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Biomedical subjects

A Pihl

Publications and source records attributed to A Pihl.

At least 37 records · Page 2Linked to original sources
PubMed1988

An immunocytochemical method for assaying oestrogen receptors in breast cancers. A comparison with the steroid binding assay.

The presence of oestrogen receptors was studied in 105 human breast carcinomas using monoclonal antibodies (Abbott ER-ICA kit). The oestrogen receptors of neoplastic cells were semiquantitatively measured and correlations were made to receptor values determined by a dextran-coated charcoal (DCC) steroid binding assay and to histological grade. Immunoreactive cells were found in about 2/3 of the tumours. Usually only a fraction of the cells within each tumour were immunoreactive, and the staining intensity varied among different cells. In general, well differentiated tumours had a greater proportion of immunoreactive cells than poorly differentiated ones. In most cases (65/98) a good agreement was found between the ER-ICA method and the DCC assay. However, in 33 cases discrepancies were demonstrated.

Breast Neoplasms↗
PubMed1988

Immunomagnetic removal of B-lymphoma cells from human bone marrow: a procedure for clinical use.

B-lymphoma cells were purged from human bone marrow by incubating the cell suspension with a cocktail of three different pan-B cell mouse IgG1 monoclonal antibodies, and then with immunobeads charged with sheep anti-mouse antibody, followed by magnetic separation. The primary antibodies used, HD37 (CD19), HD6 (CD22), and HH1 (CD37), bind to a very high percentage of the cells in non-Hodgkin's lymphomas of poor prognosis. The secondary antibody is directed against the Fc portion of the IgG antibodies. In model experiments Burkitt's lymphoma cells (Rael) were admixed to mononuclear bone marrow cells in the ratio 1/9. With a ratio of immunobeads/total antibody-binding B cells of 50/1 in a first treatment cycle and repeating the procedure with the same number of beads in a second cycle, a tumor cell depletion of more than 5 logs was achieved, as judged by a clonogenic assay. The concomitant reduction of CFU-GM and CFU-GEMM was about 20%. The purging procedure has been scaled up to clinical use. Equipment suitable for purging patients' marrow specimens, employing standard transfusion facilities, is described. With this equipment the efficacy of tumor cell removal was the same as in the model experiments, and the whole magnetic separation could be completed in 2 hours.

Animals↗
PubMed1987

Studies on the mechanism of action of abrin-9.2.27 immunotoxin in human melanoma cell lines.

Previously we have shown (Godal, A. et al., J. Natl. Cancer Inst., 77: 1247-1253, 1986) that the sensitivities of different melanoma cell lines to a conjugate of abrin with the anti-melanoma antibody 9.2.27 was correlated with their sensitivities to native abrin. To elucidate the underlying mechanism we have compared the binding and toxicity of the conjugate and of native abrin to two melanoma cell lines, FEMX and LOX, which differ in sensitivity to abrin. Abrin was linked by a disulfide bond to the monoclonal antibody 9.2.27, and the conjugate was purified by affinity chromatography to remove molecules with exposed galactose-binding sites on the toxin B-chain. Lactose had no effect on the binding of the immunotoxin (IT) to the cells but nevertheless reduced strongly the toxicity to the LOX cells. The differences in sensitivity to native abrin were much larger than the concurrent differences in binding. Lactose reduced the toxicity of abrin to a far greater extent than the associated reduction in binding to the cell surface. The toxicity of the immunotoxin to the FEMX cells could be prevented by pretreatment with excess 9.2.27 antibody, whereas the more abrin-sensitive LOX cells were protected only to a limited extent. Concurrent treatment of the LOX cells with antibody and lactose acted synergistically and afforded complete protection. It is suggested that the protective effect of lactose against the IT was exerted after internalization into vesicles of IT bound unspecifically to the cell surface and that the toxic moiety of the IT, the abrin A-chain, may be translocated from endocytotic vesicles to the cytosol by two alternative mechanisms, one mediated by the antibody and a second one facilitated by the B-chain and its lectin binding site. The relative significance of these mechanisms seems to differ in different target cell lines depending on their inherent sensitivities to native abrin which in turn largely reflects the ability of the cells to internalize and process surface-bound abrin.

Abrin↗
PubMed1987

Elimination of B-lymphoma cells from human bone marrow: model experiments using monodisperse magnetic particles coated with primary monoclonal antibodies.

