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Introduction. Hydroxycarbamide, used in therapy of hemoglobinopathies, enhances nitric oxide (NO) production both in primary human umbilical vein endothelial cells (HUVECs) and human bone marrow endothelial cell line (TrHBMEC). Moreover, NO increases γ-globin and fetal hemoglobin levels in human erythroid progenitors. Objective. In order to find out whether simple physiologic stimulation of NO production by components of hematopoietic microenvironment can increase γ-globin gene expression, the effects of NO-inducer bradykinin were examined in endothelial cells. Methods. The study was performed in co-cultures of human erythroid progenitors, TrHBMEC and HUVECs by ozone-based chemiluminescent determination of NO and real-time quantitative RT-PCR. Results. In accordance with previous reports, the endogenous factor bradykinin increased endothelial cell production of NO in a dose- and time-dependent manner (0.1-0.6 μM up to 30 minutes). This induction of NO in HUVECs and TrHBMEC by bradykinin was blocked by competitive inhibitors of NO synthase (NOS), demonstrating NOS-dependence. It has been shown that bradykinin significantly reduced endothelial NOS (eNOS) mRNA level and eNOS/Я-actin ratio in HUVEC (by twofold). In addition, bradykinin failed to increase γ-globin mRNA expression in erythroid progenitors only, as well as in co-culture studies of erythroid progenitors with TrHBMEC and HUVEC after 24 hours of treatment. Furthermore, bradykinin did not induce γ/β globin ratio in erythroid progenitors in co-cultures with HUVEC. Conclusion. Bradykinin mediated eNOS activation leads to short time and low NO production in endothelial cells, insufficient to induce γ-globin gene expression. These results emphasized the significance of elevated and extended NO production in augmentation of γ-globin gene expression. [Projekat Ministarstva nauke Republike Srbije, br. 175053]

Mesenchymal stem cells (MSCs) were isolated from human periodontal ligament (hPDL-MSCs) and characterized by their morphology, clonogenic efficiency, proliferation and differentiation capabilities. hPDL-MSCs, derived from normal impacted third molars, possessed all of the properties of MSC, including clonogenic ability, high proliferation rate and multi-lineage (osteogenic, chondrogenic, adipogenic, myogenic) differentiation potential. Moreover, hPDL-MSCs expressed a typical MSC epitope profile, being positive for mesenchymal cell markers (CD44H, CD90, CD105, CD73, CD29, Stro-1, fibronectin, vimentin, α-SMA), and negative for hematopoietic stem cell markers (CD34, CD11b, CD45, Glycophorin-CD235a). Additionally, hPDL-MSCs, as primitive and highly multipotent cells, showed high expression of embryonic markers (Nanog, Sox2, SSEA4). The data obtained provided yet further proof that cells with mesenchymal properties can be obtained from periodontal ligament tissue. Although these cells should be further investigated to determine their clinical significance, hPDL-MSCs are believed to provide a renewable and promising cell source for new therapeutic strategies in the treatment of periodontal defects. [Projekat Ministarstva nauke Republike Srbije, br. 175062 i br. III 41011]

Introduction. Mesenchymal stem cells (MSCs) are a promising tool for regenerative medicine, but due to the heterogeneity of their populations, different sources and isolation techniques, the characteristics defining MSCs are inconsistent. Objective. The aim of this study was to compare the characteristics of MSCs derived from two different human tissues: peripheral blood (PB-MSCs) and umbilical cord Wharton’s Jelly (UC-MSCs). Methods. The PB-MSC and UC-MSC were isolated by adherence to plastic after gradient-density separation or an explant culture method, respectively, and compared regarding their morphology, clonogenic efficiency, proliferating rates, immunophenotype and differentiation potential. Results. MSCs derived from both sources exhibit similar morphology, proliferation capacity and multilineage (osteogenic, chondrogenic, adipogenic and myogenic) differentiation potential. Differences were observed in the clonogenic capacity and the immunophenotype, since UC-MSCs showed higher CFU-F (colony-forming units-fibroblastic) cloning efficiency, as well as higher embryonic markers (Nanog, Sox2, SSEA4) expression. When additional surface antigens were analyzed by flow cytometry (CD44, CD90, CD105, CD33, CD34, CD45, CD11b, CD235a) or immunofluorescent labeling (vimentin, STRO-1 and α-smooth muscle actin), most appeared to have similar epitope profiles irrespective of MSC source. Conclusion. The results obtained demonstrated that both MSCs represent good alternative sources of adult MSCs that could be used in cell therapy applications. [Projekat Ministarstva nauke Republike Srbije, br. 175062]

