[Urinary bladder substitution using combined membrane based on secretions of human mesenchymal stem cells and type I collagen].

AIM: Despite the widespread use of intestinal cystoplasty, urinary bladder substitution remains a challenging problem due to the complexity of operations and the potentially high risk of complications. A promising alternative may be bio-engineered collagen-based matrices containing stem cells or their secretions. MATERIAL AND METHODS: To evaluate the effectiveness of this bladder substitution modality, an experiment was conducted on 14 male rabbits. The animals underwent resection of urinary bladder, and the formed defect was substituted with a membrane of type I collagen (series 1, 5 rabbits) or a membrane of the same composition containing a conditioned medium with secretion of mesenchymal stem/stromal cells derived from human adipose tissue (series 2, 5 rabbits). In the comparison group (4 rabbits) resection of the bladder and the closure of the defect was carried out without bladder substitution (series 3). RESULTS: At 1 month after surgery, there was a complete epithelization of the inner surface of the implant, and body tissues replaced the collagen matrix. In series 1, the collagen implant was replaced mainly by connective tissue ingrown with occasional solitary smooth muscle cells. In series 2, the newly formed bladder wall contained numerous smooth muscle cells, growing into the collagen matrix and forming the muscular coat. In series 3, the muscular layer regeneration at the scar site was also noted, but it was less intense, which was confirmed by morphometry. In series 2, more active vascularization of the collagen implant occurred due to neo-angiogenesis, which was more intense than that in series 3, and especially in series 1. Functional studies revealed a reduced bladder functional capacity in series 1 and 3, while in series 2 it was close to normal. During filling cystometry, changes in intra-vesical pressure profile in series 2 were close to normal, while in series 1 and 3 infusion of a small volume of saline resulted in a marked increase in intra-vesical pressure, showing a reduced compliance of the reconstructed bladder. Discussion The study findings show that implants based on type I collagen can be effectively used to substitute a part of the urinary bladder wall, but bio-engineered collagen matrix grafts containing cell regeneration stimulants secreted by stem cells in their culture medium seem to be more promising.

Kinetics of BMP-2 release from collagen carrier: evaluation by enzyme immunoassay in the presence of plasma proteins.

Test system ELISA-BMP-2 is developed for measuring recombinant human bone morphogenetic protein-2 in human and laboratory animal serum and plasma by sandwich ELISA. The test system has been used for studies of the kinetics of bone morphogenetic protein-2 release from collagen carrier in the presence of plasma proteins.

[Regulation of the binding of the BMP-2 growth factor with collagen by blood plasma fibronectin].

The release kinetics of recombinant human bone morphogenic factor 2 (rhBMP-2) from collageneous hydrogel in the presence of human blood plasma have been studied. The expulsion of rhBMP-2 from the collagen-BMP-2 complex by the competitive adhesion of collagen-binding proteins penetrating from plasma was firstly recognized. It was experimentally proven that that blood plasma fibronectin is the main collagen-binding protein, which is responsible for the controlled release of rhBMP-2. As a result, a new collageneous hydrogel with the incorporation of fibronectin was created which retained rhBMP-2 for a twice longer period as compared to the ordinary collageneous hydrogel. A distinctive feature of this new collagen-fibronectin matrix is the slow release of rhBMP-2 in the first three days which allows for the avoiding of adverse effects in clinics caused by the rapid release of large amounts of rhBMP-2 from collageneous hydrogel.

[Computer simulation of hydraulic flows in a human eye].

A two-dimensional computer model was developed to describe hydraulic flows inside the human eye. The flow field was described by coupled Navier-Stokes and Darcy equations. The velocity and pressure profiles in the chambers, the wall, and the vitreous body of the normal eye were obtained using the finite-element method. The model includes the filtration of fluid from the retinal capillary and its drainage through the choroid. The applications of this model include the investigation of the contribution of convection and diffusion to the transport of drugs and study of the kinetics of biodistribution in the eye.

[A new Escherichia coli strain producing human tumor necrosis factor].

An Escherichia coli strain producing human tumor necrosis factor (TNF-alpha) was obtained using a semisynthetic gene partially optimized in respect of codon composition and a phage T7 promoter. The expression product was accumulated in cells as inclusion bodies in a yield of 50-70 mg/l of culture medium. The recombinant TNF-alpha in the form of inclusion bodies was used for immunization of rats to give a polyclonal antiserum. The resulting antibodies were specific under the immunoblotting conditions to the antigen used for the immunization. A dilution-based refolding procedure was developed; it provided a yield of soluble protein exceeding 85%.

