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Biomedical subjects

I Bertoncello

Publications and source records attributed to I Bertoncello.

At least 37 records · Page 2Linked to original sources
PubMed1995

Granulocyte-macrophage colony-stimulating factor is not responsible for the correction of hematopoietic deficiencies in the maturing op/op mouse.

Osteopetrotic (op/op) mice are characterized by an autosomal recessive inactivating mutation resulting in the absence of biologically active colony-stimulating factor-1 (CSF-1). Consequently, young op/op mice have a severe deficiency of macrophages and osteoclasts resulting in excessive bone formation, occlusion of the marrow cavity, and reduced marrow hematopoietic activity. Recently, we showed that the osteopetrosis and hematopoietic deficiencies evident in young op/op mice are not permanent but are progressively corrected with age. There are increases in osteoclast activity; bone resorption; femoral marrow space; and marrow hematopoietic activity, cellularity, and macrophage content. In the present study we show that CSF-1-/- granulocyte-macrophage colony-stimulating factor (GM-CSF)(-/-)-deficient mice also undergo the same pattern of hematopoietic correction as the op/op mouse. Also, like the op/op mouse, the peritoneal cellularity and macrophage content of CSF-1/GM-CSF-deficient mice remains severely reduced. Our data show that the "knockout" of GM-CSF does not change the op/op phenotype, and that GM-CSF is not essential for the correction of the hematopoietic deficiencies in the op/op mouse. Importantly, the data also show that neither GM-CSF nor CSF-1 is an absolute requirement for the commitment of primitive hematopoietic stem cells to the macrophage lineage or for the differentiation of at least some classes of macrophages. This finding suggests that an alternate regulatory factor can be involved in macrophage and osteoclast commitment, differentiation, and function in vivo.

Age Factors↗
PubMed1995

Haematopoietic radioprotection by Cremophor EL: a polyethoxylated castor oil.

The polyethoxylated castor oil, Cremophor EL (Cremophor) is approved for human use as a vehicle for oral and intravenous administration of water-insoluble compounds. Cremophor has also previously been shown to reverse the multidrug resistance phenotype at clinically acceptable doses. This study demonstrates that doses of Cremophor in the range of 25-50 microliters/kg intravenously (i.v.) administered 1 day prior to near-lethal irradiation protected the regenerative capacity of the marrow, resulting in haematopoietic radioprotection and long-term survival of near-lethally-irradiated mice. In normal mice, Cremophor administration (1) markedly reduced the level of serum haematopoietic inhibitory activity 4-8 h following injection; (2) resulted in a transient decrease in femoral bone marrow cellularity and upregulated B220 (B cells), and 7/4 (neutrophils and activated macrophages), but not Thy-1 (T-cells) surface antigen expression in bone marrow cells within 24 h of injection; and (3) transiently elevated the incidence of both primitive and committed haematopoietic progenitor cells detected in clonal agar culture within 48 h of injection. Bone marrow progenitor cell content, and peripheral blood white cell, platelet and reticulocyte counts were unaffected. This suggests that the haematopoietic radioprotection and recovery observed in irradiated mice pretreated with Cremophor may be the result of accessory cell activation and/or modulation of accessory factors regulating haematopoietic progenitor cells. Our data suggest a potential clinical use of Cremophor as an adjunct to, or as a substitute for, cytokines to minimize myelosuppression following cytotoxic therapy.

Animals↗
PubMed1995

Developmental hematopoiesis from prenatal to young-adult life in the mouse model.

Five measurements of hematopoietic function were made in the mouse from midfetal life to young adulthood. These included two in vivo (day-8 colony-forming unit-spleen [CFU-S8] and day-12 CFU-S [CFU-S12]) and two in vitro clonal measurements of hematopoietic stem and progenitor cells (high proliferative potential colony-forming cell [HPP-CFC] and CFC of low proliferative potential [LPP-CFC]) as well as an in vitro clonal measurement of colony-forming unit-fibroblast (CFU-F). The appearance, increase, subsequent decrease, and later emergence and increase of each of these parameters in the fetal-liver, newborn, growing-infant, and young-adult bone marrow were correlated and found to be in parallel. Exceptions to this included the earlier appearance in the fetal liver of CFU-F and the relatively differentiated hematopoietic LPP-CFC. The pattern of emergence of these progenitor cell subpopulations in the fetal liver may be related, in part to the timing of the hematopoietic microenvironment development and the relative frequencies of progenitor cell types in the circulation. This developmental study in the mouse model describes additional correlations between in vivo and in vitro colony-forming stem cells and fibroblastic stromal colony-forming cells, and it suggests the dependence of hematopoietic stem cells upon the stromal microenvironment for the necessary conditions for hematopoietic stem cell lodgment, growth, and maturation.

