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IntroductionTracking the progress of universal health coverage (UHC) is typically at a country level. However, country-averages may mask significant small-scale variation in indicators of access and use, which would have important implications for policy choice to achieve UHC.MethodsWe conducted a retrospective cross-sectional household and individual-level survey in seven slum sites across Nigeria, Kenya, Bangladesh and Pakistan. We estimated the adjusted association between household capacity to pay and report healthcare need, use and spending. Catastrophic health expenditure was estimated by five different methods.ResultsWe surveyed 7002 households and 6856 adults. Gini coefficients were wide, ranging from 0.32 to 0.48 across the seven sites. The total spend of the top 10% of households was 4–47 times more per month than the bottom 10%. Households with the highest budgets were: more likely to report needing care (highest vs lowest third of distribution of budgets: +1 to +31 percentage points (pp) across sites), to spend more on healthcare (2.0 to 6.4 times higher), have more inpatient and outpatient visits per year in five sites (1.0 to 3.0 times more frequently), spend more on drugs per visit (1.1 to 2.2 times higher) and were more likely to consult with a doctor (1.0 to 2.4 times higher odds). Better-off households were generally more likely to experience catastrophic health expenditure when calculated according to four methods (−1 to +12 pp), but much less likely using a normative method (−60 to −80 pp).ConclusionsSlums have a very high degree of inequality of household budget that translates into inequities in the access to and use of healthcare. Evaluation of UHC and healthcare access interventions targeting these areas should consider distributional effects, although the standard measures may be unreliable.

This paper examines OpenStreetMap data quality at different stages of a participatory mapping process in seven slums in Africa and Asia. Data were drawn from an OpenStreetMap-based participatory mapping process developed as part of a research project focusing on understanding inequalities in healthcare access of slum residents in the Global South. Descriptive statistics and qualitative analysis were employed to examine the following research question: What is the spatial data quality of collaborative remote mapping achieved by volunteer mappers in morphologically complex urban areas? Findings show that the completeness achieved by remote mapping largely depends on the morphology and characteristics of slums such as building density and rooftop architecture, varying from 84% in the best case, to zero in the most difficult site. The major scientific contribution of this study is to provide evidence on the spatial data quality of remotely mapped data through volunteer mapping efforts in morphologically complex urban areas such as slums, the results could provide insights into how much fieldwork would be needed in what level of complexity and to what extent the involvement of local volunteers in these efforts is required.

Within the United Kingdom (UK), assisted living technologies are mostly provided through statutory health and social care services following assessment of individual need and application of eligibility criteria. This paper describes the first UK study to explore and develop business approaches and innovations required to make electronic assisted living technologies more accessible to consumers in their fifties and sixties. A robust mixed-method approach was used including a large sample size for a consumer survey, triangulation of methods and confirmation of research findings through validation workshops. This three-year study makes significant and original contributions to understanding consumer needs in this rapidly changing market and offers unique insights into the needs and wants of people aged 50–70. Analysis shows significant differences between consumer and business perceptions, indicating that marketing is not closely aligned to consumers' needs and is affecting the development of the market. New approaches to consumer-led business models are presented to improve information and marketing aimed at 50–70-year-old consumers. A ‘Broker/Independent Advisor’ business model showed most potential for meeting the needs of both consumer and business stakeholders. Findings support future development of an assisted living consumer market to meet growing levels of need and demand, and to offer greater consumer choice of mainstream technologies to enable people to age in place.

Objective To investigate the effects of culture conditions, serum and specific cytokines such as insulin-like growth factor (IGF) 1 and interleukin (IL) 1alpha on phenotype and cell survival in cultures of Syrian hamster embryonic chondrocyte-like cells (DES4(+).2). Methods Proteins and RNA extracted from subconfluent and confluent early- and late-passage DES4(+).2 cells cultured in the presence or absence of serum and IL-1alpha or IGF-1 or both cytokines together were analysed for the expression of chondrocyte-specific genes and for the chondrogenic transcription factor Sox-9 by Western and Northern blotting. Apoptosis was assessed by agarose gel electrophoresis of labelled low-molecular weight DNA extracted from DES4(+).2 cells and another Syrian hamster embryonic chondrocyte-like cell line, 10W(+).1, cultured under the different conditions and treatments. Results Early passage DES4(+).2 cells expressed chondrocyte-specific molecules such as collagen types alpha1(II) and alpha1(IX), aggrecan, biglycan and link protein and collagen types alpha1(I) and alpha1(X) mRNAs, suggesting a prehypertrophic chondrocyte-like phenotype. The expression of all genes investigated was cell density- and serum-dependent and was low to undetectable in cell populations from later passages. Early-passage DES4(+).2 and 10W(+).1 cells survived when cultured at low cell density, but died by apoptosis when cultured at high cell density in the absence of serum or IGF-1. IGF-1 and IL-1alpha had opposite and antagonistic effects on the chondrocyte phenotype and survival. Whereas IL-1alpha acting alone suppressed cartilage-specific gene expression without significantly affecting cell survival, IGF-1 increased the steady-state mRNA levels and relieved the IL-1alpha-induced suppression of all the chondrocyte-specific genes investigated; it also enhanced chondrocyte survival. Suppression of the chondrocyte phenotype by the inflammatory cytokine IL-1alpha correlated with marked down-regulation of the transcription factor Sox-9, which was relieved by IGF-1. The expression of the Sox9 gene was closely correlated with the expression of the chondrocyte-specific genes under all conditions and treatments. Conclusions The results suggest that the effects of cartilage anabolic and catabolic cytokines IGF-1 and IL-1alpha on the expression of the chondrocyte phenotype are mediated by Sox-9. As Sox-9 appears to be essential for matrix production, the potent effect of IL-1alpha in suppressing Sox-9 expression may limit the ability of cartilage to repair during inflammatory joint diseases.

