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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.

We evaluated the effect of the human immunodeficiency virus (HIV) protease inhibitor saquinavir on the imatinib-sensitive and imatinib-resistant chronic myelogenous leukemia cell lines. Saquinavir, which is also a proteasome blocker, showed dose- and time-related anti-proliferative activity, particularly on the imatinib-resistant lines and a pro-apoptotic effect. Association with imatinib caused a significant increase of activity.

Growing attention has been focused on cord blood as a source of transplantable hematopoietic stem cells. However, clinical experience is rather limited. In this study we describe a child with advanced acute lymphoblastic leukemia who received an HLA-haploidentical cord blood transplant. The patient was transplanted in third complete remission after conditioning with fractionated total body irradiation, thiotepa and cyclophosphamide. Forty-one milliliters of cryopreserved umbilical cord blood, containing 0.15 x 10(8) nucleated cells/kg and 0.25 x 10(4) CFU-GM/kg, were infused. Cyclosporine and prednisone were administered for graft-versus-host disease (GVHD) prophylaxis. The patient received G-CSF from day +1 to day +35, but no improvement in granulocyte counts was observed. Therefore, administration of GM-CSF was started on day +36 to day +59, which resulted in a significant increase in white blood cells and granulocyte counts. Sustained myeloid engraftment was evidenced by a granulocyte count >0.5 x 10(9)/1 by day +41. The presence of donor-derived cells could be documented in the peripheral blood and bone marrow of the patient by cytogenetic analysis, HCA phenotyping and DNA studies. Forty-one days after transplant, clonogenic bone marrow assays showed the presence of low frequencies of primitive hematopoietic progenitor cells (BFU-E = 19/10(5) and CFU-GM = 8/10(5)). The chimerism was complete and no host-derived cells could be detected. However, the engraftment was restricted to the myeloid lineage whereas lymphoid and megakaryocytic engraftments were inadequate. The immunophenotype of the patient's peripheral blood showed the presence of T lymphocytes expressing an immature phenotype (CD2+ CD3-) at day +21. Among the subset of T cells expressing the CD3 antigen, the majority were CD45RO+ memory cells and a significant proportion was positive for the activation markers CD25, CD69 and HLA-DR, suggesting that these T cells had been activated in vivo. On day +41, a mild cutaneous rash consistent with grade I acute GVHD resolved with steroid treatment. Unfortunately, the patient died of hepatic insufficiency and renal failure 84 days after transplantation. Autopsy showed a multiorgan failure with hepatic veno-occlusive disease (VOD). Overall, this study shows that (1) cord blood transplantation from an HLA-mismatched donor can be performed with a low risk of acute GVHD, and (2) a small volume of cord blood containing a reduced number of CFU-GM is sufficient to achieve engraftment.

Cord blood (CB) is a valuable source of stem cells. Most CB units are still cryopreserved in single bags in the world's CB banks. Thawing a single CB unit, dividing it into two parts, expanding the smaller one and refreezing the other would optimize ex vivo expansion of CB progenitors prior to transplantation: expanded and unexpanded cells could be infused together to accelerate early engraftment.The feasibility of refreezing CB samples was investigated by evaluating the effect of 3 successive cryopreservation procedures in 9 CB units. The number and viability of WBC, BFU-E, CFU-GM, CFU-MIX, LTC-IC, and the absolute CD34+ cell count were assessed at time 0 and after each thawing. The percentage of CD34 cells expressing CD38, L-selectin, VLA-4, VLA-5, H-CAM, LFA-1 and CXCR4 was also evaluated.After three freezing and thawing procedures, WBC counts decreased, while lymphocytes were unchanged. Viability was 90% of basal values after the first thawing and did not change. BFU-E decreased significantly only after the third thawing. CFU-GM and CFU-MIX did not change significantly, nor did LTC-IC, CD34+ cell counts and CAM and CXCR4 expression on CD34+/ CD38-- cells.These data show that two successive freeze-thaw procedures do not significantly affect the clonogenic potential and CAM expression of cord blood progenitors. This information could be exploited to devise new options in ex vivo expansion procedures and quality controls prior to transplantation.

Granulocyte colony-stimulating factor (G-CSF) has been shown to improve the neutropenic status of patients with bone marrow failure. The side effects in prolonged treatment still need to be determined.We have studied the efficacy and the long-term side effects of G-CSF in four patients with Fanconi's anemia and severe neutropenia.Three patients responded with an increase in their absolute neutrophil count; neither improvement in platelet count and hemoglobin concentration nor effect on transfusion requirements was seen. CFU-GM and BFU-E were undetectable before, during and after treatment. Responders showed an important reduction in number and severity of infections, with a marked improvement of clinical status. The fourth patient developed acute myeloid leukemia after 4 weeks of G-CSF treatment. During maintenance, one patient was treated with G-CSF for 18 months, until she received bone marrow transplantation, without presenting side effects. In the second responding patient G-CSF treatment was stopped because of appearance of immature cells in peripheral blood and myeloid blasts in bone marrow. The third responding patient presented immature peripheral myeloid cells during the third year of G-CSF treatment: disappearance of immature cells was observed after G-CSF reduction. In two cases FISH analysis revealed monosomy 7 after G-CSF treatment.G-CSF use results in an improvement of clinical status, but long term administration may cause adverse experiences and requires a close hematological monitoring.

Seven cord blood (CB) units were tested for their capacity to repopulate irradiated NOD/SCID mice after one or two successive cryopreservation procedures. In primary transplants with frozen or refrozen CB cells we observed equivalent human colonies and percentages of human CD45+ cells, with multilineage engraftment. In secondary transplants flow cytometry and polymerase chain reaction for the a satellite region of chromosome 17 showed equivalent levels of human engraftment. Since CB units have, to date, mainly been stored in individual bags, our results suggest new options for optimizing the timing of infusions of expanded and non-expanded progenitors in transplants.