Modulating Effect of Peptide Therapy on the Morphofunctional State of Bronchial Epithelium in Rats with Obstructive Lung Pathology.

On the model of chronic obstructive pulmonary disease, the effect of therapy with low-molecular-weight peptides on restructuring and functional activity of bronchial epithelium for restoring the immune and barrier function of the lungs and prevention of inflammatory process progression was studied. Chronic obstructive pulmonary disease was modeled in rats by 60-day intermittent exposure to NO2. Administration of tetrapeptide Bronchogen for 1 month eliminates symptoms of remodeling of the bronchial epithelium and lung tissue typical of chronic obstructive pulmonary disease (goblet cell hyperplasia, squamous metaplasia, lymphocytic infiltration and emphysema, and restoration of ciliated cells). Enhanced production of secretory IgA, a local immunity marker, attested to normalization of functional activity of bronchial epithelium, while normalization of cell composition and profile of proinflammatory cytokines in the bronchoalveolar space reflected reduction of neutrophilic inflammation.

Protective effect of fenspiride on the bronchi in rats with chronic obstructive pulmonary disease.

We studied the effect of a non-steroidal anti-inflammatory drug fenspiride on contractive activity of bronchial smooth muscles on the model of chronic obstructive pulmonary disease of rats induced by 60-day exposure to nitrogen dioxide. The administration of fenspiride during the acute stage of the disease (day 15) abolished the constricting effect of the pollutant on the bronchial smooth muscles. Dilatation effect of fenspiride in a low dose (0.15 mg/kg) was mediated by its interaction with nerve endings of bronchial capsaicin-sensitive nerve C-fibers. The interaction of drug with receptors of C-fibers prevented neurogenic inflammation, which was confirmed by the absence of structural changes in the lungs typical of this pathology. The broncholytic effect of fenspiride in a high dose (15 mg/kg) was mediated by not only afferent pathways, but also its direct relaxing action on smooth muscle cells. The observed anti-inflammatory and bronchodilatation effect of fenspiride in very low doses can be used for prevention of chronic obstructive pulmonary disease in risk-group patients contacting with aggressive environmental factors.

Experimental modelling of chronic obstructive pulmonary disease.

A method for experimental reproduction of stages of chronic obstructive pulmonary disease formation (from acute inflammation to bronchopulmonary tissue restructuring characteristic of this disease) is presented. Lung injury and inflammation were induced by nitrogen dioxide. Hyperplasia and hypersecretion of goblet cells, squamous cell metaplasia of the ciliary epithelium, emphysema, and focal fibrosis served as the morphological substrate for the formation of bronchial obstruction. The adequacy of the model is confirmed by signs characteristic of chronic obstructive pulmonary disease: hyperexpression of CD3 lymphocytes in the bronchial wall and parenchyma, manifold increased production of TNFα and TGFß, high concentrations of circulating pathogenic immune complexes. Persistence of the structural and functional shifts throughout 6 months after exposure to nitrogen dioxide indicated a chronic course of the resultant pathological process.

The TLR4 Agonist Immunomax Affects the Phenotype of Mouse Lung Macrophages during Respiratory Syncytial Virus Infection.

In the study, the effect of the TLR4 agonist Immunomax was investigated in vitro and in vivo. In particular, Immunomax was shown to polarize mouse bone marrow macrophages from the M0 and M2 states into the M1 state (ARG1 and iNOS mRNA expression levels were used to identify the mouse M1 and M2 phenotypes). Next, we investigated the prophylactic antiviral effect of Immunomax in both a model of mouse respiratory syncytial virus (RSV) infection and a model of RSV-induced bronchial asthma (BA) exacerbation. In the experiment with RSV-induced BA exacerbation, Immunomax-treated mice were characterized by a significant decrease of the viral load in lung homogenates, an increased amount of M1 macrophages in the lung, a tendency toward Th2-dependent ovalbumin-specific IgG1 antibodies decrease in blood serum, a significant increase in RSV-activated CD4+ T cells secreting IFNγ (Th1 cells), and a simultaneous significant decrease in the amount of CD4+ cells secreting IL-4 (Th2 cells) in the mouse spleen, which were detected by ELISPOT 1.5 months after experiment. These findings suggest that treatment with the TLR4 agonist Immunomax polarizes the immune response towards antiviral Th1 and may be used for short-term antiviral prophylaxis to prevent acute respiratory viral infections in asthmatics.

