Long-term bone marrow cultures in Diamond-Blackfan anemia reveal a defect of both granulomacrophage and erythroid progenitors.

The hematopoietic defect of Diamond-Blackfan anemia (DBA) results in selective failure of erythropoiesis. Thus far, it is not known whether this defect originates from an intrinsic impediment of hematopoietic progenitors to move forward along the erythroid pathway or to the impaired capacity of the bone marrow (BM) microenvironment to support proliferation and differentiation of hematopoietic cells. Reduced longevity of long-term bone marrow cultures, the most physiologic in vitro system to study the interactions of hematopoietic progenitors and hematopoietic microenvironment, is consistent with a defect of an early hematopoietic progenitor in DBA. However, stromal adherent layers from DBA patients generated in a long-term culture system, the in vitro counterpart of BM microenvironment, did not show evidence of any morphologic, phenotypic, or functional abnormality. Our major finding was an impaired capacity of enriched CD34+ BM cell fraction from DBA patients, cultured in the presence of normal BM stromal cells, to proliferate and differentiate along the erythroid pathway. A similar impairment was observed in some DBA patients along the granulomacrophage pathway. Our result points to an intrinsic defect of a hematopoietic progenitor with bilineage potential that is earlier than previously suspected as a relevant pathogenetic mechanism of the disease. The finding of impaired granulopoiesis in some DBA patients underlines the heterogeneity of this rare disorder.

Cell adhesion molecules in cord blood hematopoietic progenitors.

We investigated the expression of different cell adhesion molecules on cord blood (CB) and bone marrow (BM) CD34+/CD38+ and CD34+/CD38- cells. CD11a and CD62L were more expressed in CB than in BM CD34+/CD38- subset, suggesting a possible advantage in homing and engraftment. A short exposure to various cytokines increased CD62L expression only in the more differentiated CB and BM CD34+/CD38+ cells.

Cell adhesion molecule expression in cord blood CD34+ cells.

Self-renewal, proliferation, differentiation, homing, and mobilization of hematopoietic progenitor cells (HPCs) are regulated by a complex mechanism that involves the bone marrow (BM) microenvironment. Cell adhesion molecules (CAMs) expressed on HPCs and on endothelial and stromal cells play a pivotal role in this process. In this study, we have used three-color cytofluorometric analysis to compare CAM expression in the subsets of cord blood (CB) and BM HPCs and examined the effect of a short exposure to various cytokines on L-selectin expression. The study was carried out on unseparated samples to avoid any possible bias from positive CD34 selection. CAMs were highly expressed in both CB and BM CD34+CD38+ cells. In this population, L-selectin, H-CAM, and LFA-1 were significantly more expressed in BM than in CB. With regard to the more immature progenitors, the subsets of CD34+/CD38-/L-selectin+ and CD34+/CD38-/LFA1+ cells were significantly larger in CB than in BM. Since the expression of such CAMs has been related to the repopulating capacity of HPCs, our results suggest a possible advantage in homing and engraftment of more undifferentiated CB as opposed to BM HPCs. A 4/24-h exposure to various cytokines significantly increased the percentage of CB CD34+/CD38+/L-selectin+ cells, while HPCs were differentiated since the percentage of CD34+/CD38-/L-selectin+ cells was reduced. These data show that a short exposure to cytokines increases L-selectin expression in the more differentiated CB HPCs. This could improve their homing in a transplant setting.

Diamond-Blackfan anemia: expansion of erythroid progenitors in vitro by IL-9, but exclusion of a significant pathogenetic role for the IL-9 gene and the hematopoietic gene cluster on chromosome 5q.

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.

Expression of G1-phase cell cycle genes during hematopoietic lineage.

Characterization of proteins that control the passage through the G1 phase of the cell cycle is of particular interest because virtually all stimuli regulating cell proliferation or differentiation act primarily during this phase. We have analyzed the G1 phase proteic machinery, including cyclin D types, cyclin-dependent kinases (CDKs) and CDK inhibitors, of cell populations obtained at different stages of hematopoietic cell lineage. In particular, five cellular phenotypes, namely CD34+ cells (which contain stem cells), BFU-E, CFU-E, CFU-GM and peripheral lymphocytes were studied as representatives of distinct differentiation pathways. The results obtained indicated that all the cellular preparations express cyclin D2 and D3, while cyclin D1, which is the major cyclin D occurring in mesenchimal tissues, is not expressed. Moreover, CDK6 (but not CDK4) was detectable in all the populations investigated. Among the CDK inhibitors studied, p18INK4C and p19INK4D signals were clearly evidentiable in the various cell types. Interestingly, high levels of p15INK4B, a putative tumor suppressor protein, were detectable especially in granulocyte-monocyte precursors. Our results indicate that a specific hematopoietic G1 phase machinery occurs, which is conserved during the various steps of the different maturation processes.

