Andrzej Gamian

Effect of sepsis and cardiac surgery with cardiopulmonary bypass on plasma level of nitric oxide metabolites, neopterin, and procalcitonin: correlation with mortality and postoperative complications

Abstract Objectives: To examine the hypothesis that nitrite/nitrate, neopterin, and procalcitonin (PCT) levels can be useful predictors of sepsis-associated mortality and predictors of the postoperative complications after cardiopulmonary bypass (CPB). Design: Prospective clinical study. Setting: Intensive care unit of the Medical University Hospital. Patients: 41 patients with sepsis, 42 patients subjected to open heart surgery with CPB, and 30 healthy volunteers. Measurements and results: Nitrite/nitrate, neopterin, and PCT levels were measured in septic patients as soon as sepsis was recognized and then on the 2nd, 3rd, and 5th days of treatment. Statistically significant differences between survivors and nonsurvivors were found for neopterin and PCT. The area under receiver operating characteristic curve (AUC) for both parameters as predictors of mortality was above 0.8. The nitrite/nitrate level was also higher in nonsurvivors, but the AUC remained below 0.8, which indicates poor predictive power. The same parameters were measured in patients undergoing cardiac surgery before, during and after CPB establishment. The development of postoperative complications was correlated with increased postoperative neopterin and PCT levels. Additionally, neopterin was found as an early marker for the prognosis of postoperative complications, since patients who developed organ dysfunction had had elevated concentration of this parameter even before surgery (AUC 0.83). Measurement of NO metabolite levels was less specific and less sensitive. Conclusions: Our results confirm the value of PCT and neopterin measurement as diagnostic tools in monitoring the clinical course of patients in intensive care units.

Recognition of bacterial lipopolysaccharide using bacteriophage-adhesin-coated long-period gratings

In this paper we present a new type of highly sensitive label-free sensor based on long-period gratings (LPG) coated with T4 bacteriophage (phage) adhesin. The adhesin (gp37) binds Escherichia coli B (E. coli B) by recognizing its bacterial lipopolysaccharide (LPS). The LPG biofunctionalization methodology is based on coating LPG surface with nickel ions capable of gp37 histidine tag reversible binding. For the first time recombinant adhesive phage protein has been used as a receptor molecule in biosensing scheme. The specificity of LPS binding by adhesin has been tested with LPG-based device and confirmed using Western blot, Enzyme-Linked Immunosorbent Assay (ELISA) and BIACORE methods. The LPG-based sensor can measure bacterial contamination in real time and with a high accuracy. We show that T4 phage adhesin binds E. coli B LPS in its native or denatured form. The binding is highly specific and irreversible. The applied procedure allows for obtaining reusable biosensors.

Structural analysis of the Lactobacillus rhamnosus strain KL37C exopolysaccharide.

The exopolysaccharide from the lactic acid bacterium Lactobacillus rhamnosus strain KL37C isolated from human intestinal flora was prepared by sonication of bacterial cell mass suspended in water followed by centrifugation and cold ethanol precipitation of the supernatant. The polysaccharide material was purified by gel permeation chromatography on an TSK HW-50 column and characterised using chemical and enzymatic methods. On the basis of sugar and methylation analysis and 1H, 13C, 1D and 2D NMR spectroscopy the exopolysaccharide was shown to be composed of the following pentasaccharide repeating unit:-->3)-alpha-D-Glcp-(1-->2)-beta-D-Galf-(1-->6)-alpha-D-Galp-(1-->6)-alpha-D-Glcp-(1-->3)-beta-D-Galf-(1-->

Immunoregulatory potential of exopolysaccharide from Lactobacillus rhamnosus KL37. Effects on the production of inflammatory mediators by mouse macrophages

The ability to produce exopolysaccharides (EPS) is widespread among lactobacilli including Lactobacillus rhamnosus, the commonly used probiotic bacteria. Exopolysaccharides are a major component of the bacterial biofilm with a well‐documented impact on adherence of bacteria to host cells. However, their immunoregulatory properties are unknown. The aim of this study was to examine the immunostimulatory potential of EPS derived from L. rhamnosus KL37. We investigated the effect of EPS on the production of inflammatory mediators by mouse peritoneal macrophages and compared it with the effect of Lipopolysaccharide (LPS). Exopolysaccharides, at concentrations higher than those of LPS, stimulated production of both pro‐inflammatory (TNF‐α, IL‐6, IL‐12) and anti‐inflammatory (IL‐10) cytokines. Interestingly, analysis of the balance of TNF‐α/IL‐10 production showed a potential pro‐inflammatory effect of EPS. Furthermore, our data demonstrate that exposure of macrophages to LPS induced a state of hyporesponsiveness, as indicated by reduced production of TNF‐α after restimulation with either LPS or EPS (‘cross‐tolerance’). By contrast, EPS could make cells tolerant only to subsequent stimulation by the same stimulus. We also examined the relationship between TNF‐α production and activation of mitogen‐activated protein kinases (MAPKs) by EPS and LPS. Pretreatment of macrophages with specific inhibitors of p38 and ERK MAPKs reduced TNF‐α production induced by both stimuli to the same extent. In conclusion, these data demonstrate that EPS can effectively stimulate production of inflammatory mediators by macrophages in vitro. However, to predict whether EPS could be clinically useful as an immunomodulatory agent, further in vivo studies with highly purified EPS are necessary.