[Effect of prednisolon on trachea smooth muscle of normal rats and rats with fibrosing alveolitis].

Effect of prednisolone on isolated preparations of trachea of normal rats and rats with fibrosing alveolitis was studied. Prednisolone at a concentration of 0.4 microg/l decreased responses of smooth muscle on stimulation of preganglionar nerve fibers at trachea areas with intramural ganglia in rats with acute alveolitis by 48%, while in normal rats--by 19% of control. In trachea preparations without ganglia, prednisolone at a dose of 10 microg/l decreased responses of muscle to the nerve fiber stimulation by 21.3%. The higher prednisolone doses were less efficient: 0.1-10 microg/l glucocorticoid practically did not affect the smooth muscle responses produced by stimulation of muscle cells. In rats with fibrosing alveolitis, 10 microg/l prednisolone restored the smooth muscle responses to control values in preparations of trachea with intramural ganglia. After the prednisolone treatment, amplitude of the rat trachea muscle contraction in response to the nerve fiber electric stimulation did not differ statistically significantly from control and 0.1-10 microg/l prednisolone did not change the response value. The conclusion is made that prednisolone affected the diseased rats more efficiently than the healthy animals. The character of the glucocorticoid effect depends on the presence of intramural ganglia in the trachea wall.

[ROLE OF MAST CELLS IN BRONCHIAL CONTRACTION IN NONALLERGIC OBSTRUCTIVE LUNG PATHOLOGY].

In model of chronic obstructive pulmonary disease induced in rats by 60-day intermittent exposure to nitrogen dioxide mast cells participation in the mechanism of bronchial smooth muscle contractile activity patterns was evaluated. Since the 31st day, one group of rats was inhaled with sodium cromoglycate every day before the nitrogen dioxide exposure to stabilize the mast cell membrane. The other group (control) hasn't been treated. Isometric contraction of the bronchial isolated preparations in response to nerve or smooth muscle stimulation were determined. Inhibition of mast cell degranulation and the release of endogenous histamine by stabilizing cell membranes prevented the development of bronchial smooth muscle hyperactivity caused by prolonged inhalation of nitrogen dioxide. It is believed that a mechanism to increase the contractile activity of the bronchial wall smooth muscles is mediated by activation of the transmembrane adenosine receptor in resident mast cells, leading to their partial degranulation with release of histamine, acting on the histamine Hl-receptors with the launch of reflex pathways through intramural ganglion neurons.

[ANTIINFLAMMATORY AND REGENERATIVE EFFECT OF PEPTIDE THERAPY IN THE MODEL OF OBSTRUCTIVE LUNG PATHOLOGY].

The effect of the tetrapeptide bronchogen on the structural and functional state of the bronchial epithelium and inflammatory activity in the lungs was studied in the chronic obstructive pulmonary disease (COPD) model, created in rats by a 60-day intermittent exposure to nitrogen dioxide. The cell composition and cytokine-enzyme profile of bronchoalveolar lavage fluid (BALF), the content of secretory immunoglobulin A and surfactant protein B in BALF were determined. Following the course of peptide treatment the decreased activity of neutrophilic inflammation with the normalization of cellular composition and profile of pro-inflammatory cytokines and enzymes in the bronchoalveolar space was observed. The structure of bronchial epithelium, disturbed during formation of COPD model, was restored and accompanied by restoration of its functional activity as evidenced by an increase of secretory immunoglobulin A (local immunity marker) and surfactant protein B, responsible for reducing the alveolar surface tension.

[ENDOTHELIAL DYSFUNCTION AND OBSTRUCTIVE LUNG PATHOLOGY].

