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P Milenković

Publications and source records attributed to P Milenković.

At least 19 recordsLinked to original sources
PubMed2006

The effect of interleukin-17 on hematopoietic cells and cytokine release in mouse spleen.

To evaluate whether the response of hematopoietic cells to interleukin-17 (IL-17) depends on the tissue microenvironment in which hematopoiesis occurs, the influence of recombinant mouse IL-17 on spleen hematopoietic cells and cytokine release was assessed in normal mice in vitro and in vivo. In vitro, IL-17 did not significantly affect the growth of granulocyte-macrophage (CFU-GM) and erythroid (BFU-E and CFU-E) derived colonies. A single injection of IL-17 in vivo exhibited stimulatory effects on hematopoietic cells from both granulocytic and erythroid lineages. The increased number of metamyelocytes 48 h after treatment imply to the IL-17-induced stimulation of granulopoiesis. The number of BFU-E was increased at 24 h, while the number of CFU-E increased 6 h and 24 h after treatment. Since the same treatment in the bone marrow decreased the number of CFU-E, it may be concluded that the local microenvironment plays an important role in IL-17-mediated effects on CFU-E. IL-17 increased the release of IL-6 both in vitro and in vivo, but showed tendency to suppress the constitutive secretion of IL-10 by spleen cells. Our results suggest the complexity of target cell response and interplay of secondary induced cytokines by IL-17 in different hematopoietic organs.

Animals↗
PubMed2004

In vivo effects of interleukin-17 on haematopoietic cells and cytokine release in normal mice.

In order to gain more insight into mechanisms operating on the haematopoietic activity of the T-cell-derived cytokine, interleukin-17 (IL-17) and target cells that first respond to its action in vivo, the influence of a single intravenous injection of recombinant mouse IL-17 on bone marrow progenitors, further morphologically recognizable cells and peripheral blood cells was assessed in normal mice up to 72 h after treatment. Simultaneously, the release of IL-6, IL-10, IGF-I, IFN-gamma and NO by bone marrow cells was determined. Results showed that, in bone marrow, IL-17 did not affect granulocyte-macrophage (CFU-GM) progenitors, but induced a persistant increase in the number of morphologically recognizable proliferative granulocytes (PG) up to 48 h after treatment. The number of immature erythroid (BFU-E) progenitors was increased at 48 h, while the number of mature erythroid (CFU-E) progenitors was decreased up to 48 h. In peripheral blood, white blood cells were increased 6 h after treatment, mainly because of the increase in the number of lymphocytes. IL-17 also increased IL-6 release and NO production 6 h after administration. Additional in vitro assessment on bone marrow highly enriched Lin- progenitor cells, demonstrated a slightly enhancing effect of IL-17 on CFU-GM and no influence on BFU-E, suggesting the importance of bone marrow accessory cells and secondary induced cytokines for IL-17 mediated effects on progenitor cells. Taken together, these results demonstrate that in vivo IL-17 affects both granulocytic and erythroid lineages, with more mature haematopoietic progenitors responding first to its action. The opposite effects exerted on PG and CFU-E found at the same time indicate that IL-17, as a component of a regulatory network, is able to intervene in mechanisms that shift haematopoiesis from the erythroid to the granulocytic lineage.

Animals↗
PubMed2001

Effect of IL-17 on in vitro hematopoietic progenitor cells growth and cytokine release in normal and post-irradiated murine bone marrow.

The influence of recombinant human IL-17 on granulocyte-macrophage (CFU-GM) and erythroid (BFU-E and CFU-E) progenitors and the release of IL-1alpha/beta, IL-6 and erythropoietin (EPO) was estimated in the bone marrow cells obtained from normal and sublethally irradiated mice. In normal mice IL-17 increased CFU-GM and BFU-E and reduced CFU-E derived colonies numbers and augmented release of IL-6 and EPO. In irradiated mice the effects of IL-17 on hematopoietic progenitors were lineage-dependent, as well as dependent on their stage of differentiation and the time after the irradiation. IL-17 had no major effects on CFU-GM on day 1 and 3, but decreased their number on day 2, while enhanced both BFU-E and CFU-E on day 1 and 2 after irradiation, whereas on day 3 its effect on erythroid progenitors was again as observed in normal mice. After irradiation, IL-17 increased the release of IL-1alpha, IL-6 and EPO. The observed effects suggested the involvement of IL-17 in the regulation of hematopoiesis and indicated that its effects on both hematopoietic progenitors and cytokine release are dependent on the physiological/pathological status of the organism.

