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PubMed1989

Regulated expression of the human beta-globin gene after retroviral transfer into murine and human hematopoietic cells.

Retroviral vectors and infection protocols were developed which permit transfer in vitro of the human beta-globin gene into transformed erythroid cells and normal human and murine hematopoietic cells. In murine erythroleukemia (MEL) cells, RNA expression from the human beta-globin gene was regulated in parallel with the endogenous globin genes and this RNA directed synthesis of human beta-globin protein chains. Human BFU-E which were present in normal bone marrow samples were also infected with the globin virus. After erythroid maturation in vitro, several percent of the total beta-globin mRNA was derived from the virally transferred beta-globin gene in the erythroid progeny cells of the bursts. The initial design of the beta-globin vectors was improved after the removal of sequences which interfered with the production of high-titer retrovirus stocks. The improved vector can transfer the human beta-globin gene to pluripotential hematopoietic stem cells (PHSC) of the mouse as shown by long-term expression of human beta-globin RNA and protein in peripheral blood, and the presence of the globin provirus in reconstituted myeloid and lymphoid cell lineages in primary and secondary recipients of virus-infected bone marrow.

Animals↗
PubMed1989

Action of blood serum from rats with turpentine sterile inflammation on the development of CFU erythrocyte colonies. Possible role of erythropoietin.

Bone marrow CFUe mice cultures were prepared in Petri dishes and the number of full developed erythrocyte colonies were counted under various experimental conditions. In certain experiments, erythropoietin (EP = 0.4 U x ml-1 in the suspending medium) or sera from normal rats (100 microliters) or from animals with chronic turpentine sterile abscesses (60 or 100 microliters), were delivered to the CFUe colonies. The number of colonies in the group without EP was almost 5 times smaller than in the group with EP. Inasmuch as some few colonies are still able to develop, even in absence of added EP, the suggestion is advanced holding that the phenomenon may represent the effect of EP already bound to the group of cells initiating differentiation. Comparisons among all experimental groups indicate that the delivery of sera from turpentine rats to cultures containing added EP, reduced significantly the number of CFUe colonies seen in controls with EP but without inflammatory serum. It is suggested that the present findings could be explained assuming an inhibition of the influence of exogenous EP, subserved by the inflammatory serum, or alternatively that this kind of rat serum induces a partial blockade of EP at receptor sites whose activation is a mandatory step for the adequate and full development of cultured erythrocytes.

Abscess↗
PubMed1985

[Demonstration of a nuclear androgen receptor in erythroblasts obtained by culture of human bone marrow].

The new techniques of culture of bone marrow have shown that androgens and 5 beta steroids exert a direct effect on erythroid precursor cells from human or animal bone marrow. By contrast, the mechanisms of the intracellular actions of those compounds are poorly understood. Tritiated methyltrienolone (R 1881), a synthetic androgen that highly binds to androgen receptor, has been used for the study of a binding activity in nuclear extracts of cultured erythroblasts from human bone marrow. The nuclear extracts contain binding sites saturable at low concentrations of 3H-R 1881 (20-30 nM). Scatchard analysis revealed that the R 1881-nuclear complex has a dissociation constant (Kd) of 25-50 nM, and the number of binding sites was 235-370 fmoles/mg protein. The complex sedimented on 5-20% sucrose gradient in the 3.9 S region and 5 beta dihydrotestosterone compete strongly with R 1881 for binding sites. This binding component has characteristics of high affinity, low-capacity, sedimentation behaviour, and specificity commonly attributed to "androgen receptors".

Binding, Competitive↗
PubMed1987

Erythropoietic action of dexamethasone on the anemia associated with an experimental chronic renal failure.

Partially nephrectomized anemic uremic rats were injected with dexamethasone phosphate (10, 50 and 500 micrograms/kg/day), i.p., and erythropoietin (5 U/day), s.c., for 10 days. A marked and usually significant stimulatory effect on erythropoiesis was seen in all uremic animals treated. Administration of erythropoietin and dexamethasone produced a pronounced increment in hemoglobin, hematocrit and circulating reticulocytes. The increase in red blood cell production was also evident through the generally increased absolute numbers of nucleated erythroid cell precursors per milligram of bone marrow. The highest increases were seen in the erythropoietin treated uremic rats. A dose effect correlation was apparent in uremic rats receiving 3 different doses of dexamethasone. Dexamethasone may stimulate erythropoiesis in our anemic uremic rats through a previous augmentation of erythropoietin production in the residual renal mass. A synergistic permissive effect of dexamethasone increasing the sensitivity of the erythropoietin-responsive cells to erythropoietin in bone marrow is also quite possible.

