Biomedical subjects

Anna Czarny

Publications and source records attributed to Anna Czarny.

9 recordsLinked to original sources

PubMed2006

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.

Amino Acid Sequence↗

PubMed2006

[Activation of nuclear factor kappaB (NF-kappaB), induction of proinflammatory cytokines in vitro and evaluation of biocompatibility of the carbonate ceramic 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.

Animals↗

PubMed2005

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.

Case-Control Studies↗

PubMed2005

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.

Animals↗

PubMed2005

[The effect of polyester vascular prostheses on synthesis TNF-alpha, IFN-gamma and NO by human peripheral blood leukocytes--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.

Biocompatible Materials↗

PubMed2004

Preferences of kanamycin A towards copper(II). Effect of the resulting complexes on immunological mediators production by human leukocytes.

The widespread presence of pathogenic bacteria is a cause of permanent demand for investigating the properties of antimicrobial agents. The chemical basis of several toxic effects induced by antibiotics still remains unclear. Aminoglycosides, highly ototoxic and nephrotoxic drugs, are capable of copper(II) ions chelating. In this study we established the affinity of kanamycin A towards copper(II), in contrast with other metal ions: iron(III), nickel(II), cobalt(II) and zinc(II) by means of potentiometry. Circular dichroism spectroscopy was applied to monitor the competition of copper(II) partition between kanamycin A and human serum albumin. We show, that the drug is able to digest Cu(II) ions from HSA to some extent and comparing the stability constants for metal and antibiotic with those, obtained for the N-terminal Asp-Ala-His-Lys (DAHK) sequence, which constitutes a copper(II) binding domain within albumin, we demonstrate that the Cu(II)-kanamycin A complex formation is possible also in blood plasma. Bioassays and immunoassay were used to find out the possibility of Cu(II)-kanamycin A complexes to induce cytokines: tumor necrosis factor (TNF), interferon (IFN) and interleukin-10 (IL-10) in human peripheral blood leukocytes. The effect on the cytokines release was dose and time dependent and the interdependence between IL-10 and TNF stimulation was found. We report that Cu(II)-aminoglycoside systems can act as moderate inducers of TNF-alpha, IFN-alpha/beta and IL-10 released from human leukocytes. We have also found that these complexes are non-toxic for human A549 cells.

Anti-Bacterial Agents↗

PubMed2003

[Comparative evaluation of TNF-alpha induction in vitro and in local tissue reaction after implantation of HAP/TCP and HAP].

The aim of this work was the evaluation of biocompatibility and bioactivity of HAP/TCP as compared to HAP. This evaluation was carried out by the in vitro assessment of the induction of TNF-alpha in the rat blood after the contact with the tested HAP. The anther target was to investigate of local muscle tissue reaction of rats bones reaction of rabbits, after the implantation of tested materials. The level of TNF-alpha was estimated by biological method in the supernatants of rat blood cell cultures stimulated in vitro by tested materials. The implantation assessment was carried out on 25 rats and 15 rabbits. The muscle tissue reaction was evaluated after 2, 4, 8, 12, and 24 weeks, while the reaction of bones after 4, 12, and 26 weeks since the implantation. The level of TNF-alpha in the blood after stimulation in vitro with HAP/TCP was on the same level as control group. The implantation of HAP the level of TNF-alpha was twice as high as compared to HAP/TCP and control groups. These differences were of statically importance. The muscle tissue reaction after implantation of HAP/TCP was characterized by moderate inflammation during the early period which lead to the formation of two layer connective tissue capsule. In the capsule wall there were great cells of foreign body type and macrophages. In the cytoplasm of some the cells particles of HAP were noted, what could be regarded as the proof of its resorption. In the bone tissue surrounded HAP/TCP implants the stronger osteoconductive proprieties were observed as compared to the tissue around HAP.

Animals↗

PubMed2003

The ability of Candida spp. strains to induce production of tumor necrosis factor and interleukin-6 by whole blood cells.

The aim of this study was to investigate the ability of C. albicans, C. glabrata, C. kefyr, C. krusei, and C. parapsilosis to induce production of TNF and IL-6 by whole blood cells of healthy subjects. The highest secretion of TNF and IL-6 was obtained with the 10(7) cells mL(-1) and 10(5) mL(-1) heat killed Candida cells, respectively, for all the species of Candida tested. C. albicans was a better stimulator of TNF and C. glabrata was a better stimulator of IL-6 than other Candida spp. C. krusei was the weakest inductor of IL-6 production.

Candida↗

PubMedDate not supplied

Antimicrobial activity of N-alkoxycarbonylmethyl-N-alkyl-piperidinium chlorides.

The aim of the study was to assay antibacterial and antifungal activity of newly synthesised N-alkoxycarbonylmethyl-N-alkyl-piperidinium chlorides. The compounds tested were found to inhibit the growth of some Gram-negative bacteria, Gram-positive strains and some representatives of yeast-type Candida. From microbiological experiments two of the compounds tested, N-dodecyloxycarbonylmethyl-N-methyl-piperidinium chloride (3) and N-dodecyl-N-ethoxycarbonylmethyl-piperidinium chloride (6), emerged as more active than the other compounds. Since the resistance of biofilms to biocides should be noted during the design and testing of new antimicrobial agents therefore, we have analysed antibacterial properties of the most active compounds towards biofilms. Our study focused on strains of Pseudomonas aeruginosa and Staphylococcus aureus that served as main model organisms for the biofilm studies.

Anti-Infective Agents↗