Thrombocytopenia after autologous bone marrow transplantation in in acute lymphatic leukemia]

There is reported about the treatment of refractory thrombocytopenia in a 9 years old boy following the autologous bone marrow transplantation for acute lymphoblastic leukaemia. The megakaryocytes were found diminished in the bone marrow smears. Controls of the thrombocyte count and the kinetics with radioactively labeled platelets of a donor spoke in favour of immunothrombocytopenia. Threatening bleeding complications challenged the use of all treatment possibilities. The irradiation of the spleen was without any success. After the splenectomy the thrombocyte count increased slowly, but after a remarkable lag phase, however. A diminished reproduction capacity of the bone marrow graft for special cell sorts has to be taken into account in such cases. The usual cytodynamics after splenectomy cannot be expected at all.     

Effect of thrombopenia on bone marrow CFU-S level and its capability to modify the circulating thrombocyte number

Thrombocytopenia (rise of the thrombopoietin level) was induced by an antithrombocyte serum in mice. After 6 hours of existence of thrombocytopenia, the CFU-S and megakaryocyte-commmitted stem cell content of the bone marrow and spleen was determined by transplantation into mice pretreated with 800 cGy-rtg irradiation. Thrombocytopenia did not influence the CFU-S content. Thrombocytopoiesis of the recipient mice was better restored by bone marrow and spleen cells of thrombocytopenic animals than by cells transplanted from animals with a normal thrombocyte count.     

Evaluation of nongenotoxic and genotoxic factors modulating the frequency of micronucleated erythrocytes in the peripheral blood of mice

The hematological micronucleus test is regarded as an indicator of the clastogenic effect of chemicals and acute cytogenetic damage. The test can be carried out in red blood cells of the bone marrow and of the spleen, as well as in peripheral erythrocytes. We have determined the precise background values of micronucleated red blood cells for the peripheral blood of BALB/c, DBA/2, and NMRI mice. Bleeding, phenylhydrazine-induced hemolysis, and splenectomy generated an increase of micronucleated erythrocytes in the peripheral blood of mice. Our data thus demonstrate that such factors should be taken into consideration when the micronucleus test is used for screening the genotoxic potential of chemicals. Furthermore, the micronucleus-inducing effect of cyclophosphamide was studied in normal and splenectomized mice and, in addition, a comparison of the sensitivity of the micronucleus test was carried out in peripheral blood and bone marrow after cyclophosphamide treatment. Our data demonstrate that the kinetics of micronucleus formation were similar in normal and in splenectomized mice in which the micronucleus levels had returned to normal. The comparison of micronucleus formation in bone marrow and peripheral blood after cyclophosphamide treatment revealed the generation of similar quantities of micronucleated red blood cells in both tissues. The physiological mechanisms of micronucleus formation and removal and the potential role of chemically induced spleen damage during this process are discussed; the usefulness of the peripheral micronucleus test as a simple, rapid, and animal-saving modification of the standard bone marrow test is evaluated.Abbreviations CP cyclophosphamide - MN micronuclei - MNCE micronucleated normochromatic erythrocytes - MNPCE micronucleated polychromatic erythrocytes - MNRBC micronucleated red blood cells - NCE normochromatic erythrocytes - PCE polychromatic erythrocytes     

Antibody formation in mouse bone marrow. IX. Peripheral lymphoid organs are involved in the initiation of bone marrow antibody formation.

