Search

SUMMARY 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). RESUME Une forme de mucopolysaccharidose avec surcharge viscerale et elimination urinaire excessive de chondroitine sulfate Les auteurs decrivent un cas de mucopolysaccharidose dans lequel le principal glycosaminoglycan stocke dans le foie et elimine dans les urines etait la chondroitine sulfate. Les deux isomeres etaient presents en quantiteegaie. Les symptomes cliniques etaient semblables e ceux du syndrome de Hurler autrement dit du type I des mucopolysaccharidoses selon la classification de McKusick en 1966. ZUSAMMENFASSUNG Eine Form von Mukopolysaccharidose, bei der Chondroitinsulfat in Organen gespeichert und in hohem Maβe in Urin ausgeschieden wird Es wird ein Fall von Mukopolysaccharidose beschrieben, bei dem von Glykoaminogly-kanen hauptsachlich das Chondroitinsulfat in der Leber gespeichert und im Urin ausgeschieden wurde. Beide Isomeren waren in gleichem Mase vertreten. Das klinische Bild ahnelte dem beim Hurler Syndrom oder bei der Mukopolysaccharidose Typ I, wie auch von McKusick 1966 berichtet wurde. RESUMEN Una forma de mucopolisacaridosis con almacenamiento visceral y excesiva excrecion urinaria de condroitin sulfato Se describe un caso de mucopolisacaridosis en que el principal acumulo de glicosamino-glican en el higado y excretado en orina era el condroitin sulfato. Ambos isomeros estaban presentes en cantidades iguales. Los caracteres clinicos eran similares a los del sindrome de Hurler o mucopolisacaridosis tipo I descrita por McKusick en 1966.

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 Dulbecco's modified Eagle's medium. Peptides were added for the final 2 days of culture. Synthesis of proteoglycans and proteins was measured by the incorporation of 35 S-sulfate and 3 H-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.

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.

Previous studies have shown that a peptide identical in sequence to the N-terminal of link protein can function as a growth factor and up-regulate proteoglycan synthesis by human articular cartilage in explant culture (L. A. McKenna et al., Arthritis Rheum. 41, 157-162, 1998). The present study has extended these investigations to determine the effects of this peptide on the synthesis of collagen, another essential component of normal cartilage matrix. Explants from normal adult knee cartilage were maintained for periods of up to 8 days in medium with or without serum. Peptides were added during each day of culture. Synthesis of collagen was determined by the incorporation of [3H]proline into hydroxyproline and proteoglycans by incorporation of [35S]sulfate. The type of newly synthesized collagen was measured by SDS-polyacrylamide gel electrophoresis, fluorography, and immunoblotting. The link protein peptide stimulated synthesis of type II collagen in cartilage from a number of different subjects. Maximum up-regulation of synthesis was attained at a concentration of 100 ng/ml, similar to that observed previously for up-regulation of proteoglycan. Synthesis was up-regulated in both the presence and the absence of serum, although the overall rate of synthesis was greater when serum was added. The findings that this link peptide growth factor stimulated synthesis of proteins, including collagen, in a manner analogous to that shown previously for proteoglycans support the hypothesis that this peptide may have an important role in the feedback control of cartilage matrix synthesis.

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.

To determine the effects of a synthetic N-terminal peptide from link protein on the synthesis of proteoglycans by human articular cartilage.Explants from adult knee cartilage were maintained for 4 days in serum-free Dulbecco's modified Eagle's 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.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.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.

