A new mouse unilateral model of diffuse alveolar damage of the lung.

OBJECTIVE AND DESIGN: The existing biological models of diffuse alveolar damage (DAD) in mice have many shortcomings. To offset these shortcomings, we have proposed a simple, nonsurgical, and reproducible method of unilateral total damage of the left lung in ICR mice. This model is based on the intrabronchial administration of a mixture of bacterial lipopolysaccharide (LPS) from the cell wall of S. enterica and α-galactosylceramide (inducing substances) to the left lung. METHODS: Using computer tomography of the lungs with endobronchial administration of contrast material, we have been able to perform an operative intravital verification of the targeted delivery of the inducer. The model presented is characterized by more serious and homogeneous damage of the affected lung compared to the existing models of focal pneumonia; at the same time, our model is characterized by longer animal survival since the right lung remains intact. RESULTS: The model is also characterized by diffuse alveolar damage of the left lung, animal survival of 100%, abrupt increases in plasma levels of TNFa, INFg, and IL-6, and significant myocardial overload in the right heart. It can be used to assess the efficacy of innovative drugs for the treatment of DAD and ARDS as the clinical manifestations that are developed in patients infected with SARS-CoV-2. Morphological patterns of lungs in the noninfectious ("sterile") model of DAD induced by LPS simultaneously with α-galactosylceramide (presented here) and in the infectious model of DAD induced by SARS-CoV-2 have been compared. CONCLUSION: The DAD model we have proposed can be widely used for studying the efficacy of candidate molecules for the treatment of infectious respiratory diseases, such as viral pneumonias of different etiology, including SARS-CoV-2.

The role of platelet glycoprotein IIb / IIIa inhibitors in current treatment of acute coronary syndrome.

Current management of patients with acute coronary syndrome (ACS) includes a dual antiplatelet therapy with acetylsalicylic acid and a platelet P2Y12 receptor inhibitor. For patients without a high risk of bleeding, prasugrel and ticagrelor are preferred, since their effect is more pronounced, less dependent on metabolism of a specific patient, and occurs faster that the effect of clopidogrel. The prescription rate of platelet glycoprotein IIb/IIIa (GP IIb / IIIa) receptor inhibitors has considerably decreased. However, these drugs remain relevant in percutaneous coronary interventions in patients with a high risk of coronary thrombosis or a massive coronary thrombus, in thrombotic complications of the procedure, and in the "no-reflow" phenomenon. The intravenous route of GP IIb / IIIa inhibitor administration provides their effectiveness in patients with difficulties of drug intake or with impaired absorption of oral medications. This review presents clinical and pharmacological characteristics of various GP IIb / IIIa inhibitors and data of randomized clinical studies and registries of recent years that evaluated results of their use in patients with ACS.

[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.

[Lipoprotein-associated phospholipase A2 serum levels in patients from different categories of cardiovascular risk].

AIM: To compare levels of lipoprotein-associated phospholipase A2 (Lp-PLA2) in blood serum of patients from different cardiovascular risk categories. MATERIAL AND METHODS: Patients from Moscow prospective study database (n = 519) were divided into 4 cardiovascular risk categories according to present clinical recommendations (low, moderate, high, very high). Measurement of Lp-PLA2 concentration (mass) was performed using PLAC Test ELISA Kit. Measurement of Lp-PLA2 activity was made using PLAC Test for Lp-PLA2 Activity. Blood serum levels of total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), triglycerides (TG), lipoprotein (a) (Lp(a)), high sensitive C-reactive protein (hsCRP) and uric acid were also determined. RESULT: Preliminary analysis showed that associations between Lp-PLA2 mass and activity became more obvious in patients not treated with statins and patients without diabetes mellitus. So patients receiving statins and diabetics were excluded from final analysis. Lp-PLA2 mass and activity were lower in low cardiovascular risk category patients. There were no significant differences in Lp-PLA2 mass and activity between patients from moderate, high and very high risk categories. There was moderate correlation between Lp-PLA2 mass and Lp-PLA2 activity (r = 0.38, p < 0.00001). We did not find any correlation between Lp-PLA2 and hsCRP, Lp(a) levels, but detected moderate correlation between Lp-PLA2 mass and activity and TC, LDL-C. We also found a mild positive correlation between Lp-PLA2 mass and HDL-C levels. There was a positive correlation between Lp-PLA2 activity and TG, uric acid and negative correlation between Lp-PLA2 activity and HDL-C levels. CONCLUSION: In this group of nondiabetic patients not treated with statins both Lp-PLA2 activity and mass were similarly related to categories of cardiovascular risk.

