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L Olsson

Publications and source records attributed to L Olsson.

At least 91 records · Page 5Linked to original sources
PubMed1987

In vitro B-lymphocyte antigen priming against both non-immunogenic and immunogenic molecules requiring low amounts of antigen and applicable in hybridoma technology.

A method to efficiently antigen-prime B-lymphocytes with low amounts (less than 1 microgram/10(8) cells) of either immunogenic or non-immunogenic molecules is described. Keyhole limpet hemocyanin (KLH) and histone were used as prototypes for strongly immunogenic and for phylogenetically conserved non-immunogenic epitopes, respectively. Several modifications of previously reported methods were applied to the system and resulted in the requirement of antigen amounts sufficiently low to be obtainable by elution of proteins from electrophoretic gels. Antigen priming against highly purified antigen preparations is thereby feasible even when purified material cannot be obtained by conventional biochemical procedures. The amount of T- and B-lymphocytes and interleukin-2 production was estimated under various conditions during the priming procedure, and those optimal for generation of a high number of antigen-specific B-lymphocytes determined. In vitro antigen-primed B-lymphocytes were immortalized by conventional hybridoma technology. By fusion of lymphoid cells with myeloma cells at each day during the antigen-priming period, the optimal day of fusion to generate antigen-specific hybridomas was determined. Further, in 12 experiments with different antigens, 11 monoclonal antibodies to histones H3 and H4, two to the murine glucose transporter, 17 to trinitrophenyl-sheep red blood cells, and 20 to KLH were obtained. All specific hybridomas produced IgMs, as the antigen-priming period could not be extended for more than 9-10 days, whereafter a rapid decay in B-lymphocytes occurred.

Animals↗
PubMed1987

Glycoproteins distinguishing non-small cell from small cell human lung carcinoma recognized by monoclonal antibody 43-9F.

Cell lines derived from human squamous lung carcinoma release large amounts of a soluble glycoprotein into the culture media, having very high molecular weight (greater than 2 X 10(6] and mucin-like properties. A monoclonal antibody called 43-9F has been generated that recognizes a carbohydrate epitope on the glycoconjugate. The epitope is also present on a diverse set of smaller glycoproteins (Mr 50,000-200,000) distributed primarily on the surface of the squamous lung carcinoma cells. A sensitive assay using the 43-9F antibody in a dot blot procedure has been devised that is able to detect an amount of antigen less than that possessed by a single squamous lung carcinoma cell. This assay, and also conventional immunofluorescence and immunohistochemical assay procedures, have been used to screen different normal cells, normal tissues, cancer cells, and tumor biopsy specimens for the antigen. In the normal lung the 43-9F antigen is found only on cells of some of the seromucous glands. In the normal digestive system it is associated in certain organs only with a limited population of mucosal epithelial cells. Other organ systems lack any reactive cells. The cells of most human non-small cell lung carcinomas and their released glycoconjugates have large amounts of the 43-9F epitope, while small cell lung carcinomas and the glycoconjugates released by small cell lung cancer cells lack the epitope. The oligosaccharide recognized by the 43-9F antibody may therefore provide a useful marker to distinguish the different lung carcinomas and for investigating the different cells of origin of these tumors.

Antibodies, Monoclonal↗
PubMed1987

Identification and characterization of an antigen specific for normal erythroid precursor cells and its application in diagnosis of erythroleukemia.

A monoclonal antibody (designated K:1-6F) generated by hybridization of mouse myeloma cells with spleen cells from mice immunized with the erythroleukemic cell line K562 was found by fluorescence-activated cell sorter analysis, dot-blot assay and electroimmunoblotting to bind to a majority of cells in the K562 and HEL erythroleukemic cell lines, to a subset of cells of the erythroid lineage from normal bone marrow, to a subset of cells in all analysed cases (total 10) of erythroleukemia, and weakly to cells from patients with myeloid leukemia. The antibody did not bind to normal erythrocytes, monocytes, T- and B lymphocytes or granulocytes, as well as a panel of human malignant cell lines of hemopoietic origin (HL60, U937, Daudi, Molt-3, RH-L4 and U266). Biochemical characterization of the antigen defined by the antibody suggests that eht epitope is defined by a carbohydrate structure alone or in combination with proteins. Four molecules with Mr 100 kD, 65 kD, 45 kD and 18 kD respectively were immunoprecipitated from Triton X-100 extract of K562 erythroleukemia cells. Neuraminidase did not affect the binding of the antibody, whereas tunicamycin reduced the K:1-6F expression. The K:1-6F Mab was in normal bone marrow found to be specific for erythroid precursor cells and may therefore be useful in examination of normal and leukemic erythropoiesis.

