Effect of erythrocyte breakdown products on mast cells and erythropoietin formation]

Experiments were conducted on CBA mice and albino rats. A study was made of the effect of erythrocyte destruction products (EDP) on the content of hemopoietic colony-forming units (CFU), differentiation of stem cells and the erythropoietin production. It was shown that 3 or 4 EDP injections to normal mice or to lethally irradiated (1000 rad) mice after the transplantation of bone marrow cells caused no changes in the CFU level of stem cells differentiation. In case of a daily (for 3 days) administration of EDP to mice before the irradiation (1000 rad) and bone marrow transplantation there was observed an increase of the colonies count in the recipients' spleen on account of the erythroid colonies. EDP injection caused no changes in the erythropoietic activity of the blood serum. A possible role of erythrocyte destruction products in the mechanism of erythropoiesis autoregulation is discussed.     

Relationship between hematopoietic stem differentiation and the functional state of the thymus]

The direction of differentiation of the stem cells with respect to the physiological activity of thymus determined by the age of an animal was studied by means of histological analysis of hemopoietic colonies in the spleen of lethally irradiated mice. The immaturity of thymus of its involution are characterized by the inhibition of differentiation of the stem cell along the granuloid path. An analysis of the data on differentiation of the stem cells in mice of different age, as well as in thymectomized mice allows to draw a conclusion that the process of differentiation of the hemopoietic stem cells is thymus-dependent.     

Effect of a neutrophilia-inducing tumor on hemopoietic stem cells in mice

The influence of neutrophilic stimulation on hemopoietic stem cells was studied in mice with tumor-induced neutrophilia. Transfusions of marrow cells from normal and neutrophilic tumor-bearing mice into lethally irradiated normal and tumor-bearing mice were performed. The number and the erythroid:granuloid (E:G) ratio of day 7 colonies in the recipient spleens and bones as well as the size of spleen colonies of recipient animals were determined. The E:G ratio of spleen and bone marrow colonies between normal and tumor-bearing mouse recipients and the number of spleen colonies did not differ significantly in either experiment. However, spleen colonies which developed in tumor-bearing irradiated mice were significantly larger than those which developed in normal recipients in both experiments. These studies indicated that while the line of differentiation taken by hemopoietic stem cells was not affected by the neutrophilic influence of the tumor, the tumor-bearing host environment appeared to enhance proliferation of transfused stem cells and/or their descendants. The stimulators of granulocytopoiesis in this model of neutrophilia appear to act on a population of progenitor cells more mature than the stem cells capable of forming 7-day colonies in the spleen and bone marrow of irradiated recipient mice.     

Thymus-dependent suppression of erythroid colony formation induced by concanavalin A in the cloning of pluripotent hematopoietic stem cells of the spleen

The injection of the polyclonal activator of T-cells--concanavalin A--to normal mice is followed by a considerable decrease both in the erythroid colony-formation at the cloning of spleen CFUs, and in the number of T-cells and their helper activity in the same spleen suspensions. The suppression of erythroid colony-formation may be passively transferred by using spleen cells from mice treated with concanavalin A and irradiated lethally. Thymectomy of adult mice prevents from suppression of erythroid colony-formation induced by concanavalin A. These results suggest that the T-cells may suppress erythroid differentiation of hemopoietic stem cells.     

Effect of prolonged exposure to a magnetic field on the haematopoietic stem cell.

The authors studied the effect of prolonged exposure (3, 4 and 5 months) to the action of a magnetic field of 180-200 gauss formed by the poles of a rotating permanent magnet on the haematopoietic stem cells of mouse bone marrow donors. The effect of the field was evaluated from the ability of the donors' bone marrow cells to form haematopoietic colonies in the spleen of lethally irradiated mice. It was found that the number of stem cells was not reduced by the action of the above magnetic field and that proliferative capacity was likewide unimpaired.     