[Influence of intensive hypolipidemic therapy on blood concentration of lipoprotein-associated phospholipase A2 in patients with ischemic heart disease].

AIM: To assess the impact of combined treatment with simvastatin and ezetimibe or treatment with simvastatin only on lipoprotein-associated phospholipase A2 in patients with ischemic heart disease. METHODS: One hundred patients with angiographically documented coronary atherosclerosis took part in the investigation. Lp-PLA2 mass and cholesterol fractions were determined at baseline and after 6 months of treatment. Lp-PLA2 mass was determined by enzyme immunoassay method, using two highly specific monoclonal antibodies. RESULTS: Combined treatment with ezetimibe and simvastatin led to significantly greater declines in Lp-PLA2 and cholesterol fractions compared with treatment only with simvastatin: Lp-PLA2 decreased by 46 vs 38%, total cholesterol by 35 vs 28%, LDL cholesterol by 50 vs 40%, respectively (p<0.05). Combination therapy with ezetimibe and simvastatin 20 and 40mg/day proved to be as effective as monotherapy with simvastatin 80 mg/day on the effect on Lp-PLA2 mass and cholesterol fractions (p<0.05). Lp-PLA2 correlated positively with total cholesterol (r=0.28) and LDL-C (r=0.33). CONCLUSIONS: Combined treatment led to greater reduction of total cholesterol and LDL-C, as well as significantly reduced level of Lp-PLA2 mass. The latter can be considered as target for suppression of inflammation and achievement of stabilization of atherosclerotic plaque.

[Relationship between concentration of lipoprotein-associated secretory phospholipase A2 and markers of subclinical atherosclerotic lesion of the arterial wall in patients with low and moderate risk by SCORE scale].

UNLABELLED: AIM To show relations between a concentration of lipoprotein-associated secretory phospholipase A2 (LPPLa2) and markers of subclinical atherosclerotic lesion of the arterial wall in patients with low and moderate risk by the SCORE scale. MATERIAL AND METHODS: A total of 378 individuals with low and moderate risk of atherosclerotic lesion of the arterial wall (285 females, 93 males) were divided into groups by (1) age and sex, (2) number of atherosclerotic plaques (ASP) in the carotid arteries: OASP (n=158), ASP (n=61), more than one ASP (n=159); (3) plaque characteristics: homogeneous (n=31), heterogenous (n=189), (4) the presence of ASP in CA and level of LPPLa2 in the blood (with high content--n=137, with normal content--n=83). Duplex CA scanning was made to estimate intima-media thickness (IMT), to detect AP in the CA. Computer sphygmography estimated velocity of the pulse wave (PWV) from the carotid to femoral artery. Normal values of IMT and PWV were estimated individually with reference to gender and age. LPPLa2 was measured immunoturbodimetrically using diagnostic kits (PLAC Test Elisa Kit, dia Dexus, U.S.A.), shreshold value < 200 ng/ml. RESULTS: LPPLa2 content medians in different age groups in males and females differed insignificantly. LPPLa2 concentration in the groups of patients regarding ASP in CA was elevated in relation to the threshold value (200 ng/ml) in all the groups but did not significantly differ: 216 (179-257) ng/ml in the group OASP, 226 (190-274) ng/ml--in the group of patients 1ASP and 212 (174-254) ng/ml--in the group of patients more than one ASP (p > 0.05). In the groups with homogeneous and heterogenous ASP significant differencies were neither between the medians nor between frequency of deviation from normal (p = 0.28). 25.5% patients from the group with an elevated level of LPPLa2 had ASP with a hypoechogenic component. CONCLUSION: No significant correlation was revealed between concentration of LPPLa2, IMT PWV, number of ASP and carotid stenosis.

[Complex analsis of association of inflammation genes with myocardial infarction].

Carriage frequencies of alleles and genotypes of functionally important polymorphous loci of some inflammation genes: proinflammatory cytokines genes IL-6, LTA and TNF, anti-inflammatory cytokine gene TGFB1 and CC chemokine receptor 5 gene CCR5 were analyzed in the patients with myocardial infarction (MI) of Russian ethnic descent (199 cases) and in the control group of the same ethnic descent (142 controls). Complex analysis using APSampler algorithm revealed MI association with carriage of all polymorphous variants studied, as individual risk factors (insertion/deletion polymorphism of CCR5 and SNP G252A LTA) or only in combination with other alleles/genotypes. Carriage of bi- or triallelic combinations was associated with MI more significantly than carriage of any their subsets: single alleles or allele pairs. Protective triallelic combination d*CCR5 + 252G*LTA(+) -174C*Ll-6 was found to be most significant (p = 0.0006, OR = 0.23, CI = 0.090-0.56). Separate analysis of genetic susceptibility to MI for men and women displayed sexual dimorphism for CCR5 gene.

