Biomedical subjects

F A Anderer

Publications and source records attributed to F A Anderer.

121 records · Page 7Linked to original sources

PubMedDate not supplied

Biochemical characterization of a component in extracts of Viscum album enhancing human NK cytotoxicity.

Enhancement of human NK cytotoxicity in the presence of fresh Viscum album extract and some commercial V. album extracts Iscador correlated strictly with an increased formation of lytic effector cell/K562 tumor cell conjugates in the single-cell assay. Both activities were completely destroyed by pretreatment of V. album extracts with pectinase, hemicellulase, amyloglucosidase and alpha-glucosidase, but not with proteases and RNase, i.e., the activities are linked to a polysaccharide. The active component in V. album extract was non-dialysable at a molecular weight cutoff of 10,000. Inhibition of both activities was observed with D-galacturonic acid, poly-galacturonic acid and pectins. The site of galacturonic acid-specific interaction could be identified on the effector cells. The rate of effector cell/tumor cell conjugate formation in the presence of V. album extracts, as well as the abrogation of both activities by pretreatment of V. album extracts with exoglycosidases specific for sugars other than galacturonic acid indicated an action of the NK cytotoxicity-enhancing component on the basis of a bridging mechanism. However, no conclusive results could be obtained for the structural specificity of the site interacting with the target cells.

Binding Sites↗

PubMedDate not supplied

Chemical specificity of effector cell/tumor cell bridging by a Viscum album rhamnogalacturonan enhancing cytotoxicity of human NK cells.

The component in Viscum album extract Iscador-M enhancing the NK cytotoxicity of human CD56+CD3- NK cells (87-95% enrichment) in cocultures with K562 tumor cells and increasing the formation of NK cell/tumor cell conjugates was identified as a rhamnogalacturonan. Both activities were abolished by treatment of V. album extract with poly-alpha-D-galacturonidase and alpha-rhamnosidase and both activities were inhibited in the presence of galacturonic acid and acetylated rhamnose (6-deoxymannose). Inhibition was also observed in the presence of structurally related derivatives such as acetylated mannose or acetylated mannonic acid gamma-lactone, the latter exhibiting a 5-10-fold higher inhibitory potential. The rapid formation of NK cell/tumor cell conjugates in the presence of V. album extract was based on the bridging of NK cells with tumor cells by rhamnogalacturonan. Using a specifically adapted agglutination assay, the saccharide residues of the rhamnogalacturonan interacting with NK cells and tumor cells could be identified by the formation of homologous cell conjugates induced by acetylated rhamnose or acetylated mannose conjugated to dextran and by polygalacturonic acid: terminal acetylated rhamnose or acetylated mannose bound only to NK cells in a dose-dependent manner but not to K562 tumor cells, and terminal galacturonic acid only to K562 tumor cells but not to NK cells. This type of bridging represents a novel mechanism of enhancement of NK cytotoxicity.

Agglutination Tests↗

PubMedDate not supplied

Differences in the lipid distribution in subcellular fractions of mouse fibroblasts derived from logarithmic and stationary growth.

The lipid class compositions of subcellular fractions of SV40-transformed mouse fibroblasts derived from the logarithmic and stationary growth phase were compared. Cell populations of the stationary growth phase showed a relative decrease of the protein content and an increase of triglycerides and alkoxydiglycerides which could be located in the non-sedimenting fraction and in the nuclei and mitochondria containing fraction, respectively. Futheron, distinct shifts in the subcellular distribution of those lipid classes could be observed which exhibited no relative overall increase or decrease when the cells of both growth phases were compared. In the crude plasma membrane fraction the ratio "lipid class/protein" remained about constant with the exception of the phospholipids and alkoxydiglycerides.

Cell Division↗

PubMedDate not supplied

Highly specific characterization of tyrosine phosphoproteins in tumor cells based on monoclonal antibodies defined by conjugated phosphotyramine.

Phosphotyrosine proteins of four different tumor cell lines were characterized by monoclonal antibodies exhibiting high affinity binding to phosphotyrosine. For the preparation of the antibody-producing mouse hybridoma cell lines we used a novel kind of immunizing antigen with phosphotyramine conjugated directly to carboxylic groups of carrier proteins. Screening for high affinity binding antibodies was based on their selective reactivity in immunoprecipitation, affinity chromatography and immunofluorescence. By means of affinity chromatography we established a one-step purification of phosphotyrosine proteins yielding substantial quantities of highly pure 170kDa EGF receptor from A431 tumor cells, 210kDa bcr-abl gene product from K562 tumor cells and 120 kDa transforming protein of the Abelson murine leukemia virus from TK tumor cells. Cross-reactivity with phosphoproteins containing no phosphotyrosine was not observed.