Anemia↗
PubMed1984

Two populations of erythroid cell progenitors in paroxysmal nocturnal hemoglobinuria.

We have grown erythroid cell colonies from two patients with paroxysmal nocturnal hemoglobinuria (PNH). At 11 to 13 days, individual bursts were picked and incubated for 24 hours with 3H-leucine in order to label total cell protein (mainly hemoglobin). After appropriate washing, each burst was subjected to a miniaturized acidified serum test, and lysis was measured by the release of radioactivity. In bursts from normal controls, lysis was 19% +/- 13% SD. By contrast, of 58 bursts from PNH patients, 14 had lysis similar to that of controls (mean 15.4% +/- 10.6%), while 44 had lysis ranging from 42.2% to 85.8% (mean 70.3% +/- 10.4%). Colonies sensitive to acidified serum were acetylcholinesterase (AchE) negative, whereas normal colonies were AchE-positive. Thus, based on two independent criteria, a dual population of erythroid burst-forming units (BFU-E) can be demonstrated in PNH. These data confirm directly the somatic mutation model of the pathogenesis of PNH, and by these methods the relative sizes of the normal and the PNH cell populations can be measured at the level of the erythroid cell precursors.

Acetylcholinesterase↗
PubMed1981

Biosynthesis of the erythrocyte anion transport protein.

The biosynthesis of the erythrocyte anion transport protein (Band III) was studied in erythroid precursor cells obtained from the spleens of anemic mice. Newly synthesized Band III was inserted during or immediately after translation into rough endoplasmic reticulum membranes. The asymmetric orientation of Band III in these membranes resembled that of mature Band III in erythrocyte membranes, with the NH2-terminal portion of the molecule facing the cytoplasm. At this stage Band III contained a high mannose core oligosaccharide, which was susceptible to cleavage by endoglycosidase H. During the next 20 to 30 min, this oligosaccharide was processed to a form resistant to endoglycosidase H degradation, presumably in the Golgi complex. The processed Band III was subsequently expressed on the cell surface, at about 30 to 45 min after synthesis. In many respects, therefore, the biosynthesis of Band III resembles that of cotranslationally inserted proteins whose NH2-terminal portions are exposed on the exterior of the cell, like VSV glycoprotein, HLA-A antigens, and glycophorin.

Animals↗
PubMed1983

MBM-1, a differentiation marker of mouse hemopoietic cells defined by a rat monoclonal antibody.

Hybridoma clones were isolated after the fusion of mouse myeloma cells with spleen cells from a rat immunized with mouse bone marrow. One of them produced a monoclonal antibody reacting with a murine cell surface differentiation antigen that we have termed MBM-1 (Mouse Bone Marrow-1). This antigen is present on eosinophils, on neutrophils, and on subpopulations of lymphocytes and macrophages, but appears to be absent from erythroid cells. Precursor cell analysis, after sorting of bone marrow cells using a fluorescence-activated cell sorter, suggests that the antigen is absent from most progenitors with the exception of certain cells in the macrophage lineage.

Animals↗
PubMed1980

Increased erythropoietin sensitivity of murine CFU-E and BFU-E in in vivo cultures.

The in vivo PCDC assay was used to reassess the erythropoietin requirements for growth in culture of the erythroid precursor cells CFU-E and BFU-E. Both CFU-E and BFU-E are expressed with the physiologically low erythropoietin concentrations present in normal host mice. Moreover, the same number of CFU-E-derived colonies is observed in hypertransfused host animals. No BFU-E are observed under these conditions. Increased numbers of both CFU-E and BFU-E are expressed in anemic host animals; these values are moderately lower than those found with in vitro cultures containing large concentrations of erythropoietin. Thus, at high levels of erythropoietin, the in vivo PCDC assay appears to be somewhat less sensitive than conventional in vitro assays. However, this culture system is more sensitive at low levels of erythropoietin and demonstrates a much smaller erythropoietin requirement for the differentiation of both CFU-E and BFU-E than has hitherto been recognized. This is as should be expected if these progenitor cells play a role in normal erythropoiesis in vivo. Experiments concerning the effects of anemia or hypertransfusion in donor rather than host mice reconfirm that the population size in the marrow of CFU-E but not BFU-E is highly sensitive to erythropoietin. However, phenylhydrazine treatment of donor mice leads to the transient development of a new subclass of BFU-E which gives rise to colonies relatively late in culture, suggesting that erythropoietin does have a direct effect on the physiological status of BFU-E in the bone marrow.

Anemia↗
PubMed1980

Humoral regulation of splenic hemopoiesis in mice.

