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Biomedical subjects

H Walzel

Publications and source records attributed to H Walzel.

34 records · Page 2Linked to original sources
PubMed1991

Preparation of a neoglycoprotein using a homobifunctional reagent and its applicability for protein blotting and electron microscopy.

A new method for the preparation of a neoglycoprotein (chemically mannosylated bovine serum albumin, D-Man.BSA) is described using the homobifunctional reagent divinylsulphone.D-Man.BSA purified by affinity chromatography on ConA-Sepharose 4B shows microheterogeneity as demonstrated by immunoaffinity electrophoresis with free ConA in the first-dimension gel. The dissociation constant K for the neoglycoprotein-ConA complex has been calculated to be 2.5.10(-5) M. Biotinylated D-Man.BSA is a useful reagent to detect carbohydrate binding proteins of L1210 leukemia cells on blots. The neoglycoprotein labelled with colloidal gold may be used to demonstrate L1210 cell surface D-Man binding proteins by preembedding electron microscopy.

Animals↗
PubMed1991

Detection of mannose-binding proteins on mouse lymphocytes.

D-Mannose influences both the allogeneic stimulation of mouse spleen cells and the induction of cytotoxic T-lymphocytes. At low concentrations (10(-3) -4 x 10(-3) M), D-mannose increases [3H]thymidine incorporation and induction of cytotoxic cells. Higher concentrations lead to an inhibition of the allogeneic response. To test the involvement of mannose receptors, unstimulated and allogeneically stimulated spleen cells were lysed and the proteins electrophoretically separated. After blotting, the D-mannose-binding structures were identified by means of a biotinylated mannose-neoglycoprotein and avidin peroxidase. Two mannose-binding proteins of unstimulated mouse spleen lymphocytes with a molecular mass of 29 and 31 kDa were detected. In vitro allogeneically stimulated spleen lymphocytes show three other mannose-binding proteins with a molecular mass of 53, 65, and 76 kDa.

Animals↗
PubMed1990

Are soluble monocyte-derived HLA class II molecules candidates for immunosuppressive activity?

Supernatants of human blood monocyte cultures suppressed PHA responses (IL-2 synthesis, IL-2R expression, DNA synthesis) of autologous and allogeneic lymphocytes. The main suppressive activity was found in the 65-kDa (and 23-kDa) range. It could be incompletely neutralized by mAb specific for a non-polymorphic HLA DR determinant and could also be adsorbed to and eluted from an anti-DR immunoabsorbent column. On blots of monocyte lysates and monocyte culture supernatants, the mAb RoDR recognized antigens of nearly the same Mr. The hypothesis that soluble HLA DR alpha beta heterodimers or beta chains are likely candidates for the suppressor factor was confirmed by analogous effects of purified HLA DR molecules. We favor a model in which soluble MHC class II molecules (in contrast to surface-bound ones) may interfere with the association and cross-linking processes necessary for T cell activation by competing for CD4 binding sites.

Antibodies, Monoclonal↗
PubMed1990

Electron microscopic study of receptor mediated endocytosis of a monoclonal antibody (RoMo-1) against the surface marker CD 14 of human monocytes.

The internalization of the gold labelled monoclonal antibody RoMo-1 recognizing the CD 14 surface molecule of human monocytes was studied by electron microscopy. Monocyte enriched mononuclear cells (MNC) from the peripheral blood of healthy donors were incubated for 15, 30, 60, and 90 min with gold marked RoMo-1 in Eagle MEM at 37 degrees C. The process of internalization of RoMo-1 occurs as a receptor mediated endocytosis (RME), as described for other ligands.

Animals↗
PubMed1990

Relationship between internalization kinetics and cytotoxicity of mistletoe lectin I to L1210 leukaemia cells.

We have taken two different approaches to the study of the entry of mistletoe lectin I (MLI) into murine L1210 leukaemia cells. As detected by cellular protein synthesis and DNA synthesis inhibition, the lectin was cytotoxic to L1210 leukaemia cells. Inhibition of [3H]leucine and [3H]thymidine incorporation into L1210 cells by MLI was found dose dependent in a concentration range from 10(-16) to 10(-12) mol/ml. The kinetics of cellular protein synthesis inhibition by MLI was concentration dependent, too. Using preembedding electron microscopy, the binding and intracellular routing of the gold-labelled lectin (MLI.Au15) were studied. MLI was internalized into L1210 leukaemia cells by two different pathways: via coated pits to coated vesicles and via long enclosed invaginations of the plasma membrane.

Animals↗
PubMed1989

Selective killing of human monocytes by an immunotoxin containing partially denatured mistletoe lectin I.

The in vitro activity of an immunotoxin (IT) prepared by conjugating the monocyte specific RoMo-1 monoclonal antibody and partially inactivated Mistletoe lectin I (dMLI) containing inactivated B chain has been initially characterized. It is shown that while not affecting other mononuclear cells viability, this IT is capable of selectively destroying human monocytes after 24 h exposure thus resulting in the abrogation of monocyte support for PHA reactivity in mononuclear cell preparations. Therefore it seems to be possible to use this non-sugar binding holotoxin for immunotoxin preparation.

Antibodies, Monoclonal↗
PubMed1989

Electron microscopic visualization of receptor internalization induced by a monoclonal antibody recognizing the monocyte specific glycoprotein CD 14.