[Autoantibodies Against beta(1)-Adrenoreceptors in Patients With Cardiac Rhythm Disorders. Prevalence and Possible Role in Development of Arrhythmia]. / Autoantitela k beta(1)-adrenoretseptoram u bol'nykh s narusheniiami ritma serdtsa: chastota vyiavleniia i ikh vozmozhnaia rol' v razvitii aritmii

AIM: To assess prevalence of autoantibodies against beta(1)-adrenoreceptors (beta(1)-AR) in patients with arrhythmias of various etiology. MATERIAL: Patients with arrhythmias (n=110, including 59 patients with primary [idiopathic] electrical abnormalities, 33 - with chronic myocarditis and dilated cardiomyopathy [DCM]; 18 - with ischemic heart disease [IHD]) and healthy control subjects (n=20). METHODS: Antibodies against beta(1)-AR were measured in blood serum by direct immunoassay. Synthetic fragment containing 26 amino acids of beta(1)-AR second loop was used as antigen. RESULTS: Patients with primary electrical abnormalities and chronic myocarditis/DCM had similar prevalence of beta(1)-AR (49.1% and 54.5%, respectively), what was significantly higher than in controls (10%) and in patients with IHD (16.6%). These results provided evidence for the possible presence of an autoimmune process in the genesis of idiopathic arrhythmias. Among patients with idiopathic arrhythmias beta(1)-AR were found in 40% (10 of 25) of patients with ventricular tachycardia (VT), 63.6% (14 of 22) of patients with ventricular extrasystoles (VE), 41.6% (5 of 12) of patients with atrial fibrillation (AF). Among patients with chronic myocarditis and DCM beta(1)-AR were found in 72.2% (13 of 18) of patients with VT, 28.5% (2 of 7) of patients with VE, 37.5% (3 of 8) of patients with AF. Among patients with idiopathic arrhythmias female sex and frequent respiratory viral diseases were more common in beta(1)-AR-positive compared with beta(1)-AR-negative patients. VT and left ventricular ejection fraction <40% were more common in beta(1)-AR-positive than beta(1)-AR-negative patients among those with chronic myocarditis and DCM.

[Concentration-dependent effects of magnetic accumulation of ligand-binding microparticles and ferritin aggregates]. / Kontsentratsionnye éffekty magnitnogo osazhdeniia ligandsviazyvaiushchikh mikrochastits i aggregatov ferritina.

A method for measuring the magnetic susceptibility of microparticles of a suspension from the thickness of the layer deposited on the surface of a magnetized wire was developed. The magnetic accumulation was registered by means of a photodetector, by measuring the amplitude and spectrum of fluctuations of the intensity of scattered light. This was used as the basis for the development of a biosensor and biochips.

[Treatment of surface burns with proteolytic enzymes: mathematic description of lysis kinetics]. / Obrabotka proteoliticheskimi fermentami poverkhnosti ozhogovykh ran: matematicheskoe opisanie kinetiki lizisa.

The lysis of necrotic tissue by a proteolytic enzyme applied to the surface of a burn wound was studied. A mathematical model was proposed, which describes changes in the thickness of necrotic tissue as a function of the proteolytic activity of the enzyme. The model takes into account the inward-directed diffusion of the enzyme, the counterflow of interstitial fluid (exudates) containing specific inhibitors, and the extracellular matrix proteolysis. It was shown in terms of the quasi-stationary approach that the thickness of the necrotic tissue layer decreases exponentially with time; i.e., the lysis slows down as the thickness of the necrotic tissue layer decreases. The dependence of the characteristic time of this decrease on enzyme concentration was obtained. It was shown that, at high enzyme concentrations (more than 5 mg/ml), the entire time of lysis (after the establishment of quasi-stationary equilibrium) is inversely proportional to the concentration of the enzyme.

Structure of C-reactive protein excreted in urine during acute rejection episodes.

During acute rejection of renal allografts C-reactive protein is excreted in urine in a monomeric form. We developed an immunochemical method for detecting monomeric C-reactive protein in human physiological fluids, which is based on latex agglutination.

[Treatment of burn surfaces by proteinases: mathematical description of an enzyme distribution]. / Obrabotka proteinazami poverkhnosti ozhogovykh ran: matematicheskoe opisanie raspredeleniia fermenta.

The process of penetration of a proteolytic enzyme applied to the surface of burn wound into the depth of necrotic tissue was considered. The model approximation describes three factors by a series of mathematical equations: inward-directed enzyme diffusion, counter-flow filtration of interstitial fluid (exudates), and irreversible inactivation of the enzyme by specific inhibitors present in exudates. According to the model, a quasi-stationary distribution of enzymatic activity through the thickness of the necrotic layer is achieved within 3 h and persists as long as the enzyme concentration on the wound surface is constant. The enzyme activity diminishes linearly from the wound surface to the mid-part of the necrotic layer. No enzyme activity is retained in the inner mid-part of the necrotic layer completely protected by the prevalent inhibitor. The ratio of enzyme concentration on the wound surface to inhibitor concentration in the interstitial fluid is the same as the ratio of the depth of active enzyme area to the depth of the inhibitor-protected area through the necrotic layer. The dynamics of accumulation of the active enzyme in the necrotic zone and the rate of enzyme inactivation in the wound by inhibitors were described by formulas applicable for practical purposes.

