losartan-potassium and Fanconi-Anemia

The clinical availability of recombinant hematopoietic growth factors was initially thought to be breakthrough in the treatment of bone marrow failure syndromes. However, in most disorders of hematopoeisis, the clinical use was rather disappointing. Only in congenital neutropenias (CNs) has the long-term administration of granulocyte colony-stimulating factor (G-CSF) led to a maintained increase in absolute neutrophil count (ANC) and a reduction of severe bacterial infections. In other disorders of hematopoiesis, the use of lineage-specific growth factors is either not possible due to mutations in the growth factor receptor or leads to a transient benefit only. Initial clinical trials with multilineage hematopoietic growth factors, such as stem cell factor (SCF; c-kit ligand) were discontinued due to adverse events. It is well known that bone marrow failure syndromes are pre-leukemic disorders. So far, there is no evidence for induction of leukemia by hematopoietic growth factors. However, it has been shown in patients with CN and Fanconi anemia that hematopoietic growth factors might induce preferential outgrowth of already transformed cells. Thus, it is strongly recommended to monitor patients for clonal aberrations prior to and during long-term treatment with hematopoietic growth factors.

Topics: Anemia; Anemia, Diamond-Blackfan; Anemia, Dyserythropoietic, Congenital; Erythropoietin; Fanconi Anemia; Granulocyte Colony-Stimulating Factor; Hematopoietic Cell Growth Factors; Hematopoietic Stem Cell Transplantation; Humans; Neutropenia; Stem Cell Factor; Thrombocytopenia

The production of erythroid cells is a dynamic and exquisitely regulated process. The mature red cell is only the final phase of a complex but orderly series of genetic events that are initiated at the time a multipotent stem cell becomes committed to expressing the erythroid programme. Aberrations either in the intrinsic generation and/or amplification of functional erythroid cells or in the regulatory influences of microenvironment or cytokines form the basis for a number of blood diseases. In this review we focus upon abnormalities in red blood cell production and discuss how alterations in cytokine regulation of red blood cell production may contribute to these disease processes. We discuss clinical states in which blood red cell numbers are altered, including primary familial and congenital polycythaemia, the myeloproliferative disorder polycythaemia vera, erythroleukaemia, and Diamond-Blackfan anaemia. These disorders are briefly described and evidence supporting a potential role of specific cytokine receptor signalling defects as contributing to these phenotypes is discussed.

Topics: Erythropoietin; Fanconi Anemia; Hematologic Diseases; Hematopoiesis; Humans; Leukemia, Erythroblastic, Acute; Mutation; Polycythemia; Polycythemia Vera; Receptors, Cytokine; Receptors, Erythropoietin; Signal Transduction

Diamond-Blackfan anemia (DBA) is pleiotropic clinically and in vitro, and there is a strong suspicion that DBA is really a family of diseases that shares a common hematological phenotype. Although standard clonogenic assays of erythroid progenitors have been very informative about pathogenesis, they are not diagnostic of DBA, have no relationship to the clinical presentation and do not relate to the hemoglobin level or to the percentage of marrow erythroids at the time of study. Studies on progenitor-enriched marrow cells have furthered our understanding of DBA and have clearly shown marked differences among patients with respect to erythropoietin and "burst-promoting activity" responsiveness. In vitro addition of corticosteroids, interleukin 3 (IL-3) and/or Steel factor has produced a corrective effect on erythropoiesis in some DBA patients and has prompted clinical trials with IL-3 with variable results. It is clear that there is a disparity between the vitro data and clinical outcome, and therefore, the erythroid progenitor responsiveness to steroids and cytokines has limited predictive value clinically. Based on more than two decades of study, a model of DBA has evolved based on putative blocks at various stages along the erythropoietic differentiation pathway. These blocks likely represent a disorder of receptor-ligand interaction involving one or more growth-promoting cytokines.

Topics: Cell Differentiation; Cell Separation; Colony-Forming Units Assay; Drug Synergism; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Fanconi Anemia; Granulocyte-Macrophage Colony-Stimulating Factor; Hematopoietic Cell Growth Factors; Humans; Immunologic Factors; Interleukin-3; Recombinant Proteins; Stem Cell Factor