In UMR-106 osteosarcoma cells we found that PTH activated both the cAMP/protein kinase A and the Ca2+-dependent phosphoinositide/protein kinase C (PKC) pathways, but prostaglandin E2 (PGE2) activated only the cAMP pathway. Activation of PKC by the phorbol ester PMA had no effect on cAMP production but enhanced PTH-stimulated cAMP production by 50% or more; the effect on PGE2-induced cAMP was negligible. Inhibition of the α-subunit of the inhibitory guanine nucleotide binding protein (Gi) by pertussis toxin pretreatment also enhanced PTH-mediated cAMP production but had no effect on PGE2-induced cAMP production. These results suggest that although PTH-mediated adenylate cyclase activity is regulated via both the stimulatory (Gs) and inhibitory (Gi) guanine nucleotide binding proteins, only Gs regulates PGE2-mediated adenylate cyclase activity in UMR-106 cells. Costimulation with pertussis toxin and PMA did not increase PTH-stimulated cAMP production above that obtained with PMA alone. This implies a similar target of action for pertussis toxin and PMA, that is, the α-subunit of Gi. The α-subunit of Gi was found to be a substrate for in vitro PKC phosphorylation of membrane fractions from UMR-106 cells, seen as a ±40 kD band on SDS-PAGE. Stimulation of in situ 32P-labeled cells with either PMA or PTH also enhanced incorporation of 32P into the 40 kD band. Using the peptide antisera AS/7 and EC/2, we showed that pertussis toxin-labeled subunits of both Gi1α/Gi2α and Gi3α could be immunoprecipitated, respectively, but immuinoprecipitation of membrane proteins after in situ phosphorylation and stimulation with PMA precipitated only Gi2α. We therefore conclude that modulation of adenylate cyclase activity by phorbol esters in UMR-106 osteosarcoma cells can be ascribed, at least in part, to PKC-mediated phosphorylation of the α-subunit of the Gi2 component of the adenylate cyclase regulatory complex.

Women Physiologists , edited by Lynn Bindman, Alison Bruding and Tilli Tansey. Portland Press, 1993. £16.95. ISBN 1-85578-021-6. The Physiological Society originated as a scientific dining club in 1879 for men interested in animal physiology. It was not until 1915 that the first woman was elected as a member, and it is the 75th anniversary of this event that is marked by this book, which consists of brief biographies of 18 women physiologists whose scientific careers are outlined. As an introduction there is a short history of women and the Physiological Society. The next section is devoted to those - 8 out of the 18 - who received public recognition in the form of F.R.S. and/or D.B.E.: they are considered at greater length. All the biographies are written by women, themselves physiologists of high standing, who knew their subjects personally or through close associates. (The final section has details of each contributor.)

The chondrocyte is a specialized cell that synthesizes proteoglycans of a type found only in cartilage and nucleus pulposus. These proteoglycans are distinct in forming multiple aggregates of unique structure in which hyaluronic acid provides a central chain to which many proteoglycan molecules are bound at one end only. Chondrocytes were isolated from adult cartilage and used in suspension culture to test the effect of compounds in the medium on the synthesis of proteoglycans. Hyaluronic acid alone, among a number of compounds extracted from or analogous to those in cartilage, reduced the incorporation of [$^{35}$S]sulphate into macromolecular material. Oligosaccharides of hyaluronic acid of the size of decasaccharides and above also had this effect but hyaluronic acid already bound to proteoglycan did not. The proportion of total labelled material associated with the cells increased at the expense of that in the medium. Treatment of the cells with trypsin abolished the effect of hyaluronic acid but treatment with chondroitinase did not. It is suggested that hyaluronic acid interacts with proteoglycans at the cell surface by a specific mechanism similar to that involved in proteoglycan aggregation, as a result of which the secretion and synthesis of proteoglycans is reduced.

