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Biomedical subjects

R Grunow

Publications and source records attributed to R Grunow.

At least 19 recordsLinked to original sources
PubMed1994

Reduced number of Langerhans cells in oral mucosal washings from HIV-1 seropositives.

In order to elucidate mucosal immunity in HIV-1 seropositive individuals, we investigated oral mucosa washings from 20 HIV-1 seropositive patients for the presence of Langerhans cells (LC) and HIV-1 antigen-positive cells, and compared the results with those obtained from 20 HIV-1 seronegative healthy individuals. Monoclonal antibodies directed against CD1a, HLA-DR, CD3, and p24 were used to identify LC, T cells and HIV-1 core-antigens, respectively. In oral mucosa washings from HIV-1 seropositive patients there was a significant reduction in the number of CD1a+ cells as compared with the healthy subjects. HIV-1 antigen-positive cells were not detected. The reduction of LC in oral mucosa washings from HIV-1 seropositive patients is probably associated with HIV-1 infection. The frequent occurrence of oral mucosal disorders in HIV-1 infected patients may in part be caused by a reduced LC-number and/or function.

AIDS-Related Opportunistic Infections↗
PubMed1992

Inhibition of HIV-1 infection in vitro by murine monoclonal anti-p24 antibodies.

Murine monoclonal antibodies (Mabs) to the major core protein p24 of the human immunodeficiency virus type 1 (HIV-1) were tested for their ability to inhibit the replication and spread of the virus in permanent cell cultures (Molt4/8, K37, H9) and in the culture of II-2 stimulated T cells of healthy donors. After addition of ascitic fluid containing monoclonal anti-p24 antibodies or purified anti-p24 antibodies or the respective control to co-cultures of infected and non-infected cells, HIV-1 replication was evaluated by determining the percentage of infected cells and the activity of reverse transcriptase (RT) in cell-free supernatant. In addition, the supernatant's infectivity was determined. FACS analysis demonstrated p24 antigen in about 40% of unfixed HIV-1 infected cells at the cell membrane. Monoclonal anti-p24 antibodies of different epitope specificity added to the cells but not to the virus delayed the spread of HIV-1 infection in permanent cell culture. Furthermore, anti-p24 Mabs inhibited the release of RT-active virus particles by HIV-1 infected cell lines or II-2 stimulated T-lymphocytes, respectively, up to 60%. The mode of action of anti-p24 antibodies after HIV-1 infection is discussed on the basis of the data obtained.

Animals↗
PubMed1992

Immunoglobulin V regions and epitope mapping of a murine monoclonal antibody against p24 core protein of HIV-1.

The nucleotide sequence of a murine monoclonal antibody (CB-mab-p24/13-5) against p24 core protein of the human immunodeficiency virus (HIV-1) was determined for variable regions of the heavy and light chain, respectively. Genetic elements encoding the VDJH- and VJL-regions of the antibody were generated from RNA by the polymerase chain reaction, cloned into the vector pICEM 19R and sequenced. Synthetic peptides, 10 amino acids overlapping served for the localization of the epitope. The residues 152-156 within the p24 sequence contain the epitope.

Amino Acid Sequence↗
PubMed1992

Human MoAbs produced from normal, HIV-1-negative donors and specific for glycoprotein gp120 of the HIV-1 envelope.

Human MoAbs of IgM class were developed against three regions of the HIV-1 envelope. Uninfected donor lymphocytes were immunized in vitro with recombinant protein pB1. Four out of five antibodies were directed to different parts of the V3 region, which contains a major neutralizing site. Two out of these antibodies were directed to more than one amino acid sequence, indicating reactivity to discontinuous sites. Two of the human MoAbs inhibited viral spread between cells in tissue culture, interpreted as reactivities to conserved amino acid sequences exposed during viral maturation. No MoAb neutralized virus, which may be explained by the relatively low avidity of the antibodies. One MoAb was directed to a region containing amino acids participating in CD4 binding. This technique appears to allow formation of antibodies with fine specificities other than those obtained in infected hosts.

