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Unlabelled: After a brief review of the symptoms of Salzmann's nodular corneal dystrophy the authors report on 28 patients. As found by radiological examination, arthrosis deformans was present in all of the patients and, in nearly all cases, osteoporosis has been diagnosed. Histological examination revealed a regressive change in 3 patients' costal cartilage of identical macular character. 22 patients had cataracta and 20 an impaired hearing. Therefore, the disease belongs to the keratochondrosises., Conclusions: 1. The regressive change of the corneal stroma, the hyaline cartilage and the bones relies assumably on an abnormality of their mutual component, the mucopolysaccharides. 2. Experimental and therapeutic experiences have shown that prolonged mucopolysaccharide administration (heparin) will induce osteoporosis. This explains the relationship between osteoporosis and metabolic disorder of mucopolysaccharides. 3. The remarkably high occurrence of cataracta, in comparison to a normal population of the same age, is presumably related to the metabolic disorder of mucopolysaccharides. 4. The aforesaid seem to confirm that Salzmann's nodular corneal dystrophy, corneal dystrophy is not induced by repeated inflammations but conversely, pathological metabolism of the cornea in consequence of dystrophy is responsible for serial keratitis.

The authors performed 451 transesophageal echocardiographic (TEE) investigations over a period of three years and four months. Atrial septal aneurysm (ASA) was found in 40 cases. Of these, protrusion of the atrial septum towards the right atrium was observed in 17 cases, whilst oscillation of the atrial septum was noted in 23 cases. ASA was associated with patent foramen ovale (PFO) in ten patients, with type II. ASD in nine patients, with other congenital heart disease in six patients, and with other organic heart disease in eight patients. In three cases either an embolus or a tumor was detected in the left atrium, whilst in four cases with ASA there were no other organic cardiac disorders found. In ten patients there was a history of cerebral embolisation. Of these two had chronic atrial fibrillation, whilst the others had sinusrhythm. Of those who had cerebral embolisation, four patients had PFO, one patient had left atrial and auricular thrombi, whilst in four patients various organic heart problems (ischemic heart disease, left ventricular hypertrophy) were detected. In one patient with ASA there was no other cardiac abnormality detected. The authors conclude that ASA, which is often associated with PFO and ASD (in 25.0% and 22.5% of their cases, respectively) is detected in around eight percent of the patients who undergo TEE. ASA particularly when associated with PFO should be considered as a potential source of cerebral emboli. Indeed, cerebral embolisation occurred in 25% of their patients with ASA. It is recommended, that patients with ASA are treated with acetyl salicylic acid, whilst in patients with ASA and PFO anticoagulant therapy is the treatment of choice. In case of cerebral embolisation, or repeated cerebral ischemic attacks, operative interventions should be considered.

The authors investigated the frequency of left atrial spontaneous echo contrast in mitral valve disease. They also tested whether there was any correlation between the presence of left atrial spontaneous echo contrast and the severity of the mitral valve disease. Echocardiographic investigations were performed using both transthoracal and transesophageal echocardiographic methods employing monoplane transducer. The authors carried out 273 transesophageal investigations over a period of 2 years and found left atrial spontaneous echo contrast in 85 patients, who had mitral valve disease. Of this, in 18 cases thrombi were also detected in the left atrium and/or auricula. The diagnoses of mitral stenosis were made in 24 patients, of whom in 12 cases the stenosis were found to be severe, whilst in 12 cases to be moderate. Furthermore insufficiency of the mitral valve was detected in 35 cases. 20 patients had artificial mitral valve implanted, they received long term anticoagulant treatment. 59 patients had no spontaneous echo contrast. 14 patients had previous embolic events of which 9 were cerebral and in other cases arteries of the kidney, eye and extremities were affected. 71 patients had no history of embolism. The authors concluded that mitral valve disease, particularly mitral stenosis is frequently associated with left atrial spontaneous echo contrast. It has been also observed, that the more severe the mitral valve disease, the greater the probability of left atrial spontaneous echo contrast. In all cases where thrombi were found, left atrial echo contrast were demonstrated and the risk of embolism is high. In these cases anticoagulant therapy is suggested.

