High alloreactivity of low-dose prophylactic donor lymphocyte infusion in patients with acute leukemia undergoing allogeneic hematopoietic cell transplantation with an alemtuzumab-containing conditioning regimen.

The value of prophylactic donor lymphocyte infusion (pDLI) is unclear and differs among diseases and transplantation protocols. Experience with this approach in patients with acute leukemia undergoing hematopoietic cell transplantation (HCT) with an alemtuzumab-incorporating conditioning protocol is lacking. We conducted a single-center prospective study to investigate the applicability and efficacy of prophylactic donor lymphocyte infusion (pDLI) in patients with leukemia undergoing HCT with a low-dose alemtuzumab-containing conditioning regimen. Inclusion criteria were high-risk acute myelogenous leukemia, acute lymphoblastic leukemia, or increasing mixed chimerism. All patients included were tapered off of immunotherapy. Exclusion criteria were a history of ≥ grade II or active graft-versus-host disease (GVHD). Of the 56 consecutive patients who underwent HCT with an alemtuzumab-containing regimen, 15 patients (8 with acute myelogenous leukemia and 7 with acute lymphoblastic leukemia) met the study inclusion criteria and received prophylactic DLI (total of 45 infusions) from 7 sibling donors and 8 unrelated donors. The first infusion was given at a median of 162 days posttransplantation. The median number of DLIs was 3, and the median cumulative CD3(+) cell dose was 2 × 10(6)cells/kg. Six of the 8 patients (75%) who received pDLI while in mixed chimerism converted to stable, complete donor chimerism. Some 47% of DLI recipients developed GVHD (4 acute GVHD and 3 with chronic GVHD) after a median cumulative dose of 2 × 10(6) CD3(+) cells/kg. After a median follow-up of 575 days, 11 (73%) pDLI recipients were alive. All 4 deaths were due to GVHD-related causes. None of the patients who received pDLIs relapsed. Patients with leukemia who received low-dose pDLI after conditioning with alemtuzumab are at low risk for relapse; however, this approach is associated with a relatively high incidence of severe GVHD.

Horizontal DNA transfer from donor to host cells as an alternative mechanism of epithelial chimerism after allogeneic hematopoietic cell transplantation.

Animal and human studies have shown that after allogeneic hematopoietic cell transplantation, epithelial cells containing donor-derived genome emerge. The mechanisms underlying this phenomenon are still unclear. We hypothesized that horizontal transfer of the hematopoietic donor-DNA to the host epithelium confers a possible operating mechanism. In an in vitro model mimicking the lymphocyte-epithelial interaction, we cocultivated keratinocyte HaCaT cells (Y-chromosome negative) with nonapoptotic or apoptotic, CMFDA, or BrdU-labeled hematopoietic Jurkat cells (Y+) and looked for the emergence of HaCaT cells bearing Jurkat genome. We found that DNA can be horizontally transferred from hematopoietic to epithelial cell lines through phagocytosis of apoptotic bodies. The ingested genomic material was also found within the nuclear compartment and in isolated chromosomes obtained from HaCaT metaphases. Both lysosomal inhibition in HaCaT cells and repetitive load of HaCaT cells with apoptotic bodies increased the intercellular and intranuclear DNA delivery. Although recipient cells remained viable and showed to express the foreign DNA, this expression was transient. Taking into consideration these findings of horizontal DNA transfer between hematopoietic and epithelial cells, we evaluated by quantitative microsatellite analysis the amount of donor DNA in 176 buccal swabs obtained from 71 patients after allogeneic transplantation. We found a high amount of donor-DNA (mean 26.6%) in the majority (89.7%) of them, although no donor hematopoietic cells were evident in the samples by immunofluorescence. We propose that the incessant charge of the transplant recipient with donor-DNA and its "inappropriate" intranuclear delivery in host epithelium may explain the emergence of epithelial cells with donor-derived genome.

Disease-related anemia in chronic lymphocytic leukemia is not due to intrinsic defects of erythroid precursors: a possible pathogenetic role for tumor necrosis factor-alpha.

