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Biomedical subjects

H Katinger

Publications and source records attributed to H Katinger.

At least 55 records · Page 3Linked to original sources
PubMed1997

Human endothelial cell lines established by mutated forms of the simian virus 40 large T oncogene.

The large T oncoprotein of Simian Virus 40 is widely used to improve the growth characteristics of primary cells in culture. Beside growth stimulation and immortalization, expression of the large T protein in human cells frequently leads to a loss of differentiated characters and changes in the karyotype. We have constructed mutated forms of the large T protein by deletion of various fragments of the DNA binding domain to test, whether this region is responsible for undesired influences on cell differentiation. After transfection into human umbilical vein endothelial cells, the resulting cell lines showed no improvement in expression of the differentiation marker von Willebrand factor compared to cell lines transfected with the wild type oncogene. Changes in the karyotype were still observed. Our results contribute to the mapping of functional domains of the large T protein. The truncated large T proteins retained growth stimulating activity after removal of 111 and 241 amino acids of the DNA binding region.

3T3 Cells↗
PubMed1997

Expression of foreign proteins on the surface of Autographa californica nuclear polyhedrosis virus.

Based on the method of direct cloning into the baculovirus genome by linearizing and re-ligation in presence of the target insert, we designed viral constructs that express foreign genes on the surface of baculovirus particles. We chose the glycosylated envelope protein gp41 of human immunodeficiency virus type 1 (HIV-1) as a model for displaying recombinant proteins on budded virus. The ectodomain of the envelope protein gp41 of HIV-1 was being fused to the entire baculovirus major coat protein gp64 (Ac-cops41) and to the membrane anchor sequence of gp64 (Acmars41). Two different promoters, the "very late" polyhedrin promoter (Ac-mars41) and the "early and late" gp64 promoter (Ac-promars41) were compared. The expression of gp41 in infected cells and its presence on viral particles was confirmed by enzyme-linked immunosorbent assay (ELISA), Western blot and electron microscopy.

Animals↗
PubMed1996

Optimization of recombinant gene expression in Escherichia coli.

The major targets for improvement of recombinant expression efficiency in Escherichia coli are gene dosage, transcription and, to some extent, translation. In order to evaluate the relative importance of these factors, the kinetics of specific mRNA compared to product formation was studied for different widely used expression systems, producing recombinant human superoxide dismutase. For a system employing phage T7 RNA polymerase, where a high level of recombinant protein expression puts a high metabolic burden on the cells, it was shown that transcription is not the limiting factor. To improve the translation rate of a common vector based on the tac promoter, the Shine-Dalgarno (SD) sequence was mutated towards stronger homology to the anti-SD sequence of the E. coli 16S rRNA. A 12.2-fold increase in protein yield was accompanied by a 4.3-fold increase in specific mRNA, indicating that transcription of the recombinant gene is coupled to translation. As this coupling amplifies the detrimental effect of a low-efficiency ribosomal binding site, much attention should be paid to translation initiation when optimizing a recombinant protein production system. Finally, reasons for the high expression level before induction are discussed, and first results towards reducing it are presented.

Base Sequence↗
PubMed1996

Cytokine activity assay by means of proliferation measured in plane convex microtiter wells.

A new assay to measure cell proliferation by turbidimetric evaluation of cultures in specially designed microtiter plates was set up. The IL-2-dependent proliferation of CTLL-2 cell line was used as a model. The novel microtiter plate detection system, the General Cell Screening System (GCSS) was used for the evaluation of the cell proliferation assay. The test system utilizes the population growth rate (mu) of the cells. The tests were performed in specially designed microtiter plates which were read in a scanning spectrophotometer. These results were compared with colorimetric assays, using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) as substrates. In contrast to conventional methods, the presently described system is not an end-point method, this means that cell growth can be observed over a certain period and is not limited to a single moment during the period of cell growth. Another advantage is the reduction of manual manipulations and the avoidance of toxic or radioactive substances and organic solvents. Therefore, a higher number of samples can be analyzed compared to other methods. The range for detection was 0.015-10 units IL-2 per well, and the accuracy is in the range of < 0.005 standard deviation.

Animals↗
PubMed1996

An in vitro test system for thyroid hormone action.

