INTRODUCTION: Inflammation is a common pathological condition. On the one hand, inflammation plays a protective role, without it dangerous local processes would remain unrecognized. On the other hand, a cytokine storm can develop under conditions of systemic inflammation, which, in turn, can lead to multiple organ failure. Therefore, it is important to understand the mechanisms involved in the regulation of cytokine production in the early stages of inflammation. OBJECTIVE: The aim of this study was to evaluate the immunomodulatory properties of human defensin (HNP-1) and porcine protegrin (PG-1) antimicrobial peptides in vivo under conditions of inflammatory process initiation. METHODS: Lipopolysaccharide was administered intraperitoneally to laboratory rats to initiate an inflammatory response. Following the onset of inflammation, antimicrobial peptides were also administered intraperitoneally. The immunomodulatory effect of the peptides was evaluated by measuring the changes in gene expression of toll-like receptor 4 (TLR4) and cytokines IL1β and IL10 in the blood leukocytes of the animals using real-time PCR. RESULTS: The lipopolysaccharide-induced expression of Il1b and Il10 in leukocytes, as well as the lipopolysaccharide-induced Tlr4 expression in whole blood leukocytes and isolated mononuclear cells, was reduced to varying degrees by the introduction of HNP-1 and PG-1. CONCLUSION: It can be assumed that at the early stage of the inflammatory process, defensins can act as moderate pro-inflammatory factors, suppressing the gene expression of the anti-inflammatory cytokine IL10, but at the same time slightly reducing the gene expression of the pro-inflammatory cytokine IL1β.

Lactoferrin is a multifunctional glycoprotein of the transferrin family with a molecular mass of about 80 kDa. We studied the effect of human lactoferrin on stress- and lipopolysaccharide-induced changes in blood corticosterone levels, as well as on the gene expression of the cytokine IL4 and the pattern-recognition receptor TLR4 in rat splenocytes. Stress in rats was modeled by swimming in cold water (1-4°C) for 2 min. Lactoferrin and lipopolysaccharide (LPS) were administered intraperitoneally before the stress exposure. Corticosterone level in the plasma was determined by enzyme immunoassay, and changes in gene expression were assessed by real-time polymerase chain reaction (PCR) with reverse transcription. We showed that preventive intraperitoneal administration of lactoferrin reduced the stress and LPS-induced increase in the gene expression of both IL4 and TLR4 in rat splenocytes but did not change the concentration of corticosterone in the blood.

BACKGROUND: Severe stress causes an array of dysfunctions in the immune, neuroendocrine, cardiovascular, digestive and other systems, resulting in an emergence of various types of pathology. Common manifestations of a chronic stress are the disorders in the gastrointestinal tract, such as irritable bowel syndrome, functional dyspepsia, biliary dyskinesia, dysbiosis, inflammatory processes that determine the development of gastritis and one of the most widespread post-stress pathologies of the gastrointestinal tract — stomach ulcers. The disclosure of the molecular mechanisms of a pathogenesis of diseases associated with gastrointestinal dysfunction related to chronic stress as well as a search for new ways to correct these disorders are important tasks of fundamental and clinical medicine. The present work is focused on evaluating a participation of molecular factors of the innate immunity in intestine, such as antimicrobial peptides secreted by intestinal epithelial cells upon infection, in a response to the chronic stress. AIM: The aim of the study was to estimate the gene expression of a number of antimicrobial peptides: intestinal α- and β-defensins of laboratory animals (rats) under chronic stress conditions. MATERIALS AND METHODS: Modeling of a chronic stress was performed by daily forced swimming of laboratory animals in cold water. An expression of α- and β-defensin genes was evaluated using a real-time polymerase chain reaction. RESULTS: We found an increase in the level of expression of the rat α-defensin-5 and β-defensin-3 genes in response to chronic stress, while the expression of β-defensin-2 gene was not changed compared to the control. CONCLUSIONS: Considering that changes in the concentration and spectrum of peptides with antibacterial activity, caused by prolonged stress, can contribute to modification of the composition of the intestinal microbiota, the data obtained can expand our understanding of the molecular basis of the pathogenesis of diseases associated with disorders in the composition of microbiota under stress.