In the present study we have isolated and identified mesenchymal stem cells (MSCs) from the exfoliated deciduous teeth dental pulp (DP-MSCs), as plastic-adherent, spindle-shaped cells with a high proliferative potential. Immunophenotype analyses revealed that DP-MSCs were positive for mesenchymal cell markers (CD90, CD44, CD105, STRO-1, vimentin and α-SMA), and negative for hematopoietic stem cell markers (CD11b, CD33, CD34, CD45, CD235a). DPMSCs were also capable of differentiating into adipogenic, chondrogenic, myogenic and osteogenic lineages, fulfilling the functional criterion for their characterization. These results demonstrate that DP-MSCs offer a valuable, readily accessible source to obtain and store adult stem cells for future use.

Rats with polyvinylpyrrolidone (PVP)-induced sterile inflammation were used as a model in vivo for investigation of granulopoiesis and extramedullary-produced regulators. The data obtained demonstrated the invasion of massive numbers of granulocytes at the site of inflammation (peritoneal cavity) during the first 24 h of the acute phase of inflammation. To meet the organism's needs for granulocytes the activation of granulopoiesis in bone marrow occurred simultaneously. Accelerated production of granulocytic cells is manifested by involvement of granulocytic proliferative compartment in various stages of differentiation (CFU-GM and morphologically recognizable proliferative granulocytes--PG). Together with cellular changes within the granulocytic cells line, the changes in the content of investigated regulators influencing granulopoiesis were observed. At different time intervals the levels of interleukin-6 (IL-6), colony-stimulating activity (CSA), and granulocytic stimulating activity (GSA) were increased locally at the site of inflammation as well as in serum. The data obtained provide evidence that inducible granulopoiesis during the acute phase of inflammation is under the control of extramedullary-produced regulators, thus confirming their role in the regulation of granulocytic production in vivo. [ABSTRACT FROM AUTHOR]

Aim: The study was undertaken to extend our investigation concerning both the in vivo activity of interleukin (IL)-17 and the specific role of nitric oxide (NO) in IL-17-induced effects in the process of haematopoiesis., Methods: CBA mice were simultaneously treated with IL-17 and/or nitric oxide synthase (NOS) inhibitor, l-NAME, for 5 days and changes within various haematopoietic cell lineages in bone marrow, spleen and peripheral blood were analysed., Results: Findings showed that administration of both IL-17 and l-NAME stimulated increase in net haematopoiesis in normal mice. IL-17-enhanced myelopoiesis was characterized by stimulation of both femoral and splenic haematopoietic progenitor cells and morphologically recognizable granulocytes. Additionally, IL-17 induced alterations in the frequency of erythroid progenitor cells in both bone marrow and spleen, accompanied with their mobilization to the peripheral blood. As a consequence of these changes in the erythroid cell compartments, significant reticulocytosis was observed, which evidenced that in IL-17-treated mice effective erythropoiesis occurred. Exposure of mice to NOS inhibitor also increased the number of both granulocyte-macrophage and erythroid progenitors in bone marrow and spleens, and these alterations were followed by the mobilization of erythroid progenitors and elevated content of reticulocytes in peripheral blood. The specific role of NO in IL-17-induced haematopoiesis was demonstrated only in the IL-17-reducing effect on bone marrow late stage erythroid progenitors, CFU-E., Conclusion: The results demonstrated the involvement of both IL-17 and NO in the regulation of haematopoietic cell activity in various haematopoietic compartments. They further suggest that IL-17 effects are differentially mediated depending on the haematopoietic microenvironments.

