Increase of CALLA-positive stimulated lymphoid cells in transient erythroblastopenia of childhood.

In 1986 and 1987 11 children with TEC (transient erythroblastopenia of childhood) were referred to our hospital. Bone marrow aspirations were performed to exclude haematological malignancy. There was a marked reduction of erythropoiesis in 9 cases (1%-8%), two children had already recovered (33% and 44% erythropoiesis). Eight patients exhibited high percentages of stimulated lymphoid cells. The subsequent immunotyping revealed the expression of CALLA (common acute lymphoblastic leukaemia antigen) on these cells but there was no other sign for malignancy. The patients recovered without any specific treatment except transfusions of packed red cells. Eight patients were followed up 11-18 months after initial presentation and were all found to be in good health. A prominent increase of CALLA-positive stimulated lymphoid cells has also been found in other haematological diseases such as neutropenia and immune thrombocytopenia. The expression of CALLA in bone marrow lymphocytes is a general reactive change to various alterations.

Expression of the c-fos protooncogene by human and murine erythroblasts.

The expression of the c-fos protooncogene was investigated by in situ hybridization in normal murine bone marrow cells. A strong signal was found in murine marrow cells having the morphologic features of erythroblasts. This result was confirmed in human marrow cells using a double labeling technique (in situ hybridization and immunocytochemistry). A majority (70%) of the cells expressing c-fos mRNA were glycophorin A-positive. In contrast, granulocytic precursors (CD 15-positive) or monocytes and their precursors (CD 14-positive cells) did not significantly hybridize with the c-fos probe. In addition, c-fos mRNA (2.2Kb) was detected by Northern blotting in RNA extracted from homogeneous populations of erythroblasts obtained by immune panning from fetal liver and from adult blood BFU-E-derived colonies. Fos protein was also detected in erythroblasts by immunofluorescence. The high level of c-fos mRNA previously found in hematopoietic tissue should therefore be related to the transcription of the c-fos gene during terminal erythroid differentiation.

KU 812: a pluripotent human cell line with spontaneous erythroid terminal maturation.

A human leukemic cell line KU 812 was recently established and described as a basophilic cell line. In the present study we show that KU 812 and two of its clones are at least bipotent: in addition to a minor component of basophils, the majority of KU 812 cells belongs to the erythroid cell lineage with a significant percentage (about 15%) of mature hemoglobinized erythroblasts. This terminal differentiation is associated with the synchronized synthesis of the main erythroid proteins, including glycophorins, spectrin beta chain, band 3, and hemoglobin. The predominant hemoglobins are adult, fetal, and Bart's hemoglobin. Adult hemoglobin represented up to 75% of all hemoglobins in the KU 812 F clone in passages containing a high number of mature erythroblasts. Transcripts of all human globin chains were present with ten times less embryonic chain messenger RNA (mRNA) than alpha-, beta- or gamma-chain mRNA. Hemin slightly increased the total hemoglobin production of the cell line, especially gamma-globin chain synthesis, but did not modify the percentage of hemoglobinized cells. Phorbol myristate acetate (PMA) had a complex effect, inducing a proportion of KU 812 cells to adhere to the plastic culture flask. The adherent cell fraction expressed a very low level of specific erythroid proteins, but their ultrastructure was consistent with immature erythroid cells. In contrast, approximately 40% of the nonadherent cells were mature erythroid cells. Cell-sorting experiments showed that this paradoxic effect of PMA is mostly due to cell selection, the more mature cells being unable to adhere. In addition, KU 812 F was found to be sensitive to erythropoietin, which slightly increased its plating efficiency range (from 0% to 50%) in semisolid medium and enhanced hemoglobin accumulation twofold. In binding experiments using 125I erythropoietin, a single class of high-affinity Epo receptors (Kd: 250 pM) was detected by binding with a density of 205 receptors per cell. The KU 812 cell line is therefore a unique model for studying cell commitment toward different hematopoietic lineages and erythroid differentiation.

Proliferation and maturation of human erythroid progenitors in liquid culture.

