β-adrenoreceptor (ADRB) ligands are actively used in the therapy of bronchopulmonary and cardiovascular diseases. When using these drugs, it is important to assess changes in ADRB content in different tissues. In most cases, the direct measurement of ADRB content in lung and heart cells is not possible. ADRB2 content in peripheral blood lymphocytes (or mononuclear cells) was shown to correlate with that in myocardial cells. It has been suggested that blood lymphocytes can be used to monitor ADRB content in solid organs. However, the estimation of ADRB1 content in myocardium from its content in peripheral lymphocytes is not possible due to the low content of ADRB1 in lymphocytes. In the present study, we performed simultaneous determination of ADRB1 and ADRB2 both in the total population of PBMCs and in isolated subpopulations of monocytes, T-lymphocytes, and NK-cells from 23 healthy donors using the modified radioligand method. The highest amount of ADRB2 was detected in NK cells, followed by PBMCs, monocytes, and T cells. The content of these receptors in all blood cell subpopulations was significantly correlated with each other, suggesting the possibility of using PBMCs to monitor ADRB2 in solid organs. For the first time, ADRB1 was detected in monocytes and NK cells.

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%).

Во многих тканях и клетках человека присутствуют как β1-, так и β2-адренорецепторы, информация о содержании и динамике поведения кото- рых часто является клинически значимой. В настоящем исследовании предложена методика раздельного определения обоих типов адреноре- цепторов на основе радиолигандного анализа с использованием 125I-йодоцианопиндолола, включающая проведение трех измерений: 1) без лигандов-конкурентов; 2) в присутствии селективного лиганда ICI 118,551 (0,25 мкМ); 3) в присутствии двух селективных лигандов ICI 118,551 и CGP 20712 (по 0,25 мкМ каждого). Методика протестирована на модельной системе из двух трансгенных линий клеток с экспрессией реком- бинантных β1- и β2-адренорецепторов. При соотношении количества β1-адренорецепторов к β2-адренорецепторам 1:10 погрешность измере- ния составляет около 15%. Анализ 9 клеточных линий, представляющих различные типы клеток крови, показал наличие β2-адренорецепторов в клетках Daudi, Raji, Dami, K-562, HL-60, U-937 и THP-1 и их отсутствие в Т-лимфоцитарных клетках Jurkat и MOLT-4. β1-адренорецепторы достоверно зарегистрированы лишь в клетках THP-1 моноцитарного происхождения. В остальных клетках, за исключением линии Dami, их количество оказалось ниже порога детекции, оцениваемого на уровне 250 молекул на клетку. Измерения, выполненные на мононуклеарных клетках периферической крови здоровых доноров, продемонстрировали присутствие β2-адренорецепторов в диапазоне от 1000 до 2500 моле- кул на клетку, тогда как содержание β1-адренорецепторов во всех случаях находилось на грани или за гранью порога детекции. По-видимому, изучение β1-адренорецепторов в дальнейшем следует проводить на отдельных фракциях клеток крови, в частности на фракции моноцитовβ1- and β2-adrenergic receptors are presented in various human tissues and cells, while the information of their content and dynamic behavior is oftenly considered as clinically significant. In this study, a method for the separate determination of both types of adrenoceptors based on radioligand binding analysis using 125I iodocyanopindolol is proposed, comprising three measurements: 1) without competing ligands; 2) in the presence of selective ligand ICI 118,551 (0.25 μM); 3) in the presence of two selective ligands ICI 118,551 and CGP 20712 (0.25 μM each). The technique was tested on a model system of two transgenic cell lines with the expression of recombinant β1- and β2-adrenergic receptors. If the ratio of the number of β1-adrenergic receptors to β2-adrenergic receptors is 1:10, the measurement error is about 15%. Analysis of 9 cell lines representing different types of blood cells showed the presence of β2-adrenergic receptors in Daudi, Raji, Dami, K-562, HL-60, U-937 and THP-1 cells and their absence in Jurkat and MOLT-4 cells. β1-adrenergic receptors are reliably registered only in THP-1 cells of monocytic origin. In the remaining cell lines, with the exception of Dami, the number of β1-adrenergic receptors was found below the detection limit, estimated as 250 molecules per cell. Measurements performed on the peripheral blood mononuclear cells of healthy donors showed the presence of β2-adrenergic receptors in the range from 1000 to 2500 molecules per cell, while the content of β1-adrenergic receptors in all cases appeared to be on the border or beyond the detection limit. Apparently, further study of β1-adrenergic receptors should be performed on the blood cells isolated fractions, on monocytes in particular

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.

