four heterologous primary isolates of four various clades. Competitive and non-competitive ELISA were developed for the specific comparison of levels of antibodies against these specific epitopes in neutralizing and non-neutralizing sera from LTNP. RESULTS: We found that higher 2G12-like antibody levels were significantly associated with the broadest neutralizing activity in sera from LTNP. Levels of 2G12-like antibodies were higher in the sera that neutralized the four isolates than in the others, with a median of 5.7 microg/ml [interquartile range (IQR), 2.7-9.3 microg/ml] versus 2.3 microg/ml (IQR, 1.1-3.9 microg/ml) (Mann-Whitney test, P = 0.03). Levels of antibodies against the other targeted envelope epitopes did not differ significantly between broadly and non-broadly neutralizing sera. CONCLUSION: These results suggest that the antigenicity of the "silent face" of gp120 that exposes the 2G12 epitope should be analysed in more detail, to find ways to induce broadly neutralizing antibodies.

Antibody Specificity↗

PubMed2006

Time dependence of protective post-exposure prophylaxis with human monoclonal antibodies against pathogenic SHIV challenge in newborn macaques.

In a primate model of postnatal virus transmission, we have previously shown that 1 h post-exposure prophylaxis (PEP) with a triple combination of neutralizing monoclonal antibodies (nmAbs) conferred sterilizing protection to neonatal macaques against oral challenge with pathogenic simian-human immunodeficiency virus (SHIV). Here, we show that nmAbs can also partially protect SHIV-exposed newborn macaques against infection or disease, when given as 12 or 24 h PEP, respectively. This work delineates the potential and the limits of passive immunoprophylaxis with nmAbs. Even though 24 h PEP with nmAbs did not provide sterilizing immunity to neonatal monkeys, it contained viremia and protected infants from acute disease. Taken together with our results from other PEP studies, these data show that the success of passive immunization depends on the nmAb potency/dose and the time window between virus exposure and start of immunotherapy.

Animals↗

PubMed2006

Live attenuated influenza virus expressing human interleukin-2 reveals increased immunogenic potential in young and aged hosts.

Despite the reported efficacy of commercially available influenza virus vaccines, a considerable proportion of the human population does not respond well to vaccination. In an attempt to improve the immunogenicity of live influenza vaccines, an attenuated, cold-adapted (ca) influenza A virus expressing human interleukin-2 (IL-2) from the NS gene was generated. Intranasal immunization of young adult and aged mice with the IL-2-expressing virus resulted in markedly enhanced mucosal and cellular immune responses compared to those of mice immunized with the nonrecombinant ca parent strain. Interestingly, the mucosal immunoglobulin A (IgA) and CD8(+) T-cell responses in the respiratory compartment could be restored in aged mice primed with the IL-2-expressing virus to magnitudes similar to those in young adult mice. The immunomodulating effect of locally expressed IL-2 also gave rise to a systemic CD8(+) T-cell and distant urogenital IgA response in young adult mice, but this effect was less distinct in aged mice. Importantly, only mice immunized with the recombinant IL-2 virus were completely protected from a pathogenic wild-type virus challenge and revealed a stronger onset of virus-specific CD8(+) T-cell recall response. Our findings emphasize the potential of reverse genetics to improve the efficacy of live influenza vaccines, thus rendering them more suitable for high-risk age groups.

Animals↗

PubMed2006

Process parameter shifting: Part II. Biphasic cultivation-A tool for enhancing the volumetric productivity of batch processes using Epo-Fc expressing CHO cells.

Regulation of cell growth and protein expression potentially results in a sustainable enhancement of the volumetric productivity in a fermentation process. Following a biphasic cultivation strategy the process initially passes through a cell proliferation phase to generate a sufficiently high viable cell mass. In the subsequent production phase cells are maintained viable and productive without significant cell proliferation leading to increased viable cell days and product yields. In a previous work we have shown that the well directed alteration of the process environment based on process parameter shifting is a promising tool to regulate cell growth and protein expression. In continuation of this work we investigated process parameters which have been identified to affect cell proliferation in favor of an increased specific productivity and total product yield in a series of biphasic batch cultivation experiments. In most of these processes the integral of viable cells and the specific productivity were increased leading to a significant improvement of both final product concentration and volumetric productivity. In addition, combined parameter shifts (pH 6.90/30 degrees C and pH 6.90/33 degrees C) exerted a synergistic effect on product quality. The loss of product sialylation which occurred at reduced temperatures was prevented by simultaneously reducing the external pH. In conclusion, biphasic cultivation based on combined shifting of process parameters is a suitable tool for controlling cell proliferation and protein expression of mammalian cells in a batch bioreactor leading to enhanced volumetric productivities and therefore offers an enormous potential for bioprocess optimization.