Mouse bone marrow is barely capable of plaque-forming cell (PFC) activity during the primary response to sheep red blood cells (SRBC). However, during secondary-type responses, it becomes the major organ, containing IgM, IgG, and IgA PFC. In the present paper, the influence of splenectomy (Sx) upon the secondary bone marrow PFC response to SRBC was investigated. When previously primed mice were splenectomized just before the second intravenous (iv) injection of SRBC, the effect of Sx upon the height of the bone marrow PFC response was dependent on the booster dose. Sx just before a booster of 10⁶ SRBC iv almost completely prevented bone marrow PFC activity, whereas an iv booster dose of 4 × 10⁸ SRBC evoked a normal IgM, IgG, and IgA PFC response in Sx mice. Apparently low doses of iv administered antigen require the spleen in order to evoke antibody formation in the bone marrow. Experiments with parabiotic mice, consisting of Sx and sham-Sx mice, showed that this facilitating influence of the spleen upon bone marrow antibody formation occurs via the blood stream. In a subsequent study, it was investigated whether the spleen is required throughout the bone marrow PFC response or only during the few days of the initiation phase. Therefore, mice were splenectomized at different intervals after a booster injection of 10⁶ SRBC iv. It appeared that Sx 2 days after the booster injection could still prevent the normal bone marrow PFC activity, whereas Sx at Day 4 could no longer do so. Apparently, after an iv booster injection, the spleen is only required for initiation of the bone marrow PFC response and not for the maintenance of this PFC activity thereafter.     

Evaluation of nongenotoxic and genotoxic factors modulating the frequency of micronucleated erythrocytes in the peripheral blood of mice

The hematological micronucleus test is regarded as an indicator of the clastogenic effect of chemicals and acute cytogenetic damage. The test can be carried out in red blood cells of the bone marrow and of the spleen, as well as in peripheral erythrocytes. We have determined the precise background values of micronucleated red blood cells for the peripheral blood of BALB/c DBA/2, and NMRI mice. Bleeding, phenylhydrazine-induced hemolysis, and splenectomy generated an increase of micronucleated erythrocytes in the peripheral blood of mice. Our data thus demonstrate that such factors should be taken into consideration when the micronucleus test is used for screening the genotoxic potential of chemicals. Furthermore, the micronucleus-inducing effect of cyclophosphamide was studied in normal and splenectomized mice and, in addition, a comparison of the sensitivity of the micronucleus test was carried out in peripheral blood and bone marrow after cyclophosphamide treatment. Our data demonstrate that the kinetics of micronucleus formation were similar in normal and in splenectomized mice in which the micronucleus levels had returned to normal. The comparison of micronucleus formation in bone marrow and peripheral blood after cyclophosphamide treatment revealed the generation of similar quantities of micronucleated red blood cells in both tissues. The physiological mechanisms of micronucleus formation and removal and the potential role of chemically induced spleen damage during this process are discussed; the usefulness of the peripheral micronucleus test as a simple, rapid, and animal-saving modification of the standard bone marrow test is evaluated.Abbreviations CP cyclophosphamide - MN micronuclei - MNCE micronucleated normochromatic erythrocytes - MNPCE micronucleated polychromatic erythrocytes - MNRBC micronucleated red blood cells - NCE normochromatic erythrocytes - PCE polychromatic erythrocytes     

Splenectomy before tumor inoculation prolongs the survival time of cachectic mice

The effects of splenectomy on the development of cachexia, tumor growth and animal survival were studied in tumor-bearing CDF1 mice. Mice were inoculated with two subclones of colon 26 adenocarcinoma, clone 20 (with a potent capacity to induce cachexia) and clone 5 (without such activity), and underwent splenectomy before or after tumor inoculation. Splenectomy significantly prolonged the survival of mice bearing clone 20 when it was performed prior to tumor inoculation, although the progression of cachexia and tumor growth were not affected. The survival rate was higher in splenectomized than it was in nonsplenectomized mice 20–40 days after tumor inoculation. Such effects on survival were not observed, however, in mice splenectomized after inoculation with clone 20 or in mice that underwent splenectomy either before or after inoculation with clone 5. The decrease of peripheral blood lymphocyte count observed in mice bearing clone 20 was magnified when splenectomy was performed before tumor inoculation, but the serum levels of tumor necrosis factor and interleukin-6 were comparable. These results indicate that cancer death from cachexia is not directly attributable to enhanced catabolism. The mechanism by which splenectomy ameliorates the survival of cachectic mice remains to be studied, although several changes observed in the splenectomized mice after inoculation, including decreases in the peripheral blood L3T4⁺ cells and Lyt-2⁺ cells on the 9th day and 15th day respectively, and increase in the L3T4⁺/Lyt-2⁺ cell ratio on the 15th day suggest the involvement of the modified host's immune response.     

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.     