Syphacia obvelata is a rodent nematode parasite with high prevalence in laboratory mice. In our previous work we have demonstrated that this gut-dwelling helminth induces significant hematopoietic changes, characterized by increased myelopoiesis and erythropoiesis in infected animals, and accompanied with altered reactivity of bone marrow hematopoietic progenitors to interleukin (IL)-17. In this study we extended these investigations by demonstrating that naturally acquired S. obvelata infection induces significant alterations in murine bone marrow cells manifested at the molecular level. Namely, S. obvelata infection induced sustained phosphorylation of the members of three major groups of distinctly regulated mitogen-activated protein kinases (MAPKs), the p38, the c-Jun amino-terminal kinase (JNK) and the extracellular signal-regulated kinase (ERK), as well as enhanced expression of mRNA for the inducible nitric oxide synthase (iNOS) in the bone marrow cells of infected animals. Furthermore, the infection interfered with the IL-17-mediated effects in bone marrow cells, since in normal mice IL-17 significantly enhanced phosphorylation of p38 MAPK and upregulated the expression of iNOS and the constitutive, endothelial (e)NOS mRNA, while in S. obvelata-infected animals IL-17 did not influence the MAPKs activation, but markedly down-regulated the expression of both NOS isoforms. The data obtained demonstrating that S. obvelata is able to manipulate signal transduction pathways in the hosts' bone marrow cells, pointed to the multiple layers of immunomodulatory ability of this pinworm parasite and highlighted the importance of working under pinworm-free conditions when using experimental murine models for immunohematopoietic investigations., (Copyright 2009 Elsevier Ireland Ltd. All rights reserved.)

The effects of interleukin (IL)-17 on nitric oxide (NO) synthase (NOS) expression, as well as the participation of mitogen-activated protein kinases (MAPKs) in IL-17-mediated effects were examined in murine bone marrow cells. The results demonstrated the ability of IL-17 to upregulate the expression of mRNA for both inducible NOS and constitutive, endothelial NOS isoforms, as well as to enhance the phosphorylation of p38 MAPK. Moreover, both the NOS-inducing effect of IL-17 and the in vitro IL-17-mediated inhibition colony forming unit-erythroid (CFU-E) growth were dependent on p38 MAPK activity. The data demonstrating that the in vivo reducing effect of IL-17 on bone marrow CFU-E was prevented by co-treatment with the NOS inhibitor Nw-nitro-l-arginine methyl ester hydrochloride (L-NAME), implied that this effect is mediated through NOS activation. Besides revealing a link between the IL-17, NO, and haematopoiesis, data presented gave an insight into the mechanisms by which IL-17 exerts its modulatory effects on bone marrow cells.

Co-culture of haematopoietic cells with a stromal cell layer does not mimic the physiological, micro-environmental niche, whose major feature is a low oxygen (O2) concentration. Thus, in order to study the effects of IL-17 in a context which better approximates the physiological state, we investigated its effects on cell expansion, colony-forming ability, and the phenotypical profile of normal, human blood CD34+ cells co-cultured for five days with MSC layers at various O2 concentrations (20%, 12.5% and 3% O2. We demonstrated that IL-17 enhances CD34+ and total CFC production during the five days of MSC/CD34+ co-culture. This effect depends upon the O2 concentration, reaching its maximum at 3% O2, and is more pronounced on erythroid progenitors (BFU-E). In addition, the stimulation of IL-6 production by IL-17 in MSC cultures and co-cultures is enhanced by low O2 concentration. The expression of some differentiation markers (CD34, CD13 and CD41) on haematopoietic cells in co-cultures also depends upon the oxygen concentration. Our results strengthen the concept that physiological levels of O2 (mistakenly called hypoxia), should be considered as an important environmental factor that significantly influences cytokine activity.

