Biomedical subjects

I Bertoncello

Publications and source records attributed to I Bertoncello.

70 records · Page 4Linked to original sources

PubMed1985

Multiparameter analysis of transplantable hemopoietic stem cells: I. The separation and enrichment of stem cells homing to marrow and spleen on the basis of rhodamine-123 fluorescence.

A multiparameter cell separative procedure is described that enables normal transplantable hemopoietic stem cells that preferentially home to the marrow of lethally irradiated mice to be enriched and separated from the majority of spleen colony-forming cells that are assayed 13 days after transplantation (CFU-S13). First, bone marrow cells are centrifuged in a discontinuous bovine serum albumin gradient. Low-density cells (1.060-1.068 g/cm3) are harvested and labeled with the supravital cationic fluorochrome rhodamine 123 (Rh123). Labeled cells are analyzed using a fluorescence-activated cell sorter, and cells are sorted on the basis of relative Rh123 fluorescence within a predetermined forward versus 90 degrees red light scatter window that has been optimized for the recovery and enrichment of cells with marrow repopulating ability (MRA). Cells with MRA were characterized by relatively low Rh123 fluorescence and could be separated from a fraction that fluoresced more intensely and contained the majority of CFU-S13 but low MRA. The ratios of low-fluorescent:high-fluorescent MRA and CFU-S13 were 20:1 and 0.5:1, respectively, and the resultant ratio of MRA:CFU-S13 was 556:1 in the low-fluorescent fraction, and 48:1 in the high-fluorescent fraction. Cells with platelet repopulating ability cofractionate with MRA whereas cells with erythroid repopulating ability remain associated with CFU-S13. High-proliferative-potential macrophage colony-forming cells that form colonies in agar in the presence of the combined stimulus of pregnant mouse uterus extract plus human-spleen-conditioned medium were present in both fractions. Between 20% and 30% of transplantable hemopoietic stem cells has been recovered in approximately 1% of total nucleated cells from the starting preparation, and enrichments of 25- to 30-fold have been achieved permitting hemopoietic reconstitution of lethally irradiated host mice with as few as 400 donor cells.

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PubMed1984

The detection of subclasses of granulosa cells with differing responsiveness to EGF, FGF, and gonadotropin preparations using an anchorage-independent clonal agar assay.

Subpopulations of granulosa cells of differing responsiveness to epidermal growth factor (EGF), fibroblast growth factor (FGF), pituitary gonadotropin preparations, and rat erythrocyte suspensions (RBC) have been detected using an anchorage-independent clonal agar assay. All growth factor preparations were capable of enhancing colony formation when added alone, and elicited cloning efficiencies as high as 35% when added to the culture system at optimal concentrations in a variety of combinations. The FGF preparation was the single most effective stimulator of colony formation, augmenting both colony numbers and colony size at concentrations as low as 50 ng/ml. However, unlike the other growth factors in this assay system, a plateau in responsiveness could not be reached even at levels as high as 1 microgram/ml. NIH-FSH-P2 and NIAMMD-bLH-4 were considerably less potent than other growth factors. Both preparations were inactive at concentrations less than 1 microgram/ml and produced an optimal response at 10 micrograms/ml.

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PubMed1983

A new human breast carcinoma cell line (PMC42) with stem cell characteristics. I. Morphologic characterization.

A new human breast carcinoma cell line (PMC42) was established from a pleural effusion from a woman with metastatic breast cancer. The cells were significantly pleomorphic even after 3.5 years in continuous culture. Eight different cell types could be characterized morphologically in monolayer culture. Cells cloned in agar and replated in monolayer culture were equally heterogenous. Cells also grew in suspension in papillary clusters that structurally resembled glandular organoids. Electron microscopy confirmed the differentiated structure of these cells in which many of the features of lactating breast tissue were evident. It is proposed that this is a culture derived from a malignant breast stem that has retained its ability to differentiate in vitro.

Aged↗

PubMed1983

A new human breast carcinoma cell line (PMC42) with stem cell characteristics. II. Characterization of cells growing as organoids.