A surface-associated sulphydryl (thiol) protein (SASP) constitutively present in most nucleated cells was purified from human THP-1 monocytes and rat C6 glioma cells. The human protein was similar in mass and isoelectric point and had the same N-terminal amino acid sequence to adult T-cell leukemia-derived factor (ADF), a growth factor secreted by human lymphoid cells which is able to induce increased expression of interleukin-2 receptors. A further internal amino acid sequence, determined following cleavage of human SASP with cyanogen bromide, was also identical to the corresponding sequence deduced for ADF. Samples of SASP were able to reductively depolymerize human immunoglobulin, a property shared with thioredoxin, a ubiquitous protein, almost identical to ADF, with an essential function in many thiol-dependent reducing reactions. Furthermore, SASP purified from rat C6 glioma cells had an identical N-terminal amino acid sequence to that deduced for rat liver thioredoxin, showing that they were both members of the same family of proteins. The use of membrane-impermeable thiol reagents indicated that SASP was predominantly a cell-surface protein, and was not normally secreted. This SASP protein appeared to be a surface-associated form of thioredoxin that was constitutively present in a wide range of cells and was related to ADF, a secreted form of the same protein.

Antibodies raised to human placental beta-glucuronidase were shown to cross-react with the beta-glucuronidase secreted by mouse 3T3 fibroblasts, but did not react with other lysosomal enzymes. The beta-glucuronidase secreted by 3T3 cells was purified 15000-fold by chromatography on an affinity column made from this antibody and resolved into a single component, of Mr 68000, by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Iodinated samples of purified enzyme were taken up into mouse peritoneal macrophages by receptor-mediated endocytosis at a rate similar to that calculated previously for unlabelled enzyme, and uptake was competitively inhibited by yeast mannan. Binding of beta-glucuronidase to macrophages was saturable, with a Kd of 7 × 10(-9)l/mol, an affinity comparable with that calculated for the binding of mannosylated bovine serum albumin (Kd 1.3 × 10(-9)l/mol), a ligand specific for mannose receptors. Four times as many molecules of mannosylated albumin (12000) as of beta-glucuronidase (3000), however, bound to each cell. This purification and iodination procedure did not therefore have any adverse effect on the uptake properties of secreted beta-glucuronidase, and provides a ligand with which to investigate binding and specific endocytosis into a range of different types of cell.

We have assessed the effectiveness of transplanted histocompatible fibroblasts as a long-lived source of lysosomal enzymes for replacement therapy in three patients with Hunter's 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.

Fibroblasts grown in vitro can secrete lysosomal enzymes into the culture medium which can then be taken up by endocytosis into other fibroblasts1–4. Such cells have been used as a source of enzyme for replacement therapy in the mucopolysaccharidoses5–7, a group of related storage diseases resulting from inherited deficiencies in specific lysosomal enzymes, although the mechanism of action is unclear. Because lymphoid cells can interact directly with fibroblasts8,9 and transfer DNA to them10, we examined the possibility that a lysosomal enzyme could be transferred from normal lymphocytes to deficient fibroblasts by a mechanism other than secretion followed by endocytosis. We report here that fibroblasts from a patient with mucopolysac-charidosis type VII acquired β-glucuronidase (EC 3.2.1.31) activity as a result of direct cell-to-cell contact with lymphocytes.

Human fibroblasts totally deficient in beta-glucuronidase acquired high levels of enzyme activity when co-cultured with mouse or rabbit lymphocytes. Direct cell-to-cell contact was obligatory for this process. The enzyme acquired by the fibroblasts was shown to be identical to beta-glucuronidase from donor lymphocytes by its position of elution from DEAE-cellulose, thermal stability, mobility on polyacrylamide gels and by its antigenic determinants. The enzyme extracted from deficient fibroblasts after co-culture with lymphocytes showed no evidence of any hybridisation between human and mouse or rabbit sub-units. It is concluded that during direct cell interaction, enzymically active beta-glucuronidase is transferred directly from donor lymphocytes to deficient fibroblasts by a mechanism, previously shown not to involve normal receptor mediated endocytosis.

