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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Clinical relevance of balance between type 1 and type 2 immune responses of lymphocyte subpopulations in aplastic anaemia patients.

Immune dysfunction, which leads to the suppression of haemopoiesis by cytokines that are secreted by activated T lymphocytes, is considered to play a key role in the pathogenesis of acquired aplastic anaemia (AAA). We investigated the intracytoplasmic expression of type-1 [interferon gamma (IFN-gamma), interleukin (IL)-2] and type-2 (IL-4, IL-10) cytokines in CD4+ and CD8+ T cells before and after in vitro activation in 16 patients with AAA and 17 normal controls. Untreated or refractory patients had a significantly higher proportion of unstimulated CD4+ and CD8+ T cells that produced IFN-gamma and IL-2 whereas the IL-4 and IL-10 producing T cells did not differ from that of controls, resulting in a shift of IFN-gamma/IL-4 ratio towards a type-1 response. Patients in remission had also increased proportion of IFN-gamma-producing unstimulated CD4+ and CD8+ cells, with a parallel rise of IL-4- and IL-10-producing cells and normal IFN-gamma/IL-4 ratio. These data indicate that, in newly diagnosed and refractory patients with AAA, CD4+ cells are polarized towards a type-1 response that in turn leads to activation of cytotoxic CD8+ cells and finally to haemopoietic stem cell destruction. The type-1 response persists in patients in remission although this effect is compensated by the increase of IL-4 and IL-10 production.

Adult chronic idiopathic thrombocytopenic purpura (ITP) is the manifestation of a type-1 polarized immune response.

Derangement of cellular immunity is central in the pathophysiology of adult autoimmune/idiopathic thrombocytopenic purpura (ITP). Herein we investigated cytokine gene expression in peripheral blood mononuclear cells (PBMCs) of adult chronic ITP patients and attempted to correlate cytokine polarization with the degree of thrombocytopenia. We used semiquantitative reverse-transcriptase-polymerase chain reaction (RT-PCR) to measure the expression of type-1 (interleukin-2 [IL-2], interferon gamma [IFN-gamma]) and type-2 (IL-4, IL-5, IL-10, IL-3, IL-13) cytokines by PBMCs from 21 patients and 11 controls. Plasma transforming growth factor beta1 (TGF-beta1) levels were measured by enzyme-linked immunoassay (ELISA). T helper 1 (Th1)/Th2 ([IL-2 + IFN-gamma]/[IL-4 + IL-5]) cytokine mRNA ratios, thought to reflect the Th deviation of the pathogenic disease-specific T cells, and type-1/type-2 mRNA ratios, thought to reflect the overall immune response polarization, were significantly increased in ITP patients. The Th1/Th2 ratio was inversely correlated with platelet counts. TGF-beta1 levels appeared suppressed in patients with active disease, though not significantly. Our findings show a clear type-1 cytokine polarization of the autoimmune response in adult ITP that persists irrespective of disease status.

Prolonged administration of erythropoietin increases erythroid response rate in myelodysplastic syndromes: a phase II trial in 281 patients.

Treatment with recombinant human erythropoietin (rHuEpo) improves anaemia in approximately 20% of patients with myelodysplastic syndromes (MDS). We investigated the potential advantage of a prolonged administration of rHuEpo to achieve higher erythroid response rates (RR) in 281 MDS patients: 118 with refractory anaemia (RA), 77 with refractory anaemia and ringed sideroblasts (RARS), 59 with refractory anaemia with excess of blasts and blast count < 10% (RAEB-I), and 27 with RAEB and blast count between 11-20% (RAEB-II). rHuEpo was given subcutaneously at a dose of 150 U/kg thrice weekly, for a minimum of 26 weeks. Response to treatment was evaluated after 12 and 26 weeks of therapy. The overall RR was 45.1%; the RR for RA, RARS, RAEB-I and RAEB-II were 48.3%, 58.4%, 33.8% and 13% respectively. A significant increase in RR was observed at week 26 in RA, RARS and RAEB-I patients, as the response probability increased with treatment duration. The RR was higher in the good cytogenetic prognostic group and serum Epo level of > 150 U/l at baseline predicted for non-response. The median duration of response was 68 weeks and the overall risk of leukaemic transformation was 21.7%. These results suggest that prolonged administration of rHuEpo produces high and long-lasting erythroid RR in MDS patients with low blast counts, particularly in those with pretreatment serum Epo levels of < 150 U/l and good cytogenetic prognosis.