The Hydrophobic Stabilization of Pseudomonas aeruginosa Bacteriophage F8 and the Influence of Modified Bacteriophage Preparation on Biofilm Degradation.

The bacteriophage F8 belongs to the Myoviridae group of phages and is a pathogen of Pseudomonas aeruginosa. Since Pseudomonas aeruginosa is a multidrug-resistant opportunistic bacterium and can cause serious challenges for health services, studying the potential use of phages against them is a promising approach. Pseudomonas aeruginosa can be found on medical devices because bacteria can attach to surfaces and develop biofilms, which are difficult to eradicate because of their high resistance to environmental conditions and antimicrobial therapeutics. Phage therapy is becoming promising as an alternative for the treatment of antibiotic-resistant infections, but there is still a lack of standardized protocols approved by health organizations for possible use in the clinic. In our research, we focused on the potential use of 1-octanol, which was previously used by our team to develop a method for phage purification from bacterial lysate. 1-octanol is a fatty alcohol that is mostly used in the cosmetics industry, and its advantage is that it is approved by the FDA as a food additive. In this paper, we studied the protective properties of the addition of 1-octanol for storing phage liquid preparations. We demonstrated the stabilization effect of 1-octanol addition on F8 bacteriophage preparation during storage under various conditions. Interestingly, more effective biofilm reduction was observed after treatment with the purified bacteriophage and with 1-octanol addition compared to crude lysate.

Chemical Characteristics of Ethanol and Water Extracts of Black Alder (Alnus glutinosa L.) Acorns and Their Antibacterial, Anti-Fungal and Antitumor Properties.

The aim of this study was to identify polyphenolic compounds contained in ethanol and water extracts of black alder (Alnus glutinosa L.) acorns and evaluate their anti-cancer and antimicrobial effects. The significant anti-cancer potential on the human skin epidermoid carcinoma cell line A431 and the human epithelial cell line A549 derived from lung carcinoma tissue was observed. Aqueous and ethanolic extracts of alder acorns inhibited the growth of mainly Gram-positive microorganisms (Staphylococcus aureus, Bacillus subtilis, Streptococcus mutans) and yeast-like fungi (Candida albicans, Candida glabrata), as well as Gram-negative (Escherichia coli, Citrobacter freundii, Proteus mirabilis, Pseudomonas aeruginosa) strains. The identification of polyphenols was carried out using an ACQUITY UPLC-PDA-MS system. The extracts were composed of 29 compounds belonging to phenolic acids, flavonols, ellagitannins and ellagic acid derivatives. Ellagitannins were identified as the predominant phenolics in ethanol and aqueous extract (2171.90 and 1593.13 mg/100 g DM, respectively) The results may explain the use of A. glutinosa extracts in folk medicine.

New Insights on the Feature and Function of Tail Tubular Protein B and Tail Fiber Protein of the Lytic Bacteriophage φYeO3-12 Specific for Yersinia enterocolitica Serotype O:3.

For the first time, we are introducing TTPBgp12 and TFPgp17 as new members of the tail tubular proteins B (TTPB) and tail fiber proteins (TFP) family, respectively. These proteins originate from Yersinia enterocolitica phage φYeO3-12. It was originally thought that these were structural proteins. However, our results show that they also inhibit bacterial growth and biofilm formation. According to the bioinformatic analysis, TTPBgp12 is functionally and structurally similar to the TTP of Enterobacteria phage T7 and adopts a ß-structure. TFPgp17 contains an intramolecular chaperone domain at its C-terminal end. The N-terminus of TFPgp17 is similar to other representatives of the TFP family. Interestingly, the predicted 3D structure of TFPgp17 is similar to other bacterial S-layer proteins. Based on the thermal unfolding experiment, TTPBgp12 seems to be a two-domain protein that aggregates in the presence of sugars such as maltose and N-acetylglucosamine (GlcNAc). These sugars cause two unfolding events to transition into one global event. TFPgp17 is a one-domain protein. Maltose and GlcNAc decrease the aggregation temperature of TFPgp17, while the presence of N-acetylgalactosamine (GalNAc) increases the temperature of its aggregation. The thermal unfolding analysis of the concentration gradient of TTPBgp12 and TFPgp17 indicates that with decreasing concentrations, both proteins increase in stability. However, a decrease in the protein concentration also causes an increase in its aggregation, for both TTPBgp12 and TFPgp17.

