ABO blood group antigens on human plasma von Willebrand factor after ABO-mismatched bone marrow transplantation.

von Willebrand factor (vWF) is synthesized exclusively by endothelial cells and megakaryocytes, and stored in the intracellular granules or constitutively secreted into plasma. ABO blood group antigens are covalently associated with asparagine-linked sugar chains of plasma vWF. The effect of ABO-mismatched bone marrow transplantation (BMT) or blood stem cell transplantation (BSCT) on the expression of ABO blood group antigens on the vWF was examined to obtain information on the origin of these antigens. In ABO-mismatched (HLA-matched) groups, 8 cases of BMT and 4 cases of BSCT were examined. In all cases, the ABO blood groups on red blood cells were gradually converted to the donor's type within 80 to 90 days after the transplantation. The blood group antigens on the vWF were consistent with the recipient's blood group for the period monitored by enzyme-linked immunosorbent assay (ELISA). When vWF was isolated from normal platelets and examined for the blood group antigens using ELISA or immunoblotting, it showed few antigens. However, vWF extracted from veins expressed blood group antigens. These findings indicate that platelet (megakaryocyte)-derived vWF does not contain blood group antigens and that these antigens may be specifically associated with vWF synthesized in endothelial cells and secreted into plasma. Furthermore, it is possible that the persistence of the recipient's blood group antigens on plasma glycoproteins such as vWF, independent of the donor-derived erythrocytes, after ABO-mismatched stem cell transplantation, may influence the immunological system in the production of anti-blood group antibodies resulting in the establishment of immunological tolerance in the recipient plasma.     

Expression of adhesion antigens of human bone marrow megakaryocytes, circulating megakaryocytes and blood platelets.

There is evidence that mature megakaryocytes migrate into sinusoids, enter the blood and fragment in the vascular bed. We wondered whether differences in expression of adhesion antigens could be associated with the egress of megakaryocytes from bone marrow into the peripheral blood or the fragmentation into platelets. Megakaryocytes from human marrow were purified by counterflow centrifugal elutriation followed by a glycoprotein Ib-dependent agglutination procedure. Megakaryocytes from central venous blood and pulmonary arteries were purified by counterflow centrifugal elutriation alone. Adhesion antigens were labelled in an immunohistochemical assay. Both bone marrow megakaryocytes and platelets from healthy volunteers stained > 75% positive for CD36, CD41, CD42, Cdw49b (alpha subunit VLA2), Cdw49e (alpha subunit VLA5), Cdw49f (alpha subunit VLA6) and CD62. Circulating megakaryocytes, although > 75% positive for CD41, had, unlike platelets and bone marrow megakaryocytes, a reduced and remarkable heterogeneous (5-100% positive) labelling with antibodies against Cdw49b, Cdw49e, Cdw49f. These results could be confirmed by comparing the bone marrow megakaryocytes, circulating megakaryocytes and platelets from 7 patients that were recovered and processed at the same time. Morphologically mature, circulating megakaryocytes have, unlike bone marrow megakaryocytes, a heterogeneous expression of adhesion antigens, especially of Cdw49b, Cdw49e, and Cdw49f.     

Expression of Ia-like antigens defined by monoclonal OKIal antibody of hemopoietic progenitor cells in cord blood: a comparison with human bone marrow

Expression of Ia antigens on granulocyte/macrophage colony-forming cells (CFU-GM) in human cord blood was compared with that in bone marrow with the use of monoclonal OKIal antibody. Mononuclear cells prepared from cord blood and bone marrow were pretreated with OKIal antibody plus complement, and, thereafter, the ability of cord blood and bone marrow cells to form colonies of CFU-GM was assayed in semisolid agar culture. Consistent reduction in the number of CFU-GM in cord blood to 58.8% +/- 13.0% (mean +/- SD) of controls treated with complement alone was shown after elimination of Ia-antigen-bearing CFU- GM, but was significantly remarkable than that in bone marrow (18.0% +/- 5.6%). Although the reduction of both granulocyte (CFC-G) and macrophage colony (CFC-M) types of cord blood, characterized by the double staining for esterase activity, was shown following treatment with OKIal antibody plus complement, the relative inhibition of CFC-G weas significantly greater than that of CFC-M (p less than 0.02). These results suggest some differences in the characteristics of Ia-antigen- bearing CFU-GM between cord blood and bone marrow cells. Furthermore, it is suggested that Ia-dependent regulatory mechanisms might participate in the differentiation of CFU-GM to CFC-G and CFC-M.     

