Major Hemoglobin Research Articles

Reactive Sulphydryl Groups in Horse Carbonmonoxyhaemoglobin

Background and Objective: Reactive sulphydryl groups in haemoglobin has been related with oxygen binding. In this study, the researchers analyzed the reactive sulphydryl groups in horse (Equus ferus caballus) carbonmonoxyhaemoglobin. Materials and Methods: Hemolysate gotten from fresh horse blood was converted to yield the carbonmonoxy derivative. It was then separated via carboxymethylcellulose into major and minor fractions of haemoglobin. These fractions were titrated with Ellmanʼs reagent (DTNB) and p-hydroxymercuri(II)benzoate (p-MB) stock solutions in increasing volumes. Results: Results showed that two sulphydryl groups reacted with DTNB and p-MB in both major and minor haemoglobin fractions. p-MB is known to be more reactive with thiols than DTNB, on the other hand the reactivity is the same with horse carbonmonoxyhaemoglobin. Conclusion: This study will enable a better understanding as regards the kinetics and equilibrium behind this reaction.

Cure for thalassemia major - from allogeneic hematopoietic stem cell transplantation to gene therapy.

Allogeneic hematopoietic stem cell transplantation has been well established for several decades as gene replacement therapy for patients with thalassemia major, and now offers very high rates of cure for patients who have access to this therapy. Outcomes have improved tremendously over the last decade, even in high-risk patients. The limited data available suggests that the long-term outcome is also excellent, with a >90% survival rate, but for the best results, hematopoietic stem cell transplantation should be offered early, before any end organ damage occurs. However, access to this therapy is limited in more than half the patients by the lack of suitable donors. Inadequate hematopoietic stem cell transplantation services and the high cost of therapy are other reasons for this limited access, particularly in those parts of the world which have a high prevalence of this condition. As a result, fewer than 10% of eligible patients are actually able to avail of this therapy. Other options for curative therapies are therefore needed. Recently, gene correction of autologous hematopoietic stem cells has been successfully established using lentiviral vectors, and several clinical trials have been initiated. A gene editing approach to correct the β-globin mutation or disrupt the BCL11A gene to increase fetal hemoglobin production has also been reported, and is expected to be introduced in clinical trials soon. Curative possibilities for the major hemoglobin disorders are expanding. Providing access to these therapies around the world will remain a challenge.

Efficacy and Safety of Darbepoetin Alfa (DA) in Patients with Myelodysplastic Syndromes (MDS): A Systematic Review and Meta-Analysis

Introduction: Erythropoiesis-stimulating agents (ESAs) have been used in treating anemic MDS patients to improve erythropoiesis and reduce the risk of red blood cell (RBC) transfusion. Two previous systematic reviews found that ESA use was efficacious, but these reviews focused on short-acting epoetin alfa (EA). Since then, the results of a number of prospective interventional trials of DA have been reported. We present an updated systematic review and meta-analysis to estimate the efficacy of DA in the treatment of MDS-related anemia.Methods: We conducted a systematic review of the medical literature to identify prospective interventional trials of DA in patients with MDS. The main inclusion-exclusion (IE) criteria were: that studies had to be prospective and interventional in nature; have at least 10 adult subjects with MDS reporting either World Health Organization (WHO), French-American-British (FAB) criteria, or International Prognostic Scoring System (IPSS) status; and report results for the pre-specified primary outcome (proportion of patients with erythroid response) or one of the secondary outcomes (which included major hemoglobin response, changes from baseline in hemoglobin levels, transfusion status, and quality of life (QoL) measures). We recorded and collated all reported adverse events. Two independent reviewers identified the studies and abstracted the information and a third reviewer adjudicated. Clinical and methodological heterogeneity across studies were evaluated with respect to the study population and participant selection method (e.g. MDS diagnosis, history of ESA use, baseline erythropoietin (EPO), hemoglobin, and creatinine levels, transfusion status, and other factors), the intervention (e.g. initial and maintenance ESA dose), and the endpoints of interest including the response criteria. Forest plots with formal testing using Cochran's Q-statistic was also used to assess the heterogeneity across the studies. We used random effects methodology to generate combined estimates when warranted by the data. Subgroup analyses and/or meta-regressions were conducted by dose level, ESA-naïve status, baseline EPO level, hemoglobin level, transfusion status, and other factors.Results: Ten studies (9 single-arm, 1 randomized controlled trial [RCT]) with a total of 647 patients were included in the systematic review, with response rates ranging from 38% to 72%, and the median duration of response varying from 5 to 12 months. The overall response rate for the nine studies that used IWG 2000 response criteria could not be evaluated because of heterogeneity. However, among studies that stratified response rates by baseline endogenous EPO levels, patients with EPO <100 IU/L had an overall response of 81% (95% confidence interval [CI]: 73-90%), and on average a 39% [95% CI: 22-56%] better response than patients with EPO >100 IU/L. Across the studies that reported response stratified by prior ESA use, ESA-naïve patients had a median response rate of 75% compared to 53% for patients previously treated with an ESA. Baseline mean hemoglobin was significantly associated with response (p=0.0204), with higher baseline levels associated with improved response. We also found that response tended to improve with dose (p=0.2; Figure 2): the estimated response for a mean initial dose of 150 μg once weekly (QW) was 57% (95% CI: 49-66%) compared to 64% (95% CI: 55-74%) for 300 μg QW. Baseline transfusion independence (6 studies), and low-risk IPSS status (2 studies) were reported to be significantly associated with better response in several studies. Treatment with DA tended to show an improvement in the quality of life (QoL) measures, transfusion rates, and hemoglobin levels, but the number of studies that reported these outcomes was small. Hypertension, thromboembolism, and progression to acute myeloid leukemia were respectively reported in 2%, 1%, and 1% of the patients.Conclusions: Published studies suggest that treatment with DA yielded high hemoglobin response (38-72%) in MDS patients with anemia. The response was strongest in patients with lower baseline serum EPO level, and ESA naïve patients. [Display omitted] [Display omitted] DisclosuresPark:Hospira: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding. Fenaux:Amgen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Celgene Corporation: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Mehta:Amgen Inc.: Employment; Amgen Inc.: Equity Ownership. Callaghan:Amgen Inc.: Employment; Amgen Inc.: Equity Ownership. Kim:Amgen Inc.: Employment; Amgen Inc.: Equity Ownership. Tomita:Amgen Inc.: Employment; Amgen Inc.: Equity Ownership. Xu:Amgen Inc.: Employment; Amgen Inc.: Equity Ownership.

