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H Katinger

Publications and source records attributed to H Katinger.

At least 55 records · Page 3Linked to original sources
PubMed1994

Stable, continuous large-scale production of human monoclonal HIV-1 antibody using a computer-controlled pilot plant.

A completely automated pilot plant used for fermentation has been employed with direct digital control (DDC) technology for monitoring and regulating growth of human cells. A human hybridoma cell line (3D6) producing anti-human immunodeficiency virus (HIV)-1 antibodies was used as a model for large-scale production (300-liter airlift fermentor) in continuous culture. Parameters controlled were pH, dissolved oxygen, temperature and the flow rate of four gases used in the process. A control strategy was implemented to achieve constant fluid velocity and mixing by maintaining the rate of gas flow at a constant level. Another advantage of this approach was that the total gas flow required for optimal fluid circulation was reduced from 1 volume gas/volume fermenter/hour (vvh) to 0.3 vvh. Use of a low flow rate eliminated the serious problems of foaming, which contributed significantly to cell destruction, shorter filter-life and other considerations. Dilution rate was optimized at laboratory scale for maximum productivity, which results in relatively low viability. At a dilution rate of 0.0076 h-1, a total cell density of 6-7 x 10(5) cells/ml with a viability of approximately 75% was maintained during long-term continuous cultivation. These growth conditions resulted in a product titer stabilized in the range of 35 micrograms IgG/ml. Batchwise purification was achieved with a recovery of more than 50% and a final purification of active monoclonal antibody representing about 99% product. Results from isoelectric focusing and Western blotting demonstrated batch-to-batch consistency of the purified human monoclonal antibody to HIV-1 during the continuous growth process.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1993

Rhodamine 123 fluorescence of immortal hybridoma cell lines as a function of glucose concentration.

The fluorescence of rhodamine 123 stained cells has been described to specifically reflect the activity of mitochondria. Changes in the intensity of fluorescence observed in stimulated lymphocytes were attributed to an increased glycolytic activity of cells due to increased growth rates. Previously reported changes in mitochondrial activity observed in batch cultures were likewise attributed to changed growth rates. In this study we report that the Rh123 fluorescence of hybridoma cell lines in batch culture more closely correlates to the glucose concentration in the culture supernatant than to growth rates. When cells are transferred into glutamine free medium with defined glucose concentrations ranging from 0 to 3,000 mg/L the mean Rh123 fluorescence adapts to the respective glucose concentration within 6 hours and gives a linear correlation. This can be explained by the previously described dependence of specific glucose consumption rates on glucose availability in the medium. The importance of controlling glucose availability, especially in large scale fermentations, is discussed.

Culture Media↗
PubMed1993

HIV-1 gp41 binds to several proteins on the human B-cell line, Raji.

Based on our findings that HIV-1 gp41 independently of CD4, can bind to the helper T-cell line H9, we characterized putative binding of HIV-1 gp41 to B-cell lines, Raji, Bjab and Ramos. Using fluorescence-activated cell sorter (FACS) we examined the binding of soluble gp41 (sgp41; Env amino acid 539-684) to these B-cell lines. Using sgp41 attached to sepharose beads Raji cell lysates were absorbed. The sgp41-eluate of Raji cell lysates could inhibit the sgp41-binding to Raji cells. By SDS-PAGE of sgp41-eluate of Raji cell lysates four strong protein band, 37, 45, 49 and 62 kD, and a weak band of 92 kD were stained with Coomassie blue. By Western blot (ligand blot) analysis using sgp41 four protein bands, 37, 45, 49 and 62 kD, were observed in sgp41-eluate of Raji cell lysates. To test the individual proteins the five proteins were isolated from the sgp41-eluate of Raji cell lysates. Three proteins, 45, 49 and 62 kD, each could partially inhibit the sgp41-binding to Raji cells. The results suggest that these three proteins in Raji cell lysates are possible candidates for the putative gp41 receptor(s).

B-Lymphocytes↗
PubMed1993

Enhancement of HIV-1 gp41 binding to Raji cells by PWM, LPS, interferon-gamma and interleukin-6.

Based on our finding that HIV-1 gp41 independently of CD4 can bind to several proteins (gp41 binding protein: GBP) on the human T-cell line H9, B-cell line Raji and monocyte-cell line U937, we examined the effect of mitogens and cytokines on binding of gp41 to H9, Raji and U937 cells. Flow cytometry (FACS) analysis demonstrated that PWM and LPS, IFN-gamma and IL-6, but not Con A, IFN-alpha, -beta, -omega and IL-2, could increase gp41 binding to Raji cells. In controls, none of the regulators (IFN-alpha, -beta, -gamma, -omega, IL-2, IL-6, Con A, PWM, LPS) could modify the binding potential of H9 and U937 cells. Our data suggest that the expression of HIV-1 binding proteins is subject to regulation by PWM, LPS, IFN-gamma and IL-6 in the case of B-cells, while on T-cells and macrophages, the binding proteins may be constitutively expressed.

