Identification of link proteins in canine synovial cell cultures and canine articular cartilage.

Link proteins are glycoproteins in cartilage that are involved in the stabilization of aggregates of proteoglycans and hyaluronic acid. We have identified link proteins in synovial cell cultures form normal canine synovium using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunofluorescence, and immunolocation with specific antibodies by electrophoretic transfer. We have also found evidence for the synthesis of link proteins in these cultures by fluorography of radiolabeled synovial cell extracts. We have identified a 70,000 mol-wt protein in canine synovial cell culture extracts that has antigenic cross-reactivity with the 48,000-mol-wt link protein. Three link proteins were identified in normal canine articular cartilage. These results indicate that link proteins are more widely distributed in connective tissues than previously recognized and may have biological functions other than aggregate stabilization.

A tumor-associated fibronectin isoform generated by alternative splicing of messenger RNA precursors.

Fibronectin (FN) represents the mixture of a number of structurally different molecules (isoforms) whose make-up varies depending on the FN sources. FN from cultured transformed human cells has a very different isoform composition with respect to its normal counterpart. In fact, SV-40-transformed WI-38VAI3 human fibroblasts produce high levels of a FN isoform (B-FN) which is very poorly expressed in their normal, WI-38, counterpart. We have recently demonstrated that the B-FN isoform derives from a differential splicing pattern of the FN primary transcript which leads, in transformed cells, to a high level expression of the exon ED-B (Zardi, L., B. Carnemolla, A. Siri, T. E. Petersen, G. Paolella, G. Sebastio, and F. E. Baralle. 1987. EMBO (Eur. Mol. Biol. Organ.) J. 6:2337-2342). Here we report on the production and characterization of a monoclonal antibody (BC-1) which recognizes an epitope within the protein sequence coded for by the ED-B exon. This monoclonal antibody makes it possible to carry out immunohistochemical analysis of the distribution of the ED-B-containing FN isoform (B-FN) in human tissues. The results show that while in normal, adult, human tissues total FN has a widespread distribution, the B-FN isoform is restricted only to synovial cells, to some vessels and areas of the interstitium of the ovary, and to the myometrium. On the contrary, the B-FN isoform has a much greater expression in fetal and tumor tissues. These results demonstrate that, in vivo, different FN isoforms have a differential distribution and indicate that the B-FN isoform may play a role in ontogenesis and oncogenetic processes.

Localization of hyaluronic acid in synovial cells by radioautography.

Cultured human synovial cells secrete hyaluronic acid (HA) into the culture medium. Glucosamine-6-(3)H was shown to be a direct and relatively specific precursor of HA-(3)H by the following observations: the susceptibility of nondialyzable radioactivity in the medium to hyaluronidase, its migration with hexuronic acid on zone electrophoresis in polyvinyl chloride, its exclusion from Sephadex G-200, and the localization of radioactivity to glucosamine after hydrolysis of the labeled polysaccharide. The presence of intracellular HA-(3)H was established by sequential extraction of labeled cells and by radioautography of synovial cell cultures digested with hyaluronidase in situ. When cells were exposed to medium lacking glucose, glucosamine-(3)H-uptake was enhanced; and this made possible electron microscopic radioautographic studies. These studies demonstrate the early and continued presence of HA-(3)H within the Golgi apparatus.

Synovial cell synthesis of a substance immunologically like cartilage proteinpolysaccharide.

Fluids from joints contain a substance that reacts immunologically like one of two known antigenic components of articular cartilage proteinpolysaccharide. This newly recognized substance occurs in the lining cells of synovial membranes as shown by indirect immunofluorescence. The localization of this substance in tissue culture cells derived from synovial membranes and its identification in the culture medium supports the suggestion that it is synthesized by lining cells. Rheumatoid synovial cells contain less of this substance.

Human cartilage lysozyme.

