In order to find novel molecular markers of multiple sclerosis we developed a scheme of oligopeptides' isolation including their extraction from blood serum with 10 % trichloroacetic acid, followed by precipitation of soluble substances with acetone in ratio 6:1. Oligopeptides were dissolved in water and their characteristics was determined by gel filtration under HPLC conditions and thin layer chromatography. Obtained data have shown that blood serum of MS patients contains two oligopolypeptides with average molecular masses of 300-500 Da. We also studied biological activity of TCA-soluble peptides toward some eukaryotic and prokaryotic cells in comparison with phosphopeptides isolated from casein hydrolysates. It was found that TCA-soluble peptides are capable of effective inhibiting HeLa cells' proliferation, while their inhibitory effect was expressed toward Jurkat T-cells and was not detectable toward U373 cells. The casein's phosphopeptides were capable to stimulate proliferation of Jurkat cells and effectively inhibited growth of cells. Neither antibacterial, nor antifungal activities of these oligopeptides were detected.

The capacity of opportunistic bacteria for biofilm formation plays an important role in the development of chronic inflammatory processes, which are difficult to treat. To improve antimicrobial therapy methods, the influence of lactobacilli on the ultrastructure of biofilm-forming clinical strains of staphylococci when co-cultured was investigated. 5 biofilm-forming clinical strains of S. aureus from the skin of acne vulgaris patients (n = 24) were isolated. Using transmission electron microscopy (TEM) the morphological changes of S. aureus cells in the mixed culture with standard strains of Lactobacillus plantarum 8P-A3 and clinical strains of L. fermentum (n = 4) were studied. It was found that in 48 hours after the inoculation on the medium of samples of mixed cultures of L. plantarum 8P-A3 and S. aureus growth of staphylococci was not revealed. Only in some cases of mixed cultures of L. fermentum and biofilm-forming staphylococci was growth of S. aureus obtained. In electron diffraction patterns of control samples of 24-hour staphylococcal monocultures and 48-hour lactobacilli monocultures, natural development of the population at the cellular level was observed. Destructive changes under the influence of lactobacilli (probiotic and clinical strains) were detected in all ultrathin sections of the cells of biofilm-forming and planktonic staphylococci. Significant destructive changes in the cell wall of the staphylococci were observed: thickening, obtaining of irregular form, detachment of the cytoplasmic membrane, the complete destruction of the peptidoglycan layer and the emergence of "shadow cells". On all electron diffraction patterns fibrillar-threadlike structures of DNA could not be observed, but in some cases mesosome-like formations were poorly contrasted. It was established that the surface S-layer of lactobacilli was expressed on a significantly larger scale in the mixed culture with staphylococci. In mixed culture of clinical strains of lactobacilli with biofilm form of S. aureus, staphylococcal cells could be found in a dormant state. Thanks to an experimental model of biofilm in a mixed culture, the development of destructive changes of staphylococci under the influence of the lactobacilli both on the morphological and at the population levels has been assessed. The results obtained can be used in improving the schemes of complex antimicrobial therapy of pyoinflammatory processes with the use of biological preparations, which are composed of lactobacilli, including those in the form of local application.

