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Infantile globoid cell leukodystrophy (GLD, Krabbe disease) is a demyelinating disease caused by the deficiency of the lysosomal enzyme galactosylceramidase (GALC) and the progressive accumulation of the toxic metabolite psychosine. We showed previously that central nervous system (CNS)-directed, adeno-associated virus (AAV)2/5-mediated gene therapy synergized with bone marrow transplantation and substrate reduction therapy (SRT) to greatly increase therapeutic efficacy in the murine model of Krabbe disease (Twitcher). However, motor deficits remained largely refractory to treatment. In the current study, we replaced AAV2/5 with an AAV2/9 vector. This single change significantly improved several endpoints primarily associated with motor function. However, nearly all (14/16) of the combination-treated Twitcher mice and all (19/19) of the combination-treated wild-type mice developed hepatocellular carcinoma (HCC). 10 out of 10 tumors analyzed had AAV integrations within the Rian locus. Several animals had additional integrations within or near genes that regulate cell growth or death, are known or potential tumor suppressors, or are associated with poor prognosis in human HCC. Finally, the substrate reduction drug L-cycloserine significantly decreased the level of the pro-apoptotic ceramide 18:0. These data demonstrate the value of AAV-based combination therapy for Krabbe disease. However, they also suggest that other therapies or co-morbidities must be taken into account before AAV-mediated gene therapy is considered for human therapeutic trials. more...

Obesity is a risk factor for complications in singleton and twin pregnancies; however, there are limited data regarding maternal body mass index (BMI) in the setting of twin-twin transfusion syndrome (TTTS). We hypothesized that increased BMI in TTTS is associated with adverse perinatal outcomes and vascular pathology. A retrospective study of twin reversed arterial perfusion (n = 4), selective intrauterine growth restriction (n = 10) and TTTS (n = 33) was conducted. Treatment included fetoscopic laser photocoagulation (FLP) (n = 35) or Solomon technique (n = 12). Ex vivo placental intravascular injections, immunohistochemistry, and perinatal outcomes were compared by maternal BMI. In pregnancy complicated by TTTS, 16/33 women were obese (BMI > 30 kg/m2) and 11/33 were overweight (BMI 25–29.9 kg/m2). Women who were overweight or obese had an increased rate of premature rupture of membranes (PPROM), cesarean delivery, and/or concomitant co-morbidities when compared to the normal weight group. Duration of neonatal intensive care unit (NICU) admission was longer in neonates of overweight/obese women versus normal weight. Placental examination of FLP sites in the obese group showed larger infarcts, increased adipose triglyceride lipase, and a proangiogenic phenotype. Increased BMI is common in our TTTS cohort and it is associated with higher rate of co-morbidity, PPROM, prolonged NICU stay, and an imbalance of placental metabolic and vascular mediators. more...

Recombinant human alpha-N-acetylglucosaminidase-insulin-like growth factor-2 (rhNAGLU-IGF2) is an investigational enzyme replacement therapy for Sanfilippo B, a lysosomal storage disease. Because recombinant human NAGLU (rhNAGLU) is poorly mannose 6-phosphorylated, we generated a fusion protein of NAGLU with IGF2 to permit its binding to the cation-independent mannose 6-phosphate receptor. We previously administered rhNAGLU-IGF2 intracerebroventricularly to Sanfilippo B mice, and demonstrated therapeutic restoration of NAGLU, normalization of lysosomal storage, and improvement in markers of neurodegeneration and inflammation. Here, we studied repeated intracerebroventricular rhNAGLU-IGF2 delivery in both murine and canine Sanfilippo B to determine potential effects on their behavioral phenotypes and survival. Treated mice showed improvement in disease markers such as heparan sulfate glycosaminoglycans, beta-hexosaminidase, microglial activation, and lysosomal-associated membrane protein-1. Sanfilippo B mice treated with rhNAGLU-IGF2 displayed partial normalization of their stretch attend postures, a defined fear pose in mice (p more...

Infantile globoid cell leukodystrophy (GLD, Krabbe disease) is a fatal demyelinating disorder caused by a deficiency in the lysosomal enzyme galactosylceramidase (GALC). GALC deficiency leads to the accumulation of the cytotoxic glycolipid, galactosylsphingosine (psychosine). Complementary evidence suggested that psychosine is synthesized via an anabolic pathway. Here, we show instead that psychosine is generated catabolically through the deacylation of galactosylceramide by acid ceramidase (ACDase). This reaction uncouples GALC deficiency from psychosine accumulation, allowing us to test the long-standing “psychosine hypothesis.” We demonstrate that genetic loss of ACDase activity (Farber disease) in the GALC-deficient mouse model of human GLD (twitcher) eliminates psychosine accumulation and cures GLD. These data suggest that ACDase could be a target for substrate reduction therapy (SRT) in Krabbe patients. We show that pharmacological inhibition of ACDase activity with carmofur significantly decreases psychosine accumulation in cells from a Krabbe patient and prolongs the life span of the twitcher (Twi) mouse. Previous SRT experiments in the Twi mouse utilized l-cycloserine, which inhibits an enzyme several steps upstream of psychosine synthesis, thus altering the balance of other important lipids. Drugs that directly inhibit ACDase may have a more acceptable safety profile due to their mechanistic proximity to psychosine biogenesis. In total, these data clarify our understanding of psychosine synthesis, confirm the long-held psychosine hypothesis, and provide the impetus to discover safe and effective inhibitors of ACDase to treat Krabbe disease. more...

Low-grade fibromyxoid sarcoma (previously known as Evans tumor) is a rare soft tissue neoplasm characterized by a deceptively bland appearance despite the potential for late metastasis or recurrence. We describe a 13-year-old patient with a popliteal fossa mass initially thought to be benign that, because of array-comparative genomic hybridization findings and subsequent immunohistochemistry, was diagnosed as low-grade fibromyxoid sarcoma. The array-comparative genomic hybridization demonstrated a loss of 11p11.2p15.5 and a gain of 16p11.2p13.3 with breakpoints involving the CREB3L1 (cAMP responsive element-binding protein 3-like 1) and FUS (fused in sarcoma) genes, respectively. Subsequent fluorescence in situ hybridization analysis of a dual-labeled break-apart FUS probe on interphase cells was positive. Our case highlights the importance of using genetic information obtained via array-comparative genomic hybridization to classify accurately pediatric soft tissue tumors. more...

