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Biomedical subjects

J Caro

Publications and source records attributed to J Caro.

At least 19 recordsLinked to original sources
PubMed1992

Cellular sites of extrarenal and renal erythropoietin production in anaemic rats.

The cellular origins of erythropoietin were investigated in the rat using a probe derived from a cloned rat erythropoietin cDNA. In anaemic-hypoxic rat liver, in situ hybridization detected erythropoietin mRNA primarily in hepatocytes and less frequently in nonparenchymal sinusoidal or perisinusoidal liver cells. An RNase protection assay was used to compare the erythropoietin mRNA contents of separated rat liver cell fractions and also suggested that hepatocytes are the major source of extrarenal erythropoietin with nonparenchymal liver cells contributing less than 1% to total hepatic erythropoietin production. In kidney, in situ hybridization localized erythropoietin mRNA in nonepithelial cells, as yet of undefined lineage, in the cortical and outer medullary interstitium. These results indicate that, in the rat, the primary sources of erythropoietin in liver and kidney are different types of cells.

Anemia↗
PubMed1992

Clinical pharmacology and economics of recombinant human erythropoietin in end-stage renal disease: the case for subcutaneous administration.

The clinical pharmacology of human recombinant erythropoietin (epoetin) was studied in order to compare the effectiveness of various routes and dosing schedules in dialysis patients. Thirty-six patients received epoetin beta three times a week i.v. for at least 12 wk. The mean dose needed to achieve target hemoglobin was 225 +/- 36 U/kg per week (median dose, 180 U/kg per week). Twenty-eight of 36 patients who were converted to a once-a-week i.v. schedule increased their requirements to 429 +/- 50 U/kg per week in order to maintain a target hematocrit of 33 to 40 vol%. Twelve of 28 patients could maintain their target hematocrit when dosed once a week s.c. at 84 +/- 10 U/kg. The other 16 patients required 137 +/- 15 U/kg per week divided into two doses. In the entire group of 28 patients, the weekly requirement for epoetin was reduced by 50% when the s.c. route was used two or three times a week. Pharmacokinetic studies performed during chronic therapy indicated rapid clearance of erythropoietin (t1/2 of 6.8 +/- 0.3 h). Single i.v. doses greater than 150 U/kg were required to increase basal erythropoietin by 30 mU/mL at 44 h postdosing. With s.c. dosing, such increments in erythropoietin levels frequently persisted beyond 60 h because of prolonged and slow absorption. Pharmacokinetic simulations in conjunction with clinical correlation of the erythropoietic response suggest that the duration that the erythropoietin levels are maintained, and not the absolute peaks, is the primary determinant of efficacy. This may result from nonlinearity in the dose response. Pharmacokinetic simulation also indicated that i.v. dosing could not maintain adequate interdialytic erythropoietin levels, whereas s.c. dosing could. Cost analysis indicated that the use of s.c. dosing two or three times a week at an average total weekly dose of 110 to 120 U/kg is effective treatment of anemia in most dialysis patients.

Adolescent↗
PubMed1991

Enhancer element at the 3'-flanking region controls transcriptional response to hypoxia in the human erythropoietin gene.

Erythropoietin gene expression is greatly stimulated under conditions of hypoxia. The activation of the erythropoietin gene appears regulated primarily at the level of gene transcription. To study cis-acting elements involved in the response to hypoxia a mini-gene was constructed by an internal deletion from exon II to V of the human erythropoietin gene and used in transient transfection assays in the erythropoietin producing Hep 3B cell line. It was initially found that hypoxia responsiveness was present in an erythropoietin fragment containing 400 base pairs (bp) of 5'-flanking and 600 bp of 3'-flanking regions. Deletion analysis showed no significant effect on the response to hypoxia when highly conserved regions of 5'-flanking sequence, exon and intron I, and exon V were removed from the mini-gene construct. However, removal of a fragment containing the 3' end of the gene and 3'-flanking sequences completely eliminated hypoxia responsiveness. Reinsertion of the above fragment upstream of the 5' end of the mini-gene restored the response to hypoxia. Further analysis using hybrid erythropoietin-chloramphenicol-acetyltransferase constructs allowed the localization of enhancer-like element(s) in the 3'-flanking region, approximately 120 bp downstream of the polyadenylation site of the human erythropoietin gene. Activation by these sequences were position- and orientation-independent and stimulated 15-fold transcription of the erythropoietin gene in response to hypoxia.

Cells, Cultured↗
PubMed1991

Secondary erythrocytosis due to a cerebellar hemangioblastoma: demonstration of erythropoietin mRNA in the tumor.

