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Two cationic derivatives of γ-cyclodextrin (GCD) were synthesized by functionalization with glycidyltrimethylammonium chloride (GTMAC) and ethylenediamine (EDA). Both these derivatives (GCD-GTMAC and GCD-EDA) have been shown to interact strongly with anionic biopolymers, unfractionated heparin (UFH) and mucin, the latter showing their mucoadhesive properties. They form inclusion complexes with daidzein (DAI), an isoflavone displaying a multitude of physiological effects, much more efficiently than the unmodified GCD. It was also shown that the complexes of these GCD derivatives with DAI and Nile Red penetrate human fibroblasts and murine hippocampal neuronal cells indicating that cationic GCD derivatives can be considered as potential delivery systems for isoflavones and other poorly water soluble compounds. Moreover, it was found that DAI delivered in cationic GCD complexes decreased the level of the cellular glycosaminoglycans (GAGs) in normal fibroblasts suggesting their possible application in the control of GAGs in mucopolysaccharidoses, lysosomal storage diseases caused by pathological accumulation of GAGs in the cells., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: The mucopolysaccharidoses (MPSs) are rare genetic disorders caused by mutations in lysosomal enzymes involved in the degradation of glycosaminoglycans (GAGs). In this study, we analyzed a total of 48 patients including MPSI (n=6), MPSII (n=18), MPSIIIA (n=11), MPSIVA (n=3), and MPSVI (n=10)., Methods: In MPS patients, urinary GAGs were colorimetrically assayed. Enzyme activity was quantified by colorimetric and fluorimetric assays. To find mutations, all IDUA, IDS, SGSH, GALNS, and ARSB exons and intronic flanks were sequenced. New mutations were functionally assessed by reconstructing mutant alleles with site-directed mutagenesis followed with expression of wild-type and mutant genetic variants in CHO cells, measuring enzymatic activity, and Western blot analysis of protein expression of normal and mutated enzymes in cell lysates., Results: A total of five novel mutations were found including p.Asn348Lys (IDUA) in MPSI, p.Tyr240Cys (GALNS) in MPSIVA, and three ARSB mutations (p.Gln110*, p.Asn262Lysfs*14, and pArg315*) in MPSVI patients. In case of mutations p.Asn348Lys, p.Asn262Lysfs*14, and p.Gln110*, no mutant protein was detected while activity of the mutant protein was <1% of that of the normal enzyme. For p.Tyr240Cys, a trace of mutant protein was observed with a remnant activity of 3.6% of the wild-type GALNS activity. For pArg315*, a truncated 30-kDa protein that had 7.9% of activity of the normal ARSB was detected., Conclusions: These data further enrich our knowledge of the genetic background of MPSs., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Cardiovascular lesions, including coronary artery stenosis, are frequently associated and can cause sudden death in patients with genetic defects of glycosaminoglycan (GAG) metabolism. Early diagnosis of coronary artery lesions is difficult, although potentially lifesaving. Histopathological similarities between atherosclerotic changes in adults and in patients with genetic GAG metabolism defects have been known. Atherosclerosis is the result of a complex process involving metabolism of GAGs and proteoglycans preceded by endothelial dysfunction as a key event. Decreased nitric oxide (NO) bioavailability is considered the hallmark of endothelial dysfunction. Reduced NO synthase (NOS) has been reported in atherosclerotic arteries. Impairment in reactive hyperemia-digital peripheral arterial tonometry (RH-PAT) with EndoPAT has been validated to correlate coronary microvascular function in patients with atherosclerosis. RH-PAT is thought to reflect endothelial NO production. Immunohistological staining of endothelial NOS was performed in the stenotic lesions in the coronary artery of a 3-year-old patient with Mucopolysaccharidosis-I, showing decreased activities. This prompted a study to measure endothelial function in patients with GAG metabolism defects for early diagnosis of endothelial dysfunction in the coronary arteries as an early sign of coronary artery changes. Evaluation by RH-PAT in 30 patients with variable genetic defects in GAG metabolism revealed significantly decreased Reactive Hyperemia Indexes compared with 12 controls. Evaluation of endothelial function with RH-PAT in patients with GAG metabolism defects may detect coronary artery lesions that can be underdiagnosed by the other measures such as coronary angiography. Use of this method may prove vital in the management of patients with GAG metabolism defects.

Background: Mucopolysaccharidoses (MPSs) are rare genetic diseases caused by a deficient activity of one of the lysosomal enzymes involved in the glycosaminoglycan (GAG) breakdown pathway. These metabolic blocks lead to the accumulation of GAGs in various organs and tissues, resulting in a multisystemic clinical picture. The pathological GAG accumulation begins a cascade of interrelated responses: metabolic, inflammatory and immunological with systemic effects. Metabolic inflammation, secondary to GAG storage, is a significant cause of osteoarticular symptoms in MPS disorders., Objective and Method: The aim of this review is to present recent progress in the understanding of the role of inflammatory and immune processes in the pathophysiology of osteoarticular symptoms in MPS disorders and potential therapeutic interventions based on published reports in MPS patients and studies in animal models., Results and Conclusions: The immune and skeletal systems have a number of shared regulatory molecules and many relationships between bone disorders and aberrant immune responses in MPS can be explained by osteoimmunology. The treatment options currently available are not sufficiently effective in the prevention, inhibition and treatment of osteoarticular symptoms in MPS disease. A lot can be learnt from interactions between skeletal and immune systems in autoimmune diseases such as rheumatoid arthritis (RA) and similarities between RA and MPS point to the possibility of using the experience with RA in the treatment of MPS in the future. The use of different anti-inflammatory drugs requires further study, but it seems to be an important direction for new therapeutic options for MPS patients., (Copyright © 2013 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Mucopolysaccharidoses (MPS) are a group of lysosomal storage diseases caused by mutations in lysosomal enzymes involved in degradation of glycosaminoglycans (GAGs). Patients with MPS grow poorly and become physically disabled due to systemic bone disease. While many of the major skeletal effects in mouse models for MPS have been described, no detailed analysis that compares GAGs levels and characteristics of bone by micro-CT has been done. The aims of this study were to assess severity of bone dysplasia among four MPS mouse models (MPS I, IIIA, IVA and VII), to determine the relationship between severity of bone dysplasia and serum keratan sulfate (KS) and heparan sulfate (HS) levels in those models, and to explore the mechanism of KS elevation in MPS I, IIIA, and VII mouse models. Clinically, MPS VII mice had the most severe bone pathology; however, MPS I and IVA mice also showed skeletal pathology. MPS I and VII mice showed severe bone dysplasia, higher bone mineral density, narrowed spinal canal, and shorter sclerotic bones by micro-CT and radiographs. Serum KS and HS levels were elevated in MPS I, IIIA, and VII mice. Severity of skeletal disease displayed by micro-CT, radiographs and histopathology correlated with the level of KS elevation. We showed that elevated HS levels in MPS mouse models could inhibit N-acetylgalactosamine-6-sulfate sulfatase enzyme. These studies suggest that KS could be released from chondrocytes affected by accumulation of other GAGs and that KS could be useful as a biomarker for severity of bone dysplasia in MPS disorders.