Comparative studies on the characteristics of proliferation and differentiation of spleen colony-forming cells

Using a single spleen colony transplantation technique and sex chromosome typing as a natural cytogenetic marker, most spleen colony-forming cells (CFC) in adult bone marrow or fetal livers of inbred LACA or C57 mice re-established hemopoiesis in lethally irradiated mice when the spleen colonies were sampled at 13 days after transplantation. However, most of the spleen colony-forming cells in the peripheral blood of normal mice possess little potential for proliferation and are less efficient in the re-establishment of hemopoiesis in lethally irradiated mice. The CFC population is heterogeneous in the mice. From the subsequent retransplantation of colonies from colony-forming cells in the peripheral blood, the simple assessment of spleen colony-forming units (CFU-s) content, based on the number of splenic colonies, does not reliably represent the content of hemopoietic stem cells.     

Effect of herbal preparation, brahma rasayana, in amelioration of radiation induced damage

Oral administration of brahma rasayana (BR; 10 and 50 mg/dose/animal) for 15 days increased significantly total leukocyte count and percentage of polymorphonuclear cells in irradiated mice. Bone marrow cellularity and alpha-esterase positive cells also increased significantly in radiation-treated animals after BR administration. Number of nodular colonies on the surface of spleen on day seven increased significantly in lethally irradiated recipients receiving bone marrow cells from animals treated with BR. Oral administration of BR also enhanced in serum level of interferon-gamma (IFN-gamma), interleukin-2 (IL-2), and granulocyte macrophage-colony stimulating factor(GM-CSF) in normal and irradiated mice. These results indicated that proliferation of stem cells induced by BR in irradiated mice may be related to its stimulation of cytokine production.     

Mechanism of action of the stem-cell inhibition factor on the formation of exogenous hemopoietic colonies in the spleen of mice

It was established by previous works that thymocytes treated with antilymphocyte serum secrete soluble factor capable of inhibiting exogenous colony formation in the spleen of lethally irradiated mice injected with bone marrow cells treated with the stem cell inhibition factor (SCIF). The purpose of the present investigation was to explore possible mechanisms of SCIF action. Regeneration of erythropoiesis (measured by 59Fe incorporation) in the spleen and bone marrow of mice injected with SCIF-treated bone marrow cells was inhibited as compared with control, while CFUs started proliferating with a 3-day delay. Two hours after SCIF treatment 60% of CFUs entered S phase as judged by hydroxyurea cell kill. The CFUs fraction treated with the SCIF was found to be diminished 3-4-fold as compared with control. The data obtained suggest that SCIF treatment makes CFUs enter 3 phase, which may account for the reduced capacity of CFUs to populate the spleen and to proliferate with a 3-day delay.     

Mock retroviral infection alters the developmental potential of murine bone marrow stem cells.

Retroviral vectors were used to introduce an activated ras gene into murine pluripotent hemopoietic stem cells. We attempted to reconstitute the hemopoietic system of lethally irradiated mice with isolated spleen colonies obtained in vivo after injection of infected bone marrow cells. Spleen colonies derived from infected bone marrow were inefficient in promoting long-term survival of irradiated hosts. This loss of reconstitutive capacity of spleen colonies was not due to the retroviral infection per se but to the in vitro culture of spleen colony precursors. Incubation for 24 h in the presence of fetal calf serum and interleukin-3 without virus-producing cells was sufficient to abolish completely the reconstitutive capacity of spleen colonies while maintaining both self-renewal and pluripotential capacities of spleen colony precursors. These results show that the in vitro manipulation of stem cells that is included in current protocols for retroviral infection can modify the developmental potential of these cells. This finding clearly indicates that the use of retroviral vectors can introduce a bias in the analysis of hemopoiesis.     