The lysozyme content of human cartilage was measured by incubation of lyophilized, powdered cartilage in a variety of buffers and salt solutions, and the factors controlling the binding of lysozyme within cartilage were studied. Lysozyme was extracted from hyaline cartilage by buffers of pH greater than 9.0 by solutions 1 M in monovalent cations, and by solutions 0.12-0.40 M in divalent cations. The ability of cations to extract lysozyme from cartilage agreed with their known affinities for binding to chondroitin sulfate. The total extractable lysozyme content of five samples of human costal cartilage ranged from 1.45 to 3.36 mug lysozyme per mg of cartilage; for five samples of hyaline cartilage from peripheral joints the range was 0.80-3.03 mug lysozyme per mg of cartilage. Cartilage incubated in excess exogenous lysozyme could bind 0.053 equivalents of lysozyme per equivalent of chondroitin sulfate. Fibrocartilage and synovium from knee joints yielded no detectable lysozyme, despite the fact that synovium, a tissue rich in lysosomes, contained measurable quantities of beta-glucuronidase. Lysozyme extraction from cartilage was not augmented by incubation with streptolysin S. When incubation was carried out with mild extraction techniques, lysozyme extraction from cartilage tended to parallel uronic acid release, both as a function of time and from one specimen to another. The active material as lysozyme. Lysozyme occurs in human hyaline cartilage as a counterion to polyanionic glycosaminoglycans. Carextracted from cartilage met five criteria for identification tilage lysozyme appears to be extracellular and nonlysosomal. Degradation of cartilage may contribute to the increased serum and synovial fluid lysozyme levels often present in patients with rheumatoid arthritis.

Beta-2-microglobulin-associated amyloidosis in chronic hemodialysis patients with carpal tunnel syndrome.

The clinical manifestations of beta-2-microglobulin (beta 2M)-associated amyloidosis in chronic hemodialysis patients with carpal tunnel syndrome from a medical center hospital are presented. The predominant morbidity of beta 2M-amyloid was musculoskeletal, with deposits identified in surgical or biopsy specimens from trigger fingers, carpal tunnels, fractures, and radiolucent bone lesions. Lucent bone lesions were the characteristic radiologic finding of beta 2M-amyloidosis and were most commonly found in carpal bones, humeral heads, and femoral heads. Carpal tunnel syndrome occurred in greater than 20% of our chronic hemodialysis patients. The longer the period of time on chronic hemodialysis the greater the morbidity from beta 2M-amyloid. Although significant amounts of beta 2M-amyloid were detected in the perivascular regions of viscera, clinical compromise of internal organs from this type of amyloid was not documented. In acute studies, beta 2M clearance during hemodialysis was markedly increased using the Fresenius polysulfone dialyzers compared to cuprophane dialyzers. In summary, beta 2M-amyloid is common and causes significant morbidity in chronic hemodialysis patients. Long-term dialysis with highly permeable membranes effects greater beta 2M clearance which may result in less tissue deposition of beta 2M-amyloid, and therefore, fewer clinical complications.

Preservation of thrombomodulin antigen on vascular and extravascular surfaces.

The protein C anticoagulant system is mediated by thrombin and is highly accelerated by thrombomodulin. We studied the distribution of thrombomodulin antigen (TM Ag) in the rabbit using an affinity-purified antibody raised in a goat against rabbit thrombomodulin. The preservation of TM Ag was highly dependent on immediate fixation of the surface on which it is located. TM Ag was found on the endothelium of the entire vasculature, whereas it was absent from all connective tissue, smooth and striated muscle, secretory epithelia, cartilage, bone, neural tissue, and all parenchyma examined. A new finding was the presence of TM Ag on nonvascular surfaces of body cavities (the mesothelia of pleura, pericardium, and peritoneum, the synovial membrane, and the arachnoid enveloping the central nervous system). By use of a functional assay, TM activity was recovered in buffered saline/detergent solution which was either injected into the intraperitoneal cavity of rabbits in vivo or incubated with the surface of the arachnoid in vitro. These findings extend the importance of anticoagulant mechanisms to the systems of slowly circulating fluids, in which they might be required for maintenance of the flow, and to mesothelial cavities, in which they could be necessary for preventing adherence between the surfaces, in conditions associated with pathological exudation.

Metallothionein in cultured human epithelial cells and synovial rheumatoid fibroblasts after in vitro treatment with auranofin.