[Urokinase stimulates whereas tissue plasminogen activator attenuates blood vessel stenosis]. / Urokinaza stimuliruet, a tkanevoi aktivator plazminogena podavliaet razvitie stenoza krovenosnykh sosudov.

Periadventitial application of the urokinase-plasminogen activator (uPA) in pluronic gel to an injured artery stimulated the neointima and neoadventitia formation as well as cell migration and proliferation in vivo. In contrast, the tissue-type plasminogen activator (tPA) reduced the number of neointimal smooth muscle cells and neointimal area and attenuated the lumen stenosis after a balloon catheter injury of the rat carotid artery. This ability to stimulate the neointima and neoadbentitia formation was found to be quite specific for the uPA. The findings suggest that this uPA property provides a specific functional target for attenuating growth of the damage.

[Distribution of recombinant pro-urokinase in the rabbit blocked carotid artery]. / Raspredelenie recombinantnoi prourokinazy v zakuporennoi sonnoi arterii krolika.

Distribution of recombinant pro-urokinase (PRU) in segments of blocked carotid arteries of rabbits was measured in 10, 20 and 30 minutes after i.v. administration of the RPU. Concentration of the latter in the bloodstream taken as 100% on the 3rd minute, after the infusion decreased to 42% in 10 min., 24% in 20 min., and 13% in 30 min. The RPU concentration decreased to 2% at the artery clamp point, to 20% at 10-15 cm from the clamp point, and remained constant for 10-30 min. A thrombolytic agent accumulated at the dead-end of the blocked artery, was not subject to rapid clearance in contrast to circulating pro-urokinase.

[Pharmacokinetics of recombinant pro-urokinase ocular dosage form]. / Farmakokinetika glaznoi formy rekombinantnoi prourokinazy.

Thrombolytic enzymes are widely used in the treatment of vascular diseases of the eyes and of intraocular hemorrhages. We studied the pharmacokinetics of recombinant prourokinase (proRUK) in ocular structures after its intravitreal administration and subtenon's implantation of collagen infusion system (SICIS). Kinetic parameters of accumulation of labeled proRUK in ocular structures were obtained. After intravitreal administration, the proRUK half-life (T1/2) was 7.9 +/- 1.44 h, with proRUK losing none of its enzymatic activity while in the vitreous body. The maximum accumulation of proRUK in the vitreous administered by the SICIS is observed after 4-5 h and is 1.5-2.0% of the dose administered. In the sclera the maximum accumulation of proRUK is 10-15% (4-6 h after administration), in the vascular membrane 0.8% (4 h after administration). We believe that combination of intravitreal and SICIS-aided administration of proRUK to patients with extensive hemorrhages (subtotal and total hemopthalmia) will improve the efficacy of therapy.

[Effect of growth factors (BFGF and PDGF) on urokinase secretion by smooth muscle cells]. / Vliianie faktorov rosta (bFGF i PDGF) na sekretsiiu urokinazy gladkomyshechnymi kletkami.

The dependence of urokinase (uPA) secretion on basic fibroblast growth factor (bFGF) and platelet-deprived growth factor (PDGF BB) was investigated by using of cultured rat aortic smooth muscle cells (SMC). Growth factors stimulated secretion of urokinase with two-phase kinetics within 1-60 minutes. Namely, "apparent" concentration of uPA in the conditioned media rised to 12.5 nM within the first 1-2 min and 5.8 nM after 30 min of cell stimulation by growth factors, and decreased to basal level at 5 min after stimulation of the cells. The character of uPA-secretion kinetics was similar in response to both growth factors, but the level of secreted uPA was higher in case of PDGF BB. We have shown that this decrease of uPA content in conditioned media is not related to the binding of uPA to the cell surface receptors or extracellular matrix proteins. One can suppose that urokinase secreted within 5 minutes could bind to secretory protein which nature has to be identified. But it was established that this secretory protein, complexing urokinase in cultured media, is not identical to the plasminogen activator inhibitor type 1 (PAI-1).

[Urokinase induces adhesion of monocytes to fibrinogen]. / Urokinaza vyzyvaet adgeziiu monotsitarnykh kletok na fibrinogene.

Urokinase activates adhesion of monocytic U937 cells to fibrinogen-coated surface. This effect is due to urokinase proteolytic activity and does not depend on the urokinase binding to its receptor.

[Follow-up of fibrin clot growth in blood plasma]. / Nabliudenie za rostom fibrinovogo sgustka v plazme krovi.

The authors propose a device for following up the kinetics of the fibrin clot enlargement in the plasma. Coagulation is induced by contact activation with a fragment of an arterial wall put into the plasma. Besides the initial time of clot formation, the device helps assess the rate of clot growth. Two phases of contact-induced clotting may be distinguished. The first is slow activation of the clotting system and formation of minor amounts of fibrin, the other is rapid growth of the clot, with the linear increment of the clot size being constant during 15 min.