Animals↗
PubMed1987

Antibody-induced modulation of H-2Kb antigens on mouse tumor cells in vitro.

Modulation of H-2Kb antigens on cells of a subline of the murine Lewis lung carcinoma was induced in vitro by a monoclonal antibody with specificity for H-2Kb antigens. High antibody concentrations resulted in a more spherical morphology of the cells, in a discrete loss of all antibody-antigen complexes within 4-6 hr, and in a corresponding maximal decrease in the total cellular antigen content 6-8 hr after antibody exposure. Restoration was complete within 2 hr after removal of the complexed antigen and occurred without any visible cap formation.

Animals↗
PubMed1986

Antibody-producing human-human hybridomas. III. Derivation and characterization of two antibodies with specificity for human myeloid cells.

Peripheral blood mononuclear cells from a patient with acute myeloid leukemia (AML) and spleen cells from a patient with chronic myeloid leukemia (CML) were fused with HAT-sensitive human B lymphoma cells (RH-L4) in attempts to generate human monoclonal antibodies (Mab) against antigens with high specificity for myeloid leukemia cells. Forty-seven of 246 hybridomas secreted Ig that bound to AML cell surface constituents, as determined by FACS analysis of viable cells that were FITC-stained with the human Mab as the first-step reagent and FITC-conjugated rabbit anti-human Ig as second-step. Two of the 47 human Mab (one from each patient and designated AML-19 and CML-20, respectively) bound to both autologous and allogeneic myeloid leukemia cells. No significant binding was observed to cell surface constituents on human bone marrow cells, granulocytes, lymphocytes, erythrocytes, thymocytes, monocytes, lymphoblastic leukemia cells, fibroblasts, malignant B and T lymphocytic cell lines, and murine bone marrow cells. Both human Mab were IgG and were cytotoxic to myeloid leukemia cells in the presence of complement. About 70% of peripheral blood cell samples from 46 AML patients contained AML-19- and CML-20-positive cells, but the reactivity pattern had no correlation to the morphologic FAB classification of the samples. The promyelocytic HL60 cell line and the K562 cell line reacted with the two antibodies. Dot blot analysis of binding of AML-19 and CML-20 to cellular extracts immobilized on nitrocellulose paper showed that both human Mab in this assay also reacted with normal bone marrow cells. This was supported by microscopic immunofluorescence because both human Mab stained intracytoplasmatic structures in normal bone marrow cells, but both intracytoplasmatic and cell surface components stained in myeloid leukemia cells. Moreover, immunoblotting demonstrated that both human Mab in leukemia cells reacted with two cellular proteins with Mr approximately 14,500 and 18,000, and in normal bone marrow cells with a molecule with Mr approximately 20,000. Immunoprecipitation of cell membrane molecules with both the AML-19 and CML-20 antibody precipitated from leukemic cells only the molecule with Mr approximately 18,000 and no components from normal bone marrow cells. It is concluded that myeloid leukemogenesis may result in generation of cell surface expression of either new or abnormally processed molecules that are immunogenic in the autochthonous host. These molecules may also be useful as markers in diagnosis of myeloid leukemia.

Antibodies, Monoclonal↗
PubMed1986

On the advent and necessity of molecular biology in the clinical management of lung cancer.