Hematological studies in children with Down syndrome.

Previous studies have reported erythrocyte macrocytosis in adults and children with Down syndrome (DS), the significance of which remains unclear. We compared hematological parameters of 50 DS children aged 2 to 15 years, divided into three age groups, with those of 68 aged-matched healthy children. Patients with DS had a significantly increased mean corpuscular volume (MCV) and hemoglobin in all groups when compared with the controls. Erythrocyte creatine content, hexokinase (Hk) activity, erythrocyte and serum folates, vitamin B12, haptoglobin, serum iron, and ferritin were tested. All of these parameters were not significantly different from those of the control group. We conclude that macrocytosis may not be an expression of reduced red cell survival but rather of an altered folate remethylation pathway, secondary to enhanced cystathionine beta-synthase (CBS) activity, the gene for which is present on chromosome 21.

Mutations in the erythropoietin receptor gene are not a common cause of Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is an inherited pure red blood cell aplasia that often requires lifelong transfusional support. The origin of the imperfect erythrogenesis is not known. The existence of more than one molecular basis for DBA is indicated by its different modes of inheritance and widely variable clinical phenotypes. 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 stem cell factor or interleukin-3. The observation of elevated erythropoietin (EPO) concentrations and an impaired in vivo and in vitro response to pharmacologic doses of recombinant human EPO has suggested a defective EPO function in the pathogenesis of DBA. We have investigated the possible involvement of the EPO receptor (EPO-R) gene in 23 patients by screening its coding sequence for mutations using single-strand conformation polymorphism (SSCP). A Southern blot and hybridization with an EPO-R probe was also performed on DNA from seven patients. No causal mutations were identified. The absence of concordant segregation of the disease with the EPO-R gene in two informative families ruled out its role in their DBA children. These findings demonstrate that DBA is not commonly associated with EPO-R gene mutations.

Results of long-term iron-chelating therapy.

The improvement in survival and quality of life of iron-overloaded patients achieved by regular subcutaneous chelation has been extensively documented over the years. A review of the long-term results allows one to establish the following points: (1) with regular subcutaneous chelation, a negative iron balance can be obtained in most patients, except very young ones; (2) severe deferoxamine (DFO) toxicity may be prevented by skipping high doses and by carefully monitoring and modulating chelation, especially in patients with a low iron overload; (3) the maintenance of compliance with DFO over 0.6 and of ferritin levels below 2,000 prevents iron overloaded complications, at least for the first 20 years of life; (4) long-term chelation can reverse functional complications such as liver fibrosis, arrhythmia and echocardiographic abnormalities, but not complications due to extensive tissue alterations, such as frank diabetes, hypothyroidism and myocardiosclerosis; (5) intensive intravenous protocols can be successfully applied in heavily overloaded patients and represent the only possibility to reverse their dangerous iron burden in a relatively short period of time; (6) survival and quality of life in well-chelated patients are approaching a normal pattern, and (7) clinical outcome and prognosis are better evaluated by parameters that consider iron overload and chelation trends.

Clinical manifestations and therapy of transfusional haemosiderosis.

Long-term blood transfusions lead to the accumulation of iron that in the absence of chelation therapy causes complications such as liver cirrhosis, growth failure, hypogonadism, hypothyroidism, hypoparathyroidism, diabetes and myocardiopathy. The last still represents the most frequent cause of death in haemosiderotic transfusion-dependent patients. At the moment the only chelator widely used is desferrioxamine (DFX). The drug works best when administered as a continuous infusion, mainly by the subcutaneous route. To patients with severe iron overload, impending organ failure, or poor compliance to chelation, DFX can be administered intravenously, through an externalized central catheter or, preferably, a subcutaneous port. Several studies have shown the effectiveness of DFX in reducing the iron burden, thus preventing the complications, once considered inevitable, of iron overload, and even in reverting some, but not all, of the iron-induced dysfunctions. Practical and psychological support are necessary to ensure satisfactory compliance with a therapy that is cumbersome and difficult. Toxic effects of DFX such as growth failure, hearing impairment and bone abnormalities seem to occur mainly in patients who have received high doses of DFX despite a low iron burden. Visual loss and renal and pulmonary toxicities, on the contrary, seem to be more directly related to high DFX peak doses administered irrespective of the patient's amount of iron overload. After bone marrow transplantation, phlebotomy or erythrocytoapheresis might be necessary to reduce further the iron accumulated during years of transfusions.

