scholarly journals Feline parvovirus propagates in cat bone marrow cultures and inhibits hematopoietic colony formation in vitro

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 71-81
Author(s):  
GJ Kurtzman ◽  
L Platanias ◽  
L Lustig ◽  
N Frickhofen ◽  
NS Young
Keyword(s):  
Bone Marrow ◽  
Colony Formation ◽  
Neutralizing Antibodies ◽  
Gm Csf ◽  
Virus Inhibition ◽  
Myeloid Progenitor Cells ◽  
Bone Marrow Cultures ◽  
Infected Cells ◽  
Feline Parvovirus

Feline parvovirus (FPV) causes leukopenia in naturally infected cats. We investigated the mechanism of hematopoietic depression by this virus in feline bone marrow cultured in vitro. In suspension cultures we demonstrated FPV propagation and replication using DNA molecular hybridization. Viral RNA and DNA were observed by in situ hybridization in about 10% of marrow cells at day 3. Granulocytes and their precursors were virtually absent from infected cultures after six days. Infected cells showed viral capsid protein predominantly in nuclei by immunofluorescence. In clonal assays, FPV most efficiently inhibited hematopoietic colony formation by myeloid progenitor cells (CFU-GM), but erythroid colony formation (BFU-E and CFU-E-derived) was also depressed in the presence of virus. Inhibition of colony formation could be abrogated by physical inactivation of the virus or preincubation with specific neutralizing antibodies. Recombinant human colony stimulating factors GM-CSF and G-CSF supported feline myelopoiesis in progenitor assays, and FPV completely inhibited factor dependent colony formation.

scholarly journals Feline parvovirus propagates in cat bone marrow cultures and inhibits hematopoietic colony formation in vitro

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 71-81 ◽  
Author(s):  
GJ Kurtzman ◽  
L Platanias ◽  
L Lustig ◽  
N Frickhofen ◽  
NS Young
Keyword(s):  
Bone Marrow ◽  
Colony Formation ◽  
Neutralizing Antibodies ◽  
Gm Csf ◽  
Virus Inhibition ◽  
Myeloid Progenitor Cells ◽  
Bone Marrow Cultures ◽  
Infected Cells ◽  
Feline Parvovirus

Abstract Feline parvovirus (FPV) causes leukopenia in naturally infected cats. We investigated the mechanism of hematopoietic depression by this virus in feline bone marrow cultured in vitro. In suspension cultures we demonstrated FPV propagation and replication using DNA molecular hybridization. Viral RNA and DNA were observed by in situ hybridization in about 10% of marrow cells at day 3. Granulocytes and their precursors were virtually absent from infected cultures after six days. Infected cells showed viral capsid protein predominantly in nuclei by immunofluorescence. In clonal assays, FPV most efficiently inhibited hematopoietic colony formation by myeloid progenitor cells (CFU-GM), but erythroid colony formation (BFU-E and CFU-E-derived) was also depressed in the presence of virus. Inhibition of colony formation could be abrogated by physical inactivation of the virus or preincubation with specific neutralizing antibodies. Recombinant human colony stimulating factors GM-CSF and G-CSF supported feline myelopoiesis in progenitor assays, and FPV completely inhibited factor dependent colony formation.

scholarly journals Enhancing and suppressing effects of recombinant murine macrophage inflammatory proteins on colony formation in vitro by bone marrow myeloid progenitor cells

Blood ◽  
1990 ◽  
Vol 76 (6) ◽  
pp. 1110-1116 ◽  
Author(s):  
HE Broxmeyer ◽  
B Sherry ◽  
L Lu ◽  
S Cooper ◽  
KO Oh ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Colony Formation ◽  
Pokeweed Mitogen ◽  
Myeloid Progenitor ◽  
Hla Dr ◽  
Myeloid Progenitor Cells ◽  
Macrophage Colony Stimulating Factor ◽  
Inflammatory Proteins ◽  
Macrophage Colony