Hemopoiesis is studied in vitro mainly in semisolid culture, where hemopoietic progenitors develop into discrete colonies. We describe a liquid culture system that supports the proliferation and maturation of human erythroid progenitors. We seeded mononuclear cells from the peripheral blood (PB) of patients with beta-thalassemia in liquid medium in the presence of conditioned medium from human bladder carcinoma cells. Seven days later, RBCs, normoblasts, granulocytes, and monocytes disappeared, and the number of lymphocytes dropped considerably. In contrast, erythroid colony-forming cells increased fourfold to tenfold. The next step entailed the removal of colony-stimulating factor (CSF) and CSF-secreting cells, the exclusion of macrophages by harvesting nonadherent cells, and the lysis of T lymphocytes by treatment with monoclonal rat antihuman lymphocyte antibodies (CAMPATH-1) and complement. Reculture of the remaining cells in liquid medium supplemented with recombinant erythropoietin (EPO) resulted in the exclusive development of erythroid cells, with myeloid cells reduced to less than 2%. Stainable hemoglobin (Hb) appeared on day 3, with over 85% of the population containing hemoglobin by day 11 and the cell number increasing from 0.2 X 10(6) to 3 X 10(6) mL. By permitting the manipulation of culture conditions and components and increasing the cell yield, the liquid system may facilitate quantitative analysis of growth kinetics as well as biochemical and immunologic characterization of the developing erythroid cell.

Chromatin texture changes related to proliferation and maturation in erythrocytes.

Erythroid cell types were quantitatively assessed on the basis of the analysis of their nuclear images after Feulgen and Heidenhain blue counterstaining. An original nonsupervised classification method, developed in our laboratory, was applied to cell cycle analysis. Results obtained suggest that, in the four proliferative generation cycles, which concur with the successive morphological stages, there is a sequential flow of cells from one morphological stage to the next by cell cycling. We show that the chromatin pattern alone made it possible to set up a metric of the whole differentiation process occurring in the normal human erythroblastic lineage in which proliferating events occur.

Karyotypic evidence for the leukemic involvement of the erythroblasts in erythroleukemia.

With the use of a monoclonal anti-glycophorin A antibody and flow cytometric cell sorting, an erythroleukemic bone marrow sample was separated in highly purified erythroblast and myeloblast fractions. Similar karyotypic anomalies were found in both cell populations as in the unseparated bone marrow. This study confirms that acute nonlymphocytic leukemia can originate at the level of a multipotential hemopoietic stem cell.

Ultrastructural silver enhancement of Prussian blue-reactive iron in hematopoietic and intestinal cells.

Prussian blue has been widely used to localize iron in a variety of tissues at the light and electron microscopic level. In the present study, thin sections of human marrow and blood cells and rat duodenal cells were exposed to silver proteinate (SP) after staining en bloc with acid ferrocyanide (AF), with and without prior iron saturation using iron nitrilotriacetate (FeNTA). Silver deposition was observed over Prussian blue-reactive sites and significantly enhanced sites of minimal AF and FeNTA-AF staining. AF-SP stain deposits were present in the cytoplasmic matrix, granules, and occasionally on the surfaces of macrophages, monocytes, and erythroblasts. FeNTA-AF-SP stained additional cytoplasmic and surface sites in erythroblasts and stained neutrophil granules intensely. Duodenal epithelium from iron-loaded rats demonstrated strong AF-SP staining of ferric iron in microvilli, apical cytoplasmic matrix, and lateral membranes. Similar preparations from iron-replete rats stained sparsely; however, intense AF-SP staining was observed after iron saturation with FeNTA. SP similarly enhanced luminal ferrous iron deposits stained with acid ferricyanide in rats given intraluminal ferrous iron. AF-SP stain deposits were removed by exposure of thin sections to NH4OH, KCN, or HNO3 but were not affected by prior exposure to HIO4 or NaBH4, consistent with a silver cyanide or complex stain precipitate rather than reduced silver or silver ferriferrocyanide. SP enhancement of Prussian blue allows identification of reactive sites not readily visualized with AF or FeNTA-AF alone, and offers the potential for differentiating AF staining from other deposits or organelles of comparable density.

Endogenous polyamines associate with DNA during its condensation in mammalian tissue. A fluorescence cytochemical and immunocytochemical study of polyamines in fetal rat liver.