MPS disorders result from a deficiency or absence of glycosaminoglycan (GAG) degrading enzymes leading to an imbalance between the synthesis and degradation of GAGs and their subsequent accumulation in a range of cells. The inhibition of GAG synthesis using small chemical inhibitors has been proposed as a novel therapeutic approach to treatment. Several inhibitors have been shown to decrease heparan sulphate GAG synthesis and in this study we evaluated a novel fluorinated analog of N-acetylglucosamine (2-acetamido-1,3,6-tri-O-acetyl-4-deoxy-4-fluoro-D-glucopyranose (F-GlcNAc)) and rhodamine B for their ability to also inhibit the synthesis of chondroitin/dermatan and keratan sulphate GAGs present in bovine cartilage. Both inhibitors decreased GAG synthesis in chondrocyte monolayer culture and in cartilage chip explant culture in a dose dependent manner. Both inhibitors decreased the size of newly synthesised proteoglycans and in the case of F-GlcNAc this was due to a decrease in newly synthesised GAG chain size. Rhodamine B, however, did not affect GAG chain size, while both inhibitors decreased the amount of chondroitin/dermatan and keratan sulphate GAG equally. The expression of genes responsible for the initiation and elongation of chondroitin/dermatan sulphate and keratan sulphate GAGs were downregulated in the presence of rhodamine B but not in the presence of F-GlcNAc. Thus the 2 inhibitors appear to have differing effects on GAG synthesis, with F-GlcNAc inhibiting the epimerisation of UDP-GlcNAc to UDP-GalNAc thus decreasing the availability of monosaccharides for addition to the growing GAG chain, whereas rhodamine B is more likely to reduce the number of GAG chains. Together with previous data these 2 inhibitors are capable of non-specific inhibition of GAG synthesis, reducing the production of chondroitin/dermatan sulphate, keratan sulphate and heparan sulphate GAGs. As such they would be applicable to therapy in a range of MPS disorders., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Lysosomal storage diseases (LSD) form a group of inherited metabolic disorders caused by dysfunction of one of the lysosomal proteins, resulting in the accumulation of certain compounds. Although these disorders are among first genetic diseases for which specific treatments were proposed, there are still serious unsolved problems that require development of novel therapeutic procedures. An example is neuronopathy, which develops in most of LSD and cannot be treated efficiently by currently approved therapies. Recently, a new potential therapy, called gene expression-targeted isoflavone therapy (GET IT), has been proposed for a group of LSD named mucopolysaccharidoses (MPS), in which storage of incompletely degraded glycosaminoglycans (GAGs) results in severe symptoms of virtually all tissues and organs, including central nervous system. The idea of this therapy is to inhibit synthesis of GAGs by modulating expression of genes coding for enzymes involved in synthesis of these compounds. Such a modulation is possible by using isoflavones, particularly genistein, which interfere with a signal transduction process necessary for stimulation of expression of certain genes. Results of in vitro experiments and studies on animal models indicated a high efficiency of GET IT, including correction of behavior of affected mice. However, clinical trials, performed with soy isoflavone extracts, revealed only limited efficacy. This caused a controversy about GET IT as a potential, effective treatment of patients suffering from MPS, especially neuronopathic forms of these diseases. It this critical review, I present possible molecular mechanisms of therapeutic action of isoflavones (particularly genistein) and suggest that efficacy of GET IT might be sufficiently high when using relatively high doses of synthetic genistein (which was employed in experiments on cell cultures and mouse models) rather than low doses of soy isoflavone extracts (which were used in clinical trials). This proposal can be tested in double-blinded, placebo-controlled clinical trials., (Copyright © 2012 Wiley Periodicals, Inc.)

It has been demonstrated recently that it is possible to decrease expression of genes coding for enzymes involved in synthesis of glycosaminoglycans (GAGs) by using specific siRNAs which interfere with stability of particular mRNAs. This procedure has been proposed as a potential treatment for patients suffering from mucopolysaccharidoses, a group of inherited metabolic diseases caused by dysfunction of enzymes required for GAG degradation, and resultant storage of these compounds in cells of affected persons. Here, we asked if the simultaneous use two species of specific siRNAs aimed at silencing two genes involved in particular steps of GAG synthesis may be more effective than the use of single siRNA. We found that inhibition of GAG synthesis in cells treated with two siRNAs is generally more effective than using single siRNAs. However, the differences were not statistically significant, therefore the potential benefit from the use of two siRNAs over the use of a single siRNA is doubtful in the light of the cost-benefit ratio and possibly stronger side-effects of the putative therapy.

Mucopolysaccharidoses (MPS) are severe inherited metabolic disorders caused by storage of glycosaminoglycans (GAGs). The level of accumulated GAGs is an important parameter in assessment of the severity of the disease and the efficacy of treatment; unfortunately, biochemical methods are often unreliable and only semi-quantitative. Therefore, finding other methods for estimation of GAG levels and/or assessment of the efficacy of applied therapy is very important. Although monitoring of GAG levels during therapy is crucial, in this work it is proposed that analysis of the ultrastructure of MPS cells by electron microscopic techniques can be considered as an alternative and reliable method for assessment of lysosomal storage. The number of complex lysosomal structures was found to be significantly higher in MPS cells relative to controls, while it decreased significantly in response to either enzyme replacement therapy or substrate reduction therapy. Thus, this parameter, easily assessed by electron microscopy, appears to correspond to the physiological state of MPS cells.

The mucopolysaccharidoses (MPSs) are a series of rare genetic disorders in which progressive bone and joint disease represents a key source of morbidity for patients. The recent introduction of enzyme replacement therapy for many of the MPSs has led to a need for increased physician awareness of these rare conditions in order to ensure that treatment is initiated at a time that leads to optimal benefit for patients. In addition, the current experiences of the clinical responsiveness of patient's symptoms to enzyme replacement approaches have also fuelled an interest in the development of alternative and adjunctive therapeutic approaches directed particularly to the rheumatological aspects of disease. Understanding the underlying pathogenesis of the MPSs is a key element for advancements in both of these areas. This review highlights the current knowledge underlying the pathophysiology of disease symptoms in the MPSs and underscores the importance and role of pathogenic cascades.

Mucopolysaccharidoses (MPS) are a group of lysosomal storage diseases that are resulted from abnormal accumulation of glycosaminoglycans. Among the progressive multi-organ abnormalities often associated with MPS diseases, the deterioration of central nervous system (CNS) is the most challenging manifestations to be tackled, due to the impermeability of the blood-brain-barrier (BBB). Evolved with recent development in stem cell biotechnology and gene therapy, several novel experimental approaches have been investigated in animal models. In this review, we will address different approaches attempting to bypass the BBB for neuropathic MPS treatment using cell- and gene-based therapies. Several neurological findings in CNS pathophysiology emerged with therapeutic investigation will also be discussed.