Functional characterisation of the haemoglobins of the migratory notothenioid fish Dissostichus eleginoides

This study addresses the primary structure, the oxygen-binding properties and the CO-rebinding kinetics of the haemoglobins of the Patagonian toothfish Dissostichus eleginoides. D. eleginoides belongs to the family Nototheniidae, the most diversified of the suborder Notothenioidei, mostly exhibiting an Antarctic distribution. Some of its features are typical of Antarctic species, some are not. For instance, D. eleginoides appears not to have functional antifreeze glycoproteins (consistent with its non-Antarctic distribution). In contrast, it has a major and a minor haemoglobin (similar to many Antarctic notothenioids), and their very low oxygen affinity does not follow the trend of other non-Antarctic notothenioids and appears typical of cold-adapted species. Moreover, the amino-acid sequence reveals high identity with the globins of Antarctic notothenioids, arguing in favour of a common origin within notothenioids, and indicates that the primary structure of the major and minor haemoglobins has undergone modifications only to a limited extent. The ligand-rebinding kinetics of the major haemoglobin of D. eleginoides indicate a strong stabilisation of the quaternary T state at lower pH values.

Abstract W MP28: Safety of Parentral Antiplatelet Regimen Within 24 Hours of Intravenous tPA Administration: Challenging the Conventional Paradigm

Background and Objective: It is considered unconventional to initiate antiplatelet regimen within 24 hours of IV tPa administration in acute ischemic strokes. There has been an increasing amount of literature assessing combination therapy of IV r- tPA and IV Eptifibatide in acute ischemic stroke. Our objectives were to evaluate the safety (hemorrhagic complications) and efficacy (discharge mRS) of administering IV Eptifibatide within the first 24 hours of receiving full dose IV r-tPA respectively. Materials and Methods: All patients that presented to our university affiliated stroke center from 2010-13 with an acute ischemic stroke were included and retrospectively classified into two groups. Group A underwent full dose IV r- tPa (.9 mg/kg) (+/- Endovascular intervention). Group B underwent full dose IV r-tPa (.9mg/kg) and IV Eptifibatide (+/- Endovascular intervention). Epitafibide was administered as a bolus of 135 mcg/kg IV followed by .5 mcg/kg/min for 20 hours. The primary endpoint of bleeding is classified as major (symptomatic intracranial hemorrhage or hemoglobin decrease by &gt;5 mg/dl), minor (asymptomatic intracranial hemorrhage or hemoglobin decrease by 3-5 mg/dl) and insignificant as proposed by TIMI score. The efficacy endpoint was discharge mRS of 0-1 as favorable, with 2 and above being unfavorable. Results: We reviewed 2,016 patients with ischemic stroke, of which 170 received IV tPA. Among the group, 118 received IV r-tPa alone and 52 received combined modalities of IV r-tPa and IV Eptifibatide. In group A, there were 7 patients who had a major complication of symptomatic intracranial hemorrhage, while 1 patient had a minor complication of asymptomatic intracranial hemorrhage. In group B, there were 3 patients who had major complication of symptomatic intracranial hemorrhage and 5 with minor complications of asymptomatic intracranial hemorrhage. In group A , 9% (n=4) had a favorable outcome (OR=2.389, 95% CI 0.6645 to 8.589, p= 0.2217). Of the 52 patients in group B, 18% (n=8) had a favorable outcome. Conclusion: IV Eptifibatide, within the first 24 hours of ischemic stroke in combination with full dose IV r-tPA was found to be safe and efficacious. Further, larger prospective trials are needed to corroborate our findings.

