Prolonged administration of granulocyte colony-stimulating factor (filgrastim) to patients with Fanconi anemia: a pilot study

This report examines the effect of filgrastim (granulocyte colony- stimulating factor, [G-CSF] in 12 patients with neutropenia [absolute neutrophil count [ANC] < 1,000/mm3]) caused by Fanconi anemia (FA). Two of 14 patients who were evaluated for study entry were ineligible because of unsuspected cytogenetic abnormalities in their bone marrow (BM). G-CSF was started at 5 micrograms/kg/d. All patients had an increase in their ANC at week 8 (mean increase = 15,664/mm3). The median ANC during therapy was 5,030/mm3. Eight of 10 patients who completed 40 weeks on study maintained an ANC > 1,500/mm3 on G-CSF given every-otherday. Four patients had an increase in their platelet count by week 8 without transfusion (maximum increase = 23,000 to 45,000/mm3); however, platelet counts fell toward baseline levels as the G-CSF dose was reduced. BM CFU-MK were increased at week 8 in three of four evaluable patients. Four patients who did not receive red blood cell transfusions had an increase in their hemoglobin level of at least 2.0 g/dL. A fifth patient had a red blood cell transfusion in week 2 and then had a similar increase in hemoglobin level without subsequent transfusion. Eight of 10 patients who completed 40 weeks of treatment showed increases in the percentage of BM CD34+ cells measured by flow cytometry. The same proportion showed increases in peripheral blood CD34+ cells. Increased BM cellularity and myeloid hyperplasia were constant findings and were associated with increased expression of the proliferating cell nuclear antigen. Adverse experiences were mild fever (1 patient) and a new BM cytogenetic abnormality at week 40 (1 patient). This study shows that prolonged administration of G-CSF exerts a stimulatory effect on the BM of FA patients and may be used to maintain a clinically adequate ANC in these patients. G-CSF has beneficial effects on multiple hematopoietic lineages in some patients and may be a good candidate for use in combination cytokine protocols for FA patients with progressive aplastic anemia. G-CSF use results in an increase in circulating CD34+ cells, a finding with important implications for future gene transfer protocols.     

An essential role for lysophosphatidylcholine in the inhibition of platelet aggregation by secretory phospholipase A2

The release of secretory phospholipase A2 (sPLA2) into the mammalian circulation may contribute to the development of hemorrhagic and inflammatory diseases. sPLA2 has previously been shown to alter the behavior of platelets, leukocytes, and endothelial cells, although the molecular basis for these cellular effects has not been established. Our studies indicate that the inhibition of platelet aggregation by snake, bee venom, and pancreatic sPLA2 is dependent on a plasma cofactor. This cofactor resides within the lipoprotein fraction of plasma, with 54%, 31%, and 11% of the activity present in the high- density lipoprotein (HDL), low-density lipoprotein (LDL), and very low density lipoprotein (VLDL) fractions, respectively. Delipidation of HDL and LDL was associated with the complete loss of platelet-inhibitory activity. Incubation of purified sPLA2 with the HDL fraction of plasma resulted in the time-dependent generation of lysophosphatidylcholine (lysoPC). The formation of lysoPC correlated with the inhibition of platelet aggregation. Purified lysoPC (10 to 100 micrograms/mL) inhibited platelet aggregation and dense granule release induced by thrombin (0.05 U/mL), collagen (1 micrograms/mL), ionophore A23187 (2 mumol/L), ADP (12.5 mumol/L), and adrenaline (3.2 mumol/L). The inhibition of platelet aggregation by lysoPC was dose-dependent and correlated with decreased fibrinogen binding to glycoprotein IIb-IIIa. Our studies indicate that the enzymatic generation of lysoPC from plasma lipoproteins is essential for the sPLA2-mediated inhibition of platelet activation in the presence of albumin. These results raise the possibility that the toxic effects of circulating sPLA2 may be due in part to the generation of the bioactive lysophospholipid, lysoPC.     

