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Aims To validate a liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the measurement of glycosaminoglycans (GAGs) in plasma and serum. To establish plasma, cerebrospinal fluid (CSF) and urine reference intervals. To compare GAGs in serum with that in urine and CSF from patients with MPS I. Methods Dermatan sulfate (DS), heparan sulfate (HS), and chondroitin sulfate (CS) in serum/plasma, urine and CSF were methanolysed into dimers and analyzed using pseudo isotope dilution UPLC-MS/MS assay. Serum, CSF and urine DS and HS were quantified for 11 patients with mucopolysaccharidosis (MPS) type I before and after treatment with Aldurazyme® (laronidase) enzyme replacement therapy (ERT). Results The method showed acceptable imprecision and recovery for the quantification of serum/plasma CS, DS, and HS. The serum, urine, and CSF DS and HS concentrations were reduced after 26 weeks of ERT in 4 previously untreated patients. Serum DS and HS concentrations normalized in some patients, and were mildly elevated in others after ERT. In contrast, urine and CSF DS and HS values remained elevated above the reference ranges. Compared with serum GAGs, urine and CSF DS and HS were more sensitive biomarkers for monitoring the ERT treatment of patients with MPS I.

Central nervous system manifestations of mucopolysaccharidosis type I (MPS I) such as cognitive impairment, hydrocephalus, and spinal cord compression are inadequately treated by intravenously-administered enzyme replacement therapy with laronidase (recombinant human alpha-L-iduronidase). While hematopoietic stem cell transplantation treats neurological symptoms, this therapy is not generally offered to attenuated MPS I patients. This study is a randomized, open-label, controlled pilot study of intrathecal laronidase in eight attenuated MPS I patients with cognitive impairment. Subjects ranged between 12years and 50years old with a median age of 18years. All subjects had received intravenous laronidase prior to the study over a range of 4 to 10years, with a mean of 7.75years. Weekly intravenous laronidase was continued throughout the duration of the study. The randomization period was one year, during which control subjects attended all study visits and assessments, but did not receive any intrathecal laronidase. After the first year, all eight subjects received treatment for one additional year. There was no significant difference in neuropsychological assessment scores between control or treatment groups, either over the one-year randomized period or at 18 or 24months. However, there was no significant decline in scores in the control group either. Adverse events included pain (injection site, back, groin), headache, neck spasm, and transient blurry vision. There were seven serious adverse events, one judged as possibly related (headache requiring hospitalization). There was no significant effect of intrathecal laronidase on cognitive impairment in older, attenuated MPS I patients over a two-year treatment period. A five-year open-label extension study is underway.

Therapeutic development and monitoring require demonstration of effects on disease phenotype. However, due to the complexity of measuring clinically-relevant effects in rare multisystem diseases, robust biomarkers are essential. For the mucopolysaccharidoses (MPS), the measurement of glycosaminoglycan levels is relevant as glycosaminoglycan accumulation is the primary event that occurs due to reduced lysosomal enzyme activity. Traditional dye-based assays that measure total glycosaminoglycan levels have a high background, due to a normal, baseline glycosaminoglycan content in unaffected individuals. An assay that selectively detects the disease-specific non-reducing ends of heparan sulfate glycosaminoglycans that remain undegraded due to deficiency of a specific enzyme in the catabolic pathway avoids the normal background, increasing sensitivity and specificity. We evaluated glycosaminoglycan content by dye-based and non-reducing end methods using urine, serum, and cerebrospinal fluid from MPS I human samples before and after treatment with intravenous recombinant human alpha-l-iduronidase. We found that both urine total glycosaminoglycans and serum heparan sulfate derived non-reducing end levels were markedly decreased compared to baseline after 26 weeks and 52 weeks of therapy, with a significantly greater percentage reduction in serum non-reducing end (89.8% at 26 weeks and 81.3% at 52 weeks) compared to urine total glycosaminoglycans (68.3% at 26 weeks and 62.4% at 52 weeks, p