A new human breast carcinoma cell line (PMC42) has been further characterized. The cells can grow either as monolayers or as floating cords of cells. The cords grow in suspension for long periods but may spontaneously attach and grow out to form a typical PMC42 monolayer. Ultrastructurally, the cells resemble breast ductal cells in many respects. Both epidermal growth factor (EGF) and prolactin induce ultrastructural changes, and lipid production is stimulated markedly by both factors. EGF also promoted the attachment of the floating cords and the growth of cells from these cords as monolayer cultures. The karyotype of the cord cells is different from that previously described for the monolayer cultures. Cord cells are hypodiploid (mode 39), whereas the monolayer cultures are subtriploid (mode 66). Although the ploidy is different, the karyotypes are related with 9 marker chromosomes being common to both populations. In addition, cultures in which cords have attached and in which cells are growing out as monolayers are bimodal with 10-20% of the cells becoming pseudotetraploid with a mode of 77.

Breast Neoplasms↗

PubMed1983

Characterization of megakaryocyte spleen colony-forming units by response to 5-fluorouracil and by unit gravity sedimentation.

Properties of megakaryocyte progenitor cells in mouse bone marrow have been examined using an in vivo assay system. Perturbation with 5-fluorouracil (5-FU) and separation by unit gravity sedimentation was used to characterize the cells. Bone marrow was assayed for the presence of megakaryocyte colony-forming cells (MK CFU-S) by transplantation into lethally irradiated mice and examining spleen sections 10 days later. Donor mice were untreated or injected intravenously with 5-FU (150 mg/kg), 1 (FU-1) or 7 (FU-7) days beforehand. There was a lack of correlation between the numbers of MK CFU-S and cells giving rise to macroscopic spleen surface colonies (CFU-S10). The sedimentation profile of MK CFU-S in normal marrow was similar (modal velocity 4.16 +/- 0.05 mm/hr) to that of CFU-S10. In FU-1 marrow, MK CFU-S exhibited a bimodal sedimentation profile, with peaks at 3.26 +/- 0.06 mm/hr and 4.53 +/- 0.07 mm/hr. The marrow content of CFU-S10 was reduced to 5% of normal, while MK CFU-S numbers were only reduced to 60%. In FU-7 marrow, the sedimentation profile of MK CFU-S (modal velocity 4.86 +/- 0.16 mm/hr) differed from that of CFU-S10 (5.5 +/- 0.16 mm/hr). It was concluded MK CFU-S and CFU-S10 are different entities. The MK colonies formed from FU-1 marrow contained on average 3.8-fold more cells than those formed from normal marrow. The enhanced megakaryocyte production may be accounted for on the basis of the generation-age model for cell proliferation. It is proposed that MK CFU-S are a heterogeneous population with regard to proliferation potential and that the FU-1 marrow contains cells that survive 5-FU and have a high proliferative potential. These cells may be equivalent among megakaryocytic progenitors to the high proliferative potential colony-forming cells of the granulocyte/macrophage series. They may be responsible for the enhanced megakaryocytopoiesis seen in the marrow of mice 7 days after the injection of 5-FU.

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PubMed1982

The characteristics of an anchorage-independent clonal agar assay for primary explanted bovine granulosa cells.

Normal, primary explanted, bovine granulosa cells grow reproducibly in agar culture as anchorage-independent clones. Epidermal growth factor (EGF) and rat erythrocytes are effective stimulators of colony formation, and when both are added to the culture medium at optimal concentrations, there is an enhancement of colony numbers and colony size, indicative of an independent, and operationally additive, mode of action for the two factors. The ability of cells propagated from agar clones to secrete progesterone, and to augment progesterone secretion 4-fold in the presence of 1 mM dbcAMP is proof that colonies originate from and are composed of functional granulosa cells. Maximal colony numbers are present at day 10 of incubation, and colony forming cells undergo self-renewal as assessed by the ability of cells from primary colonies to reclone in agar. Absolute cloning efficiency, however, is dependent on a number of factors. Inherent variability exists in cloning efficiency of granulosa cells from individual follicles. Quantitative and qualitative clonal growth was improved at an osmolality of less than 300 mOsm when compared with higher osmolalities. Cl-1 medium and the alpha modification of Eagle's medium were equally effective in supporting agar clonogenic growth, whereas both Ham's F12 and NCTC 135 media exhibited poor clonogenic growth supporting properties. The substitution of agarose for agar did not affect colony numbers but colonies grown in the presence of agarose tended to be smaller and more uniform in size.