Aging↗
PubMed1995

Modified thrombopoietic response to 5-FU in mice following transplantation of Lin-Sca-1+ bone marrow cells.

An experimental murine model of bone marrow transplantation (BMT) has been used to study the mechanisms of platelet production following transplantation. A defined primitive population of hematopoietic bone marrow cells (1000 Lin-Sca-1+) was isolated and transplanted into lethally irradiated (13 Gy) syngeneic recipient mice. Platelet counts, but neither red nor white blood cell (WBC) counts, were low 30 days after transplantation. By 90 days, platelet levels had normalized in transplanted mice, but this occurred from a reduced megakaryocyte progenitor (CFU-Mk) pool, implying that altered bone marrow control was involved in platelet production. To assess the capacity of the bone marrow of these compensated mice to sustain platelet production, the rate and degree of recovery were examined following administration of 150 mg/kg of 5-fluorouracil (5-FU) 90 days after transplantation. Transplanted mice showed a delay, both in platelet recovery and rebound thrombocytosis, after 5-FU administration when compared to normal littermates treated with 5-FU. The regeneration and expansion of bone marrow CFU-Mk and mature megakaryocytes was retarded in the transplanted mice and explained the altered platelet kinetics. The onset of increased platelet and mature megakaryocyte size, however, was not different between the two groups, indicating that the transplanted mice responded normally to the mechanisms controlling megakaryocyte development and platelet formation. The data suggest that following BMT a limitation in the proliferative capacity of primitive hematopoietic cells results in a smaller pool of megakaryocyte precursors. Compensatory adjustment within the megakaryocyte lineage, nevertheless, results in normalization of megakaryocyte and platelet number. The ability of transplanted mice to sustain platelet production when challenged with increased platelet demand is not limited by megakaryocytic maturation but by a restriction in proliferation or differentiation from the stem cell pool.

Animals↗
PubMed1994

The development and establishment of hemopoiesis in fetal and newborn osteopetrotic (op/op) mice.

Previous studies of young CSF-1-less osteopetrotic (op/op) mice demonstrate a severe deficiency of both macrophages and osteoclasts, resulting in excessive bone formation, occlusion of the marrow cavity, and reduced hemopoietic activity. The accompanying splenomegaly and prolonged splenic hemopoiesis observed in these mice suggests that osteopetrosis may perturb the normal progression of fetal hemopoietic development and obstruct the seeding of hemopoietic precursors into the bone marrow. This study demonstrates that the absence of CSF-1 does not affect the progression of hemopoietic development in fetal op/op mice until after colonization of the bone marrow. Significant deficiencies in marrow cellularity and progenitor cell content in the long bones of op/op mice were not evident prior to Day 2 postnatal, suggesting that the altered hemopoietic state of young op/op mice is not a consequence of abnormal fetal hemopoietic development, but is primarily due to the lack of functional osteoclasts in op/op fetuses and hence, impaired remodeling of the marrow cavity after birth.

Animals↗
PubMed1994

Anchorage-independent culture of bovine granulosa cells: the effects of basic fibroblast growth factor and dibutyryl cAMP on cell division and differentiation.

