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V Praloran

Publications and source records attributed to V Praloran.

At least 37 records · Page 2Linked to original sources
PubMed1999

A simple, one-step clonal assay allows the sequential detection of committed (CFU-GM-like) progenitors and several subsets of primitive (HPP-CFC) murine progenitors.

Murine bone marrow (BM) cells were cultured in semisolid medium containing interleukin 3 (IL-3) and high doses of G-CSF. Colonies were counted twice, at day 7 and day 14, and the number of granulocyte/macrophage colony-forming units (CFU-GM) accurately estimated by the subtraction of day-14 from day-7 colonies, based on the principle that colonies detectable at day 7 and persisting beyond day 14 are generated by significantly more immature progenitors. The frequency of colonies relative to their size was determined and used to define subsets of high proliferative potential colony-forming cells (HPP-CFC). Two main groups of HPP-CFC were considered: those generating colonies of 0.6-1.8 mm of diameter or larger than 1.8 mm. The characterization of these groups showed that they correspond to different functional subsets of HPP-CFC. The replating ability of colonies was estimated. The percentage of clonogenic progenitors in the S phase of cell cycle was measured by cytosine arabinoside suicide assay. The sensitivity of colonies to 5-fluorouracil (5-FU) in vitro was determined and their survival after an in vivo treatment with 5-FU compared with that of colony-forming units in spleen (CFU-S). This technique allowed identification of: A) CFU-GM; B) relatively mature HPP-CFC, probably corresponding to CFU-S day12; C) more primitive HPP-CFC, relatively resistant to 5-FU in vivo and closely corresponding to CFU-S day 14, and D) very primitive HPP-CFC, resistant to 5-FU in vitro. This simple, rapid, and versatile method allows the detection of a broad range of hematopoietic progenitors in murine BM, from committed progenitors to largely quiescent, primitive stem cells, as well as the evaluation of the progenitors' self-renewal and proliferative potential.

Animals↗
PubMed1999

Lipid mediators modulate the synthesis of interleukin 8 by human bone marrow stromal cells.

Bone marrow stromal cells regulate marrow haematopoiesis by secreting interleukins (IL) such as IL-8. Lipid mediators modulate IL-8 synthesis in numerous cell types. We have investigated the effects of 5 lipid mediators (PAF, PGE(2), LTB(4), 12-HETE and 15-HETE) on the spontaneous and cytokine-induced IL-8 synthesis by human bone marrow stromal cells. By using reverse-transcriptase polymerase chain reaction (RT-PCR) we demonstrate that these cells constitutively express IL-8 transcripts. By using a specific ELISA, we found that the production of IL-8 by marrow stromal cells is enhanced after stimulation with 12-HETE (1 microM) both in serum-free and serum-containing culture medium. LTB(4)(1 microM) enhances IL-8 production only in serum-supplemented medium. PAF, PGE(2)and 15-HETE (1 microM to 0.1 nM) have no effect on the spontaneous and serum-induced production of IL-8 by human bone marrow stromal cells. PGE(2)(1 microM or 10 nM) reduces marrow stromal cell IL-8 synthesis in response to IL-1alpha or TNF-alpha. In contrast, PAF, 12-HETE, 15-HETE and LTB(4)have no effect. In conclusion, various lipid mediators modulate the spontaneous, serum- or cytokine-induced IL-8 synthesis by bone marrow stromal cells, highlighting, for the first time, their potential role in the regulation of IL-8 production within the human bone marrow.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1999

Incorporation and effect of arachidonic acid on the growth of human myeloma cell lines.