B-Lymphocytes↗
PubMed1993

The human monocyte cell line U937 binds HIV-1 gp41 by proteins of 37, 45, 49, 62 and 92 kDa.

Based on our findings that HIV-1 gp41 independently of CD4 can bind to the helper T cell line H9 and B cell line Raji, we characterized the putative binding of HIV-1 gp41 to the monocyte cell lines U937 and HL60. Using flow cytometry (FACS) we examined the binding of soluble gp41 (sgp41; env amino acids 539-684) to these monocyte cell lines. Using sgp41 attached to Sepharose beads, U937 cell lysates were absorbed. The sgp41 eluate of U937 cell lysates could inhibit sgp41 binding to U937 cells. With SDS-PAGE of sgp41 eluate of U937 cell lysates, three strong protein bands, (37, 45 and 62 kDa) and two weak bands (49 and 92 kDa) were stained with Coomassie blue. With Western blot (ligand blot) analysis using sgp41, three strong protein bands (37, 49 and 62 kDa) and a very weak band (42-45 kDa) were observed in sgp41 eluate of U937 cell lysates. The results suggest that the four proteins 37, 42-45, 49 and 62 kDa in U937 cell lysates are possible candidates for the putative gp41 receptor(s). We compared the blocking activities of sgp41 eluates from different cell lysates. Not only U937 and Raji lysate-sgp41 eluates, but also H9 and HL60 lysate-sgp41 eluates could block sgp41 binding to U937 and Raji cells. The results indicate that the sgp41-binding proteins on U937, or Raji (H9 and HL60, respectively) probably could have an identical blocking (or binding) specificity; these cell types carry very similar receptor(s) for HIV-1 gp41 binding.

B-Lymphocytes↗
PubMed1993

HIV-1 gp41 binding to human peripheral blood mononuclear cells occurs preferentially to B Lymphocytes and monocytes.

Based on our findings that HIV-1 gp41 independently of CD4 can bind to the human helper T lymphoid cell line H9, B cell line Raji and monocyte cell line U937, we characterized putative binding of HIV-1 gp41 to human peripheral blood lymphocytes (PBLs) and monocytes. Using flow cytometry (FACS), we demonstrated that the recombinant soluble HIV-1 gp41 (sgp41; Env amino acid 539-684) can bind to the normal human peripheral blood mononuclear cells (PBMCs), preferentially to B lymphocytes and monocytes independently of gp120-binding sites on CD4 molecules. This binding is dose-dependent. The HIV-1 sgp41 binds to blood B lymphocytes and monocytes more strongly than to T lymphocytes. By two-color flow cytometric analysis, we identified that sgp41 can bind 10% of CD4+ T lymphocytes, 11.9% of CD8+ T lymphocytes, 47% of CD19+ B lymphocytes and 44.2% of CD14+ monocytes.

B-Lymphocytes↗
PubMed1993

HIV-1 and HIV-2 isolates differ in their ability to activate the complement system on the surface of infected cells.

OBJECTIVE: To analyse the ability of different HIV-1 and HIV-2 isolates to activate the complement system. DESIGN: H9 cells chronically infected with various HIV isolates and the corresponding purified viruses were tested for complement activation. To identify the molecules responsible for complement activation on the surface of infected cells, the expression of complement inhibitors/regulators and viral proteins on the cell surface was analysed. METHODS: C3 deposition on the cell surface and the expression of viral and cellular antigens were determined by flow cytometry analysis. Complement activation by purified viruses was measured using a complement consumption assay and a C1 activation assay. RESULTS: H9 cells infected with different HIV-1 and HIV-2 isolates showed varying degrees of complement activation on the cell surface, ranging from strong activation and deposition of large amounts of C3 to no increased C3 deposition compared to uninfected cells. The C3 deposition was eliminated by EDTA and reduced in the presence of EGTA. In contrast, all purified viral isolates tested activated the complement system in a comparable manner. While the expression of MCP, DAF and CD59 was not modified after infection with different viral isolates, the reaction of the infected cells with a monoclonal antibody (3D6) directed against a gp41 epitope (amino acids 601-620) was found to correlate with the complement activation on the cell surface. CONCLUSIONS: Some HIV-1 as well as HIV-2 isolates activate the complement system on the surface of infected cells independent of anti-HIV antibodies, while other isolates fail to do so. Complement activation on the cell surface is mediated by the alternative and, to a lesser extent, the classical pathway. The differences in complement activation on the cell surface are not caused by a modified expression of membrane-bound complement inhibitors/regulators. C3 deposition on the cell surface correlates with the expression of an epitope lying within the major complement activating domain of gp41 (amino acids 591-620). These results suggest a role of gp41 for complement activation on HIV-infected cells as has been described previously for purified HIV.

