Effects of urinary proteins from certain leukemics upon macromolecular synthesis and enzyme levels in bone marrow cultures.

Urinary proteins from human leukemic patients have been found to alter quantitatively macromolecular synthesis in primary mouse bone marrow cultures. Urinary protein-stimulated incorporation of [3H]uridine into RNA was found after 1 day of culture. Increased levels of adenine phosphoribosyltransferase and lysozyme were demonstrable at 3 and 5 days, respectively, with urinary protein-supplemented cultures. The incorporation of 3H-labeled deoxynucleosides into DNA was higher in the presence of urinary proteins after 2 days of culture. The rate of incorporation of [3H]deoxyuridine into DNA was strongly inhibited by 10(-5) M Methotrexate and 10(-6) M 5-fluorodeoxyuridine, however, the effect of urinary proteins on incorporation of [3H]uridine into RNA and lysozyme accumulation were not inhibited. Urinary proteins also stimulated the formation of "colonies" (groups of at least 30 cells) in media containing methylcellulose. This latter phenomenon was also not inhibited by 10(-5) M Methotrexate or 10(-6) M 5-fluorodeoxyuridine. The results of these studies are consistent with the postulate that in the presence of human urinary proteins, mouse bone marrow cells in culture proceed to a phenotype characteristic of circulating peripheral white cells.     

Heterologous antiserum to rat lymphohemopoietic precursor cells.

Lymphohemopoietic precursor cells in rat bone marrow are members of a subset of lymphocyte-like cells that bears the bone marrow lymphocyte antigen (BMLA) and that lacks antigens present on peripheral B and T cells. This was demonstrated by two experimental approaches. In the first, bone marrow cells with the potential to form hemopoietic colonies in spleen (CFU-S), to repopulate lumphoid tissues and blood, and to rescue lethally irradiated recipients were enriched approximately 10-fold by a fractionation procedure designed to isolate a "null" population of bone marro lymphocytes. In the second approach, the lymphohemopoietic precursor cell activity in bone marrow was completely abrogated by opsonization with rabbit antiserum (ALSBM) raised against this "null" population of bone marrow cells. Precursor cell activity was not affected by treatment with antiserum to T and B cells. Quantitative cross-absorption studies showed that the antigen detected by ALSBM on lymphohemopoietic precursor cells had the same cellular distribution as did the previously described bone marrow lymphocyte antigen. It is likely that this antigen is present both on pluripotent stem cells and on committed progenitors of the myelocytic, erythrocytic and lymphocytic series.     

Methotrexate chemotherapy–induced damages in bone marrow sinusoids: An in vivo and in vitro study

Chemotherapeutic agents are very well evident extrinsic stimuli for causing damage to endothelial cells. Methotrexate is an antimetabolite commonly used to treat solid tumours and paediatric cancers. However, studies on the effect(s) of methotrexate on bone marrow microvascular system are inadequate. In the current study, we observed a significant bone marrow microvascular dilation following methotrexate therapy in rats, accompanied by apoptosis induction in bone marrow sinusoidal endothelial cells, and followed by recovery of bone marrow sinusoids associated with increased proliferation of remaining bone marrow sinusoidal endothelial cells. Our in vitro studies revealed that methotrexate is cytotoxic for cultured sinusoidal endothelial cells and can also induce apoptosis which is associated with upregulation of expression ratio of Bax and Bcl-2 genes and Bax/Bcl-2 expression ratio. Furthermore, it was shown that methotrexate can negatively affect proliferation of cultured sinusoidal endothelial cells and also inhibit their abilities of migration and formation of microvessel like tubes. The data from this study indicates that methotrexate can cause significant bone marrow sinusoidal endothelium damage in vivo and induce apoptosis and inhibit proliferation, migration and tube-forming abilities of sinusoidal endothelial cells in vitro.     