The substrate specificity of elastomucoproteinase (EMP), an enzyme which was first isolated from crude pancreatic elastase and described as a proteoglycan-degrading enzyme, determined on tripeptide-p-nitroanilide substrates indicates the existence of a 'new' chymotrypsin-like enzyme. EMP, however, did not cleave any glycosaminoglycans, i.e., its 'mucolytic' effect has been excluded. Activity of EMP on synthetic or protein substrates (e.g., collagen type-II and aggrecan of cartilage) was completely inhibited by serine proteinase inhibitors, which was also found when using cartilage proteoglycan monomers. EMP cleaves the core protein of proteoglycan monomer (aggrecan) into small peptides, some containing glycosaminoglycan chains resulting in an unusual elution profile on Sepharose CL-6B chromatography when compared to the effects of pancreatic and granulocyte elastases, chymotrypsin, cathepsin G and stromelysin. EMP-like activity also was detected in neutrophil granules of bovine leukocytes and polyclonal antibodies were raised against purified bovine EMP to detect the enzyme in both crude elastase preparations and the granule fraction of bovine leukocytes.

Vitamin D3 and 25-OH-D3 were tested for their effect on glycosaminoglycan (GAG) production in micro high density cultures of stage 22-24 chicken limb bud mesenchyme. Vitamin D3 (at concentration of 5 x 10(-6)M) and its biologically active metabolite, the 25-OH-D3 (at concentration of 5 x 10(-8)M) stimulated the synthesis of GAGs indicating the increase of radioactive sulfate incorporation and uronic acid content in a dose-dependent way. The findings obtained after the treatment with 25-OH-D3 are comparable with earlier studies performed on chondrocyte cultures (Corvol et al. 1978, 1980), while the results observed after the vitamin D3 treatment are the first indications that vitamin D3 can also influence the cartilage matrix composition.

In 28 dogs the distal articular cartilage of the femur was removed and the regenerating articular surface on the 70th postoperative day was studied histochemically for hexokinase, glucose-6-phosphatase, phosphohexose-isomerase, fructose-1, 6-diphosphatase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, lactate dehydrogenase isoenzymes, phosphoglucomutase, phosphorylase, glycogen synthetase, UDP--glucose dehydrogenase, and UDP-glucuronic acid-4-epimerase. The articular surface consisted of fibrous tissue and of cartilage islets. The latter contained cells differentiating into cartilage and young chondrocytes. The glycolytic enzymes reacted positively in the regenerative articular surface. Enzyme activities were higher in the cells (particularly the chondroblasts and young chondrocytes) of the cartilage islets than in the connective tissue. In the cells differentiations into cartilage, beside the LDH isoenzymes characteristic of glycolysis, a significant LDH1 and LDH2 activity was observed. At the same site the presence of fructose-1, 6-diphosphatase-activity could be assumed, but there was no glucose-6-phosphatase activity. Glycogen synthesis proceeded in the cells of the cartilage islets and UDP-glucuronic acid-4-epimerase activity was observed in the differentiated cells. UDP-glucose dehydrogenase activity was positive in every section of the articular surface.

A modification of division cell cycle under conditions of in vitro cartilage differentiation could be demonstrated. The frequency distribution of histograms showed that the G1 cells represented 50% of total cells number at zero time (i.e. in suspension before inoculation), 69% on day 2 and about 90% in following period of days 4, 6 and 14. Dextran sulfate and hyaluronic acid produced a conspicuous inhibition and the chondroitin sulfate as well as heparin exerted a moderate inhibiting effect on the cell proliferation.