Expression of major blood group antigens on human erythroid cells in a two phase liquid culture system

In order to examine the sequential expression of major blood group antigens on human erythroblasts, a selective two phase liquid culture system for erythroid progenitors was established. After mononuclear cells obtained from peripheral blood were cultured in the presence of phytohemagglutinin stimulated-leukocyte conditioned medium (PHA-LCM) for 7 days (the first phase), nonphagocytic cells were recultured under hypoxic culture conditions containing 30% fetal calf serum, 1% bovine serum albumin, 300 micrograms/mL transferrin and 2 U/mL recombinant erythropoietin (the second phase). Mature (orthochromatic) erythroblasts were observed on day 4 of the second phase, and reached 57.1 +/- 3.1% of total cells on day 8, followed by the appearance of denucleated red cells, equivalent to mature red cells in peripheral blood. Hemoglobin contents reached the level of 16.8 +/- 0.7 micrograms/10(6) cells on day 8. Flow cytometric analyses revealed that, on day 3 of the second phase, cells became blood type M-positive, corresponding to the maturation of erythroid cells. Regarding the expression of ABH blood group antigens, a small number of blood type H- positive cells were initially detected on day 0 of the second phase, while blood type A-positive cells, which essentially were not observed on day 0, increased gradually corresponding to the extent of erythroid maturation. In the present system, Lewis and P1 blood group antigens were expressed at day 5 of the second phase, although autologous plasma was required to determine the expression of Lewis blood group antigens. This culture system is beneficial for studies on normal and abnormal human red cell membranes, because the erythroid progenitors in human peripheral blood were used, and a reasonable number of erythroid cells (0.5 to 1.5 x 10(7] was obtained with good maturation.     

Expression of integrins in human bone marrow

Expression of integrins, a superfamily of glycoprotein alpha/beta heterodimers which integrate the cytoskeleton with the extracellular matrix and/or mediate cell-cell adhesive interactions, was examined on normal and leukaemic bone marrow cells by immunohistochemistry and immunotransmission electron microscopy (immuno-TEM). Among the beta 1/VLA molecules studied, VLA-2 and 6 were expressed on megakaryocytes and platelets, while VLA-4 was present on 40% of haemopoietic cells, including monocytes, erythroblasts and immature cells; this molecule was typically localized at sites of intercellular contact, as seen by immuno-TEM, suggesting it may be involved in interactions among haemopoietic cells during differentiation. In human long-term bone marrow cultures (LTBMC), VLA-1 and 3 were present respectively on 35% and 40% of the adherent cells which included fibroblasts and endothelial cells, as shown by double-labelling experiments; VLA-2 was expressed only on a subpopulation of fibroblasts. beta 2/LeuCAM molecules were absent from platelets, megakaryocytes and HLA-DR+/myeloperoxidase- early myeloid precursors, and appeared progressively during maturation in both lymphoid and myeloid cells. Expression of beta 3/cytoadhesin molecules was restricted to megakaryocytes and platelets and, in the adherent layer of LTBMC, to endothelial cells. The regulated expression and specific localization of integrins in the bone marrow suggest that these molecules may have a role in normal haemopoiesis.     

