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.

[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.

[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.

[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.

Lentivirus transduction of bone marrow hemopoietic precursor cells with Lin-c-kit+ phenotype ex vivo using a genetic construct containing green fluorescent protein gene.

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.

[Expression of human urokinase in inoculated rodent cells. Loss of introns during gene transfer using a retroviral vector]. / Ekspressiia urokinazy cheloveka v perevivaemykh kletkakh gryzunov. Utrata intronov v khode perenosa gena s pomoshch'iu retrovirusnogo vektora.

Expression of urokinase in murine and rat cells was performed by two recombinant constructs, one containing cDNA and the other--hybrid (cDNA/genome) variant of human urokinase gene conserving 7 introns of 10, in the eukaryotic retrovirus vector pPS-3-neo. DNA of both constructs was introduced into packaging cell line psi 2 by a standard Ca-phosphate transfection technique. Infection of mouse and rat fibroblasts BALB/c 3T3 and Rat I with virus particles, produced by transfected psi 2 cells, led to an integration into the host genome of one or two recombinant proviral copies. Stable expression and secretion into the culture medium of glycosylated high molecular weight human urokinase was observed for both cell types. For the hybrid gene construct, precise excision of intervening sequences was shown during transferring of genetic material from packaging to recipient cells.

[Mutual location of the rRNA operon and tuf gene in the Mycoplasma gallisepticum strain S6 genome]. / Vzaimnoe raspolozhenie operona rRNK i gena tuf v genome Mycoplasma gallisepticum, shtamm S6.

The genomic library of Mycoplasma gallisepticum was constructed and two clones, selected by hybridization on E. coli 16S rRNA, were analyzed. The restriction map of the clones indicate that both clones belong to the same region of the M. gallisepticum genome. The results of Southern hybridization with either E. coli 16S rRNA, or E. coli 23S rRNA, or oligonucleotide synthesized as a part of M. gallisepticum 5S rRNA, led to the conclusion that the unspliced rRNA operon was cloned. The order of genes in the operon is common for eubacteria: 16S-23S-5S. The tuf gene of M. gallisepticum was mapped inside the cloned region 5 kb upstream of 16S gene by hydridization on E. coli tufA gene and oligonucleotide synthesized on the basis of M. gallisepticum tuf gene sequence. The direction of transcription of the gene and the expected direction of transcription of the rRNA operon coincide.

[A new reaction catalyzed by Bollum's terminal deoxyribonucleotidyltransferase]. / Novaia reaktsiia, kataliziruemaia terminal'noi dezoksiribonukleotidiltransferazoi bolluma.

The highly purified preparations of Bollum's terminal transferase from calf thymus were shown to catalyze, along with the common reaction of nucleotide addition to the 3'-terminus of an oligonucleotide primer, a "non-common" reaction between dNTP or rNTP on one hand, and various alcohols on the other hand. This reaction was carried out with ethylene glycol, glycerol, ethanol and methanol to produce substances containing one molecule of nucleotide, one molecule of alcohol and non-organic pyrophosphate. The reaction conditions are cacodylate buffer, pH 7, 2, in the presence of Mg2+ or Co2+ ions. The structure was determined for the product of the reaction between glycerol and dATP, which appeared to be 2,3-dihydroxypropyl-ether of 2'-deoxyadenosine-5'-monophosphate.

[Specific protection of DNA by distamycin A, netropsin and bis-netropsins against the action of DNAse I]. / Spetsificheskaia zashchita DNK distamitsinom A, netropsinom i bis-netropsinami ot deistviia DNKazy I.

Interaction of netropsin, distamycin A and a number of bis-netropsins with DNA fragments of definite nucleotide sequence was studied by footprinting technique. The nuclease protection experiments were made at fixed DNA concentration and varying ligand concentrations. The affinity of ligand for a DNA site was estimated from measurements of ligand concentration that causes 50% protection of the DNA site. Distribution pattern of the protected and unprotected regions along the DNA fragment was compared with the theoretically expected arrangement of the ligand along the same DNA. The comparison led us to the following conclusions: 1. Footprinting experiments show that at high levels of binding the arrangement of netropsin molecules along the DNA corresponds closely to the distribution pattern expected from theoretical calculations based on the known geometry of netropsin--DNA complex. However, the observed differences in the affinity of netropsin for various DNA sequences is markedly greater than that expected from theoretical calculations. 2. Netropsin exhibits a greater selectivity of binding than that expected for a ligand with three specific reaction centers associated with the antibiotic amide groups. It binds preferentially to DNA regions containing four or more successive AT pairs. Among 13 putative binding sites for netropsin with four or more successive AT pairs there are 11 strong binding sites and two weaker sites which are occupied at 2 D/P less than or equal to 1/9 and 2 D/P = 1/4, respectively. 3. The extent of specificity manifested by distamycin A is comparable to that shown by netropsin although the molecule of distamycin A contains four rather than three amide groups. At high levels of binding distamycin A occupies the same binding sites on DNA as netropsin does. 4. The binding specificity of bis-netropsins is greater than that of netropsin. Bis-netropsins can bind to DNA in such a way that the two netropsin-like fragments are implicated in specific interaction with DNA base pairs. However, the apparent affinity of bis-netropsins estimated from footprinting experiments is comparable with that of netropsin for the same DNA region. 5. At high levels of binding bis-netropsins and distamycin A (but not netropsin) can occupy any potential site on DNA irrespectively of the DNA sequence. 6. Complex formation with netropsin increases sensitivity to DNase I at certain DNA sites along with the protection effect observed at neighboring sites.