ALBERT

Beschreibung: AHSP is an erythroid protein that binds the alpha globin subunit of hemoglobin (Hb) to maintain its structure and limit its oxidative activity. Biochemical and genetic studies demonstrate that AHSP is essential for normal Hb homeostasis and may act via at least two mechanisms: First, as a molecular chaperone to promote the folding and stability of alpha globin prior to its incorporation into HbA (alpha2beta2). Second, to bind and detoxify excess alpha globin that accumulates in normal erythroid precursors, and to a larger extent, in beta thalassemic ones. The existence of a potential iron responsive element (IRE) in the 3′-UTR of the AHSP mRNA raises the interesting possibility that iron homeostasis impacts on alpha globin stability via AHSP. IREs form stem-loop structures that bind cytosolic iron-sensing proteins (IRPs). Typically, IRP binding to the 3′ UTR stabilizes mRNA. Association with iron releases the IRP, enhancing mRNA degradation. Computational algorithms identified IRE-like stem-loop structures in AHSP mRNA of multiple species, yet the primary sequences deviate significantly from canonical IRE consensus sequences determined by studies of classical IREs, such as Transferrin receptor and Ferritin. Several lines of evidence now show that the AHSP IRE binds IRPs to regulate mRNA stability in an iron-dependent fashion: 1) in vitro gelshift competition assays demonstrate that human and mouse AHSP IREs bind tightly to IRPs. 2) AHSP mRNA co-immunoprecipitates with IRPs in erythroid cells in a fashion that depends on an intact IRE. 3) AHSP mRNA is destabilized by iron in both erythroid and heterologous cells; disruption of the IRE renders the mRNA constitutively unstable. To study how iron regulates AHSP expression in vivo, we treated mice with iron dextran for 10 days and then examined AHSP mRNA in Ter119+ erythroid progenitors by RT-PCR. We found that short-term iron overload reduced AHSP mRNA levels by about 50% (p 〉 0.05). Our findings indicate that AHSP mRNA stability is regulated by iron via an atypical 3′-UTR IRE. These findings extend the potential repertoire for functional IREs that do not conform to previously defined canonical consensus sequences. In addition, they provide a potential mechanism by which erythroid cells can regulate globin stability according to iron status. For example, induction of AHSP during iron deficiency might stabilize apo- alpha globin that could accumulate from lack of heme. Conversely, iron overload could destabilize alpha globin by reducing AHSP levels. As such, iron overload, which occurs in patients with beta thalassemia, might aggravate the disease by further elevating the levels of toxic free alpha globin.

Beschreibung: Acute megakaryoblastic leukemia (M7 AML) is a highly aggressive disease. We evaluated outcomes in 57 children (11 with Down syndrome) and 69 adults with M7 AML after first complete remission (CR1) following autologous or HLA-identical allogeneic transplantation. Characteristics of the recipients of autologous transplants (38 children, 37 adults) were, respectively: median age, 1.7 and 46 years; non-total body irradiation (non-TBI) conditioning regimen, 97% and 70%; bone marrow as stem cell source, 74% and 43%. Characteristics of the recipients of allogeneic transplants (19 children, 32 adults) were, respectively: median age, 2.8 and 37 years; non-TBI regimen, 63% and 42%; bone marrow as stem cell source, 95% and 69%. Autologous transplantation benefited children more; the relapse rate was high in adults. Results for autologous transplantation were (children and adults, respectively): engraftment, 90% and 100%; 3-year treatment-related mortality (TRM) rate, 3% and 8%; relapse rate, 45% and 64%; leukemia-free survival (LFS) rate, 52% and 27%; overall survival (OS) rate, 61% and 30%. After allogeneic transplantation, TRM was fairly low in children and adults, and relapse rates were lower than after autologous transplantation. Results for allogeneic transplantation were, respectively: engraftment, 95% and 90%; TRM, 0% and 26%; relapse rate, 34% and 28%; LFS, 66% and 46%; OS, 82% and 43%). We conclude that M7 AML patients in CR1 (except children with Down syndrome, who already have better outcomes) can benefit from transplantation. (Blood. 2005;105:405-409)