Identification of a potent inhibitor of type II secretion system from Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen infecting human population. The pathogen is becoming a serious health problem due to its ability to evade normal immune response of the host and multiple drug resistance to many antibiotics. The pathogen has 2 major virulence systems of which the type III secretion system (T3SS) is of major concern to humans. A third system, type 2 secretion system (T2SS), is common to bacteria and used to secrete exotoxin A (ExoA) responsible for human cell destruction. To help bypass the drug resistance, a strategy to block the T2SS based on a low similarity between human ATPases and the essential ATPases of the T3SS and T2SS of P. aeruginosa, was used. An in silico-optimized inhibitor of T3SS, made directly from the computer-optimized of previously published compounds and their combinatorial libraries, showed IC50 = 1.3 ±â€¯0.2 µM in the T2SS ExoA secretion blocking test. The compound was non-toxic to human lung epithelial cell line A549 and could block cellular destruction of those cells in a cell infection model at 200 µM for at least 24 h. The compound could be a lead candidate for the development of T2SS virulence blockers of Pseudomonas aeruginosa.

Identification of small molecule compounds active against Staphylococcus aureus and Proteus mirabilis.

Staphylococcus aureus is a human pathogen rapidly becoming a serious health problem due to ease of acquiring antibiotic resistance. To help identify potential new drug candidates effective against the pathogen, a small focused library was screened for inhibition of bacterial growth against several pathogens, including S. aureus. At least one of the compounds, Compound 10, was capable of blocking bacterial growth of S. aureus in a test tube with IC50 = 140 ±â€¯30 µM. Another inhibitor, Compound 7, was bacteriostatic against S. aureus with IC50 ranging from 33 to 150 µM against 3 different strains. However, only Compound 7 was bactericidal against P. mirabilis as examined by electron microscopy. Human cell line toxicity studies suggested that both compounds had small effect on cell growth at 100 µM concentration as examined by MTT assay. Analysis of compounds' structures showed lack of similarity to any known antibiotics and bacteriostatics, potentially offering the inhibitors as an alternative to existing solutions in controlling bacterial infections for selected pathogens.

Biological Properties of Low-Toxicity PLGA and PLGA/PHB Fibrous Nanocomposite Implants for Osseous Tissue Regeneration. Part I: Evaluation of Potential Biotoxicity.

In response to the demand for new implant materials characterized by high biocompatibility and bioresorption, two prototypes of fibrous nanocomposite implants for osseous tissue regeneration made of a newly developed blend of poly(l-lactide-co-glycolide) (PLGA) and syntheticpoly([R,S]-3-hydroxybutyrate), PLGA/PHB, have been developed and fabricated. Afibre-forming copolymer of glycolide and l-lactide (PLGA) was obtained by a unique method of synthesis carried out in blocksusing Zr(AcAc)4 as an initiator. The prototypes of the implants are composed of three layers of PLGA or PLGA/PHB, nonwoven fabrics with a pore structure designed to provide the best conditions for the cell proliferation. The bioactivity of the proposed implants has been imparted by introducing a hydroxyapatite material and IGF1, a growth factor. The developed prototypes of implants have been subjected to a set of in vitro and in vivobiocompatibility tests: in vitro cytotoxic effect, in vitro genotoxicity and systemic toxicity. Rabbitsshowed no signs of negative reactionafter implantation of the experimental implant prototypes.

Synthesis and antimicrobial properties of 4-acylaminobenzenethiosulfoacid S-esters.