Globoid cell leukodystrophy (GLD, Krabbe Disease) is a lysosomal storage disease, resulting from the genetic deficiency of galactosylceramidase (GALC). This disease is marked by accumulation of the cytotoxic lipid psychosine (Psy). Psychosine is known to induce oxidative stress in cultured cells, and this stress can be ameliorated through co-treatment with the antioxidant N-acetyl cysteine (NAC). Oxidative stress has also been observed in vivo in the mouse model of GLD, the Twitcher mouse (Twi). We hypothesized that treating oxidative stress with NAC; either alone or in combination with bone marrow transplant (BMT) would improve the course of disease. All breeding cages were maintained on water containing NAC. Once born, the pups received IP boluses of NAC three times per week, and were maintained on NAC-containing water. A separate cohort of animals received the same regimen of NAC in addition to a BMT on post-natal days 2–3. Although NAC lowers the level of oxidized proteins in the brains of Twi mice, and dramatically improves immunohistochemical markers of disease, neither treatment results in any clinical improvements in the Twi mouse. Our data suggest that oxidative stress may be sufficiently down-stream in the pathogenic cascade initiated by Psy accumulation as to be difficult or impossible to treat with standard pharmacologic agents. It is possible that NAC may synergize with other therapies or combinations of therapies. A better understanding of the initiating effects of Psy toxicity and oxidative damage may uncover treatable therapeutic targets. more...

Globoid-cell leukodystrophy (GLD) is an inherited demyelinating disease caused by the deficiency of the lysosomal enzyme galactosylceramidase (GALC). A previous study in the murine model of GLD (twitcher) demonstrated a dramatic synergy between CNS-directed adeno-associated virus 2/5 (AAV2/5) gene therapy and myeloreductive bone marrow transplantation (BMT). However, the mechanism by which these two disparate therapeutic approaches synergize is not clear. In addition, the therapeutic efficacy may have been limited since the CNS-directed gene therapy was restricted to the forebrain and thalamus. In the current study, intrathecal and intracerebellar injections were added to the therapeutic regimen and the mechanism of synergy between BMT and gene therapy was determined. Although AAV2/5 alone provided supraphysiological levels of GALC activity and reduced psychosine levels in both the brain and spinal cord, it significantly increased CNS inflammation. Bone marrow transplantation alone provided essentially no GALC activity to the CNS and did not reduce psychosine levels. When AAV2/5 is combined with BMT, there are sustained improvements in motor function and the median life span is increased to 123 d (range, 92–282 d) compared with 41 d in the untreated twitcher mice. Interestingly, addition of BMT virtually eliminates both the disease and AAV2/5-associated inflammatory response. These data suggest that the efficacy of AAV2/5-mediated gene therapy is limited by the associated inflammatory response and BMT synergizes with AAV2/5 by modulating inflammation. more...

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. more...

Sanfilippo syndrome type B (MPS IIIB) is a lysosomal storage disease resulting from a deficiency of N-acetyl-glucosaminidase (NAGLU) activity. In an attempt to correct the disease in the murine model of MPS IIIB, neonatal mice were treated with intracranial AAV2/5-NAGLU (AAV), syngeneic bone marrow transplant (BMT), or both (AAV/BMT). All treatments resulted in some improvement in clinical phenotype. Adeno-associated viral (AAV) treatment resulted in improvements in lifespan, motor function, hearing, time to activity onset, and daytime activity level, but no reduction of lysosomal storage. BMT resulted in improved hearing by 9 months, and improved circadian measures, but had no effect on lifespan, motor function, or central nervous system (CNS) lysosomal storage. AAV/BMT treatment resulted in improvements in hearing, time to activity onset, motor function, and reduced CNS lysosomal storage, but had no effect on lifespan. Combination therapy compared to either therapy alone resulted in synergistic effects on hearing and CNS lysosomal inclusions but antagonistic effects on motor function and lifespan. AAV alone is more efficacious than BMT or AAV/BMT treatment for lifespan. BMT was the least efficacious treatment by all measures. CNS-directed AAV treatment alone appears to be the preferred treatment, combining the most efficacy with the least toxicity of the approaches assessed. more...

Enzyme replacement therapy is an established means of treating lysosomal storage diseases. Infused enzymes are normally targeted to the lysosomes of affected cells by interactions with cell-surface receptors that recognize carbohydrate moieties such as mannose and mannose 6-phosphate on the enzymes. Therefore, we have investigated alternative strategies to deliver the lysosomal enzyme beta-glucuronidase in the enzyme-deficient mucopolysaccharidosis type VII mouse model. Here we summarize our recent efforts to use nontraditional ways to deliver beta-glucuronidase. First, we used a chimeric protein of the insulin-like growth factor II (IGF-II) fused to beta-glucuronidase to deliver enzyme via the IGF-II binding site on the bifunctional IGF-II/mannose 6-phosphate receptor. Second, we used the 11-amino-acid human immunodeficiency virus (HIV) Tat domain fused to beta-glucuronidase to mediate uptake by absorptive endocytosis. Interaction with heparan sulfate on the cell surface internalizes and delivers the Tat-tagged enzyme to the lysosome via plasma membrane recycling. Third, we created a chimeric beta-glucuronidase fused to the Fc portion of human immunoglobulin G (IgG) Fc, which was transported by the neonatal Fc receptor from the maternal circulation across the placenta to sites of storage in fetal tissues. Finally, periodate treatment was used to eliminate interaction with carbohydrate receptors, creating an enzyme with increased plasma half-life, resulting in transport across the blood-brain barrier and clearance of storage in neurons. These strategies for delivering lysosomal enzymes could also be used to target nonlysosomal proteins or enzymes identified for bioremediation of other conditions. more...

Infantile neuronal ceroid lipofuscinosis (INCL), also known as Santavuori-Haltia disease, is an inherited neurodegenerative disorder caused by a mutation in the gene encoding the lysosomal enzyme palmitoyl-protein-thioesterase-1 (PPT1). Fatty acid–modified proteins are not degraded and accumulate as granular osmiophilic deposits in cells in the central nervous system; patients have blindness, seizures, progressive psychomotor deterioration, and die in early childhood. Although the disease manifests clinically primarily with neurological symptoms, visceral storage also accumulates. A murine model of INCL due to PPT1 deficiency exhibits clinical findings and pathology similar to those seen in patients with INCL. Homozygous PPT1-deficient mice have a shortened life span and neurological abnormalities including seizures, blindness, and mental and motor deficits. Widespread granular osmiophilic deposits (GRODs) accumulate in lysosomes in neurons and glia in the brain, retinal cells, kidney glomerular cells, aortic smooth muscle cells, and, in lesser amounts, in the fixed-tissue macrophage system. Accumulation of GRODs in aortic smooth muscle cells is accompanied by abnormalities in cardiac function and aortic root dilatation. This PPT1-deficient murine model is a well-defined genetic system that can be used to test potential therapies for lysosomal storage disease and to study the pathophysiology of INCL. more...

