Primary hematopoietic cells from DBA patients with mutations in RPL11 and RPS19 genes exhibit distinct erythroid phenotype in vitro.

Diamond-Blackfan anemia (DBA) is caused by aberrant ribosomal biogenesis due to ribosomal protein (RP) gene mutations. To develop mechanistic understanding of DBA pathogenesis, we studied CD34⁺ cells from peripheral blood of DBA patients carrying RPL11 and RPS19 ribosomal gene mutations and determined their ability to undergo erythroid differentiation in vitro. RPS19 mutations induced a decrease in proliferation of progenitor cells, but the terminal erythroid differentiation was normal with little or no apoptosis. This phenotype was related to a G0/G1 cell cycle arrest associated with activation of the p53 pathway. In marked contrast, RPL11 mutations led to a dramatic decrease in progenitor cell proliferation and a delayed erythroid differentiation with a marked increase in apoptosis and G0/G1 cell cycle arrest with activation of p53. Infection of cord blood CD34⁺ cells with specific short hairpin (sh) RNAs against RPS19 or RPL11 recapitulated the two distinct phenotypes in concordance with findings from primary cells. In both cases, the phenotype has been reverted by shRNA p53 knockdown. These results show that p53 pathway activation has an important role in pathogenesis of DBA and can be independent of the RPL11 pathway. These findings shed new insights into the pathogenesis of DBA.

[Noninfectious febrile inflammatory syndromes in children: diagnosis and usefulness of diagnostic procedures]. / Syndromes inflammatoires fébriles non infectieux de l'enfant: diagnostic, contribution des examens complémentaires.

OBJECTIVE: To determine the causes and to quantify the benefits obtained from further diagnostic investigations in children presenting with a non infectious inflammatory fever. METHODS: The records of 62 children aged from two-months to 15 years (median: four years) admitted to a paediatric department between 1990 and 2000 for the evaluation of a fever associated to an inflammatory syndrome, defined as temperature over 38 degrees C with an increase of the erythrocyte sedimentation rate (ESR) more than 20 mm/h and/or a serum C-reactive protein level (CRP) > 20 mg/L, and excluding overt infectious diseases, were retrospectively reviewed. RESULTS: Of these patients, 79% children (49 cases) had inflammatory systemic disease, 3.2% (two cases) had malignancy, and 17.8% (11 cases) had undiagnosed disorders. The most frequent disease was Kawasaki disease (22 children), especially in young children. Increase of ESR above 100 mm/h and of CRP above 100 mg/L was present in 59% of Kawasaki disease, 71% of idiopathic juvenile arthritis, 100% of malignancies and 7% of unknown diagnoses. Increase of ESR below 50 mm/h and of CRP below 50 mg/L was present in 75% of hemophagocytic syndromes and 46% of unknown diagnosis. The polymorphonuclear count, hepatic function evaluation, triglycerides levels, abdominal ultrasound, abdominal computed tomography, echocardiography, biopsies were useful diagnosis tools. Technetium scintigraphy was helpful only when abnormalities were found on physical examination. CONCLUSION: The diagnosis of Kawasaki disease must be quickly suspected in febrile young children with inflammatory syndrome without infection. ESR and CRP values, abdominal ultrasound and echocardiography are helpful tools for the diagnostic procedure.

Autoimmune disorders of erythropoiesis.

Immune-mediated disorders of erythropoiesis can result in acquired severe anemia, low reticulocyte counts, and bone marrow exhibiting pure red cell aplasia or ineffective erythropoiesis. Erythropoiesis can be suppressed or impaired by humoral or cellular mechanisms. In vitro inhibition of erythroid colony growth by immunoglobulins or lymphocytes can be a strong argument for the immune origin of the disease. Classical etiologies are thymoma and hematologic malignancies such as chronic lymphocytic leukemia (CLL). Clonal proliferation of T cells has been incriminated. Recently, acquired circulating autoantibodies directed against erythropoietin have been detected in a case of pure red cell aplasia. Autoimmune mechanisms have also been detected or suggested in synartesis and in Fas-associated dyserythropoiesis, two distinct syndromes recently described where morphologic abnormalities specific to the erythroid lineage illustrate ineffective erythropoiesis.

