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Mucopolysaccharidoses are a group of diseases that belong to lysosomal storage disorders. A common sign of these monogenic multisystem diseases is a gene mutation leading to a deficiency of the lysosomal enzyme participating in glycosaminoglycan degradation. It results to their accumulation in the tissues and organs, where they cause a progressive damage. There is no efficient treatment available for most mucopolysaccharidoses. Moreover, the research is complicated because of the low prevalence and type of affected tissues. Animal models of these human diseases are used for an evaluation of newly developed therapeutic approaches. However, they also have many limitations due to the different pathogenesis and catabolic pathways of the accumulated substrates between humans and animals. Therefore, animal models are replaced by human cell models. In this thesis, the development of four mucopolysaccharidoses human cell models is reported (MPS IIID, MPS IVA, MPS IVB, MPS VI). Corresponding genes (GNS, GALNS, GLB1, ARSB) were inactivated using CRISPR/Cas9 technology, where plasmids containing specific inserts are delivered to the target human induced pluripotent stem cells (iPSC), using electroporation. Isolated clones, which represent iPSC disease models, were characterized by Sanger sequencing, enzyme...

The thesis is focused on a determination of heparin and chondroitin sulfate, using flow injection analysis with spectrofluorimetric detection. The determination is based on the interaction of negatively charged heparin, chondroitin sulfate resp., with a cationic dye (azure B or phenosafranine) which is manifested by the decrease in fluorescence intensity of the dye in its emission maximum. The optimal conditions for the determination in static mode were found, and calibration dependencies were measured. The conditions of FIA were optimized and following parameters were established: the volume of dispensed sample of 100 ml, the length of the reaction coil 60 cm, the flow rate 0.7 ml min-1 , the concentration of azure B 1.6×10-5 mol dm-3 , the concentration of phenosafranine 3.5×10-5 mol dm-3 . For the determination of heparin using azure B it was found: LOD = 0.023 IU ml-1 , LOQ = 0.186 IU ml-1 , and linear dynamic range 0.19-1.43 IU ml-1 . For the determination of heparin using phenosafranine it was found: LOD = 0.102 IU ml-1 , LOQ = 0.192 IU ml-1 , and linear dynamic range 0.19-1.79 IU ml-1 . For the determination of chondroitin sulfate using azure B it was found: LOD = 0.58 mg dm-3 , LOQ = 2.37 mg dm-3 , and linear dynamic range 2.37-8.32 mg dm-3 . The developed determination was applied to the...

Albuminuria (A), increased urinary excretion of glycosaminoglycans (GAG) and increased activity of N-acetyl-beta-glucosaminidase (NAG) in urine are early markers of glomerular and tubular changes in various pathological conditions at a time when renal functions do not yet display impaired function and when the changes are still reversible. The objective of the presented study was to assess to what extent these early changes may play a part in acromegaly.In a group of 24 acromegalic patients and in 18 healthy controls the authors examined the microalbuminuria (RIA Immunotech Prague), urinary excretion of glycosaminoglycans (spectrophotometrically by the carbazole method) and they assessed the NAG activity in urine (spectrophotometrically). In acromegalic patients before surgical and pharmacological treatment the authors found, as compared with healthy controls, increased urinary excretion of GAG [4.4 (0.9-22.7) g/mol creat. vs. 2.1 (0.8-5.5) g/mol creat, por = 0.001], elevated albuminuria [3.6 (0.3-37.4) g/mol creat. vs. 0.5 (0.1-2.2) g/mol creat, por = 0.001 and an enhanced NAG activity [1005 (345-2935) U/l vs. 470 (195-1135) U/l, por = 0.001]. The parameters of albuminuria and urinary GAG excretion characterize rather glomerular renal function, they correlate mutually (r = 0.64 por = 0.001), while the urinary NAG activity, depending on tubular function, does not correlate with them. No correlation of these parameters with the IGI concentration (for A: 0.3, for GAG: -0.04 and for NAG: -0.02 according to Pearson was found.In hormonally active acromegalic patients without apparent altered renal functions (normal serum creatinine, Albustix negative) the authors detected early changes of glomerular and tubular functions. They found a significant correction between albuminuria and GAG excretion.

The condition of patients after percutaneous transluminal angioplasty is influenced among others by the subsequent development of restenoses and reocclusions. The objective of the submitted work was to assess whether oral administration of heparan sulphate can influence the development of restenoses after percutaneous transluminal angioplasty in the pelvic and femoropopliteal region.102 patients (78 men and 24 women, age 42-86 years) were divided into four groups. Percutaneous transluminal angioplasty was performed either on account stenosis in the aortoiliac or femoropopliteal area. (The original number was 115 patients, 13 patients were eliminated: the reasons were technical failure of the intervention procedure, reocclusion, the patient was lost from records.) As antiaggregant the patients were given acetylsalicylic acid, 250 mg/day: patients included in the heparan group were given heparan sulphate (Hemovasal, Manetti and Roberts) 100 mg/day for a period of 3 - 4 months. As compared with controls, the patients treated with heparan sulphate had within the 3 - 4 month period a significantly longer claudication distance (p0.05), a higher Doppler index (p0.05) and maximal blood flow in the feet.Heparan sulphate administration to patients after percutaneous transluminal angioplasty on account of stenosis of the aortoiliac or femoropopliteal area improves some angiological parameters which can suggest a slighter tendency of early restenosis.