Low O(2) concentration (1%) favors the self-renewal of hematopoietic stem cells and inhibits committed progenitors (CFC). Since IL-6 influences both stem cells and committed progenitors at 20% O(2), we studied its effects in cultures at 1% O(2). The pre-CFC activity in Lin- population of mouse bone marrow was analyzed following 10 days of serum-free culture in medium (LC1) supplemented with IL-3 with and without IL-6, at 20 and 1% O(2) and phenotypic differentiation and proliferative history monitored. The IL-6 receptor expression and initiation of VEGF-A synthesis were also investigated. At 20% O(2), the effects of IL-6 on pre-CFC were negligible but effects on CFC were apparent; conversely, at 1% O(2), the IL-6 enhances activity of pre-CFC but not of CFC. Unlike at 20% O(2), at 1% O(2) a subpopulation of cells remained Lin- in spite of extensive proliferation. However, the absolute number of Lin- cells, did not correlate with pre-CFC activity. A relative increase in VEGF transcripts at 1% O(2) in presence of IL-3 alone was enhanced by the addition of IL-6. IL-6 enhanced pre-CFC activity at 1% O(2) and this was correlated to the induction of VEGF. These data reinforce the concept that physiologically low oxygenation of bone marrow is a regulator of stem cell maintenance. Since the 20% O(2) does not exist in tissues in vivo, further studies in vitro at lower O(2) concentrations should revise our knowledge relating to cytokine effects on stem and progenitor cells.

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.

Pinworm parasites commonly infect laboratory mice with high prevalence even in well-managed animal colonies. Although often considered as irrelevant, these parasites if undetected may significantly interfere with the experimental settings and alter the interpretation of final results. There are a few reports documenting the effects of pinworms on research and the effects of pinworms on the host hematopoiesis have not yet been investigated. In this study we examined the changes within various hematopoietic cell lineages in the bone marrow, spleen, peripheral blood and peritoneal space during naturally acquired Syphacia obvelata infection in inbred CBA mice. The data obtained showed significant hematopoietic alterations, characterized by increased myelopoiesis and erythropoiesis in S. obvelata-infected animals. In order to additionally evaluate if this pinworm infection modifies hematopoietic cells' reactivity, we examined the effect of murine interleukin-17, T cell-derived cytokine implicated in the regulation of hematopoiesis and inflammation, on the growth of bone marrow progenitor cells and demonstrated that bone marrow myeloid and erythroid progenitors from S. obvelata-infected mice displayed altered sensitivity to IL-17 when compared to non-infected controls. Taken together the alterations presented pointed out that this rodent pinworm is an important environmental agent that might significantly modify the hosts' hematopoietic response, and therefore interfere with the experimental settings and alter the interpretation of the final results. However, the results obtained also contributed new data concerning the activity of IL-17 on bone marrow hematopoietic cells, supporting our previous reports that depending on physiological/pathological status of the organism IL-17 exerts differential effects on the growth of progenitor cells.

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.

Recent studies have shown that the T cell-derived cytokine, interleukin-17 (IL-17), stimulates hematopoiesis, specifically granulopoiesis inducing expansion of committed and immature progenitors in bone marrow. Our previous results pointed to its role in erythropoiesis too, demonstrating significant stimulation of BFU-E and suppression of CFU-E growth in the bone marrow from normal mice. As different sensitivities of erythroid and myeloid progenitor cells to nitric oxide (NO) were found, we considered the possibility that the observed effects of IL-17 were mediated by NO. The effects of recombinant mouse IL-17, NO donor (sodium nitroprusside - SNP) and two NO synthases inhibitors (L-NAME and aminoguanidine) on erythroid progenitor cells growth, as well as the ability of IL-17 to induce nitric oxide production in murine bone marrow cells, were examined. In addition, we tested whether the inhibition of CFU-E colony formation by IL-17 could be corrected by erythropoietin (Epo), the principal regulator of erythropoiesis. We demonstrated that IL-17 can stimulate low level production of NO in murine bone marrow cells. Exogenously added NO inhibited CFU-E colony formation, whereas both L-NAME and aminoguanidine reversed the CFU-E suppression by IL-17 in a dose-dependent manner. The inhibition of CFU-E by IL-17 was also corrected by exposure to higher levels of Epo. The data obtained demonstrated that at least some of the IL-17 effects in bone marrow related to the inhibition of CFU-E, were mediated by NO generation. The fact that Epo also overcomes the inhibitory effect of IL-17 on CFU-E suggests the need for further research on their mutual relationship and co-signalling.