[Erythropoiesis disorders and other central autoimmune anemias]. / Erythroblastopénie et autres anémies centrales auto-immunes.

Immune-mediated acquired disorders of erythropoiesis can result in pure red cell aplasia or ineffective erythropoiesis. Erythropoiesis can be suppressed or impaired by humoral or cellular mechanisms. In vitro inhibition of erythroid colony growth by humoral factors or lymphocytes is a strong argument for the immune origin of the disease. Classical aetiologies are thymoma and hematological malignancies such as chronic lymphocytic leukaemia. Clonal proliferation of T cells has also been incriminated. Recently, acquired circulating autoantibodies directed against erythropoietin have been detected in a case of pure red cell aplasia. Autoimmune mechanisms have also been suggested in two dyserythropoietic syndromes recently described.

Dyserythropoiesis associated with a fas-deficient condition in childhood.

Defective lymphocyte apoptosis caused by mutations of the Fas gene can result in an autoimmune lymphoproliferative syndrome (ALPS) in humans. We report two cases of dyserythropoiesis associated with a Fas-deficient condition in childhood. In both cases, dyserythropoiesis predominated on the more mature erythroblasts, and was associated with a lymphoproliferative syndrome as well as with haemolytic anaemia, hypergammaglobulinaemia and the expansion of an unusual population of CD4- CD8- T cells that express the alpha/beta T-cell receptor. The regression of dyserythropoiesis under steroid therapy suggested that it resulted from an autoimmune mechanism, itself secondary to the lymphocyte Fas apoptosis deficiency. Fas-defective apoptosis may be a new aetiology for childhood dyserythropoiesis.

The GEN.S: a fortuitous finding of a routine screening test for hereditary spherocytosis.

As part of the evaluation of the GEN.S (Coulter), we compared the Mean Corpuscular Volume (MCV) to the Mean Spherized Corpuscular Volume (MSCV) assessed during the reticulocyte count procedure under hypo-osmotic conditions. A sub-group of patients with hereditary spherocytosis (HS) was singled out: in all of them, the MSCV became smaller than the MCV. As the cell volume normally increases in red cells derived from other patients in the same conditions, we decided to further study the reason for this particular behaviour of HS red cells. Whereas normal red cells are able to undergo an osmotic expansion, the spherocytes reach a critical osmotic volume leading to cell fragmentation consistent with the decrease of MSCV. This fortuitous finding is likely to be a reliable improvement for the routine screening of HS.

[Pachydermoperiostosis with extramedullary hematopoiesis without myelofibrosis]. / Pachydermopériostose associée à un foyer d'hématopoïèse extra-médullaire sans myélofibrose.

BACKGROUND: In three previous reports of primary hypertrophic osteoarthropathy, an associated extramedullary hematopoiesis was related to myelofibrosis. CASE REPORT: A 44-year-old male patient with primary hypertrophic osteoarthropathy diagnosed when he was 34-year-old was referred to our hospital with an abdominal mass fortuitously detected. DISCUSSION: The present case is unique for the patient developed an extramedullary hematopoïesis without associated myelofibrosis. It suggests the possible intervention of growth factors common to the skin fibroblasts and the blood progenitor cells in the pathogenesis of primary osteoarthropathy.

Characterization of a bipotent erythro-megakaryocytic progenitor in human bone marrow.

