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PubMed1983

Development of the granule population in neutrophil granulocytes from human bone marrow.

Bone marrow from hematologically healthy adults was exposed to a number of fixation procedures for investigation of the heterogeneity of the granule population in neutrophil granulocytes at the ultrastructural level. Four main cell stages were distinguished: early promyelocyte, late promyelocyte, myelocyte, and mature neutrophil granulocyte and described separately; metamyelocytes and band-form or stab cells are described together. The characteristic changes in the cytoplasm during myelopoiesis were analysed quantitatively. Special attention was given to the development of the granule population. Three types of granule arise in successive cell stages: granules which develop a sub-structure in the matrix (nucleated granules) are formed in early promyelocytes, granules with a homogeneous electron-dense matrix (azurophil granules) in late promyelocytes, and granules with a less electron-dense matrix (specific granules) in myelocytes. The three types of granule remain present during myelopoiesis. The best results in distinguishing the granule types were obtained by prefixation either in 0.1% glutaraldehyde or in 1.5% glutaraldehyde followed by washing in phosphate-buffered Ringer solution to which aminotriazole had been added. Granule counts revealed for the mature neutrophil a total number of granules of about 220 per ultrathin section. This population of granules is composed of about 12% nucleated, 11% azurophil, and 77% specific granules. When our previous findings are taken into account, the existence of three successively formed and morphologically distinguishable types of granule in heterophil (neutrophil) granulocytes has been demonstrated for three mammalian species: the guinea pig, the rat, and man. A separate term for the early promyelocyte stage is proposed: eomyelocyte.

Adult↗
PubMed1999

Large scale recovery and characterization of stromal cell-associated primitive haemopoietic progenitor cells from filter-retained human bone marrow.

Bone marrow aspirates are composed of two cellular compartments, an abundant buffy coat suspension and a minor particulate fraction. The particulate fraction is routinely removed by filtration prior to transplantation in order to reduce the risk of embolism. This study shows that the filter-retained fraction includes many multicellular complexes, previously defined as haematons. A haematon is a finely arborized stromal-web which is tightly packed with haemopoietic progenitor cells and differentiated postmitotic cells. Comparison of the pooled buffy coat and the filter-retained materials from healthy donors showed that the haematon fraction contained 8-40 x 10(6) CD34+ cells, 20-115 x 10(3) high proliferative potential colony-forming cells (HPP-CFC) and 0.49-2.67 x 10(6) granulocyte-macrophage colony-forming unit (GM-CFU) which constituted 24+/-8% (10-36; n=8) of the total GM-CFU population harvested. Similar, but more variable recoveries of GM-CFU were obtained from the haematon fractions from patients with breast cancer (21+/-13%; n=10), Hodgkin's disease (33+/-19%; n=4), non-Hodgkin's lymphoma (21+/-18; n=7), but the recovery was lower from patients with acute myelogenous leukaemia (AML) (13+/-13%; n=6). The haematon fraction was enriched in CD34+ cells (2.5-fold), long-term culture initiating cells (LTC-IC/CAFC, week 5) (3.5-fold), HPP-CFC (2.8-fold) and GM-CFU (2.3-fold) over the buffy coat. Purified CD34+ cells expanded exponentially and produced 800 to 4000-fold more nucleated cells, 300 to 3500-fold more GM-CFU and 10 to 80-fold more HPP-CFC in stroma-free suspension culture with interleukin-1 (IL-1beta), IL-3, IL-6, GM-CSF and stem cell factor (SCF), than did the starting cell input. The haematon fraction produced significantly more progenitor cells than the buffy coat in long-term liquid culture (LTC). This was due to the higher frequency of LTC-IC/CAFC and to the presence of the whole spectrum of native, stroma cell-associated CAFC in haematons. Thus, the haematon includes the most productive haematogenous compartment in human BM. This simple enrichment strategy, using filter-retained haematons, provides a rational source of BM cells for large scale experimental and/or clinical studies on haemopoietic stem cells and on critical accessory stromal cells.

Antigens, CD34↗
PubMed1998

[Application of IHW2 purged bone marrow to autologous bone marrow transplantation in the treatment of acute myeloid leukemia].

OBJECTIVE: To observe the clinical effectiveness of IHW2 purged autologous bone marrow transplantation (ABMT) in the treatment of AML patients. METHODS: Large volume of bone marrow was purged in vitro by IHW2 and then reinfused to patients for ABMT. RESULTS: All of the three patients transplanted with IHW2 purged autologous bone marrow successfully reconstituted hemopoiesis. Two patients got continuous complete remission for 36+ and 34+ months, respectively. The karyotype of one of the two patient was 46,XY,del(16)(q22) before ABMT and changed to 46,XY in 2 months after transplantation. The third patient relapsed after ABMT 19 months. CONCLUSION: The preliminary study suggests that IHW2 may have certain purging effect on leukemic cells and is safe for clinical application.