BACKGROUND/AIMS: Disease-related anemia in chronic lymphocytic leukemia (CLL) occurs when the obvious causes are excluded while its pathogenesis is still obscure. We investigated its underlying mechanisms in 56 untreated patients with CLL. METHODS: Bone marrow (BM) lymphocytic infiltration was estimated in trephine biopsies. Serum erythropoietin (EPO) and tumor necrosis factor-alpha (TNF-alpha) levels were measured by ELISA. The potential of BM CD34+ to differentiate into erythroid cells was evaluated by methylcellulose-based assays and in liquid cultures supplemented with EPO, SCF, IL-3 +/- TNF-alpha. The response of erythroid precursors to EPO +/- TNF-alpha was assessed by detecting activated key proteins of EPO-EPO receptor signalling pathway using Western Blot and EMSA. RESULTS: Bone marrow lymphocytic infiltration was not exclusively responsible for disease-related anemia and CD34+ cells were intrinsically capable of generating erythroid precursors. Also, no deficiency of serum erythropoietin (EPO) or defective intracellular response of erythroid precursors to EPO +/- TNF-alpha stimulation was observed. Serum TNF-alpha levels were found increased in anemic CLL patients and TNF-alpha appeared to directly inhibit the erythroid development in early stages of erythropoiesis. CONCLUSION: We concluded that CLL-related anemia was not due to intrinsic defects of erythroid precursors, but might result from the direct suppressive effect of TNF-alpha on the erythroid production.

Dry-reagent disposable dipstick test for visual screening of seven leukemia-related chromosomal translocations.

We report the first dry-reagent, disposable, dipstick test for molecular screening of seven chromosomal translocations associated with acute and chronic leukemia. The dipstick assay offers about 10 times higher detectability than agarose gel electrophoresis and, contrary to electrophoresis, allows confirmation of the sequence of the polymerase chain reaction (PCR) product by hybridization within a few minutes without the need of instrumentation. Biotinylated amplified DNA is hybridized with a dA-tailed probe and applied to the strip, which contains oligo(dT)-conjugated gold nanoparticles in dry form. Upon immersion of the strip in the appropriate buffer, the solution migrates and the hybrids are captured by immobilized streptavidin at the test zone generating a characteristic red line. The excess nanoparticles are captured by oligo(dA) strands immobilized at the control zone of the strip producing a second red line. We studied the: t(9;22)(q34;q11), t(15;17)(q22;q21), t(11;17)(q23;q21), t(5;17)(q32;q21), t(11;17)(q13;q21), t(8,21)(q22;q22) and inv(16)(p13;q22) that generate the BCR-ABL, PML-RARa, PLZF-RARa, NPM-RARa, NuMA-RARa, AML1-ETO and CBFbeta-MYH11 fusion genes, respectively. A single K562 cell was detectable amidst 10(6) normal leukocytes. A dipstick test was developed for actin, as a reference gene. The dipstick assay with appropriate probes can be used for identification of the fusion transcripts involved in the translocation.

A perspective for biomedical data integration: design of databases for flow cytometry.

BACKGROUND: The integration of biomedical information is essential for tackling medical problems. We describe a data model in the domain of flow cytometry (FC) allowing for massive management, analysis and integration with other laboratory and clinical information. The paper is concerned with the proper translation of the Flow Cytometry Standard (FCS) into a relational database schema, in a way that facilitates end users at either doing research on FC or studying specific cases of patients undergone FC analysis RESULTS: The proposed database schema provides integration of data originating from diverse acquisition settings, organized in a way that allows syntactically simple queries that provide results significantly faster than the conventional implementations of the FCS standard. The proposed schema can potentially achieve up to 8 orders of magnitude reduction in query complexity and up to 2 orders of magnitude reduction in response time for data originating from flow cytometers that record 256 colours. This is mainly achieved by managing to maintain an almost constant number of data-mining procedures regardless of the size and complexity of the stored information. CONCLUSION: It is evident that using single-file data storage standards for the design of databases without any structural transformations significantly limits the flexibility of databases. Analysis of the requirements of a specific domain for integration and massive data processing can provide the necessary schema modifications that will unlock the additional functionality of a relational database.

