Search PubMedSearch

Biomedical subjects

B Micheel

Publications and source records attributed to B Micheel.

At least 19 recordsLinked to original sources

Interaction of B and T lymphocyte subsets with high endothelial venules in the rat: binding in vitro does not reflect homing in vivo.

Lymphocytes continuously migrate through the body, and their efficient extravasation from the blood via high endothelial venules (HEV) is essential for initiating an appropriate immune response. Most investigations have focused on the lymphocyte/HEV interaction in vitro. However, to what extent such systems reflect the situation in vivo is not known. It is also unclear whether lymphocyte subsets immigrate into the HEV in proportion to their presence in the blood, and whether import capacity is limited by the HEV. When rat mesenteric lymph node lymphocytes were incubated in vitro on cryostat sections, the well-known preferential binding of B lymphocytes to HEV of Peyer's patches (PP) and T cells to HEV of axillary lymph nodes (axLN) was observed (axLN vs. PP: B lymphocytes 21.2 +/- 5.0% vs. 40.6 +/- 11.0%, T lymphocytes 84.6 +/- 6.3% vs. 56.5 +/- 12.9%). However, when labeled mesenteric lymph node lymphocytes were injected and their location within the HEV was analyzed 15 min later, no preferential interaction was seen. After injection of labeled thoracic duct lymphocytes, the percentage of labeled cells among B and T lymphocytes in the blood was significantly different (4.4 +/- 0.9% vs. 8.9 +/- 3.6%), whereas that in HEV of axLN (19.0 +/- 6.4% vs. 16.6 +/- 6.0%) and PP (30.6 +/- 6.1% vs. 33.9 +/- 4.4%) was comparable. Although the number of injected lymphocytes was similar in magnitude to the total blood lymphocyte pool, after injection there was no increase in lymphocyte numbers in the HEV. Thus, the adhesion assay in vitro does not completely reflect immigration into HEV in vivo. In addition, our data suggest that both the availability of lymphocyte subsets in small venules and the immigration rate into HEV are actively regulated in vivo.

Animals

The accessibility of thiophosphorylated groups in DNA fragments to the enzymatic activity of ligases and restriction endonuclease Bbs I.

The aim of this paper was to test the possibility to ligate and hydrolyse DNA sequences containing thiomodified ends and bonds. T4 DNA ligase was shown to ligate DNA fragments regardless of whether it contains phosphorylated or thiophosphorylated 5'-end. But the cleavage of an internally thiomodified phosphodiester bond was found to be totally inhibited when using the non-palindromic restrictase Bbs I. The special properties of this restriction endonuclease should allow the development of an oriented cloning strategy when combined with T4 ligase and a thiophosphorylation of DNA fragments.

Base Sequence

A flow cytometric long-term cytotoxicity assay.

A method to evaluate cytotoxic effects applicable over a wide range of incubation times has been developed. It is based on quantification by flow cytometry of dead and viable target cells stained by covalently binding the fluorescent dye fluorescein isothiocyanate (FITC). The staining with FITC did not affect cell viability and growth parameters and was stable enough to identify target cells for at least 2 days. The flow cytometric analysis of the cell mixture was performed in a test system with activated CD8+ lymphocytes as effector cells and melanoma M21 cells as targets in the presence of appropriate bispecific antibodies and revealed a rather complex pattern composed of several distinct cell subsets which were identified by use of antibodies to lymphocyte antigens. The assay compared favourably with results from a conventional 51Cr release assay obtained after 4 h and 8 h of incubation and from a target cell adherence assay obtained after 24 h of incubation. The application of the method described herein is especially advantageous for the evaluation of long-term cytotoxic effects. Furthermore, it provides valuable multi-parameter information which is useful for elucidating mechanisms of cytotoxicity.

Animals

A simple and sensitive method to study effects mediated by soluble lymphokines as demonstrated by the interaction of CD4+ and CD8+ cell subsets during T cell activation.

A method is described for the study of lymphokine-mediated cellular interactions using triple wells, which permits co-culture of cell subpopulations without direct physical contact. The triple wells are constructed by slitting the walls to half height between three adjacent wells of a 96-well microtiter plate. The cells under study are positioned in the outer two wells, whereas the middle well serves to separate the cells. The half slits permit the wells to be treated independently before filling the triple well with the culture medium and prevents cell leakage thereafter. The feasibility of the method was established by studying the interaction of isolated CD4+ and CD8+ T cell subsets during T cell proliferation induced by immobilized anti-CD3 and anti-CD28 monoclonal antibodies.

CD4-Positive T-Lymphocytes

Production of monoclonal antibodies against epitopes of the main coat protein of filamentous fd phages.

