An inherited defect of neutrophil motility and microfilamentous cytoskeleton associated with abnormalities in 47-Kd and 89-Kd proteins.

A 2-month-old male Tongan infant presented with fever, severe skin and mucosal infections, hepatosplenomegaly, thrombocytopenia, and normal neutrophil counts. While polymorphonuclear neutrophil (PMN) morphology was normal, several neutrophil motile functions were found to be altered in the patient. Furthermore, two siblings had died in infancy with a similar clinical picture, raising the possibility of an inherited neutrophil defect. Random migration and chemotaxis, assessed by the under agarose method, were profoundly impaired. Actin polymerization, as measured by flow cytometry of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phallacidin (NBD-phallacidin)-stained PMNs, showed lower basal F-actin and a 1.75-fold increase in response to 10(-7) mol/L formyl-methionyl-leucyl-phenylalanine (FMLP) compared with a 4.51-fold increase in control. Microscopic examination of NBD-phallacidin-stained PMN spread on glass showed decreased area of spreading and F-actin-rich filamentous projections distinct from control. The early phase of FMLP-induced right angle light scattering was absent, similar to the effect caused by cytochalasin-B (CB), an inhibitor of actin polymerization. Accordingly, FMLP induced secretion of elastase without the addition of CB. Staphylococcus aureus killing was 50% of control whereas superoxide production response to FMLP and surface expression of CD11b were greater than twice normal. Partial defects in actin polymerization and scatter were seen in the parents and release of elastase, in the absence of CB, was also increased in both parents. Sodium dodecyl sulfate-polyacrylamide electrophoresis of whole cell proteins from the patient showed a marked decrease in an 89-Kd protein (8% of control) and a marked increase in a 47-Kd protein (4.2-fold). Both mother and father had decreased 89-Kd (77% and 42% of control) and increased 47-Kd proteins (2- and 3.4-fold), although neither had recurrent infections or chemotactic defects. These studies describe a new inherited actin dysfunction syndrome associated with severe propensity to fungal infection and draw attention to the proteins of apparent molecular weights of 89 Kd and 47 Kd, which may be of great importance in the regulation of actin polymerization in human PMNs.

Inhibition of hematopoietic progenitor colony growth by a monoclonal antibody against the transferrin receptor: comparison of unconjugated antibody with an immunotoxin containing recombinant ricin A chain.

We studied an immunotoxin consisting of recombinant ricin A chain (rRA) conjugated to 454A12 MoAb, a monoclonal antibody which recognizes an epitope on the human transferrin receptor, and compared the ability of 454A12 MoAb-rRA immunotoxin to inhibit the growth of erythroid burst-forming units (BFU-e) and myeloid colony-forming units (CFU-c) with unconjugated 454A12 MoAb. A significant reduction in BFU-e colony growth was observed at 0.001 microgram/ml of 454A12 MoAb-rRA versus 0.1 microgram/ml of unconjugated 454A12 MoAb (p = 0.005). Comparison of the effects of 454A12 MoAb-rRA and 454A12 MoAb on myeloid colony development gave markedly different results. Unconjugated antibody had no effect on CFU-c colony growth; in contrast, 0.01 microgram/ml of 454A12 MoAb-rRA reduced the number of colonies from 139 per 1 X 10(5) to 75 per 1 X 10(5) cells plated (p = 0.0005). No myeloid progenitor colonies developed at 0.1 microgram/ml of immunotoxin. These observations suggest that 454A12 MoAb-rRA inhibits growth by a potent, ricin A chain-mediated toxic effect on any proliferating cells expressing transferrin receptors, whereas the 454A12 MoAb exerts a selective inhibitory effect primarily on erythroid progenitors by perturbing the transferrin cycle. While growth factor receptors expressed on hematopoietic cells represent promising targets for immunotoxin therapy, our data indicate that an immunotoxin could inhibit cellular proliferation by a different mechanism than the corresponding unconjugated MoAb. Depending on the antibody used, these differences may be important in trials using immunotoxins for in vivo treatment or in vitro purging of malignant hematopoietic cells.

Spontaneous resolution of primary erythrocytosis in two girls.

We report two girls with primary erythrocytosis in whom extensive diagnostic studies revealed no underlying cause. Normal growth of colonies derived from erythroid burst forming units (BFU-E) was observed, and serum erythropoietin concentrations were within or below the normal range. The absence of a rise in serum erythropoietin levels after isovolemic phlebotomy implicated the erythroid marrow as the site of the pathophysiologic abnormality in both patients. Spontaneous resolution of erythrocytosis occurred during the second decade of life. Our experience suggests that primary erythrocytosis may be self-limited in some children. In these cases, the proliferative abnormality may be sufficiently subtle as to not be detected by standard in vitro culture systems, which support the growth of colonies derived from erythroid progenitors.

Circulating erythroid progenitors in the anemia of prematurity.

We studied erythropoiesis in infants with the anemia of prematurity by counting the number of colonies derived from erythroid burst-forming units (BFU-E) in the blood of 11 premature infants before they received transfusions. Colony growth in blood from the infants was compared with growth in blood from adults and umbilical-cord blood from term infants, in the presence of erythropoietin, 0 to 2000 mU per milliliter. Addition of increasing concentrations of erythropoietin resulted in a stepwise increase in the number of colonies derived from BFU-E (P less than 0.0005) of all three groups of subjects. Cultures stimulated with 2000 mU of erythropoietin yielded 28.1 +/- 7.6, 88.0 +/- 19.4, and 121.0 +/- 22.5 bursts (mean +/- SE) per 10(5) cells plated in blood from adults, blood from premature infants, and cord blood, respectively. Although more BFU-E-derived colonies appeared when 200 or 2000 mU were present per milliliter in cultures of the infants' blood and cord blood, the intrinsic responsiveness of BFU-E to erythropoietin was similar in all groups. Although the mean hematocrit was 26 percent, mean serum erythropoietin concentrations (+/- SD) in the infants (20.7 +/- 10.0 mU per milliliter) were not significantly different from those in the adult controls (24.0 +/- 6.5). We conclude that progenitor cells committed to erythroid differentiation are present during the anemia of prematurity, and that the intrinsic responsiveness of the circulating BFU-E pool to erythropoietin is normal. These results implicate inadequate production of erythropoietin as the cause of the anemia of prematurity and suggest that recombinant erythropoietin might provide a therapeutic alternative to transfusion for symptomatic babies with this condition.