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E Goldwasser

Publications and source records attributed to E Goldwasser.

At least 73 records · Page 4Linked to original sources
PubMed1988

Anti-proliferative effect of IFN-gamma in immune regulation. II. IFN-gamma inhibits the proliferation of murine bone marrow cells stimulated with IL-3, IL-4, or granulocyte-macrophage colony-stimulating factor.

A biphasic dose response curve was observed when the bone marrow-derived cell line FDCP1, used as an indicator line for IL-3 bioassays, was exposed to supernatants from some activated T cell clones but not others. The active component which inhibited proliferation at the higher supernatant concentrations appeared to be IFN-gamma, based on the following observations. 1) Only those culture supernatants which contained IFN-gamma gave a biphasic dose response curve; 2) with these supernatants, an anti-IFN-gamma mAb augmented the proliferation of FDCP1 cells at the higher supernatant concentrations; and 3) rIFN-gamma profoundly inhibited the proliferation of FDCP1 cells stimulated with rIL-3 or rIL-4. rTNF-alpha inhibited FDCP1 proliferation only to a modest extent, yet the combination of rTNF-alpha + rIFN-gamma provided greater inhibition than each agent alone. The proliferation of a second bone marrow-derived cell line, DA1, was not inhibited by rIFN-gamma or rIFN-gamma + rTNF-alpha when stimulated with rIL-3 or recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF). Fresh bone marrow cells also showed a suboptimal proliferative response when stimulated with T cell supernatants containing IFN-gamma, and this response was augmented considerably upon the addition of anti-IFN-gamma mAb. Bone marrow cell proliferation was observed upon exposure to rIL-3, rIL-4, or rGM-CSF, and these responses were inhibited by rIFN-gamma; rTNF-alpha also produced a synergistic effect with these cells. Bone marrow cell colony formation stimulated by rIL-3 or rGM-CSF also was inhibited by rIFN-gamma. Colony formation in bone marrow cell cultures was not observed in response to rIL-4. Collectively, these results suggest that Th1 cells, which in addition to IL-3 and GM-CSF also produce IFN-gamma, may regulate hemopoietic cell proliferation and colony formation differently from the way Th2 cells do, which do not produce IFN-gamma.

Adjuvants, Immunologic↗
PubMed1988

The heterogeneity of circulating human serum erythropoietin.

The presence of more than one form of immunoreactive erythropoietin was suggested by discrepancies between the levels of immunologically detectable and biologically active erythropoietin in the sera of normal individuals and patients with end-stage renal disease. In both groups, the level of immunologically detectable hormone was significantly higher than that of bioactive hormone. Sera from end-stage renal disease patients and normal subjects were fractionated on a gel permeation column, and the immunoreactive erythropoietin in the fractions was measured by RIA. Three classes of immunoreactive erythropoietin were found in the sera: one eluting before the [125I]erythropoietin marker, one coinciding with the [125I]erythropoietin marker, and a third eluting after the labeled tracer in the region of the cytochrome c marker. The high and low mol wt components were more immunoreactive than the human urinary erythropoietin standard in the RIA. Biological activity, as determined in the in vitro rat bone marrow assay, was found only in the material eluting with the erythropoietin tracer. These studies show that immunoreactive erythropoietin in the serum consists of three components with different immunoreactivities: high and low mol wt components and a component with a mol wt similar to that of the native hormone.

Erythropoietin↗
PubMed1987

Stimulation of fetal hemoglobin synthesis by erythropoietin in baboons.

Stimulating the production of fetal hemoglobin may benefit patients with sickle cell anemia by inhibiting sickling. We gave pulsed treatments with high doses of recombinant human erythropoietin to baboons in order to test the hypothesis that the resultant rapid erythroid regeneration would stimulate F cells--i.e., cells that contain fetal hemoglobin. In normal animals, this treatment caused sharp increments in F-reticulocyte levels, which rose from 1 to 2 percent before treatment to 40 to 50 percent afterward. In two animals with chronic anemia and high levels of endogenous erythropoietin, recombinant human erythropoietin induced further increments in F-reticulocyte levels, which rose in one animal from 6 to 8 percent before treatment to 23 percent after treatment, and in the other from 20 percent before to 50 percent afterward. The time course of F-reticulocyte stimulation suggested that these cells were the products of mobilized early erythroid progenitors. These results raise the possibility that pulses of erythropoietin could be used to stimulate F-cell formation in patients with sickle cell disease.