Mucopolysaccharidoses (MPS) are inherited metabolic diseases from the group of lysosomal storage disorders (LSD). They are caused by genetic defects resulting in the absence or severe deficiency in one of lysosmal hydrolases involved in degradation of glycosaminoglycans (GAG). Partially degraded GAGs are accumulated in lysosomes, causing dysfunction of cells, tissues and organs. Last years did bring some breakthrough discoveries, which were important to understand biochemical mechanisms of MPS appearance and course, as well as to develop therapeutic procedures for these inherited metabolic disorders.

Mucopolysaccharidoses (MPS) are inherited metabolic disorders from the group of lysosomal storage diseases (LSD). They arise from mutations causing dysfunction of one of enzymes involved in degradation of glycosaminoglycans (GAGs) in lysosomes. Impaired degradation of these compounds results in their accumulation in cells and dysfunction of most tissues and organs of patients. If heparan sulfate (HS) is the sole or one of stored GAGs, brain functions are also affected. However, despite the fact that products of incomplete degradation of the same chemical, HS, are accumulated in brains of patients suffering from Hurler disease (MPS type I), Hunter disease (MPS type II), Sanfilippo disease (MPS type III) and Sly disease (MPS type VII), and obvious deterioration of brain functions occur in these patients, their behavior is considerably different between various types of MPS. Here we asked the question about biochemical reasons of these differences. We performed theoretical analysis of products of incomplete HS degradation that accumulate in tissues of patients diagnosed for these diseases. A correlation between chemical structures of incompletely degraded HS and behaviors of patients suffering from particular MPS types was found. We propose a hypothesis that particular chemical moieties occurring at the ends of incompletely degraded HS molecules may determine characteristic behavioral disturbances, perhaps due to chemical reactions interfering with functions of neurons in the brain. A possible experimental testing of this hypothesis is also proposed. If the hypothesis is true, it might shed some new light on biochemical mechanisms of behavioral problems occurring not only in MPS but also in some other diseases., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Lysosomal storage diseases (LSD) are metabolic disorders characterized by accumulation of undegraded material. The mucopolysaccharidoses (MPS) are LSDs defined by the storage of glycosaminoglycans. Previously, we hypothesized that cells affected with LSD have increased energy expenditure for biosynthesis because of deficiencies of raw materials sequestered within the lysosome. Thus, LSDs can be characterized as diseases of deficiency as well as overabundance (lysosomal storage). In this study, metabolite analysis identified deficiencies in simple sugars, nucleotides, and lipids in the livers of MPSI mice. In contrast, most amino acids, amino acid derivatives, dipeptides, and urea were elevated. These data suggest that protein catabolism, perhaps because of increased autophagy, is at least partially fulfilling intermediary metabolism. Thus, maintaining glycosaminoglycan synthesis in the absence of recycled precursors results in major shifts in the energy utilization of the cells. A high fat diet increased simple sugars and some fats and lowered the apparent protein catabolism. Interestingly, autophagy, which is increased in several LSDs, is responsive to dietary intervention and is reduced in MPSVII and MPSI mice fed a high fat diet. Although long term dietary treatment improved body weight in MPSVII mice, it failed to improve life span or retinal function. In addition, the ventricular hypertrophy and proximal aorta dilation observed in MPSVII mice were unchanged by a high fat, simple sugar diet. As the mechanism of this energy imbalance is better understood, a more targeted nutrient approach may yet prove beneficial as an adjunct therapy to traditional approaches.

Aim: To develop a method for prediction of severity and clinical course of mucopolysaccharidoses (MPS), a group of inherited metabolic diseases., Methods: Various biochemical and clinical parameters (including estimation of the level of clinical severity, presence of specific mutations, residual enzyme activity, urinary glycosaminoglycan (GAG) excretion, storage of GAG in fibroblasts and efficiency of GAG synthesis) of patients suffering from MPS types II, IIIA and IIIB were determined. Correlations between genetic, biochemical and clinical parameters were tested., Results: We found that efficiency of GAG synthesis may contribute to the level of severity of MPS. It appears that (i) combination of low or average efficiency of GAG synthesis and the presence of residual activity of the enzyme is responsible for an attenuated phenotype, (ii) a lack of detectable residual enzyme activity causes a severe phenotype, irrespective of the efficiency of GAG synthesis and (iii) high efficiency of GAG synthesis leads to a severe phenotype, even if residual enzyme activity is detected. This correlation was found to be valid in 15 out of 17 patients tested., Conclusion: Analysis of efficiency of GAG synthesis and residual activity of the enzyme may be considered for prediction of severity of MPS patients' clinical phenotypes.

Background: Mucopolysaccharidoses (MPS) are inherited metabolic disorders caused by mutations leading to dysfunction of one of enzymes involved in degradation of glycosaminoglycans (GAGs). Due to their impaired degradation, GAGs accumulate in cells of patients, which results in dysfunction of tissues and organs. Substrate reduction therapy is one of potential treatment of these diseases. It was demonstrated previously that genistein (4', 5, 7-trihydroxyisoflavone) inhibits synthesis and reduces levels of GAGs in cultures of fibroblasts of MPS patients. Recent pilot clinical study indicated that such a therapy may be effective in MPS III (Sanfilippo syndrome)., Methods: To learn on details of the molecular mechanism of genistein-mediated inhibition of GAG synthesis, efficiency of this process was studied by measuring of incorporation of labeled sulfate, storage of GAGs in lysosomes was estimated by using electron microscopic techniques, and efficiency of phosphorylation of epidermal growth factor (EGF) receptor was determined by using an ELISA-based assay with fluorogenic substrates., Results: Effects of genistein on inhibition of GAG synthesis and accumulation in fibroblasts from patients suffering from various MPS types were abolished in the presence of an excess of EGF, and were partially reversed by an increased concentration of genistein. No such effects were observed when an excess of 17beta-estradiol was used instead of EGF. Moreover, EGF-mediated stimulation of phsophorylation of the EGF receptor was impaired in the presence of genistein in both wild-type and MPS fibroblasts., Conclusion: The results presented in this report indicate that the mechanism of genistein-mediated inhibition of GAG synthesis operates through epidermal growth factor (EGF)-dependent pathway.