Bone marrow-derived mesenchymal stem cells (MSCs) are a promising platform for cell- and gene-based treatment of inherited and acquired disorders. We recently showed that human MSCs distribute widely in a murine xenotransplantation model. In the current study, we have determined the distribution, persistence, and ability of lentivirally transduced human MSCs to express therapeutic levels of enzyme in a xenotransplantation model of human disease (nonobese diabetic severe combined immunodeficient mucopolysaccharidosis type VII [NOD-SCID MPSVII]). Primary human bone marrow-derived MSCs were transduced ex vivo with a lentiviral vector expressing either enhanced green fluorescent protein or the lysosomal enzyme β-glucuronidase (MSCs-GUSB). Lentiviral transduction did not affect any in vitro parameters of MSC function or potency. One million cells from each population were transplanted intraperitoneally into separate groups of neonatal NOD-SCID MPSVII mice. Transduced MSCs persisted in the animals that underwent transplantation, and comparable numbers of donor MSCs were detected at 2 and 4 months after transplantation in multiple organs. MSCs-GUSB expressed therapeutic levels of protein in the recipients, raising circulating serum levels of GUSB to nearly 40% of normal. This level of circulating enzyme was sufficient to normalize the secondary elevation of other lysosomal enzymes and reduce lysosomal distention in several tissues. In addition, at least one physiologic marker of disease, retinal function, was normalized following transplantation of MSCs-GUSB. These data provide evidence that transduced human MSCs retain their normal trafficking ability in vivo and persist for at least 4 months, delivering therapeutic levels of protein in an authentic xenotransplantation model of human disease. Disclosure of potential conflicts of interest is found at the end of this article. more...

Enzyme replacement therapy has been used successfully in many lysosomal storage diseases. However, correction of brain storage has been limited by the inability of infused enzyme to cross the blood–brain barrier. The newborn mouse is an exception because recombinant enzyme is delivered to neonatal brain after mannose 6-phosphate receptor-mediated transcytosis. Access to this route is very limited after 2 weeks of age. Recently, several studies showed that multiple infusions of high doses of enzyme partially cleared storage in adult brain. These results raised the question of whether correction of brain storage by repeated high doses of enzyme depends on mannose 6-phosphate receptor-mediated uptake or whether enzyme gains access to brain storage by another route when brain capillaries are exposed to prolonged, high levels of circulating enzyme. To address this question, we used an enzyme whose carbohydrate-dependent receptor-mediated uptake was inactivated by chemical modification. Treatment of human β-glucuronidase (GUS) with sodium metaperiodate followed by sodium borohydride reduction (PerT-GUS) eliminated uptake by mannose 6-phosphate and mannose receptors in cultured cells and dramatically slowed its plasma clearance from a t ½ of more...

Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive disorder caused by deficiency of N -acetylgalactosamine-6-sulfate sulfatase (GALNS), required for degradation of keratan sulfate and chondroitin-6-sulfate. In order to study the effects of a missense mutation in the active site cysteine in the GALNS gene that is conserved in all mammalian sulfatases, we produced a p.C76S (an active site replacement) knock-in mouse by replacing the Cys76 with Ser in the endogenous murine Galns by targeted mutagenesis. Homozygous Galns tm(C76S)slu mice had no detectable GALNS enzyme activity. At age of 2–4 months, lysosomal storage was present primarily within reticuloendothelial cells such as Kupffer cells and spleen sinusoidal lining cells. Vacuolar change was present in glomerular visceral epithelial cells and was not present in hepatocytes or renal tubular cells. In the brain, hippocampal and neocortical neurons and meningeal cells showed lysosomal storage. Radiographs revealed no change in the skeletal bones of mice up to 12 months old. Thus, the Galns tm(C76S)slu mice had visceral storage of GAGs in organs but lacked the skeletal features of human MPS IVA. In contrast to a previously reported transgenic model ( Galns tm(hC79S·mC76S)slu ), in which the inactive human GALNS transgene was overexpressed, no reduction in other sulfatases was observed. In addition, the Galns tm(C76S)slu mice displayed milder storage. We conclude that the milder phenotype is characteristic of isolated GALNS deficiency while the more severe phenotype reflected in the Galns tm(hC79S·mC76S)slu mice was due to deficiency of other sulfatases caused by oversaturation of the sulfate modifying enzyme by the inactive human gene product. more...

Treatment of brain disease with recombinant proteins is difficult due to the blood-brain barrier. As an alternative to direct injections into the brain, we studied whether application of high concentrations of therapeutic enzymes via intrathecal (IT) injections could successfully drive uptake across the ependyma to treat brain disease. We studied IT enzyme replacement therapy with recombinant human iduronidase (rhIDU) in canine mucopolysaccharidosis I (MPS I, Hurler syndrome), a lysosomal storage disorder with brain and meningeal involvement. Monthly or quarterly IT treatment regimens with rhIDU achieved supranormal iduronidase enzyme levels in the brain, spinal cord, and spinal meninges. All regimens normalized total brain glycosaminoglycan (GAG) storage and reduced spinal meningeal GAG storage by 58-70%. The improvement in GAG storage levels persisted three months after the final IT dose. The successful use of enzyme therapy via the CSF represents a potentially useful approach for lysosomal storage disorders. more...

Globoid-cell leukodystrophy (GLD) is a rapidly progressing inherited neurodegenerative disorder caused by a deficiency in galactosylceramidase activity. Previous studies in the murine model of GLD (Twitcher mouse) have shown that both bone marrow transplantation (BMT) and central nervous system (CNS)-directed gene therapy can be moderately effective at ameliorating certain aspects of GLD. As BMT and CNS-directed gene therapy target fundamentally different tissues, we tested the hypothesis that combining these disparate therapies would be more efficacious than either therapy alone. Mice receiving myeloreductive conditioning at birth followed by syngeneic BMT had approximately 25–35% donor chimerism. Untreated Twitcher mice, Twitcher mice treated with BMT alone, AAV2/5 alone, or a combination of BMT and AAV2/5 had mean lifespans of 39, 44, 49, and 104 days, respectively. Twitcher mice treated with a combination of BMT and AAV2/5 also had significantly improved performance in several behavioral tests and greater reduction in demyelination, astrocytosis, and macrophage infiltration compared to untreated Twitcher mice or mice that received either therapy alone. These data suggest that CNS-directed gene therapy synergizes with BMT. The combination of these disparate therapeutic approaches may form the basis for more effective treatment of this inherited neurodegenerative disorder. more...

Granular cell tumor is a benign tumor of likely neural or neuroectodermal origin that occurs most commonly in the subcutaneous tissues of the trunk, breast, and extremities of adults. Congenital gingival lesions comprise the majority of the pediatric granular cell tumors. Granular cell tumors are generally small and asymptomatic, and while 1 in 10 patients has multiple tumors, recurrence and malignancy are very rare. Mediastinal granular cell tumors have been reported, most occurring in young adult or middle-aged women. We present a case of a 16-year-old asymptomatic boy with a large mediastinal granular cell tumor incidentally identified after a motor vehicle accident, and we review the intraoperative, microscopic, and ultrastructural features of this tumor. Both the patient's age and anatomical location are unusual for this tumor, which presented technical and diagnostic challenges to the patient care team. more...

Quadruple synchronous primary neoplasms are exceedingly rare with only one case reported in the English literature. We herein report a case of synchronous quadruple primary neoplasms in a 70-year-old Arabic male with a history of prostate cancer who presented to our hospital for work-up of a brain mass found at an outside hospital. Subsequent (18)Fluorodeoxyglucose (FDG) positron emission tomography demonstrated a 5.9-cm temporoparietal mass and three additional lesions, each with increased maximum standardized uptake value (SUV(max)). Histologic examination, immunohistochemistry and cytogenetic analyses of the lesional tissue revealed four primary neoplastic lesions: primary glioblastoma, inguinal schwannoma, well-differentiated neuroendocrine tumor of the terminal ileum and an appendiceal sessile serrated adenoma/polyp. This case is unique among previous reports as our patient presented with four primary neoplasms synchronously. To the best of our knowledge, this combination of synchronous multiple primary neoplasms has not been reported in the English literature. more...