A consistent chondrogenesis takes place in high-density microcultures derived from bud mesenchymal cells of 4-day-old chicken embryos in a serum-supplemented medium. In serum-free medium DNA level and uronic acid content in the cultures were low, as well as the 35SO4 uptake and release, and only a small mass of cartilage was formed. With the addition of 0.025-10 micrograms/ml insulin to serum-free medium the uronic acid and DNA content in the cultures increased considerably in a dose-dependent way. The intensity of 35SO4 uptake and release exceeded the values measured in serum-containing medium, more cartilage tissue was formed in them also in a dose-dependent manner. With the use of 20-80 micrograms/ml insulin, the increment in DNA content proved to decrease, and with the use of 80 micrograms/ml insulin the uronic acid content and the cartilage mass decreased to a greater extent than in the case of lover doses.

The distal articular surface of the femur was surgically removed in 57 dogs. Succinate dehydrogenase and cytochrome oxidase activities were assayed on postoperative days 7, 20, 26, 33 and 70 in the regenerating, chondrifying articular surface and in the granulation tissue adhering to the capsule. In the 70-day samples, the cyanide-induced inhibition of oxygen consumption was determined and enzyme histochemical reactions (cytochrome oxidase, monoamine oxidase, xanthine oxidase, peroxidase and "catalase") were performed. The succinate dehydrogenase activity was the highest in the early postoperative stage in both tissues. This was followed by a definite decrease and a subsequent significant increase in activity when chondrification took place. Measurement of cytochrome oxidase activity could not reveal any convincing result, presumably because of the properties of the tissues studied. The oxygen consumption by the chondrifying articular surface at 70 days was inhibited to about 50% by cyanide, and about 90% inhibition was observed in the tissue adhering to the capsule. The cells of the regenerating articular surface possess cytochrome oxidase and a cyanide- (and sodium azide-) resistant oxidase activity. The enzyme activity of the cartilaginous islets exceeded that of their connective tissue environment. The cytochrome oxidase activity increased in the cells during cartilage differentiation. Presumably, some further cyanide-sensitive and cyanide-resistant oxidases are present in chondroblasts and young chondrocytes.

cAMP and cGMP levels were measured in micro high-density cultures of chick limb bud mesenchyme cells stages 22-24 after 1, 2, 4 and 6 days of culturing. In these cultures, a consistent cartilage differentiation proceeds parallel to the progressive accumulation of cells in the G0 phase. The cAMP level increased by 45% by the time of the onset of cartilage phenotype expression, and significantly decreased thereafter. The cGMP level gradually diminished by a total of 39% during the period examined. It is suggested that the decrease in cell proliferation may be the consequence of the reduction of the cGMP level, and that a short-term marked elevation of cAMP level induces cartilage differentiation in the limb mesenchymal cells which are able to select only from a limited number of differentiation programmes.

Effects of hyaluronate, heparin and chondroitin-6-sulfate were studied on micromass cultures of chick limb bud mesenchyme (Hamburger and Hamilton stages 23-24). Histochemical, electron microscopical, biochemical and radiochemical investigations of day 4 cultures revealed dose-dependent inhibitory effects of these glycosaminoglycans on chondrogenesis, cyclic AMP level and growth of cells. In addition, hyaluronate with 100 micrograms/ml dose caused a displacement of newly formed proteoglycan from cultures into the medium. It is supposed that exogenous glycosaminoglycans influence ionic equilibrium in the immediate vicinity of cells and disturb the organization of the prechondrogenic extracellular matrix resulting in alterations of cell membrane--cytoskeleton associations. These alterations may provoke a reduction in cyclic AMP level and DNA synthesis. It is suggested that a reduction in cyclic AMP level preceding the expression of cartilage phenotype results in the inhibition of chondrogenesis.

