Synthetic antimicrobial and LPS-neutralising peptides suppress inflammatory and immune responses in skin cells and promote keratinocyte migration

The stagnation in the development of new antibiotics and the concomitant high increase of resistant bacteria emphasize the urgent need for new therapeutic options. Antimicrobial peptides are promising agents for the treatment of bacterial infections and recent studies indicate that Pep19-2.5, a synthetic anti-lipopolysaccharide (LPS) peptide (SALP), efficiently neutralises pathogenicity factors of Gram-negative (LPS) and Gram-positive (lipoprotein/-peptide, LP) bacteria and protects against sepsis. Here, we investigated the potential of Pep19-2.5 and the structurally related compound Pep19-4LF for their therapeutic application in bacterial skin infections. SALPs inhibited LP-induced phosphorylation of NF-κB p65 and p38 MAPK and reduced cytokine release and gene expression in primary human keratinocytes and dermal fibroblasts. In LPS-stimulated human monocyte-derived dendritic cells and Langerhans-like cells, the peptides blocked IL-6 secretion, downregulated expression of maturation markers and inhibited dendritic cell migration. Both SALPs showed a low cytotoxicity in all investigated cell types. Furthermore, SALPs markedly promoted cell migration via EGFR transactivation and ERK1/2 phosphorylation and accelerated artificial wound closure in keratinocytes. Peptide-induced keratinocyte migration was mediated by purinergic receptors and metalloproteases. In contrast, SALPs did not affect proliferation of keratinocytes. Conclusively, our data suggest a novel therapeutic target for the treatment of patients with acute and chronic skin infections

Physicochemical characterization of the endotoxins from Coxiella burnetii strain Priscilla in relation to their bioactivities

BACKGROUND: Coxiella burnetii is the etiological agent of Q fever found worldwide. The microorganism has like other Gram-negative bacteria a lipopolysaccharide (LPS, endotoxin) in its outer membrane, which is important for the pathogenicity of the bacteria. In order to understand the biological activity of LPS, a detailed physico-chemical analysis of LPS is of utmost importace. RESULTS: The lipid A moiety of LPS is tetraacylated and has longer (C-16) acyl chains than most other lipid A from enterobacterial strains. The two ester-linked 3-OH fatty acids found in the latter are lacking. The acyl chains of the C. burnetii endotoxins exhibit a broad melting range between 5 and 25°C for LPS and 10 and 40°C for lipid A. The lipid A moiety has a cubic inverted aggregate structure, and the inclination angle of the D-glucosamine disaccharide backbone plane of the lipid A part with respect to the membrane normal is around 40°. Furthermore, the endotoxins readily intercalate into phospholipid liposomes mediated by the lipopolysaccharide-binding protein (LBP). The endotoxin-induced tumor necrosis factor α (TNFα) production in human mononuclear cells is one order of magnitude lower than that found for endotoxins from enterobacterial strains, whereas the same activity as in the latter compounds is found in the clotting reaction of the Limulus amebocyte lysate assay. CONCLUSIONS: Despite a considerably different chemical primary structure of the C. burnetii lipid A in comparison with enterobacterial lipid A, the data can be well understood by applying the previously presented conformational concept of endotoxicity, a conical shape of the lipid A moiety of LPS and a sufficiently high inclination of the sugar backbone plane with respect to the membrane plane. Importantly, the role of the acyl chain fluidity in modulating endotoxicity now becomes more evident

Biophysical Mechanisms of the Neutralization of Endotoxins by Lipopolyamines

Endotoxins (lipopolysaccharides, LPS) are one of the strongest immunostimulators in nature, responsible for beneficial effects at low, and pathophysiological effects at high concentrations, the latter frequently leading to sepsis and septic shock associated with high mortality in critical care settings. There are no drugs specifically targeting the pathophysiology of sepsis, and new therapeutic agents are therefore urgently needed. The lipopolyamines are a novel class of small molecules designed to sequester and neutralize LPS. To understand the mechanisms underlying the binding and neutralization of LPS toxicity, we have performed detailed biophysical analyses of the interactions of LPS with candidate lipopolyamines which differ in their potencies of LPS neutralization. We examined gel-to-liquid crystalline phase behavior of LPS and of its supramolecular aggregate structures in the absence and presence of lipopolyamines, the ability of such compounds to incorporate into different membrane systems, and the thermodynamics of the LPS:lipopolyamine binding. We have found that the mechanisms which govern the inactivation process of LPS obey similar rules as found for other active endotoxin neutralizers such as certain antimicrobial peptides

