Review of methods in "breast augmentation: a risk factor for breast cancer?".
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Biomedical subjects
Publications and source records attributed to J M Turc.
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It is currently impossible to isolate or identify human hematopoietic progenitor cells from the bone marrow, yet the biophysical properties of these cells are important for the development of techniques to isolate and preserve stem cells for transplantation. Osmotic permeability properties of human bone marrow stem cells were estimated from the kinetics of cell damage in a hypotonic solution measured using in vitro colony assays for multipotential (CFU-GEMM) and committed (BFU-E, CFU-GM) progenitor cells. Cells exposed to a hypotonic solution swell as a result of water influx, and the rate of change of volume is proportional to the hydraulic conductivity of the plasma membrane. Cell damage occurs when the cell volume exceeds the maximum tolerable volume, so the hydraulic conductivity can be estimated from the kinetics of cell damage. For all the progenitor cells studied, the mean value of the hydraulic conductivity was 0.283 micron3/micron2/min/atm at 20 degrees C, with an Arrhenius activation energy of 6.41 kcal/mole. No significant differences were observed in the osmotic properties of the various progenitor cells. These data were used to predict the osmotic responses of human bone marrow stem cells at subzero temperatures during freezing.
The murine lymphoblastic cell line DA-1 has been characterized as dependent upon both interleukin-3 (IL-3, multicolony-stimulating factor [multi-CSF]) and granulocyte-macrophage colony-stimulating factor (GM-CSF) for survival and growth. Here we demonstrate that it is responsive to a third hematopoietic factor, the erythroid-specific hormone, erythropoietin (Epo). DA-1 cells are stimulated to proliferate by partly purified natural murine and human Epo, and pure recombinant human Epo. Antibody to Epo specifically blocks Epo-stimulated growth. Maximal growth stimulated by Epo and GM-CSF is similar, and considerably less than that stimulated by multi-CSF. Proliferation stimulated by Epo and GM-CSF is transient, decreasing within 24 to 48 hours of exposure. However, Epo acts cooperatively with GM-CSF to sustain proliferation. With or without GM-CSF, no obvious erythroid differentiation of DA-1 cells occurs after exposure to Epo for up to 72 hours. This is the first report of a growth factor-dependent cell line also responsive to Epo for survival and growth. The availability of this cell line model should greatly facilitate biochemical analysis of the mechanism of Epo growth-stimulating action.
K562 is a human leukemic cell line used as model of hematopoietic differentiation. A variety of differentiation-inducing agents was used in this study, and the expression of surface membrane antigens associated with specific lineages of differentiation and changes in the cytochemistry of the induced cells were monitored. Sodium butyrate, hemin, retinoic acid, dimethyl sulfoxide (DMSO), phorbol myristate acetate (PMA), and interferon induced unique alterations in the binding of monoclonal antibodies specific for erythroid, granulocytic, monocytic, and megakaryocytic lineages. Hemoglobinization, Sudan Black B, glycogen content, nonspecific esterase, alkaline phosphatase, and 5'-nucleotidase staining were also altered. K562 cells were terminally differentiated with PMA to nitroblue tetrazolium-(NBT) positive macrophages. Expression of 3-fucosyl-N-acetyl lactosamine, previously thought to be myeloid specific but found on all early hematopoietic progenitors, was modulated during differentiation to nonmyeloid lineages. Lineage infidelity was noted during functional differentiation along all hematopoietic lineages. The presence of multiple lineage surface markers and cytoplasmic characteristics in leukemic cells is not indicative of lack of potential to differentiate. K562 cells cannot be compared to any normal stage of hematopoietic differentiation, but they do have the capacity to differentiate along erythroid, macrophage, and megakaryocytic lineages.
The storage of fresh frozen plasma (FFP) for short periods at -20 degrees C for 6 weeks, -30 degrees C for 12 weeks, or -40 degrees C for 12 weeks, did not result in significant deterioration in factor VIII: coagulant (factor VIII:C) activity in the primary packs. In studies examining whether plasma segments could be used for quality control purposes, the mean factor VIII:C activity of the primary pack was found to be identical to that of the attached segments of plasma for units of FFP which were thawed within 2 h after preparation. This was also true for FFP units stored at -40 and -60 degrees C for up to 12 weeks. There was, however, a loss in factor VIII:C activity in the segments of FFP units stored at either -20 or -30 degrees C for 6 and 12 weeks, respectively. Thus for units of FFP stored at temperatures colder than -40 degrees C, segments are suitable for assessing the factor VIII:C activity in the primary pack but not for FFP units stored at -20 or -30 degrees C.