Antibodies, Monoclonal↗

PubMedDate not supplied

Dialysable factors from human leukocyte extracts activating human NK cytotoxicity against tumor cells: identification of factor-producing leukocyte subpopulations.

Dialysable human leukocyte extracts (10 kDa molecular weight cutoff) contain at least three cytotoxicity-stimulating factors CySF-L1, CySF-L2 and CySF-L3, activating natural killer (NK) cytotoxicity of human peripheral blood mononuclear cell (PBMC) against tumor cells when PBMC were pre-incubated with the factors for 72h prior to cytotoxicity assays. The factors could be separated by ion exchange chromatography on Dowex 50WX2. The leukocyte subpopulations producing these factors could be identified: CySF-L1 was detected in granulocyte extracts and T lymphocyte extracts, CySF-L2 in granulocyte extracts and monocyte extracts, CySF-L3 in T lymphocyte extracts and in B lymphocyte/NK cell extracts. All three factors were also present with low concentrations in fresh human plasma. The effector cells activated during pre-incubation of PBMC with the factors were partly identical: CySF-L1 and L2 activated the NK cytotoxicity of monocytes and of NK cells whereas CySF-L3 only stimulated the cytotoxicity of NK cells. All three factors stimulated LAK-like cytotoxicity since the effector cells showed enhanced activity not only against standard NK targets like K562 tumor cells but also against NK-insensitive targets like DAUDI cells and HT29 tumor cells.

Biological Factors↗

PubMedDate not supplied

Expression of LDL receptor on tumor cells induced by growth factors.

For the determination of LDL receptor expression on living human cells two monoclonal antibodies specific for the extracellular domain of LDL receptor were established using affinity-purified LDL receptor and carrier-conjugated LDL receptor peptide 163-174 as immunizing antigens. The 125I-labeled antibodies were used to quantify increases in LDL receptor expression on human cells grown in the presence of increasing concentrations of various growth factors. Growth factor-mediated increase of LDL receptor expression was entirely different in various cell lines with respect to a distinct growth factor and for different growth factors when tested with one and the same cell line. An increased LDL receptor expression was observed on A431 epidermoid carcinoma cells of the vulva in the presence of epidermal growth factor (EGF) or insulin but not with platelet-derived growth factor (PDGF), on HUV-EC primary endothelial cells in the presence of insulin or PDGF but not with EGF, and on MRC-5 diploid fetal lung cells only in the presence of PDGF. HEP-3B hepatoma cells did not respond to any of the three growth factors essentially maintaining the original level of LDL receptor expression.

Amino Acid Sequence↗

PubMedDate not supplied

Activation of antitumor cytotoxicity of human blood mononuclear cells by a basic factor from dialysable human-leukocyte extract.

Dialysable human leukocyte extract (10 kDa molecular weight cutoff) contained a basic factor stimulating natural killer (NK) cytotoxicity of human peripheral blood mononuclear cells (PBMC) against human K562 tumor cells when PBMC were pre-incubated with the factor for 72h prior to cytotoxicity assays. This cytotoxicity-stimulating factor (CySF-L2) could be enriched by adsorption to ion exchange resin Dowex 50WX2 (H-form) eluting at pH 9.0-9.6 when a pH gradient between pH 3 and pH 10 was used. Further purification was achieved by chromatography on DEAE-Sepharose. The factor has a molecular weight of approximately 1000 Da and is insensitive to protease and exopeptidase treatment but sensitive against treatment with endoglycosidase F. Using immunomagnetic cell sorting, complement-mediated cell depletion and depletion by planning, the cytotoxic effector cells activated during pre-incubation of PBMC with CySF-L2 could be identified as CD16+ CD14+ monocytes/macrophages and as Leu7+ Leu19+ CD16+ CD3- CD8- NK cells.

Adult↗

PubMedDate not supplied

[Quantification of SS-B autoantibodies in patient sera using highly purified human SS-B antigens and their clinical interpretation].

