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 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.

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.

Culture of mouse macrophages (RAW 264.7 ATCC strain) in wells of a 6-well plate was infected with M. tuberculosis in proportion of 15 mycobacteria per one macrophage and then treated with a lytic strain of mycobacteriophage D29. Antibacterial efficacy of mycobacteriophages was studied using D29 phage (activity 108 plaque-forming units/ml) previously purified by ion exchange chromatography. After single and double 24-h treatment, the lysed cultures of macrophages were inoculated onto Middlebrook 7H10 agar medium. The number of mycobacterial colonies in control and test wells (at least 3 wells in each group) was 300.178±12.500 and 36.0±5.4, respectively (p<0.01).

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 role of beta2-integrins CD11b/CD18 and CD 11c/CD 18 in adhesion and migration of leukocytes on fibrinogen was studied. The monoclonal antibodies against CD11b inhibited the spontaneous adhesion of monocytic THP-1 cells on fibrinogen, whereas antibodies to CD11c more effectively inhibited the adhesion stimulated by chemokine MCP-1. By the RNA-interference method the clones of THP-1 with reduced expression of CD11b and general beta2-subunit CD18 were obtained. MCP-I stimulated the adhesion to fibrinogen of THP-1 cells of wild-type and mutant cells with reduced expression of CD11b (THP-1-CD11b-low), but not of cells with low expression of CD18 (THP-1-CD18-low). THP-1-CD18-low cells were also characterized by the impaired chemotaxis in presence of MCP-1. The data obtained suggest that spontaneous cell adhesion to fibrinogen is mediated to a greater extent by CD11b/CD18 integrins, while chemokine-stimulated adhesion and migration is mostly dependent on CD11c/CD18 molecules.

In the present work, we labeled human epidermal keratinocytes and dermal papilla cells in order to study their behavior after intradermal transplantation. The cells were transduced by lentiviral vectors that bore a marker gene that encodes green fluorescent protein (copGFP) or red fluorescent protein (DsRed). A portion of the transgene expressing cells was evaluated by flow cytometry. The proposed genetic constructions have allowed one to achieve high efficiency (>95%) of the transduction of hair follicle cells. The in vitro transduced cells were injected under epidermis of human skin fragments, after which these fragments were transplanted under the skin of immunodeficient mice. The injected epidermal keratinocytes were found mainly in hair follicles and partially in the zone of interfollicular epidermis, while dermal papilla cells were found in the papilla of the derma. The results of the present study have shown that the chosen genetic constructions obtained based on human immunodeficiency lentivirus are capable of the effective and stable transduction of human skin cells. The injected cells survived and were found in the corresponding skin structures.

In the present study, human keratinocytes and dermal papilla cells were labeled to investigate their behaviour after intradermal transplantation. Cells were transduced by lentiviral vectors that bore marker gene encoding green fluorescent protein (copGFP) or red fluorescent protein (DsRed). A portion of transgene expressing cells was evaluated by flow cytometry. Genetic constructions that we used provided high level (> 95 %) of transduction of hair follicle cells. In vitro transduced cells were injected under the epidermis of human skin fragments, and these fragments were then transplanted under the skin of immunodeficient mice. Injected epidermal keratinocytes were found, mainly, in hair follicles and partially in a zone of interfollicular epidermis, while dermal papilla cells were found in papilla derma. The results of the present research show that the chosen genetic constructions obtained on a basis of human immunodeficiency lentivirs are capable of effective and stable transduction of human skin cells. Injected cells survived and were found in the corresponding structures of the skin.

The effect of the suppression of expression of the actin-binding protein caldesmon on the motility of nonmuscle cells has been studied. A more than fivefold decrease in the content of this protein in cells by RNA interference led to the disturbance of the formation of actin stress fibrils and acceleration of cell migration to the zone of injury of the monolayer. A stimulation of stationary cells by serum induced a more than 1.5-fold accumulation of stress fibrils only in control cells but not in caldesmon-deficient cells. Similarly, the accumulation of actin filaments was observed in actively migrating cells of only wild type but not in cells with a low caldesmon content. These changes occurred mainly at the leading edge of the migrating cell where the distinct structure of actin filaments was not seen in the absence of caldesmon. It was assumed that caldesmon inhibits cell migration due to the stabilization of actin in filaments and a decrease in the dynamics of monomeric actin at the leading edge of the migrating cell.