造血干细胞移植条件对小鼠造血重建的影响

通过同种基因型小鼠构建造血干细胞移植模型,将预处理的全骨髓单个核细胞或c-Kit⁺造血干细胞移植至致死剂量照射的受体小鼠体内,动态监测移植2～16周后受体小鼠体内供体来源细胞造血重建以及嵌合情况,以期揭示不同群体的供体细胞以及预处理等因素对小鼠造血干细胞移植后造血重建的影响。实验结果显示,移植后早期(2周)全骨髓单个核细胞组髓系比例要高于c-Kit⁺细胞移植组,但全骨髓移植组受体小鼠呈现出较大的移植后不良反应,出现脱毛、食欲不振以及体重减轻的症状。c-Kit⁺细胞移植组在淋系重建上要早于全骨髓移植组,供体细胞的嵌合植入也早于全骨髓移植组,但两组实验组最终均能完成造血重建过程。实验结果表明c-Kit⁺细胞移植组在移植后能够较快地实现供体细胞植入,进而开始造血重建,且c-Kit⁺细胞移植组的不良反应要低于全骨髓移植组。结果说明在整体造血重建效果上c-Kit⁺细胞移植组要优于全骨髓移植组。     

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.     

Studies on the maturation of immune responsiveness in the mouse. II. Role of the spleen.

Experiments were designed to investigate the role of the spleen in the development of the murine immune system. By using mice splenectomized within 24 hr of birth, as well as mice with a hereditary, congenital absence of the spleen, the primary immune response to sheep erythrocytes was examined. The immunocompetence of lymph node cells from spleenless or control mice was assessed in vitro, in organ and in cell suspension cultures, and in vivo, by transfer into lethally irradiated syngeneic recipients followed by antigenic stimulation. The immunologic capacities of thymus and bone marrow cells were similarly tested by injection separately or in combination into irradiated syngeneic mice. Lymph node cells from spleenless animals appeared fully competent both in vitro and in transfer experiments. Neither neonatal splenectomy nor congenital absence of the spleen significantly reduced the capacity of bone marrow or thymus cells to participate in the immune response to sheep erythrocytes.     

脾加骨髓细胞移植治疗小鼠大肠癌的实验研究

目的:探讨半相合脾加骨髓细胞移植治疗小鼠大肠癌的效果及其对嵌合体水平和移植物抗宿主病(GVHD)的影响。方法:以接种CT26大肠癌细胞的BALB/c×C57BL/6杂交Fl代雌性小鼠为受鼠,以健康雌性Fl、雄性C57BL/6、雄性C3H小鼠为MHC全相合、半相合、不相合供鼠,观察移植后的抑瘤情况;另设5只化疗联合半相合脾加骨髓细胞移植的小鼠为监测组,用来作嵌合体的分析,观察各组GVHD的情况。结果:经化疗预处理的脾加骨髓细胞移植组小鼠肿瘤明显缩小,与单纯化疗未进行移植组比较,差异具有统计学意义(P0.05);化疗联合半相合脾加骨髓细胞移植的小鼠于移植3周后达完全供者植入,于移植后第10天左右出现纳差、倦怠、步态不稳、脱毛、腹泻、体重明显下降等GVHD的症状。结论:化疗预处理联合脾加骨髓细胞移植能对CT26大肠癌细胞产生GVT效应,并伴随着GVHD及嵌合率的变化。     

B-lymphocyte differentiation in lethally irradiated and reconstituted mice. III. The influence of splenectomy on the recovery of the B-cell populations.

The recovery of the B-cell population was studied in irradiated and fetal liver-reconstituted mice. Since in irradiated and reconstituted mice the B-cell population in the spleen recovers much more rapidly than in the other lymphoid organs, we assessed the role of the spleen in the recovery of the B-cell compartment in the other organs. It was found that the absence of the spleen did not delay or diminish the recovery of the immunoglobulin (Ig)-bearing (B)-cell population in the bone marrow, lymph nodes, Peyer's patches, and peripheral blood. Throughout the recovery period the number of B lymphocytes in the lymphoid organs of splenectomized mice was even greater than in the same organs of sham-operated mice. B cells obtained from the bone marrow of splenectomized, irradiated, and reconstituted mice appeared to be fully immunocompetent, as shown by their ability to cooperate with thymocytes in an adoptive plaque-forming cell response to sheep red blood cells. The compensatory effect of the increased numbers of B cells in the bone marrow and peripheral lymphoid organs of splenectomized mice was reflected in the level of the serum immunoglobulins. Apart from a lower IgM concentration in the serum of splenectomized mice, no significant differences were found in IgG₁, IgG_2b, and IgA levels between splenectomized and sham-splenectomized mice. It is concluded that the spleen is not essential for both normal B-lymphocyte differentiation and maturation after irradiation and reconstitution.     

