ABSTRACT
A half-volume quadrature head transverse electromagnetic (TEM) coil has been constructed for 4 T imaging applications. This coil produces a sufficiently large homogeneous B(1) field region for the use as a volume coil. It provides superior transmission efficiency, resulting in significantly lower power deposition, as well as greater sensitivity and improved patient comfort and accessibility compared with conventional full-volume coils. Additionally, this coil suppresses the RF penetration artifact that distorts the RF magnetic field profile and alters the intensity in high-field images recorded with linear surface and volume coils. These advantages make it possible to apply this device as an efficient transmit/receive coil for high-field imaging with a restricted field of view.
Subject(s)
Magnetic Resonance Imaging/instrumentation , Brain/anatomy & histology , Electromagnetic Phenomena , Humans , Image Enhancement/methodsABSTRACT
Sickle cell disease (SCD) is caused by a single point mutation in the human betaA globin gene that results in the formation of an abnormal hemoglobin [HbS (alpha2betaS2)]. We designed a betaA globin gene variant that prevents HbS polymerization and introduced it into a lentiviral vector we optimized for transfer to hematopoietic stem cells and gene expression in the adult red blood cell lineage. Long-term expression (up to 10 months) was achieved, without preselection, in all transplanted mice with erythroid-specific accumulation of the antisickling protein in up to 52% of total hemoglobin and 99% of circulating red blood cells. In two mouse SCD models, Berkeley and SAD, inhibition of red blood cell dehydration and sickling was achieved with correction of hematological parameters, splenomegaly, and prevention of the characteristic urine concentration defect.
Subject(s)
Anemia, Sickle Cell/therapy , Genetic Therapy , Genetic Vectors , Globins/genetics , HIV-1/genetics , Anemia, Sickle Cell/genetics , Animals , Disease Models, Animal , Erythrocytes/metabolism , Gene Expression , Globins/metabolism , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/metabolism , Hemoglobin, Sickle/metabolism , Humans , Lentivirus/genetics , Locus Control Region , Mice , Mice, Inbred C57BL , Mice, Transgenic , Oxyhemoglobins/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Thalassemia/genetics , Thalassemia/therapy , Transduction, Genetic , Transgenes , beta-GlobinsABSTRACT
The solubility of deoxygenated hemoglobin S (HbS), which is the concentration of fully deoxygenated HbS in equilibrium with polymer (C(SAT)), is a factor that determines in vivo polymer formation. However, measurement of C(SAT) is usually performed under conditions that are far from physiological. In solution studies of HbS by Benesch et al, it was demonstrated that p50, the point at which hemoglobin is half-saturated with oxygen, is proportional to the amount of polymer formed and that it may be used to measure C(SAT). This method has been extended to measure C(SAT) in intact red cells by varying extracellular osmolarity, which, in turn, varies intracellular hemoglobin concentration. This method measures intracellular C(SAT) under physiological conditions with intact red cell contents and can be applied to human and transgenic mouse red cells. The principle is demonstrated by measuring p50 as a function of extracellular osmolarity for AA, SS, and AS red cells. (Blood. 2001;98:883-884)
Subject(s)
Hemoglobin, Sickle/analysis , Anemia, Sickle Cell/blood , Buffers , Dimerization , Erythrocytes/chemistry , Hemoglobin, Sickle/chemistry , Hemoglobin, Sickle/metabolism , Hemoglobins/metabolism , Humans , Osmolar Concentration , Polymers/analysis , Solubility/drug effects , Solutions/chemistry , Solutions/pharmacologyABSTRACT
K:Cl cotransport (KCl) was examined in transgenic mice expressing exclusively human hemoglobin C. In contrast to previous studies in early transgenic mice expressing human alpha and beta(S) and residual mouse globins, we found significant volume and pH stimulation and sensitivity to. Exposure to physiological levels of also blocked a significant fraction of KCl cotransport.