The author presents his experience assembled in a short-term (two-week) therapeutic trial with Mobilisin ointment, which contains flufenamic acid, in 50 patients with primary localized periarticular and muscular fibrositis. The preparation was used locally three times per day-2-3 cm of ointment pressed from a tube. The patients were given a form in which they recorded every night the effect classified in four grades from the best to zero effect. In 49 the tolerance of the ointment was very good. In 45 patients a therapeutic effect was achieved (90%), comprising 21 (42%) where the effect was excellent, in 24 it was good or satisfactory (48%) and only in 5 patients no effect was recorded or the patents' statement was vague. In the first group of 21 patients 16 were able to abandon analgetic and anti-inflammatory treatment with non-steroid antirheumatics. Patients with the muscular form of fibrositis practically agreed in favour of the ointment. Gels according to these patients tissues. The authors remind that according to their experience gels, incl. Mobilisin, are suited for periarticular forms of fibrositis. It is also useful to rub the ointment in micro-massage to achieve a greater initial hyperaemia and better resorption. Mobilisin ointment may prove useful also in other disciplines, in particular orthopaedics, surgery, traumatology, neurology, rehabilitation and sports medicine.

Contemporary views on the treatment of thromboembolic disease comprise the possibility to use preparations with an antithrombotic effect in indications, where it is not necessary to administer anticoagulants or thrombolytic drugs or combined treatment. Sulodexide can due to its effects, easy administration (in particular the oral form) and unpretentious monitoring be used where so far only more pretentious therapeutic procedures could be applied. Our initial experience in fourteen patients with deep venous thrombosis seems to be very encouraging.

Chronic congestive heart failure is one of the risk groups of acquired hypercoagulant and hyperviscous state. In a group of patients with medium and severe cardiac failure the administration of sulodexide led to an increased activation of the fibrinolytic potential--a drop of PAI-1 and fibrinogen, to an increased activation of anticoagulatory potential--an increase of AT III and reduced plasma viscosity. Global coagulation and biochemical screening parameters were not affected by treatment. Administration of the preparation did not exert important undesirable effects and was well tolerated.

The authors examined the glycosaminoglycan level in the peritoneal fluid of 54 infertile women with or without endometriosis. The peritoneal fluid was collected during a routine laparoscopic examination. Glycosaminoglycans were assayed in complexes with Alcian blue. There was a higher concentration of glycosaminoglycans in peritoneal fluid during the follicular phase of women with endometriosis. The authors did not prove a statistically significant difference between women with and without endometriosis.

A total of nine patients with diabetes type I and II and with claudications due to macro- and microangiopathies of the lower extremities took sulodexide (Vessel Due F) by the i.m. route for a period of 20 days and then as capsules for 90 days. In all a favourable therapeutic effect was recorded, in particular as regards delay of claudications. The authors recorded lower blood sugar values, lower levels of cholesterol, triglycerides, fibrinogen and plasma viscosity. In the authors' view sulodexide is an important advance in the treatment of diabetics with claudications.

In the submitted review the authors summarize briefly knowledge on the mechanism of action of chondroprotective substances and the present state of their use in clinical practice. The authors mention also the chondroprotective action of some non-steroid anti-rheumatic drugs.

The authors describe their own modification of the ELISA method for the assessment of the epitope of keratate sulphate (KS) in synovial fluid. Considerably elevated values of KS were found in patients with gouty arthritis and reactive arthritis, as compared with patients with rheumatoid arthritis. The KS levels in the exudate of patients with osteoarthritis were significantly higher than in patients with rheumatoid arthritis. In patients with rheumatoid arthritis a negative relationship was found between the KS level in the exudate and the activity of the disease. A relationship was found between KS in the exudate and the interleukin-1 level in the exudate. Finally the authors discuss the importance of assessment of cartilage fragments in body fluids by immunochemical methods and future development trends of this discipline.

On 10 experimental animals (20 eyes of bastard rabbits) the authors tested the possibility to influence by cryostimulation the increase of the acid mucopolysaccharide content (glycosaminoglycans or proteoglycans) after damaging the eye with 6.0% = 1.4 N sulphuric acid. Eight corneae were subjected to cryotherapeutic treatment 4-6 hours after corrosion, by the method of contact freezing with a cryoapparatus OKG 2, using a technique which in the first part of the work had a convincing cryostimulating effect: working temperature -70 degrees C, 10 spots with a diameter of 2 mm for 7 seconds placed directly in the cornea surrounding the damaged area. As controls for both groups (corroded and cryotreated and corroded not treated) served four normal intact corneae (two animals as above). Collection of samples of the cornea was made on the 4th and 10th day after corrosion. The corneae were processed by histological methods stained with toluidine and alcian blue. It was revealed that on the 4th as well as 10th day after cryostimulation the acid mucopolysaccharide content was higher than without cryostimulation. On the 10th day after the injury and cryotherapy the acid mucopolysaccharide content was normal. The results were evaluated by Fischer's test. The increase of acid mucopolysaccharides on the cornae was more rapid than at the 1% level of significance, as compared with corneae without cryotherapy. Cryotherapy thus promoted restoration of acid mucopolysaccharides in the corroded corneae.