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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.

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.

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.

To study and compare cytogenetic abnormalities in the bone marrow (BM) and peripheral blood (PB) CD34+ hematopoietic progenitor cells and in the BM mesenchymal stromal cells (MSCs) in patients with myelodysplastic syndrome (MDS). Subjects and methods. The results of a cytogenetic analysis of the total population of BM cells (BMC), CD34+ hematopoietic progenitor cells from BM and PB, and BM MSCs were analyzed in 35 patients (29 patients with MDS and 6 with MDS transformed into acute myeloid leukemia (AML)) and 7 healthy BM donors. Cytogenetic examinations were performed by G-banding of chromosomes and fluorescence in situ hybridization (FISH).The BMC karyotype was abnormal in 17 (49%) of the 35 patients (13 with MDS and 4 with AML); the others were found to have a normal BM karyotype. The FISH analysis confirmed the same cytogenetic abnormalities in the BM and PB CD34+ hematopoietic progenitor cells in all the examinees with an abnormal karyotype. The mean abnormal clone sizes in the total population of BMCs and BM and PB CD34+ progenitor cells did not differ and constituted 65.8, 73.1, and 74.8%, respectively. The patients with a normal BM karyotype had no chromosome abnormalities in the CD34+ cells either. The karyotype of MSCs was analyzed in 23 (19 with MDS and 4 with AML) of the 35 patients. No karyotype abnormalities were revealed in the patients with MDS transformed into AML. There were structural chromosome aberrations in 2 (11%) of the 19 patients with MDS (one with constitutional inv(9)(p1 3q21) was found to have non-clonal translocation t(2;22)(p10;q1 1) and the other had a clone with an additional segment of the long arm of chromosome 2 in 35% of the cells. No numerical MSC karyotype abnormalities were detected. A normal MSC karyotype was defined in 7 healthy BM donors.The cytogenetic analysis of hematopoietic and mesenchymal progenitor cells showed that the chromosome abnormalities revealed in these cell populations were different in the patients with MDS. The isolated CD34+ cells displayed the same cytogenetic abnormalities as in a total population of BMC. Examination of the latter could reveal no cytogenetic abnormalities in the majority of the patients. A normal BMC karyotype was detectable in the patients with AML. Two (11%) patients with MDS were found to have structural chromosome abnormalities that differed from those detected in the total population of BMC and in isolated CD34+ cells. The differences of chromosome abnormalities in the hematopoietic and mesenchymal progenitor cells point to the fact that the stromal microenvironment is not part of the abnormal clone in MDS, however, it may be of great importance in the pathogenesis of the disease.

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.

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.

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.

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.

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.