An in vitro bioassay for the biological action of thyroid hormones has been developed. The test is based on a dose-dependent increase in growth rate and cell activation of the rat pituitary tumor cell line GH3. This cell line is dependent on thyroid hormones for cell division when plated at low density in serum-free medium. The measurement of the cell-stimulating effect of L-triodothyronine (T3) is based on the capacity of mitochondrial enzymes of viable cells to transform the MTT tetrazolium salt into MTT formazan. A dose-dependent response of the MTT signal can be observed within a range from 0.075 to 1 nM T3 in the culture medium. The test is performed in 96-well microplates and allows screening of compounds which may interfere with the action of thyroid hormones.

Animals↗
PubMed1996

Restricted antigenic variability of the epitope recognized by the neutralizing gp41 antibody 2F5.

OBJECTIVE: To investigate whether variations of the conserved gp41 amino-acid sequence ELDKWA affect its binding or neutralization by monoclonal antibody (MAb) 2F5. DESIGN AND METHODS: Neutralization assays were performed with primary isolates from different HIV-1 subtypes and the sequences corresponding to the 2F5 epitope region were analysed. Studies of MAb 2F5 peptide reactivity were performed by spot analysis, using peptides immobilized on cellulose. The frequency of emergence of neutralization-resistant virus variants was determined by immune selection experiments in the presence of MAb 2F5. RESULTS: Primary isolates from clades A, B and E were neutralized by MAb 2F5. Neutralization sensitivity correlated with the presence of the LDKW motif. A K-to-N change in the core sequence was identified in a neutralization-resistant patient isolate. Neutralization resistant virus variants that were selected in the presence of MAb 2F5 were found to contain D-to-N, D-to-E, or K-to-N changes within the LDKW sequence. Neither in natural isolates nor in variants obtained under immune selection conditions in the laboratory were changes in the L and W positions observed. Studies of MAb 2F5 binding to variations of the ELDKWA peptide confirmed that the changes at the first and last positions did not significantly reduce binding capacity, whereas amino-acid changes from D to N, D to E, and K to N almost completely abrogated binding of MAb 2F5. CONCLUSION: Sequence analysis of a variety of primary isolates suggests that the major determinant of MAb 2F5 binding corresponds to the amino-acid sequence LDKW. Naturally occurring and in vitro selected neutralization-resistant viruses contained changes in the D and K positions of the ELDKWA motif.

Antibodies, Monoclonal↗
PubMed1996

Immunogenic presentation of a conserved gp41 epitope of human immunodeficiency virus type 1 on recombinant surface antigen of hepatitis B virus.

In view of the high antigenic variability of human immunodeficiency virus type 1 (HIV-1), a vaccine against AIDS must induce an immune response to epitopes as invariable as possible among the various virus strains and clones. Previously the highly conserved six amino acid sequence Glu-Leu-Asp-Lys-Trp-Ala (ELDKWA) from gp41, defining the epitope of the human MAb 2F5, was shown to elicit HIV-1-neutralizing antibodies when presented on haemagglutinin of influenza virus. We investigated the immunogenic potential of the MAb 2F5 epitope and two of its major escape epitopes as internal fusions to the hepatitis B virus (HBV) surface antigen (HBsAg). Recombinant HBsAg-HIV proteins produced in the methylotrophic yeast Pichia pastoris self-assembled into 22 nm lipoprotein particles. Mice immunized with these particles developed an anti-HBsAg immune response in a range that is considered to be protective against HBV infection in humans. More importantly, antisera had extremely high titres of antibodies reactive with a structurally flexible form of the HIV-1 epitope, whereby strong cross-reactivity with the escape variants of the epitope was observed. Although HIV-1 gp 160 and the ectodomain of gp41 containing the epitope were significantly recognized, the antisera failed to neutralize HIV-1 in vitro. These data, together with those on the haemagglutinin-ELDKWAS fusion suggest that the ability of the MAb 2F5 epitope to induce neutralizing antibodies depends on the molecular context in which it is presented. Therefore, further characterization of secondary and tertiary structure requirements of the epitope is indispensable for the full exploitation of its potential as a vaccine component.

AIDS Vaccines↗
PubMed1996

The consequence of passive administration of an anti-human immunodeficiency virus type 1 neutralizing monoclonal antibody before challenge of chimpanzees with a primary virus isolate.