Adult↗
PubMed2002

Bone formation following implantation of fibrous calcium compounds (beta-Ca(PO3)2, CaCO3(aragonite)) into bone marrow.

Bone formation around three types of fibrous calcium-containing crystals has been examined histologically using rats. The implanted materials are (i) calcium metaphosphate (beta-Ca(PO(3))(2)) fibers having aspect ratios of 15-80 with 2-20 microm in diameter, (ii) beta-Ca(PO(3))(2)) fibers surface-modified using dilute NaOH and (iii) calcium carbonate (CaCO(3); aragonite phase) whiskers having aspect ratios of 15-40 with 0.5-3 microm in diameter. Beta-Ca(PO(3))(2) fibers show a mechanically high strength with a low modulus of elasticity, and the surface-modified fibers have a thin layer consisting of a calcium orthophosphate phase. CaCO(3) whiskers were used for comparison reasons. The materials were implanted for 4, 8, and 12 weeks into bone defects created in the bone marrow of rat tibiae. Cancellous bone formation was observed around beta-Ca(PO3)2 fibers, the surface-modified fibers and CaCO(3) whiskers after implantation for 12, 4 and 4 weeks, respectively. CaCO(3) whiskers were scarcely observed after 12 weeks for resorbing. The calcium phosphate fibrous materials show combined advantages of mechanically high strength for toughening a matrix phase and biological activities; thus, these materials may prove to be useful for novel applications in the biomedical field.

Journal Article↗
PubMed1989

The differential diagnosis of polycythaemia--a bone marrow study (the bone marrow in polycythaemia).

Bone marrow sections from posterior iliac crest aspiration and/or trephine specimens have been examined in 39 patients with true polycythaemia, along with a variety of other clinical and laboratory data. The emphasis has been on objective assessment of cellularity and megakaryocyte concentration in a prospective four year study. In patients with untreated primary polycythaemia mean cellularity was 87.0% and 86.4% for aspirate and trephine specimens compared with 55.5% and 48.7% for secondary cases and 48.3% and 45.5% for controls. Eighty per cent of primary polycythemia patients had moderate to marked megakaryocytic hyperplasia. We conclude that, in the presence of an elevated red cell volume, marrow cellularity of greater than 75%, particularly when accompanied by megakaryocytic hyperplasia, of iliac crest aspirate or trephine specimens is sufficient per se to establish a diagnosis of polycythaemia rubra vera.

Bone Marrow Cells↗
PubMed1995

In vitro LAK (lymphokine activated killer) activity following autologous peripheral blood stem cell is significantly greater than that following autologous bone marrow and allogeneic bone marrow transplantation.

LAK activity is known to increase following autologous and allogeneic bone marrow transplant and peripheral blood stem cell transplant (PBSCT). The aim of this study was to directly compare the 3 types of transplant and the LAK activity generated. LAK activity following PBSCT is significantly greater than that following autologous bone marrow transplantation and even allogeneic transplantation up to 8 weeks. The type of killer cells generated is similar for the different types of transplants, with most killing activity following PBSCT due to CD56+ cells, though CD3+ cells also contribute. This study would suggest that attempts to augment the graft-versus-leukaemia effect is more likely to be successful following PBSCT than autologous bone marrow transplantation.

Adolescent↗
PubMed1986

The significance of host haemopoietic cells detected by cytogenetic analysis of bone marrow from recipients of bone marrow transplants.

Bone marrow from 39 patients who received a bone marrow transplant (BMT) from a matched donor of different sex were studied by chromosome analysis for evidence of mixed haemopoietic chimaerism (MC). Recipient metaphases were detected in the bone marrow of 10 patients after BMT. Patients in whom MC was detected within 6 weeks of BMT did not all have a poor outcome. Two of seven are disease-free survivors at greater than 470 and greater than 632 d. All three patients in whom MC was diagnosed more than 6 weeks after BMT subsequently relapsed. Four factors appear to be important in determining the probability of relapse when MC is detected in a patient after BMT: the timing of detection of residual recipient cells; the proportion of these cells in the bone marrow; persistence of these cells in increasing proportions; and the karyotype of the recipient metaphases detected. Cytogenetic assessment may provide the earliest indication of relapse in these patients. In addition, this study provides further evidence that cyclophosphamide and total body irradiation, as used in these patients, may be inadequate conditioning therapy for BMT.

