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Mary E Fabry

Publications and source records attributed to Mary E Fabry.

18 recordsLinked to original sources
PubMed2004

Decreased cerebral perfusion correlates with increased BOLD hyperoxia response in transgenic mouse models of sickle cell disease.

Neurological complications such as stroke are known consequences of sickle cell disease (SCD). In order to improve methods for the evaluation of stroke risk in SCD, MRI was used to evaluate cerebrovascular function in transgenic mouse models of human SCD. It is hypothesized that oxygen-sensitive imaging in the brain will reveal areas of excess deoxygenation that are either at risk of or the result of vaso-occlusion. Arterial spin labeling (ASL) perfusion was performed in order to correlate BOLD results with microvascular cerebral blood flow. Upon comparison with control animals, there was a relative increase in BOLD hyperoxia response of 42-67% (P < 0.001) in the transgenic mice while cerebral blood flow during normoxia was reduced by 30-40% (P < 0.02). Hyperoxia caused cerebral blood flow to decrease in control mice, whereas blood flow increased in the sickle transgenic mice. These results indicate impairment in brain autoregulation in the sickle cell transgenic mice leading to increased cerebral deoxyhemoglobin. Increased deoxyhemoglobin coupled with reduced perfusion may further increase the risk of vaso-occlusion and stroke. This may reflect polymer reduction or reduced cell adhesion during hyperoxia. The MRI protocol is noninvasive and thus directly applicable to a clinical population.

Anemia, Sickle Cell↗
PubMed2004

Expression of an anti-sickling beta-globin in human erythroblasts derived from retrovirally transduced primitive normal and sickle cell disease hematopoietic cells.

OBJECTIVE: Recent improvements in human beta-globin vector design have fueled interest in gene therapy approaches to the treatment of human thalassemia and sickle cell disease (SCD). The present study was undertaken to determine whether human beta-globin mRNA and protein could be obtained in the erythroid progeny of more primitive human target cells transduced with a retrovirus containing murine stem cell virus long terminal repeats, a phosphoglycerate kinase promoter driving the expression of a green fluorescence protein (GFP) cDNA, and an anti-sickling beta-globin (beta87(+)) gene under the control of an HS2, HS3, HS4 enhancer cassette. MATERIALS AND METHODS: A two-step pseudotyping strategy was devised to obtain useful preparations of this virus. Primitive cells present in normal human cord blood (CB) and adult SCD patients' blood samples were infected and the level of gene transfer (% GFP(+) cells) and erythroid-specific beta87(+)-globin expression assessed. RESULTS: Analysis of the proportion of infected cells that became GFP(+) showed that this virus transduced approximately 50% of initial CD34(+) CB and SCD cells and up to 23% of cells able to regenerate both lymphoid and myeloid cells in sublethally irradiated primary and secondary NOD/SCID mice. beta87(+)-globin transcripts were readily detected in erythroblasts generated from primitive transduced CB cells and SCD progenitors. Evidence of beta87(+)-derived protein in transduced CB cell-derived erythroblasts also was obtained. CONCLUSION: These findings demonstrate that retroviral vector-based gene transfer approaches can be used to achieve human beta-globin protein expression in the erythroid progeny of transplantable human precursors.

Anemia, Sickle Cell↗
PubMed2004

Arginine supplementation improves rotorod performance in sickle transgenic mice.

Patients with sickle cell disease (SCD) have been shown to have impaired visual-motor speed and coordination. Sensorimotor deficits in mice can be investigated by motor coordination tests that require whole body movements such as the rotorod. A sickle transgenic mouse model (S+S-Antilles) that expresses human alpha, human beta(S) and human beta(S-Antilles), is homozygous for the mouse beta(major) deletion, and has low plasma arginine was compared to control C57BL/6J mice and S+S-Antilles mice supplemented with 5% arginine on the rotorod. The rotorod consists of a 2.5 cm diameter, grooved rod turning at constant acceleration, requiring postural adjustments on the part of the mice to maintain equilibrium. C57BL mice on Purina mouse chow had an average latency to fall of S+S-Antilles mice on Purina mouse chow had an average of 127+/-56 s S+S-Antilles mice after 5% arginine supplementation had a mean latency of Arginine may improve rotorod performance in sickle transgenic mice by increasing NO synthesis thereby improving vasodilatation and blood flow with reversal of ischemia in brain and/or muscle. In conclusion, impaired rotorod performance in sickle transgenic mice presents an opportunity to apply this simple task to provide an efficient method to screen some types of therapeutic regimens for efficacy in SCD.