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PubMed1982

Limitations of the clonal agar assay for the assessment of primary human ovarian tumour biopsies.

114 biopsy specimens from 70 patients with ovarian carcinoma at all stages of disease were submitted for assessment of clonogenic capacity in agar. A highly significant correlation was found between agar clonogenicity and patient survival after biopsy. However, problems related to inherent tumour heterogeneity, quality of sample and tissue disaggregation indicate that this technique may have limited applicability in the routine assessment of patients. Only 41 biopsy specimens (36%) from 31 patients (44.3%) complied with the prerequisite criteria for agar clonogenic assessment, namely: (a) the confirmed presence of malignant cells in the biopsy, (b) the ability to prepare a single-cell suspension, and (c) adequate viable cell numbers for assay. Furthermore, although the dominant patterns of agar clonogenic growth could be identified and correlated with stage of disease, the heterogeneity in both initial clonogenic capacity and "self-renewal" capacity assessed by the ability of primary clones to propagate in liquid culture and reclone in agar was too inconsistent for the assay to be used as a prognostic index for the individual patient.

Agar↗

PubMed1982

A comparison of counterflow centrifugation and unit gravity sedimentation in the separation of mouse bone marrow progenitor cells.

Both Unit Gravity Sedimentation and Counterflow Centrifugation separate cells primarily on the basis of size. However, when performed concurrently on the same normal mouse bone marrow cell suspensions, Unit Gravity Sedimentation was found to be far superior in terms of the recovery of total nucleated cell load and the recovery and enrichment of two classes of functional progenitors: (1) those responsive to the combined stimulus of pregnant mouse uterus extract (PMUE) plus human spleen conditioned medium (HUSPCM), and (2), those responsive to PMUE alone. Neither technique was able to resolve PMUE from PMUE + HUSPCM progenitors. The major limitation of Counterflow Centrifugation for the separation of sub-populations of marrow cells is related to the complexity and range of cell sizes in marrow cell suspensions which lead to significant pellet formation within the elutriator chamber. Furthermore, cells undergoing Counterflow Centrifugation are subjected to forces not encountered in Unit Gravity Sedimentation. Therefore, the basis of the separations is not identical and this is reflected in cell volume distributions of comparable fractions separated by each method.

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PubMed1981

Clonal agar culture of normal primary explanted bovine granulosa cells.

When seeded at low cell densities (500 to 2500 cells per dish), normal primary explanted bovine granulosa cells grow in a double layer semi-solid agar culture system as anchorage-independent clones composed of well differentiated cells with vacuolated granular cytoplasm with a cloning efficiency of between 2% and 4%. The addition of growth promoting factors to the culture medium such as epidermal growth factor (EGF), fibroblast growth factor (PGF) or rat erythrocyte suspensions resulted in a 5 to 6-fold increase in colony numbers and this was accompanied by an increase in colony size. Cells from both small (3--7 mm diameter) and large (less than 1 cm diameter) ovarian follicles exhibited similar patterns of responsiveness to growth factors but the cloning efficiency of cells from large follicles was approximately double that of cells from small follicles.

Agar↗

PubMed1981

Characterization and enrichment of macrophage progenitor cells from normal and 5-fluorouracil treated mouse bone marrow by unit gravity sedimentation.

