[Enrichment of extracellular DNA from the cultivation medium of human peripheral blood mononuclears with genomic CpG rich fragments results in increased cell production of IL-6 and TNF-a via activation of the NF-kB signaling pathway]. / Obogashchenie vnekletochnoi DNK sredy kul'tivirovaniia mononuklearov perifericheskoi krovi cheloveka CpG-bogatymi fragmentami genoma privodit k uvelicheniiu produktsii kletkami IL-6 i TNF-a putem aktivatsii signal'nogo puti NF-kB.

Previously, it was found that blood plasma extracellular DNA (ecDNA) of patients with rheumatoid arthritis (RA) is enriched with CpG-rich genomic DNA fragments, which contain TLR9 ligands (Veiko et al., 2006). In this study we have demonstrated that ecDNA of a RA patient and model fragments added to a cultivation medium of peripheral blood mononuclear cells (PBMC) of healthy donors stimulate expression of genes for the TLR9-MyD88-NF-kB signaling pathway; this leads to a significant increase in concentrations of the proinflammatory cytokines IL-6 and TNF-a in the cultivation medium. Human genomic DNA non-enriched with the CpG sequences did not stimulate IL-6 and TNF-a synthesis in PBMC. A scheme explaining the potential role ecDNA in the induction and maintenance of increased levels of the proinflammatory cytokines under conditions damaging the human cells has been proposed.

[CpG-DNA inhibits cell reactions accompanied with the development of the adaptive response in human lymphocytes after low-dose X-ray exposure].

The lymphocytes of peripheral blood of healthy donors were influenced by X-ray radiation (10 cGy) or a fragments of the transcribed region of rDNA (TRrDNA) transmitted to the incubation medium of non-irradiated cells. Both factors induced transposition of the loci 1q12 of homologous chromosomes from the membrane to the centre of the nucleus in lymphocytes; produced the activation of the genes TLR9 and MyD88 expression, the chromosomal nucleolus-forming regions, TNF-alpha and caspase-3; and also increased nuclease activity and synthesis RNA of the cells. However all the investigated reaction in the cells did not developed during the synergetic radiation and TRrDNA but the activity level of the cytokine TNF-alpha was increasing. The reactions of human lymphocytes on the induced influence are discussed herein.

Peripheral blood serum from healthy donors contains antibodies against the fragment of transcribed region of ribosomal repeat.

Enzyme immunoassay showed that blood serum from healthy donors contains specific high-affinity antibodies (apparent association constant > or = 5 x 10(9) M(-1)) against a fragment of transcribed region of ribosomal DNA repeat of human serum, which are present in a free form or are bound to extracellular DNA. Preheating of the serum at 55 degrees C and high ionic strength (1.5 M NaCl) had no effect on the interaction of antibodies with this fragment. Competitive binding assay showed that these antibodies recognize DNA epitopes, which differ from the epitopes recognized by most anti-DNA antibodies in systemic lupus erythematosus.

[Stress signaling between human lymphocytes after induction of bystander effect by exposure to ionizing radiation in adaptive doses].

We previously reported that the consequence of human lymphocytes irradiation by the adaptive doses (X-rays, 10 cGy) was a transposition of the homologous chromosome loci in the cell nucleus (FISH method); this phenomenon was mediated by the increase of nucleolus activity. They both are transmited to non-irradiated cells by the bystander effect (BE). We shown that the reaction of stress signaling is induced by the DNA fragments of irradiated lymphocytes. The study shows that after the inhibition of caspase 3 activity in irradiating lymphocytes or the blockade TLR9 in bystander cells the transposition was not observed. A signaling way of BE from irradiated lymphocytes apoptosis to bystander cells receptors is discussing.

[Conservative treatment of Peyronie's disease in the light of new pathogenetic data].