Antigens, CD↗
PubMed1993

A conserved neutralizing epitope on gp41 of human immunodeficiency virus type 1.

Vaccination against human immunodeficiency virus type 1 (HIV-1) requires an immunogen which will elicit a protective immunity against viruses that show a high degree of genetic polymorphism. Therefore, the identification of neutralizing epitopes which are shared by many strains would be useful. In previous studies, we established a human monoclonal antibody (2F5) that neutralizes a variety of laboratory strains and clinical isolates of HIV-1. In the present report, we define the amino acid sequence Glu-Leu-Asp-Lys-Trp-Ala (ELDKWA) on the ectodomain of gp41 as the epitope recognized by this antibody. The sequence was found to be conserved in 72% of otherwise highly variable HIV-1 isolates. Escape mutants were not detected in cells infected with HIV-1 isolates MN and RF in the presence of antibody 2F5. Since sequence variability of neutralizing epitopes is considered to be a major obstacle to HIV-1 vaccine development, the conserved B-cell epitope described here is a promising candidate for inclusion in a vaccine against AIDS.

Amino Acid Sequence↗
PubMed1992

Amino-acid sequence comparison of nepovirus coat proteins.

The amino acid sequence of the coat proteins of several nepoviruses was determined by a combination of peptide and nucleic acid sequencing (grapevine fanleaf virus, arabis mosaic virus, tomato blackring virus, grapevine chrome mosaic virus). These sequences were compared and showed homologies ranging from 10% to 69%, and 96.7% for the two arabis mosaic virus strains. 10% homology does not reflect any relationship between viruses, and our results implicate, that nepoviruses, considering the homology of the coat protein sequences of viruses as a parameter for virus taxonomy, may be divided into several subgroups.

Amino Acid Sequence↗
PubMed1992

Flow cytometry and two-dimensional electrophoresis (2-DE) for system evaluation of long term continuous perfused animal cell cultures in macroporous beads.

Immobilization of r-CHO cells at high density using macroporous polyethylene carriers in a modular fluidized bed reactor is demonstrated. Specific growth rates of the cells are measured by incorporation of BrdU. At a cell density of about 10(8) cells/ml a stable growth rate of 0.004 h-1 was established. Total release of proteins into the culture supernatant during protein-free perfusion was analyzed by 2-DE in various phases of the long-term culture showing very similar patterns indicating a constant pattern of gene expression.

Animals↗
PubMed1992

Determination of division rates of rCHO cells in high density and immobilized fermentation systems by flow cytometry.

As most high density and immobilized fermentation systems do not allow the direct quantitative determination of cell density, two flow cytometric methods (the determination of incorporation of bromodeoxyuridine into newly synthesized DNA and the increase in mitotic cells by colchicine blockage) were evaluated as to their suitability to measure true division rates of cells in bioreactors. The BrdU method gave division rates identical to the growth rates measured by cell count, while the colchicine block method gave values that were lower and varied with the cell line. This is due to the cytotoxicity of colchicine and makes a calibration of the method for each cell line necessary. Both methods have been successfully used to measure division rates of rCHO cells immobilized in an alginate matrix as well as in macroporous carriers in a fluidised bed system and in dialysis culture.

Animals↗
PubMed1992

Two-dimensional electrophoresis as a tool for control of quality and consistency in production systems using animal cells. Two-dimensional electrophoresis in animal cell culture technology.

A nonrecombinant human melanoma cell line and recombinant chinese hamster ovary (CHO) cells were used as examples for long-term in vitro cultivation in protein-free media. The method used to monitor the consistency of protein release by these mammalian cells was two-dimensional electrophoresis with immobilized pH gradient. Secreted proteins from a melanoma cell line cultivated in a continuous fermentation system over a period of 22 months were monitored. Two-dimensional patterns of secreted proteins were compared and the stability of their composition was determined over a period of nearly 14 months, with significant pattern variation being observed after 14 months. The protein pattern from this extended in vitro culture was compared to those of the very same melanoma cell line recultivated after being frozen in liquid nitrogen for more than 2 years. Due to the high resolution of complex polypeptide mixtures and the possibility to detect even minor differences in the composition of protein patterns, we propose the two-dimensional electrophoresis as a tool for quality assessment in animal cell culture technology.

Animals↗
PubMed1992

Stability of von Willebrand factor secretion in different human endothelial hybrid cell lines.