The widespread resistance to antibiotics in pathogenic bacteria makes the development of a new generation of antimicrobials an urgent task. The development of new antibiotics must be accompanied by a comprehensive study of all of their biological activities in order to avoid adverse side-effects from their application. Some promising antibiotic prototypes derived from the structures of arenicins, antimicrobial peptides from the lugworm Arenicola marina, have been developed. Previously, we described the ability of natural arenicins -1 and -2 to modulate the human complement system activation in vitro. In this regard, it seems important to evaluate the effect of therapeutically promising arenicin analogues on complement activation. Here, we describe the complement-modulating activity of three such analogues, Ar-1[V8R], ALP1, and AA139. We found that the mode of action of Ar-1[V8R] and ALP1 on the complement was similar to that of natural arenicins, which can both activate and inhibit the complement, depending on the concentration. However, Ar-1[V8R] behaved predominantly as an inhibitor, showing only a moderate increase in C3a production in the alternative pathway model and no enhancement at all of the classical pathway of complement activation. In contrast, the action of ALP1 was characterized by a marked increase in the complement activation through the classical pathway in the concentration range of 2.5–20 μg/mL. At the same time, at higher concentrations (80–160 μg/mL), this peptide exhibited a complement inhibitory effect characteristic of the other arenicins. Peptide AA139, like other arenicins, exhibited an inhibitory effect on complement at a concentration of 160 μg/mL, but was much less pronounced. Overall, our results suggest that the effect on the complement system should be taken into account in the development of antibiotics based on arenicins.

The aim of present study was to investigate the hBD-3 gene expression in the surface epithelium of mucosa in ORL organs. We have studied a total of 210 mucosal samples, obtained at the most frequent surgical intervantions from 5 different anatomical functional areas: nose and paranasal sinuses, middle ear, nasopharynx, oropharynx, larynx. The inferior turbinate mucosa (1) and the normal middle nasal passage mucosa (2) served as controls. Estimation of hBD-3 and β-actin gene expression was performed by reverse transcription and realtime PCR. In the nasal and sino-nasal mucosa, only negligible expression levels were detected in 14.29-33.33% of samples, most often in the specimens from the middle nasal passage and ethmoid labyrinth polyps (53.84%), being absent in hypertrophic inferior turbinate. In the middle ear cavity, the frequency detection of the hBD-3 gene expression varied from 7.69% in the stapes superstructures mucosa to 53.85% of the mucosal samples in the presence of cholesteatoma. hBD-3 gene expression was detected in most tissue samples with high microbial contamination: palatine tonsils (100%); adenoid hypertrophy (84.62%); adenoids in hypertrophic states of adenoids and palatine tonsils (87.5%); laryngeal fibrous-vascular polyps (87.5%); other laryngeal pathology (77.78% of the samples). The highest levels of hBD-3 gene expression were found in laryngeal fibrous-vascular polyps. The findings presumed two functionally different types of immune response in mucosa of the ORL organs. In the anatomical-functional areas lined with ciliated epithelium (middle and inferior nasal passages, maxillary and ethmoid sinuses, middle ear), significantly lower frequencies (Fisher's exact test, p < 0.05 to p < 0.001) and levels (Mann-Whitney test, p < 0.05 to p < 0.001) of hBD-3 gene expression were detected, except of polyps of the middle nasal passage and ethmoid labyrinth, and mucosa of the tympanic cavity in cholesteatoma, which may be related to the nature of the pathological process. In the areas lined with squamous epithelium or a combination of squamous and ciliated epithelium, hBD-3 gene expression was detected almost everywhere and at significantly higher levels. In the context of chronic inflammation and infection-related diseases of the ORL organs, in addition to the direct microbicidal activity of hBD-3 as the first line of immune response, one may suggest peptide dysregulation and, even, pathogenetic effects of hBD-3, e.g., increased sensitivity to infections, pathological changes in the composition of the commensal bacteria, fibrous remodeling.

The review presents data on the history of the discovery of the first endogenous antibiotic compounds, on the contribution of Russian scientists, in particular, researchers from the Institute of Experimental Medicine (St. Petersburg), in the study of the structural and functional properties of antimicrobial proteins and peptides important molecular factors of innate immunity that can act as an alternative to conventional antibiotics in the fight to control pathogenic microorganisms.

Neutrophilic granulocytes are one of the key cellular factors of innate immunity. The review presents data on the morphology, migration and utilization of neutrophilic granulocytes, phagocytosis and degranulation processes, neutrophilic extracellular traps, plasticity of neutrophils, their role in systemic inflammatory reactions and regulation of adaptive immunity.

The effect of various structural variants of human lactoferrin on stress-induced changes in the corticosterone level in the blood in rats was studied. A model of combined emotional and physical stress - swimming in cold water (1-4 °C) for 2 minutes. The level of corticosterone in plasma was determined by enzyme immunoassay. We have previously found that preventive intraperitoneal administration of native lactoferrin reduces the stress-induced increase in corticosterone concentration in the blood of rats 30 minutes after stress. This work shows that transgenic human lactoferrin, which lacks four arginine residues at the N-terminus, does not have this effect. The obtained results allow us to conclude about the key role of N-terminal amino acid residues in the implementation of the corticostatic activity of lactoferrin.

