Interactions of linear dicationic molecules with lipid A: structural requisites for optimal binding affinity

The structural determinants of the binding affinity of linear dicationic molecules toward lipid A have been examined with respect to the distance between the terminal cationic functions, the basicity, and the type of cationic moieties using a series of spermidine derivatives and pentamidine analogs by fluorescence spectroscopic methods, The presence of two terminal cationic groups corresponds to enhanced affinity, A distinct sigmoidal relationship between the intercationic distance and affinity was observed with a sharp increase at 11 Angstrom, levelling off at about 13 Angstrom. The basicity (pK) and nature of the cationic functions are poor correlates of binding potency, since molecules bearing primary amino, imidazolino, or guanido termini are equipotent, The interaction of pentamidine, a bisamidine drug, with lipid A, characterized in considerable detail employing the putative intermolecular excimerization of the drug, suggests a stoichiometry of 1:1 in the resultant complex, The binding is driven almost exclusively by electrostatic forces, and is dependent on the ionization states of both lipid A and the drug, Under conditions when lipid A is highly disaggregated, pentamidine binds specifically to bis-phosphoryl- but not to monophosphoryl-lipid A indicating that both phosphate groups of lipid A are necessary for electrostatic interactions by the terminal amidininium groups of the drug, Based on these data, a structural model is proposed for the pentamidine-lipid A complex, which may be of value in designing endotoxin antagonists from first principles.

Characterization of the interactions of a polycationic, amphiphilic, terminally branched oligopeptide with lipid A and lipopolysaccharide from the deep rough mutant of Salmonella minnesota

The lipid A and lipopolysaccharide (LPS) binding and neutralizing activities of a synthetic, polycationic, amphiphilic peptide were studied. The branched peptide, designed as a functional analog of polymyxin B, has a six residue hydrophobic sequence, bearing at its N-terminus a penultimate lysine residue whose alpha- and epsilon-amino groups are coupled to two terminal lysine residues. In fluorescence spectroscopic studies designed to examine relative affinities of binding to the toxin, neutralization of surface charge and fluidization of the acyl domains, the peptide was active, closely resembling the effects of polymyxin B and its nonapeptide derivative; however, the synthetic peptide does not induce phase transitions in LPS aggregates as do polymyxin B and polymyxin B nonapeptide. The peptide was also comparable with polymyxin B in its ability to inhibit LPS-mediated IL-l and IL-6 release from human peripheral blood mononuclear cells. The synthetic compound is devoid of antibacterial activities and did not induce conductance fluxes in LPS-containing asymmetric planar membranes. These results strengthen the premise that basicity and amphiphilicity are necessary and sufficient physical properties that ascribe endotoxin binding and neutralizing activities, and further suggest that antibacterial/membrane perturbant and LPS neutralizing activities are dissociable, which may be of value in designing LPS-sequestering agents of low toxicity.

The role of polar and facial amphipathic character in determining lipopolysaccharide-binding properties in synthetic cationic peptides

Two series of peptides, designated K and NK were synthesized and tested for lipid A binding and neutralizing properties. K-2, which has an 11-residue amphiphilic core, and a branched N-terminus bearing two branched lysinyl residues does not bind lipid A, while NK2, also with an 11-residue amphiphilic core comprised entirely of non-ionizable residues, and a similarly branched, cationic N-terminus, binds lipid A very weakly. Both peptides do not inhibit lipopolysaccharide (LPS) activity in the Limulus assay, nor do they inhibit LPS-induced TNF-alpha and NO production in 5774 cells. These results are entirely unlike a homologous peptide with an exclusively hydrophobic core whose LPS-binding and neutralizing properties are very similar to that of polymyxin B [David SA, Awasthi SK, Wiese A et al. Characterization of the interactions of a polycationic, amphiphilic, terminally branched oligopeptide with lipid A and lipopolysaccharide from the deep rough mutant of Salmonella minnesota. J Endotoxin Res 1996; 3: 369-379]. These data suggest that a clear segregation of charged and apolar domains is crucial in molecules designed for purposes of LPS sequestration and that head-tail (polar) orientation of the cationic/hydrophobic regions is preferable to molecules with mixed or facial cationic/amphipathic character.