During ovarian folliculogenesis granulosa cells divide while in contact with stromal cells and other granulosa cells. Following ovulation, however, they cease dividing and differentiate into large luteal cells. When cultured in monolayer, granulosa cells spontaneously differentiate into luteal cells, thus confounding the study of the follicular functions of granulosa cells in vitro, such as cell division. We have found that bovine granulosa cells were able to divide in an anchorage-independent culture system consisting of soft agar and an overlay of methylcellulose. The cells grew in colonies and retained the ultrastructural features of follicular granulosa cells. They also secreted an extracellular matrix with features of basal lamina. The granulosa cells responded to basic fibroblast growth factor (bFGF) in a dose- and time-dependent manner, with a three- to five-fold increase (at 50 ng bFGF/ml for 14 days) in the level of DNA per dish. This mitogenic effect was inhibited by dibutyryl cAMP (dbcAMP) (1 mM). In the presence of dbcAMP the cells hypertrophied considerably, did not secrete extracellular matrix, and developed the ultrastructural features of luteal cells. They also secreted threefold more progesterone. This system offers the ability to study the follicular functions of granulosa cells in culture.

Animals↗
PubMed1994

The relationship between different high proliferative potential colony-forming cells in mouse bone marrow.

Previous work has shown that part of the hierarchical structure of the hematopoietic system can be described by HPP-CFC-1 (primitive high proliferative potential colony-forming cells responding to colony-stimulating factor-1 [CSF-1] + interleukin-3 [IL-3] + IL-1), HPP-CFC-2 (more mature HPP-CFC responding to CSF-1 + IL-3), and mature HPP-CFC responding to the single factors, CSF-1, granulocyte-macrophage colony-stimulating factor (GM-CSF), or IL-3. In this study, we have attempted to relate the murine HPP-CFC, stimulated by various combinations of growth factors (GFs)--CSF-1, GM-CSF, IL-3, IL-6, IL-1, stem cell factor (SCF), and transforming growth factor-beta (TGF-beta)--and by CSF-1, GM-CSF, and IL-3 on their own, to these known progenitors. Studies involving regeneration of the bone marrow after 5-fluorouracil (5-FU) treatment, generation of progenitors in liquid cultures in response to different GF combinations, and the HPP-CFC content of lineage-negative rhodamine-sorted bone marrow (BM) fractions have indicated that: 1. the combinations CSF-1 + IL-3 + IL-1 + SCF and CSF-1 + IL-3 + IL-1 + IL-6, and possibly CSF-1 + GM-CSF + IL-3 + IL-1, stimulate pre-HPP-CFC-1; 2. the combinations CSF-1 + IL-1 + GM-CSF, CSF-1 + IL-1 + IL-6, CSF-1 + IL-1 + SCF, CSF-1 + IL-3 + SCF, CSF-1 + IL-6 + SCF, and IL-3 + SCF, appear to overlap with the CSF-1 + IL-3 + IL-1 combination to stimulate the more mature cells of the HPP-CFC-1 compartment; 3. the combinations CSF-1 + GM-CSF, CSF-1 + IL-1, CSF-1 + IL-6, and CSF-1 + SCF may stimulate the more mature cells of the HPP-CFC-2 population, while the single factors CSF-1, GM-CSF, and IL-3, as suggested in other reports, may stimulate HPP-CFC that are more mature than the HPP-CFC-2; 4. the combinations IL-3 + IL-6 and SCF + IL-6 appear to stimulate HPP-CFC that overlap with the HPP-CFC-1 population, while those responding to the combination GM-CSF + TGF-beta overlap with the HPP-CFC-2 population within the hematopoietic hierarchy; and 5. CSF-1 and GM-CSF appear to be interchangeable in the combinations studied.

Animals↗
PubMed1994

Classes of primitive murine megakaryocytic progenitor cells.

We describe high proliferative potential colony-forming cells-megakaryocyte (HPP-CFU-Mk) from mouse bone marrow preparations using known cytokine combinations. These primitive precursors, which generate at least 80 megakaryocytes per colony, were detected from bone marrow populations enriched for primitive cells, either following treatment with 5-fluorouracil (5-FU) or after enrichment from normal bone marrow using immunological procedures. HPP-CFU-Mk were most reproducibly grown in the presence of interleukin-1 (IL-1) plus IL-3 plus IL-6, and mostly grew as a single large aggregate, rarely forming multiple foci. Cell separation studies showed that the HPP-CFU-Mk have membrane properties that characterized these cells as being intermediate between high proliferative myeloid primitive progenitors (high proliferative colony-forming cells [HPP-CFC]) and committed megakaryocyte progenitors (CFU-Mk). The data show that HPP-CFU-Mk can be designated as primitive cells on the basis of their being spared in vivo after 5-FU treatment, their proliferative potential to produce megakaryocytes, and their cofractionation with a proportion of primitive myeloid progenitor cells.