Anemia, Sickle Cell↗
PubMed2004

Effect of fetal hemoglobin on microvascular regulation in sickle transgenic-knockout mice.

In sickle cell disease, intravascular sickling and attendant flow abnormalities underlie the chronic inflammation and vascular endothelial abnormalities. However, the relationship between sickling and vascular tone is not well understood. We hypothesized that sickling-induced vaso-occlusive events and attendant oxidative stress will affect microvascular regulatory mechanisms. In the present studies, we have examined whether microvascular abnormalities expressed in sickle transgenic-knockout Berkeley (BERK) mice (which express exclusively human alpha- and beta(S)-globins with <1% gamma-globin levels) are amenable to correction with increased levels of antisickling fetal hemoglobin (HbF). In BERK mice, sickling, increased oxidative stress, and hemolytic anemia are accompanied by vasodilation, compensatory increases in eNOS and COX-2, and attenuated vascular responses to NO-mediated vasoactive stimuli and norepinephrine. The hypotension and vasodilation (required for adequate oxygen delivery in the face of chronic anemia) are mediated by non-NO vasodilators (i.e., prostacyclin) as evidenced by induction of COX-2. In BERK mice, the resistance to NO-mediated vasodilators is associated with increased oxidative stress and hemolytic rate, and in BERK + gamma mice (expressing 20% HbF), an improved response to these stimuli is associated with reduced oxidative stress and hemolytic rate. Furthermore, BERK + gamma mice show normalization of vessel diameters, and eNOS and COX-2 expression. These results demonstrate a strong relationship between sickling and microvascular function in sickle cell disease.

Acetylcholine↗
PubMed2004

High-level beta-globin expression and preferred intragenic integration after lentiviral transduction of human cord blood stem cells.

Transplantation of genetically corrected autologous hematopoietic stem cells is an attractive approach for the cure of sickle-cell disease and beta-thalassemia. Here, we infected human cord blood cells with a self-inactivating lentiviral vector encoding an anti-sickling betaA-T87Q-globin transgene and analyzed the transduced progeny produced over a 6-month period after transplantation of the infected cells directly into sublethally irradiated NOD/LtSz-scid/scid mice. Approximately half of the human erythroid and myeloid progenitors regenerated in the mice containing the transgene, and erythroid cells derived in vitro from these in vivo-regenerated cells produced high levels of betaA-T87Q-globin protein. Linker-mediated PCR analysis identified multiple transgene-positive clones in all mice analyzed with 2.1 +/- 0.1 integrated proviral copies per cell. Genomic sequencing of vector-containing fragments showed that 86% of the proviral inserts had occurred within genes, including several genes implicated in human leukemia. These findings indicate effective transduction of very primitive human cord blood cells with a candidate therapeutic lentiviral vector resulting in the long-term and robust, erythroid-specific production of therapeutically relevant levels of beta-globin protein. However, the frequency of proviral integration within genes that regulate hematopoiesis points to a need for additional safety modifications.

Animals↗
PubMed2004

In vivo studies of sickle red blood cells.

The defining clinical feature of sickle cell anemia is periodic occurrence of painful vasoocclusive crisis. Factors that promote trapping and sickling of red cells in the microcirculation are likely to trigger vasoocclusion. The marked red cell heterogeneity in sickle blood and abnormal adhesion of sickle red cells to vascular endothelium would be major disruptive influences. Using ex vivo and in vivo models, the authors show how to dissect the relative contribution of heterogeneous sickle red cell classes to adhesive and obstructive events. These studies revealed that (1) both rheological abnormalities and adhesion of sickle red cells contribute to their abnormal hemodynamic behavior, (2) venules are the sites of sickle cell adhesion, and (3) sickle red cell deformability plays an important role in adhesive and obstructive events. Preferential adhesion of deformable sickle red cells in postcapillary venules followed by selective trapping of dense sickle red cells could result in vasoocclusion. An updated version of this 2-step model is presented. The multifactorial nature of sickle red cell adhesion needs to be considered in designing antiadhesive therapy in vivo.