The effect of the suppression of expression of the actin-binding protein caldesmon on the motility of nonmuscle cells has been studied. A more than a fivefold decrease in the content of this protein in cells by RNA interference led to the disturbance of the formation of actin stress fibers and acceleration of cell migration to the zone of injury of the monolayer. A stimulation of stationary cells by serum induced more than 1,5-fold accumulation of stress fibers only in control cells, but not in caldesmon-deficient cells. Similarly, the accumulation of actin filaments was observed in actively migrating cells of only wild type, but not in the cells with low caldesmon content. These changes occurred mainly at the leading edge of the migrating cell where the distinct structure of actin filaments was not seen in the absence of caldesmon. It was assumed that caldesmon inhibits cell migration due to the stabilization of actin in filaments and a decrease in the dynamics of monomeric actin at the leading edge of the migrating cell.

We developed a technology of labeling bone marrow precursor cells with the Lin—c-kit+ phenotype in culture by green fluorescent protein gene using a lentivirus vector. The proposed system provides effective transduction of bone marrow precursor cells and high transgene expression level in vitro (27%). The integration of the transgene into the transduced cell genome in vivo was verified by the method of splenic colonies.

To investigate the effect of lectin-like ox-LDL receptor-1 (LOX-1) on oxidized low-density lipoprotein (ox-LDL)-induced apoptosis and the involvement of the endoplasmic reticulum (ER) stress response pathway.Human umbilical vein endothelial cells were treated with 50, 100, or 200 μg/ml ox-LDL and cultured for 12, 24, or 48 h for concentration- and time-dependent studies. Cells were transfected with LOX-1 or Nox-4 shRNAs, and target proteins were inhibited with the corresponding antibodies for mechanistic studies. Active proteins and mRNAs were analyzed by Western blotting and RT-PCR, respectively. Cell apoptosis was analyzed by Annexin and Hoechst staining assays. Ox-LDL induced both apoptosis and protein expression of LOX-1 and Nox-4 through activation of ER stress sensors IRE1 and PERK, and nuclear translocation of ATF6 and their subsequent pathways were indicated by JNK, eukaryotic initiation factor 2 phosphorylation, XBP-1, and chaperone GRP78 expression; up-regulation of proapoptotic proteins CHOP and Bcl-2; and caspase-12 activity. LOX-1 gene silencing and treatment with an anti-LOX-1 antibody attenuated the effects of ox-LDL. Pretreatment with irestatin 9389, salubrinal, or AEBSF also blocked ox-LDL-induced expression of CHOP and Bcl-2 and activation of caspase-12 activity, leading to an attenuation of endothelial cell apoptosis. Furthermore, Nox-4 siRNA attenuated the up-regulated expression of GRP78, PERK, IRE1, and XBP-1 to reduce ox-LDL-induced endothelial cell apoptosis.LOX-1 plays a critical role in ox-LDL-induced endothelial cell apoptosis via the ER stress pathway.

To investigate the effect of lectin-like ox-LDL receptor-1 (LOX-1) on oxidized low-density lipoprotein (ox-LDL)-induced apoptosis and the involvement of the endoplasmic reticulum (ER) stress response pathway.Human umbilical vein endothelial cells were treated with 50, 100, or 200 μg/ml ox-LDL and cultured for 12, 24, or 48 h for concentration- and time-dependent studies. Cells were transfected with LOX-1 or Nox-4 shRNAs, and target proteins were inhibited with the corresponding antibodies for mechanistic studies. Active proteins and mRNAs were analyzed by Western blotting and RT-PCR, respectively. Cell apoptosis was analyzed by Annexin and Hoechst staining assays. Ox-LDL induced both apoptosis and protein expression of LOX-1 and Nox-4 through activation of ER stress sensors IRE1 and PERK, and nuclear translocation of ATF6 and their subsequent pathways were indicated by JNK, eukaryotic initiation factor 2 phosphorylation, XBP-1, and chaperone GRP78 expression; up-regulation of proapoptotic proteins CHOP and Bcl-2; and caspase-12 activity. LOX-1 gene silencing and treatment with an anti-LOX-1 antibody attenuated the effects of ox-LDL. Pretreatment with irestatin 9389, salubrinal, or AEBSF also blocked ox-LDL-induced expression of CHOP and Bcl-2 and activation of caspase-12 activity, leading to an attenuation of endothelial cell apoptosis. Furthermore, Nox-4 siRNA attenuated the up-regulated expression of GRP78, PERK, IRE1, and XBP-1 to reduce ox-LDL-induced endothelial cell apoptosis.LOX-1 plays a critical role in ox-LDL-induced endothelial cell apoptosis via the ER stress pathway.