Animals↗

PubMed2006

Process parameter shifting: Part I. Effect of DOT, pH, and temperature on the performance of Epo-Fc expressing CHO cells cultivated in controlled batch bioreactors.

The impact of process environment changes on process performance is one of the most crucial process safety issues when cultivating mammalian cells in a bioreactor. In contrast, directed shifting of process parameters can also be used as an optimization tool providing higher cell and product yields. Compared to other strategies that also aim on the regulation of cell growth and protein expression process parameter shifts can be easily performed without reagent addition or even genetic modification of the host cell line. However, a successful application of changing process conditions implies a profound understanding of the provoked physiological changes within the cells. In a systematic approach we varied the dissolved oxygen tension (DOT), pH, and temperature of CHO cultures in controlled bioreactors and investigated the impact on growth, productivity, metabolism, product quality and cell cycle distribution using a recombinant CHO cell line expressing the highly glycosylated fusion protein Epo-Fc. We found the reduction of cultivation temperature and the reduction of (external) pH to exert the most significant effects on process performance by mainly reducing cell growth and metabolism. With respect to the cell line used we identified a set of parameters capable of affecting cell proliferation in favor of an increased specific productivity and total product yield. The well directed alteration of the process environment has emerged as a tool adequate for further process optimization applying a biphasic cultivation strategy.

Animals↗

PubMed2006

Long-term stability of marker gene expression in Prunus subhirtella: a model fruit tree species.

Transgenic trees currently are being produced by Agrobacterium-mediated transformation and biolistics. Since trees are particularly suited for long-term evaluations of the impact of the technology, Prunus subhirtella autumno rosa (PAR) was chosen as model fruit tree species and transformed with a reporter gene (uidA) under the control of the 35S promoter. Using Southern and GUS fluorometric techniques, we compared transgene copy numbers and observed stability of transgene expression levels in 34 different transgenic plants, grown under in vitro, greenhouse and screenhouse conditions, over a period of 9 years. An influence of grafting on gene expression was not observed. No silenced transgenic plant was detected. Overall, these results suggest that transgene expression in perennial species, such as fruit trees, remains stable in time and space, over extended periods and in different organs, confirming the value of PAR as model species to study season-dependent regulation in mature stone fruit tissues. While the Agrobacterium-derived Prunus transformants contained one to two copies of the transgenes, 91% of the transgenic events also contained various lengths of the bacterial plasmid backbone, indicating that the Agrobacterium-mediated transformation is not as precise as previously perceived. The implications for public acceptance and future applications are discussed.

Base Sequence↗

PubMed2006

Biochemical characterization of rhEpo-Fc fusion protein expressed in CHO cells.

One challenge in biotechnology industry is to produce recombinant proteins with prolonged serum half-life. One strategy for enhancing the serum half-life of proteins includes increasing the molecular weight of the protein of interest by fusion to the Fc part of an antibody. In this context, we have expressed a homodimer fusion protein in CHO cells which consists of two identical polypeptide chains, in which our target protein, recombinant human erythropoietin (rhEpo), is N-terminally linked with the Fc part of a human IgG(1) molecule. In the present study, culture supernatant of a stable clone was collected and purified by affinity chromatography prior characterization. We emphasized product quality aspects regarding the fusion protein itself and in addition, post-translational characterization of the subunits in comparison to human antibodies and rhEpo. However, overproduction of recombinant proteins in mammalian cells is well established, analysis of product quality of complex products for different purposes, such as product specification, purification issues, batch to batch consistency and therapeutical consequences, is required. Besides product quantification by ELISA, N-acetylneuraminic acid quantification in microtiterplates, quantitative isoform pattern and entire glycan profiling was performed. By using these techniques for the characterization of the recombinant human Epo-Fc (rhEpo-Fc) molecule itself and furthermore, for the separate characterization of both subunits, we could clearly show that no significant differences in the core glycan structures compared to rhEpo and human antibody N-glycans were found. The direct comparison with other rhEpo-Fc fusion proteins failed, because no appropriate data were found in the literature.

Animals↗

PubMed2006

The HIV-neutralizing monoclonal antibody 4E10 recognizes N-terminal sequences on the native antigen.