Relationship of endothelial dysfunction and obstructive pulmonary diseases is a complex and poorly understood. Vascular endothelium is a multifunctional autonomous endocrine organ. The review discusses the various functions of the endothelium, causes, mechanisms and possible markers of endothelial dysfunction. The contribution of pulmonary vessel endothelial dysfunction in the initiation and progression of chronic obstructive pulmonary disease and asthma is considered. The existing approaches to the restoration of the structural and functional integrity of the vascular endothelium are discussed.

[EFFECT OF MAST CELL DEGRANULATION BLOCKADE ON THE INFLAMMATION OUTCOME IN THE MODEL OF OBSTRUCTIVE LUNG PATHOLOGY].

Effect of mast cell degranulation blockade on the inflammatory response and character of the lung tissue structure-functional changes were evaluated in the chronic obstructive pulmonary disease model produced in rats by 60-day intermittent exposure to nitrogen dioxide. The membrane stabilizer sodium cromoglicate was used to blockade of mast cell degranulation. Lung tissue sections were stained with toluidine blue to identify mast cells. Bronchoalveolar lavage fluid (BALF) cytogram was determined. The levels of mast cell tryptase and chymase, proinflammatory cytokine TNF-α, surfactant protein B were measured in BALF. Suppression of mast cell degranulation prevented the release of proteases in the bronchoalveolar space and reduced activity of the inflammatory process. The influx of inflammatory cells and TNF-α concentration decreased. There was no interstitial inflammatory infiltration. Bronchoalveolar epithelium structure was recovered that is the basis of its functional usefulness. The results confirm the active involvement of mast cells in the development of the inflammatory process in obstructive pulmonary diseases and allow us to consider them as a possible therapeutic target.

[MODIFICATION OF ALVEOLAR MACROPHAGE POOL UNDER INFLUENCE OF PEPTIDE THERAPY IN THE BLEOMYCIN PNEUMOFIBROSIS MODEL].

Effect of peptide therapy on morphological and functional characteristics of alveolar macrophages and role of their phenotypic reprogramming in modulation of pulmonary fibrosing process were evaluated on the rat's model of pulmonary fibrosis, initiated by intratracheal administration of bleomycin. Status of alveolar macrophages was evaluated on the basis of electron microscopic studies and phagocytic activity. In lung tissue of control animals widespread diffuse interstitial fibrosis was determined. Alveolar macrophage cytoplasm was filled geterophagosomes with surfactant fragments, lipid droplets and cholesterol crystals; foam cells were a third of macrophage pool. After the course of peptide therapy the young cell with rare geterophagosomes and lipid droplets, without cholesterol crystals and increased phagocytic activity prevailed in macrophage population. There were rare sites of fibrosis in lungs; connective tissue contained much less collagen fibers than in the control; there was a growing proliferation of the bronchial epithelium. It may be assumed that under the influence of the peptide therapy a certain balance in the alveolar macrophage population was established with a predominance of M2 phenotype for the formation of the optimal ratio of cellular and humoral immune response, providing effective remedy of bronchial epithelium and prevention of lung tissue remodeling with the interstitial fibrosis formation.

[Characteristics of circulation, ventilation and gas exchange in intact and anesthetized dogs]. / Kharakteristika krovoobrashcheniia, ventiliatsii i gazoobmena u intaktnykh i narkotizirovannykh sobak.

Light hexenal anesthesia was found not to affect the mean values of hemodynamics and O2 tension in the blood but increased the CO2 tension in arterial blood and decreased the oxyhemoglobin content and pH of the blood in dogs.

[Functional alterations in the pulmonary respiratory system in dogs during alveolar hypoxia with hypercapnia]. / Funktsional'nye izmeneniia sistemy legochnogo dykhaniia u sobak pri al'veoliarnoi gipoksii s giperkapniei.

In acute experiments on 25 dogs, interrelationships of parameters of the small circulation, ventilation, and diffusion were studied during alveolar hypoxia with hypercapnia. Two types of responses of the lung breathing system were revealed. The 1st one involved an increase of the small circulation pressure (by 84%), of volume blood flow (by 42%), of the lung blood volume (by 24%), and of the lung diffusion ability (by 47%). The 2nd type of responses involved an increase of the small circulation pressure on the average by 70%, while the other three parameters practically did not change at all. The combined action of such hemodynamic factors as the increased intravascular pressure and blood flow seem to involve previously inactive vascular areas of the lungs in the circulation. The lung vascular bed of animals with the 1st type of responses is more plastic which can provide a better adaptation of the organism for extreme conditions.