The objectives of this work are to investigate the incorporation of arachidonic acid (AA) in the human myeloma cell lines OPM2, U266 and IM9, and to assess the effect of AA and lipoxygenase products of AA on their growth. The kinetics of acylation of [3H]AA indicates that myeloma cells incorporate AA into their membrane phospholipids and triglycerides. PLA2-treatment and base hydrolysis experiments confirm that [3H]AA is incorporated unmodified in U266, IM9 and OPM2 phospholipids, and is linked by an ester bond. Prelabeling-chase experiments indicate no trafficking of labeled AA among the various phospholipid species. Addition of AA and lipoxygenase products of AA (leukotriene B4 and C4, lipoxin A4 and B4, 12- and 15-hydroxyeicosatetraenoic acid) have no effect on U266, IM9 and OPM2 proliferation assessed by [3H]thymidine incorporation into DNA. In conclusion, while human myeloma cells readily incorporate AA in their membrane phospholipids and triglycerides, AA and lipoxygenase products are not important modulators of their proliferation.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1999

Arachidonic acid and freshly isolated human bone marrow mononuclear cells.

Arachidonic acid (AA), a fatty acid found in the human bone marrow plasma, is the precursor of eicosanoids that modulate bone marrow haematopoiesis. To further our understanding of the role of AA in the bone marrow physiology, we have assessed its incorporation in human bone marrow mononuclear cells. Gas chromatography analysis indicates the presence of AA in their fatty acid composition. In bone marrow mononuclear cells, [3H]-AA is incorporated into triglycerides and is later delivered into phospholipids, a result not observed with blood mononuclear cells. Prelabelling-chase experiments indicate a trafficking of labelled AA from phosphatidylcholine to phosphatidylethanolamine. Stimulation of prelabelled bone marrow mononuclear cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) results in the release of a part of the incorporated labelled AA. Finally, exogenous AA (up to 1 microM) has no significant effect on cell growth. In conclusion, human bone marrow mononuclear cells participate to the control of marrow AA concentrations by incorporating AA into phospholipids and triglycerides. In turn, bone marrow mononuclear cells can release AA in response to the potent haematopoietic growth factor GM-CSF.

Arachidonic Acid↗
PubMed1999

Whole blood production of monocytic cytokines (IL-1beta, IL-6, TNF-alpha, sIL-6R, IL-1Ra) in haemodialysed patients.

BACKGROUND: The production of monocytic cytokines by isolated mononuclear cells after stimulation by phytohaemagglutinin (PHA) and lipopolysaccharide (LPS) is generally increased in haemodialysed (HD) patients. We performed whole blood (WB) cultures to evaluate cytokine production by blood cells inside their complex cellular and humoral network. METHODS: Diluted whole blood from HD patients (collected before dialysis) and controls was cultured alone with PHA (2.5 microg/ml) or LPS (1 and 3 microg/ml). Supernatants were collected after 24 and 48 h of culture, and concentrations of IL-1 beta, IL-6, TNF-alpha, sIL-6R and IL-1Ra were determined by ELISA. RESULTS: The low spontaneous production of IL-1beta, IL-6 and TNF-alpha in both patients and controls was not significantly modified by PHA. The lower dose of LPS (1 microg/ml) induced a significant but lower increase in production of IL-1beta, IL-6 and TNF-alpha in patients than in controls. In contrast, while it did not further increase their production in controls, the higher concentration of LPS (3 microg/ml) still increased their production in patients to the same level than in controls. The plasma concentrations of sIL-6R were higher in patients than in controls. In both groups, the sIL-6R concentration did not vary during the culture period whether the cells were stimulated or not with LPS or PHA. This suggests that the increased plasma levels of sIL-6R were not produced by blood cells. Despite a similar significant LPS and PHA induced production of IL-1Ra, the IL-1Ra/IL-1beta ratio was always higher in patients than in controls. CONCLUSION: Monocytes from HD patients in WB cultures are hyporesponsive to PHA and LPS for their IL-1beta, TNFalpha and IL-6 production in contrast to isolated monocytes that demonstrate signs of activation. If it reflects the in vivo situation it could partly explain the immune defect in uraemic and haemodialysed patients. Higher sIL-6R/IL-6 and IL-1Ra/IL-1beta ratios could also participate to the complex immune disturbances of HD patients by reducing the biological activity of two cytokines playing a major role in the immune and inflammatory network.

Blood Cells↗
PubMed1998

Arachidonic acid and human bone marrow stromal cells.