Recombinant cytokines were used to investigate the pathophysiology of Diamond-Blackfan anemia (DBA) and to treat patients with Fanconi's anemia (FA) and amegakaryocytic thrombocytopenic purpura (AMT). We compared the erythroid burst forming units (BFU-E) colony growth of six DBA patients with four normal controls. BFU-E showed erythropoietin (Epo) dose dependence in all patients, although colony numbers were reduced in comparison with normals. The number and size of BFU-E were increased with the addition to Epo of interleukin 3 (IL-3) or Steel factor (SF), but IL-3 + SF was not synergistic. SF increased the nonadherent cell production in DBA long-term bone marrow cultures, and stromal cells from DBA patients showed normal SF mRNA transcripts. These data suggest that SF is not involved in the pathogenesis of DBA, although it may be useful in treatment. A small group of patients with FA and bone marrow failure were treated with daily s.c. granulocyte-macrophage colony stimulating factor (GM-CSF). Toxicity was minimal, and the majority of the patients responded with significant, sustained increase in neutrophils. Multilineage response was rare. GM-CSF may thus be palliative in patients with FA. Five patients with AMT were treated with IL-3 or IL-3 followed by GM-CSF in a phase I/II study. There was minimal toxicity, and IL-3, but not GM-CSF, resulted in improved platelet counts in two patients and decreased platelet transfusion requirements in the other three. Prolonged IL-3 treatment resulted in platelet increases in two of the latter patients. Thus, IL-3 may contribute to the treatment of patients with AMT.

Topics: Adult; Bone Marrow; Cells, Cultured; Child, Preschool; Drug Synergism; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Fanconi Anemia; Granulocyte-Macrophage Colony-Stimulating Factor; Hematopoietic Cell Growth Factors; Humans; Immunologic Factors; Infant; Interleukin-3; Leukocyte Count; Platelet Count; Purpura, Thrombocytopenic; Recombinant Proteins; Stem Cell Factor; Treatment Outcome

This report describes the response of 18 Diamond-Blackfan anemia (DBA) patients to recombinant human interleukin 3 (rhIL-3). rhIL-3 was administered s.c. once daily on an escalating dose schedule (0.5-10 micrograms/kg/day). The rhIL-3 dose was escalated every 21 days until erythroid response was attained, grade III or IV nonhematologic toxicity was observed, or the maximal rhIL-3 dose was reached. Four patients experienced clinically significant erythroid responses. Two of the responders were steroid-dependent and transfusion-independent, while two were steroid-independent and transfusion-dependent. Baseline clinical or laboratory parameters, in particular in vitro bone marrow erythroid progenitor assays, were not useful in predicting rhIL-3 response. Two of the responding patients remain on maintenance rhIL-3 without diminution of effect at 490 and 855+ days. rhIL-3 was discontinued in the other two responders because of the development of deep venous thrombi.

Topics: Animals; Blood Cell Count; Child; Eosinophilia; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Fanconi Anemia; Female; Hematopoietic Cell Growth Factors; Humans; Hypotension; Immunologic Factors; Interleukin-3; Male; Mice; Recombinant Proteins; Stem Cell Factor; Thrombophlebitis; Treatment Outcome

Graft failure following allogeneic HCT in Fanconi anemia is associated with significant mortality. Retransplantation may be considered; however, the limited toxicity profile of HGFs also makes them an option for the treatment of graft failure. We describe a five-yr-old female diagnosed with Fanconi anemia and marrow failure treated with HCT. The course was complicated by secondary graft failure treated successfully with HGFs including G-CSF, EPO, and romiplostim. The outcome could be related to the intervention, but could also be the natural course of recovery, including recovering from a recent CMV infection treated with ganciclovir. We found the use of HGFs to be an effective and safe alternative to the potential complications as well as morbidity and mortality associated with the use of retransplantation.

Topics: Child, Preschool; Cord Blood Stem Cell Transplantation; Drug Therapy, Combination; Erythropoietin; Fanconi Anemia; Female; Graft Rejection; Granulocyte Colony-Stimulating Factor; Hematologic Agents; Humans; Receptors, Fc; Recombinant Fusion Proteins; Thrombopoietin; Transplantation, Homologous

Transient treatment with cytokines appears to improve hematopoietic function in Fanconi anemia; however, the effectiveness or adverse effect of long-term treatment is not known. The mitomycin C-treated Fancc(-/-) mouse provides a valuable model to address long-term efficacy of such treatment. Fancc(-/-) mice injected with granulocyte colony-stimulating factor, erythropoietin, or both cytokines showed a delay in mitomycin C (MMC)-induced bone marrow (BM) failure compared to untreated mice. However, long-term cytokine exposure followed by MMC challenges did not protect mice from the reduction of peripheral blood counts or the number of early myeloid progenitors. These results suggest that cytokine treatment may be beneficial only in the short-term, while long-term treatment is not protective for BM aplasia.