Natural protection against severe Plasmodium falciparum malaria due to impaired rosette formation.

Genes for two lethal diseases, thalassemia and sickle cell anemia, are favored by evolution because, in their heterozygous form, they protect against cerebral malaria. Rosette formation, the binding of uninfected red cells (RBCs) to Plasmodium falciparum-infected RBCs (PRBCs), has previously been found to be associated with cerebral malaria, the most important severe manifestation of P falciparum malaria. We show here that thalassemic RBCs and, under certain conditions, even hemoglobin S (HbS)-containing RBCs possess an impaired ability to bind to PRBCs, forming small and weak erythrocyte rosettes compared with rosettes formed by normal RBCs. This decreased rosetting ability is associated with the small size of the thalassemic RBCs and with distortion of the mechanical properties of HbS-containing RBCs. The impairment of rosette formation may hinder the development of cerebral malaria by abatement of sequestration.

Effect of desferrioxamine and hydroxypyridones on hemopoietic progenitors and neuroectodermal tumor cells.

The iron chelator desferrioxamine (DFO) has been shown to inhibit the proliferation of hemopoietic progenitors and several tumor cell lines. We have compared the in viro hemopoietic inhibitory effect of desferrioxamine (DFO) and hydroxypyridones (HPOs) on hemopoietic progenitors and two human neuroectodermal (NE) tumor cell lines, NB 100 and SKNMC. Both DFO and HPOs showed a direct dose-related inhibitory effect on BFU-E and CFU-GM obtained from purified human non-T MNAC (T-lymphocyte-depleted nonadherent mononuclear cells) and CD34+ cells. DFO and HPOs displayed both an inhibitory and a cytotoxic effect on NE cell lines. We calculated the ratio between NE cell and hemopoietic cell growth inhibition for a range of concentrations of chelators. DFO showed the most satisfactory ratio. This suggests that DFO is still the most preferable chelating agent for the treatment of neuroblastoma, since it combines the highest antineuroblastoma effect with the lowest hematopoietic toxicity.

Interaction of rare illegitimate recombination event and a poly A addition site mutation resulting in a severe form of alpha thalassemia.

The clinical diversity of thalassemia depends on interaction of diverse genetic defects. We have characterized a severe form of alpha thalassemia caused by coinheritance of a rare alpha-globin gene deletion and a nondeletional defect in a southern Italian family. The proband, a 7-year-old girl, exhibited an abnormal hemoglobin electrophoresis pattern with hemoglobin H and hemoglobin Barts, indicating inheritance of H and hemoglobin Barts, indicating inheritance of a severe form of alpha thalassemia. Southern blot analysis of DNA showed normal as well as aberrant alpha-globin gene fragments indicating heterozygosity for a deletional form of alpha thalassemia in the proband and her mother. The coinheritance of a nondeletional form of alpha thalassemia (alpha alpha T) was suspected because of the severity of the proband's phenotype and the presence of normal alpha-globin gene fragments in the father. Selective polymerase chain reaction of the paternal alpha 1- and alpha 2-globin genes in the proband followed by DNA sequence analysis showed an AATAAA to AATGAA mutation in the polyadenylation signal sequence of the alpha 2-globin gene. Genomic DNA mapping and sequence analysis of a unique polymerase chain reaction product generated across the deletion breakpoint of the maternal allele showed a 5,201-bp deletion extending from 870 nucleotides 5' of the alpha 2-globin gene to nucleotide +519 in the alpha 1-globin gene. This deletion is similar to that previously suggested by blotting studies in a Greek family (Pressley et al, Nucleic Acids Res 8:4889, 1980) and removes the entire alpha 2-globin gene and a portion of the 5' end of the alpha 1-globin gene. Sequence characterization of the resultant aberrant truncated alpha 1-globin gene from the proband showed a 27 nucleotide duplication corresponding to the 3' end of the alpha-globin gene IVS-2 region separated by the insertion of a tetranucleotide (GGTT), suggesting that this deletion is caused by an illegitimate recombination event.

Expansion of cord blood progenitors and use for hemopoietic reconstitution.