A series of esters of 4-acetyl, 4-trifluoroacetyl- and 4-(3-chloropropionyl)aminobenzenethiosulfoacids (twenty-four compounds) were synthesized and characterized by elemental analysis, 1H NMR and IR spectroscopy. The antibacterial activity of the novel candidates has been screened using the agar diffusion or serial dilution methods against representative Gram-positive (Staphylococcus aureus, Bacillus subtilis, Bacillus mesentericus, Mycobacterium sp., Mycobacterium luteum), Gram-negative (Aeromonas sp., Burkholderia cepacia, Alcaligenes faecalis, Pseudomonas aeruginosa, Escherichia coli, Proteus vulgaris) bacteria and fungi (Candida albicans, Candida tenuis, Candida glabrata, Verticillium dahliae, Trichophyton gypseum, Aspergillus niger, Aspergillus fumigatus, Penicillium chrysogenum). Particular potency has been discovered against all tested pathogenic bacteria and fungi by compounds 1l and 3l at nanomolar concentrations. Some appropriate effect of thiosulfoesters structure upon their antimicrobial activity was determined.

Activity of JAK/STAT and NF-kB in patients with axial spondyloarthritis.

BACKGROUND: The etiology of axial spondyloarthritis (axSpA) is not fully elucidated. Research continues in determining the mechanisms responsible for initiation of the disease process, its maintenance and development. OBJECTIVES: The aim of this study was to evaluate the expression of transcription factors STAT (signal transducer and activator of transcription) and NF-κB (nuclear factor kappa B) as well as Janus kinase3 (JAK3) in the peripheral blood leukocytes. We also analyzed the connection between the degree of activation of transcription factors and the disease activity. MATERIAL/METHODS: The study involved 46 patients with axSpA and 19 healthy individuals who comprised the control group. The expression of NF-κB, STAT1, STAT3, STAT4, STAT5, STAT6, and JAK3 in peripheral blood leukocytes was assessed. To determine the degree of activation of transcription factors STAT-s and NF-κB and JAK3 kinase, the immunocytochemistry method was used. For location of the factors, the primary monoclonal anti-NF-κB, anti-JAK3 and polyclonal anti-STAT-s antibodies were used (Chemicon International, USA, Abcam, Cambridge, UK), and the set of antibodies Novocastain Super ABC Kit (Novocastra, UK). RESULTS: Expression of STAT1, STAT3, STAT4, STAT5, STAT6, NF-κB and JAK3 was statistically higher in the group of patients with axSpA than in the control group. There was a positive correlation with ESR values and expression of STAT4. There was no correlation between STAT, NF-κB, and JAK3 expression and ASDAS, BASDAI, and BASFI. Nine patients were treated with TNF-α inhibitors. The expression of NF-κB and STAT6 was higher in the group treated with TNF-α inhibitors, even though disease activity in these patients was shown to be lower than in those not receiving such treatment (ASDAS = 1.34±0.51 vs. 3.52±0.90, BASDAI = 2.34±1.92 vs. 5.51±2.41). CONCLUSIONS: In the group of patients with axSpA compared with the control group, higher expression of the transcription factors STAT and NF-κB as well as JAK3 was observed. Due to its crucial roles in inflammation and autoimmunity, STAT4 may have promise as an effective therapeutic target for axSpA.

Antimicrobial properties of substituted quino[3,2-b]benzo[1,4]thiazines.

Our previous studies demonstrated that among phenothiazines several derivatives could be found showing strong antiproliferative actions and the property of inhibiting inducible tumor necrosis factor alpha (TNF a) production in human blood cultures. The aim of this investigation was to determine potential antimicrobial actions of forty four new phenothiazine derivatives with the quinobenzothiazine structure. The compounds showed differential antibacterial and antifungal activities against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans depending on the compound structures, concentrations and bacterial strains. More specifically, 6-(1-methyl- 2-piperidylethyl) quinobenzothiazine displayed strongest actions against S. aureus and E. coli whereas 6-methanesulfonylaminobutyl-9-methylthioquinobenzothiazine exhibited the most universal antimicrobial properties. The correlation between antimicrobial activity and the chemical structure of quinobenzothiazines was discussed.

Comparison of microbiological and physicochemical methods for enumeration of microorganisms.

