bluuebunny/crossref_metadata_2025_split huggingface dataset:
Gene Expression Profiling of Hematopoietic Stem and Progenitor Cells in Zebrafish.
Authors: Weber Gerhard J. , Chiang Ken , Mayhall Elizabeth A. , Zhou Yi , Zon Leonard I. 
Published: April 2020

Abstract
The differentiation of hematopoietic stem cells (HSCs) into distinct blood lineages is orchestrated by a tight regulation of critical genes during development. To describe the genetic program during the early steps of blood and blood vessel formation in greater detail, we analyzed the expression profiles of zebrafish cloche mutants (clo), in which blood and endothelial cells are completely abrogated, and of isolated hematopoietic and vascular progenitor cell populations from FAC-sorted transgenic lmo2-GFP zebrafish embryos. Consistent with a defect in hemangioblast differentiation, clo mutants at the 5 somite stage were characterized by a prominent downregulation of transcription factors, such as scl and lmo2. As a novel finding, clo mutants at this stage strongly upregulated muscle-related genes, such as myosin, actin, or creatine kinase. FAC-sorted lmo2-GFP cells from 14 somite stage embryos expressed similar hematopoietic transcription factors including scl, lmo2, and gata1. In addition, socs genes, tgfb, fos and jun related genes, and heat shock proteins were also strongly expressed. Comparison with the expression profile of human and mouse fetal liver HSCs revealed a similar regulation between fish and mammals: Expression of 102/168 genes from lmo2-GFP positive cells were also present in mammalian fetal HSCs, indicating an evolutionary highly conserved genetic program of blood formation. In sum, gene expression profiling of zebrafish mutants and of sorted progenitor cells from transgenic zebrafish represent a powerful and versatile tool to define the genetic regulation of vertebrate hematopoietic development.
A Stem-Loop in the 3′-UTR Mediates Iron-Dependent Regulation of Alpha Hemoglobin Stabilizing Protein mRNA Stability.
Authors: dos Santos Camila O. , Duarte Adriana , Dore Louis C. , Saad Sara , Weiss Mitchell J. , Costa Fernando F. 
Published: April 2020

Abstract
Alpha-hemoglobin stabilizing protein (AHSP) acts as a molecular chaperone that binds and stabilizes alpha globin, minimizing the formation of cytotoxic alpha-globin precipitates in red blood cells. It has been proposed that variations in AHSP protein levels could influence disease severity in patients with beta-thalassemia. A number of genomic sequences that could be involved with AHSP transcriptional regulation have been studied. Multiple consensus GATA-1 binding motifs and a single CACCC-binding site have been identified and characterized. In vivo studies demonstrated that AHSP gene transcription is dependent on both EKLF and GATA-1 transcription factors. Here, we identify an IRE (iron response-like element) in the AHSP 3′ untranslated region that is predicted to regulate mRNA stability. IREs are regulatory elements that encode a phylogenetically defined sequence-structure motif that provide a specific recognization site for the IRE-binding proteins (IRE-BP1 or IRE-BP2). Iron starvation of cells induces high affinity binding of cytoplasmic IRE-BP to an IRE, which has at least two distinct known biological consequences. IRE in the 5′-UTR of an mRNA provides for iron-dependent regulation of translation, whereas an IRE in the 3′-UTR confers iron-dependent regulation of mRNA stability. Computer modeling identified a potential IRE stem-loop structure near the 3′-end of murine and human AHSP mRNAs. In vitro binding studies indicate that this structure recognizes an IRE-BP. Moreover, the affinity of this interaction was reduced by mutations that disrupt base pairing within the stem-loop. Iron supplementation, which is predicted to decrease IRE-BP affinity for the IRE, destabilizes AHSP in human and mouse erythroleukemia cells. These results indicate that AHSP expression is regulated by RNA stability in an iron-dependent fashion. Our findings have implications for the control of AHSP expression in beta-thalassemia.
AHSP Is a Quantitative Trait Gene That Modifies the Phenotype of β Thalassemia.
