Missing band 7 membrane protein in two patients with high Na, low K erythrocytes.

We investigated the erythrocyte membrane proteins of two patients with congenital hemolytic anemia due to increased permeability of the erythrocyte membrane to Na and K (hereditary stomatocytosis and cryohydrocytosis). One-dimensional sodium dodecyl sulfate (SDS) gel electrophoresis resolved the band 7 erythrocyte membrane proteins into three components with approximate molecular weights of 30,000, 28,000, and 26,000. The 28,000-dalton component was decreased in both patients with permeability disorders. Two-dimensional electrophoresis (nonequilibrium pH gradient electrophoresis in the first dimension combined with SDS gel electrophoresis in the first dimension combined with SDS gel electrophoresis in the second dimension) resolved the 28,000-dalton component from normal erythrocyte membranes into two proteins with different isoelectric points, designated 22 x 8 and 60 x 8. In the patients with hereditary stomatocytosis and cryohydrocytosis, 22 x 8 was completely absent, whereas 60 x 8 was detected as usual. In contrast, all the band 7 proteins (including 22 x 8) were invariably present in a survey of normal subjects and reticulocytosis controls. The unique finding of a missing band 7 protein in the patients with hereditary stomatocytosis and cryohydrocytosis raises the possibility that the absence of this protein is responsible for the increased Na and K permeability in these disorders.

Modulation of erythrocyte membrane mechanical stability by 2,3-diphosphoglycerate in the neonatal poikilocytosis/elliptocytosis syndrome.

To explain the transient anemia and poikilocytosis seen during infancy in hereditary elliptocytosis (HE), we resealed erythrocyte (RBC) ghosts from affected children or their elliptocytic parents with 2,3-diphosphoglycerate (DPG) (0-8 mM), a compound that dissociates membrane skeletons, then measured ghost mechanical stability in the ektacytometer. Without added 2,3-DPG, ghost mechanical stability was subnormal in infantile poikilocytosis (IP) and HE but was even more abnormal in hereditary pyropoikilocytosis (HPP). Addition of 2,3-DPG (2.55 mM) to IP or HE ghosts, decreased their stability to that of HPP ghosts (without 2,3-DPG). Nonphysiological 2,3-DPG levels (6-8 mM) were required to elicit a similar effect in normal ghosts. The data suggest that free 2,3-DPG, present in neonatal RBC as a consequence of diminished binding to HbF, may render HE susceptible to in vivo fragmentation. The developmental switch from fetal to adult hemoglobin, by diminishing available free 2,3-DPG, may explain the abatement of poikilocytosis and hemolytic anemia that accompanies maturation.

Unique alpha-spectrin mutant in a kindred with common hereditary elliptocytosis.

We report here a unique variant of alpha spectrin in a kindred with hereditary elliptocytosis. This novel red blood cell-membrane protein migrated to a position between the normal alpha- and beta-spectrin subunits in SDS polyacrylamide gel electrophoresis. It was identified as an alpha spectrin by its binding to anti-alpha spectrin antibodies, by the absence of a phosphorylation site, and by the normal 1:1 stoichiometry between total alpha- and beta-spectrin molecules. The quantity of the alpha-spectrin mutant, expressed as a percentage of the total alpha spectrin, varied from 9.9-45.2% among six affected individuals. Two-dimensional electrophoretic analysis of spectrin tryptic digests was qualitatively normal but showed a decreased quantity of a normal alpha IV fragment. The variable quantity of alpha-spectrin mutant among family members correlated directly with the increased percentage of spectrin dimers in cold low ionic strength spectrin extracts (r = 0.92) and inversely with red blood cell ghost mechanical stability (r = -0.98). The data suggest that this new alpha-spectrin mutant is responsible for decreased spectrin dimer-dimer association and for red cell instability in affected individuals.

Modification of hemoglobin H disease by sickle trait.