Characterization of the epitope recognized by the broadly neutralizing anti-HIV Ab 4E10 has, heretofore, focused on a linear sequence from the gp41 pretransmembrane region (PTMR). Attempts to generate neutralizing Abs based on this linear epitope sequence have been unsuccessful. We have characterized the antigenic determinants on recombinant glycosylated full-length Ags, and nonglycosylated and truncated Ags recognized by 4E10 using epitope extraction and excision assays in conjunction with MALDI mass spectrometry. The mAb recognized the peptides (34)LWVTVYYGVPVWK(46) and (512)AVGIGAVFLGFLGAAGSTMGAASMTLTVQAR(542) located at the N-terminal region of gp120 and gp41, respectively. Immunoassays verified AV(L/M)FLGFLGAA as the gp41 epitope core. Recognition of the peptide from the gp41 PTMR was detected only in constructs in which the N termini of the mature envelope proteins were missing. In this region, the epitope core is located in the sequence (672)WFDITNWLWY(681). We hypothesize that the hydrophobic surface of the paratope functions as a "trap" for the viral sequences, which are responsible for insertion into the host cell membrane. As the N-terminal region of gp120, the fusogenic peptide of gp41, and the PTMR of gp41 show high sequence homology among various HIV strains, this model is consistent with the broadly neutralizing capabilities of 4E10.

Amino Acid Sequence↗

PubMed2006

A novel strategy for quantitative isoform detection directly performed from culture supernatant.

Currently, one of the most used techniques for the determination of isoform pattern analysis is isoelectric focusing. Routinely, this is performed by immunoblotting. Blotting of proteins after isoelectric focusing on IPG gels may cause several problems, such as protein loss by the blotting itself and band broadening, in some cases the immunostaining with antibodies might be problematic. In the present study, an alternative isoform prestaining method with CyDye fluors is presented. For this approach, a highly glycosylated fusion protein, Epo-Fc, was used consisting of two recombinant human erythropoietin attached to the Fc part of a human IgG(1) molecule. By using CyDye fluors, up to three samples can be focused on the same lane under identical electrophoretic conditions. A fundamental benefit of this technique is the ability to perform quantitative isoform pattern analysis directly from serum-free culture supernatant.

Animals↗

PubMed2006

Validated method for quantification of genetically modified organisms in samples of maize flour.

Sensitive and accurate testing for trace amounts of biotechnology-derived DNA from plant material is the prerequisite for detection of 1% or 0.5% genetically modified ingredients in food products or raw materials thereof. Compared to ELISA detection of expressed proteins, real-time PCR (RT-PCR) amplification has easier sample preparation and detection limits are lower. Of the different methods of DNA preparation CTAB method with high flexibility in starting material and generation of sufficient DNA with relevant quality was chosen. Previous RT-PCR data generated with the SYBR green detection method showed that the method is highly sensitive to sample matrices and genomic DNA content influencing the interpretation of results. Therefore, this paper describes a real-time DNA quantification based on the TaqMan probe method, indicating high accuracy and sensitivity with detection limits of lower than 18 copies per sample applicable and comparable to highly purified plasmid standards as well as complex matrices of genomic DNA samples. The results were evaluated with ValiData for homology of variance, linearity, accuracy of the standard curve, and standard deviation.

DNA↗

PubMed2006

SNEV overexpression extends the life span of human endothelial cells.

In a recent screening for genes down regulated in replicatively senescent human umbilical vein endothelial cells (HUVECs), we have isolated the novel protein SNEV. Since then SNEV has proven as a multifaceted protein playing a role in pre-mRNA splicing, DNA repair, and the ubiquitin/proteosome system. Here, we report that SNEV mRNA decreases in various cell types during replicative senescence, and that it is increased in various immortalized cell lines, as well as in breast tumors, where SNEV transcript levels also correlate with the survival of breast cancer patients. Since these mRNA profiles suggested a role of SNEV in the regulation of cell proliferation, the effect of its overexpression was tested. Thereby, a significant extension of the cellular life span was observed, which was not caused by altered telomerase activity or telomere dynamics but rather by enhanced stress resistance. When SNEV overexpressing cells were treated with bleomycin or bleomycin combined with BSO, inducing DNA damage as well as reactive oxygen species, a significantly lower fraction of apoptotic cells was found in comparison to vector control cells. These data suggest that high levels of SNEV might extend the cellular life span by increasing the resistance to stress or by improving the DNA repair capacity of the cells.

Adult↗

PubMed2006

Evaluation of a genomics platform for cross-species transcriptome analysis of recombinant CHO cells.

Microarray technology for mammalian cells has been utilized mainly for humans, mouse, and rat gene expression analysis. In this approach the feasibility of cross-species hybridization experiments using Chinese hamster ovary (CHO) cells was evaluated. Sequence alignments of available data for CHO were performed against mouse and rat transcripts to determine the homology between the investigated species. We implemented a probability model based on this homology in order to estimate the chance for successful hybridization using Agilent's 60-mer oligonucleotide platform. Heat-shock expression data from CHO, mouse 3T3, and rat A10 cells were generated to determine intraspecies variability, reproducibility, and specificity in order to assess the accuracy of this method. Detected signature genes, in particular from studies with the mouse arrays, showed a reliable similarity between these two rodents and were confirmed by quantitative RT-PCR. Our findings provide evidence that cross-species analysis can be a useful tool to study gene expression profiles of related organisms for which species-specific microarrays are not available.

