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The genes p53, mdm2, p21, c-myc, bcr/abl, bcr, bcl2, bax, and gapdh participate in the regulation of cell proliferation and differentiation, apoptosis and cell distribution for the cell cycle ex vivo in the Ph +cells of chronic myeloid leukemia containing the Ph chromosome and bcr/abl oncogene. Expression of these genes correlates with regulation of cell proliferation and differentiation by alternating proliferation and maturation stages for three main Ph +cell types that occur under chronic myeloid leukemia. The p53, p21, mdm2, and gapdh genes overexpress in active proliferating myeloid cells in the cell cycle S+G2/M phases and when the phases are coincident with the proliferation stage. Expression of these genes decreases to a considerable level under alternation of the Ph +cell proliferation and maturation stages and whenever the expression is greatly diminished under significant neutrophil accumulation and especially under repeated alternation of the stages. In the course of neutrophil maturation, gene expression levels decrease in the range of gapdh actin c-myc, bcr/abl,p21 p53 bcl2 bax. The expression levels of these genes in neutrophils are lower than those in myelocytes and lower by an order of magnitude than that in the cells with a prolonged proliferation stage. The Bcr/abl expression gene under prolonged maturation and neutrophil accumulation is inhibited; however it is enhanced by 2-3 times for the proliferation stage with myelocyte accumulation. Minimal bcr/abl expression is observed under overexpression of p53, mdm2, p21, c-myc, as well as under cell maximum at the S and G2/M phases. Bcr/abl overexpression is observed under low expression of the p53, p21, mdm2 genes. In the Ph + cells with a high P/D efficiency index (5-20), overexpression of the genes in the range of bcr gapdhbcr/abl, as well as a decreased expression of the p53, bcl2, mdm2, p21 gapdh genes is observed for Ph + cells from the CML blast crisis and CML acceleration phase. Low control of cell proliferation and cell cycle by gene-regulators presumably promotes bcr/abl overexpression and activates the production of bcr/abl + cells. Apoptosis in the Ph + cells is induced by expression of the bax bcl2, p53, p21, c-myc and gapdh genes. The blocking of Ph + cell apoptosis, neutrophil accumulation, and decrease in the expression of the p53, mdm2 and p21, c-myc, bcr/abl genes occur at the maturation stage.

Increased PRV-1 mRNA expression and the presence of Jak2(V617F) mutation in peripheral blood granulocytes are specific markers for chronic myeloproliferative disorders (MPD), which facilitate the differential diagnosis between polycythemia vera (PV) and secondary erythrocytosis (SE) and may be helpful for monitoring treatment efficacy in MPD patients. We evaluated the presence of the Jak2V617F mutation and increased PRV-1 mRNA expression along with previously established markers - erythropoietin (EPO) independent colony formation (EEC) and erythropoietin level for diagnosis of PV and assessment of treatment efficiency. Increased PRV-1 expression was found in 37 out of 46 patients diagnosed with PV (80%), in 4 out of 15 patients diagnosed with essential thrombocythemia (ET) (27%) and in 4 out of 8 patients with chronic idiopathic myelofibrosis (CIMF) (50%), and increased PRV-1 expression plus EEC formation was observed in 19 of 36 examined MPD patients indicating the superiority of PVSG and WHO bone marrow criteria for the diagnosis of ET, PV and CIMF. We could confirm a very high sensitivity, specificity and utility of the Jak2(V617F) mutation for differential diagnosis between PV and SE. Spontaneous EEC, serum EPO levels, PRV-1 expression was evaluated in 22 PV patients who carried the Jak2(V617F) mutation. A concordance of increased PRV-1 expression and presence of Jak2(V617F) mutation in 19/22 (85%); of increased PRV-1/Jak2/EEC in 14/22 (63%); and of Jak2/PRV-1/EEC/low Epo level in 10/22 (45%) patients was found indicating the superiority of the presence of Jak2(V617F) mutation for the diagnosis of PV. IFN-alpha therapy in patients with PV was more effective then hydroxyurea treatment and significantly reduced increased PRV-1 expression together with higher levels of Jak2(V617F) mutation (50-100%) in PV patients treated with hydroxy urea (HU) and lower levels of Jak2(V617F) mutation (35-90%) in PV patients treated with IFN-alpha. Normal PRV-1 expression level was observed in 44% of PV patients who achieved clinical remission and only in 3% of patient who did not. These preliminary observations indicate that the Jak2(V617F) mutation in particular and PRV-1 overexpression appear to be suitable markers for monitoring treatment efficiency in prospective randomised clinical studies comparing pegylated interferon and hydroxyurea in well defined PV patients with a clear indication for cytoreductive therapy.