Conditions for removing B-lymphoma cells from human bone marrow using "immunobeads" (IBs) were investigated. The IBs were prepared by coupling monoclonal antibodies directly to a new type of monodisperse magnetic polymer particles (M 450). Two monoclonal immunoglobulin M antibodies, AB-1 (CD 19), a B-cell-specific antibody, and AB-4, an HLA-DR-specific antibody, were used. The IBs were incubated with Rael Burkitt lymphoma cells admixed to fresh, mononuclear human bone marrow cells. After incubation for 30 min at 4 degrees C, the IBs were removed using cobalt samarium magnets. The number of remaining clonogenic tumor cells was assayed by the Courtenay and Mills soft agar procedure, and the clonogenic capacity of the bone marrow progenitor cells was measured by granulocyte-monocyte and granulocyte-erythroid-monocyte-megakaryocyte assays. With a ratio of tumor cells to normal bone marrow cells of 0.1 or 0.01 and a ratio of immunobeads to tumor cells in excess of 75, a tumor cell depletion of more than 3 logs was achieved with the AB-4 IBs and slightly less with the AB-1 beads. After two consecutive cycles of purification with the AB-4 beads, no colonies were found, corresponding to more than 6 logs of purification. In the case of the AB-1 beads, 4 to 5 logs of purification were achieved. The concomitant reduction in clonogenic bone marrow progenitor cells was only 30 to 40%. Flow cytometric studies showed that the tumor cell population contained appreciable proportions of cells binding only small amounts of the antibodies used. The results indicate that the IB procedure is highly efficient and capable of removing tumor cells expressing low levels of antigen. Compared to other purging methods in use the procedure described seems to offer several advantages with respect to efficacy, speed, and simplicity. By the use of a panel of suitable antibodies the new immunobead procedure may be potentially useful in autologous bone marrow transplantation of B-lymphomas and non-T-leukemias with poor prognosis.

Antibodies, Monoclonal↗
PubMed1987

Selective localisation of two radiolabelled anti-sarcoma monoclonal antibodies in human osteosarcoma xenografts.

Two mouse monoclonal antibodies (MoAbs), TP-1 and TP-3, previously shown in immunohistochemical studies to react with osteosarcomas, were labelled with 125I or 131I and evaluated for their ability to localise to human osteogenic sarcoma xenografts after intravenous injection. The radiolabelled TP-1 and TP-3 MoAbs had immunoreactive fractions of 70% and 67%, respectively, and bound to target cells with binding constants of 8.5 X 10(8) M-1 and 4.0 X 10(9) M-1, respectively. After injection of labelled TP-3 IgG, approximately 16% of the dose X g-1 tissue was found in the tumour after 24 hours. Maximum tumour/blood radioactivity ratios of 6-7 were achieved 3-4 days after antibody injection, while the ratios for the normal tissues were less than 1. The tumours could be clearly visualised by whole-body gamma scintigraphy without the need for subtraction techniques. The TP-1 IgG accumulated to a large extent also in the spleen. Hence, with this antibody the tumour was less well delineated from the adjacent normal tissues. However, the F(ab')2 fragments, derived from the TP-1 IgG, gave tumour/blood ratios up to approximately 40 after 3-4 days and yielded sharp gamma scintigrams of the tumour. Specificity of the antibody localisation was indicated by the lack of accumulation in a contralateral melanoma xenograft and the failure of 2 isotype-matched irrelevant MoAbs to localise to the sarcomas. With the F(ab')2 fragments satisfactory images could be obtained already after 16 hours. The results suggest that this preparation may be useful in clinical radioimmunodetection of osteogenic sarcomas.

Animals↗
PubMed1986

New monoclonal antibodies specific for human sarcomas.

