Extracellular Nucleoside Diphosphate Kinase NDPK/Nm23 Protein Isoforms A and B Support Erythropoiesis and Have a Burst-Promoting Activity.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1625-1625
Author(s):  
Zwi N. Berneman ◽  
Roel Willems ◽  
Griet Nijs ◽  
Marc Lenjou ◽  
Dirk R. Van Bockstaele
Keyword(s):  
Physiological Role ◽  
Nucleoside Diphosphate Kinase ◽  
Fold Increase ◽  
Protein Isoforms ◽  
Bone Marrow Mononuclear Cell ◽  
Gm Csf ◽  
Low Concentrations ◽  
High Concentrations ◽  
Macrophage Colony

Abstract We previously demonstrated the in vitro hematopoietic effects of different nucleoside diphosphate kinase NDPK/Nm23 proteins, i.e. increase of burst-forming units erythroid (BFU-E) and decrease of colony-forming units macrophage (CFU-M) (Willems R et al. Experimental Hematology2002;30: 640–648). The effect was especially pronounced in the marrows that had a particular low BFU-E count. Since these experiments were performed under optimal erythroid growth factor stimulation including adequately high concentrations of erythropoietin (Epo) and stem cell factor (SCF 100 ng/mL), subtle effects of NDPK/Nm23 proteins on erythropoiesis could have been missed. In the present series of experiments on 7 normal bone marrow mononuclear cell suspensions, we deliberately created suboptimal in vitro conditions for early (BFU-E) erythropoiesis: i.e. serum free conditions with a limited number of cytokines (only Epo and SCF) and a low concentration of burst-promoting activity (SCF at 5 ng/ml). The results show that addition of NDPK A/Nm23-H1 and NDPK B/Nm23- H2 (but not of NDPK C) at high (40 μg/mL), but not low, extracellular concentration is sustaining and improving in vitro erythropoiesis in a statistically significant manner (P<0.05). Red cell colonies increased by a factor of 1.9 and 1.4 following addition of NDPK A/Nm23-H1 and NDPK B/Nm23- H2 respectively. Especially with NDPK A/Nm23-H1, this approaches levels attained under optimal conditions for erythropoiesis, i.e. at SCF concentration of 100 ng/mL, where a 2.1-fold increase was seen, as compared to cultures with low concentrations of SCF (5 ng/mL) without addition of extracellular NDPK/Nm23. In cultures without Epo no erythroid colonies were seen, even following addition of NDPK A/Nm23-H1 or NDPK B/Nm23-H2. This study strongly suggests that NDPK/Nm23 has a burst-promoting activity. The fairly high concentrations of NDPK/Nm23 constitutively present in plasma supports the physiological role of this protein in stimulating early erythropoiesis. Of the (known) cytokines or proteins with burst-promoting activity (eg. SCF, interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), NDPK/Nm23 (this study)), only SCF and NDPK/Nm23 are produced in a constitutive fashion, strongly supporting their role in the steady-state support of early erythropoiesis.

scholarly journals Combinations of recombinant colony-stimulating factors are required for optimal hematopoietic differentiation in serum-deprived culture

Blood ◽  
1989 ◽  
Vol 73 (3) ◽  
pp. 688-693 ◽  
Author(s):  
CA Sieff ◽  
SC Ekern ◽  
DG Nathan ◽  
JW Anderson
Keyword(s):  
Fetal Calf Serum ◽  
Calf Serum ◽  
Gm Csf ◽  
Colony Forming Units ◽  
Macrophage Colony Stimulating Factor ◽  
High Concentrations ◽  
Macrophage Colony ◽  
Stimulating Factor ◽  
Cell Conditioned Medium