The aim of the present study was to determine if the human erythroid (E) and megakaryocytic (MK) lineages were closely linked to the existence of a bipotent burst-forming unit (BFU) E/MK progenitor. In methylcellulose cultures, BFU-E/MK colonies were observed at day 12 and closely resembled mature BFU-E with the exception that the erythroid component was surrounded by MK. These colonies were quite different from the colony forming unit (CFU)-GEMM-derived colonies, which were composed of a larger number of erythroblasts and which developed later in culture. The existence of these bilineage colonies composed of 100 to 1,000 erythroblasts intermingled with a few MK and without granulocytic cells was confirmed by the plasma clot technique and immunoalkaline phosphatase labeling of the MK. To investigate if this bipotent progenitor belonged to the compartment of primitive progenitors, CD34+ marrow cells were subfractionated according to expression of the CD38 antigen. The bipotent BFU-E/MK progenitor as well as a large fraction of MK progenitors were found in the CD34+ CD38+/- or in the CD34+ CD38- cell fractions. Growth of this bipotent BFU-E/MK progenitor required the combination of stem cell factor (SCF), Interleukin-3 (IL-3), and Epo in serum free conditions. Addition of IL-6 had only a marginal effect, whereas megakaryocyte growth and development factor (MGDF) was not an absolute requirement, but slightly increased the plating efficiency of CFU-MK and of BFU-E/MK progenitors when combined with SCF, IL-3, and Epo. In contrast, when these cultures were performed in the presence of 30% fetal calf serum, no BFU-E/MK colonies were observed irrespective of the combination of growth factors used, including the presence of MGDF; however, inclusion of the MS-5 cell line restored the growth of this bipotent progenitor. In contrast, in cultures performed in the presence of human normal or aplastic plasma, MS-5 had only a slight effect on the cloning efficiency but improved MK cytoplasmic maturation and MK size, suggesting that the main effect of MS-5 is to diminish the inhibitory effect of the fetal calf serum on the MK differentiation. The clonal origin of bipotent BFU-E/MK colonies was demonstrated in liquid culture of single CD34+ CD38low cells by immunophenotyping individual clones. At day 12, 30% of the clones contained erythroblasts (glycophorin A+) and some MK (CD41+) without granulocytes (G) or macrophages (M) (CD14+ and CD15+). At day 20, clones containing erythroblasts and MK were rare (5%). In contrast multilineage clones could be frequently detected at this time without passage from BFU-E/MK clones at day 12 to GEMM at day 20. These results suggest that a bipotent BFU-E/MK progenitor may be a nonrandom step in the hierarchical development of stem cells.

Characterization of hematopoietic progenitors from human yolk sacs and embryos.

Hematopoiesis first arises in the extraembryonic yolk sac, and it is generally believed that yolk sac-derived stem cells migrate and seed the fetal liver at approximately week 6 of development in humans. Recently, the identification at day 8.5 to 9 of multipotential stem cells in intraembryonic sites different from the liver suggests that the establishment of hematopoiesis might be more complex than initially believed. In an attempt to understand initial steps of hematopoiesis during human ontogeny, we characterized clonogenic myeloid progenitor cells in human yolk sacs and corresponding embryos at 25 to 50 days of development. Most erythroid colonies derived from the yolk sacs differed from adult marrow-derived progenitors in that they also contained cells of the granulomacrophagic lineage, suggesting that they were pluripotent and exhibited a different response to cytokines. Furthermore, a subclass of nonerythroid progenitors generated very large granulomacrophagic colonies, some of which generated secondary erythroid colonies on replating. Analysis of the distribution of progenitors revealed that in contrast to erythroid progenitors, whose numbers were equally distributed between the yolk sac and the embryo, 80% of the nonerythroid progenitors were found in the embryo at stages II and III. Interestingly, a high proportion of nonerythroid progenitors (including high proliferative potential cells) was present in colony assays initiated with cells remaining after the liver has been removed. These findings were validated in colony assays established with CD34+ cells purified from extraembryonic yolk sacs and intraembryonic tissues. Increased knowledge about the biology of hematopoietic stem cells early in life may help to further understanding of the mechanisms associated with the restriction in proliferative and differentiative potential observed in the adult hematopoietic stem cell compartment.

Hydrocortisone differentially affects the ability of murine stromal cells and human marrow-derived adherent cells to promote the differentiation of CD34++/CD38- long-term culture-initiating cells.