The study of pathogenetic mechanisms of Peyronie's disease (PD) in respect of raising efficacy of complex conservative therapy was made in 41 patients with PD. The control group consisted of 266 persons. Immunological and virusological investigations were made using enzyme immunoassay. HLA typing of class I antigens was made, antinuclear antibodies and antibodies to DNA were investigated. Effects of intron A and verapamil on proliferation of penile tunica albuginea were studied in vitro. Conservative therapy was given to 32 patients with acute PD. Patients with PD were found to carry chronic infection with type II herpes significantly more frequently. There was a significant association of PD with HLA-antigen B8 with high percentage of diagnostic titers of antinuclear antibodies. In vitro effect of intron A and verapamil was found to be dose- and time-dependent. Verapamil has a narrow range of dose-dependence and cytotoxicity in high concentrations. Combined treatment raised the proportion of good results while that of satisfactory outcomes decreased. Viral infection may be involved in pathogenesis of PD. This infection may alter mechanisms of immune regulation and start of autoimmune process in predisposed patients. Combination of magnetolaser therapy with intron A injections is an effective method of acute PD treatment. The addition of specific antiviral therapy raises treatment efficacy by action on one of pathogenetic mechanisms of the disease.

Stimulatory effect of fragments from transcribed region of ribosomal repeat on human peripheral blood lymphocytes.

Fragments from the transcribed region of the ribosomal repeat include considerable amounts of unmethylated CpG DNA motifs. These motifs activate immune cells via the interaction with Toll receptors. In vitro experiments confirmed the stimulatory effect of transcribed region of ribosomal repeat on human lymphocytes. Culturing of lymphocytes in a medium containing 2-20,000 ng/ml fragments from transcribed region of ribosomal repeat was accompanied by structural changes in the nucleus in a considerable number of cells. These changes manifested in translocation of pericentromeric heterochromatin from the membrane to the center of the nucleus and activation of the nucleolus and were accompanied by a significant increase in interleukin-6 production and slight stimulation of tumor necrosis factor-a synthesis. The transcribed region of the ribosomal repeat and E. coli DNA had various effects on quantitative parameters of lymphocytes. Our results suggest the existence of mechanisms of stimulation not mediated by the interaction of CpG DNA motifs with Toll receptors.

[Antiphospholipid syndrome in the structure of hematogenic thrombophilia in young and middle-aged patients with venous thrombosis].

AIM: To specify detectability and clinical presentation of antiphospholipid syndrome (APS) in young and middle aged patients with phlebothromboses (PT). MATERIAL AND METHODS: Enzyme immunoassays for lupus anticoagulant, PCR determination of G1691A mutation in the gene of coagulation factor V, mutation G20210A in prothrombin gene, mutation C677T in methylenetetrahydrofolate reductase (MTHFR) gene were made in 97 patients (57 males and 40 females) with venous thrombosis as well as estimation of external and internal coagulation, antithrombin III activity, protein C activity, plasma fibrinogen, stimulated platelet aggregation, blood and plasma viscosity. RESULTS: APS was detected in 20.6% young and middle-aged patients with venous thrombosis, in 18.5% of them--primary APS. In APS patients acquired risk factors of thrombosis occurred significantly less frequently than in patients with venous thrombosis free of APS (30 and 70%, respectively). Recurrent pulmonary artery thromboembolism (TEPA) prevailed in APS patients. In patients with combined hemostatic disturbances (APS+mutation) TEPA was diagnosed more frequently than in APS patients and in the absence of markers of genetic thrombophilia. Plasma viscosity is most important diagnostically among rheological indices.

Comparison of cytotoxicity of aminoglycoside antibiotics using a panel cellular biotest system.

The cytotoxicity of four aminoglycoside antibiotics was studied by estimation of the dose-effect relationship using a panel cellular biotest system including cell cultures for test objects. The cultures represented 4 differentiation types: normal human fibroblasts and myoblasts, human or Syrian hamster hepatoma cells, and mouse/mouse hybridoma cells. It was found that three widely used antibiotics gentamicin, kanamycin, and neomycin exhibit similar, but not identical cytotoxicity parameters and differ distinctly from geneticin. Hence, the proposed panel biotest system helps to quantitatively evaluate and differentiate the effects of bioactive substances with similar chemical structure.