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.

A detailed study of the biochemistry of each of the lower lumbar intervertebral discs from 3 spines aged 8, 16, and 44 years has shown progressive changes down the spine in a number of biochemical parameters. These were most apparent in the 44-year-old spine. The chemical composition of proteoglycans of the nucleus pulposus and of its constituent proteoglycans differed from those of the corresponding annulus fibrosus of all three spines. The interaction of proteoglycans with collagen, as assessed by extractability, changed markedly with advancing age, while the molecular size of the proteoglycans from both regions decreased and their keratan sulphate content increased. These changes would be expected to affect the mechanical properties of the disc.

An electric field causes partial alignment of macromolecules in a dilute solution. The accompanying changes in the solution birefringence offer a sensitive and quick means of monitoring the rates of particle orientation and hence the size of the solute molecules. Such measurements are reported for dilute solutions of proteoglycans in the absence and presence of added hyaluronic acid. The proteoglycan molecules are shown to be some 580 nm long. In the presence of hyaluronic acid they form aggregates that appear to be consistent with the model previously proposed in which the proteoglycans attach radially to the extended hyaluronic acid chain. The electric-birefringence relaxation rates indicate aggregates of similar length to that of the extended hyaluronic acid chain, with the proteoglycans spaced on average at 29nm intervals. A proteoglycan sample the cystine residues of which had been reduced and alkylated showed no evidence of aggregation with hyaluronic acid up to the concentrations of the acid corresponding to 1% of the total uronic acid content. The electric-birefringence method is shown to have a large potential in the study of associating polysaccharide solutions.

Low molecular mass proteoglycans (PG) were isolated from human articular cartilage and from pig laryngeal cartilage, which contained protein cores of similar size (Mr 40-44 kDa). However, the PG from human articular cartilage contained dermatan sulphate (DS) chains (50% chondroitinase AC resistant), whereas chains from pig laryngeal PG were longer and contained only chondroitin sulphate (CS). Disaccharide analysis after chondroitinase ABC digestion showed that the human DS-PG contained more 6-sulphated residues (34%) than the pig CS-PG (6%) and both contained fewer 6-sulphated residues than the corresponding high Mr aggregating CS-PGs from these tissues (86% and 20% from human and pig respectively). Cross-reaction of both proteoglycans with antibodies to bovine bone and skin DS-PG-II and human fibroblasts DS-PG suggested that the isolated proteoglycans were the humans DS-PG-II and pigs CS-PG-II homologues of the cloned and sequenced bovine proteoglycan. Polyclonal antibodies raised against the pig CS-PG-II were shown to cross-react with human DS-PG-II. SDS/polyacrylamide-gel analysis and immunoblotting of pig and human cartilage extracts showed that some free core protein was present in the tissues in addition to the intact proteoglycan. The antibodies were used in a competitive radioimmunoassay to determine the content of this low Mr proteoglycan in human cartilage extracts. Analysis of samples from 5-80 year-old humans showed highest content (approximately 4 mg/g wet wt.) in those from 15-25 year-olds and lower content (approximately 1 mg/g wet wt.) in older tissue (greater than 55 years). These changes in content may be related to the deposition and maintenance of the collagen fibre network with which this class of small proteoglycan has been shown to interact.

Experimental intervertebral disc degeneration produced in rabbits by ventral nuclear herniation reliably produces vertebral osteophytes. Osteophytes arise from proliferating inner annular fibers which undergo metaplasia into cartilage, calcify, and proceed through an endochondral ossification sequence. Proteoglycans extracted from the osteophytes reveal that the degree of aggregation, molecular size of the monomer, and the chondroitin sulfate/keratin sulfate ratio are directly related to the cartilage state of the tissue.

1. Dissociation of purified proteoglycan aggregates was shown to release an interacting component of buoyant density higher than that of the glycoprotein-link fraction of Hascall & Sajdera (1969). 2. This component, which produced an increase in hydrodynamic size of proteoglycans on gel chromatography, was isolated by ECTEOLA-cellulose ion-exchange chromatography and identified as hyaluronic acid. 3. The effect of pH of extraction showed that the proportion of proteoglycan aggregates isolated from cartilage was greatest at pH4.5. 4. The proportion of proteoglycans able to interact with hyaluronic acid decreased when extracted above or below pH4.5, whereas the amount of hyaluronic acid extracted appeared constant from pH3.0 to 8.5. 5. Sequential extraction of cartilage with 0.15m-NaCl at neutral pH followed by 4m-guanidinium chloride at pH4.5 was shown to yield predominantly non-aggregated and aggregated proteoglycans respectively. 6. Most of the hyaluronic acid in cartilage, representing about 0.7% of the total uronic acid, was associated with proteoglycan aggregates. 7. The non-aggregated proteoglycans were unable to interact with hyaluronic acid and were of smaller size, lower protein content and lower keratan sulphate content than the disaggregated proteoglycans. Together with differences in amino acid composition this suggested that each type of proteoglycan contained different protein cores.