Antibodies to proteoglycan (PG) and glycoprotein of bovine nasal cartilage were conjugated with fluorescein isothiocyanate and with horseradish peroxidase. Hyaluronidase digestion of cartilage tissue-specimens increased the intensity of immune reactions; pronase digestion or extraction with 4 M guanidinium chloride abolished the staining. In the intercellular matrix fine filaments beaded with small granules were seen forming an irregular network. The interstices of the network are filled with collagen fibers linked together by the filaments and granules. In view of the linear conformation of core proteins of PGs and the globular conformation of glycoproteins (link proteins), it may be supposed that the granules and filaments represent these two protein components of PG-aggregates. In chondrocytes a homogeneous staining was recorded in the endoplasmic reticulum, in the juxtanuclear areas and in several smooth-walled vesicles and elongated areas situating subjacent to the cell membrane. In contrast to the extracellular immune reactions, this homogeneous intracellular staining was never enhanced by hyaluronidase digestion. This is interpreted in the sense that conformation changes of molecules secreted, and the aggregation of PGs, occur extracellularly.

A consistent chondrogenesis takes place in micro high-density cultures derived from limb mesenchymal cells of chick embryos of stages 23-24. Flow-cytometric measurements of DNA content showed that cells in the phase of G1 or G0 made up 51% of the dispersed cell suspensions. The proportion of these cells increased to 71% by the onset of cartilage differentiation in day-2 cultures. This ratio was 84% when the voluminous matrix formation began on the 4th day of culturing. Thereafter, it increased to 90% by the 6th day, and to 93% by the 14th day. The results suggest that cartilage differentiates from G0 mesenchymal cells of the limb. In our measurements, however, the G0 phase includes all non-proliferative cell population which have identical DNA content with G1 cells. Therefore, the G0 phase contains also an increasing number of chondroblasts and chondrocytes as the chondrogenesis proceeds.

The articular surface of the distal part of the femur was removed operatively in dogs, and the regenerating articular surface and the GTC were investigated at different stages from the 7th to the 70th postoperative days. During this period cartilage islets arose in the GTAS, while the GTC transformed to connective tissue. At 7 days the lipid content of the tissue was markedly higher than at the other stages studied. Lipids, predominantly triglycerides, were present in extracellular form as well. From the 20th to the 70th day the PL fraction became predominant and, in addition to the pre-existing lecithin, relatively large quantities of lysolecithin, sphingomyelin, phosphatidyl-ethanolamine, phosphatidyl-serine and phosphatidyl-inositol could be gradually demonstrated. Differences were noted in the time of appearance and binding of PLs between the two types of granulation tissue. As time proceeded, the proportion of saturated fatty acids decreased in favour of unsaturated ones. At 70 days, the GTAS contained fatty acids up to C18. About 50% of the fatty acids consisted of C16:1, C18:2 and C18:1. At the same stage, in the GTC C16:1, C18:1 and C20:1 were present in larger amounts. Of the free fatty acids C16:1, C16 and C18 were in predominance in the GTAS and the proportion of fatty acids having more then one double bonds increased with time. In the GTC C16 and C18:1 were in great majority. According to histochemical evidence, the tissues did not contain extracellular lipids from the 20th postoperative day. In the cells, the presence of glycerides, PLs, lipoproteins and cholesterol was demonstrated. In addition, in cartilage precursors of more advanced maturity, a considerable fatty acid positivity was noted.

In micro-mass cultures of stage 23-24 chicken embryos the expression of cartilage phenotype was density dependent and it always took place practically to the same extent at the density of 2 X 10(5) cells per culture. The cells formed aggregates on day 1 and cartilage nodules appeared on day 2. By day 3, 17% of the culture surface consisted of areas showing cartilage differentiation and these regions represented more than 50% on day 6. By day 14, the centre of th cultures contained a mass of cartilage. The progression of cartilage differentiation was indicated by the increasing amount of toluidine blue fixed by the cultures. By day 14 the DNA and protein contents increased 8.7 fold and 16.7 fold, respectively. Uronic acid and OH-proline were detected from the 2nd day. From day 3 to 14, the microgram uronic acid and microgram OH-proline per microgram DNA gradually increased 25 fold and 14 fold, respectively. A decreased density of the inoculum reduced the probability of cartilage formation and it failed to occur at the density of 1.25 X 10(4) cells per culture. Stage 19-20 limb cultures with identical density had the same morphological characteristics. Aggregates appeared but nodules failed to do so in cell cultures of stage 28 limb bud soft tissue region. It is supposed that an oxygen gradient may be present in the cell aggregates.