Abstract Previous in vitro investigations on enriched human hematopoietic progenitors have led to the conclusion that the purified recombinant multipoietins, interleukin 3 (IL-3) and granulocyte-macrophage colony- stimulating factor (GM-CSF) can alone induce the formation of colonies from a variety of multipotent and lineage committed progenitors. Since fetal calf serum was included in these cultures and itself might contain growth factors or other cofactors, we re-examined the actions of the CSFs in serum-deprived conditions. Results show that both the multipoietins are inadequate stimuli of colony formation. At maximal concentrations IL-3 alone induces only 25% of the granulocyte and macrophage colony-forming units (CFU-G and CFU-M) produced by a T-cell conditioned medium that contains a mixture of CSFs. When IL-3 was added at the initiation of the cultures and erythropoietin (ep), G-CSF, or M- CSF added on day 3, almost full recovery of erythroid, granulocytic, and monocytic colonies, respectively, was obtained. Similar results were obtained with GM-CSF except that fewer erythroid colonies were recovered at high concentrations, and almost maximal CFU-M proliferation could be induced. These results show that in serum- deprived conditions, the multipoietins must be combined with lineage specific CSFs for full progenitor expression.

scholarly journals Granulocyte-macrophage colony-stimulating factor induces the expression of the CD11b surface adhesion molecule on human granulocytes in vivo

Blood ◽  
1988 ◽  
Vol 72 (2) ◽  
pp. 691-697
Author(s):  
MA Socinski ◽  
SA Cannistra ◽  
R Sullivan ◽  
A Elias ◽  
K Antman ◽  
...  
Keyword(s):  
Fold Increase ◽  
Low Density ◽  
Colony Stimulating Factor ◽  
Cd11b Expression ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

The CD11b (Mol) molecule is a member of a family of surface glycoproteins that are essential for adhesion-dependent granulocyte functions. Brief exposure of granulocytes to human granulocyte- macrophage colony-stimulating factor (GM-CSF) in vitro increases the surface expression of CD11b and increases granulocyte adhesiveness. To assess the possible in vivo significance of these observations we studied the effect of GM-CSF on CD11b, CD11a (LFA-1), and CD11c (gp 150, 95) expression on granulocytes from nine adult patients with sarcoma who were receiving GM-CSF as part of a phase I trial. GM-CSF was administered as a continuous infusion at a dose of 32 or 64 micrograms/kg/d. Granulocyte CD11b, CD11a, and CD11c expression was determined by indirect immunofluorescence staining of whole blood, thereby minimizing in vitro manipulation. A transient leukopenia developed within 15 minutes of initiation of GM-CSF treatment that was associated with a marked increase in the surface antigen density of CD11b. A mean 1.7-fold increase (P = .001) in the percentage of CD11b- positive granulocytes and a mean 2.1-fold increase (P = .002) in CD11b surface antigen density was noted after 12 hours of treatment. No change in CD11a or CD11c expression was observed over the first 12 hours. The level of CD11b expression was followed in six patients for up to 5 days of treatment with GM-CSF. Compared with the 12-hour value, three of six patients showed a subsequent decrease in CD11b expression, two remained constant, and one showed a continued increase in CD11b surface density. Fluorescence-activated cell sorting of granulocytes into high- and low-density CD11b-positive groups revealed a preponderance of immature myeloid forms in the low-density CD11b fraction, which suggests that the late decrease in CD11b expression in some patients may be related to a greater proportion of circulating immature myeloid forms in the peripheral blood. This study suggests that GM-CSF administered as a continuous infusion rapidly upregulates the expression of granulocyte CD11b in vivo. The influence of this phenomenon on in vivo granulocyte aggregation may be clinically relevant with regard to the toxicity of GM-CSF and deserves further investigation.

scholarly journals Granulocyte-macrophage colony-stimulating factor induces the expression of the CD11b surface adhesion molecule on human granulocytes in vivo