Subject(s)
Bicarbonates/pharmacology , Carrier Proteins/antagonists & inhibitors , Erythrocytes/metabolism , Hemoglobin C/biosynthesis , Symporters , Animals , Carrier Proteins/metabolism , Erythrocytes/pathology , Humans , Mice , Mice, Knockout , Mice, Transgenic , Models, Animal , K Cl- CotransportersABSTRACT
PURPOSE: Patients with sickle cell disease have elevated circulating levels of cytokines including tumor necrosis factor (TNF) alpha. TNF-alpha stimulates expression by endothelial cells of adhesion molecules, including vascular cell adhesion molecule (VCAM) 1. Others have demonstrated that VLA-4 (alpha(4)beta(1)), a ligand for VCAM-1 or fibronectin, is present on a fraction of sickle reticulocytes. The intent of this study was to determine, using a rat model, if TNF-alpha increases retention of sickle erythrocytes in retina and if that retention can be inhibited. METHODS: TNF-alpha was given intraperitoneally to rats 5 hours before IV administration of FITC-labeled, density-separated sickle erythrocytes. After 5 minutes, rats were exsanguinated, and retinas were excised and incubated for ADPase activity, permitting the determination of the number and location of retained cells. RESULTS: TNF-alpha caused a three- to fourfold increase in retention of sickle erythrocytes in retinal capillaries (P < 0.05) but not of normal human erythrocytes. Preincubation of sickle erythrocytes with TBC772, a peptide that blocks the binding of alpha(4)beta(1) and alpha(4)beta(7), or a monoclonal antibody against VLA-4 (19H8), significantly inhibited the TNF-alpha-induced retention (P < or = 0.02), whereas a control cyclic peptide and antibody had no effect. IV TBC772 also inhibited sickle erythrocyte retention (P = 0.01). Two intravenously administered anti-fibronectin antibodies inhibited sickle cell retention as well, but an anti-rat VCAM-1 antibody did not inhibit retention. CONCLUSIONS: The authors conclude that TNF-alpha stimulates retention of sickle erythrocytes in the retinal vasculature. This increased retention can be blocked by a VLA-4 antagonist, suggesting that the cells retained after cytokine stimulation are reticulocytes. The counter-receptor for VLA-4 in this rat retina model appears to be fibronectin and not VCAM-1, based on data obtained using antibodies against these molecules.
Subject(s)
Anemia, Sickle Cell/blood , Erythrocytes, Abnormal/metabolism , Integrins/antagonists & inhibitors , Receptors, Lymphocyte Homing/antagonists & inhibitors , Retinal Vessels/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Animals , Antibodies, Monoclonal/pharmacology , Cell Adhesion/drug effects , Endothelium, Vascular/drug effects , Humans , Injections, Intraperitoneal , Integrin alpha4beta1 , Integrins/immunology , Integrins/metabolism , Male , Microscopy, Fluorescence , Peptides, Cyclic/pharmacology , Rats , Rats, Sprague-Dawley , Receptors, Lymphocyte Homing/immunology , Receptors, Lymphocyte Homing/metabolism , Reticulocytes/drug effects , Vascular Cell Adhesion Molecule-1/metabolismSubject(s)
Anemia, Sickle Cell , Disease Models, Animal , Globins/genetics , Mice, Transgenic , Animals , Humans , Mice , Mice, KnockoutABSTRACT
Sickle transgenic mice expressing exclusively human globins are desirable for studying pathophysiology and testing gene therapy strategies, but they must have significant pathology and show evidence of amelioration by antisickling hemoglobins. Mice were generated that expressed exclusively human sickle hemoglobin with 3 levels of HbF using their previously described sickle constructs (cointegrated human miniLCRalpha2 and miniLCRbeta(S) [PNAS 89:12150, 1992]), mouse alpha- and beta-globin-knockouts, and 3 different human gamma-transgenes. It was found that, at all 3 levels of HbF expression, these mice have balanced chain synthesis, nearly normal mean corpuscular hemoglobin, and, in some cases, F cells. Mice with the least adult HbF expression were the most severe. Progressive increase in HbF from less than 3% to 20% to 40% correlated with progressive increase in hematocrit (22% to 34% to 40%) and progressive decrease in reticulocyte count (from 60% to 30% to 13%). Urine concentrating ability was normalized at high HbF, and tissue damage detected by histopathology and organ weight were ameliorated by increased HbF. The gamma-transgene that produces intermediate levels of HbF was introduced into knockout sickle mice described by Pàszty and coworkers that express the miniLCRalpha1(G)gamma(A)gammadeltabeta(S) transgene and have fetal but not adult expression of HbF. It was found that the level of HbF required to ameliorate low hematocrit and normalize urine concentrating defect was different for the miniLCRalpha2beta(S) and miniLCRalpha1(G)gamma(A)gammadeltabeta(S) mice. We conclude that knockout mice with the miniLCRalpha2beta(S) transgene and postnatal expression of HbF have sufficiently faithful sickle pathology to serve as a platform for testing antisickling interventions.