Anemia, Sickle Cell↗
PubMed2003

Expression of HbC and HbS, but not HbA, results in activation of K-Cl cotransport activity in transgenic mouse red cells.

Elevation of K-Cl cotransport in patients with homozygous hemoglobin (Hb) S or HbC increases red cell mean corpuscular hemoglobin concentration (MCHC) and contributes significantly to pathology. Elucidation of the origin of elevated K-Cl cotransport in red cells with mutant hemoglobins has been confounded by the concomitant presence of reticulocytes with high K-Cl cotransport. In red cells of control mice (C57BL), transgenic mice that express only human HbA, and transgenic mice that express both mouse globins and human HbS, volume stimulation is weak and insensitive to NO3- and dihydroindenyl-oxy-alkanoic acid (DIOA). DIOA and NO3- are inhibitors in all other mammalian red cells. In contrast, in knock-out mice expressing exclusively human hemoglobin HbC or HbS+ gamma, replacement of isotonic Cl- media by hypotonic Cl- resulted in strong volume stimulation and sensitivity to DIOA, okadaic acid, and NO3-. In summary, we find that HbC, under all conditions, and HbS+ gamma, in the absence of mouse globins, have significant quantitative and qualitative effects on K-Cl cotransport in mouse red cells and activate mouse K-Cl. We conclude that human globins are able to stimulate the activity and/or regulation of K-Cl cotransport in mouse red cells. These observations support the contention that HbS and HbC stimulate K-Cl cotransport in human red cells.

Animals↗
PubMed2003

The paradox of hemoglobin SC disease.

Homozygous HbC gene results only in mild hemolytic anemia. In HbSC disease red cells contain equal levels of HbS and HbC. It is a paradox that HbSC exhibit a moderately severe phenotype in spite of being a mixture of HbS trait and HbC trait, neither of which has significant pathology. Why does the combination of these two Hbs result in a serious disease? The short answer is that HbC enhances, by dehydrating the SC red cell, the pathogenic properties of HbS, resulting in a clinically significant disorder, but somewhat milder that sickle cell anemia (SCA). Nevertheless, retinnitis proliferans, osteonecrosis, and acute chest syndrome have equal or higher incidence in HbSC disease compared to SCA. This pathogenic trick is accomplished by HbC inducing, by mechanisms not fully understood, an increase in the activity of K:Cl cotransport that induces the lost of K(+) and consequently of intracellular water. This event creates a sufficient increase of MCHC, so that the lower levels of HbS found in SC red cells can polymerize rapidly and effectively. This situation offers a unique opportunity: if we could inhibit the effect of HbC on K(+) transport we can cure the disease.

Anemia, Sickle Cell↗
PubMed2003

Hemoglobinopathies.

The outlook for patients with sickle cell disease has improved steadily during the last two decades. In spite of these improvements, curative therapies are currently available only to a small minority of patients. The main theme of this chapter is to describe new therapeutic options that are at different stages of development that might result in further improvements in the outlook for patients with these disorders. Dr. Joseph DeSimone and his colleagues had previously made the important observation that the hypomethylating agent 5-azacytidine can reverse the switch from adult to fetal hemoglobin in adult baboons. Although similar activity was demonstrated in patients with sickle cell disease and beta-thalassemia, concern about the toxicity of 5-azacytidine prevented its widespread use in these disorders. In Section I, Dr. DeSimone discusses the role of DNA methylation in globin gene regulation and describe recent clinical experience with decitabine (an analogue of 5-azacytidine) in patients with sickle cell disease. These encouraging studies demonstrate significant fetal hemoglobin inducing activity of decitabine in patients who fail to respond to hydroxyurea. In Section II, Dr. George Atweh continues the same theme by describing recent progress in the study of butyrate, another inducer of fetal hemoglobin, in patients with sickle cell disease and beta-thalassemia. The main focus of his section is on the use of a combination of butyrate and hydroxyurea to achieve higher levels of fetal hemoglobin that might be necessary for complete amelioration of the clinical manifestations of these disorders. Dr. Atweh also describes novel laboratory studies that shed new light on the mechanisms of fetal hemoglobin induction by butyrate. In Section III, Dr. Ronald Nagel discusses the different available transgenic sickle mice as experimental models for human sickle cell disease. These experimental models have already had a significant impact on our understanding of the pathophysiology of sickle cell disease. Dr. Nagel describes more recent studies in which transgenic sickle mice provide the first proof of principle that globin gene transfer into hematopoietic stem cells inhibits in vivo sickling and ameliorates the severity of the disease. Although stroke in adult patients with sickle cell disease is not as common as in children, adult hematologists, like their pediatric colleagues, need to make management decisions in adult patients with a stroke or a history of stroke. Dr. Robert Adams has led several large clinical studies that investigated the role of transfusions in the prevention of stroke in children with sickle cell disease. Much less is known, however, about the prevention of first or subsequent strokes in adult patients with sickle cell disease. In Section IV, Dr. Adams provides some general guidelines for the management of adult patients with stroke while carefully distinguishing between recommendations that are evidence-based and those that are anecdotal in nature.

