Mucopolysaccharidosis type IIIB (MPS IIIB, Sanfilippo syndrome type B) is a lysosomal storage disease characterized by profound intellectual disability, dementia, and a lifespan of about two decades. The cause is mutation in the gene encoding α-N-acetylglucosaminidase (NAGLU), deficiency of NAGLU, and accumulation of heparan sulfate. Impediments to enzyme replacement therapy are the absence of mannose 6-phosphate on recombinant human NAGLU and the bloodbrain barrier. To overcome the first impediment, a fusion protein of recombinant NAGLU and a fragment of insulin-like growth factor II (IGFII) was prepared for endocytosis by the mannose 6-phosphate/ IGFII receptor. To bypass the blood-brain barrier, the fusion protein ("enzyme") in artificial cerebrospinal fluid ("vehicle") was administered intracerebroventricularly to the brain of adult MPS IIIB mice, four times over 2 wk. The brains were analyzed 1-28 d later and compared with brains of MPS IIIB mice that received vehicle alone or control (heterozygous) mice that received vehicle. There was marked uptake of the administered enzyme in many parts of the brain, where it persisted with a half-life of approximately 10 d. Heparan sulfate, and especially disease-specific heparan sulfate, was reduced to control level. A number of secondary accumulations in neurons [β-hexosaminidase, LAMP1(lysosome-associated membrane protein 1), SCMAS (subunit c of mitochondrial ATP synthase), glypican 5, β-amyloid, P-tau] were reduced almost to control level. CD68, a microglial protein, was reduced halfway. A large amount of enzyme also appeared in liver cells, where it reduced heparan sulfate and β-hexosaminidase accumulation to control levels. These results suggest the feasibility of enzyme replacement therapy for MPS IIIB. M ucopolysaccharidosis type III (MPS III, Sanfilippo syndrome) is a heritable lysosomal disorder of heparan sulfate degradation, divided into four types (A-D), depending on the enzyme deficiency (1, 2). All four MPS III types are characterized by severe neurologic problems and relatively mild somatic ones. Profound intellectual disability that progresses to dementia, behavioral disturbances, and death in the second or third decade bring untold suffering to the MPS III patients and their families. Despite the dire need, treatment for the MPS III disorders has lagged behind other MPS diseases. Hematopoietic stem cell transplantation, an effective procedure for MPS I patients with CNS involvement (3), is not effective for MPS III (4). Enzyme replacement therapy has been available for some years for several MPS with extensive somatic involvement [MPS I (5, 6), II (7), and VI (8)], or is newly approved (MPS IVA), or in clinical trial (MPS VII). However, development of enzyme replacement for MPS III did not seem promising because access to therapeutic enzyme to brain parenchyma would be limited by the blood-brain barrier. With respect to MPS IIIB, a deficiency of α-N-acetylglucosaminidase, EC 3.2.1.50) (NAGLU), there is an additional difficulty in that, in contrast to ...

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(Received 11 September 2001 ; revised 11 January 2002 ; accepted 4 February 2002)  A multiplex allele specific polymerase chain reaction (MAS-PCR) based on the Cryptosporidium parvum dihydrofolate reductase (dhfr) gene sequence differentiates genotype 1 (' Human ') from 2 (' Cattle ') in a 1-step reaction. The MAS-PCR was validated on a panel of 34 microscopically positive C. parvum faecal samples of human and animal origin in comparison with 2 published PCR-restriction fragment length polymorphism (RFLP) methods targeting dhfr and the oocyst wall protein (cowp) genes. A validation panel of 37 negative faecal samples of human and animal origin was also tested in comparison with the cowp PCR-RFLP. MAS-PCR was found to be as sensitive for species detection as the most sensitive of the other tests, and detected more mixed genotype infections than the two other tests combined. In addition the MAS-PCR showed equivalent detection sensitivity in comparison with a published nested RFLP targeting the SSU rRNA gene, on a panel of prepared mixed genotype samples. The 1-step reaction is simpler and less expensive to perform than the RFLP methods, while the C. parvum specific amplicons and those for genotypes 1 and 2 (575, 357 and 190 bp respectively) can be easily distinguished on agarose gel.

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