Interrelation between malonyl dialdehyde-dependent modification and cholesterol content in low-density lipoproteins.

Epidemiological study of an independent representative sample of population revealed a strong positive correlation between the content of oxidized (MDA-modified) LDL and concentration of atherosclerosis biomarkers (total cholesterol and LDL cholesterol) in blood plasma from 348 probands. The correlation between these parameters was more significant in atherosclerotic patients, but was less pronounced in probands with diabetes mellitus. The correlation between the concentration of atherosclerosis markers and content of MDA was absent in probands with diabetes mellitus. These data attest to the presence of LDL-modifying agents differing from MDA (e.g., glyoxal and methylglyoxal) in the blood of diabetes mellitus patients. We conclude that the content of MDA-modified LDL can serve as an additional biomarker of atherosclerosis.

[Cultivated human hair follicle cells are capable of integrating to skin structure in vivo].

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.

[Polymorphism C1444T of C-reactive protein gene and C-reactive protein concentration in blood serum of healthy people and patients with myocardial infarction].

UNLABELLED: CRP level is a risk factor of development of ischemic heart disease (IHD) and acute myocardial infarction (MI) in healthy people, while in patients with cardiovascular diseases it is a marker of unfavorable prognosis. It has been shown in recent investigations that individual variations of plasma CRP levels to a great extent are genetically determined. These data constitute a basis for the study of associations of polymorphic variants of the CRP gene with risk of MI in healthy people as well as with unfavorable prognosis in IHD patients. MATERIAL AND METHODS: We included into the study 232 Russian patients aged 52.3 +/- 10.3 years, 175 men (50.1 +/- 10.6 years) and 57 women (55.2 +/- 10.1 years). Control group comprised 159 Russians without history of cardiovascular diseases and other serious severe concomitant diseases (age 60.5 +/- 14 years), 76 men (age 57.3 +/- 13.9 years ) and 83 women (age 63.1 +/- 14 years). CRP concentration was measured initially (at the moment of hospitalization), on days 3, at discharge, in 1 and 6 months, 1 year after onset of infarction. For genomic typing of C1444T polymorphism of CRP gene we used restriction fragment length analysis of products of polymerase chain reaction (PCR). RESULTS: Distribution of genotypes of C1444T polymorphism of CRP gene: C/C 51.8%, C/T 35.8%, T/T 12.4% in patients with MI; C/C 55.2%, C/T 40.2%, T/T 4.6% in control group. We found significant difference (p = 0.006, relative risk [RR] 0.3, 95% confidence interval [CI] 0.15-0.74) in frequency of carriers of C1444 allele (sum of C/C and C/T genotypes), which was higher in control group. Correspondingly in the group of patients with MI T/T genotype was met significantly more frequently than in control (p = 0.006, RR 3.0, 95% CI 1.3-6.5), and can be looked upon as risk factor of MI. We found no relation between carriage of CRP alleles/genotypes of CRP and one year prognosis in patients with MI. Analysis of association of the C1444T polymorphism with CRP concentration revealed significant relationship between T/T genotype and higher CRP level.

[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.

[Caldesmon changes the structure of actin at the leading edge and suppresses cell migration].

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.

[Phospholipase A2, group IIa, as an earlier unknown immunohistological marker of cardiac myxoma].

Phospholipase A2, group IIA, gene expression has been analyzed in primary heart tumors. High expression has been demonstrated through several ways: reverse-transcriptase chain polymerase chain, Northern blotting hybridization at the RNA level and immunoblotting, immunohistochemical assay at the protein level. Human cardiac myxoma exhibits highly positive phospholipase A2, group IIA, immunophenotype (100% positive cases). The immunophenotype is unique among human primary cardiac tumors. Phospholipase A2, group IIA, can be proposed as a tissue marker for pathological examination after heart tumor resection.

Expression level of lipoprotein lipase and dystrophin genes predict survival in B-cell chronic lymphocytic leukemia.