Novel Bacterial Polar Lipids Containing Ether-linked Alkyl Chains, the Structures and Biological Properties of the Four Major Glycolipids from Propionibacterium propionicum PCM 2431 (ATCC 14157T)

Propionibacterium propionicum belongs to the “acnes group” of propionibacteria, which is currently considered as clinically important because of its growing potential in infections, in particular with those connected with immune system dysfunctions. Propionibacteria are thought to be actinomycete-like microorganisms and may still cause diagnostic difficulties. The chloroform-methanol extracts of the cell mass ofP. propionicum (type strain) gave in TLC analysis the characteristic glycolipid profile containing four major glycolipids, labeled G1 through G4. These polar lipids were found to be useful chemotaxonomic markers to differentiate P. propionicum from other cutaneous propionibacteria, in particular from strains of the acnes group. Glycolipids G1–G4 were isolated and purified using gel-permeation chromatography, TLC, and high performance liquid chromatography, and their structures were elucidated by compositional and methylation analyses, specific chemical degradations, MALDI-TOF mass spectrometry, and 1H NMR and 13C NMR spectroscopy, including HMBC, TOCSY, HMQC, and NOESY experiments. Glycolipids G2 and G3 possess as backbone α-d-Glcp-(1 → 3)-α-d-Glcp-(1 → 1)-Gro (Gro, glycerol), in which position O-2 of the glycerol residue is acylated by a fatty acid (mainly C15:0) while O-3 is substituted by an alkyl ether chain. In glycolipid G3, an additional fatty acyl chain was linked to O-6 of the terminal glucose residue. Glycolipid G4 was structurally related to G2 but devoid of one glucose residue. Glycolipid G1 was isolated in small amounts, and its structure was therefore deduced from MALDI-TOF-MS experiments alone, which revealed that it possessed the structure of G2 but was lacking one fatty acid residue. In studies on the biological properties of P. propionicum glycolipids, the anti-P. propionicumrabbit antisera reacted in dot enzyme-immunoblotting test with G2 and G3. Glycolipid G3 was able to induce the delayed type of hypersensitivity. The results indicated that these novel ether linkage-containing polar glycolipids are immunogenic and possibly active in hypersensitivity, and thus, in pathogenesis.

The structure of the sialic acid-containing Escherichia coli O104 O-specific polysaccharide and its linkage to the core region in lipopolysaccharide

Mild acid hydrolysis of Escherichia coli O104 lipopolysaccharide released an O-specific polysaccharide, a tetrasaccharide repeating unit, the corresponding dimer, and a disaccharide fragment of the repeating unit. Complete and incomplete cores, and oligosaccharides comprising fragments of the repeating unit and the core region, were also obtained. On the basis of sugar and methylation analysis, FAB-mass spectrometry and NMR spectroscopy of the hydrolysis products, the repeating unit of the O-specific polysaccharide was shown to be the tetrasaccharide:-->4)-alpha-D-Galp-(1-->4)-alpha-Neup5,7,9Ac3++ +-(2-->3)-beta-D- Galp-(1-->3)-beta-D-GalpNAc (1-->. The linkage between the O-specific polysaccharide chain and the core region, which appeared to be of the R2 type, was established. These results indicate that N-acetylneuraminic acid, located in the O-specific polysaccharide, is an inherent lipopolysaccharide component.

Lactobacillus rhamnosus exopolysaccharide ameliorates arthritis induced by the systemic injection of collagen and lipopolysaccharide in DBA/1 mice

Oral administration of some probiotic bacteria (e.g. Lactobacillus rhamnosus) attenuates various types of experimental arthritis, including collagen-induced arthritis (CIA) and inhibits arthritogenic autoantibodies. Much less is known about the possible anti-arthritogenic properties of exopolysaccharide (EPS), the major component of lactic bacteria biofilm. In this study, we asked the question whether systemic administration of EPS derived from L. rhamnosus KL37 depresses the production of anti-collagen IgG and affects the development of CIA in DBA/1 mice. Arthritis was induced employing two models of active CIA, in which mice were immunized with type II collagen (CII) either in the presence of lipopolysaccharide (LPS; mild arthritis with moderate CII-specific IgG production) or with Complete Freund’s Adjuvant and LPS (severe arthritis with massive CII-specific IgG production). Passive CIA was induced by intravenous injection of CII-specific monoclonal antibodies and LPS. Disease progression, the incidence and severity of arthritis, were determined. Serum concentration of CII-specific IgG was measured by enzyme-linked immunosorbent assay. Systemic administration of EPS markedly reduced CII-specific antibody production. Moreover, EPS significantly ameliorated arthritis in the active models of CIA, especially, when LPS alone was used as an adjuvant. In contrast, when arthritogenic antibodies were injected to mice in high amounts, the effect of EPS on the development of passive CIA was negligible and transient. These results show that EPS can suppress active CIA by the inhibition of arthritogenic antibodies production. Therefore, we suggest that EPS or EPS-producing probiotics may be promising agents for the supporting therapy of patients with rheumatoid arthritis.