Amino Acid Sequence↗
PubMed1992

Oligonucleotide fingerprinting as a means to identify and survey long-term cultured B cell hybridomas and T cell lines.

Common problems encountered during cell culture are cross-contamination, instability, and inadvertent exchange of cells. Here we report on the application of oligonucleotide fingerprinting as a simple and efficient method to screen hybridomas and T cell lines. Among the fingerprint probes tested, the simple repetitive oligonucleotide (CAC)5/(GTG)5 proved to be most useful for obtaining many fragments specific for each cell line. Because of variable loss of chromosomes, cloned hybridoma cells from one fusion exhibit different fingerprint patterns. Thus, antibody-secreting B cell hybridomas can be distinguished easily even when they originate from the same fusion. Furthermore, we were able to monitor the genomic integrity of myelomas and hybridomas over a period of more than 2 years, thereby highlighting long-term stability. Another application of the method is the control of T cell lines requiring irradiated or mitomycin-treated feeder cells for continuous growth or cloning. There cell lines are always threatened to be overgrown by feeder cells that have escaped from the lethal pretreatment. T cell clones of one individual, known to display differently rearranged T cell receptors, did not show differences in their fingerprint patterns. However, in EBV-transformed cloned B cells, slight differences between clones from the same donor were identified.

B-Lymphocytes↗
PubMed1991

Comparison of four anti-HIV screening assays which belong to different test generations.

There are three different test generations of enzyme-linked immunosorbent assays (ELISA) for the detection of human immunodeficiency virus (HIV) infection, depending on whether virus lysate, recombinant proteins or synthetic peptides are used as solid phase antigen. Four different assays, i.e., three sandwich ELISAs and one competitive test, were used to demonstrate differences between the three systems with regard to the content of different diagnostically relevant virus proteins. The sensitivities and specificities of these assays were compared by using 312 anti-HIV positive sera and 500 sera of healthy blood donors. The highest sensitivity and specificity were achieved by the competitive ELISA based on recombinant proteins, and by the sandwich ELISA based on synthetic peptides.

Blotting, Western↗
PubMed1990

Production and characterization of monoclonal antibodies against human Cu/Zn superoxide dismutase and the establishment of a super-rapid enzyme-linked immunosorbent assay (SURALISA).

Murine monoclonal IgG1 antibodies directed against four different epitopes of human Cu/Zn superoxide dismutase (SOD) were produced by immunization with recombinant Cu/Zn SOD. The antibodies reacted well with the recombinant protein and Cu/Zn SOD purified from human erythrocytes, with binding constants ranging from 8.8 X 10(9) to 2.2 X 10(10) l/mol. When mixed, these antibodies completely prevented the binding of rabbit and sheep polyclonal antibodies raised against erythrocyte Cu/Zn SOD. Whereas one antibody was directed against a common homology region of bovine and human Cu/Zn SOD, all the other antibodies reacted exclusively or preferentially with human Cu/Zn SOD. Only one epitope on the human Cu/Zn SOD molecule was accessible at two different sites as demonstrated in a homologous two-site assay with one and the same antibody used as both capture and indicator antibody. In the indirect two-site assay with unlabelled monoclonal antibodies, and additive effect with a steeper dose-response curve was obtained by mixing antibodies against different epitopes. A super-rapid one-step two-site enzyme immunoassay (overall duration 20 min) was established with antibodies against two different epitopes. Its detection limit was 0.5 micrograms SOD/l.

Antibodies, Monoclonal↗
PubMed1990

[Use of fermenter-dialysis culture for the cultivation of medically relevant microbes. III. Candida albicans].

For mass cultivation of Candida albicans (serotype A), a fermenter dialysis culture technique is described and compared with shaking culture and fermenter batch culture techniques. Important growth parameters such as yeast dry weight and viable cell counts demonstrate the advantage of the fermenter dialysis culture. Mannan, the major antigen from Candida albicans prepared by phenol-water extraction followed by gel chromatography was tested with the monoclonal IgM antibody H5.