Effect of splenectomy on the development of neonatal lymphoid organs transplanted to animals of varying age]

The effect of splenectomy on the development of newborn thymus and spleen grafted under the kidney capsule of young and old mice has been investigated. Preliminary splenectomy is shown to increase cell counts in grafted spleen that is more conspicuous in young recipients as compared with old ones. This result suggests a decrease with age in the inhibitory effect of the host spleen on the maturation of spleen grafted from newborn donor. Combined transplantation of newborn thymus and spleen has revealed a decrease of cell counts in the donor spleen grafted to the young splenectomized recipients and, on the contrary, increase of this parameter in old ones. Immune response in donor spleen with combined transplantation of the thymus to the old splenectomized recipients is much higher as compared with the same parameter in recipient without splenectomy. It is concluded that partial destruction of the old immune system is essential for its correction.     

Natural killer cells, bone, and the bone marrow: studies in estrogen-treated mice and in congenitally osteopetrotic (mi/mi) mice.

Mice lose natural killer cells after 6 weeks of treatment with 17 beta-estradiol. We here demonstrate that the same protocol leads to loss of genetic resistance to bone marrow transplantation and to significant osteoproliferation with loss of bone marrow. We also show that mice with reduced marrow because of congenital osteopetrosis are deficient in natural killing. These findings are consistent with previous evidence that natural killing and genetic resistance to bone marrow transplantation are dependent upon the marrow. Temporal studies of bone histology and radiology during and after treatment with estrogen reveal that alterations in natural killing proceed more rapidly than changes in bone marrow volume. These studies also demonstrate that estrogens induce osteoproliferation only at endosteal surfaces that are adjacent to hematopoietic marrow. From these observations, we conclude that estrogens do not reduce natural killer cells simply by reducing the volume of bone marrow. Estrogens may instead have an effect on bone marrow. Estrogens may instead have an effect on bone marrow cells that leads both to osteoproliferation and to a deficiency of marrow-dependent cells.     

The effects of repeated bleedings on bone marrow and blood morphology in adult laboratory rats

There were slight and rapidly disappearing changes in the bone marrow and blood morphology of laboratory rats after repeated bleedings on consecutive days, except marked increases in the counts of circulating reticulocytes. However, the significantly elevated reticulocyte count was not accompanied by the similar increase in the count of marrow erythroid nucleated cells. Marked increments could be found in the counts of splenic erythroid nucleated cells and, therefore, it is possible for rat circulating reticulocytes to originate following repeated bleedings from the spleen. The validity of investigations of bone marrow and blood morphology for detecting haemorrhage is discussed with a view to preclinical safety drug evaluations performed on adult laboratory rats.     