Topics: Animals; Bone Marrow; Erythrocyte Count; Erythropoietin; Fanconi Anemia; Granulocyte Colony-Stimulating Factor; Hematopoiesis; Hematopoietic Stem Cells; Leukocyte Count; Mice; Mice, Inbred BALB C; Mice, Knockout; Mitomycin; Pancytopenia; Survival Rate

We have characterized the proliferation kinetics of hematopoietic cells in long-term marrow cultures (LTMC) from five normal children and seven children with congenital bone marrow failure (four with Fanconi anemia [FA] and three with congenital pure red cell aplasia [PRCA]). Total nonadherent and adherent cells, as well as nonadherent progenitors, were determined weekly in the presence or in the absence of rhGM-CSF (10 ng/ml) or rhEPO (3 U/ml). As compared to normal LTMC, hematopoiesis was drastically reduced in cultures from FA patients. Myeloid and erythroid progenitor cells reached undetectable levels after only 3 and 1 weeks of culture, respectively. This was observed even in cultures supplemented with rhGM-CSF, in which no response to this cytokine occurred. In LTMC from PRCA children, the growth of erythroid and multipotent progenitors was also drastically reduced. Myelopoiesis, on the other hand, showed normal levels during the first three weeks of culture; however, from week 4, there was a significant decrease in the levels of both progenitor and mature cells, reaching undetectable levels several weeks before normal cells did. Response to rhGM-CSF and rhEPO was transient and deficient. Our results suggest that in FA, alterations at the level of primitive progenitor cells are so severe that myeloid, erythroid and multipotent progenitors are unable to proliferate in LTMC, even in the presence of rhGM-CSF. In patients with PRCA the erythroid arm of hematopoiesis is preferentially affected and addition of rhGM-CSF and/or rhEPO to these cultures had little or no effect on erythroid cell production. Interestingly, myelopoiesis in this culture system was deficient as well and response to rhGM-CSF was defective, suggesting that the myeloid lineage is also altered in congenital PRCA.

Topics: Bone Marrow Cells; Bone Marrow Diseases; Cell Adhesion; Cell Division; Cell Nucleus; Cells, Cultured; Child; Child, Preschool; Erythropoietin; Fanconi Anemia; Female; Granulocyte-Macrophage Colony-Stimulating Factor; Hematopoietic Stem Cells; Humans; Kinetics; Male; Recombinant Proteins; Red-Cell Aplasia, Pure

Diamond-Blackfan anemia (DBA) is a congenital pure red blood cell aplasia that often requires lifelong transfusional therapy. Autosomal dominant and recessive inheritance have both been reported, suggesting genetic heterogeneity, but most cases occur sporadically. The origin of impaired erythropoiesis is unknown. Several erythroid growth factors have been thought to have a role in the pathogenesis of DBA. However, there is neither molecular nor clinical evidence for the involvement of erythropoietin (EPO), its receptor, stem cell factor (SCF), or interleukin (IL)-3, even if the addition of SCF to IL-3 and EPO does significantly increase the growth of erythroid progenitors in in vitro cultures in most patients. In this work we evaluated the possible role of another early-acting erythroid growth factor, IL-9. We found that the addition of IL-9 to SCF, IL-3, and EPO further increases burst-forming unit-erythroid growth in in vitro cultures of those DBA patients who responded to SCF. To investigate the role of the IL-9 gene, we evaluated its segregation in 22 families with members who have DBA by using a polymorphic microsatellite located within its intron 4. Lod score analysis ruled out any statistically significant involvement of the IL-9 gene in the pathogenesis of DBA. Moreover, linkage analysis with 11 highly polymorphic markers spanning 5q31.1-q33.2 excluded this region, which is included in the major cluster of genes active in hematopoiesis of the human genome.

Topics: Adolescent; Child; Chromosome Mapping; Chromosomes, Human, Pair 5; Erythropoietin; Fanconi Anemia; Female; Genetic Linkage; Hematopoiesis; Hematopoietic Cell Growth Factors; Humans; Infant; Interleukin-3; Interleukin-9; Male; Microsatellite Repeats; Stem Cell Factor

The congenital disorder of erythropoiesis Diamond Blackfan anaemia (DBA) exhibits a defect in the stem/progenitor cell compartment, located at the erythroid progenitor level (CFU-GEMM, BFU-E, CFU-E). Treatment of DBA with interleukin-3 (IL-3) has had limited effect, despite in vitro studies suggesting that progenitor cells were capable of responding to IL-3. Whether IL-3 is not reaching the appropriate defective target cell, the cells cannot respond, or the marrow humoral inhibitory system is overriding it, is not clear. To investigate humoral inhibitory activities we examined the response of 15 DBA bone marrows in vitro to the inhibitory chemokine macrophage inflammatory protein 1-alpha (MIP1-alpha) in the presence of the stimulatory cytokines erythropoietin, granulocyte-macrophage colony-stimulating factor, IL-3, and stem cell factor. In vitro data agreed with our previous work showing that our patients formed three statistically different groups in response to stimulatory cytokines (type I DBA erythroid colony numbers approximately normal > type II DBA > type III DBA). Addition of MIP1-alpha to cultures caused average erythroid and myeloid suppression, which sequentially increased with DBA type (type I inhibition < type II < type III). The differential level of inhibition shown by MIP1-alpha in these DBA patients lends further evidence for the presence of distinct subgroups in this disorder.