Mucopolysaccharidosis type I (Hurler syndrome) is caused by a deficiency of the enzyme alpha-L-iduronidase (IDUA), and is characterized by widespread lysosomal glycosaminoglycan (GAG) accumulation. Successful treatment of central nervous system (CNS) diseases is limited by the presence of the blood-brain barrier, which prevents penetration of the therapeutic enzyme. Given that the brain capillary endothelial cells that form this barrier express high levels of the transferrin receptor (TfR), we hypothesized that the coupling of IDUA to transferrin (Tf) would facilitate IDUA delivery to the CNS. A plasmid bearing a fusion gene consisting of Tf and IDUA was constructed which, when delivered in vivo, resulted in the production of high levels of an enzymatically active protein that was transported into the CNS by TfR-mediated endocytosis. Short-term treatment resulted in a decrease in GAGs in the cerebellum of mucopolysaccharidosis type I (MPS I) mice. This approach, therefore, represents a potential strategy for the delivery of therapeutic enzyme to the CNS.

The mucopolysaccharidoses represent a devastating group of lysosomal storage diseases affecting approximately 1 in 25 000 individuals. Advances in biochemistry and genetics over the past 25 years have resulted in the identification of the key hydrolases underlying the mucopolysaccharidoses, with subsequent isolation and characterisation of the genes involved. Ultimately these advances have led to the recent development of specific treatment regimens for some of the mucopolysaccharidoses, in the form of direct enzyme replacement. Direct replacement of the defective gene product has been attempted for very few genetic disorders, and thus the experience gained in the lysosomal storage diseases by the development, evaluation and integration of treatment regimens into healthcare is instructive for other rare genetic disorders. This review focuses on the pathophysiology of the mucopolysaccharidoses and highlights the complex biochemical and physiological perturbations that underlie the disease phenotype.

We have previously shown that glycosaminoglycan (GAG) storage in animal models of the mucopolysaccharidoses (MPS) leads to inflammation and apoptosis within cartilage. We have now extended these findings to synovial tissue and further explored the mechanism underlying GAG-mediated disease. Analysis of MPS rats, cats, and/or dogs revealed that MPS synovial fibroblasts and fluid displayed elevated expression of numerous inflammatory molecules, including several proteins important for lipopolysaccharide signaling (eg, Toll-like receptor 4 and lipoprotein-binding protein). The expression of tumor necrosis factor, in particular, was elevated up to 50-fold, leading to up-regulation of the osteoclast survival factor, receptor activator of nuclear factor-kappaB ligand, and the appearance of multinucleated osteoclast-like cells in the MPS bone marrow. Treatment of normal synovial fibroblasts with GAGs also led to production of the prosurvival lipid sphingosine-1-phosphate, resulting in enhanced cell proliferation, consistent with the hyperplastic synovial tissue observed in MPS patients. In contrast, GAG treatment of normal chondrocytes led to production of the proapoptotic lipid ceramide, confirming the enhanced cell death we had previously observed in MPS cartilage. These findings have important implications for the pathogenesis and treatment of MPS and have further defined the mechanism of GAG-stimulated disease.

Mucopolysaccharidoses (MPS) are inherited, severe, progressive, metabolic disorders caused by deficiencies in different enzymes involved in degradation of glycosaminoglycans (GAGs). Although enzyme replacement therapy (ERT) has recently been available for MPS type I, and clinical trials have been performed in ERT for MPS II and MPS VI, there is little chance that this kind of treatment may be effective for neurodegenerative forms of MPS (due to inefficient delivery of enzymes to central nervous system through the blood-brain barrier), hence currently there is no effective therapy available for them. Therefore, we aim to develop an alternative therapy for these diseases. We found that genistein (4',5,7-trihydroxyisoflavone or 5,7-dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one) inhibits synthesis of GAGs considerably in cultures of fibroblasts of MPS patients (types I, II, IIIA and IIIB were tested). Prolonged cultivation of these cells in the presence of genistein resulted in reduction of GAG accumulation and normalization of cells as estimated by biochemical tests and electron microscopic analysis, respectively. As genistein inhibits kinase activity of epidermal growth factor receptor, which is required for full expression of genes coding for enzymes involved in GAG production, we propose to consider a substrate reduction therapy for MPS, which is referred to as 'gene expression-targeted isoflavone therapy'.

The 2A-like sequences from members of the picornavirus family were utilized to construct a tricistronic vector bearing the human iduronidase (IDUA) gene along with the firefly luciferase and DsRed2 reporter genes. The 2A-like sequences mediate a cotranslational cleavage event resulting in the release of each individual protein product. Efficient cleavage was observed and all three proteins were functional in vitro and in vivo, allowing for supratherapeutic IDUA enzyme levels and the coexpression of luciferase and DsRed2 expression, which enabled us to track gene expression.

Mucopolysaccharidosis type VI (MPS VI, Maroteaux-Lamy syndrome, McKusick #253200) is a lysosomal storage disorder that is caused by a deficiency in the lysosomal exohydrolase N-acetylgalactosamine-4-sulphatase (4-sulphatase, EC 3.1.6.1). We report a patient with no obvious clinical signs of MPS VI that has 5% of normal 4-sulphatase catalytic capacity. This patient represents an index case for the attenuated end of the MPS VI clinical spectrum.

The mucopolysaccharidoses (MPS) is characterized by accumulation of glycosaminoglycans (GAGs), and mucolipidosis (ML) by accumulation of GAGs and sphingolipids. Each type of MPS accumulates specific GAGs. The lysosomal enzymes N-acetylgalactosamine-6-sulphate sulphatase and beta-galactosidase involve the stepwise degradation of keratan sulphate (KS). Deficiency of these enzymes results in elevation of KS levels in the body fluids and in tissues, leading to MPS IV disease. In this study, we evaluated blood and urine KS levels in types of MPS and ML other than MPS IV. Eighty-five plasma samples came from MPS I (n = 18), MPS II (n = 28), MPS III (n = 20), MPS VI (n = 3), MPS VII (n = 5) and ML (n = 11) patients while 127 urine samples came from MPS I (n = 34), MPS II (n = 34), MPS III (n = 32), MPS VI (n = 7), MPS VII (n = 9) and ML (n = 11) patients. KS levels were determined using the ELISA method. Plasma KS levels varied with age in both control and patient populations. In all age groups, the mean values of plasma KS in MPS and ML patients were significantly higher than those in the age-matched controls. Plasma KS values in four newborn patients were above the mean + 2SD of the age-matched controls (mean, 41 ng/ml). Overall, 85.9% of individual values in non-type IV MPS and ML patients were above the mean + 2SD of the age-matched controls. For urine KS levels, 24.4% of individual values in patients were above the mean + 2SD of the age-matched controls. In conclusion, KS in blood is elevated in each type of non-type IV MPS examined, in contrast to the conventional understanding. This finding suggests that measurement of KS level provides a new diagnostic biomarker in a wide variety of mucopolysaccharidoses and mucolipidoses in addition to MPS IV.

