Lentiviral-mediated gene correction of mucopolysaccharidosis type IIIA

BACKGROUND: Mucopolysaccharidosis type IIIA (MPS IIIA) is the most common of the mucopolysaccharidoses. The disease is caused by a deficiency of the lysosomal enzyme sulphamidase and results in the storage of the glycosaminoglycan (GAG), heparan sulphate. MPS IIIA is characterised by widespread storage and urinary excretion of heparan sulphate, and a progressive and eventually profound neurological course. Gene therapy is one of the few avenues of treatment that hold promise of a sustainable treatment for this disorder. METHODS: The murine sulphamidase gene cDNA was cloned into a lentiviral vector and high-titre virus produced. Human MPS IIIA fibroblast cultures were transduced with the sulphamidase vector and analysed using molecular, enzymatic and metabolic assays. High-titre virus was intravenously injected into six 5-week old MPS IIIA mice. Three of these mice were pre-treated with hyperosmotic mannitol. The weight of animals was monitored and GAG content in urine samples was analysed by polyacrylamide gel electrophoresis. RESULTS: Transduction of cultured MPS IIIA fibroblasts with the sulphamidase gene corrected both the enzymatic and metabolic defects. Sulphamidase secreted by gene-corrected cells was able to cross correct untransduced MPS IIIA cells. Urinary GAG was found to be greatly reduced in samples from mice receiving the vector compared to untreated MPS IIIA controls. In addition, the weight of treated mice became progressively normalised over the 6-months post-treatment. CONCLUSION: Lentiviral vectors appear promising vehicles for the development of gene therapy for MPS IIIA

Plasma and dried blood spot lysosphingolipids for the diagnosis of different sphingolipidoses: a comparative study.

Abstract Background Lysosphingolipids, the N-deacylated forms of sphingolipids, have been identified as potential biomarkers of several sphingolipidoses, such as Gaucher, Fabry, Krabbe and Niemann-Pick diseases and in GM1 and GM2 gangliosidoses. To date, different methods have been developed to measure various lysosphingolipids (LysoSLs) in plasma. Here, we present a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for a simultaneous quantification of LysoSLs (HexSph, LysoGb3, LysoGM1, LysoGM2, LysoSM and LysoSM509) in dried blood spot (DBS). This LC-MS/MS method was used to compare the levels of LysoSLs in DBS and plasma in both affected patients and healthy controls. Methods Lysosphingolipids were extracted from a 3.2 mm diameter DBS with a mixture of methanol:acetonitrile:water (80:15:5, v/v) containing internal stable isotope standards. Chromatographic separation was performed using a C18 column with a gradient of water and acetonitrile both with 0.1% formic acid in a total run time of 4 min. The compounds were detected in the positive ion mode electrospray ionization (ESI)-MS/MS by multiple reaction monitoring (MRM). Results The method was validated on DBS to demonstrate specificity, linearity, lowest limit of quantification, accuracy and precision. The reference ranges were determined in pediatric and adult populations. The elevated levels of LysoSLs were identified in Gaucher disease (HexSph), Fabry disease (LysoGb3), prosaposin deficiency (HexSph and LysoGb3) and Niemann-Pick disease types A/B and C (LysoSM and LysoSM509). The correlation in the levels between DBS and plasma was excellent for LysoGb3 and HexSph but poor for LysoSM and LysoSM509. Conclusions Despite the fact that plasma LysoSLs determination remains the gold standard, our LC-MS/MS method allows a rapid and reliable quantification of lysosphingolipids in DBS. The method is a useful tool for the diagnosis of different sphingolipidoses except for Niemann-Pick type C

Neonatal screening for MPS disorders in Latin America : a survey of pilot initiatives

Newborn screening enables the diagnosis of treatable disorders at the early stages, and because of its countless benefits, conditions have been continuously added to screening panels, allowing early intervention, aiming for the prevention of irreversible manifestations and even premature death. Mucopolysaccharidoses (MPS) are lysosomal storage disorders than can benefit from an early diagnosis, and thus are being recommended for newborn screening. They are multisystemic progressive disorders, with treatment options already available for several MPS types. MPS I was the first MPS disorder enrolled in the newborn screening (NBS) panel in the USA and a few other countries, and other MPS types are expected to be added. Very few studies about NBS for MPS in Latin America have been published so far. In this review, we report the results of pilot studies performed in Mexico and Brazil using different methodologies: tandem mass spectrometry, molecular analysis, digital microfluidics, and fluorimetry. These experiences are important to report and discuss, as we expect to have several MPS types added to NBS panels shortly. This addition will enable timely diagnosis of MPS, avoiding the long diagnostic odyssey that is part of the current natural history of this group of diseases, and leading to a better outcome for the affected patients