Topics: Adolescent; Adult; Antibody Formation; Cell Division; Chemokine CCL3; Chemokine CCL4; Child; Child, Preschool; Colony-Forming Units Assay; Erythroid Precursor Cells; Erythropoietin; Fanconi Anemia; Female; Granulocyte-Macrophage Colony-Stimulating Factor; Hematopoietic Stem Cells; Humans; Infant; Interleukin-3; Macrophage Inflammatory Proteins; Male; Monokines; Stem Cell Factor

Interleukin-3 (IL-3) therapy as a treatment for Diamond Blackfan anaemia (DBA) patients has been largely disappointing despite early hope it would be suitable for stimulating arrested erythropoiesis. Initial hope came from in vitro discoveries that IL-3 (+EPO) generated well-haemoglobinized BFU-E colonies in some patients, but was soon tempered by the realization that in vitro and in vivo IL-3 response did not, in the majority of cases, correlate. Nevertheless in vitro testing has been the main focus in analysing the abnormality in the stem and progenitor cell compartment in DBA. Here we report in vitro analysis of a DBA patient who responded once to IL-3 therapy, but not a second time following relapse, using short-term culture, long-term culture and c-kit analysis. Progenitor numbers before and after the first therapy were in the high normal range, but after relapse were much reduced below normal levels. Long-term cultures suggested some arrested progenitors had been reactivated into normal cycle by the first therapy, but may not have been replaced by more immature progenitors. c-kit analysis revealed increased expression in all tested cell populations. These results imply that the first IL-3 therapy reactivated some erythroid progenitors but left the progenitor pool depleted when more immature cells remained arrested.

Topics: Cell Count; Cells, Cultured; Child, Preschool; Colony-Forming Units Assay; Erythroid Precursor Cells; Erythropoietin; Fanconi Anemia; Female; Humans; Interleukin-3; Proto-Oncogene Proteins c-kit; Treatment Failure

Culture of bone marrow from patients with Diamond-Blackfan anaemia (DBA) has previously shown a variable progenitor response to growth factor stimulation. An extensive standardized study has now been undertaken to investigate the presence of distinct sub-groups in this disorder. In vitro response of bone marrow progenitors to recombinant human growth factors, including stem cell factor, was examined in 18 DBA patients and five normal donors, assessing BFU-E, CFU-GM and CFU-GEMM development. In 16 of the DBA patients a synergistic response to combinations of growth factors was observed with optimal growth in cultures containing erythropoietin, interleukin-3 and stem cell factor. Growth factor induced erythroid response formed three distinct groups, based on BFU-E numbers: type I (mean age 4.87 years) showed > 70% normal erythroid response; type II (mean age 13.87 years) showed < 70% normal; and type III (mean age 15.29 years) < 5% normal. CFU-GM response also followed the trigrouping. The results suggest more than one pathogenic mechanism for the erythroid failure in DBA, indicating DBA may be composed of more than one distinct disorder, and further suggest the defect in DBA may not be confined to the erythroid series.

Topics: Adolescent; Adult; Bone Marrow; Cells, Cultured; Child; Child, Preschool; Colony-Forming Units Assay; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Fanconi Anemia; Female; Hematopoietic Cell Growth Factors; Hematopoietic Stem Cells; Humans; Infant; Interleukin-3; Male; Middle Aged; Recombinant Proteins; Stem Cell Factor

In most patients with Diamond-Blackfan anaemia (DBA) the addition of IL-3 to in vitro cultures increases the number of erythroid progenitors. However, a small proportion of patients show an erythroid response to in vivo administration of IL-3. We treated two adult patients with corticosteroid-refractory DBA, who had shown a clear in vitro erythroid response to IL-3, with increasing doses of IL-3 (from 2.5 to 10 micrograms/kg/d) by subcutaneous injections for 7 and 9 weeks. Monitoring of marrow and circulating progenitors showed a sustained elevation of BFU-E and CFU-E as well as an increase of other progenitors and CD34+ cells during therapy. However, this effect did not translate into a clear clinical response, although transfusion requirements decreased transiently in one patient. This report shows that a sustained elevation of erythroid progenitors induced by in vivo IL-3 administration may not translate into an increase of mature red cells production in DBA patients.

