[Autoantibodies to M2-cholinoreceptors as a potential development factor of arrhythmia in patients with paroxysmal atrial fibrillation].

AIM: We aimed to assess autoantibodies to M2-cholinoceptors (M2-CR) in patients with paroxysmal lone atrial fibrillation (AF) and in patients with AF and arterial hypertension (AH). MATERIALS AND METHODS: 100 patients with lone AF and 84 patients with AF and AH were included. Patients underwent clinical blood and urinalysis, assessment of biochemistry blood panel, 12-lead ECG, 24-hour Holter monitoring, echocardiography and stress - testing (treadmill or stress - echocardiography). Assessment of IgM and IgG autoantibodies to M2-CR was performed by indirect immunoenzyme assay. The following peptide molecules were used as epitopes for detection of autoantibodies: M1 - amino acid sequence YTVIGYWPLGVVCDL (83-98) of the first extracellular loop of M2-CR; M2 - sequence VRTVEDGECYIQFFSNAAVTFGTAI (168-192) of the second extracellular loop of M2-CR; M3 - sequence NTFCAPCIPNTV (410-421) of the third extracellular loop of M2-CR; M4 - short sequence VEDGECYIQFFS (171-182) of the second extracellular loop of M2-CR; M1+M4 - chimeric molecule formed by sequences of the first and the second extracellular loops of M2-CR connected by disulfide bound YTVIGYWPLGVVCDL + VEDGECYIQFFS (83-98 + 171-182). RESULTS: Autoantibodies to M2-CR were found in 45% patients with lone AF and in 35% patients with AF and AH. In patients with lone AF prevalence of increased IgG to M2-CR were greater than in patients with AF and AH (32% vs 20%; p.

[Role of Autoantibodies Against 1-Adrenergic Receptor in Cardiovascular Disease].

According to current knowledge, autoantibodies against 1-adrenergic receptors may be involved in pathogenesis of different cardiovascular diseases and are mostly studied in patients with Chagas disease, dilated cardiomyopathy and heart rhythm disorders. They may play an important role in cardiomyocyte apoptosis, alteration of their chrono- and inotropic effects and electrophysiological characteristics. Their effects are transduced via 1-adrenergic receptors and depend on multiple factors as ligand properties, durability of its coupling with the receptor, amount of receptors on the cell surface, their affinity and conformation. Up to the present moment, reasons for autoimmune response and clinical significance of autoantibodies against 1-adrenergic receptors are not thoroughly understood. Autoantibodies against 1-adrenergic receptors can be removed from the bloodstream by immunoadsorption and thus development of validated methods of their identification is relevant.

[Detection of Autoantibodies Against the 1-Adrenergic Receptor in the Sera of Patients via the Competitive cell-Based Enzyme Linked Immunosorbent Assay].

OBJECTIVE: This study aimed to assess the level of anti-1-adrenergic receptor autoantibodies in patients with ventricular arrhythmias with no signs of organic heart disease and with presence of cardiovascular pathology in comparison with a group of healthy volunteers. MATERIAL AND METHODS: The study included 44 patients with ventricular arrhythmias with no signs of organic heart disease ("idiopathic"), 34 patients with diagnosed dilated cardiomyopathy (DCM) of inflammatory origin, 35 patients with coronary heart disease and ventricular arrhythmias, 12patients with coronary heart disease with no ventricular arrhythmias, and 19 healthy volunteers (control group). The level of autoantibodies against the 1-adrenergic receptor was determined by the developed competitive cell-based enzyme-linked immunosorbent assay (ELISA) and by the standard ELISA using peptides corresponding to the second extracellular loop of the 1-adrenergic receptor. RESULTS: Elevated level of autoantibodies detected by a competitive cell-based ELISA was observed in 62% of patients with DCM compared to 21% of healthy volunteers (p=0.0006). In patients with "idiopathic" ventricular arrhythmias, the level of 1-adrenergic receptor autoantibodies was lower than in healthy subjects (p=0.003). Coronary heart disease patients with or without ventricular arrhythmias exhibited no differences from the control group. The number of significantly positive signals in peptide-based ELISA did not exceed 10% in any of the groups. No correlation between the data from competitive cell-based ELISA and peptide-based ELISA was found. CONCLUSIONS: This study demonstrated that competitive cell-based ELISA technique can be applied for detection of 1-adrenergic receptor autoantibodies. The results in DCM patients generally correspond to the expected. Decreased level of autoantibodies in patients with "idiopathic" ventricular arrhythmias indicates that this disease is related to changes in the immune system. Such relation is not observed in the case of coronary heart disease patients.