CD4 Expression by erythroid precursor cells in human bone marrow

Flow cytometry was used to assess CD4 expression in 62 consecutive bone marrow specimens from patients with a variety of clinical conditions. Using a lysed-whole-blood technique for labeling with monoclonal antibodies, two populations of CD4+ cells were identified within the lymphocyte/blast-cell fraction in 58 (94%) of these specimens. These consisted of (1) a population of T helper cells with high density expression of CD4 and (2) a second population of cells with low-density expression of CD4, which ranged from 1% to 36% of the gated cells. This latter population was present regardless of age, sex, or clinical condition including 21 of 21 specimens (100%) categorized as unremarkable bone marrows both morphologically and by flow cytometry and in four of four patients (100%) with human immunodeficiency virus- type 1 (HIV-1) infection. Coexpression of the erythroid lineage marker, glycophorin A, with the majority of cells in this second population was demonstrated in all 11 randomly selected samples using two-color flow cytometric analysis. These cells also expressed low levels of the myeloid markers, CD13 and CD33, but CD34 expression could not be demonstrated. These results provide evidence for expression of CD4 on cells of erythroid lineage in human marrow, and offer a potential mechanism for direct infection of erythroid precursor cells and deranged erythropoiesis in patients with HIV-1 infection.     

The distribution of histochemically demonstrable glycogen in human blood and bone marrow cells

By applying Schiff’s reagent after periodic acid treatment to blood and bonemarrow films, a cytoplasmic staining reaction is seen in some cells of the myeloidseries, as well as in megakaryocytes and platelets. The intensity of the staining reaction in the myeloid cells increases with their maturation. The staining reactioncan be prevented altogether in alcohol-fixed films by salivary digestion, but onlyincompletely in air-dried films. The staining reaction is due to the presence of glycogen in some chemical association, possibly with protein.

     

HLA-class II antigens on hemopoietic and stromal cells in human micro long-term bone marrow cultures

Using a complement-dependent cytotoxicity assay (CDC), we analyzed the presence of HLA-class II antigens on both stromal and hemopoietic cells in a miniaturized human long-term bone marrow culture system. 4-Hydroperoxycyclophosphamide (4-HC)-resistant hemopoietic stem cells capable of restoring in vitro hemopoiesis on irradiated stromal cell layers were HLA-DR, -DP, and -DQ negative. In addition, these cells failed to bind the monoclonal antibody (mAb) Tü 39, previously proposed as a candidate for the recognition of a novel class II antigen, "-DY." On the other hand, the formation of confluent stromal cell layers was inhibited by HLA-DR- or -DP-specific mAbs, but not by the HLA-DQ-specific mAb Tü 22. This suggests the presence of HLA-DR- and/or HLA-DP-positive, but HLA-DQ-negative stromal precursor cells.     

Confirmation of bone marrow engraftment by detection of M/N blood group antigens on erythroblasts in erythroid bursts

In order to confirm erythroid engraftment, we examined the changes in blood group antigens expressed on erythroblasts in the haematopoietic colonies of a bone marrow transplant recipient. A 44-year-old female with acute myelogenous leukaemia underwent an allogeneic bone marrow transplantation from her sister donor. Prior to the transplantation, the blood groups of both recipient and donor were analysed, and their M/N groups found to differ, the former being MN and the latter N. Bone marrow mononuclear cells were obtained from the patient on day 21 after bone marrow transplantation and cultured in semi-solid medium. Erythroblasts were collected from the erythroid bursts that had formed, and were subjected to flow cytometry using monoclonal anti-M and anti-N. Anti-N reactive cells accounted for 99.2% of those in the erythroid bursts, while only 0.3% of these cells were anti-M reactive. MN type erythrocytes in the recipient's peripheral blood had been replaced by N type and only 2.0% of erythrocytes were anti-M reactive on the 70th day after BMT. These erythroblasts and erythrocytes were phenotypically N, and originated from the donor haemopoietic stem cells with the success of bone marrow engraftment. From our findings, it appears that the M/N blood group antigens were produced by the erythroid cells themselves.     

ABO blood group system and bone marrow transplantation.

