Interaction of ristocetin and bovine plasma with guinea pig megakaryocytes: a means to enrich megakaryocytes based on membrane rather than physical characteristics

We have investigated whether megakaryocytes can be aggregated by ristocetin and bovine plasma and whether such aggregation can be used as a step in the purification of megakaryocytes from marrow cell suspensions. Guinea pig marrow cell suspensions were first enriched for megakaryocytes by density equilibrium centrifugation in continuous Percoll density gradients. The megakaryocyte-enriched marrow was stirred in a platelet aggregometer to which ristocetin or bovine plasma was added. Megakaryocytes were aggregated by both ristocetin and bovine plasma with the proportion aggregated being related to the concentration of ristocetin or bovine plasma. Maximal aggregation (greater than 90% of megakaryocytes) was achieved with 2.0 mg/mL ristocetin or 5% bovine plasma and required five minutes. All maturation stages of morphologically recognizable megakaryocytes were aggregated. The megakaryocyte aggregates were separated from the marrow suspension by sedimentation at 1 g and the megakaryocytes disaggregated by dilution with media (ristocetin aggregated) or addition of dextran sulfate (bovine plasma aggregated). Megakaryocyte purity and recovery were higher with bovine plasma than with ristocetin. A mean of 92% of the megakaryocytes in the bovine plasma aggregated cell suspensions were recovered with megakaryocytes constituting an average of 76% of the final cell suspensions. The viability as well as the diameters and DNA content distribution of these megakaryocytes were similar to those of the starting population. We conclude that guinea pig megakaryocytes behave like platelets in that they can be aggregated with ristocetin or bovine plasma and that megakaryocyte aggregation induced by ristocetin or bovine plasma provides a means to enrich these cells based on membrane rather than physical characteristics. This approach yields purified megakaryocyte populations that are representative of those in unfractionated marrow.     

Stromal cell progeny of murine bone marrow fibroblast colony-forming units are clonal endothelial-like cells that express collagen IV and laminin

Studies of human and murine bone marrow explants have demonstrated the existence of stromal cell precursors that give rise to colonies of adherent cells in short-term cultures. Because previous data suggested that these colonies were composed of fibroblasts, the precursor cells were termed fibroblast colony-forming units (CFU-F). However, we have recently shown that the stromal cells which support hematopoiesis in murine long-term bone marrow cultures (LTBC) express collagen IV and laminin, markers associated with an endothelial cell lineage, but are negative for collagen I and III, markers associated with a fibroblast cell lineage. Because these conflicting results suggest major functional differences between the stromal cells observed in long-term cultures and the short-term assay, we re-examined the lineage of CFU-F- derived stromal cells. Using two-color immunofluorescence, we characterized virtually all of the cells comprising individual "CFU-F" colonies derived from mouse radiation chimeras. Identification of donor (hematopoietic) or host (stromal) origin was based on surface staining for strain-specific H-2 surface antigens, and, for endothelial or fibroblast properties, on cytoplasmic staining for laminin and collagen IV, or collagens I and III, respectively. The results demonstrate that a large proportion of the cells in CFU-F colonies are donor-derived and fail to stain with any of the antisera specific for nonhematopoietic cells. In addition, these donor-derived cells exhibit marked phagocytic capacity and stain positively with monoclonal antibodies characteristic of the monocyte-macrophage hematopoietic cell lineage (anti-T200, anti- Mac-1, F4/80). However, the remainder of the cells are host-derived cells that stain positively with antisera to collagen IV and laminin. In contrast, stains for collagen types I and III were negative under conditions that allowed for strong staining of control skin fibroblasts. In separate studies, using mixtures of two genetically distinct bone marrows, the cells expressing collagen IV were further shown to be clonal in origin within individual colonies, directly demonstrating that the CFU-F assay provides a quantitative measure of the numbers of marrow stromal cell precursors. Thus, the current studies establish a remarkable similarity between the hematopoietic microenvironment in the short-term CFU-F assay and the long-term culture system: the majority of adherent cells are hematopoietic cells of the monocyte-macrophage lineage, while the remainder are stromal cells whose precise lineage remains uncertain, but whose pattern of collagen expression is more consistent with an endothelial rather than a fibroblast cell origin.     