The very rapidly expanding knowledge and technologies of molecular biology are reviewed with special reference to problems in the clinical management of lung cancer. Genetic events, tumor-associated antigens, production of murine and human monoclonal antibodies, culture of cell lines, intratumoral phenotypic diversity and squamous-lung-cancer-associated antigens are discussed and related to possible therapeutical approaches. A monoclonal antibody with high specificity for squamous cell lung cancer reacted positively in blood samples and tissue extracts in about 80%. Its use as a marker during follow-up after surgical treatment is demonstrated by examples. It is concluded that there will be limiting factors in the therapeutic use of monoclonal antibodies, such as intratumoral phenotypic diversity. Genetic analysis might be a method for selecting a high risk group of individuals in whom exposure to carcinogenic factors, such as cigarette smoking, would be fatal. Murine monoclonal antibodies can be used in vitro for screening, for histological examination and for prognostic studies. Human monoclonal antibodies should be used for in vivo purposes as well as for the screening of primary tumor and metastases for the therapy. To achieve usable results, the monoclonal antibodies should be raised against the cell membranes that, in particular, are expressed on the stem cells of the neoplastic cell population.

Forecasting↗
PubMed1986

The major histocompatibility complex class I heavy chain as a structural subunit of the human cell membrane insulin receptor: implications for the range of biological functions of histocompatibility antigens.

Monoclonal antibodies against some of the monomorphic determinants of major histocompatibility complex (MHC) class I molecules reduce insulin binding and precipitate 125I-labeled insulin receptor preparations. A monoclonal antibody with specificity for the insulin binding site on the cell membrane insulin receptor of human cells was used to precipitate insulin receptors from human cell lines and resulted in distinct bands of Mr approximately 130,000, 90,000, and 45,000. The Mr 45,000 molecules thus precipitated were subjected to NaDodSO4/PAGE, eluted from the gels, and found to react with monoclonal antibodies against monomorphic and a polymorphic MHC class I determinant known to be expressed on the cell line used as receptor source. Moreover, a murine thymoma line (RI) with MHC class I expression bound significant amounts of insulin, whereas a MHC class I-negative variant had low insulin binding capacity. Reduction in the density on human cells of the MHC class I heavy chain was obtained by capping with antibodies to beta 2-microglobulin or to the MHC class I heavy chain and resulted in decreased insulin binding, whereas down-regulation of insulin receptors induced increased density of MHC class I molecules. It is concluded that the MHC class I heavy chain and the tetrameric insulin receptor are structurally associated in the cell membrane and suggested that this association may occur by displacement of beta 2-microglobulin by the insulin receptor.

Antibodies, Monoclonal↗
PubMed1986

Specificity and diagnostic implications of the reactivity pattern of a panel of monoclonal antibodies against myeloid leukemia cells.

The immunological phenotypes of leukemia cell samples from 60 patients, of whom 54 had acute myeloid leukemia (AML), were assessed with a panel of monoclonal antibodies (Mabs) with specificity for the following epitopes: Epitopes associated with myeloid leukemia cells, Epitopes expressed only on immature myeloid cells (or subsets) and on monocytes, Epitopes only expressed on granulocytes or on granulocytes and mature myeloid cells (promyelocytes, myelocytes and monocytes), Epitopes on HLA-class II (DR) and HLA-class I molecules and on insulin receptors. This panel of Mabs proved useful to identify leukemia cells in blood and to assess their myeloid origin. The panel of Mabs was found also to be useful for immunophenotyping of leukemia cells. Furthermore, the analysis revealed considerable variations in the immunological phenotype of AML cells, reflecting antigenic heterogeneity within the individual leukemia cell population as well as abnormal or no expression of histocompatibility antigens and insulin receptors in some samples. Some of the Mabs bound preferentially to subgroups in the French-American-British (FAB) classification.

Antibodies, Monoclonal↗
PubMed1986

Morphological features of established cultures of human squamous lung carcinoma cells and the cellular distribution of tumor-specific glycoproteins.

Phenotypic characteristics of a cloned cell line, RH-SLC-L11, established from a human squamous lung carcinoma, were studied. The line has maintained its morphologically characteristic growth pattern for over 3 years. Settling cells exhibited extensive surface blebbing during spreading and established small cell islands that eventually expanded by mitosis to confluent cultures. Cell islands and confluent cultures presented three cell types: (i) small, polygonal cells, (ii) polygonal cells of intermediary size and (iii) very large, extremely flattened, degenerating cells. Mitotic activity was present predominantly in type (i) and the sequence (i)--(iii) is presumed to represent the lines' cycle. Previous work has demonstrated that the SLC-L11 line releases tumor-associated glycoproteins and glycolipids. These could be identified with a murine Mab (43-9F). The specific epitope was determined by carbohydrate residues and was shown to have growth factor-like properties. Mab 43-9F bound heterogeneously to the surface of SLC-L11 cells: Most large cells were unreactive while both type (i) and (ii) cells showed conspicuous differences in immunostaining intensity. Immunocytochemical analysis also indicated redistribution, shedding and internalization of antigen-Mab complexes, which may have significant impact on the use of the epitope as tumor marker in diagnosis and therapy. No definite clue was obtained as to the release of the antigenic carbohydrate epitope itself.