Enzyme replacement therapy with laronidase (recombinant human alpha-l-iduronidase) is successfully used to treat patients with mucopolysaccharidosis type I (MPS I). However, the intravenously-administered enzyme is not expected to treat or prevent neurological deterioration. As MPS I patients suffer from spinal cord compression due in part to thickened spinal meninges, we undertook a phase I clinical trial of lumbar intrathecal laronidase in MPS I subjects age 8 years and older with symptomatic (primarily cervical) spinal cord compression. The study faced significant challenges, including a heterogeneous patient population, difficulty recruiting subjects despite an international collaborative effort, and an inability to include a placebo-controlled design due to ethical concerns. Nine serious adverse events occurred in the subjects. All subjects reported improvement in symptomatology and showed improved neurological examinations, but objective outcome measures did not demonstrate change. Despite limitations, we demonstrated the safety of this approach to treating neurological disease due to MPS I.

Niemann-Pick C, type 1 (NPC1) is a progressive autosomal recessive neurologic disease caused by defective intracellular cholesterol and lipid trafficking. There are currently no United States Food and Drug Administration approved treatments for NPC1. We undertook a study evaluating the safety, efficacy, and biomarker response of intrathecal 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) in a 12-year old subject with mildly symptomatic NPC. The subject received 200mg intrathecal HP-β-CD administered biweekly via lumbar puncture. To date the subject has received 27 intrathecal HP-β-CD injections. Intrathecal HP-β-CD has been generally safe and well tolerated in this subject. There has been an improvement in vertical gaze. The subject has developed subclinical hearing loss at high frequency that is likely HP-β-CD related. Plasma 24-(S)-hydroxycholesterol, a pharmacodynamic biomarker for cholesterol redistribution in the central nervous system, was significantly increased in response to each of the first 5 drug administrations. Further dosing as well as dose escalations are needed to more completely ascertain the safety and efficacy of intrathecal HP-β-CD.

Mucopolysaccharidosis type I (MPS I) is an inherited lysosomal storage disease that seriously affects the brain. Severity of neurocognitive symptoms in attenuated MPS subtype (MPS IA) broadly varies partially, due to restricted permeability of blood‐brain barrier (BBB) which limits treatment effects of intravenously applied α‐L‐iduronidase (rhIDU) enzyme. Intrathecal (IT) rhIDU application as a possible solution to circumvent BBB improved brain outcomes in canine models; therefore, our study quantifies effects of IT rhIDU on brain structure and function in an MPS IA patient with previous progressive cognitive decline. Neuropsychological testing and MRIs were performed twice prior (baseline, at 1 year) and twice after initiating IT rhIDU (at 2nd and 3rd years). The difference between pre‐ and post‐treatment means was evaluated as a percentage of the change. Neurocognitive performance improved particularly in memory tests and resulted in improved school performance after IT rhIDU treatment. White matter (WM) integrity improved together with an increase of WM and corpus callosum volumes. Hippocampal and gray matter volume decreased which may either parallel reduction of glycosaminoglycan storage or reflect typical longitudinal brain changes in early adulthood. In conclusion, our outcomes suggest neurological benefits of IT rhIDU compared to the intravenous administration on brain structure and function in a single MPS IA patient. © 2017 The Authors. American Journal of Medical Genetics Part A Published by Wiley Periodicals, Inc.

Intrathecal enzyme replacement therapy has been proposed to treat central nervous system (CNS) disease due to mucopolysaccharidosis type I. Our research has shown that repeated injections of recombinant enzyme into the spinal fluid corrects enzyme deficiency and normalizes lysosomal storage in the canine model. The challenge is to translate the success in the animal where there are fewer study limitations to human patients where studies are more restricted. This review will explore what is known about the measurement of clinically-relevant outcomes of intrathecal enzyme replacement therapy for MPS I (including ongoing clinical trials), the challenges in translating therapies for the CNS in rare diseases, and new outcome measures that could aid translation of CNS therapies for MPS disorders.

