Death Mechanisms of Pulmonary Alveolocytes in Mice Infected with Influenza Viruses A/H1N1/California/04/2009 and A/H5N1/Goose/Krasnoozerskoye/627/05.

In CBA mice infected with influenza viruses A/H1N1/California/04/2009 and A/H5N1/Goose/Krasnoozerskoye/627/05 in a dose of 10 MLD50, the mechanisms of death of pulmonary alveolocytes over 10 postinfection days were studied by light microscopy, immunohistochemistry, and morphometry. In mice infected with A/H1N1, alveolocytes died predominantly via necrosis, while apoptosis mostly employed the mitochondrial pathway. In mice infected with A/H5N1, apoptosis was the dominant mechanism of alveolocyte death proceeded via membrane receptor signaling followed by switching to FAS-mediated pathway via activation of FADD, the apoptotic signal transduction protein.

[Effect of a gel based on recombinant human angiogenin on the healing of donor palate wounds]. / Vliianie gelia na osnove rekombinantnogo angiogenina cheloveka na zazhivlenie donorskikh ran tverdogo neba.

The aim of the study was to study the effect of the gel on the basis of recombinant human angiogenin on the rate of regeneration of donor palatal wounds. The study involved 20 patients (8 men and 12 women) aged 32 to 55 years. Patients were divided into two groups: the 1st group is a study group (n=10), whose patients in the postoperative period used a gel based on recombinant human angiogenin, the 2nd group is a control group (n=10) in which a gel based on recombinant human angiogenin was not used. Patients in both study groups underwent vestibuloplasty with simultaneous plasty of the attached keratinized gingiva with a free gingival graft from the area of the hard palate. The operations were carried out at the stage of disclosing dental implants, simultaneously with the installation of healing abatements or 4 weeks before dental implantation. For histological examination, tissue samples were obtained from the region of the edge of the donor's wounds of the palate at the 7th and 14th days after surgery. As a result of the study, significant differences were found in the comparison groups when assessing the processes of inflammation, angiogenesis and epithelization. The local application of the gel containing recombinant human angiogenin resulted in a rapid decrease in the intensity of inflammation in lamina propria mucosae and a significant decrease in the bulk density of cell infiltrates, accelerating regeneration. This is primarily due to the stimulation of the development of epidermal growth factor (EGF), vascular endothelial growth factor (VEGF) and increased blood supply to the affected area, as well as an increase in the proportion of fibroblasts. The most important observation was the increase in the rate of epithelialization of donor wounds of the hard palate.

Changes in the Structure of Mouse Kidney in the Acute Period after Infection with Influenza Viruses A/H5N1 and A/H1N1.

Changes in the kidney structure in outbred and inbred male BALB/c mice were analyzed in the acute period after infection with influenza viruses A/H5N1 (10 MLD50; 10 days) and A/H1N1 (1 MLD50; 30 days). Antibodies to influenza viruses of both strains were most often expressed by endothelial cells of the glomeruli and arterioles and were rarely expressed by mesangiocytes and tubule epithelial cells. In the kidney, destructive processes induced by viruses and by ischemia due to massive blood vessel thrombosis. Mesangiocytes expressed factors, indicating that they could be qualified as M1 and M2 macrophages. Kidney destruction was more significant after infection of mice with the A/H5N1 virus, but in both experiments cell infiltrates were actually absent, probably due to blood vessel thrombosis and limited possibility of migration of mononuclear phagocytes and lymphocytes to the kidney.

Cell Death and Development of Fibrotic Alterations in Lung Granuloma of BALB/c Mice during Chronic BCG-Induced Granulomatosis.

Light microscopy, immunohistochemistry, and morphometric examinations established that cell death in lung granulomas of BCG-infected mice resulted mainly from activation of receptor-mediated apoptosis, which did not prevent the persistence of the causative agent in macrophages of the granulomas and promoted the formation of pronounced fibrosis in granulomas and pulmonary interstitium.

