Monique P. Gelderman

Adeno-associated viral vector-mediated gene transfer results in long-term enzymatic and functional correction in multiple organs of Fabry mice

Fabry disease is a lysosomal storage disorder caused by a deficiency of the lysosomal enzyme α-galactosidase A (α-gal A). This enzyme deficiency leads to impaired catabolism of α-galactosyl-terminal lipids such as globotriaosylceramide (Gb3). Patients develop painful neuropathy and vascular occlusions that progressively lead to cardiovascular, cerebrovascular, and renal dysfunction and early death. Although enzyme replacement therapy and bone marrow transplantation have shown promise in the murine analog of Fabry disease, gene therapy holds a strong potential for treating this disease in humans. Delivery of the normal α-gal A gene (cDNA) into a depot organ such as liver may be sufficient to elicit corrective circulating levels of the deficient enzyme. To investigate this possibility, a recombinant adeno-associated viral vector encoding human α-gal A (rAAV-AGA) was constructed and injected into the hepatic portal vein of Fabry mice. Two weeks postinjection, α-gal A activity in the livers of rAAV-AGA-injected Fabry mice was 20–35% of that of the normal mice. The transduced animals continued to show higher α-gal A levels in liver and other tissues compared with the untouched Fabry controls as long as 6 months after treatment. In parallel to the elevated enzyme levels, we see significant reductions in Gb3 levels to near normal at 2 and 5 weeks posttreatment. The lower Gb3 levels continued in liver, spleen, and heart, up to 25 weeks with no significant immune response to the virus or α-gal A. Also, no signs of liver toxicity occurred after the rAAV-AGA administration. These findings suggest that an AAV-mediated gene transfer may be useful for the treatment of Fabry disease and possibly other metabolic disorders.

Carbon nanotubes activate blood platelets by inducing extracellular Ca2+ influx sensitive to calcium entry inhibitors.

To elucidate a mechanism of prothrombotic effects of carbon nanotubes (CNTs), we report here that multiwalled CNTs activate blood platelets by inducing extracellular Ca(2+) influx that could be inhibited by calcium channel blockers SKF 96365 and 2-APB. We also demonstrate platelet aggregating activity of different single-walled and multiwalled CNTs. In addition, we show that CNT-induced platelet activation is associated with a marked release of platelet membrane microparticles positive for the granular secretion markers CD62P and CD63.

Long-term correction of globotriaosylceramide storage in Fabry mice by recombinant adeno-associated virus-mediated gene transfer

Fabry disease is an X-linked recessive inborn metabolic disorder characterized by systemic and vascular accumulation of globotriaosylceramide (Gb3) caused by a deficiency of the lysosomal enzyme α-galactosidase A (α-gal A). The condition is associated with an increased morbidity and mortality due to renal failure, cardiac disease, and early onset of stroke. Hemizygous males are primarily affected clinically with variable expression in heterozygous females. Gene-therapy trials have been initiated recently in α-gal A knockout mouse models of Fabry disease by using a variety of viral vectors. In the present investigation we administered single i.v. injections of 1 × 1010 genomes of recombinant adeno-associated virus (rAAV) encoding the human α-gal A gene driven by a modified chicken β-actin (CAG) promoter to α-gal A knockout (Fabry) mice. Transgenic mice were analyzed for expression of α-gal A activity and Gb3 levels in liver, kidney, heart, spleen, small intestine, lung, and brain. Administration of the rAAV-CAG-hAGA vector resulted in stable expression of α-gal A in organs of the Fabry mice for >6 months. α-Gal A activity in the organs became equal to or higher than that of wild-type mice. Accumulated Gb3 in the liver, heart, and spleen was reduced to that of wild-type mice with lesser but significant reductions in kidney, lung, and small intestine. Injection of the rAAV-CAG-hAGA construct into skeletal muscle did not result in expression of α-gal A in it or in other tissues. This study provides a basis for a simple and efficient gene-therapy approach for patients with Fabry disease and is indicative of its potential for the treatment of other lysosomal storage disorders.