Divalent cations affect chain mobility and aggregate structure of lipopolysaccharide from Salmonella minnesota reflected in a decrease of its biological activity

AbstractThe physicochemical properties and biological activities of rough mutant lipopolysaccharides Re (LPS Re) as preformed divalent cation (Mg2+, Ca2+, Ba2+) salt form or as natural or triethylamine (Ten+)-salt form under the influence of externally added divalent cations were investigated using complementary methods: Differential scanning calorimetry (DSC) and Fourier-transform infrared spectroscopic (FT-IR) measurements for the β↔α gel to liquid crystalline phase behaviour of the acyl chains of LPS, synchrotron radiation X-ray diffraction studies for their aggregate structures, electron density calculations of the LPS bilayer systems, and LPS-induced cytokine (interleukin-6) production in human mononuclear cells. The divalent cation salt forms of LPS exhibit considerable changes in physicochemical parameters such as acyl chain mobility and aggregate structures as compared to the natural or monovalent cation salt forms. Concomitantly, the biological activity was much lower in particular for the Ca2+- and Ba2+-salt forms. This decrease in activity results mainly from the conversion of the unilamellar/cubic aggregate structure of LPS into a multilamellar one. The reduced activity also clearly correlates with the higher order – lower mobility – of the lipid A acyl chains. Both effects can be understood by an impediment of the interactions of LPS with binding proteins such as lipopolysaccharide-binding protein (LBP) and CD14 due to the action of the divalent cations

Comparative analysis of selected methods for the assessment of antimicrobial and membrane-permeabilizing activity: a case study for lactoferricin derived peptides

<p>Abstract</p> <p>Background</p> <p>Growing concerns about bacterial resistance to antibiotics have prompted the development of alternative therapies like those based on cationic antimicrobial peptides (APs). These compounds not only are bactericidal by themselves but also enhance the activity of antibiotics. Studies focused on the systematic characterization of APs are hampered by the lack of standard guidelines for testing these compounds. We investigated whether the information provided by methods commonly used for the biological characterization of APs is comparable, as it is often assumed. For this purpose, we determined the bacteriostatic, bactericidal, and permeability-increasing activity of synthetic peptides (n = 57; 9–13 amino acid residues in length) analogous to the lipopolysaccharide-binding region of human lactoferricin by a number of the most frequently used methods and carried out a comparative analysis.</p> <p>Results</p> <p>While the minimum inhibitory concentration determined by an automated turbidimetry-based system (Bioscreen) or by conventional broth microdilution methods did not differ significantly, bactericidal activity measured under static conditions in a low-ionic strength solvent resulted in a vast overestimation of antimicrobial activity. Under these conditions the degree of antagonism between the peptides and the divalent cations differed greatly depending on the bacterial strain tested. In contrast, the bioactivity of peptides was not affected by the type of plasticware (polypropylene vs. polystyrene). Susceptibility testing of APs using cation adjusted Mueller-Hinton was the most stringent screening method, although it may overlook potentially interesting peptides. Permeability assays based on sensitization to hydrophobic antibiotics provided overall information analogous – though not quantitatively comparable- to that of tests based on the uptake of hydrophobic fluorescent probes.</p> <p>Conclusion</p> <p>We demonstrate that subtle changes in methods for testing cationic peptides bring about marked differences in activity. Our results show that careful selection of the test strains for susceptibility testing and for screenings of antibiotic-sensitizing activity is of critical importance. A number of peptides proved to have potent permeability-increasing activity at subinhibitory concentrations and efficiently sensitized <it>Pseudomonas aeruginosa </it>both to hydrophilic and hydrophobic antibiotics.</p

Оптимизация системы материально-технического обеспечения как фактор повышения производительности производства

Объектом исследования является система материально-технического обеспечения ЗАО "Сибирская сервисная компания". Цель работы–анализ, оценка и оптимизация деятельности компании в сфере материально-технического обеспечения на примере ЗАО "Сибирская сервисная компания".The object of the research is the logistics system of the Siberian Service Company CJSC. The purpose of the work is the analysis, evaluation and optimization of the company's activities in the field of logistics through the example of CJSC Siberian Service Company