Effect of lipopolysaccharide on alteration of phospholipids and their fatty acid composition in spleen and thymus by in vitro metabolic labeling

Lipopolysaccharide (LPS) is an endotoxin, a potent stimulator of immune response and induction of LPS leads to acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). ARDS is a life-threatening disease worldwide with a high mortality rate. The immunological effect of LPS with spleen and thymus is well documented; however the impact on membrane phospholipid during endotoxemia has not yet been studied. Hence we aimed to investigate the influence of LPS on spleen and thymus phospholipid and fatty acid composition by 32P]orthophosphate labeling in rats. The in vitro labeling was carried out with phosphate-free medium (saline). Time course, LPS concentration-dependent, pre- and post-labeling with LPS and fatty acid analysis of phospholipid were performed. Labeling studies showed that 50 mu g LPS specifically altered the major phospholipids, phosphatidylcholine and phosphatidylglycerol in spleen and phosphatidylcholine in thymus. Fatty acid analysis showed a marked alteration of unsaturated fatty acids/saturated fatty acids in spleen and thymus leading to immune impairment via the fatty acid remodeling pathway. Our present in vitro lipid metabolic labeling study could open up new vistas for exploring LPS-induced immune impairment in spleen and thymus, as well as the underlying mechanism.

Bactericidal/permeability increasing protein: A multifaceted protein with functions beyond LPS neutralization

Bactericidal permeability increasing protein (BPI), a 55-60kDa protein, first reported in 1975, has gone a long way as a protein with multifunctional roles. Its classical role in neutralizing endotoxin (LPS) raised high hopes among septic shock patients. Today, BPI is not just a LPS-neutralizing protein, but a protein with diverse functions. These functions can be as varied as inhibition of endothelial cell growth and inhibition of dendritic cell maturation, or as an anti-angiogenic, chemoattractant or opsonization agent. Though the literature available is extremely limited, it is fascinating to look into how BPI is gaining major importance as a signalling molecule. In this review, we briefly summarize the recent research focused on the multiple roles of BPI and its use as a therapeutic.

Vascular endothelial growth factor regulates melanoma cell adhesion and growth in the bone marrow microenvironment via tumor cyclooxygenase-2

Background: Human melanoma frequently colonizes bone marrow (BM) since its earliest stage of systemic dissemination, prior to clinical metastasis occurrence. However, how melanoma cell adhesion and proliferation mechanisms are regulated within bone marrow stromal cell (BMSC) microenvironment remain unclear. Consistent with the prometastatic role of inflammatory and angiogenic factors, several studies have reported elevated levels of cyclooxygenase-2 (COX-2) in melanoma although its pathogenic role in bone marrow melanoma metastasis is unknown. Methods: Herein we analyzed the effect of cyclooxygenase-2 (COX-2) inhibitor celecoxib in a model of generalized BM dissemination of left cardiac ventricle-injected B16 melanoma (B16M) cells into healthy and bacterial endotoxin lipopolysaccharide (LPS)-pretreated mice to induce inflammation. In addition, B16M and human A375 melanoma (A375M) cells were exposed to conditioned media from basal and LPS-treated primary cultured murine and human BMSCs, and the contribution of COX-2 to the adhesion and proliferation of melanoma cells was also studied. Results: Mice given one single intravenous injection of LPS 6 hour prior to cancer cells significantly increased B16M metastasis in BM compared to untreated mice; however, administration of oral celecoxib reduced BM metastasis incidence and volume in healthy mice, and almost completely abrogated LPS-dependent melanoma metastases. In vitro, untreated and LPS-treated murine and human BMSC-conditioned medium (CM) increased VCAM-1-dependent BMSC adherence and proliferation of B16M and A375M cells, respectively, as compared to basal medium-treated melanoma cells. Addition of celecoxib to both B16M and A375M cells abolished adhesion and proliferation increments induced by BMSC-CM. TNF alpha and VEGF secretion increased in the supernatant of LPS-treated BMSCs; however, anti-VEGF neutralizing antibodies added to B16M and A375M cells prior to LPS-treated BMSC-CM resulted in a complete abrogation of both adhesion-and proliferation-stimulating effect of BMSC on melanoma cells. Conversely, recombinant VEGF increased adherence to BMSC and proliferation of both B16M and A375M cells, compared to basal medium-treated cells, while addition of celecoxib neutralized VEGF effects on melanoma. Recombinant TNFa induced B16M production of VEGF via COX-2-dependent mechanism. Moreover, exogenous PGE2 also increased B16M cell adhesion to immobilized recombinant VCAM-1. Conclusions: We demonstrate the contribution of VEGF-induced tumor COX-2 to the regulation of adhesion-and proliferation-stimulating effects of TNFa, from endotoxin-activated bone marrow stromal cells, on VLA-4-expressing

