OPTIMIZATION OF THE CULTIVATION CONDITIONS AND THE BASIC MOLECULAR TOOLS FOR ROSEOFLAVIN PRODUCER STREPTOMYCES DAVAWENSIS.

Roseoflavin (RoF), and its metabolic precursor 8-dimethylaminoriboflavin (AF), produced by Gram-positive bacteria Streptomyces davawensis and Streptomyces cinnabarinus, reveal a strong antibiotic effect against Staphyloccus aureus, Bacillus subtilis, Bacillus cereus, Micrococcus luteus and other Gram-positive bacteria and could be promising for developing the new effective antibacterial drugs. In this work, we present the optimization of basic molecular tools for S. davawensis including total DNA preparation, PCR amplification of target gene, cell transformation. In addition, the compositions of cultivation media were studied and the optimal cultivation conditions for increased of RoF production have been developed.

Expression of CCX CKR in pulmonary sarcoidosis.

OBJECTIVE: CCX CKR is a decoy chemokine receptor that specifically binds the chemokines CCL19, CCL25 and CCL21. CCL19 was previously found to be upregulated in pulmonary sarcoidosis. We have, therefore, investigated CCX CKR expression in this inflammatory disease. METHODS AND RESULTS: CCX CKR mRNA was semiquantitated using RT-PCR in unseparated bronchoalveolar (BAL) cells from sarcoidosis patients (S, n = 29) and healthy control subjects (C, n = 9). CCX CKR transcripts were upregulated in patients (mean +/- SEM); S, 0.82 +/- 0.10; C, 0.44 +/- 0.04; p = 0.01; this upregulation paralleled the disease course as assessed by the chest radiographic stage (p < 0.02). Immunocytochemistry localised the CCX CKR protein to ciliated bronchial cells. Flow cytometric fluorescent ligand uptake assay showed that these cells are able to internalize its ligand. CONCLUSION: These observations implicate CCX CKR in the modulation of the inflammatory response in sarcoidosis.

In vitro studies of activation of phagocytic cells by bioactive peptides.

The effect of formyl chemotactic peptide (fCTP, fMet-Leu-Phe), beta-amyloid peptides (beta-AP, 1-42, 1-16 and 25-35), and bradykinin (BK) on functional activity of phagocytic cells has been investigated. Wheat germ agglutinin (WGA) was also used as a model membrane binding agent of polypeptide nature. Murine monocyte-macrophage cell line J774.2 and normal human blood polymorphonuclear (PMN) cells were used as target phagocytic cells. Their activity was quantitatively estimated by measuring phagocytosis of killed yeast cells. Beta-AP (1-41) maximally stimulated phagocytosis at 0.1 microg/ml, BK--at 1.0 microg/ml, and fCTP--at 2.0 microg/ml. Beta-AP (1-16) and beta-AP (25-35) were inactive in used test-systems. Phagocytosis-inducing activity of beta-AP (1-42) and BK reached maximal levels in 2 h and decreased after 4-6 h of incubation. Phagocytosis numbers were compared with the indicators of phagocytic cell activation, such as absorption of neutral red dye, glucose utilization, production of super-oxide anion (NBT-test) and nitrite accumulation (indicator of NO production). NBT-test, which may be related to the killing ability of phagocytic cells towards the ingested objects, was positive only in stimulated PMN leukocytes, while the nitrite accumulation was detected only in stimulated macrophages. Nitrite accumulation in macrophages was markedly induced by lipopolysaccharide and to a lower extent by 0.5 microg/ml beta-AP (1-42). In high dose (5.0 microg/ml) beta-AP suppressed nitrite accumulation in macrophages stimulated by lipopolysaccharide. Other studied peptides were inactive in inducing nitrite accumulation. Transforming growth factor type beta suppressed phagocytic activity of PMN cells activated by beta-AP or WGA. The anti-inflammatory drugs (indomethacin and L-lysine aescinate) inhibited beta-AP (1-42)-induced phagocytosis. The interrelations between the regulatory pathways of BK, beta-AP and fCTP are discussed.

In vitro response of phagocytic cells to immunomodulating agents.

BACKGROUND: Phagocytes (polymorphonuclear cells and monocyte-macrophages) are the first line of defence of the host against infectious microorganisms and other foreign antigens. Agents which participate in activation of phagocytic cells possess a potential immunomodulating action. Thus, search for convenient in vitro test-systems and study of mechanisms of action of these agents are of practical interest. MATERIAL AND METHODS: Human blood polymorphonuclear (PMN) cells and murine macrophages (line J774.2) were used as cellular test-systems for study of phagocytosis-stimulating action of immunomodulating agents. Indexes of phagocytic activity were estimated by the phagocyte ingestion of yeast cells. NO-synthase activity, nitrite production, and nitroblue tetrazolium test were determined after phagocyte stimulation. RESULTS: It was revealed that indexes of phagocytic activity can be used as quantitative indicators for measurement immunomodulating activity. Zymosan A-induced phagocytosis in almost 100% PMN cells and macrophages and thus can be used as a positive control. Wheat germ agglutinin (WGA, 0.5-1.0 microg/ml) stimulated phagocytosis in PMN cells 1.8 times after 2-3 h incubation, although in higher concentrations (5-10 microg/ml) it strongly inhibited phagocytosis. TGF-b1 (10 ng/ml) suppressed phagocytosis in WGA-stimulated PMN cells. Mistletoe agglutinin-1 stimulated phagocytosis in PMN cells, although its effect in macrophages was weak, while concanavalin A stimulation of phagocytosis in macrophages was well expressed. Vasodilating peptide bradykinin increased phagocytosis 2.5 times in macrophages. We did not reveal changes in NO-synthase activity and nitrite production in macrophages and PMN cells activated by different immunomodulatig agents. Only lipopolysacharide stimulated such activity in macrophages. CONCLUSIONS: Cultured macrophages and PMN cells can provide reproducible quantitative results in screening phagocytic activity of different immunomodulating agents. Both positively and negatively acting immunomodulators might be studied using these test cells.