[Transfection of eucaryotic cells using cytochrome CYP450 2B6 gene: sensitivity to cyclophosphamide]. / Transfektsiia éukarioticheskikh kletok genom tsitokhroma CYP450 2B6: poiavlenie chuvstvitel'nosti k tsiklofosfamidu.

Sensitivity of 293 human epithelial kidney cells transfected by human cytochrome CYP450 gene to cyclophosphamide was investigated. Transfection was carried out by plasmid DNA containing CYP2B6 gene complexed with cationic liposomes. Liposomes were prepared from mixture of cationic lipids and cholesterol at different molar ratios. Experimental protocol included the following steps: transfection of epithelial kidney cells by complexes of plasmid DNA-cationic liposomes, clone selection in the medium with antibiotic Geneticin G418, selected clone harvesting and their treatment by cyclophosphamide as following cytotoxicity evaluation. It was shown that addition of 0.25 mM of cyclophosphamide resulted in death of 40-45% transfected cell population.

[Prevention of postoperative scars in otorhinolaryngology: experimental rationale]. / Eksperimental'noe obosnovanie profilaktiki posleoperatsionnykh rubtsovykh protsessov v otorinolaringologii.

It is demonstrated that in vitro inhibition of fibroblasts proliferation is feasible with the use of 5-fluorouracil. In stimulated growth of fibroblasts, the antiproliferative effect of 5-fluorouracil manifests even in a minimal concentration (2 mcg/ml). Recovery of fibroblast proliferative activity after elimination of 5-fluorouracil occurs in concentration under 63 mcg/ml.

Improvement of nutrient absorption may enhance systemic oxidative stress in cystic fibrosis patients.

BACKGROUND: The life expectancy of patients with cystic fibrosis (CF) is largely dependent on the pulmonary disease severity and progress. Malnutrition may be an important complicating factor in active and chronic lung disease. AIMS: The focus of this study was to investigate several inflammatory markers in pancreatic-insufficient CF patients with different enzyme treatment regimens. METHODS: CF patients with pancreatic insufficiency were examined at a time of symptomatic exacerbation of their lung disease. Group A (n = 11) regularly received microspheric enzymes. Group B (n = 8) were treated with enzymes during the hospitalization period only and demonstrated the presence of malnutrition. Inflammatory markers in the sputa (neutrophil elastase activity, interleukin-8 and tumour necrosis factor-alpha levels) and in the peripheral blood (plasma malondialdehyde (MDA), lymphocyte response to PHA, and the cell sensitivity to steroid suppression) have been investigated. RESULTS: During acute lung exacerbation, group B demonstrated reduced levels of lymphocyte proliferation. This parameter was normalized after combined antibiotic and pancreatic enzyme therapy. Simultaneously, plasma MDA in group B markedly increased following treatment. For this group, a significant positive linear association between values of plasma MDA and lymphocyte proliferation has been observed. For group A, neither the same correlation nor changes in MDA levels and lymphocyte proliferation have been found. CONCLUSIONS: Our data indicate that acute lung exacerbation in malnourished CF patients may be associated with alteration in T-lymphocyte activity. Adequate therapy normalizes lymphocyte function but results in systemic oxidative stress.

[Prevention of postoperative scar processes in otorhinolaryngology experimental rationale]. / Eksperimental'noe obosnovanie profilaktiki posleoperatsionnykh rubtsovykh protsessov v otorinolaringologii.

It is shown in vitro that fibroblast proliferation may be inhibited by 5-fluorouracil (5-Fu). In stimulated growth of the fibroblasts the 5-Fu antiproliferative effect takes place even in minimal concentration (2 mcg/ml). Fibroblastic proliferative activity recovers after elimination of 5-Fu from the medium at concentration under 63 mcg/ml.

Effect of semisynthetic analog of alpha(1)-acid glycoprotein on immunomodulatory and antiinflammatory activity of natural glycoprotein.