Topics: Adult; Bone Marrow; Dose-Response Relationship, Immunologic; Erythroid Precursor Cells; Erythropoietin; Fanconi Anemia; Female; Hematopoietic Stem Cells; Humans; Interleukin-3; Middle Aged; Red-Cell Aplasia, Pure

Fanconi anemia (FA) is a recessively inherited disease characterized by bone marrow failure, congenital anomalies, chromosomal instability and hypersensitivity to crosslinking agents. Some of the cellular defects of FA are known to be responsive to the ambient oxygen concentration. We examined the responsiveness of the FA complementation group C (FAC) gene to changes in oxygen concentration using two types of human cell lines, hypoxia-responsive Hep3B hepatoma cells and Epstein-Barr virus-immortalized lymphoblasts (normal and FA complementation groups B and C). Although the expression of erythropoietin in Hep3B cells was induced in response to the hypoxia-mimicking agent CoCl₂, there was no concomitant induction in FAC expression as assessed by mRNA levels and immunoprecipitable protein, and no detectable change in the cytoplasmic location of the FAC polypeptide as determined by indirect immunofluorescence. In human lymphoblasts we examined the effect of oxygen (0.1% -95% O₂) on cell proliferation and FAC expression. FA lymphoblasts had a normal sensitivity to the cytostatic effect of hyperoxia, while in both control and FA lymphoblasts FAC mRNA levels were unaffected by oxygen. Our results indicate that ambient oxygen is not a regulator of the FAC gene.

Topics: Carcinoma, Hepatocellular; Cell Cycle Proteins; Cell Hypoxia; Cell Line, Transformed; Cobalt; DNA-Binding Proteins; Erythropoietin; Fanconi Anemia; Fanconi Anemia Complementation Group C Protein; Fanconi Anemia Complementation Group Proteins; Gene Expression Regulation; Herpesvirus 4, Human; Humans; Liver Neoplasms; Lymphocytes; Nuclear Proteins; Oxygen; Protein Biosynthesis; Proteins; RNA, Messenger; Tumor Cells, Cultured

In two previous studies, we observed that recombinant human interleukin-3 (IL-3) induced an increase in marrow burst-forming unit-erythroid-derived colonies in vitro in some patients with Diamond-Blackfan anemia (DBA). To determine whether a similar erythropoietic response could be induced in vivo, we treated 13 patients with DBA (aged 4 to 19 years) with two preparations of IL-3. All patients had absent absolute reticulocyte counts and markedly reduced to absent recognizable bone marrow erythroid elements; patients with circulating reticulocytes in the previous 12 months were excluded from study. All patients except 1 had failed steroid therapy and had been transfusion-dependent since infancy; 1 patient was maintained on high-dose prednisone at the time of enrollment. On the first arm of the study, IL-3 (Immunex Corp, Seattle, WA) was administered subcutaneously using a dose escalation regimen of 125 to 500 micrograms/m2/day in divided dosage at 12-hour intervals, coadministered with 1.5 mg/kg/d of oral ferrous sulphate. Of the 13 patients that entered the trial, 4 stopped prematurely because of adverse side effects. In the other 9 evaluable cases, reticulocytes increased transiently in 1 patient from 0 to 65 x 10(9)/L after 35 days of IL-3 therapy at 250 micrograms/m2, but transfusion dependency persisted. One transient peak in absolute reticulocyte count was noted in 6 other patients, but no erythroid response was observed after completion of a full course of IL-3. Oral prednisone at 0.5 mg/kg/d was then coadministered with IL-3 at 500 micrograms/m2 to 5 of the patients without effect, and treatment was stopped. In 2 patients, a second preparation of IL-3 (Sandoz Canada Inc, Dorval, Quebec, Canada) was initiated in a dose escalation regimen of 2.5 to 10 micrograms/kg and was coadministered with ferrous sulphate. No erythroid response was observed in either patient, and in one of the two, alternate-day subcutaneous recombinant erythropoietin at 300 U/kg was administered for 3 weeks in combination with daily IL-3 at 10 micrograms/kg, but no increased erythropoiesis was seen. Significant increases in white blood cell and eosinophil counts during administration of both preparations of IL-3 were observed in all patients. These data show that the response of DBA patients to IL-3 in vivo is heterogeneous and cannot be predicted from in vitro studies. The absence of a corrective effect of IL-3 in these patients with DBA indicates that a deficiency of the cytok

Topics: Adolescent; Bone Marrow; Child; Child, Preschool; Erythrocyte Count; Erythropoiesis; Erythropoietin; Fanconi Anemia; Female; Ferrous Compounds; Humans; Interleukin-3; Male; Prednisone; Recombinant Proteins; Reticulocytes