Very primitive human hematopoietic progenitor cells are identified indirectly by their ability to give rise to clonogenic progenitors in the presence of either human or murine stromal cells. These long-term culture-initiating cell (LTC-IC) assays are usually performed in the presence of hydrocortisone based on the initial observation that hydrocortisone was required for prolonged hematopoiesis in standard long-term bone marrow cultures. In this report, we investigated the role of hydrocortisone in LTC-IC assays initiated with CD34++/CD38- cells seeded onto either human bone marrow LTC-derived adherent cells or a murine marrow-derived stromal cell line, MS-5. It was found that weekly addition of hydrocortisone to the cultures reduced the frequency of LTC-IC (from 1/5 to 1/20) calculated from limiting dilution experiments and also reduced fivefold to 10-fold the number of their progeny clonogenic cells detected after 4 to 5 weeks. In contrast, the frequency and differentiative potential of CD34++/CD38- grown in the presence of human marrow feeders was unaltered by the addition of glucocorticoids. Data are consistent with the hypothesis that hydrocortisone inhibited LTC-IC differentiation by downregulating the expression of a synergistic factor produced by MS-5 cells. (1) In the absence of hydrocortisone, the number of clonogenic progenitors generated by LTC-IC was much higher in cultures seeded on MS-5 than in cultures seeded on human marrow adherent cells, which was also true when cytokines were added to the cocultures. However, based on the phenotype of the colonies, progenitors produced in MS-5 cocultures were more mature than those generated on human marrow adherent cells. (2) Hydrocortisone counteracted the stimulatory effect of recombinant human cytokines (interleukin-3, interleukin-6, and steel factor) in assays performed on MS-5 but not on human marrow feeders. (3) Hydrocortisone led to a 50% decrease in the numbers of colony-forming units-granulocyte-macrophage found in methycellulose colony assays of CD34++/CD38- cells performed in the presence of MS-5 cells. Taken together, our results indicate that hydrocortisone acts differently on a murine stromal cell line and on marrow-derived human stromal cells and may suppress the expression by MS-5 cells of an activity selectively promoting amplification of clonogenic cells derived from primitive LTC-IC.

Age-related alterations in erythroid and granulopoietic progenitors in Diamond-Blackfan anaemia.

Mechanisms involved in the erythroid failure characterizing Diamond-Blackfan anaemia (DBA) remain unidentified. The general consensus is that the defect is intrinsic to the marrow erythroid progenitor, but the target progenitor cell has not been precisely identified, and in vitro studies have revealed considerable heterogeneity between patients. In order to understand better the meaning of such a biological heterogeneity, we examined the in vitro response of erythroid progenitors CFU-E (colony-forming unit-erythroid) and BFU-E (burst-forming unit-erythroid) to erythropoietin (Epo), interleukin-3 (IL-3) and stem cell factor (SCF) in a large series of 24 patients from 1 month to over 20 years of age. Results of colony assays revealed a striking correlation between the age of the patient and the extent of the abnormalities detected in vitro. Therefore, despite profound anaemia, 80% (7/10) of the patients studied within 1 year of diagnosis had normal numbers of both CFU-E and BFU-E which exhibited a normal response to cytokines. In contrast, 12/14 patients followed up for more than 3 years had decreased numbers of erythroid progenitors, in seven cases associated with decreased colony-forming unit granulocyte-macrophage (CFU-GM). The number of CFU-E and BFU-E was not normalized even by the addition of high concentrations of combined Epo, IL-3 and SCF.(ABSTRACT TRUNCATED AT 250 WORDS)

Refractory anaemia and mitochondrial cytopathy in childhood.

