Erythropoietic kinetics in sheep studied by means of induced changes in hemoglobin phenotype.

This investigation is concerned with the kinetics of the reciprocal relationship between sheep hemoglobin (Hb) A and Hb C formation in response to anemia. The relative synthesis of the hemoglobin types was assessed at various times in bone marrow erythroid cells incubated in vitro with (59)Fe. The changeover from Hb A to Hb C formation lagged by about 3 days behind the development of anemia and was complete within about 11 days. After recovery from anemia the reciprocal change back to preanemic conditions proceeded at a much slower rate, Hb C formation gradually declining to unmeasurable levels over about 25 days. Infusions of plasma with high erythropoietin titre induced the formation of relatively large quantities of Hb C in erythroid cells of nonanemic sheep, demonstrating the central importance of a humoral mechanism in the change of expression of the hemoglobin genes. THE FOLLOWING CONCLUSIONS WERE DRAWN: hemoglobin phenotype is determined at a stem cell level. Erythroid stem cells appear to undergo gradual renewal. The identity of the plasma factor which induces Hb C formation is not yet known; it is not present in plasma from nonanemic sheep, and its production is not dependent upon hemoglobin genotype. If the plasma factor turns out to be erythropoietin, then this hormone must have an important influence on the pool of erythroid stem cells.

Glucocorticoid receptors in peripheral blood lymphocytes from bovine leukemia virus-infected cows with persistent lymphocytosis.

Bovine leukemia virus-infected cows with persistent lymphocytosis have an expanded population of B-lymphocytes in the peripheral blood that is sensitive to glucocorticoids in vitro and in vivo. We examined peripheral blood lymphocytes from cows with persistent lymphocytosis for the presence of specific glucocorticoid receptors. Steroid binding in intact cells was determined by a whole cell competitive binding assay using [3H]dexamethasone. The binding of the glucocorticoid to receptor was characterized in terms of affinity, specificity, and kinetics of the reaction. We found that peripheral blood lymphocytes from three cows with persistent lymphocytosis had 5000 to 6600 specific glucocorticoid-binding sites/cell. Compared with that reported for human lymphoid cells, glucocorticoid receptors in the bovine lymphocytes were found to have a greater affinity for the steroid with an association rate that was three times faster and a dissociation rate that was less than one-half of the former. We examined the biological half-life of hydrocortisone in the normal cow and found it to be 69.3 min, which is shorter than that reported for other domestic species and humans. The kinetics and affinity of the steroid binding may explain why in vivo glucocorticoid sensitivity was demonstrated in these animals despite the fact that elevated levels of plasma corticoids were not maintained. These results suggest that glucocorticoid sensitivity may be influenced by the nature of the binding reaction between steroid and receptor.

Altered glucose metabolism in metastatic carcinoma.

To evaluate the possible role of altered glucose metabolism in malignant cachexia, metabolic parameters including total glucose turnover, glucose oxidation, and Cori cycle activity were measured in fourteen patients with metastatic carcinoma. Eight patients with progressive weight loss (PWL) were compared to 6 without (controls). Cori cycle activity was significantly increased (p less than 0.02) in PWL patients, 90 mg/kg/hr (range, 22 to 193) compared to 18 mg/kg/hr (range, 13 to 24) in controls. Total glucose turnover was moderately increased in PWL patients, 196 mg/kg/hr compared to 110 mg/kg/hr in controls. Glucose oxidation was 62 mg/kg/hr versus 48 mg/kg/hr, and total caloric expenditure was 36 kcal/sq m/hr compared to 33 Kcal/sq m/hr. PWL patients were metabolically heterogenous and mean values are skewed by four patients with increased glucose turnover, oxidation, and markedly high recycling rates that were equivalent to total endogenous glucose turnover of a normal subject. Total caloric expenditure was greatest in three of the four patients with a marked increase in Cori cycle activity. Energy loss associated with a high rate of gluconeogenesis from lactate has been suggested as an explanation for increased energy expenditure in some cancer patients, thus contributing to mechanisms that promote weight loss.

