Hematopoiesis in 5-fluorouracil-treated adenosine A(3) receptor knock-out mice.

The purpose of the study was to describe and compare normal and 5-fluorouracil (5-FU)-suppressed hematopoiesis in adenosine A(3) receptor knock-out (A(3)AR KO) mice and their wild-type (WT) counterparts. To meet the purpose, a complex hematological analysis comprising nineteen peripheral blood and bone marrow parameters was performed in the mice. Defects previously observed in the peripheral blood erythrocyte and thrombocyte parameters of the A(3)AR KO mice were confirmed. Compartments of the bone marrow progenitor cells for granulocytes/macrophages and erythrocytes were enhanced in the control, as well as in the 5-FU-administered A(3)AR KO mice. 5-FU-induced hematopoietic suppression, evaluated on day 2 after the administration of the cytotoxic drug, was found to be significantly deeper in the A(3)AR KO mice compared with their WT counterparts, as measured at the level of the bone marrow progenitor cells. The rate of regeneration, as assessed between days 2 and 7 after 5-FU administration, was observed in the population of the granulocyte/macrophage progenitor cells to be higher in the A(3)AR KO mice in comparison with the WT ones. The increased depth of 5-FU-induced suppression in the compartments of the hematopoietic progenitor cells in the A(3)AR KO mice represents probably a hitherto undescribed further consequence of the lack of adenosine A(3) receptors and indicates its synergism with the pharmacologically induced cytotoxic action of 5-FU.

Erythropoiesis- and thrombopoiesis-characterizing parameters in adenosine A3 receptor knock-out mice.

Influence of the regulatory system mediated by adenosine A(3) receptors on the functioning of erythropoiesis and thrombopoiesis was studied by means of evaluation of the numbers and attributes of peripheral blood erythrocytes and platelets, as well as of erythroid bone marrow progenitor cells in adenosine A(3) receptor knock-out (Adora3(tm1Jbsn)/Adora3(tm1Jbsn), A(3)AR((-/-))) mice and their wild-type C57BL/6 counterparts, both males and females. Minor but statistically significant disturbances in the properties of erythrocytes, namely in the parameters of mean erythrocyte volume and mean erythrocyte hemoglobin were observed in A(3)AR((-/-)) mice. In addition, adenosine A(3) receptor knock-out mice were found to exhibit an expressive, statistically significant decrease of their blood platelet count, amounting to 17 % and 21 % in males and females, respectively. This decrease in platelet levels was accompanied by a significant 17 % decline in the plateletcrit in both sexes. The obtained data can help to define therapeutic applications based on the principle of adenosine receptor signaling.

IB-MECA, an adenosine A(3) receptor agonist, does not influence survival of lethally γ-irradiated mice.

In our previous studies, IB-MECA, an adenosine A(3) receptor agonist, was found to stimulate proliferation of hematopoietic progenitor and precursor cells in mice. This property of IB-MECA was considered to be responsible for its ability to support regeneration of suppressed hematopoiesis after irradiation with sublethal doses of γ-rays when the drug was given in a post-irradiation treatment regimen. This study was aimed at assessing the ability of IB-MECA to influence a 30-day survival of lethally irradiated mice. In a series of experiments, IB-MECA was administered following various lethal radiation doses in various numbers of drug doses and various administration routes. Though in some of these experiments a moderate increase in 30-day survival was observed in IB-MECA-treated mice, the differences in comparison with the controls were not significantly different. It can be inferred from these results and those of previous studies assessing the effects of IB-MECA after sublethal radiation doses that IB-MECA can probably influence only a substantially preserved hematopoiesis like that remaining after sublethal irradiation. Future studies should be aimed at evaluation of the abilities of IB-MECA to influence post-irradiation survival when administered as a part of combined treatment regimens.

Expression of mRNA for adenosine A(1), A(2a), A(2b), and A(3) receptors in HL-60 cells: dependence on cell cycle phases.

The present studies investigated changes in expression of mRNA for adenosine A(1), A(2a), A(2b), and A(3) receptors in samples of HL-60 promyelocytic cells differing in the actual presence of cells in various phases of the cell cycle induced by the double thymidine block method. Real-time PCR technique was used for obtaining data on mRNA expression. Statistical analysis of the data revealed that the mRNA expression of adenosine A(1), A(2a), and A(3) receptors is dependent on the cell cycle phase. G(0)/G(1) and G(2)/M phases were characterized by a higher mRNA expression of adenosine A(1) receptors and a lower one of adenosine A(2a) and A(3) receptors whereas the opposite was true for the S phase. Interestingly, expression of mRNA of the adenosine A(2b) receptors was independent on the cell cycle phase. The results indicate the plasticity of mRNA expression of adenosine receptors in the investigated promyelocytic cells and its interaction with physiological mechanisms of the cell cycle.

