Isolation and characterization of an age-related antigen present on senescent human red blood cells

Autologous membrane-bound IgG was isolated from a subpopulation of human red blood cells (RBC) with specific density greater than 1.110, by affinity chromatography of purified RBC membrane glycoprotein preparations using immobilized wheat germ agglutinin and immobilized anti-human immunoglobulin (Ig) as immunoabsorbents. The Ig-containing population thus obtained, when further separated by chromatography on Sephadex G-200 in the presence of chaotropic agents, yielded four peaks (Ia, Ib, II, and III). Double immunodiffusion revealed the presence of Ig in the first three peaks (IgM in peak Ia, IgA in Ib, and IgG in II) but not in peak III. Peak III was precipitated by the Ig-containing peaks (Ia, Ib, and II) in immunodiffusion assays, suggesting that the antigenic membrane determinants responsible for the binding of autologous Ig to senescent human RBC were contained in this peak (III). Peaks Ia, Ib and II precipitate purified asialoglycophorin; peak III was reactive with purified autoantibodies directed against asialoglycophorin. These results suggest that an age-related antigenic determinant(s) present on senescent human RBC is exposed by desialylation of the major sialoglycoprotein component of the RBC membrane.     

Recombinant alpha-2-interferon for hairy cell leukemia

Twenty-two patients with hairy cell leukemia were treated with biosynthetic (recombinant) alpha-2-interferon in an open-label, single- arm efficacy study. Patients received 2 X 10(6) U/m2 recombinant alpha- 2-interferon three times weekly. Therapy was well tolerated subjectively with minimal short-term hematologic toxicity. Two patients had bacterial infections during the period of study, and one patient experienced a short-lived readily reversible rejection of a corneal transplant. Statistical comparison of the mean hematologic indices at study entry and after three to six months of therapy with recombinant alpha-2-interferon indicates a significant improvement in hemoglobin, granulocyte, and platelet counts. Bone marrow biopsies in six of 14 patients after six months of therapy showed a greater than 50% decrease in the infiltration of leukemia cells. We conclude that recombinant alpha-2-interferon is highly effective therapy for hairy cell leukemia.     

Demonstration of burst-promoting activity of recombinant human GM-CSF on circulating erythroid progenitors using an assay involving the delayed addition of erythropoietin

We demonstrate through the use of an in vitro assay involving the delayed addition of erythropoietin that human recombinant GM-CSF, cloned from a mature T cell line, Mo, clearly has burst-promoting activity (BPA) on peripheral blood erythroid progenitors at picomolar concentrations. Delay for up to 72 hours of the addition of erythropoietin to semi-solid methylcellulose cultures of concentrated peripheral blood progenitors minimizes or eliminates BPA-independent erythroid colony formation with little loss of BPA-dependent erythroid colony formation. This assay will prove useful in accurately detecting sources of BPA.     

BCR-ABL protein expression in peripheral blood cells of chronic myelogenous leukemia patients undergoing therapy

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder associated with the Philadelphia chromosome (Ph1) in more than 95% of these patients. The Ph1 and the resulting BCR-ABL fused genes are markers for this type of leukemia. In CML, the product of the fused BCR- ABL gene is typically a protein of approximately 2,000 amino acids termed P210 BCR-ABL. We have developed an assay for the BCR-ABL protein involving Western blotting of circulating white blood cells (WBC) with an anti-ABL monoclonal antibody that can detect P210 BCR-ABL and P145 ABL in peripheral blood cells from chronic phase Ph1-positive leukemia patients. This assay was used to analyze the BCR-ABL protein content of circulating WBC from CML patients before and after various treatments. In parallel to changes in percentages of Ph1-positive blood cells as determined by cytogenetic analyses of bone marrow samples, BCR-ABL protein expression in blood cells decreased or increased as patients entered remission or underwent relapse. Of interest, six Ph1-negative CML patients were BCR-ABL protein-positive. All except one had a rearrangement in the major breakpoint cluster region and that patient expressed P185 BCR-ABL and not P210. Our results indicate that the BCR- ABL Western blotting assay has clinical applications for both diagnosis and prospective evaluation of Ph1-positive and Ph1-negative CML patients.     

Characteristics of bone marrow fibroblast colony-forming cells (CFU-F) and their progeny in patients with myeloproliferative disorders

Chronic myeloproliferative disorders (MPD) are clonal diseases of the pluripotent hematopoietic stem cell frequently associated with myelofibrosis (MF). There is only indirect evidence indicating that the increased deposition of collagen in bone marrow matrix is a secondary phenomenon. A liquid culture system for cloning and growing bone marrow fibroblasts has permitted us to approach more directly the understanding of the pathogenesis of myelofibrosis by comparing the biophysical, growth, and functional characteristics of fibroblasts from normals, MPD patients without MF, and those with MF. In patients with MF, marrow fibroblast colony (CFU-F) formation could not be studied; fibroblasts were grown from marrow explants. CFU-E from normals and MPD patients exhibited similar cell density distribution and similar cell sedimentation rates. These similarities contrasted sharply with the differences seen when the erythroid and granulocyte-macrophage progenitors were studied by the same methods. There was a marked light density shift and a rapidly sedimenting shift of MPD hematopoietic colony-forming cells. Marrow fibroblasts from MPD patients with and without MF displayed the same in vitro growth characteristics as fibroblasts from normals. Both types of fibroblasts exhibited anchorage and serum dependence, and contact inhibition of growth. Marrow fibroblasts were also characterized for the presence and distribution of fibronectin and collagen types by immunofluorescent staining using monospecific antibodies. Extracellular matrix, membrane-, and cytoplasm- associated fibronectin, type I, type III, and type V collagen showed a similar staining pattern in both normal and myelofibrotic marrow fibroblasts. Plasminogen-dependent fibrinolytic activity elicited from normal and myelofibrotic marrow fibroblasts were equivalent. Chromosomal analysis of hematopoietic cells and marrow fibroblasts from Philadelphia chromosome positive chronic myelocytic leukemia patients with and without MF showed that the Philadelphia chromosome was present only in hematopoietic cells. The results of these studies taken together demonstrate that bone marrow collagen-producing cells from MPD patients with and without MF behave in vitro as do those from normals. These findings support the hypothesis that that the marrow fibrosis observed in patients with MPD results from a reactive process rather than from a primary disorder affecting the marrow collagen-producing cells.     