Blood ◽  
1988 ◽  
Vol 72 (2) ◽  
pp. 691-697 ◽  
Author(s):  
MA Socinski ◽  
SA Cannistra ◽  
R Sullivan ◽  
A Elias ◽  
K Antman ◽  
...  
Keyword(s):  
Fold Increase ◽  
Low Density ◽  
Colony Stimulating Factor ◽  
Cd11b Expression ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract The CD11b (Mol) molecule is a member of a family of surface glycoproteins that are essential for adhesion-dependent granulocyte functions. Brief exposure of granulocytes to human granulocyte- macrophage colony-stimulating factor (GM-CSF) in vitro increases the surface expression of CD11b and increases granulocyte adhesiveness. To assess the possible in vivo significance of these observations we studied the effect of GM-CSF on CD11b, CD11a (LFA-1), and CD11c (gp 150, 95) expression on granulocytes from nine adult patients with sarcoma who were receiving GM-CSF as part of a phase I trial. GM-CSF was administered as a continuous infusion at a dose of 32 or 64 micrograms/kg/d. Granulocyte CD11b, CD11a, and CD11c expression was determined by indirect immunofluorescence staining of whole blood, thereby minimizing in vitro manipulation. A transient leukopenia developed within 15 minutes of initiation of GM-CSF treatment that was associated with a marked increase in the surface antigen density of CD11b. A mean 1.7-fold increase (P = .001) in the percentage of CD11b- positive granulocytes and a mean 2.1-fold increase (P = .002) in CD11b surface antigen density was noted after 12 hours of treatment. No change in CD11a or CD11c expression was observed over the first 12 hours. The level of CD11b expression was followed in six patients for up to 5 days of treatment with GM-CSF. Compared with the 12-hour value, three of six patients showed a subsequent decrease in CD11b expression, two remained constant, and one showed a continued increase in CD11b surface density. Fluorescence-activated cell sorting of granulocytes into high- and low-density CD11b-positive groups revealed a preponderance of immature myeloid forms in the low-density CD11b fraction, which suggests that the late decrease in CD11b expression in some patients may be related to a greater proportion of circulating immature myeloid forms in the peripheral blood. This study suggests that GM-CSF administered as a continuous infusion rapidly upregulates the expression of granulocyte CD11b in vivo. The influence of this phenomenon on in vivo granulocyte aggregation may be clinically relevant with regard to the toxicity of GM-CSF and deserves further investigation.

scholarly journals Influence of Mycobacterium bovisBacillus Calmette Guérin on In Vitro Induction of CD1 Molecules in Human Adherent Mononuclear Cells

2001 ◽  
Vol 69 (12) ◽  
pp. 7461-7470 ◽  
Author(s):  
Anna Giuliani ◽  
Salvatore P. Prete ◽  
Grazia Graziani ◽  
Angelo Aquino ◽  
Alessandra Balduzzi ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Mononuclear Cells ◽  
Gm Csf ◽  
Protein Levels ◽  
High Concentrations ◽  
Macrophage Colony ◽  
Nonpeptide Antigens ◽  
Mycobacterial Antigens ◽  
Antigen Presenting

ABSTRACT Nonpeptide antigens (including glycolipids of microbial origin) can be presented to T cells by CD1 molecules expressed on monocyte-derived dendritic cells. These HLA unrestricted responses appear to play a role in host immunity against Mycobacterium tuberculosis and other pathogenic bacteria. It is known that vaccination withMycobacterium bovis bacillus Calmette-Guérin (BCG) has limited efficacy in many clinical settings, although the reasons for its inadequacy remain unclear. Here we have investigated the influence of BCG on the induction of CD1b on human monocytes by granulocyte-macrophage colony-stimulating factor (GM-CSF), which is believed to be the principal inducer of this antigen-presenting molecule. Although BCG alone led to a slight induction of CD1b expression, this agent reduced markedly the ability of GM-CSF to induce high levels of CD1b that were typically observed in uninfected cells. Inhibition of CD1b expression in BCG-infected monocytes was apparent at both the mRNA transcript and CD1b protein levels. Down-regulation of CD1b expression by BCG was mediated, at least in part, by one or more soluble factors and could not be reversed with high concentrations of GM-CSF or a variety of other cytokines. The present results suggest that BCG could diminish the efficiency of CD1-restricted T-cell responses against nonpeptide mycobacterial antigens by reducing CD1 expression on antigen-presenting cells. These findings have potential implications for understanding the nature of the immune response elicited by BCG in humans and suggest potential strategies that could be important for the development of better vaccines for the prevention of tuberculosis.