Analysis of the knee-joint cartilage of pigs at five ages (namely foetuses from the second half of pregnancy and animals 10 weeks, 25 weeks, 3 years and 5 years old) showed that the composition approached that of adult cartilage by 25 weeks of age, the most marked differences being between foetal and 10 week-old cartilage. Protein–polysaccharides were extracted sequentially, first by brief low-speed homogenization with iso-osmotic sodium acetate, then by two extractions with 2m-CaCl2 for 24h with gentle agitation interspersed with brief low-speed homogenization and agitation for another 24h. About half of the protein–polysaccharides were removed from foetal cartilage by the first extraction and the remainder by the second. The proportion in the first extract declined sharply with the age of the animal, but that in the first CaCl2 extract was similar at all ages other than 10 weeks. The amount left in the residue increased approximately with the collagen content from about one-fifth at 10 weeks of age to one-third in adult and old cartilage. The proportion of medium-sized protein–polysaccharides in the extracts changed little with age after birth, but the glucosamine content increased about fivefold and the protein content almost doubled between 10 weeks and 5 years of age. Other analytical values changed little. These results cannot be explained solely by changes in the proportion of ‘link-glycoprotein’ in the protein–polysaccharides. Since major changes in most parameters had taken place by 25 weeks of age, the first weeks after birth may be a critical period for cartilage development in the pig.

Proteoglycans in the matrix of adult cartilage are metabolised quite rapidly [ 1,2], nevertheless adult cartilage has a limited capacity for repair. Embryonic cartilage on the other hand, is able to replace the proteoglycans of the matrix within a few days after they have been largely depleted by the action of enzymes [3,4], which suggests that the macromolecules in the environment exert some influence over the synthetic or secretory functions of chondrocytes. The possible influence of a variety of macromolecules on the biosynthesis of sulphated glycosaminoglycans has therefore been examined using suspensions of nondividing adult chondrocytes, obtained from pig laryngeal and articular cartilage. Cartilage contains small amounts of hyaluronic acid [5], low concentrations of which have important biological effects [6]. The effect of low concentrations of hyaluronic acid on the incorporation of 35SOiinto material precipitable by cetylpyridinium chloride (CPC) is reported here.

The kinetics of incorporation of [35S]sulphate into slices of pig laryngeal cartilage in vitro was linear with time up to 6h. The specific radioactivities of the extracted proteoglycans (containing about 80% of the uronic acid of the cartilage) and the glycosaminoglycans remaining in the tissue after extraction were measured after various times of continuous and ‘pulse–chase’ radioactivity incorporation. Radioactivity was present in the isolated chondroitin sulphate after 2 min, but there was a 35min delay in its appearance in the extractable proteoglycan fraction. Fractionation of the proteoglycans by gel chromatography showed that the smallest molecules had the highest specific radioactivity, but ‘pulse–chase’ experiments over 5h did not demonstrate any precursor–product relationships between fractions of different size. Equilibrium density-gradient centrifugation in 4m-guanidine hydrochloride showed that among the proteoglycan fractions the specific radioactivity increased as the chondroitin sulphate content decreased, but with preparations from ‘pulse–chase’ experiments there was again no evidence for precursor–product relationships between the different fractions. Differences in radioactive incorporation would seem to reflect metabolic heterogeneity within the proteoglycans extracted from cartilage. This may be due either to a partial separation of different types of proteoglycans or to differences in the rates of degradation of the molecules of different size and composition as a result of the nature and specificity of the normal degrading enzymes. The results suggest that molecules of all sizes were formed at the same time.

1. Protein–polysaccharides of chondroitin 4-sulphate were extracted with neutral calcium chloride from pig laryngeal cartilage that was not completely homogenized. The protein–polysaccharides were purified by precipitation with 9-aminoacridine. On zone electrophoresis in compressed glass fibre at pH7·2 it was separated into two fractions, although two distinct zones were not obtained. These fractions, which had already been shown to differ in their antigenic determinants, also differed considerably in amino acid composition, total protein, hexose and glucosamine contents. 2. The fraction of higher mobility contained approx. 2% of protein and only traces of glucosamine. Serine and glycine accounted for over half the total amino acid residues, but aromatic, basic and sulphur-containing amino acids were not detected. The weight-average molecular weight, determined by sedimentation, was 230000. 3. Assuming that there was the same sequence of neutral sugars at the linkage points as in PP-L fraction (protein–polysaccharide light fraction), the approximate molar ratio of hexose to serine suggested that most of the serine residues were linked to chondroitin sulphate chains. Support for this was derived from the agreement between the weight-average molecular weight of the chondroitin sulphate–peptide after proteolysis, and the chain weight calculated from its serine content. The chain weight based on the serine content of the fraction of higher electrophoretic mobility was approximately similar. 4. In contrast, the fraction of lower electrophoretic mobility resembled PP-L fraction in its amino acid composition, protein and glucosamine contents. The presence of glucosamine, together with the higher hexose content, suggested that this fraction contained some keratan sulphate. 5. The relatively low molecular weight of the fraction of higher mobility enabled it to be extracted without complete disintegration of the cartilage. The unlikelihood of its being produced by autolytic enzymes is discussed.