We studied the expression of VCAM-1 adhesion molecules on stromal cells from the bone marrow of patients with myelodysplastic syndromes, healthy donors, and patients with chronic myeloproliferative diseases and acute leukemias. Expression of adhesion molecule on mesenchymal stromal cells from the bone marrow of patients and healthy donors was evaluated after 2-4 passages by the methods of immunoprecipitation and electrophoresis. VCAM-1 expression in the majority of patients with myelodysplastic syndromes was lower than in healthy donors. At the same time, VCAM-1 expression was not identified on mesenchymal cells from acute leukemia patients. VCAM-1 expression on cells from patients with chronic myeloproliferative diseases did not differ from that in healthy donors. We conclude that VCAM-1 synthesis in bone marrow stromal cells is impaired in patients with myelodysplastic syndromes and acute leukemias. These changes can be followed by the loss of relationships between hemopoietic cells and stromal microenvironment in bone marrow niches. Hemopoietic cells gain the ability for uncontrolled growth, which results in progression of the disease.

Human myeloid cells with Ph chromosome (Ph+ cells) from chronic myeloid leukemia (CML) in the course of proliferation and differentiation ex vivo are regulated under alternation of cell proliferation and neutrophil maturation stages by consecutive blocking and inducing apoptosis with of neutrophils participation as well bcr/abl, bax and bcl2 genes expression. Apoptosis regulation of three main Ph+ cells types from CML patients depends on alternation sequences of proliferation (1) and maturation (2) cell stages and realized by two ways. The first one is performed by consecutive blocking and inducing apoptosis under 2/1/2 stage alternation. The way is not described early. Neutrophils accumulation correlates with apoprosis blocking. Apoptosis level enhances under neutrophils exhausted. Apoptosis blockage allows cells to proliferate and, thus, to form new portion of neutrophils with consecutive regular their death as well a consequent alternation of apoptosis blocking and inducing. This way regulates proliferation efficiency indexes P/D that reflect Ph+ cells proliferating potential and performs cycle completion for proliferation and differentiation. The second way of apoptosis regulation starts from proliferation stage and performs for 1/2/1 alternations under diminished content of neutrophils and a little increase under next maturation. It leads to resistant depressed apoptosis levels that, at maximal points, are 3-8 times lower than those under alternation 2/1/2. Resistant apoptosis blocking is observed in the Ph+ cells with prolong proliferation or maturation stages, when blasts and myelosytes are accumulated under enhanced bcr/abl and bcl2box gene expression and remain under next maturation. Stable apoptosis blocking is accompanied by increasing amounts of blasts and myelocytes and enhancing bcr/abl and bcl 2bax expression. This is observed under CML progression. Ph+ cells cultivation may be useful for more distinct diagnostics of CML phases of individual CML patients and optimization of the treatment.

We studied functional disturbances in hemopoietic microenvironment and cytokine production by stromal sublayer in long-term bone marrow cultures and peripheral blood macrophages from patients with various forms of myelodysplastic syndrome. Production of factors stimulating the growth of normal erythroid and granulocytic precursors by cells of the stromal sublayer from patients with refractory sideroblast anemia and refractory anemia with excess blasts is impaired compared to cells from healthy donors. The medium conditioned by macrophages from patients with chronic myelomonocytic leukemia displayed a higher ability to stimulate the growth of granulocytes and macrophages compared to media conditioned by cells from donors and patients with refractory sideroblast anemia and refractory anemia with excess blasts. Cultured stromal cells and macrophages produced tumor necrosis factor-α and interleukin-6. Their content in media conditioned by cells from patients with myelodysplastic syndrome surpassed that in healthy donors. Our results suggest that production of cytokines by stromal microenvironmental cells is impaired in patients with various forms of myelodysplastic syndrome.