Subject(s)
Anemia, Sickle Cell , Disease Models, Animal , Mice, Knockout/genetics , Mice, Transgenic/genetics , 2,3-Diphosphoglycerate/blood , Age Factors , Anemia, Sickle Cell/blood , Anemia, Sickle Cell/metabolism , Anemia, Sickle Cell/pathology , Animals , Chromatography, High Pressure Liquid , Erythrocytes/drug effects , Erythrocytes/metabolism , Erythrocytes/pathology , Fetal Hemoglobin/pharmacology , Globins/biosynthesis , Globins/drug effects , Hematocrit , Hemoglobin, Sickle/drug effects , Hemoglobin, Sickle/genetics , Humans , Kidney/drug effects , Kidney/pathology , Kidney Concentrating Ability/drug effects , Liver/drug effects , Liver/pathology , Mice , Mice, Inbred C57BL , Reticulocyte Count , Spleen/drug effects , Spleen/pathology , Thalassemia/blood , Thalassemia/metabolism , Thalassemia/pathologyABSTRACT
The purpose of this study was to develop an in vivo, noninvasive method to assess the velocities of normal and sickle red blood cells (RBCs) in the retinal and choroidal vasculatures of rats. Human and rat RBCs were isolated from whole blood, labeled with fluorescein isothiocyanate (FITC), and administered intravenously to anesthetized rats. A Rodenstock scanning laser ophthalmoscope (SLO) was used to image the FITC-labeled RBCs as an NTSC video signal. Video sequences of RBC transit in the retinal (pigmented rats) and choroidal (albino rats) vessels were captured directly to digital format. Following in vivo angiography, the animals were sacrificed, the eyes enucleated, and retinas prepared by our adenosine diphosphatase vascular labeling technique for viewing by conventional optical microscopy. Although rat and normal human RBCs differ slightly in size, their velocities were similar in the retinal arteries and capillaries (within 4%). Velocities of RBCs from sickle cell patients (sRBCs) were slower by 12 and 9% in arteries and by 38 and 25% in capillaries, compared to rat and normal human RBCs, respectively. Compared to velocities in retinal capillaries, the velocities in choroidal capillaries were much slower for rat RBCs (77%), normal human RBCs (79%), and sRBCs (67%). In contrast to normal human RBCs, sRBCs were often retained transiently in retinal capillaries at preferred sites, but in choroidal capillaries large numbers of cells were retained for extended periods. SLO imaging of FITC-labeled RBCs in rat retina and choroid provided a reliable method for evaluating normal and abnormal hemodynamics.
Subject(s)
Anemia, Sickle Cell/blood , Anemia, Sickle Cell/physiopathology , Choroid/blood supply , Erythrocytes, Abnormal/physiology , Erythrocytes/physiology , Retinal Vessels/physiology , Animals , Blood Flow Velocity , Fluorescein-5-isothiocyanate , Fluorescent Dyes , Humans , Male , Ophthalmoscopy , Rats , Rats, Inbred BN , Rats, Sprague-DawleyABSTRACT
When present in the homozygous form, hemoglobin C (HbC, CC disease) increases red cell density, a feature that is the major factor underlying the pathology in patients with SC disease (Fabry et al., JCI 70, 1315, 1982). The basis for the increased red cell density has not yet been fully defined. We have generated a HbC mouse in which the most successful founder expresses 56% human alpha and 34% human beta(C). We introduced knockouts (KO) of mouse alpha- and beta-globins in various combinations. In contrast to many KO mice, all partial KOs have normal MCH. Full KOs that express exclusively HbC and no mouse globins have minimally reduced MCH (13. 7 +/- 0.3 pg/cell vs 14.5 +/- 1.0 for C57BL/6) and a ratio of beta- to alpha-globin chains of 0.88 determined by chain synthesis; hence, these mice are not thalassemic. Mice with beta(C) > 30% have increased MCHC, dense reticulocytes, and increased K:Cl cotransport. Red cell morphology studied by SEM is strikingly similar to that of human CC cells with bizarre folded cells. We conclude that red cells of these mice have many properties that closely parallel the pathology of human disease in which HbC is the major determinant of pathogenesis. These studies also establish the existence of the interactions with other gene products that are necessary for pleiotropic effects (red cell dehydration, elevated K:Cl cotransport, morphological changes) that are also present in these transgenic mice, validating their usefulness in the analysis of pathophysiological events induced by HbC in red cells.
Subject(s)
Hemoglobin C/genetics , Alanine Transaminase/blood , Animals , Biological Transport , Bone Marrow/pathology , Breeding , Calcium/pharmacology , Cations/metabolism , Chromatography, High Pressure Liquid , Erythrocyte Indices , Erythrocytes/cytology , Erythrocytes/metabolism , Erythrocytes/ultrastructure , Female , Founder Effect , Gene Expression , Genotype , Globins/genetics , Globins/metabolism , Hemoglobin C/metabolism , Humans , Liver/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microscopy, Electron, Scanning , Potassium/metabolism , Reticulocytes/cytology , SplenectomyABSTRACT
Priapism is a common complication of sickle cell anemia. We report a little known sequela of priapism: painless megalophallus, with significant penile enlargement. The patient had had an intense episode of priapism 9 years previously and his penis remained enlarged. Blood oxygen level-dependent magnetic resonance imaging revealed enlarged, hypoxic corpora cavernosa. Megalophallus probably resulted from permanent loss of elasticity of the tunica albuginea due to severe engorgement during the episode of priapism. This sequela needs to be recognized by physicians because no intervention is necessary and sexual function seems to remain intact.