Treatment of preleukemic syndromes with marrow transplantation

Thirty patients with advanced preleukemic syndromes were treated with marrow transplantation. Most cases were diagnosed by the presence of peripheral pancytopenia and a diagnostic marrow examination but in 6 of the 30 patients pretransplant chromosome studies were instrumental in establishing the diagnosis. Three patients prepared for transplantation with cyclophosphamide alone recurred with their disease within 6 months of transplantation. The other 27 patients were treated with cyclophosphamide and total body irradiation. Twenty of these 27 patients had preleukemia not associated with prior therapy or severe marrow fibrosis. Thirteen of these 20 are alive and well 9 to 56 months from transplant and 7 died, 4 of interstitial pneumonia, 2 of candida septicemia, and 1 of disseminated zoster. There have been no disease recurrences in this group. The remaining preleukemic patients, which include 3 patients transplanted for preleukemia secondary to prior therapy and 4 patients transplanted for preleukemia associated with severe marrow fibrosis, have all died. Major problems in these patients included disease recurrence (2 cases) and, in those with severe marrow fibrosis, graft failure (2 cases). These results suggest that for patients with life-threatening pancytopenia due to spontaneous preleukemia without severe marrow fibrosis, marrow transplantation can prolong disease-free survival and may result in cure of the disease.     

The familial occurrence of polycythemia vera: report of a father and son, with consideration of the possible etiologic role of exposure to organic solvents, including tetrachloroethylene

The occurrence of polycythemia vera in a father and son, both of whom had intermittent exposure to organic solvents, including tetrachloroethylene and Stoddard solvent, is reported. Only three other well substantiated familial occurrences of polycythemia vera, none encompassing successive generations, were found among many reported instances.     

Analysis of interphase cells for the Philadelphia translocation using painting probe made by inter-Alu-polymerase chain reaction from a radiation hybrid

Fluorescence in situ hybridization (FISH) probe for the identification of the Philadelphia (Ph) translocation [t(9;22) (q34;q11)] in chronic myelogenous leukemia cells was developed by inter-Alu-polymerase chain reaction of DNA from an interspecific somatic cell hybrid containing approximately 5 Mb of human DNA covering the ABL gene region on human chromosome 9q34. This probe was large enough to be effective in identifying the genomic domains yet small enough to resolve them in more than 90% of bone marrow interphase cells. Combination of the probe with a cosmid contig probe for the BCR region of chromosome 22 in two- color FISH reduced the frequency of false-positive identification of the Ph chromosome to less than 1%. The procedure allows detection of as few as 1% Ph+ cells independent of the cycling status or BCR/ABL expression level of cells, and the quantitation of non-Ph chromosome- containing interphase nuclei in the marrow of patients judged 100% Ph+ by standard cytogenetics.     

Long-term leukemia-initiating capacity of a CD34-subpopulation of acute myeloid leukemia

Acute myeloid leukemia (AML) proliferation in vivo is maintained by a small fraction of progenitor cells. These cells have been assumed to express an immature phenotype and to produce most colony-forming units (CFU-AML). For one case of AML (French-American-British [FAB] M1, normal cytogenetics), we examined the capacity of the CD34+ (25% of unseparated AML cells) and CD34- fractions to initiate leukemia in severe combined immunodeficient (SCID) mice. In addition, the production of CFU-AML and nucleated cells (NC) of these subsets was investigated in long-term bone marrow culture (LTBMC). The frequencies of cobblestone area-forming cells (CAFC) were also estimated; early appearing cobblestone areas (CAs) are indicative of relatively mature progenitors and late CAs represent the progeny of primitive progenitors. In mice transplanted with CD34- (98% pure) or CD34+ (98% pure) grafts, similar AML cell growth was seen throughout an observation period of 106 days. The capacity to establish long-term growth from the CD34- cells was confirmed by renewed outgrowth after retransplantation. In vitro, the CD34- fraction contained both immature and mature CAFCs and produced high numbers of CFU-AML and NC in LTBMC. The CD34+ fraction produced only small numbers of CFU-AML, NC, and mature CAFCs. Therefore, the expression of CD34 and the content of CFU- AML were not associated with long-term growth of AML. However, similar frequencies of primitive CAFCs were observed in both fractions. Thus, both CD34- and CD34+ subsets of this AML sample contained immature progenitors with the capacity to initiate long-term AML growth as characterized in vivo (in SCID mice) as well as in vitro (in CAFC assay), indicating asynchrony between functional and immunophenotypical maturation of AML progenitor cell compartments.