The mucopolysaccharidoses (MPSs) are a complex family of lysosomal storage disorders characterized by failure to degrade heparan sulfate (HS) and/or other types of glycosaminoglycans (GAGs) secondary to the absence of specific lysosomal enzymes. An accompanying storage of glycosphingolipids (GSLs), most notably GM2 and GM3 gangliosides, has also been documented to occur in many types of MPS disease and is believed to be caused by secondary inhibition of GSL-degradative enzymes by intracellular GAG accumulation. We have documented the presence of secondary ganglioside accumulation in mouse models of several MPS disorders (types I, IIIA, IIIB, and VII) and report that this storage is accompanied by sequestration of free cholesterol in a manner similar to that observed in primary gangliosidoses. Using confocal microscopy, we evaluated the cellular distribution of cholesterol, GM2 and GM3 gangliosides, and HS in brains of mice with MPS IIIA disease. Unexpectedly, we found that although both gangliosides often accumulated in the same neurons, they were consistently located in separate populations of cytoplasmic vesicles. Additionally, GM3 ganglioside only partially co-localized with the primary storage material (HS), and cholesterol likewise only partially co-localized with the GM2 and GM3 gangliosides. These findings raise significant questions about the mechanism(s) responsible for secondary accumulation of storage materials in MPS disease. Furthermore, given that GSLs and cholesterol are constituents of membrane rafts believed critical in signal transduction events in neurons, their co-sequestration in individual neurons suggests the presence of defects in the composition, trafficking, and/or recycling of raft components and thus possible new mechanisms to explain neuronal dysfunction in MPS disorders., (Copyright 2004 Wiley-Liss, Inc.)

Lysosomal storage disorders represent a group of over 45 distinct genetic diseases, each one resulting from a deficiency of a particular lysosomal protein or, in a few cases, from non-lysosomal proteins that are involved in lysosomal biogenesis. A common biochemical feature of this group of disorders is the accumulation within lysosomes of undegraded or partially degraded substrates that are normally degraded within, and transported out of the lysosome. The particular substrates stored and the site(s) of storage vary with disease type and enzyme/protein deficiency. The nature of the substrate can be used to group the disorders into broad categories including the mucopolysaccharidoses, lipidoses, glycogenoses and oligosaccharidoses. These categories show many clinical similarities within groups as well as significant similarities between groups. For most lysosomal storage disorders the relationship between the stored substrates (type, amount and location) and the disease pathology is not well understood. The use of mass spectrometry and in particular tandem mass spectrometry provides a powerful tool for the investigation of stored substrates in this group of disorders. In this review we will describe the use of mass spectrometry for the analysis of stored substrates. We will discuss progress in the field, limitations of current methods, and summarise issues relating to the diagnosis and treatment of some of the more prevalent lysosomal storage disorders.

Studies were carried out to determine the changes in glycosaminnoglycan (GAG) metabolism in rats fed cassava with varying cyanoglucoside levels and two levels of protein. Results indicated that there was an enhancement in the level of total and individual GAG with a corresponding reduction in the activity of enzymes involved in the degradation of glycosaminoglycan. These changes were significant for rats given a cassava diet (raw and boiled cassava) and low protein. The changes in total and individual GAG and the decrease in the activity of degrading enzymes was more for high cyanide (raw cassava) groups compared with other groups showing that consumption of untreated cassava is an additive factor for the promotion of mucopolysaccharidosis.

Unlabelled: A series of dicationic amphiphilic drugs, most of them immunomodulatory agents, are known to induce generalised lysosomal storage of sulphated glycosaminoglycans (GAGs) in rats and in cultured cells of several species including man. The present study deals with the cytological effects of two experimental immunomodulatory acridine derivatives upon the retina of rats. The animals were treated orally with compound CL-90.100 (3,6-bis[2-(diethylamino)ethoxy]acridine) or an analogue for periods up to 22 weeks at a dose range of 60-90 mg/kg body weight and the retinae examined by light and electron microcopy. ERG measurements were done initially and after 16 weeks of treatment. All types of retinal cells developed abnormal cytoplasmic vacuoles which represented the ultrastructural counterpart of lysosomal GAG storage as demonstrated by histochemical and cytochemical staining experiments. The retinal pigment epithelium and the Müller cells were most prominently affected, photoreceptor cells to a lesser degree, and retinal neurons to varying degrees. The topographical distribution of the drug as detected by fluorescence microscopy closely resembled the distribution of the GAG accumulation in the retinal layers. After treatment for 16 weeks, the a-and b-wave amplitudes in the ERG were significantly reduced compared with the controls., Conclusion: the glycosaminoglycan storage in pigment epithelium is reminiscent of that seen in some inherited mucopolysaccharidoses of humans. When a given cell type shows lysosomal accumulation of glycosaminoglycans as a consequence of impaired degradation, it can be assumed to be engaged in the turnover of glycosaminoglycans under normal conditions. Thus the present results suggest that not only the retinal pigment epithelium but also Müller cells, photoreceptor cells, and, to variable degree, retinal neurons are normally involved in the catabolism of sulphated glycosaminoglycans. We believe that the lysosomal storage of glycosaminoglycans caused secondary cellular disturbance responsible for the functional changes shown by electroretinography.

A longitudinal study of cranial magnetic resonance imaging (MRI) was carried out in 23 patients with mucopolysaccharidoses (MPS); 1 each of types IH, VI, and VII; 2 of type IS; 10 of type II; and 4 each of types IIIB and IVA. Six types of distinct abnormalities were 1) cribriform changes or spotty changes in the corpus callosum, basal ganglia, and white matter; 2) high-intensity signal in the white matter on T2-weighted image; 3) ventriculomegaly; 4) diffuse cerebral cortical atrophy; 5) spinal cord compression; and 6) megacisterna magna. The cribriform changes that corresponded to dilated perivascular spaces were found in the patients with MPS IS, II, and VI. The patchy and diffuse intensity changes were found in the patient with MPS II and IIIB, respectively. MPS IH and the severe type of MPS II showed marked ventriculomegaly. Marked cerebral atrophy was observed in all MPS IIIB patients and in the severe type of MPS II patients. Spinal cord compression was a feature usually observed in MPS IH, IVA, VI, and VII. Megacisterna magna was frequent in the patients with MPS II (6/10). In two of five patients, the therapeutic effect of bone marrow transplantation (BMT) was remarkable. Both the cribriform changes and the intensity change of the white matter in a MPS VI patient disappeared eight years after the BMT. Slight improvement of cribriform change was noted in one patient with MPS II three years after the BMT. MRS was not sufficient to estimate the accumulation of glycosaminoglycans but was useful for evaluating neuronal damages.