The Species and Heme Pocket Properties of Sturgeon Hemoglobins Upon Interaction with N-dodecyl Trimethylammonium Bromide

The variations in fish hemoglobin (Hb) structures play a vital role in their respiratory performance under various environmental conditions and are impacted by their physiological properties. The major hemoglobins from two species of sturgeon were studied upon interaction with n-dodecyl trimethyl ammonium bromide (DTAB) using the UV-vis absorption, circular dichroism (CD), fluorescence spectroscopy, and oxygen affinity measurement methods as well as chemometric analysis. The UV-Vis absorption spectra between 500 and 650 nm was used to identify each species of hemoglobin, and to show that the concentration of oxyHb and metHb decreases, while that of deoxyHb increases upon interaction with DTAB. Both reduced oxyHb and oxidized hemichrome of the two Hbs were studied to obtain information about the DTAB efects on their structural features. The circular dichroism (CD) was utilized to obtain secondary structure and compactness for Hb upon interaction with DTAB. Binding of DTAB molecules induced the unfolding of Hb, and was accompanied with exposure of the heme pocket facilitating its oxidation. The differences between unfolding processes for the two Acipenser species were indicated by fluorescence spectroscopy. The chemometric analysis of Hbs was investigated upon interaction with DTAB under titration, using fluorescence spectra allowing determination of the number of components and mole fractions of the oxidized Hb. Our data showed that Acipenser persicus Hb had a more hyperchromic character, more surface area, more loosely folded structure, and therefore, exposed region of heme group compared with Acipenser stellatus oxyHb. In addition, with increasing DTAB the transition of Acipenser stellatus oxyHb to the state of hemichrome occurred at a slower speed than Acipenser persicus oxyHb, and finally more oxygen affinity and compactness. Our results suggest that these differences aroused from the inherent differences between the heme groups which fulfil a potentially important physiological role in these fish.

Genetics and Epigenetics of Fetal Hemoglobin Control

Expression of fetal hemoglobin (HbF, α2γ2) greatly ameliorates the severity of the major hemoglobin disorders, sickle cell disease (SCD), and the β-thalassemias. Efforts to reactivate HbF in adults with these disorders have relied on empirical observations or therapeutic modalities that are indirect. A major goal for the field is the development of targeted reactivation of HbF through relief of γ-globin gene silencing. The regulatory factors that participate in the switch from HbF to HbA in ontogeny and in γ-gene silencing in the adult have been elusive, therefore precluding mechanism-based reactivation of HbF. Recent findings, largely derived from genome-wide association studies (GWAS), have transformed the current understanding of globin switching. This presentation will review recent evidence supporting direct involvement of the zinc-finger repressor protein BCL11A in both developmental switching of globins and HbF silencing in the adult. These studies include the impact of naturally occurring genetic variation at the BCL11A locus on HbF levels, proof-of-principle experiments in genetically engineered mice suggesting that interference with BCL11A action alone may be sufficient to provide therapeutic elevation of HbF, and the nature of protein partners of BCL11A that may mediate some aspects of BCL11A function. Recent findings on the manner in which genetic variation within the BCL11A locus influences BCL11A expression provide special insight into quantitative aspects of HbF regulation and raise the possibility of new strategies to cripple BCL11A. The opportunities and challenges for the development of mechanism-based reactivation of HbF will be discussed in the context of ongoing efforts to exploit small molecule and genetic approaches. The tools are in hand to translate an improved understanding of globin gene regulation for the benefit of patients with the major hemoglobin disorders. Disclosures:No relevant conflicts of interest to declare.