We studied hematopoiesis in bone marrow and blood of CBA mice following infection with Toxoplasma gondii. Our data showed that acute infection with the virulent RH strain was associated with leucopenia, thrombocytopenia and bone marrow hypoplasia while, in spite of the infection-induced damage of the granulocyte cell lineage, in bone marrow stimulated production of granulocytes was revealed. In peripheral blood, T. gondii infection caused a significant decrease in the total number of white blood cells, reticulocytes and platelets. However, the relative proportion of granulocytes and lymphocytes was changed in favor of granulocytes, as compared to pre-infection levels. The functional activity of granulocytes was also increased. The bone marrow alterations were characterized by a decrease in the total number of nucleated cells due to the reduced numbers in all cell compartments of erythroid and megakaryocytic lineage, as well as in the number of mature granulocytes and lymphocytes. In contrast, femoral granulocytic proliferative compartments, colony forming unite granulocyte-macrophage (CFU-GM) and morphologically recognizable proliferative granulocytes (PG), exhibited stimulated granulopoiesis, while the number of mature monocytes was close to the control value. In summary, we have shown that acute T. gondii infection results in profound alterations of the hematopoietic system that markedly contribute to the clinical onset of the disease and the, ultimately lethal, outcome.

Contact hypersensitivity (CHS) reaction is a classic example of a cell-mediated reaction. As the afferent phase of the reaction includes inflammation, CHS is a suitable model for investigating non-specific immunity. Some aspects of granulocyte activity in the afferent phase of experimentally induced CHS to dinitrochlorobenzene (DNCB) in two genetically different rat strains, AO and DA were examined in this study. A shift in the ratio of granulocytes to lymphocytes in favour of granulocytes and an increase in granulocyte survival were noted in DA rats. Granulocytes from both strains demonstrated increased levels of NBT reduction and an increase in their adhesion to plastic. Decreased granulocyte adhesion in the presence of monoclonal antibodies to beta2 integrins (anti-CD11b/c and anti-CD18) points to the contribution of these molecules to granulocyte adhesiveness during the sensitization phase of CHS. Stimulation of adhesion in the presence of anti-CD11a antibody, points to a differential modulation of adhesion molecule activity during the afferent phase of CHS. Changes in functional activity of granulocytes demonstrated in this study might contribute to the development of CHS in rats.

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.

Donor leukocyte infusions are an effective therapy for patients who relapse with leukemia after bone marrow transplantation. We report the case of 14-year-old boy who relapsed 34 months after sibling donor bone marrow transplant for Philadelphia-positive chronic myeloid leukemia. Subsequently, he received three infusions of donor mononuclear cells (DMNC) harvested in steady state hematopoiesis and one G-CSF mobilized-peripheral blood mononuclear cells (PBMC) infusion. Simultaneously, test named as--"Test of Mixed Progenitors" (TMP) was performed for the assessment whether the outcome of donor leukocyte infusion treatment could be predicted. Prior to DMNC infusions, the CFU-GM and BFU-E colony assays were performed for donor's and recipient's PBMC individually, as well as for the mixture of these cells at 1:1 ratio. The cells were plated either directly in the semisolid medium or after 24 h preincubation treatment. Significantly lower values for CFU-GM derived colonies were determined in TMP in comparison to the CFU-GM values obtained for the recipient's cells. The reduced number of CFU-GM was determined both in TMP performed without preincubation treatment, app. 80% and after the 24 h preincubation, app. 55%. The reduced number of BFU-E derived colonies (app. 44%) was observed only related to recipient's cells and after the preincubation treatment of the cells. The patient did not develop GVHD and currently (40 months after the first infusion). He remained well in complete hematological, cytogenetic, molecular and clinical remission, which was the most direct evidence of the GVL effect. The novel in vitro TMP test in which the specific contribution of donor's leukocytes to the growth of recipient's hematopoietic precursor cell growth was determined, correlated with the clinical outcome.