Human bone marrow stromal cells regulate the growth of marrow hematopoietic progenitors by secreting cytokines. Arachidonic acid (AA) is the fatty acid precursor of prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) that modulate the growth of human bone marrow progenitors. We have investigated the incorporation of AA in human bone marrow stromal cell cultures, their production of PGE2 and LTB4 and the effect of AA on their growth. Gas chromatography analysis reveals the presence of AA in the human bone marrow plasma and in bone marrow stromal cell cultures. In stromal cells, [3H]-AA is incorporated into triglycerides and is later delivered into phospholipids. Prelabeling-chase experiments indicate a preferential incorporation of AA into phosphatidylethanolamine and no trafficking of labeled AA between phospholipid species. Bone marrow stromal cells release PGE2 and LTB4 in response to phorbol myristic acetate (PMA) (1 microM) and tumor necrosis factor alpha (TNF-alpha) (10 ng/ml). Exogenous AA (up to 1 microM) has no significant effect on cell growth. In conclusion, human bone marrow stromal cells capt exogenous AA and, thus, may participate to the control of marrow AA concentrations. They may also regulate human marrow hematopoiesis by secreting AA metabolites such as PGE2 and LTB4.

6-Ketoprostaglandin F1 alpha↗
PubMed1998

M-CSF: haematopoietic growth factor or inflammatory cytokine?

Macrophage colony-stimulating factor (M-CSF), initially described as a growth factor of the mononuclear phagocytic lineage, also participates in immunological and inflammatory reactions, bone metabolism and pregnancy. All its biological activities are mediated by a tyrosine kinase receptor (M-CSF-R) that is encoded by the c-fms protooncogene. After a brief overview on the synthesis, structure, metabolism and signalling of M-CSF and its receptor, we present with more details the major in vitro and/or in vivo biological activities of this cytokine. A particular attention has been devoted to the results suggesting that the various M-CSF isoforms (i.e. soluble, cell-associated and matrix anchored forms) play different specific roles on target cells bearing M-CSF-R at their surface. Infectious, inflammatory and neoplastic diseases in which M-CSF is involved and could participate to their physiopathology are mentioned. Finally, the role that the various isoforms of M-CSF could play in the regulation of "physiological and pathological cytokine networks" during inflammatory and immune responses is discussed.

Animals↗
PubMed1998

IL-6 and IL-8 production by human bone marrow stromal cells.

By using a specific enzyme-linked immunosorbent assay, the authors demonstrated that human bone marrow stromal cells produce IL-6 and IL-8. Their synthesis is enhanced in a dose-dependent manner after stimulation with lipopolysaccharide (LPS) and phorbol myristate acetate (PMA). Interleukin 6 (IL-6) and IL-8 production in response to PMA were markedly diminished by the PKC inhibitor staurosporine. IL-6 (10 ng/ml) stimulated IL-8 production with 0% and 10% fetal calf serum (FCS) in the culture medium. In similar conditions, IL-8 (10 ng/ml) enhanced IL-6 production. IL-1 alpha, IL-1 beta, and IL-3, tumour necrosis factor alpha (TNF-alpha), Stem cell factor (SCF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (at 10 ng/ml) stimulated IL-6 and IL-8 production in 0% and 10% FCS. G-CSF stimulated and IL-4 inhibited IL-8 production in 10% FCS. IL-2, IL-4 and bFGF stimulated IL-6 production in 0% FCS. These results suggest that bone marrow stromal cells might represent a major source for the cytokine-regulated local production of IL-6 and IL-8 inside human bone marrow.

Bone Marrow Cells↗
PubMed1998

Effects of lipid mediators on the synthesis of leukaemia inhibitory factor and interleukin 6 by human bone marrow stromal cells.

