The detection of autoantibodies against the β1-adrenergic receptor (ADRB1 Ab) in the blood of patients and the monitoring of the levels of these antibodies is an urgent need in clinical practice. The solid-phase enzyme-linked immunosorbent assay (ELISA), using ADRB1 in native conformation as antigen, seems to be the most suitable for this task. We have previously tested various amphipathic polymers for their ability to solubilize ADRB1 in the form of nanodiscs so that ADRB1 retains its antigenic properties. The aim of the present work was to investigate the ligand binding properties of ADRB1 in nanodiscs prepared with amphipathic polymers such as UltrasoluteTM Amphipol (UA17) and AASTY 11-45 and to determine the size of the nanodiscs by dynamic light scattering. The binding of the ligands isoproterenol (agonist) and cyanopindolol (antagonist) was assessed by their ability to compete with recombinant hAB2367 antibodies specific for the second extracellular loop of ADRB1 in ELISA. It was found that ADRB1 solubilized with UA17 and AASTY 11-45 retained its ligand-binding properties. This fact supports the assumption that ADRB1 retains its native structure in nanodiscs. The size of nanodiscs prepared with UA17 was determined for the first time by dynamic light scattering. In the range of polymer concentrations from 0.0625% to 0.5%, no significant differences were observed in the size of the nanodiscs, which varied between 10 and 16 nm.

Введение. Фибронектин (ФН) — белок внеклеточного матрикса, который определяет клеточную адгезию, распространение, миграцию, пролиферацию и апоптоз, и подразделяется на 2 формы — растворимую в плазме крови и клеточную нерастворимую, которая является основным компонентом внеклеточного матрикса. Цель исследования: разработка способа получения препарата аутофибронектина (аутоФН), содержащего тромбоциты, из обогащенной тромбоцитами плазмы крови пациента и оценка эффективности этого препарата в лечении трофических поражений кожи. Материалы и методы. В исследование включены 86 пациентов (37 мужчин и 49 женщин) в возрасте от 23 до 89 лет с трофическими дефектами кожных покровов различной этиологии. Препарат, содержащий аутоФН и тромбоциты, получали двумя способами: на аппарате автоматического донорского плазмафереза PCS2 Haemonetics (Haemonetics Corporation, США) и центрифужным методом с использованием рефрижераторной центрифуги Roto Silenta RP 3100 (Helling, Германия). В полученную этими способами обогащенную тромбоцитами аутоплазму пациента для активирования плазменного ФН вводили гепарин в дозе 15000–20000 ЕД/л. Полученный препарат содержал 1,5–1,8 мг/мл ФН и тромбоциты исходной плазмы пациента. Препарат наносили тонким слоем на трофический дефект кожи, предварительно обработанный 3%-м раствором перекиси водорода 3 раза в день. Результаты. В результате проведенного лечения полное заживление трофических язв и кожных дефектов наблюдалось у 81 (94,2%) пациента в течение 5–12 дней. Заключение. Препарат аутоФН с тромбоцитами оказался высокоэффективным при лечении трофических дефектов кожных покровов различной этиологии в 94,2% случаев. Эффективность разработанного в настоящем исследовании препарата аутоФН с тромбоцитами оказалась выше, чем клинический эффект предложенного нами ранее препарата аутоФН, полученного методом криофракционирования. Introduction. Fibronectin (FN) is an extracellular matrix protein that plays a major role in cell adhesion, distribution, migration, proliferation, and apoptosis. There are two FN forms: soluble plasma fibronectin and insoluble cellular fibronectin that is the major component of the extracellular matrix. Objective: to develop a method to obtain autofibronectin (autoFN) containing patient’s platelets from platelet-rich blood plasma and to assess autoFN effectiveness in trophic skin lesions. Materials and Methods. The study included 86 patients (37 men and 49 women) aged 23–89 years with trophic skin lesions of various etiologies. The medication containing autoFN and platelets was obtained in two ways: on the PCS2 Haemonetics automatic donor plasmapheresis apparatus (Haemonetics Corporation, USA) and by the centrifugal method on Roto Silenta RP 3100 refrigerated centrifuge (Helling, Germany). To activate plasma FN, heparin at a dose of 15,000–20,000 U/L was injected into the patient’s platelet-rich autoplasma obtained by these methods. The resulting medication contained 1.5–1.8 mg/mL of FN and platelets from the patient’s original plasma. A thin layer of the medication was applied on a trophic skin lesion pre-treated with a 3% hydrogen peroxide solution 3 times a day. Results. Complete healing of trophic ulcers and skin lesions was observed in 81 (94.2%) patients within 5–12 days. Conclusion. In 94.2% of cases autoFN with platelets was highly effective in trophic skin lesions of various etiologies. The effectiveness of autoFN with platelets developed in this study was higher than the clinical effect of autoFN obtained by cryofractionation that we previously proposed.