Prevalence of anti-HAV antibodies in multitransfused patients with beta-thalassemia.

AIM: To detect the prevalence of anti-HAV IgG antibodies in adult multitransfused beta-thalassemic patients. METHODS: We studied 182 adult beta-thalassemic patients and 209 controls matched for age and sex from the same geographic area, at the same time. Anti-HAV IgG antibodies, viral markers of hepatitis B virus (HBV) and hepatitis C virus (HCV) infection were evaluated. RESULTS: Anti-HAV IgG antibodies were detected more frequently in thalassemic patients (133/182; 73.1%) than in healthy controls (38/209; 18.2%, P < 0.0005). When we retrospectively evaluated the prevalence of anti-HAV IgG antibodies in 176/182 (96.7%) thalassemic patients, whose medical history was available for the previous ten years, it was found that 83 (47.2%) of them were continuously anti-HAV IgG positive, 16 (9.1%) acquired anti-HAV IgG antibody during the previous ten years, 49 (27.8%) presented anti-HAV positivity intermittently and 28 (15.9%) were anti-HAV negative continuously. CONCLUSION: Multitransfused adult beta-thalassemic patients present higher frequency of anti-HAV IgG antibodies than normal population of the same geographic area. This difference is difficult to explain, but it can be attributed to the higher vulnerability of thalassemics to HAV infection and to passive transfer of anti-HAV antibodies by blood transfusions.

Influence of donor/recipient sex matching on outcome of allogeneic hematopoietic stem cell transplantation for aplastic anemia.

BACKGROUND: Increased risk of transplant related mortality in male recipients of female hematopoietic stem cell grafts and in vitro reactivity of lymphocytes against H-Y encoded gene products in females with rejected male grafts have been documented. An increased rejection of male grafts in female recipients is not reported for solid organ or stem cell transplants and the role of H-Y as transplantation antigen has been controversial. METHODS: Data from 1481 patients with a hematopoietic stem cell transplant for aplastic anemia reported from 154 centers in 28 countries were analyzed. Outcome was compared between patients with donors of the same or opposite sex. RESULTS: Survival at 5 years was significantly better in patients with donors from the same sex: 68% vs. 60% (P = 0.001). Male patients with female donors had a decreased survival (relative risk of death 1.52, P < 0.001) and an increased risk of severe graft-versus-host disease (relative risk 1.33, P = 0.03) compared to recipients of sex-matched grafts. Female patients with male donors had a decreased survival (relative risk of death 1.44, P = 0.01) and an increased risk of rejection (relative risk 2.20, P = 0.01) compared to recipients of sex-matched grafts. In a subgroup analysis, the negative effects of donor/recipient sex-mismatching appeared confined to patients receiving conditioning regimens not containing antithymocyte globulin. CONCLUSIONS: These data confirm H-Y as a clinically relevant transplantation antigen, in both the graft-versus-host and the host-versus-graft direction. Wherever possible, donor-recipient sex-matching should be integrated into donor selection algorithms.

A probable role for trail-induced apoptosis in the pathogenesis of marrow failure. Implications from an in vitro model and from marrow of aplastic anemia patients.

Aplastic anemia (AA) is a syndrome of hematopoietic failure involving increased apoptosis of stem cells. In order to investigate the molecular mechanisms participated in the process of marrow failure, we created an in vitro model of hematopoietic cell suppression, by continuous addition of TNF-alpha and IFN-gamma in an vitro long-term bone marrow culture system. An up-regulation of Fas expression was observed in CD34+ cells in cytokine treated cultures, compared to controls. This was accompanied by significant TRAIL and decreased caspase 3 mRNA expression, whereas the expression of Bcl-2 family members was low (Bcl-xl) or absent (Bcl-2, Bax). The expression of these apoptotic genes was also investigated in aplastic anemia patients. Apart from Fas mRNA expression in total marrow and/or CD34+ cells, TRAIL mRNA expression was found only in CD34+ cells in active disease while in total marrow cell compartment this remains a constant finding even in patients in remission. The above data are in agreement with previous studies proposing a major role for the extrinsic apoptosis pathway in the pathogenesis of aplastic anemia and additionally introduce TRAIL as a probable important molecule in the process.