The mucopolysaccharidosis are hereditary diseases. The neurological attack constitutes the principal factor of gravity of these affections. We conducted a retrospective study over a period of 12 years (1988-1999) in the pediatric department of Sfax University Hospital. This study allowed us to observe 11 cases of mucopolysaccharidosis confirmed by an enzymatic proportioning, with 3 cases of Hurler disease (IH), 3 cases of the disease of sanfilippo, (two II A and one III B), 3 cases of the disease of Morquio A (type IVA) and 2 cases of the disease of Maroteaux Lamy (type VI). A sex ratio of 1.75. The parents were cousins in 90% of the cases. The age of revelation ranged between 6 months to 4 years. The clinical examination has found a staturo-pondral delay in 81.8% of the cases, a craniofacial dysmorphy in 100%, deformations of the rachis in 63.6% of the cases, a psychomotor regression in 54.5% of the cases, a medullary compression in 18% of the cases, hepatosplenomegaly in 36.4%, and corneal opacities in 45.4% of the cases. The therapeutic treatment was limited to the symptomatic measures with genetic consulting and antenatal diagnosis.

In seven patients with mucopolysaccharidoses (1 Hurler, 1 Hurler-Scheie, 4 Hunter, 1 Sly), cranial (1)H-magnetic resonance spectroscopy was performed to evaluate the accumulation of mucopolysaccharides and biochemical changes in the CNS in vivo before and after bone marrow transplantation (BMT). In two of seven patients, (1)H-magnetic resonance spectroscopy was performed before and after BMT. Nuclear magnetic resonance spectra of dermatan sulfate and chondroitin sulfate-C and magnetic resonance spectroscopy of chondroitin sulfate-C and urine from patients with mucopolysaccharidoses showed resonance higher than the chemical shift of myoinositol in the brain (3.7 ppm). The resonance was considered to contain signals from mucopolysaccharide molecules. The resonance was measured as presumptive mucopolysaccharides (pMPS). In white matter lesions detected by magnetic resonance imaging, pMPS/creatine ratios and choline/creatine ratios were consistently higher than control ratios. In white matter without lesions, choline/creatine ratios were higher than control ratios. Patients with higher developmental quotient or intelligence quotient tended to show higher N:-acetylaspartate/creatine ratios and lower pMPS/creatine ratios in basal ganglia. After BMT, the pMPS/creatine ratio in white matter lesions of patient 3, with Hunter syndrome, was slightly decreased, but in none of the patients was the ratio ever below the control ratios, even 7 y after BMT. In white matter without lesions, the pMPS/creatine ratio in patient 3 was decreased to the control ratios after BMT, but although the choline/creatine ratios were gradually decreased, they remained higher than the control ratio, 2 y after BMT. These results suggest that evaluation of pMPS, choline, and N:-acetylaspartate by (1)H-magnetic resonance spectroscopy is an important technique that may provide useful biochemical information in vivo on the neurologic process and the efficacy of BMT in patients with mucopolysaccharidoses.

The energy needed by cardiac muscle to maintain proper function is supplied by adenosine Ariphosphate primarily (ATP) production through breakdown of fatty acids. Metabolic cardiomyopathies can be caused by disturbances in metabolism, for example diabetes mellitus, hypertrophy and heart failure or alcoholic cardiomyopathy. Deficiency in enzymes of the mitochondrial beta-oxidation show a varying degree of cardiac manifestation. Aberrations of mitochondrial DNA lead to a wide variety of cardiac disorders, without any obvious correlation between genotype and phenotype. A completely different pathogenetic model comprises cardiac manifestation of systemic metabolic diseases caused by deficiencies of various enzymes in a variety of metabolic pathways. Examples of these disorders are glycogen storage diseases (e.g. glycogenosis type II and III), lysosomal storage diseases (e.g. Niemann-Pick disease, Gaucher disease, I-cell disease, various types of mucopolysaccharidoses, GM1 gangliosidosis, galactosialidosis, carbohydrate-deficient glycoprotein syndromes and Sandhoff's disease). There are some systemic diseases which can also affect the heart, for example triosephosphate isomerase deficiency, hereditary haemochromatosis, CD 36 defect or propionic acidaemia.

To evaluate whether in vivo accumulations of heparan sulfate caused by inborn errors in the metabolism of glycosaminoglycans lead to the formation of neurofibrillary tangles and/or senile plaques, as seen in Alzheimer disease (AD), we studied postmortem brains from 9 patients, ages 1 to 42 years, with mucopolysaccharidosis (MPS). The brains of patients with Hurler's syndrome (MPS I: n = 5) and Sanfilippo's syndrome (MPS III; n = 4) as well as from caprine MPS IIID and murine MPS VII models were evaluated by thioflavine-S staining and by immunohistochemistry using antibodies directed against heparan sulfate proteoglycans, hyperphosphorylated tau, amyloid-beta peptide precursor proteins (APP), and amyloid-beta peptides (A beta [1-40], and A beta [1-42]). A two-site sandwich enzyme-linked immunosorbent assay (ELISA) was also utilized to compare levels of total soluble and insoluble A beta (1-40) and A beta (1-42) obtained from temporal cortex of MPS patients. Although no neurofibrillary tangles, senile plaques, or tau-positive lesions were detected in any of the MPS brains studied here, antibodies directed against A beta (1-40) intensely and diffusely stained the cytoplasm of cells throughout the brains of the MPS patients and the caprine MPS model. The ELISA assay also demonstrated a significant 3-fold increase in the level of soluble A beta (1-40) in the MPS brains compared with normal control brains. Thus, at least some of the metabolic defects that lead to accumulations of glycosaminoglycans in MPS also are associated with an increase in immunoreactive A beta (1-40) within the cytoplasmic compartment where they could contribute to the dysfunction and death of affected cells in these disorders, but not induce the formation of plaques and tangles. Models of MPS may enable mechanistic studies of the role A beta and glycosaminoglycans play in the amyloidosis that is a neuropathological feature of AD.

A combination of anion-exchange chromatography and 30-40% gradient polyacrylamide gel electrophoresis (gradient-PAGE) was used to purify and characterize urinary glycosaminoglycans from various mucopolysaccharidoses (MPS). The urinary glycosaminoglycans from the different MPS displayed distinct patterns on gradient-PAGE and further confirmation of MPS types and subtypes was demonstrated by an electrophoretic shift in the banding pattern after digestion with the appropriate MPS enzyme. Thus each of the MPS accumulates a unique spectrum of glycosaminoglycans with a nonreducing terminal consisting of the substrate specific for the deficient enzyme in that particular MPS disorder. The absolute correlation of the nonreducing terminal structure with a particular MPS and the availability of recombinant lysosomal enzymes provide the means for a rapid and accurate diagnosis of individual MPS. Analysis of tissue glycosaminoglycans in one MPS type (feline MPS VI) indicated a tissue-specific pattern of glycosaminoglycan accumulation. Undegraded glycosaminoglycans had distinct banding patterns on gradient-PAGE and although dermatan sulfate was predominantly excreted in MPS VI urine, some tissues were observed to accumulate predominantly chondroitin sulfate glycosaminoglycans, e.g., bone and kidney. The spectrum of glycosaminoglycans excreted in the urine is therefore most likely a combination of glycosaminoglycans from various tissues., (Copyright 1998 Academic Press.)