The development of a reliable and easily used diagnostic test for measuring autoantibodies to ?1-adrenergic receptor (?1ADR Ab) in patient blood is an unmet clinical need. The enzyme-linked immunosorbent assay (ELISA) is considered as the most appropriate method for this task. In ELISA, the use of peptides corresponding to various fragments of amino acid sequence of ?1ADR as antigens leads to inadequate results as β1ADR Ab appear to recognize conformationally dependent epitopes that are generated during the formation of unique tertiary structure of the receptor. Isolation of ?1ADR preserving the native conformation and functional characteristics is a quite challenging task. A promising approach to address this task is the use of amphipatic polymers capable of forming nanodiscs, it permits to successfully solubilize membrane proteins. In order to obtain the preparations of solubilized β1ADR that can be used as antigens in ELISA we have tested 17 various amphipatic polymers. The best relative solubilization values (RSV) were obtained using UltrasoluteTM Amphipol 17 (87%) and 18 (62%), as well as by AASTY 11-45 (76%), 11-50 (77%) and 6-50 (78.5%).

Plasma fibronectin is a high molecular weight adhesive glycoprotein. There are two types of fibronectin: plasma (soluble) and cellular derived (insoluble). Electron microscopy revealed two types of structural organization of fibronectin: compact and expanded. In solution, fibronectin has a compact conformation, and after binding to certain substrates (collagen, fibrin, heparin), it is expanded. Plasma fibronectin is one of the main opsonins of blood plasma in relation to the “targets” of phagocytosis of a predominantly non-bacterial nature, as well as to some types of bacteria. For the treatment of septic processes, as well as respiratory distress syndrome of adults with severe fibronectin deficiency, plasma cryoprecipitate is used – a donor plasma preparation containing a large amount of plasma fibronectin (more than 2 mg/ml). It was proposed to replenish the level of fibronectin in patients with sepsis and other conditions that cause plasma fibronectin deficiency with the help of donor freshly frozen plasma. Transfusion of large volumes of freshly frozen plasma (up to 1000–1500 ml) to patients effectively eliminates the deficiency of plasma fibronectin. The concentration of plasma fibronectin in the blood significantly decreases after the addition of severe infectious processes to hematological diseases, as well as acute DIC syndrome. Extracorporeal methods of blood purification – selective plasmapheresis – have been developed to correct immunocomplex and fibronectin-complex pathology. Two variants of selective plasmapheresis have been proposed: the method of heparinocryoprecipitation of plasma proteins and the method of heparinocryofractionation. In 1987, a plasma heparin precipitate was proposed as a source of fibronectin for the treatment of patients with trophic skin lesions. In 1992, a new method was proposed for obtaining blood preparations with a high concentration of plasma fibronectin from patients themselves (heparin cryofractionation). Autofibronectin preparations obtained by such methods are effective in the local treatment of trophic ulcers in 90–93% of cases. The proposed drugs are safe against infection of patients with infectious diseases transmitted through the blood.

