Effect of enzyme replacement therapy on bone formation in a feline model of mucopolysaccharidosis type VI.

A range of skeletal abnormalities are evident in mucopolysaccharidosis type VI (MPS VI, Maroteaux-Lamy syndrome) including short stature and dysostosis multiplex, resulting from a deficiency in the lysosomal hydrolase N-acetylgalactosamine-4-sulphatase (4S). In this article, bone pathology was assessed in a feline model of MPS VI to evaluate the efficacy of enzyme replacement therapy (ERT) as a treatment modality for this genetic disorder. Osteopenia is clearly evident in MPS VI animals, with bone mineral volume (BV/TV) falling well below that of normal animals (4.39% vs. 20.11%, respectively). Trabecular bone architecture was also affected in MPS VI with fewer, thinner, and more widely spaced trabeculae apparent. Bone formation rate (BFR/BS) was also lower in MPS VI animals than controls (0.0011 mm3/mm2 per day vs. 0.008 mm3/mm2 per day, respectively). Vertebral and tibial bone length in MPS VI animals progressively fell behind normal values with increasing age, as did cortical bone thickness. Vertebral body shape was also altered. ERT with recombinant human 4S (rh4S) resulted in a vertebral BV/TV of 8.23% in animals treated with an intravenous enzyme dose of 1 mg/kg and a BV/TV of 14.33% in animals treated with a dose of 5 mg/kg. BFR/BS also increased to 0.0034 mm3/mm2 per day in animals treated with enzyme doses of either 1.0 or 5.0 mg/kg rh4S. All other affected histomorphometric parameters also improved with ERT to a level intermediate between MPS VI untreated animals and normals. However, individual animals treated with 0.2 mg/kg rh4S intravenously or 1.0 mg/kg rh4S administered subcutaneously did not exhibit an improvement over untreated MPS VI animals. Vertebral and tibial bone lengths, tibial cortical bone thickness, and vertebral body shape also responded to ERT, with a trend away from the untreated group. Thus, ERT had a positive effect on bone development in MPS VI animals that was dependent upon the dose of enzyme administered and the route of administration.

Enzyme replacement therapy in a feline model of MPS VI: modification of enzyme structure and dose frequency.

Enzyme replacement therapy (ERT) in the MPS VI cat is effective at reducing or eliminating pathology in most connective tissues. One exception is that cartilage and chondrocytes remained distended with extensive lysosomal vacuolation after long-term, high-dose ERT. In this study, we demonstrate that recombinant human N-acetylgalactosamine-4-sulphatase (4S) is taken up by chondrocytes via a mannose-6-phosphate-dependent mechanism and is effective at removing MPS storage. In vitro, the penetration of 4S into articular cartilage is low (partitioning coefficient = 0.06) and i.v. administered enzyme does not distribute significantly into articular cartilage in vivo. To alter the tissue distribution of 4S, the enzyme was coupled to ethylene diamine or poly-L-lysine, increasing its overall charge and diffusion into cartilage, and the dosing frequency of unmodified 4S was increased. Modification resulted in active 4S that maintained its ability to correct MPS storage and increased the partitioning coefficient of 4S into cartilage by 77% and 50% for ethylene diamine and poly-L-lysine, respectively. However, in vivo ERT studies demonstrated that response to therapy was not significantly improved by either the enzyme modifications or change to the dosing regimen, when compared with ERT with unmodified enzyme. Distribution experiments indicated the majority of enzyme is taken up by the liver irrespective of modification. To optimize therapy and improve the amount of enzyme reaching cartilage and other tissues demonstrating poor uptake, it may be necessary to bypass the liver or prolong plasma half-life so that proportionately more enzyme is delivered to other tissues.

Histomorphometric analysis of the tibial growth plate in a feline model of mucopolysaccharidosis type VI.

Mucopolysaccharidosis type VI (MPS VI) is a genetically inherited lysosomal storage disorder. Severely affected children exhibit a range of skeletal abnormalities including short stature, facial dysmorphia, and dysostosis multiplex. Naturally occurring and transgenic animal models of MPS VI are also found which exhibit pathology similar to the human disorder. In this paper we have characterized the formation of trabecular bone from growth plate cartilage in a feline model of MPS VI. Tibial trabecular bone was shown to be osteopenic in MPS VI animals with a bone mineral volume (BV/TV) of 4.51% compared with a BV/TV of 15.64% in normal animals. In addition to osteopenia, a rearrangement of trabecular bone architecture was also observed in MPS VI tibiae, with fewer, thinner trabeculae noted; bone formation rate was also decreased. These observations support those previously made in the L5 vertebrae of MPS VI animals. When the sequential formation of growth plate cartilage structural elements, their transition into primary bone spongiosa, and remodeling into secondary bone spongiosa was characterized, no difference between normal and MPS VI could be detected in the number of cartilage septae and their arrangement in the proliferative and hypertrophic regions of the growth plate or trabecular elements in the primary spongiosa. However, a deviation from normal was observed in the resting zone of the growth plate and in the secondary spongiosa of bone. Thus, the osteopenia observed in MPS VI bone appears to arise primarily from a defect in bone production within the metaphysis and diaphysis rather than the creation of an abnormal template in the preceding growth plate cartilage.

Molecular detection of blood-borne epithelial cells in colorectal cancer patients and in patients with benign bowel disease.

In colorectal cancer (CRC), a proportion of patients with early stage disease still die of metastatic or recurrent disease within 5 years of "curative" resection. Detection of carcinoma cells in the peripheral circulation at presentation may identify a subgroup of patients with micro-metastatic disease who may benefit from adjuvant chemotherapy or radiotherapy. Our aim was to determine the presence and clinical significance of colon carcinoma cells in peripheral blood at the time of surgery. Preoperative peripheral blood samples were collected from 94 patients with CRC and 64 patients undergoing bowel resection for benign conditions (adenoma, diverticular disease or Crohn's colitis). Blood was also obtained from 20 normal donors not undergoing bowel surgery. Immunomagnetic beads were used to isolate epithelial cells followed by reverse transcription-polymerase chain reaction (RT-PCR) analysis of expression of cytokeratin (CK) 19, CK 20, mucin (MUC) 1 and MUC 2. Nineteen of 94 (20%) CRC patients were positive for epithelial cells in preoperative blood, including 6 with early stage disease. Kaplan-Meier survival analysis showed that detection of epithelial cells in preoperative blood was associated with reduced disease-free and overall survival (log-rank test, p = 0.0001). Surprisingly, circulating epithelial cells were detected in 3/30 (10%) patients resected for adenoma, and in 4/34 (12%) patients resected for benign inflammatory conditions, suggesting that cells from nonmalignant colonic epithelium may also gain entry into the bloodstream in the presence of bowel pathology. All 20 normal control bloods were negative for epithelial cells.