Authors: Lai Mei I. , Jiang Jie , Silver Nicholas , Best Steve , Menzel Stephan , Garner Chad , Weiss Mitchell J. , Thein Swee Lay 
Published: April 2020

Abstract
Alpha hemoglobin stabilizing protein (AHSP) was identified in a screen for genes that are activated by the erythroid transcription factor, GATA-1. Studies have shown that:
AHSP binds specifically to the α chain of hemoglobin (Hb) but not to β Hb or Hb A; AHSP knock-out mice demonstrated pathological features similar to β thalassemia; and loss of AHSP exacerbates severity of disease in β thalassemia mice.
The evidence suggests that AHSP acts as a chaperone for free α Hb and that altered AHSP expression levels could modify the severity of β thalassemia in humans.
To assess variation of AHSP expression, mRNA levels were measured in peripheral blood reticulocytes of 103 healthy individuals by quantitative real-time RT-PCR. The sample was approximately 90% (91/103) female with an average age of 52 years (SD=0.14, min=18, max=76). AHSP expression relative to GAPDH, varied up to 3-fold, and did not correlate with age or sex. A systematic survey of the genomic region encompassing the AHSP locus revealed 8 sequence variants of which six were common - five single nucleotide polymorphisms (SNPs), and one homopolymer (Tn) at position 160 bp upstream of exon 1. Four variants (c.-69–237A, c.-69–160 T18, c.-4–27G, and c. 337T) showed strong association with AHSP expression and had nearly equal frequencies. The four variants are in near complete linkage disequilibrium with the minor alleles in coupling. The haplotype consisting of the four minor alleles termed clade B, was associated with relatively lower expression of AHSP. Relative expression of AHSP and AHSP/α globin ratio were both significantly higher (p&lt;0.001) in homozygotes for clade A haplotypes compared to heterozygotes. A potential functional role of one of the variable sites, a T-homopolymer (the T18 variant being part of clade A) in the promoter was investigated in-vitro using luciferase reporter assays in K562 cells. Luciferase activity was 1.30±0.08 times higher in the T18 promoter compared to T14, consistent with genetic studies. We investigated if a shorter homopolymer could have an adverse effect on β thalassemia. Nine patients with thalassemia intermedia and a genotypic combination of heterozygous β thalassemia and 5 α globin genes (aaa/aa) were studied. Based on the allele frequencies in the healthy population, 6–7 of the 9 patients are expected to be homozygous for T18 but only two were observed. In contrast, while 2–3 patients are expected to be heterozygous for the shorter homopolymer, 3 patients were homozygous and 4 heterozygous.
In conclusion, AHSP gene expression is variable, with cis control accounting for some of its variance. The subtle altered expression might precipitate a phenotype of thalassemia intermedia in β thalassemia heterozygotes with α globin overload.
Dysregulated Arginine Metabolism and Elevated Arginase Activity in Thalassemia.
Authors: Morris Claudia R. , Kuypers Frans A. , Kato Greg , Gardner Jennifer , Larkin Sandy , Singer Titi , Vichinsky Elliott 
Published: April 2020

Abstract
Pulmonary hypertension (PHT) is associated with high mortality rate and is a leading factor in heart failure and death in thalassemia (Thal) intermedia. However, data on PHT and Thal major is limited. While there are several risk factors for PHT in Thal major, ongoing chronic hemolysis, despite transfusion, supports the development of hemolysis-associated PHT. Recently, we reported in sickle cell disease a novel paradigm whereby hemolysis liberates vasoactive hemoglobin and erythrocyte arginase, resulting in impaired NO bioavailability, endothelial dysfunction, PHT and death. Since arginase activity is also elevated in Thal red cells, we hypothesize that elevated arginase contributes to dysregulated arginine (Arg) metabolism in Thal. Pilot data was analyzed from transfused Thal patients (8 Thal-major, 4 E-beta Thal, 2 Hb H alpha Thal). Echocardiogram were performed in steady state in 10 of the patients; 70% (7/10) demonstrated PHT (tricuspid regurgitant jet velocity ≥ 2.5 m/s). Plasma Arg trended lower in patients with Thal (19.5 to 122mM, median 50 mM) compared to controls. Ornithine (Orn) levels were higher, and Arg/Orn ratio was lower in Thal vs. contrls. Plasma arginase activity was significantly elevated (median 0.83 mmol/cc/hr, range 0.06 – 1.17). Proline, a downstream metabolite of arginase causing pulmonary vascular remodeling was also elevated. Exhaled nitric oxide (NO) levels are significantly elevated in Thal vs. controls (49±41 parts per billion vs.18±8 ppb, p=0.02), suggesting upregulation of NO synthase in the lungs in addition to higher plasma arginase activity. Recent findings indicate diverse origins of PHT share similar pathophysiologic abnormalities, particularly decreased Arg bioavailability and a shift of metabolism towards ornithine-dependent pathways. Hemolysis is most likely a trigger for these pathologic pathyways in hemoglobinopathies. Therapies that maximize Arg and NO bioavailability may benefit patients with thalassemia.