Protection of erythrocyte membrane amino groups from reaction with methyl acetimidate by pyridoxal 5'-phosphate Schiff base formation.

Pre-equilibration of erythrocytes with the membrane-impermeable aldehyde, pyridoxal 5'-phosphate, for 30 min at 22 degrees C, prior to the addition of methyl acetimidate to the incubation mixture has been shown to prevent agglutination of acetamidinated cells which were resuspended in immune serum (Chao, T.L. and Berenfeld, M.R. (1981) J. Biol. Chem. 256, 5324-5326). This observation led to the possibility that the immune reaction, observed in some sickle cell anemia patients to reinfused cells which had been reacted with methyl acetimidate, could be prevented. The present communication further evaluates that reaction sequence and shows that while the pre-equilibration of cells with pyridoxal 5'-phosphate does protect membrane amines from reaction with methyl acetimidate, the protection is not extensive enough to prevent an immune response in a sickle cell anemia patient who had already been sensitized against acetamidinated cells. It is apparent that the design of antisickling agents which covalently modify hemoglobin must take into account protection of functional groups in the erythrocyte membrane, modification of which could produce an immunogenic response.

Normal serum ferritin levels in a patient with HEMPAS syndrome and iron overload.

Serum ferritin levels in a patient with HEMPAS syndrome (hereditary erythroblastic multinuclearity associated with positive acidified serum test) were correlated with body iron stores directly measured on spleen and liver biopsy specimens as well as by quantitative serial phlebotomy. Normal serum ferritin concentrations were found in the presence of a moderate excess in iron stores (approximately 6-12 times normal). They temporarily increased after transfusion and splenectomy with a prompt return to the normal range. As repeated phlebotomies over a period of nine months depleted the excess iron stores, the serum ferritin ultimately decreased to a subnormal concentration. The serum ferritin concentration was not a reliable index of increased body iron stores in this iron overloaded patient, but did reflect their depletion by serial phlebotomy.

Symmetric myositis and fasciitis: a complication of sickle cell anemia during vasoocclusion.

Repeated episodes of symmetric myositis and fasciitis occurred in 2 males with sickle cell anemia after prolonged painful crises. Drugs and infection could not be implicated as a cause. Histologically and therapeutically these cases differ from all other known types of myositis/fasciitis. Microscopic evidence of vasoocclusion compels consideration of an ischemic basis for this previously undescribed entity.

Current status of methyl acetimidate as an extracorporeal antisickling agent.

Methyl acetimidate has been shown to be an effective in vitro antisickling agent with few detrimental effects on the red cell. 51Cr-survival of red cells that had been incubated in vitro with methyl acetimidate was prolonged to near normal levels in sickle cell anemia patients. However, some patients developed an immune response following multiple reinfusions of the acetamidinated cells. Pre-equilibration of erythrocytes with the membrane-impermeable aldehyde, pyridoxal 5'-phosphate, prior to the addition of methyl acetimidate to the reaction mixture, has been shown to prevent agglutination of acetamidinated cells which were resuspended in immune serum. However, the protection was not extensive enough to prevent an immune response in a sickle cell anemia patient who had already been sensitized against acetamidinated cells. It is apparent that further consideration of imidoesters as extracorporeal antisickling agents will require complete protection of membrane amino groups against reaction with the imidoester.

Glucocorticoid effects on peripheral blood lymphocytes in cows infected with bovine leukemia virus.

We examined the effects of glucocorticoids on peripheral blood lymphocytes (PBL) in lymphoproliferative conditions associated with bovine leukemia virus (BLV): persistent lymphocytosis (PL) and lymphosarcoma cell leukemia (BLSL). The effects of hydrocortisone 21-sodium succinate (HSS) on spontaneous incorporation (SI) and mitogen-stimulated incorporation of radiolabeled-thymidine and the effects of intramuscular administration of prednisolone acetate were studied. An expanded population of B lymphocytes in cows with PL was remarkable sensitive to glucocorticoids in vitro and in vivo. SI was markedly inhibited by concentrations of HSS as low as 10(-7) M. These results correlated well with in vivo observations, where an 80%-90% decrease in PBL occurred during the course of glucocorticoid administration. The decrease in total lymphocytes was accounted for almost entirely by a decrease in the expanded B lymphocyte population. Steroid-sensitive lymphocytes together with steroid-resistant cells were observed in cows with BLSL. The reduction in the steroid-sensitive lymphocytes was associated with rapid disease progression in cows with lymphosarcoma. Steroid-sensitive lymphocyte populations in cows with BLSL may include the same reactive B-cell population found in cows with PL. Glucocorticoids may prove to be a useful tool for study of the immune response to the oncogenic virus and lymphoma in BLV-infected cattle.