Collagen-free extracts were prepared from bovine, porcine and canine hyaline, elastic and fibrous cartilages, articular capsule, tendon, aorta, cortical bone and regenerating articular surfaces. The extracts were investigated with antisera to bovine nasal septal cartilage, dog articular cartilage and non-collagenous protein fraction of bovine cortical bone. Immunodiffusion, immunoelectrophoresis, and immunohistochemical methods were used. In the different supporting tissues of the three animal species a common antigen, probably of proteoglycan origin, was demonstrated. The finer differences in antigenicity between the different tissues are probably due to the variations in proteoglycan composition of the given supporting tissues. Owing to the wide-spread occurrence of the antigen, the authors suggest the term "species-common connective tissue antigen" instead of the "species-common cartilage antigen" used so far.

A consistent chondrogenesis takes place in micro-mass cultures of stage 23-24 chicken limb bud mesenchymal cells. In these cultures a short, marked elevation of cAMP level was detected at the time of the onset of cartilage phenotype expression. On the other hand, exogeneous glycosaminoglycans which inhibited chondrogenesis caused a reduction in the cAMP level of the cells. These correlations between cAMP level and phenotypic characteristics suggest that, among other things required in chondrogenesis, cAMP level may be a prominent factor.

From powdered bovine bone cortex an extract has been prepared with 0.15 M NaCl and its collagen-free fraction obtained on DEAE-cellulose column was used for the immunization of rabbits. The non-collagenous protein fraction (NCP) produced against immune serum two precipitation arcs both on agarose double gel-diffusion and with immune electrophoresis. After decalcination in 10% EDTA PH 7.4, direct and indirect immune histochemical examinations were performed with fresh, freshly lyophilized, as well as with bones kept for three and six months at 20 degrees C, and the antigens were localized. Prolonged storage increased the antibody-binding capacity of the bones, whereas lyophilization decreased it. Fixation in cold acetone was suited best for immunohistochemical study of the cortical bones.

Binding of fibronectins (FN) to collagen types I-IV were studied using polyclonal antibodies against human and chicken FNs, proteoglycan monomers, collagen type II and monoclonal antibodies reacting with both soluble and insoluble forms of human FN. Plasma fibronectin and type II collagen were shown to interact specifically in a homologous system. Type II collagen, however, proved to be less effective in inhibition assays compared to other types of collagen. In high density cultures of chicken limb bud cells, fibronectin was first localized within the fibroblast-like cells of 4 hr cultures and an extensive extracellular filamentous network developed by the end of day 1. Fibronectin was present in the newly formed cartilage nodules although it seemed to disappear by day 6, when the proteoglycan accumulation became more intensive. Enzyme treatments (testicular hyaluronidase, chondroitinase ABC) helped to localize FN at this stage of development of chicken cartilage, in microdroplet high density cultures of human fetal chondrocytes and in articular cartilage. Fibronectin was localized only in the pericellular ring of intact human articular cartilage using monoclonal antibodies with the biotin-avidin system.

The distal articular surface of the femur was removed operatively in 36 dogs. In the regenerating chondrifying articular surface and in the granulation tissue adhering to the capsule glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities were determined 7, 33 and 70 days after operation. In both tissues the activity of these enzymes characteristic of the pentose phosphate cycle ws the highest in the early postoperative stage. This initial increase in activity was followed by a marked reduction in the regenerating articular surface and by a moderate decrease in the tissue adhering to the capsule. For the loss in activity occurring in the chondrifying articular surface, the connective tissue cells (fibroblasts) are responsible. Cartilage precursors and young chondrocytes show a high glucose-6-phosphate dehydrogenase and 6-phosphogluconate activity. Presumably, in the given case of the functions of the pentose-phosphate cycle the NADPH generation and supply of building stones prevail. The activity of these enzymes ws determined in the articular cartilage and in the synovial membrane of the knee joint in further 18 dogs. The activity in the articular cartilage was very slight as compared to that in the synovial membrane.