Abstract Purified recombinant (r) macrophage inflammatory proteins (MIPs) 1 alpha, 1 beta, and 2 were assessed for effects on murine (mu) and human (hu) marrow colony-forming unit-granulocyte-macrophage (CFU-GM) and burst-forming unit-erythroid (BFU-E) colonies. Recombinant MIP-1 alpha, -1 beta, and -2 enhanced muCFU-GM colonies above that stimulated with 10 to 100 U natural mu macrophage-colony-stimulating factor (M-CSF) or rmuGM-CSF, with enhancement seen on huCFU-GM colony formation stimulated with suboptimal rhuM-CSF or rhuGM-CSF; effects were neutralized by respective MIP-specific antibodies. Macrophage inflammatory proteins had no effects on mu or huBFU-E colonies stimulated with erythropoietin (Epo). However, natural MIP-1 and rMIP-1 alpha, but not rMIP-1 beta or -2, suppressed muCFU-GM stimulated with pokeweed mitogen spleen-conditioned medium (PWMSCM), huCFU-GM stimulated with optimal rhuGM-CSF plus rhu interleukin-3 (IL-3), muBFU- E and multipotential progenitors (CFU-GEMM) stimulated with Epo plus PWMSCM, and huBFU-E and CFU-GEMM stimulated with Epo plus rhuIL-3 or rhuGM-CSF. The suppressive effects of natural MIP-1 and rMIP-1 alpha were also apparent on a population of BFU-E, CFU-GEMM, and CFU-GM present in cell-sorted fractions of human bone marrow (CD34 HLA-DR+) highly enriched for progenitors with cloning efficiencies of 42% to 75%. These results, along with our previous studies, suggest that MIP-1 alpha, -1 beta, and -2 may have direct myelopoietic enhancing activity for mature progenitors, while MIP-1 alpha may have direct suppressing activity for more immature progenitors.

scholarly journals Inhibin Suppresses and Activin Stimulates Osteoblastogenesis and Osteoclastogenesis in Murine Bone Marrow Cultures

Endocrinology ◽  
2002 ◽  
Vol 143 (1) ◽  
pp. 74-83 ◽  
Author(s):  
D. Gaddy-Kurten ◽  
J. K. Coker ◽  
E. Abe ◽  
R. L. Jilka ◽  
S. C. Manolagas
Keyword(s):  
Bone Marrow ◽  
Alkaline Phosphatase ◽  
Bone Morphogenetic Protein ◽  
Colony Formation ◽  
Tartrate Resistant Acid Phosphatase ◽  
Murine Bone Marrow ◽  
Morphogenetic Protein ◽  
Bone Marrow Cultures ◽  
Marrow Cultures

Abstract Using primary murine bone marrow cell cultures, we demonstrate that inhibin suppresses osteoblastogenesis and osteoclastogenesis. In contrast, activin supports osteoblast formation (by alkaline phosphatase-positive and mineralized colony formation); and activin also stimulates osteoclast formation (as measured by staining tartrate-resistant acid phosphatase-positive multinucleated cells). Inhibin, the activin antagonist follistatin, and the bone morphogenetic protein antagonist noggin can all suppress endogenous activin accumulation in bone marrow cultures. Associated with this decrease in activin is the loss of mineralized osteoblastic colony formation (colony forming unit-osteoblast; CFU-OB). However, exogenous activin administration, even in the presence of noggin, permits both alkaline phosphatase-positive and CFU-OB colony formation in vitro. In contrast, the stimulatory effects of locally produced activin on osteoblast and osteoclast development are not likely to be dominant over the suppressive effects of gonadally derived inhibin. The suppressive effect of inhibin is maintained in the presence of either activin or bone morphogenetic protein, suggesting the presence of a distinct inhibin-specific receptor. Taken together, the direct regulation of osteoblastogenesis and osteoclastogenesis by inhibin and activin in vitro suggest that changes in the inhibin/activin ratio detected by bone marrow cells, during the perimenopausal transition, contribute to altered cell differentiation and may be associated with the increased bone resorption observed at this time.

scholarly journals Recombinant human macrophage colony-stimulating factor (M-CSF) requires subliminal concentrations of granulocyte/macrophage (GM)-CSF for optimal stimulation of human macrophage colony formation in vitro.