The bcl-2 oncogene in Hodgkin's disease arising in the setting of follicular non-Hodgkin's lymphoma

Expression of the bcl-2 proto-oncogene on chromosome 18 is deregulated by the 14; 18 chromosomal translocation, an abnormality that is consistently associated with follicular non-Hodgkin's lymphomas (NHL). Because bcl-2 is believed to function by prolonging cell survival rather than by increasing proliferation, the presence of t(14; 18) in Hodgkin's disease (HD) would have profound implications for the pathogenesis of this neoplasm. We evaluated 32 cases of HD for t(14; 18) by polymerase chain reaction (PCR). These results were correlated with expression of bcl-2 oncogenic protein by Hodgkin cells and with the presence of Epstein-Barr virus (EBV), as determined by immunohistochemistry or in situ hybridization. PCR provided evidence of t(14; 18) in only 2 HD cases (6%), both of which were associated with a prior history of follicular lymphoma, and both of which were among the 7 cases (22%) with strong bcl-2 expression in Hodgkin cells. In at least 1 of the cases, the translocation involved identical chromosomal breakpoints in both types of lymphoma. Furthermore, 7 additional cases of combined follicular NHL and HD showed strong bcl-2 staining in Hodgkin cells. Although EBV was detected in 6 of 30 cases, it was not associated with t(14; 18) and usually not with strong bcl-2 expression. These results suggest that a small proportion of HD cases might evolve from follicular NHL, possibly through molecular events superimposed on the t(14; 18). High-level bcl-2 expression in Hodgkin cells is a potentially useful but not definitive marker for these cases.     

Rapid detection of deletions causing delta beta thalassemia and hereditary persistence of fetal hemoglobin by enzymatic amplification

A considerable number of deletions of variable size and position that involve the beta-globin gene complex on chromosome 11 are associated with the clinical entities of hereditary persistence of fetal hemoglobin (HPFH) and delta beta thalassemia. Specific deletions appear to be associated with consistent phenotypes and some are known to be recurrent. To facilitate the molecular diagnosis of uncharacterized patients with HPFH and delta beta thalassemia, oligonucleotide primers have been designed to enzymatically amplify deletion-specific products for nine known deletions, which include those responsible for HPFH-1, HPFH-2, HPFH-3, Spanish (delta beta)zero thalassemia, hemoglobin (Hb) Lepore, Sicilian (delta beta)zero thalassemia, Chinese G gamma(A gamma delta beta)zero thalassemia, Asian-Indian inversion-deletion G gamma(A gamma delta beta)zero thalassemia, and Turkish inversion-deletion (delta beta)zero thalassemia. Using this approach, we have successfully characterized the molecular basis for delta beta thalassemia in 23 individuals from 16 families of diverse ethnic origins. Thirteen individuals from this group were shown to be heterozygous for the 13.4- kb Sicilian deletion, two were heterozygous for the 100-kb Chinese G gamma(A gamma delta beta)zero deletion, four were heterozygous for the Turkish form of inversion-deletion delta beta thalassemia, and three were heterozygous for the Asian-Indian form of inversion-deletion G gamma(A gamma delta beta)zero thalassemia. One Vietnamese subject was heterozygous for a 12.6-kb deletion, which we have fully characterized at the molecular level. Sequence analysis of the breakpoint regions of the Chinese deletion and the Turkish rearrangement indicates that, in each case, the mutation is likely to have arisen from a single origin. This hypothesis is supported by the evident geographical clustering of the various deletions described here.     