A high number of stem cells migrate in fetal blood and, at birth, the number of progenitors in cord blood equals or exceeds that of adult bone marrow. Recently hemopoiesis has been successfully reconstituted with the infusion of cord blood cells. It is important to clearly define the quantity and quality of cord blood totipotent and multilineage progenitors to evaluate the possibility of their utilization in transplants. Our first aim was to study the growth characteristics of cord blood progenitors. We have evaluated the number of cycling cells with the thymidine suicide technique and the production, by phytohemagglutinin (PHA) stimulated cord blood mononuclear cells, of some cytokines involved in the proliferation of progenitor cells, such as granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 6 (IL-6) and leukemia inhibitory factor (LIF). We have also studied by flow cytometry the CD34+CD33-, CD34+CD33+ cell subsets and the presence of the c-kit receptor in order to quantitate the number of earlier progenitors. Our second aim was to elucidate whether the cord blood totipotent stem cell population or the committed progenitors could be expanded in vitro. Our results showed that in cord blood the number of early progenitors, as evaluated by the number of mixed lineage colony forming units (CFU-Mix), by the CD34+CD33- subsets and the expression of the c-kit, is higher than in bone marrow. We have also demonstrated the possibility in vitro of increasing the number of progenitors by more than 30-fold by utilizing stem cell factor (SCF) in association with other cytokines.(ABSTRACT TRUNCATED AT 250 WORDS)

Human cord blood progenitors: kinetics, regulation and their use for hemopoietic reconstitution.

The high number of cord blood (CB) hematopoietic progenitors made possible CB transplantation in children with hematologic diseases. We collected and cryopreserved CB from siblings of 11 hematologic pediatric patients for possible transplant. The number of BFU-E+CFU-GEMM and CFU-GM was adequate for engraftment. However the possibility of CB transplantation in adolescents and adults is limited by the amount of CB that can be collected. We studied the possibility of expanding the pool of CB hematopoietic progenitors by preincubation of CB CD 34+ cells with several cytokines. After pre-exposition to rhIL-3, IL-1 beta, rIL-6, GM-CSF and stem cell factor (SCF) and cloning with SCF the number of hematopoietic progenitors increased 14-18 fold compared to basal values.

In vitro growth and regulation of bone marrow enriched CD34+ hematopoietic progenitors in Diamond-Blackfan anemia.

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.

A newly-characterized alpha-thalassaemia-1 deletion removes the entire alpha-like globin gene cluster in an Italian family.

We describe a new deletional form of alpha thalassaemia which encompasses the entire alpha-like globin gene cluster in a 15-year-old boy of Southern Italian descent. The deletion removes approximately 31 kb, the 5'-end point is located approximately 4 kb upstream of the xi gene, while the 3'-end point maps between the alpha 1- and theta 1-globin genes. The interaction of this deletion with the common-alpha 3.7 form gives origin to a classical form of haemoglobin (Hb) H disease in the propositus of this study. Deletional forms of xi alpha-thalassaemia are uncommon in the Mediterranean basin; as for other unusual xi alpha-thalassaemia forms, heterozygotes for this mutation may escape detection in population surveys based on zeta and alpha probes.

Current therapy for thalassemia in Italy.

Care and life quality of thalassemic patients in Italy have greatly improved over the last years thanks to cooperation between many clinical centers. The achievement of the following points played an important role: 1. A national treatment protocol was adopted in 1980; it has been widely accepted and regularly updated. 2. A standardized format for recording clinical data and a computerized clinical record (Computhal) were adopted. 3. A national registry was set up in order to evaluate the patient age distribution and birth and death rates both at the regional and national level. 4. A quality assurance program (Cooley Care) was devised after key indicators for evaluating treatment were identified and a central data base was set up. 5. Cooperative clinical trials provided information on many pathological and therapeutical aspects of the disease (incidence of complications, causes of death). 6. Pilot studies were carried out on emerging problems (intensive chelation, desferrioxamine (DFO) pharmacokinetics and toxicity). 7. Attention was paid to psychological and social problems.

Hearing loss and desferrioxamine in homozygous beta-thalassemia.

The authors present the results obtained during an audiometric screening of 153 children aged 5-18 years, affected by beta-thalassemia and treated with regular blood transfusions and iron overload chelation by means of desferrioxamine. Thirty-eight percent of the patients showed a significant sensorineural hearing loss at high frequencies with recruitment. Younger patients had a greater hearing loss, indicating that cochlear damage was not due to the disease itself. Furthermore, hearing loss appeared to be correlated with the mean and peak desferrioxamine doses administered and was higher in subjects with lower iron load. Thus, the ototoxic effect seems to have been higher when a good iron chelation had been obtained. Among our patients, conductive hearing loss was not more frequent than in patients without beta thalassemia.