The effects on splenic hemopoiesis were investigated of injecting the bacterial cell wall component, lipid A, or post-lipid A serum (PLAS) into mice. Both lipid A and syngeneic PLAS caused an increase in splenic numbers of multipotential hematopoietic stem cells (CFUS), committed hemopoietic progenitor cells (CFC) of different hemopoietic lineages and morphologically recognizable hemopoietic cells of various lineages. C57BL/6 Sld/Sld PLAS had a lower stimulating effect on hemopoiesis than C57BL/6 +/+ PLAS. PLAS from pre-irradiated mice was as active as normal PLAS in elevating splenic CFC levels. Serum from mice which received irradiation only, had no stimulating effect on splenic hemopoiesis. Medium conditioned by the myelomonocytic leukemia cell line WEHI-3B elevated splenic CFC numbers of similarly to PLAS, but supernatants from long-term marrow cultures or serum of mice treated with latex or phenylhydrazine did not have such an effect. Despite its marked effect on splenic numbers of nucleated erythroid cell precursors, PLAS did not contain elevated levels of erythropoietin.

Animals↗
PubMed1981

Purification and crystallization of the polypeptide hormone human chorionic somatomammotropin.

We have purified the large polypeptide hormone human chorionic somatomammotropin to near electrophoretic homogeneity by a new purification scheme. The hormone crystallized from polyethylene glycol in a form suitable for high resolution x-ray analysis; the crystals are monoclinic, space group C2, a = 123.9, b = 30.3, c = 53.8 A, beta = 119 degrees 30 min, with 1 molecule/asymmetric unit. The growth-promoting activity of homogeneous hormone and of dissolved crystals was measured through their effect on committed erythroid precursor cells in tissue culture assays. Both homogeneous and redissolved crystalline hormone had activities comparable to that of hormone purified by standard procedures. The latter preparations yielded either no crystals, or disordered crystals unsuitable for x-ray analysis.

Biological Assay↗
PubMed1995

In vitro human immunodeficiency virus-1 infection of purified hematopoietic progenitors in single-cell culture.

Uni- or multi-lineage suppression of hematopoiesis is observed in the majority of acquired immunodeficiency syndrome (AIDS) patients. The mechanism(s) underlying these abnormalities is not understood: particularly, the human immunodeficiency virus (HIV) infection of hematopoietic progenitor and stem cells (HPCs/HSCs) is highly controversial. We report that CD34+ HPCs from adult peripheral blood (PB) are in part CD4+ and susceptible to in vitro HIV infection. Primitive CD34+ HPCs were approximately 80% purified from PB. Double labeling for CD34 and CD4 membrane antigens was shown for 5% to 20% of the purified cells, thus suggesting their potential susceptibility to HIV-1 infection. The enriched HPC population, challenged with purified or unpurified HIV-1 strains, was cloned in unicellular methylcellulose culture. The single colonies generated by erythroid burst-forming units (BFU-E), granulocyte-macrophage colony-forming units (CFU-GM), and granulocyte-erythroid-macrophage-megakaryocyte colony-forming units (CFU-GEMM) were analyzed for the presence of HIV, ie, for gag DNA, tat mRNA, and p24 protein by PCR, reverse transcription PCR (RT-PCR), and enzyme-linked immunosorbent assay, respectively. In the first series of experiments incubation of HPCs with HIV-1 at multiplicities of infection (MOI) ranging from 0.01 to 10 TCID50/cell consistently yielded an 11% to 17% infection efficiency of BFU-E-generated colonies, thus indicating the sensitivity of HPCs to in vitro HIV infection. An extensive series of experiments was then performed on HPCs challenged with HIV at 0.1 MOI level. In the initial studies proviral gag sequences were detected in 9.2% of 121 analyzed CFU-GM colonies. In further experiments tat mRNA was monitored in 17% and 23% of BFU-E and CFU-GM colonies, respectively, but never in CFU-GEMM clones. Finally, 12% of CFU-GM clones and rare erythroid bursts were shown to be positive for the p24 viral protein. In control studies, purified HPCs grown in liquid suspension culture were induced to terminal unilineage erythroid, monocytic, or granulocytic differentiation: monocytes were consistently HIV-infected, whereas mature-terminal erythroblasts and granulocytes were not. Our observations indicate that a minority of primitive HPCs, but not of the multipotent type, is susceptible to in vitro HIV infection. These observations may reflect on the in vivo hematopoietic impairment in AIDS patients; more important, they provide an experimental model for studies on HIV hematopoietic infection and in vitro tests for anti-HIV HSC gene therapy.