The role of the ABO blood group system in determining the outcome of bone marrow transplantation was investigated in 53 patients with aplastic anemia and acute leukemia grafted from HLA-identical siblings. There was no correlation between ABO compatibility and marrow engraftment, graft rejection, or graft-versus-host disease. In 5 recipients with antibodies prior to transplantation to antigens of the ABH system present on the cells of their donors, plasma exchange and antibody absorption in vivo were effective in permitting engraftment of ABO-incompatible bone marrow. These findings indicate that the ABO system is not a clinically significant barrier to successful bone marrow transplantation in otherwise histocompatible individuals.     

Plasma blood group glycosyltransferase activities after bone marrow transplantation

Human blood groups (ABO) are known to be determined by the terminal glycosyl residues attached to common carbohydrate chains of the red cell surface. N-acetylgalactosaminyltransferase (A-enzyme) in blood group A persons and galactosyltransferase (B-enzyme) in blood group B persons are responsible for producing A and B substances on the red cell surface, with both enzymes absent in blood group O persons. The plasma transferase (A - and B-) activity was assayed after the complete replacement of the bone marrow of patients with acute leukemia or aplastic anemia by transplantation bone marrow from donors with ABO blood group differing from the recipient. The patient's blood type completely changed from the recipient's type to the donor's type. However, the A- and B-enzyme activities of the patients changed only slightly after bone marrow transplantation. The results indicate that most of the A- and B-enzymes in the circulatory plasma is not derived from the bone marrow, lymphoid, or macrophage tissue. Other tissues must be the primary source of the enzymes in plasma.     

Plastic-adherent progenitor cells in human bone marrow

Human bone marrow contains plastic-adherent hemopoietic progenitor cells whose plating efficiency is increased by brief (2 h) exposure to methylprednisolone (MP). When subsequently covered with methylcellulose medium, they form colonies of monoblastoid cells. Colony size, but not number, and mature cell production are increased by erythropoietin (epo) and granulocyte-macrophage colony-stimulating factor (GM-CSF). However, colonies do not grow under serum-free conditions. The resistance of plastic-adherent progenitors to treatment with 5-fluorouracil (5FU), their growth pattern, and their capacity to produce granulocytic and erythroid colonies on replating, suggest that they may be similar to the primitive, 5FU-resistant, plastic-adherent progenitor cells (HPP-CFC) in murine marrow.     

A and B blood group antigen expression on mixed colony cells and erythroid precursors: relevance for human allogeneic bone marrow transplantation

Using anti-A and anti-B blood group monoclonal antibodies and fluorescent activated cell sorting of human bone marrow, A (or B) blood group antigen was shown to be on 5.2 ± 5.9 (meanfSD) % of CFU-GEMM and 12 ± 5 ± 19.6% of the erythroid burst forming cells (designated BFU-GEMM) as defined by the mixed colony assay, and 49.5±20% of the BFU-E and 83.5±9.9% of the CFU-E as defined by the erythroid colony assay. This antigen expression on the BFU-GEMM is consistent with the concept that erythroid bursts stimulated by leucocyte conditioned medium are less mature, and are closer in development to the pluripotent stem cell than the BFU-E. These results help to explain the delayed erythropoiesis, and perhaps impaired engraftment of all cell lineages, that may occur in some recipients of ABO incompatible bone marrow transplants, with persistent and high anti-A titres.     

Basic fibroblast growth factor expression in human bone marrow and peripheral blood cells