Variable NL Control (n=36) Thalassemia (n=14) p* Arginine (μM) 67±18 57±26 (50) 0.15 Ornithine (μM) 62±22 85±68 0.05 Arg/Orn ratio 1.2±0.5 0.79±0.4 &lt;0.01 Proline (μM) 161±48 258±116 &lt;0.001 Citrulline (μM) 25±11 42±17 &lt;0.001 Arginase (μmol/cc/hr) 0.33±0.2 (n=45) 0.71±0.3 &lt;0.001
A Randomized, Placebo Controlled, Double Blind Trial of the Effect of Combined Therapy with Deferoxamine and Deferiprone on Myocardial Iron in Thalassaemia Major Using Cardiovascular Magnetic Resonance.
Authors: Tanner Mark A. , Galanello Renzo , Dessi Carlo , Westwood Mark A. , Smith Gillian C. , Khan Mohammed , Nair Sunil V. , Walker J. M. , Pennell Dudley J. 
Published: April 2020

Abstract
Introduction: Beta thalassaemia major (TM) is a hereditary anaemia affecting 60 000 births worldwide each year. Survival is dependent upon lifelong blood transfusions with cardiac failure secondary to myocardial iron loading being the commonest cause of death. Conventional treatment with the parenteral iron chelator deferoxamine improves mortality but prognosis remains poor and it has been reported that approximately two-thirds of patients maintained on this treatment have myocardial iron loading. More recently, the oral iron chelator, deferiprone has been demonstrated to remove myocardial iron and it has been proposed that in combination with deferoxamine it may have an additive or synergistic effect. Myocardial iron can now be rapidly and reproducibly quantified using the validated cardiac magnetic resonance (CMR) T2* technique. CMR is therefore well suited to assess the efficacy of new therapies for the removal of myocardial iron in TM.
Purpose: To report the primary outcome measure (heart T2*) from a randomized placebo controlled trial testing the hypothesis that the combined therapy of deferiprone and deferoxamine is superior in the removal of myocardial iron than the standard therapy of deferoxamine alone.
Methods: A mobile CMR scanner (1.5T Siemens Sonata) was transported to Cagliari, Italy on 3 occasions. The myocardial T2* was assessed in 167 patients with TM. 65 patients (male 27, female 38, age 30+/−5.2years) with mild-moderate myocardial iron loading (T2* 8–20ms) were randomized to receive either deferoxamine and placebo, or deferoxamine and deferiprone. The primary outcome measure of myocardial T2* was assessed at baseline, 6 and 12 months. Normal myocardial T2* is greater than 20ms.
Results: In the combined treatment group myocardial T2* improved (reducing myocardial iron) from a baseline of 11.7ms to (at 6 months) 14.6ms (+25%; CV 11.9%; p=0.007) and at 12 months 16.8ms (+40%; CV 14.2%; P&lt;0.001). In the placebo controlled group myocardial T2* improved from a baseline of 12.4ms to (at 6 months) 14.2ms (+14%; CV 14%; p=0.15) and at 12 months 15.2ms (+21%; CV 16%; p=0.02). Analysis of covariance showed a significant difference between groups (p=0.017) with the combined group showing greater effects in improving myocardial T2* (figure 1).
Conclusion: In mild-moderate cardiac iron loading the combined therapy of deferiprone and deferoxamine is superior to deferoxamine alone in the removal of myocardial iron in TM.
