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John J Hopwood

Publications and source records attributed to John J Hopwood.

At least 19 recordsLinked to original sources
PubMed2006

Injection of recombinant human sulfamidase into the CSF via the cerebellomedullary cistern in MPS IIIA mice.

At present, there is no widely available, safe and effective treatment for lysosomal storage disorders (LSD) that affect the brain. We have used a naturally occurring mouse model of mucopolysaccharidosis type IIIA (MPS IIIA) or Sanfilippo syndrome, to evaluate the effect of repeated injection of recombinant human sulfamidase (rhSGSH) into the cerebrospinal fluid via the cisterna magna (CM) on central nervous system (CNS) pathology and behavioral function. Mice received up to seven injections of rhSGSH (5-20 microg rhSGSH per injection) or vehicle on a fortnightly or monthly basis. A dose-dependent reduction in the level of a heparan sulfate-derived monosulfated disaccharide was observed within the brain (up to 62% reduction compared with vehicle-treated MPS IIIA mice) and spinal cord (up to 71% reduction). Ultrastructural examination revealed a reduction in lysosomal vesicle formation in various cell types and fewer (ubiquitin-positive) axonal spheroids were observed in several brain regions. The biochemical changes were accompanied by improved behavior, particularly in mice-treated more frequently. A humoral immune response to rhSGSH was observed in treated animals. Intra-CM injection of lysosomal enzyme may therefore represent an immediately applicable method of treating the CNS effects of this and potentially other LSD that affect the brain.

Animals↗
PubMed2006

Mutational analysis of mucopolysaccharidosis type VI patients undergoing a phase II trial of enzyme replacement therapy.

Mucopolysaccharidosis type VI (MPS VI; Maroteaux-Lamy syndrome) is a lysosomal storage disorder caused by mutations in the N-acetylgalactosamine-4-sulfatase (ARSB) gene. These mutations result in a deficiency of ARSB activity. Ten MPS VI patients were involved in a phase II clinical study of enzyme replacement therapy. Direct sequencing of genomic DNA from these patients was used to identify ARSB mutations. Each individual exon of the ARSB gene was amplified by PCR and subsequently sequenced. Thirteen substitutions (c.215T>G [p.L72R] c.284G>A [p.R95Q], c.305G>A [p.R102H], c.323G>T [p.G108V], c.389C>T [p.P130L], c.511G>A [p.G171S], c.904G>A [p.G302R], c.944G>A [p.R315Q], c.1057T>C [p.W353R], c.1151G>A [p.S384N], c.1178A>C [p.H393P], c.1289A>G [p.H430R] and c.1336G>C [p.G446R]), one deletion (c.238delG), and two intronic mutations (c.1213+5G>A and c.1214-2A>G) were identified. Nine of the 16 mutations identified were novel (R102H, G108V, P130L, G171S, W353R, H430R, G446R, c.1213+5G>A and c.1214-2A>G). The two common polymorphisms c.1072G>A [p.V358M] and c.1126G>A [p.V376M] were identified in some of the patients, along with the silent mutations c.972A>G and c.1191A>G. Cultured fibroblast ARSB mutant protein and residual activity were determined for each patient and, together with genotype information, used to predict the expected clinical severity of each patient.

Adolescent↗
PubMed2006

N-acetylgalactosamine-6-sulfatase protein detection in MPS IVA patient and unaffected control samples.

BACKGROUND: Mucopolysaccharidosis type IVA (MPS IVA; Morquio syndrome) is a lysosomal storage disorder caused by a deficiency in the activity of the lysosomal hydrolase N-acetylgalactosamine-6-sulfatase (GALNS). MPS IVA patients can present with severe myelopathy, hearing loss, heart valve involvement, short trunk/dwarfism and corneal clouding. Early diagnosis of MPS IVA will allow potential treatments to be implemented before the onset of irreversible pathology. METHODS: We have developed a sensitive immune-quantification assay for the accurate detection of GALNS protein in skin fibroblasts, blood and plasma from unaffected control and MPS IVA patients. RESULTS: MPS IVA patient fibroblast extracts (n=11) had non-detectable (ND)-10 ng/mg of 6-sulfatase protein compared to 3-82 ng/mg for normal controls (n=19). Dried blood-spots from MPS IVA patients (n=4) contained ND-1.3 ng/L of 6-sulfatase protein compared to 18-145 ng/L for normal controls (n=49). Plasma from MPS IVA patients (n=7) contained ND 6-sulfatase protein compared to 1-9 ng/L for normal controls (n=49). CONCLUSIONS: The immune assay described here had the capacity to accurately measure the amount of GALNS protein in various biological samples, providing the basis of an assay that could be further developed to enable newborn and high-risk population screening for MPS IVA patients.