Subject(s)
Anemia, Sickle Cell/complications , Penis/pathology , Priapism/complications , Adult , Humans , Hypertrophy/etiology , Hypertrophy/pathology , Magnetic Resonance Imaging , MaleABSTRACT
Transgenic sickle mice expressing human beta(S)- and beta(S-Antilles)-globins show intravascular sickling, red blood cell adhesion, and attenuated arteriolar constriction in response to oxygen. We hypothesize that these abnormalities and the likely endothelial damage, also reported in sickle cell anemia, alter nitric oxide (NO)-mediated microvascular responses and hemodynamics in this mouse model. Transgenic mice showed a lower mean arterial pressure (MAP) compared with control groups (90 +/- 7 vs. 113 +/- 8 mmHg, P < 0.00001), accompanied by increased endothelial nitric oxide synthase (eNOS) expression. N(G)-nitro-L-arginine methyl ester (L-NAME), a nonselective inhibitor of NOS, caused an approximately 30% increase in MAP and approximately 40% decrease in the diameters of cremaster muscle arterioles (branching orders: A2 and A3) in both control and transgenic mice, confirming NOS activity; these changes were reversible after L-arginine administration. Aminoguanidine, an inhibitor of inducible NOS, had no effect. Transgenic mice showed a decreased (P < 0.02-0.01) arteriolar dilation in response to NO-mediated vasodilators, i.e., ACh and sodium nitroprusside (SNP). Indomethacin did not alter the responses to ACh and SNP. Forskolin, a cAMP-activating agent, caused a comparable dilation of A2 and A3 vessels ( approximately 44 and 70%) in both groups of mice. Thus in transgenic mice, an increased eNOS/NO activity results in lower blood pressure and diminished arteriolar responses to NO-mediated vasodilators. Although the increased NOS/NO activity may compensate for flow abnormalities, it may also cause pathophysiological alterations in vascular tone.
Subject(s)
Anemia, Sickle Cell/physiopathology , Nitric Oxide/physiology , Vasodilation , Animals , Arginine/pharmacology , Blood Pressure/drug effects , Colforsin/pharmacology , Cyclooxygenase Inhibitors/pharmacology , Endothelium, Vascular/physiology , Enzyme Inhibitors/pharmacology , Hemoglobin, Sickle/analysis , Hemoglobin, Sickle/genetics , Humans , Indomethacin/pharmacology , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic/genetics , Microcirculation/drug effects , NG-Nitroarginine Methyl Ester/pharmacology , Nitric Oxide Synthase/metabolism , Vasodilator Agents/pharmacologySubject(s)
Phosphodiesterase Inhibitors/adverse effects , Piperazines/adverse effects , Priapism/chemically induced , Sickle Cell Trait/complications , 3',5'-Cyclic-GMP Phosphodiesterases , Adult , Cyclic GMP/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 5 , Humans , Male , Muscle, Smooth, Vascular/drug effects , Penis/blood supply , Penis/drug effects , Penis/enzymology , Phosphoric Diester Hydrolases/metabolism , Priapism/etiology , Purines , Sildenafil Citrate , SulfonesABSTRACT
To test the hypothesis that HbOARAB induces an increase in red cell mean corpuscular haemoglobin concentration (MCHC), we studied members of four Tunisian families who were either homo- or heterozygous for HbOARAB or were double heterozygotes for HbS and HbOARAB. The alpha-gene status was also tested. The findings included: (1) Distinctive variation in red cell density (MCHC) as determined by separation of red cells on isopycnic gradients: (a) All red cells from patients homozygous for HbOARAB were denser than normal red cells, as is observed for homozygous HbC patients. (b) In patients heterozygous for HbOARAB, red cell density was strongly influenced by the presence of alpha-thalassaemia. The coexistence of -alpha/alphaalpha resulted in an average red cell density slightly greater than normal (AA) red cells. Patients heterozygous for HbOARAB with a normal complement of four alpha genes had denser red cells similar to sickle cell disease with some cells of normal density but with most cells very dense. (c) Finally, the double heterozygotes for HbS and HbOARAB had significant haemolytic anaemia and red cells denser than normal with some as dense as the densest cells found in sickle cell anaemia. (2) Reticulocytes in patients homozygous for HbOARAB were found in the densest density fraction of whole blood. (3) Cation transport in patients homozygous for HbOARAB was abnormal, with K:Cl cotransport activity similar to that of HbS-Oman and only somewhat lower than in sickle cell anaemia red cells. The activity of the Gardos channel was indistinguishable from that found in HbS, HbC and HbS-Oman cells. We conclude that the erythrocytic pathogenesis of HbOARAB involves the dehydration of red cells due, at least in part, to the K:Cl cotransport system. The similarity of the charge and consequences of the presence of both HbC and HbOARAB, which are the products of mutations at opposite ends of the beta-chain, raises the possibility that this pathology is the result of a charge-dependent interaction of these haemoglobins with the red cell membrane and/or its cytoskeleton and that this abnormality is present early in red cell development.
