Michael F. Dean

Enzyme replacement therapy by fibroblast transplantation: long-term biochemical study in three cases of Hunter's syndrome.

We have assessed the effectiveness of transplanted histocompatible fibroblasts as a long-lived source of lysosomal enzymes for replacement therapy in three patients with Hunters syndrome, over periods ranging from 2.5 to 3.75 yr. The level of Hunter corrective factor excreted by all three patients increased after transplantation, as did the activity of alpha-L-idurono-2-sulfate sulfatase in serum, when measured directly with a radioactive disulfated disaccharide substrate. Sulfatase activity was also raised in leukocyte homogenates from the two patients that we were able to assess. These increases in enzyme activity were accompanied by corresponding increases in catabolism of heparan and dermatan sulfates, as shown by (a) a decrease in sulfate:uronic ratios of urinary oligosaccharides, (b) an increase in iduronic acid monosaccharide, and (c) a normalization of Bio-Gel P-2 gel filtration profiles. Both the increase in enzyme activity and increased catabolism were maintained during the period of study and were not affected by either a gradual decrease or total withdrawal of immunosuppressive therapy.

Identification of a thioredoxin — related protein associated with plasma membranes

A low molecular weight membrane associated sulphydryl protein was detected on a wide range of nucleated cells when [14C]-iodoacetamide was used as a probe. This protein was extracted from THP-1 monocytes, purified to homogeneity and its isoelectric point, Mr and N-terminal amino acid sequence determined. These were shown to be almost identical to the corresponding values for both human thioredoxin and a Tac interleukin-2 receptor activator, indicating that the protein may be a member of this family and function as an essential growth factor.

AN N-terminal peptide from link protein stimulates proteoglycan biosynthesis in human articular cartilage in vitro

OBJECTIVE To determine the effects of a synthetic N-terminal peptide from link protein on the synthesis of proteoglycans by human articular cartilage. METHODS Explants from adult knee cartilage were maintained for 4 days in serum-free Dulbeccos modified Eagles medium. Peptides were added for the final 2 days of culture. Synthesis of proteoglycans and proteins was measured by the incorporation of 35S-sulfate and 3H-serine. The sizes, sulfation patterns, and serine: sulfate ratios of newly synthesized glycosaminoglycans were measured by gel chromatography, high performance liquid chromatography, and ion-exchange chromatography. RESULTS The N-terminal peptide stimulated proteoglycan synthesis in cartilage from a wide age range of patients of both sexes. The newly synthesized glycosaminoglycans were identical in size and composition to those of control tissues, and their serine:sulfate ratios remained unchanged. CONCLUSION This N-terminal peptide, which can be liberated from proteoglycan aggregates by proteolysis, potently stimulated the synthesis of proteoglycans with normal glycosaminoglycan chains. The results suggest that the N-terminal peptide may have a regulatory role in maintaining the integrity of human cartilage matrix.

Enzyme replacement therapy by fibroblast transplantation in a case of Hunter syndrome

SUPPLEMENTATION of deficient enzymes essential for complete catabolism of glycosaminoglycans (GAG) has been used with limited success in several types of mucopolysaccharidosis1–4. The beneficial effects and concomitant changes in urinary GAG after this form of treatment, however, have been only transient, presumably because of the short life in vivo of the enzymes involved5–7. Because of this limitation, we recently tried, by means of skin transplantation, to provide a more permanent source of corrective enzymes in a patient with Hunter syndrome8. Although two HLA antigens from each donor were incompatible with those of the patient and both grafts had been visibly rejected within 3 months, there was a marked increase in breakdown and excretion of GAG subsequent to treatment, which lasted for more than 9 months. In addition, the activity of Hunter corrective enzyme isolated from the patients urine, was also significantly increased. We attributed the effectiveness of the skin transplant to the release of Hunter corrective factor by donor cells and its uptake by host cells, in a manner analogous to that described for fibroblasts in vitro9–11. We have now attempted to increase further both the effectiveness and longevity of replacement therapy, using fully histocompatible skin fibroblasts injected sub-cutaneously as a source of corrective enzyme. An advantage of this procedure is that surgery is not required and it would in principle be applicable to other genetic deficiency diseases of lysosomal enzymes.

THE MACROMOLECULAR CHARACTERISTICS OF CARTILAGE PROTEOGLYCANS DO NOT CHANGE WHEN SYNTHESIS IS UP-REGULATED BY LINK PROTEIN PEPTIDE

Previous studies have shown that a synthetic, unglycosylated analogue of the N-terminal peptide from link protein can function as a growth factor and up-regulate proteoglycan biosynthesis in explant cultures of normal human articular cartilage from a wide age range of subjects (McKenna et al., Arthritis Rheum. 41 (1998) 157-162). The present work further shows that link peptide increased proteoglycan synthesis by cartilage cultured in both the presence and absence of serum, suggesting that the mechanism of up-regulation may be different from that of insulin-like growth factors. The proteoglycans synthesised during stimulation with link peptide were of normal hydrodynamic size and the ratio of core protein to glycosaminoglycan side chains and the proportions of the large proteoglycan aggrecan to the small proteoglycans, decorin and biglycan, remained constant. Aggrecan molecules were equally capable of forming aggregates as those from control tissues and the relative proportions of decorin and biglycan were unchanged showing that both were co-ordinately up-regulated. These results confirmed that this novel peptide is a potent stimulator of proteoglycan synthesis by articular cartilage and showed that the newly synthesised proteoglycans were of normal composition.