Lactoferrin — multifunctional glycoprotein of the transferrin family with a molecular mass of about 80 kDa. The review presents data on the physicochemical properties and localization of the protein, on antimicrobial properties, antitumor and anti-inflammatory effects, participation in neuroendocrinoimmune interactions, and on possible mechanisms for the realization of its functional manifestations.

Aim. Study the functional properties of cationic antimicrobial peptides 0-defensins isolated from baboon Papio hamadryas blood leukocytes. Materials and methods. Baboon 0-defensins were extracted from leukocyte mass using ultrafiltration, preparative electrophoresis and reverse phase high performance liquid chromatography. The test microorganisms used were Gram-negative bacteria Escherichia coli, Gram-positive bacteria Listeria monocytogenes and Staphylococcus aureus, and fungi Candida albicans. Minimal inhibitory concentrations (MICs) were determined by testing serial dilutions of the test peptides by radial diffusion in agarose gel. Microbicidal action was evaluated by counting surviving colony forming units after incubation microorganisms with the peptides. The 0-defensins influence on E.coli membrane permeability was assessed using chromogenic markers o-nirofenil- β-D-galactopyranoside and nitrocefin. Results. The analysis of the 0-defensins antimicrobial properties showed that they produce antimicrobial activity against test microorganisms, exhibiting bactericidal and fungicidal effects at micromolar concentrations. We studied the influence of different environmental conditions (low and high ionic strength, blood serum in medium) for antimicrobial activity. It is shown that 0-defensins have the ability to increase the outer membran of E.coli permeability, however, in contrast to 0-defensins have no noticeable influence on the inner membrane permeability. Conclusion. Baboon 0-defensins isolated from blood leukocytes are effective antimicrobial agents with a broad spectrum of microbicidal action. 0-Defensins baboon unlike 0-defensins exhibit antimicrobial activity in environments with high ionic strength. 0-Defensins increase the outer membran of E.coli permeability but not the cytoplasmic membrane, suggesting that the mechanism of antimicrobial effect of these peptides other than 0-defensins.

The aim of this study was to investigate the TLR2 gene expression in the surface epithelium of the upper airway mucosa. 210 surgical samples of nasal and sinonasal mucosa (inferior turbinate mucosa, the mucosa of the middle nasal passage, polyps), tympanic and mastoid cavity and laryngeal mucosa, adenoids, palatine tonsils were investigated. Estimation of TLR2 and beta-actin gene expression by levels of the corresponding mRNA synthesis was performed by real-time PCR. The expression of TLR2 gene was detected in the all mucosal tissue specimens. The lowest levels of TLR2 gene expression was detected in the samples of adenoids and tonsillar epithelium (p < 0.01; Mann-Whitney test), laryngeal mucosa (p < 0.02). The highest levels of TLR2 gene expression was detected in sterile middle ear pathology — otosclerosis (p < 0.02). Probably, low levels of TLR2 gene expression in the surface epithelium of the highly colonized upper respiratory mucosa serve as one of the ways to protect against the development of excessive local inflammation.

© Коллектив авторов, 2016 Хронический риносинусит (ХРС) рассматривают как многофакторный и гетерогенный процесс с отклоняющимися от нормы реакциями иммунной защиты организма [1—3]. С ХРС часто ассоциированы системные и местные факторы: мукоцилиарные дисфункции, астма, аллергия [4], непереносимость аспирина, иммунодефициты, гастроэзофагеальная рефлюксная болезнь, анатомические вариации, формирование биопленок [5], остеиты. В патогенез ХРС вовлечены персистирующее воспаление слизистой оболочки и микробный дисбиоз, а также сниженное бактериальное разнообразие [3, 6, 7]. Эпителиальная поверхность носа и околоносовых пазух является областью активного взаимодействия между проникающими в дыхательные пути патогенами и молекулярно-клеточными факторами иммунной системы [6]. Эпителиальные клетки помимо функции физического барьера несут на себе Toll-подобные рецепторы (TLR), которые распозна-