Subject(s)
Erythrocytes/pathology , Hemoglobinopathies/blood , Hemoglobins, Abnormal/physiology , Symporters , Adolescent , Adult , Carrier Proteins/metabolism , Child , Erythrocyte Count , Female , Hemoglobinopathies/genetics , Hemoglobinopathies/metabolism , Heterozygote , Humans , Male , Middle Aged , Reticulocytes/pathology , alpha-Thalassemia/blood , alpha-Thalassemia/metabolism , K Cl- CotransportersABSTRACT
Our current strategy for gene therapy of sickle cell anemia involves retroviral vectors capable of transducing "designer" globin genes that code for novel anti-sickling globins (while resisting digestion by a ribozyme), coupled with the expression of a hammerhead ribozyme that can selectively cleave the human beta s mRNA. In this report, we have tested in vivo an anti-beta s hammerhead ribozyme embedded within a cDNA coding for the luciferase reporter gene driven by the human beta-globin promoter and hyper-sensitive sites 3 and 4 of the locus control region. We have created mice transgenic for this luciferase-ribozyme construct and bred the ribozyme transgene into mice that were already transgenic for the human beta s gene. We then measured expression of the beta s transgene at the protein and RNA levels by HPLC and primer extension. The presence of the ribozyme was associated with a statistically significant reduction in the level of beta s mRNA in spleen stress reticulocytes (from 60.5 +/- 4.1% to 52.9 +/- 4.2%) and in the percentage of beta s globin chains in very young mice (from 44.5 +/- 0.6% to 40.8 +/- 0.7%). These results demonstrate that it is possible to decrease the concentration of beta s chains and mRNA with the help of a hammerhead ribozyme. While the enormous amount of globin mRNA in reticulocytes is a challenge for ribozyme technology, the exquisite dependence of the delay time for formation of Hb S nuclei on the concentration of Hb S in red blood cells suggests that even a modest reduction in Hb S concentration would have therapeutic value.
Subject(s)
Hemoglobin, Sickle/genetics , RNA, Antisense/genetics , RNA, Catalytic/genetics , RNA, Messenger/genetics , Amino Acid Sequence , Anemia, Sickle Cell/genetics , Anemia, Sickle Cell/therapy , Animals , Base Sequence , Chromatography, High Pressure Liquid , DNA Primers , Female , Genetic Engineering , Genetic Therapy , Globins/genetics , Globins/metabolism , Hemoglobin, Sickle/analysis , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Molecular Sequence Data , RNA/genetics , RNA/metabolism , RNA, Antisense/physiology , RNA, Catalytic/physiology , RNA, Messenger/metabolism , Time Factors , TransgenesABSTRACT
BACKGROUND: In a previous study, nitric oxide synthases (NOS) were found to be strongly expressed in the tubular epithelium of kidneys of a transgenic mouse model of sickle cell disease (alphaHbetaS[betaMDD]). Because NOS activity is often associated with peroxynitrite formation when superoxide radical (.O-2) is present in abundance, we examined the kidneys of sickle cell mice for nitrotyrosine, considered to be a footprint of ONOO-. METHODS: Western blot and immunohistochemistry for nitrotyrosine was carried out. Since peroxynitrite and other reactive oxygen radicals are capable of causing apoptosis, we also performed agarose gel electrophoresis of kidney DNA and TUNEL staining of nuclei, indicators of apoptosis. RESULTS: Nitration of tyrosine residues of three proteins (kD 66, 57 and 22) was found on Western blot of kidney protein extracts of the sickle cell mice. The degree of tyrosine nitration of the 66 kD protein was not significantly different in the control versus transgenic mice, whereas tyrosine nitration of the 57 and 22 kD proteins was clearly increased in transgenic mice. Strong immunostaining for nitrotyrosine was seen in tubular epithelial cells of the sickle cell mice, in close proximity to positive immunostaining of iNOS. Neither iNOS nor nitrotyrosine was expressed in the control mice. DNA "laddering" was found localized to the same zones of the kidney as nitrotyrosine and iNOS immunostaining. TUNEL assay on mouse kidney tissue sections showed minimal tubular cell apoptosis in normal mouse with hypoxia, mild tubular cell apoptosis in sickle cell mouse in room air, and moderate tubular cell apoptosis in sickle cell mouse with hypoxia. CONCLUSIONS: The observations suggest that ONOO- and perhaps other reactive oxygen species are being produced in the sickle cell kidney. The mechanism may be ischemia/reperfusion due to intermittent vascular occlusion by sickle cells. The resulting hypoxia could result in iNOS activation, superoxide radical and peroxynitrite formation. Two consequences of these reactions appear to be nitration of tyrosine residues of some renal proteins and enhanced apoptosis.