Adult↗
PubMed1995

Murine spleen stromal cell line SPY3-2 maintains long-term hematopoiesis in vitro.

The hematopoietic microenvironment (HIM) of mouse spleen predominantly induces the differentiation of hematopoietic progenitors into erythroid lineage in vivo. However, the mechanisms of this phenomenon have not been fully explored because of the lack of an adequate in vitro system mimicking the spleen hematopoiesis. To reconstruct the HIM of mouse spleen in vitro, we established spleen stromal cell lines from a three-dimensional (3D) spleen primary culture in collagen gel matrix. Of these, SPY3-2 cells were negative for preadipocytic and endothelial markers, had a fibroblastoid morphology, and were not converted to adipocytes in the presence of 1 mumol/L hydrocortisone. They supported the maintenance and multilineal differentiation of hematopoietic progenitor cells for more than 8 weeks in vitro. The differentiated hematopoietic cells in the coculture medium were predominantly monocytes rather than granulocytes. Furthermore, erythropoiesis was predominantly induced in the presence of 2 U/mL erythropoietin and continued for more than 12 weeks. The number of burst-forming units-erythroid (BFU-E) was increased 10 times after 3 weeks of coculture, which was followed by pronounced production of erythroid cells in the coculture after week 4. SPY3-2 expressed high levels of c-kit ligand and low levels of granulocyte macrophage colony-stimulating factor and interleukin-3, and these molecules were all involved in this long-term erythropoiesis. Thus, the clonal SPY3-2 cell line will provide a novel HIM in vitro analogous to that of mouse spleen in vivo. These results suggest that 3D collagen gel culture may facilitate the establishment of functioning stromal cell lines of hematopoietic organ.

Adipose Tissue↗
PubMedDate not supplied

[In vitro studies of anemia accompanying a chronic inflammatory processes and neoplasms. Effect of cachectin (TNF-alpha) and lymphotoxin (TNF-beta) on human erythropoiesis].

The influence of tumor necrosis factor alpha and beta, on the clonal growth of human erythroid progenitors, from different stages of development--early BFU-E, late BFU-E, and CFU-E--in vitro cultures was evaluated. In the cultures of human bone marrow cells enriched in clonogenic progenitors the inhibitory effect on erythroid colony formation, was confirmed. The influence of TNF-alpha was more profound in comparison to TNF-beta and also visible at early BFU-E level. We did not however, observe the inhibitory effect, of both inflammatory cytokines, on granulocyte-monocytic colony formation. These data demonstrated that TNF-alpha and beta inhibit selectively the growth of the erythroid progenitors. Simultaneously it was found that early erythroid progenitors did not respond, to the kit ligand, in the presence of TNF-alpha.

Anemia↗
PubMed1993

[Ferrokinetics and bone marrow scanning in patients with myelodysplastic syndrome, hypoplastic myelodysplastic syndrome and aplastic anemia].

Ferrokinetic measurement were performed in a total of 48 patients with myelodysplastic syndrome (MDS), 13 with hypoplastic MDS and 25 with aplastic anemia (AA). Forty seven % of patients with hypoplastic MDS progressed to acute leukemia, however none of the patients with AA progressed to acute leukemia. Sixty-nine% of the patients with MDS showed increased erythron transferrin uptake (ETU) caused by ineffective erythropoiesis. On the other hand, 85% of the patients with hypoplastic MDS and all patients with AA showed decreased ETU caused by reduced erythropoiesis. Positive correlation was observed between ETU and the erythroid precursor cells in the bone marrow. Bone marrow scintigraphy utilizing 99mTc-sulphur colloid showed peripheral expansion of active marrow in MDS patients and islands-like distribution in hypoplastic MDS and AA patients. Ferrokinetics and bone marrow scintigraphy demonstrated the difference between typical MDS and hypoplastic MDS. Hypoplastic MDS appears to be a distinct clinicopathologic entity.

Anemia, Aplastic↗
PubMed1995

Proliferation of individual hematopoietic progenitors purified from umbilical cord blood.