Cells, Cultured↗
PubMed2006

Immunochemistry of lysosomal storage disorders.

BACKGROUND: Lysosomal storage disorders are a group of genetic diseases, each with a broad spectrum of clinical presentation that ranges from attenuated to severe. The immunochemical analysis of patient samples is aimed at several key aspects of patient management, including early detection of the disorder, prediction of clinical severity, determining the most appropriate therapeutic regimen, and monitoring of patients on therapy. METHODS: In this study, we review the current and emerging technology available to achieve these assessments. RESULTS: Immune assays have direct practical application for the early detection, diagnosis and prognosis of lysosomal storage disorder patients. Multiplexing of these assays may provide a platform to allow newborn screening for multiple lysosomal storage disorders. CONCLUSIONS: We have reviewed the immunochemical techniques available for the analysis of lysosomal storage disorder patient samples and advise that these may be used in conjunction with other technologies for effective patient management.

Biomarkers↗
PubMed2006

Stabilising normal and mis-sense variant alpha-glucosidase.

alpha-Glucosidase (EC 3.2.1.3) is a lysosomal enzyme that hydrolyses alpha-1,4- and alpha-1,6-linkages of glycogen to produce free glucose. A deficiency in alpha-glucosidase activity results in glycogen storage disorder type II (GSD II), also called Pompe disease. Here, d-glucose was shown to be a competitive inhibitor of alpha-glucosidase and when added to culture medium at 6.0 g/L increased the production of this protein by CHO-K1 expression cells and stabilised the enzyme activity. D-Glucose also prevented alpha-glucosidase aggregation/precipitation and increased protein yield in a modified purification scheme. In fibroblast cells, from adult-onset GSD II patients, D-glucose increased the residual level of alpha-glucosidase activity, suggesting that a structural analogue of d-glucose may be used for enzyme enhancement therapy.

Animals↗
PubMed2006

Characterization of a C57BL/6 congenic mouse strain of mucopolysaccharidosis type IIIA.

The original mucopolysaccharidosis type IIIA (MPS IIIA) mice were identified in a mixed background with contributions from four different strains. To ensure long-term stability and genetic homogeneity of this lysosomal storage disease (LSD) model, the aim of this study was to develop and characterize a C57BL/6 congenic strain. The B6.Cg-Sgsh(mps3a) strain compares favorably with the original mixed donor strain, exhibiting low liver sulfamidase activity and significant brain heparan sulfate-derived disaccharide elevation from birth. A rapid increase in brain disaccharide levels occurred after birth, with a plateau reached by 13 weeks of age at 110x the levels observed in brains of age-matched unaffected mice. Typical lysosomal inclusions were observed in cerebral cortical and cerebellar neurons and in liver hepatocytes and Kupffer cells. Ubiquitin-positive spheroids and GM(2)-ganglioside were also detected in brain. Using the Morris water maze in male mice, impaired memory and spatial learning was evident at 20 weeks of age in B6.Cg-Sgsh(mps3a) MPS IIIA mice. Other behavioral changes include motor, cognitive and sensory deficits, and aggression. Male B6.Cg-Sgsh(mps3a) MPS IIIA mice exhibited more behavioral abnormalities than B6.Cg-Sgsh(mps3a) MPS IIIA females, as observed previously in the original mixed background strain. Affected mice generally survive to 9 to 12 months of age, before death or euthanasia for humane reasons. Overall, minor differences were apparent between the new congenic and previously described mixed MPS IIIA strains. Availability of an in-bred strain will ensure more reproducible experimental outcomes thereby assisting in our goal of developing effective therapies for LSD with central nervous system disease.

Age Factors↗
PubMed2006

Characterization of sulfated oligosaccharides in mucopolysaccharidosis type IIIA by electrospray ionization mass spectrometry.