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.

The influence of five different cryopreservation protocols on the quality and/or quantity of frozen cells was investigated on mouse bone marrow cells and human peripheral blood mononuclear cells (MNC). The efficiency of the protocols was evaluated on the basis of the recovery of very primitive pluripotent hematopoietic stem cells (MRA), pluripotent progenitors (CFU-Sd12), committed granulocyte-monocyte progenitors (CFU-GM) of mouse cells after thawing. The recovery of MRA, CFU-Sd12 and CFU-GM varied depending on the type of freezing procedure and cryoprotector (DMSO) concentrations used. It was shown that the controlled-rate protocol was more efficient, enabling better recovery of all categories of progenitor cells in frozen samples. The most efficient was the controlled-rate protocol of the cryopreservation designed to compensate for the release of fusion heat, which enabled the best recovery of CFU-GM (73.0 +/- 8.8%) and CFU-Sd12 (90.0 +/- 15.9%) when combined with 5% DMSO concentration (protocol 4). On the contrary, a better recovery (79.8 +/- 13.5%) of very primitive stem cells (MRA) was achieved only when the higher concentration (10%) DMSO was used in combination with a five-step protocol of cryopreservation (protocol 1). These results pointed out the adequately used controlled-rate freezing to be 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 MRA, but not 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 MRA cells, which is not the case in less primitive progenitors. For human MNC, the recovery and viability of the cells, as well as the engraftment potential of cryopreserved cells after thawing were investigated. Cryopreservation protocol 1 resulted in better MNC recovery (82.7 +/- 10.4%) than protocol 3 (49.9 +/- 15.1%). The mean recovery of MNCs (collected from patients for autologous transplantation) was 78.5 +/- 7.3% (protocol 1) and 53.1 +/- 26.2% (protocol 3). The obtained favorable recovery of thawed cells and rapid reconstitution of hematopoiesis (on the day 11th following the transplantation) in patients confirmed that the controlled-rate freezing in combination with optimal DMSO concentration was able to obtain sufficient progenitor cryoprotection.

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.

In this study we have demonstrated that the bone marrow of the anemic Belgrade laboratory (b/b) rat, as the primary site of erythropoiesis, has a decreased globin polypeptide synthesis in total protein cell extracts. Therefore, it is the source of red blood cells containing decreased amounts of globin mRNAs and polypeptides. In spite of the fact that the b/b rat shows a splenomegaly, the spleen is not capable of compensating for the anemic state. Spleen erythroid cells are defective in differentiation and contain a decreased share of globin polypeptides in total protein cell extracts compared to the control. Spleen cells are also characterized by a drastic imbalance of alpha- to beta-globin resulting in the beta-globin chains surplus.