Autologous transplantation of a bone marrow graft manipulated by chemoseparation to eliminate residual tumor cells

An autologous bone marrow transplantation (ABMT) was performed in a 45-year-old male patient with AML in therapy-resistant first relapse including CNS-disease. The marrow graft was harvested 18 months before, at the beginning of first remission, and was subjected to an in-vitro incubation with 4-hydroperoxycyclophosphamide (4-HC) prior to cryopreservation to eliminate residual clonogenic tumor cells. After myeloablative therapy with high-dose Cyclo-phosphamide (CY), total body irradiation (TBI) and intrathecal application of Methotrexate (MTX), the manipulated marrow graft was reinfused. The myelopoietic reconstitution started already 6 days after ABMT and was completed 40 days thereafter. There is evidence for a complete remission in marrow and spinal fluid observed over a period of 4 months.     

Exogenous bone marrow cells do not rescue non-irradiated mice from acute renal tubular damage caused by HgCl2, despite establishment of chimaerism and cell proliferation in bone marrow and spleen

Abstract.   Objective : Various studies have shown that bone marrow stem cells can rescue mice from acute renal tubular damage under a conditioning advantage (irradiation or cisplatin treatment) favouring donor cell engraftment and regeneration; however, it is not known whether bone marrow cells (BMCs) can contribute to repair of acute tubular damage in the absence of a selection pressure for the donor cells. The aim of this study was to examine this possibility. Materials and methods : Ten-week-old female mice were assigned into control non-irradiated animals having only vehicle treatment, HgCl₂-treated non-irradiated mice, HgCl₂-treated non-irradiated mice infused with male BMCs 1 day after HgCl₂, and vehicle-treated mice with male BMCs. Tritiated thymidine was given 1 h before animal killing. Results : Donor BMCs could not alleviate non-irradiated mice from acute tubular damage caused by HgCl₂, deduced by no reduction in serum urea nitrogen combined with negligible cell engraftment. However, donor BMCs could home to the bone marrow and spleen and display proliferative activity. This is the first report to show that despite no preparative myeloablation of recipients, engrafted donor BMCs can synthesize DNA in the bone marrow and spleen. Conclusions : Exogenous BMCs do not rescue non-irradiated mice from acute renal tubular damage caused by HgCl₂, despite establishment of chimerism and cell proliferation in bone marrow and spleen.     

Cytotoxic and noncytotoxic mechanisms involved in the in vitro anti-leukaemia effects of T cell clones established from a chronic myelogenous leukaemia patient during treatment in vivo with interferon α

Summary T cell clones derived from a chronic myelogenous leukaemia (CML) patient during interferon (IFN, Wellferon) biotherapy preferentially lysed autologous rather than allogeneic CML target cells in an apparently MHC-unrestricted fashion, but also lysed bone marrow cells from certain normal donors regardless of whether or not they shared HLA antigens with the patient. Although T cell clones inhibited both CML and normal bone marrow in the colony-forming assay, they blocked proliferation of CML cells more efficiently than bone marrow cells. This inhibitory effect was mediated at least in part by the tumour necrosis factor (TNF) and IFN secreted by the clones. Antisera to these cytokines partially prevented inhibition. Involvement of additional factors is also suggested in blocking CML cell proliferation because this was not 100% inhibited even by a combination of TNF and IFN. In addition, most clones failed strongly to block the proliferation of normal bone marrow cells, which were susceptible to inhibition by these cytokines.This work was supported in part by the Deutsche Forschungsgemeinschaft (SFB 120)     

Mechanism of induction of micronuclei and chromosome aberrations in mouse bone marrow by multiple treatments of methotrexate.

Methotrexate (MTX), an inhibitor of dihydrofolate reductase (DHFR), slightly induced micronuclei and this induction of micronuclei was enhanced by multiple treatments with the drug (Yamamoto et al., 1981; Hayashi et al., 1984; CSGMT/JEM.MMS, 1990). More micronuclei and chromosomal aberrations in mouse bone marrow cells were induced by multiple than by single treatment. The MTX level in mouse plasma and bone marrow showed little (or no) differences between single and quadruple treatments several hours after the injection(s). On the other hand, the DHFR activity in bone marrow cells 3 h after one and four injections was decreased to approximately 38 and 0%, respectively, of that in non-treated mice. Furthermore, the intracellular MTX level in the bone marrow cells (but not in total bone marrow) after four injections was about 10-fold higher than that after one injection. The amount of MTX bound to protein 3 h after four injections, as assayed by gel filtration (Sephadex G-25), was approximately 8-fold greater than after one injection. Therefore, the multiple-dose effects of MTX on the induction of micronuclei and chromosomal aberrations may be explained by the intracellular accumulation of MTX resulting in an enhancement of enzyme inhibition.     