Cerebellar hemangioblastoma is a rare cause of secondary erythrocytosis. Although the erythrocytosis is a result of erythropoietin (Ep) stimulation, direct evidence of Ep synthesis by the tumor has been lacking. In an erythrocytotic patient with a cerebellar hemangioblastoma we found elevated levels of Ep in the tumor cyst fluid and for the first time demonstrated Ep mRNA in the tumor by Northern blotting. This finding confirms cerebellar hemangioblastoma as a site of ectopic Ep production.

Adult↗
PubMed1991

The importance of N- and O-linked oligosaccharides for the biosynthesis and in vitro and in vivo biologic activities of erythropoietin.

Erythropoietin (EPO) plays a critical role in stimulating the proliferation and differentiation of erythroid precursor cells. EPO is heavily glycosylated with three asparagine (N)-linked tetraantennary oligosaccharides that may contain N-acetyl-lactosamine repeats and a single serine (O)-linked oligosaccharide. EPO expressed in Chinese hamster ovary cells exhibits biologic properties and amino acid and carbohydrate composition similar to natural urinary EPO. The importance of the complex N-linked and the O-linked carbohydrate was studied by expressing EPO in cells that are deficient in UDP-galactose/UDP-N-acetylgalactosamine 4-epimerase activity. In these cells, the ability to add galactose and N-acetylgalactosamine to glycoproteins can be controlled by the addition of these sugars to the culture medium. The results demonstrate that a block in O-linked glycosylation and/or the ability to process N-linked carbohydrate to completion does not alter EPO secretion. EPO produced without O-linked carbohydrate exhibits normal in vitro and in vivo biologic activity and in vivo clearance. However, EPO produced with incompletely processed N-linked oligosaccharides exhibits normal in vitro activity but is at least 500-fold less effective in stimulating erythropoiesis in vivo. Studies on the survival of bioactive EPO remaining in the circulation demonstrated that EPO with incomplete N-linked oligosaccharides exhibits a sevenfold increased rate of clearance. However, this increased clearance may not fully account for the 500-fold loss of in vivo activity. These results suggest a potentially important unique requirement for appropriate complex N-linked oligosaccharides for the intrinsic biologic activity of EPO in vivo.

Acetylgalactosamine↗
PubMed1991

Erythropoietin life span in rats with hypoplastic and hyperplastic bone marrows.

The metabolic fate of erythropoietin (EPO) remains unknown. Urinary excretion does not appear to play a major role and liver catabolism has been shown to occur only after terminal sugars on the hormone have been removed. However, it has been proposed that EPO is eliminated by consumption in the bone marrow. In order to examine the extent of such consumption we measured the half-life of radioidinated recombinant EPO injected intravenously (IV) to rats with bone marrows suppressed by cyclophosphamide or hypertransfusion and marrows stimulated by phenylhydrazine or bleeding. The mean half-life or erythropoietin in normal rats was 179 +/- 16 min, with similar half-lives found in the other rats regardless of decreased or increased bone marrow activity. The results indicate that it is unlikely that erythroid activity determines EPO life span and catabolism.

Animals↗
PubMed1990

Enhancement by hypoxia of human erythropoietin gene transcription in vitro.

Erythropoietin (Epo) gene transcription is stimulated in Hep3B cells under hypoxic conditions. We have prepared transcriptionally active nuclear extracts from normal and hypoxia-induced Hep3B cells and shown that the hypoxic extracts produce a consistent increase in the level of Epo transcription in vitro, relative to control Hep3B cells. Hypoxic treated HeLa cells failed to express the endogenous Epo gene in vivo, and extracts prepared from them did not show increased Epo transcription in vitro. The Epo transcript which is induced in vitro is initiated at the same site as Epo RNA synthesized in intact Hep3B cells and in human kidney adenocarcinoma cells. This system will facilitate the purification and analysis of factors and sequences required for Epo gene transcription in response to changes in tissue oxygen tension.

Blotting, Northern↗
PubMed1990

Pharmacokinetics and erythropoietic response to human recombinant erythropoietin in healthy men.

To assess the safety, pharmacokinetics, and erythropoietic responses to human recombinant erythropoietin (epoetin beta), single intravenous doses (10, 50, 150, and 500 IU/kg) were administered at monthly intervals to 16 healthy subjects in a two-panel, placebo-controlled, double-blind ascending-dose trial. A 1000 IU/kg dose was subsequently administered in an open manner. Epoetin concentrations were determined in serum and urine by radioimmunoassay. Reticulocyte, hemoglobin, and hematocrit values were serially measured after each dose. Mean epoetin apparent half-lives ranged from 4.42 to 11.02 hours. The apparent volume of distribution was between 40 and 90 ml/kg, consistent with plasma water, and the apparent clearance values ranged from 4 to 15 ml/kg/hr, with both parameters having the highest values at the 10 IU/kg dose level. Clearance tended to decrease as a function of dose. Maximum reticulocyte counts were dose-dependent and occurred 3 to 4 days after the epoetin dose. Epoetin was well tolerated, and no antibodies were detected.

