Characterization of a thrombocytopoietic-stimulating factor from kidney cell culture medium.

The chemical characteristics of a thrombocytopoietic-stimulating factor (TSF or thrombopoietin) found in serum-free kidney cell culture medium were further delineated by subjecting the TSF-rich medium to varying temperatures, different pH, and trypsin digested; the ability of TSF to bind lectins on affinity chromatography was also determined. After treatment, the TSF was assayed in immunothrombocythemic mice by its ability to increase the incorporation of 35S-sodium sulfate into newly formed platelets. TSF appeared to be relatively heat stable; incubation of TSF for 16 h at temperatures of 4, 37, and 56 degrees C showed no loss of TSF activity. However, after incubation at 85 degrees C, TSF was completely inactivated TSF in culture medium was stable of pH 1-8. Above these pH values, the potency of the TSF material decreased sharply. Digestion of TSF with trypsin completely destroyed the thrombocytopoietic-stimulating activity. For TSF purification, two different lectin-agarose derivatives were used; i.e., wheat germ agglutinin (WGA) and concanavalin A (Con A). Both lectins bound TSF, and the hormone was eluted by the sugars specific for the particular lectin. lectins, therefore, can be used to partially purify the hormone; a further 10 to 200-fold purification was achieved by these techniques. Since other workers have shown that TSF from plasma of thrombocytopenic rabbits will bind WGA and Con A, TSF from kidney cell culture medium and TSF from animal sources appear to have similar carbohydrate compositions.

Regulation of erythropoiesis in diabetes mellitus.

The regulation of erythropoiesis in 40 subjects with diabetes mellitus was investigated to test the hypothesis that increased blood concentrations of glycosylated hemoglobin would produce a reduced p50 and a compensatory increase in erythropoietin. Little evidence to support this hypothesis was observed. However, diabetes did produce complex changes in hemoglobin-oxygen releasing capacity which were compensated for in the expected way, i.e. oxygen releasing capacity showed an inverse correlation with erythropoietin. We conclude that the effects of diabetes on erythropoiesis are minor and probably have no pathological significance in most patients suffering from this disease.

Animal & computer investigations into the murine erythroid response to chronic hypoxia.

During chronic hypoxia, the number of splenic erythroid progenitor cells in mice, particularly CFU-E, increased dramatically but transiently. Since all three classes of erythroid progenitors in the femoral bone marrow were suppressed, a large part of this increase might be attributed to migration of CFU-E and/or their progenitors from the medullary cavity. The changes in CFU-E were preceded 48-72 hours earlier by an increase in serum erythropoietin (Ep) titers which, in turn, had been preceded by a rapid and marked "shift-to-the-right" in the hemoglobin oxygen dissociation curve. During hypoxia, the mice lost a considerable fraction of their body weight. Computer simulations, using a mathematical model of erythropoietic regulation, suggest that this weight loss, either indirectly by reducing the need for red cells in a smaller-than-control animal or by directly altering the sensitivity of the Ep-producing mechanism, is the major cause of the falling Ep titers despite continuation of the hypoxic stress. Because of high endogenous 59Fe incorporation levels, it was not possible to confirm the thesis that animals with an expanded Erythropoietin Responsive Cell (ERC) compartment would be more sensitive to exogenous erythropoietin than are mice with a normal or reduced ERC population.

Translation of messenger RNA from a renal tumor into a product with the biological properties of erythropoietin.

The renal tumor RCC-3-JCK, when transplanted into immunodeficient mice, caused an erythrocytic polycythemia. When grown in culture, the tumor cells secreted a substance into the culture medium that chromatographed by size-exclusion high-performance liquid chromatography similarly to purified human erythropoietin (Ep) and was positive when assayed for Ep by its ability to stimulate erythropoiesis in fetal mouse liver cells (the FMLC assay). The poly(A) + RNA was extracted from the tumor cells and injected into Xenopus oocytes, inducing the appearance of Ep(FMLC) in the oocyte culture medium. Both the tumor cells and oocyte culture media were fractionated by size-exclusion high-performance liquid chromatography, and two fractions with Ep(FMLC) activity were found in the tumor-cell culture medium. Three active fractions were found in the medium from the mRNA-injected oocytes. The largest component from both culture media had the same elution time as a human standard (Ep). The poly(A) + RNA was fractionated by sucrose density-gradient centrifugation and the 8S and 10S fractions were found to induce Ep(FMLC) synthesis when they were injected into the oocytes. We conclude that poly(A) + RNA isolated from the Ep-producing tumor RCC-3-JCK included mRNA for Ep and that the Ep was a translational product of Xenopus oocytes injected with this mRNA.