A single dose of an inhibitor of cyclooxygenase 2, meloxicam, administered shortly after irradiation increases survival of lethally irradiated mice.

This study extends earlier findings of the authors demonstrating that meloxicam, a selective inhibitor of cyclooxygenase 2, supports hematopoietic recovery in sublethally irradiated mice and is radioprotective when given before irradiation. We report here that when meloxicam was administered in a single dose 1 h after a lethal 9-Gy whole-body dose, an increased 30-day survival was achieved. Additional studies showed that administration of meloxicam 24 h after lethal irradiation is ineffective and its repeated administration deleterious. Possible mechanisms of the therapeutic effects of meloxicam administered early after irradiation are discussed.

Products of radiation removal of lead from aqueous solutions.

The influence of typical *OH radical scavengers as potassium formate and isopropanol on the radiation-induced removal of lead was individually studied. The lead can be completely removed from aqueous solutions containing 1x10(-2) mol/L of formate already at the dose of 2.5 kGy. With increasing concentration of formate (5x10(-5)-1x10(-2) mol/L) increases the amount of Pb(formate)(+) species in the solution before irradiation. The radiation product is metallic lead at low concentration of formate to PbCO(3) at higher concentration of scavenger. In the system with 10% isopropanol dominates the species Pb(2+) and the product of radiation reduction is then metallic lead.

Adenosine A(1), A(2a), A(2b), and A(3) receptors in hematopoiesis. 2. Expression of receptor mRNA in resting and lipopolysaccharide-activated mouse RAW 264.7 macrophages.

Expression of mRNA for adenosine receptor subtypes A(1), A(2a), A(2b), and A(3) in normal and lipopolysaccharide (LPS)-activated murine RAW 264.7 macrophages has been investigated using the method of quantitative real-time polymerase chain reaction. The results have shown a very low, unquantifiable expression of adenosine A(1) receptor mRNA in both normal and LPS-activated macrophages. The other three adenosine receptor mRNAs have been found to be expressed at various but always quantifiable levels. Activation of the macrophages by LPS induced upregulation of the expression of adenosine receptor A(2a) and A(2b) mRNA, whereas the expression of adenosine receptor A(3) mRNA was downregulated. Unstimulated macrophages exhibited a high expression of the A(2b) adenosine receptor mRNA. The findings are discussed from the point of view of the antiinflammatory and hematopoiesis-stimulating roles of the adenosine receptor signaling.

Adenosine A(1), A(2a), A(2b), and A(3) receptors in hematopoiesis. 1. Expression of receptor mRNA in four mouse hematopoietic precursor cells.

Four mouse bone marrow or thymus cell populations, namely granulopoietic/monocytopoietic, erythropoietic, B-lymphopoietic, and T-lymphopoietic precursor cells have been assayed by RT-PCR technique for the presence and relative amounts of adenosine A(1), A(2a), A(2b), and A(3) receptor mRNA. It has been found that (i) all four populations studied express all four adenosine receptor subtypes, (ii) the A(1), receptor is the least expressed in all populations studied, (iii) the A(3) receptor is markedly expressed in the populations of granulopoietic/monocytopoietic and erythropoietic cells, (iv) the A(2a) receptor is markedly expressed in the populations of B-lymphopoietic and T-lymphopoietic cells, and v) the A(2b) receptor does not predominate in any of the precursor cells studied. Our data offer a new possibility for the assessment of the readiness of these cells to respond, by receptor-mediated mechanisms, to adenosine or its analogs present in the tissues as a result of endogenous processes and/or following their administration.

Fluorescence of reduced charge montmorillonite complexes with methylene blue: Experiments and molecular modeling.

The intensity of fluorescence of montmorillonites fully saturated by methylene blue (MB) is very poor due to energy dissipation in MB aggregates. A series of reduced charge montmorillonites (RCM) were prepared from Na-homoionic SWy and Ca homoionic SAz with the aim to decrease the MB aggregation. Fine tuning MB adsorption degree by charge reduction and MB concentration enabled controlled production of different dye species from aggregates via dimers to monomers. It was shown that the intensity of the fluorescence of low-loaded MB-RCM complexes is enhanced by several orders of magnitude with respect to dye-saturated original montmorillonites. XRD analyses, molecular modeling, and diffuse reflectance spectroscopy revealed that low MB-loaded RCMs are very probably adsorbed mainly on the external montmorillonite surface as isolated dye molecules. Such a state cannot be achieved in the solid state without very careful tailoring of the host-guest interaction.