Função pulmonar, estresse oxidativo e marcadores inflamatórios na lesão pulmonar aguda induzida por lipopolissacarídeo: diferentes efeitos da atorvastatina, pravastatina e simvastatina

Nosso objetivo foi determinar que tipo de estatina pode atenuar a lesão pulmonar aguda (LPA) induzida por lipopolissacarídeo (LPS) em camundongos da linhagem C57Bl/6. Trinta camundongos machos ( 23 g) foram divididos em 5 grupos (n=6 cada): grupo LPS (10 mg/kg) administrado intraperitonealmente (i.p.), LPS mais atorvastatina (10 mg/kg/dia; grupo LPS+A), LPS mais pravastatina (5 mg/kg/dia; grupo LPS+P) e LPS mais sinvastatina (20 mg/kg/dia; grupo LPS+S). O grupo controle recebeu salina i.p.. Em um grupo separado de camundongos (n=5), a soma das pressões pulmonares resistivas e viscoelásticas (DeltaPtot) e elastância estática (E[st]) foram medidas. Um dia após a administração de LPS os camundongos foram sacrificados (24 h) por deslocamento cervical e logo em seguida foi realizado lavado broncoalveolar (LBA). Os pulmões foram removidos para análise histopatológica e homogeneizados para análises bioquímicas (ELISA, catalase, superóxido dismutase, mieloperoxidase, substâncias reativas ao ácido tiobarbitúrico, carbonilação de proteínas e método de Griess). A quantidade de leucócitos foi menor no grupo LPS+P (p<0,01) e LPS+S (p<0,05) em comparação ao grupo LPS. Os níveis de MCP-1 e IL-6 reduziram no grupo LPS+P (p<0,01), enquanto o grupo LPS + S mostrou redução apenas nos níveis de IL-6 (p<0,05) em comparação ao grupo LPS. Marcadores redox (superóxido dismutase e catalase) foram menores no grupo LPS+A (p<0,01) em comparação ao grupo LPS. A peroxidação lipídica (malondialdeído e hidroperóxidos) diminuiu em todos os grupos tratados (p<0,05) quando comparados ao grupo LPS. A mieloperoxidase foi menor no grupo LPS+P (p<0,01) quando comparado ao grupo LPS. DeltaPtot e E(st) foram, significativamente, maiores no grupo LPS do que nos outros grupos. Nossos resultados sugerem que atorvastatina e pravastatina, mas não a sinvastatina, exibiram ações anti-inflamatórias e antioxidantes na LPA induzida por LPS.