1987 ◽  
Vol 166 (6) ◽  
pp. 1851-1860 ◽  
Author(s):  
D Caracciolo ◽  
N Shirsat ◽  
G G Wong ◽  
B Lange ◽  
S Clark ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Colony Formation ◽  
Human Macrophage ◽  
Colony Stimulating Factor ◽  
Colony Assay ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Human macrophage colony-stimulating factor (M-CSF or CSF-1), either in purified or in recombinant form, is able to generate macrophagic colonies in a murine bone marrow colony assay, but only stimulates small macrophagic colonies of 40-50 cells in a human bone marrow colony assay. We report here that recombinant human granulocytic/macrophage colony stimulating factor (rhGM-CSF) at concentrations in the range of picograms enhances the responsiveness of bone marrow progenitors to M-CSF activity, resulting in an increased number of macrophagic colonies of up to 300 cells. Polyclonal antiserum against M-CSF did not alter colony formation of bone marrow progenitors incubated with GM-CSF at optimal concentration (1-10 ng/ml) for these in vitro assays. Thus, GM-CSF at higher concentrations (nanogram range) can by itself, elicit macrophagic colonies, and at lower concentrations (picogram range) acts to enhance the responsiveness of these progenitors to M-CSF.

scholarly journals Effects of testosterone and erythropoietin on erythroid colony formation in human bone marrow cultures

Blood ◽  
1975 ◽  
Vol 45 (5) ◽  
pp. 665-670 ◽  
Author(s):  
Y Moriyama ◽  
JW Fisher
Keyword(s):  
Bone Marrow ◽  
Colony Formation ◽  
Methyl Cellulose ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Erythroid Cell ◽  
Bone Marrow Cultures ◽  
Human Bone Marrow Cultures ◽  
Marrow Cultures

Abstract The effects of testosterone and erythropoietin (ESF) on erythroid colony formation in normal human bone marrow cultures were studied in vitro using a methyl cellulose gel system. Testosterone was found to produce a significant increase in erythroid colony formation at concentrations of 10–4–10-4M in vitro. In this system, the numbers of erythroid colonies formed per plate increased in direct proportion to the increase in the number of erythroid precursors inoculated as well as to the increase in the dose of ESF in vitro. In addition, a synergistic effect of a combination of testosterone and ESF on erythroid colony formation was seen when ESF was present at high concentrations. These data suggest that a greater number of erythropoietin-responsive cells are available for ESF to differentiate into the nucleated erythroid cell line in the presence of testosterone, indicating that the effect of a combination of testosterone and ESF is greater in enhancing erythropoiesis than the additive effects of either agent alone.

scholarly journals In vitro myelosuppressive effects of the parvovirus minute virus of mice (MVMi) on hematopoietic stem and committed progenitor cells

Blood ◽  
1991 ◽  
Vol 77 (5) ◽  
pp. 980-988 ◽  
Author(s):  
JC Segovia ◽  
A Real ◽  
JA Bueren ◽  
JM Almendral
Keyword(s):  
Bone Marrow ◽  
Protein A ◽  
Myeloid Cells ◽  
Minute Virus Of Mice ◽  
Hematopoietic Stem ◽  
Reliable Parameter ◽  
Bone Marrow Cultures ◽  
Infected Cells ◽  
Minute Virus

Abstract The interaction of two strains of the parvovirus minute virus of mice (MVM) with the mouse hematopoietic system has been studied. The immunosuppressive strain MVMi, but not the prototype virus MVMp, inhibited hematopoiesis in vitro, as judged by colony-forming assays of the erythroid burst-forming unit and granulocyte-monocyte colony- forming unit (CFU-GM) progenitors. Interestingly, primitive hematopoietic cells of the stem compartment (CFU-S12d), were equally susceptible to the MVMi cytotoxic infection, unravelling an unprecedented feature of virus-hematopoiesis interactions. The replication of both strains of MVM virus was evaluated in primary myeloid cells of long-term bone marrow cultures. A high viral DNA synthesis and maturation was observed in MVMi-infected myeloid cells, but it was undetectable in MVMp infections; moreover, the expression of the cytotoxic nonstructural NS-1 protein, a more reliable parameter of cell permissiveness to MVM infection, was only detected in MVMi- infected cells. Correspondingly, MVMi was propagated to high titers of infectious virus and it mediated an acute myelosuppression in these cultures. We conclude that MVMi has a wider tropism than was previously suspected and it is proposed that cytotoxic infection of hematopoietic stem cells, besides that of committed progenitors, may provide an additional basis to understand the pathogenesis of certain animal and human bone marrow failures of viral etiology.

scholarly journals Role of cytokines in sustaining long-term human megakaryocytopoiesis in vitro