Die Milz bei nicht-malignen hämatologischen Erkrankungen

The spleen functions as a filter of the circulating blood, removing aging or abnormal red blood cells, intraerythrocyte inclusions as well as foreign particals. As the spleen is composed of lymphocytic tissue, circulatory elements and mononuclear phagocytic cells it plays an important role in the nonspecific as well as the specific immune response. Additionally, the spleen serves as a reservoir for circulating blood cells, especially platelet sequestration by the spleen is well do cumented. The spleen produces blood cells during fetal development and in certain haematological disorders such as myelofibrosis. The destruction of red blood cells within the splenic cords releases iron in the circulation, which is recycled and used to manufacture new erythrocytes in the bone marrow. In several non-malignant haematological disorders antibody-coated cells are cleared from the circulation by phagocytic cells of the spleen. This involves erythrocytes in autoimmunhaemolytic anaemias, platelets in immunthrombocytopenia and neutrophils in Felty syndrome. In hereditary spherocytosis the spleen destroys the resulting defective, spherical red cells. In pyruvate kinase deficiency impaired production of adenosine triphosphate leads to destruction of red blood cells in the spleen or in the liver. In sickle cell anaemia the defective erythrocytes cause sludging and thrombosis in small vessels with infarcts for instance in the spleen, which over time can result in autosplenectomy. In thalassaemia major abnormal haemoglobin forms protein precipitates in the red cells with development of a severe hypochromic anaemia with haemolysis and intramedullary inef fective erythropoiesis. Therapeutic splenectomy can be an option in all of these mentioned non-malignant haematological disorders. The rationale and the pathophysiology of its role in thrombotic thrombocytopenic purpura is probably at least well understood. The use of new and effective drugs such as the monoclonal antibody rituximab or thrombopoietin agonists in immunthrombocytopenia as well as the option of a laparoscopic rather than an open splenectomy have changed our view on benefits and risks of splenectomy in recent years. To minimize these risks such as overwhelming postsplenectomy infection or abdominal vein thrombosis, prophylactic vaccination, postoperative thromboembolism prophylaxis and patient education is mandatory.

Abstract TP123: Open-label Phase I Clinical Study to Assess the Safety and Efficacy of Cilostazol in Patients Undergoing Carotid Artery Angioplasty and Stent Placement

Background: One month dual antiplatelet therapy, with Aspirin and Clopidogrel, following intra- and/or extra-cranial stent placement is the standard of care to prevent in- stent thrombosis. Cilostazol, a novel antiplatelet drug, with vasodilatory effects and smooth muscle cell proliferation inhibition, has recently been demonstrated to be safe and efficacious in secondary stroke prevention and has a safety profile comparable to both Aspirin and Clopidogrel. Objective: To evaluate the safety and clinical efficacy of Cilostazol and Aspirin therapy following internal carotid angioplasty and stent placement prior to and one month post-procedure. Methods: We conducted a Phase I open label, non-randomized single center prospective study. All patients received Aspirin (325 mg/day) and Cilostazol (200 mg/day) for at least 3 days before internal carotid stenting. The two anti-platelet agents were continued for one month after the procedure and then patients were continued on aspirin daily. The primary efficacy end point was the 30-day composite occurrence of death, stroke, TIA, and unplanned endovascular revascularization. The primary safety end point was bleeding. Bleeding complications were classified as major (hemoglobin decrease &gt;5 g/dl), minor (hemoglobin decrease 3-5 g/dl), or insignificant. Results: Twelve patients (mean age 66±12 years; 10 men) were enrolled and underwent internal carotid angioplasty and stent placement. One patient discontinued Cilostazol after the first dose, prior to stent placement, secondary to non-specific dizziness. Another patient did not follow study protocol and continued anticoagulation dose enoxoparin with Aspirin and Cilostazol resulting in symptomatic intracerebral hemorrhage 15 hours following successful stent placement; ultimately leading to withdrawal of care. None of the patients that successfully completed the study, and followed protocol, experienced any complications at 1-month and 3-month follow up. Conclusion: The use of Cilostazol and Aspirin for carotid angioplasty and stent placement appears to be safe but protocol compliance needs to be emphasized. Further studies are required to analyze the effectiveness and role of Cilostazol in neurointerventional procedures.

High thermal sensitivity of blood enhances oxygen delivery in the high-flying bar-headed goose

The bar-headed goose (Anser indicus) crosses the Himalaya twice a year at altitudes where oxygen (O2) levels are less than half those at sea level and temperatures are below -20°C. Although it has been known for over three decades that the major hemoglobin (Hb) component of bar-headed geese has an increased affinity for O2, enhancing O2 uptake, the effects of temperature and interactions between temperature and pH on bar-headed goose Hb-O2 affinity have not previously been determined. An increase in breathing of the hypoxic and extremely cold air experienced by a bar-headed goose at altitude (due to the enhanced hypoxic ventilatory response in this species) could result in both reduced temperature and reduced levels of CO2 at the blood-gas interface in the lungs, enhancing O2 loading. In addition, given the strenuous nature of flapping flight, particularly in thin air, blood leaving the exercising muscle should be warm and acidotic, facilitating O2 unloading. To explore the possibility that features of blood biochemistry in this species could further enhance O2 delivery, we determined the P50 (the partial pressure of O2 at which Hb is 50% saturated) of whole blood from bar-headed geese under conditions of varying temperature and [CO2]. We found that blood-O2 affinity was highly temperature sensitive in bar-headed geese compared with other birds and mammals. Based on our analysis, temperature and pH effects acting on blood-O2 affinity (cold alkalotic lungs and warm acidotic muscle) could increase O2 delivery by twofold during sustained flapping flight at high altitudes compared with what would be delivered by blood at constant temperature and pH.