Heparan sulfate is a linear glycosaminoglycan with considerable structural diversity that binds a myriad of growth factors and proteins that play pivotal roles in a variety of biological processes. We have investigated the structural complexity of partially degraded fragments of heparan sulfate in mucopolysaccharidosis type IIIA in which there is a defect in heparan sulfate catabolism. Mono- to hexadecasaccharides were isolated from the urine of a mucopolysaccharidosis IIIA patient and shown to have non-reducing end glucosamine N-sulfate residues, reflecting the catabolic deficiency in heparan N-sulfatase (sulfamidase) activity. The use of nitrous acid digestion (pH 1.5) combined with separation by reverse-phase high-performance liquid chromatography and analysis by electrospray ionization-mass spectrometry identified multiple forms of these oligosaccharides with some N-acetylated glucosamine residues and one to three sulfates per disaccharide. Furthermore, we demonstrated that each oligosaccharide existed in multiple sulfated forms. Many structural isomers were present, suggesting a complex mixture of oligosaccharides present in the urine as a consequence of a defect in heparan sulfate degradation.

Animals↗
PubMed2006

Enzyme replacement therapy in alpha-mannosidosis guinea-pigs.

alpha-Mannosidosis is a lysosomal storage disorder caused by deficient activity of lysosomal alpha-mannosidase and is characterised by massive accumulation of mannose-containing oligosaccharides in affected individuals. Patients develop behaviour and learning difficulties, skeletal abnormalities, immune deficiency and hearing impairment. Disease in alpha-mannosidosis guinea-pigs resembles the clinical, histopathological, biochemical and molecular features of the human disease. We have used the guinea-pig model to investigate efficacy of enzyme replacement therapy as a treatment for alpha-mannosidosis. Intravenous recombinant human lysosomal alpha-mannosidase, administered at a dose of 1mg/kg, was cleared from circulation with a half-life of 53 h, with significant enzyme activity (1.4x normal levels) detected in circulation one week post-injection. alpha-Mannosidase administered to alpha-mannosidosis guinea-pigs at 1mg/kg (onset at birth or approximately 30 days) and 10mg/kg (at birth) was distributed widely amongst tissues, including to capillary depleted brain. By monitoring with tandem mass spectrometry, enzyme replacement therapy was found to be effective in reducing stored substrates in peripheral tissues at both dose rates, and in brain by up to 39% at the 10mg/kg dose, compared with untreated alpha-mannosidosis controls. Reductions of up to 60% of urinary mannose containing oligosaccharides were also observed. No histological improvements were seen in the brain at either dose, however marked decreases in lysosomal vacuolation in liver, kidney, spleen and endocrine pancreas, as well as a significant reduction in trigeminal ganglion neurons were observed. Multiple injections of 1mg/kg recombinant enzyme in alpha-mannosidosis guinea-pigs induced a very rapid humoral immune response precluding long-term intravenous treatment.

Animals↗
PubMed2006

Stop-codon read-through for patients affected by a lysosomal storage disorder.

Lysosomal storage disorders are a group of inherited diseases that can result in severe and progressive pathology due to a specific lysosomal dysfunction. Current treatment strategies include bone-marrow transplantation, substrate reduction, chemical-chaperone and enzyme-replacement therapy. However, each of these treatments has its limitations. Enhanced stop-codon read-through is a potential alternative or adjunct therapeutic strategy for treating lysosomal-storage-disorder patients. Premature stop-codon mutations have been identified in a large cohort of patients with a lysosomal storage disorder, making stop-codon read-through a possible treatment for this disease. In lysosomal-storage-disorder cells (mucopolysaccharidosis type I, alpha-L-iduronidase deficient), preclinical studies have shown that gentamicin induced the read-through of premature stop codons, resulting in enzyme activity that reduced substrate storage.

Codon, Nonsense↗
PubMed2006

Newborn screening for lysosomal storage disorders.