Expression and inducibility of cytoprotective heat shock proteins in the bone marrow of patients with myelodysplastic syndrome: correlation with disease progression.

We determined the intracellular expression and inducibility of heat shock proteins (Hsps) 72, 73 and 27 in the bone marrow of patients with myelodysplastic syndrome (MDS) and controls. Hsps were overexpressed in MDS marrow especially in advanced disease, providing resistance to induction of apoptosis. These data suggest that Hsps could be implicated in the progression of MDS to acute myeloid leukemia.

Heart failure in beta-thalassemia syndromes: a decade of progress.

The thalassemias are common monogenic disorders of hemoglobin synthesis. beta-thalassemias are the most important among the thalassemia syndromes and have become a worldwide clinical problem due to an increasing immigrant population. In beta-thalassemia major, regular blood transfusions are necessary early in life. Beta-thalassemia intermedia refers to a less severe phenotype, whereas beta-thalassemia/hemoglobin E disease encompasses a broad phenotypic spectrum. Blood transfusions and increased gastrointestinal iron absorption result in iron overload and tissue damage. Among patients with beta-thalassemia major, biventricular, dilated cardiomyopathy remains the leading cause of mortality. In some patients, a restrictive type of left ventricular cardiomyopathy or pulmonary hypertension is noted. The clinical course, although variable and occasionally fulminant, is more benign in recent than in older series. Myocarditis has been described as a cause of left-sided heart failure in younger patients. Pulmonary arterial hypertension is the principal cause of heart failure in beta-thalassemia intermedia. Chelation therapy has improved prognosis in beta-thalassemia major both by reducing the incidence of heart failure and by reversing cardiomyopathy. Estimation of the patient's cardiac risk is mainly based on clinical criteria and serial echocardiography. A new cardiovascular magnetic resonance technique will probably fulfill the need for more precise risk stratification in beta-thalassemia syndromes. By increasing the proportion of patients on optimal chelation, survival in beta-thalassemia major may further improve. Recent advances in gene therapy are expected to result in the long-awaited cure of this disease.

Serum lipid profile and hepatic steatosis of adult beta-thalassaemia patients with chronic HCV infection.

OBJECTIVES: The aim of this study was to evaluate the serum lipid profile and to assess the prevalence of hepatic steatosis in adult beta-thalassaemic patients with chronic hepatitis C virus (HCV) infection. METHODS: Thirty-five adult HCV infected, multi-transfused, beta-thalassaemia patients (beta-HCV patients), 63 otherwise normal patients with chronic HCV infection (HCV patients) and 54 beta-thalassaemia patients without chronic viral hepatitis (beta patients) were studied. Total cholesterol, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglycerides, viral markers and liver histology were evaluated. RESULTS: Serum total cholesterol, HDL-C and LDL-C were found at significantly lower levels in beta-HCV and beta patients than in HCV patients. Triglyceride levels were significantly lower in the HCV group compared with the beta group. Nine (25.7%) of the 35 beta-HCV patients had mild hepatic steatosis. Thirteen (23.6%) of 55 HCV patients presented mild and 4/55 (7.3%) moderate hepatic steatosis. None of the beta group presented steatosis. When we compared beta-HCV and HCV patients with steatosis, we found that beta-HCV patients had a lower degree of steatosis (11.1+/-7% vs 22.9+/-17.2%, P=0.021). Multivariate logistic regression analysis showed that the only independent predictor associated with hepatic steatosis in beta-HCV and HCV patients was genotype 3a (OR, 3.61; 95% CI, 1.22-10.71, P=0.021). CONCLUSIONS: Adult beta-thalassaemia patients, compared to other patients with chronic HCV infection, present lower cholesterol levels (total cholesterol, HDL, LDL) and similar frequency but a lower degree of hepatic steatosis. This difference in the degree of steatosis is most likely due to the higher prevalence of genotype 3a in the non-beta-thalassaemia group.