Intrathecal (IT) recombinant human α-L-iduronidase (rhIDU) has been shown to reduce mean brain glycosaminoglycans (GAGs) to normal levels in MPS I dogs. In this study, we examined storage in neuroanatomical regions of the MPS I dog brain, including frontal lobe, cerebellum, basal ganglia, thalamus, hippocampal formation, and brainstem, to determine the response of these functional regions to treatment with IT rhIDU. GAG storage in untreated MPS I dogs was significantly different from normal dogs in all examined sections. GAG levels in normal dogs varied by region: frontal lobe (mean 2.36 ± 0.54 µg/mg protein), cerebellum (2.67 ± 0.33), basal ganglia and thalamus (3.51 ± 0.60), hippocampus (3.30 ± 0.40), and brainstem (3.73 ± 1.10). Following intrathecal treatment, there was a reduction in GAG storage in each region in all treatment groups, except for the brainstem. Percent reduction in GAG levels from untreated to treated MPS I dogs in the deeper regions of the brain was 30% for basal ganglia and thalamus and 30% for hippocampus, and storage reduction was greater in superficial regions, with 61% reduction in the frontal lobe and 54% in the cerebellum compared to untreated MPS I dogs. Secondary lipid storage in neurons was also reduced in frontal lobe, but not in the other brain regions examined. Response to therapy appeared to be greater in more superficial regions of the brain, particularly in the frontal lobe cortex.

OBJECT Automated eye movement tracking may provide clues to nervous system function at many levels. Spatial calibration of the eye tracking device requires the subject to have relatively intact ocular motility that implies function of cranial nerves (CNs) III (oculomotor), IV (trochlear), and VI (abducent) and their associated nuclei, along with the multiple regions of the brain imparting cognition and volition. The authors have developed a technique for eye tracking that uses temporal rather than spatial calibration, enabling detection of impaired ability to move the pupil relative to normal (neurologically healthy) control volunteers. This work was performed to demonstrate that this technique may detect CN palsies related to brain compression and to provide insight into how the technique may be of value for evaluating neuropathological conditions associated with CN palsy, such as hydrocephalus or acute mass effect. METHODS The authors recorded subjects' eye movements by using an Eyelink 1000 eye tracker sampling at 500 Hz over 200 seconds while the subject viewed a music video playing inside an aperture on a computer monitor. The aperture moved in a rectangular pattern over a fixed time period. This technique was used to assess ocular motility in 157 neurologically healthy control subjects and 12 patients with either clinical CN III or VI palsy confirmed by neuro-ophthalmological examination, or surgically treatable pathological conditions potentially impacting these nerves. The authors compared the ratio of vertical to horizontal eye movement (height/width defined as aspect ratio) in normal and test subjects. RESULTS In 157 normal controls, the aspect ratio (height/width) for the left eye had a mean value ± SD of 1.0117 ± 0.0706. For the right eye, the aspect ratio had a mean of 1.0077 ± 0.0679 in these 157 subjects. There was no difference between sexes or ages. A patient with known CN VI palsy had a significantly increased aspect ratio (1.39), whereas 2 patients with known CN III palsy had significantly decreased ratios of 0.19 and 0.06, respectively. Three patients with surgically treatable pathological conditions impacting CN VI, such as infratentorial mass effect or hydrocephalus, had significantly increased ratios (1.84, 1.44, and 1.34, respectively) relative to normal controls, and 6 patients with supratentorial mass effect had significantly decreased ratios (0.27, 0.53, 0.62, 0.45, 0.49, and 0.41, respectively). These alterations in eye tracking all reverted to normal ranges after surgical treatment of underlying pathological conditions in these 9 neurosurgical cases. CONCLUSIONS This proof of concept series of cases suggests that the use of eye tracking to detect CN palsy while the patient watches television or its equivalent represents a new capacity for this technology. It may provide a new tool for the assessment of multiple CNS functions that can potentially be useful in the assessment of awake patients with elevated intracranial pressure from hydrocephalus or trauma.