Treatment of nonmalignant childhood disorders by bone marrow transplantation (BMT) is limited by toxicity from preparatory regimens and immune consequences associated with engraftment of allogeneic donor cells. Using costimulatory blockade (anti-CD40L mAb and CTLA-4Ig) combined with high-dose BMT in nonablated neonates, we obtained engraftment and established tolerance using both partially MHC mismatched (H2g7 into H2b) and fully mismatched BM (H2s into H2b). Recipients were mucopolysaccharidosis type VII (MPS VII) mice with lysosomal storage disease in order to assess therapeutic outcome. Recipients treated with donor lymphocyte infusion (DLI) amplified microchimerism to full donor. Recipients without DLI maintained long-term engraftment, tolerance, and had extended life spans. DLI increased donor cell mediated replacement of beta-glucuronidase (GUSB) activity in all tissues and maintained clearance of lysosomes better than in non-DLI-treated mice. DLI amplification of partially mismatched BM and fully mismatched BM caused late onset chronic GvHD in 56% and 100% of recipients, respectively. more...

Enzyme replacement therapy (ERT) effectively reverses storage in several lysosomal storage diseases. However, improvement in brain is limited by the blood-brain barrier except in the newborn period. In this study, we asked whether this barrier could be overcome by higher doses of enzyme than are used in conventional trials. We measured the distribution of recombinant human β-glucuronidase (hGUS) and reduction in storage by weekly doses of 0.3-40 mg/kg administered i.v. over 1-13 weeks to mucopolysaccharidosis type VII mice immunotolerant to recombinant hGUS. Mice given up to 5 mg/kg enzyme weekly over 3 weeks had moderate reduction in meningeal storage but no change in neo-cortical neurons. Mice given 20-40 mg/kg three times over 1 week showed no reduction in storage in any area of the CNS except the meninges. In contrast, mice receiving 4 mg/kg per week for 13 weeks showed clearance not only in meninges but also in parietal neocortical and hippocampal neurons and glia. Mice given 20 mg/kg once weekly for 4 weeks also had decreased neuronal, glial, and meningeal storage and averaged 2.5% of wild-type hGUS activity in brain. These results indicate that therapeutic enzyme can be delivered across the blood-brain barrier in the adult mucopolysaccharidosis type VII mouse if administered at higher doses than are used in conventional ERT trials and if the larger dose of enzyme is administered over a sufficient period. These results may have important implications for ERT for lysosomal storage diseases with CNS involvement. more...

Globoid-cell leukodystrophy (GLD) is an autosomal recessive lysosomal storage disorder caused by mutations in the galactosylceramidase (GALC) gene. Infantile GLD has a lethal course with severe cerebral demyelination that progresses to death by 2 years of age. In the current study twitcher mice, an authentic murine model of infantile GLD, were given intracranial injections of either recombinant adeno-associated virus serotype 2 encoding the murine Galc cDNA (AAV2-GALC) or the same genome pseudotyped with AAV5 capsid proteins (AAV2/5-GALC) on day 3 of age. The group injected intracranially with AAV2/5-GALC had approximately 25-fold greater than normal Galc levels in the brain, while AAV2-GALC-injected animals had 28% normal levels. The average life expectancy of twitcher mice ( approximately 38 days) was significantly (P < 0.0001) increased to 48 and 52 days for the AAV2-GALC- and AAV2/5-GALC-treated groups, respectively. The AAV2/5-GALC group performed significantly better in a battery of behavioral tests compared to untreated, AAV2-GFP-treated, or AAV2-treated twitcher animals. This longitudinal study demonstrated that AAV2/5-GALC-mediated gene therapy resulted in higher levels of Galc expression and slowed the neurologic deterioration more completely than AAV2-GALC in the murine model of globoid-cell leukodystrophy. However, the clinical improvements, as assessed by behavioral tests and life span, were only modest. more...

We used recombinant forms of human β-glucuronidase (GUS) purified from secretions from stably transfected CHO cells to compare the native enzyme to a GUS–Tat C-terminal fusion protein containing the 11-amino-acid HIV Tat protein transduction domain for: (1) susceptibility to endocytosis by cultured cells, (2) rate of clearance following intravenous infusion, and (3) tissue distribution and effectiveness in clearing lysosomal storage following infusion in the MPS VII mouse. We found: (1) Native GUS was more efficiently taken up by cultured human fibroblasts and its endocytosis was exclusively mediated by the M6P receptor. The GUS–Tat fusion protein showed only 30–50% as much M6P-receptor-mediated uptake, but also was taken up by adsorptive endocytosis through binding of the positively charged Tat peptide to cell surface proteoglycans. (2) GUS–Tat was less rapidly cleared from the circulation in the rat ( t 1/2 = 13 min vs 7 min). (3) Delivery to most tissues of the MPS VII mouse was similar, but GUS–Tat was more efficiently delivered to kidney. Histology showed that GUS–Tat more efficiently reduced storage in renal tubules, retina, and bone. These studies demonstrate that Tat modification can extend the range of tissues corrected by infused enzyme. more...

Enzyme replacement therapy (ERT) has been developed for several lysosomal storage disorders, including mucopolysaccharidosis I (MPS I), and is effective at reducing lysosomal storage in many tissues and in ameliorating clinical disease. However, intravenous ERT does not adequately treat storage disease in the central nervous system (CNS), presumably due to effects of the blood-brain barrier on enzyme distribution. To circumvent this barrier, we studied whether intrathecal (IT) recombinant human alpha-L-iduronidase (rhIDU) could penetrate and treat the brain and meninges. An initial dose-response study showed that doses of 0.46-4.14 mg of IT rhIDU successfully penetrated the brain of normal dogs and reached tissue levels 5.6 to 18.9-fold normal overall and 2.7 to 5.9-fold normal in deep brain sections lacking CSF contact. To assess the efficacy and safety in treating lysosomal storage disease, four weekly doses of approximately 1 mg of IT rhIDU were administered to MPS I-affected dogs resulting in a mean 23- and 300-fold normal levels of iduronidase in total brain and meninges, respectively. Quantitative glycosaminoglycan (GAG) analysis showed that the IT treatment reduced mean total brain GAG to normal levels and achieved a 57% reduction in meningeal GAG levels accompanied by histologic improvement in lysosomal storage in all cell types. The dogs did develop a dose-dependent immune response against the recombinant human protein and a meningeal lymphocytic/plasmacytic infiltrate. The IT route of ERT administration may be an effective way to treat the CNS disease in MPS I and could be applicable to other lysosomal storage disorders. more...

