Eugene Goldwasser

Immune responses to transgene-encoded proteins limit the stability of gene expression after injection of replication-defective adenovirus vectors.

The use of replication–defective adenoviruses (RDAd) for human gene therapy has been limited by host immune responses that result in transient recombinant gene expression in vivo. It remained unclear whether these immune responses were directed predominantly against viral proteins or, alternatively, against foreign transgene–encoded proteins. In this report, we have compared the stability of recombinant gene expression in adult immunocompetent mice following intramuscular (i.m.) injection with identical RDAd encoding self (murine) or foreign (human) erythropoietin. Our results demonstrate that immune responses directed against foreign transgene–encoded proteins are the major determinants of the stability of gene expression following i.m. injection of RDAd. Moreover, we demonstrate long–term recombinant gene expression in immunocompetent animals following a single i.m. injection of RDAd encoding a self protein. These findings are important for the design of future preclinical and clinical gene therapy trials.

Studies on erythropoiesis. III. Factors controlling erythropoietin production.

Summary and Conclusion 1. We studied the erythropoietic response to anemic plasma of a variety of experimental conditions in the rat. Rats subjected to hypophysectomy, an atmosphere of high O2, starvation, and transfusion-induced polycythemia have a decreased rate of erythropoiesis and an exaggerated response to the administration of anemic plasma. 2. Treatment with dinitrophenol increases the rate of erythropoiesis and decreases the response to anemic plasma. 3. These findings are in agreement with the hypothesis that the rate of erythropoiesis is determined by the amount of erythropoietin, the production of which is regulated by the relationship between O2 supply and demand, not by either factor alone.

On the mechanism of erythropoietin-induced differentiation

Abstract A rapid method for assessment of erythropoietin-stimulated glucosamine incorporation is described. The effect of erythropoietin on the incorporation of glucosamine is inhibited by puromycin but not by colchicine. Actinomycin D has a dose-dependent effect which suggests that there are two processes involved in erythropoietin action which require DNA-dependent RNA synthesis. Using actinomycin inhibition, the lifetime of the messenger RNA species concerned with stroma synthesis is estimated to be between 3 and 4 h. Data are presented which suggest that erythropoietin-induced differentiation occurs in a wave-like manner.

On the mechanism of erythropoietin-induced differentiation: II. The effect on RNA synthesis

1. 1. An increase in RNA synthesis in bone marrow cells in vitro within 15 min after the addition of an erythropoietin extract has been demonstrated. 2. 2. The high purity of the hormone preparation used and the inhibition of this effect with trypsin make it probable that the effect is due to erythropoietin itself. 3. 3. This effect is not apparent in the undifferentiated Murphy-Sturm tumor. 4. 4. The increased RNA synthesis due to erythropoietin is abolished by actinomycin D, indicating that the effect of the hormone is on DNA-dependent RNA synthesis. 5. 5. The rapid labeling, high specific activity, DNA dependence, and sucrose gradient fractionation suggest that the component responsible for the effect is messenger RNA.

Erythropoietin Radioimmunoassay in Evaluating Patients with Polycythemia

We measured serum concentrations of erythropoietin in 59 patients with polycythemia using a sensitive and specific radioimmunoassay. The mean concentration was 17.5 +/- 8.4 mU/mL (+/- SD) in 26 patients with polycythemia vera and 14.9 +/- 4.2 mU/mL in 26 normal persons. In contrast, the average concentration was 94.3 +/- 101.2 mU/mL in 33 patients with secondary polycythemia, representing a highly significant elevation (p < 0.0001) compared to both normal and polycythemia vera groups. The average hematocrit value did not differ between the polycythemia vera and the secondary polycythemia patients, and both groups had higher values (median, 55%) than the normal donors (median, 41%). Erythropoietin concentrations ascertained by radioimmunoassay helped discriminate between polycythemia vera and secondary polycythemia. Ninety-two percent of polycythemia vera patients had concentrations less than 30 mU/mL (the concentration used as a cut off point), and 94% of secondary polycythemia patients had concentrations greater than 30 mU/mL. This represents an overall correct classification of 93% of the patients. Serum erythropoietin levels as ascertained by radioimmunoassay can distinguish between most polycythemia vera and secondary polycythemia patients and should prove useful in the differential diagnosis of polycythemia.

Studies on Erythropoiesis. Part IV. Reticulocyte Response of Hypophysectomized and Polycythemic Rodents to Erythropoietin

Summary and Conclusions 1. Following hypophysectomy of rats, reticulocytes in the peripheral blood fall to a minimal level within a period of 3 to 4 weeks. This reduction represents a 5- to 10-fold decrease from the prehypophysectomy control. This reticulocyte decline is more rapid in 8-week-old rats than in younger animals. 2. Administration of anemic plasma to hypophysectomized rats produces a reticulocyte rise that is many fold greater than that produced by normal plasma or saline. 3. Erythropoiesis, as measured by the number of reticulocytes in peripheral blood, falls to zero in 6 days in mice made polycythemic by repeated intraperitoneal injections of homologous red cells. Anemic plasma obtained from rats or rabbits, when injected intravenously or intra-peritoneally into polycythemic mice, produces a reticulocytosis many times greater than that observed from normal plasma. Normal saline produces no increase in reticulocytes. 4. In polycythemic mice and hypophysectomized rats, reticulocyte determinations in the peripheral blood and Fe59 red cell incorporation studies are of about equal sensitivity in measuring the response to anemic plasma.