Human bone marrow stromal cells regulate haematopoiesis by releasing cytokines such as leukaemia inhibitory factor (LIF) and interleukin 6 (IL-6). We have investigated the effects of 5 lipid mediators (i.e. 12-HETE, 15-HETE, platelet-activating factor (PAF), LTB4 and prostaglandin E2 (PGE2)) on their LIF and IL-6 synthesis. 12-HETE, 15-HETE and LTB4 (both at 1 microM) stimulate the LIF production by human bone marrow stromal cells grown in 10% fetal calf serum (FCS). In contrast, PAF and PGE2 have no effect. 12-HETE (1 microM) enhances LIF synthesis in serum free medium 7.7-fold and stimulates IL-1 induced LIF production. 12-HETE, 15-HETE, PAF and LTB4 have no effect on the spontaneous, serum- and cytokine-induced IL-6 synthesis by bone marrow stromal cells. In contrast PGE2 significantly stimulates serum-induced IL-6 synthesis. This study reports for the first time that lipid mediators may act on human haematopoiesis by modulating LIF and IL-6 synthesis by bone marrow stromal cells. Particularly, 12-HETE enhances LIF but not IL-6 synthesis. The different regulation of IL-6 and LIF synthesis in response to lipid mediators highlights the complexity of the cytokine regulation into the human bone marrow.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1998

In vivo modifications of AcSDKP metabolism and haematopoiesis in mice treated with 5-fluorouracil and Goralatide.

BACKGROUND: The tetrapeptide acetyl-Ser-Asp-Lys-Pro (AcSDKP), a physiological inhibitor of the proliferation of haematopoietic stem cells, is degraded by the angiotensin-I-converting enzyme (ACE). Whereas synthetic AcSDKP (Goralatide) protects normal mice from the haematological toxicity of chemotherapy, it has a lower beneficial effect in humans. This discrepancy could be dependent on Goralatide administration schedules, as well as on the endogenous concentrations of AcSDKP and ACE, which vary during chemotherapy. METHODS: We investigated the effect of one myelotoxic dose of 5-fluorouracil (5-FU, 200 mg kg-1) administered without or with Goralatide on blood, bone marrow (BM) and spleen AcSDKP concentrations, ACE activity, nucleated cell counts and survival of the primitive haematopoietic progenitors high proliferative potential colony-forming cells (HPP-CFCs). RESULTS: The 5-FU treatment dramatically decreased the BM concentrations of AcSDKP by 73% and increased the ACE activity in plasma by 50% during the period of active BM regeneration. Repeated injections of Goralatide from 24 h before to 36 h after the i.p. injection of 5-FU spared BM HPP-CFCs. As an injection of 10 mg of Goralatide induced a short peak of plasma AcSDKP without modifying its BM concentrations, we suggest that its protective effect on HPP-CFCs could be mediated by its interference with other plasma molecules targeting to the BM. CONCLUSION: By improving our knowledge of the biology of AcSDKP in vivo during chemotherapy, our results could help to better define the therapeutic use of Goralatide.

Animals↗
PubMed1998

Effect of platelet-activating factor on the growth of human erythroid and myeloid CD34+ progenitors.

We have assessed the effect of platelet-activating factor (PAF), a biologically active phospholipid present in the human marrow, on the growth of human marrow and blood CD34+ progenitors. While the metabolization rate of PAF by CD34+ cells is low (weak acetylhydrolase and acylation processes) it is readily catabolized by the acetylhydrolase activity present in the growth medium (10% fetal calf serum + 10% 5637-conditioned medium). Treatment of marrow CD34+ cells with the non-metabolizable PAF agonist C-PAF (1 nM to 100 nM) immediately before semi-solid culture significantly (P < 0.01) decreased the number of BFU-E but not of CFU-GM colonies. Treatment of marrow or blood CD34+ cells with C-PAF (10-100 nM) for 3 days in liquid medium before semi-solid culture significantly (P < 0.01) decreased the number of BFU-E and CFU-GM colonies. Treatment of blood CD34+ cells with the two PAF receptor antagonists CV 3988 and BN 52021 (1 microM) had no significant effect on the number of BFU-E and CFU-GM colonies suggesting no role of endogenous PAF in these processes. These results show that exogenous PAF downregulates human erythropoiesis and myelopoiesis, a result that might be of importance during inflammatory states.