Hemopoiesis in sublethally irradiated mice is provided by a succession of clones of hemopoietic cells containing unique radioactive markers. Dozens of clones with short life-span (not more than 4 months) are functioning during mouse life. Ten percent of these clones are long-lived and can be revealed during 20 months. These clones are formed exclusively by bone marrow stem cells in the G0-period of the cell cycle. A new structure of hemopoietic stem cell population is discussed.

Using clonal methods for assessment of hemopoietic and stromal cells and long-term bone marrow cell cultures, we have demonstrated heterogeneity of myelodysplastic syndrome. Low content of stromal precursor cells in native bone marrow, peculiarities in the formation of the stromal layer and its hemopoiesis-stimulating capacity in long-term cultures, and altered properties of stromal precursor cells in long-term cultures indicate defect in the stromal microenvironment in myelodysplastic syndrome.

Ph+, bcr/abl+ cells arise due to t(9,22) chromosome translocation and Ph+ chromosome formation in hematopoietic stem cells. The cells show appreciable apoptosis suppression but retain their ability to differentiate and maturate. Ph chromosome, bcr/abl oncogene and Ph+, bcr/abl+ cells themselves are the hallmark of chronic myeloid leukemia. Under leukemia progression differentiating Ph+, bcr/abl+ cells transform into leukemic malignant cells with differentiation block. It is assumed to be a result of subsequent mutations or activation of proliferation of long silent Ph+ cells arisen previously in the stem cells because of the translocation. Real mechanism underlying the cell transformation remains unknown. This work was performed to develop a proper cell model allowing us to study functioning of differentiating Ph+, bcr/abl+ cells and their real transformation into malignant cells with block of differentiation. For this purpose we have investigated kinetics of Ph+, bcr/abl+ cells proliferation, differentiation, cell death and transcription of antiapoptotic genes in cultured 14-day of Ph+ mononuclear cells isolated from peripheral blood of a patient in chronic phase of chronic myeloid leukemia before treatment. The results obtained revealed that Ph+ cell differentiation proceeded in accord with characteristic scheme of chronic myeloid leukemia in vivo. Myeloid cells of hematopoietic cell lineage amounted to 3/4 of live Ph+ mononuclear cells undergoing accumulation and subsequent consumption in the course of differentiation. 95% myeloid cells were differentiating Ph+ granulocytes. The most deal of differentiating Ph+ cells was myelocytes. The rate ratio of myelocyte accumulation to its subsequent consumption showed that the rate of transformation into metamyelocytes was significantly decreased at this differentiation stage. Ph+ cells cultivation curves characterized cell death at different differentiation stages. There were observed the cell death of proliferating Ph+ cells and Ph+ myelocytes, and intensive death of mature cells as well. P/D index, that is ratio of immature Ph+ granulocytes differentiated by cell dividing (blasts, promyelocytes and myelocytes) to the cells differentiated without dividing (metamyelocytes and mature neutrofiles), revealed active of proliferation at the beginning of cultivation and unexpected new proliferative activity at the end of cultivation in the presence of growth factor. The peaks of antiapoptotic bcr/abl gene transcription activity coincided with the observed active proliferation at the beginning and at the end of cultivation. Cell proliferation, differentiation and apoptosis were noticeably accelerated by growth factor treatment. Thus, the study of the Ph+ cells cultivation kinetics is rather informative approach to investigation of continuous regulation of cellular and molecular processes in vitro in the case of chronic myeloid leukemia and allows more complete consideration of Ph+, bcr/abl+ cells hematopoiesis.

Some peripheral blood parameters and serum erythropoietic activity were measured in neonatal, young and aged normal mice. There were low level of hemoglobin and red blood cells but increased percentage of reticulocytes and serum erythropoietin level in neonatal mice. The increased serum erythropoietic level were found in aged mice, while peripheral blood parameters were normal. Thus this study demonstrates some changes in erythropoiesis and its regulation during postnatal ontogenesis.