Candida albicans↗
PubMed1990

Immunochemical quantification of Cu/Zn superoxide dismutase in prenatal diagnosis of Down's syndrome.

Cu/Zn superoxide dismutase (SOD) was quantified by enzyme immunoassay for prenatal diagnosis of Down's syndrome. Overall, 154 samples of amniotic fluid, 72 samples of amniotic cells and 31 samples of chorionic tissue were investigated. Due to the large biological variance of the SOD concentrations in normal pregnancies (range for amniotic fluid 10.5-154.9, for amniotic cells 40.0-338.8, and for chorionic tissue 132.2-649.5 g SOD/g protein) the cases of Down's syndrome detected by karyotype analysis were not reliably identified by Cu/Zn SOD quantification. As in erythrocytes obtained from patients with Down's syndrome, a trisomy 21 was easily and accurately detected in the erythrocytes from very small quantities (about 50 microliters) of umbilical blood. The SOD concentrations in normal cases (n = 40) varied between 11.4 and 17.3 and in the cases of trisomy 21, as confirmed by karyotyping (n = 4), between 22.5 and 23.2 ng/one million cells. SOD quantification in fetal erythrocyte is a helpful additional method in prenatal Down syndrome diagnosis under certain conditions, which are discussed.

Amniotic Fluid↗
PubMed1990

Binding behaviour of antibodies reacting specifically with an immunodominant region of the transmembrane protein gp41 of HIV-1.

A highly conserved region of the transmembrane protein gp41 of the HIV-1 was found in which the amino acid sequence 606 to 620 was characterized as an immunodominant region. The binding behaviours of a human monoclonal antibody and of polyclonal specific antibodies in sera of HIV-infected persons to this particular sequence have been studied. In two sandwich-type ELISAs with synthetic peptides as antigen representing this region, sera of 312 HIV-infected persons at all stages could be clearly discriminated against 500 anti-HIV antibody negative sera of healthy blood donors.

Amino Acid Sequence↗
PubMed1990

Immortalization of magnetically separated human lymphocytes by electrofusion.

Human lymphocytes from peripheral blood (MNC) were separated on magnetic beads for the presence of different surface markers. Cells from positive and negative fractions were successfully immortalized by electrofusion with the heteromyeloma line CB-Fu2. B cells, which were separated on anti-CD 19 coated beads, could be immortalized at a rate between 10(-5) and 10(-4) even if the fusion was conducted with just a few hundred thousand cells. Comparison of the frequency of Ig-positive hybridomas in B cell, T cell, and unseparated MNC fusions indicated that also non-B cells may give rise to HAT resistant hybridoma clones, although the fusion frequency was low.

Animals↗
PubMed1990

The hybridization of EBV-immortalized human B-lymphocytes with a human-mouse heteromyeloma cell line.

The combination of Epstein-Barr-Virus (EBV)-permitted immortalization and somatic hybridization (fusion with a myeloma partner) may be the method of choice to produce human monoclonal antibodies. We show here that the fusion of EBV-infected human B-lymphocytes to the HAT-sensitive, ouabain-resistent heteromyeloma (human x mouse) fusion line CB-F7, resulted in stable growing hybridomas producing much more immunoglobulin than the parental lymphoblastoid lines. A more efficient clonability was shown for hybridoma cultures too. The loss of B cell markers (HLA-class II antigen, CD-22, CD-37) was detected. Limiting dilution experiments showed a better fusionability of IgM-producing EBV-transformed B cells in comparison to IgG-secreting counterparts.

Animals↗
PubMed1990

Strategies in the development of human monoclonal antibodies.