Impaired clonogenic growth of myelodysplastic bone marrow progenitors in vitro is irrelevant to their apoptotic state.

Excessive intramedullary apoptosis has been considered to account for the paradox of hypercellular marrow and refractory cytopenias in myelodysplastic syndrome (MDS). However, a causative relationship of apoptosis to the progenitor's defective clonogenic growth has not been sufficiently demonstrated. We investigated the degree of apoptosis and its contribution to ineffective hematopoiesis in MDS, by assessing the differential clonogenic capacity of purified "apoptotic" and "non-apoptotic" bone marrow progenitors in a short-term semisolid culture system. Although increased apoptosis was indeed detected in MDS bone marrow progenitors, there was no correlation between the existence of apoptosis and culture performance. Non-apoptotic as well as apoptotic CD34+ cells gave similar patterns of growth, both defective compared to normal. The ability of "apoptotic" CD34+ cells to proceed in colony formation as well as the abnormal growth of "non-apoptotic" progenitors are probably pointing towards the need to reconsider the role of apoptosis in the defective clonogenicity of MDS.

Markers of endothelial and in vivo platelet activation in patients with essential thrombocythemia and polycythemia vera.

We investigated endothelial and in vivo platelet activation in a cohort of 52 patients with essential thrombocythemia (ET) and polycythemia vera (PV) before and after cytoreductive treatment, 22 healthy controls, and 17 patients with acute cerebrovascular ischemia (ACVI) and normal platelet counts. We measured platelet expression of CD62P and CD63 antigens and levels of soluble vascular cell adhesion molecule 1 (sVCAM-1). We found increased in vivo platelet activation in all patients with ET and PV, both before and after cytoreductive treatment, compared with controls. In patients with arterial thrombosis, platelet expression of CD62P, and in patients with erythromelalgia, expression of both markers was higher compared with expression in patients without thrombotic complications. In patients with ET and PV before and after treatment, sVCAM-1 expression was increased compared with expression in controls but also compared with expression in patients with ACVI and normal platelet counts. In patients with arterial thrombosis and erythromelalgia, in vivo platelet activation correlated with the level of sVCAM-1. Our findings indicated that in vivo platelet activation reflects intrinsic platelet defects in patients with ET and PV, persists after cytoreductive treatment, and results in endothelial damage, probably through release of angiogenic factors and/or activation of white blood cells.

The functional role of S/MARs in episomal vectors as defined by the stress-induced destabilization profile of the vector sequences.

The scaffold/matrix attachment regions (S/MARs) are chromosomal elements that participate in the formation of chromatin domains and have origin of replication support functions. Because of all these functions, in recent years, they have been used as part of episomal vectors for gene transfer. The S/MAR of the human beta-interferon gene has been shown to support efficient episome retention and transgene expression in various mammalian cells. In Jurkat and other cells, DNA plasmid vectors containing Epstein-Barr virus origin of replication (EBV OriP) and the EBV nuclear antigen-1 gene mediate prolonged episome retention in the host cell nucleus, which, however, diminishes over time. In order to enhance retention, we combined this system with an S/MAR element. Unexpectedly, this completely eliminated the capacity of episomes to replicate. Calculation of the stress-induced DNA duplex destabilization profile of the vectors suggested that the S/MAR element had created an increase in molecular stability at the OriP site that may have disturbed replicative potential. In contrast, introduction of an alternative initiation of replication region from the beta-globin gene locus, instead of EBV OriP and the EBV nuclear antigen-1 gene, restored replicative capacity and enhanced episome retention mediated by the S/MAR. These effects were associated with a destabilization profile at the initiation of replication region. These data demonstrate a correlation between S/MAR-mediated vector retention and the presence of an unstable duplex at a replication origin, in this particular setting. We consider that the calculation of stress-induced duplex destabilization may be an informative first step in the design of units that replicate extrachromosomally, particularly as the latter present a safer and, therefore, attractive alternative to integrating viral vectors for gene therapy applications.

Cardiac mortality in {beta}-thalassemia major: resting but not dobutamine stress echocardiography predicts mortality among initially cardiac disease-free patients in a prospective 12-year study.