[The immune-enzyme analysis based on chimeric molecule and oligopeptide fragmentations to detect autoantibodies to beta-adrenergic receptor in patients with dilation cardiomyopathy].

The article deals with specification of technique of immune-enzyme analysis to detect autoantibodies to beta-adrenergic receptors (beta1-AP) using compound of oligopeptids representing the fragmentations of extracellular sites beta1-AP and chimeric molecule of extracellular section of receptor This technique significantly exceeds the analogues defined in publications by its sensitivity and correlation with diagnosis.

[Novel conformational peptide antigen, which simulates an immunodominant epitope of the 2nd extracellular loop of ß1-adrenoceptor. Computer simulation, synthesis, spatial structure].

By means of computer simulation has been built polypeptide antigen conformational structure that imitates the immunodominant epitope of the 2nd extracellular loop of ß1-adrenoreceptor. A linear 25-membered peptide corresponding to calculated sequence was synthesized by means of solid-phase methoyd using Fmoc-technology, then directed by the closure ofdisulfide bridges was obtained original bicyclic polypeptide corresponding to the proposed structure of the conformational antigen. With the help of high-resolution NMR spectroscopy 3D structure of synthetic conformational antigen was investigated. It was shown that the structure of the bicyclic polypeptide similar to that of building computer model. Bicyclic conformational antigen was suitable for the detection of autoantibodies in the blood serum of patients with rhythm and conductivity violation without evidence of organic disease of the cardiovascular system.

[Synthetic conformational antigen, simulating the extracellular part of M2-muscarinic receptor and its reactivity with sera from patients with idiopathic arrhythmias].

Linear peptides corresponding to fragment 83-98 of the first loop and fragments 168-192 and 171-182 of the second extracellular loops of M2-muscarinic receptor (marker of early cardiac disorders and arrhythmias) were synthesized by Fmoc-SPPS method. A new conformational antigen was synthesized by method of selective ligation of linear peptides by disulfide bond with native localization. Peptides were studied in reaction with sera from patients with idiopathic arrhythmias. A new conformational antigen was recognized by sera from patients with idiopathic arrhythmias with high reactivity.

[Development and use of a domestic test system for diagnosis of tuberculous infection on the basis of quantitative analysis of interferon-gamma induction in whole blood samples in vitro, by using specific recombinant antigens].

A test system was developed to detect tuberculous infection by qualitative analysis of interferon-gamma (IFN-gamma) in the plasma samples after 20-24-hour incubation of whole blood samples in the presence of Mycobacterium tuberculosis (MBT) antigens: tuberculin PPD and a mixture of the MBT-specific recombinant antigens ESAT-6 and CFP-10. The analysis used 3 test tubes each containing 1 ml of heparinized venous blood, one of which served as a control; the other two test tubes were employed to measure antigen-induced IFN-gamma production. Whether this test system might be used to determine primary tuberculous infection was studied in 277 children and adolescents. The threshold diagnostic IFN-gamma induction level determined in the test tube containing a mixture of the antigens ESAT-6 and CFP-10 was ascertained. Postvaccine allergy was detectable if there was IFN-gamma induction in the test tube containing tuberculin and if there was no diagnostic IFN-gamma level in that containing the antigens ESAT-6 and CFP-10. The diagnostic sensitivity of detection of primary tuberculous infection was 97.6% with 94.4% specificity, which enabled this condition to be differentiated from postvaccine allergy. The level of antigen-induced IFN-gamma may be lower in relatively disseminated forms of pulmonary tuberculosis.

[In vitro interferon-gamma induction in whole blood samples is a test for tuberculous infection in children and adolescents].

By applying the interferon-gamma (IFN-gamma) induction technique in the whole blood samples exposed to short-term (22-24-hour) incubation in the presence of Mycobacterium tuberculosis antigens--PPD tuberculin and specific recombinant ESAT-6 lacking in the cells of vaccine BCG and other non-tuberculous mycobacteria, the authors studied the groups of children and adolescents with a negative Mantoux test (n = 31), with postvaccine BCG allergy (n = 40), as well as patients with primary tuberculous infection (n = 84) and those with pulmonary tuberculosis (n = 44). Patients with primary tuberculous infection and a high sensitivity (94%) and a high specificity (97%) may be differentiated from children and adolescents with postvaccinal allergy when the recombinant ESAT-6 antigen and the critical IFN-gamma level (greater than 70 pg/ml) detectable in the plasma samples after incubation with the antigen. It has been also shown that in adolescents with local forms of pulmonary tuberculosis specific IFN-gamma induction may be suppressed in number of cases, which is ascribed to decreased specific immunity.