Programmed cell death, also known as apoptosis, is frequently initiated when cells are deprived of specific trophic factors. To investigate if accelerated apoptosis contributes to the pathogenesis of Diamond-Blackfan anemia (DBA), a rare pure red blood cell aplasia of childhood, we studied the effect of erythropoietin (epo) deprivation on erythroid progenitors and precursors from the bone marrow of DBA patients as compared with hematologically normal controls. Apoptosis in response to epo deprivation was evaluated by enumeration of colony-forming unit-erythroid (CFU-E)- and burst-forming unit-erythroid (BFU-E)-derived colonies in plasma clot semisolid culture and by the identification of typical DNA oligosomes by gel electrophoresis from marrow mononuclear cells in liquid culture. In all DBA patients there was a marked decrease in CFU-E- and BFU-E-derived colony formation compared with normal controls at comparable time points of epo deprivation, with a complete loss of CFU-E-derived colonies in semisolid culture by 9 hours of epo deprivation versus 48 hours in controls. The BFU-E-derived colony response to epo deprivation displayed a similar pattern of decrement. Apoptotic changes assessed by the presence of characteristic DNA fragmentation began in the absence of epo deprivation and were readily detected within 3 hours of epo deprivation in DBA cultures versus 9 hours in controls. We conclude that DBA is characterized by accelerated apoptosis as measured by the loss of erythroid progenitor clonogenicity and increased progenitor and precursor DNA fragmentation leading to the formation of characteristic oligosomes, consistent with an intrinsic erythroid-progenitor defect in which increased sensitivity to epo deprivation results in erythroid failure.

Topics: Adolescent; Adult; Apoptosis; Child, Preschool; DNA; Erythroid Precursor Cells; Erythropoietin; Fanconi Anemia; Female; Humans; Infant; Male

Stem cell factor (SCF) enhances normal hematopoiesis. We examined its effect in vitro on bone marrow and blood progenitors from patients with inherited bone marrow failure syndromes, including 17 patients each with Diamond-Blackfan anemia (DBA) and Fanconi's anemia (FA), 3 with dyskeratosis congenita (DC), and 1 each with amegakaryocytic thrombocytopenia (amega) and transient erythroblastopenia of childhood (TEC). Mononuclear cells were cultured with erythropoietin (Ep) alone or combined with SCF or other factors. SCF increased the growth of erythroid progenitors in cultures from 50% of normal controls, 90% of DBA, 70% of FA, 30% of DC, and the amega and TEC patients; normal numbers were reached in 25% of DBA studies. Improved in vitro erythropoiesis with SCF in all types of inherited marrow failure syndromes does not suggest a common defect involving kit or SCF, but implies that SCF may be helpful in the treatment of hematopoietic defects of varied etiologies.

Topics: Adolescent; Adult; Blood Transfusion; Bone Marrow; Bone Marrow Diseases; Cells, Cultured; Child; Child, Preschool; Colony-Forming Units Assay; Erythropoiesis; Erythropoietin; Fanconi Anemia; Female; Hematologic Diseases; Hematopoietic Cell Growth Factors; Hematopoietic Stem Cells; Humans; Infant; Male; Recombinant Proteins; Reference Values; Stem Cell Factor

Diamond-Blackfan anaemia (DBA) is a congenital macrocytic anaemia. To investigate whether DBA is due to hyporesponsiveness to or hypoproduction of Steel factor (SF), we compared the in vitro responsiveness of the BFU-E contained in the Ficoll-Hypaque non-adherent cell fraction of six DBA marrows with that of four normal marrows and one transient erythroblastopenia of childhood (TEC) marrow. In addition, we studied the effect of soluble SF on long-term marrow cultures (LTMC) and analysed the stromal cells from these cultures for SF mRNA transcripts. All the patients showed an erythropoietin dose-related increase of small BFU-E. The number and size of BFU-E was increased with the addition to the epo of IL-3 or SF; IL-3+SF was not synergistic. The addition of soluble SF to LTMC of DBA patients was associated with a small but consistent increase in non-adherent cell production and an increase in the number of progenitors. Messenger RNA from immortalized stromal cell lines of three patients and from primary bone marrow stromal cells of one patient showed the presence of expected SF transcripts by PCR analysis. These results demonstrate that this group of DBA patients responds to SF and produces SF mRNA normally, indicating that SF itself is not involved in DBA pathophysiology. The effects observed suggest that, despite the lack of evidence for a causative role, SF may prove to be effective treatment for such patients.