Flow Cytometric Analysis of Cell Membrane Microparticles

Cell membrane microparticles (MPs) are phospholipid microvesicles shed from the plasma membrane of most eukaryotic cells undergoing activation or apoptosis. The presence of MPs is common in healthy individuals. However, an increase in their release is a controlled event and is considered a hallmark of cellular alteration. Microparticles display cell surface proteins that indicate their cellular origin. In addition, they may also express other markers, e.g., markers of cellular activation. Elevated levels of circulating MPs are associated with various vascular pathologies and their pathogenic potential has been widely documented. MPs have been analyzed in plasma and cell cultures by means of flow cytometry or solid phase assays. Here we present a three-color flow cytometric assay for immunophenotyping of MPs in plasma. This assay has been used to study elevated counts of different phenotypes of circulating endothelial MPs in several hematological and vascular diseases. A modified version of this assay can also be used for MP analysis in blood products and cell cultures.

T Cell Tolerance to a Neo-Self Antigen Expressed by Thymic Epithelial Cells: The Soluble Form Is More Effective Than the Membrane-Bound Form

We have previously shown that transgenic (Tg) mice expressing either soluble or membrane-bound hen egg lysozyme (sHEL or mHEL, respectively) under control of the αA-crystallin promoter develop tolerance due to thymic expression of minuscule amounts of HEL. To further address the mechanisms by which this tolerance develops, we mated these two lines of Tg mice with the 3A9 line of HEL-specific TCR Tg mice, to produce double-Tg mice. Both lines of double-Tg mice showed deletion of HEL-specific T cells, demonstrated by reduction in numbers of these cells in the thymus and periphery, as well as by reduced proliferative response to HEL in vitro. In addition, the actual deletional process in thymi of the double-Tg mice was visualized in situ by the TUNEL assay and measured by binding of Annexin V. Notably, the apoptosis localized mainly in the thymic medulla, in line with the finding that the populations showing deletion and increased Annexin V binding consisted mainly of single- and double-positive thymocytes. Interestingly, the thymic deletional effect of sHEL was superior to that of mHEL in contrast to the opposite differential tolerogenic effects of these HEL forms on B cells specific to this Ag. Analysis of bone marrow chimeras indicates that both forms of HEL are produced by irradiation-resistant thymic stromal cells and the data suggest that sHEL is more effective in deleting 3A9 T cells due mainly to its higher accessibility to cross-presentation by dendritic APC.

Upregulation of phagocytosis and candidicidal activity of macrophages exposed to the immunostimulant, acemannan

Previous studies by these investigators have shown that mannosylated bovine serum albumin (m-BSA) enhances the respiratory burst (RB), phagocytosis, and killing of Candida albicans by resident murine peritoneal macrophages (MO). Upregulation of the above MO functions was associated with binding of m-BSA to the MO-mannose receptor. The present study was done to determine if the immunostimulant, acemannan prepared from aloe vera, could stimulate MO in a similar manner. Resident peritoneal MO collected from C57BL/6 mice were exposed to acemannan for 10 min. The RB was measured using chemiluminescence and demonstrated approximately a two-fold increase above the media controls. In studies involving phagocytosis, MO were exposed to acemannan, washed and exposed to Candida at a ratio of 1:5. The percent phagocytosis and Candida killing were determined using fluorescence microscopy. There was a marked increase in phagocytosis in the treated cultures (45%) compared to controls (25%). Macrophages exposed to acemannan for 10 min resulted in ca 38% killing of Candida albicans compared with 0-5% killing in controls. If MO were incubated with acemannan for 60 min, 98% of the yeast were killed compared to 0-5% in the controls. The results of the present study indicate that short term exposure of MO to acemannan upregulates the RB, phagocytosis and candidicidal activity. Further studies are needed to clarify the potential use of this immunostimulant as an anti-fungal agent.

Exposure of macrophages to an enzymatically inactive macrophage mannose receptor ligand augments killing of Candida albicans.