A high-efficiency, HAT-sensitive heteromyeloma fusion line CB-F7 has been developed from an 8-Azaguanin-treated Ig-non-secreting human X mouse heterohybridoma. The use of this line allowed us to produce human hybridomas more successfully by fusion of cell material from blood, lymph node or spleen. A polyspecific repertoire of IgM isotype was detected among the hybridomas obtained from the spleen. These IgM antibodies reacted with autoantigens as well as with foreign material. This naturally occurring repertoire may be of interest since it has anti-bacterial activity. The frequency of the occurrence of polyspecific antibody-producing hybridomas was high in the spleen. Apart from the detection of polyspecific IgM antibodies we did not find IgG-secreting hybridomas with anti-bacterial reactivity among thousands of initial lines derived from non-immunized persons. We therefore tried to fuse lymphocytes from donors, who were boosted with Tetanus Toxoid (TTd). A short and limited optimum time period at which the blood should be taken from the donors after boosting, was detected. Seven days after in vivo immunization high yields of IgG-anti-TTd-producing lines (239 out of 731 IgG-producers) were found. The methods of developing more efficient production of human monoclonal antibodies of a pre-defined specificity were discussed.

Animals↗
PubMed1989

Application of a human monoclonal antibody in a rapid competitive anti-HIV ELISA.

The ELISA is the established screening technique for the detection of antibodies directed against HIV. The first generation assays, mostly based on the sandwich principle, employed purified virus from cell culture and gave both false-positive and false-negative results. Sandwich-type assays preferentially detect IgG antibodies, require a high serum dilution and are two-step procedures. In order to detect an immune response as early as possible after infection anti-HIV antibodies of the IgM class should also be measured. To this end a competitive ELISA has been developed using a solid phase-adsorbed recombinant HIV envelope protein and an enzyme-labelled human monoclonal antibody. This detects both IgM and IgG antibodies, the results are available within 1 h and a serum predilution is not necessary.

Antibodies, Monoclonal↗
PubMed1989

[A fermenter dialysis cultivation technic for encapsulated pathogenic bacteria. II. Group B streptococci].

For mass cultivation of group B streptococci (B-III and B-variant streptococci), a fermenter dialysis culture technique is described and compared to conventional shaking culture and fermenter batch culture techniques. The influence of two kinds of cultivation media on the bacterial yield is demonstrated. The growth rate of the bacteria and the yield of the microbes is higher in modified POPE medium than that observed with Nährbouillon I. The type-specific polysaccharide of B-III-streptococci prepared by phenol-water extraction followed by gel-chromatography can be used as a screening antigen for the production of monoclonal antibodies against B-III-streptococci.

Antigens, Bacterial↗
PubMed1989

[Chemical characterization of Escherichia coli K 1 capsular polysaccharide].

The exact chemical characterization of the prepared Escherichia coli K 1 capsule polysaccharide is necessary and a prerequisite for using this antigen as a screening antigen in the production of monoclonal anti-K 1-antibodies. The K 1-antigen, prepared by phenol-water-extraction, was analyzed by protein, RNA, and neuraminic acid determination. An addition, the antigen was subjected to elementary analysis, infrared- and 13C-NMR-spectroscopy, and gelchromatography. After testing the serological specificity, the K 1-antigen was identified as a high molecular polyneuraminic acid.

Antigens, Bacterial↗
PubMed1989

Development of specific IgG-producing human hybridomas by fusions of lymphocytes from actively immunized persons.

More than 13,500 initial hybridoma lines derived from fusions of lymphocytes from non-boosted persons were tested for IgG production against Tetanus Toxoid. However, only 2 were found to produce IgG monoclonal antibodies of the desired specificity. Peripheral blood lymphocytes were then taken from actively immunized donors 3, 7, 14 or 60 days after boosting and fused to the HAT-sensitive heteromyeloma cell line CB-F7. A strong enhancement of both IgG-producing hybridomas and specific IgG-producers was detected in fusions of lymphocyte material derived 7 days after booster injection (239 of 731 IgG-producing lines showed anti-TT-specificity). Two months after boosting no more specific IgG-producing hybridomas could be established from the peripheral blood of the donors. Data were discussed regarding an optimized human monoclonal antibody production against bacterial antigens.

Antibodies, Bacterial↗