Studies of delayed hypersensitivity to L. Monocytogenes in mice: nature of cells involved in passive transfers

C3Hf/Umc mice were immunized by an intravenous injection of a sublethal dose of live Listeria monocytogenes. The animals developed delayed-type hypersensitivity (DH) concomitant with infectious immunity to this organism. Delayed hypersensitivity could be transferred to normal lethally irradiated mice with spleen cells from immune animals. The immune cells cells responsible for transfer of adoptive immunity were susceptible to in vitro cytolytic action of anti-theta iso-antibody and complement, since such treatment rendered these cells incapable of further passive transfer of specific immunity to Listeria. The acquired DH to Listeria persisted in mice after 900 R lethal irradiation, provided normal syngeneic bone marrow cells were also administered, thus indicating the persistance of a cell population in the immune irradiated mice, resistant to effects of radiation. The radio resistant nature of this immune cell population was further demonstrated by passive transfer with spleen cells, derived from preimmunized lethally irradiated mice to normal syngeneic mice or to lethally irradiated nonimmunized hosts reconstituted with normal bone marrow which then responded to antigenic challenge with DH.Treatment of the immune radio resistant spleen cells in vitro with anti-theta and complement eliminated passive transfers of DH by these cells; however, this effect was less obvious than similar treatment of the immune, nonirradiated, spleen cells.     

Smad3基因剔除对小鼠造血功能的影响

研究Smad3基因剔除对小鼠造血功能的影响。实验小鼠分为 5组 ,每组有Smad3基因剔除小鼠(Smad3 - - )和其同窝孪生的野生型小鼠 (Smad3 + + )各 1只。小鼠的造血功能用 14天形成的脾结节 (CFU S1 4 )、多系祖细胞 (CFU GEMM)、粒 单系祖细胞 (CFU GM)、红系祖细胞 (BFU E)测定及外周血象、骨髓象等实验血液学指标来确定。每组小鼠取尾血作白细胞、红细胞和血小板计数 ,涂片作白细胞分类计数。将一侧股骨的骨髓冲出 ,制成单细胞悬液 ,计数其中有核细胞数 ,测定CFU GM、BFU E、CFU GEMM值。将每只小鼠的 4× 10 4个骨髓有核细胞 ,经尾静脉注入 3只 8～ 10周经致死量射线照射的同系雌性小鼠体内 ,测定 14天的CFU S。取一部分胸骨、肝脏、脾脏固定做病理切片 ,其余胸骨冲出骨髓 ,涂片作分类计数。结果Smad3 - - 小鼠外周血白细胞和血小板计数明显高于Smad3 + + 小鼠 ,红细胞数无显著差异。外周血白细胞分类结果也表明粒细胞显著增高。骨髓有核细胞数无显著差异 ,CFU GM显著增高 ,BFU E无显著差异 ,CFU GEMM明显减少 ,CFU S显著减少。病理形态学观察发现骨髓增生极度活跃 ,以粒系为主 ,肝脾无显著差别。骨髓涂片分类表明粒系增多 ,粒系 :红系比例增高。因此得出结论Smad3基因剔除使小鼠造血干祖细胞数目     

Effect of various factors on the differentiation of hematopoietic stem cells]

The treatment in vitro of bone marrow cells in mice by phytohemagglutinin, concanavaline, or antilimphocytic globulin resulted in the suppression of exogenous hemopoietic colonies in the spleen of lethally irradiated (830r) syngenic recipients, whereas lipopolysaccharide, tuberculin, anti-theta serum or nati-gamma-globulin serum exerted no influence on the colony-forming function of hemopoietic stem cells. The morphological analysis of the ratio and cell composition of hemopoietic colonies has revealed no marked differences between the experimental and control groups. The suppression of hemopoietic stem cells by mitogens might be due both to their direct effect and indirect one, possibly, through a humoral factor.     

Therapeutic efficiency of spleen or bone marrow CFU in X-irradiated 89Sr marrow-ablated mice.

Experiments were carried out to compare the therapeutic efficiency (TE: number of CFU required to reduce the mortality from 100 to 50 per cent) of spleen or marrow (BM) stem cells (CFU) grafted into lethally irradiated mice (807 rad) which had been previously treated with 89Sr or splenectomized. It was found that during the reconstitution of the haemopoietic organs, the spleen does not provide more than 10 per cent of the functional cells necessary for survival. Besides, the BM-derived CFU growing in 89Sr marrow-ablated mice remain twice as efficient as the spleen-derived ones. Similarly, spleen-derived CFU transplanted into splenectomized mice are half as efficient as BM-derived ones. It may therefore be assumed that haemopoietic stem cells grafted into a foreign microenvironment retain their original kinetics of growth and differentiation during 7 to 10 days after their transplantation.     