Potential causes of mortality for horseshoe crabs (Limulus polyphemus) during the biomedical bleeding process

Biomedical companies catch and bleed horseshoe crabs for the production of Limulus amebocyte lysate (LAL), a product used for protecting public health (Berkson and Shuster, 1999). LAL is a clotting agent, derived solely from horseshoe crab blood cells, which is used to detect the presence of pathogenic gramnegative bacteria in injectable drugs and implantable medical and dental devices (Mikkelsen, 1988; Novitsky, 1991). In addition, LAL is used in many diagnostic tests for such illnesses as gram-negative bacterial meningitis and typhoid fever (Ding and Ho, 2001). Because the LAL test allows one to detect femtogram levels of endotoxin (Ding and Ho, 2001), it is the most effective test for detecting endotoxin contamination, and its increasing use in medical and pharmaceutical laboratories makes it a highly valued product.

Preparation and performance of cellulose acetate/polyethyleneimine blend microfiltration membranes and their applications

A modified microfiltration membrane has been prepared by blending a matrix polymer with a functional polymer. Cellulose acetate (CA) was blended with polyethyleneimine (PEI), which was then crosslinked by polyisocyanate, in a mixture of solvents. In the membrane, PEI can supply coupling sites for ligands in affinity separation or be used as ligands for metal chelating, removal of endotoxin or ion exchange. The effects of the time of phase inversion induced by water vapor, blended amount of PEI and amount of crosslinking agent on membrane performance were investigated. The prepared blend membranes have specific surface area of 12.04-24.11 m(2)/g and pure water flux (PWF) of 10-50 ml/cm(2) min with porosity of 63-75%. The membranes, made of 0.15 50 wt.% PEI/CA ratio and 0.5 crosslinking agent/PEI ratio, were applied to adsorbing Cu2+ and bovine serum albumin (BSA) individually. The maximum adsorption capacity of Cu2+ ion on the blend membrane is 7.42 mg/g dry membrane. The maximum adsorption capacities of BSA on the membranes with and without chelating Cu2+ ion are 86.6 and 43.8 mg/g dry membrane, respectively. (C) 2004 Elsevier B.V. All rights reserved.

Cloning and expression of tumor necrosis factor (TNF alpha) cDNA from red seabream Pagrus major

A fragment of TNFalpha cDNA sequence from red seabream was cloned by homology cloning approach with two degenerated primers which were designed based on the conserved regions of other animals' TNF sequences. The sequence was elongated by 3' and 5' RACE to get the full length CDS sequence. This sequence contained 1264 nucleotides that included a 5' UTR of 85 bp, a 3' UTR of 514 bp and an open reading frame (ORF) of 666 bp which could encode 222 amino acids propeptide. In 3' UTR, there were several mRNA instability motifs and three endotoxin-responsive sequences, but the sequence lacked the polyadenylation signal. The deduced peptide had a clear transmembrane domain, a TNFalpha family signature and a TNF2 family profile. The cell attachment sequence and the glycosaminoglycan attachment sites were also found in the sequence. The red seabream TNF sequence shared relatively high similarity with both mammalian TNFalpha and TNFbeta by multiple sequence alignments. Phylogenetic analysis showed that the piscine TNFalpha were located independently in a different branch compared with mammalian TNFalpha and TNFbeta. Based on the primary and secondary structure analysis and gene expression study, we could concluded that the red seabream TNF should be a TNFalpha, not TNFbeta. RT-PCR was used to study TNFa transcript expression. 24 h after the red seabream was challenged by Vibrio anguillarum, the RS TNFalpha transcript expression were detected in blood, brain, gill, heart, head kidney, kidney, Ever, muscle and spleen. Results showed that TNFalpha mRNA was constitutively expressed in parts of the tissues both in stimulated and unstimulated fish and the expression could be enhanced after the pathogen infection.