In previous studies, we described long-term cultures of subpopulations of CD34+ cord blood cells that were fractionated using antibodies specific for CD45RA and CD71. In the present study, we plated the most primitive CD34+CD45RAlowCD71low cells individually and analyzed the proliferation and expansion kinetics of the various hematopoietic progenitors included in this subpopulation. The proliferation capacity of single progenitors was assessed by the total number of cells produced, and their expansion capacity was assessed by the production of CD34+ and colony-forming cells. Monolineage progenitors (erythroid, granulocyte, and macrophage progenitors) showed the lowest expansion capacity, which correlated with a relatively short life span (< 37 days) of the generated colonies. Among these cells, erythroid progenitors showed the highest proliferative potential. Based on their relative cell content and proliferation/expansion capacities, two types of both granulomacrophagic (GM) and multipotent (Mix) progenitors were identified. GM progenitors producing colonies with > 70% granulocytes had a low expansion capacity and gave rise to colonies that were sustained for < 42 days. A second type of GM progenitors, giving rise to colonies with > 90% macrophages, had a higher expansion capacity and the colonies they produced were sustained for > 72 days. On the other hand, Mix progenitors producing colonies with > 70% erythroid cells had a lower expansion capacity and life span (< 46 days in culture), than Mix progenitors producing colonies with > 90% myeloid cells (life span of > 72 days). These results demonstrate, at the single-cell level, a correlation between the type of progenitor cell and its expansion capacity. In turn, the expansion capacity of the progenitors was correlated with the life span of the colonies produced. Furthermore, our results suggest that in cord blood, at least two types of bipotential myeloid (GM) progenitors and two types of multipotent (Mix) progenitors can be distinguished, that have respectively low and high proliferative and expansion capacity.

Antigens, CD↗
PubMed1997

Influence of leukemia inhibitory factor (LIF) on the survival, proliferation and differentiation of human erythroid progenitor cells. In vitro studies under serum free conditions.

Different inflammatory cytokines are involved in the pathogenesis of the anemia of chronic disease (ACD), by inhibiting the proliferation of erythroid progenitors. Leukemia inhibitory factor (LIF), is an important inflammatory cytokine, and the purpose of this study was to evaluate its effect on the proliferation and viability of human erythroid progenitors. We have found that LIF slightly increased the survival of human early progenitor cells cultured under serum free conditions. LIF also slightly costimulated in vitro growth of human erythroid colonies. This last effect seems to be a direct one, because we found that LIF receptor (LIF-R) is expressed in cells isolated from growing in vitro human erythroid BFU-E colonies. These data, and the data reported by others in in vivo models, where LIF administration to experimental animals did not change the values of erythropoietic parameters, demonstrate that this inflammatory cytokine itself is not involved in the pathogenesis of ACD.

Antigens, CD34↗
PubMedDate not supplied

Congenital parvovirus infection.

Congenital parvovirus infection was diagnosed in two liveborn premature infants born at 24 and 35 weeks of gestational age. The illnesses were associated with placentomegaly, petechial rash, edema, hepatomegaly, anemia and thrombocytopenia, respiratory insufficiency, and death at 5 and 6 days of age. The syndromes exhibited by these cases shared common but nonspecific features with other life-threatening congenital infections. Serological studies in one case supported the diagnosis of parvoviral infection. Postmortem examination of both revealed nuclear inclusions in erythroid precursor cells characteristic of parvovirus infection. Use of the polymerase chain reaction confirmed the presence of parvovirus DNA in one of the cases. Intrauterine parvovirus B19 infection is most commonly associated with hydrops fetalis, "transient" hydrops, or a favorable outcome in infants found to be viremic after birth. These and previously reported examples of congenital B19 disease exemplify an exceptional form of human parvovirus infection.

DNA, Viral↗
PubMed1981

Isolation of murine fetal hemopoietic progenitor cells and selective fractionation of various erythroid precursors.

Hemopoietic progenitor cells (colony- and cluster-forming cells in semisolid agar) were purified from light density CBA murine fetal liver cells using fluorescein-conjugated pokeweed mitogen (PWM) and a rhodamine-conjugated antineutrophil serum sandwich (alpha N) and three-parameter fluorescence-activated cell sorting. All clonable progenitor cells were highly enriched (36-50-fold) in PWM-positive (greater than channel 15), alpha N-negative (less than channel 30) fractions with relatively high intensity (greater than 100) low angle light scatter. No separation was achieved between different types of progenitor cells (granulocyte-macrophage and erythroid colony-forming cells). The enriched fraction was a pure population of large, basophilic, undifferentiated blast cells, and in agar cultures stimulated with colony-stimulating factors, up to 90% of the enriched cells were hemopoietic progenitor cells capable of varying levels of clonal proliferation. Further fractionation based on increasing fluorescence with PWM separated into discrete populations, nonproliferative morphologically recognizable erythroid cells, late erythroid progenitor cells (day 2 CFU-E), and cells forming pure or mixed erythroid burst colonies. In addition, the majority of pluripotential hemopoietic stem cells (CFU-SS) were clearly separated from progenitor cells forming colonies in vitro. The present techniques provide suitable numbers of enriched progenitor cells for a variety of biological and biochemical studies.

Animals↗