Hemopoietic and stromal precursor cells were studied in mice deficient for tumor necrosis factor or lymphotoxin-alpha. In normal hemopoiesis the main characteristics of hemopoiesis in knockout mice did not differ from those in wild-type mice. Implantation of bone marrow cells from mice deficient for tumor necrosis factor onto irradiated sublayer of a long-living bone marrow culture led to a notable increase in the number of mature cells and granulocytic-macrophage precursor cells. This can be due to the fact that tumor necrosis factor inhibits proliferation of hemopoietic precursor cells, while in the absence of this factor precursor cells actively proliferate. On the other hand, cell composition and number of colony-forming units of granulocytes-macrophages are significantly decreased in cultures onto which bone marrow cells from lymphotoxin-alpha-deficient mice were implanted. This can be explained by impaired expression of adhesion molecules in these animals. In addition, the number of stromal precursor cells was changed in mice deficient by genes of the tumor necrosis factor cluster.

We studied functional disturbances in hemopoietic microenvironment and cytokine production by stromal sublayer in long-term bone marrow cultures and peripheral blood macrophages from patients with various forms of myelodysplastic syndrome. Production of factors stimulating the growth of normal erythroid and granulocytic precursors by cells of the stromal sublayer from patients with refractory sideroblast anemia and refractory anemia with excess blasts is impaired compared to cells from healthy donors. The medium conditioned by macrophages from patients with chronic myelomonocytic leukemia displayed a higher ability to stimulate the growth of granulocytes and macrophages compared to media conditioned by cells from donors and patients with refractory sideroblast anemia and refractory anemia with excess blasts. Cultured stromal cells and macrophages produced tumor necrosis factor-alpha and interleukin-6. Their content in media conditioned by cells from patients with myelodysplastic syndrome surpassed that in healthy donors. Our results suggest that production of cytokines by stromal microenvironmental cells is impaired in patients with various forms of myelodysplastic syndrome.

The low plasma erythropoietic (Epo) activity which is non-adequate to manifestation of anemia, and the lack of correlation between Epo activity and the degree of anemia and hypoxia were found in children with sepsis. The lowest Epo activity was determined in plasma of patients after repeatedly blood transfusion and in emaciated children. The non-specific Epo activity inhibitor was determined in acute period of sepsis in majority of patients. We suppose that the low Epo activity was due to the violation of Epo synthesis regulation mechanism or was connected with the presence of non-specific inhibitors. These results suggest recombinant Epo for the treatment of anemia in children with sepsis.

The level of plasma erythropoietic activity was determined in healthy and iron deficiency anemia children by bioassay in vitro. The erythropoietic activity in healthy children was 13.6 +/- 2.9 (5.0-24.8) mU/ml and the lowest activity was in two month (6.3 +/- 0.7) and three month (6.9 +/- 1.4) mU/ml healthy children. The erythropoietic activity was increased from 40.5 to 715.0 mU/ml in iron deficiency anemia children and its level distinctly depends on anemia stage and iron deficiency. From this study we suggest, that erythropoietin is produced in response of anemia. The results also demonstrate the necessity of erythropoietin treatment in iron deficiency anemia in early infants.

Some peripheral blood parameters and serum erythropoietic activity were measured in neonatal, young and aged normal mice. There were low level of hemoglobin and red blood cells but increased percentage of reticulocytes and serum erythropoietin level in neonatal mice. The increased serum erythropoietic level were found in aged mice, while peripheral blood parameters were normal. Thus this study demonstrates some changes in erythropoiesis and its regulation during postnatal ontogenesis.