Inherited genetic deficiency of lysosomal acid alpha glucosidase or acid maltase (GAA) results in the autosomal recessive glycogen storage disease type II (GSD II). To investigate whether we could generate a functional recombinant human GAA (rhGAA) for enzyme replacement therapy, we subcloned the cDNAs for human GAA and mouse dihydrofolate reductase (DHFR) into DHFRneg Chinese hamster ovary cells and established a stable cotransformant that expressed rhGAA. We cultured the recombinant cells in media with progressively increasing concentrations of methotrexate and found that human GAA enzyme activity increased to over 2,000 IU per gram protein. Importantly, the human GAA enzyme activity correlated to equivalent amounts of human GAA protein by rocketimmunoelectrophoresis. We confirmed that the human GAA enzyme activity corresponded to an amplification in human GAA mRNA by Northern analysis and human GAA cDNA copy number by Southern analysis. Exposing the rhGAA to human GSDII fibroblast cells or patient's lymphocytes or monocytes resulted in uptake of the rhGAA and reversal of the enzymatic defect. Mannose-6-phosphate in the media blocked uptake. GAA −/− mice were treated with the rhGAA at 1 mg/kg, which resulted in heterozygous levels of GAA in tissues, most notably skeletal muscle, heart and diaphragm after two infusions. More importantly, after multiple infusions, hind, and fore-limb muscle weakness was reversed. This rhGAA would be ideal for enzyme replacement therapy in GSD II.

CD23, the low affinity receptor for IgE (FcvarepsilonRII), is involved in regulation of IgE synthesis by B-lymphocytes. Five monoclonal antibodies to human CD23 were generated from cynomolgus macaques immunized with purified soluble CD23 (sCD23). Four of the five primate antibodies blocked the binding of IgE complexes to CD23 positive cells and also inhibited the production of IgE in vitro by IL-4 induced human peripheral blood mononuclear cells (PBMC). The variable domains of several primate antibodies were utilized to construct chimeric macaque/human (PRIMATIZED((R))) monoclonal antibodies. PRIMATIZED((R)) p5E8G1, containing human gamma 1 constant region, inhibited IgE production in vitro as efficiently as the parent primate antibody, but the human gamma 4 constant version, PRIMATIZED((R)) p5E8G4, was not as effective in IgE inhibition. An F(ab')(2) of p5E8G1 did not inhibit IgE production but did interfere with IgE inhibition by the intact anti-CD23 antibody in a dose dependent fashion. The murine monoclonal antibody MHM6 recognizes human CD23 at a different epitope than primate antibody 5E8, and inhibits IgE production by IL-4 induced PBMC. As with the F(ab')(2) of p5E8G1, the F(ab')(2) of MHM6 also failed to inhibit IgE production. These data imply that the mechanism by which anti-CD23 antibodies inhibit IgE production requires cross-linking of CD23 to an IgG receptor. These data also imply that neither bivalent cross-linking of CD23 alone or inhibition of CD23 binding to its natural ligands is sufficient to inhibit IgE production.

BackgroundIntrathecal (IT) enzyme replacement therapy with recombinant human α-L-iduronidase (rhIDU) has been studied to treat glycosaminoglycan storage in the central nervous system of mucopolysaccharidosis (MPS) I dogs and is currently being studied in MPS I patients.MethodsWe studied the immune response to IT rhIDU in MPS I subjects with spinal cord compression who had been previously treated with intravenous rhIDU. We measured the concentrations of specific antibodies and cytokines in serum and cerebrospinal fluid (CSF) collected before monthly IT rhIDU infusions and compared the serologic findings with clinical adverse event (AE) reports to establish temporal correlations with clinical symptoms.ResultsFive MPS I subjects participating in IT rhIDU trials were studied. One subject with symptomatic spinal cord compression had evidence of an inflammatory response with CSF leukocytosis, elevated interleukin-5, and elevated immunoglobulin G. This subject also complained of lower back pain and buttock paresthesias temporally correlated with serologic abnormalities. Clinical symptoms were managed with oral medication, and serologic abnormalities were resolved, although this subject withdrew from the trial to have spinal decompressive surgery.ConclusionIT rhIDU was generally well tolerated in the subjects studied, although one subject had moderate to severe clinical symptoms and serologic abnormalities consistent with an immune response.

We identified the presumably rare event of de novo mutation in an autosomal recessive disorder, glycogen storage disease type II (GSDII). GSDII results from inherited deficiency of acid alpha-glucosidase (acid maltase) and both the expressed and structural gene (designated GAA) have been isolated. The mutation was a deletion of 13 nt of coding sequence (delta nt 1456-1468) on the paternally derived allele of the proband. The delta nt 1456-1468 results in a reading frameshift and a premature termination signal upstream of the enzyme catalytic site. Paternity was confirmed by presence of two downstream, uncommon amino acid substitutions (E689K, W746C) in both proband and father and by comparison of nine short tandem repeats. The maternal allele carried a newly identified deleterious C647W missense mutation in a highly conserved area of the protein. The C647W mutation was also found in a second unrelated proband, heteroallelic with a deletion extending from IVS17 to IVS18.

