Formation of a heterotopic hemopoiesis focus from the administration of a cryptococcal heteropolysaccharide to bone marrow donors and recipients

The effect of Cryptococcus luteolus (strain 228) heteropolysaccharide on heterotopic bone marrow organ formation was studied. Heteropolysaccharide (Hp) injections were initiated on the next day after bone marrow transplantation and were performed weekly for 30 days. This resulted in the increase in both the cellularity and the weight of the ossicle. The injections performed twice weekly produced the decrease of cellularity while ossicle weight did not change. Higher dose of Hp produced no changes in heterotopic bone marrow organ. Hp injections to donor mice did not result in any changes in heterotopic organ in bone marrow recipients. Thus, this kind of Hp was capable both of stimulating and inhibiting heterotopic bone marrow organ formation in relation to the dose and scheme of administration.     

Effect of a deficiency in the mononuclear phagocyte system and the administration of yeast polysaccharide on the development of a heterotopic source of hematopoiesis

Mononuclear phagocyte system (MPS) deficiency was induced by repeated peritoneal lavage in (C57Bl x CBA) F1 mice. The animals were then used as donors or recipients in heterotopic bone marrow transplantation. Yeast polysaccharide (YP) produced by Cryptococcus luteolus strain 228 was injected weekly (25 mg/kg) during 30 days after bone marrow transplantation under the kidney capsule. Bone marrow transplantation from MPC-deficient mice to intact mice 30 days later resulted in no variations from the control in cellularity and ossicle weight. YP produced an increase in cellularity, but not in ossicle weight. In the opposite experimental scheme (transplantation from intact mice to MPS-deficient mice) an increase in both cellularity and weight was not noticed. YP injections in this case resulted in the reduction of heterotopic organ size to the control level. Possible mechanisms of this phenomenon are discussed.     

Interrelation of the osteogenic and hemopoietic tissues of the bone marrow during the development of estrogenic myelofibrosis

By means of estrogenic myelofibrosis, using the method of heterotopic transplantation of the bone marrow, interrelations of osteogenic and hemopoietic tissues have been studied. Under estrone effect disorders in stromal microenvironment take place at the level of stem osteogenic cells of the bone marrow. Deficiency in cells of monocytic-macrophagal line, inhibiting proliferation of the bone marrow connective tissue cells, contributes to development of myelofibrosis. The hormone acts mainly in committed hemopoietic cells, singly launching them from G0- into S-period of the cycle, and then--into differentiation, accompanied with an enhanced discharge of cells from the bone marrow. There is neither direct, nor indirect dependence between the osteogenic mass and the cellularity of the hemopoietic tissue of the bone marrow. The changes, that take place in the bone marrow under estrone effect, are reversible.     

Role of stromal microenvironment in the regulation of bone marrow hemopoiesis after curantyl administration

Role of the stromal microenvironment in regulation of bone marrow hemopoiesis at the administration of the thrombocyte disaggregant curantyl was studied by the method of heterotopic transplantation of the mice bone marrow. It is shown that the action of curantyl on hemopoiesis is realised through the stem stromal cells of the bone marrow. It is noted that the inhibitory action of the preparation on proliferation of osteogenic precursor-cells is followed by activation of bone resorption processes in regenerating ectopic hemopoietic organ. Under the action of curantyl at low bone marrow cellularity in the focus of heterotopic hemopoiesis and femur an increase of mitotic activity in hemopoietic elements is noted. It is revealed that a phenomenon of ineffective megakaryocytopoiesis with intramedullary destruction of megakaryocytes leads to the local excretion of the thrombocyte released growth factor (TRGF) which has a compensatory character.     

Development of hematopietic and lymphoid tissues in conjoint bone marrow and thymus transplants]

The model of heterotopic transplantation of the mixture of bone marrow and thymus fragments was used to study the interaction of hemopoietic and lymphoid tissues under their direct contact. The bone marrow and thymus fragments of adult mice F1 (CBAXXC57BL) were transplanted separately or in the mixture under the kidney capsule of mice of the same strain. During the whole period of observation (from 10 days up to 14 months), the development of bone marrow and thymus fragments in the joint transplants proceeded independently, no "mixed" stroma appeared, and the stroma of each organ ensured the differentiation characteristic of its organ. The development of joint transplants somewhat differs from that of isolated transplants: on the 10th day a greater amount of hemopoietic tissues was noted in the former; the bone marrow component increases continuously up to 6 months (vs. 1--2 months in the isolated transplants); the bone and hemopoietic tissues predominate in the joint transplants by 14 months, the amount of thymic tissue markedly decreases but it does not disappear completely.     