Topics: Adult; Cell Division; Cells, Cultured; Erythroid Precursor Cells; Erythropoietin; Fanconi Anemia; Hematopoietic Cell Growth Factors; Humans; Interleukin-3; Polymerase Chain Reaction; RNA, Messenger; Stem Cell Factor; Time Factors

In patients with idiopathic aplastic anaemia (n = 34) and Fanconi's anaemia (n = 8), sampled once or on several occasions, serum erythropoietin (Epo) increased with increasing severity of anaemia with apparently similar rates of increase in each group. However, after adjustment for Hb, log Epo values for the Fanconi's anaemics tended to be greater than those for the idiopathic aplastic anaemics (P < 0.01). Erythropoietin concentrations in serum samples from patients with Fanconi's and idiopathic aplastic anaemias tended to be greater than in samples from patients with anaemias from protein energy malnutrition, myelodysplasia and iron deficiency. The results suggest that there is no deficiency of erythropoietin in Fanconi's and idiopathic aplastic anaemias and that if exogenous erythropoietin is of any benefit it would need to be administered in doses large enough to induce a significant increase in log Epo. Results of the study illustrate the need to take account of the assumptions which underlie interpretation of the statistical analysis. Use of erythropoietin values in place of log Epo gives misleading conclusions demonstrable as invalid as the conditions for normality of distribution of the data and homogeneity of variances were not satisfied.

Topics: Adolescent; Adult; Aged; Anemia, Aplastic; Blood Component Transfusion; Child; Child, Preschool; Erythropoietin; Fanconi Anemia; Female; Hemoglobins; Humans; Male; Middle Aged; Oxymetholone; Sex Factors

To provide insights into the pathogenesis of Diamond-Blackfan anemia, we examined the in vitro response of erythroid progenitors to the recently isolated ligand for c-kit (stem cell factor, SCF). For these studies, marrow or blood mononuclear cells from 10 Diamond-Blackfan patients were cultured with erythropoietin (Ep), Ep and interleukin-3, Ep and granulocyte-macrophage colony-stimulating factor, or Ep and lymphocyte conditioned media (LCM). These combinations were tested in the presence or absence of SCF. The mean number of cells per erythroid burst increased 5 to 50-fold in cultures containing SCF. Furthermore, many additional erythroid bursts were seen (mean increment 3.2 x baseline values). Although burst-forming unit-erythroid (BFU-E) from all patients responded, there were differences among individuals in the sensitivity of their BFU-E to SCF. In six patients and all control studies, plateau frequencies of erythroid bursts were achieved with less than or equal to 10 ng/mL SCF, whereas in studies from the other four patients, over 50 ng/mL SCF was required. These data invite speculation that the c-kit receptor/ligand axis is involved in the pathogenesis of Diamond-Blackfan anemia. More importantly and regardless of whether the observed patterns of response reflect the primary defect or an epiphenomenon, our data strongly support a therapeutic trial of SCF in patients with Diamond-Blackfan anemia.

Topics: Adolescent; Adult; Bone Marrow; Cells, Cultured; Child, Preschool; Colony-Forming Units Assay; Culture Media; Erythroid Precursor Cells; Erythropoietin; Fanconi Anemia; Female; Granulocyte-Macrophage Colony-Stimulating Factor; Hematopoietic Cell Growth Factors; Humans; Interleukin-3; Lymphocytes; Male; Stem Cell Factor

Diamond-Blackfan anemia (DBA) is a congenital red blood cell aplasia. No clear explanation has been given of its defective erythropoiesis, although different humoral or cellular inhibitory factors have been proposed. To clarify the nature of this defect we studied the effect of several human recombinant growth factors on an enriched CD34+ population obtained from the bone marrow of 10 DBA patients. We observed a defect underlying the early erythroid progenitors, which were unresponsive to several growth factors (erythropoietin, interleukin-3 [IL-3], IL-6, granulocyte-macrophage colony-stimulating factor [GM-CSF], erythroid potentiating activity), either alone or in association. The production of cytokines was not impaired, and high levels of IL-3 and GM-CSF were found in phytohemagglutinin-leukocyte-conditioned medium (PHA-LCM) when tested with a sensitive biologic assay on the M-07E cell line. Hematopoietic stem cells in DBA patients may be induced to differentiate to the granulocyte megakaryocyte, but not the erythroid compartment, as shown after CD34+ cell preincubation with IL-3. Addition of the stem cell factor to IL-3 and erythropoietin induces a dramatic in vitro increase in both the number and the size of BFU-E, which also display a normal morphologic terminal differentiation.