Pseudo-alpha(1)-acid glycoprotein with carbohydrate chain ratio typical of native form was synthesized by a previously developed original technique of quantitative transfer of alpha(1)-acid glycoprotein carbohydrate chains to other polymeric carrier. Similarly to native glycoprotein, the semisynthetic analog inhibited lymphocyte proliferation and stimulated the production of antiinflammatory cytokines by peripheral blood mononuclear leukocytes. However, it possessed no antioxidant activity and did not inhibit complement activation by the alternative pathway. The role of carbohydrate and protein components of alpha(1)-acid glycoprotein molecule in the realization of its biological effects is discussed.

[Immunologic monitoring of patients with cystic fibrosis: value of different laboratory findings]. / Immunologicheskii monitoring bol'nykh mukovistsidozom, znachenie razlichnykh laboratornykh pokazatelei.

Cystic fibrosis (CF) is a common, serious, and frequently fatal autosomal recessive genetic disorder associated with the poor function of chloride channels. Chronic endobronchial inflammation and bacterial infection are main causes of morbidity and mortality due to CF. The study dealt with a relationship between progression and inflammation markers. Twenty one CF children with acute pulmonary exacerbation were examined. The signs of peripheral blood inflammation (responses of lymphocytes to PHA and their sensitivity to the antiproliferative effect of glucocorticoids) and in situ inflammation markers (sputum elastase activity, IL-8 and TNF-alpha, and protein concentrations in the same sputum specimens). These laboratory findings were used to calculate a "laboratory index" (LI). The clinical status of each patient was evaluated with a "clinical index" (CI), a parameter that includes respiratory secretion cultures, pulmonary function test results, nutritional status, and the presence of disease-related complications. There was a positive linear correlation between LI and CI. The presence of P. aeruginosa was strongly associated with the changes of inflammatory markers. CF patients with prolonged P. aeruginosa infection demonstrated extremely enhanced elastase activity and elevated amounts of sputum IL-8 and TNF-alpha as compared to uninfected subjects. The lung elastase activities, sputum protein contents, and TNF-alpha levels in individuals with short-term colonization were at or below those without P. aeruginosa infection. In patients with or without short-term colonization, the normalization of laboratory parameters was strongly related to evident clinical improvement. At the same time, antibiotic treatment failed to suppress an excessive inflammatory response in the lungs of patients with prolonged P. aeruginosa infection. The importance of individual inflammation markers is discussed in the paper.

[Increased proinflammatory cytokine production by human peripheral lymphocytes treated with glucocorticoids]. / Stimuliatsiia produktsii provospalitel'nykh tsitokinov mononuklearami perifericheskoi krovi cheloveka pod deistviem gliukokortikosteroidov.

Glucocorticosteroids (GC) are the true immunomodulators that can depress and enhance immune reactions. Hormone-activated GC receptors (GCR) change the transcription of many genes, resulting in modified immune responses. The direction of immunomodulation under which GC act depends on their level, the quantity and state of GCR, the amount of different cytokines and cytokine receptors and other immunoactive molecules. The modulation of proinflammatory (IL-1 beta, IL-6, TNF-alpha) cytokine production by human peripheral lymphocytes treated with a wide range of dexamethasone (10(-6)-10(-12) mol) in the serum-free culture medium was observed in the present study. Enhanced or suppressed cytokine release depends on GC doses, intermittent or continuous contact with the hormone and cell environment. The magnitude of changes in IL-1 beta and TNF-alpha production was similar (parallel stimulation or depression) while diminished TNF-alpha was observed simultaneously with enhanced IL-6 production and vice versa. The suppression of proinflammatory cytokine production by GC is well documented. The experimental conditions of increased cytokine release with dexamethasone in the serum-free culture medium can serve as a model of investigation in false results of steroid immunosuppression.

Inflammatory markers in cystic fibrosis patients with lung Pseudomonas aeruginosa infection.