Animals↗
PubMed1994

Age-related changes in extramedullary hematopoiesis in the spleen of normal and perturbed osteopetrotic (op/op) mice.

Occlusion of the marrow cavity by excessive bone formation in young osteopetrotic (op/op) mice results in a significant reduction in the space available for hematopoiesis. At this time, splenomegaly is evident, and the spleen is a site of significant extramedullary hematopoiesis. In vitro clonal assays of spleen cell suspensions in young op/op mice demonstrated a 22-fold elevation in the content of high proliferative potential colony-forming cells (HPP-CFC) (4 weeks of age) and a 14-fold elevation in the content of committed progenitors responsive to colony-stimulating factor-1 (CSF-1) (6 weeks of age). Flow-cytometric analysis also demonstrated a shift in the myeloid:lymphoid cell ratio in the spleens of young op/op mice, with a 35% reduction in the number of B220+ cells, and a two-fold increase in myeloid cells expressing the hematopoietic cell surface lineage antigens Mac-1 and Gr-1. However, the hematopoietic deficiencies of op/op mice are not permanent. An age-related progressive remodeling of the bone marrow cavity results in the correction of bone marrow parameters by 22 weeks of age. This correction in marrow hematopoietic activity is accompanied by a resolution of the splenomegaly, a progressive decrease in splenic hematopoietic activity at both the primitive and committed progenitor cell levels, and a correction of the lymphoid:myeloid cell ratio. Negative immunomagnetic selection of splenic hematopoietic progenitor cells from op/op and control littermate mice, followed by analysis of their expansion in liquid culture, demonstrated that primitive hematopoietic progenitor cells of high proliferative potential continued to reside in the spleen of old op/op mice. The response of these mice to a 5-fluorouracil (5-FU) cytotoxic challenge suggested that this pool of primitive progenitor cells acted as a hematopoietic reserve capable of rapidly responding to hematopoietic perturbation.

Aging↗
PubMed1993

Identification and cloning of a protein kinase-encoding mouse gene, Plk, related to the polo gene of Drosophila.

We have determined the nucleotide sequence of a cDNA encoding a protein kinase that is closely related to the enzyme encoded by the Drosophila melanogaster mutant polo and that we have designated Plk (polo-like kinase). Plk is also related to the products of the Saccharomyces cerevisiae cell cycle gene MSD2 (CDC5) and the recently described early growth response gene Snk. Together, Plk, polo, Snk, and MSD2 define a subfamily of serine/threonine protein kinases. Plk is expressed at high levels in a number of fetal and newborn mouse tissues but is not expressed in the corresponding adult organs. With the exception of adult hemopoietic tissues, the only adult tissues in which we could detect Plk expression were ovaries and testes. Taken together, the patterns of Plk expression suggest an association with proliferating cells. Since polo is required for mitosis in Drosophila it is possible that Plk is involved in some aspect of cell cycle regulation in mammalian cells.

Amino Acid Sequence↗
PubMed1993

Delayed hematopoietic development in osteopetrotic (op/op) mice.

Changes in structure, cellularity, hematopoietic progenitor cell and macrophage content, and osteoclast activity were investigated in the hematopoietic organs of the colony-stimulating factor 1(CSF-1)-less osteopetrotic (op/op) mouse. The data indicated that op/op mice undergo an age-related hematopoietic recovery and resolution of osteopetrosis, suggesting that the hematopoietic system has the capacity to use alternative mechanisms to compensate for the absence of an important multifunctional growth factor, CSF-1. In young animals, op/op femurs were heavily infiltrated with bone, and marrow cellularity was significantly reduced. After 6 wk of age, there was an increase in the marrow space available for hematopoiesis. The femoral cavity of op/op mice progressively enlarged, and by 22 wk of age its appearance and marrow cellularity was comparable to that of controls. The percentage of op/op mononuclear phagocytes, defined by F4/80 antigen expression, progressively increased to normal levels by 35 wk of age. There was no difference in the incidence of both primitive and mononuclear phagocyte-committed, CSF-1-responsive progenitor cells in op/op marrow, but their femoral content was significantly reduced in young mice. During the period of reduced hematopoiesis in the marrow of young op/op mice, splenic hematopoietic activity was elevated. This mutant mouse represents a system for the study of the CSF-1-independent regulatory mechanisms involved in hematopoietic regulation.

