The diagnostic significance of autoantibodies against cartilage components or their counterparts in destructive articular diseases.

In this work four different types of antibodies to collagens, membrane protein of chondrocytes (MP), cartilage matrix proteins (CMP) and heat shock proteins (HSP-s) were tested in order to find an available clinical laboratory method for diagnostic and monitoring purposes in patients suffering from osteoarthritis. From the point of view of diagnostic efficiency, the estimation of antibody level to MP seemed to be the best and as a supplementary method the determination of rheuma factor was recommended. The anticollagen antibody estimation is less sensitive, anti CMP antibodies are not detectable. In spite of immunological crossreaction between HSP and cartilage matrix component the antibody level against HSP has no correlation with osteoarthritis. The appearance of humoral reaction, antibodies against different cartilage specific collagens and chondrocyte membrane proteins, is an epiphenomenon, however as the supposed immune complexes, trapped in a cartilage, play an important role in the damage of cartilage which may explain the self-perpetuating and chronic nature of cartilage degradation on osteoarthritis.

The clinical diagnostic significance of the investigation of receptor mediated pathway of LDL.

Some types of LDL-receptor investigations have been demonstrated which can also be carried out in routine clinical laboratory. Special attention has been layed on the testing of lymphocytes and monocytes which are available easier than tissue samples but, which are, however, according to our experiences appropriate for the LDL receptor analysis.

Estimation of serum anticollagen and the antibodies against chondrocyte membrane fraction: their clinical diagnostic significance in osteoarthritis.

The serum anticollagen antibodies to collagen types II, IX, and XI, as well as the antibody level to chondrocyte membrane extract were investigated in patients suffering from severe osteoarthritis (n = 86) in comparison with patients free of primary arthritis (n = 33) and with control healthy patients (n = 44), respectively. Isolation and purification of cartilage antigens and their relevance to ELISA reaction have been outlined. Although the method for anticollagen antibodies to types IX and XI was more sensitive than that of type II, its sensitivity was very low (52%). The determination of the specific IgG fraction by affinity chromatography seemed to be more sensitive: in osteoarthritic patients the percentage of the "arthritogen" IgG rose to 10% of the total IgG. The determination of antibody level against chondrocyte membrane extract was adapted to human diagnostic purposes. In osteoarthritic patients the serum antibody level was significantly higher than in healthy controls. The specificity of this new test was proved by the facts that: (a) only the collagen-binding fraction of the membrane extract reacts with the patient's sera; (b) the ELISA reaction could be totally inhibited by the antigen; (c) patients suffering from noninflammatory joint diseases were characterized by low antibody levels.

The clinical diagnostic significance of serum antichondrocyte membrane antibodies in osteoarthritis.

In osteoarthritic patients significantly elevated level of antibodies against human chondrocyte membrane extract was found by double solid phase ELISA which seemed to be more specific for the disease than the antibody level of collagen types II, IX and XI. Altogether 86 patients with a mean age of 53.6 years and a ratio of 25% men and 75% women suffering from severe hip joint osteoarthritis were studied in comparison with 44 control persons with a mean age of 35.8 years and identical sex distribution. The specificity of reacting antibodies was proved partly by inhibition test and partly by the fact that the ELISA reaction could be produced only by proteins of the membrane extract bound to collagen (II) after affinity chromatography. The diagnostic importance of the methods is striking, in view of the lack of any other objective laboratory tests for the disease.

Effects of glycosaminoglycan polysulphate in the treatment of chondrocalcinosis.

The effect of intra-articular injections of glycosaminoglycan polysulphate (Arteparon) on pain, joint mobility, inflammatory reactions, cartilage calcification and the urinary excretion of inorganic pyrophosphate was studied in 12 patients with chondrocalcinosis. All cases were bilateral and the less affected homologous joint served as an untreated control. The patients were followed over a one-year period. The glycosaminoglycan polysulphate treatment brought about a significant reduction of pain (p less than 0.01) and improvement of joint mobility (p less than 0.001). This effect continued for the whole one-year follow-up period, but could not be seen in the control joints. After the treatment period of 2-7 weeks there was a marked decrease in cartilage calcification paralleled with an increase in the excretion of inorganic pyrophosphate.

[Effect of arteparon on the formation of calcium phosphate and calcium pyrophosphate crystals--comparative experimental study]. / Wirkung von Arteparon auf die Bildung von Calciumphosphat- und Calciumpyrophosphatkristallen--Vergleichende experimentelle Untersuchung.