Abstract Macrophages (Mφ) are involved in host defenses against opportunistic pathogens. Previous studies by the present investigators indicate that Mφ exposed to enzymatically active myeloperoxidase (MPO), exhibited both increased phagocytosis and killing of Candida albicans. The purpose of this study was to determine if enzymatically inactive Mφ-mannose receptor (MMR) ligands could function similarly. Resident murine peritoneal Mφ were exposed to the MMR ligands, mannosylated bovine serum albumin (mBSA), and enzymatically inactive myeloperoxidase (iMPO), followed by exposure to opsonized C. albicans. Both mBSA and iMPO induced a slight increase in the number of phagocytizing cells; however, candidacidal activity was significantly higher in treated cultures compared to controls (P ≤ 0.001). The production of reactive oxygen intermediates (ROI) was detected using chemiluminescence. After employment of ROI scavengers, a decrease in candidacidal activity was observed. The data suggest that MMR-ligand interaction alone is sufficient to significantly enhance the candidacidal activity of Mφ via ROI, and that iMPO which is released at a site of inflammation induces Mφ-mediated killing of microorganisms. These findings indicate a previously unrecognized role of iMPO.

Elevated Endothelial Microparticles in Fabry Children Decreased After Enzyme Replacement Therapy

To the Editor: Fabry disease (FD), an X-linked metabolic disorder, is caused by insufficient activity of the lysosomal enzyme alpha galactosidase A (α-gal A). This results in impaired catabolism of globotriaosylceramide ([Gb3] also called ceramidetrihexoside) and its subsequent accumulation leading to endothelial dysfunction and other anomalies.1 At present, enzyme replacement therapy (ERT) is the only available therapeutic approach for patients with Fabry disease. However, in adult patients with advanced FD, ERT has its limitations.2 Therefore, studies have been initiated to analyze the efficacy of ERT started at an earlier stage of FD.3,4 In general, the identification of markers that could serve as indicators of disease severity, prognosis, and possible effects of specific therapies is highly desirable in genetic disorders. Because it has been documented that elevated counts of endothelial cell derived membrane microparticles can be present in the circulation of patients with a variety of diseases with a vascular injury component, we explored whether microparticles (MPs) could serve as a potential marker.5 We investigated the …

Genomic abnormalities of the murine model of Fabry disease after disease-related perturbation, a systems biology approach

Fabry disease is a disorder of α-d-galactosyl-containing glycolipids resulting from a deficiency of α-galactosidase A. Patients have a poorly understood vascular dysregulation. We hypothesized that disease-related perturbation by using enzyme replacement therapy in the murine model of Fabry disease would provide insight into abnormal biological processes in Fabry disease. Gene expression analyses of the heart, aorta, and liver of male α-galactosidase A knockout mice 28 weeks of age were compared with that of WT mice. Microarray analyses were performed before and after six weekly injections of α-galactosidase A. Alteration of Rpgrip1 ranked highest statistically in all three organs when knockout mice were compared with WT, and its splice variants responded in a unique way to α-galactosidase A. Enzyme replacement therapy tended to not only normalize gene expression, e.g., reduce the overexpression of securin, but also specifically modified gene expression in each tissue examined. Following multiple comparison analysis, gene expression correlation graphs were constructed, and a priori hypotheses were examined by using structural equation modeling. This systems biology approach demonstrated multiple and complex parallel cellular abnormalities in Fabry disease. These abnormalities form the basis for informed, in a Bayesian sense, sequential, hypothesis-driven research that can be subsequently tested experimentally.

Fabry disease and vascular risk factors: future strategies for patient-based studies and the knockout murine model.

UNLABELLED Fabry disease is secondary to deficiency of the lysosomal enzyme alpha-galactosidase A, leading to altered glycosphingolipid metabolism and accumulation that is often associated with endothelial dysfunction. Current evidence suggests that there is impairment of the vascular nitric oxide pathway, with abnormalities evident in the cerebral circulation and in the dermal vasculature of patients with Fabry disease. Some of these findings have been confirmed in a mouse model of Fabry disease. The murine model, however, allows investigation of Fabry disease at a non-clinical level and a near complete investigation of biological processes within an affected tissue. This is of particular utility in allowing gene expression analysis of clinically inaccessible tissues such as the aorta. CONCLUSION Future developments in array technology for proteins and DNA single nucleotide polymorphism analysis, together with gene expression microarray analysis, may open a new chapter in our understanding of the biology of lysosomal storage disorders.