For the identification of SS-B autoantibodies in the sera of patients with rheumatic diseases (n = 319) a sensitive and specific enzyme-linked immunosorbent assay was developed using highly purified human SS-B antigen as a reference antigen. In 30/319 patients' sera SS-B autoantibodies were detected. In this group, the ratio female : male patients was outstandingly high (29:1), the serum of the single male patient exhibiting the lowest titer of SS-B autoantibodies. The highest portion of anti-SS-B positive sera was observed in the group of patients with systemic Lupus erythematosus (48%) followed by the groups with undifferentiated connective tissue disease (17%), Sjögren-syndrome (13%) and rheumatoid arthritis (6%). The protein components reacting with human SS-B autoantibodies were characterized in permanent cell lines of various species. In each of 9 human cell lines only one component with a molecular weight of 49 kD reacted; in cell lines of other species analogously, only one component reacted, showing slightly different molecular weights depending on the species. The SS-B antigen isolated from human cells contained at least 7 isoelectric variants, all reacting with human SS-B autoantibodies.

Arthritis, Rheumatoid↗

PubMedDate not supplied

Lack of expression of a 31/33kD surface protein on human colon carcinoma cells is a marker for metastasizing potential.

Two highly metastatic human tumor cell lines, SLU-M1 SLU-M2, were established by in vivo selection in Balb/c-nu/nu mice of SLU-1 xenotransplants derived from an adenocarcinoma of the sigmoid colon. Metastatic spread was screened by transplantation of tissues from various organs of s.c.-tumor-bearing nu/nu mice. A monoclonal antibody, mab ME6H2, prepared against a membrane fraction of HT29 cells, also derived from an adenocarcinoma of the colon, showed high 125I-mab ME6H2 binding only to HT29 and SLU-1 cells, whereas hardly any binding was recorded for SLU-M1 and SLU-M2 cells. All cells of the HT29 and SLU-1 populations exhibited a positive immunofluoresence (IF) but only 1-5% of the SLU-M2 and 10-15% of the SLU-M1 subpopulation. A number of other tumor cell lines did not express the ME6H2 target antigen except for line MCF7, derived from an adenocarcinoma of the breast, which showed an IF positive reaction of 100% of the cells but only 25% of mab binding compared to HT29 and SLU-1 cells. The data indicate that expression of the ME6H2 target antigen is adenocarcinoma-specific and lack of expression is a marker for the metastatic potential of these cells. Mab ME6H2 was rapidly internalized upon binding to viable HT29 cells, resulting in an enhancement of cell growth in vitro and tumor growth in vivo. The mab ME6H2-defined target antigen was isolated from cell lysates by antibody affinity chromatography and was identified as a double band in SDS-PAGE with 31kD and 33kD molecular mass usually present in equal amounts.

Adenocarcinoma↗

PubMedDate not supplied

Signal requirement for induction of MHC-unrestricted antitumor cytotoxicity of human T cell CD4+/CD8+ subpopulations.

The role of cosignalling in the generation of MHC-unrestricted cytotoxicity of T cells was studied with CD4+ and CD8+ sub-populations highly purified (> 98%) by immunomagnetic cell sorting using OKT4 mab, Dynal anti-CD4 mab, OKT8 mab, Dynal anti-CD8 mab, and OKT3 mab. Cytotoxicity was determined in 4 h cytotoxicity assays against K562 tumor cells known to lack expression of MHC class 1 and class 2 antigens, thus avoiding interference with anti-CD4- or anti-CD8-mediated signalling. Signal transfer was induced via CD4, CD8, CD3, IL-2 receptor and RG receptor specifically interacting with a plant rhamnogalacturonan (RG). In CD8+ cells, the first signal delivered by the sorting mab (immobilized OKT8 or Dynal anti-CD8 or OKT3) only induced low MHC-unrestricted cytotoxicity but committed the cells to develop largely enhanced cytolytic potential upon stimulation with a second (IL-2 or RG) or third (OKT3, IL-2, RG) signal. The highest cytolytic potential was achieved by cumulative signalling via CD8, CD3, IL-2 receptor and RG receptor. The generation of MHC-unrestricted cytotoxicity of CD8+ cells correlated with increased effector cell/target cell conjugate formation. In CD4+ cells, OKT4 as sorting mab induced very low cytolytic potential, and a moderate committment to IL-2 signals but a stronger one to RG signals, yielding further cytotoxicity enhancement. The highest cytolytic potential was obtained by cumulative signalling via CD4, IL-2 receptor and RG receptor. Dynal anti-CD4 mab was inefficient and OKT3, as sorting mab of CD4+ cells from CD8-depleted PNAC, appeared to block subsequent OKT4-induced generation of MHC-unrestricted cytotoxicity by delivering a negative signal. Immobilized OKT3 as second signal present in cultures of OKT4-sorted CD4+ cells was inefficient. Surprisingly, soluble OKT3 together with IL-2 delivered a positive signal in cultures of OKT4-sorted CD4+ cells.