We report two cases of childhood myelodysplasia (MDS) related to a mitochondrial (mt) cytopathy that illustrate the difficulty in recognizing such disorders in patients with solely haematological signs. Both patients have refractory anaemia with ring sideroblasts and vacuolization of haemopoietic precursors. These cytological features are similar to those observed in Pearson's disease, recently identified as a mitochondrial disease, and are strongly suggestive of a mitochondrial enzyme defect. The diagnosis of mitochondrial cytopathy was established on Southern blotting of mt DNA, showing a mt DNA deletion, or on the impairment of the respiratory chain enzyme activities. The absence of cytogenic abnormalities, and the polyclonal pattern of peripheral neutrophil and lymphocyte fractions, suggest that, in mt cytopathies, MDS cannot be considered as a truly malignant disorder.

A murine stromal cell line allows the proliferation of very primitive human CD34++/CD38- progenitor cells in long-term cultures and semisolid assays.

Analysis of molecular mechanisms associated with stem cell commitment and differentiation requires an in vitro assay that identifies the most primitive hematopoietic stem cells in human bone marrow. Such primitive stem cells usually do not form colonies in short-term semisolid assays and are best identified by their ability to initiate sustained hematopoiesis when they are cocultured with competent stromal cells. In this study, we investigated whether a murine marrow stromal cell line (MS-5) that supports colony-forming unit-spleen (CFU-S) maintenance would permit, both in short-term colony assays and long-term cultures, the development of primitive human stem cells sorted on the basis of their high expression of CD34 and lack of expression of CD38 antigen. In short-term colony assays, this population included almost exclusively primitive progenitor cells. MS-5 cells synergized with any combination of interleukin-3, Steel factor, granulocyte colony-stimulating factor, agar-leukocyte conditioned medium, and erythropoietin and increased at least twofold both the cloning efficiency of CD34++/CD38- cells and the size of the colonies. Furthermore, MS-5 cells triggered the development of multipotent blast cell progenitors with a high proliferative potential, which in these conditions represented 1% to 2% of CD34++/CD38- cells. When MS-5 cells were substituted by human stromal cells or when growth factor combinations were used in the absence of stromal cells, much lower numbers of CFU-blast were detected. This selective action of MS-5 on early progenitors was also observed when MS-5 cells were used as feeders in long-term cultures of CD34++/CD38- cells. Murine cells promoted the expansion of high proliferative potential primitive progenitor cells up to 3 months, although they did not support their differentiation in mature clonogenic progenitors or terminally differentiated cells. Sustained hematopoiesis in these longterm cultures was accounted for by 2% to 5% of initial CD34++/CD38- cells as estimated by limiting dilution experiments. Mechanisms by which murine stromal cells act specifically on human primitive stem cells are unclear, but from our data this effect is unlikely to be explained solely by known species cross-reactive growth factors. Further manipulation of this long-term coculture system should prove useful in identifying stromal molecules regulating commitment and differentiation of early human progenitor cells.

Diamond Blackfan anaemia: apparent relapse due to B19 parvovirus.

A spontaneous remission occurred after 3 years of regular transfusions in a cortico resistant Diamond Blackfan anaemia. However after 1 year of transfusion independence an apparent relapse was observed which was transient and could be attributed to a B19 parvovirus primary infection. Previous inapparent impairment of erythropoiesis can be transiently spotlighted by B19 parvovirus primary infection.

Automated quantitation of cell density distribution and hyperdense cell fraction in RBC disorders.

An altered state of cell hydration is a hallmark of a number of RBC disorders. The two parameters most often used to characterize the state of cell hydration are (a) the complete cell density distribution profile, and (b) the fraction of cells with densities greater than a defined value. Buoyant density cell fractionation with a variety of polymers has long been the preferred method for obtaining data on cell density distribution. Although this method provides accurate and quantitative information on the state of RBC hydration, its applicability has been limited due to the time-consuming experimental procedure involved in generating the data. A recently developed light-scattering method has been used in the present study to quantitate RBC density distribution. This new method accurately quantitates both the cell density distribution profile and the fraction of dense, dehydrated cells in various RBC disorders. The ability of the automated method to generate this information rapidly makes possible objective testing of different hypotheses concerning the contributions of cell hydration and dehydration to the pathophysiology of various RBC disorders.