Bone marrow transplantation. Part II--autologous.

Autologous bone marrow transplantation provides an effective form of "rescue" following high-dose therapy used for treating certain malignant diseases. The high doses of radiotherapy or chemotherapy, or both, should allow for greater tumor cell kill if dose-response to therapy exists for that tumor. The use of autologous bone marrow obviates the need for an HLA-identical donor, and the need for pretransplant immunosuppression; no graft-versus-host disease would ensue. We review in part II the history and background, methods of obtaining autologous stem cells, and details of the results achievable with this type of therapy. We discuss potential difficulties with autologous transplantation, as well as possible future areas of research.     

Simultaneous analysis of DNA content and surface antigens in human bone marrow

In order to identify when cellular expansion occurs during hematopoietic maturation, a method was developed for the simultaneous analysis of one or two cell-surface antigens and DNA content on bone marrow cells while preserving their light-scatter properties. Proliferation in a population defined by light-scatter and surface-antigenic characteristics was assessed by measuring the percentage of cells in this population having more than 2C amount of DNA ("proliferation index"). Viable, low-density (1.077 g/cm3), bone marrow cells, stained with monoclonal antibodies conjugated with fluorescein or phycoerythrin, were fixed with paraformaldehyde and subsequently treated with the detergent, Tween 20. The UV-excitable DNA stain Hoechst 33342 was used to quantify DNA content in the cells without interference with immunofluorescence. A FACS IV flow cytometer was used, equipped with the first laser at 488 nm emitting for light scattering and immunofluorescence measurements and the second laser emitting at 360 nm for the Hoechst excitation. The Hoechst uptake was the same for all bone marrow populations, yielding a tight coefficient of variation (CV) (average 5.0%) for the G0/G1 DNA peak. This permitted high sensitivity of cell detection in S, G2, and M phases of the cell cycle, while preserving light-scattering properties of the cells and maintaining cell surface immunofluorescence. The lowest "proliferation index" detected using this technique was 0.08% in a sample obtained from a patient with chronic lymphocytic leukemia. Normal helper T lymphocytes in marrow had approximately 0.5% of the cells in S, G2, or M phase. We show that the erythroid lineage, in the adult normal bone marrow, is the most active in proliferation among all hematopoietic lineages.     

Lymphohemopoiesis in culture is prevented by interaction with adherent bone marrow cells from mutant viable motheaten mice

Mice with the recessive "motheaten" (me) or "viable motheaten" (mev) mutations have severe immunologic disturbances and die at an early age. The function of hemopoietic progenitor cells and microenvironmental elements that regulate their growth and differentiation were studied in mev mice with two types of long term bone marrow cultures. Cells from bone marrow of homozygous defective mev/mev mice were non-productive under conditions that normally support replication of stem cells and production of neutrophil granulocytes. Similarly, in a different culture system, lymphocytes were produced from normal littermate, but not mev/mev bone marrow. Initial overgrowth of cells having macrophage-like characteristics occurred in both culture systems with marrow from defective mice. Co-cultures of normal and defective bone marrow cells were always non-productive. In contrast, supernatants of mev/mev bone marrow cultures did not have a detrimental effect on cultures of normal cells, implying that the suppression was cell-associated. Furthermore, there was no evidence for abnormal release of granulocyte or macrophage growth factors in mev bone marrow cultures. A small population of cells in mev/mev bone marrow cultures were morphologically similar to "stromal" cells that support lymphohemopoiesis. Certain culture strategies could be used to enrich for these. mev/mev stromal cells had affinity for normal lymphocytes; however, they did not support lymphocyte growth. The long term bone marrow cultures thus reveal an apparent imbalance in the regulatory mechanisms affected by these single gene mutations. This is manifested by preferential or aberrant growth of one type of adherent cell and a possible functional abnormality of stromal cells. mev mice could provide an ideal model for investigating cell-associated molecules that normally limit progenitor cell replication.     