Determination of the number of cultured bacteria is essential for scientific and industrial practice. A spread plate technique is the most common and accurate method for counting of microorganisms. However, time consuming incubation does not allow for a quick estimation of the number of bacteria in a growing culture. In the present study, the results of photometric measurements: direct optical density method (OD at 585 nm), UV absorbance at 260 and/or 280 nm of separated and lysed bacteria by sodium hydroxide and surfactant with the spread plate technique were compared. The linear regression model for bacterial strains Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli was used to compare these three methods. The UV measurement method enabled determination of the number of bacteria with similar precision. The procedure for solubilized bacteria UV measurement is robust, and is not influenced by dispersions in the original culture medium.

Antioxidant and Protective Effects of the Polyphenolic Glycoconjugate from Agrimonia eupatoria L. Herb in the Prevention of Inflammation in Human Cells.

Herein, structural and biological studies of a complex biopolymer (polyphenolic glycoconjugate) isolated from the flowering parts of Agrimonia eupatoria L. (AE) are presented. Spectroscopic analyses (UV-Vis and 1H NMR) of the aglycone component of AE confirmed that it consists mainly of aromatic and aliphatic structures characteristic of polyphenols. AE showed significant free radical elimination activity, i.e., ABTS+ and DPPH·, and was an effective copper reducing agent in the CUPRAC test, eventually proving that AE is a powerful antioxidant. AE was nontoxic to human lung adenocarcinoma cells (A549) and mouse fibroblasts (L929) and was nongenotoxic to S. typhimurium bacterial strains TA98 and TA100. Moreover, AE did not induce the release of proinflammatory cytokines such as interleukin 6 (IL-6) and tumor necrosis factor (TNF-α) by human pulmonary vein (HPVE-26) endothelial cells or human peripheral blood mononuclear cells (PBMCs). These findings correlated with the low activation of the transcription factor NF-κB in these cells, which plays an important role in the regulation of the expression of genes responsible for inflammatory mediator synthesis. The AE properties described here suggest that it may be useful for protecting cells from the adverse consequences of oxidative stress and could be valuable as a biomaterial for surface functionalization.

Evaluation of Antimicrobial Efficacy of UVC Radiation, Gaseous Ozone, and Liquid Chemicals Used for Disinfection of Silicone Dental Impression Materials.

Effective disinfection of dental impressions is an indispensable requirement for the safety of dental personnel and patients. The ideal method should be not only effective but also convenient, cheap, and environmentally friendly. This study aimed to reliably evaluate the efficacy of ultraviolet C (UVC) radiation, gaseous ozone, and commercial liquid chemicals used for silicone dental impressions disinfection. These methods were applied to two types of elastomeric impression materials: condensation silicones and addition silicones of various consistency (putty, medium, and light). The antimicrobial effectiveness against Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans was evaluated in vitro by counting colony-forming units (CFU) on the surface of samples. The one-way ANOVA with a Tukey HSD test or the Kruskal-Wallis with a Dunn's test was performed. The results obtained revealed the efficacy of the proposed methods for disinfection of both C-silicones and A-silicones in most of the studied groups. Only one material (Panasil initial contact Light) was not effectively disinfected after UVC irradiation or ozone application. In conclusion, the potential of each disinfection method should be evaluated separately for each material. Moreover, in further research, the possible influence of the proposed methods on the physical properties of the impression materials should be thoroughly investigated.

Cytotoxicity and Antibacterial Activity of Mineral Trioxide Aggregate Cement with Radiopacity Introduced by ZrO2.

The article presents the results of in vitro studies on cytotoxicity and antibacterial activity of new MTA-type cements, developed on the basis of the sintered tricalcium silicate enriched with ZnO, along with an agent introducing the radiopacity in the form of ZrO2. The new materials have been developed to ensure that their physical and chemical properties are suited for endodontic applications. The cements were evaluated via characterisation of setting time, compressive strength, as well as translucency on X-ray images, and bioactivity in the simulated body fluid (SBF). The µCT was used to test the influence of the ZrO2 grains in the powder component on the microstructure of the produced cement. Then, the cytotoxic action of the cements was evaluated by applying a reference L-929 cell line. The conditions of the culture upon contact with the tested materials or with extracts from the cements were assessed using image analysis or an MTT colorimetric assay. Two strains of streptococci, Streptococcus mutans and Streptococcus sanguinis, were used to study the antibacterial activity of the tested cements with ZrO2 acting as the agent introducing the radiopacity. The new cements are characterised by appropriate properties as far as retrograde root canal filling is concerned.