Beschreibung: The initial analysis of the oral combination melphalan, prednisone, and thalidomide (MPT) in newly diagnosed patients with myeloma showed significantly higher response rate and longer progression-free survival (PFS) than did the standard melphalan and prednisone (MP) combination and suggested a survival advantage. In this updated analysis, efficacy and safety end points were updated. Patients were randomly assigned to receive oral MPT or MP alone. Updated analysis was by intention to treat and included PFS, overall survival (OS), and survival after progression. After a median follow-up of 38.1 months, the median PFS was 21.8 months for MPT and 14.5 months for MP (P = .004). The median OS was 45.0 months for MPT and 47.6 months for MP (P = .79). In different patient subgroups, MPT improved PFS irrespective of age, serum concentrations of β2-microglobulin, or high International Staging System. Thalidomide or bortezomib administration as salvage regimens significantly improved survival after progression in the MP group (P = .002) but not in the MPT group (P = .34). These data confirm activity of MPT for PFS but failed to show any survival advantage. New agents in the management of relapsed disease could explain this finding. The study is registered at www.clinicaltrials.gov as #NCT00232934.

Beschreibung: Introduction: Screening for multiple myeloma requires both serum and urine protein electrophoresis, because in about 20% of patients with myeloma, monoclonal free light chain (FLC) is the only paraprotein found, and it is commonly missed by serum protein electrophoresis. However, many requests for testing do not include a urine sample (〉80% of requests in our experience). This risks missing clinically significant disease. Recent availability of serum FLC assays has raised the possibility that these assays may replace testing for urinary FLC in screening for monoclonal gammopathies, and that the serum kappa:lambda light chain ratio (LCR) may be more sensitive for detecting monoclonal FLC than serum and urine protein gel electrophoresis. Aims: To identify how many additional patients with monoclonal gammopathies would be detected if serum FLC assays were incorporated into the routine myeloma screen. To evaluate the ability of serum FLC assays to identify all patients identified by urine protein electrophoresis. Method and Setting: We analysed data from a consecutive cohort of 753 serum blood samples submitted for myeloma screening to Hull Royal Infirmary Immunology Laboratory between 03/23/07 and 05/31/07. During this period all myeloma screen requests received serum capillary zone protein electrophoresis (CE) (SEBIA Capillarys 2, Analytical Technologies) and serum FLC analysis using a latex-enhanced immunoassay (The Binding Site, Birmingham, UK on a Beckman-Coulter IMMAGE nephelometric analyzer). When available, urine protein CE was also perfomed (SEBIA Capillarys 2). Samples with an abnormal serum CE or serum LCR were tested by immunofixation (SEBIA Hydrasys, Analytical Technologies). Repeat samples were requested from patients with LCR outside the reference interval (0.26–1.65) before referral, but an immediate hematology referral was recommended if LCR 〉3.5 sd from the mean (ie 0.18–2.01). Results: Of 753 patients, 118 had features on serum CE requiring immunofixation. Of these, 76 had a monoclonal paraprotein identified. A further 46 samples had normal serum CE with abnormal LCR and 25 of these had LCR outide mean±3.5 sd. Of 6 patients so far referred as a result of abnormal LCR but normal serum CE, 4 (67%) had a lymphoproliferative disease (free kappa myeloma, free kappa MGUS, free lambda MGUS, and a chronic lymphocytic leukaemia). Urine samples were received from 128 (17%) patients, of whom 8 (6%) had a monoclonal FLC identified in the urine. All of these patients had an abnormal serum LCR, though in one patient with acute renal failure and raised kappa and lambda results the LCR was borderline abnormal (1.75), with a very small band in the urine, visible only by agarose gel immunofixation. For the 2 patients with normal serum CE, but with urinary monoclonal FLC present, serum LCR was abnormal (LCR= 33 and 1.75). Discussion and Conclusions: Use of serum FLC assays increased the detection of monoclonal paraproteins (by 5% so far but further follow up is required to quantify this exactly). Serum FLC analysis had a sensitivity of 100% for identifying patients with urinary FLC.