Radioimmunoassay (RIA) and reversed-phase high-pressure liquid chromatography (HPLC) were used to investigate gold-binding proteins of possible metallothionein (MT) nature occurring upon auranofin exposure of cultured human cells. An epithelial cell line (HE) and two sub-strains were examined. The HEAF sub-strain had been made resistant to 2 mumole auranofin/l culture medium. The resistance was associated with the appearance of gold-binding substances with gel filtration characteristics like MT. The HE100 sub-strain had been made resistant to 100 mumole CdCl2/l and contained high amounts of cytosolic Cd-induced MT. In addition, cultured synovial fibroblasts, derived from normal (SN) and rheumatoid (SRA) synovial tissues, were investigated. Evidence was obtained by RIA that the low molecular weight (mol.wt. 6000-7000) gold-binding proteins occurring in the HEAF cells and SRA cells following auranofin exposure, were of MT nature. The relative amounts of MT in the epithelial cell lines were: HE:HEAF:HE100 = 1:18:100. The relative amounts in the synovial fibroblasts were: SN:SRA:SRA treated with auranofin = 1:3:10. The HPLC methods used were found suitable for isolation of Cd-MT in the HE100 cells, but not for the Au-MT in the HEAF cells. By HPLC, the Cd-MT in the HE100 cells was resolved into 3 MT-1 and 1 MT-2 iso-proteins exhibiting the amino acid composition typical of MT. Judged by HPLC, the MT in these cells constituted 0.4% of the cytosolic proteins.

Amyloid localized to tenosynovium at carpal tunnel release. Natural history of 124 cases.

One hundred fifty-two patients with amyloid in the tenosynovium who had carpal tunnel release were identified. Twenty-eight patients were excluded because of systemic amyloidosis: primary systemic amyloidosis (AL) in 24, secondary amyloidosis (AA) in 3, and familial amyloidosis (AF) in 1. The remaining 124 patients (82%) had carpal tunnel syndrome with local deposition of amyloid and no evidence of systemic amyloidosis. Median survival of the 124 patients from diagnosis of amyloidosis was 12 years. Only two patients had systemic amyloidosis develop--9 and 10 years after recognition of tenosynovial amyloid. Of particular interest were 12 patients who had an M-protein in the serum or urine. None of the 12 patients have had evidence of systemic amyloidosis or multiple myeloma during the median follow-up of 14 years. The authors conclude that amyloid may be localized to the tenosynovium and that systemic amyloidosis rarely develops during long-term follow-up.

Clinicopathological importance of deposits of amyloid in the femoral head.

The pattern of amyloid deposits in the femoral head is described in four cases, two of which had deposits of amyloid related to age and two of which had generalised systemic amyloidosis (one of primary amyloidosis, one of multiple myeloma). The deposition of amyloid in the articular cartilage of the femoral head was similar in all four cases. Heavy deposits of synovial amyloid were identified in the case with primary amyloidosis and in one of the cases with amyloidosis related to age. Both cases of generalised systemic amyloidosis showed abundant deposits of amyloid in the bone marrow. Amyloid was not present in the bone marrow of either case with amyloidosis related to age. The importance of these findings is discussed in relation to the pathogenesis of the arthropathy syndrome of a rheumatoid type described in cases of primary amyloidosis and multiple myeloma.

Cathepsin B and cysteine protease inhibitors in human osteoarthritis.