Endothelial cells form a highly differentiated tissue on the inner surface of blood vessels. One of the typical characteristics is the expression of von Willebrand Factor, a protein that participates in blood coagulation. The in vitro cultivation of endothelial cells is limited by the fact that primary cells become senescent after 40 generation doublings. We have immortalized human endothelial cells by somatic cell hybridization. Primary cells were fused to different tumor cell lines of murine and human origin. The degree of differentiation of the resulting hybrids was analyzed by characterizing the expression of von Willebrand Factor. This protein was identified intracellularly and in the culture supernatant. During long-term cultivation the hybrid cells showed a tendency to lose this differentiated property even after several subcloning steps. However by fusing them with primary endothelial cells a second time, cell lines expressing von Willebrand Factor for more than 180 population doublings were generated.

Animals↗
PubMed1992

Growth and production kinetics of human x mouse and mouse hybridoma cells at reduced temperature and serum content.

The growth and production kinetics of a mouse hybridoma cell line and a human-mouse heterohybridoma were analyzed under conditions of reduced temperature and serum content. The mouse hybridoma P24 had a constant cell specific production rate and RNA content, while the heterohybridoma 3D6-LC4 showed growth associated production kinetics and an increased RNA content at higher growth rates. This behaviour of 3D6-LC4 cells can be explained by the unusual cell cycle kinetics of this line, which can be arrested in any phase under growth limiting conditions, so that a low growth rate does not result in a greater portion of high producing G1-phase cells. Substrate limitation changes the cell cycle distribution of this cell line to a greater extent than low temperature or serum content, which indicates that this stress factor exerts a greater physiological control than assumed.

Animals↗
PubMed1992

Influence of transfected SV40 early region on growth and differentiation of human endothelial cells.

Endothelial cells isolated from human umbilical veins show a limited in vitro life span of about 40 population doublings. Cell division is dependent on the presence of endothelial cell growth factor in the culture medium. We have transfected primary endothelial cells with a plasmid containing the early region of SV40 virus. Large T positive cells were obtained which grew in the absence of endothelial cell growth factor at low serum concentrations and showed a prolonged lifespan. Expression of von Willebrand factor and SV40 large T antigen was detected simultaneously in transfected cells.

Antigens, Polyomavirus Transforming↗
PubMed1992

HIV-1 gp41 contains two sites for interaction with several proteins on the helper T-lymphoid cell line, H9.

OBJECTIVE: To characterize putative binding sites for HIV-1 gp41 to H9 cells. DESIGN: Based on accumulating evidence in the literature that HIV-1 can bind to cell surfaces independent of CD4, we attempted to clarify whether gp41, the transmembrane HIV-1 protein, contributes to CD4-independent binding. We therefore tested binding of gp41 to cells. METHODS: Using fluorescence-activated cell-sorter analysis, we examined the binding of recombinant gp160 (gp160) and soluble gp41 (sgp41; Env amino acids 539-684) to H9 cells, and located the putative binding site(s) of gp41 by inhibition using 16 HIV-1 Env peptides. Putative HIV-1 receptor proteins in H9 cell lysates were Western blotted and precipitated using sgp41. RESULTS: sgp41 bound to the CD4+ H9 cells and rgp160 bound to H9 cells independent of gp120-binding sites on CD4 molecules. Two gp41 peptides (Env amino acids 591-605 and 651-665) inhibited the binding of sgp41 to H9 cells. Four bands, of 37, 40, 55 and 97 kD, were blotted in H9 cell lysates, and three bands, 40, 97 and 108 kD, were observed in the precipitation analysis using lysates of 125I-surface-labelled H9 cells and sgp41 attached to sepharose beads. CONCLUSIONS: HIV-1 gp41 contains two putative binding sites to H9 cells. These sites may be located within Env amino acids 591-605 (ERYLKDQQLLGIWGC) and 651-665 (TLLEESQNQQEKNEQ). Using two different techniques, five proteins (37, 40, 55, 97 and 108 kD) were identified in H9 lysates as possible candidates for gp41 binding.

Amino Acid Sequence↗
PubMed1992

Selective inhibition of in vitro cell growth by the anti-tumour drug Ukrain.

The inhibitory effect of Ukrain on malignant cells and on normal cells, in vitro, has been compared. To obtain a 50% inhibition of cell growth, a tenfold concentration had to be used with normal endothelial cells compared to a human osteosarcoma cell line. Hybrids of the two cell types showed nearly the same sensitivity as normal cells. A laser scanning microscope showed a high uptake of Ukrain in malignant cells, while the content in normal cells under the same experimental conditions was substantially lower.

Alkaloids↗