Functional and structural characterization of Spl proteases from Staphylococcus aureus.

Staphylococcus aureus is the major cause of nosocomial infections world-wide, with increasing prevalence of community-acquired diseases. The recent dramatic increase in multi-antibiotic resistance, including resistance to the last-resort drug, vancomycin, together with the lack of an effective vaccine highlight the need for better understanding of S.aureus pathogenicity. Comparative analysis of available bacterial genomes allows for the identification of previously uncharacterized S.aureus genes with potential roles in pathogenicity. A good example is a cluster of six serine protease-like (spl) genes encompassed in one operon, which encode for putative proteases with similarity to staphylococcal glutamylendopeptidase (V8 protease). Here, we describe an efficient expression system for the production of recombinant SplB and SplC proteases in Escherichia coli, together with structural and functional characterization of the purified enzymes. A unique mechanism of cytoplasm protection against activity of misdirected SplB was uncovered. Apparently, the co-translated signal peptide maintains protease latency until it is cleaved by the signal peptidase during protein secretion. Furthermore, the crystal structure of the SplC protease revealed a fold resembling that of the V8 protease and epidermolytic toxins. Arrangement of the active site cleft and substrate-binding pocket of SplC explains the mechanism of enzyme latency and suggests that some Spl proteases possess restricted substrate specificity similar to that of the V8 protease and epidermolytic toxins.

[Activation of nuclear factor kappaB (NF-kappaB), induction of proinflammatory cytokines in vitro and evaluation of biocompatibility of the carbonate ceramic in vivo]. / Aktywacja jadrowego czynnika transkrypcyjnego NF-kappaB, indukcja cytokin prozapalnych in vitro oraz badania biozgodnosci ceramiki weglanowej in vivo.

UNLABELLED: Ceramic materials based on calcium carbonate have been prepared in response for the demand for resorbable materials for use in bone surgery. Calcium carbonate in the form of crystalline aragonite or calcite with various amount of lithium fluoride was the raw material. Material CC-1FA from crystalline aragonite (99% CaCO3 and 1% LiF), CC-5FA - from crystalline aragonite (95% CaCO3 and 5% LiF) and CC-1FK -material from crystalline calcite (99% CaCO3 and 1% LiF) was studied. To evaluate their biocompatibility and inflammatory effect we investigated the activity of nuclear factor kappaB (NF-kappaB) and proinflammatory cytokine concentrations: tumor necrosis factor TNF-alpha, interleukins 11-6 and IL-8 in peripheral human leukocytes (PBL) after stimulation in vitro with tested calcite materials. Evaluation of local soft tissue reaction after implantation was also the aim of our study. Proinflammatory cytokines take part in inflammatory reaction caused by biomaterials. Expression of these proteins is controlled by proinflammatory regulatory transcription factors including the commonly appearing NF-kappaB (Nuclear Factor kappaB). In a quiescent cell NF-kappaB resides in the cytosol in an inactive form which its activated under the influence of kinases. The activated NF-kappaB protein translocates from cytosol to nucleus of cell and binding to specific DNA sequence it initiates transcriptions. Thanks to the quick regulation of immunological response it ensures the survival of cells in unfavorable reactions of environmental factors. It regulates the expression of many genes mainly connected with the course of the inflammatory process (of some cytokines, proteins of acute phase, collagenasis, stromilozine other enzymes decomposing the elements of matrix) and with proliferation and differentiating of cells. However its excessive activity can lead to unfavorable reactions, for example uncontrollable division of cells, appearance of giant cells of foreign body type. In our study protein expression of NF-kappaB in PBL were assessed using anti-c-Rel-antibody (PBL expressing c-Rel in the nucleus = labelled NF-kappaB (+) cells). The NF-kappaB activation in PBL was expressed as: the percentage of NF-kappaB (+) cells. The level of cytokines: IL-6, IL-8 and TNF-alpha level in the supernatants from leukocytes culture with tested materials was determined by an enzyme-linked immunoabsorbent assay (ELISA) after 24 and 72 hours incubation. The local tissue reaction in vivo was evaluated 1 and 3 months after implantation calcite devices into dorsal muscles of rats. The achieved results showed that the tested calcite material incubated 24 and 72 hours with PBL culture didn't synthesized higher amounts of the cytokines IL-6 and IL-8 in comparison with the untreated cells. All tested materials stimulated after 24 and 72 hours PBL culture to produce a significant level of TNF-alpha (p < 0.05) (higher level after CC-5FA and CC-1FA materials and lower after CC-1FK). Activation of transcription factor nuclear factor NF-kappaB in leukocytes after 24 hours incubation with CC-5FA, CC-1FA was higher than CC-1FK but significantly was only after CC-5FA stimulating. The expression of NF-kappaB after 72 hours of incubation decreased and was on the level comparable with control group. Histological observation 1 month after implantation showed that the carbonate ceramic devices were surrounded with connective tissue surrounding implants capsule with presence of macrophages. Additionally after 3 month in the dense connective tissue cartilaginous and calcification focus was visible. Soft tissue reaction showed chronic inflammatory reaction connected with resorbtion of biomaterials, the most increased after implantation of CC-5FA, than CC-1FA and CC-1FK. CONCLUSION: Transcription nuclear factor NF-kappaB was activated after in vitro stimulation in short time by the tested calcite material CC-5FA. This activity was correlated with induction in vitro of TNF-alpha in short and long time. The most increased soft tissue reaction 1 and 3 month after implantation of CC-5FA material was found. Our study showed that calcite materials can activate NF-kappaB and demonstrated differences in biocompatibility of tested materials.