Analysis of oxygen affinity in aquatic amphibian; homology modelling of the major Haemoglobin component HbA1 from the African clawed frog (Xenopus laevis, Anura)

The homology model of major haemoglobin component HbA1 of the African Clawed Frog was predicted using the pigeon (Columba livia) haemoglobin as a template. The model was built with the help of MODELLER9v8. The models were evaluated with ProSA and PROCHECK. In X. laevis Gln38α is unable to form a hydrogen bond with β97His or β99Asp, which is responsible for the increase in oxygen affinity of the Xenopus HbA1. The hydrogen bond between α34Thr and β124Pro, which stabilises the deoxy state of the haemoglobin, was absent in X. laevis. Hence it is predicted that the HbA1 component of X. laevis has higher oxygen affinity.

Synthesis and Insight into the Structure–Activity Relationships of Chalcones as Antimalarial Agents

Licochalcone A (I), isolated from the roots of Chinese licorice, is the most promising antimalarial compound reported so far. In continuation of our drug discovery program, we isolated two similar chalcones, medicagenin (II) and munchiwarin (III), from Crotalaria medicagenia , which exhibited antimalarial activity against Plasmodium falciparum . A library of 88 chalcones were synthesized and evaluated for their in vitro antimalarial activity. Among these, 67, 68, 74, 77, and 78 exhibited good in vitro antimalarial activity against P. falciparum strains 3D7 and K1 with low cytotoxicity. These chalcones also showed reduction in parasitemia and increased survival time of Swiss mice infected with Plasmodium yoelii (strain N-67). Pharmacokinetic studies indicated that low oral bioavailability due to poor ADME properties. Molecular docking studies revealed the binding orientation of these inhibitors in active sites of falcipain-2 (FP-2) enzyme. Compounds 67, 68, and 78 showed modest inhibitory activity against the major hemoglobin degrading cysteine protease FP-2.

Method for noninvasive determination of hemoglobin content in biological tissues

We have developed a simple and efficient method for determining the hemoglobin concentration in biological tissues, based on spatially resolved measurements of the diffuse reflectance of the tissue at λ = 524, 578, and 662 nm (or 773 nm) using multiple regressions between the measured parameters and the hemoglobin concentrations. The method takes into account the presence in the blood of the major hemoglobin derivatives (oxyhemoglobin, deoxyhemoglobin, carboxyhemoglobin, and methemoglobin) and eliminates the effect of the parameters of the top layer of the biological tissue (for example, the epidermis of the skin) and the bulk scattering coefficient of the tissue on the measurement results. Based on numerical Monte Carlo experiments, we have estimated the uncertainties in determining the hemoglobin concentration in skin tissues when its structural and biochemical parameters are all variable.

Kinetics of reaction of 5,5′-dithiobis(2-nitrobenzenzoate) with CysF9[93]β sulphydryl group of sheep haemoglobins at neutral and physiological pH ranges

The kinetics of the reaction of 5,5′-dithiobis(2-nitrobenzoate) (DTNB) with CysF9[93]β sulphydryl group of the sheep haemoglobins were studied at neutral and physiological pH (6.8⩽pH⩽7.6) ranges under pseudo first order conditions. Each reaction is of first order with respect to the DTNB concentration. The pH dependence of the observed rate constant of the reaction at neutral and physiological ranges gave a complex trend. The observed rate shows that at neutral pH, the presence of inositol hexakisphossphate (IHP) leads to a relative lowering of the pseudo first order rate constant. Overall, the rate varied among the derivatives as aquomet>oxy>carbomonoxy and the major haemoglobin show a higher reactivity than the minor haemoglobin.

Methylphenidate (MP) and nursing telephone intervention (NTI) for cancer-related fatigue (CRF) in advanced cancer patients: A double-blind randomized phase II trial.

