[Incidence of septal aneurysm and its clinical significance]. / Az atrialis septalis aneurysma elöfordulása és klinikai jelentösége.

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 frequency of left atrial spontaneous echo contrast and its clinical significance in mitral valve insufficiency]. / Bal pitvari spontán echo kontraszt elöfordulsa és klinikai jelentösége mitralis vitiumban.

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.

Substrate specificity of 'elastomucoproteinase': an enzyme which can degrade cartilage aggrecan.

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.

Interrelationship between cyclic AMP level and chondrogenesis in vitro.

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.

Exogenous glycosaminoglycans modulate chondrogenesis, cyclic AMP level and cell growth in limb bud mesenchyme cultures.

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.

[Effect of insulin on cartilage differentiation in vitro]. / Vliianie insulina na differentsirovku khriashcha in vitro.

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.

Effect of vitamin D3 and 25 hydroxyvitamin D3 on glycosaminoglycans in micro high density culture.

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.

Proteoglycan biosynthesis is stimulated by D-penicillamine in chondrifying high density cell cultures.

Chondrifying high density cell cultures of stage 22-24 chick embryo limb bud mesenchyme were treated with 5, 10 and 15 mmol/l D-penicillamine (DPA) for 4 and 6 days. The cultures were analyzed with morphological and biochemical techniques to learn more about the effect of DPA on the metabolism of cartilage glycosaminoglycans (GAGs). Using light and electron microscopic histochemical reactions for GAG, a considerable increase in the intensity of staining of the cartilage matrix could be detected in cultures treated with DPA as compared to the untreated controls. The uronic acid content of the treated cultures was higher than that of the controls. Liquid scintillation measurements and autoradiography revealed that DPA treatment increased the 35S-sulfate into the cultures. These data suggest that DPA - besides its well known inhibitory effect on collagen crosslink formation - alters the metabolism of sulfated GAGs in differentiating cartilage. It is supposed that DPA stimulates the biosynthesis of these macromolecules.

The effect of OH(.) radicals generated by Fenton reaction on the growth and cartilage differentiation in limb bud cell culture.

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.

Appearance and persistence of fibronectin in cartilage. Specific interaction of fibronectin with collagen type II.

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.

Changes in cyclic AMP and cyclic GMP levels during in vitro chondrogenesis.

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.

In vitro cartilage differentiation: modification of division cycle and effect of some polyanions.

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.

Modification of the cell cycle of limb bud mesenchyme during in vitro cartilage differentiation.

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.

Cartilage differentiation in micro-mass cultures of chicken limb buds.

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.