Beschreibung: We performed a prospective, randomized study of single (arm A) versus double (arm B) autologous stem-cell (SC) transplantation (ASCT) for younger patients with newly diagnosed multiple myeloma. A total of 321 patients were enrolled in the study and were randomly assigned to receive either a single course of SC-supported melphalan, 200 mg/m2 (MEL-200) (n=163 patients) or MEL-200 followed, 3 to 6 months apart, by melphalan, 120 mg/m2, and busulfan, 12 mg/kg (n=158 patients). As compared with arm A, randomization to receive double ASCT significantly increased the probability to attain at least a near (n) complete response (CR) (33% vs 47%, respectively; P=0.008). Patients who responded to induction therapy had a CR or nCR rate following either single or double ASCT of 73% and 52%, respectively (P=0.010). Both these values were much higher than those observed among patients who failed to respond to induction therapy, whose posttransplantation ≥ nCR rate was 11% in arm A and 12% in arm B (P=0.20). On multivariate analysis, randomization to double ASCT (P

Beschreibung: The nuclear factor GATA-1 controls erythropoiesis by regulating transcription of numerous protein-encoding genes. Here we show that GATA-1 also activates the expression of a critical erythroid microRNA (miR) locus. MicroRNAs regulate gene expression and tissue differentiation by binding the 3′ untranslated regions of mRNA transcripts to inhibit their translation or stability. We used an oligonucleotide-based microarray to analyze miR expression in G1E cells, a GATA-1-null erythroid line that undergoes terminal maturation when GATA-1 activity is restored. We identified 8 miRs that are significantly upregulated during GATA-1-induced erythroid maturation. Two of the most highly induced miRs, 144 and 451, are encoded in tandem on mouse chromosome 11 and are transcribed on the same primary microRNA transcript. Northern blot studies showed that the miR144/451 locus is strongly induced by GATA-1 and specifically expressed at high levels in human and murine erythroid precursors. Bioinformatic analysis of the miR144/451 locus identified a conserved region containing three closely-spaced GATA binding motifs located about 2.7 kb upstream of the transcriptional start. In erythroid cells, this region exhibits enhancer activity and binds GATA-1 and its cofactor FOG-1, as determined by chromatin immunoprecipitation studies. MicroRNAs 144 and 451 are conserved between mammals and zebrafish, facilitating further in vivo studies. Whole mount in situ hybridization of zebrafish embryos showed both miRs to be exclusively expressed in the developing blood islands (intermediate cell mass) at 24h post-fertilization (hpf), colocalizing with gata-1 and β-globinE1 expression. Both miRs are deficient in the gata-1 null mutant, vlad tepes, consistent with their epistatic relationship to gata-1. Transient inactivation of miR-451 function using anti-sense morpholino oligomers selectively ablated hemoglobin staining cells in embryos at 48 hpf. These morphant embryos also exhibited dramatically reduced β-globinE1 mRNA expression. In contrast, there were no quantitative effects on thrombocytes (platelet-equivalent cells) in morphant embryos. Together, our data define a new regulatory axis through which GATA-1 promotes red cell development by directly activating the transcription of miR144/451, a critical erythroid-specific microRNA locus.