The substrateO-(α-L-idopyranosyuronic acid-2-sulphate)-(1 → 4)-2,5-anhydro-D-(3H-1) mannitol-6-sulphate was used at a final concentration of 50 mmol/l to measure theα-L-idurono-2-sulphate sulphatase activities of cell extracts, serum and amniotic fluid. Activities were measured after dialysis against water, to avoid the inhibitory effect of sodium chloride and the reaction products separated by ion-exchange chromatography on ECTEOLA cellulose. The enzyme present in normal serum had an apparentK m of 0.12 mmol/l. The mean activities of normal serum, fibroblasts and leukocytes were 0.61, 16.63 and 18.75 nmol/mg protein per hour respectively, while corresponding ‘Hunter’ tissues contained between 1% and 3% of normal activity. Cultured normal amniotic cells had a mean activity of 8.46 nmol/mg protein per hour, while those cultured from ‘Hunter’ patients contained about 6% of normal activity. Hunter disease can thus be readily diagnosed in a wide variety of tissues. Samples from three obligate heterozygotes were found to have iduronate sulphatase activities amounting to 41%, 57% and 63% of normal values.

The subcellular distribution of beta-glucuronidase acquired by deficient human fibroblasts during co-culture with peritoneal macrophages was compared with that taken up by receptor-mediated endocytosis. Labelled enzyme taken up via receptors was located initially in a low-density endosomal fraction and was transferred to lysosomes within a few minutes. The beta-glucuronidase acquired during 24 h of co-culture was present almost entirely within lysosomes and had a distribution profile identical with that of endogenous beta-hexosaminidase. Monensin prevented transfer of radiolabelled enzyme from endosomes to lysosomes and had a similar effect on the distribution of enzyme acquired by direct transfer, causing beta-glucuronidase to accumulate within endosomes. When the temperature was lowered from 37 degrees C to 19 degrees C, the rate of transfer of enzyme from endosomes to lysosomes was decreased during both direct transfer and indirect receptor-mediated endocytosis. These results show that a lysosomal enzyme acquired by direct transfer during cell-to-cell contact follows a similar intracellular route and has a similar distribution to that of enzymes taken up via cell-surface receptors.

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.

Oxide films were stripped from a series of stressed Fe-Ni-Cr alloys exposed to boiling 42 percent MgCl2. Evidence of dissolution tunnels was found in Types 304 and 310 stainless steels and Incoloy 800. No tunnels were observed in Incoloy 825 or Inconel 600. The frequency of observed tunnels was proportional generally to the susceptibility of these alloys to stress corrosion cracking in MgCl2. The mechanism for formation of the tunnels appears to be related to the interaction between dislocations and prior irregularities in initial dissolution trenches resulting from rapid dissolution at emergent slip steps. Evidence from this work suggests that the tunnels are a special case of local slip step activated dissolution. Tunneling itself appears not to be an integral part of the mechanism of stress corrosion cracking but can occur as an associated or parallel process.

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 patient's 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.