Generation and characterization of IL-2-activated bone marrow cells as a potent graft vs tumor effector in transplantation

The potential of bone marrow transplantation as an immunotherapeutic modality, using biomodulation of the marrow cells has been ignored in autologous transplantation. Furthermore, many common cancers such as lung, colon, prostate, and pancreas are resistant to even transplant doses of conventional agents and hence require novel approaches such as biomodulation. This study shows that we can generate cytotoxic killer cells similar to lymphokine-activated killer cells capable of lysing NK-resistant tumor cells in vitro if we incubate human or murine bone marrow in IL-2. This was accomplished without affecting the ability of the bone marrow to fully reconstitute mice similar to that of fresh nonactivated bone marrow. Studies evaluating the IL-2 activated human bone marrow in vitro also indicated that these activated bone marrow have similar CFU to that of fresh human marrow. Furthermore, in murine in vivo studies, the activated bone marrow (ABM) caused significant tumor regression in tumor-bearing mice. Also, these ABM cells had similar or higher tumoricidal activity and longer kinetics than spleen lymphokine-activated killer cells in vitro. Also, the ABM had purging ability in vitro. Therefore this IL-2 ABM could be used as an active therapeutic tool and not just as a passive rescue element in the autologous bone marrow transplantation setting.     

The influence of therapeutic radiation on the patterns of bone marrow in ovary-intact and ovariectomized mice

Background

The functional components of bone marrow (i.e., the hematopoietic and stromal populations) and the adjacent bone have traditionally been evaluated incompletely as distinct entities rather than the integrated system. We perturbed this system in vivo using a medically relevant radiation model in the presence or absence of ovarian function to understand integrated tissue interaction.

Methodology/Principal Findings

Ovary-intact and ovariectomized mice underwent either no radiation or single fractional 16 Gy radiation to the caudal skeleton (I±R, OVX±R). Marrow fat, hematopoietic cellularity, and cancellous bone volume fraction (BV/TV %) were assessed. Ovariectomy alone did not significantly reduce marrow cellularity in non-irradiated mice (OVX−R vs. I−R, p = 0.8445) after 30 days; however it impaired the hematopoietic recovery of marrow following radiation exposure (OVX+R vs. I+R, p = 0.0092). The combination of radiation and OVX dramatically increases marrow fat compared to either factor alone (p = 0.0062). The synergistic effect was also apparent in the reduction of hematopoietic marrow cellularity (p = 0.0661); however it was absent in BV/TV% changes (p = 0.2520). The expected inverse relationship between marrow adiposity vs. hematopoietic cellularity and bone volume was observed. Interestingly compared with OVX mice, intact mice demonstrated double the reduction in hematopoietic cellularity and a tenfold greater degree of bone loss for a given unit of expansion in marrow fat.

Conclusions/Significance

Ovariectomy prior to delivery of a clinically-relevant focal radiation exposure in mice, exacerbated post-radiation adipose accumulation in the marrow space but blunted bone loss and hematopoietic suppression. In the normally coupled homeostatic relationship between the bone and marrow domains, OVX appears to alter feedback mechanisms. Confirmation of this non-linear phenomenon (presumably due to differential radiosensitivity) and demonstration of the mechanism of action is needed to provide strategies to diminish the effect of radiation on exposed tissues.     

Spontaneous growth of colonies with mature T lineage phenotype from T-cell-depleted bone marrow precursors in a patient with a lymphoproliferative disorder of the large granular lymphocytes

Bone marrow cells from a patient with pancytopenia and a lymphoproliferative disorder of large granular lymphocytes (LGL) were cultured and tested for their hemopoietic colony-forming potential. Neither erythroid nor granulocyte-macrophage colony formation could be obtained from unfractionated, or LGL-depleted bone marrow cell preparations. However, a spontaneous growth of lymphoid colonies was observed after culturing LGL-depleted (T3-) bone marrow cell suspensions for 25 days. Pooled colonies expanded with recombinant interleukin-2 yielded a population composed predominantly of mature T cells (T3+, Leu 6-). These findings suggest that some (T3-) T cell precursors may mature in the bone marrow and that, in our patient, LGL may have exerted a suppressor effect on this maturational process.     