Three monoclonal antibodies (MAbs) were produced which react with epitopes of the main structural coat protein (pVIII) of filamentous fd phages as demonstrated by solid-phase fluorometric enzyme immunoassays and by immunoelectron microscopy. The antibodies are of the IgG1, IgG2a and IgG2b immunoglobulin subclasses. Since they also react with recombinant phages expressing antigen fragments in their pIII region they may be suitable reagents for the demonstration and isolation of filamentous phages used in recombinant protein technology.

Animals

Monoclonal antibodies to human chorionic gonadotropin (HCG) and their use in two-site binding enzyme immunoassays.

A panel of mouse monoclonal antibodies (MABs) was produced against human chorionic gonadotropin (HCG) and its isolated beta-subunit (beta-HCG). According to their binding specificities the antibodies could be divided into HCG-specific and cross-reactive MABs. The HCG-specific antibodies reacted with antigenic sites on holo-HCG or holo-HCG and beta-HCG, or exclusively with the non-associated beta-HCG chain. The cross-reactive antibodies reacted with either HCG and luteinizing hormone (LH) or with HCG, LH, follicle-stimulating hormone (FSH) and thyroid-stimulating hormone (TSH). According to the binding specificities of the MABs and their reciprocal inhibition detected in two-site binding enzyme immunoassays (EIA), altogether 13 epitopes (including the 3 hidden epitopes detectable only on free non-associated beta-HCG) were distinguished by the antibodies described here. Antibody combinations resulting in most effective and specific HCG- or beta-HCG-determination were used as clinical assays and proved their reliability and correctness for monitoring patients with HCG- and/or beta-HCG-producing tumors before and after therapy.

Animals

Bispecific IgA/IgM antibodies and their use in enzyme immunoassay.

Two hybrid hybridomas secreting polymeric bispecific antibodies to human chorionic gonadotropin and calf intestinal alkaline phosphatase were produced by fusion of IgA- and IgM-secreting mouse hybridomas. Both hybrid antibodies were purified from ascitic fluid by size exclusion chromatography. An IgM-like fraction was shown to exhibit bispecific activity. Bispecificity was completely lost following mild reduction and alkylation. Both bispecific antibodies were used to develop a sensitive enzyme immunoassay for hCG.

Alkaline Phosphatase

Immunoaffinity purification of human alpha-fetoprotein (AFP) using monoclonal antibodies.

A two-step immunoaffinity isolation procedure for human alpha-fetoprotein (AFP) was developed resulting in highly purified AFP at high yield. A monoclonal antibody immunoadsorbent was used in the first step. Elution of AFP was carried out at alkaline pH by a solution of 0.5 mol/l ammonia containing 0.5 mol/l sodium chloride. To remove impurities caused by the first step, an anti-mouse immunoglobulin antibody immunoadsorbent was applied in the second step.

Antibodies, Monoclonal

Production and characterization of monoclonal antibodies against carcinoembryonic antigen (CEA).

In the experiments presented here 22 monoclonal antibodies (MoAbs) were produced which reacted with the tumor marker carcinoembryonic antigen (CEA). Eleven of the MoAbs reacted neither with peripheral blood granulocytes nor with purified spleen NCA-60 kDa and were therefore regarded as "CEA-specific". Only three antibodies of this group reacted exclusively with CEA-180 kDa. Eight MoAbs reacted with CEA-180 kDa and with CEA-like substances of lower molecular mass (of 160 kDa and/or 120 kDa) present in colon carcinoma cells as determined by immunoblotting. These molecules seem to be different from the classical non-specific cross-reacting antigens (NCAs) present in peripheral blood granulocytes. In contrast to that, the other 11 anti-CEA MoAbs recognized in addition to CEA-180 kDa also NCAs on granulocytes. Six of them were reactive with a purified spleen NCA-60 kDa preparation. These MoAbs bound also to reduced and alkylated CEA-180 kDa (CEA r/a), i.e. they recognize sequential epitopes. All 22 MoAbs reacted with CEA expressed in different human tumor cell lines as determined by immunocytological analysis. But six of them did not bind to the surface of these cells when tested in a radioimmuno-binding assay. It was concluded that the epitope(s) recognized by these antibodies are involved in cell membrane anchoring of the CEA-molecules.

Antibodies, Monoclonal

An experimental immunoscintigraphic study with anti-CEA monoclonal antibody (DG2).