CD4 Antigens↗

PubMedDate not supplied

Induction of NK-like activity in T cells by IL-2/anti-CD3 is linked to expression of a new antitumour receptor with specificity for acetylated mannose.

The generation of MHC-unrestricted cytotoxicity of highly enriched human CD3+ T cells (95-99%) by treatment with IL-2 and/or anti-CD3 antibodies was studied. T cells obtained by positive immunomagnetic sorting (anti-CD3) developed comparable specific cytotoxicities against K562 and Daudi cells when cultured with IL-2 and anti-CD3 for 96 h (80% of donors; n = 25). This increase of MHC-unrestricted cytotoxicity correlated fairly well with an increased formation of T cell/tumour cell conjugates. Moreover, simultaneous expression of a rhamnogalacturonan-binding receptor on activated T cells could be demonstrated. Rhamnogalacturonan was reported to enhance cytotoxicity of CD56+ NK cells by effector cell/target cell bridging. Untreated CD3+ cells hardly reacted with rhamnogalacturonan and IL-2-activated T cells showed only a moderate enhancement of cytotoxicity in the presence of rhamnogalacturonan. However, when CD3+ T cells had interacted with anti-CD3 antibodies during cell-sorting or during subsequent culturing with IL-2, enhancement in cytotoxicity and increased formation of lytic effector cell/tumour cell conjugates in the presence of rhamnogalacturonan could be readily demonstrated, indicating a bridging effect analogous to CD56+ NK cells. The conjugate formation of activated T cells with tumour cells as well as the additional rhamnogalacturonan-mediated bridging must be based on the expression of receptors with acetyl mannose specificity, since enhancements of MHC-unrestricted T cell cytotoxicity and conjugate formation were inhibited in a dose-dependent manner when acetylated mannose was present in the assays.

Antibodies, Monoclonal↗

PubMedDate not supplied

Human monoclonal antibody developed against a metastasizing paraganglioma: preparation and preclinical studies.

A tumor cell line, named HS, was established from a bone metastasis of a patient with metastasizing paraganglioma. In vitro immunization of normal human peripheral blood mononuclear cells by coculturing with viable HS cells, followed by fusion with mouse myeloma cells, yielded a stable human/murine heterohybridoma producing the highly specific monoclonal anti-body KM-155. This MAB KM-155 is a member of the IgG3 subclass and shows no alpha GAL glycosylation that is specific for mouse but not for human antibodies. In pilot preclinical studies it could be demonstrated that MAB KM-155 is highly efficient in targeting a KM-155 antigen-expressing human tumor developing in nu/nu mice after xenografting. Moreover, the growth of KM-155 antigen-expressing human tumors in nu/nu mice was largely inhibited when the concentration of circulating MAB KM-155 was maintained at a high enough level by serial injections.

Animals↗

PubMedDate not supplied

IL-12-mediated activation of MHC-unrestricted cytotoxicity of human PBMC subpopulations: synergic action of a plant rhamnogalacturonan.

IL-12 mediated activation of human MHC-unrestricted cytotoxicity was studied with freshly isolated, highly enriched CD56 +CD3- NK cells (95.98%), monocytes/macrophages (90-95%) and CD3 + T cells (95-98%). Activation of NK cell cytotoxicity and monocyte cytotoxicity by IL-12 were independent of exogenous IL-2 and IFN gamma. Activation of CD3+T cells to MHC-unrestricted cytotoxicity required coactivation by anti-CD3 antibody. The enhanced cytotoxicities were directed against NK-sensitive as well as NK-resistant target cells and coincided with enhancement of effector cell/target cell conjugate formation. The specific cytotoxicity of all three activated effector cell populations was further increased in the presence of rhamnogalacturonan. These increases were based on an additional increase of effector cell/target cell conjugate formation that is based on rhamnogalacturonan-mediated bridging between effector cells and target cells. Simultaneous enhancement of cytotoxicity indicates involvement of receptors on effector cells cross-reacting with acetylrhamnose (6-deoxymannose) that might play an important role in human MHC-unrestricted cytotoxicity against tumor cells.

Adjuvants, Immunologic↗