A Form of Mucopolysaccharidosis with Visceral Storage and Excessive Urinary Excretion of Chondroitin Sulphate

A case of mucopolysaccharidosis is described, in which the principal glycosaminoglycan stored in the liver and excreted in the urine was chondroitin sulphate. Both isomers were present in equal amounts. The clinical features were similar to those of the Hurler syndrome or mucopolysaccharidoses type I (McKusick 1966).

Receptor-mediated endocytosis of fibroblast β-glucuronidase by peritoneal macrophages

beta-Glucuronidase secreted by mouse 3T3 fibroblasts in vitro was taken up into mouse peritoneal macrophages and into human fibroblasts by a process which was rapid and saturable. High concentrations of mannose-containing compounds inhibited uptake into macrophages but had no effect on uptake into fibroblasts. Mannose-6-phosphate inhibited uptake into both types of cell, reducing uptake into macrophages by 34% and abolishing uptake into fibroblasts completely at a concentration of 5 mM. Fructose-1-phosphate was almost equally as effective at inhibiting uptake into fibroblasts but had no effect on macrophages. Pre-treatment of beta-glucuronidase with alkaline phosphatase totally prevented its uptake into fibroblasts but had no effect on its uptake into macrophages. These results indicate that fibroblasts can secrete a lysosomal enzyme in a form recognised as a high uptake ligand not only by other fibroblasts but also by peritoneal macrophages and that endocytosis appears to be mediated by different receptors present on each type of cell. This has important implications for the potential treatment of mucopolysaccharidoses by fibroblast transplants.

Inhibition of Macrophage Migration by a Proteoglycan Extracted from Kurloff Cells of the Guinea-Pig

Proteoglycan extracted from the Kurloff cells of the guinea-pig spleen and its corresponding glycosaminoglycan inhibits the migration of macrophages from capillary tubes in vitro, but the proteoglycan has no effect on lymphocyte transformation in vitro. A proteoglycan, chemically and spectrally similar to Kurloff cell proteoglycan, extracted from human spleen, also inhibits macrophage migration. In contrast, proteoglycan from cartilage and its corresponding glycosaminoglycan had no significant effect. The differences between Kurloff cell proteoglycan and migration inhibitory factor (MIF) are discussed.

The effect of link peptide on proteoglycan synthesis in equine articular cartilage

The basal rate of in vitro proteoglycan (PG) synthesis in explants of equine articular cartilage was subject to considerable variation in animals of the same age but was greater in younger than older animals. Synthesis of PGs in explant cultures was stimulated by a synthetic link peptide, identical in sequence to the N-terminus of the link protein (LP) of PG aggregates, in a similar manner to that demonstrated previously for human articular cartilage [Biochem. Soc. Trans. 25 (1997) 427; Arthritis Rheum. 41 (1998) 157]. Stimulation occurred in tissue from animals ranging from 1 to 30 years old but older animals required higher concentrations of peptide to produce a measurable response. Synthesis of PGs increased in a concentration-dependent manner and was paralleled by increases in the ability of aggrecan monomers to form aggregates with hyaluronan (HA). In addition to its effect on synthesis of PGs, link peptide also increased synthesis of both aggrecan and LP mRNA. Cartilage explant and chondrocyte cultures secreted small amounts of biologically active interleukin 1 (IL 1) and secretion of this cytokine was reduced considerably by the addition of link peptide. Reduction in the activity of this catabolic cytokine coupled with the increased synthesis of mRNA for aggrecan and link peptide may be the mechanism by which link peptide exerts its positive effect on the rate of PG synthesis in articular cartilage.

Proteoglycans from sheep, pig, rat and human spleens having chemical and biological resemblances to that in Kurloff cells

Kurloff cells appear to be lymphocytes which contain characteristic inclusions [ 1, 21 that have been shown to consist mainly of a chondroitin sulphate protein [3,4]. The infrared spectrum of this proteoglycan shows prominent unidentified bands absorbing at 805 cm-i and 1260 cm-’ [4, 51 in addition to those bands at 720 cm-‘, 850 cm-’ and 928-r characteristic of chondroitin 4sulphate [6] , while the material also shows strong ultraviolet absorption with maxima at 257 nm in acid medium and at 265 nm in alkaline medium. Samples of the proteoglycan and of the corresponding glycosaminoglycan derived from it by proteolysis have been shown to be specifically toxic to macrophages in vitro at high dilution [7]. The spleens of pregnant or oestrogen treated guineapigs are a rich source of Kurloff cells and have been used as a starting material for the preparation of proteoglycans [3,4,8]. Although Kurloff cells, identifiable histologically by their characteristic inclusion bodies, have not been described in species other than the guinea-pig, this report describes. the purification and characterization of proteoglycans from sheep, pig, rat and human spleens obtained during pregnancy. 2. Experimental