Monoclonal antibody D11-DG2 (DG2) against carcinoembryonic antigen (CEA) was examined for suitability for radioimmunodetection of human tumors grown in nude mice. Antibodies DG2 and a control antibody of the same IgG1 subclass were labeled with 131I and injected into mice bearing one of three types of CEA-containing tumors (cell lines LS 174T, HT-29 and Rec S) and/or a CEA-negative tumor (Rec R). Gamma-camera imaging and distribution studies revealed that CEA-containing tumors selectively accumulate DG2 but Rec R does not. As the tumors differ in CEA-content, the highest accumulation of 131I-DG2 (corresponding to the best scintigraphic imaging) was found in LS 174T tumors, intermediate in Rec S and lowest in HT-29 tumors. The mean tumor-to-blood ratios on the sixth day after antibody administration were 4.6, 3.2, and 2.1, respectively, in the control experiments the value of this parameter was always lower than 1. The results showed the applicability of DG2 for immunoscintigraphic studies in patients. Furthermore, a positive correlation was found between the uptake of anti-CEA antibody and CEA-content in the tumors.

Animals

[Immunoscintigraphy in experiments and clinical practice: its possibilities, advantages and limitations].

The authors present their experience gained in preparing, isolating and labeling antibodies with radionuclides for the purpose of using them in immunoscintigraphy. The experimental part includes results obtained with different labeled antibodies and their F/ab/2 fragments in distribution studies, involving also immunoscintigraphic imaging of tumors. The clinical part presents results of immunoscintigraphy obtained with the commercial antibody kits Iodomab and Imacis in patients with tumors of the digestive tract.

Animals

Production and ELISA application of bispecific monoclonal antibodies against fluorescein isothiocyanate (FITC) and horseradish peroxidase (HRP).

Hybrid hybridomas producing bispecific monoclonal antibodies reacting with both horseradish peroxidase (HRP) and fluorescein isothiocyanate (FITC) were obtained by fusing two hybridoma lines and selecting the fused cells using a fluorescence activated cell sorter (FACS). FITC was used to label different monoclonal antibodies and the bispecific antibodies acted as a linking agent between FITC-labelled antibody and the marker enzyme HRP. This system was used in enzyme immunoassays for the detection of different antigens. The results suggest a wide application of bispecific anti-FITC/anti-HRP antibodies as a detection system in EIA.

Animals

Bispecific antibody-producing hybrid hybridomas selected by a fluorescence activated cell sorter.

Hybrid hybridomas (tetradomas) producing bispecific monoclonal antibodies reacting with both horseradish peroxidase (HRP) and human alpha-fetoprotein (AFP) were obtained by fusing two hybridoma lines and selecting the fused cells by a fluorescence activated cell sorter (FACS III). The hybridoma cells were labelled before fusion with fluorescein isothiocyanate (FITC) and tetramethyl rhodamine isothiocyanate (TRITC) respectively and heterofluorescent cells were sorted out after fusion. Several clones were found to produce bispecific antibodies, and one clone, designated T1, was subjected to growth in ascitic fluid in mice in order to obtain large quantities of hybrid antibodies. Bispecific antibodies could be separated from the monospecific antibody populations by one-step hydroxylapatite chromatography. SDS-polyacrylamide gel electrophoresis demonstrated that the hybrid antibody molecules contained the heavy chains of both anti-HRP and anti-AFP origin. The bispecific antibodies were used to build up a sensitive two-site binding enzyme immunoassay.

Animals

Differences of K562 leukemia cell clones in the pattern of monoclonal antibody binding and NK cell susceptibility.

Fifty-three K562-clones were tested against three monoclonal anti-K562-antibodies (MoAbs). The individual NK-cell sensitivity of thirty-one K562-clones was evaluated simultaneously. It was found that considerable differences exist in the antibody binding between single clones and the parental cell line indicating antigenic heterogeneity in the K562 line. Some K562-clones showed increased NK-cell sensitivity in comparison with the uncloned cell line. However, no correlation was observed between both investigated parameters.

Antibodies, Monoclonal

Search for CEA-like molecules in polymorphonuclear leukocytes of non-human primates using monoclonal antibodies.

The monoclonal anti-CEA antibody ZIK-A42-A/C1 which reacts with NCA of human polymorphonuclear leukocytes was found to bind also to polymorphonuclear blood leukocytes of the following non-human primates tested: hamadryas baboon (Papio hamadryas), stump-tailed monkey (Macaca arctoides), pig-tailed monkey (Macaca nemestrina), and rhesus monkey (Macaca mulata). No binding was observed to mononuclear blood leukocytes. It was concluded that non-human primates contain CEA-like substances in their polymorphonuclear leukocytes as humans do and that these substances carry some identical epitopes.

Animals