The rarity of hemoglobin (Hb) H disease in combination with sickle trait may be due in part to the absence of actual Hb H in individuals who, nonetheless, have inherited the deletion of three alpha-globin genes. We describe here a boy with persistent microcytic, hypochromic anemia despite adequate iron stores, who exhibited splenomegaly with a normal reticulocyte count and only rare inclusions in circulating erythrocytes. Starch gel electrophoresis and isoelectric focusing at age 5 yr showed 21% Hb S, persistent Hb Bart's, but no Hb H. Recticulocyte alpha/non-alpha globin chain synthesis ratio was 0.58 at age 5. The mother (Asian) had laboratory evidence of alpha-thalassemia trait and the father (Black) had sickle trait. The nature of alpha-thalassemia in this patient was investigated both by liquid hybridization and by the Southern method of gene mapping, in which DNA is digested with restriction endonucleases and the DNA fragments that contained the alpha-globin structural gene identified by hybridization with complementary DNA. The patient had only one alpha-globin structural gene, located in a DNA fragment shorter than that found in normal or alpha-thalassemia trait individuals, but similar to that present in other patients with Hb H disease. Morphologic studies of bone marrow by light and electron microscopy revealed erythroid hyperplasia with inclusions in polychromatic and orthochromatic erythroblasts, suggesting early precipitation of an unstable hemoglobin. The lack of demonstrable Hb H may be the result of both diminished amounts of beta(A) available for Hb H formation (since one beta-globin gene is beta(S)) and the greater affinity of alpha-chains for beta(A) than beta(S)-globin chains leading to the formation of relatively more Hb A than Hb S. The presence of a beta(S) gene may thus modify the usual clinical expression of Hb H disease.

Selective reticulocyte destruction in erythrocyte pyruvate kinase deficiency.

Radioisotope studies of bilirubin turnover, ferrokinetics, and red cell survival ((51)Cr) in a patient with erythrocyte PK deficiency have provided evidence for prompt reticulocyte sequestration and destruction by the reticuloendothelial system. More mature erythrocytes appeared to survive well despite their deficiency of PK. PK-deficient reticulocytes, dependent upon oxidative phosphorylation for ATP production, are exquisitely sensitive to cyanide- or nitrogen-induced mitochondrial inhibition. If oxidative phosphorylation is unavailable, ATP levels decline rapidly, producing alterations in the cell membrane which allow massive losses of potassium and water. The result is a shrunken, spiculated, viscous cell whose rheologic properties would favor its sequestration by the reticuloendothelial system. Those reticulocytes with particularly low levels of PK exhibit very low glycolytic rates and thus are uniquely reliant upon oxidative phosphorylation. Other reticulocytes, better endowed with PK activity, can meet the increased ATP requirements of young erythrocytes. Upon reaching maturity, such cells have diminished ATP needs and can, therefore, survive despite their enzyme deficiency.

Molecular and functional changes in spectrin from patients with hereditary pyropoikilocytosis.

The structural and functional properties of spectrin from normal and hereditary pyropoikilocytosis (HPP) donors from the two unrelated families were studied. The structural domains of the spectrin molecule were generated by mild tryptic digestion and analyzed by two-dimensional electrophoresis (isoelectric focusing; sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The alpha I-T80 peptide (Mr 80,000) is not detectable in two related HPP donors; instead, two new peptides (Mr 50,000 and 21,000) are generated and have been identified as fragments of the normal alpha I-T80. A third sibling has reduced levels of both the normal alpha I-T80 and the two new peptides. A similar analysis of spectrin from another HPP family indicates that their spectrins contain reduced amounts of the alpha I-T80 and the 50,000 and 21,000 fragments of the alpha I domain. The HPP donor also has other structural variations in the alpha I, alpha II, and alpha III domains. The alpha I-T80 domain of normal spectrin has been shown to be an important site for spectrin oligomerization (J. Morrow and V.T. Marchesi. 1981. J. Cell Biol. 88: 463-468), and in vitro assays indicate that HPP spectrin has an impaired ability to oligomerize. Ghost membranes from HPP donors are also more fragile than membranes from normal erythrocytes when measured by ektacytometry. In both the oligomerization and fragility assays, the degree of impairment is correlated with the amount of normal alpha I-T80 present in the spectrin molecule. We believe that a structural alteration in the alpha I-T80 domain perturbs normal in vivo oligomerization of spectrin, producing a marked decrease in erythrocyte stability.

Two-dimensional electrophoretic analysis of human erythrocyte cylindrin.

Cylindrin, a macromolecule isolated from the human erythrocyte, and the band 7 proteins of the erythrocyte membrane were analyzed by one- and two-dimensional electrophoresis. Cylindrin was recovered from both the cytosol and cell membranes of hypotonically lysed erythrocytes, and its identity was confirmed by electrophoresis and transmission electron microscopy. Cylindrin from either source produced eight bands on one-dimensional SDS gels, and seventeen spots on two-dimensional gels, revealing a more complex composition than previously reported. It is unlikely that this complexity was due to proteolysis, since preparations of cylindrin with various protease inhibitors gave the same electrophoretic patterns. Mixing experiments showed that the polypeptide subunits of the cylindrin complex are distinct from the band 7 proteins of the erythrocyte membrane. This finding failed to support a role for the cylindrin macromolecule in the permeability disorders of the erythrocyte membrane associated with a missing band 7 protein.

Recombinant human erythropoietin stimulates erythropoiesis and reduces erythrocyte transfusions in very low birth weight preterm infants.