The interplay of osteogenesis and hematopoiesis: expression of a constitutively active PTH/PTHrP receptor in osteogenic cells perturbs the establishment of hematopoiesis in bone and of skeletal stem cells in the bone marrow

The ontogeny of bone marrow and its stromal compartment, which is generated from skeletal stem/progenitor cells, was investigated in vivo and ex vivo in mice expressing constitutively active parathyroid hormone/parathyroid hormone-related peptide receptor (PTH/PTHrP; caPPR) under the control of the 2.3-kb bone-specific mouse Col1A1 promoter/enhancer. The transgene promoted increased bone formation within prospective marrow space, but delayed the transition from bone to bone marrow during growth, the formation of marrow cavities, and the appearance of stromal cell types such as marrow adipocytes and cells supporting hematopoiesis. This phenotype resolved spontaneously over time, leading to the establishment of marrow containing a greatly reduced number of clonogenic stromal cells. Proliferative osteoprogenitors, but not multipotent skeletal stem cells (mesenchymal stem cells), capable of generating a complete heterotopic bone organ upon in vivo transplantation were assayable in the bone marrow of caPPR mice. Thus, PTH/PTHrP signaling is a major regulator of the ontogeny of the bone marrow and its stromal tissue, and of the skeletal stem cell compartment.     

2 populations of bone marrow cells transfering the hematopoietic environment]

A bone marrow fragment transplanted under the kidney capsule created a focus of ectopic hemopoiesis, whose isze, measured by the number of hemopoietic cells, was proportional to the implant size. Dimensions of the focus proved to be 11/2--21/2 greater in the irradiated than in the intact recipients. Cells building up the focus of heterotopic hemopoiesis had a different radiosensitivity in the intact and irradiated recipients--their Do constituted about 160 and 350 rad, respectively. In this connection it is supposed that two cell populations of precursors took part in the creation of the focus. Their possible relations with the determined and inducible osteogenic precursor cells are discussed.     

Osteogenic properties of adhesive cells in Dexter culture of the mouse bone marrow

Disaggregated cell suspensions obtained by mouse bone marrow fermentative digestion as well as stromal tissue obtained by marrow mild mechanical destruction were explanted. Both methods yield the cultures in which the hematopoiesis duration is comparable with dexter cultures. Adhesive cells from all of these three culture types were resuspended and in the porous gelatin sponges heterotopically transplanted under the kidney capsule of syngenic recipients. In the transplantation site there develops the hemopoietic organ containing reticular stroma, hemopoietic cells, and in most cases the well developed bone tissue. Thus, the adherent layers of mouse bone marrow dexter and similar cultures contain for a long period (not less than 2-3.5 months) the stromal fibroblast population which maintains its osteogenic and hemopoietic microenvironment transfer capacities.     

The precursor hematopoietic cell: its origin in ontogeny, proliferative activity and proliferative potential

Cells responsible for repopulation of irradiated longterm cultures of murine bone marrow and capable of generating CFUs for at least 4-5 weeks after seeding referred here to as primitive hemopoietic stem cells (P-HSC) were assayed by limiting dilution analysis. During development of mice P-HSC can be detected for the first time in the liver of 12-13-day-old embryos and their number is about 10 per organ. At day 17-18 of gestation the number of P-HSC increases ten-fold; however, we could not detect the proliferation of these cells using the technique of hydroxyurea suicide. In the adult mouse P-HSC content is about 100 precursors per femur and their concentration is one P-HSC per 1-2 x 10(5) bone marrow cells. P-HSC content in the spleen is 0.5 per 10(6) cells. In vivo treatment with 5-fluorouracil or hydroxyurea (six injections every 6 h) does not alter significantly the number of P-HSC, although either treatment kills about 99% of CFUs. Several months after reconstitution of lethally irradiated mice with a "small" inoculum of bone marrow cells (0.20-0.35 x 10(6)) the number of bone marrow P-HSC was reduced as compared to that in animals reconstituted by injection of a "large" cell dose (20-35 x 10(6)). These data suggest that P-HSC have limited proliferative potential and are incapable of self-maintenance.     

Kinetics of elemental content changes of bone tissue of mice during evolution under hypokinetic stress

Concentration of 13 elements in bone tissue of mice held in tightly spaced cages for 3 wk, which caused an acute stress reaction, was determined by means of neutron activation analysis. Functionally different bone tissues of mice skeletons—the femur, accomplishing both supporting and dynamic functions; the parietal bone, being practically immovable; and the ectopic bone, newly formed under kidney capsule in the place of syngeneic bone marrow implantation—were analyzed. Similar dynamics of the elemental composition of investigated bones was found: the progressive demineralization owing to the loss of Ca, P, Mg, and Fe is accompanied by the compensatory inclusion of Sr in the bone tissues. In the ectopic bone, it was not as high. During evolution under hypokinetic stress, the microelement concentrations (Zn, Cr, Rb, Ru, Br, Co, Sb) change significantly. Results obtained form the evidence for some system character of osteoporosis at limited mobility.     