[Modification of the FF neuropeptide enhances its hypertensive effect].

Neuropeptide FF (H-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2) injected intravenously temporarily enhanced the arterial pressure (AP) and the heart rate (HR). However, its role in the regulation of blood circulation is obscure. To study the properties of the molecule, its analogue was synthesized, in which proline in position 7 was substituted with glycine, and leucine in the position 2 with norleucine. Modified neuropeptide FF (FFm) also temporarily and in a dose-dependent manner increased the AP and HR; however, the equal degree of increase was reached at doses of FFm being 5-7 times lesser as compared with the natural peptide. The application of the FFm at hemorrhagic shock excluded mortality of animals during the experiment, considerably increased the degree of AP and HR restoration in the remaining experiments, and improved the survival of animals in 24 hours. It has been found that the level of antibodies to the fragment of hFF1 receptor in the serum is lower in spontaneously hypertensive rats SHR as compared with Wistar rats, but it is increased in patients of cardiological profile as compared with donors. The findings suggest involvement of neuropeptide FF in the regulation of blood circulation; however, the precise mechanisms remain to be determined.

[Significance of determination of gamma-interferon in the diagnosis of tuberculous exudative pleurisy].

The pleural fluid concentration of gamma-interferon (gamma-IFN) was studied in 44 patients with exudative pleurisy. The tuberculous nature of exudative pleurisy was established in 25 patients on the basis of X-ray study, a follow-up, microbiological study of pleural exudate specimens, and morphological studies of biopsy specimens obtained at video-assisted thoracoscopy or surgery. The the pleural exudate concentrations of gamma-IFN were over 300 pg/ml (mean 1019 +/- 161 pg/ml) in 19 out of 21 patients with exudative pleuritis in a phase of active inflammation. The patients with the fibrous outcome of pleurisy of tuberculous etiology and those with exudative pleurisy of non-tuberculous etiology, including that in malignancies and papapneumonic pleurisy, were observed to have lower concentrations of gamma-IFN (mean 118 +/- 16 pg/ml). With the discriminating level of above 300 pg/ml, the sensitivity and specificity of the test were 90.5 and 100%, respectively.

[Role of certain neurohumoral factors in development of pulmonary hypertonia in patients with interstitial lung diseases]. / Rol' nekotorykh neirogumoral'nykh faktorov v razvitii legochnoi gipertonii u bol'nykh interstitsial'nymi bolezniami legkikh.

AIM: To ascertain the role of some neurohumoral factors--nitric oxide (NO), ACE, histamine--in development of pulmonary hypertension (PH) in patients with interstitial lung diseases (ILD). MATERIAL AND METHODS: A total of 32 ILD patients were examined. Of them 14 had idiopathic fibrosing alveolitis (IFA), 6 had exogenous allergic alveolitis (EAA) and 12 patients had ILD in diffuse disease of the connective tissue (ILD-DDCT). In addition to routine tests, those for NO, ACE, histamine, serotonin in plasma were performed; Doppler echocardiography and high-resolution computed tomography were conducted. In 9 patients the diagnosis was verified at thoracoscopic biopsy of the lung. The control group consisted of 16 healthy subjects. RESULTS: The highest mean pressure in the pulmonary artery (PmPA) was registered in IFA vs EAA and ILD-DDCT patients (p < 0.001). NO concentration in plasma was higher in ILD-DDCT than in control patients. In IFA and EAA the level of NO was like in controls. Concentration of NO in plasma of IDL patients correlated with high activity of the process. No correlation was found between ACE in plasma and PmPA, ACE levels were much higher in controls than in the ILD patients (p < 0.05). Histamine levels were higher in ILD patients than in controls being the highest in ILD-DDCT. Serotonin was insignificantly higher in ILD patients than in controls. CONCLUSION: Damage to the endothelium of the pulmonary arteries and imbalance of neurohumoral factors may be considered as a mechanism of development and stabilization of PH in ILD patients.

[Plasmapheresis in combine therapy of hypercoagulation syndrome in hematogenic thrombophilia]. / Plazmaferez v kompleksnoi terapii giperkoaguliatsionnogo sindroma pri gematogennykh trombofiliiakh.