Blood ◽  
1992 ◽  
Vol 79 (2) ◽  
pp. 332-337 ◽  
Author(s):  
RA Briddell ◽  
JE Brandt ◽  
TB Leemhuis ◽  
R Hoffman
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Interleukin 1 ◽  
Gm Csf ◽  
Liquid Suspension ◽  
Bone Marrow Cultures ◽  
Macrophage Colony

An in vitro liquid suspension culture system was used to determine the role of cytokines in sustaining long-term human megakaryocytopoiesis. Bone marrow cells expressing CD34 but not HLA-DR (CD34+DR-) were used as the inoculum of cells to initiate long-term bone marrow cultures (LTBMC). CD34+DR- cells (5 x 10(3)/mL) initially contained 0.0 +/- 0.0 assayable colony-forming unit-megakaryocytes (CFU-MK), 6.2 +/- 0.4 assayable burst-forming unit-megakaryocytes (BFU-MK), and 0.0 +/- 0.0 megakaryocytes (MK). LTBMCs were recharged every 48 hours with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin- 1 alpha (IL-1 alpha), IL-3, and/or IL-6, alone or in combination. LTBMCs were demidepopulated weekly or biweekly, the number of cells and MK enumerated, and then assayed for CFU-MK and BFU-MK. LTBMCs receiving no cytokine(s) contained no assayable CFU-MK or BFU-MK and no observable MK. LTBMCs receiving GM-CSF, IL-1 alpha, and/or IL-3 contained assayable CFU-MK and MK but no BFU-MK for 10 weeks of culture. The effects of GM-CSF and IL-3, IL-1 alpha and IL-3, but not GM-CSF and IL-1 alpha were additive with regards to their ability to augment the numbers of assayable CFU-MK during LTBMC. LTBMCs supplemented with IL-6 contained modest numbers of assayable CFU-MK for only 4 weeks; this effect was not additive to that of GM-CSF, IL-1 alpha, or IL-3. The addition of GM-CSF, IL-1 alpha, and IL-3 alone or in combination each led to the appearance of significant numbers of MKs during LTBMC. By contrast, IL-6 supplemented cultures contained relatively few MK. These studies suggest that CD34+DR- cells are capable of initiating long-term megakaryocytopoiesis in vitro and that a hierarchy of cytokines exists capable of sustaining this process.

scholarly journals In vitro myelosuppressive effects of the parvovirus minute virus of mice (MVMi) on hematopoietic stem and committed progenitor cells

Blood ◽  
1991 ◽  
Vol 77 (5) ◽  
pp. 980-988 ◽  
Author(s):  
JC Segovia ◽  
A Real ◽  
JA Bueren ◽  
JM Almendral
Keyword(s):  
Bone Marrow ◽  
Protein A ◽  
Myeloid Cells ◽  
Minute Virus Of Mice ◽  
Hematopoietic Stem ◽  
Reliable Parameter ◽  
Bone Marrow Cultures ◽  
Infected Cells ◽  
Minute Virus

The interaction of two strains of the parvovirus minute virus of mice (MVM) with the mouse hematopoietic system has been studied. The immunosuppressive strain MVMi, but not the prototype virus MVMp, inhibited hematopoiesis in vitro, as judged by colony-forming assays of the erythroid burst-forming unit and granulocyte-monocyte colony- forming unit (CFU-GM) progenitors. Interestingly, primitive hematopoietic cells of the stem compartment (CFU-S12d), were equally susceptible to the MVMi cytotoxic infection, unravelling an unprecedented feature of virus-hematopoiesis interactions. The replication of both strains of MVM virus was evaluated in primary myeloid cells of long-term bone marrow cultures. A high viral DNA synthesis and maturation was observed in MVMi-infected myeloid cells, but it was undetectable in MVMp infections; moreover, the expression of the cytotoxic nonstructural NS-1 protein, a more reliable parameter of cell permissiveness to MVM infection, was only detected in MVMi- infected cells. Correspondingly, MVMi was propagated to high titers of infectious virus and it mediated an acute myelosuppression in these cultures. We conclude that MVMi has a wider tropism than was previously suspected and it is proposed that cytotoxic infection of hematopoietic stem cells, besides that of committed progenitors, may provide an additional basis to understand the pathogenesis of certain animal and human bone marrow failures of viral etiology.

scholarly journals Separation of functionally distinct human granulocyte-macrophage colony- stimulating factors