Subject(s)
Anemia, Sickle Cell/metabolism , Apoptosis , Kidney/metabolism , Nitrates/metabolism , Animals , Blotting, Western , Immunohistochemistry , Mice , Mice, Inbred C57BL , Mice, Transgenic , Nitric Oxide Synthase/metabolism , Nitric Oxide Synthase Type II , Tyrosine/analogs & derivatives , Tyrosine/analysisABSTRACT
In sickle cell anemia (SS), some red blood cells dehydrate, forming a hyperdense (HD) cell fraction (>1.114 g/mL; mean corpuscular hemoglobin concentration [MCHC], >46 g/dL) that contains many irreversibly sickled cells (ISCs), whereas other SS red blood cells dehydrate to an intermediate density (ID; 1.090 to 1.114 g/mL; MCHC, 36 to 46 g/dL). This study asks if the potassium-chloride cotransporter (K:Cl) and the calcium-dependent potassium channel [K(Ca2+)] are participants in the formation of one or both types of dense SS red blood cells. We induced sickling by exposing normal density (ND; 1.080 to 1.090 g/mL; MCHC, 32 to 36 g/dL) SS discocytes to repetitive oxygenation-deoxygenation (O-D) cycles in vitro. At physiologic Na+, K+, and Cl-, and 0.5 to 2 mmol/L Ca2+, the appearance of dense cells was time- and pH-dependent. O-D cycling at pH 7.4 in 5% CO2-equilibrated buffer generated only ID cells, whereas O-D cycling at pH 6.8 in 5% CO2-equilibrated buffer generated both ID and HD cells, the latter taking more than 8 hours to form. At 22 hours, 35% +/- 17% of the parent ND cells were recovered in the ID fraction and 18% +/- 11% in the HD fraction. Continuous deoxygenation (N2/5% CO2) at pH 6.8 generated both ID and HD cells, but many of these cells had multiple projections, clearly different from the morphology of endogenous dense cells and ISCs. Continuous oxygenation (air/5% CO2) at pH 6.8 resulted in less than 10% dense cell (ID + HD) formation. ATP depletion substantially increased HD cell formation and moderately decreased ID cell formation. HD cells formed after 22 hours of O-D cycling at pH 6.8 contained fewer F cells than did ID cells, suggesting that HD cell formation is particularly dependent on HbS polymerization. EGTA chelation of buffer Ca2+ inhibited HD but not ID cell formation, and increasing buffer Ca2+ from 0.5 to 2 mmol/L promoted HD but not ID cell formation in some SS patients. Substitution of nitrate for Cl- inhibited ID cell formation, as did inhibitors of the K:Cl cotransporter, okadaic acid, and [(dihydroindenyl) oxy]alkanoic acid (DIOA). Conversely, inhibitors of K(Ca2+), charybdotoxin and clotrimazole, inhibited HD cell formation. The combined use of K(Ca2+) and K:Cl inhibitors nearly eliminated dense cell (ID + HD cell) formation. In summary, dense cells formed by O-D cycling for 22 hours at pH 7.4 cycling are predominately the ID type, whereas dense cells formed by O-D cycling for 22 hours at pH 6.8 are both the ID and HD type, with the latter low in HbF, suggesting that HD cell formation has a greater dependency on HbS polymerization. A combination of K:Cl cotransport and the K(Ca2+) activities account for the majority of dense cells formed, and these pathways can be driven independently. We propose a model in which reversible sickling-induced K+ loss by K:Cl primarily generates ID cells and K+ loss by the K(Ca2+) channel primarily generates HD cells. These results imply that both pathways must be inhibited to completely prevent dense SS cell formation and have potential therapeutic implications.