The hematopoietic stem cell antigen, CD34, is not expressed on the malignant cells in multiple myeloma

Autologous stem cell transplantation has become an important therapy in multiple myeloma (MM). To develop adequate autograft purging methods, it is necessary to determine whether antigens expressed on early hematopoietic progenitors exist on malignant cells. The Ig heavy chain produced by the MM cells shows evidence of prior somatic mutation without intraclonal diversity. As a result, this sequence can be used as a specific marker to detect all members of the malignant clone. The Ig heavy chain sequence expressed by the MM cells was obtained in five patients with advanced disease. Patient specific oligonucleotide primers were designed based on the complementarity determining regions (CDR) of each MM Ig sequence and used to amplify DNA by polymerase chain reaction for the detection of malignant cells. A highly purified collection of CD34+ cells was obtained after passage of the initial bone marrow cells through an immunoadsorption column and fluorescence- activated cell sorting. Despite an assay sensitivity of 1 tumor cell in 2,500 to 44,000 normal cells, none of the CD34+ samples showed product with the myeloma-specific CDR primers. Therefore, positive selection for cells bearing this antigen should yield a tumor-free autograft capable of providing hematopoietic recovery after myeloablative chemotherapy.     

In vivo synergy between recombinant human stem cell factor and recombinant human granulocyte colony-stimulating factor in baboons enhanced circulation of progenitor cells

Recombinant human stem cell factor (rhSCF) and recombinant human granulocyte colony-stimulating factor (rhG-CSF) are synergistic in vitro in stimulating the proliferation of hematopoietic progenitor cells and their precursors. We examined the in vivo synergy of rhSCF with rhG-CSF for stimulating hematopoiesis in vivo in baboons. Administration of low-dose (LD) rhSCF (25 micrograms/kg) alone did not stimulate changes in circulating WBCs. In comparison, administration of LD rhSCF in combination with rhG-CSF at 10 micrograms/kg or 100 micrograms/kg stimulated increases in circulating WBCs of multiple types up to twofold higher than was stimulated by administration of the same dose of rhG-CSF alone. When the dose of rhG-CSF is increased to 250 micrograms/kg, the administration of LD rhSCF does not further increase the circulating WBC counts. Administration of LD rhSCF in combination with rhG-CSF also stimulated increased circulation of hematopoietic progenitors. LD rhSCF alone stimulated less of an increase in circulating progenitors, per milliliter of blood, than did administration of rhG-CSF alone at 100 micrograms/kg. Baboons administered LD rhSCF together with rhG-CSF at 10, 100, or 250 micrograms/kg had 3.5- to 16-fold higher numbers per milliliter of blood of progenitors cells of multiple types, including colony-forming units granulocyte/macrophage (CFU-GM), burst-forming unit-erythroid (BFU-E), and colony-forming and burst-forming units-megakaryocyte (CFU- MK and BFU-MK) compared with animals given the same dose of rhG-CSF without rhSCF, regardless of the rhG-CSF dose. The increased circulation of progenitor cells stimulated by the combination of rhSCF plus rhG-CSF was not necessarily directly related to the increase in WBCs, as this effect on peripheral blood progenitors was observed even at an rhG-CSF dose of 250 micrograms/kg, where coadministration of LD rhSCF did not further increase WBC counts. Administration of very-low- dose rhSCF (2.5 micrograms/kg) with rhG-CSF, 10 micrograms/kg, did not stimulate increases in circulating WBCs, but did increase the number of megakaryocyte progenitor cells in blood compared with rhG-CSF alone. LD rhSCF administered alone for 7 days before rhG-CSF did not result in increased levels of circulating WBCs or progenitors compared with rhG- CSF alone. Thus, the synergistic effects of rhSCF with rhG-CSF were both dose- and time-dependent. The doses of rhSCF used in these studies have been tolerated in vivo in humans.(ABSTRACT TRUNCATED AT 400 WORDS)     

Anti-CD38-blocked ricin: an immunotoxin for the treatment of multiple myeloma [see comments]

