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.     

HSP10 selective preference for myeloid and megakaryocytic precursors in normal human bone marrow

Heat shock proteins (HSPs) constitute a heterogeneous family of proteins involved in cell homeostasis. During cell life they are involved in harmful insults, as well as in immune and inflammatory reactions. It is known that they regulate gene expression, and cell proliferation, differentiation and death. HSP60 is a mitochondrial chaperonin, highly preserved during evolution, responsible of protein folding. Its function is strictly dependent on HSP10 in both prokaryotic and eukaryotic elements. We investigated the presence and the expression of HSP60 and HSP10 in a series of 20 normal human bone marrow specimens (NHBM) by the means of immunohistochemistry. NHBM showed no expression of HSP60, probably due to its being below the detectable threshold, as already demonstrated in other normal human tissues. By contrast, HSP10 showed a selective positivity for myeloid and megakaryocytic lineages. The positivity was restricted to precursor cells, while mature elements were constantly negative. We postulate that HSP10 plays a role in bone marrow cell differentiation other than being a mitochondrial co-chaperonin. The present data emphasize the role of HSP10 during cellular homeostasis and encourage further investigations in this field.     

Humoral detection of leukaemia-associated antigens in presentation acute myeloid leukaemia

The serological analysis of recombinant cDNA expression libraries (SEREX) technique was used to immunoscreen a testes cDNA expression library with sera from newly diagnosed acute myeloid leukaemia (AML) patients. We used a testis cDNA library to aid our identification of cancer-testis (CT) antigens. We identified 44 antigens which we further immunoscreened with sera from AML, chronic myeloid leukaemia (CML), and normal donors. Eight antigens were solely recognised by patient sera including the recently described CT antigen, PASD1, and the cancer-related SSX2 interacting protein, SSX2IP. RT-PCR analysis indicated that we had identified three antigens which were expressed in patient bone marrow (BM) and peripheral blood (PB) but not in normal donor samples (PASD1, SSX2IP, and GRINL1A). Real-time PCR (RQ-PCR) confirmed the restricted expression of PASD1 in normal donor organs. Antigen presentation assays using monocyte-derived dendritic cells (mo-DCs) showed that PASD1 could stimulate autologous T-cell responses, suggesting that PASD1 could be a promising target for future immunotherapy clinical trials.     

Immunodiffusion analysis of water-soluble rat bone marrow antigens

Antisera were obtained against six electrophoretic fractions of the rat bone marrow extract. With their help, 18 tissular antigens and 11 antigens immunologically similar to the blood serum proteins were revealed in the rat bone marrow. All tissular antigens are divided in five groups by the degree of organ specificity: 1) bone marrow organospecific antigens (4 antigens), 2) antigens present in the bone marrow, spleen and lung extracts (2), 3) "granulocytic" antigens (4), 4) antigens common for many rat organs, but not found in the extracts of blood formed elements, skeletal muscle, heart, brain, eyes (3), 5) antigens present in all the organs studied (5). The bone marrow organospecific antigens may be specific antigens of hemopoietic cell precursors. The possibility of utilization of antisera against the bone marrow water soluble proteins for labelling hemopoietic cells of different lines of differentiation is discussed.     

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. Download video file.^{(65M, mp4)}     

Brain Ia antigens have a bone marrow origin

Our results, using radiation-induced bone marrow chimeras, demonstrate that the Ia antigen found in the brains of such animals is produced by cells having precursors in the bone marrow. These cells are not immediately blood borne since no IgM is detected in these brains. This rules out the obvious possibility of B-lymphocyte contamination as the source of Ia in the brain cell preparations. It thus appears that the central nervous system, like many other nonlymphoid organs, has a source of Ia-positive cells that are derived from bone marrow precursors.     

Insights into the expression of ABH and Lewis antigens through human bone marrow transplantation.

Twelve information bone marrow transplants, with at least one difference in ABO and/or Lewis types between donor and recipient, were retrospectively studied. ABH and Lewis antigens were determined in plasma, erythrocytes, and lymphocytes. Donor lymphocytes acquired the ABH and Lewis antigens from the recipient's plasma in the same way that donor erythrocytes acquired the Lewis antigens from it. Lymphocytotoxicity detected type 1 ABH and Lewis antigens only, providing evidence for the existence of combined ABH and Lewis antigens on lymphocytes. This was in contrast with the ABH antigens on type 2 chains of red cells, which are devoid of Lewis specificities. The differences in genetic control, probable chemical structure, and cellular origin of these two types of ABH antigens are presented in a theoretical model that accounts for most of the known data.     