1. Protein–polysaccharides from pig laryngeal cartilage extracted by two procedures described in the preceding paper (Tsiganos & Muir, 1969) were shown to consist of macromolecules of various sizes as assessed by gel filtration in 4% and 6% agarose. 2. A larger proportion of the smaller molecules was present in the preparation obtained by brief extraction in iso-osmotic sodium acetate (procedure I) than in that obtained by more prolonged extraction in 10% (w/v) calcium chloride (procedure II). 3. Two fractions were separated by gel filtration in 6% agarose and by electrophoresis in compressed glass fibre. These fractions differed in chemical composition and in antigenic determinants. The gel-retarded fraction R and that of higher electrophoretic mobility possessed the same single antigen, whereas the gel-excluded fraction E and the slower electrophoretic fraction contained all the antigens of the starting material including that of fraction R. 4. Five N-terminal amino acid residues were identified in preparation I and fraction E, only two of which were present in fraction R. 5. The relative proportions of gel-excluded and gel-retarded fractions did not change when solutions of high ionic strength, urea or guanidine hydrochloride were used for elution. 6. The differences in chemical and amino acid composition between fractions R and E showed that the latter was not a simple aggregate of the former. Fraction E contained more basic and aromatic amino acids, and some methionine and cystine; the last two were absent from fraction R. Hydroxyproline was not detected in either fraction. 7. The number of glycosidic linkages in both fractions was estimated by alkaline β-elimination. Appreciable amounts of threonine as well as serine were destroyed in both fractions. An average chain length for chondroitin sulphate was calculated from the galactosamine content of both fractions and the amounts of hydroxy amino acid destroyed. Average chain lengths were also calculated from the xylose and galactosamine content of each fraction. Each independent method gave a value of approximately 28 disaccharide units for the chain length in both fractions and hence their difference in size could not be explained by differences in the length of carbohydrate chains. 8. All fractions contained glucosamine, which was attributed to keratan sulphate. Content of both protein and keratan sulphate increased with the size of the macromolecules. 9. It is suggested, from these results, that chondroitin sulphate–protein complexes normally exist as a heterogeneous population of macromolecules in cartilage, and that keratan sulphate is involved in the formation of larger molecules.

Protein–polysaccharides of knee-joint cartilage of 9-month-old pigs were extracted sequentially with neutral iso-osmotic sodium acetate after five repeated homogenizations. One-third of the uronic acid originally present in the tissue was brought into solution, about half being in the first extract. The protein–polysaccharides, which were purified by precipitation with 9-aminoacridine, were heterogeneous in size on gel chromatography. The smallest (retarded by 6% agarose) were the most easily extracted since they were most prevalent in the initial extracts and absent from later ones, whereas the proportion of larger molecules increased progressively in successive extracts. Nevertheless a small proportion of the largest molecules (excluded from Sepharose 2B) was present even in the first extract. None of the protein–polysaccharide preparations contained hydroxyproline, and the analyses of their constituent sugars were the same, although there was a progressive increase in the protein content and in the glucosamine/galactosamine molar ratio of successive extracts. In each preparation this molar ratio was invariably greater in larger than in smaller molecules separated by gel filtration. From galactosamine/pentose molar ratios it appeared that the chondroitin sulphate chains were on average about 29 disaccharide units in length in the protein–polysaccharides of each extract, although gel-chromatography and cetylpyridinium chloride elution profiles showed that a somewhat higher proportion of shorter chondroitin sulphate chains occurred in the larger protein–polysaccharides. In the last extract, where the largest molecules predominated, about half could be reversibly dissociated by urea, whereas this had no effect on the protein–polysaccharides of earlier extracts even though these contained some large molecules.