Adolescent↗
PubMed2004

Intra-bone marrow injection of bone marrow and cord blood cells: an alternative way of transplantation associated with a higher seeding efficiency.

OBJECTIVE: Intravenous (IV) injection is currently the normal method for transplanting hematopoietic cells. However, the problem of seeding efficiency and homing is relevant especially when a limited number of stem cells is available. Intra-bone marrow (IBM) injection of bone marrow cells (BMCs) may overcome this problem. MATERIALS AND METHODS: Irradiated (750 cGy) C57BL/6J mice were transplanted with 1 x 10(5) BMCs harvested from transgenic mice expressing an enhanced version of the green fluorescent protein (EGFP+) via IBM or with 1 x 10(6) EGFP+ BMCs via IV. Irradiated (320 cGy) NOD/SCID mice were transplanted with 1 x 10(6) human cord blood (CB) cells via IBM or with 1 x 10(7) human CB cells via IV. RESULTS: In C57BL/6J mice after 90 days, the fraction of EGFP+ cells harvested was 37% and 53% in IV-treated and IBM-treated (contralateral tibia and femur in the IBM) mice, respectively: the expansion folds were 114 and 1760, respectively. In NOD/SCID mice, the percentages of CD45+ cells and CD45+/CD34+ cells were, at 30 days, 3.3% and 0.3% in IV-treated mice, and 4.4% and 1.1% in IBM-treated mice. At 60 days, the percentages of CD45+ cells and CD45+/CD34+ cells were 2.1% and 0.3% in IV-treated mice and 1.4% and 0.4% in IBM-treated mice. At day 90 the percentages of CD45+ cells and CD45+/CD34+ cells were 3% and 0.3% in IV-treated mice and 2.3% and 0.4% in IBM-treated mice. CONCLUSION: Our data demonstrate that IBM transplantation is associated with a seeding efficiency 15 times greater than IV transplantation. IBM transplantation may improve the results of transplant and may be useful in several settings: 1) when a limited number of hematopoietic progenitors are available; and 2) in experiments aiming to place in the bone marrow stem cells of other lineages (CNS, muscle, etc.).

Animals↗
PubMed1998

Long-term cryopreservation: successful trilineage engraftment after autologous bone marrow transplantation with bone marrow cryopreserved for seven years.

Successful autologous bone marrow transplantation (ABMT) and peripheral blood stem cell transplantation depend on safe hematopoietic stem cell (HSC) cryopreservation and storage. Several successful methods for cryopreservation and storage have been established and are commonly used all over the world. However, little is known about the effects of long-term cryopreservation on the capacity to sustain a complete immunohematopoietic engraftment. Several authors have investigated stem cell viability after cryopreservation and storage for more than 5 years and reported preclinical good viabilities in terms of dye-exclusion or colony-forming capability in vitro. Only two studies using BM cryopreserved for more than 5 years for transplantation are reported, but they did not provide proof of trilineage engraftment. In February 1997 at our institution, a patient with relapsed non-Hodgkin's lymphoma underwent ABMT with BM harvested in February 1990. He achieved a granulocyte count > 500 x 10(6)/L on day 21 and a self-supporting platelet count > 20 x 10(9)/L on day 30. After day 29, his hemoglobin level was satisfactory without need of transfusion support. This successful trilineage engraftment with cryopreserved BM that had been stored for 7 years suggests that HSC are able to maintain their capability to reconstitute hematopoiesis for a long time.

Bone Marrow↗
PubMed1982

Generation of leukotriene C4 from a subclass of mast cells differentiated in vitro from mouse bone marrow.

Bone marrow-derived mast cells, differentiated in vitro, demonstrate surface IgE, receptors and contain histamine in metachromatic granules, which are composed of chondroitin sulfate E proteoglycan rather than heparin proteoglycan. Activation of this subclass of mast cells with calcium ionophore A23187 resulted in generation of immunoreactive C-6-sulfidopeptide leukotriene in a dose- and time-dependent fashion. Isolation of immunoreactive C-6-sulfldopeptide leukotriene by reverse-phase high-performance liquid chromatography (RP-HPLC) revealed a retention time and a specific biologic activity identical to those of synthetic leukotriene C4 (LTC4). Neither radiolabeled nor immunoreactive conversion products of [3H]LTC4/LTC4 were recognized during RP-HPLC resolution of the supernatants. The failure of fresh bone marrow cultures to generate C-6-sulfidopeptide leukotriene in response to ionophore indicates that leukotriene generation is dependent upon cellular differentiation into a mast cell population. The amount of LTC4 generated during optimal ionophore stimulation, 90.9 +/- 7.5 ng per 10(6) cells, contrasts with the relatively low amounts of C-6-sulfidopeptide leukotriene generated by the conventional heparin-containing rat mast cells or mouse mastocytoma cells.