Beschreibung: Mutations in the tyrosine kinase receptor FLT3 are the most common molecular abnormality in acute myeloid leukemia (AML) being detected in about 30% of AML cases. According to the protein domain altered FLT3 mutations may be classified as juxtamembrane or activation loop. The former are caused by internal tandem duplications (ITD) in exons 14 and 15 and is detected in 20–27% of AML patients. Mutations in the activation loop are mainly due to point mutations in exon 20 and is present in 5–7% of AML patients. Most of these mutations lead to changes in the aspartate in position 835 (D835), which have been detected in about 7% of AML cases. Both types of mutations cause the constitutive activation of FLT3 and are associated with bad prognosis. AML characteristics in Latin America are different from those in Europe and US. Namely, there is a higher frequency of acute promyelocytic leukemia (APL) and the clinical outcome of adult patients with other subtypes of AML treated with standard protocols is poorer. The worse prognosis seems to be related to the biology of the disease, rather than socio-economic features, based on studies of other hematological malignancies. In order to test if a higher frequency or different FLT3 mutations might explain these observations, we performed a screening for mutations in FLT3 using PCR and single strand conformation polymorphism (SSCP) techniques to evaluate exons 12 to 20, which encode for the intracytoplasmatic domains of the protein. Ninety-nine consecutive patients with AML (90 adults and 9 children) and 55 blood donors (controls) were analyzed. Two synonyms mutations that have not been previously described were detected: one in exon 12 (T526T) and the other in exon 17 (G697G). ITD mutations were detected in 23 (23.2%) patients with AML, therefore within the expected frequency based on the studies in developed countries. On the other hand, D835 mutations were absent, and except for a mutation detected in one patient causing the deletion of the aminoacid in the position 836 no other abnormality was detected in exon 20. No mutations were detected in exons 13, 16, 18 and 19. In adults, mutations were more frequent in acute promyelocitic leukemia and in women. There were no associations between FLT3 mutations and CBC values. Interestingly, FLT3 mutations were less frequently detected in patients whose leukemic cells expressed the CD56 marker. As described for other countries, overall survival was worse in patients with FLT3 mutations. In conclusion, this study demonstrates that the frequency of ITD mutations in FLT3 gene in Brazilian patients with AML was similar to the frequency described in the European and North American populations, whereas activation loop mutations were rarely detected, especially those in D835. Thus, our results suggest that Brazilian patients with AML have a distinct profile of genetic abnormalities. In addition, this is the first study to demonstrate a negative association between FLT3 mutations and the expression of CD56 by leukemic blasts. This is a relevant observation, since CD56 expression per se has been identified as prognostic factor by other investigators. Finally, our study demonstrates that the SSCP method may be useful for screening mutations of FLT3 gene.

Beschreibung: MicroRNAs (miRNAs) regulate tissue development by destabilizing cellular mRNAs or blocking their translation. We previously described two conserved erythroid miRNAs, miR144 and miR451, which are encoded by a single gene locus that is regulated by the essential hematopoietic transcription factor GATA-1. Morpholino-induced inhibition of miR451 impairs erythropoiesis in zebrafish. To gain further insight into miR144/451, we deleted the locus in mice. Mutant animals are born at the normal Mendelian ratio and exhibit no obvious structural defects. At baseline, loss of miR 144/451 causes mild anemia and reticulocytosis with moderate abnormalities in erythrocyte morphology. However, compared to littermate controls, miR144/451 null animals exhibit 33% greater depletion of circulating erythrocytes after phenylhydrazine-induced oxidant stress. At baseline, the mutant erythrocytes exhibit increased levels of reactive oxidant species, exquisite sensitivity to hydrogen peroxide induced hemolysis, and 50% reduced catalase activity compared to controls. Catalase is a downstream effector in an erythroid anti-oxidant program regulated by the forkhead transcription factor Foxo3a. In the mutant erythroblasts, mRNAs encoding catalase and glutathione peroxidase 1, another Foxo3a-regulated anti-oxidant, were reduced 2 and 1.7 fold, respectively. Messenger RNAs encoding several additional Foxo3a target genes, including Cdkn1b and Btg1 were also significantly reduced. Quantitative confocal fluorescence microscopy demonstrated that although total cellular Foxo3a protein was similar in wt and miR144/451 null erythroblasts, nuclear Foxo3a was reduced by 40% in miR144/451 null erythroblasts with a corresponding relocation of the protein to the cytoplasm. To explain this, we showed that miR451 (but not miR144) directly blocks translation of Ywhaz mRNA encoding 14-3-3 zeta, a cytoplasmic protein that sequesters Foxo3a away from the nucleus. In agreement, 14-3-3 zeta protein is upregulated by approximately 2-fold higher levels in miR144/451 null erythroblasts. Together, our findings suggest a model in which miR451 represses 14-3-3 zeta expression, which releases Foxo3a for translocation to the nucleus, thereby activating an anti-oxidant program of gene expression. This illustrates a new miRNA-regulated pathway through which erythroid cells are protected against oxidant stress.