We report the development of a potent anti-CD38 immunotoxin capable of killing human myeloma and lymphoma cell lines. The immunotoxin is composed of an anti-CD38 antibody HB7 conjugated to a chemically modified ricin molecule wherein the binding sites of the B chain have been blocked by covalent attachment of affinity ligands (blocked ricin). Conjugation of blocked ricin to the HB7 antibody has minimal effect on the apparent affinity of the antibody and no effect on the ribosome-inactivating activity of the ricin A-chain moiety. Four to six logs of CD38+ tumor cell line kill was achieved at concentrations of HB7-blocked ricin in the range of 0.1 to 3 nmol/L. Low level of toxicity for normal bone marrow (BM) granulocyte-macrophage colony- forming units (CFU-GM), burst-forming units-erythroid (BFU-E), colony- forming units-granulocyte/erythroid/monocyte/macrophage (CFU-GEMM) cells was observed. Greater than two logs of CD38+ multiple myeloma cells were depleted from a 10-fold excess of normal BM mononuclear cells (BMMCs) after an exposure to HB7-blocked ricin under conditions (0.3 nmol/L) that were not very toxic for the normal BM precursors. HB7- blocked ricin was tested for its ability to inhibit protein synthesis in fresh patients' multiple myeloma cells and in normal BMMCs isolated from two healthy volunteers; tumor cells from four of five patients were 100-fold to 500-fold more sensitive to the inhibitory effect of HB7-blocked ricin than the normal BM cells. HB7 antibody does not activate normal resting peripheral blood lymphocytes, and HB7-blocked ricin is not cytotoxic toward these cells at concentrations of up to 1 nmol/L. The potent killing of antigen-bearing tumor cells coupled with a lack of effects on peripheral blood T cells or on hematopoietic progenitor cells suggests that HB7-blocked ricin may have clinical utility for the in vivo or in vitro purging of human multiple myeloma cells.     

Reconstitution of normal megakaryocytopoiesis and immunologic functions in Wiskott-Aldrich syndrome by marrow transplantation following myeloablation and immunosuppression with busulfan and cyclophosphamide

Three patients with Wiskott-Aldrich syndrome received transplants of marrow from their HLA-A, B, C, D identical siblings after myeloablation with busulfan, 2 mg/kg/day x 4 days, followed by immunosuppression with cyclophosphamide, 50 mg/kg/day x 4. Sustained engraftment of lymphoid and hematopoietic elements was documented in each case. Platelet counts in excess of 100,000/cu mm were restored 20--50 days posttransplant and remain in the normal range 6--12 mo later. Platelets exhibit normal size and in vitro aggregation. The patients produce isoagglutinins and antibodies to other polysaccharides. The use of busulfan in moderate dosages as a myeloablative agent, coupled with cyclophosphamide, may offer an improved alternative to the use of lethal total body irradiation as a preparative regimen for complete correction of Wiskott- Aldrich syndrome by marrow transplantation.     

Progressive dysfunction of monocytes associated with iron overload and age in patients with thalassemia major

We evaluated phagocytic and lytic activities of peripheral blood monocytes (PBMo) from patients with thalassemia major (ThP) using C pseudotropicalis as the target. PBMo from ThP showed decreased lytic activity (P less than .001), whereas the phagocytic activity did not differ from that of the controls. Significant inverse correlations were found between lytic activity of PBMo and age of patients (r2 = .47; P less than .01) and also between lytic activity and serum ferritin levels (r2 = .65; P less than .001). No association was found between lytic activity and other variables (blood transfusion regimens, therapy with desferrioxamine, liver damage, and the presence of sHBAg). Splenectomy showed no positive effect on PBMo functions from ThP. Our results suggest that PBMo from ThP have an intracellular defect in their microbicidal mechanisms associated with iron overload. This cell dysfunction could be responsible, at least in part, for the increased susceptibility to infections reported in ThP.