The influence of Arteparon on crystal formation in vitro has been examined in calcium orthophosphate and in calcium pyrophosphate mixtures. Similar factors may be operative in the cartilage calcification processes, and thus in chondrocalcinosis. Authors have ascertained that Arteparon - like chondroitin sulphate - inhibits crystal separation. The optimal concentration for this inhibition lies in the range of the proteoglycan content of normal cartilage. Where there are physiological phosphate-pyrophosphate ratios and a low magnesium concentration, it is certain that Arteparon has an inhibiting effect. Apart from the spatial structure of the substance, the importance of negative charge density for the inhibition of separation of calcium phosphate and pyrophosphate crystals is thought to contribute to this effect of Arteparon.

The importance of minor collagenous chains in the articular cartilage.

This is a review of the present knowledge on the newly-described minor cartilage collagens. The authors deal with the nomenclature, methodology, structure and function of the minor collagens based on both literary data and their own results. The authors were the first to describe the occurrence of minor collagens in adult human articular cartilage and the change of the minor collagen content with age and osteoarthrosis [25]. The methods used by different groups for fractionation of minor collagens are different. As the recommended pepsin digestion used for solubilization of these collagens gives different results for the size of one of the minor collagens called M-collagen, special attention should devoted to the method used. The differential salt fractionation of pepsin-solubilized collagen was found the best. In this case the molecular structure of the M-collagen remained intact. In the neutral salt extraction technique recommended recently by Burgeson et al. [5], yield of collagen from sample is high, M-collagen is degraded. The greatest solubilization of collagen was achieved by the cyanogen bromide technique. The mixture of peptides from different proteins, however, requires further fractionation. The authors propose to investigate minor collagens in tissue samples obtained from biopsies and in synovial fluid.

Minor collagens in arthrotic human cartilage. Change in content of 1 alpha, 2 alpha, 3 alpha and M-collagen with age and in osteoarthrosis.

The content and distribution of 1 alpha, 2 alpha, 3 alpha and M-collagens in human articular cartilage were studied. As controls, normal femoral heads and costal cartilage of autopsy material from newborn to 91-year-old persons were used. The osteoarthrotic cartilage was obtained from patients undergoing total hip replacement aged 45-80. The pepsin-digested cartilage collagen was fractionated by differential salt fractionation. The collagen content of the fractions was determined, and the fractions were separated by polyacrylamide slab gel electrophoresis. In the extracted collagen, the type II collagen varied from 82 to 97 per cent with increasing age. The 1 alpha, 2 alpha and 3 alpha chains decreased. M-collagen, especially of the high molecular weight components, disappeared with age. In osteoarthrosis three types of change - degeneration, new fibrocartilage formation on the surface of osteophyte and reparative cartilage - were separately studied. In all types of osteoarthrosis, an increase of minor collagens was found. In newly formed fibrocartilage, the reappearance of M-collagen was conspicuous. It is proposed that the three types of osteoarthrotic cartilage may be characterized on the basis of content and distribution of minor collagens.

The effect of proteoglycans of cartilage and over-sulphated polysaccharides on the development of calcium-hydroxy-apatite (CHA) crystal formation in vitro.

A coulometric model system is described which facilitates the quantitative study of the kinetics of transformation of amorphous calcium phosphate (ACP) into calcium-hydroxy-apatite (CHA) crystals. Proteoglycans of high molecular weight and over-sulphated polysaccharides (Arteparon, dextran sulphate) delayed CHA crystal formation. The results have enabled us to characterize the structure activity relationship of inhibitors of CHA formation, and to postulate a general structural requirement for molecules with inhibitory effect. As working mechanism, binding of calcium ions by sulphate groups of polyanions was supposed, which might reversibly impair "the critical nuclei formation", and/or further deposition of calcium ions in the CHA crystals. The clinical, therapeutical significance of the determination of the threshold concentration of different compounds is discussed.

The effect of inorganic pyrophosphate (PPi) anions on the in vitro collagen fibril formation.

This paper presents a study on the effect of sodium salt of pyrophosphate (Na-PPi) in different concentrations (1-10 mMol/l) on the in vitro collagen fibril formation. Collagen was mixture of type I and III collagen dissolved in neutral buffer containing sodium chloride of 1.0 mol/l. Na-PPi added to collagen delayed the fibrillary precipitation of collagen from the solution. Electronmicroscopically the fibrils are of native collagen type, but they are thicker and show a pattern of fine substriation. Calcium and other divalent cations showed differences in their influence on the effect of Na-PPi. This experimental model was developed in order to interpret the supposed role of PPi in the inhibition of apatite crystal formations as well as the fibrogenic effect of its calcium salt in PPi-arthropathy. The effect of PPi on the collagen fibril formation should also be taken into consideration.