Explants of cartilage from tibiae of 11–12 days chick embryos were grown in organ culture. To one group hyaluronidase was added to the medium during the first 2 days of culture; the treated tissue was then cultured in medium without enzyme for a further 4 days. Control explants grown in hyaluronidase-free medium for 6 days grew rapidly in size and the total hexosamine content more than doubled during this time. After exposure to hyaluronidase, much of the hexosamine was lost from treated cartilage and appeared in the culture medium, but it was mostly replaced in the tissue during the subsequent recovery period. Analysis of cartilage and medium showed that net synthesis of hexosamine increased greatly in treated cartilage. The proteoglycans were extracted by two procedures from control and treated cartilage after 2, 4 and 6 days in culture. The hydrodynamic sizes of the purified proteoglycans were compared by gel chromatography and the composition of the gel-chromatographic fractions was determined. The proteoglycans from controls did not change during culture, but after exposure to hyaluronidase the proteoglycans from treated cartilage were of much smaller size and lower chondroitin sulphate content. During recovery, even though new proteoglycans were formed, they were nevertheless of smaller size and lower chondroitin sulphate content than control proteoglycans. They gradually became more like control proteoglycans during recovery from treatment, but even after 4 days they were not yet the same. After 2 days of treatment with the enzyme, the chondroitin sulphate in the cartilage was of shorter chain length than in controls but during recovery after 4 and 6 days in culture, the chain lengths in control and treated cartilage were similar. It is concluded that the proteoglycans formed in embryo cartilage in response to their depletion by enzyme treatment contained fewer chondroitin sulphate chains attached to the protein moiety of proteoglycans. This may have resulted from a failure under stress to glycosylate the protein moiety to the usual extent; alternatively the synthesis of normal proteoglycans of low chondroitin sulphate content may have increased, thus changing the proteoglycan population.

Pig articular cartilage, from which protein–polysaccharides soluble in iso-osmotic sodium acetate had been removed, was extracted in three further stages with 8m-urea in 2m-sodium acetate and with tris–HCl buffer after bacterial collagenase digestion, followed by the same urea–sodium acetate solution, thus leaving only 2% of the original uronic acid in the tissue. The histological appearance of the cartilage was unaltered until after collagenase digestion. The collagenase used did not affect the viscosity or molecular size of a protein–polysaccharide preparation obtained previously. The protein–polysaccharides in each extract differed in size, amino acid composition and protein content, but protein and keratan sulphate contents were not related to hydrodynamic size, in contrast with protein–polysaccharides extracted previously before collagenase digestion. Hydroxyproline could not be removed from those obtained by the first urea–sodium acetate extraction until degraded by heat. The galactosamine/pentose molar ratio agreed closely with the galactosamine/serine molar ratio that was destroyed on treatment with 0.5m-sodium hydroxide, showing that chondroitin sulphate was attached only to serine residues. From these molar ratios the chondroitin sulphate chains were calculated to be of the same average length in protein–polysaccharides in all three extracts although somewhat shorter than in protein–polysaccharides extracted previously. Some threonine residues were also destroyed on alkali treatment suggesting that keratan sulphate may be attached to threonine. These findings together with previous results show that differences in size, composition and physical state extend to all the protein–polysaccharides in cartilage.

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.

The glycosaminoglycans in the menisci of beagles 5--7 years old were analyzed at various times after osteoarthritis was induced by sectioning the anterior cruciate ligament of one knee; the unoperated knee served as control. In the first month after induction, there were signs of inflammation in the operated joint. After 1 week, the water content was elevated and the glycosaminoglycan content (per dry weight) was reduced. The content of keratan sulfate decreased more than that of chondroitin sulfate, but the hyaluronic acid content did not change consistently. The relative proportions of chondroitin-4-sulfate, chondroitin-6-sulfate, and dermatan sulfate remained unchanged. After 3--18 months, the glycosaminoglycan levels reverted to normal, and there was some evidence that after 15--18 months, they were elevated above normal. These results, together with results obtained from single examples of mild and severe osteoarthritis in working foxhounds, suggest that, in contrast to articular cartilage, the meniscus is capable of some regeneration in response to injury.

The structure of the proteoglycans from normal pig nucleus pulposus and relatively normal human annulus fibrosus and nucleus pulposus was investigated in detail and the results were compared with the current structural model of proteoglycans of hyaline cartilage. Like proteoglycans of cartilage, those of intervertebral disc contain keratan sulphate and chondroitin sulphate attached to a protein core; they are able to aggregate to hyaluronic acid; the protein core likewise has three regions, one lacking glycosaminoglycans, another rich in keratan sulphate and a third region rich in chondroitin sulphate. However, disc proteoglycans contain more keratan sulphate and protein and less chondroitin sulphate and are also considerably smaller than cartilage proteoglycans. In proteoglycans of human discs, these differences appeared to be due principally to a shorter region of the core protein bearing the chondroitin sulphate chains, whereas in proteoglycans of pig discs their smaller size and relatively low uronic acid content were due to shorter chondroitin sulphate chains. There were subtle differences between proteoglycans from the nucleus and annulus of human discs. In the latter a higher proportion of proteoglycans was capable of binding to hyaluronate.