Preventive Effects of Oxidized Dextran on Functional Activity of Pulmonary Macrophages in Mice Infected with Influenza A Virus.

We analyzed cytokine profile of pulmonary macrophages in mice infected with highly pathogenic influenza A/H5N1 virus after preventive injections of oxidized dextran. Light microscopy, immunohistochemistry, and morphometric examinations showed that preventive injections of oxidized dextran led to more effective virus elimination, modulation of the proinflammatory cytokine response, and host antiviral response and reduce animal mortality. Our findings allow recommending oxidized dextran for further studies in order to create a vaccine with antiviral and adjuvant potencies.

Study of SMAD-Dependent Signal Pathway in the Development of Early Pulmonary Fibrosis in Mice Infected with Influenza A/H1N1 Virus.

Early fibrosis of the visceral organs is one of the main complications of infection caused by influenza A virus. Structural manifestations and molecular regulators of the epithelialmesenchymal transformation as a possible mechanism of fibrosis progression were studied in mice infected with influenza A/H1N1 A/Tomsk/13/2010 virus. We found early fibrosis of the lungs against the background of minor changes in fibroblast count. However, enhanced expression of TGF-ß and SMAD-2 by macrophages and alveolocytes attested to possible development of epithelial-mesenchymal transformation and its contribution to activation of fibrogenesis process in the lungs.

Studies of Influenza A/H1N1 A/Tomsk/13/2010 Virus Topology during Development of Infectious Process in Mammals.

Influenza A/H1N1 A/Tomsk/13/2010 virus registered in Siberia in 2010 proved to be an extremely pathogenic strain. Dynamic study of the topology of this influenza virus strain in the lungs, liver, kidneys, lymph nodes, and great vessels of infected mice was carried out. Influenza A virus was detected by immunohistochemical methods in cells of different histogenesis in all the studied organs throughout the observation period (days 1-30 postinfection), which indicated effective replication and long persistence of influenza A/H1N1 A/Tomsk/13/2010 virus in mammalian cells.

Effects of preventive administration of oxidized dextran on liver injury and reparative regeneration in mice infected with influenza A/H5N1 virus.

Intranasal infection of outbred male mice with influenza A/H5N1 A/goose/Krasnoozerskoye/627/05 virus led to high (85%) mortality of animals. Morphological studies of liver specimens showed destructive changes in the parenchyma (93.5% hepatocytes), caused by long persistence of the virus in the liver. The virus persistence was conjugated with activation of cellular immunity, manifesting by an increase in the counts of cells with high expression of proinflammatory cytokines (TNF-α) and lysosomal enzymes (lysozyme, cathepsin D). Injections of oxidized dextran 3 and 1 days before infection reduced mortality and 2-fold attenuated destructive changes in the liver, presumably due to prevention of virus penetration into the target cells, modulation of immune reactions, and stimulation of reparative plastic processes.

Experimental study of the efficiency of oxidized dextran for prevention of influenza A/H5N1.

Oxidized dextran is suggested for prevention of infection induced by influenza A/H5N1 viruses, methods of its use and doses are determined. Two intravenous injections of dextran 3 and 1 days before experimental infection of outbred mice by influenza A/H5N1 A/goose/Krasnoozerskoye/627/05 virus resulted in a high preventive dose-dependent effect: the mean lifespan was 25% prolonged, the mortality decreased 3-fold.

Study of fibrotic complications and hydroxyproline content in mouse liver at different stages of generalized BCG-induced granulomatosis.

Generalized BCG-induced granulomatous was simulated in BALB/c male mice. The number of tuberculous granulomas in the liver and their size as well as the number of hepatocytes showing vacuolar degeneration increased from day 3 to 180 postinfection. Necrotic changes in hepatocytes were most pronounced at the acute phase of inflammation (days 3 to 30). Proliferative processes in the liver parenchyma in the experimental group were less marked than in the control. Increased content of collagen fibers in the liver was determined by excessive collagen synthesis in necrotic areas as well as increased amount of granulomas and fibroblasts. Enhanced proliferative and fibroplastic activity of fibroblasts in granulomas and liver parenchyma was evidently determined by activated granuloma macrophages. These shifts determined changes in the liver content of hydroxyproline during the acute and chronic periods of the disease.