Antibodies, Monoclonal↗
PubMed1986

Generation of stable cellular phenotypes in a human malignant cell line conditioned by alterations in the cellular microenvironment.

Microenvironmental conditions may result in phenotypic changes of malignant cells and/or selection of preexisting variants with a growth advantage under the new growth conditions. The present study was initiated to evaluate the stability of changes in crucial cellular attributes as induced in vivo by nonimmunological mechanisms. Expression of major histocompatibility antigens, membrane immunoglobulin, in vitro growth rate, chromosome complement, and modal chromosome number were thus examined in a human B-lymphoma line (RH-L4) prior to and after short-term passage through the peritoneal cavity or spleen of newborn mice. Seven sublines (four from spleen and three from peritoneal cavity) of RH-L4 cells were established after the passage. These lines were found to differ phenotypically from the original line in respect to several or all of the attributes that were studied. These differences were stable for greater than 100 cell generations. Analyses of major histocompatibility complex Class II antigen expression indicated that the modulation of this antigen was independent of the immunological competence of the mice and unrelated to cell cycle-dependent variations. The emergence of the variant sublines seemed not to reflect a microenvironmentally determined selection of minority subpopulation in the original RH-L4 lymphoma line but, instead, induced by the growth conditions in the cellular microenvironment in the mouse. It is suggested that inductive processes may be of importance in the generation of phenotypic diversity within individual tumor cell populations, including the generation of phenotypic variants with high metastatic activity.

Animals↗
PubMed1986

Non-specific binding of protein-stabilized gold sols as a source of error in immunocytochemistry.

The observation that protein-A conjugated gold sols bound to fibronectin-collagen (FNC) fibres in human fibroblast cultures prompted a series of studies on the binding of gold particles stabilized in various ways (Staphylococcal protein A, bovine serum albumin, avidin, streptavidin, gelatin, hemoglobin, polyethylene glycol (MW 20 000), methylcellulose and the nonionic detergent Tween 20) to cell and tissue components, to protein dot blots and SDS-PAGE blots on nitrocellulose paper. We found that binding of gold particles to certain cell and tissue components and to various immobilized proteins did occur irrespective of the stabilizing agent. We argue that, albeit gold sols are stabilized against salt coagulation by adsorption of proteins and other stabilizing agents, "naked areas" are (constantly or intermittently) present on particle surfaces, available for interaction with cell and tissue components that have a high electrostatic affinity for the charged gold surface under prevailing experimental conditions. Non-specific binding may be reduced or abolished by competing proteins (i.e. proteins with a higher affinity for gold than any component in the object studied) provided the proteins and the gold conjugate are present concomitantly during incubation. We found gelatin (Bloom number 60-100) to be an effective competitive protein probably due to its high affinity for gold over a wide pH range. Further, gelatin did not appreciably inhibit the specific interaction in dot blots between SpA and IgG except at very low IgG concentrations. A protocol for the use of gold-protein conjugates to circumvent the hazards of unspecific gold binding is suggested.

Cell Line↗
PubMed1985

Modulatory effects of 5-azacytidine, phorbol ester, and retinoic acid on the malignant phenotype of human lung cancer cells.