Introduction. The term “cryoglobulinemia” is currently used to identify immunoglobulins in vitro in the blood serum that precipitate at temperatures below 37 °C; in vivo they form immune complexes that can be deposited in small vessels and activate the complement system with the development of leukocytoclastic vasculitis. Cryoglobulinemia may develop in various lymphoproliferative, autoimmune and infectious diseases. Aim of study. To develop the technique of plasma proteins cryofraction (selective plasmapheresis with the use of heparin as a stimulant of fibronectin opsonic activity and purified autoplasma to compensate for the removed volume), to evaluate the effectiveness and tolerability of the developed technique in the treatment of patients with cryoglobulinemia. Materials and methods. 159 patients were treated (120 women and 39 men aged 21 to 83 years). Research results. Heparinocryofraction technique is a highly effective method of extracorporeal blood purification, which allows to selectively remove from the patients’ plasma such pathological components as cryoglobulins (up to 100% of the initial content), adhesive proteins (up to 84% of the initial content), fibronectin and immune complexes (up to 7% of the initial content). It is possible to reduce significantly and reliably the level of cryoglobulins, circulating immune complexes, non-specific markers of inflammation, daily proteinuria, as well as to normalize the initially reduced concentration of complement components and hemoglobin in the blood of patients with cryoglobulinemia before and after the procedure of cryofractionation. Purified by the proposed method autoplasma is a solution of albumin and normal immunoglobulins, which allows to use it for plasma substitution during a course of cryofractionation procedures, on average 7 procedures with an interval of 1–2 days. Conclusion. The technique of cryofractionation using heparin and purified autoplasma can and should be widely used in the complex treatment of patients with cryoglobulinemia. Carrying out 6–-7 sessions of plasma cryofractionation allows to remove cryoglobulins from plasma effectively and selectively. Application of purified autoplasma allows to avoid using of blood preparations in plasmapheresis. The proposed method allows to significantly improve the efficiency and tolerance of medication therapy and increase the duration of disease remission.

Autoantibodies to 1-adrenergic receptor may play a role in the development of certain cardiovascular system diseases.Synthetic peptide-based enzyme-linked immunosorbent assay (eliSA), traditionally used for the detection of such antibodies, does not allow to obtain adequate data, probably due to the poor similarity of peptide conformations to the spatial structure of the extracellular regions.Aim of the study: to improve the peptide-based eliSA method for the detection of autoantibodies to 1 adrenergic receptor by a separate determination of the igG1, igG2, igG3, igG4, igM and igA autoantibody isotypes.Material and methods: a modification of the eliSA method with peptides corresponding to the sequences of the 1-adrenergic receptor first and second extracellular loops, and secondary antibodies specific to the abovementioned human immunoglobulin isotypes.the method has been tested on a panel of samples obtained from patients with dilated cardiomyopathy (dcM), arrhythmia and coronary heart disease.ResultS: small excess of the signal mean values of individual antibody isotypes have been recorded for some groups of patients in comparison with the group of healthy volunteers.however, these differences were not statistically significant.the proportion of signals «above the 1 ооо «МонА», ул.кастанаевская, 38, стр. 1, Москва, 121108, российская федерация; 2 фГБу «национальный медицинский исследовательский центр кардиологии» Минздрава россии, ул.3-я черепковская, 15а, Москва, 121552, российская федерация Резюме Аутоантитела к 1-адренорецептору могут играть важную роль в развитии некоторых заболеваний сердечно-сосудистой системы.иммуноферментный анализ (ифА) с использованием синтетических пептидов, традиционно применяемый с целью детекции таких антител, не позволяет получать адекватные данные, вероятно, вследствие слабого сходства конформаций пептидов с пространственной структурой внеклеточных участков 1-адренорецептора.Цель исследования.усовершенствовать метод ифА для детекции аутоантител к 1-адренорецептору за счет раздельного определения аутоантител изотипов igG1, igG2, igG3, igG4, igM и igA.Материал и методы.предложена модификация метода ифА с использованием пептидов, соответствующих последовательностям первой и второй внеклеточных петель 1-адренорецептора, и вторых антител, специфических к вышеуказанным изотипам иммуноглобулинов человека.Метод опробован на панели образцов сыворотки крови, полученных от пациентов с дилатационной кардиомиопатией (дкМп), желудочковой аритмией и ишемической болезнью сердца.Результаты. для некоторых групп больных зафиксированы небольшие превышения средних значений сигналов отдельных изотипов антител по сравнению с группой здоровых добровольцев, однако эти отличия не являются статистически значимыми.доля сигналов «выше уровня нормы» в группе больных дкМп составила 21% для изотипа igG1 и от 6 до 16% для остальных изотипов, что весьма далеко от частоты встречаемости аутоантител к 1-адренорецептору (70-80%), наблюдаемой у больных дкМп при использовании функциональных методов анализа.в других группах доля таких сигналов не превысила 30% ни для одного изотипа антител.