Monoclonal antibodies (MAbs) against sarcoma-associated cell membrane antigens were prepared by immunizing BALB/c mice with tumor cells from a human osteosarcoma, TPX, grown as a xenograft in athymic BALB/c nude mice. Spleen cells from immunized mice were hybridized with X-63 Ag. 8.653 mouse myeloma cells which yielded 260 growing hybridomas. Seven of these produced antibodies that bound to TPX cells and to cells from another osteosarcoma, but not to autologous skin fibroblasts. MAbs from 2 (TP-1 and TP-3) of these 7 clones did not cross-react with non-sarcomatous tumor cells or peripheral blood lymphocytes. Immunohistochemical studies on frozen tissue sections showed that the TP-1 (IgG-2a) and TP-3 (IgG-2b) antibodies had characteristic and identical specificity profiles. Binding of TP-1 (TP-3) was demonstrated to 15/15 (15/15) osteosarcomas, 3/3 (2/2) synovial sarcomas, 7/9 (6/8) malignant fibrous histiocytomas, 2/2 (1/1) malignant hemangiopericytomas, 1/2 (1/2) chondrosarcomas and 3/6 (1/3) unclassified sarcomas. The antibodies did not bind to any of 16 sarcomas belonging to other histological subtypes, including liposarcomas and leio- and rhabdomyosarcomas. Moreover, they failed to bind to sections of 66 different non-sarcomatous malignancies, or to any of a range of normal adult and fetal tissues, although some weak staining of proximal kidney tubules was seen. The restricted specificity of these antibodies to some major subtypes of human sarcomas makes them promising tools for identification and subclassification of sarcomas.

Animals↗
PubMed1986

Characteristics of a cell line established from a patient with multiple osteosarcoma, appearing 13 years after treatment for bilateral retinoblastoma.

An osteosarcoma cell line, OHS, was established from a patient with multiple skeletal manifestations of osteosarcoma, developing after bilateral retinoblastoma. The tumor cells expressed sarcoma-associated antigens and showed rapid growth in monolayers and as multicellular spheroids. They formed distinct colonies in soft agar, and subcutaneous tumors in nude mice. Morphological studies indicated that OHS cells had retained important characteristics of the cells of origin. No deletion of the retinoblastoma genes on chromosome 13q14 could be demonstrated with the banding techniques used. However, cytogenetic studies revealed double minute chromosomes, as evidence of gene amplification, as well as translocations involving chromosomes 1,6,11 and 13. The OHS line can be used to study the genetic basis of tumor initiation and growth, and to elucidate factors predisposing for second primary cancers in retinoblastoma patients.

Adolescent↗
PubMed1986

Chemosensitivity profiles of human cancers assessed by the 6-day SRC assay on serially xenografted tumors.

To assess the potential value of the 6-day subrenal capsule (SRC) assay in preclinical evaluation of new drugs using serially xenografted human tumors as source of tumor tissue, we studied the response of 31 human tumor lines (8 malignant melanomas, 12 sarcomas, 9 lung carcinomas and 2 colon carcinomas) to relevant standard drugs and to a new imidazotetrazine, Mitozolomide. Mitozolomide was found to be the most active drug tested in 50% of the lung carcinomas and as active as CCNU in melanomas. The activity of the standard anticancer drugs against subrenal grafts closely resembled the patterns seen with the same tumors in the clinic. In further attempts to validate the procedure, sensitivity profiles of some tumors were concurrently determined in the subcutaneous (s.c.) nude mouse model. In 11 out of the 12 tumors, the two assays selected the same drug as being the most active and in most of these tumors the two procedures gave the same ranking for the different drugs. Also, when the relative sensitivities of a series of melanoma xenografts to each of two drugs (DTIC and CCNU) were tested, the two assays gave the same ranking of the xenografts for each drug. The concordance between the two assays and the fact that the s.c. nude mouse assay reflects the chemosensitivity of the parent tumor in patients, suggest that the application of the 6-day SRC assay to xenografted tumors is a valid and useful procedure permitting rapid preclinical evaluation of new drugs to be carried out at relatively low cost.

Animals↗
PubMed1986

Human melanoma cell lines showing striking inherent differences in sensitivity to immunotoxins containing holotoxins.

The in vitro sensitivity of human melanoma cell lines to conjugates of whole abrin or ricin linked through disulfide bonds to the monoclonal antimelanoma antibody 9.2.27 was studied. After passage of the conjugates through a Sepharose 4B column to remove molecular species with exposed binding sites on their B-chains, toxicity of the conjugates to different melanoma cell lines and nonmelanoma tumor lines was assessed by measuring their ability to inhibit cellular protein synthesis. The abrin conjugate was far more toxic to the target cells than the corresponding ricin conjugate. The 8 melanoma cell lines studied differed widely in their sensitivities to the abrin conjugate. The differences were associated with concomitant large differences in the sensitivities of the cells to the native toxins, and the significance of the level of the antigen expression became apparent only when the sensitivities of the different cell lines were normalized with respect to their sensitivity to native abrin. The observed relationship could not be accounted for by unspecific binding via the B-chain binding site of the immunotoxin. The differential sensitivity of the melanoma cell lines to the immunotoxin seems to be related to inherent differences between the cells in their ability to internalize and to process immunotoxins and toxins. The findings may have considerable practical implications.