The Activity of JAK/STAT and NF-κB in Patients with Rheumatoid Arthritis.

BACKGROUND: Research is still being conducted in order to determine the mechanisms responsible for the initiation of rheumatoid arthritis (RA) as well as for its persistence and progression. OBJECTIVES: The aim of this work was to establish the expression of the signal transducer and activator of transcription (STAT) transcription factors and the nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) transcription factor in peripheral blood leukocytes and synovial fluid cells. The correlations between the activation level of the transcription factors and the activity of the disease were also analyzed. MATERIAL AND METHODS: In total, the study included 34 RA patients and 19 healthy individuals as controls. The expression of NFκB, STAT1, STAT3, STAT4, STAT5 and STAT6 in peripheral blood leukocytes and synovial fluid cells was established. The immunocytochemistry method was used to determine the degree of activation of STAT and NF-κB transcription factors. For the location of the factors, primary polyclonal anti-STATs and monoclonal anti-NF-κB antibodies were used. RESULTS: The expression of STAT1, STAT3, STAT4, STAT5, STAT6 and NFκB was significantly higher in the group of RA patients than in the controls. No statistically significant differences were found between the expression of STATs in peripheral blood leukocytes and synovial fluid cells. CONCLUSIONS: In comparison with the control group, the expression of the STAT and NFκB transcription factors in RA patients was higher, which may be helpful in better understanding the etiopathogenesis of the disease in the future, and may potentially have important therapeutic implications.

Activation of the NF-kappaB system in peripheral blood leukocytes from patients with chronic heart failure.