Fibrogenesis in granulomas and lung interstitium in tuberculous inflammation in mice.

The study in mouse model of BCG-induced granulomatous inflammation showed that early pulmonary fibrosis (day 3-30 postinfection) in tuberculous inflammation was primarily determined by increased number of fibroblasts in the lung interstitium and granulomas and enhanced fibroplastic activity. Fibroplastic processes are initiated via an increase in secretory activity of activated granuloma macrophages caused by the persistence of the pathogen in the cells of the mononuclear phagocytic system. The dynamics of hydroxyproline concentration under these conditions is determined by changes in the number and differentiation degree of fibroblasts in granulomas and lung interstitium at various stages of tuberculous inflammation.

Expression of profibrotic growth factors and their receptors by mouse lung macrophages and fibroblasts under conditions of acute viral inflammation in influenza A/H5N1 virus.

Morphological signs of early interstitial fibrosis, developing under conditions of acute viral inflammation (postinfection days 1-14), were observed in C57Bl/6 mice infected with influenza A/H5N1 A/goose/Krasnoozerskoye/627/05 virus. The development of fibrosis was confirmed by an increase in the number of lung cells expressing TNF-α. These changes were recorded in the presence of a many-fold increase in the counts of macrophages and fibroblasts expressing FGF, EGF, and their receptors.

Role of matrix metalloproteinases and their inhibitor in the development of early pulmonary fibrosis in mice infected with influenza A/H5N1 A/goose/Krasnoozerskoye/627/05 virus.

High levels of macrophages and fibroblasts expressing MMP-2, MMP-9, and MMP-10 against the background of progressing early fibrosis of the lungs (manifesting in an increase in volume density of type I, III, IV, and VI collagens) were found in C57Bl/6 mice infected with influenza A/H5N1 A/goose/Krasnoozerskoye/627/05 virus. Progressing fibrosis of the lungs in infected mice was associated with imbalance of collagen synthesis and degradation processes conjugated with high levels of macrophages and fibroblasts expressing TIMP-2.

Morphofunctional changes in the lung vascular endotheliocytes in mice with influenza A/H5N1 A/Goose/Krasnoozerskoye/627/05.

The lung vessels of male C57Bl/6 mice were studied by immunohistochemical and stereometric methods on days 1, 3, 6, and 10 after intranasal infection with influenza A/H5N1 A/Goose/Krasnoozerskoye/627/05 virus. Influenza virus replicates in mouse lung vascular endotheliocytes and persists in these cells until the beginning of convalescence (day 10 after infection). This indicates high pathogenic activity of this strain. Active proliferation and apoptosis of endotheliocytes are detected early after infection; the counts of endotheliocytes expressing lysosomal hydrolases and NO-synthases increase many-fold.

Hydroxyproline content and fibrogenesis in mouse liver and lungs during the early stages of BCG granulomatosis.

In generalized BCG granulomatosis, fibrosis starts early (on day 3) and not only around the granulomas, but also in the organs. The severity of organ fibrosis is apparently determined by the concentration of granulomas, in particular their macrophages inducing proliferation of fibroblasts in organs and granulomas as well as activation of fibrogenesis. On day 30 after infection, the degree of fibrosis in the lungs was by 6 times higher than in the liver. The increase in hydroxyproline concentration in organs in early period of infection was determined by acute stress, while on day 30 it resulted from its enhanced synthesis by granuloma fibroblasts and resident fibroblasts in organs.