Granulocyte-macrophage colony stimulating factor (GM-CSF): one of a family of epithelial cell-derived cytokines in the preimplantation uterus

1992 ◽  
Vol 4 (4) ◽  
pp. 435 ◽  
Author(s):  
SA Robertson ◽  
RF Seamark
Keyword(s):  
Nk Cell ◽  
Fold Increase ◽  
Pregnant Mouse ◽  
Leukaemia Inhibitory Factor ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Granulocyte-macrophage colony stimulating factor (GM-CSF) is one of a number of lympho-haemapoietic cytokines, including CSF-1, interleukin-6 (IL-6) and leukaemia inhibitory factor (LIF) now known to be synthesized by epithelial cells in the murine uterus. GM-CSF synthesis is regulated primarily by the ovarian steroid hormone oestrogen, but is also subject to modulation by factors including a seminal component of seminal vesicle origin which stimulates a 20-fold increase in luminal fluid content at mating, and bacterial lipopolysaccharide (LPS) and the T-lymphocyte and natural killer (NK) cell product interferon-gamma (IFN gamma). In the non-pregnant mouse GM-CSF synthesis peaks at oestrus. Synthesis is maintained at comparable or moderately higher levels during the preimplantation period of pregnancy and in the non-decidualized endometrium during mid gestation. An embryotrophic activity is suggested by studies in vitro that indicate that GM-CSF stimulates attachment and outgrowth of blastocysts. It is postulated that GM-CSF is of major importance to the physiology of pregnancy through its role as a component of a local cytokine circuit acting to recruit and regulate function of endometrial leukocytes, and by its action as interlocutor and important effector arm in embryo-maternal interactions during gestation.

scholarly journals Combinations of recombinant colony-stimulating factors are required for optimal hematopoietic differentiation in serum-deprived culture

Blood ◽  
1989 ◽  
Vol 73 (3) ◽  
pp. 688-693 ◽  
Author(s):  
CA Sieff ◽  
SC Ekern ◽  
DG Nathan ◽  
JW Anderson
Keyword(s):  
Fetal Calf Serum ◽  
Calf Serum ◽  
Gm Csf ◽  
Colony Forming Units ◽  
Macrophage Colony Stimulating Factor ◽  
High Concentrations ◽  
Macrophage Colony ◽  
Stimulating Factor ◽  
Cell Conditioned Medium

Previous in vitro investigations on enriched human hematopoietic progenitors have led to the conclusion that the purified recombinant multipoietins, interleukin 3 (IL-3) and granulocyte-macrophage colony- stimulating factor (GM-CSF) can alone induce the formation of colonies from a variety of multipotent and lineage committed progenitors. Since fetal calf serum was included in these cultures and itself might contain growth factors or other cofactors, we re-examined the actions of the CSFs in serum-deprived conditions. Results show that both the multipoietins are inadequate stimuli of colony formation. At maximal concentrations IL-3 alone induces only 25% of the granulocyte and macrophage colony-forming units (CFU-G and CFU-M) produced by a T-cell conditioned medium that contains a mixture of CSFs. When IL-3 was added at the initiation of the cultures and erythropoietin (ep), G-CSF, or M- CSF added on day 3, almost full recovery of erythroid, granulocytic, and monocytic colonies, respectively, was obtained. Similar results were obtained with GM-CSF except that fewer erythroid colonies were recovered at high concentrations, and almost maximal CFU-M proliferation could be induced. These results show that in serum- deprived conditions, the multipoietins must be combined with lineage specific CSFs for full progenitor expression.