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

An animal model of intervertebral disc degeneration produced by surgical ventral disc herniation in the rabbit is described. Histologic studies showed proliferation of cells on the inner third of the annular wound, with metaplasia into fibrocartilage in the first 2 weeks following injury. Progressive fibrocartilaginous change occurred, reproducing the morphologic changes of disc degeneration over the first 6 weeks involving nearly the entire disc. Proteoglycans (total and newly synthesized) were studied qualitatively and quantitatively for periods of 1 to 200 days after herniation. There were two periods of time during the early course of degeneration when the ability of the proteoglycans to aggregate by interaction with hyaluronic acid was recovered, but this decreases progressively after 6-7 weeks. There was an immediate loss of water content from the injured disc which was restored only transiently during the first 2 days after herniation. Thereafter the water content progressively decreased. The uronic acid content of the disc changed in parallel with the changes in water content. The hyaluronic acid content decreased rapidly after herniation. However, the size of the proteoglycan monomers did not change with degeneration. The biochemical and morphologic changes are correlated, and an early repair mechanism is postulated to exist after injury.

The binding of hyaluronate oligosaccharide fractions to proteoglycans from pig laryngeal cartilage has been studied by equilibrium dialysis in dilute solution. It has been shown that: (1) each proteoglycan monomer binds only one hyaluronate oligosaccharide molecule [containing about eighteen saccharide residues (HA approximately 18) and of number-average molecule weight (Mn) 37501]; (2) the dissociation constant, Kd, for interaction between proteoglycan monomer and oligosaccharide HA approximately 18 is 3 × 10(-8) M at 6 degrees C at I 0.15-0.5, pH 7.4; (3) the dissociation constant has little dependence on temperature, so that Kd at 54 degrees C is 3 × 10(-7) M under the same conditions; (4) the aggregatability is high at 6 degrees C, falls significantly at 54 degrees C, but much of it can be recovered on cooling to 6 degrees C again, demonstrating reversible denaturation; (5) a method for determining the proportion of the proteoglycan molecules capable of binding to hyaluronate by equilibrium dialysis was compared with gel-chromatographic and ultracentrifugal methods; (6) a hyaluronate oligosaccharide, HA approximately 56 (Mn 11 000), could bind more than one proteoglycan molecule; (7) consideration of ultracentrifugal data shows that when proteoglycans bind to a hyaluronate of larger size (mol.wt. 670 000), an average Kd of 12 × 10(7) M fits the data in 0.5 M-guanidine hydrochloride at 20 degrees C.

The biosynthesis of proteoglycans in the menisci and articular cartilages of the knee (stifle) of mature beagles was studied in the early stages of experimental osteoarthritis. The rate of proteoglycan synthesis, determined by systemic labeling in vivo at 21, 42, and 84 days after sectioning of the anterior cruciate ligament, was generally found to be 1.5-2.5-fold higher than control in articular cartilages and 3-10-fold higher than control in menisci. The medial meniscus was more stimulated than the lateral meniscus, and the medial tibial plateau under the meniscus was more stimulated than the adjacent tibial area. This area-specific stimulation suggests the involvement of mechanical factors in the cellular response. The rate of proteoglycan synthesis determined in vitro at 7, 14, and 21 days after operation was also about 2-fold higher than control in articular cartilages and about 3-fold higher in menisci. This increase in biosynthetic activity in vitro was confirmed by 35S-autoradiography and appeared to be due to general stimulation of existing chondrocytes, particularly in the middle and deep zones of the articular cartilage and throughout the meniscal cartilage. The rate of proteoglycan synthesis determined in vitro in cartilages from 2-week and 3-week sham operated joints was also increased relative to controls, suggesting that humoral as well as mechanical factors are involved in stimulating chondrocyte activity.

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.

THE guinea pig lymphocyte has long been known to contain a special inclusion body. Such lymphocytes, called Kurloff cells after their discoverer, increase greatly in number in pregnancy and were shown by Ledingham1 to be produced in both malesand females by administration of œstrogens. Marshall and Swettenham2, by histochemical methods, have shown that the inclusion body is composed of a mucoprotein–sulphated mucopolysaccharide complex. Further work on the chemistry of a mucopolysaccharide fraction by extraction of spleens of œstrogen-treated guinea pigs gave the following results

1. Protein–polysaccharides of chondroitin sulphate were extracted from fresh laryngeal cartilage at pH6·8 by two procedures. Procedure I consisted of brief low-speed homogenization in 0·15m (iso-osmotic) sodium acetate and procedure II consisted of longer homogenization followed by prolonged extraction in 10% calcium chloride solution. 2. The protein–polysaccharides in both extracts were isolated and purified by precipitation with 9-aminoacridine hydrochloride. They were free from serum proteins, collagen and nucleic acids and also of degradative enzymes. The absence of such enzymes was shown by viscosity measurements on solutions of protein–polysaccharides incubated for up to 24hr. at pH4 and 6·8. 3. Mannose, glucose or fucose were not detected by paper chromatography and only traces of sialic acid were present. 4. The yield with procedure II was twice that with procedure I and the products differed in their protein and glucosamine contents. 5. Hyaluronic acid was unlikely to have been precipitated at an acid pH, so the glucosamine was attributed to keratan sulphate, as serum proteins were absent. There was no free keratan sulphate in the preparation. 6. Both preparations were heterogeneous in the ultracentrifuge, showing at least three components.