Lysosomal storage disorders (LSD) are chronic progressive diseases that have a devastating impact on the patient and family. Most patients are clinically normal at birth but develop symptoms early in childhood. Despite no curative treatment, a number of therapeutic options are available to improve quality of life. To achieve this, there is a pressing need for newborn screening to identify affected individuals early, before the onset of severe irreversible pathology. We have developed a multiplexed immune-quantification assay of 11 different lysosomal proteins for the identification of individuals with an LSD and evaluated this assay in a retrospective study using blood-spots from; newborns subsequently diagnosed with an LSD (n=19, six different LSD), individuals sampled after diagnosis of an LSD (n=92, 11 different LSD), newborn controls (n=433), and adult controls (n=200). All patients with mucopolysaccharidosis type I (MPS I), MPS II, MPS IIIA, MPS VI, metachromatic leukodystrophy, Niemann-Pick disease type A/B, and multiple sulfatase deficiency could be identified by reduced enzyme levels compared to controls. All mucolipidosis type II/III patients were identified by the elevation of several lysosomal enzymes, above the control range. Most Fabry, Pompe, and Gaucher disease patients were identified from either single protein differences or profiles of multiple protein markers. Newborn screening for multiple LSD is achievable using multiplexed immune-quantification of a panel of lysosomal proteins. With further validation, this method could be readily incorporated into existing screening laboratories and will have a substantial impact on patient management and counseling of families.

Adult↗
PubMed2006

Detection of mucopolysaccharidosis type II by measurement of iduronate-2-sulfatase in dried blood spots and plasma samples.

BACKGROUND: Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disorder related to a deficiency in the enzyme iduronate-2-sulfatase (IDS). Clinical trials of enzyme replacement therapy are in progress, but effective treatment will require screening assays to enable early detection and diagnosis of MPS II. Our study evaluated the diagnostic accuracy of IDS protein and enzyme activity measurements in dried blood spots and plasma. METHODS: We collected dried-blood-spot and plasma samples from unaffected control individuals and from MPS II patients. We measured IDS protein concentration with a 2-step time-delayed dissociation-enhanced lanthanide fluorescence immunoassay. To measure enzyme activity, we immobilized anti-IDS antibody on microtiter plates to capture the enzyme and measured its activity with the fluorogenic substrate 4-methylumbelliferyl sulfate. RESULTS: Dried-blood-spot samples from MPS II patients showed an almost total absence of IDS activity (0-0.075 micromol x h(-1) x L(-1)) compared with control blood spots (0.5-4.7 micromol x h(-1) x L(-1)) and control plasma (0.17-8.1 micromol x h(-1) x L(-1)). A dried-blood-spot sample from only 1 of 12 MPS II patients had detectable concentrations of IDS protein (24.8 microg/L), but no IDS protein was detected in plasma from MPS II patients. Ranges for IDS protein in control samples were 25.8-153 microg/L for blood spots and 22.8-349.4 microg/L for plasma. CONCLUSION: Measurement of the IDS protein concentration and enzyme activity (as measured by a simple fluorogenic assay with an immune capture technique) enables identification of the majority of MPS II patient samples from both dried blood spots and plasma samples.

Adolescent↗
PubMed2006

Galsulfase.

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Animals↗
PubMed2006

Intra-articular enzyme administration for joint disease in feline mucopolysaccharidosis VI: enzyme dose and interval.

Degenerative joint changes have been reported in human mucopolysaccharidosis VI (MPS VI) and are a prominent feature of feline MPS VI. Joint disease has proven refractory to intravenous enzyme replacement therapy (ERT) in the MPS VI cat because enzyme is unable to reach cells in cartilage. In this study, enzyme was infused directly into the intraarticular space to determine whether joint tissues are able to respond to replacement enzyme. Clearance of glycosaminoglycans from chondrocytes was observed at a dose of 10 microg recombinant human N-acetylgalactosamine-4-sulfatase (rh4S), but greater clearance was observed with higher doses. The chondrocytes at the articular surface were cleared preferentially. Lysosomal vacuolation in cruciate ligament and synovial cells also decreased upon addition of rh4S. One month after injection of rh4S, a slight reaccumulation of storage was observed at the surface of the joint, but extensive reaccumulation was observed 2 mo after injection. These results indicate that by bypassing the synovium using intraarticular ERT, significant reduction in storage material in joint tissues can be achieved. Localized ERT in the joint space provides a mechanism for delivering enzyme directly to the articular cartilage and a potential therapy for joint pathology in MPS VI.

Animals↗
PubMed2006

Directed differentiation and characterization of genetically modified embryonic stem cells for therapy.

Lysosomal storage disorders are rare, inherited diseases caused by a deficiency of a specific, lysosomal enzyme. In the case of mucopolysaccharidosis type IIIA, a lack of active sulfamidase enzyme results in heparan sulfate accumulation, severe and progressive neurological deficits, and usually premature death. Embryonic stem cells can be genetically modified to overexpress lysosomal enzymes, providing a renewable reservoir of cells that can be readily expanded in culture. Screening clonal lines of embryonic stem cells for desirable properties such as high levels and maintenance of enzyme activity throughout terminal differentiation to neural phenotypes theoretically provides a reproducible population of cells that can be fully characterized in vitro before implantation within the central nervous system in animal models of lysosomal storage disorders.