Erythropoietic islands located in the yolk sac (YS) mesoderm are known to be the first sites of hemopoiesis in the ontogeny of mice. Committed precursors colony-forming unit — granulocyte-macrophage (CFU-GM) and burst-forming unit-erythroid (BFU-E), as well as pluripotent progenitors (CFUmix) were found in murine YS as early as 8 days of gestation; in embryos proper (bodies) and in the circulation, committed precursors appeared 1 day later than in YS [1–3]. As for CFU-spleen (CFU-S), there are few communications and they are contradictory. Moore and Metcalf [1] reported that CFU-S initially appeared in murine YS on day 8 of gestation, and they were evident in the embryonic circulation by day 10 of development. However, other investigators failed to find macroscopic spleen colonies after the injection of day-9 YS cells [4, 5]. After the cultivation of a day-9 YS in organ culture for 24–96 h, CFU-S were readily found [4]. In the present study we tested the accurate stages — in somite pair (SP) numbers – of embryonic development when CFU-S and CFU-GM appeared for the first time in the YS, circulation, embryos proper, and liver rudiments (LR). We have used the organ culture system to examine whether pre-CFU-S, capable of differentiating into CFU-S in vitro, exist in the 9-days YS and embryo proper.

It is known that anemia occurs in normal young and elderly animals [1, 2]. As a rule it is accompanied with some changes in hemopoietic tissues and disturbances in regulation of erythropoiesis. However mechanisms of erythropoietic regulation may differ significantly in young and elderly animals. The nature of senescent anemia is not clear [3, 4]. Some authors deny any changes in basal hemopoiesis in aged mice [5, 6].

Transgenic mice were obtained inheriting the human erythropoietin gene under the control of viral regulatory elements. The reliable difference in haematocrit, the content of haemoglobin and percentage of reticulocytes in peripheral blood were not revealed. The level of serum erythropoietin in transgenic mice is several fold higher than in control mice. The increased pool of erythroid cells was observed in the bone marrow of transgenic mice, especially of normoblasts (3-fold) and reticulocytes (4,5-fold).

Effects of unpurified murine erythropoietin and unpurified human recombinant erythropoietin on the growth of erythroid--BFU-E and granulocyte--macrophage progenitor cells--CFU--GM from the mouse bone marrow were compared using a methylcellulose culture system. Average erythropoietin titers for murine serum and supernatant human recombinant erythropoietin were 16 U/ml and 33 U/ml, respectively. The maximal stimulation was observed at 1-2 U/ml culture recombinant erythropoietin and 0.5 U/ml culture murine erythropoietin. Murine erythropoietin was more effective then human one. Murine and human recombinant erythropoietin had no significant effect on the number of CFU-GM colonies. But human recombinant erythropoietin could be preferentially used when studying the mechanism of erythropoiesis in man and animals because there were erythropoiesis inhibitors in mouse serum.

As many as ten patients with terminal uremia on maintenance hemodialysis (MH) performed from 11 to 52 months (28 +/- 4 on the average) were treated with recombinant human erythropoietin (rhERP) manufactured by Cilag (Switzerland). The preparation was injected i.v. in the initial dose 65 U/kg bw thrice a week after each session of MH. If the effect was lacking for 2 weeks, the doze was augmented by 25 U/kg (maximally up to 218 U/kg). Provided the Hb content increased to 100-120 g/l, the maintenance therapy was initiated (108 +/- 13 U/kg). The treatment lasted 17 +/- 2 weeks. As a result, the Hb content in the patients rose from 63.0 to 102.0 g/l, the hematocrit index from 21.2 to 34.4%, the red blood cell count from 2.09 to 3.18 x 10(12)/l; there was a transitory increase of the reticulocyte and platelet counts (from 2.2 to 3.3% and from 172.0 to 284.0 x 10(9)/l) whereas the leukocyte count remained unchanged. It has been demonstrated by the bicycle ergometry data that in addition to the amelioration of the hematological parameters, the patients showed an increase of oxygen consumption at rest, rise of the economy of energy losses at exercise, and a decline of oxygen "cost" of work. According to echocardiography, the patients manifested a reduction of the size of the left heart, of the minute and stroke volumes. In men, the treatment with rhERP brought about libido elevation in the absence of significant alterations in the blood gonadotrophin and testosterone concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Two types of erythroid precursors were isolated during culture of mouse bone marrow in a plasma clot in the presence of mouse serum and without the addition of exogenous erythropoietin to the medium. The first type of precursor was more similar in its characteristics to the erythroid colony-forming unit described previously. The second type of precursor was an erythroid burst-forming unit, similar in its properties to that described previously. The optimal concentration of mouse serum in the culture medium was 10–15%. The clonal nature of the colonies and bursts described is confirmed by the linear relationship between their number and the cell concentrations in culture.