The effect of glycosaminoglycans (GAG) on the intramolecular bindings of collagen.

The subfractions of collagen (alpha, beta and gamma components) precipitated from neutral collagen solution by gelation corresponded to those of collagen precipitated in the same conditions (ion environments) and in the presence of chondroitin-4-sulphate. Collagen fibrils precipitated delayed in the presence of heparin poor in beta chains and rich in alpha chains. The delaying effect of oversulphated GAG on the in vitro fibril formation can be explained by the release or prevention of ion-type intramolecular bonds of collagen. The possible in vivo significance of the above bonds is discussed.

The effect of glycosaminoglycans on the in vitro fibril formation of collagen type I and type III.

The effect of glycosaminoglycans (GAG) on the in vitro collagen fibril formation of type I and type III collagen extracted from rat skin was studied by measuring the optical absorbancy change of collagen-GAG solution during the fibrillary precipitation and following the electronmicroscopic structure of collagen fibrils. The main difference between type I and type III collagen was found in the kinetic of fibrillary precipitation mainly during the logarithmic growth ("log") period of fibril formation. Chondroitin sulphate-A (CSA) shortened this phase and affected especially type III collagen which has a longer "log" period. No difference was found in the electronmicroscopic structure of collagen fibrils. The supposed biological significance of the results is briefly discussed.

The influence of inorganic phosphate and citrate anions on the effect of glycosaminoglycans during collagen fibril-formation.

Some anions like phosphate, sulfate and citrate delay the fibril-formation. The interaction of phosphate and citrate with chondroitin sulfate-A (CSA) in binding to collagen was investigated in different environmental conditions. By changing the concentration of phosphate from 5 to 50 mM/l and that of CSA from 0.5 to 16 mM/l some kind of competition of the anions was discovered. When different equilibration, systems were used the affinity of CSA to collagen was found to be 10 times greater than that of phosphate at pH 7.2 and I=0.15. As phosphate anions bind to collagen at physiological concentration. phosphate anions may be supposed to influence the biological fibril-formation. On the other hand the CSA exchanges phosphate for collagen. Therefore, glycosamino-glycans (GAG) in connective tissue ground substance in fibrillogenesis may regulate the ion binding and through this the tendency of aggregation of the collagen molecules. The binding of citrate to collagen and its effect and possible role in fibril-formation was also evaluated.

The role of sulphation of glycosaminoglycans in their structural and functional characteristics.

The CD spectra in the far ultraviolet and the CD and ORD spectra of methylene blue-complexes (MB-complex) of glycosaminoglycans (GAG) with different sulphate contents were studied in comparison with their effect on the in vitro formation of collagen fibres. The following polyanions were investigated: Chondroitin sulphate-A with different sulphate contents, oversulphated chondroitin sulphate, heparin and dextran sulphate and their desulphated derivatives. It was observed that the MB-complex of polysaccharides with the same backbone structure, but with different sulphate contents might show ORD and CD spectra of exciton type, but of inverse sign. This phenomenon was interpreted on the basis of different types of aggregation of dye molecules bound to the polysaccharides. The biological effect of GAG changed also with the sulphate content. This suggests that it is the extent of sulphation rather than the glycosidic structure of GAG which determines their chiroptical and functional properties.

Secondary structure of glycosaminoglycans.

It has been shown by X-ray diffraction and chiroptic spectroscopy that the glycosaminoglycans in connective tissue develop a helical structure; this induces the coordination of polycations and scleroproteins in their environment polycations and promotes or inhibites fibrillary aggregation. Analysis of the ORD and CD spectra of various glycosaminoglycans, as well as those of oversulphated and desulphated preparations allowed the following conclusions concerning the secondary structure. 1. For the helical structure of glycosaminoglycans a chain-length of 120 to 150 A and the presence of at least one sulphate group per two disaccharides are required. The helical structure is not influenced by binding to the protein skeleton, e.g. in proteoglycans. 2. Chiroptical properties of the glycosaminoglycan--either in themselves, or if their methylene blue complexes are studied--are determined by the degree of their sulphatation. 3. The degree of sulphatation determines the secondary structure of the molecule and thus also its biological properties. Fibrillogenesis in vitro and probably also in vivo are determined by the strength of the bond between collagen and glycosaminoglycans, in addition to the effect inducing the coordination. Structural analysis of the polysaccharides supports the coordinated fibrillary structure of GAG, as already assumed on the basis of polarization microscopic methods.