Blood ◽  
1979 ◽  
Vol 54 (3) ◽  
pp. 614-627
Author(s):  
NA Nicola ◽  
D Metcalf ◽  
GR Johnson ◽  
AW Burgess
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Colony Formation ◽  
Bone Marrow Cells ◽  
Molecular Size ◽  
The Other ◽  
Adherent Cell ◽  
Colony Stimulating Factors ◽  
Gm Csf

Human placental conditioned medium (HPCM) contans colony-stimulating factors (CSFs) required for the growth in vitro of neutrophilic granulocyte-macrophage (GM) and eosinophilic (EO) progenitor cells from human bone marrow. Fractionation of CSFs in HPCM was achieved by manipulation of the elution conditions on a column of phenyl-Sepharose. After equilibration of the phenyl-Sepharose column at high ionic strength (1 M ammonium sulfate), all of the CSF bound; one species of GM-CSF (alpha) and all of the elutable EO-CSF were eluted from the column simply by reducing the salt concentration, whereas the second species of GM-CSF (beta) was free of EO-CSF and was eluted only by increasing the concentration of tehylene glycol in the elution buffer. The two GM-CSFs were functionally distinct. GM-CSF alpha preferentially stimulated colony formation by day 14 of culture, and there was a decreased proportion of neutrophil colonies and increased proportion of macrophage colonies as the strength of the stimulus was decreased; GM- CSF beta, on the other hand, preferentially stimulated colony formation by day 7 of culture, and the proportion of neutrophil colonies was high (average 80%) and independent of the concentration of GM-CSF beta. GM- CSF alpha and GM-CSF beta were indistinguishable on the basis of apparent molecular size on tel filtration columns (molecular weight 30,000), charge properties on isoelectric focusing beds (isoelectric point, 4.9), and were not related to each other as a sialoglycoprotein is related to its asialo form. Adherent cell removal of the target bone marrow cells (to remove colony-stimulating cells) suggested that both GM-CSFs acted directly rather than by stimulating the production of GM- CSF. Mixing and titration experiments indicated that the differences in functional specificities of the two GM-CSFs (and the lack of EO-CSF associated with GM-CSF beta) were not due to the presence of specific inhibitory molecules or lower absolute levels of CSF in one fraction relative to the other. These two species of GM-CSF should be useful in separately enumerating subpopulations of different GM-progenitor cells inhuman hemopoietic disorders.

scholarly journals Stromal growth factor production in irradiated lectin exposed long-term murine bone marrow cultures

Blood ◽  
1987 ◽  
Vol 69 (4) ◽  
pp. 1120-1127
Author(s):  
TA Alberico ◽  
JN Ihle ◽  
CM Liang ◽  
HE McGrath ◽  
PJ Quesenberry
Keyword(s):  
Bone Marrow ◽  
Growth Factor ◽  
Stromal Cells ◽  
Colony Formation ◽  
Murine Bone Marrow ◽  
Gm Csf ◽  
Bone Marrow Cultures ◽  
Marrow Cultures ◽  
Marrow Cells

Hematopoietic regulatory factors produced by adherent (stromal) cells in long-term murine bone marrow cultures have been investigated. Using an in situ double layer agar overlay system, we demonstrated that exposure of the stromal cells to 1,100-rad irradiation increased their activities in stimulating colony formation of FDC-P1, an interleukin 3 (IL 3)-responsive cell line. The colony-stimulating activities (CSAs) of the irradiated stroma also stimulated normal marrow cells to form granulocyte-macrophage, megakaryocyte, and mixed lineage colonies. Addition of the lectin pokeweed mitogen to the irradiated stroma increased the level of CSAs. The FDC-P1 CSA of the irradiated stroma was inhibited by antibodies directed against murine granulocyte- macrophage colony stimulating factor (GM-CSF) but not by those against murine IL 3. Stromal-derived CSA for marrow cells was also partially blocked by anti-GM-CSF antibodies, probably reflecting the presence of other CSAs such as CSF-1. This latter growth factor has been found to be present in conditioned media from Dexter stroma, but levels are not increased after irradiation or lectin exposure. Partially purified GM- CSF, like IL 3, stimulated FDC-P1 proliferation and granulocyte, macrophage, and megakaryocyte colony formation. These results indicate that the major terminal differentiating hormone elicited by irradiation or lectin exposure of murine marrow stromal cells is GM-CSF. This growth factor, along with CSF-1, can account for the differentiated progeny produced in this system: macrophages, granulocytes, and megakaryocytes.

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