AIMS: Cardiac death remains the principal cause of mortality in beta-thalassemia major (beta-TM). Echocardiography may provide additional information, incremental to haematological profile, both for guiding chelation therapy and to assess prognosis. METHODS AND RESULTS: Between 1993 and 1995, 36 patients with beta-TM and normal cardiac function and 25 normal volunteers underwent evaluation using resting and dobutamine stress echocardiography (DSE). Dobutamine stress echocardiography was performed at baseline and repeated after 2 years. The primary endpoint was cardiac mortality. During a 12-year observation period, seven patients (19%) died from heart failure. All seven deaths occurred among the cohort of 12 patients with median ferritin concentrations >or= 2800 ng/mg. In addition, a resting left ventricular ejection fraction (LVEF) < 60% was also associated with increased late mortality. In multivariate analysis, increased serum ferritin levels and reduced LVEF but not DSE or other haematological variables were independent survival determinants. CONCLUSION: Resting LVEF provides prognostic information that is additional to ferritin levels among patients with beta-TM.

Global vasomotor dysfunction and accelerated vascular aging in beta-thalassemia major.

BACKGROUND: Patients with beta-thalassemia major (beta-TM) demonstrate an increased incidence of vascular complications, which are thought to result from a procoagulant/proinflammatory environment. We investigated the arterial vasorelaxing capacity and sought for early carotid atherosclerosis and underlying pathophysiological correlates in these transfusion-dependent patients. METHODS AND RESULTS: The vasodilatory properties of the brachial artery and the carotid intima-media thickness (IMT) were examined with ultrasonography in 35 non-diabetic young adults with beta-TM (patient group) and 35 control subjects (control group). Among thalassemic patients, both endothelium-dependent (FMD) and -independent dilatation (FID) as well as their ratio was impaired, whereas IMT was increased (p<0.01). Patients on optimal, as compared with those on non-optimal chelation treatment had a non-significantly lower IMT. Vasodilatory capacity in the patient group was inversely correlated with IMT and independently associated either with the quality of chelation therapy (FMD) or serum ferritin levels (FID). Plasma concentrations of D-dimers, circulating markers of endothelial activation, inflammation and apoptosis were higher, while plasma cholesterol and fibrinogen levels were lower-than-normal in the patient group. Independent predictors of IMT among thalassemic patients were tumor necrosis factor-alpha levels and age. CONCLUSIONS: Young adults with beta-TM exhibit both a global impairment of arterial vasorelaxation and early carotid atherosclerosis. A procoagulant/proinflammatory state in these transfusion-dependent patients may overwhelm atheroprotective mechanisms, including an optimal chelation regimen, and promote vascular injury and atherogenesis.

Inappropriately low erythropoietin response for the degree of anemia in patients with noninsulin-dependent diabetes mellitus.

We investigated erythropoietin (Epo) response in a cohort of diabetic patients with various types of anemia to approach the pathogenesis of some cases of "unexplained" anemia encountered among diabetics. Serum Epo levels were determined totally in 747 evaluable subjects with normal renal and hepatic function, of whom 694 had anemia. Among anemic patients, 237 were diabetics, while among the 53 nonanemic persons, there were also 21 diabetics. Diabetic and nondiabetic subjects were uniformly balanced in relation to their demographic features and were categorized according to the etiology of their anemia. Hemoglobin (Hb) did not differ between diabetic and nondiabetic subjects in all the etiological groups and in the whole population. Diabetic patients had significantly lower serum Epo levels as compared to nondiabetics (36.5+/-61 vs 69.4+/-191 IU/ml, p<0.0001), and this was true for all etiologic groups of anemia with the exception of patients with myeloproliferative disorders and those with megaloblastic anemia. The natural logarithmic (ln)-EpoxHb component was used as an index of response to anemia and was found to be significantly decreased in almost all subgroups of diabetic patients. Serum Epo levels were also negatively correlated with the percentage of glycosylated Hb, HbA1(C) (r=-0.446), and the correlation was stronger with the ln of serum Epo (r=-0.638, p<0.001). Inappropriately low serum Epo level is a uniform feature in patients with type II diabetes mellitus and may represent a constitutive blunted response to anemia or an altered metabolic rate of Epo, probably as a result of abnormal glycosylation of the cytokine.