Comparison of in vitro and in vivo modulation of myelopoiesis by biological response modifiers

Summary In vitro growth and differentiation of granulocyte-macrophage progenitor cells (GM-CFU-C) requires colony-stimulating factors (CSF), and an in vivo role for CSF has also been proposed. Prostaglandins of the E series (PGE) have been reported to serve as negative feedback regulators of myelopoiesis. Here, we report evidence of augmented CSF secretion by mouse peritoneal Mo (macrophages) and bone marrow cells in vitro upon stimulation with various biological response modifiers (BRMs). Optimal induction of CSF secretion occurred after in vitro treatment of peritoneal Mo and mononuclear bone marrow cells with 50 g/ml poly ICLC (polyriboinosinic-polycytidylic acid poly-L-lysine), 5 g/ml lipopolysaccharide (LPS), or 500 U/ml interferon (IFN_,) for 2 days. The in vitro stimulation of CSF secretion was paralleled by an increase in PGE secretion by Mo and bone marrow cells. The PGE secretion could, however, be selectively blocked by preincubating the cells for 3 h with indomethacin (10^–7 Mol) leaving CFS production intact. In vivo treatment of mice with either maleic anhydride divinyl ether copolymer (MVE-2; 25 mg/kg) or poly ICLC (2 mg/kg) significantly increased levels of CSF in serum, as well as in culture supernatants of in vivo-treated peritoneal Mo and bone marrow cells. The increase in serum CSF levels and in secretion of CSF by peritoneal Mo and bone marrow cells was followed by a dose-dependent increase in GM-CFU-C, in nucleated bone marrow cells, and in peripheral blood leukocytes. The same BRMs also stimulated the secretion of PGE by in vivo-activated peritoneal Mo, but not by bone marrow cells. Pretreatment of the mice with indomethacin (4 mg/kg) almost completely suppressed PGE secretion by peritoneal Mo, but did not change the CSF secretion by peritoneal Mo or bone marrow cells and had no significant effect on bone marrow cellularity. Therefore, MVE-2 and poly ICLC, in addition to their immunomodulatory activity, can also have stimulatory effects on myelopoiesis, presumably mediated through secretion of CSFs. Protection and/or restoration of bone marrow function could thus either provide the opportunity for more extensive chemotherapy or could increase the number of Mo effector cells available for activation against tumor targets.     

Choice of fixatives for the immunohistochemical study of antigens using immunoadsorption

The effect of fixatives on the rat bone marrow antigens was studied by a method combining immunoadsorption and immunodiffusion. This method allows to estimate rapidly and reliably the effect of fixation on immunochemical properties of the antigen under study, using polyvalence antisera. Acetone and ethanol proved to be the best fixatives for the rat bone marrow antigens since they preserved their immunochemical properties. Aldehydes (formaldehyde and glutaraldehyde) "inactivated" the bone marrow antigens.     

Population balance model of in vivo neutrophil formation following bone marrow rescue therapy

In this paper, we develop a simple four parameter population balance model of in vivo neutrophil formation following bone marrow rescue therapy. The model is used to predict the number and type of neutrophil progenitors required to abrogate the period of severe neutropenia that normally follows a bone marrow transplant. The estimated total number of 5 billion neutrophil progenitors is consistent with the value extrapolated from a human trial. The model provides a basis for designing ex vivo expansion protocols. This revised version was published online in August 2006 with corrections to the Cover Date.     

Failure of alglucerase infused into Gaucher disease patients to localize in marrow macrophages.