SUMMARY Infusion of a dextran solution of equivalent osmolality to plasma into a patient with the Sanfilippo syndrome caused an increased excretion of uronic acid. This increase was less than that obtained previously, when an equal amount of plasma was infused into the same patient. Moreover, there were no significant changes in the ratio of larger to smaller glycosaminoglycan fragments or in the sulphate/uronic acid ratios, in contrast to the changes noted subsequent to plasma infusion. It is concluded, therefore, that in this case infusion of dextran was not as effective as plasma in inducing a temporary increased mobilisation of pathological glycosaminoglycans. RESUME Effet de perfusion de dextrane sur l'excretion des glucosaminoglycuroniques dans le syndrome de Sanfilippo La perfusion de solution de dextrane isotonique au plasma chez un malade presentant un syndrome de Sanfilippo a provoque une excretion accrue d'acides uroniques. Cet accroissement a ete moindre que celui obtenu anterieurement, apres perfusion d'une quantiteequivalente de plasma chez le meme sujet. De plus, il n'y a pas eu de modifications significa-tives dans le rapport des fragments glucosaminoglycuroniques lourds et legers, ou dans le rapport sulfate-acide uronique, comme cela avait ete note anterieurement lors de la perfusion de plasma. Les auteurs concluent alors que, dans ce cas, la perfusion de dextrane n'a pas ete aussi efficace que celle de plasma pour provoquer un accroissement temporaire de mobilisation des glucosaminoglycuroniques pathologiques. ZUSAM MENFASSUNG Die Wirkung von Dextraninfusionen auf die Glycosaminoglycanausscheidung beim Sanfilippo-Syndrom Die Infusion einer Dextranlosung mit plasmaaquivalenter Osmolalitat steigert bei einem Patienten mit Sanfilippo-Syndrom die Ausscheidung von Harnsaure. Dieser Anstieg war geringer als er bisher durch Infusion gleicher Mengen Plasma bei demselben Patienten erreicht wurde. Daruberhinaus gab es keine signifikante Anderungder Rate vongroszlig;erenzu kleineren Glycosaminoglycanfragmenten oder im Sulfat/Harnsaureverhaltnis wie sie im Anschlus an Plasmainfusionen beobachtet wurden. Die Autoren stellen daher fest, das in diesem Fall die Infusion von Dextran nicht so wie die von Plasma die zeitweise verstarkt auftretende Mobilisation pathologischer Glycosaminoglycane bewirkte. RESUMEN Efecto de perfusiones de dextrano sobre la excrecion de glucosaminogliean en el sindrome de Sanfilippo La perfusion de una solution de dextrano de una osmolaridad equivalente a la del plasma a un paciente con sindrome de Sanfilippo causo una excrecion aumentada de acido uronico. Este aumento fue menor del obtenido previamente, al perfundir una cantidad igual de plasma en el mismo paciente. Ademas no hubo cambios significativos en la relacion entre los fragmentos mas grandes y mas pequenos de glycosaminoglican, o en la relacion sulfato/acido uronico, como los observados tras una perfusion de plasma. Los autores concluyen, por lo tanto, que en este caso la perfusion de dextrano no fue tan efectiva como la de plasma para inducir una movilizacion temporal aumentada de glycosaminoglicanos patologicos.

Fibroblasts deficient in β-glucuronidase acquired high levels of this enzyme when they were co-cultured with concanavalin A-stimulated lymphocytes. Acquired enzyme activity, determined using a single-cell cytochemical assay, was directly proportional to the number of lymphocytes added and persisted for several days in fibroblasts maintained at high density. Lymphocytes did not secrete significant levels of β-glucuronidase into their culture medium, and did not release other substances able to induce synthesis of the enzyme by the deficient fibroblasts. Nor did β-glucuronidase acquisition result from concanavalin A-mediated uptake of enzyme, since a-methylmannoside did not reduce acquired activity. Moreover, lymphocytes from various sources, whether unstimulated or activated by a different mitogen, bacterial lipopolysaccharide, were equally effective in promoting the appearance of β-glucuronidase. Deficient fibroblasts did not acquire β-glucuronidase by active endocytosis when co-cultured with lymphocytes, since enzyme extracted from lymphocytes was not itself effective in this respect. Furthermore, mannose 6-phosphate, which did inhibit endocytosis by deficient fibroblasts of exogenous β-glucuronidase prepared from 3T3 cells, had no effect on enzyme acquisition by fibroblasts during their co-culture with lymphocytes. Conversely, inhibitors of protein synthesis and energy metabolism, which did not interfere with endocytosis of exogenous enzyme, abolished the acquisition of βglucuronidase during co-culture. Deficient fibroblasts did not acquire β-glucuronidase when they were cultured together with lymphocytes but separated from them by Millipore membranes permeable to exogenous enzyme. Thus, although the mechanism of acquisition is still unclear, the present results suggest that β-glucuronidase is transferred from lymphocytes to deficient fibroblasts by a process in which direct cell-to-cell contact is obligatory.

The first inborn error of metabolism to be recognized was alcaptonuria, described by Garrod in 19081. He developed the concept that some diseases occur because an enzyme which controls a single metabolic step is entirely absent or has a greatly reduced activity. More than 330 such disorders have now been described2, more than 30 of which are lysosomal storage diseases.