Unit gravity sedimentation has been used to characterize and enrich cells from normal, post-5-fluorouracil (FU) and post 5-fluorouracil plus endotoxin (FUEt) regenerating mouse bone marrow with respect to two classes of macrophage progenitor cells. The two classes of progenitor cells assayed were (1) those responsive to the combined stimulus of pregnant mouse uterus extract (PMUE) plus human spleen conditioned medium (HUSPCM), and (2) those responsive to PMUE alone. In contrast to the bimodal nucleated cell distribution of normal marrow, 7 day post-FU marrow exhibited a unimodal nucleated cell profile. In marrow from 7 day post-FUEt treated mice, in ;which marrow regeneration was accelerated, there was a reemergence of a second nucleated cell peak. In addition, in 7 day post-FU and 7 day post-FUEt marrow there was a shift in the modal sedimentation velocities of both PMUE responsive and PMUE + HUSPCM responsive populations to higher values. The combined effect of these changes resulted in a marked increase in plating efficiencies of the peak enrichment fractions, reaching 12.5% in 7 day post-FUEt marrow. However, the highest yield of progenitor cells responsive to the combined stimulus of PMUE + HUSPCM compared to those responsive to PMUE alone were obtained in 7 day post-FU marrow.

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PubMed1980

Characterization of early leukemic cells in spleen and bone marrow of Friend virus infected mice.

Nonidentity of leukemic cell populations of DBA/w mice (TCFU and CRVEI) early after Friend Visus infection has been demonstrated for spleen by different growth kinetics of these cell populations during leukemogenesis and for spleen and bone marrow of DBA/w mice by the velocity sedimentation technique. In addition the velocity sedimentation data indicate a heterogeneity of the TCFU population. Stem cell analysis of TCFU and CFUS derived spleen colonies showed that TCFU colonies contain significantly more CFUEI than the surrounding tissue. The erythroid nature of TCFU colonies is confirmed by the almost complete absence of CFUC from these colonies as compared to CFUS derived spleen colonies.

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PubMed1978

Erythroid stem cells in Friend-virus infected mice.

The erythropoietic stem cell compartment was studied in Friend-virus (polycythemic strain, FV-P) infected DBA/2 and NMRI mice with the CFUE and BFUE technique. Early after infection there was a depression in CFUE number in bone marrow and spleen, followed by an increase of the CFUE concentration, earlier and more pronounced in the spleen than in the marrow. Three days after FV-P infection an erythropoietin (Ep) independent CFUE population started to grow and replaced the normal Ep-dependent population within 8 to 12 days. The shift to Ep independency was not gradual. CFUE colonies of FV-P infected bone marrow cells were two to three times larger than control colonies after three days in vitro incubation. BFUE colonies increased in number during the first days of infection, but were totally lost after more than ten days. After velocity sedimentation of bone marrow cells of FV-P infected animals, however, the BFUE containing fractions showed normal BFUE colony growth and normal Ep sensitivity. In unfractionated bone marrow cell cultures BFUE colony growth could be observed later than ten days post infection when the cultures were refed with medium. It was therefore concluded that the loss of BFUE colony growth after FV-P infection was an in vitro artefact due to inadequate culture conditions.

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PubMed1977

The physicochemical properties of erythrocyte derived activity which enhances murine bone marrow colony growth in agar culture.

A stable preparation which retains enhancement activity for the growth of murine granulocyte-macrophage colonies in agar culture has been extracted from human and rat erythrocytes by osmotic lysis and high speed centrifugation. Trypsin digestion followed by Sephadex G-25 column chromatography has resulted in the removal of haemoglobin and the isolation of an active fraction with an apparent molecular weight of 900 to 1000 daltons. Enhancement activity was abolished by n-ethylmaleimide (NEM) treatment, suggesting that the enhancement is dependent on the presence of free sulphydryl groups.

Agar↗

PubMed1976

Sensitivity of renin secretion to volume depletion in the anaesthetized dog: comparison between urinary drainage and slow haemorrhage.