9023 Background: CRF is the most common and distressing symptom in advanced cancer patients. Preliminary studies support MP and NTI for CRF (Bruera et al. JCO 2006). The primary objective of our study was to determine the effect of MP as compared to placebo (P). A secondary objective was to investigate the role of NTI as compared to control telephone intervention (CTI). Methods: Advanced cancer patients with fatigue ≥4/10 on the Edmonton Symptom Assessment Scale (ESAS), normal cognition, no evidence of major depression and hemoglobin ≥8 were eligible. Patients were randomized to 4 groups in a 2x2 factorial design (MP+NTI, P+NTI, MP+CTI and P+CTI). Primary endpoint was Functional Assessment of Chronic Illness-Fatigue (FACIT-F) subscale scores between day 15 and baseline. The dose and duration of methylphenidate was 5 mg every two hours, as needed, up to 20 mg/day. We tested the median difference in FACIT-F subscale scores between the groups using the Kruskal Wallis test and Wilcoxon signed rank test. Longitudinal regression analysis was conducted with a mixed model. Results: Total accrual was 197. Mean (SD) age was 58 (12), female 67% (N=148), white 72% (N=136), gastrointestinal cancers were the most common 22% (N=41). Baseline FACIT-F subscores were similar among the 4 groups. The median FACIT-F subscores showed significant improvement between Day 15 and baseline for all four groups except for P+CTI (Table): MP+NTI (4.5, P=0.004), P+NTI (8, P&lt;0.001), MP+CTI (7, P&lt;0.001), and P+CTI (5, P=0.06), with no statistically significant difference between MP and P (6 vs. 6, P=0.89). Longitudinal regression analysis showed a time effect (P&lt;0.001) and group differences for NTI vs. CTI with FACIT-F (P=0.13) and ESAS (P=0.03). Grade 3 toxicities were similar between the MP and P arms (34/93 vs. 24/97, P=0.09). Conclusions: MP was not effective as compared to P for CRF in advanced cancer patients. NTI may be effective and should be further studied. [Table: see text]

ATP regulation of the ligand-binding properties in temperate and cold-adapted haemoglobins. X-ray structure and ligand-binding kinetics in the sub-Antarctic fish Eleginops maclovinus

The major haemoglobin of the sub-Antarctic fish Eleginops maclovinus was structurally and functionally characterised with the aim to compare molecular environmental adaptations in the O(2)-transport system of sub-Antarctic fishes of the suborder Notothenioidei with those of their high-latitude relatives. Ligand-binding kinetics of the major haemoglobin of E. maclovinus indicated strong stabilisation of the liganded quaternary T state, enhanced in the presence of the physiological allosteric effector ATP, compared to that of high-Antarctic Trematomus bernacchii. The activation enthalpy for O(2) dissociation was dramatically lower than that in T. bernacchii haemoglobin, suggesting remarkable differences in temperature sensitivity and structural changes associated with O(2) release and exit from the protein. The haemoglobin functional properties, together with the X-ray structure of the CO form at 1.49 Å resolution, the first of a temperate notothenioid, strongly support the hypothesis that in E. maclovinus, whose life-style varies according to changes in habitat, the mechanisms that regulate O(2) affinity and the ATP-induced Root effect differ from those of high-Antarctic Notothenioids.

Correction of Murine Sickle Cell Disease Through Interference with Fetal Hemoglobin Silencing