The anti-gp41 virus neutralizing monoclonal antibody 2F5 was infused into chimpanzees, which were then given an intravenous challenge with a primary human immunodeficiency virus type I (HIV-1) isolate. In two control animals, the infection was established immediately, as evidenced by positive cell-associated DNA PCR and serum RNA PCR tests within 1 week, seroconversion by 4 weeks, and development of lymphadenopathy in this acute phase. Serum RNA PCR tests were negative in one of the two antibody-infused animals until week 8 and in the other antibody-infused animal until week 12; both animals seroconverted at week 14. The peak of measurable virus-specific serum RNA was delayed until week 16 in one antibody-infused animal. Virus-specific RNA in the other animal did not reach levels comparable to those in the other animals through 1 year of follow-up studies. Virus was isolated from the week 16 blood sample from one infused animal. Virus was not isolated from peripheral blood of the second animal but was isolated from lymph node cells taken at week 36. The infection of untreated chimpanzees with this primary isolate appears robust. Use of this isolate should widen the scope of possible experiments in the chimpanzee model. This antibody infusion study indicates that neutralizing antibody, when present at the time of challenge, affects the timing and level of infection and remains influential after it can no longer be detected in the peripheral circulation. It is possible that preexisting, neutralizing antibodies (passively administered or actively elicited) affect the course of acute-phase virus replication in humans. It remains to be established whether these immunologically mediated early effects will influence the course of HIV-1 disease.

Amino Acid Sequence↗
PubMed1996

Human monoclonal antibody 2G12 defines a distinctive neutralization epitope on the gp120 glycoprotein of human immunodeficiency virus type 1.

We have isolated and characterized human monoclonal antibody 2G12 to the gp120 surface glycoprotein of human immunodeficiency virus type 1 (HIV-1). This antibody potently and broadly neutralizes primary and T-cell line-adapted clade B strains of HIV-1 in a peripheral blood mononuclear cell-based assay and inhibits syncytium formation in the AA-2 cell line. Furthermore, 2G12 possesses neutralizing activity against strains from clade A but not from clade E. Complement- and antibody-dependent cellular cytotoxicity-activating functions of 2G12 were also defined. The gp120 epitope recognized by 2G12 was found to be distinctive; binding of 2G12 to LAI recombinant gp120 was abolished by amino acid substitutions removing N-linked carbohydrates in the C2, C3, V4, and C4 regions of gp120. This gp120 mutant recognition pattern has not previously been observed, indicating that the 2G12 epitope is unusual. consistent with this, antibodies able to block 2G12 binding to recombinant gp120 were not detected in significant quantities in 16 HIV-positive human serum samples.

Amino Acid Sequence↗
PubMed1995

Localization of fruit tree viruses by immuno-tissue printing in infected shoots of Malus sp. and Prunus sp.

Immuno-tissue printing protocols for the localization of apple chlorotic leaf spot virus (ACLSV), stem grooving virus (SGV) and plum pox virus (PPV) in shoots of Prunus and Malus in vitro have been established for routine diagnosis in a virus elimination program. Since these viruses belong to different virus genera, the protocols were adapted according to the properties of the virus under investigation. Accumulation of ACLSV was highest in the base of the stem and decreased towards the apex of the shoots. ACLSV was found in the epidermis, the cortex, in the vascular bundles, but seldom in the pith tissue of in vitro apple shoots. ACLSV immuno-tissue printing was as sensitive as ELISA and the intensity of color signals in immuno-tissue prints correlated with absorbance values by two-step ELISA. SGV could be detected by immuno-tissue prints at infectivity levels, where it reacted negative in ELISA. SGV accumulated in the vascular bundles, occurred locally in the parenchymatic tissue, was found in high amounts in young leaves near the meristem, but not within the meristem. PPV was detected in all tissue types of stem sections with an irregular pattern reflecting the in vivo situation causing problems with detection. Discrimination of poorly and heavily infected shoots was possible with the naked eye.

Culture Techniques↗
PubMed1995

Cross-clade neutralization of primary isolates of human immunodeficiency virus type 1 by human monoclonal antibodies and tetrameric CD4-IgG.