The aim of this study was to determine the involvement of cathepsin B and its inhibitors in the proteolytic degradation of human osteoarthritic (OA) tissue. The characteristics of the cathepsin B found in both normal and OA cartilage and synovium were similar to those of the lysosomal cathepsin B. Two inhibitors of cysteine proteases were found with a molecular weight of 67,000 and 16,000 Da. The cartilage cathepsin B level of OA specimens (54.8 +/- 7.3 units/micrograms of DNA) was greater than the controls (39.8 +/- 3.2 units/micrograms of DNA). Mild-moderate graded samples (78.1 +/- 12.0 units/micrograms of DNA) had significantly higher levels of enzyme activity than the severely graded ones (31.4 +/- 3.9 units/micrograms of DNA, p less than 0.001) and controls (p less than 0.01). Compared to controls (2.3 +/- 0.4 units/mg of tissue w.w.), cysteine protease inhibitory activity in OA cartilage was decreased in specimens with severe lesions (1.5 +/- 0.2 units/mg of tissue). This was particularly noted in patients who had not received steroid injections (1.2 +/- 0.3 units/mg of tissue, p less than 0.05). In OA synovia, the cathepsin B level was greater (40.7 +/- 7.4 units/mg of tissue w.w., p less than 0.02) than in the controls (13.6 +/- 3.7 units/mg of tissue). The cysteine protease inhibitory activity was similar in OA synovium (1.7 +/- 0.2 units/mg of tissue w.w.) and in controls (1.5 +/- 0.3 units/mg of tissue). This data demonstrated an imbalance between the levels of cathepsin B and cysteine protease inhibitors in OA tissue. A decrease of specific inhibitors could be an important contributing factor, particularly in more severe lesions.

Amyloid arthropathy: characterization of the amyloid protein.

An 82 year-old man was referred for joint pain and numbness of his hands. Physical examination revealed limitation of movement of the PIP's, MCP's, wrists, shoulders and knees. There was marked synovial thickening of the wrists and atrophy of the thenar muscles of both hands due to arpal tunnel syndrome. The patient was operated on both hands, the median nerves were released and a synovectomy of the wrist was performed. Two months later, a synovectomy of the right shoulder was performed. Histological examination of tissues from the wrists and shoulder demonstrated large deposits of amyloid in the synovia. Amyloid fibrils were extracted, solubilized in 6M and were fractionated on a Sepharose 6B. All three proteins that were purified from the amyloid fibrils proved to be derived from VkI light chain by their amino terminal sequences. This is the first amyloid protein to be characterized from amyloid arthropathy.

Polymeric debris in synovium after total joint replacement: histogical identification.

In studying a series of synovial biopsy specimens from patients with loose joint replacements, we were able to differentiate reliably between polymethylmethacrylate and ultra high-molecular-weight polyethylene by observing alterations in their birefringence with changes in temperature. The glass transition temperature of polymethylmethacrylate (when it softens from a glass-like to a rubber-like state) is about 105 degrees centigrade. Polyethylene melts at 135 degrees centigrade. The birefringence in polyethylene returns after it has been melted and then cooled; this is not true of methylmethacrylate fragments heated above their glass transition temperature. When cooled, methacrylate fails to regain its birefringence. In addition, free particles of the two plastics were studied in vitro to validate this method of differentiation. We recommend its use routinely in histological studies of patients with loosening.

Metallic wear in failed titanium-alloy total hip replacements. A histological and quantitative analysis.

We conducted extensive histological examination of the tissues that were adjacent to the prosthesis in nine hips that had a failed total arthroplasty. The prostheses were composed of titanium alloy (Ti-6Al-4V) and ultra-high molecular weight polyethylene. The average time that the prosthesis had been in place in the tissue was 33.5 months (range, eleven to fifty-seven months). Seven arthroplasties were revised because of aseptic loosening and two, for infection. In eight hips cement had been used and in one (that had a porous-coated implant for fifty-two months) no cement had been utilized. Intense histiocytic and plasma-cell reaction was noted in the pseudocapsular tissue. There was copious metallic staining of the lining cells. Polyethylene debris and particles of cement with concomitant giant-cell reaction were present in five hips. Atomic absorption spectrophotometry revealed values for titanium of fifty-sic to 3700 micrograms per gram of dry tissue (average, 1047 micrograms per gram; normal, zero microgram per gram), for aluminum of 2.1 to 396 micrograms per gram (average, 115 micrograms per gram; normal, zero micrograms per gram), and for vanadium of 2.9 to 220 micrograms per gram (average, sixty-seven micrograms per gram; normal, 1.2 micrograms per gram). The highest values were found in the hip in which surgical revision was performed at fifty-seven months. The concentrations of the three elements in the soft tissues were similar to those in the metal of the prostheses. The factors to which failure was attributed were: vertical orientation of the acetabular component (five hips), poor cementing technique on the femoral side (three hips), infection (two hips), and separation of a sintered pad made of pure titanium (one hip). A femoral component that is made of titanium alloy can undergo severe wear of the surface and on the stem, where it is loose, with liberation of potentially toxic local concentrations of metal debris into the surrounding tissues. It may contribute to infection and loosening.