Infantile neuronal ceroid lipofuscinosis (INCL) is the earliest onset form of a class of inherited neurodegenerative disease called Batten disease. INCL is caused by a deficiency in the lysosomal enzyme palmitoyl protein thioesterase-1 (PPT1). Autofluorescent storage material accumulates in virtually all tissues in INCL patients, including the brain, and leads to widespread neuronal loss and cortical atrophy. To determine the efficacy of viral-mediated gene therapy, we injected a recombinant adeno-associated virus 2 vector encoding human PPT1 (rAAV–PPT1) intracranially (I.C.) into a murine model of INCL. INCL mice given four I.C. injections of rAAV–PPT1 as newborns exhibited PPT1 activity near the injection sites and decreased secondary elevations of another lysosomal enzyme. In addition, storage material was decreased in cortical, hippocampal, and cerebellar neurons, and brain weights and cortical thicknesses were increased. These data demonstrate that an adeno-associated virus 2 (AAV2)-mediated gene therapy approach may provide some therapeutic benefit for INCL. more...

Mucopolysaccharidosis VII (MPS VII) is a lysosomal storage disease caused by deficient beta-glucuronidase (GUSB) activity. Accumulation of glycosaminoglycans (GAGs) in bone, cartilage, and synovium likely contributes to reduced mobility in untreated MPS VII individuals. We previously reported that neonatal intravenous injection of a retroviral vector (RV) expressing canine GUSB resulted in hepatocyte transduction in mice and dogs, and secreted GUSB was taken up from blood by other organs. Here we report the effect of this therapy on bone, cartilage, and joint disease. Osteocytes and bone-lining cells from RV-treated MPS VII mice had GUSB activity, resulting in a marked reduction, as compared with untreated MPS VII mice, in lysosomal storage in bone and at the bone:growth plate interface where bone elongation occurs. Although chondrocytes did not have detectable GUSB activity and had little reduction in lysosomal storage, the thickness of the growth plate was reduced toward normal. These pathological changes were likely responsible for improvements in facial morphology and long bone lengths. The synovium had reduced hyperplasia and lysosomal storage, and the thickness of the articular cartilage was reduced. Similarly, RV-treated MPS VII dogs had improved facial morphology and reduced lysosomal storage in osteocytes and synovium, but not chondrocytes. Nevertheless, the internal area of the trachea was increased, and erosions of the femoral head were reduced. We conclude that neonatal gene therapy can improve bone and joint disease in MPS VII mice and dogs. However, better delivery of GUSB to chondrocytes will be necessary to achieve more profound effects in cartilage. more...

Enzyme-replacement therapy is an established means of treating lysosomal storage diseases. Infused therapeutic enzymes are targeted to lysosomes of affected cells by interactions with cell-surface receptors that recognize carbohydrate moieties, such as mannose and mannose 6-phosphate, on the enzymes. We have tested an alternative, peptide-based targeting system for delivery of enzymes to lysosomes in a murine mucopolysaccharidosis type VII (MPS VII) model. This strategy depends on the interaction of a fragment of insulin-like growth factor II (IGF-II), with the IGF-II binding site on the bifunctional, IGF-II cation-independent mannose 6-phosphate receptor. A chimeric protein containing a portion of mature human IGF-II fused to the C terminus of human β-glucuronidase was taken up by MPS VII fibroblasts in a mannose 6-phosphate-independent manner, and its uptake was inhibited by the addition of IGF-II. Furthermore, the tagged enzyme was delivered effectively to clinically significant tissues in MPS VII mice and was effective in reversing the storage pathology. The tagged enzyme was able to reduce storage in glomerular podocytes and osteoblasts at a dose at which untagged enzyme was much less effective. This peptide-based, glycosylation-independent lysosomal targeting system may enhance enzyme-replacement therapy for certain human lysosomal storage diseases. more...

Cardiovascular manifestations of lysosomal storage disease (LSD) are a significant health problem for affected patients. Infantileonset cardiac disease, because of its rapid progression, is usually treated symptomatically. Therapy in older patients includes valve replacement and bone marrow (BM) transplantation, both of which are life threatening in the already debilitated patients. Enzyme replacement therapy has potential benefit but has not yet been demonstrated to provide long-term relief for cardiac disease. Here, we demonstrate prevention of severe cardiac manifestations in β-glucuronidase (GUSB) null mice BM-transplanted i.v. as neonates without myeloablative pretreatment. The mice, a model of mucopolysaccharidosis type VII (MPSVII, Sly syndrome), develop progressive LSD unless provided with GUSB early in life. The BM recipients retained GUSB+ donor cells in the peripheral blood and heart until necropsy at >11 months of age. The enzyme β-hexosamindase increased in tissues of GUSB null MPSVII mice was reduced significantly ( P = 0.001) in treated MPSVII hearts. Electrocardiography demonstrated normalization of heart rate, PR, PQ, and QRS intervals in BM recipients. Storage was markedly reduced in the stroma of heart valves, adventitial cells of the aortic root, perivascular and interstitial cells of the myocardium, and interstitial cells of the conduction tissue. Heart/body weight ratio normalized. The aortic root was still grossly distended, and the conductive myocytes retained storage, suggesting neither plays a major role in ECG normalization. We conclude that transplantation of MPSVII neonates without toxic intervention can prevent many of the cardiovascular manifestations of LSD. more...

Mucopolysaccharidosis VII (MPS VII, Sly syndrome) is an autosomal recessive lysosomal storage disease caused by beta-glucuronidase (GUS) deficiency. A naturally occurring mouse model of that disease has been very useful for studying experimental approaches to therapy. However, immune responses can complicate evaluation of the long-term benefits of enzyme replacement or gene therapy delivered to adult MPS VII mice. To make this model useful for studying the long-term effectiveness and side effects of experimental therapies delivered to adult mice, we developed a new MPS VII mouse model, which is tolerant to both human and murine GUS. To achieve this, we used homologous recombination to introduce simultaneously a human cDNA transgene expressing inactive human GUS into intron 9 of the murine Gus gene and a targeted active site mutation (E536A) into the adjacent exon 10. When the heterozygote products of germline transmission were bred to homozygosity, the homozygous mice expressed no GUS enzyme activity but expressed inactive human GUS protein highly and were tolerant to immune challenge with human enzyme. Expression of the mutant murine Gus gene was reduced to about 10% of normal levels, but the inactive murine GUS enzyme also conferred tolerance to murine GUS. This MPS VII mouse model should be useful to evaluate therapeutic responses in adult mice receiving repetitive doses of enzyme or mice receiving gene therapy as adults. Heterozygotes expressed only 9.5-26% of wild-type levels of murine GUS instead of the expected 50%, indicating a dominant-negative effect of the mutant enzyme monomers on the activity of GUS tetramers in different tissues. Corrective gene therapy in this model should provide high enough levels of expression of normal GUS monomers to overcome the dominant negative effect of mutant monomers on newly synthesized GUS tetramers in most tissues. more...