Simultaneous flow cytometric measurement of K-562 megakaryocytic differentiation and CD56+ large granular lymphocyte cytotoxicity.

K-562 cells have the capacity to undergo multi-lineage differentiation, which may be crucial to their ability to serve as target reservoirs for CD56+ large granular lymphocytes (LGL). Conventional techniques using chromium release assays to measure lymphocyte-mediated cytotoxicity suffer from disadvantages, including radioactive contamination and the inability to simultaneously determine K-562 and/or CD56+ lymphocyte phenotypes. We illustrate here a three-color flow cytometric method providing for the simultaneous evaluation of K-562-CD56+ LGL binding, K-562 cell viability, and the status of K-562 cell differentiation. Phorbol 12-myristate 13-acetate (PMA) engenders megakaryocytic differentiation in K-562 cell populations, as measured by presentation of the beta(3) integrin (gpIIIa, CD61), while maintaining a negative expression of MHC-I and MHC-II molecules. Using the auto-fluorescence of K-562 cells, flow cytometry can be used to demonstrate a significant decrease in CD56+ LGL activity against K-562 cells in populations pre-incubated with PMA. The capacity of three-color flow cytometry to measure lymphocyte-target cell binding and cell death kinetics, while simultaneously determining target cell phenotype, permits the specific localization of CD61-expressing K-562 cells to areas inconsistent with CD56+ LGL-mediated patterns of lysis.

Canine cyclic hematopoiesis: blood gas and 2,3-diphosphoglyceric acid studies.

Cyclic hematopoiesis in gray Collies was first described in 1967. These dogs are anemic in comparison with the healthy littermates, and their erythropoiesis is abnormal. Although the basic disorder appears to be an as yet unidentified abnormality of hematopoietic progenitor cells, an inherent difference in responses to blood gas control mechanisms remains as a possible cause. In a study of these mechanisms in dogs with cyclic hematopoiesis, the P50 and 2,3-diphosphoglyceric acid concentrations were increased. Differences in pH, PCO2, PO2, and oxygen saturation were not observed.

Rosette formation of human erythrocytes on canine transmissible venereal sarcoma cells.

Human erythrocytes have been shown to form spontaneous rosettes on nonlymphoid canine transmissible venereal sarcoma cells. The tumor cell would attach on glass surface, and morphologic studies by both electron microscopy and light microscopy on Giemsa-stained rosetting tumor cells showed that they could be differentiated from the rosetting T lymphocytes in the tumor.

Some immunological and haematological aspects of human cyclic neutropenia.

In addition to standard peripheral blood cell counts, sequential studies have been made of changes in the T-lymphocyte population and in the serum titres of the presumptive humoral regulators of haematopoiesis, Colony Stimulating Activity (CSA) and Erythroid Stimulating Activity (ESA), in a young woman with cyclic neutropenia (CN). In addition, serum immunoglobulins, C3 and total complement levels and serum protein concentrations were determined on several occasions during the study. Similar tests were done concomitantly on a haematologically normal, age and sex-matched control. Cell counts on peripheral blood from the subject with CN demonstrated a clearly defined periodicity in neutrophil and monocyte concentrations and equivocal fluctuations in reticulocyte numbers. There was no evidence of periodicity in the lymphocyte concentrations and the T-lymphocyte population appeared functionally normal. Spontaneous incorporation of tritiated thymidine into peripheral blood cells showed a highly significant correlation with the monocyte count, suggesting that these cells were responsible for the radioisotope uptake. CSA titres were elevated on all occasions tested and showed no evidence of periodicity. ESA showed some evidence of cycling with elevated levels being observed during the periods of neutropenia. Serum complement levels were within the normal range but all classes of immunoglobulins were elevated and albumin levels were depressed.

Preparation of purified erythropoietin by high performance liquid chromatography.

