We have demonstrated that viability of preimplantation mouse embryos F1 (C57B1/6 x CBA) after cryoconservation at the stage of four blastomeres improves after pretreatment with serotonin (5 HT, 5 microM) or total gangliosides of bovine brain gangliosides (TG, 3 microM) added to the cultivation medium. After thawing, 64% of embryos preincubated with total brain gangliosides and 49% of embryos preincubated with serotonin developed to the stage of blastocyst during the cultivation in vitro; in the control, no more than 25% of embryos reaches this stage, and all these embryos were abnormal. Possible mechanisms of protective action of these compounds is discussed. We conclude that mouse embryos subjected to freezing-thawing procedure can be used to examine the role of serotonin and gangliosides in the regulatory processes of mammalian preimplantation development.

Bone marrow cells of CBA males were transplanted to irradiated syngeneic females and after 13 days the mean frequency of blood erythrocytes with micronuclei, cloning efficiency of haemopoietic stem cells (HSC), and variability range of frequency of cells with micronuclei in spleen colonies were studied in 44 primary recipients. A wide range of variability has been shown for the indicators of mutability and cloning efficiency (0.1-1.8% for frequency of erythrocytes with micronuclei, 200-300-1500 for cloning efficiency, 0.1-0.5% for frequency of cells with micronuclei in spleen colonies of individual transplants). A weak negative correlation between the frequency of erythrocytes with micronuclei and the cloning efficiency was observed. We conclude that the difference in frequency of erythrocytes with micronuclei between HSC subpopulations is sufficiently high to carry out an artificial selection for high and low mutability in the course of transplantations. The cloning efficiency can be increased when spleen colonies are studied by micronuclear analysis. Data on the importance of such a selection in studying the behavior of "mutability" and "cloning efficiency" characters, immortalization, "aging", and death of HSC populations of CBA mice have been reported.

Erythropoesis activation, realizing by microenvironmental cells, was noted in hepar of the tailless amphibians R. Lessonae and R. Ridibunda, living in the Chernobyl AES alienation zone. Destructive changes, and the ribosome-protein complexes, fibrillar structures synthesis intensification was revealed in hepatocytes. Ribosome-protein complexes and fibrillar-granular material of hepatocytes enter the blood stream, where they incorporate into the erythroblasts cytoplasm. Focal hyperplasia and macrophages hypertrophy were revealed. Macrophages, transporting the hepatocytes cytoplasm components into erythroblasts and supplementing them with their cytoplasm synthesis products, take an active part in supply of differentiating red blood cells with different materials.

Cytophotometry and population analysis of rat bone marrow erythroid cells, during anaemia induced by phenylhydrazin (4-8 days after the beginning of infections), revealed that in the period of restoration of erythron after the rats acute anaemia in the bone marrow blood formation the reciprocally complementary processes of microcytosis, macrocytosis and normal erythropoiesis occur. These processes are based on the normally functioning regulation mechanisms and on the reserve mechanisms, including DNA hyper-replication, which are turned on in the extreme conditions. The various paths of the bone marrow erythroid cells development during anaemia, the means and rates of the micro- and macrocytes formation and part of each line of the erythrocyte development in the restoration of blood cells quantity and haemoglobin homeostasis are analysed. It is suggested that mechanisms of the reserve erythropoiesis, activated during the acute anaemia of adult animals, could function also during the process of normal primary erythropoiesis of embryos.

Cytophotometry of rat blood erythroid cells during anaemia, induced by phenylhydrazin (4-8 days from the beginning of injections), revealed that all forms of bone marrow containing haemoglobin were thrown into the blood. On its peak (4th day), the greatest contribution in blood haemoglobinization (50%) is made by microcytes. From the 5th day and up to the end of the restoration period the important role in this process is played by macrocytes. From the 6th day the role of normocytes increases, whose contribution by 8th day reaches 70% of the whole haemoglobin amount in blood. In contrary to anaemizated birds, whose erythroid cells ripen in blood, in rats all the transformations of erythron during anaemia are accomplished in bone marrow.

Passaged bone marrow fibroblasts (PBMF) fail to maintain long-term hemopoiesis in culture unlike stromal adhesive layers of long-term culture of the bone marrow (LTCBM). What differs PBMF from LTCBM stromal cells in terms of the above ability we attempted to find out using immunocytochemical microscopy and flow cytometry. There were no differences as to protein production of the extracellular matrix, production of granulocytic-macrophagal colony-stimulating factor, stem cell factor, transforming growth factor beta, interleukin-1 beta and interleukin-6. The difference lies in inability of PBMF to produce granulocytic colony-stimulating factor (GCSF). It is evident that production of GCSF by stromal cells of the bone marrow is essential for maintenance of myelopoiesis in LTCBM.

Eighteen patients with relapsed or refractory Hodgkin's disease (HD) have been treated with high-dose chemotherapy (BEAM regimen) followed by autologous peripheral stem cells and/or bone marrow rescue. There were no treatment-related deaths. Overall response rate was 82%. With a median follow-up of 10 months (3-24 months) overall survival and freedom from progression were 100 and 94% (95% confidence interval 58-97%), respectively. The use of peripheral stem cells in addition to bone marrow resulted in a significant shortening of the time to engraftment (p < 0.01). The BEAM regimen is an effective conditioning schedule which is well tolerated.