Cloned human cell lines of squamous-cell lung carcinoma and small-cell lung carcinoma were treated with 5-azacytidine (5-azaC), 12-0-tetradecanoyl-phorbol-13 acetate (TPA), retinoic acid (RA), or a combination of these drugs, and the effects on cellular morphology, in vitro growth properties, antigenicity, tumorigenicity and metastatic activity in nude mice were studied. Antigenicity was measured by the expression of major histocompatibility antigens (MHC) and of lung-tumor-associated antigens. 5-AzaC treatment resulted in subclones with shorter population doubling times (from 40-50 hr down to 14-20 hr) and increased cloning efficiencies (from less than 1% to 5-50%). TPA and RA induced loss of proliferative activity in vitro and tumorigenicity in vivo of both lines. This was morphologically associated with the appearance of an abundance of large vaculated cells which by DNA-analysis were all found to be in G0-G1 phase. However, 2 of the 5-azaC-treated subclones were insensitive to both TPA and RA, whereas the remaining subclones (20) all responded like untreated lines to TPA and RA. One of the sublines insensitive to TPA and RA formed metastases in nude mice in contrast to all the other lines used. The density of lung-tumor-associated antigens was significantly reduced by TPA-RA treatment, whereas the expression of MHC antigens was unaffected. In contrast, 5-azaC resulted in some cases in increased density of MHC antigens. The effects of 5-azaC on cellular phenotypes were not directly correlated to the total genomic content of 5-methylcytosine. The data suggest that this experimental system is suitable for studies of phenotypic features of malignant cells as related to cellular differentiation.

Animals↗
PubMed1985

Analysis of insulin receptors on heterogeneous eukaryotic cell populations with fluorochrome-conjugated insulin and fluorescence-activated cell sorter. Advantages and limitations to the 125I-labelled insulin methodology.

The diversity in insulin receptor expression within eukaryotic cell populations can be studied with fluorochrome conjugated reagents with high affinity to the insulin receptor in combination with flow cytometry. We studied the optimal conditions for application of fluorescein isothiocyanate (FITC) conjugated insulin in combination with the fluorescence activated cell sorter (FACS) to analyse insulin receptor expression, and also studied the feasibility of this method for identifying and isolating viable subsets with differences in insulin receptor expression within a cell population. Semisynthetic human insulin was conjugated to FITC, which resulted in at least four types of FITC-insulin molecules with different affinities to the insulin receptor. Each type of FITC-insulin was isolated by semipreparative reverse phase high pressure liquid chromatography. The preparation with a fluorescein/protein ratio of approximately 1.0 was found to have the highest affinity to the receptor, the highest biological activity (approximately 50% of native insulin), and similar antigenicity as native insulin. The optimal staining conditions with respect to pH, time of incubation, and cell number were determined, and were different in some aspects from labelling with 125I-insulin. The binding of FITC-insulin to cells was saturable and could be displaced with unlabelled insulin. The fluorescence signal could be converted to absolute numbers of fluorescein molecules by a calibration curve, and the absolute number of specifically bound FITC-insulin molecules calculated from a F/P ratio approximately 1.0. The FITC-insulin/FACS method permits estimation of the total number of insulin receptors (high plus low affinity), and the data obtained correlate well with the results from Scatchard plot of 125I-insulin binding data.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1985

A monoclonal antibody (NAT-9 II:3F-6F) that identifies a differentiation antigen on human myeloid cells.

A monoclonal antibody, designated NAT-9 II:3F-6F (IgM), was generated by hybridization of mouse myeloma cells with spleen cell from mice immunized with normal human bone marrow cells. The antibody reacted with 40-60% of bone marrow cells as analysed on samples from 40 normal individuals and only with a subpopulation of human acute myeloid leukemia (AML) cells of the M2 class (20/20 tested) and M4 class (12/12 tested) (subclasses of the French-American-British (FAB) classification), but not with leukemic cells of the M1 (0/12 tested) and M5 (0/12 tested) FAB subclasses. This is in contrast to many other myeloid-specific monoclonal antibodies. Fluorescence-activated cell sorter (FACS) analyses and morphological examination of cells stained with peroxidase as based on the NAT-9 II:3F-6F monoclonal antibody showed that this antibody reacted with a distant differentiation antigen which is absent on myeloblasts, but expressed on promyelocytes, myelocytes, metamyelocytes, band neutrophils, and on a minority of mature granulocytes. NAT-9 II:3F-6F did not bind to circulating monocytes, T and B cells, erythrocytes and a variety of different human cell culture lines. Immunoblotting demonstrated that the antibody bind to a cellular component with a Mr approximately 97.400 dalton. The antibody may be useful in immunological subclassification of non-lymphoid leukemias and in studies on hematopoiesis.

Antibodies, Monoclonal↗