Figure 1 Figure 1.
Reduced Intensity Conditioning Regimen for Unrelated Cord Blood Transplantation in Adults. A Multicentric Phase I-II Trial.
Authors: Rio Bernard , Belhocine Ramdane , Renaud Marc , François Sylvie , Gluckman Eliane , Blaise Didier , Rocha Vanderson 
Published: April 2020

Abstract
Curative potential of allogeneic transplantation from related or unrelated donors is limited by tranplant related mortality (TRM) and donor compatibility. After reduced intensity conditioning regimens (RIC) transplantation can be extended to older patients, those with poor performance status, organ dysfunction or intensive prior therapy. In absence of HLA-matched donor, unrelated cord blood transplant (UCBT) is a possible source of hematopoietic stem cells. In order to confirm previous report of RIC after UCBT in adults we conducted a phase I–II trial. Primary endpoint was neutrophil recovery and chimerism. Secondary endpoints were early TRM, acute GVHD incidence, early relapse and survival. Between October 2003 and March 2005, 18 patients (pts) were enrolled: median age: 46y (19–64), weight: 62.5 kg(45–90), gender: 13 females and 5 males, CMV serpositivity in 8 pts, ABO major incompatibility in 8 pts. The median of follow-up was 4.5 mo (0.5–16). Disease status were 2 ALL (CR1: 1; CR2: 1), 10 AML (CR1: 3, CR2: 6, CR3: 1), 1 AP CML, 1 CLL, 1 Waldenström disease, 3 follicular lymphoma relapsed after autologous transplantation. Infused nucleated and CD34+ cells number were respectively: 2.3x107/kg (1.7–3.7) and 0.7x105/kg (0.4–1.2). Only a single cord blood was used. HLA disparity was 0/6 in 1, 1/6 in 6 and 2/6 in 11. RIC regimen consisted of cyclophosphamide 50 mg/kg D-6, fludarabine 200 mg/m2 and 2Gy TBI at D-1. CsA and MMF from D-3 were used in GVHD prophylaxis. Pts received GCSF until 5 G ANC/l.
Results: Median day for 0.5 G/l ANC recovery was 14 days (0–28) and for 20 G/l platelets was 28 days (0–48). Mixt chimerism was present until D+56 and full chimerism at D+80/100 in all pts except 2 who relapsed. Mucositis was not observed, CMV reactivation in 2 pts, bacteremia in 3, SRV pneumonitis in 1 pt, bone aspergillosis in 1 pt. Regressive limbic encephalitis in 2 pts. Two pts recovered normal ovarian function 5 mo and 12 mo after UCBT. Grade 2 acute GVHD was observed in 2 pts and limited chronic GVHD in 3 out of 8 pts at risk. TRM was 0%. Two pts relapsed and died at D+100 and D+171. EFS was 80+/−13%. In conclusion, in spite of the short follow-up and few patients enrolled in this trial, these results confirm those reported by Barker J et al. Importantly there was a rapid neutrophil recovery with acquired late full chimerism and absence of early TRM. The encouraging results justify the use of cord blood cells in RIC and prospective trials in order to compare phenoidentical peripheral blood stem cell transplantation and UCBT with the same RIC regimen.
Efficacy of Performing a Second Non-Myeloablative Allogenic Stem Cell Transplant.
Authors: Byrne Brian J. , Antin Joseph H. , Alyea Edwin P. , Choa Nelson J. , Rizzieri David A. 
Published: April 2020

Abstract
Introduction: Non-myeloablative stem cell transplants are being performed for a variety of benign and malignant hematologic conditions. The reported graft failure rates have been as high as 20%. Little is reported on the safety and efficacy of a second attempt at a nonmyeloablative allogeneic transplant.
Methods: Patients who underwent at least 2 nonmyeloablative allogenic transplants at either Duke University or Dana Farber Cancer Centers were included.