Subject(s)
Anemia, Sickle Cell/pathology , Erythrocytes/pathology , Symporters , Adenosine Triphosphate/deficiency , Adenosine Triphosphate/physiology , Buffers , Calcium/pharmacology , Carbon Dioxide/metabolism , Carrier Proteins/physiology , Centrifugation, Density Gradient , Chlorides/physiology , Enzyme Inhibitors/pharmacology , Erythrocytes/drug effects , Erythrocytes/metabolism , Fetal Hemoglobin/analysis , Humans , Hydrogen-Ion Concentration , Ion Transport/drug effects , Oxygen/metabolism , Potassium/physiology , Specific Gravity , Time Factors , K Cl- CotransportersABSTRACT
Hemoglobin (Hb) S-Oman has two mutations in the beta-chains. In addition to the classic betaS mutation (beta6 Glu --> Val), it contains a second mutation in the same chain (beta121 Glu --> Lys) identical to that of HbOARAB. We have studied a pedigree of heterozygous carriers of HbS-Oman that segregates into two types of patients: those expressing about 20% HbS-Oman and concomitant -/ thalassemia and those with about 14% of HbS-Oman and concomitant -/- thalassemia. The higher expressors of S-Oman have a sickle cell anemia (SS) clinical syndrome of moderate intensity, while the lower expressors have no clinical syndrome, and are comparable to the solitary case first described in Oman. In addition, the higher expressors exhibit a unique form of irreversibly sickled cell reminiscent of a "yarn and knitting needle" shape, in addition to folded and target cells. The CSAT of S-Oman is identical to that of S-Antilles, another supersickling hemoglobin, whose carriers express the abnormal hemoglobin at 40% to 50%, with a very similar clinical picture to HbS-Oman. Because the level of expression is so different and the clinical picture so similar, and based on the hemolysates CSAT's, we conclude that HbS-Oman produces pathology beyond its sickling tendencies. A clue for this additional pathogenesis is found in the fact that homozygous HbOARAB, which has the same second substitution as S-Oman, has a moderately severe hemolytic anemia; when HbOARAB is combined with HbS, it makes the phenotype of this double heterozygote as severe as SS. Properties of HbS-Oman red blood cells (RBCs) include reticulocytes that are much denser than normal (similar to those of SC and CC disease), a decrease in the Km for Ca2+ needed to activate the Gardos' channel (making this transporter more sensitive to Ca2+), increased association of HbS-Oman with the RBC membrane, the presence of dense cells by isopycnic gradient, the presence of folded cells, and abundant nidus of polymerization under the membrane. Other properties include a clear increase in volume and N-ethylmaleimide-stimulated K:Cl cotransport in RBCs expressing more than 20% HbS-Oman. We conclude that the pathology of heterozygous S-Oman is the product of the sickling properties of the beta6 Val mutation which are enhanced by the second mutation at beta121. In addition, the syndrome is further enhanced by a hemolytic anemia induced by the mutation at beta121. We speculate that this pathology results from the abnormal association of the highly positively charged HbS-Oman (3 charges different from normal hemoglobin) with the RBC membrane.
Subject(s)
Hemoglobin, Sickle/genetics , Hemoglobins, Abnormal/genetics , Mutation , Syndrome , Adolescent , Adult , Anemia, Sickle Cell/genetics , Child , Female , Heterozygote , Humans , Male , Pedigree , alpha-Thalassemia/geneticsABSTRACT
Studies in vitro by Pasvol et al (Nature, 270:171, 1977) have indicated that the growth of Plasmodium falciparum in cells containing fetal hemoglobin (HbF = alpha2gamma2) is retarded, but invasion is increased, at least in newborn cells. Normal neonates switch from about 80% HbF at birth to a few percent at the end of the first year of life. Carriers of beta-thalassemia trait exhibit a delay in the normal HbF switch-off, which might partially explain the protection observed in populations with this gene. To study this hypothesis in vivo, we used transgenic (gamma) mice expressing human Agamma and Ggamma chains resulting in 40% to 60% alpha2Mgamma2 hemoglobin, infected with rodent malaria. Two species of rodent malaria were studied. P chabaudi adami causes a nonlethal infection, mainly in mature red blood cells (RBC). P yoelii 17XNL is a nonlethal infection, invading primarily reticulocytes, whereas P yoelii 17XL is a lethal variant of P yoelii 17XNL and causes death of mice in approximately 1 to 2 weeks. Data indicate that this strain may cause a syndrome resembling cerebral malaria caused by P falciparum (Am J Trop Med Hyg, 50:512, 1994). In gamma transgenic mice infected with P chabaudi adami, the parasitemia rose more quickly (in agreement with Pasvol) than in control mice, but was cleared more rapidly. In mice infected with P yoelii 17XNL, a clear reduction in parasitemia was observed. Interestingly, splenectomy before this infection, did not reverse protection. The most striking effect was in lethal P yoelii 17XL infection. Control mice died between 11 to 13 days, whereas gamma mice cleared the infection by day 22 and survived, a phenomenon also observed in splenectomized animals. These results suggest that HbF does indeed have a protective effect in vivo, which is not mediated by the spleen. In terms of mechanisms, light microscopy showed that intraerythrocytic parasites develop slowly in HbF erythrocytes, and electron microscopy showed that hemozoin formation was defective in transgenic mice. Finally, digestion studies of HbF by recombinant plasmepsin II demonstrated that HbF is digested only half as well as hemoglobin A (HbA). We conclude that HbF provides protection from P falciparum malaria by the retardation of parasite growth. The mechanism involves resistance to digestion by malarial hemoglobinases based on the data presented and with the well-known properties of HbF as a super stable tetramer. In addition, the resistance of normal neonates for malaria can now be explained by a double mechanism: increased malaria invasion rates, reported in neonatal RBC, will direct parasites to fetal cells, as well as F cells, and less to the approximately 20% of HbA containing RBC, amplifying the antimalarial effects of HbF.