Аутоантитела к β1-адренорецептору (АДРБ1 АТ) часто обнаруживаются в сыворотке пациентов с хронической сердечной недостаточностью, обусловленной различными этиологическими факторами. АДРБ1 АТ выявляются также и у некоторых здоровых доноров, однако они отличаются по своим функциональным свойствам от антител крови больных. Патогенетическая роль АДРБ1 АТ была продемонстрирована в различных модельных экспериментах на животных. Рассмотрены предполагаемые патогенетические механизмы действия АДРБ1 АТ. Приводятся различные данные, свидетельствующие о предполагаемой особой роли аутоантител подкласса IgG3 в развитии сердечно-сосудистых патологий. В клинической практике наличие АДРБ1 АТ в крови больных играет прогностическую роль , а так же может являться маркером желудочковых нарушений ритма сердца. Анализируются различные методы выявления АДРБ1 АТ: иммуноферментный анализ с использованием пептидов в качестве антигена, а также методы с использованием в качестве антигена нативной молекулы адренорецептора. Наиболее информационно значимыми являются функциональные методы определения АДРБ1 АТ, в которых исследуется их биологическая активность. Такими методами авляются определение хронотропного эффекта антител на культурах кардиомиоцитов, а также детектирование подъема концентрации цАМФ на линиях клеток, экспрессирующих молекулу адренорецептора. Autoantibodies to β1-adrenergic receptor (β1ADR Ab) are often found in the serum of patients with congestive heart failure due to various etiologic factors. β1ADR Abs also occur in some healthy donors, however, such antibodies differ by their functional characteristics from those discovered in the blood of diseased subjects. Pathogenetic role of β1ADR Abs has been demonstrated in various animal models. Presumed pathogenetic mechanisms of β1ADR Ab action have been reviewed. Different evidences for putative specific role of IgG3 autoantibodies in the development of cardiovascular disorders are presented. In clinical practice, the presence of β1ADR Abs in patients’ blood has a prognostic value and can also be a marker of ventricular cardiac rhythm disturbances. Different methods of β1ADR Abs detection have been reviewed, including enzyme-linked immunosorbent assay using peptides as antigens and methods utilizing a native molecule of adrenergic receptor as an antigen. The highest informative value have the functional methods of β1ADR Abs detection, which are based on the evaluation of their biologic activity. These methods include the measuring of the chronotropic effect of antibodies in cultured cardiomyocytes and detection of increase in intracellular cAMP levels in cell lines expressing the molecules of adrenergic receptor.

Abstract Background Takotsubo cardiomyopathy (TTC) is an acute, life-threatening condition which is typically induced by stress and manifested by chest pain and ECG changes. The TTC prevalence among patients (pts) with acute coronary syndrome is 1.7–2.2%. The mortality rate from TTC is up to 8%. TTC is clinically indistinguishable from acute myocardial infarction (AIM). Differential diagnosis of TTC and AIM remains an unresolved problem. Recent studies have shown the differences in profiles of plasma microRNAs inTTC and AIM. Purpose To evaluate the possibility of differentiating TTC and AIM using a PCR-based semi-quantitative analysis of mRNAs, the plasma levels of which had been shown to be increased in patients with AIM (miR-1, miR-208a, miR-133a, miR-499a) and TTC (miR-16, miR-26a). Methods Plasma from 38 pts was used: 13 pts with confirmed TTC (12 women, 1 man), 25 pts with AIM (9w, 16m). For 10 pts with AIM, blood was collected twice: at 6 and 24 hours after the heart attack. The control arm comprised 40 healthy people from the same age group (12w, 28m). Plasma was obtained using the Cell-Free DNA blood collection tubes. Nucleic acids were separated using a modified Boom method. A semi-quantitative assessment of the mRNA levels was performed using dCq method with stem-loop qRT-PCR. Normalization for spiked synthetic cel-miR-39 and endogenous miR-23a was performed. Control of hemolysis was performed by measuring the ratio of miR-451 (specific for RBCs) and miR-23a (absent from RBCs). The statistical significance of differences between mRNA levels was assessed using Mann–Whitney test. Results No significant differences in the levels of miR-16, miR-26a and miR-208a in TTC group and control group have been found. Pts with AIM significantly differed from pts with TTC (p=0.0038 and 0.0002, respectively) and control pts (p=3.1x10–9 and 2,66x10–10) with the increased levels of cardiac-specific miR-1 and miR-133a. As compared to plasma levels at 6 hours after the heart attack, at 24-hour point the levels of these mRNAs were markedly reduced in 9 of 10 pts (mean reduction was 22.3-fold for miR-133a and 7.5-fold for miR-1). The correlation between changes in the levels of these mRNAs was high (Spearman correlation coefficient=0.89). Significant differences in plasma levels of miR-133a between pts with AIM and TTC were maintained even if blood was collected after 24 hours (p=0.007). For miR-16 and miR-26a, no significant differences between pts with AIM and TTC were found. The results of analysis of these mRNAs are affected to a substantial degree by the residual hemolysis due to their high content in blood cells. Conclusions It was shown that measuring the plasma levels of mRNAs miR-1 and miR-133a allows to distinguish TTC from AIM by excluding the diagnosis of TTC. The differential diagnosis is possible only within several hours after acute clinical symptoms and requires proper normalization and full compliance with the technical specifications.

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.

Efficacy of critical limb ischemia gene therapy can be improved by application of novel plasmid vectors with higher transgene expression. The goal of this study is to evaluate in vitro and in vivo expression of angiogenic growth factors after gene transfer using a novel plasmid vector PC4W. Plasmid constructs with genes of human VEGF185 CpC4W-hHGFopt), HGF CpC4W-hHGFopt) and angiopoietin-1 (pC4W-hAng-1optJ were tested in vitro in a HEK293T cell culture. Cells were subjected to calcium-phosphate transfection and conditioning medium samples were assayed for transgene levels using Western blot and ELISA. Results were compared with commercially available pcDNA3 based vectors encoding the same growth factors. Reverse transcription PCR was used to assay transgene expression in BALB-c mice ischemic muscle. ELISA and Western blotting data suggest that PC4W based constructs give a higher protein output of about 2-2,5 fold compared with pcDNA3 based plasmids. Optimization of nucleotide sequence in growth factors cDNA results in additional increase in transgene expression. RT-PCR data shows that expression of human HGF persists in murine ischemic skeletal muscle up to 14 days after gene transfer. Our results indicate that novel plasmid constructs for angiogenic growth factors expression have a good efficacy in vitro and in vivo and can be used for VEGF1B5, HGF and angiopoietin-1 expression in human cell culture and in experimental animals' tissue. At the moment all developed constructs pass through a series of experiments in animal ischemia models and will be used for combined gene therapy development.