We have tested three human monoclonal antibodies (MAbs) IgG1b12, 2G12, and 2F5) to the envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1), and a tetrameric CD4-IgG molecule (CD4-IgG2), for the ability to neutralize primary HIV-1 isolates from the genetic clades A through F and from group O. Each of the reagents broadly and potently neutralized B-clade isolates. The 2F5 MAb and the CD4-IgG2 molecule also neutralized strains from outside the B clade, with the same breadth and potency that they showed against B-clade strains. The other two MAbs were able to neutralize a significant proportion of strains from outside the B clade, although there was a reduction in their efficacy compared with their activity against B-clade isolates. Neutralization of isolates by 2F5 correlated with their possession of the LDKW motif in a segment of gp41 near the membrane-spanning domain. The other two MAbs and CD4-IgG2 recognize discontinuous binding sites on gp120, and so no comparison between genetic sequence and virus neutralization was possible. Our data show that a vaccine based on the induction of humoral immunity that is broadly active across the genetic clades is not impossible if immunogens that express the epitopes for MAbs such as 2F5, 2G12, and IgG1b12 in immunogenic configurations can be created. Furthermore, if the three MAbs and CD4-IgG2 produce clinical benefit in immunotherapeutic trials in the United States or Europe, they may also do so elsewhere in the world.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Expression of SV40 tumour antigens enables human endothelial cells to grow independently from foetal calf serum and exogenous growth factors.

Human endothelial cells were transfected with a plasmid containing the coding sequence of the large T protein of simian virus 40. Transfected cells were selected for their ability to grow in defined medium (DM). Several cell lines were derived and characterized in their response to endothelial cell growth supplement (ECGS), epidermal growth factor (EGF) and insulin (INS). In addition to cell lines that were dependent on these additives, others growing without any exogenous growth factor could be selected. No evidence of autocrine growth stimulation was found. For growth studies, a simple assay was used based on the acid phosphatase activity as a parameter for the cell number. Cell lines in defined medium showed less chromosome aberrations than those grown in serum-containing medium. Because of their long in vitro life span of about 100 generation doublings and defined medium requirements these cells represent valuable test material for all kinds of investigations on the vascular endothelium.

Animals↗
PubMed1994

Purification of human recombinant superoxide dismutase by isoelectric focusing in a multicompartment electrolyzer with zwitterionic membranes.

Human recombinant superoxide dismutase (SOD), purified to homogeneity, is resolved by both conventional isoelectric focusing and immobilized pH gradients into three bands, with isoelectric points (pIs) in the pH range 4.8 to 5.1, the pI 4.80 form representing the minor component. Due to the fact that this enzyme is expressed in E. coli, N-terminal acetylation or glycosylation should be ruled out. When purified by small-scale preparative isoelectric focusing in immobilized pH gradient gels, it was found that, upon subsequent analysis, the pI 5.07 form would band in the same position, but the intermediate pI 4.92 band would split into the upper (pI 5.07) and the lower (pI 4.80) species, in nearly the same amounts, whereas the lowest pI component would always generate both the intermediate and upper forms. Enzymatic essays pointed out that these three isoforms had nearly the same specific activity, slightly higher than that of the starting material. Metal analysis indicated that all three forms contained the same metal/protein ratio, approaching the value Cu2Zn2-SOD, as reported in the literature. Circular dichroism spectra of the pI 4.80 and 5.07 forms showed the same profile in the 190-240 nm range, but marked differences in the 250-350 nm region. Treatment with EDTA produces 1-2 additional, slightly higher pI isoforms, whereas treatment with KCN generates a number of higher pI components, reaching pI values as high as pH 7, with nearly complete disappearance of the three major SOD isoforms. It is concluded that these three isoforms could represent interconvertible species, the highest pI component representing the most stable conformer.

Circular Dichroism↗
PubMed1994

Two-dimensional gel electrophoresis for controlling and comparing culture supernatants of mammalian cell culture productions systems.