Abrin↗
PubMed1985

Properties of volkensin, a toxic lectin from Adenia volkensii.

Volkensin, a highly toxic protein from the roots of Adenia volkensii (kilyambiti, kinoria), was purified by affinity chromatography on acid-treated Sepharose 6B. The toxin is a glycoprotein (Mr 62,000, neutral sugar content 5.74%) consisting of an A subunit (Mr 29,000) and of a B subunit (Mr 36,000) linked by disulfide and noncovalent bond(s). The amino acid, amino sugar, and neutral sugar composition of the protein were determined. Volkensin is a galactose-specific lectin and is a potent inhibitor of eukaryotic protein synthesis in whole cells as well as in a cell-free system (a rabbit reticulocyte lysate). The inhibitory and the lectin activities are functions of the A and B subunits, respectively. Volkensin can be included amongst the ricin-like toxins and resembles most closely modeccin, the toxin of Adenia digitata.

Amino Acids↗
PubMed1985

The use of multicellular spheroids in establishing human sarcoma cell lines in vitro.

By a new procedure stable monolayer cultures were derived from spheroids in 8 out of 17 different human sarcomas (16 soft-tissue and I osteogenic sarcoma). Eleven of the sarcomas were obtained from patients undergoing surgery, and 6 from BALB/c nude mice carrying s.c. growing xenografts. The new procedure involves aggregation of single-cell suspensions into spheroids and cultivation of these in agar-coated flasks until the growth rate levels off, at which time the spheroids are transferred to uncoated flasks. Cells proliferating from the rim of adhering spheroids are trypsinized and aggregated to form new spheroids. By 3 to 5 such alternations, monolayer cultures were obtained that have now been subcultured for about 6 months. The cell lines all gave rise to colonies in a clonogenic soft-agar system, and upon s.c. injection into athymic nude mice 3 lines tested formed growing tumors. The histology of spheroids formed from late monolayer passages closely resembled that of the original tumors. That the new procedure is superior to other methods of establishing sarcoma cell lines is indicated by the fact that a stable monolayer culture could be obtained directly from the tumors in only 1/8 cases where the above procedure was successful, and in only 2 instances from soft-agar colonies derived from the tumors.

Adolescent↗
PubMed1985

Chemosensitivity measurements of human tumour cells by soft agar assays are influenced by the culture conditions.

To investigate the influence of culture conditions on the in vitro responses of tumour cells to anticancer drugs, the sensitivities observed with the soft agar methods of Hamburger & Salmon (1977) (H-S) and of Courtenay & Mills (1978) (C-M) were compared. In all cases the ID50 values were determined from dose-response curves. Six human tumour cell lines exposed to 10 different agents, and 9 patients' melanomas exposed to 5 different agents, were examined. In the studies of cell lines the H-S method gave higher sensitivity values than the C-M method in 38 out of 52 cases, whereas in 14 cases the results were the same. In the patients' tumours the H-S method gave higher sensitivity in 21 of 35 cases, equal sensitivity in 11, and lower sensitivity in 3 cases. In many instances the ID50 values obtained with the two test systems differed by factors of 10 or more, both in the case of cell lines and tumour specimens. Systematic alterations in the culture conditions indicated that the presence or absence of rat erythrocytes is the most important factor responsible for the differences observed. Also, other factors, such as supplements (in the H-S method) and the use of different serum types, appeared to influence both colony growth and chemosensitivity.

Agar↗
PubMed1985

Selection of tumour cell subpopulations occurs during cultivation of human tumours in soft agar. A DNA flow cytometric study.