To understand the emergence and evolutionary selection of the efficient mechanisms of innate immunity it is necessary to accumulate knowledge about the structural and functional properties of antimicrobial peptides in different animal species. The cationic antimicrobial peptides, α-defensins, were isolated from leukocytic extracts of the lower narrow-nosed monkey, hamadryas baboon Papio hamadryas, using ultrafiltration, preparative electrophoresis and reverse-phase highperformance liquid chromatography. Analysis of the antimicrobial properties of α-defensins showed that they display a wide spectrum of antimicrobial activity, comparable with that of human α-defensin HNP1, and exert bactericidal and fungicidal effects at micromolar concentrations. A study of the influence of different medium conditions on antimicrobial activity of α-defensins revealed that a higher ionic strength or the presence of blood serum leads to a marked decrease in antimicrobial activity of α-defensins, while pH has no appreciable effect on it. We found that hamadryas baboon α-defensins are able to increase the permeability of the outer and inner membranes of E. coli, suggests that the bacterial membrane is one of the major targets of the antimicrobial effects of these peptides. The revealed differences in antimicrobial activity of α-defensins may result from their structural heterogeneity, which reflects different pathways of evolution of α-defensins in primates and underlies the selectivity of their antimicrobial effect.

Lactoferrin - multifunctional glycoprotein of the transferrin family with a molecular mass of about 80 kDa. We studied the effect of human lactoferrin on stress-induced changes in the level of corticosterone and adrenocorticotropic hormone (ACTH) and redistribution of leukocytes in the blood of rats. The used model of stress - swimming in cold water (1-4 °C) for 2 minutes. Corticosterone and ACTH levels in the plasma were determined by enzyme immunoassay. It has been established that preventive intraperitoneal administration of lactoferrin has reduced stress-induced increase in the concentration of corticosterone in 30 minutes after the stress and has normalized stress-inducing changes in the number of neutrophils in the blood after 30 minutes and 3 hours after exposure of stress, but has not affected the level of ACTH. The results of this study suggest that lactoferrin can act as an adaptogen during experimental stress.

We studied an action of the immunomodulator Glutoxim, lipopolysaccharide and N-formyl-methionyl-leucyl-phenylalanine on the degranulation of neutrophils, both isolated and in the composition of whole blood. It was established that Glutoxim stimulates the secretion of peptides and proteins from the granules of neutrophils. Glutoxim as well as lipopolysaccharide and N-formyl-methionyl-leucyl-phenylalanine demonstrate higher degranulation activity for neutrophils in whole blood, than for isolated neutrophils. It is shown that a longer incubation time of whole blood with Glutoxim and lipopolysaccharide is associated with decreased concentration of neutrophil peptides and proteins in the plasma, which may indicate that stimulation by Glutoxim and lipopolysaccharide, unlike stimulation by N-formyl-methionyl-leucyl-phenylalanine, can lead to activating of the receptors of blood cells and binding of secreted compounds by the cells. Secretory activity Glutoxim depends on phosphodiesterases, since this activity can be blocked by the addition of the phosphodiesterases inhibitor pentoxifylline.

Разработана и оптимизирована иммуноферментная тест-система для количественного определения дефенсина RNP-3 из нейтрофильных гранулоцитов крысы, одного из катионных антимикробных пептидов, являющихся ключевыми молекулами врожденного иммунитета. Дизайн разработанной тест-системы – «сэндвич» на основе поликлональных антител в качестве первичных антител и их конъюгатов с пероксидазой хрена, используемых в качестве детектирующих антител. Полученная система имеет чувствительность 0,1 нг/мл и диапазон измерений 0,1–8 нг/мл. Данная тест-система выявляет также дефенсин RNP-2 (с эффективностью 3 % от значений для RNP-3) и не выявляет дефенсины RNP-1 и RNP-4. Установлено, что среднее значение концентрации дефенсина RNP-3 в плазме крови у интактных животных составляет 36 ± 6 нг/мл (среднее ± ошибка среднего), а относительное содержание – 9,82 ± 3,02 нг /1 млн нейтрофилов.

Two cyclic theta-defensin peptides were isolated from leukocytes of the hamadryas baboon, Papio hamadryas, and purified to homogeneity by gel electrophoresis and reversed-phase high-performance liquid chromatography. Both peptides had high in vitro activity against Escherichia coli, Listeria monocytogenes, methicillin-resistant Staphylococcus aureus (MRSA) and Candida albicans. Here, we report their de novo sequencing by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). This was accomplished by combining conventional enzymatic digestion with N-terminal derivatization by 2-sulfobenzoic acid cyclic anhydride (SACA) or 4-sulfophenylisothiocyanate (SPITC) to facilitate the interpretation of fragment ion spectra. In addition to the two cyclic theta-defensins (PhTDs) we also sequenced a novel Papio hamadryas alpha-defensin, PhD-4, which showed high sequence homology to rhesus alpha-defensin RMAD-1 and human alpha-defensin HNP-1.