We present the imaging findings in a patient with mucopolysaccharidosis (MPS) type VI (Maroteaux-Lamy syndrome) who developed holocord syringomyelia. This represents the only reported case of syrinx formation in a child with MPS VI. Clinical, neurologic and spinal magnetic resonance imaging findings are presented. The patient has maintained a stable clinical and neurologic course over the period following allogeneic bone marrow transplant.

Several immunomomodulatory drugs, all of them symmetrically substituted dicationic amphiphilic compounds, are known to cause lysosomal storage of sulfated glycosaminoglycans (GAGs) in intact animals and cultured fibroblasts. The storage is due to impaired GAG degradation. The standard compound is tilorone (2,7-bis[2-(diethylamino)ethoxy]fluoren-9-one). In the present study two bis-aminomethyl anthrachinones were examined for their ability to induce lysosomal GAG storage in cultured bovine corneal fibroblasts. For reference, a bis-aminoethoxy-anthrachinone compound (RMI-10.024) was included, which is known to be a potent inducer of lysosomal GAG storage. The present morphological, radiochemical, and biochemical results show that the bis-aminomethyl anthrachinone compounds investigated cause lysosomal storage of GAGs, although with significantly lower potencies than the bis-aminoethoxy anthrachinone. Dermatan sulfate contributed approximately 90% to the drug-induced increment of intracellular GAGs. The present results suggest that the length of the side chains, i.e., the distance between the aromatic ring system and the protonizable nitrogen of the side chains, and the position of the side chains relative to the aromatic ring system are important molecular features influencing the potency of inducing lysosomal GAG storage.

This investigation deals with a drug side-effect. The immunomodulatory drug tilorone (2,7-bis[2-(diethylamino)ethoxy]fluoren-9-one) and congeners induce lysosomal storage of sulphated glycosaminoglycans (GAGs) in animals and in cultured cells. At high tilorone concentrations, GAG storage in cultured fibroblasts was previously reported to be accompanied, and presumably caused by, disturbance of intracellular targeting of lysosomal enzyme precursors, which leads to enhanced secretion and thus loss of lysosomal enzymes. The purpose of the present study was to examine whether the GAG storage induced in cultured bovine fibroblasts by low tilorone concentrations is also accompanied by enhanced lysosomal enzyme release. Enhanced secretion of beta-hexosaminidase (EC 3.2.1.52) was taken as indicating the intracellular mistargeting of lysosomal enzyme precursors. Dose-response curves were established for (a) the intracellular accumulation of 35S-GAGs and (b) the release of beta-hexosaminidase after exposure (72 hr) to tilorone (1-35 microM). For positive controls, the classical lysosomotropic agents NH4Cl (1-30 mM) and chloroquine (1-60 microM) were used. With NH4Cl, 35S-GAG storage was accompanied by enhanced enzyme release throughout the concentration range (EC50 at 3.3 mM for either effect). With chloroquine, low concentrations (< or = 5 microM) caused a small increase in 35S-GAG accumulation without abnormal enzyme secretion; at higher concentrations both drug effects were produced (EC50 around 15 microM for either effect). With tilorone, low concentrations (< or = 5 microM) caused marked 35S-GAG accumulation without enhancement of enzyme release. The EC50 for tilorone-induced 35S-GAG storage was 3 microM, as opposed to 15 microM for enzyme release. The results indicate that GAG storage induced by low concentrations of tilorone is due to mechanisms other than mistargeting and loss of lysosomal enzymes. On the basis of previous results it may be hypothesized that tilorone and other symmetrically substituted dicationic compounds form complexes with the polyanionic GAG chains and thereby impair their enzymic degradation.

The contents of total dolichol were measured in the cerebral cortex of various patients with lysosomal storage disorders, including mucopolysaccharidosis. Strikingly high levels of dolichol were demonstrated in GM1-gangliosides, Sanfilippo B syndrome, and a severe type of Hunter syndrome as well as neuronal ceroid-lipofuscinosis. An increased level of dolichol in cerebral cortex in neuronal ceroid-lipofuscinosis (NCL) was once regarded as pathognomonic for NCL. Our data, however, suggest that an increased level of dolichol in cerebral cortex is a nonspecific phenomenon related to some lysosomal dysfunction secondary to various neurodegenerative disorders.

Two weeks after a single injection of suramin, the secretory and post-secretory ameloblasts of the rat incisor were filled with large lysosome-like vacuoles. At the light-microscope level, these vacuoles were positively stained with Alcian blue when MgCl2 was used at a critical electrolyte concentration varying between 0.1 and 0.3 M, whereas no staining appeared when MgCl2 varied between 0.7 and 0.9 M. Hyaluronidase digestion markedly reduced but did not totally abolish the staining, indicating that glycosaminoglycans were accumulated inside these vacuoles. Examination of these cells with the electron microscope revealed a polymorphic population of large vesicles, filled to various degrees with cetylpyridinium chloride (CPC)-positive and malachite green aldehyde (MGA)-positive material. The same pattern was observed in secretory odontoblasts but to a lesser extent. In the extracellular matrix, suramin-induced alterations appeared as large defects occurring during enamel formation. In predentin and dentin, the number and/or size of electron-dense aggregates resulting from CPC and MGA fixation, were enhanced in the suramin-injected rats. These aggregates were largely reduced or suppressed respectively by hyaluronidase digestion and chloroform/methanol treatment of the sections. The accumulation of glycosaminoglycans and phospholipids reported here inside ameloblasts and odontoblasts and in predentin and dentin supports the occurrence of suramin-induced mucopolysaccharidosis and lipidosis in this experimental animal model.

Serial liver biopsies were obtained in 20 patients undergoing bone marrow transplantation (BMT) for mucopolysaccharidosis (MPS). The 13 patients with MPS I, one with MPS II, four with MPS III, and two with MPS VI underwent liver biopsy prior to and from 1 to 37 months after BMT. The amount of accumulated glycosaminoglycan (GAG) was assessed by semiquantitation of Kupffer cell staining with colloidal iron and by counting the number of hepatocellular GAG-containing lysosomes in electron micrographs. Eleven of 13 patients with MPS I achieved engraftment, and 10 of the 11 cleared the Kupffer cells and hepatocytes of GAG by 3 to 19 months post-BMT. Two patients with autologous recovery demonstrated persistent hepatocyte inclusions. The three patients with MPS II and MPS VI engrafted and showed clearance of hepatocyte and Kupffer cell GAG by 7 months after BMT. All four patients with MPS III engrafted. Although the Kupffer cells in these patients were cleared of GAG by 12 months after BMT, hepatocellular inclusions persisted in all four. For MPS I, II and VI, donor engraftment was associated with resolution of lysosomal storage material in donor-derived Kupffer cells and untransplanted hepatocytes, indicative of transcellular metabolic correction. Failure of hepatocyte clearance in one case of MPS I and all patients with MPS III suggested a diminished capacity of the graft-derived enzyme to enter the hepatocyte lysosomes in these patients.