Distribution of 3-Hydroxy Fatty Acids in Tissues after Intraperitoneal Injection of Endotoxin

BACKGROUND 3-Hydroxy fatty acids (3-OH FAs) with 10- to 18-carbon chain lengths are constituents of the endotoxin [lipopolysaccharide (LPS)] of gram-negative bacteria. We investigated whether these FAs may be used as chemical markers in measuring endotoxin concentrations in mammalian tissue samples. METHODS We used gas-liquid chromatography-tandem mass spectrometry to measure 3-OH FAs in serum and tissues (heart, liver, and skeletal muscles) of rats after intraperitoneal injection of Escherichia coli LPS. One group of rats (group I) received a single LPS dose of 20 mg/kg of body weight; group II rats received the same total dose but over the course of 10 days (2 mg/kg each day). Rats receiving saline (group III) were used as controls. RESULTS 3-OH FAs with chain lengths of 10, 12, 14, 16, and 18 carbons were detected in all studied types of samples. Group I rats had 50-fold and group II rats had 3-fold higher serum concentrations of 3-hydoxytetradecanoic acid (3-OH 14:0, the predominant 3-OH FA of E. coli LPS) than group III rats. Concentrations of 3-OH 14:0 in livers from group I and II rats were similar and fourfold higher than in group III rats, whereas concentrations of the same acid in skeletal and heart tissues did not differ among the three groups of rats. 3-OH 14:0 dominated in heart and liver of group III rats, whereas 3-OH 16:0 (followed by 3-OH 14:0) dominated in skeletal muscles and blood. CONCLUSIONS 3-OH FAs 10-18 carbons in length, probably originating from endotoxin and mitochondrial beta-oxidation, are abundant in rat liver, skeletal muscles, and heart and can also be detected in blood. The widespread presence of these compounds in mammals limits their usefulness as LPS markers for endotoxin in clinical samples.

Protein Fraction of Barley Spent Grain as a New Simple Medium for Growth and Sporulation of Soil Actinobacteria.

A cheap value-added product, the protein fraction of barley spent grains is proposed as a source of a potential and economical cultivation medium. We showed that medium composed of protein fraction extract allows the isolation of actinobacteria, especially Streptomyces, from soil samples, and enhances the sporulation. It was used for the screening and production of the biologically active substances from actinobacteria.

Midkine, a multifunctional cytokine, in patients with severe sepsis and septic shock: a pilot study.

The objective of the study was to evaluate whether severe sepsis and septic shock are related to alterations in midkine concentrations, to identify disease-related factors associated with these alterations, and to initially appraise whether midkine might serve as a biomarker in sepsis. Prospective observational cross-sectional study with 5-day follow-up. Circulating midkine was measured (enzyme-linked immunosorbent assay) in 38 septic (13 with severe sepsis, 25 with septic shock), 82 active inflammatory bowel disease (IBD) (26 with systemic inflammatory response syndrome [SIRS]) patients, and 87 healthy subjects. Midkine significantly increased along with a sequence: health-inflammation (IBD)-systemic inflammation (IBD-SIRS)-severe sepsis/septic shock. High midkine levels (>1,000 ng/L) were found in 63% of septic and in 19% of IBD-SIRS patients, whereas extremely high concentrations (>5,000 ng/L) were found in 16% vs. 4%. Although not different at admission, midkine gradually decreased in severe sepsis and remained high in shock. Similarly, persistently high midkine was observed in patients with cardiovascular insufficiency (CVI) and in mechanically ventilated as compared with normalizing levels in patients without CVI and not requiring ventilation. The differences in devised simple rates (&Dgr;5th-1st) were significant in all these cases. Accordingly, admission midkine was higher in patients with metabolic acidosis. Concerning pathogen, gram-positive infections were associated with the highest midkine levels. In conclusion, sepsis and septic shock are associated with midkine elevation, substantially more pronounced than in inflammation, even systemic, revealing a new potential mediator of deregulation of neutrophil migration. Sepsis-related global hypoxia seems to contribute to midkine elevation. Our results substantiate further research on possible midkine application as a sepsis biomarker: in differentiating SIRS from sepsis and identifying gram-positive sepsis and septic patients at risk of CVI and shock.