Anemia, Hypochromic↗
PubMed1987

Binding and receptor-mediated endocytosis of erythropoietin in Friend virus-infected erythroid cells.

The binding of labeled erythropoietin (EP) to cell surface receptors and subsequent processing of the hormone within the cell was studied in erythroid cells procured from the spleens of mice infected with the anemia strain of Friend virus. These immature erythroid cells respond to EP in culture to differentiate into reticulocytes and erythrocytes. Radiolabeled EP (both iodinated and tritiated) binds to 800-1000 cell surface receptors on these cells at 4 degrees C. Using 125I-EP, we found that 300 of these cell surface receptors have a higher affinity for EP (Kd = 0.09 nM) than the remaining receptors (Kd = 0.57 nM). The number of molecules of EP bound per cell increased about 2-fold when binding was carried out at 37 degrees C. Treatment of the cell surface with pronase or removal of surface-bound EP with a low pH wash revealed that radiolabeled EP is internalized by the cells at 37 degrees C. Pulse chase experiments showed that degradation products of radiolabeled EP are released into the medium with a corresponding loss of label from the interior of the cell. Inhibitors of lysosomal function greatly reduced this degradation of 125I-EP. Since 180 of the 300 high affinity receptors and very few of the low affinity receptors are occupied at the concentration of EP which elicits the maximum biological response in these cells, we suggest that interaction of EP with the high affinity receptors are necessary for the full biological effect of the hormone. A different murine erythroleukemia cell line which does not differentiate in response to EP was found to have only the lower affinity binding sites for the hormone.

Ammonium Chloride↗
PubMed1987

Blunted erythropoietin response to anaemia in rheumatoid arthritis.

The relationship of serum immunoreactive erythropoietin to haemoglobin concentration was defined for 54 patients with rheumatoid arthritis (RA) and 41 patients with anaemia of varying aetiology (excluding pregnancy and renal insufficiency), not associated with RA. Significant inverse correlations between the logarithm of serum immunoreactive erythropoietin and the haemoglobin concentration were noted for the anaemic patients in both groups. The regression line for the RA patients had a similar slope, but a significantly lower y-intercept as compared to that for the non-RA patients. Erythropoietin levels were also significantly lower for the group of RA patients than for the group of non-RA patients when matched for comparable haemoglobin concentrations. These studies suggest that the erythropoietin response to anaemia in RA is intact but blunted relative to that for anaemia of other aetiologies. Lower levels of serum erythropoietin in anaemic RA patients may contribute to the pathogenesis of their anaemia.

Adolescent↗
PubMed1987

Rearrangement and expression of erythropoietin genes in transformed mouse cells.

The erythroleukemia cell line IW32, derived by transformation with the Friend murine leukemia virus, has been shown previously to produce erythropoietin (EPO) constitutively. Here we demonstrate that, in addition to the normal mouse EPO locus, this cell line has another EPO locus which has undergone rearrangement and amplification. Both loci were cloned, and the rearrangement breakpoint of the second EPO locus was located within a 1.1-kilobase region upstream of an otherwise apparently normal EPO gene. There are no viral sequences present in the immediate vicinity of the rearranged EPO gene. DNase I digestion studies suggest that the rearranged gene is in a region where the chromatin is more sensitive to DNase hydrolysis than is the site of the normal gene. We conclude, tentatively, that the rearranged EPO locus is probably the transcriptionally active one and that either proviral sequences are acting at a distance to activate the EPO gene or the rearrangement itself has served to activate the gene.

Animals↗
PubMed1987

Expression of the erythropoietin gene.

Comparison of maps of the human and mouse erythropoietin (EPO) genes shows overall general conservation. A cloned mouse EPO gene was used to study EPO gene expression by the Northern blot method. Ten hours after bleeding to induce stress erythropoiesis in the rat, the only tissue to show detectable message was the kidney. Other preliminary studies on EPO expression in cells transformed by FMuLV that secrete EPO constitutively showed no detectable viral sequences close to the gene regardless of high levels of EPO expression. Further studies on regulation of EPO gene expression are now possible with the probes developed from the cloned gene.

Animals↗
PubMed1987

Induction of globin mRNA transcription by erythropoietin in differentiating erythroid precursor cells.