The kinetics of hematopoietic, stem cells was studied in organ culture of a suspension of mouse embryonic liver grown directly on filters and on a previously grown embryonic thymic feeder. In the first two weeks of culture, hematopoiesis in the suspension was virtually indistinguishable from hematopoiesis in fragments of the same age and the same time of culture, whereas later hematopoiesis was more active in the suspension grown on thymic feeder. The possible role of the thymus in hematopoiesis is discussed.

A study was made of the kinetics of hemopoietic stem cells in the organic culture of a suspension of mouse embryonic liver cultivated directly on the filters and on the preliminarily grown embryonic thymic feeder. It was shown that stem hemopoietic cells were retained in the course of one month of cultivation in the embryonic liver suspensions; during the first two weeks hemopoiesis in the suspension practically failed to differ from such in the fragments of the same age and cultivation period, whereas during the subsequent periods hemopoiesis proved to be more active in the suspension cultivated on thymic feeder. A possible participation of the thymus in the hemopoiesis is discussed.

The simple, specific and sensitive erythropoietin bioassay in serum and plasma from phenylhydrazine treated mice is described, based on H3-thymidine incorporation into divided hemopoietic cells. Spleen cells taken from mice on the third day following the second of 2 daily injections of phenylhydrazine were cultured in 24 hours in the presence of test material. Following incubation for 2 hours with H3-thymidine solution radioactivity was measured.

Lymphoblastoid cell line was obtained from long-term human bone marrow culture. The cell line was characterized using methods of immunological phenotyping, cytochemistry and cytogenetics. This cell line represents different stages of B-cell differentiation, karyotype 46 XX. The cells of this line were able to support their growth during more than 10 months without exogenous stimulation.

To study the reasons for the failure of erythroid differentiation in a long-term organ culture of mouse embryonal liver, the development of erythroid colony-forming progenitors was examined. The "early" (BFUei) and "late" (CFUei) erythropoietin-independent erythroid progenitors were present in washes from organ cultures for at least 56 days and in the "rests" of the cultures for 46 days. The mean concentration and the correlation of the "early" and "late" progenitors were similar to those in the bone marrow and initial embryonal liver. The data suggest that the stopping of erythroid differentiation in organ culture of embryonal liver occurs after CFUei formation, interfering with their maturation to morphologically recognizable erythroid cells.

Two types of erythroid precursors were found by cultivation of the mouse bone marrow in the plasma clot with mouse serum and without adding exogenic erythropoietin to the culture medium. The first precursor had properties similar to the erythroid colony-forming unit (CFUe) previously described while the second resembles in its properties the erythroid burst-forming unit (BFUe). Optimal concentration of mouse serum in the culture medium was 10-15%. Clone nature of the colonies and bursts described is confirmed by linear dependence of their number on the cell concentration in the culture.

The properties of early (BFUen) and late (CFUen) erythropoietin-independent progenitors were studied in bone marrow plasma clot cultures of mice. Syngeneic serum was used as a stimulant of colony formation. It was revealed that serum from polycytemic mice did not alter the efficacy of cloning the erythroid progenitors. Varying effects produced on erythropoiesis in vivo did not significantly change the number of CFUen and BFUen, with the exception of posttransfusion polycytemia where the number of CFUen showed a statistically significant rise. Assessment of radiosensitivity and proliferative activity of the erythroid progenitors showed that for CFUen Do=206 rad and n=1.3; for BFUen Do=118 rad and n=1.4; the proportion of proliferating CFUen was 42% +/- 12.5 and that of BFUen 46% +/- 4.5. The data obtained confirm the hypothesis of the erythropoietin-independent nature of the erythroid progenitors expressed under the culture conditions used.