1. An experimental technique utilizing 'denervation diuresis' from one kidney with measurement of renin release from the contralateral innervated kidney was developed to study the sensitivity of renin secretion to volume depletion. 2. With urine excretion, release of renin increased progressively from the innervated kidney. The increase was significant at a sodium deficit of 0-23 mole.kg-1. At a sodium deficit of 0-6 m-mole.kg-1 renin release had doubled. 3. Bilateral vagotomy did not alter this response. 4. Precise replacement of sodium loss with isotonic saline but without replacement of other urinary components returned renin release to control levels. 5. Slow haemorrhage causing a rate of volume and sodium loss equivalent to urinary drainage did not alter the rate of renin release. 6. With a single denervated kidney and contralateral nephrectomy, renin release fell progressively to minimal levels despite sodium deficits up to 2-6 m-mole.kg-1. 7. It is concluded that renin secretion is sensitive to at most a 0-5% change in body fluid volume and should be considered a primary response to volume depletion. The sensitivity of the response depends upon normal renal innervation but is not mediated via vascular volume receptors nor via receptors innervated by the vagus. 8. It is proposed that the extreme sensitivity of the renin-secreting system in these experiments results from the combination of volume depletion and slight hypotonicity of extracellular fluid acting on the renal afferent arteriole without the mediation of the macula densa,

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PubMedDate not supplied

Defective stem cell factor expression in c-myb null fetal liver stroma.

High levels of c-Myb are observed in immature precursor myeloid and lymphoid cells, while downregulation of c-myb accompanies terminal differentiation to a mature phenotype. This has established c-Myb as a crucial transcription factor for hematopoiesis. Further evidence for this is the embryonic death of the c-myb homozygous mutant mouse at ED15 due to defective fetal liver erythropoiesis. Cells from fetal liver of wild-type and c-myb-/- embryos were examined in detail for their hematopoietic potential and the capacity of the stroma to support wild-type hematopoiesis. The c-myb-/- fetal liver was shown to harbor sevenfold fewer spleen focus-forming cells and a similarly lower number of cells with long-term repopulating capacity (high proliferative potential cells). However, shorter term repopulating cells were not substantially reduced. c-myb-/- stromal cells were unable to support the proliferation of wild-type bone marrow lineage-negative cells. This was found to be partly due to a decrease in stem cell factor (SCF) expression while partial rescue of the stromal cell cultures was achieved through the addition of exogenous SCF. DNA binding studies for two sites within the SCF promoter demonstrated an in vitro interaction between the SCF promoter and c-Myb and transient transfection studies demonstrated that c-Myb could substantially transactivate the SCF promoter in HEK293 cells. These data explain why the c-myb-/- embryos are so impaired in their ability to establish hematopoiesis.

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PubMedDate not supplied

Abolition of the renin-releasing action of frusemide by acute renal denervation in dogs.

1. The effects of infusions of frusemide at low (0.05-0.1 mg.kg-1.min-1) and high (0.5-2.0 mg.kg-1.min-1) rates were studied on renin secretion and urinary outputs of sodium and potassium in anaesthetized dogs in which one kidney was removed and the remaining kidney was either innervated or denervated. 2. When the kidney was innervated, low rates of infusion of frusemide did not significantly affect renin secretion if urinary volume and sodium losses were replaced. Without replacement of urinary losses, renin secretion increased at sodium deficits of 0.7-0.9 mmol.kg-1 in the presence of elevated rates of sodium and potassium excretion. 3. High rates of infusion of frusemide caused an immediate increase in renin secretion from innervated kidneys which was not related to urinary losses. 4. Denervation of the kidney increased the urinary outputs of sodium and potassium while it decreased the rate of renin secretion to one-tenth of the resting value. 5. Denervation of the kidney abolished the renin-releasing action of frusemide at both low and high infusion rates even when the sodium deficit amounted to 4.3 mmol.kg-1. 6. Constriction of the aorta producing a fall of 10-30 mmHg in perfusion pressure raised the rate of renin secretion from denervated kidneys to control levels and partially restored the renin-releasing action of frusemide at high infusion rates. 7. The findings indicate that frusemide has a site of action apart from the macula densa in mediating renin release.

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