Purification and quantities of immunoglobulins of rheumatoid synovial tissues.

Synovial tissue of rheumatoid arthritic origin was incubateed with radio-labelled amino acids and the immunoglobulin fraction of the products isolated using either a diethylaminoethyl (DEAE)-cellulose column or an affinity column of antihuman immunoglobulin coupled to Sepharose. The affinity column method provided a simple, one-step procedure for obtaining quantitative yields of substantially pure immunoglobulin. The elutions from the affinity columns utilized guanidine-HCl allowing the elutions to be performed quickly and under conditions which apparently did not denature the immunoglobulins except perhaps immunoglobulin M. Other solvent conditions for dissociating antibody-antigen complexes were shown to be not suitable and resulted in large volumes of eluates. The affinity columns could be reutilized many times without apparent loss of capacity to bind immunoglobulins.

The pathogenesis of chronic haemophilic arthropathy.

Specimens of tissue from haemophilic synovium and articular cartilage were collected from 39 patients during reconstructive surgery. They were studied by histochemistry, electron microscopy and microprobe analysis. The detailed findings are presented and discussed. It is suggested that haemophilic arthropathy is the result of a number of mechanisms affecting the synovial lining which becomes progressively fibrotic and the hyaline cartilage which disintegrates and is eventually lost. Mechanical and chemical processes cause degeneration of cells but enzymatic processes appear to be primarily responsible for the degradation of the matrix of the articular cartilage.

Nuclei of cultured synovial fibroblasts studied by immunofluorescence and microdensitometry.

Two techniques which might be expected to detect alterations in DNA have been used in a comparison of cultured non-rheumatoid and rheumatoid synovial fibroblasts. Microdensitometry showed no alteration in staining affinity for methyl green when two pairs of cultures were compared. There was, however, a minor difference in the predominant staining pattern to a fluorescein conjugated anti-IgM serum when five rheumatoid cultures were compared with five non-rheumatoid cultures after exposure to a serum containing anti-nuclear antibodies.

The technique of electron probe x-ray analysis and the atomic composition of aurosomes.

Electron probe x-ray analysis is an exciting new method of elemental analysis at the ultrastructural level. This is a relatively non-destructive form of analysis by which elements from Na onwards (i.e. above Z number 10) can be simultaneously detected and displayed as a spectrum. The best spatial resolution that can be achieved is of the order of 20 nm. The sensitivity of detection varies with the elements but about 10-18 g of an element can be detected. With the available computer programs elemental ratios are readily determined without the need of standards. The special appeal of this technique stems from the fact that it is the only way of analyzing minute inclusions in situ within the cell and so correlate morphology with atomic composition. With this method, gold has been demonstrated in aurosomes produced in various sites after the administration of soluble gold salts to experimental animals and man. A more detailed analysis of aurosomes found in the synovial membrane 3 days and 18 months after injection of gold sodium thiomalate into the rabbit knee joint revealed that the aurosome contains gold, phosphorus, and sulphur, and that the atomic ratios of these elements do not alter over the above mentioned time interval.

[Electrophoretic separation of glycosaminoglycans from different types of connective tissue and biological fluids]. / Metod elektorforeticheskogo razdeleniia glikozaminoglikanov razlichnykh vidov soedinitel' noi tkani i biologicheskikh zhidkostei

Separation by means of electrophoresis on paper of glycoseaminoglycans from different types of human and animals connective tissue (bone tissue, synovial membrane of joints, skin and others) and from biological fluids (blood serum, urine, joint liquid) enabled to determine the ratio between sulphatated and nonsulphatated glycoseaminoglycans in normal conditions, in different pathological states and impairments of connective tissue structures. The method was found to be adequate for studies on quantitative and qualitative composition of glycoseaminoglycans and their metabolic products in pathologically altered connective tissue and biological fluids.