[Role of changes in the hemodynamics of the pulmonary circulation in pulmonary gas exchange]. / Rol' izmenenii gemodinamiki v malom kruge krovoobrashcheniia v legochnom gazoobmene.

High pressure in the lung artery was maintained with the aid of i. v. serotonin administration (0.5 mg/ml) in dogs during 10 min. The simultaneous increase of the intravascular pressure in minor circulation and of the lung volume circulation was followed by an increasing blood volume in lungs, diffusion capacity of lungs, oxygen tension in the arterial blood, and satiation of the latter with oxygen. The combined action of the above two hemodynamic factors aids to opening of latent vascular areas, to a more regular distribution of perfusion over the lung areas according to the ventilation level. This mechanism seems to be one of the first compensatory responses to disturbances in the lung gas exchange.

[Content of trace elements and steroid hormones in blood and tissues of internal organs in experimental pulmonary fibrosis induced by intratracheal administration of bleomycetin]. / Soderzhanie mikroélementov i steroidnykh gormonov v krovi i tkaniakh vnutrennikh organov pri ékperimental'nom fibroze legkikh, vyzvannom vnutritrakheal'nym vvedeniem bleomitsetina.

Pulmonary fibrosis was produced in rats by intratracheal administration of bleomycetin. The development of the fibrous process in the lungs was attended by marked reduction of the zinc content in the tissues of the viscera, plasma, and formed elements of the blood, as well as of corticosterone and testosterone in blood plasma. Respiratory insufficiency is a probable cause of the developing zinc-deficiency which leads to disturbed synthesis of steroid hormones. Injection of exogenous testosterone normalizes partly the level of steroid hormones in the blood, improves the general condition of the animals, and facilitates an increase of body weight.

[Content of trace elements and steroid hormones in blood and tissues of internal organs of rats during an inflammatory process in the bronchi]. / Soderzhanie mikroélementov i steroidnykh gormonov v krovi i tkaniakh vnutrennikh organov krys pri vospalitel'nom protsesse v bronkhakh.

Prolonged interrupted inhalation of nitrogen dioxide in a dose of 36.0 +/- 2.2 mg/m3 was attended by definite morphological signs of inflammation in the lungs of rats (hyperplasia of goblet cells, thickening of the basilar membrane, differentiation of ciliated epithelium, etc.), reduction of the zinc content in all studied tissues and testosterone levels in blood. Intramuscular injection of 1% testosterone propionate normalized the blood level of the hormone and influenced the synthetic processes in the cells of the bronchial epithelium and the rate of its regeneration. To raise the efficacy of the correction of the pathological process in the lungs it is suggested that hormonal therapy should be combined with correction of zinc deficiency in the organism.

[Effect of endotelioprotectors on the tone of pulmonary arteries and bronchi in the model of obstructive lung pathology].

In the model of chronic obstructive pulmonary disease (COPD), produced in rats by 60-day exposure to nitrogen dioxide, the effect of drugs with endotelioprotector properties (sulodexide and rosuvastatin) on the functional state of small pulmonary arteries and bronchi was studied. We evaluated the contractile activity of smooth muscle strips of the bronchi caused by stimulation of the nerves or muscles, and changes in tone of isolated pulmonary artery rings at the application of reagents-vasodilators. The use of sulodexide promoted restoration NO-dependent mechanism of vasodilatation and improved ß-adrenergic regulation of the pulmonary artery tone. The use of rosuvastatin had no effect on the dilator activity of pulmonary arteries. Both drugs improved the functional status of the bronchial smooth muscles and intrabronchial nervous system that controls the contractile activity of smooth muscle structures of the airways. The results of the study suggest that the one-way relaxing effect of sulodexide on pulmonary arterial and bronchial smooth muscles enables the recovery of coordinated regulation of the tone of these structures, which is essential for maintaining the optimal ratio of ventilation and pulmonary blood flow for efficient gas exchange.