The mucopolysaccharidoses (MPSs) are lysosomal storage diseases resulting from impaired catabolism of sulfated glycosaminoglycans. MPS VII mice lack lysosomal beta-glucuronidase (GUSB) activity, leading to the accumulation of partially degraded chondroitin, dermatan, and heparan sulfates in most tissues. Consequently, these mice develop most of the symptoms exhibited by human MPS VII patients, including progressive visual and cognitive deficits. To investigate the effects of reducing lysosomal storage in nervous tissues, we injected recombinant adeno-associated virus encoding GUSB directly into the vitreous humor of young adult mice. Interestingly, GUSB activity was subsequently detected in the brains of the recipients. At 8-12 weeks after treatment, increased GUSB activity and reduced lysosomal distension were found in regions of the thalamus and tectum that received inputs from the injected eye. Lysosomal storage was also reduced in adjacent nonvisual regions, including the hippocampus, as well as in the visual cortex. The findings suggest that both diffusion and trans-synaptic transfer contribute to the dissemination of enzyme activity within the CNS. Intravitreal injection may thus provide a means of delivering certain therapeutic gene products to specific areas within the CNS. more...

A novel murine system was developed to study the in vivo localization of xenotransplanted human cells and assess their therapeutic effect in an authentic model of disease. The β-glucuronidase (GUSB) mutation of the mucopolysaccharidosis type VII (MPSVII) mouse was backcrossed onto the nonobese diabetic/severe combined immunodeficient (NOD/SCID) xenotransplantation strain. The resulting NOD/SCID/MPSVII mice displayed the characteristic features of lysosomal storage disease because of GUSB deficiency and were also capable of engrafting human cells. Human CD34+hematopoietic progenitor cells from healthy, GUSB+donors engrafted NOD/SCID/MPSVII mice in a manner similar to that of standard NOD/SCID mice. Six to 12 weeks following transplantation, 1% to 86% of the host bone marrow was positive for human CD45. By using a GUSB-specific histochemical assay, human engraftment was detected with single-cell sensitivity not only in well-characterized hematopoietic tissues like bone marrow, spleen, lymph node, and thymus, but also in other nonhematopoietic organs like liver, kidney, lung, heart, brain, and eye. Quantitative measurements of GUSB activity confirmed this expansive tissue distribution. The GUSB-specific assays were validated for their accuracy in identifying human cells through colocalization of human CD45 expression with GUSB activity in tissues of mice receiving transplants. An analysis of the therapeutic effects of engrafted human cells revealed a reduction of pathologic storage material in host organs, including the bone, spleen, and liver. Such xenotransplantation experiments in the NOD/SCID/MPSVII mouse represent a powerful approach to both study the in vivo biology of human cells and gather preclinical data regarding treatment approaches for a human disease. more...

Lymphocyte-directed gene transfer has been proposed as potential therapy to treat certain congenital immunological deficiencies as well as other genetic diseases such as lysosomal storage diseases (LSDs). To understand better the extent to which adoptively transferred peripheral T lymphocytes (PTLs) are able to ameliorate LSDs we utilized the beta-glucuronidase-deficient mouse as a model system. PTLs (1 x 10(7)) isolated from the spleen of syngeneic mice overexpressing ( approximately 8-fold) human beta-glucuronidase (GUSB) were injected intravenously into young adult beta-glucuronidase-deficient mice without myeloablative conditioning. Using biochemical and histochemical assays, we were able to track the donor lymphocytes in vivo. Donor lymphocytes were detected in relatively high numbers in liver, spleen, small intestine, mesenteric lymph node, and thymus for at least 5 months, the last time point of analysis. Although liver and spleen had the highest total GUSB activity, histopathologic analysis demonstrated minimal to no correction of lysosomal distention at all time points studied. By contrast, we have shown in earlier studies that administration of similar numbers of macrophages reduced lysosomal storage in several organs, including liver and spleen. To understand this difference in efficacy, we compared the relative level of GUSB released into the medium by nonactivated and activated PTLs as well as by macrophages. Macrophages released >50-fold excess enzyme compared to either activated or nonactivated PTLs. These data suggest that a LSD can be more effectively treated by directing a gene therapy approach to a hematopoietic lineage other than T lymphocytes. more...

Human mucopolysaccharidosis VII (MPS VII, Sly syndrome) results from a deficiency of β-glucuronidase (GUS) and has been associated with a wide range in severity of clinical manifestations. To study missense mutant models of murine MPS VII with phenotypes of varying severity, we used targeted mutagenesis to produce E536A and E536Q, corresponding to active-site nucleophile replacements E540A and E540Q in human GUS, and L175F, corresponding to the most common human mutation, L176F. The E536A mouse had no GUS activity in any tissue and displayed a severe phenotype like that of the originally described MPS VII mice carrying a deletion mutation ( gus mps/mps ). E536Q and L175F mice had low levels of residual activity and milder phenotypes. All three mutant MPS models showed progressive lysosomal storage in many tissues but had different rates of accumulation. The amount of urinary glycosaminoglycan excretion paralleled the clinical severity, with urinary glycosaminoglycans remarkably higher in E536A mice than in E536Q or L175F mice. Molecular analysis showed that the Gus mRNA levels were quantitatively similar in the three mutant mouse strains and normal mice. These mouse models, which mimic different clinical phenotypes of human MPS VII, should be useful in studying pathogenesis and also provide useful models for studying enzyme replacement therapy and targeted correction of missense mutations. more...

deviation of both eyes, absent oculocephalic responses, marked bifacial weakness, absent corneal reflexes, and bilateral tongue atrophy, as well as persistence of the previously noted hypertonia. Laboratory evaluation included normal computed tomography and magnetic resonance imaging of the brain. Sequential electroencephalogram examinations were appropriate for age, except for 1 study that showed a less mature pattern than expected. Electromyography and nerve conduction studies were normal. Ophthalmologic examination showed immature retina in zone 2 and exotropia. Brainstemevoked potentials performed between 11 and 12 weeks showed no transmission through the brainstem. Karyotype was normal female (46,XX). Because of the striking impairment in brainstem function, the diagnosis of Mobius syndrome was considered. The infant’s condition did not improve during a 3-month period. The neurologic condition was considered fatal regardless of underlying cause, and, after consultation with the infant’s parents, life support was withdrawn. The infant died at approximately 3 months of age. An unrestricted postmortem examination was performed. more...