Animals↗
PubMed2000

Incubation of murine bone marrow cells in hypoxia ensures the maintenance of marrow-repopulating ability together with the expansion of committed progenitors.

We developed previously a hypoxic culture system in which progenitors endowed with marrow-repopulating ability (MRA), unlike committed progenitors, were selected and maintained better than in air. We report here an improvement to this system targeted at combining the maintenance of progenitors sustaining MRA with the numerical expansion of multipotent and committed progenitors. Murine bone marrow cells were incubated at 1% oxygen in liquid medium supplemented with stem cell factor, granulocyte colony-stimulating factor, interleukin-6 and interleukin-3. In day 8 hypoxic cultures, the numbers of high proliferative potential and granulocyte/macrophage colony-forming cells (HPP-CFC and CFU-GM) were increased with respect to time zero. Colonies generated by HPP-CFC derived from hypoxic cultures exhibited a high replating ability, whereas colonies generated by HPP-CFC derived from control cultures exhibited a low replating ability. MRA was fully maintained in hypoxia and markedly reduced in air. Thus, severe hypoxia is able to ensure a full maintenance of progenitors sustaining MRA, together with a significant expansion of in vitro-detectable clonogenic progenitors, including those endowed with replating ability. This system could contribute to the improvement of current techniques for the in vitro treatment of human haematopoietic cell populations before transplantation.

Animals↗
PubMed2000

Effects of treatment with interleukin-1 receptor antagonist on endogenous interleukin-1 levels in normal and irradiated mice.

The in vivo effects of recombinant human interleukin-1 receptor antagonist (rhIL-1Ra) administration on endogenous IL-1 levels in the circulation and conditioned media (CM) from different immunohematopoietic organ/tissues were studied in CBA mice under steady state and postirradiation conditions. In normal mice, constitutive IL-1 levels were demonstrated in the plasma, CM of peritoneal exudate cells and full-thickness skin explants with low or undetectable levels in CM of splenic and bone marrow cell suspensions. In irradiated mice (2 Gy, X rays) on day 3 post exposure a significant increase of IL-1 levels was seen in the circulation and CM of peritoneal exudate cells, with no significantly different levels in postirradiation bone marrow, spleen and skin. After rhIL-1Ra treatment of the animals (2 x 50 microg/mouse, i.p.), significantly elevated IL-1 levels were observed in the skin and CM of peritoneal exudate cells in normal mice, whereas slightly increased levels were detected in CM of splenic cells. The rhIL-1Ra administration in irradiated mice led to decreased IL-1 concentrations in the circulation, and CM of peritoneal exudate cells and skin. The results pointed out the importance of IL-1 secretion and receptor expression in the maintenance of homeostasis in steady state, as well as during recovery after irradiation. Modulatory effects of IL-1Ra on IL-1 production were dependent on basic endogenous IL-1 concentration.

Animals↗
PubMed1999

The cryopreservation protocol optimal for progenitor recovery is not optimal for preservation of marrow repopulating ability.

The efficiency of five different cryopreservation protocols (our original controlled-rate and noncontrolled-rate protocols) was evaluated on the basis of the recovery after thawing of very primitive pluripotent hemopoietic stem cells (MRA(CFU-GM), pluripotent progenitors (CFU-Sd12) and committed granulocyte-monocyte progenitors (CFU-GM) in mouse bone marrow. Although the nucleated cell recovery and viability determined immediately after the thawing and washing of the cells were found to be similar, whether controlled-rate or noncontrolled-rate cryopreservation protocols were used, the recovery of MRA(CFU-GM), CFU-Sd12 and CFU-GM varied depending on the type of protocol and the cryoprotector (DMSO) concentrations used. It was shown that the controlled-rate protocol was more efficient, enabling better MRA(CFU-GM), CFU-Sd12 and CFU-GM recovery from frozen samples. The most efficient was the controlled-rate protocol of cryopreservation designed to compensate for the release of fusion heat, which enabled a better survival of CFU-Sd12 and CFU-GM when combined with a lower (5%) DMSO concentration. On the contrary, a satisfactory survival rate of very primitive stem cells (MRA(CFU-GM)) was achieved only when 10% DMSO was included with a five-step protocol of cryopreservation. These results point to adequately used controlled-rate freezing as essential for a highly efficient cryopreservation of some of the categories of hematopoietic stem and progenitor cells. At the same time, it was obvious that a higher DMSO concentration was necessary for the cryopreservation of very primitive stem cells, but not, however, for more mature progenitor cells (CFU-S, CFU-GM). These results imply the existence of a mechanism that decreases the intracellular concentration of DMSO in primitive MRA cells, which is not the case for less primitive progenitors.