Generation and characterisation of biologically active milk-derived protein and peptide fractions

In recent years, extensive research has been carried out on the health benefits of milk proteins and peptides. Biologically active peptides are defined as specific protein fragments which have a positive impact on the physiological functions of the body; such peptides are produced naturally in vivo, but can also be generated by physical and/or chemical processes, enzymatic hydrolysis and/or microbial fermentation. The aims of this thesis were to investigate not only the traditional methods used for the generation of bioactive peptides, but also novel processes such as heat treatment, and the role of indigenous milk proteases, e.g., in mastitic milk, in the production of such peptides. In addition, colostrum was characterised as a source of bioactive proteins and peptides. Firstly, a comprehensive study was carried out on the composition and physical properties of colostrum throughout the early-lactation period. Marked differences in the physico-chemical properties of colostrum compared with milk were observed. Various fractions of colostrum were also tested for their effect on the secretion of pro- and anti-inflammatory cytokines from a macrophage cell line and bone marrow dendritic cells, as well as insulin secretion from a pancreatic beta cell line. A significant reduction in the secretion of the pro-inflammatory cytokines, TNF-α, IL-6, IL-1β and IL-12, a significant increase in the secretion of the anti-inflammatory cytokine, IL-10, as well as a significant increase in insulin secretion were observed for various colostrum fractions. Another study examined the early proteomic changes in the milk of 8 cows in response to infusion with the endotoxin lipopolysaccharide (LPS) at quarter level in a model mastitic system; marked differences in the protein and peptide profile of milk from LPS challenged cows were observed, and a pH 4.6-soluble fraction of this milk was found to cause a substantial induction in the secretion of IL-10 from a murine macrophage cell line. Heat-induced hydrolysis of sodium caseinate was investigated from the dual viewpoints of protein breakdown and peptide formation, and, a peptide fraction produced in this manner was found to cause a significant increase in the secretion of the anti-inflammatory cytokine, IL-10, from a murine macrophage cell line. The effects of sodium caseinate hydrolysed by chymosin on the gut-derived satiety hormone glucagon-like peptide-1 (GLP-1) were investigated; the resulting casein-derived peptides displayed good in vitro and in vivo secretion of GLP-1. Overall, the studies described in this thesis expand on current knowledge and provide good evidence for the use of novel methods for the isolation, generation and characterisation of bioactive proteins and/or peptides.

Fiber type-specific nitric oxide protects oxidative myofibers against cachectic stimuli.

Oxidative skeletal muscles are more resistant than glycolytic muscles to cachexia caused by chronic heart failure and other chronic diseases. The molecular mechanism for the protection associated with oxidative phenotype remains elusive. We hypothesized that differences in reactive oxygen species (ROS) and nitric oxide (NO) determine the fiber type susceptibility. Here, we show that intraperitoneal injection of endotoxin (lipopolysaccharide, LPS) in mice resulted in higher level of ROS and greater expression of muscle-specific E3 ubiqitin ligases, muscle atrophy F-box (MAFbx)/atrogin-1 and muscle RING finger-1 (MuRF1), in glycolytic white vastus lateralis muscle than in oxidative soleus muscle. By contrast, NO production, inducible NO synthase (iNos) and antioxidant gene expression were greatly enhanced in oxidative, but not in glycolytic muscles, suggesting that NO mediates protection against muscle wasting. NO donors enhanced iNos and antioxidant gene expression and blocked cytokine/endotoxin-induced MAFbx/atrogin-1 expression in cultured myoblasts and in skeletal muscle in vivo. Our studies reveal a novel protective mechanism in oxidative myofibers mediated by enhanced iNos and antioxidant gene expression and suggest a significant value of enhanced NO signaling as a new therapeutic strategy for cachexia.

Human dendritic cell maturation and cytokine secretion upon stimulation with Bordetella pertussis filamentous haemagglutinin.