Algorithms↗

PubMed2006

GMP production of liposomes--a new industrial approach.

A new scalable liposome production system is presented, which is based on the ethanol injection technique. The system permits liposome manufacture regardless of production scale, as scale is determined only by free disposable vessel volumes. Once the parameters are defined, an easy scale up can be performed by just changing the process vessels. These vessels are fully sterilizeable and all raw materials are transferred into the sanitized and sterilized system via 0.2 microm filters to guarantee an aseptic production. Liposome size can be controlled by the local lipid concentration at the injection point depending on process parameters like injection pressure, lipid concentration and injection rate. These defined process parameters are furthermore responsible for highly reproducible results with respect to vesicle diameters and encapsulation rates Compared to other technologies like the film method which is normally followed by size reduction through high pressure homogenization, ultrasonication or extrusion, no mechanical forces are needed to generate homogeneous and narrow distributed liposomes. Another important advantage of this method is the suitability for the entrapment of many different drug substances such as large hydrophilic proteins by passive encapsulation, small amphiphilic drugs by a one step remote loading technique or membrane association of antigens for vaccination approaches.

Liposomes↗

PubMed2006

Membrane association and epitope recognition by HIV-1 neutralizing anti-gp41 2F5 and 4E10 antibodies.

It has been proposed that HIV-1-neutralizing 2F5 and 4E10 monoclonal antibodies recognize gp41 ectodomain pretransmembrane sequences in the context of the membrane interface. With the aim of evaluating lipid-bilayer surface effects on recognition, we use phospholipid model systems to investigate the ability of 2F5 and 4E10 to transfer into membrane interfaces and bind epitope sequences immersed therein. The experimental data support the predicted tendencies for partitioning and recognition of membrane-bound epitopes, albeit with lower affinity in the case of 2F5. Our findings support the existence of two different recognition mechanisms at membrane surfaces: the association of 2F5 with the interface possibly prevents epitope immersion into the bilayer, and 4E10 membrane association might allow recognition of the membrane-bound epitope. We discuss the relevance of these observations for the design of immunogens aimed at eliciting 2F5- and 4E10- like humoral responses.

AIDS Vaccines↗

PubMed2006

Long-term multiple-dose pharmacokinetics of human monoclonal antibodies (MAbs) against human immunodeficiency virus type 1 envelope gp120 (MAb 2G12) and gp41 (MAbs 4E10 and 2F5).

While certain antibodies directed against the human immunodeficiency virus (HIV) envelope have the potential to suppress virus replication in vitro, the impact of neutralizing antibodies in vivo remains unclear. In a recent proof-of-concept study, the broadly neutralizing monoclonal antibodies 2G12, 4E10, and 2F5 exhibited inhibitory activities in vivo, as exemplified by a delay of the viral rebound following the interruption of antiretroviral therapy. Unexpectedly, the antiviral effect seen was most prominently due to 2G12 activity. To further investigate whether differential HIV-inhibitory activity was due to different pharmacokinetic properties of the antibodies, we performed a formal pharmacokinetic analysis with 14 patients. Repeated infusions at high dose levels were well tolerated by the patients and did not elicit an endogenous immune response against the monoclonal antibodies. The pharmacokinetic parameters of all three antibodies correlated with each other. Mean estimates were 0.047, 0.035, and 0.044 liter/kg for the central volume of distribution of 2G12, 4E10, and 2F5, respectively, and 0.0018, 0.0058, and 0.0077 liter/kg . day for the systemic clearance of 2G12, 4E10, and 2F5, respectively. Monoclonal antibody 2G12 had a significantly longer elimination half-life (21.8 +/- 7.2 days [P < 0.0001]) than monoclonal antibodies 4E10 (5.5 +/- 2.2 days) and 2F5 (4.3 +/- 1.1 days). The comprehensive pharmacokinetic data from this long-term multiple-dose phase II study were coherent with those from previous short-term phase I studies, as assessed by compartmental and noncompartmental techniques. The anti-HIV type 1 antibodies studied showed distribution and elimination kinetics similar to those seen for other human-like antibodies. Further studies examining tissue concentrations to explain the differential in vivo activity of the anti-gp120 antibody compared with those of the two anti-gp41 antibodies are warranted.

Adult↗