We have carried out a study of the bone marrow status in both irradiated and non-irradiated zones of 56 patients with stage I-II Hodgkin's disease in complete 9-12 (33 patients, group 1) and 18-23 (23 patients, group 2) year remission after therapeutic irradiation of the supradiaphragmatic lymphatic collectors at a dose of 40 Gy with irradiation of the spleen (33 patients) or splenectomy (23 patients). The total count of myelokaryocytes, myelogram, a relative and absolute content of lymphoid cells, immature granulocytes and elements of erythroid series were calculated in the aspirates from the exposed to radiotherapy sternum and non-irradiated upper portion of the ileum. The number of granulocyte-macrophage (CFU-GM) and stromal (CFU-F) precursor cells were defined using in vitro culture technique. There was a complete annihilation of the bone marrow in the irradiated zones, when the dose exceeded 35 Gy in 3-4 weeks. The concentration of myelokaryocytes, immature granulocytes, erythronormoblasts, CFU-GM, CFU-F in non-exposed bone marrow were significantly lower in all patients of group 2 than in normal subjects and in group 1 patients. Absolute lymphoid count in patients with 18-23 year remission was found to be normal but was considerably reduced in comparison to patients of group 1. These changes may be the result of the previous hyperactivity of the non-irradiated bone marrow which could be a cause of stem cell compartment depletion. The differential calculation of compact and diffuse subpopulations of CFU-F revealed a significant reduction of compact colony-forming CFU-F in both irradiated and unexposed bone marrow. Almost all the stromal precursor cells from irradiated zone formed diffuse colonies in cultures. These results confirm experimental data concerning greater radiosensitivity and proliferative potential of CFU-F, forming compact colonies versus diffuse colony-forming CFU-F. Aplasia of the irradiated bone marrow and hypoplasia of the non-irradiated bone marrow 18-23 years after radiotherapy completion coexisted with normal circulating CFU-GM and granulocyte blood count suggesting a compensatory mechanism involving a mitotic amplification between the progenitor cell and the final differentiated cell.

Effects of stromal environment on human leukemic cell apoptosis and proliferation have been investigated. The MS-5 cell line (kindly provided by Dr.Itoh) capable of supporting human hemopoietic cells and the K562 human erythromyeloid leukemic cell line were used. The total increment of cultured leukemic cells was lower than in control due to their co-cultivation with stromal cells, 96% of cells remaining viable. Colony formation by K562 cells appeared to be significantly lower, too (7.2 vs. 22 colonies per 10(3) cells). Inhibition by stromal cells was reproduced when stromal and leukemic cells were separated by a 0.5% agar layer as well as when leukemic cells were cultured on an agar layer which contained a 50% condition medium. Morphological signs of apoptosis in 78.75 +/- 7.64% of leukemic cells, as a result of serum deprivation, were in evidence after 72 hr. while only 14.33 +/- 3.69% of cells co-cultured with MS-5 died. Only 4% of adherent cells showed morphological signs of programmed death. Since protein bcl-2 was not detected in K562 cells it may be suggested that apoptosis of leukemic cells co-cultured with stromal ones does not use the bcl-2 gene pathway. Stromal cell-mediated inhibition of proliferation may be effected through humoral mechanisms.

Local and distant hemopoietic regulatory mechanisms were studied under various emergencies (immobilization, cytostatic injection, radiation, etc.). There were common processes (cell migration, destruction, T-lymphocyte accumulation, activation of the microenvironment and hemopoietic precursors) forming the local basis for compensatory and adaptive reactions of bone marrow hemopoietic tissue. Emphasis is laid on the association of local mechanisms with the neuroendocrine system which inevitably becomes activated under extreme exposures. On exposure to irritants having no myeloinhibitory effect, neurotransmitters and hormones, mainly glucocorticoids and catecholamines cause the development of erythro- and granulocytopoietic hyperplasia due to stimulation of the hemopoiesis-inducing microenvironment (HIM). Under hemopoiesis-suppressing influences, catecholamine-induced activations of HIM's resistant elements is accompanied by simultaneous inhibition of hemopoietic cells and HIN's factors damaged by an irritant. Under these conditions hemopoietic separation caused by the heterogeneous effect of transmitters on the cell leads to imbalance of hemopoietic precursor proliferation and differentiation and, finally, to delayed hemopoietic tissue regeneration. At the same time, the neuroendocrine system does not seem to play a leading role as compared to the control intact hemopoiesis.

The capacity of CFU-S-11, which formed colonies in the spleen on day 11 after transplantation to an irradiated recipient for self-maintenance, was studied. The indices for self-renewal of individual CFU-S from the spleen, bone marrow, and peripheral blood were compared in the same sexually mature donor mice by retransplanting the colonies isolated from the spleen parenchyma to secondary recipients. The number of secondary CFU-S-11 per primary CFU-S-11 originating from the liver hardly differed from that originating from the peripheral blood, but is almost twice less than for the bone marrow CFU-S-11. The conclusion was drawn that compartment CFU-S of the liver of adult animals contains the cells migrating from blood flow, which, apparently, are not dormant embryonic cells.

The authors propose their own system of assessment of high-dose polychemotherapy toxicity. The system was applied to toxic complications of high-dose polychemotherapy in 31 patients with hematological malignancies subjected to allogenic, autologous bone marrow transplantation and transplantation of stem cells from peripheral blood within the scope of different protocols of high-dose polychemotherapy in conditioning regimen. A special scale developed in the Belarus Center for Bone Marrow Transplantation basing on the above system provides prediction of survival in early post-transplantation period.