Evidence for the existence of multipotential lympho-hematopoietic stem cells in adult rat

Rats were given near-lethal doses of x-ray to produce clones of hemic cells marked by radiation-induced chromosome abnormalities. Subsequently, bone marrow from these rats was injected into lethally irradiated mice to form erythropoietic spleen colonies; and peripheral blood lymphocytes from the same rats were stimulated to proliferate in a mixed lymphocyte interaction (MLI), an immunological response to histocompatibility isoantigens. Chromosome markers indicated that in several instances the cells of an erythroid spleen colony and a proportion of the lymphocytes reacting in the MLI were progeny of the same stem cell in the donor rat. In addition, lymphocytes of the same radiation-marked clone were shown to proliferate in response to several different histocompatibility isoantigens. The data indicate the presence in the adult rat of a primitive lymphohematopoietic stem cell capable of yielding both erythroid and lymphoid progeny. The findings also suggest that immunological specificity is determined during lymphoid differentiation, subsequent to the stem cell stage.     

Persistence of lpr/lpr-derived IgG2a secreting B cells in normal----lpr/lpr adult radiation chimeras or lpr/lpr----normal kidney capsule chimeras.

Lethally irradiated MRL/lpr mice reconstituted with bone marrow stem cells from a normal mouse strain develop a state of split hematopoietic chimerism; erythrocytes, granulocytes, and macrophages are derived from the normal stem cell inoculum while the peripheral T lymphocytes are derived from radioresistant lpr host cells. Moreover, these mice have normal levels of serum IgM and IgG2a produced by radioresistant host B cells, even though they have relatively few sIgM+ B cells. In order to better understand the differentiation and regulation of B cells present in these chimeric mice, the current study was undertaken to localize and to assess the functional capacity of the lpr B cells producing the serum antibodies. Surface IgG2a+ cells could not be found in the spleen or lymph nodes of these mice, but large lymphocytes containing cytoplasmic IgG2 of host (lpr) allotype could be readily detected, even though they constituted less than 1% of the total spleen population. The host-derived serum IgG2 and IgG2+ cells were even present in the spleens of "leaky" mice that had relatively normal numbers of donor-derived sIgM+ B cells. These lpr B cells secreted IgG2a antibody that bound ssDNA, but they could not respond to immunization with SRBC. These results indicate that the lpr-derived radioresistant B cells have a limited capacity for proliferation and are already committed to the memory lineage. The presence of similar B cells in normal mice transplanted with neonatal lpr/lpr spleen fragments suggests that lpr/lpr B cell development is inherently abnormal.     

B lymphocyte differentiation in lethally irradiated and reconstituted mice. I. The effect of Strontium-89 induced bone marrow aplasia on the recovery of the B cell compartment in the spleen.

The influence of ⁸⁹Sr-treatment on the recovery of the B cell compartment in lethally irradiated, fetal liver reconstituted mice was studied by means of membrane fluorescence. ⁸⁹Sr is a bone-seeking radio-isotope which causes in a dose of 3 μCi ⁸⁹Sr/g body weight a depletion of all nucleated cells, including immunoglobulin-bearing (B) cells, of the bone marrow.Treatment of irradiated and fetal liver reconstituted mice with 3 μCi ⁸⁹Sr/g body weight immediately and at 17 days after irradiation and reconstitution prevented recovery of the nucleated cell population, including B cells, in the bone marrow. In the spleen of such mice both nucleated cells and B cells reappeared at day 7 and 14 respectively. The B cell population in the spleen did not recover up to normal values during the experimental period of 45 days. It is concluded that B cell differentiation in lethally irradiated, fetal liver reconstituted mice can take place outside the bone marrow. The efficiency of this extra-medullary differentiation is discussed. The conclusion was drawn that mice with a ⁸⁹Sr-induced bone marrow aplasia are able to generate B lymphocytes. Consequently the bone marrow microenvironment seems not to be obligate to the differentiation of B lymphocytes. The peripheral lymphoid organs of such mice were found to be unable to compensate completely for the absence of B lymphocyte production in the bone marrow.     