The effects of erythropoietin (epo) on the proliferation of late erythroid progenitor cells (CFU-E) and on the formation of hemoglobin and of globin mRNA in these cells are described. CFU-E were isolated from thiamphenicol-pretreated anemic mice by elutriation and Percoll density gradient methods. These CFU-E are restricted in their capacity to proliferate in vitro without added epo. The epo dependence in vitro was not absolute. With no epo in the culture medium the first cell division was unimpaired, whereas the third division was only 1%-2% of the control. In the absence of epo the synthesis of hemoglobin is very low in CFU-E, but is increased significantly after about 5 h of incubation with epo present. In epo deprived cells there was considerable hemoglobin formed at about 14 h, but not earlier. The presence as detected by the Northern blot technique of globin mRNA, isolated from CFU-E, was variable, probably depending on the presence of some more mature erythroid cells. By an extrapolation method we show evidence that pure CFU-E would have virtually no detectable globin mRNA. The production of globin mRNA is rapidly (2 h) induced in cells incubated with epo. We conclude that epo, besides having a mitogenic effect on CFU-E, induces the rapid expression of the globin genes.

Animals↗
PubMed1986

Structural characterization of human erythropoietin.

Erythropoietin is the primary regulator of red blood cell formation in mammals. Because of its extreme scarcity, very little information is available regarding structural features of this important glycoprotein. We report here the primary structure of human urinary erythropoietin, determined by protein sequencing. In addition, the sites of glycosylation, assignment of disulfide bonds, and the circular dichroism of the hormone analyzed for secondary structure in comparison with the prediction from the sequence are presented.

Amino Acid Sequence↗
PubMed1986

Cloning, sequencing, and evolutionary analysis of the mouse erythropoietin gene.

The gene for mouse erythropoietin was cloned and sequenced. We present here a preliminary analysis of the overall genomic organization of the coding portions and the two flanking regions of the gene. This is the third mammalian erythropoietin for which the sequence is available, but it represents the first from a nonprimate species. We investigated the evolutionary divergence of sequence and structure of the three erythropoietins and identified specific regions of the molecules that are apparently under various degrees, and perhaps different types, of functional constraint.

Amino Acid Sequence↗
PubMed1986

Expression of the erythropoietin gene.

Injection of cobalt into rats resulted in erythropoietin (EPO) mRNA accumulation in the kidney. The same response was obtained upon bleeding. No EPO mRNA was detected in the spleen, salivary gland, or thymus following cobalt injection or bleeding. In some animals, but not in others, EPO mRNA was also expressed in the liver in response to cobalt injection. Time course studies showed that message appearance begins sometime between 3 and 6 h after cobalt injection. This correlated very well with the EPO concentration in the circulation; EPO levels in the circulation were the same as those of controls at 3 h but increased to six- to sevenfold that of controls by 6 h after cobalt injection. The mature EPO mRNA in the rat and mouse comigrated with the 18S rRNA, indicating that it is about 1,850 nucleotides in length.

Animals↗
PubMed1986

Sickle cell anemia patients have low erythropoietin levels for their degree of anemia.

We have studied serum immunoreactive erythropoietin (SIE) levels in 28 patients with sickle cell anemia (SCA) without renal insufficiency and in 17 patients with nonhemoglobinopathy anemias of comparable severity using a sensitive radioimmunoassay procedure. An exponential relationship between SIE level and degree of anemia was noted in all patients. However, in nonhemoglobinopathy anemia, a sharp rise in the SIE level occurred as hemoglobin (Hb) levels fell below about 12 g/dL, whereas in sickle cell patients the increase was not marked until hemoglobin fell to about 9 g/dL. The response was more blunted in older SCA patients than in younger ones. A linear regression model relating SIE level to Hb level, presence/absence of SCA, and age explained 63% of the variation in SIE. We conclude that the serum erythropoietin levels in SCA increased at a lower hemoglobin concentration and are of a lower magnitude than that of the other anemias.

Adolescent↗
PubMed1985

The regulation of heme biosynthesis during erythropoietin-induced erythroid differentiation.