The major purpose of this investigation was to ascertain through the use of confirmatory maximum likelihood factor analysis (CM-LFA) (Joreskog and Sorbom, 1989b) how accurately each of several hypothesized combinations of first-order and higher-order factors reflecting creativity in social intelligence for a sample of 192 high school students in a middle-class suburban community could describe the covariation within selected submatrixes taken from the total correlation matrix originally analyzed by Hendricks, Guilford, and Hoepfner (1969). Exploratory factor analyses involving both orthogonal (varimax) and oblique (promax) solutions were also completed and compared with the original orthogonal factor analysis of the total correlation matrix by Hendricks et al. The exploratory factor analyses showed a moderate degree of correspondence with the solution offered by Hendricks et al. It was concluded that CMLFA lent support to hypothesized first- and higher-order factors within the context of Guilford's structure-of-intellect model.

Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disease caused by loss of activity of α-l-iduronidase and attendant accumulation of the glycosaminoglycans dermatan sulfate and heparan sulfate. Current treatments are suboptimal and do not address residual disease including corneal clouding, skeletal deformities, valvular heart disease, and cognitive impairment. We treated neonatal dogs with MPS I with intravenous recombinant α-l-iduronidase replacement therapy at the conventional 0.58 mg/kg or a higher 1.57 mg/kg weekly dose for 56 to 81 weeks. In contrast to previous results in animals and patients treated at a later age, the dogs failed to mount an antibody response to enzyme therapy, consistent with the induction of immune tolerance in neonates. The higher dose of enzyme led to complete normalization of lysosomal storage in the liver, spleen, lung, kidney, synovium, and myocardium, as well as in the hard-to-treat mitral valve. Cardiac biochemistry and function were restored, and there were improvements in skeletal disease as shown by clinical and radiographic assessments. Glycosaminoglycan levels in the brain were normalized after intravenous enzyme therapy, in the presence or absence of intrathecal administration of recombinant α-l-iduronidase. Histopathological evidence of glycosaminoglycan storage in the brain was ameliorated with the higher-dose intravenous therapy and was further improved by combining intravenous and intrathecal therapy. These findings argue that neonatal testing and early treatment of patients with MPS I may more effectively treat this disease.

Genetic deficiency of acid α-glucosidase (GAA) results in glycogen storage disease type II. To investigate whether we could generate a functional recombinant human GAA protein for future enzyme replacement therapy, we subcloned the GAA cDNA into the bacterial expression plasmid pMaI and analyzed the recombinant protein produced. This nonglycosylated recombinant human GAA was found to be antigenic by reacting with polyclonal rabbit antibody to human placental GAA using ELISA and Western techniques. However, the protein was not enzymatically active, suggesting that glycosylation may play a role in enzymatic function.

Autosomal recessive deficiency of lysosomal acid maltase (GAA) or glycogen storage disease type II (GSDII) results in a spectrum of phenotypes including a rapidly fatal infantile disorder (Pompe's), juvenile, and a late-onset adult myopathy. The infantile onset form presents as hypotonia with massive accumulation of glycogen in skeletal and heart muscle, with death due to cardiorespiratory failure. Adult patients with the slowly progressive form develop severe skeletal muscle weakness and respiratory failure. Particle bombardment is a safe, efficient physical method in which high-density, subcellular-sized particles are accelerated to high velocity to carry DNA into cells. Because it does not depend on a specific ligand, receptor, or biochemical features on cell surfaces, particle-mediated gene transfer can be readily applied to a variety of systems. We evaluated particle bombardment as a delivery system for therapy of GSDII. We utilized a vector carrying the CMV promoter linked to the human GAA cDNA. Human GSDII cell lines (fibroblasts and lymphoid) as well as ex vivo with adult-onset peripheral blood cells (lymphocytes and monocytes) were transiently transfected by bombardment with a Helios gene gun delivering gold particles coated with the GAA expression plasmid. All cell types showed an increase in human GAA activity greater than 50% of normal activity. Subsequently, GAA -/- mice were treated every 2 weeks for 4 months by particle bombardment to the epidermis of the lower back and hind limbs. Muscle weakness in the hind and forelimbs was reversed. These data suggest that particle delivery of the GAA cDNA by the Helios gene gun may be a safe, effective treatment for GSDII.