Erythrocytosis in Patients on Long-term Hemodialysis

Erythrocytosis was seen in two men during maintenance hemodialysis therapy for end-stage renal disease secondary to apparent chronic glomerulonephritis. Nonrenal causes of erythrocytosis such as polycythemia vera, chronic hypoxemia, high-oxygen affinity hemoglobin, and hepatoma were excluded by appropriate clinical studies. A computed tomographic scan of the abdomen showed numerous renal cysts in each patient consistent with acquired cystic disease of end-stage kidneys. Peripheral serum erythropoietin levels were elevated as measured by sensitive radioimmunoassay. The findings suggest that the erythrocytosis is caused by an erythropoietic mechanism related to the diseased kidneys. A review of the literature failed to show previous reports of this clinical association.

Studies of erythropoietin: the hormone regulating red cell production.

There is mounting evidence that erythropoiesis is controlled by a humoral factor or factors. Since Carnot and DCflandrel first published this thesis in 1906, many investigators have attempted to substantiate their work, but it has been largely in the last twenty years that such investigators as Reissmann,Z Erslevj3 Gordon and his co-~orkers ,~ Borsook et u Z . , ~ and Stohlman et aL6 have succeeded in bringing the problem to the point of serious study and acceptance. In 1952 Grant and Root7 wrote a comprehensive review of the literature on the subject. The first convincing support of Carnot’s hypothesis is found in the work of Reissmann,2 and Stohlman et uL6 Reissmann demonstrated that increased erythropoiesis occurred in both parabionts even though only one of the pair was maintained in low O2 tension. Stohlman and Rath found that erythroblastic hyperplasia was not confined to the areas of regional hypoxia in a human being with regional hypoxia secondary to a patent ductus arteriosus. Hyperplasia was also observed in the bone marrow that was supplied by blood with a normal 0 2 saturation. Of necessity, the relationship of the humoral factor that is present in anemic plasma to the maintenance of a normal amount of red blood cells and hemoglobin has been one of conjecture until recently, when Gurney and his associates demonstrated its presence in normal plasma.* The exact relationship of arterial oxygen saturation to the production of circulatory erythropoietint and, thus, to erythropoiesis has likewise not been established. During the past several years we have been concerned with: (1) applying the technique of Fe69 incorporation as a measure of red cell production; (2) finding more sensitive assay preparations for erythropoietin; (3) elucidating the role of erythropoietin in maintaining the dynamic equilibrium of erythropoiesis; (4) determining the basic conditions that control erythropoietin blood levels and, thus, the rate of erythropoiesis; (5) studying the effect of cobaltous ion on erythropoietin production; (6) searching for the site of erythropoietin production; and, of course, (7) using the information obtained to solve certain clinical problems. A brief discussion of our research and some interpretations of the data are the subjects of this paper.

Erythropoiesis. II. Assay of erythropoietin in hypophysectomized rats.

Summary and Conclusions Using incorporation of Fe59 into newly-formed red cells as index of red cell production, it has been shown that erythropoiesis is gradually reduced, reaching a minimum in rats at 8 to 13 days after hypophysectomy. A factor of 10 exists between incorporation of Fe59 into red cells of normal control (37 ± 4) and the hypophysectomized rat (4 ± 2) at this interval. We have also found that the hypophysectomized rat is an extremely sensitive preparation for assay of factor(s) in anemic plasma that stimulates or mediates erythropoiesis. Our observations may be summarized as follows: 1. Administration of anemic plasma to hypophysectomized assay animal increases incorporation of Fe59 3- to 7-fold. 2. Plasma from hypophysectomized animals and un-operated controls made anemic by repeated phlebotomy increases Fe59 red cell incorporation to the same extent when administered to the hypophysectomized assay animal. 3. A single injection of 2 ml of anemic plasma to the hypophysectomized assay animal elicits a 2- to 3-fold increase in Fe59 incorporation. The mechanism of rapid reduction of erythropoiesis that follows hypophysectomy in rats and its relationship to increased sensitivity of the hypophysectomized animal to anemic plasma are discussed briefly.

HEMOGLOBIN SYNTHESIS IN MARROW CELL CULTURE: THE EFFECT OF RAT PLASMA ON RAT CELLS

In cultures of rat marrow cells essentially all of the labeled iron which was incorporated into heme was shown to be in hemoglobin. Iron bound to rat plasma was incorporated into hemoglobin to a greater extent than was iron bound to fetal calf serum. Incorporation of iron bound to rat plasma was greater when some fetal calf serum was present than when rat plasma provided the total protein. These data suggest preferential use of homologous iron-binding proteins of the plasma.