Animals↗
PubMed1999

Pluripotent haemopoietic progenitor cells (CFU-Sd8) in peripheral blood of hereditarily anaemic Belgrade (b/b) rats.

The unique anaemic syndrome of the Belgrade laboratory (b/b) rat is due to an intracellular iron deficiency which is induced by a not yet defined mutation, resulting in impairment of haemopoiesis. We investigated the CFU-Sd8 number and concentration in the peripheral blood of b/b rats to study the relationship between medullary and extramedullary haemopoiesis in this anaemic syndrome. The results show normal concentration of CFU-Sd8 in the peripheral blood of b/b rats. This finding was unexpected in the state of severe anaemia and disturbed growth factor production in b/b rats, where the mobilization of CFU-Sd8 from bone marrow to blood is expected. The results suggest that severe anaemia is not regularly accompanied by the mobilization of pluripotent progenitors from bone marrow to the blood.

Anemia↗
PubMed1998

Alpha- and beta-globins of the anemic Belgrade laboratory rat. II. The effect of hemin and iron-dextran treatment.

We have studied the changes in bone marrow and spleen globin chain levels after in vivo hemin and iron-dextran treatment of hereditarily anemic Belgrade laboratory (b/b) rats. The increase of globin chains was detected in the bone marrow and in the spleen when b/b animals were treated with either iron or hemin. The analysis of changes in alpha- and beta-globin chain ratios revealed the distinctive role of these molecules in regulating globin chain status. Iron-dextran, as expected, ameliorated the imbalance of alpha- and beta-globin chains in the b/b rat spleen. On the other hand, hemin, as we have hypothesized in the accompanying paper, leads to a surplus of beta-globin chains in the bone marrow, similar to the one detected in the b/b rat spleen. Therefore, an iron-rich microenvironment has a stimulatory effect, while a hemin-rich microenvironment has an inhibitory effect on erythropoiesis.

Anemia↗
PubMed1996

The inhibitory effect of human macrophage inflammatory protein-1 alpha (LD78) on acute myeloid leukemia cells in vitro.

Macrophage inflammatory protein-1 alpha (MIP-1 alpha) has recently been shown to inhibit proliferation of immature hemopoietic progenitors. In addition, significant inhibition of early and mature leukemic progenitors in acute myeloid leukemia (AML) has been obtained with MIP-1 alpha. We performed a study of 25 AML patients at diagnosis to evaluate the effect of a human homolog of MIP-1 alpha (LD78) on bone marrow (BM) and peripheral blood (PB) leukemic progenitors (colony-forming unit-AML [CFU-AML]) and AML cell proliferation. A methylcellulose culture system was used for CFU-AML and incorporation of 3H-TdR for AML cell proliferation. We found that LD78 inhibits CFU-AML colony formation up to 100% for the BM in 14/16 samples studied with the average maximal inhibition of 62.7 +/- 9.1% and up to 100% for the PB in 12/13 samples studied with the average maximal inhibition of 71.4 +/- 9.9%. In addition to this, LD78 inhibited AML cell proliferation up to 60% for the BM in 10/18 samples studied with the average maximal inhibition of 17.8 +/- 3.5%, and up to 87.1% for the PB cell proliferation in 10/16 samples studied with the average maximal inhibition of 27.5 +/- 6.8%. Our results have shown that LD78 is more active on AML progenitors than on AML cell proliferation. Inhibition of the AML cells, although less than that of the progenitors, indicates that more limited activity of LD78 on more mature leukemic cells is present in AML.

Adolescent↗
PubMed1996

The effect of IL-1 receptor antagonist on the proliferation of hematopoietic progenitor cells in regenerating bone marrow.

To evaluate the involvement of IL-1 on bone marrow granulocyte-macrophage (CFU-GM) and erythroid (BFU-E and CFU-E) progenitor cell regeneration during the recovery of hematopoiesis after sublethal irradiation of CBA mice, we examined the effects of IL-1 receptor blockade by recombinant human IL-1 receptor antagonist (rhIL-1ra). The actual number of progenitors and proportion of these cells in S phase of the cell cycle were determined in regenerating bone marrow cells obtained 3 days after 2 Gy irradiation both following the in vivo administration of rhIL-1ra, as well as after the in vitro preincubation with increasing amounts of rhIL-1ra. The results revealed that rhIL-1ra decreased the number and the proportion of CFU-GM in the S phase in regenerating bone marrow. As concerning erythroid progenitors, rhIL-1ra treatment suppressed BFU-E and enhanced CFU-E-derived colony growth, indicating that the biological effects of IL-1 might be different depending on the stage of differentiation. The observed effects pointed to the importance of the basal levels of IL-1, as well as IL-1 receptor expression during the recovery of hematopoiesis.