Activation of adenosine A(3) receptors potentiates stimulatory effects of IL-3, SCF, and GM-CSF on mouse granulocyte-macrophage hematopoietic progenitor cells.

Adenosine A(3) receptor agonist N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA) has been tested from the point of view of potentiating the effects of hematopoietic growth factors interleukin-3 (IL-3), stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte colony-stimulating factor (G-CSF) on the growth of hematopoietic progenitor cells for granulocytes and macrophages (GM-CFC) in suspension of normal mouse bone marrow cells in vitro. IB-MECA alone induced no GM-CFC growth. Significant elevation of numbers of GM-CFC evoked by the combinations of IB-MECA with IL-3, SCF, or GM-CSF as compared with these growth factors alone has been noted. Combination of IB-MECA with G-CSF did not induce significantly higher numbers of GM-CFC in comparison with G-CSF alone. Joint action of three drugs, namely of IB-MECA + IL-3 + GM-CSF, produced significantly higher numbers of GM-CFC in comparison with the combinations of IB-MECA + IL-3, IB-MECA + GM-CSF, or IL-3 + GM-CSF. These results give evidence of a significant role of selective activation of adenosine A(3) receptors in stimulation of the growth of granulocyte/ macrophage hematopoietic progenitor cells.

Inhibition of cyclooxygenase 2 in mice increases production of g-csf and induces radioprotection.

Meloxicam, a selective inhibitor of cyclooxygenase 2, was tested to determine its ability to modulate hematopoiesis and to influence survival of mid-lethally gamma-irradiated mice. A single dose of meloxicam (20 mg/kg) administered to mice intraperitoneally 1 h before irradiation was shown to enhance serum levels of granulocyte colony-stimulating factor (G-CSF) during the first 24 h after irradiation, to elevate numbers of granulocytic precursor cells in bone marrow and granulocyte counts in peripheral blood on day 10 after irradiation, and to increase 30-day survival of these mice. The results provide new evidence for the protective ability of meloxicam administration to mice irradiated with mid-lethal doses and contribute to the understanding of the mechanisms of this meloxicam action by drawing attention to the possible role of increased endogenous G-CSF production.

Meloxicam, an inhibitor of cyclooxygenase-2, increases the level of serum G-CSF and might be usable as an auxiliary means in G-CSF therapy.

Hematopoiesis-modulating action of meloxicam, a cyclooxyge-nase-2 inhibitor, has been evaluated in mice. Increased serum level of granulocyte colony-stimulating factor (G-CSF) after meloxicam administration has been found in sublethally gamma-irradiated animals. In further experiments hematopoiesis-stimulating effects of meloxicam and G-CSF given alone or in combination have been investigated. Granulocyte/macrophage progenitor cells counts were used to monitor these effects. Meloxicam and exogenous G-CSF did not act synergistically when given in combination, but could be mutually substituted during their repeated administration. The results suggest a promising possibility of using meloxicam as an auxiliary drug reducing the high costs of G-CSF therapy of myelosuppression.

Molecular modeling of layered double hydroxide intercalated with benzoate, modeling and experiment.

The structure of Zn4Al2 Layered Double Hydroxide intercalated with benzencarboxylate (C6H5COO-) was solved using molecular modeling combined with experiment (X-ray powder diffraction, IR spectroscopy, TG measurements). Molecular modeling revealed the arrangement of guest molecules, layer stacking, water content and water location in the interlayer space of the host structure. Molecular modeling using empirical force field was carried out in Cerius(2) modeling environment. Results of modeling were confronted with experiment that means comparing the calculated and measured diffraction pattern and comparing the calculated water content with the thermogravimetric value. Good agreement has been achieved between calculated and measured basal spacing: d(calc) = 15.3 A and d(exp) = 15.5 A. The number of water molecules per formula unit (6H2O per Zn4Al2(OH)12) obtained by modeling (i.e., corresponding to the energy minimum) agrees with the water content estimated by thermogravimetry. The long axis of guest molecules are almost perpendicular to the LDH layers, anchored to the host layers via COO- groups. Mutual orientation of benzoate ring planes in the interlayer space keeps the parquet arrangement. Water molecules are roughly arranged in planes adjacent to host layers together with COO- groups.