In addition to antibodies, Th1-type T cell responses are also important for long-lasting protection against pertussis. However, upon immunization with the current acellular vaccines, many children fail to induce Th1-type responses, potentially due to immunomodulatory effects of some vaccine antigens, such as filamentous haemagglutinin (FHA). We therefore analysed the ability of FHA to modulate immune functions of human monocyte-derived dendritic cells (MDDC). FHA was purified from pertussis toxin (PTX)-deficient or from PTX- and adenylate cyclase-deficient Bordetella pertussis strains, and residual endotoxin was neutralized with polymyxin B. FHA from both strains induced phenotypic maturation of human MDDC and cytokine secretion (IL-10, IL-12p40, IL-12p70, IL-23 and IL-6). To identify the FHA domains responsible for MDDC immunomodulation, MDDC were stimulated with FHA containing a Gly→Ala substitution at its RGD site (FHA-RAD) or with an 80-kDa N-terminal moiety of FHA (Fha44), containing its heparin-binding site. Whereas FHA-RAD induced maturation and cytokine production comparable to those of FHA, Fha44 did not induce IL-10 production, but maturated MDDC at least partially. Nevertheless, Fha44 induced the secretion of IL-12p40, IL-12p70, IL-23 and IL-6 by MDDC, albeit at lower levels than FHA. Thus, FHA can modulate MDDC responses in multiple ways, and IL-10 induction can be dissociated from the induction of other cytokines.

LPS-binding protein and IL-6 Mark paradoxical tuberculosis immune reconstitution inflammatory syndrome in HIV patients

Background: Tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) remains a poorly understood complication in HIV-TB co-infected patients initiating antiretroviral therapy (ART). The role of the innate immune system in TB-IRIS is becoming increasingly apparent, however the potential involvement in TB-IRIS of a leaky gut and proteins that interfere with TLR stimulation by binding PAMPs has not been investigated before. Here we aimed to investigate the innate nature of the cytokine response in TB-IRIS and to identify novel potential biomarkers. Methods: From a large prospective cohort of HIV-TB co-infected patients receiving TB treatment, we compared 40 patients who developed TB-IRIS during the first month of ART with 40 patients matched for age, sex and baseline CD4 count who did not. We analyzed plasma levels of lipopolysaccharide (LPS)-binding protein (LBP), LPS, sCD14, endotoxin-core antibody, intestinal fatty acid-binding protein (I-FABP) and 18 pro-and anti-inflammatory cytokines before and during ART. Results: We observed lower baseline levels of IL-6 (p = 0.041), GCSF (p = 0.036) and LBP (p = 0.016) in TB-IRIS patients. At IRIS event, we detected higher levels of LBP, IL-1RA, IL-4, IL-6, IL-7, IL-8, G-CSF (p ≤ 0.032) and lower I-FABP levels (p = 0.013) compared to HIV-TB co-infected controls. Only IL-6 showed an independent effect in multivariate models containing significant cytokines from pre-ART (p = 0.039) and during TB-IRIS (p = 0.034). Conclusion: We report pre-ART IL-6 and LBP levels as well as IL-6, LBP and I-FABP levels during IRIS-event as potential biomarkers in TB-IRIS. Our results show no evidence of the possible contribution of a leaky gut to TB-IRIS and indicate that IL-6 holds a distinct role in the disturbed innate cytokine profile before and during TB-IRIS. Future clinical studies should investigate the importance and clinical relevance of these markers for the diagnosis and treatment of TB-IRIS. Copyright: © 2013 Goovaerts et al.

Recovery of Bacillus thuringiensis in vegetative form from the phylloplane of clover (Trifolium hybridum) during a growing season

Two media were developed which specifically allow the cultivation of Bacillus thuringiensis while it is in the vegetative as opposed to the spore form. Using these media B. thuringiensis was shown conclusively for the first time to exist in an active form on the phylloplane. The profile of its appearance in vegetative and spore form was followed over a growing season on clover (Trifolium hybridum) in the field. Three simultaneous and sudden rises and declines of both spore and vegetative cell densities were observed. The most common other spore-former on these leaves was Bacillus cereus but the fluctuations in appearance of these two very closely related species were not co-incident. Using specific PCR primers a considerable diversity of cry toxin gene types was found in isolates that had been recovered in vegetative form ([`]vegetative isolates') with the majority possessing multiple [delta]-endotoxin genes while some had only one of those tested. Bioassays against a lepidopteran insect of purified [delta]-endotoxins showed that they were no more potent than those from a laboratory-adapted strain. PCR primers for an internal region of the vip3A gene produced amplification in 70% of the vegetative isolates compared to 25% of the laboratory-adapted strains tested.