Animals↗
PubMed2002

Permanent and panerythroid correction of murine beta thalassemia by multiple lentiviral integration in hematopoietic stem cells.

Achieving long-term pancellular expression of a transferred gene at therapeutic level in a given hematopoietic lineage remains an important goal of gene therapy. Advances have recently been made in the genetic correction of the hemoglobinopathies by means of lentiviral vectors and large locus control region (LCR) derivatives. However, panerythroid beta globin gene expression has not yet been achieved in beta thalassemic mice because of incomplete transduction of the hematopoietic stem cell compartment and position effect variegation of proviruses integrated at a single copy per genome. Here, we report the permanent, panerythroid correction of severe beta thalassemia in mice, resulting from a homozygous deletion of the beta major globin gene, by transplantation of syngeneic bone marrow transduced with an HIV-1-derived [beta globin gene/LCR] lentiviral vector also containing the Rev responsive element and the central polypurine tract/DNA flap. The viral titers produced were high enough to achieve transduction of virtually all of the hematopoietic stem cells in the graft with an average of three integrated proviral copies per genome in all transplanted mice; the transduction was sustained for >7 months in both primary and secondary transplants, at which time approximately 95% of the red blood cells in all mice contained human beta globin contributing to 32 +/- 4% of all beta-like globin chains. Hematological parameters approached complete phenotypic correction, as assessed by hemoglobin levels and reticulocyte and red blood cell counts. All circulating red blood cells became and remained normocytic and normochromic, and their density was normalized. Free alpha globin chains were completely cleared from red blood cell membranes, splenomegaly abated, and iron deposit was almost eliminated in liver sections. These findings indicate that virtually complete transduction of the hematopoietic stem cell compartment can be achieved by high-titer lentiviral vectors and that position effect variegation can be mitigated by multiple events of proviral integration to yield balanced, panerythroid expression. These results provide a solid foundation for the initiation of human clinical trials in beta thalassemia patients.

3T3 Cells↗
PubMed2002

Expression of a human beta-globin transgene in erythroid cells derived from retrovirally transduced transplantable human fetal liver and cord blood cells.

Transfer of therapeutic genes to human hematopoietic stem cells (HSCs) using complex vectors at clinically relevant efficiencies remains a major challenge. Recently we described a stable retroviral vector that sustains long-term expression of green fluorescent protein (GFP) and a human beta-globin gene in the erythroid progeny of transduced murine HSCs. We now report the efficient transduction of primitive human CD34(+) fetal liver or cord blood cells with this vector and expression of the beta-globin transgene in the erythroid progeny of these human cells for at least 2 months. After growth factor prestimulation and then a 2- to 3-day exposure to the virus, 35% to 55% GFP(+) progeny were seen in assays of transduced colony-forming cells, primitive erythroid precursors that generate large numbers of glycophorin A(+) cells in 3-week suspension cultures, and 6-week long-term culture-initiating cells. In immunodeficient mice injected with unselected infected cells, 5% to 15% of the human cells regenerated in the marrow (including the erythroid cells) were GFP(+) 3 and 6 weeks after transplantation. Importantly, the numbers of GFP(+) human lymphoid and either granulopoietic or erythroid cells in individual mice 6 weeks after transplantation were significantly correlated, indicative of the initial transduction of human multipotent cells with in vivo repopulating activity. Expression of the transduced beta-globin gene in human cells obtained directly from the mice or after their differentiation into erythroid cells in vitro was demonstrated by reverse transcriptase-polymerase chain reaction using specific primers. These experiments represent a significant step toward the realization of a gene therapy approach for human beta-globin gene disorders.