Antigens, CD34↗
PubMed1998

Effects of lipoxygenase metabolites of arachidonic acid on the growth of human mononuclear marrow cells and marrow stromal cell cultures.

The effects of various lipoxygenase metabolites of arachidonic acid (AA) were investigated on the growth of freshly isolated human bone marrow mononuclear cells and marrow stromal cell cultures. LTB4, LXA4, LXB4, 12-HETE and 15-HETE (1 microM) decreased [3H]-thymidine incorporation on marrow stromal cell cultures without affecting cell number. Only 12-HETE showed a dose-response effect on [3H]-thymidine incorporation. While LTB4 (1 microM) decreased thymidine incorporation on marrow mononuclear cells, LTC4, LXA4, LXB4, 12-HETE and 15-HETE had no effect. The lipoxygenase inhibitor NDGA had no effect on both cell types suggesting no role of endogenous lipoxygenase metabolites on cell growth. These results suggest no important role of lipoxygenase metabolites of AA on the proliferation of human marrow mononuclear cells and marrow stromal cell cultures.

Arachidonic Acid↗
PubMed1998

Use of collagen for standardization of PBSC graft quality evaluation: a multicenter comparative analysis of commercial collagen-based and methylcellulose-based colony-forming unit (CFU) assay kits.

The colony-forming unit-granulocyte-macrophage (CFU-GM) assay, an essential test in evaluation of the quality of autologous grafts of hematopoietic stem cells, has yet to be standardized. With this aim in view, we carried out a multicenter study of five commercially available culture kits for CFU-GM evaluation. Four kits were methylcellulose-based (H4431, H4434, H4435, StemBio1d) and one was collagen-based (EasyClone-Multi). Using fresh and frozen samples of PBSC grafts, we compared CFU-GM and burst-forming unit-erythrocytes (BFU-E) growth using the EasyClone kit to each of the methylcellulose kits. BFU-E and CFU-GM clonogenicity of both fresh and frozen PBSC was clearly inferior with the H4431 kit, which provides conditioned medium only. CFU-GM numbers obtained with fresh and frozen PBSC samples were significantly higher with the EasyClone kit than with the H4434 and StemBio kits. BFU-E numbers were also higher with the EasyClone kit, but only when colonies were scored after May-Grünwald-Giemsa (MGG) staining. Finally, although the H4435 kit provides higher doses of recombinant cytokines than the EasyClone kit, CFU-GM and BFU-E numbers obtained for fresh or frozen PBSC with both kits were similar. In addition, CFU-GM and BFU-E numbers correlated well with CD34+ cell numbers for all five kits for both fresh and frozen PBSC. In summary, our study shows that the EasyClone-Multi and H4435 kits provide the best CFU-GM growth. The collagen-based EasyClone kit has the additional advantage of allowing gel staining and storage, which facilitates colony identification and, more importantly, makes gel exchange possible for standardization of the CFU-GM assay.

Adolescent↗
PubMed1998

B cell compartments in multiple myeloma: flow cytometric analysis of their labelling indices and Bcl-2 protein expression.

Because recent reports have suggested that non plasmacytic tumor B cells are very rare in Multiple Myeloma (MM), we tried to characterize the B lineage in this disease by comparing by flow cytometry in the PB and BM of MM patients and of controls the proliferative activity (BrdU incorporation) and the Bcl-2 expression of different B cell subsets defined by cytoplasmic light chain, CD19 or CD10 antigen expression. The labelling indices (LI) of CD19+ and CD10+ BM cells in treated patients were higher than in controls and untreated patients. Plasma cell LI (PCLI) were close to previously published values of PCLI flow assays and did not correlate with the LI of BM B cells. Bcl-2 expression by BM CD19+ and CD10+ cells in patients was inferior to controls. These results agree with previously published data about the likely polyclonal nature of most pre PC B cells in MM.

Antigens, CD↗