Chronic endobronchial inflammation and bacterial infection are the main causes of morbidity and mortality in cystic fibrosis (CF), an autosomal recessive genetic disorder associated with improper function of chloride channels. Inflammation in CF lung is greatly amplified after Pseudomonas aeruginosa infection. In this study the relationship between P. aeruginosa status and inflammatory markers has been investigated. Seventeen CF children in acute lung exacerbation were examined. CF patients without P. aeruginosa infection were characterized by elevated activity of sputum elastase, reduced response of peripheral blood lymphocytes to PHA and significant resistance to the antiproliferative action of glucocorticoids. These parameters were normalized after antibiotic treatment. The patients with prolonged P. aeruginosa infection demonstrated extremely high levels of elastase activity and elevated amounts of sputum IL-8 and TNF-alpha. Although antibiotic treatment resulted in clinical improvement, it failed to suppress excessive immune response in the lung. The data indicate that CF patients with prolonged P. aeruginosa need the modified treatment, which should include immunomodulating drugs and protease inhibitors as well as antibacterial therapy.

Immunomodulating activities of a natural alpha1-acid glycoprotein and its carbohydrate chains attached to the protein-free polymer.

Immunomodulating effects of a neoglycoconjugate created on the basis of alpha1-acid glycoprotein (AGP) carbohydrate chains and synthetic protein-free carrier have been investigated. It was demonstrated that this pseudo-AGP suppressed PHA- or anti-CD3 antibody-induced lymphocyte proliferation in a dose-dependent manner. Pseudo-AGP revealed a similar antiproliferative effect as the natural AGP samples. Stimulation of the LPS-induced proinflammatory cytokine production by mononuclear cells treated with both natural and pseudo-AGP has been also demonstrated. These data show that carbohydrate chains of AGP play a crucial role in the studied biological effect realization.

[Plasma contents of cytokines (TNF-alpha, IL-1 beta, IL-6) and their clearance during continuous hemofiltration in patients with sepsis and multiple organ failure]. / Soderzhanie v plazme tsitokinov (TNF-alfa, IL-1beta, IL-6) i ikh klirens pri postoiannoi gemofil'tratsii u bol'nykh s sepsisom i poliorgannoi nedostatochnost'iu).

The total-systems inflammatory response was assessed in patients with sepsis and multiple organ failure by measuring plasma cytokines TNF-alpha, IL-1 beta, and IL-6 and their clearance and total elimination in the course of permanent hemofiltration (PHF). Sepsis and multiple organ failure were found to involve a stable circulation of numerous cytokines, their levels reaching the peaks in some cases. No correlation between the content of individual cytokines in the plasma were detected. Appreciable amounts of TNF-alpha and IL-1 beta were eliminated during PHF, their clearance being approximately 15 ml/min, whereas elimination of IL-6 was negligible. Hence, PHF affects the mediator component in the pathogenesis of sepsis and the multiple organ failure syndrome.

[Tumor necrosis factor-alpha kinetics during renal replacement therapy in patients with sepsis and multiple organ failure]. / Kinetika faktora nekroza opukholi al'fa pri zamestitel'noi pochechnoi terapii u patsientov sepsisom i s poliorgannoi nedostatochnost'iu.

Peptide TNF-alpha (tumor necrosis factor alpha) secreted by monocytes and resident macrophages is a key proinflammatory mediator. It can generate response systemic inflammation leading to shock and polyorganic insufficiency. Elimination of circulating TNF-alpha is pathogenetically perspective in respect to therapy of septic shock. Plasma level of TNF-alpha and its elimination with the filter/dialysate was traced in 23 patients with sepsis and polyorganic insufficiency receiving substitute renal therapy (continuous hemodiafiltration, intermittent hemodialysis) for acute renal failure. Sepsis and polyorganic insufficiency was associated with elevated plasma levels of TNF-alpha correlating in many cases with the disease severity. TNF-alpha was for the most part eliminated with the filter/dialysate. The degree of this elimination was related to the technique of blood perfusion and characteristics of the procedure.