Introduction. The term “cryoglobulinemia” is currently used to identify immunoglobulins in vitro in the blood serum that precipitate at temperatures below 37 °C; in vivo they form immune complexes that can be deposited in small vessels and activate the complement system with the development of leukocytoclastic vasculitis. Cryoglobulinemia may develop in various lymphoproliferative, autoimmune and infectious diseases. Aim of study. To develop the technique of plasma proteins cryofraction (selective plasmapheresis with the use of heparin as a stimulant of fibronectin opsonic activity and purified autoplasma to compensate for the removed volume), to evaluate the effectiveness and tolerability of the developed technique in the treatment of patients with cryoglobulinemia. Materials and methods. 159 patients were treated (120 women and 39 men aged 21 to 83 years). Research results. Heparinocryofraction technique is a highly effective method of extracorporeal blood purification, which allows to selectively remove from the patients’ plasma such pathological components as cryoglobulins (up to 100% of the initial content), adhesive proteins (up to 84% of the initial content), fibronectin and immune complexes (up to 7% of the initial content). It is possible to reduce significantly and reliably the level of cryoglobulins, circulating immune complexes, non-specific markers of inflammation, daily proteinuria, as well as to normalize the initially reduced concentration of complement components and hemoglobin in the blood of patients with cryoglobulinemia before and after the procedure of cryofractionation. Purified by the proposed method autoplasma is a solution of albumin and normal immunoglobulins, which allows to use it for plasma substitution during a course of cryofractionation procedures, on average 7 procedures with an interval of 1–2 days. Conclusion. The technique of cryofractionation using heparin and purified autoplasma can and should be widely used in the complex treatment of patients with cryoglobulinemia. Carrying out 6–-7 sessions of plasma cryofractionation allows to remove cryoglobulins from plasma effectively and selectively. Application of purified autoplasma allows to avoid using of blood preparations in plasmapheresis. The proposed method allows to significantly improve the efficiency and tolerance of medication therapy and increase the duration of disease remission.

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.

Abstract Introduction Atrial fibrillation (AFib) is the most frequenly encountered arrhythmia. In the majority of cases AFib occurs in patients with cardiovascular diseases such as arterial hypertension (AH). In 10–15% of cases AFib occurs in the absense of comorbidities in structurally normal heart. It is reffered to as “lone AFib” and the cause of this arrhythmia remains unknown. Activation of cardiac M2-actylcholinoreceptors (M2-CR) leads to decrease in duration of atrial refractory periods that may contribute to development of AFib. Autoantibodies against M2-CR has cholinomimetic properties, but role of these autoantibodies in development and maintenance of AFib has not been studied. Purpose To assess autoantibodies against M2-CR in patients with paroxysmal lone AFib, in patients with AFib and AH and healthy people. Methods 100 patints with lone Afib, 100 patients with Afib and AH and 25 healthy people were included. Patients underwent clinical blood and urinlysis, assessment of biochemistry blood panel, 12-lead ECG, 24-hour Holter monitoring, echocardiography and stress-testing (treadmill or stress-echocardiography). Assesment 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 extacellular loops of M2-CR connected by disulfide bound YTVIGYWPLGVVCDL+VEDGECTIQFFS (83–98+171–182). Results IgG to M2-CR were found in 32% of patients with AFib and in 12% of healthy subjects (p<0,05). IgM to M2-CR were found in 25% of patients with AFib and in 32% of healthy subjects (p<0,05). In patients with lone AFib prevalence of IgG to M2-CR were greater than in patients with AFib and AH (39% vs 25%; p<0,05). Patients with lone AFib and AFib and AH had higher prevalence of IgG to all amino-acid sequences and difference was statistically significant for M2, M4, M1+M4 epitopes (see fig.). Prevalence of IgG to M2-CR epitopes Conclusion Higher prevalence of IgG to M2-CR in patients with AFib than in healthy subjects may suppose an active role of autoimmune processes in arrhythmogenesis.