Composition, structure, and luminescence of montmorillonites saturated with different aggregates of methylene blue.

The distribution of various aggregates (dimers, trimers, and tetramers) of methylene blue (MB) formed in aqueous solution at various concentrations of dye has been calculated using the equilibrium aggregation constants betaq. Two montmorillonite samples with different cation exchange capacities, surface areas, and interlayer distances d001, Na-SWy, and Ca-Cheto, were saturated with methylene blue (MB) solutions with various ratios between monomers and higher aggregates of dye. The total amount of MB in the intercalated montmorillonite samples (MB-SWy and MB-Cheto) increases with increasing concentration of dye in water solutions, i.e., with increasing aggregates/monomers ratio of MB in water solution. In all intercalated montmorillonite samples with methylene blue except guest qth aggregate cations [MBqq+] low contents of Na+ (in MB-SWy) and Ca2+ (in MB-Cheto) cations were also determined. A very good positive correlation between the basal spacing d001 and the MB/montmorillonite molar ratio was revealed for saturated MB-montmorillonite samples. Structural analysis using a combination of diffraction data with molecular modeling revealed the differences in the interlayer arrangement of MB guests in MB-SWy and MB-Cheto intercalates. Also, fluorescence measurements showed the strong effect of the silicate layer charge on the spectroscopic behavior of MB guests intercalated in montmorillonite. Methylene blue exhibits a certain luminescence in MB-SWy samples with cation exchange capacity 0.80 meq g-1 and almost no luminescence in MB-Cheto samples with higher cation exchange capacity 1.50 meq g-1.

Drugs elevating extracellular adenosine administered in vivo induce serum colony-stimulating activity and interleukin-6 in mice.

Our previous studies have shown that the combined administration of drugs elevating extracellular adenosine, i.e. dipyridamole (DP) and adenosine monophosphate (AMP), enhances murine hematopoiesis and potentiates the action of granulocyte colony-stimulating factor (G-CSF). In this study, colony-stimulating activity (CSA) of blood serum of mice treated with DP+AMP, G-CSF or all these drugs in combination, i.e. the ability of the sera to stimulate the growth of GM-CFC colonies, was assayed in vitro. Furthermore, the concentration of GM-CSF and IL-6 in the sera was determined. Administration of DP+AMP was found to enhance significantly serum CSA at all time intervals of serum sampling including 24 h after the last injection of the tested drugs. Additive effects of DP+AMP and G-CSF on serum CSA were noted at early intervals after administration of the drugs. Furthermore, IL-6 levels were significantly elevated in the sera of mice which were administered DP+AMP either alone or in combination with G-CSF. Our results show that the effects of DP+AMP are indirect, mediated through the induction of some cytokine(s) and/or growth factor(s) and that extracellular adenosine can act in cooperation with G-CSF. These findings contribute to the further elucidation of the role of adenosine in hematopoiesis.

Meloxicam, a cyclooxygenase 2 inhibitor, supports hematopoietic recovery in gamma-irradiated mice.

Meloxicam, a selective inhibitor of cyclooxygenase 2, a nonsteroidal anti-inflammatory drug with an improved side-effects profile in terms of gastrointestinal toxicity, has been found to stimulate hematopoiesis in whole-body gamma-irradiated mice. A distinct corroboration of this positive action of meloxicam is an enhancement of the recovery of hematopoietic progenitor cells committed to granulocyte-macrophage and erythroid development, which has been demonstrated in sublethally irradiated animals treated with meloxicam at a dose of 20 mg/kg administered intraperitoneally either singly 1 h before irradiation or repeatedly after radiation exposure. The results suggest that meloxicam can be added to the list of biological response modifiers that can be used in the treatment of hematopoietic damage induced by ionizing radiation.

Structure of montmorillonite cointercalated with stearic acid and octadecylamine: modeling, diffraction, IR spectroscopy.