Animals↗
PubMed2002

Arginine supplementation of sickle transgenic mice reduces red cell density and Gardos channel activity.

Nitric oxide (NO), essential for maintaining vascular tone, is produced from arginine by nitric oxide synthase. Plasma arginine levels are low in sickle cell anemia, and it is reported here that low plasma arginine is also found in our sickle transgenic mouse model that expresses human alpha, human beta(S), and human beta(S-Antilles) and is homozygous for the mouse beta(major) deletion (S+S-Antilles). S+S-Antilles mice were supplemented with a 4-fold increase in arginine that was maintained for several months. Mean corpuscular hemoglobin concentration (MCHC) decreased and the percent high-density red cells was reduced. Deoxy K(+) efflux is characteristic of red cells in sickle cell disease and contributes to the disease process by increasing the MCHC and rendering the cells more susceptible to polymer formation. This flux versus the room air flux was reduced in S+S-Antilles red cells from an average value of 1.6 +/- 0.3 mmol per liter of red cells x minute (FU) in nonsupplemented mice to 0.9 +/- 0.3 FU (n = 4, P < .02, paired t test) in supplemented mice. In room air, V(max) of the Ca(++)-activated K(+) channel (Gardos) was reduced from 4.1 +/- 0.6 FU (off diet) to 2.6 +/- 0.4 FU (n = 7 and 8, P < .04, t test) in arginine-supplemented mice versus clotrimazole. In conclusion, the major mechanism by which arginine supplementation reduces red cell density (MCHC) in S+S-Antilles mice is by inhibiting the Ca(++)-activated K(+) channel.

Anemia, Sickle Cell↗
PubMed2002

Feasibility of using autologous transplantation to evaluate hematopoietic stem cell-based gene therapy strategies in transgenic mouse models of human disease.

Histoincompatibility between murine donors and recipients of bone marrow (BM) transplants reduces engraftment, and this compromises assessment of hematopoietic stem cells (HSCs) in certain transgenic mice. To study HSCs in the S+S-Antilles mouse model of human sickle cell disease (SCD), we developed an autotransplant protocol. Initial experiments showed no differences between S+S-Antilles mice and normal C57BL/6 (+/+) mice in their radiosensitivity or baseline hematopoietic progenitor numbers. The kinetics of red blood cell (RBC) replacement post-transplant in +/+ recipients of mixtures of transgenic and +/+ BM cells also showed no competitive advantage of the +/+ cells. BM cells were then aspirated from mice 4 days after 5-fluorouracil treatment, transduced with a green fluorescent protein (GFP)-encoding retrovirus, and transplanted into the same recipients that, just before transplant, were irradiated with 800 cGy. We subsequently detected high levels of GFP(+) RBCs (21-79%) and white blood cells (WBCs; 35-88%) in the blood for 11 months and showed that transduced HSCs regenerated in the primary mice also repopulated secondary mice. These findings provide a generally applicable protocol for performing autotransplants in mice and forecast the potential utility of this approach in assessing HSC-based gene therapy protocols in transgenic mouse models of many human diseases.

Anemia, Sickle Cell↗
PubMed2002

Mechanisms for sickle red blood cell retention in choroid.