Animals↗
PubMed1993

Expansion of hemopoietic activity in long-term culture of human bone marrow by c-kit ligand (stem cell factor).

The response of hemopoietic activity in long term culture of human bone marrow (LTMC) to exogenous c-kit ligand has been investigated. Addition of c-kit ligand as recombinant Stem Cell Factor after the adherent stromal cell layer had become confluent failed to increase the content of hemopoietic cells in the culture system. Production of hemopoietic progenitors and differentiated progeny was on the other hand amplified by addition of c-kit ligand at regular intervals from the initiation of the culture, and resulted in net formation of these cells in the culture system. The characteristics of the response were consistent with consequences of increased survival of hemopoietic progenitors during a vulnerable initial phase in which the availability of endogenous c-kit ligand was limited by the initially low number of stromal cells. The increase in differentiated progeny was essentially due to increased formation of neutrophil series, and the lack of increased basophil production under these circumstances presumably reflected the nature of the contributory effects of endogenously generated growth regulatory molecules which acted in a synergistic manner with added c-kit ligand.

Animals↗
PubMed1993

Accelerated establishment of murine long-term bone marrow cultures by addition of stem cell factor.

The effect of stem cell factor (SCF) on the establishment of hematopoietic activity in murine long-term bone marrow cultures (LTBMC) was investigated by addition of SCF to (a) normal LTBMC from the onset of culture and (b) pre-established irradiated bone marrow stroma inoculated with lineage negative (Lin-) primitive hematopoietic progenitor cells enriched on the basis of low rhodamine-123 uptake (Rh-dull). Hematopoietic activity was established more rapidly in LTBMC grown in the presence of SCF (70 ng/mL), and the typical decline in cellularity and progenitor cell content during the first weeks of culture was not observed. SCF also promoted the rapid expansion of progenitor cells derived from Lin-, Rh-dull primitive hematopoietic cells inoculated onto irradiated preestablished bone marrow stroma. The data demonstrate that exogenous SCF augments hematopoietic activity in LTBMC, and that the levels of endogenous SCF elaborated in LTBMC may be suboptimal for expansion of hematopoietic cells.

Animals↗
PubMed1993

Temporal thrombocytopenia after engraftment with defined stem cells with long-term marrow reconstituting activity.

Delayed platelet recovery and altered marrow megakaryocytopoiesis was observed in highly irradiated mice following transplantation with 1000 lineage-negative, stem cell antigen-positive (Lin-Sca-1+) cells. Thirty days after transplantation, the mice were thrombocytopenic. Normal platelet levels were reestablished by 90 days after transplantation. Platelet levels were established from the bone marrow with altered megakaryocytopoiesis, however. Megakaryocyte progenitor numbers were found at reduced levels in the reconstituted marrow at all time points assessed. Bone marrow function was also different in that marrow reserve was also diminished. While the transplantation regime did give long-term reconstitution of blood cells, the quality of the marrow reserve was significantly impaired as revealed by response to further hematopoietic challenge. The data indicate that mice transplanted with a population of highly defined stem cells have perturbed marrow function, one characteristic being an altered process of megakaryocytopoiesis.

Animals↗
PubMed1992

Recombinant rat stem cell factor stimulates the amplification and differentiation of fractionated mouse stem cell populations.