Proteoglycan from Kurloff cells, when present in the medium in low concentrations, increased the incorporation of (3-H)leucine into trichloracetic acid-precipitable material by macrophages from peritoneal exudates, in addition to inhibiting their migration from capillary tubes, as observed previously. After treatment with 0.5 M-NaOH, followed by dialysis or ultrafiltration, material with the distinctive u.v. and i.r. spectra of the whole proteoglycan appeared in the diffusate, and biological activity was lost from the proteoglycan which remained in the dialysis residue. The diffusible material absorbed near 260 nm and had i.r. bands at 805 cm-minus-1 and 1260 cm-minus-1, but did not display the i.r. bands characteristic of chondrotin 4-sulphate. It contained little sulphate, no hexosamine and less than 1% of the uronic acid present in the whole proteoglycan, and there were only trace amounts of amino acids, xylose and galactose. However, significant amounts of ribose and organic phosphate were present, each representing about 1% of the whole proteoglycan. After proteolysis and chondroitanase digestion of the proteoglycan, a fraction with absorbance at 260 mn was eluted from Dowex 1 with water which stimulated the incorporation of (3H)leucine by macrophages and inhibited their migration from capillary tubes.

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.

The mucopolysaccharidoses are a group of connective tissue disorders, classified by McKusick (1966), in which specific recessively inherited enzyme deficiencies result in an inadequate catabolism of glycosaminoglycans (GAGs), leading to an excessive accumulation within the tissues and a greatly increased urinary excretion. In pregnancies where it is suspected that the foetus may have inherited one of these disorders, amniocentesis is often carried out in order to ascertain the GAG composition of the amniotic fluid as a diagnostic aid to prophylactic abortion. A number of instances have now been reported (Matalon, Dorfman, Nadler and Jacobson, 1970; Matalon and Dorfman, 1972a; Crawfurd et al., 1973) in which qualitative and quantitative GAG abnormalities have been detected in the amniotic fluid of suspected cases, presumably due to excretion by the foetus. We have carried out further studies of foetal tissue from two cases of Hurler’s syndrome in which GAG abnormalities have been detected following amniocentesis and, in both cases, the GAG abnormalities in the amniotic fluids reflected the abnormalities in the foetal tissues.

Enzyme Replacement Therapy by Transplantation of HLA-Compatible Fibroblasts in Sanfilippo A Syndrome

Although radiological involvement of the lower dorsal and upper lumbar vertebrae is common in the severe form of Hunter's disease (MPS II), there are reports in the literature that clinical kyphosis does not occur. We report a boy with marked clinical kyphosis in whom the diagnosis of MPS II was proved by demonstrating a severe deficiency of serum and leucocyte iduronate-sulphate sulphatase and an accelerated incorporation of radiosulphate into his cultured fibroblast glycosaminoglycans, which could not be corrected by the product of other typed reference MPS II cells. The existence of several other genetic diseases, sometimes complicated by kyphosis, was excluded by assay of fibroblast lysosomal enzymes.

Changes in the activities of several lysosomal enzymes were studied during transformation of mouse spleen cells in vitro. The activity of beta-glucuronidase increased during culture in the presence of T or B-cell mitogens, and lymphoblasts contained higher levels of activity than did small, non-transformed lymphocytes. Moreover, lymphoblasts in well-transformed cultures had higher activities than those in poorly-transformed cultures. The activities of other lysosomal enzymes (N-acetyl-beta-glucosaminidase, alpha-mannosidase, beta-glucosidase) also increased during mitogenic stimulation, but each at different rates, although aryl sulphatase was unaffected. Such differences may be of importance when lymphocytes are used for diagnosis of inherited lysosomal deficiency diseases.

ATTEMPTS to treat the mucopolysaccharidoses with infusions of normal plasma or leukocytes have produced mixed results; some workers1–3 reported little or no positive effect, while others4–6 noted both clinical and biochemical changes after treatment. Among the more obvious reasons for these conflicting findings are differences in the responsiveness to treatment of genetically distinct forms of the disorders7, in the volumes of plasma administered and in the activities of the corrective factors in the plasma.