Combinations of recombinant colony-stimulating factors are required for optimal hematopoietic differentiation in serum-deprived culture

Previous in vitro investigations on enriched human hematopoietic progenitors have led to the conclusion that the purified recombinant multipoietins, interleukin 3 (IL-3) and granulocyte-macrophage colony- stimulating factor (GM-CSF) can alone induce the formation of colonies from a variety of multipotent and lineage committed progenitors. Since fetal calf serum was included in these cultures and itself might contain growth factors or other cofactors, we re-examined the actions of the CSFs in serum-deprived conditions. Results show that both the multipoietins are inadequate stimuli of colony formation. At maximal concentrations IL-3 alone induces only 25% of the granulocyte and macrophage colony-forming units (CFU-G and CFU-M) produced by a T-cell conditioned medium that contains a mixture of CSFs. When IL-3 was added at the initiation of the cultures and erythropoietin (ep), G-CSF, or M- CSF added on day 3, almost full recovery of erythroid, granulocytic, and monocytic colonies, respectively, was obtained. Similar results were obtained with GM-CSF except that fewer erythroid colonies were recovered at high concentrations, and almost maximal CFU-M proliferation could be induced. These results show that in serum- deprived conditions, the multipoietins must be combined with lineage specific CSFs for full progenitor expression.     

Enzyme histochemistry and immunohistochemistry on biopsy specimens of pathologic human bone marrow

We have systematically investigated a variety of fixation and plastic embedding procedures and arrived at a method that allows processing of approximately 2-micron sections of bone marrow biopsies for examination by light microscopy. More importantly, this method permits the use of enzyme histochemical and immunohistochemical procedures that are rapidly becoming mandatory in the diagnosis of hematologic malignancies. Over 200 full-length bone marrow biopsy specimens were fixed in a mixture of paraformaldehyde, glutaraldehyde, and acrolein, dehydrated in acetone, and embedded in a mixture of methyl and glycolmethacrylate. All procedures were carried out at 4 degrees C. Decalcification was unnecessary. Sections 2-micron thick were cut and incubated for peroxidase, naphthol AS-D chloroacetate esterase, alpha- naphthyl butyrate esterase, acid phosphatase (with and without tartrate), or alkaline phosphatase and then examined by light microscopy. Specimens could be prepared for examination within 48 hr. This approach, which provides definitive markers for various hematopoietic cell lines in intact tissues, is invaluable when aspirated material is unavailable. It is also useful in the analysis of focal lesions of bone marrow due to inflammation or neoplasia and shows potential as an investigative tool. For example, we have discovered that early myelofibrosis is accompanied by a marked increase in the number of alkaline-phosphatase-positive reticulum cells.     

Regulation of early human hematopoietic (BFU-E and CFU-GEMM) progenitor cells in vitro by interleukin 1-induced fibroblast-conditioned medium

Stimulators of human erythroid burst-forming units (BFU-E) and multipotential colony-forming cells (CFU-GEMM) can be produced by a number of different cell types. A product of human peripheral blood monocytes, interleukin 1 (IL-1), was evaluated for its ability to stimulate fibroblast cultures to produce stimulators of human bone marrow BFU-E and CFU-GEMM colony formation. BFU-E and CFU-GEMM colony formation was evaluated using low-density, nonadherent low-density, and T lymphocyte-depleted nonadherent low-density human bone marrow cells cultured in the presence of a source of pure human erythropoietin. Both human monocyte conditioned medium (MCM) and human recombinant IL-1 (hrIL-1) induced fibroblasts to produce stimulators of BFU-E and CFU- GEMM in a dose-dependent fashion with maximal colony formation occurring when fibroblasts were stimulated by 25% MCM or 140 ng/mL ROO6B hrIL-1, or 1.25 to 5 ng/mL ROO6T hrIL-1. Preincubation of MCM and hrIL-1 with an antibody to IL-1 inactivated the ability of MCM and hrIL- 1 to induce the release of erythroid and multipotential colony stimulating activity from fibroblasts. These results suggest that monocyte-derived IL-1 is involved in regulating the production of humoral stimulators of early human hematopoietic progenitors.     