Monoclonal antibodies to carbohydrate antigens in autologous bone marrow transplantation

Normal and malignant myeloid cells express a highly immunogenic oligosaccharide, lacto-n-fucopentaose-III (LNF-III), that has been identified by numerous monoclonal antibodies (MoAb). We have been interested in the use of a particular monoclonal antibody to LNF-III, PM-81, in the treatment of patients with acute myelogenous leukemia using the antibody to treat bone marrow in vitro. Following in vitro treatment of bone marrow with PM-81 and another MoAb, AML-2-23, the remaining cells are used as an autograft in a patient treated with high-dose chemotherapy and radiotherapy. In order to enhance the ability of the MoAb to lyse leukemic cells in the remission bone marrow, we have explored the effect of neuraminidase treatment on leukemia cells. In this paper we describe that myeloid leukemia cells expressing low levels of LNF-III by immunofluorescence can be shown to have high levels of LNF-III after neuraminidase treatment. In addition, we show that normal bone marrow progenitor cells do not have cryptic LNF-III antigen, thus allowing the application of this finding to the clinical setting. Moreover, we have shown that leukemia colony-forming cells from one patient with acute myelogenous leukemia express cryptic LNF-III and that after exposure to neuraminidase there was an increased ability of PM-81 in the presence of complement to eliminate these colony forming cells. These data indicate that the LNF-III moiety is almost universally expressed on myeloid leukemia cells and their progenitors but not expressed on normal progenitors. Thus, it may be possible to enhance leukemia cell kill in vitro by neuraminidase treatment of bone marrow.     

Fetuin in human bone marrow: detection in foetal tissue and patients with mastocytosis

Fetuin, a foetal protein of unknown function, has been shown to be expressed in both the immune and nervous systems, especially during development. Here, we show for the first time, that fetuin is abundantly present in many cells of the foetal human bone marrow, but is restricted to cells of the monocytic lineage in the adult. Fetuin's immunoreactivity increased considerably in adult human bone marrow in some pathological conditions, particularly in mastocytosis and was also increased in bone marrows in some cases of acute leukaemias, especially in acute myeloid leukaemia. This increase in the presence of fetuin in neoplastic bone marrows is not reflected by an increased level of circulating fetuin. This last observation contradicts earlier suggestions that fetuin is specifically reduced in cancer patients. A consistent increase in fetuin immunoreactivity in bone marrow of most cases of mastocytosis, as demonstrated in this paper, could become a useful tool in the diagnosis of this disease.     

A technique for the separation and cryopreservation of myeloid stem cells from human bone marrow.

We have developed a technique for the cryopreservation of large volumes of human bone marrow, which reduces cell losses due to clumping and release of lysosomal enzymes from mature granulocytes. Mononuclear cells were separated from whole bone marrow by a large-scale Ficoll-Hypaque procedure. The agar colony assay for myeloid stem cells (CFU-C) was used to assess each step of the isolation and cryopreservation procedure. Conditions of varied cell and cryoprotectant concentrations and freezing and thawing rates were compared to obtain optimal recovery of mononuclear cells and CFU-C. This technique has been used to store bone marrow from 45 patients with hematologic and non-hematologic neoplasms. Up to 750 ml of marrow was obtained from each patient and separated by step-gradient centrifugation, and the cell fraction containing myeloid stem cells was cryopreserved. The mean recoveries following separation, cryopreservation, and thawing for 18 marrow storages from patients with hematological neoplasms were 8.8 ± 2.9% for mononuclear cells and 47.8 ± 20.8% for CFU-C. In comparison, values for 27 marrows from patients with non-hematological neoplasms were 14.5 ± 5.5% for cells and 57.7 ± 13.7% for CFU-C.     