BACKGROUND: Gaucher disease is a common glycolipid storage disease, caused by a deficiency of lysosomal beta-glucosidase (glucocerebrosidase). Alglucerase is a form of glucocerebrosidase enriched with terminal mannose moieties, so as to "target" the preparation to the high-affinity macrophage receptor in patients with Gaucher disease. Our earlier in vitro studies indicated that alglucerase was bound by cells other than macrophages by a widely distributed, low-affinity mannose receptor. MATERIALS AND METHODS: Bone was removed at surgery from six patients with Gaucher disease; in three cases, bone was obtainable both when the patient was untreated and after receiving an infusion of alglucerase. Four samples of bone were obtained from patients without Gaucher disease and served as controls. A bone marrow aspirate was obtained from another patient with Gaucher disease immediately after enzyme infusion. Marrow beta-glucosidase activity and chitotriosidase (a macrophage marker) was determined on all samples. RESULTS: Even with the large bolus doses used for the treatment of Gaucher disease by some, scarcely any beta-glucosidase activity was found in marrow samples; the amount of the enzyme was much less than would have been anticipated had the enzyme been evenly distributed to all body cells. CONCLUSIONS: Alglucerase is not targeted to marrow macrophages. Its unquestioned therapeutic effectiveness must be due either to its activity at some site other than marrow macrophages or to the fact that the doses administered are so enormous that even a small fraction is sufficient to achieve a therapeutic effect.     

Characterization of zinc effect to inhibit osteoclast-like cell formation in mouse marrow culture: Interaction with dexamethasone

The inhibitory effect of zinc compounds on osteoclast-like cell formation in mouse marrow culture in vitro was characterized. The bone marrow cells were cultured for 7 days in -minimal essential medium containing a well-known bone resorbing agent, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] or prostaglandin E2 (PGE2). Osteoclast-like cell formation was estimated by staining for tartrate-resistant acid phosphatase (TRACP), a marker enzyme of osteoclasts. The presence of 1,25(OH)2D3 (10-8 M) or PGE2 (10-6 M) induced a remarkable increase in osteoclast-like multinucleated cells. These increases were enhanced by the presence of dexamethasone (10-9 to 10-6 M). The dexamethasone (10-7 M)-enhanced osteoclast-like cell formation was not inhibited by the presence of zinc sulfate (10-6 M) or zinc-chelating dipeptide (-alanyl-L-histidinato zinc; 10-6 M), although the zinc compounds had an inhibitory effect on osteoclastic formation in the absence of the steroid. The effect of dexamethasone was not seen, when the steroid was added at the later stage of culture with bone-resorbing agents. In this case, the inhibitory effect of zinc compounds was clearly revealed. This effect of zinc compounds disappeared in the presence of Ca2+-chelating agent (0.5 mM EGTA). The present study suggests that zinc compounds have an inhibitory effect at the stage of differentiation of preosteoclastic cells in bone marrow cell culture system. (Mol Cell Biochem 166: 145-151, 1997)     

Prevention of Liver Fibrosis by Intrasplenic Injection of High-Density Cultured Bone Marrow Cells in a Rat Chronic Liver Injury Model

Endothelial progenitor cells (EPCs) from bone marrow have proven to be functional for the prevention of liver fibrosis in chronic liver injury. However, expansion of EPCs in culture is complicated and expansive. Previously, we have established a simple method that could enrich and expand EPCs by simple seeding bone marrow cells in high density dots. The purpose of this study is to evaluate whether cells derived from high-density (HD) culture of rat bone marrow cells could prevent the liver fibrosis in a chronic liver injury rat model, induced by carbon tetrachloride (CCl₄). Flow cytometric analysis showed that cells from HD culture were enriched for EPCs, expressing high levels of EPC markers. Intrasplenic injection of HD cultured bone marrow cells in the CCl₄-induced liver injury rat showed an enhanced antifibrogenic effect compared with animals treated with cells from regular-density culture. The antifibrogenic effect was demonstrated by biochemical and histological analysis 4 weeks post-transplantation. Furthermore, cells from HD culture likely worked through increasing neovascularization, stimulating liver cell proliferation, and suppressing pro-fibrogenic factor expression. HD culture, which is a simple and cost-effective procedure, could potentially be used to expand bone marrow cells for the treatment of liver fibrosis.