Lipoid granulornatosis (Erdheim-Chester disease) is a rare but distinct form of histiocytosis. This disease has characteristic radiologic findings involving the musculoskeletal system that are critical to the diagnosis: symmetric sclerosis of the metaphysis and diaphysis of long bones with relative sparing of the epiphysis as depicted on conventional radiography. However, it is a systemic disease that involves multiple organ systems. This pictorial essay is of a single patient imaged over multiple years, using various pulse sequences with both low and high field strength MR scanners. It depicts many of the characteristic findings encountered in this rare systemic disorder.

Infantile patients with acid maltase deficiency have severe hypertrophic cardiomyopathy, left ventricular outflow obstruction, and generalized muscle weakness and die before 1 year of age. We identified 12 infants with acid maltase deficiency who had a similar clinical presentation but less severe cardiomyopathy and absence of left ventricular outflow obstruction, and 9 of 12 had longer survival with assisted ventilation and supplemental intubation.

Two newly identified splice site mutations (IVS1 -13T-->G and IVS10 +1GT-->CT) were found in a patient with adult onset of the autosomal recessive disorder glycogen storage disease type II (GSDII). The IVS1 -13T-->G transversion in the acceptor splice site was found on one allele in over two thirds of adult onset GSDII patients studied (28/41), but was not seen in 58 normal or 12 infantile onset GSDII chromosomes. Molecular analysis of cDNA from the index patient and four additional, ethnically different, individuals carrying the IVS1 -13T-->G transversion showed splicing out of the first coding exon as well as rare utilization of a cryptic splice site in the exon. An IVS10 +1GT-->CT transversion, unique to the index patient, was detected on the second chromosome. The IVS10 +1GT-->CT results in splicing out of exon 10 including part of the enzyme catalytic site. Additionally, a large deletion encompassing exon 18, previously described in four unrelated patients, was also detected in three unrelated adult GSDII patients, two of whom carried the IVS1 -13T-->G transversion. The frequency of the IVS1 splice site mutation suggests that detection of this mutation could potentially aid in the diagnosis of the phenotypically variable syndrome of adult onset GSDII. The finding that the -13T-->G mutation is a very common mutation in adult onset GSDII patients of varying ethnic and racial backgrounds, suggests that it is either an ancient mutation or confers a selective advantage. Although to our knowledge these are the first splice site mutations to be reported for GSDII, additional splice site mutations are likely and could provide the basis for later onset disease in GSDII.

Murine monoclonal antibodies (mAbs) were raised against human, polyclonal, anti-gp120 antibodies (Ab1) and were selected for binding to broadly neutralizing anti-gp120 antibodies in sera positive for human immunodeficiency virus (HIV). One anti-idiotype mAb (Ab2), 3C9, was found to be specific for human anti-gp120 antibodies directed against an epitope around the conserved CD4 attachment site of gp120. The 3C9 reactive human anti-gp120 antibodies (3C9+ Ab) neutralized MN, IIIB, RF, and four primary isolates of HIV type 1 (HIV-1). Cynomolgus monkeys were immunized with 3C9 in adjuvant to test whether this anti-idiotype mAb could induce neutralizing anti-gp120 antibodies. The results show that purified anti-anti-idiotype antibodies (Ab3) from 3C9 immune sera bind to an epitope around the CD4 attachment site of gp120SF and gp120IIIB. Furthermore, purified gp120-specific Ab3 neutralize MN, IIIB, and RF isolates. These results demonstrate that primates immunized with an anti-idiotype mAb produce broadly neutralizing anti-HIV-1 antibodies. Since this anti-idiotype mAb was selected by identifying a clonotypic marker, its biological activity can be explained as the results of clonotypic B-cell stimulation.