Behavior and therapeutic efficacy of beta-glucuronidase-positive mononuclear phagocytes in a murine model of mucopolysaccharidosis type VII.

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 beta-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 beta-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.

Enzyme replacement therapy for murine mucopolysaccharidosis type VII leads to improvements in behavior and auditory function.

Mucopolysaccharidosis type VII (MPS VII; Sly syndrome) is one of a group of lysosomal storage diseases that share many clinical features, including mental retardation and hearing loss. Lysosomal storage in neurons of the brain and the associated behavioral abnormalities characteristic of a murine model of MPS VII have not been shown to be corrected by either bone marrow transplantation or gene therapy. However, intravenous injections of recombinant beta-glucuronidase initiated at birth reduce the pathological evidence of disease in MPS VII mice. In this study we present evidence that enzyme replacement initiated at birth improved the behavioral performance and reduced hearing loss in MPS VII mice. Enzyme-treated MPS VII mice performed similarly to normal mice and significantly better than mock- treated MPS VII mice in every phase of the Morris Water Maze test. In addition, the auditory function of treated MPS VII mice was dramatically improved, and was indistinguishable from normal mice. These data indicate that some of the learning, memory, and hearing deficits can be prevented in MPS VII mice if enzyme replacement therapy is initiated early in life. These data also provide functional correlates to the biochemical and histopathological improvements observed after enzyme replacement therapy.

Gene therapy for murine mucopolysaccharidosis type VII.

Mucopolysaccharidosis type VII (MPS VII) is caused by a deficiency in the lysosomal enzyme beta-glucuronidase resulting in the accumulation of undegraded glycosaminoglycans in many tissues. A murine model of MPS VII shares many of the clinical, biochemical and histopathological features of human MPS VII and has provided an opportunity to study novel therapeutic approaches in a system with a uniform genetic background. Retroviral mediated gene therapy directed to the hematopoietic system or to artificial neo-organs resulted in low levels of enzyme in several tissues and reduced lysosomal storage in the liver and spleen. Partial correction of the disease in the eye was observed following an intravitreal injection of recombinant adenovirus. Neither retroviral nor adenoviral mediated gene transfer techniques resulted in a systemic reduction of lysosomal storage. Here we discuss several novel gene transfer approaches designed to increase the systemic levels of beta-glucuronidase in the MPS VII mouse.

FimH adhesin of type 1 pili is assembled into a fibrillar tip structure in the Enterobacteriaceae.

Type 1 pili are heteropolymeric mannosebinding fibers produced by all members of the Enterobacteriaceae family. The bulk of the fiber is composed of FimA. Two macromolecular complexes responsible for mediating an interaction with mannose-containing receptors were purified from fimA- Escherichia coli by mannose affinity chromatography and ion-exchange chromatography. One complex contained only the mannose-binding adhesin, FimH, associated with FimG, a minor component of the type 1 pilus. In the other complex the FimG-FimH moiety was loosely associated with a chaperone-minor subunit complex (FimC-FimF), possibly representing an intermediate in tip fibrilla assembly. The FimC chaperone has also been shown to form a preassembly complex with FimH that has been purified and characterized previously. Purified FimC did not bind to the FimG-FimH complex but did recognize FimH dissociated from the FimG-FimH complex. Quick-freeze deep-etch electron microscopy revealed that the FimG-FimH complex had a thin fibrillar architecture. High-resolution electron microscopy of type 1 pili revealed that a 16-nm fibrillar tip structure with an architecture identical to that of the FimG-FimH complex was joined end-to-end to the pilus rod. In a fimH- deletion mutant, the tip fibrillae joined to pilus rods were approximately 3 nm in length. The full-length tip fibrilla was restored by complementation with the fimH gene in trans. The bipartite nature of the type 1 pilus was also demonstrated on pili purified from clinical isolates of members of the Enterobacteriaceae family arguing that it is a conserved feature of the type 1 pilus.

FimC is a periplasmic PapD-like chaperone that directs assembly of type 1 pili in bacteria.

Biogenesis of the type 1 pilus fiber in Escherichia coli requires the product of the fimC locus. We have demonstrated that FimC is a member of the periplasmic chaperone family. The deduced primary sequence of FimC shows a high degree of homology to PapD and fits well with the derived consensus sequence for periplasmic chaperones, predicting that it has an immunoglobulin-like topology. The chaperone activity of FimC was demonstrated by purifying a complex that FimC forms with the FimH adhesion. A fimC1 null allele could be complemented by the prototype member of the chaperone superfamily, PapD, resulting in the production of adhesive type 1 pili. The general mechanism of action of members of the chaperone superfamily was demonstrated by showing that the ability of PapD to assemble both P and type 1 pili was dependent on an invariant arginine residue (Arg-8), which forms part of a conserved subunit binding site in the cleft of PapD. We suggest that the conserved cleft is a subunit binding feature of all members of this protein family. These studies point out the general strategies used by Gram-negative bacteria to assemble adhesins into pilus fibers, allowing them to promote attachment to eukaryotic receptors.