Protostellar collapse and fragmentation using an MHD GADGET

Although the influence of magnetic fields is regarded as vital in the star formation process, only a few magnetohydrodynamics (MHD) simulations have been performed on this subject within the smoothed particle hydrodynamics (SPH) method. This is largely due to the unsatisfactory treatment of non-vanishing divergence of the magnetic field. Recently smoothed particle magnetohydrodynamics (SPMHD) simulations based on Euler potentials have proven to be successful in treating MHD collapse and fragmentation problems, however these methods are known to have some intrinsical difficulties. We have performed SPMHD simulations based on a traditional approach evolving the magnetic field itself using the induction equation. To account for the numerical divergence, we have chosen an approach that subtracts the effects of numerical divergence from the force equation, and additionally we employ artificial magnetic dissipation as a regularization scheme. We apply this realization of SPMHD to a widely known setup, a variation of the 'Boss & Bodenheimer standard isothermal test case', to study the impact of the magnetic fields on collapse and fragmentation. In our simulations, we concentrate on setups, where the initial magnetic field is parallel to the rotation axis. We examine different field strengths and compare our results to other findings reported in the literature. We are able to confirm specific results found elsewhere, namely the delayed onset of star formation for strong fields, accompanied by the tendency to form only single stars. We also find that the 'magnetic cushioning effect', where the magnetic field is wound up to form a 'cushion' between the binary, aids binary fragmentation in a case, where previously only formation of a single protostar was expected.Comment: 18 pages, 11 figures. Final version (with revisions). Accepted to MNRA

Brucella abortus and its closest phylogenetic relative, Ochrobactrum spp., differ in outer membrane permeability and cationic peptide resistance

The outer membrane (OM) of the intracellular parasite Brucella abortus is permeable to hydrophobic probes and resistant to destabilization by polycationic peptides and EDTA. The significance of these unusual properties was investigated in a comparative study with the opportunistic pathogens of the genus Ochrobactrum, the closest known Brucella relative. Ochrobactrum spp. OMs were impermeable to hydrophobic probes and sensitive to polymyxin B but resistant to EDTA. These properties were traced to lipopolysaccharide (LPS) because (i) insertion of B. abortus LPS, but not of Escherichia coli LPS, into Ochrobactrum OM increased its permeability; (ii) permeability and polymyxin B binding measured with LPS aggregates paralleled the results with live bacteria; and (iii) the predicted intermediate results were obtained with B. abortus-Ochrobactrum anthropi and E. coli-O. anthropi LPS hybrid aggregates. Although Ochrobactrum was sensitive to polymyxin, self-promoted uptake and bacterial lysis occurred without OM morphological changes, suggesting an unusual OM structural rigidity. Ochrobactrum and B. abortus LPSs showed no differences in phosphate, qualitative fatty acid composition, or acyl chain fluidity. However, Ochrobactrum LPS, but not B. abortus LPS, contained galacturonic acid. B. abortus and Ochrobactrum smooth LPS aggregates had similar size and zeta potential (-12 to -15 mV). Upon saturation with polymyxin, zeta potential became positive (1 mV) for Ochrobactrum smooth LPS while remaining negative (-5 mV) for B. abortus smooth LPS, suggesting hindered access to inner targets. These results show that although Ochrobactrum and Brucella share a basic OM pattern, subtle modifications in LPS core cause markedly different OM properties, possibly reflecting the adaptive evolution of B. abortus to pathogenicity

Thermodynamic analysis of the lipopolysaccharide-dependent resistance of gram-negative bacteria against polymyxin B

Cationic antimicrobial cationic peptides (CAMP) have been found in recent years to play a decisive role in hosts' defense against microbial infection. They have also been investigated as a new therapeutic tool, necessary in particular due to the increasing resistance of microbiological populations to antibiotics. The structural basis of the activity of CAMPs has only partly been elucidated and may comprise quite different mechanism at the site of the bacterial cell membranes or in their cytoplasm. Polymyxin B (PMB) is a CAMP which is effective in particular against Gram-negative bacteria and has been well studied with the aim to understand its interaction with the outer membrane or isolated membrane components such as lipopolysaccharide (LPS) and to de. ne the mechanism by which the peptides kill bacteria or neutralize LPS. Since PMB resistance of bacteria is a long-known phenomenon and is attributed to structural changes in the LPS moiety of the respective bacteria, we have performed a thermodynamic and biophysical analysis to get insights into the mechanisms of various LPS/PMB interactions in comparison to LPS from sensitive strains. In isothermal titration calorimetric (ITC) experiments considerable differences of PMB binding to sensitive and resistant LPS were found. For sensitive LPS the endothermic enthalpy change in the gel phase of the hydrocarbon chains converts into an exothermic reaction in the liquid crystalline phase. In contrast, for resistant LPS the binding enthalpy change remains endothermic in both phases. As infrared data show, these differences can be explained by steric changes in the headgroup region of the respective LPS