Animals↗
PubMed1993

[Investigation of meropenem levels in the human bone marrow blood, bone, joint fluid and joint tissues].

A solution of 0.5 g of meropenem (MEPM) in 100 ml of saline was administered to adult patients before the orthopaedic surgery via intravenous drip infusion for 30 minutes. Eleven samples of bone marrow blood, 13 bone, 8 joint fluid and 8 joint tissues obtained from 15 clinical cases were analyzed for MEPM levels. At the same time, blood samples were taken from peripheral veins and serum served as control was analyzed. The concentration of MEPM in the marrow blood at 30 minutes after administration of MEPM reached 15.4 micrograms/ml, which was above 50% of the maximum concentration in serum. Ratios of concentration in bone marrow to that in serum were between 93% and 105% at that time. MEPM levels were 5.74-0.40 micrograms/g in bones, 20.3-4.55 micrograms/ml in joint fluid and 18.2-4.05 micrograms/g in joint tissues at 30 to 75 minutes after administration. In joint fluid and joint tissues, the concentration above 50% of maximum level in serum were maintained for more than an hour. MEPM is thought to be useful for the treatment of bone and joint infections and also prophylaxis of infections in cases of major surgery of skeletal tissue in the field of orthopaedic surgery.

Adult↗
PubMed2000

Bone marrow pathology after bone marrow transplantation.

Trephine biopsy evaluation in patients who have undergone high dose chemotherapy with stem cell infusion performed by an experienced pathologist is important in evaluation of hematopoietical restitution, evaluation of complications such as graft rejection, infections, relapse or secondary neoplasms. In the first week after high dose chemotherapy or radiotherapy bone marrow aplasia is evident. In the case of accompanying myelofibrosis, fragmentation of reticular and collagen fibers is noticeable only following stem cell transplantation. The first precursors, which appear after the second or third week after transplantation are erythroid precursors found in small groups. Promyelocytes and myelocytes are observed between the second and fourth week, the ration off erythroid to myeloid populations at that time is 1:1. Megakaryocytes ate the last precursors to appear. Trephine biopsy evaluation is useful in diagnosis of graft rejection, especially in case of a discrepancy between the blood morphology examination and bone marrow cellularity. Granuloma formation is an unspecific finding, which requires diagnosis of infections. Residual disease can be recognized in histopathologic examination, or sooner, with immunophenotyping, cytogenetic examination using FISH or PCR. Secondary neoplasms to high dose radiotherapy or chemotherapy are usually lymphoproliferative processes connected with latent EBV infections, myelodysplastic syndromes and acute myeloblastic leukaemia.

Biopsy↗
PubMed1991

Separation of allostimulatory and natural suppressor/stem cell functions of murine bone marrow--implications for bone marrow transplantation.

Natural suppressor (NS) activity is defined by the ability of a null cell population to inhibit antigenic and mitogenic activation of lymphoid cells. We had previously demonstrated that murine and human bone marrow was a rich source of NS activity and could be enriched in a population of large, low-density cells after counterflow centrifugal elutriation (CCE). In this report we confirm previous findings in the mixed lymphocyte reaction (MLR) that NS activity is an endogenous function of BM and is not present in peripheral immune tissues. Furthermore, suppression by BM NS cells in the MLR is not MHC restricted, which distinguishes NS activity from veto activity also associated with BM. To enrich for NS activity, BM cells were separated into 3 fractions according to size and density by CCE. Fraction (F) 1 contained small, high-density cells; F2 contained cells of intermediate size and density; and F3 comprised large, low-density cells. Addition of CCE-enriched fractions to the MLR revealed potent NS activity associated with F3, consistent with our previous findings in the Ab response. CCE-enriched fractions of BM cells were also used as stimulators in an MLR to determine which fraction, if any, could support alloantigen-induced proliferation. The use of unfractionated C57Bl/6 (H-2b) BM cells as stimulators for BALB/c (H-2d) splenic responders resulted in little detectable proliferation as compared with that induced by C57Bl/6 splenic stimulators. However, when elutriated fractions were used as stimulators, F1 BM cells induced a significant proliferative response, albeit to a lesser magnitude than spleen cells. In order to determine which fraction contained hematopoietic progenitors, CCE-separated cells were assayed for granulocyte/macrophage colony formation (CFU-GM). F3 BM was enriched five-fold for CFU-GM progenitors as compared with unfractionated BM, while progenitors were virtually absent in F1 and F2 BM. The results suggest that CCE may represent an effective way of removing potential alloantigen-presenting cells (F1) from BM, while retaining hematopoietic progenitors and NS activity (F3). The implications of the findings as they relate to allogeneic BM transplantation are discussed.