SUMMARY Two sisters are described who were suffering from an unusual and clinically distinct variant of mucopolysaccharidosis. One sister presented with a slowly progressing paraparesis which was found to be caused by compression of the cervical spinal cord by ligamentous thickening, in association with stenosis of the bony spinal canal. There was rapid and almost complete recovery after decompressive laminectomy. The importance of this finding is discussed in relation to the occurrence of gait disorders in the mucopolysaccharidoses. It is suggested that these two sisters may represent a genetic compound of the Hunter and Scheie syndromes. RESUME Compression de la moelle cervicale et mucopolysachcaridose Les auteurs ont observe deux soeurs souffrant d'une forme inhabituelle et cliniquement distincte de mucopolysaccharidose. L'une des soeurs a presente une paraparesie lentement progressive dont la cause s'est revelee etre une compression de la moelle cervicale par epaississement ligamentaire associe a une stenose du canal rachidien. II y eut recuperation rapide et presque complete apres laminectomie decompressive. L'importance de cette observation est discutee en relation avec la frequence des troubles de la demarche dans les mucopolysaccharidoses. II est suggere que ces deux soeurs pouvaient presenter une combinaison genetique des syndromes de Hunter et de Scheie. ZUSAMMENFASSUNG Halsmarkkompression bei Mukopolysaccharidose Es werden zwei Schwestern beschrieben, die eine ungewohnliche klinisch eindeutige Form der Mukopolysaccharidose hatten. Eine Schwester hatte eine langsam fortschreitende Paraparese bedingt durch die durch ligamentare Verdickungen und Stenose des knochernen Spinalkanals hervorgerufene Kompression des Halsmarks. Nach Dekompression durch Laminektomie trat eine schnelle und fast vollstandige Heilung ein. Dieser Befund wird im Zusammenhang mit Gangschwierigkeiten bei Mukopolysaccharidosen diskutiert. Es wird angenommen, das diese beiden Schwestern eine genetische Mischform des Hunter- und Scheie-Syndroms aufweisen. RESUMEN Compresion de la medula cervical en mucopolisacaridosis Se describen dos hermanas que padecian una variante clinicamente distinta de mucopolisacaridosis. Una hermana presentaba una paraparesia lentamente progiesiva que se hallo que era causada por una compresion de la medula cervical debida a un engrosamiento ligamentoso, junto con estenosis del canal oseo raquideo. Hubo una recuperacion rapida y casi completa despues de una laminectomia descompresiva. La importancia de este hallazgo se discute en relacion con la aparicion de alteracion en la marcha en las mucopolisacaridosis. Se sugiere que estas dos hermanas pueden representar un componente genetico de los sindromes de Hunter y Scheie.

Mucopolysaccharidosis Type I (Hurler's syndrome) was diagnosed in an 18 week old fetus of a woman with 1 affected child by direct assay of glycosaminoglycans in amniotic fluid. Subsequently the fetus was aborted by hysterotomy and diagnosis was confirmed by analyzing glycosaminoglycans in fetal liver, electron micrographs isozymes of galactosidases, and fetal skin sulfate turnover. In this case the amnio tic fluid contained 78% dermatan and heparan sulfates. The fetal liver contained 28.8% dermatan sulfate and 62.4% heparan sulfate among the soluble glycosaminoglycans. Some translucent lysosomal vacuoles with osmiophilic inclusions were evident in liver but not in central nervous system. Isozymes A and B of alpha-galactosidase and beta-galactosidase- B were very low in fetal tissues compared with age-matched controls. 5 other lysosomal enzymes were slightly elevated upon assay with fluorescent substrates. Sulfate turnover studies in cultured fetal skin cells showed excessive accumulation but enhanced release rates in presence of pooled normal human serum (Hurler corrective factor). When these studies were done, the enzyme defect in Hurler's syndrome was known to be alpha-L-iduronidase, but no substrate was available commercially.