We have shown previously that basic fibroblast growth factor (bFGF) is a mitogen for human bone marrow (BM) stromal cells and that bFGF stimulates myelopoiesis in primary BM cultures. In this article, we demonstrate the presence of bFGF in two cell lineages in human BM and peripheral blood as well as the deposition of bFGF into the extracellular matrix of BM stromal cell cultures. In immunofluorescence experiments on BM and peripheral blood smears, megakaryocytes and platelets stained strongly for bFGF, whereas weaker staining was observed in immature and mature cells of the granulocyte series. The presence of bFGF in platelets was confirmed by enzyme-linked immunosorbent assay as well as by immunoprecipitation followed by immunoblotting. bFGF was synthesized by BM stromal cell cultures and was found either cell associated or localized in the nucleus and the nucleoli, and its location was dependent on the fixation procedure used. Addition of exogenous bFGF to stromal cells showed the presence of extracellular binding molecules for this cytokine. bFGF could be released from these sites by soluble heparin or phosphatidylinositol- specific phospholipase C. This study supports the role of bFGF as a stromal cell mitogen and stimulator of myelopoiesis. The data indicate that the stromal cells produce bFGF and that their extracellular matrix can serve as a reservoir for this growth factor. In addition, the results suggest a possible involvement of bFGF in platelet function as well as in megakaryocytopoiesis.     

A simple method for culturing myeloma cells from human bone marrow aspirates and peripheral blood in vitro

A double layer agar technique has been developed to grow myeloma colonies (MY-CFUc) from human bone marrow aspirates and peripheral blood. Heavily irradiated HL60 cells (5 x 10(5)/plate) are added to an agar underlay in growth medium containing 0.5% agar. Mononuclear cells from the test bone marrow or blood are overlayered in either 0.2 ml HL60-conditioned medium (HL60-CM) or in 0.5 ml growth medium containing 0.23% agar, and the cultures are incubated at 37 degrees C in an atmosphere of 5% CO2, 10% O2 and 85% N2. Colonies (greater than 50 cells) form between 2 and 3 weeks. Using this method 60/68 samples of bone marrow and 7/12 samples of blood from 54 patients have produced colonies in soft agar and in liquid on an agar underlay. The cells which form these colonies are of two distinct sizes, the larger cells being plasmacytoid and the smaller lymphoid. The two cell types are usually, but not always, present in separate colonies. Both plasmacytoid and lymphoid cells carry the isotype of the respective patient's myeloma protein and the plasma cell marker (HAN PC1). This technique has enabled us to culture myeloma cells from patients with as few as 2% plasma cells in the bone marrow but it does not permit the growth of normal B, T or granulocyte-macrophage colonies (GM-CFUc). The drug sensitivity of myeloma cells (MY-CFUc) compared with normal haemopoietic cells (GM-CFUc) can be measured using dose-response curves in individual patients. Furthermore, this method can detect resistant subpopulations within a given myeloma sample.     

Expression of blood group A and B antigens in gastric cancer examined by monoclonal antibodies

Fourty-eight patients with gastric carcinoma were histologically examined and classified according to Laurén as intestinal or diffuse type. Employing monoclonal antibodies, in all samples the expression of blood group A and B antigens was studied using the method of indirect immunoperoxidase reaction. In patients with blood groups A and AB the comparison of expression revealed marked differences between the intestinal and diffuse type carcinomas. In the intestinal type, the expression of the blood group antigens was highly heterogenous, while diffuse type carcinomas displayed a homogenous expression. The obtained differences support Laurén's classification. The staining of supranuclear regions of tumor cells by the monoclonal antibody HE 10 (which reacts with antigenic determinants A and H of type 3 and 4) was found more frequently in intestinal type carcinomas. The possible prognostic significance of these findings is discussed.     

Expression of the major sialoglycoprotein (glycophorin) on erythroid cells in human bone marrow

The major sialoglycoprotein of human erythrocyte membranes (glycophorin) is one of the most-studied membrane proteins. Although the structure is relatively well known, almost nothing is known about its expression in erythroid cells. To study this we raised an antiserum that reacted specifically with this protein. This was accomplished by immunization of rabbits with a preparation of glycophorin followed by absorption with En(a-) erythrocyte membranes, which lack glycophorin. By use of this antiserum and a staphylococcus protein A technique we could establish that only bone marrow cells of erythrocyte lineage express glycophorin at the cell surface. This occurs in basophilic normoblasts and later stages of erythrocyte differentiation, whereas pronormoblasts do not seem to contain glycophorin.