Gene therapy using recombinant adeno-associated virus vectors (rAAV) is generally considered safe. During the course of a study designed to determine the long-term efficacy of rAAV-mediated gene therapy initiated in newborn mice with the lysosomal storage disease, mucopolysaccharidosis type VII (MPSVII), a significant incidence of hepatocellular carcinomas and angiosarcomas was discovered. A hepatocellular carcinoma was first detected in a 35-week-old mouse and by 72 weeks of age, three out of five rAAV-treated MPSVII mice had similar lesions. These types of tumors had not been seen previously in long-term studies of MPSVII mice using recombinant enzyme or bone marrow transplantation. In an attempt to ascertain whether mouse strain or GUSB expression confers susceptibility to tumor formation, we histopathologically examined untreated normal mice of the same strain, untreated MPSVII mice, and normal mice overexpressing human GUSB for the presence of tumors and increased hepatocyte replication. The results of these studies do not indicate that MPSVII mice or mice overexpressing human GUSB are susceptible to tumor formation; however, the number of animals examined is too small to draw definitive conclusions. Results from quantitative PCR performed on the tumor samples suggest that the tumors are probably not caused by an insertional mutagenesis event followed by the clonal expansion of a transformed cell. In a separate study, a relatively large group of mice injected with varying doses and types of rAAV vectors had no evidence of hepatic or vascular tumors. Although the mechanism of tumor formation is currently unknown, the tumorigenic potential of rAAV vectors must be rigorously determined in long-term in vivo studies. more...

For many inborn errors of metabolism, early treatment is critical to prevent long-term developmental sequelae. We have previously shown that systemic treatment of neonatal mucopolysaccharidosis type VII (MPS VII) mice with recombinant adeno-associated virus (AAV) vectors results in relatively long-term expression of beta-glucuronidase (GUSB) in multiple tissues, and a reduction in lysosomal storage. Here, we demonstrate that therapeutic levels of enzyme persist for at least 1 year following a single intravenous injection of virus in neonatal MPS VII mice. The level and distribution of GUSB expression achieved is sufficient to prevent the development of many aspects of clinical disease over the life of the animal. Following treatment, bone lengths, weights and retinal function were maintained at nearly normal levels throughout the life of the animal. In addition, significant improvements in survival and auditory function were seen in AAV-treated MPS VII mice when compared with untreated mutant siblings. These data suggest that AAV-mediated gene transfer in the neonatal period can lead to prevention of many of the clinical symptoms associated with MPS VII in the murine model of this disease. more...

Mucopolysaccharidosis type VII (MPS VII) is a lysosomal storage disease caused by the lack of beta-glucuronidase (GUSB) activity. GUSB deficiency leads to the progressive accumulation of undegraded glycosaminoglycans (GAGs) in cells of most tissues, including the brain, and is associated with mental retardation. Reduction of lysosomal storage in the central nervous system and prevention of cognitive dysfunction may require intracranial delivery of a therapeutic agent during the newborn period that provides a continuous source of GUSB. Therefore, we injected recombinant adeno-associated virus encoding human GUSB into both the anterior cortex and the hippocampus of newborn MPS VII mice. Total GUSB activity in the brain approached normal levels by 18 weeks. Although GUSB activity was concentrated near the injection sites, lysosomal distension was reduced in most areas of the brain. In addition to histopathologic evidence of GAG reduction, the previously undescribed accumulation of GM2 and GM3 gangliosides in the brain was also prevented. Furthermore, GUSB expression and reduced lysosomal distension correlated with improvements in cognitive function as measured in the Morris Water Maze test. These findings indicate that localized overexpression of GUSB has positive effects on the pathology and cognitive function and does not have overt toxicity. more...

We describe the clinical and pathologic findings in a murine model of mucopolysaccharidosis VII (Sly disease) that arose spontaneously in the C3H/HeOuJ mouse strain. Affected gus(mps2J)/gus(mps2J) mice are deficient in beta-glucuronidase because of insertion of an intracisternal A particle element into intron 8 of the gus structural gene. This is the first model of a human lysosomal storage disease caused by an intracisternal A particle element insertion. Mice with the gus(mps2J)/gus(mps2J) genotype have < 1% of normal beta-glucuronidase activity and secondary elevations of other lysosomal enzymes. The phenotype includes shortened life-span, dysmorphic features, and skeletal dysplasia. Lysosomal storage of glycosaminoglycans is widespread and affects the brain, skeleton, eye, ear, heart valves, aorta, and the fixed tissue macrophage system. Thus the phenotypic and pathologic alterations in gus(mps2J)/gus(mps2J) mice are similar to those in patients with mucopolysaccharidosis VII. The finding of antibodies to beta-glucuronidase in some older gus(mps2J)/gus(mps2J) mice suggests the mice produce sufficient enzyme to elicit an immune response. The gus(mps2J)/gus(mps2J) model provides another well-defined genetic system for the study of the pathophysiology of mucopolysaccharidosis and for evaluation of experimental therapies for lysosomal storage diseases. The disease in gus(mps2J)/gus(mps2J) mice is less severe than that seen in the previously characterized B6.C-H2(bm1)/ByBir-gus(mps)/gus(mps) mouse model. Furthermore, unlike gus(mps)/gus(mps) mice, gus(mps2J)/gus(mps2J) mice are fertile and breed to produce litters, all of which are mucopolysaccharidosis VII pups. This feature makes them extremely useful for testing intrauterine therapies. more...

The toxicity of preparative regimens render neonatal bone marrow transplantation (BMT) for progressive childhood diseases a controversial treatment. Ablative BMT in neonatal mice with or without the lysosomal storage disease mucopolysaccharidosis type VII (MPS VII) show high morbidity and developmental disruption of both brain and bone structure. In this investigation, BMT was performed with a high dose of congenic, normal bone marrow into nonablated newborn mice. Recipients had lifelong, multilineage, peripheral blood chimerism with the donor β-glucuronidase-positive (GUS+) cells that was both well tolerated and therapeutic. Three daily injections of normal adult marrow increased the average life span by at least 6 months and corrected the functional breeding deficits typical of the MPS VII mice. Twelve months after injection, several structural features of femurs were more like that of normal mice than of untreated MPS VII mice. Periosteal circumference and bone cortical thickness were significantly improved in males and cortical density did not differ significantly from values in normal females. Significant reduction of lysosomal glycosaminoglycan storage corresponded directly with GUS enzyme activity and percentage of histochemically GUS+ cells in visceral organs and hematopoietic tissues such as thymus, spleen, peripheral blood, and bone marrow. By all criteria tested, BMT into neonatal MPS VII mice in the absence of any preparative regimen is a successful therapy. more...

Summary: Pathologic examination of the placenta is of clinical importance in the evaluation of pregnancies with a less than perfect outcome. Morphologic alterations of the placenta can mirror disorders of the fetus and the mother and evaluation of the placenta can identify clinically significant lesions, allow understanding of a child's disability and may have a role in resolving medical-legal disputes. Pathologic findings in the placenta can provide information on the pathogenesis of cerebral palsy, mental retardation, or neurodevelopmental disorders. This review will cover a variety of frequently encountered, clinically important, and morphologically distinct disorders of the placenta. The current understanding of the clinical implications of lesions for the mother, infant, and for future pregnancies will also be considered. more...