Kurloff cells of guinea pigs increase in number and accumulate in the spleen on oestrogen treatment. Because they contain metachromatic inclusions and are considered to be lymphocytes they were examined as a possible model for mucopolysaccharidoses like Hurler's syndrome, where some lymphocytes are also metachromatic. Oestrogen treatment produced a large increase in a glycosaminoglycan resembling chondroitin 4-sulphate in chemical analysis, chromatographic behaviour and i.r. spectrum but with an additional strong band at 805cm−1. Material isolated without proteolysis behaved on gel chromatography as a multiple-chain protein–polysaccharide whose molecular size was decreased by proteolysis. It contained xylose and galactose in molar proportions with serine, compatible with the presence of the same linkage region as in cartilage chondroitin 4-sulphate proteins and which likewise underwent alkaline β-elimination. Kurloff glycosaminoglycan chains were significantly longer than chondroitin sulphate chains of cartilage protein–polysaccharides as assessed by gel chromatography and the molar ratios of galactosamine to xylose or to serine. Kurloff cells thus contain intact rather than partially degraded protein–polysaccharide and hence are not analogous to Hurler cells, and their electron micrographs were also different. The purified Kurloff protein–polysaccharide and glycosaminoglycan isolated here has been shown by Marshall, Swettenham, Vernon-Roberts & Revell (1970) to be toxic specifically to macrophages at extremely low concentrations in vitro, unlike chondroitin sulphate of protein–polysaccharides from cartilage. The toxic constituent may account for the i.r.-absorption band at 805cm−1. Although active incorporation of [35S]sulphate occurs at early stages of Kurloff-cell induction (Marshall et al. 1970), the fully developed Kurloff cell studied here showed very low incorporation in vitro and in vivo, suggesting that the inclusions are specialized for the storage of the toxic material.

Glycosaminoglycans were isolated from the urine of three patients with Hurler's, Hunter's and Morquio's syndromes and also from the liver and spleen of the case of Hurler's syndrome by a procedure avoiding further degradation. A method of determining the proportions of dermatan sulphate, heparan sulphate and chondroitin sulphate in each preparation is described. The relative proportions of these glycosaminoglycans in the urine and organs of the case of Hurler's syndrome were very similar. Glycosaminoglycans from the organs were of much lower molecular weight than normal, consisting of single chains of molecular weight about 5000 together with multiples of up to four such chains attached to peptide moieties. The linkage region normally attaching glycosaminoglycan chains to protein in whole protein–polysaccharides of connective tissue was degraded progressively towards serine. The total output and relative proportions of abnormal glycosaminoglycans in the urine were compared in two brothers with Hunter's syndrome examined on two occasions 4 years apart. At comparable ages they excreted about the same amount, and the relative proportions of each glycosaminoglycan remained essentially constant. The composition and chromatographic behaviour of the glycosaminoglycan in the urine from the case of Morquio's syndrome indicated that it consisted of material containing about one-third keratan sulphate and two-thirds chondroitin sulphate as part of the same molecule, as in proteoglycans of cartilage. The total output of glycosaminoglycans, although higher than normal, was considerably less than in other types of Mucopolysaccharidoses.

Letter to the Editor: Effectiveness of HLA-Compatible Fibroblasts for Enzyme Replacement Therapy in the Mucopolysaccharidoses

A 3-year old boy with the severe form of Hunter syndrome (MPS IIA), was treated by the infusion of plasma from 6 pints of pooled blood over a period of 2 days. Total 24hr uronic acid excretion increased fourfold immediately following infusion. This increase was almost entirely due to glycosaminoglycan fragments not precipitated with 9-aminoacridine although there was also a small increase in the amount of precipitated (polymeric) material. There was an increase in the proportion of glycosaminoglycan fragments of lowest molecular wt. coupled with a decrease in intermediate sized fragments shown by Sephadex gel filtration. Degree of sulphation was stable before infusion, then fell and rose again in a cyclical pattern after treatment. Larged sized precipitable glycosaminoglycan fragments had a noticably higher content of glucosamine than the smaller supernatant fragments suggesting that the heparan sulphate glycosaminoglycans were less readily degradable than dermatan & chondroitin sulphates. This pattern of response differed qualitatively from those we have found in 2 types of (MPS III) (Dean et al, 1973.)