Abstract 351The switch from fetal (HbF, α2γ2) to adult (HbA, α2β2) hemoglobin is critical to the pathogenesis of the major hemoglobin disorders, sickle cell disease (SCD) and β-thalassemias. Increased HbF expression ameliorates the clinical severity of these conditions, forming the basis for the goal of elucidating mechanisms of globin switching and developing target-based therapies to enhance HbF production. The rationale for this therapeutic approach rests on the hypothesis that manipulation of a single target would lead to sufficient HbF reactivation for clinical benefit. Despite recent advances in understanding the complex processes of developmental globin switching and HbF silencing, this hypothesis is untested. The transcription factor BCL11A is a critical mediator of HbF switching and silencing. Previously we showed that BCL11A controls globin switching in mice and silencing of HbF expressed from human β-globin locus transgenes in mouse fetal liver, and contributes to HbF silencing in primary human erythroid cells. Furthermore, we demonstrated that BCL11A is required in vivo for HbF silencing in adult animals, yet dispensable for red cell production. These findings illustrate favorable features of BCL11A as a potential target for reactivation of HbF in the β-hemoglobinopathies.Here, we test whether impairment of BCL11A is sufficient to ameliorate disease symptoms in a relevant preclinical model of a major β-hemoglobin disorder, the humanized SCD mouse model (“Berkeley” SCD model, Paszty et al. Science 278:876–878, 1997). These mice express exclusively human sickle hemoglobin and faithfully recapitulate the principal hematologic and histopathologic phenotypes of patients with SCD. We introduced BCL11A-null alleles into the “Berkeley” SCD model (SCD Bcl11afl/fl EpoR-Cre+, hereafter called SCD/Bcl11a−/− mice). Loss of BCL11A in SCD animals restored the hematologic deficits to normal values. Compared with sickle animals, SCD/Bcl11a−/− mice have marked increases in RBC counts (6.4 ± 0.5 to 9.8 ± 0.4 × 1012/L; non-sickle control 10.1 ± 0.2 × 1012/L ) and hemoglobin level (7.8 ± 0.6 to 13.6 ± 0.7 g/L; non-sickle control 13.1 ± 0.3 g/L), accompanied by a significant reduction in reticulocyte counts (38.2% ± 3.9% to 7.0% ± 0.3%; non-sickle control 3.1% ± 0.6%). Consistent with correction of hematologic parameters, sickled erythrocytes were absent in the peripheral blood of SCD/Bcl11a−/− mice and red cell lifespan was substantially normalized. In addition, tissue damage in multiple organs was prevented in SCD/Bcl11a−/− mice. Urine osmolality of SCD/Bcl11a−/− mice was restored (from 1037 ± 82 mOsm in SCD mice to 2133 ± 333 mOsm in SCD/Bcl11a−/− mice), indicating improved renal function.To assess the extent to which these phenotypic findings were related to HbF induction, we determined the level of γ-globin expression and cellular distribution of HbF in SCD/Bcl11a−/− mice. Compared with SCD mice, expression of γ-globin genes was markedly elevated in adult SCD/Bcl11a−/− mice (1.3% to 28.3% of total β-like human globins; P = 0.00017). Peripheral blood of control and SCD mice contained few F cells (3.6% and 7.2%, respectively). In contrast, peripheral blood of SCD/Bcl11a−/− mice displayed strong pancellular staining of HbF, and F cells contributed to 85.1% of total RBCs (P = 3.9 × 10−5). The level of HbF expression achieved in the absence of BCL11A is well beyond that needed to impede HbS polymerization.In conclusion, we demonstrate that inactivation of a single component involved in HbF regulation, the transcription factor BCL11A, provides phenotypic correction of SCD in a mouse model. Our findings provide a crucial “proof of principle” for targeted reactivation of HbF as a realistic therapeutic goal. Disclosures:No relevant conflicts of interest to declare.

Renal Iron Load in Sickle Cell Disease Correlates with Hemolysis and Transfusion History, but Not with Hepatic Iron

Abstract 2129 Background:Sickle cell disease (SCD) is characterised by chronic hemolysis and frequent, painful vaso-occlusive episodes, eventually leading to organ damage. Overall, renal failure is found in approximately 11% of SCD patients, and incidence increases with age. Both the vaso-occlusive and hemolytic aspects of SCD have been implicated in renal failure, as assessed by glomerular filtration rate (GFR) and microalbuminurea. Additionally, the increasing use of blood transfusion therapy in SCD has resulted in a sub-population with secondary iron overload, also leading to organ damage. Differential organ iron loading has been noted in the two major hemoglobin disorders; cardiac iron toxicity is common in β-thalassemia patients with high liver iron concentration (LIC), whereas this is uncommon in SCD patients for an equivalent LIC. Relatively limited research has been carried out regarding renal iron loading in SCD. This study investigates the clinical significance and etiology of renal iron accumulation in SCD patients. Participants and Methods:41 patients from the specialist hematology clinic at King’s College Hospital, London had already had an assessment of hepatic iron loading by spin density projection assisted R2-MRI (FerriScan®) as part of their clinical care programme and/or as part of another study approved by the NHS Research Ethics Committee (REC 05/Q0703/21). One patient was excluded due to prior chelation therapy; the remaining 40 were chelation naïve. Owing to the retrospective nature of the study, the REC confirmed that informed consent was not required of patients for this study. Renal R2 (R-R2) values were derived from monoexponential fits to kidney image data obtained during LIC assessment in order to assess kidney iron concentration. R-R2 data were also collected from 17 healthy control participants to provide a reference range. Control data were acquired with approval from the Fremantle Hospital Human Research Ethics Committee (08/404) and the University of Western Australia Human Research Ethics Committee.Clinical data were collected retrospectively for the 2 year period prior to the MRI scan. Thresholds for renal hyperfiltration and microalbuminurea were MDRD eGFR ≥140 ml/min/1.73m2 and albumin:creatinine ratio ≥4.5 mg/mmol, respectively. Lifetime blood transfusion history (top-up units) was recorded up to the date of the R2-MRI scan. Results:Mean R-R2 (expressed in units of s−1) was significantly higher (p<0.0001) in the SCD patients (26.87 ± SD 8.89 s−1) compared with healthy controls (17.77 ± SD 2.94 s−1). R-R2 values correlated with transfusion history (r2=0.23, p=0.004) but not with LIC. Highly significant correlations were seen between R-R2 and bilirubin (r2=0.39, p<0.0001) and lactate dehydrogenase levels (r2=0.37, p<0.0001), known markers of hemolysis. SCD patients with co-inherited α thalassemia had a lower mean R2 (23.45 ± SD 6.1 s−1) than those without (28.94 ± 9.6 SD s−1), which approached significance (p=0.070).Patients were stratified into those with evidence of renal hyperfiltration and those without. Mean R-R2 was significantly higher (p=0.019) in patients with renal hyperfiltration (according to the described MDRD eGFR criteria) than those without (30.48 ± SD 6.6 s−1vs. 23.3 ± SD 10.3 s−1, respectively). A weak but significant negative correlation was observed between renal R2 and age (r2=0.12, p=0.03). The renal hyperfiltration group were also significantly younger (p=0.003) than those with normal filtration rates (29.8 ± SD 11.26 vs. 42.3 ± SD 11.24 years). No significant relationship was observed between R-R2 and microalbuminurea. Conclusion:We showed that R-R2 is not related to liver iron concentration, but does correlate with transfusion history and markers of hemolysis. Given the lack of correlation with LIC, we believe that the correlation with transfusion history is not causative, but rather reflects the increased clinical severity; those patients with more severe disease and anemia requiring more frequent transfusions. Though not significant, mean renal R2 was noticeably lower in patients who have co-inherited α-thalassemia, in keeping with the reduced hemolysis and protective effect of α-thalassemia on renal impairment in SCD. Disclosures:St Pierre:Resonance Health: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Research Funding, Speakers Bureau.