Structural analysis of Na-montmorillonite co-intercalated with octadecylamine and stearic acid was carried out using combination of experiment: X-ray powder diffraction and IR spectroscopy with molecular modeling (force field calculations) in Cerius(2) modeling environment. Results of structure analysis revealed the chemical reaction of guest compounds leading to the formation of octadecylammonium stearate. This reaction may occur even before the intercalation out of the interlayer space of montmorillonite. The presence of octadecylammonium stearate in the samples was clearly confirmed by IR spectroscopy and X-ray diffraction. Present results also showed that: (1) Stearic acid itself does not intercalate into Na-montmorillonite; (2) cointercalation with octadecylamine led to the formation of octadecylammonium stearate, which was successfully intercalated into the interlayer space of montmorillonite, and (3) Na-montmorillonite intercalated with octadecylammonium stearate does not create a stable structure. Intercalated samples in ambient conditions undergo gradual decomposition, accompanied by the release of octadecylammonium stearate from the interlayer space and rearrangement of the interlayer structure. Co-intercalation of STA and ODA to lower the octadecylamine content and consequently to suppress the unfavorable effect of amine groups on the polymer matrix in nanocomposite, was investigated.

Adenosine potentiates stimulatory effects on granulocyte-macrophage hematopoietic progenitor cells in vitro of IL-3 and SCF, but not those of G-CSF, GM-CSF and IL-11.

The aim of the studies was to ascertain if adenosine is able to co-operate with selected hematopoietic growth factors and cytokines, namely with granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF), interleukin-3 (IL-3), and interleukin-11 (IL-11), in inducing the growth of colonies from hematopoietic progenitor cells for granulocytes and macrophages (GM-CFC) from normal bone marrow cells in vitro. Adenosine was found not to produce any colonies when present in the cultures as the only potential stimulator. All the tested cytokines and growth factors were observed to induce the growth of distinct numbers of GM-CFC colonies, with the exception of IL-11. When suboptimal concentrations of the evaluated cytokines and growth factors were tested in the cultures in which various concentrations of adenosine were concomitantly present, mutually potentiating effects were found in the case of IL-3 and SCF. These results confirm the role of adenosine in regulation of granulopoiesis and predict IL-3 and SCF as candidates for further in vivo studies of their combined administration with adenosine.

Effects of stable adenosine receptor agonists on bone marrow hematopoietic cells as inferred from the cytotoxic action of 5-fluorouracil.

The aim of the study was to investigate the effects of stable adenosine receptor agonists on bone marrow hematopoiesis by utilizing the model of hematopoietic damage induced by 5-fluorouracil (5-FU), a cycle-specific cytotoxic agent. Effects of a non-selective agonist NECA activating all the known adenosine receptors (A1, A2A, A2B, A3) and of the selective agonists for A1 (CPA), A2A (CGS 21680), and A3 (IB-MECA) adenosine receptors were investigated. Experiments were performed with B10CBAF1 mice under in vivo conditions. Adenosine receptor agonists were given in single injections before 5-FU administration and the effects were determined 4 days later. The numbers of femoral marrow nucleated cells and hematopoietic progenitor cells (CFC-GM and BFU-E) were taken as indices of the effects. The non-selective agonist NECA given at a dose of 200 nmol/kg induced biphasic time-dependent effects, i.e. protection and sensitization, when given 10 h and 22 h before 5-FU administration, respectively. The use of isomolar doses of selective receptor agonists indicated that the protective effects of NECA were induced by activation of A2A and A2B receptors, while the sensitizing action of NECA was mediated via A3 receptors. In addition, it was observed that A1 receptors induced protection when activated by administration of CPA 22 h before 5-FU. These findings are discussed with respect to the action of adenosine receptor agonists on the cell cycle state and on the cell cycle-independent cellular protective mechanisms.

Structure analysis of intercalated layer silicates: combination of molecular simulations and experiment.

Na(+)-montmorillonite type Wyoming, cloisite Na(+) from Southern Clay Products, Inc., was intercalated (i) with octadecylammonium cations and subsequently intercalated with octadecylamine molecules, (ii) with dodecylamine molecules, and (iii) with octylamine molecules to determine the applicability of these intercalates for nanocomposite materials on the base of polymer/clay. The structures were determined on the basis of a combination of results from X-ray diffraction and molecular simulations. The calculated values of basal spacings are in good agreement with experimental basal spacings when experimental samples were prepared. The interlayer space of intercalated montmorillonite shows a monolayer or bilayer arrangement of alkyl chains in dependence on the concentration of the intercalation solution. The values of the total sublimation energy, interaction energy, and exfoliation energy were calculated for all investigated samples. Low values of exfoliation energies lead to better exfoliation of intercalated silicate layers and this material appears suitable for use as a precursor for polymer/clay nanocomposites. The values of exfoliation energy for the investigated samples show that montmorillonite intercalated with dodecylamine or octadecylamine molecules is suitable for exfoliation of silicate layers.