Topics: Antigens, CD; Antigens, CD34; Bone Marrow; Cell Differentiation; Cell Division; Culture Media; Erythroid Precursor Cells; Erythropoietin; Fanconi Anemia; Female; Granulocyte-Macrophage Colony-Stimulating Factor; Growth Substances; Hematopoietic Stem Cells; Humans; Infant; Interleukin-3; Interleukin-6; Leukocytes; Male

Diamond-Blackfan anemia is a congenital disorder of erythropoiesis in humans, characterized by a macrocytic anemia often associated with physical anomalies. Mutations at either the W or Steel loci in the mouse also leads to a severe macrocytic anemia, as well as other developmental abnormalities. The W locus encodes the proto-oncogene c-kit, a member of the receptor tyrosine kinase family, while the Steel locus encodes a potent hematopoietic growth factor that is the ligand for c-kit. Growth of clonogenic marrow erythroid progenitor cells in vitro in the presence of the recombinant hematopoietic growth factors interleukin-3 (IL-3) and Steel was used to characterize this disease at the cellular level. Three patterns of in vitro marrow response to both recombinant IL-3 or Steel were observed among 10 Diamond-Blackfan patients: those that responded quantitatively and qualitatively almost as well as cells from normal marrow, those that responded at an intermediate level, and those that did not respond at all. These results provide evidence for cellular heterogeneity underlying the pathogenesis of this disorder and therefore raise the possibility that there may be more than one underlying molecular basis for the disease. No gross abnormalities in the structure of either the c-kit or Steel loci were observed in these patients. The normal response in culture of the progenitor cells from at least some patients to Steel with or without IL-3 raises the possibility of using this novel growth factor as a therapeutic agent in Diamond-Blackfan anemia.

Topics: Adolescent; Adult; Blotting, Southern; Cells, Cultured; Child; Child, Preschool; Colony-Forming Units Assay; DNA; Erythroid Precursor Cells; Erythropoietin; Fanconi Anemia; Female; Hematopoietic Cell Growth Factors; Humans; Interleukin-3; Male; Proto-Oncogene Mas; Recombinant Proteins; Stem Cell Factor

Topics: Anemia, Aplastic; Child, Preschool; Erythropoietin; Fanconi Anemia; Fanconi Syndrome; Humans; Recombinant Proteins

The erythropoietin response to anaemia was compared in 30 children with haemolytic anaemia and in 5 children with Fanconi's hypoproliferative anaemia. Serum erythropoietin was measured by radioimmunoassay. In children with haemolytic anaemia the serum erythropoietin concentration increased exponentially with decreasing haematocrit values (r = 0.74; p less than 0.001). Serum erythropoietin levels also correlated with reticulocyte counts (r = 0.62; p less than 0.001). Children with Fanconi's hypoproliferative anaemia had considerably higher serum erythropoietin levels than children with haemolysis for the same degree of anaemia. These data indicate that erythropoietin production in Fanconi's anaemia may be dependent on other factors in addition to the degree of anaemia and relative hypoxaemia.

Topics: Adolescent; Anemia, Aplastic; Anemia, Sickle Cell; Cell Count; Child; Child, Preschool; Erythropoietin; Fanconi Anemia; Female; Hematocrit; Humans; Male; Radioimmunoassay; Reticulocytes; Sickle Cell Trait

Topics: Anemia, Dyserythropoietic, Congenital; Cell Differentiation; Erythropoiesis; Erythropoietin; Fanconi Anemia; Fetal Hemoglobin; Hematopoietic Stem Cells; Humans; Models, Biological; Thalassemia

The results of the erytropoietin level determination in serum and urine of patients with congenital anemias are presented and compared to the results obtained in children with acute aplastic anemias. Three patients with congenital hypoplastic anemia Diamond-Blackfan, two with Fanconi's anemia, one with congenital pancythopenia with hyperplastic marrow and five patients with acute aplastic anemia were studied. The increased serum erythropoietin level was found in every patient whose blood hemoglobin was less than 12g%. Erythropoietin was detected in nonconcentrated urine when serum erythropoietin level was higher than 0,5 units/ml. The statistically significant negative correlation between the serum erythropoietin level and blood hemoglobin concentration was found. In two patients suffering from congenital anemias, in whome the significantly increased erythropoietin level (about 1.0 units/ml) was detected, increased ammount of hemoglobin F in peripheral blood as well as increased MCV--signs of so called "stress" erythropoiesis-were noted. The results presented, together with the results obtained by other authors, indicate that congenital anemias studied here are not due to the disturbance in erythropoietin production.

Topics: Adolescent; Adult; Anemia, Aplastic; Child; Child, Preschool; Erythrocytes, Abnormal; Erythropoietin; Fanconi Anemia; Female; Humans; Infant; Male; Syndrome