Animals↗
PubMed2005

A defect in exodegradative pathways provides insight into endodegradation of heparan and dermatan sulfates.

Within cells, dermatan sulfate (DS) and heparan sulfate (HS) are degraded in two steps. The initial endohydrolysis of these polysaccharides is followed by the sequential action of lysosomal exoenzymes to reduce the resulting oligosaccharides to monosaccharides and inorganic sulfate. Mucopolysaccharidosis (MPS) type II is a lysosomal storage disorder caused by a deficiency of the exoenzyme iduronate-2-sulfatase (I2S). Consequently, partially degraded fragments of DS and HS have been shown to accumulate in the lysosomes of affected cells and are excreted in the urine. Di- to hexadecasaccharides, isolated from the urine of a MPS II patient using anion exchange and gel filtration chromatography, were identified using electrospray ionization-tandem mass spectrometry (ESI-MS/MS). These oligosaccharides were shown to have non-reducing terminal iduronate-2-sulfate residues by digestion with recombinant I2S. A pattern of growing oligosaccharide chains composed of alternating uronic acid and N-acetylhexosamine residues was identified and suggested to originate from DS. A series of oligosaccharides consisting of hexosamine/N-acetylhexosamine alternating with uronic acid residues was also identified and on the basis of the presence of unacetylated hexosamine; these oligosaccharides are proposed to derive from HS. The presence of both odd and even-length oligosaccharides suggests both endo-beta-glucuronidase and endo-N-acetylhexosaminidase activities toward both glycosaminoglycans. Furthermore, the putative HS oligosaccharide structures identified indicate that heparanase activities are directed toward regions of both low and high sulfation, while the N-acetylhexosaminidase activity acted only in regions of low sulfation in this polysaccharide.

Carbohydrate Conformation↗
PubMed2005

Common antigenicity for two glycosidases.

Enzyme replacement therapy (ERT) has proven to be an effective therapy for some lysosomal storage disorder (LSD) patients. A potential complication during ERT is the generation of an immune response against the replacement protein. We have investigated the antigenicity of two distantly related glycosidases, alpha-glucosidase (Pompe disease or glycogen storage disease type II, GSD II), and alpha-L-iduronidase (Hurler syndrome, mucopolysaccharidosis type I, MPS I). The linear sequence epitope reactivity of affinity purified polyclonal antibodies to recombinant human alpha-glucosidase and alpha-L-iduronidase was defined, to both glycosidases. The polyclonal antibodies exhibited some cross-reactive epitopes on the two proteins. Moreover, a monoclonal antibody to the active site of alpha-glucosidase showed cross-reactivity with a catalytic structural element of alpha-L-iduronidase. In a previous study, in MPS I patients who developed an immune response to ERT, this same site on alpha-L-iduronidase was highly antigenic and the last to tolerise following repeated enzyme infusions. We conclude that glycosidases can exhibit cross-reactive epitopes, and infer that this may relate to common structural elements associated with their active sites.

Animals↗
PubMed2005

Profiling oligosaccharidurias by electrospray tandem mass spectrometry: quantifying reducing oligosaccharides.

A method to semiquantify urinary oligosaccharides from patients suffering from oligosaccharidurias is presented. 1-Phenyl-3-methyl-5-pyrazolone has been used to derivatize urinary oligosaccharides prior to analysis by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). Disease-specific oligosaccharides were identified for several oligosaccharidurias, including GM1 gangliosidosis, GM2 gangliosidosis, sialic acid storage disease, sialidase/neuraminidase deficiency, galactosialidosis, I-cell disease, fucosidosis, Pompe and Gaucher diseases, and alpha-mannosidosis. The oligosaccharides were referenced against the internal standard, methyl lactose, to produce ratios for comparison with control samples. Elevations in specific urinary oligosaccharides were indicative of lysosomal disease and the defective catabolic enzyme. This method has been adapted to enable assay of large sample numbers and could readily be extended to other oligosaccharidurias and to monitor oligosaccharide levels in patients receiving treatment. It also has immediate potential for incorporation into a newborn screening program.

Carbohydrate Sequence↗