Shear-dependent changes in the three-dimensional structure of human von Willebrand factor

The three-dimensional tertiary structure of human von Willebrand Factor (vWF) on a hydrophobic surface under aqueous conditions and different shear stress regimes was studied by atomic force microscopy (AFM). vWF was imaged by AFM at molecular level resolution under negligible shear stress, under a local applied shear force (7.4 to 19 nN) using the AFM probe in contact mode scanning, and after subjecting vWF to a range of shear stress (0 to 42.4 dyn/cm2) using a rotating disk system. The results demonstrate that vWF undergoes a shear stress-induced conformational transition from a globular state to an extended chain conformation with exposure of intra-molecular globular domains at a critical shear stress of 35 +/- 3.5 dyn/cm2. The globular vWF conformation (149 nm by 77 nm and height 3.8 nm) is representative of native vWF after simple diffusion to the hydrophobic surface, followed by adhesion and some spreading. In a shear stress field above the critical value, protein unfolding occurs and vWF is observed in extended chain conformations oriented in the direction of the shear stress field with molecular lengths ranging from 146 to 774 nm and 3.4 nm mean height. The shear stress-induced structural changes to vWF suggest a close conformation-function relationship in vWF properties for thrombogenesis in regions of high shear stress.     

Evaluation of the iron status of a population

The iron status of a population of 1564 subjects living in the northwestern United States was evaluated by measurements of transferrin saturation, red cell protoporphyrin, and serum ferritin. The frequency distribution of these parameters showed no distinct separation between normal and iron-deficient subjects. When only one of these three parameters was abnormal (transferrin saturation below 15%, red cell protoporphyrin above 100 mug/ml packed red blood cells, serum ferritin below 12 ng/ml), the prevalence of anemia was only slightly greater (10.9%) than in the entire sample (8.3%). The prevalence of anemia was increased to 28% in individuals with two or more abnormal parameters, and to 63% when all three parameters were abnormal. As defined by the presence of at least two abnormal parameters, the prevalence of iron deficiency in various populations separated on the basis of age and sex ranged from 3% in adolescent and adult males to 20% in menstruating women. It is concluded that the accuracy of detecting iron deficiency in population surveys can be substantially improved by employing a battery of laboratory measurements of the iron status.     

Role of transferrin in determining internal iron distribution

The behavior in vivo of transferrin in loading and unloading iron from its two sites was examined in rats. Radioiron entering the plasma from the gastrointestinal tract in iron-deficient, normal and iron-loaded rats did not differ in its subsequent tissue distribution between erythroid marrow and liver of normal recipients from a second isotope added to the same plasma in vitro. Loading studies in vitro were then carried out employing a reticulocyte incubation model designed to place one isotope predominantly on one site of transferrin, more available to the erythron, and the second isotope on the other site, more available to the liver. In 15 groups of animals in which 3 different iron salts were employed to load transferrin with iron, the mean isotope ratio in the erythron was 1.03 (+/-0.06 SD) and the mean liver ratio was 0.75 (+/-0.21 SD). It was found that the incubation of plasma with reticulocytes resulted in contamination of the plasma by radioactive hemoglobin. After allowance was made for hepatic uptake of radiohemoglobin in the 13 groups in which proper correction could be made, the isotope ratio in the liver became 0.97 (+/-0.17 SD). It is concluded that iron atoms from the two sites of transferrin have similar tissue distributions in vivo in the experimental situations examined.     