Animals↗
PubMed2000

Correlation of CD34+ cell counts with volume of bone marrow collected for allogeneic bone marrow harvests.

The quantity of bone marrow collected for allogeneic bone marrow transplantation is based on collecting 10 to 15 cc of bone marrow/kg of recipient weight. We hypothesized that the percentage of CD34+ cells collected during a bone marrow harvest decreased at the end of the harvest because of increasing amounts of peripheral blood contamination. We performed a prospective, blinded study in which we measured CD34+ percentages and cell counts at 200-cc intervals during bone marrow harvests from 11 consecutive human leukocyte antigen (HLA)-matched sibling bone marrow donors. We observed that the percentage of CD34+ cells in aspirated bone marrow did not vary significantly from the start to the end of the bone marrow harvest, and the total number of CD34+ cells/kg increased in a linear fashion, thus disproving our original hypothesis. In conclusion, the percentage of CD34+ cells in aspirated bone marrow will remain constant throughout a bone marrow harvest.

Adolescent↗
PubMed1990

Nude rat model for studying metastasis of human tumor cells to bone and bone marrow.

Bone metastases reproducibly developed in nude rats after an injection of LOX human malignant melanoma cells into the left ventricle, with hind leg paralyses appearing in all animals within approximately 2 weeks. Manifest metastases were present exclusively in the skeletal system, predominantly in the lumbar portion of the spine, the long bones, and occasionally in the skull. Intracardially injected 125I-labeled tumor cells and monodisperse microspheres were distributed in parallel to the various tissues. Moreover, because the levels of radioactivity were significantly lower in bone than in lung, kidney, and liver, the pattern of metastases could not be explained solely by hemodynamic factors. In chemotherapy experiments, the survival time of rats given left ventricular injections of LOX cells increased in a dose-dependent manner after the animals were treated with dacarbazine. Researchers may find the model useful for studying the biology of bone metastases and for testing the sensitivity of these lesions to drugs.

Age Factors↗
PubMed1984

Strong natural killer (NK) cell activity in bone marrow of myeloma patients: accelerated maturation of bone marrow NK cells and their interaction with other bone marrow cells.

Bone marrow natural killer (NK) cell activity was studied in patients with multiple myeloma. Bone marrow mononuclear cells from myeloma patients expressed considerable levels of cytotoxicity against K562 in a 4 h 51Cr-release assay, which was comparable to that of blood lymphocytes. In contrast, NK-cell activity was markedly low or absent in bone marrow of normal donors and control patients. Fractionation of bone marrow cells from myeloma patients on linear bovine serum albumin gradients enriched NK effector cells in the upper, lymphocyte-enriched fraction, with no reactivity in the middle fraction containing mainly myeloma cells. Treatment with either Leu-7 or OKMI monoclonal antibody plus complement reduced or abrogated bone marrow NK-cell activity of myeloma patients as well as blood NK-cell activity. However, OKMI plus complement failed to reduce control bone marrow NK-cell activity, although the activity was reduced by Leu-7 plus complement. Overnight exposure to interferon (IFN) of bone marrow cells resulted in an augmentation of NK-cell activity in myeloma patients, but not in controls. Furthermore, adherent bone marrow cells from controls suppressed IFN-induced enhancement of NK-cell activity of blood lymphocytes, whereas bone marrow of myeloma patients did not contain such suppressor cells. No cytotoxicity was induced in control bone marrow cells by cocultivation with bone marrow myeloma cells. Bone marrow NK cells from myeloma patients were able to lyse control bone marrow cells in a 18 h assay and their lytic activity was augmented by IFN treatment. However, neither bone marrow cells nor blood lymphocytes were cytotoxic to autologous and allogeneic fresh myeloma cells even after activation with IFN. These results suggest that NK precursor cells differentiate into HNK-I- and OKMI-positive mature NK cells in bone marrow of myeloma patients, but not in control bone marrow, and that these functional bone marrow NK cells may interact with other bone marrow elements.

Bone Marrow↗