A consistent chondrogenesis takes place in micro high-density cultures of chick limb bud mesenchyme cells stage 22-24. The effect of an increased generation of OH(.) free radicals by Fenton reaction was tested in these cultures. Components of Fenton reaction (i.e., ferrous iron in form of ADP-Fe2+ complex in 0.1 mM concentration, or 0.2 mM H2O2, or the combination of these components with each other, as well as with 0.2 mM ascorbate) were supplemented to the culture medium after the first 24 h. ADP-Fe2+ complex resulted in a drastic decrease of the frequency and confluence of the cartilage nodules seen in light microscope, accompanied electron microscopically by a strikingly increased frequency of occurrence of lipofuscin-like, residual bodies in the cells. Biochemical methods revealed a significant decrease of both the DNA and glycosaminoglycan contents (to 48.6 and 20.7% of the controls, respectively), in day 6 cultures. H2O2 alone caused similar alterations of the cultures, whereas the combination of it with ADP-Fe2+ complex proved to be lethal for the cells. Ascorbate when added to the ADP-Fe2+-treated cultures displayed a slight protective effect for the glycosaminoglycan content but not for DNA. The results are interpreted in terms of free radical theory of aging.

Complete removal of the articular cartilage in dogs is followed by regeneration of the articular surface. At the site of the bone wound, granulation tissue develops, which later differentiates into cartilage. The O2 and glucose supply of the regenerating articular surface is ensured by the synovial fluid, by the large exposed surface of the medullary cavity, and by the capillary network of the granulation tissue. Oxygen and glucose supply of the articular surface in different stages of differentiation has been statistically analyzed. It is suggested that in the early stage of regeneration O2 supply comes predominantly from the capillaries of the granulation tissue. Later on, as capillarization regresses, the oxygen supply, originating from the synovia and medullary cavity, assumes a more important role. In the stage of cartilage regeneration an oxygen-deficient state can be supposed in the entire articular surface, but areas differing in oxygen supply may be formed owing to local differences (due mainly to the extent of vascularization and degree of generation of the subchondral bone layer). At the site of chondrogenesis, conditions allowing aerobic metabolism of cells with reduced O2 requirements seem to be ensured. Glucose supply deriving from the above-mentioned sources satisfies the highest glucose requirements of the cells in the regenerating articular surface.

The cartilaginous articular surface of the distal part of the femur of adult dogs was removed and the composition of GAGs was determined in the granulation tissue adhering to the bone wound and in that adhering to the articular capsule 7, 33, and 70 days after operation. The articular cartilage and the synovial layer of the articular capsule of intact adult dogs were also studies. The materials were digested with papain and the released GAGs were fractionated according to Svejcar and Robertson's method. The articular cartilage of non-operated dogs contained, on the average, 65.3% ChS, 13% KS, 5.8% HA and 15.8% GAG of lower molecular weight. The synovial layer of the capsule contained 41.1% HA, 15.3% Ch4-S and Ch6-S, 13.7% DS, 21.7% KS, 2% H and 6% GAG of lower molecular weight. The granulation tissue of the articular surface and that adhering to the capsule show a different developmental course. The former differentiates into cartilage, whereas the latter is simply added to the tissue of the capsule. The two tissues are different in GAG composition as early as on the 7th postoperative day. With time an increase of Ch4-S, Ch6-S and KS can be observed in the tissue of the articular surface. The tissue adhering to the capsule is characterized by a high HA and an increasing DS content. From the study of the composition of GAG's (proportion of GAG building stones) a deeper insight can be obtained into the details of GAG biosynthesis characteristic of cartilage than from the analysis of quantitative data of ChS. In the development of GAG composition characteristic of the tissue, the epimerase reactions participating in GAG biosynthesis, and the mechanisms regulating their activities seem to play a primary role.