Partially differentiated ex vivo expanded cells accelerate hematologic recovery in myeloablated mice transplanted with highly enriched long- term repopulating stem cells

The ability of an infusion of ex vivo expanded hematopoietic cells to ameliorate cytopenia following transplantation of hematopoietic stem cells (HSCs) is controversial. To address this issue, we measured the recovery of circulating leukocytes, erythrocytes, and platelets in lethally irradiated mice transplanted with 10(3) enriched HSCs, with or without their expanded equivalent (EE) generated after 7 days of culture in interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor and Steel Factor. Two HSC populations differing in their content of short-term repopulating progenitors were evaluated. Thy-1loLIN-Sca- 1+ (TLS) bone marrow (BM) is enriched in colony-forming cells (CFCs), day 8 and day 12 spleen colony-forming units (CFU-S) (435 +/- 19, 170 +/- 30, and 740 +/- 70 per 10(3) cells, respectively), and stem cells with competitive long-term repopulating potential (> or = 1 per 43 cells). Thy-1loSca-1+H-2Khl cells (TSHFU) isolated from BM 1 day after treatment of donor mice with 5-fluorouracil (5-FU) are also highly enriched in competitive repopulating units (CRU, > or = 1 per 55 cells), but are depleted of CFCs, day 8 and day 12 CFU-S (171 +/- 8, 0 and 15 +/- 4 per 10(3) cells, respectively). Recipients of 10(3) TLS cells transiently recovered leukocytes to > or = 2,000/microL in 12 days, but sustained engraftment required 25 days. Platelets recovered to > or = 200,000/microL in 15 days, and erythrocytes never decreased below 50% of normal. Mice transplanted with 10(3) TSHFU cells recovered leukocytes in 15 days, and platelets and erythrocytes in 18 days. Recipients of unseparated normal or 5-FU-treated BM cells (containing 10(3) TLS or TSHFU cells) recovered safe levels of blood cells in 9 to 12 days, suggesting that unseparated marrow contains early engrafting cells that were depleted by sorting. Upon ex vivo expansion, total cells, CFCs and day 12 CFU-S were amplified 2,062-,83- and 13-fold, respectively, from TLS cells; and 1,279-, 259- and 708-fold, respectively, from TSHFU cells. Expanded cells could regenerate the majority of lymphocytes and granulocytes in primary (17 weeks) and secondary (26 weeks) hosts and were only moderately impaired compared to fresh HSCs. The EE of TSHFU cells was more potent than that of TLS cells, suggesting that more highly enriched HSCs are more desirable starting populations for this application. When mice were transplanted with 10(3) TSHFU cells and their EE, the duration of thrombocytopenia was shortened from 18 to 12 days, and anemia was abolished. Leukocytes were also elevated on days 9 to 12, although sustained recovery was not accelerated. Anemia was also abrogated in recipients of 10(3) TLS cells and their EE. Early platelet counts were slightly higher than with TLS cells alone, but leukocyte recovery was not improved. These data confirm that TLS cells contribute to early and sustained hematopoiesis, and demonstrate a benefit of ex vivo expanded cells in accelerating engraftment of more primitive TSHFU stem cells depleted of progenitors.     

The p53 gene in pediatric therapy-related leukemia and myelodysplasia

We investigated the frequency of p53 mutations in 19 pediatric cases of therapy-related leukemia or myelodysplastic syndrome. Eleven children presented with acute myeloid leukemia, one with mixed-lineage leukemia, two with acute lymphoblastic leukemia, and five with myelodysplasia at times ranging from 11 months to 9 years after a primary cancer diagnosis. The primary cancers, which included 11 solid tumors and eight leukemias, were treated with various combinations of DNA topoisomerase II inhibitors, alkylating agents, or irradiation. Leukemic or myelodysplastic marrows were screened for possible mutations by single-strand conformation polymorphism (SSCP) analysis of p53 exons 4 to 8. The only observed mutation was an inherited 2- basepair deletion at codon 209 in exon 6 that would shift the open reading frame, create a premature termination codon, and foreshorten the resultant protein. Prior therapy in this patient included DNA topoisomerase II inhibitors, alkylating agents, and irradiation. The secondary leukemia presented as myelodysplasia with monosomies of chromosomes 5 and 7 and abnormalities of chromosome 17. Although the primary cancer was an embryonal rhabdomyosarcoma and there was a family history of cancer, the case did not fulfill the clinical criteria for Li-Fraumeni syndrome. This study suggests that germline p53 mutations may predispose some children to therapy-related leukemia and myelodysplasia, but that p53 mutations otherwise are infrequent in this setting.