Marrow cells induced toward erythroid differentiation by treatment with erythropoietin respond by increasing the rates of iron uptake and hemoglobin synthesis. Study of the enzymes of heme biosynthesis during erythroid differentiation suggests that induction of heme synthesis in these cells is regulated by synthesis of porphobilinogen deaminase. The activities of delta-aminolevulinic acid synthase, gamma, delta-dioxovaleric acid transaminase, delta-aminolevulinic acid dehydratase, and ferrochelatase were not affected significantly by treatment of suppressed marrow cells with erythropoietin over a period of 4 days, whereas that of porphobilinogen deaminase was increased by as much as 3.5-fold by the 3rd day of incubation. The time course of increase in porphobilinogen deaminase activity was parallel to that of the increase in heme synthesis. Moreover, when porphobilinogen deaminase activity was compared in marrow cells exposed to increased levels of erythropoietin in vivo (hyperplastic marrow) and marrow cells exposed to lowered levels of erythropoietin in vivo (suppressed marrow), the activity in the former case was greater than that in normal cells and for the latter type of cell it was lower than normal. Experiments using actinomycin D and cycloheximide suggest that transcription is required for the erythropoietin-induced porphobilinogen deaminase activity, indicating that induction is probably at the level of de novo synthesis of enzyme.

5-Aminolevulinate Synthetase↗
PubMed1985

The frequency of bone marrow cells that bind erythropoietin.

The frequencies of rat and mouse bone marrow cells capable of binding erythropoietin were studied by both direct fluorescence and indirect immunofluorescence. We found that between 1-2% of the cells bound erythropoietin, that the binding was specific, and that the number of cells that bound erythropoietin was, in part, a function of the erythropoietic state of the donor animal. A statistical method for evaluating the data obtained is included.

Animals↗
PubMed1985

Some chemical properties of human erythropoietin.

Erythropoietin (epo) is the glycoprotein hormone that induces normal red cell differentiation. Reaction of native or denatured epo with either [3H]iodoacetic acid or N-ethyl-2-[3H]maleimide did not result in the incorporation of any significant amount of radioactivity. Radiolabeling took place only if the protein were denatured before reduction and alkylation. When reduction was carried out in the presence of 6 M guanidine HCl, about 3.7 mol N-ethyl-2[3H]maleimide were covalently linked per mol epo. These results show that there are no free, accessible sulfhydryl groups in epo; there are two internal disulfide bonds. When epo was reduced in the presence of 6 M guanidine HCl and then reoxidized and the guanidine removed, about 85% of the biological activity was regenerated. The biological activity was lost irreversibly if the sulfhydryl groups were alkylated. Limited proteolysis of [3H]epo (labeled at sialic acid residues of the oligosaccharide chains) showed that it consists of two rather trypsin-resistant domains, each having a mol wt of about 16,000, connected by a small region of protein that is trypsin sensitive. The two large fragments contain most of the label. Biological activity and immunoreactivity are lost after limited tryptic proteolysis. Complexing epo with a neutralizing antibody protects its activity from proteolysis.

Chemical Phenomena↗
PubMed1985

The role of carbohydrate in erythropoietin action.

The carbohydrate composition of human erythropoietin (epo) was determined by micro-GLC. Enzymic removal of most of the sugars results in aggregation of glycosidase-treated epo, loss of biological activity when assayed in mice, and retention of activity when assayed in marrow cell cultures or by RIA. Endoglycosidase F causes the removal of most of the carbohydrates indicating that the oligosaccharides are asparagine linked. The lack of O-linked sugar is confirmed by the absence of N-acetylgalactosamine. These findings indicate that the oligosaccharide portion of epo, although required for action in vivo, is not required for interaction with the target cells of the blood-forming system.

Carbohydrates↗
PubMed1985

Primary myeloproliferative disorder and hepatic vein thrombosis. A prospective study of erythroid colony formation in vitro in 20 patients with Budd-Chiari syndrome.

We assessed the prevalence of overt and latent primary myeloproliferative disorders in hepatic vein thrombosis. Cultures of bone marrow or peripheral blood mononuclear cells were done in 20 patients with Budd-Chiari syndrome. Erythroid colony formation in the absence of erythropoietin, which is a reliable indicator for a primary myeloproliferative disorder, was seen in 16 patients in whom Budd-Chiari syndrome was due to hepatic vein thrombosis, including 13 women aged 18 to 45 years. Among these 16 patients, the conventional criteria for the diagnosis of a primary myeloproliferative disorder were met in only 2. Primary myeloproliferative disorder, often without peripheral blood changes, is a major cause of hepatic vein thrombosis in young women.

Adolescent↗