Patients with inherited cystic kidney diseases have progressive cystic dilation of nephrons with concomitant loss of functional renal parenchyma and renal failure. Animal models of inherited cystic kidney disease are useful for study of the pathogenesis and molecular basis of cystic renal diseases. This article describes the clinical and pathologic features in two spontaneously occurring murine models of inherited polycystic kidney disease due to independent allelic mutations on mouse chromosome 8. The mutations, designated kat and kat2J, affect a chromosomal segment homologous to a region of human chromosome 4q35; the altered gene has not yet been identified. An allelism test showed that the mutations are at the same locus. The phenotype, inherited as an autosomal recessive, is more severe in kat2J/kat2J mice. Their kidneys are morphologically normal at birth, but by 3 mo of age, cysts affect all levels of the nephron. Adult males have testicular hypoplasia and they are sterile. A few of the oldest kat2J/kat2J mice have focal portal bile duct proliferation and dilation. kat2J/kat2J mice develop anemia and uremia and die before 1 yr of age. In kat/kat mice, the renal cystic disease progresses more slowly but is morphologically similar to that of kat2J/kat2J mice. The progressive cystic transformation of the kidneys in these allelic murine models resembles that seen in humans with autosomal dominant polycystic kidney disease. more...

Bone marrow transplantation (BMT) is relatively effective for the treatment of lysosomal storage diseases. To better understand the contribution of specific hematopoietic lineages to the efficacy of BMT, we transplanted β-glucuronidase–positive mononuclear phagocytes derived from either the peritoneum or from bone marrow in vitro into syngeneic recipients with mucopolysaccharidosis type VII (MPS VII). Cell surface marking studies indicate that the bone marrow-derived cells are less mature than the peritoneal macrophages. However, both cell types retain the ability to home to tissues rich in cells of the reticuloendothelial system after intravenous injection into MPS VII mice. The half-life of both types of donor macrophages is approximately 7 days, and some cells persist for at least 30 days. In several tissues, therapeutic levels of β-glucuronidase are present, and histopathologic analysis demonstrates that lysosomal storage is dramatically reduced in the liver and spleen. Macrophages intravenously injected into newborn MPS VII mice localize to the same tissues as adult mice but are also observed in the meninges and parenchyma of the brain. These data suggest that macrophages play a significant role in the therapeutic efficacy of BMT for lysosomal storage diseases and may have implications for treatments such as gene therapy. more...

We have previously shown that mucopolysaccharidosis type VII (MPS VII) mice receiving six weekly injections of recombinant beta-glucuronidase from birth had improved cognitive ability and reduced central nervous system lysosomal storage. However, a single beta-glucuronidase injection at 5 wk of age did not correct neuronal storage. We define the age at which central nervous system storage in MPS VII mice becomes resistant to beta-glucuronidase therapy and determine the effect of enzyme on other tissues by comparing the histology of mice begun on therapy at various times after birth. MPS VII mice received injections on the day of birth and then weekly for 5 wk with 16,000U/g beta-glucuronidase had reduced lysosomal storage in brain. The same therapy begun on d 14 of life or thereafter failed to correct neuronal storage, even when treatment was continued for six doses. Glial responsiveness or accessibility to enzyme also depended on early treatment. In contrast, leptomeningeal, osteoblast, and retinal pigment epithelial storage reduction depended on enzyme dose rather than age at initiation of therapy. Fixed tissue macrophage storage was reduced in all treated MPS VII mice, even those receiving a single dose. These observations indicate that fixed tissue macrophages in MPS VII mice remain sensitive to enzyme replacement therapy well into adulthood although neurons are responsive or accessible to enzyme therapy early in life. Because early initiation of enzyme replacement is important to achieve a central nervous system response, these studies emphasize the importance of newborn screening for lysosomal storage diseases so that early treatment can maximize the likelihood of a favorable therapeutic response. more...

the feet was appreciated. Bilateral hydroceles were noted. Results of neurologic examination were normal. A chest x-ray film showed marked cardiomegaly and normal pulmonary vascularity. Sinus tachycardia was apparent from the electrocardiogram with a heart rate of 160/min, QRS axis 120°. The PR interval was shortened to 0.08 seconds; there was severe biventricular hypertrophy with ST-T wave changes. The echocardiogram revealed severe left and right ventricular hypertrophy with compressed ventricular cavities (Fig 1). The left ventricular end-diastolic dimension was 18 mm (normal, 20 mm), and the end-systolic dimension was 6 mm (normal 13 mm). The left ventricular end-diastolic wall thickness was 12 mm (normal, 3.5 mm). The left ventricular shortening fraction was increased to 50%. There was anterior motion of the mitral valve during systole, and Doppler study showed mild subaortic obstruction. There was no aortic valve stenosis or coarctation of the aorta. Results of a skeletal survey were normal. Initial laboratory evaluation revealed: creatine kinase, 17.10 microkatal (μkat)/L (normal, 0.50 to 3.67 μkat/L [1026 U/L; normal, 30 to 220 /U/L]); serum total carnitine, 11 nmol/mL (normal, 17 to 46 nmol/mL); free carnitine, 8 nmol/mL (normal, 10 to 29 nmol/mL); urine catecholamines, 8 μg/24 h (normal more...

Toxoplasmosis can cause polymyositis either by reactivation or by recent infection. Inconsistent response to antiprotozoal therapy has been the strongest argument against toxoplasmic polymyositis as a separate entity. We report a biopsy-proven case of toxoplasmic polymyositis in a cardiac transplant patient presenting with a severe proximal weakness, myopathic, electromyographic changes and ten-fold increase of anti-Toxoplasma antibodies. An early antiprotozoal therapy and plasmapheresis led to recovery. A review of previously reported cases of toxoplasmic polymyositis suggests that an early antiprotozoal therapy is the most important variable affecting the outcome of this disease. We propose that toxoplasmic polymyositis has two phases: acute, responsive to antiprotozoal therapy, and chronic, manifested by altered immune response requiring steroids. We suggest that all patients presenting with polymyositis should have serological tests for toxoplasmosis as a part of their initial evaluation and an early trial of antiprotozoal therapy in case of positive findings. more...

Mucopolysaccharidosis type VII (Sly syndrome) is a lysosomal storage disease caused by inherited deficiency of the lysosomal enzyme beta-glucuronidase. A murine model of this disorder has been well characterized and used to study a number of forms of experimental therapies, including gene therapy. We produced recombinant adenovirus that expresses human beta-glucuronidase and administered this recombinant adenovirus to beta-glucuronidase-deficient mice intravenously. The beta-glucuronidase activities in liver and spleen were elevated to 40% and 20%, respectively, of the heterozygote enzymatic level at day 16. Expression persisted for at least 35 days. Pathological abnormalities of these tissues were also improved, and the elevated levels of urinary glycosaminoglycans were reduced in treated mice. However, the beta-glucuronidase activity in kidney and brain was not significantly increased. After administration of the recombinant adenovirus directly into the lateral ventricles of mutant mice, the beta-glucuronidase activity in crude brain homogenates increased to 30% of heterozygote activity. Histochemical demonstration of beta-glucuronidase activity in brain revealed that the enzymatic activity was mainly in ependymal cells and choroid. However, in some regions, the adenovirus-mediated gene expression was also evident in brain parenchyma associated with vessels and in the meninges. These results suggest that adenovirus-mediated gene delivery might improve the central nervous system pathology of mucopolysaccharidosis in addition to correcting visceral pathology. more...