In Vitro Effects of Ethanol on Rabbit Platelet Aggregation, Secretion of Granule Contents, and Cyclic AMP Levels in the Presence of Prostacyclin

1989 ◽  
Vol 61 (02) ◽  
pp. 254-258 ◽  
Author(s):  
Margaret L Rand ◽  
Peter L Gross ◽  
Donna M Jakowec ◽  
Marian A Packham ◽  
J Fraser Mustard
Keyword(s):  
Cyclic Amp ◽  
Inhibitory Effect ◽  
Rabbit Platelet ◽  
Inhibitory Effects ◽  
Low Concentrations ◽  
Rabbit Platelets ◽  
High Concentrations ◽  
In Vitro Effects ◽  
Aggregation Of Platelets

SummaryEthanol, at physiologically tolerable concentrations, inhibits platelet responses to low concentrations of collagen or thrombin, but does not inhibit responses of washed rabbit platelets stimulated with high concentrations of ADP, collagen, or thrombin. However, when platelet responses to high concentrations of collagen or thrombin had been partially inhibited by prostacyclin (PGI2), ethanol had additional inhibitory effects on aggregation and secretion. These effects were also observed with aspirin- treated platelets stimulated with thrombin. Ethanol had no further inhibitory effect on aggregation of platelets stimulated with ADP, or the combination of ADP and epinephrine. Thus, the inhibitory effects of ethanol on platelet responses in the presence of PGI2 were very similar to its inhibitory effects in the absence of PGI2, when platelets were stimulated with lower concentrations of collagen or thrombin. Ethanol did not appear to exert its inhibitory effects by increasing cyclic AMP above basal levels and the additional inhibitory effects of ethanol in the presence of PGI2 did not appear to be brought about by further increases in platelet cyclic AMP levels.

Aggregation of Cat Platelets in Vitro

1970 ◽  
Vol 23 (03) ◽  
pp. 601-620 ◽  
Author(s):  
Th. B Tschopp
Keyword(s):  
Adenosine Monophosphate ◽  
Blood Collection ◽  
Base Line ◽  
Reversible Aggregation ◽  
Low Concentrations ◽  
Irreversible Aggregation ◽  
High Concentrations ◽  
Two Phases ◽  
Improved Technique

SummaryAggregation of cat platelets in the citrated plasma is examined by means of Born’s absorptiometer. A marked tendency of the platelets of this species to spontaneous aggregation necessitated first of all the development of an improved technique of blood collection.A hypothesis according to which 5-HT is released from the platelets, explains the absence of oscillations on the base line of the absorptiometer, the absence of platelet swelling, when ADP is added, and the effect of stirring on the aggregation curves in cat PRP. The average volume of cat platelets amounts to 10.46 μ3 when directly fixed in the blood, when fixed from PRP to 12.17 μ3, when fixed from stirred PRP to 13.51 μ3.In low concentrations (0.3-2 μM) ADP produce reversible aggregation; in narrowly restricted, individually dissimilar mean concentrations irreversible aggregation in two phases and in high concentrations, irreversible aggregation in one phase. Like ADP serotonin produces 2 phase irreversible aggregation in concentrations of 3-10 μM, but unlike ADP, the aggregation velocity decreases again with high 5-HT concentrations (>100 μM). Adrenaline does not produce aggregation and it is likely that adenosine and adenosine monophosphate inhibit the aggregation by serotonin but not by ADP. Species differences in the aggregation of human, rabbit and cat platelets are discussed.

scholarly journals THE RATE OF BACTERICIDAL ACTION OF PENICILLIN IN VITRO AS A FUNCTION OF ITS CONCENTRATION, AND ITS PARADOXICALLY REDUCED ACTIVITY AT HIGH CONCENTRATIONS AGAINST CERTAIN ORGANISMS

1948 ◽  
Vol 88 (1) ◽  
pp. 99-131 ◽  
Author(s):  
Harry Eagle ◽  
A. D. Musselman
Keyword(s):  
Bacterial Species ◽  
Fold Increase ◽  
Bactericidal Effect ◽  
Penicillin G ◽  
Bacterial Suspension ◽  
Maximal Effect ◽  
Maximal Rate ◽  
Effective Level ◽  
High Concentrations