AIM: To evaluate the activation of transcriptional nuclear factor kappa-B (NF-kappaB) in peripheral blood leukocytes (PBL) from patients with chronic heart failure (CHF). In vitro experiments were used to elucidate the role of lipopolysaccharide (LPS) as a stimulus for the NF-kappaB system in PBL. METHODS AND RESULTS: We examined 46 CHF patients (age: 62+/-1 years, LVEF: 31+/-1%, NYHA class: 2.7+/-0.1), 11 coronary artery disease (CAD) patients without CHF, and 13 healthy young subjects. The immunocytochemical localisation of NF-kappaB in PBL was assessed using a polyclonal rabbit IgG anti-c-Rel-subunit antibody. NF-kappaB activation was expressed as the percentage of PBL nuclei stained positively for c-Rel (NF-kappaB+cell). PBL from healthy controls were exposed in vitro to the following concentrations of LPS from Escherichia coli (strain O111:B4): 0.1, 10 and 5000 ng/mL. CHF patients demonstrated the highest NF-kappaB activation in PBL (NF-kappaB+cells [%]: 37.1+/-1.5) as compared to CAD patients (29.1+/-3.0%) and controls (12.6+/-1.5%) (all p<0.05). There were three main clinical determinants of NF-kappaB activation in PBL from CHF patients: peak oxygen consumption (r=0.53, p=0.025), presence of peripheral oedema (r=0.37, p<0.05) and serum C-reactive protein (r=0.40, p=0.02). In PBL from healthy subjects, LPS at all concentrations increased NF-kappaB activity towards the pattern detected in CHF. CONCLUSIONS: The NF-kappaB system is highly overactive in PBL from CHF patients. LPS at low concentrations in peripheral blood may be involved in NF-kappaB activation in PBL, and is a potential target for future therapeutic applications.

Major structural proteins of type 1 and type 3 Klebsiella fimbriae are effective protein carriers and immunogens in conjugates as revealed from their immunochemical characterization.

Fimbriae are filamentous structures present on the cell surface of many bacteria, including genus Klebsiella. The use of fimbriae as protein carriers in conjugates may allow to formulate effective multivalent vaccines and suitable diagnostics. However, the evidences have been reported that fimbriae may enhance the inflammatory response. This prompted us to examine the degree of cytokine induction by the type 1 and type 3 Klebsiella fimbriae and their conjugates. Fimbriae were assessed as carrier proteins for Escherichia coli K12 endotoxin core oligosaccharide. MALDI-MS revealed the molecular mass of fimbrial monomer major protein, which was 15,847 Da for type 1 and 18,574 Da for type 3 fimbriae of Klebsiella. These two types of fimbriae were moderate inductors of IL-6 and interferon and almost inactive with regard to the stimulation of TNF when tested in human whole blood assay. Coupling of fimbriae with E. coli K12 core oligosaccharide gave immunogenic conjugates with respect to a saccharide ligand and protein carrier, although only 10% of the pilin monomers possessed the attached oligosaccharide. Rabbit antiserum reacted with a broad spectrum of lipopolysaccharides, as measured by ELISA and immunoblotting assays. The antibodies against glycoconjugates were bactericidal for the wild, S-type bacteria of some species. Regarding the induction of cytokines by conjugates only the TNF level was noticeably elevated. These results prompt for the practical use of fimbriae, as effective protein carriers for conjugates to obtain broad-spectrum antisera for diagnostic applications.

[The effect of polyester vascular prostheses on synthesis TNF-alpha, IFN-gamma and NO by human peripheral blood leukocytes--in vitro]. / Wplyw poliestrowych protez naczyniowych na synteze TNF-alpha, IFN-gamma oraz NO przez ludzkie leukocyty krwi obwodowej--in vitro.

Vascular prostheses produced on the basis of polyester are today commonly used in vascular surgery. To improve their biofuncionality special technologies are used, among them double knit and hydrophile feature enrichment. These modifications could cause the local activation of leukocytes to produce the mediators of inflammatory reaction, which in turn leads to hyperplasia of endothelium and other dangerous complications. In our study we used two kinds of polyester prostheses: double velour knitted hydrophilic Dallon H and standard double velour knitted prosthesis Dallon as control. The aim of this work was to compare in vitro the levels of cytokines TNF-alpha, interferon (IFN) and nitric oxide (NO) found in the supernatants of human blood leukocyte cultures after stimulation by both these above-mentioned vascular prostheses materials which are designed for use in direct blood contact. Tested implant materials Dallon H had no influence on synthesis of production of IFN, TNF-alpha and NO by human blood leukocytes. These results allow to made the initial selection of biomaterials before their in vivo evaluation.