Animals↗
PubMed1995

Modulation of acute myeloblastic leukemia (AML) cell proliferation and blast colony formation by antisense oligomer for IL-1 beta converting enzyme (ICE) and IL-1 receptor antagonist (IL-1ra).

In the present study we investigated the effects of IL-1 antagonism on the autonomous growth of cells in acute myeloblastic leukemia (AML). To examine the role of pro-IL-1 processing, antisense technology was employed with 16-mer phosphorothioate oligodeoxynucleotide directed against human IL-1 beta converting enzyme (ICR) in 7 randomly selected AML cases. The addition of 10-75 microM of antisense oligonucleotide (but not of control oligonucleotide) significantly inhibited spontaneous proliferation of bone marrow- (BM) and peripheral blood- (PB) derived low density leukemic cells in a dose-dependent way. Similarly, spontaneous as well as induced CFU-AML colony formation was inhibited by human ICE antisense oligonucleotide with sample-to-sample variability. In separate experiments, in order to examine the effects of blockade of endogenously produced IL-1 to IL-1 receptors, the functional activity of human recombinant IL-1 receptor antagonist (IL-1ra) was tested. Continuous exposure to high concentrations of IL-1ra (up to 100 micrograms/ml) produced dose-dependent inhibition of spontaneous proliferation of the BM-derived blast cells from 9 of the 14 patients and of the PB-derived cells from 10 of the 14 patients. However, in some of these patients, the lower IL-1ra doses (down to 100 ng/ml) induced potentiation of spontaneous proliferation, suggesting a novel regulatory pathway for IL-1 receptor engagement. Similar results were obtained on CFU-AML colony formation, showing inhibition at higher IL-1ra doses, but in a few AML cases stimulatory effect at lower IL-1ra doses.(ABSTRACT TRUNCATED AT 250 WORDS)

Antineoplastic Agents↗
PubMed1995

Hematopoietic stem cells in the hereditarily anemic Belgrade laboratory (b/b) rat.

The Belgrade (b/b) rat has hereditary hypochromic microcytic anemia as the consequence of intracellular iron deficiency. Studies in the b/b rat have also demonstrated alteration in hematopoiesis at the progenitor cell level. In the present study, investigations were extended to the bone marrow hematopoietic stem cells as determined by measurements of marrow repopulating ability (MRA) and day 8 spleen colony-forming units (CFU-S-8). A reduced number of CFU-S-8 per femur was found, together with a low incidence per 10(5) bone marrow cells and a nondetectable proliferation rate. The proliferation rate did not increase after treating b/b rat bone marrow cells in vitro with a stimulator for CFU-S proliferation, indicating a proliferative block. The treatment of b/b rats with iron enhanced the proliferation rate and partially increased the number of CFU-S-8 in bone marrow. Chronic transfusion of b/b rats with washed RBC from non-anemic animals restored both the number and the proliferative response of bone marrow CFU-S-8. The MRA of b/b rats was reduced, but in proportion to the decrease in the bone marrow cellularity of these animals. MRA (pre-CFU-S) of b/b rats recovered completely after both iron treatment and chronic transfusions, suggesting that changes in the pre-CFU-S pool are secondary rather than directly induced by the genetic defect. These results indicate the importance for stem cell proliferation of normal oxygenation--the arterial oxygen content in b/b rats is six times lower than in (+/+, b/+) rats--which recovered after the iron treatment and was completely restored after chronic transfusions. High-dose iron therapy abrogated the proliferative block of CFU-S-8, but number of CFU-S-8 was not completely recovered in spite of normalized oxygenation, indicating a possible suppressive effect of iron overload on the marrow microenvironment.

Anemia↗
PubMed1993

Stimulator of proliferation of spleen colony-forming cells in acute sterile inflammation.