Many different cell types, coordinated by proinflammatory mediators, take part in the acute inflammatory reaction, but there is a lack of evidence regarding the role of erythroid cells in such conditions. In this study, changes in bone marrow, splenic, and peripheral blood erythroid cells and in erythropoietin (Epo) blood levels were investigated up to 72 hours after polyvinylpyrrolidone (PVP)-induced sterile inflammation in male Wistar rats (two intraperitoneal injections of 15 mL 3.5% PVP at 18-hour intervals). Transient changes within progenitor erythroid cells were observed in the bone marrow. Significant increases in the number of splenic immature erythroid progenitors (BFU-E) 6 hours and mature erythroid progenitors (CFU-E), erythroblasts, and orthochromatic erythroblasts 48 and 72 hours after the induction of inflammation pointed to stimulated splenic erythropoiesis. This was confirmed by semiquantitative assessment of splenic smears, which demonstrated expansion of erythroid cells at hours 48 and 72. The changes observed in the bone marrow and spleen indicated that during acute inflammation erythropoiesis was stimulated and that the spleens of PVP-treated rats were favorable to erythroid development. The significant increase in the percentage of peripheral blood reticulocytes 48 and 72 hours after PVP-induced inflammation provided evidence that effective erythropoiesis occurred. In spite of the stimulated erythropoiesis, serum levels of Epo remained unchanged, implying that other non-Epo regulatory molecules may be responsible for erythroid cellular changes.

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.

The aim of this study was to clarify the nature of the abnormalities of granulopoiesis in anemic b/b rats. The size of different granulocytic cell compartments (granulocytic and macrophage precursor cells, proliferative and nonproliferative morphologically recognizable granulocytic cells) in the bone marrow, spleen, and peripheral blood was determined, and activity of granulocytic stimulators (colony-stimulating activities derived from lung-conditioned medium of endotoxin-treated rats and from spleen cells stimulated in vitro with pokeweed mitogen) was investigated. Depressed bone marrow granulopoiesis and expanded granulopoiesis in the spleen, together with the sustained production of granulocytic stimulators, was found in anemic b/b rats. It is suggested that the changes within the bone marrow microenvironment are the essential cause of disturbances of granulopoiesis in anemic b/b rats. The increased proliferation of granulocytic cells in iron-treated b/b rats indicates a role of iron in the cellular proliferation.

The model of experimentally induced inflammation demand for granulocytes was used to study the changes in granulocytic cell compartments and the level of humoral regulators of granulopoiesis. The experimental inflammation in rats induced by i.p. injection of 3.5% solution of polivinylpyrrolidone (PVP) provoked during 24 hours intensive mobilisation of granulocytes into peritoneal exudate and the intensive proliferation of granulocytic cells in the bone marrow between 18-24 hours. When the bone marrow of CBA mice was cultivated in diffusion chambers implanted into rats with experimentally induced inflammation, stimulation of granulopoiesis was noted during the period of five days. The results indirectly indicate that the level of granulopoiesis stimulating factor was increased in the diffusion chambers host animals. It was demonstrated also that rat's serum obtained 20 hours after injection of PVP (leukophoretic serum) stimulates the proliferation of granulocytes in the bone marrow of normal CBA mice cultivated in diffusion chambers. The level of stimulation was confirmed to be dose dependent. Presented data indicate that in the conditions of increased demand for granulocytes, the level of humoral regulator--stimulator of proliferation of granulocytic cells--is increased in the circulation.

Leucophoretic rat serum (LS) obtained from rats previously treated by i.p. injection of polyvinylpyrrolidone (PVP), specifically stimulates proliferation of morphologically recognizable granulocytic cells in vivo. In this work the effect of the same serum on CFU-c and CFU-s was studied. It was demonstrated that a single injection of LS induced firstly (after 12 hr) an increase in CFU-c and afterwards, at 24 and 48 hr, increase in CFU-s. It was therefore concluded that the humoral factor present in LS, stimulating primarily the proliferating granulocytic cell compartment, has some effect on CFU-c, while the effect on the CFU-s cell compartment is secondary to the changes in more mature cell compartments.

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.