DESIGN AND METHODS: We hypothesized that treatment with recombinant human erythropoietin (r-HuEPO) would stimulate erythropoiesis and would thereby reduce the need for erythrocyte transfusions in preterm infants. We treated 157 preterm infants born at 26.9 +/- 1.6 weeks of gestation who weighed 924 +/- 183 g at birth with either subcutaneous r-HuEPO (100 U/kg/d, 5 days per week) or placebo for 6 weeks in a randomized, double-blind, controlled clinical trial. All patients received oral iron and were managed according to uniform conservative transfusion guidelines. RESULTS: Treatment with r-HuEPO was associated with fewer erythrocyte transfusions (1.1 +/- 1.5 per infant in the r-HuEPO group versus 1.6 +/- 1.7 per infant in the placebo group; P = .046) and with a reduction in the volume of packed erythrocytes transfused (16.5 +/- 23.0 mL versus 23.9 +/- 25.7 mL per infant; P = .023). Overall, 43% of the infants in the r-HuEPO group and 31% of placebo-treated infants were transfusion-free during the study (P = .18). The volume of blood removed for laboratory tests and the need for respiratory support at the start of treatment had major effects on transfusion requirements independent of r-HuEPO. Reticulocyte counts were higher during treatment in the r-HuEPO group (P = .0001), and r-HuEPO-treated infants had higher hematocrit values at the end of the study (32% versus 27.3% in the placebo group; P = .0001). We found no differences in the incidence of major complications of prematurity between the treatment groups. CONCLUSION: We conclude that treatment with r-HuEPO at a weekly dose of 500 U/kg stimulates erythropoiesis, moderates the course of anemia, is associated with a reduction in erythrocyte transfusions, and appears safe in very low birth weight preterm infants who are receiving iron supplements. Conservative transfusion criteria, minimization of phlebotomy losses, and treatment with r-HuEPO are complementary strategies to reduce erythrocyte transfusions in these infants.

Low number of antibody producing cells in patients with sickle cell anemia.

B cell function is impaired in patients with sickle cell anemia. Although the number of surface IgM positive cells was similar in sickle cell patients and controls, in vitro spontaneous IgM, and PWM stimulated IgA, IgM, and IgG synthesis was significantly lower in the patients than in controls. The number of PWM induced and antigen specific immunoglobulin producing cells after immunization with Pneumovax, containing 21 serotypes of Streptococcus pneumoniae, was about 100-fold lower in the patients as compared with controls. Finally, the ability of the patients' peripheral blood mononuclear cells to proliferate in response to mitogens (PWM, SAC, PHA) was diminished. Because of the observed impairments in both nonspecific and antigen specific immunoglobulin synthesis and cell proliferation assays in the patients, we determined serum concentrations of IL-4 and IL-6, two cytokines associated with antibody production. IL-4 concentrations appeared low in sickle cell patients, and correlated with that of serum IgM. We hypothesize that B cell maturation in sickle cell patients is arrested at an IL-4 dependent stage.

Total body irradiation and bone marrow transplantation for immunodeficiency disorders in young children.

Congenital immunodeficiency disorders such as severe combined immunodeficiency disease (SCID), Wiskott-Aldrich syndrome, and Chediak-Hegashi syndrome are almost uniformly fatal with most children dying before age one. Allogeneic bone marrow transplant (BMT) is the treatment of choice. Few of these children have matched donors. We use bone marrow processing techniques that allow us to utilize marrow from the parents. Children who lack HLA-identical donors are offered haploidentical, T-cell depleted parental BMTs. Some of these children do not have an immune deficiency severe enough to allow durable engraftment of processed mismatched bone marrow. Successful engraftment may necessitate the use of immunosuppression. Total body irradiation (TBI) is part of our intensive conditioning regimen for children with Wiskott-Aldrich and Chediak-Hegashi syndrome and most children with SCID who have undergone an unsuccessful prior mismatched, T-cell depleted BMT, or who have a high likelihood of donor marrow rejection based on pre-transplant immune function testing. TBI is considered extremely toxic therapy in infancy, with little information available on the acute and chronic effects. The 10 children presented in this report are among the youngest to have received TBI. Five patients were 2 to 6 months of age when they received TBI. The conditioning regimen for all patients was; antithymocyte globulin (25 mg/kg/day, x 3 days), cyclophosphamide (60 mg/kg/day, x 2 days), and TBI. 7.0 Gy TBI was given as a single dose AP-PA at approximately 15 cGy/min. Half value blocks shielded the brain, eyes and lungs. Six of 10 children were alive from 7 to 72 months post transplant.(ABSTRACT TRUNCATED AT 250 WORDS)

Prenatal diagnosis of chronic granulomatous disease.