The polyamines spermidine and spermine are essential for cell proliferation and differentiation. By two independent fluorescence cytochemical methods as well as by immunocytochemistry, we have studied the distribution of these molecules in fetal rat liver. Strong reactions for polyamines were found in highly condensed chromatin, present in chromosomes in mitotic cells, and in condensed nuclei in late erythropoietic cells. Moreover, polyamines were so closely associated with DNA in condensed chromatin that DNase pretreatment was necessary for making them available for reaction with antibodies. In other cells, polyamines were mainly localized to the cytoplasm. Studies of cells at different stages in erythropoiesis revealed that polyamines become associated with DNA during its condensation and inactivation. Our data strongly indicate that polyamines participate in the condensation of DNA.

Isolation and characterization of stable nuclear matrix preparations and associated DNA from avian erythroblasts.

A novel procedure for isolation of nuclear matrices from chicken erythroblast cells was elaborated. The influence of variations in the isolation procedure on structural integrity and morphology of nuclear matrices as well as on properties of the nuclear matrix-associated DNA fractions was investigated. The incubation of isolated nuclei in the presence of Cu2+ ions provided significant stabilization of the nuclear matrix. Copper treatment of nuclei did not affect the properties of the nuclear skeleton-associated DNA fraction. In both copper-stabilized as well as unstabilized nuclei, nuclear matrix-attached DNA was digested to the same extent with nucleolytic enzymes, and could be totally removed from nuclear matrices by 2 M NaCl-2 M urea treatment. The fine morphology of the nuclear matrix did not change after extraction of nuclear skeleton-associated DNA fragments. In the presence or absence of copper ions, matrix DNA was found to be qualitatively different compared with total DNA, in particular with respect to the representation of specific repetitive sequences of the chicken beta globin gene domain.

Erythromyeloid lineage fidelity is conserved in erythroleukaemia.

Blast cells from eight patients with erythroleukaemia and one with erythroid blast crisis of chronic myeloid leukaemia were studied for the co-expression of cell surface myeloid and erythroid markers, and the phenotype compared with that of erythroblasts from two patients with megaloblastic anaemia. The technique of dual indirect immunofluorescence was used with a panel of seven mouse monoclonal antibodies against well-defined myeloid antigens (CD11b, 13, 14, 15, 33 and HLA-DR) and a rat antibody, YTH89.1, specific for glycophorin A. No dual fluorescence, emanating from myeloid or erythroid lineage markers, was found to occur in either the neoplastic or non-neoplastic erythroid cells studied. These data support the hypothesis that lineage fidelity is conserved in leukaemia.

Evidence for a highly nuclease resistant RNA fragment in prosomes.

Prosomes, small cytoplasmic particles of mouse erythroblasts were found to contain low molecular weight RNA molecules in the range of 80 nucleotides. Nuclease digestion of prosomes suggests that prosomal proteins cover and protect almost the whole length of their RNA(s). Our results demonstrate clearly that RNA is an intrinsic component of prosomes.

Two 3' sequences direct adult erythroid-specific expression of human beta-globin genes in transgenic mice.

Previous experiments have demonstrated that the human beta-globin gene is correctly regulated in transgenic mice. The beta-globin gene is not expressed in yolk sac-derived erythroid cells in early embryonic development but is expressed concomitantly with the adult mouse beta-globin genes in 14- to 16-day fetal liver and adult reticulocytes. In an attempt to localize sequences that direct erythroid-specific expression, fragments of the human beta-globin gene were inserted in the opposite orientation 200 base pairs (bp) upstream of an intact human A gamma marker gene, which is not expressed on its own in mouse fetal liver. In the experiments reported here, two beta-globin 3' sequences activated the marker gene specifically in fetal liver. One sequence is located in a 250-bp Pst I fragment 550-800 bp downstream from the poly(A) site; the other is located near an EcoRI site in the third exon. These two sequences are active individually, and their combined effect is greater than their effects alone. beta-Globin 5' sequences from -815 to -50 were also analyzed for activity in this assay. The 5' sequences did not activate the marker gene when tested alone but did stimulate expression that was already directed to adult erythroid tissue by the two 3' sequences. These results suggest that three separate sequences are involved in human beta-globin gene regulation. The two 3' sequences act as adult erythroid enhancers and the 5' sequence stimulates expression that is already determined to be erythroid specific.

A unique gene for 5-aminolevulinate synthase in chickens. Evidence for expression of an identical messenger RNA in hepatic and erythroid tissues.