Methylcellulose culture assay was used to detect committed haemopoietic stem cells, CFU-C and CFU-E, in aplastic anaemia patients with autologous haemopoietic reconstitution. Severe diminution of CFU-C was found in all the patients studied and the absence of a dose-response to colony stimulating factor (CSF) was demonstrated. A reduced number of CFU-E and lower erythropoietin (Ep) sensitivity of those progenitors was detected as well. Autologous serum added to the bone marrow cultures of these patients enhanced the growth of CFU-C but inhibited CFU-E growth. According to the results presented, some residual damage at the stem cell level is suggested.

The role of T cells in regeneration of CFU-S(d8) and GM-CFU was investigated in adult Cy treated mice. CBA mice were deprived of T cells by adult thymectomy and irradiation (TIR) and treated with 200 mg/kg Cy. The type of CFU-S(d8) and GM-CFU regeneration curve after Cy was not different from that found in nonthymectomized, irradiated (NIR) mice. The activity of a stimulator of CFU-S(d8) proliferation was increased to the same degree in the bone marrow of both TIR and NIR mice. However CFU-S(d8) recovered to a higher level in TIR than in NIR mice. The results presented demonstrate that T-cell depletion in adult mice influences CFU-S(d8) growth.

Serum obtained from rats with polyvinylpyrrolidone-induced sterile inflammation contains granulopoiesis-stimulating activity (GSA). In triple-set diffusion chambers (DC) with GSA in the middle chamber a stimulation of morphologically recognizable granulocytic cells (MRG) was found in mouse bone marrow cells cultivated in side chambers for 6 and 24 h. GSA also induced an increase in the number of CFU-GM when DC were inplanted into the peritoneum of normal rats. Under the same experimental conditions, colony-stimulating factor (CSA) from mouse-lung-conditioned medium did not cause such changes either in MRG or in CFU-GM compartments. The data obtained indicate the different stimulating action of GSA and CSA on granulopoiesis.

The effect of leucophoretic serum (LS), obtained from rats with polyvinylpyrrolidone (PVP)-induced inflammation, on granulopoiesis in the bone marrow of normal CBA mice was studied. The following test systems were used: short term cultures (4 hr), diffusion chambers (8, 24, 48 and 72 hr) and in vivo assays (12, 24 and 48 hr). The results indicate that LS stimulates the proliferations of granulocytic cells by increasing the number of proliferative granulocytes in mitosis, as well as increasing the total number of proliferative granulocytes. LS did not appear to effect monocytes and other cell lines. It is concluded that a factor present in LS specifically stimulates the proliferation of granulocytic cells, both in vitro and in vivo.

In posthypoxic polycythemic mice (P) expanded granulopoiesis was found. After aplasia induced by a sublethal dose of cyclophosphamide (Cy), more granulocytic cells both in the bone marrow and spleen were found in P than in normal (N) mice treated in the same way. Spontaneous regeneration of granulocytic precursors during the first three days after Cy was more intensive in P mice, and a significant role of the spleen in CFU-GM regeneration was observed. It is suggested that P animals could be used as an appropriate model for investigation of the cell specificity of humoral regulators of granulopoiesis and their action, as well as in studies on the competition of granulocytic and erythroid cells.

Sublethally irradiated mice were used as a model for an in vivo study of the effect of the stimulating factor present in rat leukophoretic serum (LS). LS was obtained from rats with inflammation induced by polyvinylpyrrolidone. Irradiated mice were injected i.v. with 0.2 ml LS, normal rat serum or Parker 199 medium, respectively. Animals were sacrificed 24, 48 and 72 h later. The obtained data indicated that LS stimulated the proliferation of granulocytic cells in the period observed.

The cell extracts prepared from leucocytes and erythrocytes of normal rats were applied to C57B1 mice exposed to 300 Rad in 5 injections of 0.5 ml each, during the period of 50-80 hours after irradiation. Significant inhibition in the proliferative compartments of both granulocytic and erythrocytic cells were found in mice submitted to the corresponding cell extract. Our results indicate that cell extracts tested in an animal system demonstrated the same type of inhibitory action as previously reported for the bone marrow cells cultivated in vitro.