Adult↗
PubMed1990

Erythropoietin fails to reverse the anemia in mice continuously exposed to tumor necrosis factor-alpha in vivo.

Tumor necrosis factor-alpha (TNF) is a monokine produced by activated macrophages that has cytotoxic and cytostatic effects on erythroid progenitor cells. We have recently shown that Chinese hamster ovary cells transfected with the human TNF gene and which constitutively express TNF induced a hypoproliferative anemia, mild thrombocytopenia, and mild leukocytosis when injected into nude mice. We have used this murine model to determine if treatment with recombinant human erythropoietin can prevent or ameliorate the anemia seen with long-term continuous exposure to high concentrations of TNF. Mice bearing TNF-producing tumors became anemic with hematocrits ranging from 30% to 32%. Treatment with recombinant human erythropoietin (100-1000 U/kg body weight three times per week) increased the reticulocyte counts initially in mice bearing TNF-producing tumors but failed to reverse the anemia seen in these animals. Erythropoietin did not significantly increase the number of marrow erythroid colony-forming units (CFU-E) or erythroid burst-forming units (BFU-E) in mice bearing TNF-producing tumors. The data suggest that erythropoietin could not sufficiently overcome the decreased number of erythroid progenitors in mice bearing tumors producing high levels of TNF to correct their anemia.

Anemia↗
PubMed1989

Quantitation of erythropoietin-producing cells in kidneys of mice by in situ hybridization: correlation with hematocrit, renal erythropoietin mRNA, and serum erythropoietin concentration.

In situ hybridization was used to quantitate the cells that produce erythropoietin (EP) in the renal cortices of mice with varying severities of acute anemia and of mice recovering from severe, acute anemia. The number of EP-producing cells in the renal cortex increased in an exponential manner as hematocrit was decreased. Individual EP-producing cells had very similar densities of silver grains in autoradiograms regardless of whether they were from normal mice or from slightly, moderately or severely anemic animals. With increasingly severe anemia, total renal EP mRNA levels and serum EP concentrations showed increases that correlated with the number of renal EP-producing cells. These results indicate that as mice become more anemic, additional cells are recruited to produce EP rather than the cells already producing EP being stimulated to increase their individual production. In mildly and moderately anemic animals, small clusters of EP-producing cells were found in the inner cortex with large areas of cortex containing no EP-producing cells. In severely anemic mice, EP-producing cells were found throughout the inner cortex with only a very few found scattered in the outer cortex and outer medulla. The data indicate that only a subset of total renal interstitial cells produce EP. During recovery from severe, acute anemia, the numbers of EP-producing cells decreased exponentially as hematocrits rose and correlated with decreases in total renal EP mRNA and serum EP concentrations. These results suggest that following an acute blood loss and during the recovery from a blood loss, the capacity to deliver oxygen, as represented by hematocrit, is the major regulator of EP production.

Acute Disease↗
PubMed1989

Inappropriate increase in erythropoietin titers during chemotherapy.

Serial erythropoietin measurements by RIA were performed in six patients with acute leukemia treated by intensive chemotherapy. In all cases erythropoietin titers increased after the onset of treatment, although the hemoglobin concentration remained at stable values. Subsequently the erythropoietin titers gradually returned to baseline levels. In same patients this reduction occurred at the end of chemotherapy, in others coincident with infections and antibiotic therapy. In four patients this decrease occurred at the time of bone marrow recovery. The explanation for this inappropriate increase in erythropoietin titers is not clear but may be related to a direct or indirect effect of a suppressed marrow on sites of erythropoietin production or catabolism.

Adult↗
PubMed1989

Erythropoiesis in cancer patients undergoing immunotherapy.

We studied ten patients with various types of cancer who were being treated with Interleukin-2 (IL-2) and lymphokine activated killer cells (LAK). All patients developed a reticulocytopenic, normochromic, normocytic anemia. We noted some variability but no significant suppression of circulating erythroid progenitors. The levels of erythropoietin were lower than expected for the hemoglobin/hematocrit values. We could not detect Interferon or Tumor Necrosis Factor (TNF) in the serum of these patients; however, the supernatant of LAK cells did contain Interferon and TNF which could be neutralized with appropriate antibodies. These results suggest that the etiology of this anemia is multi-factorial. Administration of recombinant erythropoietin (Ep) may be of benefit in some of these patients.

Anemia↗