Reports to date have led to the conclusion that there are isozymes for 5-aminolevulinate synthase in the liver and erythroid tissue of chicken. Indeed, the existence of a multigene family for chicken 5-aminolevulinate synthase has been proposed. We find no evidence to support these proposals. In this work we show that 5-aminolevulinate synthase mRNA from chicken liver and reticulocytes is identical as determined by RNase mapping and primer extension studies and that the 5-aminolevulinate synthase protein from these tissues is the same size as judged by immunoblot analysis. We also show that a single mRNA species for 5-aminolevulinate synthase is present in chicken liver, reticulocytes, brain, and heart and an avian erythroblastosis virus-transformed chicken erythroblast cell line. Southern analysis shows the presence of only one gene copy for 5-aminolevulinate synthase in the chicken haploid genome. Overall, these results lead to the conclusion that in chickens 5-aminolevulinate synthase is encoded by a unique gene and is expressed as a single mRNA species in all tissues.

Demonstration of retroviral proteins associated with erythroid progenitors of cats with feline leukemia virus-induced erythroid aplasia.

Feline erythroid aplasia is a fatal retrovirus-induced disease related to infection with feline leukemia virus of subgroup C. When bone marrow cells from cats inoculated with FeLV were incubated with polyvalent antibody to FeLV and cultured for erythroid colony formation, a complement-dependent inhibition of erythroid progenitors was demonstrated with virtually complete suppression of erythroid colony-forming units (CFU-E) and burst-forming units (BFU-E) evident from post-inoculation week 4 until termination in cats with progressive infection. When bone marrow cells from cats with regressive infection were treated similarly, CFU-E were inhibited on post-inoculation week 4 (98% inhibition) when the cats were viremic, and on week 6 (77% inhibition) when the leukocytes were negative for FeLV by immunofluorescence. These data suggest that the retroviral proteins are associated with erythroid progenitors or critical accessory cells and may play a role in selective suppression of erythropoiesis.

Human embryonic hemopoiesis. Kinetics of progenitors and precursors underlying the yolk sac----liver transition.

Human embryonic development involves transition from yolk sac (YS) to liver (L) hemopoiesis. We report the identification of pluripotent, erythroid, and granulo-macrophage progenitors in YS, L, and blood from human embryos. Furthermore, comprehensive studies are presented on the number of hemopoietic progenitors and precursors, as well as of other cell types, in YS, L, and blood at precisely sequential stages in embryos and early fetuses (i.e., at 4.5-8 wk and 9-10 wk postconception, respectively). Our results provide circumstantial support to a monoclonal hypothesis for human embryonic hemopoiesis, based on migration of stem and early progenitor cells from a generation site (YS) to a colonization site (L) via circulating blood. The YS----L transition is associated with development of the differentiation program in proliferating stem cells: their erythroid progeny shows, therefore, parallel switches of multiple parameters, e.g., morphology (megaloblasts----macrocytes) and globin expression (zeta----alpha, epsilon----gamma).

Identification and characterization of the chicken transferrin receptor.

Among a panel of monoclonal antibodies that recognize chicken haematopoietic-differentiation antigens, one, JS 8, was found to immunoprecipitate a 95000-Mr cell-surface protein from chicken erythroblasts transformed with avian-erythroblastosis virus. This protein was shown, by affinity chromatography on immobilized chicken transferrin (conalbumin), to be the chicken transferrin receptor. Although immunologically unrelated to the human transferrin receptor, biochemical comparison of the chicken transferrin receptor to the human receptor showed similarities with respect to the pattern of biosynthesis, degree of glycosylation, dimerization in the absence of reducing agents and subcellular localization. The present report contrasts with recent ones describing the chicken transferrin receptor isolated from embryonic tissues as a 58000-Mr protein.

Influence of histone H5 on mononucleosome structure during differentiation in the avian erythroid series.

Changes in nucleosome repeat length during avian erythroid development have been previously correlated with changes in H5 content. In order to determine the effects of H5 on the length of DNA in mononucleosomal particles as a function of differentiation, a two-dimensional electrophoretic system was used to analyse DNA and histones of particles generated by micrococcal nuclease digestion of nuclei from several stages of erythroid development. Although the relative proportions of H5- to H1-containing mononucleosomes increased during development, only in mature erythrocytes did H5 protect a greater length of linker DNA from micrococcal nuclease digestion than did H1. These results suggest that changes in average nucleosome repeat length during erythroid development can be attributed only partially to an increase in the proportion of H5-containing nucleosomes which contribute to this average.