A recombinant Chinese hamster ovary cell line, producing human erythropoietin, was cultivated in a continuous mode in a stirred tank reactor applying different dilution rates. In order to monitor the stability of this expression system, product and non-product proteins of the cell culture supernatant were analyzed by two-dimensional electrophoresis. The consistency of the isoforms of the recombinant product was determined by western blot combined with specific staining. The same cell line was propagated in a high cell density cultivation system based on macro-cell-aggregates. The patterns of secreted proteins of the cell line cultivated in the different systems were compared in order to detect modifications in protein expression of the product and of non product proteins relevant for cell culture supernatant. Hardly any alterations in two-dimensional pattern were detectable. The isoforms of erythropoietin, as well as the overall pattern of secreted proteins, detectable with the two-dimensional electrophoresis method were remarkably stable under different cultivation conditions.

Animals↗
PubMed1994

HIV-1 gp41 binding proteins and antibodies to gp41 could inhibit enhancement of human Raji cell MHC class I and II expression by gp41.

Based on our findings, that HIV-1 soluble gp41 could bind to several proteins on the human, T, B and monocyte cells independently of CD4, we examined the effect of HIV-1 soluble gp41 (sgp41;Env amino acids 539-684) on surface expression of MHC I and II, ICAM-1 and CD21 molecules on human Raji cells. Flow cytometry (FACS) analysis demonstrated that sgp41 could selectively enhance MHC class I and II expression on Raji cells, but did not increase expression of other cell surface antigens, such as, CD21 and CD54 (ICAM-1). Soluble gp41 could also enhance MHC class I and II expression on another human B cell line, Bjab. The sgp41-dependent enhancement of the MHC class I and II expression on Raji cells is time- and dose-dependent. The sgp41 enhancement effect on the MHC antigen expression could be inhibited by the sgp41-binding proteins of 45, 49 and 62 kD (isolated from Raji-lysate) which could inhibit the spg41-binding to Raji cells. Interestingly, this sgp41-dependent enhancement of the MHC class I and II expression could also be inhibited by two mAbs to HIV-1 gp41, but not by a third mAb binding to a different site on gp41. These results demonstrate that HIV-1 sgp41 can selectively enhance the human Raji cell MHC class I and II antigen expression and this enhancement effect could be inhibited by the sgp41-binding proteins and anti-gp41 antibodies, and suggest that the sgp41-dependent enhancement is mediated by its binding to Raji membrane proteins of 45, 49 and 62 kD.

Antibodies, Monoclonal↗
PubMed1994

HIV-1 gp41 shares a common immunologic determinant with human T, B and monocyte cell lines.

Several examples of molecular mimicry between HIV-1 and human proteins are reported in the literature. Here we report on yet another example. The monoclonal antibody 3D6 recognized a 17-amino-acid region in HIV-1 gp41 (amino acids 602-618) and could bind to the human T-cell lines H9 and Molt4, B cell lines Raji and Bjab, and monocyte cell lines U937 and HL60. By Western blot using 3D6, a strong band of 43 kDa and a very weak band of 80 kDa were detected in lysates of H9, Molt4, Raji and Bjab cell lines, but only a strong band of 43 kDa was observed in case of U937 and HL60 cells, under reducing or non-reducing conditions. The results indicate the presence of a common immunologic determinant between HIV-1 gp41 and membrane proteins of these human T, B and monocyte cells.

Amino Acid Sequence↗
PubMed1994

HIV-1 gp41 binds to two proteins in cell culture supernatant of human B cell line Raji and monocyte cell line U937.

Based on our findings that HIV-1 gp41 can bind independently of CD4 to the human T cell line H9, B cell line Raji and monocytic cell line U937, as well as human peripheral blood mononuclear cells preferentially to B lymphocytes and monocytes, we examined whether soluble protein for HIV-1 gp41 binding exists in the cell culture supernatant of Raji and U937. Using HIV-1 recombinant soluble gp41 (sgp41; Env amino acid 539-684) attached to sepharose beads, the cell culture supernatants of Raji and U937 were absorbed. By SDS-PAGE of sgp41-eluates of these cell culture supernatants three protein bands of 70, 75 and 92kD were stained with Coomassie blue. By Western blot (ligand blot) analysis using sgp41, two protein bands of 70 and 75kD were observed in sgp41-eluates from Raji and U937 cell culture supernatants. These sgp41-eluates could inhibit the sgp41 binding to Raji and U937 cells. Our data indicate that Raji and U937 cells produce two soluble binding proteins for HIV-1 gp41.

B-Lymphocytes↗