Current state and innovations in newborn screening: continuing to do good and avoid harm

In 1963, Robert Guthrie’s pioneering work developing a bacterial inhibition assay to measure phenylalanine in dried blood spots, provided the means for whole-population screening to detect phenylketonuria in the USA. In the following decades, NBS became firmly established as a part of public health in developed countries. Technological advances allowed for the addition of new disorders into routine programmes and thereby resulted in a paradigm shift. Today, technological advances in immunological methods, tandem mass spectrometry, PCR techniques, DNA sequencing for mutational variant analysis, ultra-high performance liquid chromatography (UPLC), iso-electric focusing, and digital microfluidics are employed in the NBS laboratory to detect more than 60 disorders. In this review, we will provide the current state of methodological advances that have been introduced into NBS. Particularly, ‘second-tier’ methods have significantly improved both the specificity and sensitivity of testing. We will also present how proteomic and metabolomic techniques can potentially improve screening strategies to reduce the number of false-positive results and improve the prediction of pathogenicity. Additionally, we discuss the application of complex, multiparameter statistical procedures that use large datasets and statistical algorithms to improve the predictive outcomes of tests. Future developments, utilizing genomic techniques, are also likely to play an increasingly important role, possibly combined with artificial intelligence (AI)-driven software. We will consider the balance required to harness the potential of these new advances whilst maintaining the benefits and reducing the risks for harm associated with all screening

The 2003 Tracker Inner Barrel Beam Test

Before starting the CMS Silicon Strip Tracker (SST) mass production, where the quality control tests can only be done on single components, an extensive collection of activities aiming at validating the tracker system functionality has been performed. In this framework, a final component prototype of the Inner Barrel part (TIB) of the SST has been assembled and tested in the INFN laboratories and then moved to CERN to check its behaviour in a 25~ns LHC-like particle beam. A set of preproduction single-sided silicon microstrip modules was mounted on a mechanical structure very similar to a sector of the third layer of the TIB and read out using a system functionally identical to the final one. In this note the system setup configuration is fully described and the results of the test, concerning both detector performance and system characteristics, are presented and discussed

KLOE results in kaon physics and prospects for KLOE-2

The phi-factory DAPHNE offers a possibility to select pure kaon beams, charged and neutral ones. In particular, neutral kaons from phi->KS KL are produced in pairs and the detection of a KS (KL) tags the presence of a KL (KS). This allows to perform precise measurements of kaon properties by means of KLOE detector. Another advantage of a phi-factory consists in fact that the neutral kaon pairs are produced in a pure quantum state (J^(PC) = 1^(--)), which allowsto investigate CP and CPT symmetries via quantum interference effects, as well as the basic principles of quantum mechanics.A review of the most recent results of the KLOE experiment at DAPHNE using pure kaon beams or via quantum interferometry is presented together with prospects for kaon physics at KLOE-2.Comment: 5 pages, 4 figures, From Phi To Psi 2011 conference, to be published in Nuclear Physics B (Proceedings Supplements

Prostaglandin D2 in Inflammatory Arthritis and Its Relation with Synovial Fluid Dendritic Cells

Prostaglandin (PG)D2 has been shown to be an active agent in the resolution of experimentally induced inflammation. This study was undertaken to determine the presence of PGD2 in chronic joint effusions and to explore the potential contributions of dendritic cells (DC) and monocytes to the intra-articular synthesis of PGD2. Synovial fluid (SF) was obtained from patients with inflammatory arthritis and knee effusions. PGD2 and PGE2 were detected in SF by ultrahigh-performance tandem mass spectrometry. Cellular fractions in SF were separated by density-gradient centrifugation and flow cytometry. The expression of hematopoietic prostaglandin D-synthase (hPGDS) and PGE-synthase (PGES) mRNA was determined by RT-PCR. Both PGD2 and PGE2 were detected in blood and SF, with PGD2 being more abundant than PGE2 in SF. mRNA for hPGDS was more abundant in SF mDCs than SF monocytes (P<0.01) or PB monocytes (P<0.001). SF mDC expressed significantly more hPGDS than PGES. Expressions of PGD2 and hPGDS were inversely associated with serum C-reactive protein (P<0.01) and erythrocyte sedimentation rate (P<0.01). The findings suggest that synovial DCs may be an important source of hPGDS and that systemic disease activity may be influenced by actions of PGD2 in RA and other arthropathies

Multiple acyl-coenzyme A dehydrogenase deficiency: diagnosis by acyl-carnitine analysis of a 12-year-old newborn screening card

Poplawski, Nicola K. ; Ranieri, Enzo ; Harrison, J.Rodney ; Fletcher, Janice M