P<0.05) as well as severity of arrhythmic episodes (AS 36.5 6 27.0 vs 8.3 6 12.3 in RVA 60, P<0.05).Conclusion: Prevention of acute bradycardia decreases the probability of arrhythmia development as well as the severity of induced arrhythmic events.P1022

The P26 peptide corresponding to the 197–222 sequence of the second extracellular loop of the β 1 -adrenoreceptor (β 1 -AR) was synthesized by solid-phase fragment condensation on the Wang polymer. Pentapeptide fragments were prepared on the 2-chlorotrityl resin. The racemization degree of the C-terminal alanine residue of the pentapeptide was experimentally evaluated for the synthetic H-Glu-Ser-Asp-Glu-Ala-Arg-OH hexapeptide β 1 -АR-(202–207) which was prepared by the 5 + 1 fragment condensation with the use of various condensing agents. A content of the diastereoisomeric peptide in the products of the fragment condensation was determined by HPLC on a reversed phase. The D-alanine-containing hexapeptide was specially synthesized and used for a comparison. The minimum racemization degree of the C -terminal alanine residue was observed if complex F was applied to the synthesis of the hexapeptide.

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.

Aim. To identify the most promising epitopes that simulate various sites β1-adrenergic and M2-cholinergic receptors, and to evaluate their possible contribution to the development and maintenance of cardiac arrhythmias, particularly idiopathic ventricular arrhythmia. Material and methods. Patients with ventricular arrhythmias without organic cardiovascular disease (the study group; n=70) were included in the study. The control group consisted of 20 healthy volunteers. Evaluation of levels of antibodies to antigenic determinants, modeling various sites β1-adrenergic and M2-cholinergic performed in all patients. Causal treatment with clarithromycin and valacyclovir performed in part of patients. Results. Antibodies to different peptide sequences of β1-adrenergic and M2-cholinergic receptors have been identified in 25% of main group patients. A direct correlation between the frequency of episodes of ventricular tachycardia and IgG levels to MRI-MRIV (p=0.02) revealed. Increase in titre of antibodies to β1-adrenoceptors, to a peptide sequence β8 (p=0.02), and lower titers of antibodies to the M2 acetylcholine receptor — chimera MRI-MRIV IgM (p=0.06) and ARI-MRIV IgM (p=0.07) were observed when assessing the efficacy of the therapy in the causal dynamics in the group of "untreated" patients. IgG titer reduction of ARI-MRIV (p=0.02), which is 4 times out of 10 with reduction of ventricular ectopic activity , recorded after valacyclovir therapy. Clarithromycin therapy on the level of antibodies exerted no significant effect. Conclusion. Possible involvement of antibodies to β1-adrenoceptor and M2-cholinergic receptors in the development of idiopathic ventricular arrhythmias demonstrated. The relationship between the frequency of episodes of ventricular tachycardia and levels of antibody titers to M2-cholinergic receptors found. Attempt of causal treatment, depending on the possible mechanisms of the autoimmune process is executed. Further studies to confirm or refute the results to the larger sample of patients are needed.

In 35 patients (pts) with dilated cardiomyopathy (DCM) the cardiovascular magnetic resonance (CMR) data in combination with the assessment of the level of autoantibodies to beta(1)-adrenergic receptor (beta(1)-AAbs) and M-2-muscarinic receptor (M-2-AAbs) was analyzed. beta(1)-AAbs (IgG) were detected in serum of 68.6% patients, 31.4% of patients were M-2-AAbs (IgG) positive. CMR revealed late gadolinium enhancement (LGE) in 43% of patients. Presence of focal fibrosis in myocardium (persistence of LGE according to CMR) and detection of beta(1)-AAbs was closely associated with presence of ventricular arrhythmias. These findings allow using CMR data and the level of beta(1)-AAbs in determination of risk of ventricular arrhythmias in patients with left ventricular dysfunction including DCM.