1. The concentrations of penicillin G which (a) reduced the net rate of multiplication, (b) exerted a net bactericidal effect, and (c) killed the organisms at a maximal rate, have been defined for a total of 41 strains of α- and ß-hemolytic streptococci, Staphylococcus aureus and Staphylococcus albus, Diplococcus pneumoniae, and the Reiter treponoma. 2. The concentration which killed the organisms at a maximal rate was 2 to 20 times the minimal effective level ("sensitivity" as ordinarily defined). With some organisms, even a 32,000-fold increase beyond this maximally effective level did not further increase the rate of its bactericidal effect. However, with approximately half the strains here studied (all 4 strains of group B ß-hemolytic streptococci, 4 of 5 group C strains, 5 of 7 strains of Streptococcus fecalis, 2 of 4 other α-hemolytic streptococci, and 4 of 9 strains of staphylococci), when the concentration of penicillin was increased beyond that optimal level, the rate at which the organisms died was paradoxically reduced rather than increased, so that the maximal effect was obtained only within a relatively narrow optimal zone. 3. There were marked differences between bacterial species, and occasionally between different strains of the same species, not only with respect to the effective concentrations of penicillin, but also with respect to the maximal rate at which they could be killed by the drug in any concentration. Although there was a rough correlation between these two factors, there were many exceptions; individual strains affected only by high concentrations of penicillin might nevertheless be killed rapidly, while strains sensitive to minute concentrations might be killed only slowly. 4. Within the same bacterial suspension, individual organisms varied only to a minor degree with respect to the effective concentrations of penicillin. They varied strikingly, however, in their resistance to penicillin as measured by the times required to kill varying proportions of the cells.

In vitro p53 and/or Rb antisense oligonucleotide treatment in association with growth factors induces the proliferation of peripheral hematopoietic progenitors

1995 ◽  
Vol 108 (3) ◽  
pp. 1287-1293
Author(s):  
T. Mahdi ◽  
A. Brizard ◽  
C. Millet ◽  
P. Dore ◽  
J. Tanzer ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Cell Types ◽  
Suppressor Gene ◽  
Antisense Oligodeoxynucleotide ◽  
Signal Pathways ◽  
Semisolid Medium ◽  
Gm Csf ◽  
Colony Forming Units ◽  
Macrophage Colony

In this work we intended to determine whether p53 and/or retinoblastoma (Rb) tumor suppressor genes are involved at specific stages in the process of in vitro human peripheral stem cell hematopoiesis. Mononuclear peripheral blood cells were depleted of adherent cells and T lymphocytes (A-T-PMCs). Cells were then cultured in semisolid medium, under conditions that favor the growth of specific progenitor cell types. A-T-PMCs were exposed to p53 and/or Rb sense, scrambled DNA and antisense oligodeoxynucleotides. p53 and/or Rb antisenses (but not their senses or scrambled DNA) treatment of A-T-PMCs resulted in a significantly increase in the number of granulocyte/macrophage colony-forming units (CFU-GM) in the presence of interleukin-3 (IL-3) and/or granulocyte/macrophage colony-stimulating factor (GM-CSF). After antisense treatment, blast forming units/erythroblasts (BFU-E) derived from A-T-PMCs cultured in the presence of IL-3 + erythropoietin (Epo) were also increased whereas colony forming units/erythroblasts (CFU-E) were not markedly affected in the presence of Epo only. Megakaryocytic colony (CFU-Meg) formation from A-T-PMCs in the presence of interleukin-6 (IL-6) + IL-3 + Epo was also increased after antisense oligodeoxynucleotide treatment. These results are consistent with the hypothesis that p53 and Rb tumor suppressor gene products are involved in the control of distinct signal pathways in different peripheral progenitor cells.

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