Недостатня ефективність антимікробної терапії багатьох інфекційних запальних процесів нерідко обумовлена наявністю мікроорганізмів, які утворюють біоплівкові форми. Досліджено здатність до плівкоутворення клінічних штамів S. aureus і S. epidermіdis, виділених при вугровій хворобі (acne vulgaris), а також референтного штаму L. рlantarum 8P-A3. Для оцінки плівкоутворення використовували спектрофотометричне визначення оптичної щільності структури на дні пластикової чашки Петрі. Стан плівкової структури контролювали методом мікроскопування. Встановлено, що оптична щільність моновидової біоплівки, утвореної S. aureus і S. epidermіdis, становить 1,69±0,77 та 1,50±0,60 од. Відповідно, оптична щільність планктонної форми референтного штаму S. aureus ATCC 25923 (F-49) становила 0,09±0,06 од. Біоплівкова структура L. рlantarum 8P-A3 мала оптичну щільність 1,79±1,07 од. Використання люмінесцентної мікроскопії дало змогу оцінити частку життєздатних клітин у біоплівках: через 48 год у біоплівці зі змішаної культури L. рlantarum 8P-A3 та S. aureus немає життєздатних клітин стафілокока. Місцеве застосування препаратів лактобактерій сприятиме підвищенню ефективності лікування запальних процесів, спричинених біоплівковими формами стафілококів.Ключові слова: біоплівка, Lactobacillus, Staphylococcus aureus, Staphylococcus epidermіdis. ВСТУПОстанніми роками низьку ефективність протимікробної терапії багатьох хронічних захворювань пояснюють утворенням мікроорганізмами біоплівкових форм. Метаболічні процеси, що відбуваються у біоплівці, відрізняються від процесів у планктонних бактеріальних монокультурах [13,17].Біоплівка -ідеальне середовище для здійснення генетичних обмінів між окремими мікроорганізмами: тут підвищуються кон'югативна активність та інші види генетичних рекомбінацій, що сприяє швидкому поширенню серед представників популяції генів резистентності до антибіотиків і детермінант інших видів біологічної активності (здатності протистояти фагоцитозу, дії антитіл) [2,3]. Особливо складно Biol. Stud. 2015: 9(3-4); 89-98 • DOI: https://doi.org/10.30970/sbi.0903.458 www.http://publications.lnu.edu.ua/journals/index.php/biology 2. S. epidermіdis (n= 21) 1,50±0,60 Змішана культура 3. S. aureus + L. рlantarum 8P-A3 (n = 3) 1,82±0,82 4. S. epidermidis + L. рlantarum 8P-A3 (n = 3) 1,56±0,62 Контролі 5. S. aureus ATCC 25923 (F-49) 0,09±0,06* 6. L. рlantarum 8P-A3 1,79±1,07 Примітка: * Статистично достовірна різниця порівняно з показником контрольного штаму Р<0,05 Comment: * Тhe difference is statistically significant, compared to control group (Р<0.05)Для мікроскопічного дослідження плівкоутворення відібрано один із штамів S. aureus зі значною оптичною щільністю біоплівки (рис. 1).

The mechanisms of development of the inflammatory process of the pilosebaceous apparatus in patients with acnе vulgaris are not fully understood, and variations in bacterial colonization are one of the key elements of the inflammatory process. Under the pathological conditions caused by pus-forming cocci which induce the production of proinflammatory cytokines, there is an increase in arginase expression. The capacity for film formation in selected strains was determined by the cultural properties (increased viscosity of the colony biomass) and by differential interference contrast microscopy using a dark field condenser and fluorescence microscopy. Arginase activity (μmole urea/min•mg of protein) was determined spectrophotometrically at 520 nm on saponin-perimabilized lymphocytes of peripheral blood by the rate of urea formation. The cultures of film-forming and planktonic forms of Staphylococcus epidermidis and Staphylococcus aureus were isolated from purulent pustules of 44 patients, aged 18–30. 63.6% of clinical strains of film-forming staphylococci were isolated, out of which 15 strains (53.6%) were S. aureus and 13 strains (46.4%) S. epidermidias. It was found that the arginase activity in patients (film-forming S. aureus) was significantly higher than in practically healthy donors (control) and was equal to 0.262 ± 0.006 and 0.279 ± 0.005 (planktonic form of S. aureus) versus 0.087 ± 0.009 μmole urea / min∙mg of protein in the control. The arginase activity in patients (film-forming S. epidermidis) was significantly higher than in practically healthy donors and was equal to 0.281 ± 0.009 and 0.297 ± 0.006 (planktonic form of S. epidermidis) versus 0.087 ± 0.009 μmol urea / min∙mg of protein in the control. After the auto-vaccine therapy and the administration of the probiotic Lacidofil (Institut Rosell Inc., Canada), enzyme activity decreased significantly in both experiments, however it had not attained control levels. The enzyme activity decreased through the administration of a vaccine, which in turn has an immunomodulating and immunostimulating effect. In addition, comparing the data of the arginase activity after treatment in patients with S. epidermidis, there was a slight decrease in the enzyme activity. This result is probably due to the formed tolerance of the immune system to commensal microorganisms. It was found that all patients had a moderate dysbiosis, which was accompanied by a deficiency of the main normal symbionts of the intestine. After treatment, all patients experienced significant improvementst in the microbiocenosis of the intestine in the direction of normalization of parameters and improvement of the skin condition. Increase in arginase activity in patients with acne vulgaris indicates the competition of this enzyme with NO-synthases for the substrate L-arginine and the alteration of physiological reactions in the organism caused by staphylococci which induce the phagocytic response and the cytokines production of the humoral system.