[Cell-molecular mechanisms of nitrogen dioxide-induced damaging effect on bronchial epithelium].

Nitrogen dioxide and reactive nitrogen species play a key role in the development environment related diseases by initiation of cell death and damage of bronchoalveolar epithelium. This review examines the possible cell-molecular mechanisms of nitrogen dioxide-induced damaging effect on bronchial epithelium.

[Effect of different therapy options on bronchial contraction in rats with modeled obstructive pulmonary disease].

In the model of chronic obstructive pulmonary disease, produced in rats by 60-day exposure to nitrogen dioxide, the effect of different options of combination therapy (corticosteroids, anticholinergics, adrenergic agonists) on the functional state of the bronchi was studied. The contractile activity of strips of the bronchi caused by nerve or smooth muscle stimulation was evaluated. Corticosteroid monotherapy resulted in deterioration of the functional state of the bronchial wall neuromuscular apparatus due to corticosteroid resistance, evolving under the influence of long-term exposure to nitrogen dioxide. Application of M-anticholinergic tiotropium had a beneficial effect on the functional state of the bronchi smooth muscles, leading to the full restoration of the bronchial wall contractile activity and removal the morphological manifestations of inflammatory lung tissue remodeling. Most effective in terms of impact on the functional state of the bronchial wall neuromuscular apparatus was corticosteroid therapy combined with M-cholinolytik or beta2-adrenoagonist.

Regulation of the target protein (transgene) expression in the adenovirus vector using agonists of toll-like receptors.

Replication-defective adenoviral vectors are effective molecular tools for both gene therapy and gene vaccination. Using such vectors one can deliver and express target genes in different epithelial, liver, hematopoietic and immune system cells of animal and human origin. The success of gene therapy and gene vaccination depends on the production intensity of the target protein encoded by the transgene. In this work, we studied influence of Toll-like receptors (TLR) agonists on transduction and expression efficacy of adenoviral vectors in animal and human antigen-presenting cells. We found that agonists of TLR2, 4, 5, 7, 8 and 9 significantly enhance a production of the target protein in cells transduced with adenoviral vector having the target gene insert. The enhancement was observed in dendritic cells and macrophages expressing cytoplasmic (GFP), membrane (HA) or secretory (SEAP) proteins encoded by the respective rAd-vectors. Experiments in mice showed that enhancement of the transgene expression can be achieved in the organism of animals using a pharmaceutical-grade TLR4-agonist. In contrast to other TLR-agonists, the agonist of TLR3 substantially suppressed the expression of transgene in cells transduced with adenoviral vectors having insert of GFP or SEAP target genes. We propose that the enhancement of transgene expression is linked to the activation of MyD88→ NF-kB, while the inhibition of transgene expression depends on TRIF→ IRF signaling pathways. Both of these pathways jointly exploited by TLR4-agonists lead to the enhancement of transgene expression due to the dominant role of the MyD88→ NF-kB signaling.

[Changes in pulmonary arteries dilatation reserve during chronic obstructive pulmonary disease model formation].

Model of chronic obstructive pulmonary disease (COPD) was induced in rats by nitrogen dioxide inhalation for 60 days. The effect of reagents-vasodilators on the isolated pulmonary arteries with a diameter less than 0.5 mm was studied in 15, 30 and 60 days of COPD induction. All vasodilators (beta-adrenoreceptor agonist izopreterenol, nitric oxide donor nitrosorbid, acetylcholine, activator of C-fibers capsaicin, corticosteroid beclometasone) dose-dependently decreased vascular tone of pulmonary arteries isolated from intact rats. On extension nitrogen dioxide exposure pulmonary arteries responded to the impact of all vasodilators by smaller relaxation. Dose-dependence of dilatation reaction disappeared. In the process of COPD model formation functioning of almost all pulmonary arterial wall neurotransmitter systems were broken. This led to decrease in vasodilators influence on vascular tone and could facilitate the development of pulmonary hypertension which is typical of COPD.