KINETICS OF ERYTHROPOIETIN STIMULATED PROLIFERATION OF ERYTHROID PROGENITORS IN THE SPLEEN

The effect of RBC transfusion and erythropoietin (EPO) on the proliferation of immature erythrocyte progenitors was studied in the spleens of RBC transfused, lethally irradiated mice injected with bone marrow. Transfusion decreased expansion of the progenitors and slowed their proliferation: the mean cycle time as measured by per cent labelled mitosis (PLM) on the third day after injection of bone marrow was 10.7 hr in transfused as compared to 5.6 hr in non-transfused mice. One injection of five units of erythropoietin on day 2 decreased the mean cycle time to 7.3 hr in transfused mice and increased expansion of the progenitor cells. The effects of erythropoietin on cell proliferation were prompt: a significant increase of incorporation of ³H-TdR into DNA occurred within 2 hr of injection. Erythroblasts were absent from the spleens of transfused, irradiated bone marrow injected mice; however, erythroblasts appeared by 72 hr and 48 hr following EPO injection either 2 days or 5 days after transplantation respectively. Increased uptake of radioactive iron in spleen after erythropoietin injection preceded the appearance of erythroblasts by 2 and 1 days when erythropoietin was injected either 2 or 5 days after marrow transplantation respectively. The increase in cellular proliferation induced by erythropoietin in transfused irradiated mice injected with bone marrow equivalent to 0.35 femoral shaft was manifested as an increase of the total DNA content in the spleen by 119 μg (11.9 × 10⁶ cells) within 48 hr of injection. The cellular increment produced by EPO injection on day 5 to mice given 0.05 femoral shaft consisted mainly of undifferentiated mononuclear cells, most of which were labelled, with erythroblasts comprising only one quarter of the increment. Erythropoietin inactivated by mild acid hydrolysis failed to increase cellular proliferation.     

Quantitative determination of transplantable hemopoietic stem cells by limiting dilution method

The concentration of hemopoiesis restoring units (HRU) in bone marrow of mice was assayed by using the limiting dilution method in transplantation to lethally irradiated mice. 7 to 12.7 HRU were found in 10(6) bone marrow cells of CBF1 mice and 19.2 to 50.6 HRU in BCT6F1 mice when the survival of the recipients was registered in 4 weeks after transplantation. The proportion of not surviving recipients increased with time when marrow doses were low (2.5 X 10(4) to 2 X 10(5) cells or 0.5-2.5 HRU per mouse) and remained stable when middle or high marrow doses (10(6)-10(7) cells) were used.     

MULTIPLICATION OF HAEMOPOIETIC COLONY FORMING UNITS (CFU) IN MICE AFTER X-IRRADIATION AND BONE MARROW TRANSPLANTATION

Kinetics of the multiplication of haemopoietic CFUs was studied in lethally irradiated mice receiving various numbers of syngeneic bone marrow cells. After transplantation of a small number of bone marrow cells, the growth rate of CFU in femoral bone marrow appeared to decrease after about 10 days after transplantation, before the normal level of CFU in the femur was attained. In the spleen it was found that the overshoot which was observed about 10 days after transplantation of a large number of bone marrow cells is smaller or absent when a small number of cells is transplanted. Experiments dealing with transplantation of 50 x 10⁶ bone marrow cells 0, 4 or 10 days after a lethal irradiation indicated that the decline in growth rate of CFUs about 10 days after irradiation could not be attributed to environmental changes in the host.
The results are explained by the hypothesis that a previous excessive proliferation of CFUs diminishes the growth rate thereafter. This hypothesis is supported by experiments in which 50 x 10⁶ bone marrow cells derived from normal mice or from syngeneic chimaeras were transplanted. The slowest growth rate was observed when bone marrow that had been subjected to the most excessive proliferation in the weeks preceding the experiment was transplanted.     

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.     

骨髓动员对骨髓造血干细胞在心肌梗死后心脏中定植及分化的影响

目的观察小鼠心肌梗死后骨髓造血干细胞在心脏内的分化及细胞因子的影响。方法C57/BL6小鼠60只分为骨髓动员组和对照组,先后行脾切除、骨髓移植（骨髓供体为增强绿色荧光蛋白转基因小鼠）、骨髓动员及建立心肌梗死模型。心肌梗死后3周将小鼠心脏取出并切片行组织学及激光共聚焦显微镜免疫荧光检查。结果骨髓动员可以增加EGFP阳性细胞在心脏中梗死区和边缘区的定植,但绝大多数EGFP阳性细胞都同时表达CD45。仅发现有极少数骨髓来源的心肌细胞、成纤维细胞及血管内皮细胞,且与骨髓动员无相关性。结论骨髓动员能够明显促进骨髓来源细胞定植入小鼠心脏的梗死区;极少数骨髓造血干细胞可以分化为心肌细胞,其数量远不足以修复梗死心肌及改善心功能;骨髓造血干细胞不参与梗死区疤痕形成的病理过程。