PURPOSE: Although sickle (SS) red cell-mediated vaso-occlusion in retina and resultant retinopathy is well documented, the effects of SS red cells on choroidal vasculature are poorly understood. The intent of this study was to determine, using a rat model, the conditions under which retention of sickle erythrocytes in choroid occur and if that retention can be inhibited. METHODS: Sickle red cells were density separated into high density (SS4) or normal density, reticulocyte-enriched fractions (SS2). Red cells were labeled with FITC and administered IV to anesthetized Sprague Dawley rats. Rats were made either hypoxic or were given TNF-alpha intraperitoneally 5 hours before intravenous administration of red cells. Five minutes after administration of red cells, rats were exsanguinated, the retinas removed, and choroids prepared as flatmounts. The number of red cells retained in five high power fields of choroid was then determined. In other experiments, SS red cells were preincubated with the cyclic peptide TBC772 [inhibits binding of alpha4beta1 (VLA-4) and alpha4beta7 to their ligands], a control peptide TBC1194, or a VLA-4 neutralizing antibody before administration to the rat or antibodies against VLA-4 ligands were delivered IV before administration of SS red cells. RESULTS: Hypoxic conditions before administration of SS red cells significantly stimulated retention of SS4 cells (P = 0.0003), but did not significantly increase retention of SS2 cells. Administration of TNF-alpha significantly increased retention of all types of SS red cells (P < 0.001). Preincubation of cells with anti-VLA-4 or TBC 772 inhibited retention of SS red cells in choriocapillaris of TNF-alpha-treated rats (P < 0.0001). Complete inhibition of cytokine-stimulated retention was also accomplished by IV administration of monoclonal antibodies against fibronectin or its CS-1 domain, a ligand for VLA-4. CONCLUSIONS: The mechanisms for retention of SS red cells in retina and choroid appear identical: hypoxia-mediated retention of dense red cells and adherence of red cells in reticulocyte-rich fractions after cytokine stimulation. TNF-alpha-stimulated retention of SS red cells in choroid appears to be mediated by VLA-4, presumably on the surface of some reticulocytes. This increased retention was inhibited by a VLA-4 antagonist (TBC772), a VLA-4 neutralizing antibody or by blocking one of VLA-4's ligands, the CS-1 portion of fibronectin.

Anemia, Sickle Cell↗
PubMedDate not supplied

Differential gene expression in the kidney of sickle cell transgenic mice: upregulated genes.

The S+S-Antilles transgenic mouse used in this study has renal defects similar to those seen in sickle cell anemia patients: congested glomeruli, medullary fibrosis, renal enlargement, vasoocclusion, and a urine concentrating defect. We used gene expression microarrays to identify genes highly up-regulated in the kidneys of these mice and validated their expression by real-time PCR. Kidney hypoxia, as demonstrated by the presence of deoxyhemoglobin, was detected by blood oxygen dependent magnetic resonance imaging (BOLD-MRI). Some of the up-regulated genes included cytochrome P450 4a14, glutathione-S-transferase alpha-1, mitochondrial hydroxymethylglutaryl CoA synthase, cytokine inducible SH-2 containing protein, retinol dehydrogenase type III, arginase II, glycolate oxidase, Na/K ATPase, renin-1, and alkaline phosphatase 2. An increase in enzyme activity was also demonstrated for one of the up-regulated genes (arginase II). These genes can be integrated into several different pathophysiological processes: a hypoxia cascade, a replacement cascade, or an ameliorating cascade, one or all of which may explain the phenotype of this disease. We conclude that microarray technology is a powerful tool to identify genes involved in renal disease in sickle cell anemia and that the identification of various metabolic pathways may open new avenues for therapeutic interventions.

Anemia, Sickle Cell↗
PubMedDate not supplied

Generation of transgenic mice expressing human hemoglobin E.

Hemoglobin E (HbE, beta26 Glu-->Lys) is the most common abnormal Hb variant in the world, and found in greatest frequency in Southeast (SE) Asia. In the United States, HbE is the third most prevalent variant (after HbS and HbC); and its now increasing frequency is due to immigration from SE Asia. HbE homozygotes present a benign clinical picture, but when HbE is coupled with beta0-thalassemia or HbS, variably severe hemoglobinopathies arise. To date, there are no transgenic animal models of HbE-related diseases. We report here the creation of transgenic mice expressing human HbE as a step toward creating animal models for HbE-related diseases. The betaE mice exhibit red blood cell hypochromia and target cells consistent with those observed in human patients exhibiting HbE trait. Furthermore, the transgenic HbE hemolysates contain increased amounts of Hb oxidation products.

Animals↗