Cloning stem cells in the bone marrow of irradiated mice]

Different amount of intact or irradiated bone marrow from syngenous donors was administered to mice irradiated with a lethal dose. There was revealed a linear dependence of the number of the 8-9-day colonies grown in the bone marrow of the femur on the amount of the administered cells, and an exponential dependence on the irradiation dose. Regularity of the stem cell cloning in the bone marrow was analogous to such in the spleen. Radiosensitivity of the colony-forming units (CFU) differed depending on the site (the spleen, the bone marrow) of their colony formation. The CFU settling in the marrow proved to be more radioresistant (D(0) equalled 160-200 P) in comparison with the CFU settling in the spleen (D(0) constituted 80-100 P). It is supposed that a different radiosensitivity of the CFU was caused by the presence of heterogenic population of the stem cells and also by specific peculiarities of the organ (the spleen, the bone marrow) in which the colonies formed.     

The role of cells sensitive to anti-mouse brain serum in the regulation of CFU-S proliferation

CFU-S differentiation and regeneration kinetics in the spleen and femur was studied after treatment of bone marrow cells with RAMB serum. The effect of thymocytes on the rate of CFU-S regeneration was also investigated. It was found that CFU-S regeneration in the spleen was similar in RAMBS-treated and intact cell populations on days 4-14 after transplantation. On the contrary, the rate of CFU-S regeneration in the femur was slower in RAMBS-treated than in intact bone marrow cells. However, the growth rate in the femur could be restored to the normal level by the administration of freshly isolated syngeneic thymocytes to mice pre-injected with RAMBS-treated CFU-S population. The treatment of bone marrow suspension with RAMB serum did not affect the differentiation of spleen colonies. It is suggested that RAMBS eliminates cell population regulating CFU-S proliferation, without affecting its differentiation.     

Effect of dipyridamole on the interrelations of the stromal and hematopoietic tissues of the bone marrow in experimental studies]

By means of heterotopic transplantation of the bone marrow interrelations of the stromal and hemopoietic tissues of the mice bone marrow have been studied at administration of dipiridamol. Effect of the drug to the hemopoiesis is realized via stem stromal cells of the bone marrow. Under the influence of dipiridamol a focus of heterotopic hemopoiesis the osteogenic component in it is present only in 30% of cases in comparison with the control. Inhibition of the stromal component proliferation is accompanied with increasing mitotic activity of the hemopoietic elements against the background of the bone marrow cellularity decrease both in the femoral bone and in the focus of heterotopic hemopoiesis. At administration of dipiridamol a phenomenon of noneffective megakaryocytopoiesis with the intrabone marrow destruction of megakaryocytes, resulting in local release of thrombocyte growth factor, which has a compensatory character.     

ADAPTATION OF HEMOPOIETIC TISSUE RESULTING FROM ESTRONE-INDUCED OSTEOSCLEROSIS IN MICE

The redistribution of hemopoietic tissue resulting from estrone-induced osteosclerosis in the mouse was studied. As the marrow was gradually replaced by bone, extramedullary hematopoiesis in the spleen increased at a rate sufficient to maintain hemopoietic homeostasis. The total numbers of colony forming units (CFU) in the tibia and spleen as well as the proportion of CFU in cycle was assessed. After five injections of estrone, tibial CFUs decreased to 2% of control values whereas splenic CFUs increased approximately nine-fold. The proliferative capacity of the splenic CFU was also increased in the estrone-treated animals. The increased numbers of splenic CFUs as well as the increased proliferative capacity of this compartment are probably related to the ability of extramedullary hematopoiesis in the spleen to compensate for a marrow that has been replaced by bone.     

An experimental analysis of factors affecting the localization of embryonic bone marrow

Summary The present investigations have been concerned with factors which determine and influence the localization and development of hemopoietic bone marrow in the embryo mouse and the adult. These studies, which have employed organ cultures and the transplantation of mouse embryo femur and tail rudiments, indicate that the surrounding mesenchyme is required for the normal development of the cartilage rudiment and its ossification, and for the formation and colonization of the marrow cavity. It was suggested that hemopoiesis results from the colonization of the “prepared” marrow cavity by stem cells arising from sources external to the rudiment. The addition of erythropoietin and L-thyroxine produced distinct erythropoietic differentiation in the normally myelocytic embryonic marrow cavity. The significance of the microenvironment present in developing bone rudiments and the initiation of hemopoiesis in stem cells was discussed. A hypothesis was developed to explain marrow localization in adults based on the colonization of bone rudiments which are developing their marrow sites at a time when the blood contains large numbers of colony-forming units.     