Morphofunctional characteristics of fibroblasts and fibrogenesis in the lungs of mice infected with influenza A/H5N1 A/goose/Krasnoozerskoye/627/05 virus.

Mice C57Bl/6 were intranasally infected with influenza A/H5N1 A/goose/Krasnoozerskoye/627/05 virus. The expression of influenza A virus antigen in the lung interstitium fibroblasts was recorded during all periods of the study (on days 1-14 after infection), the maximum expression on days 1 and 3. On day 1, the volume density of collagen fibers in the lungs was 3-fold higher than in intact animals; by day 14 it increased almost 15-fold. The numerical density of PCNA(+)fibroblasts increased almost 2-fold from day 1 to day 10; "fibroplastic activity" of fibroblasts increased more than 3-fold.

Structural and functional changes in pulmonary macrophages and lungs of mice infected with influenza virus A/H5N1 A/goose/Krasnoozerskoye/627/05.

C57Bl/6 mice were intranasally infected with influenza virus A/H5N1 A/goose/Krasnoozerskoye/627/05. The mortality rate of animals reached 70% on day 14 of the disease. The lungs of animals were characterized by necroses, destruction of vessels, hemorrhagic and thrombotic complications, edematous syndrome, and early fibrosis of the interstitium. On days 6-10 after infection, fibrosis was found in the zones of postnecrotic inflammatory infiltration. The expression of lysozyme and myeloperoxidase by pulmonary macrophages was initially increased, but decreased on day 10 of the study. The number of cathepsin D-expressing macrophages was elevated up to the 10th day of examination.

Morphological changes in the brain of mice with systemic candidiasis treated with composition of amphotericin B and oxidized dextran.

We observed morphological manifestation of encephalitis 3, 7, 10 and 28 days after intravenous infection of adult male CBA mice with Candida albicans. Compounds were administered intraperitoneally every other day starting from the next day postinfection. Untreated animals (100%) died over the period between days 18 and 20 postinfection; 60% animals receiving oxidized dextran alone survived by day 28 of observation. All animals treated with amphotericin B and composition of amphotericin B and oxidized dextran survived. On day 3 postinfection, the count of macrophage infiltrates and granulomas in the cerebral interstitium of mice treated with amphotericin B was equal to that in untreated mice, but was sufficiently lower in animals treated with the composition or oxidized dextran alone. On day 10, this index was similar in all groups and was approximately 5 times lower than in untreated animals on day 3. On day 28, macrophage infiltrates and granulomas were absent in the brain of all treated mice. These data suggest that oxidized dextran produced a therapeutic effect, which manifested earlier than the effect of amphotericin B and potentiated its effect, probably due to its competition with Candida albicans for mannose receptors on the brain-blood barrier endothelium.

Structural and functional peculiarities of mast cells in undifferentiated connective tissue dysplasia.

We performed an immunomorphological study of mast cells from undamaged skin in women with phenotypical evidence of undifferentiated connective tissue dysplasia syndrome, patients of cosmetological clinics. It was found that the numerical density of mast cells containing chymase granules in this condition 1.7-fold surpassed the corresponding parameter in patients without signs of connective tissue dysplasia syndrome, which probably was a result of compensatory and adaptive reaction aimed at activation of the synthesis of the connective tissue extracellular matrix components. It was hypothesized that increased content of chymase-positive mast cells in the skin of patients with connective tissue dysplasia syndrome contributed to the formation of associated arterial hypertension.

Preventive efficacy of oxidized dextran and pathomorphological processes in mouse lungs in avian influenza A/H5N1.

Oxidized dextran (60 kDa) exerted a pronounced preventive effect in laboratory mice infected with avian influenza subtype H5N1 A/Goose/Krasnoozerskoye/627/05 virus, which manifested in a significant increase in mouse lifetime (by 24.4%) and a decrease in mortality rate (3.3-fold). This was probably related to significant alleviation of pathological changes in the lungs and severity of hemodynamic and inflammatory complications and early fibrosis [corrected].