Granulocyte factors as potential regulators of hemopoiesis. I. Localisation of hemopoietic activity within granulocytes

Granulocytes stimulate GFU-S in lethally irradiated mice. A factor responsible for this stimulation was found to be contained within granulocyte granules. The factor is released from intact granulocytes during adherence. Granulocyte extract from adherent cells is devoid of this GFU-S-stimulatory activity. The obtained results indicate that factor inducing secretion of granule products indirectly affect hemopoiesis. This may be of particular importance in the course of inflammation.     

Characterization of a cycling murine pluripotent stem cell population

In the present report we describe the properties of pluripotent stem cells isolated with elutriation and Percoll density gradient centrifugation techniques from spleens of recovering thiamphenicol pretreated anemic mice. Cells with a diameter between 7.2 and 8.4 microns and sedimenting at a density of 1.065 g/ml had a high capacity to repopulate lethally irradiated mice with all the hemopoietic precursor cells. The spleen colonies formed on day 8 and day 12 after inoculation in irradiated recipients showed no morphological differences in mixed and lineage-specific composition. Day 12 colonies were much larger than day 8 colonies. Pluripotent stem cells isolated from regenerating spleens were very susceptible to 3'-thymidine; 65% of the cells were killed. Only 2% kill was found in stem cells present in vivo 4 days earlier in the bone marrow. The purified stem cells were probably all in cycle and possibly partly synchronized.     

Plasmodium chabaudi: Adoptive transfer of immunity with different spleen cell populations and development of protective activity in the serum of lethally irradiated recipient mice

Groups of lethally X-irradiated NIH mice were injected with either glass wool-filtered (g.w.) immune spleen cells or nylon wool enriched immune T cells from syngeneic mice immune to Plasmodium chabaudi, or g.w. normal spleen cells. After cell recipients were infected with P. chabaudi the three groups reached similar mean peak parasitaemias on Day 11. In passive transfer tests serum obtained from mice sacrificed at this time gave little protection compared to normal serum. On Day 14 g.w. immune spleen cell recipients had subpatent infections and enriched immune T-cell recipients had a lower mean parasitaemia than g.w. normal spleen cell recipients. Serum obtained on Day 14 from g.w. immune spleen cell recipients gave better protection after passive transfer than sera from enriched immune T-cell or g.w. normal spleen cell recipients. Day 14 serum from enriched immune T-cell recipients, but not from g.w. normal spleen cell recipients, produced some initial protection after passive transfer. These results suggest that the transferred immune spleen cells contributed to the observed humoral immunity in lethally irradiated recipient mice.     

Developments in modern hematology.

In the past 40 years our concepts about hemopoiesis have been changed dramatically. The results of bone marrow transplantation into lethally irradiated mice since the mid-fifties suggested the existence of a hemopoietic stem cell, which was initially identified as a spleen colony forming cell (CFU-S). Later experiments showed that the stem cell compartment is rather heterogeneous and that the most primitive stem cell, unlike the CFU-S, has the ability for long-term engraftment of an irradiated recipient. Daughter cells of such primitive quiescent stem cells lose their capacity for self-generation gradually with each mitosis and become more and more committed to a specific differentiation lineage. In vitro culture techniques in a serum-free semi-solid medium enabled the establishment and analysis of specific hemopoietic growth factors. Such factors, which are essential for the maintenance, proliferation and differentiation of progenitor cells and the functional activity of mature cells can now be produced with recombinant DNA techniques in pure form and large quantities. Hemopoiesis requires an appropriate microenvironment, consisting of various stromal cell types and an extracellular matrix. Intercellular contacts, adhesion of cells and growth factors to the matrix molecules seem essential in the regulating action of this hemopoietic microenvironment. In long-term bone marrow cultures the development of a stromal hemopoietic microenvironment can facilitate long-term maintenance of stem cells and hemopoietic differentiation. For bone marrow transplantation and infusion of hemopoietic growth factors many clinical indications are well established and our possibilities to interfere in the regulation of hemopoiesis are still growing.