Beschreibung: The pathogenesis of osteonecrosis of the jaws (ONJ) in patients treated with intravenous bisphosphonates is still not completely understood, and likely involves both reduction in blood supply and the activity of oral bacteria. We present an animal model of ONJ, which will significant contribute to the understanding of the aetiopathogenesis of this condition. Five skeletally-mature female Wistar rats (weight 450 g) were given intravenous zoledronic acid 0.04 mg in saline solution (0.2 mg/ml) once a week for 5 weeks. After 2 weeks, the animals underwent the extraction of the upper right first molar, followed by the creation of a 4 mm-diameter bone defect on the same site under general anaesthesia. After 7 weeks from the extraction, the animals were clinically examined and a static bone-scintigraphy using 99mTc-MDP methylene diphosphonate was performed with a gamma camera equipped with pinhole collimator (Siemens-Ecam). After an additional week, the rats were sacrificed and a Computed Tomography (CT) was carried out. Samples obtained from the bone defect were decalcified and prepared for histological assessment. Five rats, not treated with zoledronic acid and exposed to the same surgical treatment, were used as controls. Histological and imaging features were assessed blindly. At 7 weeks after the extraction, all the rats treated with zoledronic acid showed impair healing, expansion of the defect and bone exposure. These features were confirmed by the scintigraphy, which showed abnormal localized activity in comparison with the surrounding tissues. On clinical examination, the rats of the control group demonstrated epithelialization of the bone defect and a normal uptake of the contrast medium during the scan. The CT scan disclosed irregularity of the cortical margin and destruction of the cortical bone, which were not evident in the control group. The histology showed sheets of necrotic bone, with loss of osteocytes from their lacunae and peripheral resorption. No inflammatory infiltrate was observed. The control group instead demonstrated normal bone healing. All animals were treated according with the guiding principles for experimental procedures found in the Declaration of Helsinki of the World Medical Association. Based on this study, the rat treated with zoledronic acid can be considered a novel, reliable and reproducible model to better understand the pathophysiology of ONJ and to develop a therapeutic approach.

Beschreibung: Alpha Hemoglobin Stabilizing Protein (AHSP, Eraf) is an abundant erythroid protein that binds and stabilizes alpha globin and alpha hemoglobin (Hb). In mice, loss of AHSP causes hemolytic anemia, with elevated levels of reactive oxygen species and Hb precipitation in erythrocytes. Loss of AHSP exacerbates beta thalassemia phenotypes in mice, presumably by enhancing the toxicity of excessive free alpha Hb. Based on these findings, AHSP is a candidate modifier gene for beta thalassemia in humans. No mutations in the AHSP coding region have been identified in patients to date. However, several groups reported an inverse correlation between beta thalassemia severity and erythroid AHSP expression levels, raising the possibility that AHSP is a quantitative trait modifier of beta thalassemia. To address this possibility, it is important to define the mechanisms that control expression of the AHSP gene. Transcripts of murine Ahsp are inducible by GATA-1. The goals of the current studies are to investigate the mechanisms of this induction and to define the DNA domain that regulates the locus. Using phylogenetic comparisons, we identified a hotspot for mammalian chromosomal rearrangement just downstream of the Ahsp gene. This hotspot is located at the end of a syntenic block of approximately 350 kb that is conserved in mammals and likely marks the 3′ end of the gene regulatory domain. We focused our initial functional studies on a 7 kb genomic region bounded at the 5′ (centromeric) end of Ahsp by the nearest adjacent gene, an EST expressed in multiple tissues, and at the 3′ (telomeric) end by the rearrangement hotspot. In transient transfection assays, the Ahsp promoter region conferred erythroid-specific expression to a linked reporter gene. In heterologous cells, GATA-1 transactivated the Ahsp promoter in a dose-dependent fashion. To examine GATA-1 binding and its subsequent effects on the Ahsp gene in vivo, we used G1E-ER4 cells, a GATA-1 null erythroblast line that undergoes terminal erythroid maturation after activation of an estradiol-inducible form of GATA-1. We made several findings with regards to the role of GATA-1 in Ahsp gene regulation. First, GATA-1 and its cofactor, Friend of GATA-1 (FOG-1), bind directly to the Ahsp locus at regions that contain conserved GATA consensus motifs and are predicted to be important erythroid regulatory elements by our bioinformatic studies. Second, GATA-1 induces epigenetic changes in chromatin structure that are associated with gene activation, including formation of a DNase I hypersensitive site, hyperacetylation of histones H3 and H4, and methylation of histone H3 lysine-4. Together, these findings begin to establish the DNA region and mechanisms that control Ahsp transcription, allowing for further studies to map the cis elements responsible for population variations in gene expression.