The role of recombinant rat stem cell factor (rrSCF) was studied on defined primitive bone marrow cell populations. In agar culture of 500 lineage-negative/Sca-1-positive (Lin-/Sca-1+) cells, rrSCF alone stimulates small colonies of predominantly granulocytic cells. The combinations of rrSCF plus interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or macrophage CSF (CSF-1) stimulated primitive progenitor cells defined as high proliferative potential colony-forming cells (HPP-CFC). Synergistic increases in total colony numbers were obtained with rrSCF plus GM-CSF, granulocyte CSF (G-CSF), CSF-1, or IL-6, but not IL-1 or IL-3. Lin-/Sca-1+ cells were incubated in liquid culture at 3,000 cells/mL for 6 days in the presence of rrSCF alone or in combination with other growth factors. The total number of cells was increased twofold in the presence of rrSCF, with the progeny primarily myeloid in nature. The greatest increase in cell number was obtained with rrSCF plus IL-3, where the cell number increased 40-fold. These factors also stimulated an increase in HPP-CFC (10-fold) and GM-CFC (500-fold). To determine if these interactions were direct, single Lin-/Sca-1+ cells were sorted into microtiter wells and the cell proliferation scored 6 days later. RrSCF synergized with IL-3, IL-6, and G-CSF to stimulate the proliferation of single cells. The cells in positive wells were subcultured into colony-forming assays and up to 400 CFC per well were obtained after 14 days incubation of the secondary cultures. These data demonstrate that rrSCF acts in combination with various growth factors to directly stimulate the amplification potential of hematopoietic primitive precursors, resulting in differentiation of these precursors.

Animals↗
PubMed1992

The role of interleukin 6 in megakaryocyte formation, megakaryocyte development and platelet production.

Megakaryocytopoiesis is the cellular amplification and differentiation of precursors into immature megakaryocytes, and the cytoplasmic maturation of these megakaryocytes, a process terminating in the release of platelets into the circulation. Interleukin 6 (IL-6) stimulates megakaryocytopoiesis in the bone marrow, increasing platelet numbers in the circulation. IL-6 alone is poorly active on the growth of stem cell populations, but acts in synergy with stem cell factor (c-kit ligand) to expand the committed myeloid progenitor compartments but not the megakaryocyte progenitors. IL-6 has a direct action on megakaryocyte progenitors but only in synergy with low doses of interleukin 3 (IL-3), increasing the number of immature megakaryocytes and enhancing the processes of development into mature megakaryocytes. IL-6 is about 10 times more active on megakaryocytes than on megakaryocyte progenitors in cell culture. It is active alone and will stimulate increases in cell size and DNA content. IL-6 does not appear to stimulate the process of platelet release. IL-6 is found in bone marrow, in both macrophage subsets and megakaryocytes, indicating that it may be an important physiological regulator of both paracrinal (microenvironmental) and autocrinal mechanisms controlling megakaryocyte development in bone marrow.

Animals↗
PubMed1992

Transforming growth factor beta inhibits the action of stem cell factor on mouse and human hematopoietic progenitors.

In agar culture of post 5-fluorouracil mouse bone marrow cells (FUBM), recombinant rat stem cell factor (rrSCF) synergizes with granulocyte colony-stimulating factor (G-CSF), interleukin-3 (IL-3) or interleukin-6 (IL-6) to stimulate primitive progenitor cells (HPP-CFCs). The addition of recombinant human transforming growth factor beta (rhTGF-beta) to cultures of FUBM containing rrSCF plus rhG-CSF, rrSCF plus recombinant murine (rm)IL-3, or rrSCF plus rhIL-6 resulted in 100% inhibition of colony formation. Highly enriched populations of primitive bone marrow cells were obtained by isolating lineage negative (Lin-), Sca-1-positive (Sca-1+) cells from normal mouse bone marrow. RhTGF-beta inhibited 90% of colony formation stimulated by rrSCF plus rmIL-3 in agar culture of the Sca-1+ cells. RhTGF-beta also inhibited colony formation in agar culture of post FU human bone marrow cells. The synergistic increase in colony formation obtained with recombinant human SCF (rhSCF) plus rhGM-CSF and rhSCF plus rhIL-3 was inhibited by rhTGF-beta (approx. 60% and 87% inhibition, respectively). RhTGF-beta also totally inhibited the erythroid colony formation stimulated by rhSCF plus recombinant human erythropoietin (rhEpo). These data demonstrate that TGF-beta inhibits SCF-stimulated colony formation of mouse and human BM. This inhibition on progenitor cells appears to be a direct action of TGF-beta and is consistent with the target cells of SCF being more primitive progenitors than the CFCs stimulated by the CSFs alone.

Animals↗