Livers from normal cats and dogs, cats with mucopolysaccharidoses (MPS) I and VI, and dogs with MPS VII were analyzed biochemically and morphometrically to determine the lysosomal storage of glycosaminoglycans (GAG) in these animal models of human genetic disease. Analyses were performed on liver samples from seven normal cats ranging in age from 13 weeks to 15 months; six MPS I-affected cats ranging in age from 10 weeks to 26 months; four MPS VI-affected cats ranging in age from 9 months to 32 months; four normal dogs ranging in age from 1 month to 47 months; and three MPS VII-affected dogs, 5 days, 11 days, and 14 months of age. All of the animals were from the breeding colony at the University of Pennsylvania School of Veterinary Medicine and were maintained in accordance with national standards for the care and use of laboratory animals. Each GAG subclass was quantitated, and total GAG concentration was determined. Liver from cats with MPS I had the highest total GAG concentration (5.7 times that of the control), followed by liver from dogs with MPS VII (1.8 times) and cats with MPS VI (1.5 times). These data were very closely correlated (R2 = 0.982) with the results of the morphometric analyses of hepatocyte and Kupffer cell vacuolation associated with lysosomal storage and support the validity of both methods. This is particularly important for the quantification of total and individual GAG concentrations in tissue preparations. The values obtained should prove useful in future assessments of therapeutic regimes, such as enzyme replacement, bone marrow transplantation, and gene therapy, for these genetic diseases.

The gusmps/gusmps mouse has no beta-glucuronidase activity and develops murine mucopolysaccharidosis type VII (MPS VII). The clinical and pathologic abnormalities are similar to those found in humans with severe MPS VII. Mutant mice are dysmorphic, dwarfed, and have a shortened life span. Pathologic findings include widespread lysosomal storage. To determine whether bone marrow transplantation (BMT) corrects these abnormalities, genetically identical mutant animals were given syngeneic bone marrow transplants using cells from +/+ mice. Initial experiments showed that levels of beta-glucuronidase activity in recipient tissues correlated with the amount of radiation administered before BMT. Two groups of mice given BMT therapy were observed for periods of 1 and 2 years, respectively. These mice were evaluated using a combination of clinical, biochemical, histochemical, and pathologic analyses. Spleen, liver, cornea, and glomerular mesangial cells showed essentially complete correction at all radiation doses. Storage was partially corrected in meninges and perivascular cells in brain, and in renal tubular epithelial cells at the higher radiation doses. Life span in BMT-treated animals was increased approximately three-fold, approaching that seen in normal mice after BMT. These results support the position that BMT has a place in the therapeutic regimen for MPS VII.

Patients with certain systemic deficiencies in the degradation of glycosaminoglycans (GAGs) often suffer from a retinal degeneration similar to that seen in retinitis pigmentosa. This applies to mucopolysaccharidosis (MPS) types I, II, and III, but not to type VI. The retinal pigment epithelium (RPE) is thought to contribute significantly to the synthesis and degradation of proteoglycans in the interphotoreceptor matrix. This raises the possibility that a defect in the synthesis or degradation of GAGs by the RPE may be related to some forms of retinal degeneration. In the present work, RPE from normal and RP donors was investigated for the capacity to correct deficiencies in GAG degradation by cultured skin fibroblasts from patients with different forms of MPS. A cross-correction technique was used in which abnormal increases in the incorporation of 35S-sulfate into GAGs by MPS fibroblasts was measured in the absence or presence of RPE cultures. RPE from normal donors corrected the defects in GAG degradation of fibroblasts from patients with MPS I, II, and III, but not MPS VI. The RPE from four donors with retinitis pigmentosa (one autosomal dominant, one sex-linked, and two isolated cases) and one donor with an unclassified isolated retinal degeneration demonstrated the same capacities to correct the MPS deficiencies as did normal RPE. Therefore, although retinitis pigmentosa is a heterogeneous disorder with several possible etiologies, no evidence was found in these five patients for a defect in GAG degradation that resembles the deficiencies of MPS patients.

1. Drug accumulation without a concomitant elevation of blood level may occur if the capacity of the tissue to bind drug increases during chronic treatment. 2. This special type of accumulation is found with cationic-amphiphilic drugs, which induce the formation of lysosomal inclusion bodies containing undergraded lipids or mucopolysaccharides (drug-induced lipidosis or mucopolysaccharidosis, respectively); the stored material provides the additional binding sites for the drug. 3. Factors determining the potential for inducing lipidosis or mucopolysaccharidosis are: (a) affinity of the drugs to phospholipid layers (governed by hydrophobicity) or mucopolysaccharides (drug-induced lipidosis or mucopolysaccharidosis, respectively); the free intra-lysosomal concentration, which is elevated compared with the blood level due to lysosomal trapping (especially with dicationic drugs); (c) the therapeutically required drug concentration in the blood: the therapeutic concentrations are high with drugs that do not act via binding to specific high-affinity receptors.

A 68-year-old man and a 66-year-old woman had diffuse corneal stromal deposits that stained with alcian blue and colloidal iron but did not react with periodic acid-Schiff stain and lipid stains. Similar deposits were found within postmortem sclera in one case, but not in other ocular or extraocular tissues. The abnormal material was sensitive to testicular hyaluronidase and chondroitinase. The material reacted with monoclonal antibody 9-A-2 after digestion by chondroitinase AC in one case and ABC in both cases, which is consistent with the identification of the glycosaminoglycans chondroitin 4-sulfate and dermatan sulfate. Electron microscopic examination of the cornea in both cases disclosed granular material in vacuoles dispersed extracellularly and, rarely, in keratocytes. Results of blood and skin fibroblast enzyme assays for clinically relevant mucopolysaccharidoses and mucolipidoses were normal in both patients, and there were no somatic abnormalities suggesting a storage disease.

The turnover of proteoglycans in the extracellular matrix was studied in fibroblasts cultures derived from patients with mucopolysaccharidosis (MPS) and healthy donors. The cells were labelled with 35S-sulfate and 14C-glucosamine and it was found that in MPS-fibroblasts the rate of extracellular matrix turnover was hardly affected, where as the intracellular turnover was severely inhibited. Similar results were obtained with normal fibroblasts treated with 20 mM ammonia. Autoradiography revealed that MPS fibroblasts have a preferential accumulation of 35S-sulfate labelled material in the nuclear area, indicating that the nucleus may also be affected in MPS pathology. It is suggested that, although lysosomal enzymes are an important factor in intracellular proteoglycan turnover, they do not play a crucial role in the turnover of extracellular matrix proteoglycans.

The case is reported of a diabetic female aged 32 years with lichen myxedematosus. Skin biopsy showed enormous amounts of mast cells (59,000/mm3) and heavy deposition of acid mucopolysaccharides.