Engraftment of NOD/SCID-beta2 microglobulin null mice with multilineage neoplastic cells from patients with myelodysplastic syndrome.

The development of immunodeficient mouse xenograft models has greatly facilitated the investigation of some human hematopoietic malignancies, but application of this approach to the myelodysplastic syndromes (MDSs) has proven difficult. We now show that cells from most MDS patients (including all subtypes) repopulate nonobese diabetic-severe combined immunodeficient (scid)/scid-beta2 microglobulin null (NOD/SCID-beta2m(-/-)) mice at least transiently and produce abnormal differentiation patterns in this model. Normal marrow transplants initially produce predominantly erythroid cells and later predominantly B-lymphoid cells in these mice, whereas most MDS samples produced predominantly granulopoietic cells. In 4 of 4 MDS cases, the regenerated cells showed the same clonal markers (trisomy 8, n = 3; and 5q-, n = 1) as the original sample and, in one instance, regenerated trisomy 8(+) B-lymphoid as well as myeloid cells were identified. Interestingly, the enhanced growth of normal marrow obtained in NOD/SCID-beta2m(-/-) mice engineered to produce human interleukin-3, granulocyte-macrophage colony-stimulating factor, and Steel factor was seen only with 1 of 7 MDS samples. These findings support the concept that human MDS originates in a transplantable multilineage hematopoietic stem cell whose genetic alteration may affect patterns of differentiation and responsiveness to hematopoietic growth factors. They also demonstrate the potential of this new murine xenotransplant model for future investigations of MDS.

The beta-globin C-->G mutation at 6 bp 3' to the termination codon causes beta-thalassaemia by decreasing the mRNA level.

We have studied the expression of the silent beta-thalassaemia term+6 (C-->G) mutation, at nucleotide 6 after the stop codon within the human beta-globin 3' untranslated regions (3'UTR), by stable transfection in murine erythroleukaemia (MEL) cells. Steady state mRNA levels from transfected MEL cells containing the term+6 mutant allele were reduced by 52-60%, compared with those obtained from the normal beta-globin gene, in both total and cytoplasmic RNA fractions, showing that the mutation itself is responsible for the similar data obtained from patients. Upon analysis of nuclear RNA, the term+6 mutation was found to also lower the ratio of cleaved/uncleaved transcripts by 22-30%, thus revealing that it interferes with correct 3'-end formation of beta-globin mRNA. The term+6 mutation lies within a polypyrimidine track, similar to that in the beta-intervening sequence II (beta-IVSII), which is known to be an important contributor to the promotion of premRNA 3'-end formation. We propose that the two polypyrimidine tracks flanking the translated region of exon III of the human beta-globin gene may co-operate during beta-globin mRNA biogenesis.

Defective tumor necrosis factor alpha-induced maturation of monocyte-derived dendritic cells in patients with myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are clonal stem cell disorders, characterized by ineffective and dysplastic hematopoiesis. MDS patients have a defective immune response manifested by increased susceptibility to bacterial infections, autoimmune phenomena, and high incidence of lymphoid malignancies. Presently, we investigated the phenotype and function of monocyte-derived dendritic cells (MoDC) in 23 MDS patients and 15 controls at different stages of differentiation using the maturation stimuli tumor necrosis factor-alpha (TNF-alpha) and LPS. Monocytes from MDS patients showed low potential to differentiate into dendritic cells (DC), as determined by low cell yield and CD1a expression. MDS-MoDCs exhibited low expression of mannose receptor and reduced endocytic capacity. MDS-MoDCs showed a diminished response to TNF-alpha with low CD83, CD80, and CD54 expression and allostimulatory capacity. In patients with 5q syndrome, monocytes and MoDCs were positive for the 5q deletion, suggesting their origin from the malignant clone. Our data indicate that MoDCs in MDS display quantitative and functional abnormalities that may contribute to the defective immune response of these patients.