In vivo survival studies of 51Cr-labeled methyl acetimidate treated erythrocytes in patients with sickle cell disease.

Studies of the survival time of 51Cr labeled erythrocytes treated in vitro with methyl acetimidate (MAI) were conducted in 13 patients with sickle cell disease in order to assess the suitability of this antisickling agent for more extensive clinical testing. In comparison with previously measured control values (average t1/2 8.4 +/- 1.1 days a), the survival time of the treated erythrocytes in 10 of the patients who were not transfused was initially prolonged (average t1/2 24.4 +/- 4.6 days). However, 5 of the 13 patients studied developed circulating antibody against the MAI treated erythrocytes, markedly reducing the survival time of MAI treated erythrocytes in subsequent studies. Two patients, each challenged 3 times with infused MAI treated erythrocytes, failed to show evidence of antibody production, suggesting that not all subjects become immunized even after repeated exposure. In spite of many other promising properties of MAI as an antisickling agent of potential value, consideration of its use in further clinical testing must depend on successful avoidance of immunization in patients receiving infusions of treated erythrocytes.

The effects of oxygen affinity and gelation of hemoglobin S crosslinked by reaction with methyl acetimidate.

The contribution of the high molecular weight hemoglobin (HMW Hb) to the antisickling effect produced by treatment of sickle cell erythrocytes with methyl acetimidate (MAI) was investigated. Erythrocytes obtained from sickle cell anemia and normal individuals were incubated with varying concentrations of MAI. The presence of intermolecularly crosslinked HMW Hb was detected by gel filtration of dialyzed hemolysates obtained from the incubated cells. HMW Hb has an increased oxygen affinity and a decreased Hill constant. Intermolecular crosslinking per se was shown to have no additional effect on Hb oxygen affinity other than that due to Hb amino group modification. Modified deoxy Hb S of normal molecular weight has a higher minimum gel concentration (MGC) than control deoxy Hb S. Deoxy HMW Hb did not gel at intracellular Hb concentrations. Therefore, the intracellular concentration of deoxy Hb S that gels necessarily decreases as the HMW Hb concentration increases. This "excluded volume effect" of the non-gelling Hb would be expected to increase the MGC of completely deoxygenated hemolysate obtained from the treated cells. The HMW Hb contributes to the inhibition of sickling resulting from treatment of sickle erythrocyte with MAI by increasing both MGC and oxygen affinity of the modified Hb.

Beta-carotene content of certain organs from two patients receiving high doses of beta-carotene.

We measured carotene in the livers of two patients receiving beta-carotene therapy for photosensitivity. In both patients, the amount of carotene was within reported ranges for patients on normal diets; no abnormal amounts of beta-carotene had accumulated in the livers after oral administration of large amounts of beta-carotene. Light-microscopic examination of the liver (specimen obtained at cholecystectomy) of one patient with erythropoietic protoporphyria and cholesterosis showed findings characteristic of these conditions. Electron-microscopically, semicrystalline inclusions were seen in the mitochondria of the hepatocytes; such inclusions have been reported in several conditions, including erythropoietic protoporphyria and porphyria cutanea tarda, and in control subjects. Microscopic examination of the liver of the other patient, who had Rothmund-Rhomson syndrome and died of acute bacterial meningitis, revealed no abnormalities. The cerebrum of the latter patient contained 13 mug of carotene per kilogram (wet weight) of brain. No previous report of analysis of human brain for carotenoids could be found.