Growth of human bone marrow in vitro

Attempts were made to establish human bone marrow (HBM) culture. Cell proliferation was triggered by medium conditioned by acute myelocytic leukemic (AML) cells exposed to 12-0-tetradecanoyl-phorbol-13-acetate (TPA). A 6-15 fold multiplication of myelomonocytic cells was obtained in tissue culture from HBM cells. Proliferation of granulocyte-macrophage colony forming cells (CFC-GM) occurred up to 30-fold. The morphology of the cells and cytochemical markers were investigated at all stages of the culture.     

Adipogenesis in a myeloid supporting bone marrow stromal cell line.

The bone marrow stroma contains pre-adipocyte cells which are part of the hemopoietic microenvironment. Cloned stromal cell lines differ both in their ability to support myeloid and lymphoid development and in their ability to undergo adipocyte differentiation in vitro. These processes have been examined in the +/+2.4 murine stromal cell line and compared to other stromal and pre-adipocyte cell lines. In long-term cultures, the +/+2.4 stromal cells support myeloid cell growth, consistent with their expression of macrophage-colony stimulating factor mRNA. However, despite the presence of mRNA for the lymphoid supportive cytokines interleukins 6 and 7, +/+2.4 cells failed to support stromal cell dependent B lineage lymphoid cells in vitro, suggesting that these stromal cells exhibit only a myelopoietic support function. The +/+2.4 cells differentiate into adipocytes spontaneously when cultured in 10% fetal bovine serum. The process of adipogenesis can be accelerated by a number of agonists based on morphologic and gene marker criteria. Following induction with hydrocortisone, methylisobutylxanthine, indomethacin, and insulin in combination, a time dependent increase in the steady state mRNA and enzyme activity levels of the following adipocyte specific genes was observed: adipocyte P2, adipsin, CAAT/enhancer binding protein, and lipoprotein lipase. In contrast, adipogenesis was accompanied by a slight decrease in the signal intensity of the macrophage-colony stimulating factor mRNA level, similar to that which has been reported in other bone marrow stromal cell lines. These data demonstrate that although the lympho-hematopoietic support function of pre-adipocyte bone marrow stromal cell lines is heterogeneous, they share a common mechanism of adipogenesis.     

Lipid composition of human bone marrow

1. A modified method for the analysis of phospholipid mixtures by selective hydrolysis is described. 2. The phospholipid compositions of normal human bone marrow and of the bone marrows of patients who died with anaemia or various forms of leukaemia were investigated. 3. Phospholipids from normal bone marrow comprised about 44% of lecithin, 4% of choline plasmalogen, 7% of glyceryl ether phospholipid (choline base), 10% of sphingomyelin, 22% of phosphatidylethanolamine plus phosphatidylserine, 8% of ethanolamine plasmalogen and 5% of glyceryl ether phospholipid (ethanolamine base). 4. The proportion of kephalin (i.e. phosphatidylethanolamine plus phosphatidylserine) in the pathological bone marrows tended to be lower than normal. No other consistent differences were observed between the normal and pathological samples. 4. A ceramide dihexoside was isolated from normal bone marrow.     

An overview of bone marrow transplantation for chronic myeloid leukemia.

The use of intensive therapy together with transplantation of marrow from a suitable donor is the only established curative treatment for patients with chronic myeloid leukemia (CML). However, marrow transplantation is hazardous, costly and applicable to relatively few patients. Therefore, we evaluated the results and limitations of marrow transplantation for CML and discussed new treatment strategies. We decided to select a limited number of papers that focused on the relevant issues rather than to undertake an exhaustive comparison of treatment results from different centres. Patients with CML in the chronic phase who receive marrow from a sibling with the same human leukocyte antigen type can expect to have a long-term disease-free survival rate of 50%. However, the procedure is associated with a mortality rate of 30%, mainly because of graft-versus-host disease (GVHD) and interstitial pneumonitis. Moreover, because of the requirements for age and histocompatibility only 10% of patients with chronic-phase CML are currently eligible. Transplantation earlier in the chronic phase (within 1 year after diagnosis), the use of marrow from matched, unrelated donors and the development of improved methods for reducing the incidence of GVHD all hold promise. In addition, the preliminary results of intensive therapy followed by transplantation with cultured autologous marrow have been encouraging. If further progress is to be made, continued optimism coupled with carefully developed and executed studies will be necessary.