Subject(s)
Fetal Hemoglobin/physiology , Malaria/prevention & control , Plasmodium chabaudi/pathogenicity , Plasmodium yoelii/pathogenicity , Animals , Aspartic Acid Endopeptidases/metabolism , Chromatography, High Pressure Liquid , Disease Susceptibility , Erythrocytes/parasitology , Female , Fetal Hemoglobin/biosynthesis , Fetal Hemoglobin/chemistry , Fetal Hemoglobin/genetics , Globins/biosynthesis , Globins/genetics , Host-Parasite Interactions , Mice , Mice, Inbred C57BL , Mice, Transgenic , Parasitemia/blood , Parasitemia/prevention & control , Plasmodium falciparum/enzymology , Protein Conformation , Protozoan Proteins/metabolism , Recombinant Proteins/metabolism , Substrate SpecificityABSTRACT
PURPOSE: To determine if vascular occlusion and nonperfusion is associated with the outer retinal atrophy, retinopathy, and choroidopathy (chorioretinopathy) that occurs in the alpha H beta S[beta MDD] and alpha H beta S [alpha MD beta MDD] transgenic mouse models of sickle cell disease. METHODS: Mice from the alpha H beta S[beta MDD] and alpha H beta S[alpha MD beta MDD] transgenic mouse lines that express high levels of human beta S globin were anesthetized and administered horseradish peroxidase (HRP) intracardially. After 1 min, the animals were sacrificed, and the retina from one eye was excised, fixed, and developed in diaminobenzidine (DAB). The contralateral eye was fixed, embedded whole in glycol methacrylate, and HRP developed in 2.5 microns sections. RESULTS: HRP reaction product (HRP-RP) and stained erythrocytes (RBCs) (due to endogenous peroxidase) were diffusely distributed within all vascular lumens in flatmount retinas from control animals (littermates homozygous for the mouse Beta Major deletion not expressing the beta S transgene). In 42.5% of the transgenic mice expressing beta S without any proliferative retinopathy, many blood vessels contained RBC plugs and lacked lumenal HRP-RP. In addition to packed RBCs, fibrin was sometimes present at sites of occlusion. In sections from whole eyes of the same animals, foci of photoreceptor degeneration were associated with areas of choriocapillaris nonperfusion (lumen that lacked HRP-PR). In areas with normal photoreceptors, the choriocapillaris appeared perfused (HRP-RP was present). In animals with proliferative chorioretinopathy, some neovascular formations lacked luminal HRP-RP, suggesting autoinfarction. CONCLUSIONS: Nonperfused retinal and choroidal vessels were observed in mice from the alpha H beta S[beta MDD] and alpha H beta S[alpha MD beta MDD] lines without retinal and choroidal neovascularization, whereas, all mice with neovascularization had nonperfused areas. Furthermore, small foci of PR loss were associated with areas of nonperfused choriocapillaris. These results suggest that sickle cell-mediated vaso-occlusions are an initial event in the chorioretinopathy and outer retinal atrophy that occurs in these models.
Subject(s)
Anemia, Sickle Cell/pathology , Choroid Diseases/pathology , Retinal Diseases/pathology , Anemia, Sickle Cell/genetics , Animals , Choroid/blood supply , Disease Models, Animal , Horseradish Peroxidase , Humans , Mice , Mice, Transgenic , Perfusion , Retinal Artery Occlusion , Retinal Neovascularization , Retinal Vein OcclusionABSTRACT
We show here that alpha2(G-Phila.) beta2(C) has an increased rate of crystal nucleation compared to alpha2 beta2(C) (HbC). We conclude from this finding that position alpha68, the mutation site of alpha2(G-Phila.) beta2 (HbG(Philadelphia)), is a contact site in the crystal of HbC. In addition, that HbS enhances HbC crystallization (additive to the effect of alpha(G-Phila.) as shown here) and that alpha(G-Phila.) inhibits polymerization of HbS are pathogenically relevant previously known facts. All of these findings help explain the phenotype of an individual simultaneously heterozygous for the betaS, betaC, and the alpha(G-Phila.) genes (SC alpha-G Philadelphia disease). This disease is characterized by a mild clinical course, abundant circulating intraerythrocytic crystals, and increased folded red cells. This phenotype seems to be the result of increased crystallization and decreased polymerization brought about by the opposite effects of the gene product of the alpha(G-Phila.) gene on the betaC and betaS gene products. Some of the intraerythrocytic crystals in this syndrome are unusually long and thin, resembling sugar canes, unlike those seen in SC disease. The mild clinical course associated with increased crystallization implies that, in SC disease, polymerization of HbS is pathogenically more important than the crystallization induced by betaC chains. The SC alpha-G Philadelphia disease is an example of multiple hemoglobin chain interactions (epistatic effect among globin genes) creating a unique phenotype.