The excretion and degradation was studied of 35S-labelled 4-chondroitin sulphate injected into guinea pigs in the form of proteoglycan isolated from cartilage and in the form of free chondroitin 4-sulphate prepared from the same proteoglycan by proteolysis. When the proteoglycan was injected there was a delay of about 15–20min before significant amounts or radioactivity were excreted, whereas after injection of chondroitin 4-sulphate a considerable amount of radioactivity was excreted within 10min and a much higher proportion of the radioactive dose was excreted in 1h or 24h compared with the proteoglycan. In both cases, however, a major part of the radioactivity was not excreted even in 24h. Sterile conditions were used to collect the radioactive material directly from the bladder. When chondroitin 4-sulphate was injected, the molecular sizes of injected and excreted materials were similar, as assessed by gel chromatography on Sephadex G-200, whereas when proteoglycan was injected the molecular size of the excreted labelled material was similar to that of the chondroitin 4-sulphate chains in the original proteoglycan. In neither case did the size of the excreted labelled material change with time over 1h, and low-molecular-weight labelled material was virtually absent. In contrast, when urine was collected for 24h without preservative the labelled material in it was extensively degraded after either the proteoglycan or chondroitin 4-sulphate had been given. Chondroitin 4-sulphate became similarly degraded when incubated with non-sterile urine, but not when the urine was passed through a bacterial filter, suggesting that degradation was caused by contaminating micro-organisms in the experiments in which urine was collected for 24 h. It is concluded that chondroitin 4-sulphate chains of about 18000 molecular weight can be excreted readily as such, whereas intact proteoglycans must be degraded to free glycosaminoglycans first, although both are taken up by the tissues more rapidly than they are excreted.

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.

Protein–polysaccharides of femoral articular cartilage from pigs of ages 9 months and 5 weeks were compared after extraction at pH6·8 with iso-osmotic sodium acetate followed by 0·63m-calcium acetate. The cartilage from the younger animals had a higher moisture content and contained considerably larger amounts of protein–polysaccharide, but less than half as much collagen/g. dry weight, than cartilage from the older pigs. There was notably less keratan sulphate in the fractions from the less mature animals. After gel filtration on 6% agarose, elution profiles of the calcium acetate extracts were similar to those of the sodium acetate extracts of the same tissue. Chemical analyses, however, showed that in both age-groups the extraction procedure had achieved a sequential solubilization of protein–polysaccharides in that the initial extracts contained a higher proportion of keratan sulphate than those that were extracted subsequently. Both extracts from the older animals contained up to 25% of a relatively small protein–polysaccharide that was retarded on 6% agarose and that had a lower protein content and less keratan sulphate than the larger protein–polysaccharides. In contrast, in extracts from the less mature cartilage only about 5% of the protein–polysaccharides were small enough to be retarded by 6% agarose, suggesting that the small components may not be precursors of the larger. The average length of chondroitin sulphate chains, as calculated from the analytical data, was the same in the smaller protein–polysaccharides as in the larger.

Intensive rearing, and restricted activity, induce rapid growth in pigs, but they often become lame. Groups of normal and lame pigs reared intensively were killed when 10 or 25 weeks old. Although there were no differences in the overall composition of the knee-joint cartilage of lame and sound animals, the proteoglycans in the cartilage of the lame pigs were extracted more easily by a standardized sequential procedure and contained a higher proportion of molecules of smaller size as assessed by gel chromatography on 6% agarose and Sepharose 4B. These increased at the expense of both the larger and mediumsized molecules. Differences were most evident at 10 weeks of age, when there was twice as much of the smaller proteoglycans in the cartilage of lame pigs. Despite these size-differences, the compositions of the proteoglycans in corresponding sequential extracts of cartilage of lame and normal groups were the same, as were the changes in chemical composition that accompany development. Proteoglycans from lame animals may have undergone limited proteolysis, thus decreasing their size without changing their composition detectably. As the differences between normal and lame groups were greater at 10 weeks than at 25 weeks of age, the first weeks after birth (when the greatest changes occur in the proteoglycans and in the cartilage) may be a critical period in the maturation of articular cartilage in this species. At this time, rapid gain in weight produced by intensive rearing may be too great for the immature cartilage to bear.

Oligosaccharides derived from hyaluronic acid were shown to inhibit proteoglycan–hyaluronic acid interaction, as measured in a viscometer. The relative inhibition increased with the size of the oligosaccharide and the results suggested that decasaccharides were the smallest fragments able to bind strongly to the proteoglycan.