Origin of stromal bone marrow mechanocytes according to the results of typing them by isoantigens and chromosomal markers]

The bone marrow of radiochimaeras and heterotopic bone marrow transplants were used to study the origin of precursors of the fibroblasts growing in the monolayer cultures of hemopoietic tissue. In the bone marrow explants of the (C57BL/6 X CBA) F1 mice, in which the CBA bone marrow was transplanted following the lethal irradiation, the fibroblasts grown in the colonies were of recipient origin judging by isoantigens in the reaction of indirect immunofluorescence with the anti-C57BL/6-serum. At the same time in the bone marrow explants from heterotopic transplants (CBA leads to CBA X C57BL/6) the fibroblasts grown in colonies were of donor origin. The cultures of hemopoietic cells of the bone marrow of females heterotopically transplanted in the singenic male (guinea pigs Huston) contained only fibroblasts which were of donor origin judging by sex chromosomes in the metaphase plates of dividing cells. Hence, the bone marrow precursors of fibroblasts do not depend histogenetically on hemopoietic cells and are not replaced at the expense of repopulating cells of the second partner.     

Induction of hydroxyapatite resorptive activity in bone marrow cell populations resistant to bafilomycin A1 by a factor with restricted expression to bone and brain, neurochondrin

Bone, one of the favored sites for tumor metastasis, is a dynamic organ undergoing formation and resorption. We found bone metastasis with osteolytic lesion in the bone marrow of the femur by injecting BW5147 T-lymphoma cells into the tail vein of AKR mice. To understand this bone destruction, we constructed a cDNA library from BW5147 with a cloning vector that allowed in vitro synthesis of mRNAs, and then identified a particular cDNA clone by adding the conditioned medium from Xenopus oocytes following injection of the mRNA synthesized in vitro to primary bone marrow heterogeneous cell populations on hydroxyapatite thin films. By means of this method, we isolated a factor with 16% leucine residues, termed neurochondrin, that induces hydroxyapatite resorptive activity in bone marrow cells resistant to bafilomycin A1, an inhibitor of macrophage- and osteoclast-mediated resorption. Expression of the gene was localized to chondrocyte, osteoblast, and osteocyte in the bone and to the hippocampus and Purkinje cell layer of cerebellum in the brain. This may provide insights into the molecular mechanisms underlying bone resorption with potential implications for the activation of cells other than macrophages and osteoclasts in bone marrow cells.     

Corticosteroids and the humoral immune response of mice. II. Enhancement of bone marrow antibody formation to lipopolysaccharide by high doses of corticosteroids.

The effect of injection of the synthetic corticosteroid dexamethasone sodium phosphate upon the primary response to Escherichia coli lipopolysaccharide (LPS) was studied in mouse spleen and bone marrow. Daily corticosteroid injections, starting 1 day before immunization with LPS, could suppress the anti-LPS plaque-forming cell (PFC) response in the spleen. The higher the dose of corticosteroids, the more the splenic PFC response was suppressed. On the other hand, the bone marrow PFC response showed a dose-dependent enhancement after corticosteroid injections. This effect was maximal when tested 7 days after antigen injection, and constituted a 3- to 15-fold increase after daily injection of 16 mg dexamethasone/kg body wt. The same effect was found in genetically athymic nude mice, showing that the corticosteroid-mediated enhancement of the anti-LPS PFC response in the bone marrow is not due to elimination of T suppressor cells. Probably the differential effect of corticosteroids upon antibody formation in spleen and bone marrow is due to a redistribution of B-lineage cells, with a resulting accumulation in the bone marrow.     

The action of dipyridamole on megakaryocytopoiesis in regenerating and stationary bone marrow cell populations]

The effect of dipyridamole on megakaryocytopoiesis in regenerating and stationary populations of mouse bone marrow cells has been studied by heterotopic transplantation of the bone marrow using histological, electron microscopic and biochemical techniques. It is shown that drug administration induced destruction of megakaryocytes. In megakaryocytic cytoplasm giant lipid granules were found whose growth and number increase resulted in megakaryocytes kill. Gas-liquid chromatography was used to evaluate the effect of dipyridamole on distribution of lipid fatty acids of the stationary and regenerating populations of the bone marrow cells. A marked increase of the percentage of docosahexaenoic acid was found in lipids of the stationary population. Chronic dipyridamole administration caused an increase of percentage of myristic, palmitic oleic acids, and decrease of percentage of arachidonic and eicosapentaenoic acids in lipids of regenerating bone marrow cells population.     

The dosimetry of 224Ra in mouse bone

Calculations are described, based on experimental findings, which show the variation of absorbed dose from 224Ra in bone marrow of CBA/H mice. These calculations indicate that, following an injection of a leukaemogenic amount of 16 kBq 224Ra into these mice, most marrow cells in the cancellous bone of femur ends are killed but most marrow cells in the femur shaft survive. The calculations also suggest that the mean leukaemogenic absorbed dose of about 1.5 Gy is received by a population of marrow cells about 30 microns from bone surface in the femur shaft.