Inositol-P6 induced structural changes in duck major haemoglobin

The reactivities of 5,5’-Dithiobis (2-nitrobenzoate) with the sulphydryl groups of major haemoglobin of duck ( Anas platyrhinchos ) have been carried out in the presence of inositol hexakisphosphate, inositol-P 6 . The time course of the reaction is biphasic. The addition of inositol-P 6 changed the pH dependence profile of the apparent second-order rate constant of both oxy- and carbonmonoxyhaemoglobin for the reaction with CysF9(93)β. Inositol-P 6 reduced the rate of reaction with CysF9(93)β of the carbonmonoxy derivative by about 2 to 9-fold, depending on the pH. In contrast, the addition of inositol-P 6 increased the rates of reaction of the oxy- and aquomet derivatives. This difference might have risen from the fact that the salt bridge that is normally formed in human haemoglobins between HisHC3(146)β and AspFG(94)β was not formed in the presence of inositol-P 6 in liganded derivatives whose pH profiles were changed. Consequently, HisHC3(146)β comes sufficiently close to the CysF9(93)β sulphydryl group to interact with it electrostatically and so influences its reactivity directly. However, inositol-P 6 changed only that of carbonmonoxyhaemoglobin for CysB5(23)β. For the reaction with CysB5(23)β, inositol-P6 reduced the rate in the carbonmonoxy derivative with increasing pH up to about 15-fold at pH 9.0, and increased the rates of reaction of the oxy- and aquomet derivatives by about 2 to 3-fold. Key words : 5,5‟-Dithiobis (2-nitrobenzoate), duck, haemoglobin, inositol-P 6 , sulphydryl group.

Inositol-P 6 induced structural changes in duck major haemoglobin

The reactivities of 5,5’-Dithiobis (2-nitrobenzoate) with the sulphydryl groups of major haemoglobin of duck ( Anas platyrhinchos ) have been carried out in the presence of inositol hexakisphosphate, inositol-P 6 . The time course of the reaction is biphasic. The addition of inositol-P 6 changed the pH dependence profile of the apparent second-order rate constant of both oxy- and carbonmonoxyhaemoglobin for the reaction with CysF9(93)β. Inositol-P 6 reduced the rate of reaction with CysF9(93)β of the carbonmonoxy derivative by about 2 to 9-fold, depending on the pH. In contrast, the addition of inositol-P 6 increased the rates of reaction of the oxy- and aquomet derivatives. This difference might have risen from the fact that the salt bridge that is normally formed in human haemoglobins between HisHC3(146)β and AspFG(94)β was not formed in the presence of inositol-P 6 in liganded derivatives whose pH profiles were changed. Consequently, HisHC3(146)β comes sufficiently close to the CysF9(93)β sulphydryl group to interact with it electrostatically and so influences its reactivity directly. However, inositol-P 6 changed only that of carbonmonoxyhaemoglobin for CysB5(23)β. For the reaction with CysB5(23)β, inositol-P6 reduced the rate in the carbonmonoxy derivative with increasing pH up to about 15-fold at pH 9.0, and increased the rates of reaction of the oxy- and aquomet derivatives by about 2 to 3-fold. Key words : 5,5‟-Dithiobis (2-nitrobenzoate), duck, haemoglobin, inositol-P 6 , sulphydryl group.