A luminol enhanced chemiluminescence micromethod has been adapted for use in prenatal diagnosis of chronic granulomatous disease (CGD). After validation of the assay in normal adults, newborns, fetuses, CGD carriers, and CGD patients, the fetuses of two pregnant CGD carriers were tested after fetoscopic aspiration of fetal blood. Normal neutrophil chemiluminescence and nitroblue tetrazolium slide tests were followed by delivery of two healthy infants whose normality was confirmed. Amniocytes proved useless for the prenatal diagnosis of CGD. They were found to have negligible nitroblue tetrazolium reduction, oxygen metabolism, and oxygen dependence.

Prenatal diagnosis of unusual hemoglobinopathies.

While analyzing 280 hemoglobinopathy kindreds with prescribed molecular tests, 3 unusual mutations were observed that required additional characterization. In the first case, the hypervariable region flanking the alpha-globin genes generated an intermediate length 8.2 kb psi zeta-globin gene fragment on a Southeast Asian chromosome with two deleted alpha-globin genes. Rehybridization of the Southern blot with alpha-globin probe distinguished the mutation unambiguously. In the second case, restriction enzyme analysis of a PCR amplified black beta-globin gene detected a novel beta-83 point mutation adjacent to a promoter element. In the third case, which was uninformative with available allele specific oligonucleotides (ASOs), total genomic PCR amplification and sequencing identified a single basepair insertion in codon 36/37 of an Iranian beta-globin gene that shifted the reading frame and obliterated gene activity. Developing additional region-specific ASOs will further diminish the number of cases that must be characterized by genomic PCR sequencing.

AUR memorial Award. Induced alignment of flowing sickle erythrocytes in a magnetic field. A preliminary report.

Deoxygenated sickle erythrocytes in static suspension align perpendicular to a magnetic field. To assess the importance of this observation to MRI of sickle-cell disease, an in vitro flow apparatus was devised and the orientation of sickle erythrocytes flowing through a 0.38 T magnetic field was investigated. We showed a significant perpendicular alignment of fully deoxygenated sickle erythrocytes flowing at 3 to 4 mm/minute (P less than .001). These results suggest that deoxygenated erythrocytes in a sickle-cell patient could orient perpendicular to a magnetic field, and therefore that MRI of such patients could possibly result in worsening of vaso-occlusive complications. Further studies are needed to assess the possible hazards of MRI of sickle-cell disease, especially at high field strengths.

Elevated resting energy expenditure in adolescents with sickle cell anemia.

OBJECTIVE: To assess the reliability of standard prediction equations in estimating resting energy expenditure (REE) values in adolescents with sickle cell anemia. SUBJECTS/DESIGN: Body composition and metabolic measurements were performed in 8 adolescents, aged 11 to 18 years, with homozygous sickle cell anemia. REE was measured by indirect calorimetry under standard conditions, and measurements were compared with 4 prediction formulas (Harris-Benedict, Schofield, Mayo Clinic, and Food and Agriculture Organization/World Health Organization/United Nations University). Fat-free mass was measured to assess REE per unit of actively metabolizing tissue. Fat-free mass was expressed as a mean of values obtained by densitometry, deuterium dilution, 40K-counting, and total body electrical conductivity. STATISTICAL ANALYSES: Repeated measures analysis of variance was performed to determine whether measured REE values and predicted values differed. The Fischer test was used to identify which predicted values differed significantly from the measured REE. RESULTS: All 4 prediction formulas significantly underestimated REE. Group mean values for the prediction formulas ranged from 83% to 89% of the measured value. REE averaged 47.7 +/- 10.0 kcal/kg fat-free mass per day, which is 30% to 50% higher than reported values in healthy adolescent populations. CONCLUSIONS: These data suggest that REE is elevated in adolescents with sickle cell anemia. Standard equations used to predict REE are unreliable in these patients. APPLICATIONS: REE in patients with sickle cell anemia is best determined by indirect or direct measurement of energy expenditure. Clinically useful formulas to estimate REE should be developed for patients with conditions, including sickle cell anemia, where the metabolic rate may be altered.

Nontraumatic fat embolism syndrome in sickle cell anemia.

A 14-year-old girl with sickle cell disease and nephrotic syndrome developed bone pain, followed by pulmonary edema, seizures, coma, and bilateral flaccid paralysis. Fat embolism syndrome was diagnosed by cranial magnetic resonance imaging and an exchange transfusion was performed. Within 3 months, all symptoms had resolved. It is concluded that fat embolism syndrome must be considered as a possible cause of acute neurologic deterioration in patients with sickle cell anemia.