Highly purified erythropoietin was not available in quantities needed to carry out planned investigations and, therefore, the use of high performance liquid chromatography was explored. This technique permits the separation of proteins with high efficiency and resolution. Three types of chromatography were used. Size exclusion or gel permeation, reversed phase, and ion exchange columns were utilized with different solvent systems. The chromatographic fractions were assayed either by an exhypoxic polycythemic mouse assay or by the fetal liver cell assay. In addition, selected fractions were tested for their capability to stimulate CFU-E and BFU-E colony formation in methyl cellulose. The results of the techniques of size exclusion and ion exchange chromatography were found to be rapid and reproducible. Although reversed phase chromatography gave excellent resolution, the results were somewhat variable. Using different chromatographic combinations, erythropoietin with a specific bioactivity in the range of 50,000 u/mg protein was isolated. Although the erythropoietin gene has now been cloned and the hormone purified by utilizing monoclonal antibodies, high performance liquid chromatography may be useful in the removal of unwanted contaminants, as a tool in chemical characterization, and as a possible method for the hormone's identification and measurement in clinical laboratories.

Effect of thiopental on neurologic outcome following coronary artery bypass grafting.

To determine if thiopental reduces the incidence of neurologic sequelae after coronary artery surgery, we prospectively studied 300 patients undergoing coronary artery bypass grafting. Patients who had no history of neurologic or psychiatric illness were randomly assigned to receive either a thiopental infusion or a saline placebo infusion beginning with the administration of heparin and ending just after aortic decannulation. The patients received an opioid-relaxant anesthetic administered by an anesthesiologist who was not involved in this investigation and who was blinded to the test infusion. One of the investigators infused either saline or thiopental to produce an isoelectric electroencephalogram with 30-45 s between bursts. Standardized neurologic examinations were performed preoperatively and on the 2nd and 5th postoperative days by one of the blinded investigators. The group of patients receiving thiopental required a longer time for awakening (6.4 +/- 3.9 vs. 4.0 +/- 2.4 h, mean +/- SD, P less than 0.05) and for tracheal extubation (22.4 +/- 18.4 vs. 17.4 +/- 9.6 h, P less than 0.05), and a greater number of these patients were lethargic on the 2nd postoperative day. More patients receiving thiopental required vasoconstrictors during the thiopental loading and cardiopulmonary bypass (CPB) periods, while a greater number of patients receiving placebo required vasodilators. A greater number of patients receiving thiopental required inotropic drugs during separation from CPB. Despite the above differences, only 2 of the 151 patients in the placebo group (1.3%) and 5 of the 149 patients in the thiopental group (3.3%) experienced strokes (P = 0.2535).(ABSTRACT TRUNCATED AT 250 WORDS)

Secretion of ceruloplasmin by a human clear cell carcinoma maintained in nude mice.

Ceruloplasmin is the best known but least understood copper protein. Studies preliminary to investigating the control of ceruloplasmin synthesis have utilized a human renal cell carcinoma maintained in nude mice for 73 passages over a 5-year period. In vitro cultures of these cells were accomplished and the mRNAs were extracted prior to microinjection into Xenopus oocytes. The media examined by SE-HPLC and immunological techniques demonstrated that (1) after in vitro culture, ceruloplasmin was secreted as an uncleaved polypeptide chain with a MW of 135,000; (2) the translational product of ceruloplasmin mRNA injected into Xenopus oocytes was cleaved into fragments with MWs of 110,000, 67,000, and 50,000. The results indicate that mRNA for human ceruloplasmin can be obtained to serve as a template for the synthesis of a cDNA probe to investigate the control of human ceruloplasmin's synthesis.

Hematological measurements in rats flown on Spacelab shuttle, SL-3.

Previous studies have shown that a decrease in red cell mass occurs in astronauts, and some studies indicate a leukocytosis occurs. A life science module housing young and mature rats was flown on shuttle mission Spacelab 3 (SL-3), and the results of hematology studies of flight and control rats are presented. Statistically significant increases in the hematocrit, red blood cell counts, and hemoglobin determinations, together with a mild neutrophilia and lymphopenia, were found in flight animals. No significant changes were found in bone marrow and spleen cell differentials or erythropoietin determinations. Clonal assays demonstrated an increased erythroid colony formation of flight animal bone marrow cells at erythropoietin doses of 0.02 and 1.0 U/ml but not 0.20 U/ml. These results agree with some but vary from other previously published studies. Erythropoietin assays and clonal studies were performed for the first time.