We produced a monoclonal antibody against human monocytes (ROMO 1) which was identified to be directed against the monocyte specific glycoprotein CD 14. For electron microscopic studies, this antibody was labelled with colloidal gold. By using a preembedding technique, we investigated the binding and internalization of gold labelled ROMO 1. The binding is highly specific. No other cells than monocytes bind it. Suspensions of monocytes, prepared from human blood, were incubated for 15 to 90 min with the marked antibody. The cells were labelled on the whole surface. Already after an incubation period of 15 min, the internalization of the antibody receptor-complex is demonstrable. This process is similar to that of receptor-mediated endocytosis (RME), described for different ligands such as cell nutrients, growth factors, hormones, and others.

Animals↗
PubMed1989

Detection and quantification of ligand leakage from lectin affinity columns.

The usefulness of lectin affinity chromatography for the preparation of glycoproteins is impaired by ligand release. Ligand leakage from mistletoe lectin (MLI) Sepharose 4B column was detected by 24 h skin reaction in mice and by immunoblotting. Immunoaffinity chromatography was found to be an efficient method for the separation of lectin traces from the glycoprotein fraction. A sugar concentration dependent increase of lectin release from MLI-Sepharose 4B column was detected by a solid phase enzyme immunoassay.

Animals↗
PubMed1988

Human monocyte activation induced by an anti-CD14 monoclonal antibody.

An anti-CD14 mAb RoMo-1 rapidly induces in human monocytes a transient oxidative burst activity as detected by chemiluminescence assay. Pretreatment of these cells with the mAb markedly suppresses the monocyte chemiluminescence response to opsonized zymosan. In addition, the antibody induces a significant increase of IL-1 production and secretion by mononuclear cells, comparable to a similar effect of rIFN-gamma or LPS. Electron microscopy demonstrates internalization of the CD14 molecules after interaction with the mAb in a characteristic receptor-like manner.

Antibodies, Monoclonal↗
PubMed1988

[A monoclonal antibody against peripheral human blood monocytes (RoMo-1)].

The monoclonal IgG2a-antibody RoMo-1 binds to peripheral human blood monocytes but does not react with other blood cells, tissue macrophages, HL-60, K 562 or U 937. It seems possible, that the antibody defines an antigen expressed on the premonocytic and monocyte stages of cells from the mononuclear phagocytic system only. The antibody is cytotoxic.

Antibodies, Monoclonal↗
PubMed1984

[Modification of granulocyte phagocytosis by extracts from tumor antigens].

The results with the granulocyte phagocytosis test adding various tumor antigen preparations are represented. In comparison with clinically healthy probands and patients having benign diseases there could not be proved any malignancy and organ specificity of granulocyte phagocytosis in patients with malignant tumors of different stages and various localization of organs.

Antigens, Neoplasm↗
PubMed1984

Immunological reactivity of tumor associated glycoproteins prepared by affinity chromatography.

Crude 3M KCl extracts prepared from tumor tissue and ascitic tumor cells of methylnitrosourea-induced fibrosarcoma of CBA mice or of a human stomach adenocarcinoma were gelfiltrated on Ultrogel AcA 34. Single fractions of these preparations were incubated with spleen cells of tumor bearing mice or blood peripheral lymphocytes. The response was detected by MEM-technique. Antigenic activities were found in molecular weight ranges from 200 to 300 kD (P1) and from 40 to 70 kD (P2). Single glycoproteins of the P1- and P2-fractions could be separated by affinity chromatography on immobilized D-galactose specific mistletoe lectin I, as detected by electrophoresis on microgradient gels (1.2-40%) or on SDS polyacrylamide slab gels. Especially the isolated glycoproteins of the P1-fractions are responsible for the observed antigenic reactivity, assayed in vitro by the MEM-technique and in vivo by foot pad swelling test.

Adenocarcinoma↗
PubMedDate not supplied

The immunomodulatory beta-galactoside-specific lectin from mistletoe: partial sequence analysis, cell and tissue binding, and impact on intracellular biosignalling of monocytic leukemia cells.

Nanogram quantities of the beta-galactoside-specific lectin from mistletoe (ML-I) that is composed of two different types of subunits exhibit immunomodulatory potency and enhance cytokine secretion in vitro and in vivo. Partial sequence analysis of the carbohydrate-binding B chain revealed a ragged N-terminus and overall homologies to the B subunit of Ricin D and Ricin E. Two evolutionarily neutral substitutions were apparent in the otherwise identical N-terminal sequences of the two toxic chains within the lectin preparation. On the basis of the influence of chemical modification by group-specific reagents on ligand binding, the lectin was biotinylated with biotinyl-N-hydroxysuccinimide ester to allow monitoring of cell binding. Monocytic leukemia cells (THP-1) specifically bound the lectin with positive cooperativity at low lectin concentrations. Radiolabelled lectin could be found in several organs and in an experimental solid tumor in biodistribution in mice. Its presence in a notable amount in spleens is especially noteworthy with respect to the already reported immunomodulation. To determine intracellular responses that precede the lectin-dependent augmentation of cytokine secretion, phosphorylation of proteins and phospholipids as well as Ca(2+)-mobilization were assessed in THP-1 cells. Quantitative increases of [32P]-phosphate incorporation were determined for a 28 kDa protein and for phosphatidylinositol-4,5-biphosphate. Similarly, the fluorescence activity of the intracellular Ca(2+)-indicator fluo-3 is elevated by approximately 25% after lectin treatment. Apparently, cell binding of the lectin is followed by modulation of biosignalling processes.

Amino Acid Sequence↗