Mutational status of immunoglobulin variable region genes (VH-genes) is known as the strongest predictor of long term prognosis in B-CLL. However, applications in the routine clinical practice are time consuming, and therefore some other predictions are required. In this study, we have compared prognostic values of real time PCR quantification of the expression levels of four genes previously shown to be differentially expressed in V(H)-unmutated and mutated B-CLL subtypes: ZAP-70, ZBTB20, DMD and LPL. The study included 134 B-CLL patients. Expression levels of LPL and DMD genes were significantly correlated to mutational status, while expression levels of of ZAP-70 gene correlated only in CD19+ selected cases (N = 40). No correlation was observed for ZBTB20 gene. Expression levels of LPL and DMD predicted overall survival in the entire cohort of patients. Prognostic values of LPL gene expression levels were significant even for CLL patients with stage A. Quantitative RT-PCR assays for measuring LPL gene expression are robust enough to be introduced into routine clinical practice.

Identification of 130 kDa cell surface LDL-binding protein from smooth muscle cells as a partially processed T-cadherin precursor.

Atypical cell surface lipoprotein-binding proteins of 105 kDa and 130 kDa are present in membranes of vascular smooth muscle cells. We recently identified the 105 kDa protein from human aortic media as T-cadherin, an unusual glycosylphosphatidylinositol (GPI)-anchored member of the cadherin family of cell adhesion proteins. The goal of the present study was to determine the identity of 130 kDa lipoprotein-binding protein of smooth muscle cells. We applied different approaches that included protein sequencing of purified protein from human aortic media, the use of human T-cadherin peptide-specific antisera, and enzymatic treatment of cultured cells with trypsin and GPI-specific phospholipase C. Our results indicate that the 130 kDa protein is a partially processed form of T-cadherin which is attached to the membrane surface of smooth muscle cells via a GPI anchor and contains uncleaved N-terminal propeptide sequence. Our data disclose that, in contrast to classical cadherins, T-cadherin is expressed on the cell surface in both its precursor (130 kDa) and mature (105 kDa) forms.

[Expression patterns of the human and mouse IFGP family genes].

The IFGP family is a recently identified group of human and mouse genes structurally related to the genes of leukocyte Fc-receptors. In this study we compared expression patterns of six human and four mouse IFGP genes. With the exception of mouse IFGP2, the genes of the family were found to be predominantly expressed in haemopoietic cells. Expression of human IFGP1-IFGP5 and mouse IFGP3 was B cell-specific. Mouse IFGP1 transcripts were mainly found in B cells, but this gene may be either expressed by nonlymphoid cells. Expression of the human IFGP6 was detected in CD8+ T cells and NK cells. We further demonstrated that alternative splicing of human IFGP1 and IFGP6 mRNA may generate transcripts coding for the previously unknown isoforms. The novel IFGP1 isoform lacks transmembrane domain, whereas the IFGP6 isoform has altered cytoplasmic tail. The data obtained indicate that the receptors of family may contribute to the regulation of development and/or functions of three effector types of lymphocytes, namely B cells, CD8 T cells and NK cells.

[Results of clinical trials of a novel glycoprotein IIb-IIIa antagonist framon in high-risk coronary angioplasty].

New glycoprotein (GP) IIb-IIIa antagonist preparation framon (Monafram), is the F(ab')(2) fragment of a monoclonal antibody FRaMon directed against GP IIb-IIIa. This preparation blocks GP IIb-IIIa binding with fibrinogen and inhibits platelet aggregation both in vitro and upon intravenous administration. Safety and ability of framon to prevent thrombotic complications in high risk coronary angioplasty (CA) was evaluated in the present study. FRAMON was injected intravenously into 153 patients just before the start of procedure as a single bolus at the dose of 0.25 mg/kg. Control group was formed of 126 patients who underwent angioplasty without GP IIb-IIIa blockers. After framon administration there were no allergic reactions or major bleedings, deep thrombocytopenia (< 50000/microl) developed in 1 patient (< 1%), and antibodies against framon were detected in less than 5% of patients. Number of unfavorable outcomes (cardiovascular death, myocardial infarction, angina recurrence) within 1 month after CA was 3 times higher in control group than in the group of patients treated with framon (11.4% and 3.3%, respectively, p = 0.018). The effect of framon was most strongly pronounced within the first day after procedure -- administration of the drug reduced number of acute thromboses from 6.5% to 0.7% (p = 0.013). Significant differences between numbers of end points was still preserved at 6 months after procedure (25.7 and 14.2% in control and framon groups, respectively, p = 0.023). The data obtained proved safety and clinical efficacy of framon administration in coronary angioplasty with high risk of thrombotic complications.