Results: 9 patients were identified who underwent at least 2 preparatory regimens with stem cell infusion. 7 patients underwent related donor stem cell infusions and 2 patients had an umbilical cord donor. 4 patients, including the 2 with an umbilical cord donor, had primary graft failure. The remaining had secondary graft failure within 1–9 months from viral disease/therapy or disease progression. 5 of 9 received a 5 day regimen of fludarabine 125mg/m2, campath100mg and cyclophosphomide 2 gm/m2 total for a secondary preparatory regimen (FCC). Two patients received a one-day prep with fludarabine 30mg/m2, 200cGyTBI, cyclophosphamide 2gm/m2 and campath 20mg (FCC/TBI). The two umbilical cord patients received a one day prep that included TBI, cyclophosphamide 500mg/m2, fludarabine 30 mg/m2, and campath 30 mg (FCC/One). One patient with refractory CML failed to engraft, though 8/9 did (1 required a 3rd attempt), including 4 patients who had the same donor as their first transplant. Long-term follow-up revealed the graft was intact in 5 patients at 6, 7, 8, 21, 39, and 63 months. Three patients died.
Conclusion: Patients who have graft failure can be transplanted with a second nonmyeloablative allotransplant. Response and long-term engraftment is possible, even using the same donor.
Table 1: Patients who have undergone greater than one mini allogeneic transplant. Disease Prep #1 Donor Engraftment Prep #2 Donor Engraftment Response Key: FCC: Fludarabine, campath, cyclophosphamide; ATG FM: Fludarabine, melphalan, ATG FCC/TBI: TBI, fludarabine, campath, cyclophosphamide, FCC/One: TBI, fludarabine, cyclophosphamide, campath Thalaseemia FCC 6/6 Yes FCC 6/6 RFLP 75% CR Aplastic anemia FCC 4/6 No FCC/TBI same RFLP 99% CR Myelofibrosis FCC/TBI 6/6 RFLP 63% FCC same RFLP 98% CR CML FCC 4/6 RFLP 61% FCC 4/6 No PD Renal Cell FCC 3/6 RFLP 30% FCC same RFLP 94% PD AML/MDS FCC 4/6 RFLP 5% FCC/TBI same RFLP 89% PR AML FCC/TBI 3/6 RFLP 44% FCC 3/6 RFLP 98% CR AA/MDS ATG,FM UCB 4/6 No FCC/One UCB 4/6 RFLP 100% CR AA/MDS ATG,FM UCB 4/7 No FCC/One UCB 4/7 RFLP 100% CR
Kinetics of Early Donor Chimerism after Nonmyeloablative Haematopoietic Stem Cell Transplantation Monitored with High-Resolution Real Time Quantitative PCR.
Authors: Masmas Tania N. , Petersen Soren L. , Madsen Hans O. , Ryder Lars , Dickmeiss Ebbe , Svejgaard Arne , Vindelov Lars 
Published: April 2020

Abstract
Purpose: From May 2003 to March 2005, 37 patients with haematological malignancies were transplanted with peripheral blood stem cells after nonmyeloablative conditioning with fludarabine 30 mg/m2 i.v. once daily on day −4 to −2 and 200 cGY of total body irradiation on day 0. Post-transplant immunosuppression consisted of oral cyclosporin 12.5 mg/kg/day and mycophenolate mofetil 30 mg/kg/day. Three patients were not eligible for follow-up due to lack of informed consent or informative markers for chimerism measurement. The purpose of this study was to evaluate kinetics of early chimerism of CD4+, CD8+, and, CD15+ cells immediately post transplant and furthermore to identify factors associated with rejection of graft.
Methods: Blood samples were collected post transplant on day +1–3, +5–7, and then weekly. Samples were separated with immunomagnetic beads and DNA was salt extracted. Chimerism analysis was performed by automated high-resolution real-time quantitative PCR based on short insertion or deletion polymorphisms or single nucleotide polymorphisms.