The presence and activity of a spleen colony-forming cell (CFU-S) proliferation stimulator was investigated in rat bone marrow after induction of sterile inflammation. Wistar rats were treated intraperitoneally with two 15 ml injections of 3.5% polyvinylpyrrolidone (PVP) at 18 h intervals, and the presence of CFU-S proliferation stimulator determined in bone marrow 6, 20 and 24 h after the first injection. The marrow of these mice was used to condition medium which was then fractionated using Amicon Diaflo ultrafiltration membranes. The 30-50 kDa fraction, taken from 20h post-PVP-bone marrow extract, was found to induce cycling of d8-CFU-S in normal mouse bone marrow. This activity was not related to the presence of interleukins-1, -2 or -6 like activities in the material tested. The results demonstrate the existence of CFU-S proliferation stimulator in the bone marrow of rats with sterile inflammation (i.e. in an in vivo tissue response to non-specific cell stimulation), similar to that originally described as a macrophage product in mouse bone marrow after treatment with a variety of cytotoxic agents.

Animals↗
PubMed1991

Stimulator of proliferation of spleen colony-forming cells in T-cell deprived mice treated with cyclophosphamide or irradiation.

A role for T-cells in the regulation of CFU-S proliferation was investigated by determining the presence and activity of CFU-S proliferation stimulator (CFU-S stimulator) in adult mouse bone marrow after irradiation or cyclophosphamide (Cy) treatment. CBA mice previously deprived of T-cells by thymectomy, irradiation and bone marrow reconstitution (TIR) were thereafter treated with 4.5 Gy irradiation or 200 mg/kg Cy. Regenerating bone marrow cells of TIR and corresponding control mice after irradiation or Cy treatment produced CFU-S stimulator. The dose dependent increase in cytosine arabinoside cell death of normal bone marrow day 8 CFU-S was found when both CFU-S stimulators obtained after irradiation of TIR or corresponding control animals were tested. CFU-S stimulator activity in the bone marrow of TIR-Cy treated mice was also detected, but the effect was not dose-dependent. This was not related to the presence of an inhibitor of CFU-S proliferation. It appears that the CFU-S stimulator activity is not related to IL-6, IL-1 or IL-2, or to an inhibitor of IL-6 or IL-1 activity. The results demonstrate the existence of CFU-S proliferation stimulator unrelated to the two major monokines in the bone marrow of immunosuppressed mice.

Animals↗
PubMed1988

The effect of serum containing granulocytic stimulating activity and colony stimulating activity on regenerating hemopoiesis in mice.

The influence of postinflammatory rat serum containing granulocytic stimulating activity (GSA) on the process of spontaneous regeneration of hemopoietic tissue was studied and compared to the effect of colony stimulating activity (CSA) in CBA mice in the regenerative phase of hemopoiesis after aplasia induced by a sublethal dose of cyclophosphamide (Cy). One hour after Cy application, mice were injected with 0.2 ml of GSA or CSA. Then 6, 12, 24 and 48 hr later the changes within bone marrow and spleen CFU - GM, morphologically recognizable hemopoietic cells and white blood cells (WBC) were followed. GSA stimulated spontaneous regeneration of granulocytic cells, specifically increasing the number of morphologically recognizable proliferative granulocytes continuously during 48 hr. The number of bone marrow (at the 6th hr) and splenic (until the 24th hr) CFU - GM were also increased under the influence of GSA. Under the same experimental conditions in vivo, CSA did not induce any changes within bone marrow granulocytic cells, but the increase in splenic CFU - GM was observed until the 12th hr. The differences in the action of GSA and CSA on the process of spontaneous regeneration of granulocytic cells in vivo in Cy treated mice indicate that these 2 factors play different roles in the regulation of granulopoiesis.

Animals↗
PubMed1988

Erythrocytosis in spontaneously hypertensive rats.

Spontaneously hypertensive rats (SH) with an increased number of red blood cells (RBC), microcytosis, and normal hemoglobin (Hb) concentration were used to study the effect of different manipulations of the erythron on erythropoietin production and on erythroid progenitor proliferation by bone marrow cells in order to gain insight regarding the regulation of erythropoiesis. The serum erythropoietin (Ep) level was increased in untreated SH rats. After stimulation by either bleeding, hemolysis, or acute hypoxia, both the erythropoietin level and erythroid colony-forming unit (CFU-E) proliferation by bone marrow cells increased in SH rats to levels that were similar to those of normotensive Wistar (W) rats. Exposure to chronic hypoxia induced an increase in Hb concentration in SH rats concomitantly with the increase in RBC. The results obtained in SH rats raise the possibility of a defect in nonEp stimulators of erythropoiesis that may alter Hb synthesis.

Animals↗