AIM: To specify indications to plasmapheresis (PA) and to assess its efficiency in patients with hypercoagulatory syndrome in hematogenic thrombophilia (HT). MATERIAL AND METHODS: 18 patients (11 males, 7 females, age 26-50 years) with various forms of HT received standard antiaggregation, anticoagulatory therapy plus therapeutic PA. By PA technique the patients were divided into two groups. RESULTS: A positive trend in clinical, instrumental and laboratory indices was observed in all the patients. CONCLUSION: Therapeutic PA in hypercoagulation syndrome in HT patients leads to fast normalization of the clinical picture and reestablishment of patency of thrombus-affected vessels.

[Antibodies to low density oxidated lipoprotein and ischemic cerebrovascular disorders in young patients]. / Antitela k okislennomu lipoproteinu nizkoi plotnosti i ishemicheskie narusheniia mozgovogo krovoobrashcheniia v molodom vozraste.

AIM: The study of the detection rate and potential pathogenetic significance of antibodies to low density oxidated lipoprotein (LDOL) in patients who had ischemic cerebral circulation disorder (ICCD) at young age. MATERIALS AND METHODS: The examination (enzyme immunoassay, general and neurological investigations, laboratory and instrumental tests) covered 148 patients who survived ICCD at young age (mean age 37.2 years). RESULTS: 48 of 108 (44%) patients had LDOL antibodies. Antibodies to cardiolipin, lupus anticoagulant were recorded in 33 (31%) patients. LDOL antibodies were higher and occurred more frequently in patients with Sneddon's syndrome (35% of patients, mean LDOL antibodies-44 units) and primary antiphospholipid syndrome (17% of patients, 45 units) than in atherosclerotic affection of the major head arteries (4%, 29 units) or occlusion of cerebral arteries of unclear genesis (8% of patients, 29 units). CONCLUSION: ICCD were not related to fast development of cerebral atherosclerosis or periaortitis due to production of LDOL antibodies as no relationships were found between their rise and atherosclerotic lesions of cerebral major arteries or periaortitis as shown by ultrasonic dopplerography or cerebral angiography. Feasibility of LDOL antibodies participation in the coagulation cascade and induction of hypercoagulatory condition causing ICCD needs special investigation.

A simple direct enzyme immunoassay of antibodies based on the differences in diffusion rates in a gel of a synthetic antigen and of its complex with antibodies.

A simple direct enzyme immunoassay for semiquantitative detection of antibodies is suggested. It is based on the difference in diffusion rates in a gel for a synthetic low-mol-wt antigen and of its complexes with antibodies to be detected. Sensitivity and specificity of the developed assay are equal to an ELISA method. The assay has been tested with antibodies against HIV protein gp41 in rabbit serum. Possible applications and limitations of the method are discussed.

[Circulating complexes of plasma fibronectin (fibronectin-fibrin) in human diseases]. / Tsirkuliruiushchie kompleksy plazmennogo fibronektina--fibronektin-fibrin pri nekotorykh zabolevaniiakh cheloveka.

The study of circulating complexes fibronectin-fibrin in 21 patients with immunocomplex affections revealed their high levels which occurred in inverse relationships with fibronectin concentrations in the blood, but in direct correlation with indicators of inflammation (fibrinogen, cialic acid). It is suggested that the complexes are involved in the development of fibrosis at the sites of chronic inflammation in immune disorders.

[Preparation and use of monoclonal antibodies against seal alkaline phosphatase]. / Poluchenie i primenenie monoklonal'nykh antitel proteiv shchelochnoi fosfatazy tiulenia.

Monoclonal antibodies (termed as APP.1 and related to subclass IgG1) against seal alkaline phosphatase, have been obtained. APP.1 did not influence the enzymatic activity of alkaline phosphatase. The dissociation constant for the APP.1 interaction with Greenland seal alkaline phosphatase was equal to 8.5 x 10(-10) M. It was found that APP.1 interact with intestinal isoenzymes of common and fur seal, calf and deer alkaline phosphatases. An APP.1 complex with seal alkaline phosphatase was obtained and successfully applied in immunoenzymatic analysis. The use of this complex made it possible to diminish the limit of detectability of antibodies against peptide fragments of HIV-1 and HIV-2 proteins. Moreover, this complex allowed the identification of cytokeratin-8 and vimentin in human kidney slices and embryonic fibroblast-like cells, respectively.