To examine whether selection of tumour cell subpopulations occurs during cultivation in soft agar, we compared in 23 human tumours of different histological types the DNA content of cells from colonies formed in soft agar (method of Courtenay and Mills, 1978) with that of the original tumour cells. The ploidy as well as the fraction of cells in S phase were determined from DNA histograms after staining of the nuclei with a propidium-iodide procedure and flow cytometric recordings. In 8 of 17 aneuploid tumours analysed, specific aneuploid subpopulations disappeared during cultivation or new aneuploid populations, not demonstrable in the original cell suspensions, appeared in the colonies. In 9 cases identical aneuploid populations were found in the colonies and the tumours. In one of 6 diploid tumours examined, aneuploid cell populations not revealed in the original cell suspension, were found in addition to diploid cells, whereas 5 tumours gave rise to colonies containing a purely diploid population. The results show that in a variety of human malignant tumours cultivation in soft agar may select specific aneuploid tumour cell populations.

Agar↗
PubMed1985

Characteristics of human tumour xenografts transplanted under the renal capsule of immunocompetent mice.

Human tumour lines established in athymic nude mice were grafted under the renal capsule of immunocompetent mice. Grafts from 27 human tumour lines comprising 9 malignant melanomas, 10 sarcomas, 2 colon carcinomas, 4 lung carcinomas and 2 mammary carcinomas, grew well under the renal capsule of the immunocompetent mice and retained morphological and functional characteristics of the parent tumours, as judged by light and electron microscopy and immunohistochemical examinations. Numerous mitoses were detected. Granulation tissue and necrosis were not predominant features. After Day 4, the grafts became infiltrated from the periphery by mouse inflammatory cells. The infiltration could be prevented by pretreatment of the animals with cyclophosphamide. Anti-human antibodies were detected after Day 3. Single cell suspensions from the subrenal grafts were able to form colonies in soft agar. and upon reimplantation in nude mice, subcutaneous tumours were formed showing that the grafted tumour tissue had also retained its malignant character. Altogether the results support the view that human tumour xenografts grow well under the renal capsule of immunocompetent mice and that the grafts retain important characteristics of the original tumour.

Animals↗
PubMed1985

The six-day subrenal capsule assay (SRCA) for testing the response of human tumours to anticancer agents. Validity and usefulness in cancer research and treatment.

To assess the value of the 6-day subrenal capsule assay (SRCA), the growth of human tumours under the kidney capsule of immunocompetent mice as well as the response of the grafts to chemotherapy were studied and compared with the results obtained when the same tumours were grown subcutaneously (SC) in athymic, nude mice. Human tumours serially transplanted in athymic mice were used as test material. The subrenal grafts increased rapidly in size and showed individual, characteristic and reproducible growth rates and they retained the morphological and functional characteristics of the parent tumours, including their malignant character. The infiltration of the grafts by host cells, demonstrable already after day 4, contributed some 15-35% of the total graft volume on day 6 but did not seriously interfere with the growth measurements and the assay. When the chemosensitivity of different tumours was tested both in the 6-day SRCA and in the subcutaneous nude mouse model, the same ranking of drugs was obtained with the two procedures. The results show that the 6-day SRCA on tumour xenografts permits reliable and meaningful measurements of the response of human tumours to drugs. The data indicate that the assay deserves a wider use in cancer chemotherapy and research.

Animals↗
PubMed1985

Activity of mitozolomide (NSC 353451), a new imidazotetrazine, against xenografts from human melanomas, sarcomas, and lung and colon carcinomas.

The chemosensitivity of human tumor xenografts to mitozolomide, 8-carbamoyl-3-(2-chloroethyl)imidazo[5-1-d]-1,2,3,5-tetrazin-4(3H) -one, was studied in 3 different assay systems. In concentrations of 1 to 500 micrograms/ml, mitozolomide completely inhibited the colony-forming ability in soft agar of cell suspensions from sarcomas, melanomas, lung and colon cancers, and a mammary carcinoma. When a panel of tumors of the different histological types was tested for its sensitivity to mitozolomide in vitro, in the 6-day subrenal capsule assay in conventional mice, and, in some cases, as s.c. growing tumors in nude mice, good agreement between the different assay systems was seen. In most cases, a very pronounced antitumor effect was observed. The efficacy of mitozolomide was as good or better than that of the drugs clinically used against the tumor types tested. Tumor size measurements and histological examinations indicated that nude mice carrying a melanoma, a small cell lung cancer, and an osteosarcoma were cured of their tumors. The approach here used for evaluating the effect of a new drug on human cancers may be useful for selecting the tumor types which primarily should be studied in clinical trials. The results indicate that clinical responses to mitozolomide may be anticipated in sarcoma, melanoma, small cell lung cancer, and possibly in colon cancer.

Animals↗