The immune response appears to be partially regulated by genes linked to the IgGH chain gene complex. Immunoglobulin allotypic markers Gm groups are distributed in Caucasians largely in 5 relatively well defined phenotypes. We examined the prevalence of these phenotypes in 119 patients with ankylosing spondylitis, Reiter's syndrome or acute anterior uveitis. No significant deviation from the control frequencies was noted.
A murine monoclonal antibody (82H5, IgM class) has been developed that detects an antigenic determinant expressed by greater than 90% of normal granulocytes and 60-80% of light-density normal bone-marrow cells, including human pluripotential progenitors (colony-forming-unit-granulocyte, erythroid, macrophage, megakaryocyte; CFU-GEMM) and committed progenitors: granulocyte-macrophage (CFU-GM), erythroid (BFU-E), and megakaryocytic (CFU-MK). This antibody did not react with erythrocytes, monocytes, platelets, lymphocytes from normal peripheral blood, lymphoblasts from patients with acute lymphoblastic leukaemia, or with lymphoid cells lines. The 82H5-defined antigenic determinant was expressed on greater than 90% of leukaemic cells of promyelocytic, myelomonocytic and monocytic morphology, and cell lines KG.1, ML.1, HL.60, K562 and U.937. Cortical thymocytes were unreactive with 82H5. Treatment of human bone-marrow cells with granulocytic-specific monoclonal antibody 82H5 plus complement significantly inhibited colony formation (48-74%; P less than 0.05) of CFU-GEMM, CFU-GM, BFU-E, CFU-MK, whereas treatment with control monoclonal anti-Ia antibody plus complement caused 79-89% inhibition. This antibody reacted strongly with 3-fuc-NAc lactosamine when tested with a panel of synthetic carbohydrate structures. We conclude that 82H5 may be a useful probe for phenotypic analysis of leukaemic cells and investigation of haematopoiesis.
Cryopreservation of BB rat embryos would ensure perpetuation, at greatly reduced cost, of this model of nonobese, insulin-dependent, juvenile-onset diabetes in man. Immature prediabetic female BB rats were ovulated hormonally. The embryos were removed at the 8- to 16 cell and blastocyst stages and cryopreserved, and after thawing were assayed for viability in vitro or transplanted into pseudopregnant foster mothers of a nondiabetic strain. The highest rate of viability in vitro (100%) and development into normal fetuses (31%) was obtained with blastocysts transplanted into day-4 foster mothers. The results indicate the feasibility of low-temperature banking of this clinically important strain of rats.
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Monoclonal antibodies (MCA) were obtained by immunizing BALB/c mice with 99% pure granulocytes from normal donors or with a whole leukocyte suspension obtained from a chronic myelogenous leukemia (CML) patient, and then fusing the mouse spleen cells with a 315-43 myeloma cell clone. Four MCA were selected and studied using ELISA, immunofluorescence, cytotoxicity assays, and FACS analysis. Antibodies 80H.1, 80H.3, and 80H.5 (from normals) and 81H.1 (from CML) detected antigens expressed on neutrophils. Antibodies 80H.1 and 80H.3 (IgG) also reacted with monocytes but not with other blood cell subsets. Antibodies 80H.5 and 81H.1 (IgM) were cytotoxic and reacted strongly with most of the cells of the neutrophil maturation sequence, i.e., myeloblasts, promyelocytes, myelocytes, and mature granulocytes. Antibodies 80H.5 and 81H.1 also inhibited CFU-GM growth stimulated by leukocyte feeder layers or placental conditioned media, but did not inhibit BFU-E and CFU-E. Antigens recognized by 80H.3, 80H.5, and 81H.1 were expressed both on a proportion of cells from HL.60, KG.1, ML.1, and K562 myeloid cell lines, and on a proportion of blast cells isolated from patients with acute myelogenous leukemia. They were not found on lymphoid cell lines or lymphoid leukemia cells. These MCA recognize either late differentiation antigens expressed on mature neutrophils and monocytes (80H.1 and 80H.3) or early differentiation antigens (80H.5 and 81H.1) specific to the granulocytic lineage. They may be useful for a better definition of those antigens specific to hematopoietic stem cells and their relationship with normal or neoplastic hematopoiesis.
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Cell surface charge, assessed by the analytical electrophoresis of fresh and cryopreserved human peripheral blood lymphoctes, is changed in any perceivable manner by freezing and thawing. This was confirmed by different membrane markers (E, EA and EAC rosettes).
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A reliable technique for cryopreservation of lymphocytes which conserves mitogenic response and marker (E-Rosettes, immunofluorescence and peroxydase) capability is presented. The data which is presented shows no significant difference between tests involving fresh and cryopreserved lymphocytes.
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