Results: The kinetics of CD15+ chimerism differed from that of CD4+ and CD8+ chimerism (Figure). CD15+ donor chimerism was low with a median of 0.9% donor cells on day +1–3, and remained low until around day +14. Subsequently the donor chimerism increased rapidly towards 100%. The CD4+ and CD8+ donor chimerism in contrast started higher with a median of 16.3% and 15.1% respectively on day +1–3, increased slowly, and took months to reach 100%. CD4+, CD8+ or CD15+ donor chimerism on day +1–3 did not predict later development of acute graft versus host disease. Two patients had primary rejection of the donor stem cells, while four patients had secondary rejection. The patients with primary rejection had no measurable donor chimerism in any cell lineages from day +1–3. No patient with CD15+ donor chimerism above the median (0.9%) on day +1–3 rejected, while 40% (6/15) of the patients with CD15+ donor chimerism below the median rejected (LogRank test, p=0.01). No differences were found in total number of nucleated cells, CD34+, CD3+, CD4+, or CD8+ cells in the donor harvest between patient who rejected and those who did not.
Conclusions: The kinetics of early CD15+ donor chimerism differed from the kinetics of early CD4+ and CD8+ donor chimerism. Early CD15+ donor chimerism was markedly lower than CD4+ and CD8+ donor chimerism. CD15+ donor chimerism increased toward 100% donor chimerism more rapidly than the CD4+ and CD8+ chimerism. Primary rejection of donor stem cells can be identified early, as these patients have no measurable donor CD4+ or CD8+ chimerism on day +1–3. Furthermore, patients with CD15+ donor chimerism above the median on day +1–3 have a low risk for rejection.
Figure Figure
Non-Myeloablative Allogeneic Hematopoietic Stem Cell Transplantation (SCT) with Pre-Transplant Targeted Immune Depletion Results in Rapid Full Donor Engraftment in Pediatric Patients with Malignancy.
Authors: Fry Terry J. , Cardesa-Salzman Teresa , Hakim Frances , Love Cynthia , Layton Paula , Gress Ronald , Fowler Daniel , Bishop Michael R. , Mackall Crystal L. , Wayne Alan S. 
Published: April 2020

Abstract
Background: Although myeloablative SCT is curative for a number of pediatric malignancies, it is associated with significant acute toxicity and late effects. Non-myeloablative SCT can result in successful engraftment of adults with diminished toxicity, but there is limited experience in pediatric pts where increased thymic function might confer a greater risk of graft rejection or mixed chimerism. Prolonged periods of mixed chimerism might in turn impair the graft-vs-tumor response and increase the risk of relapse in highly proliferative pediatric cancers.
Methods: Twenty-one pediatric pts (age 7–21, median 14 years) with high-risk malignancies (Hodgkins, ALL, AML, CML, rhabdomyosarcoma, Ewings sarcoma) were enrolled. One to three cycles (C#) of fludarabine-based induction chemotherapy were administered for disease control and host immune depletion (targeted CD4 count reduction). Pre-transplant conditioning consisted of cyclophosphamide (1,200 mg/m2/day) and fludarabine (30 mg/m2/day) x 4 days plus melphalan (100 mg/m2 x 1 dose in sarcoma pts). Unmodified, G-CSF mobilized peripheral blood stem cells collected from 5–6/6 HLA-matched first degree relatives were infused (median CD34 dose 11.4 x 106/kg, range 4.44–19.12; median CD3 dose 414 x 106/kg, range 228–815). Cyclosporine was used as GVHD prophylaxis.
Results: CD4 counts decreased from 332 +/− 65 at entry to 176 +/− 30 (mean +/− SEM/μL) after induction (Fig 1). Treatment was well tolerated and there was no transplant-related mortality. Median time to ANC &gt; 500 cells/μL was post-transplant day (D+) 9 (range 7–11). Complete donor lymphoid chimerism (&gt; 95% by VNTR-PCR on CD3 sorted peripheral blood) was achieved in all pts (18/22 by D+14 and 21/22 by D+28) (Fig 2). Acute GVHD developed in 21/22 patients, but was low grade (18/21 grades 1–2, 3/21 grade 3) and responsive to immunosuppression.
Conclusions: Rapid, complete donor engraftment and chimerism can be achieved in pediatric pts with cancer using targeted pre-transplant immune depletion and a non-myeloablative preparative regimen. This approach may reduce regimen-related toxicity, and thus has the potential to improve the long-term outlook for childhood cancer survivors.
Fig 1: CD4 Depletion Fig 1:. CD4 Depletion Fig 2: Donor Chimerism (CD2) Fig 2:. Donor Chimerism (CD2)