Endotoxin removal : a critical step in vaccine production

A major drawback to the immunological potency of conventional vaccines, resulting in reduced level of immune responses, tissue injury, shock and high cytotoxicity, thus making their applications contraindicated in immunodeficiency diseases, is the presence of high contaminant concentrations in vaccine titers. Vaccine contamination arises from the simultaneous occurrence of competitive pathways resulting in the formation of other bio-products during cellular metabolism aside the pathways necessary for the production of vaccine molecules. One of such vaccine contaminating molecules is endotoxins which are mainly lipopolysaccharides (LPS) complexes found in the membrane of bacterial cell wall. The structural dynamics of these molecules make their removal from vaccine titers problematic, thus making vaccine endotoxin removal a major research endeavour. This presentation will discuss a novel technique for reducing the endotoxin level of vaccines. The technique commences with the disentanglement of endotoxin-vaccine molecular bonding and then capturing the vaccine molecules on an affinity monolith to separate the vaccine molecules from the endotoxins.

The O-specific polysaccharide structure and biosynthetic gene cluster of Yersinia pseudotuberculosis serotype O:11

In the Yersinia pseudotuberculosis serotyping scheme, 21 serotypes are present originating from about 30 different O-factors distributed within the species. With regard to the chemical structures of lipopolysaccharides (LPSs) and the genetic basis of their biosynthesis, a number, but not all, of Y. pseudotuberculosis strains representing different serotypes have been investigated. In order to present an overall picture of the relationship between genetics and structures, we have been working on the genetics and structures of various Y. pseudotuberculosis O-specific polysaccharides (OPSs). Here, we present a structural and genetic analysis of the Y. pseudotuberculosis serotype O:11 OPS. Our results showed that this OPS structure has the same backbone as that of Y. pseudotuberculosis O:1b, but with a 6d-l-Altf side-branch instead of Parf. The 3′ end of the gene cluster is the same as that for O:1b and has the genes for synthesis of the backbone and for processing the completed repeat unit. The 5′ end has genes for synthesis of 6d-l-Altf and its transfer to the repeating unit backbone. The pathway for the synthesis of the 6d-l-Altf appears to be different from that for 6d-l-Altp in Y. enterocolitica O:3. The chemical structure of the O:11 repeating unit is [Figure]

The Wzy O-antigen polymerase of Yersinia pseudotuberculosis O:2a has a dependence on the Wzz chain-length determinant for efficient polymerization

Lipopolysaccharide is a major immunogenic structure for the pathogen Yersinia pseudotuberculosis, which contains the O-specific polysaccharide (OPS) that is presented on the cell surface. The OPS contains many repeats of the oligosaccharide O-unit and exhibits a preferred modal chain length that has been shown to be crucial for cell protection in Yersinia. It is well established that the Wzz protein determines the preferred chain length of the OPS, and in its absence, the polymerization of O units by the Wzy polymerase is uncontrolled. However, for Y. pseudotuberculosis, a wzz mutation has never been described. In this study, we examine the effect of Wzz loss in Y. pseudotuberculosis serotype O:2a and compare the lipopolysaccharide chain-length profile to that of Escherichia coli serotype O111. In the absence of Wzz, the lipopolysaccharides of the two species showed significant differences in Wzy polymerization. Yersinia pseudotuberculosis O:2a exhibited only OPS with very short chain lengths, which is atypical of wzz-mutant phenotypes that have been observed for other species. We hypothesise that the Wzy polymerase of Y. pseudotuberculosis O:2a has a unique default activity in the absence of the Wzz, revealing the requirement of Wzz to drive O-unit polymerization to greater lengths.

Soft-particle model analysis of effect of LPS on electrophoretic softness of Acidithiobacillus ferrooxidans grown in presence of different metal ions

The effect of Surface lipopolysaccharides (LPS) on the electrophoretic softness and fixed charge density in the ion-penetrable layer of Acidithiobacillus ferrooxidans cells grown in presence of copper or arsenic ions have been discussed, The electrophoretic mobility data were analyzed using the soft-particle electrophoresis theory. Cell surface potentials of all the strains based on soft-particle theory were lower than those estimated using the conventional Smoluchowski theory, Exposure to metal ions increased the Surface electrophoretic softness with decrease in the fixed charge density. Effect of cell surface lipopolysaccharides on the model parameters are investigated and discussed.

Interaction of cationic amphiphilic drugs with lipid A: Implications for development of endotoxin antagonists

This report presents evidence for the interactions of several classes of cationic amphiphilic drugs including the phenothiazines, aminoquinolines, biguanides, and aromatic diamidines, with lipid A, the endotoxic principle of lipopolysaccharides. The interactions of the drugs were quantitatively assessed by fluorescence methods. The affinities of the drugs for lipid A parallel their endotoxin-antagonistic effects in the Limulus gelation assay. Dicationic compounds bind lipid A with greater affinity; the affinity of such molecules increases exponentially as a function of the distance between the basic moieties. The bis-amidine drug - pentamidine - examined in greater detail, binds lipid A with high affinity (apparent K-d: 0.12 mu M), and LPS, probably due to simultaneous interactions of the terminal amidine groups with the anionic phosphates on lipid A. The sequestration of endotoxin by pentamidine reduces its propensity to bind to cells, and the complex exhibits attenuated toxicity in biological assays. These results have implications in the development of therapeutic strategies against endotoxin-related disease states.

Differential binding of peanut agglutinin with lipopolysaccharide of homologous and heterologous Rhizobium

To establish the crucial role of lipopolysaccharide in the initial recognition event of symbiotic peanut-Rhizobium system the ability of various surface polysaccharides isolated from Bradyrhizobium arachis to inhibit the precipitin reaction between peanut agglutinin and asialoganglioside: deoxycholate (1:1) micelles was estimated. It was compared with that of nonsymbiotic systems e.g. Bradyrhizobium japonicum, Bradyrhizobium ciceris and Escherichia coli. Peanut agglutinin was found to interact more strongly with the lipopolysaccharide of Bradyrhizobium arachis than the exopolysaccharide or capsular polysaccharide. The inhibitory capacity of lipopolysaccharides from homologous and heterologous Bradyrhizobium as measured in terms of the concentration necessary for 50 percent inhibition of precipitin reaction were 1428, 500, 410, and 277 times less than that of lactose for Bradyrhizobium arachis, B. japonicum, B. ciceris and Escherichia coli, respectively. These results support that host lectin peanut agglutinin can recognize homologous Bradyrhizobium lipopolysaccharide by virtue of its binding specificity of higher magnitude.

Interaction of melittin with endotoxic lipid

Several amphipathic and cationic substances are known to bind lipid A, the toxic component of bacterial lipopolysaccharides. In this report, we have characterized, by fluorescence methods, the interaction of melittin, an amphipathic and basic 26-residue polypeptide isolated from bee venom, with lipid A. The stoichiometry of the complex appears to be two molecules of melittin to one of lipid A with a dissociation constant of 2.5 × 10 −6 M. The binding of melittin not only modifies the endotoxic properties of lipid A in a number of biological assays, but also results in abrogation of the hemolytic activity of melittin. A model of the complex is proposed based on the known structures of lipid A and melittin, and the observed stoichiometry of binding.

Role of outer membrane exopolymers of Acidithiobacillus ferrooxidans in adsorption of cells onto pyrite and chalcopyrite

Bacterial surface polymers play a major role in the adhesion of bacterial cells to solid surfaces. Lipopolysaccharides (LPS) are essential constituents of the cell walls of almost all Gram-negative bacteria. This paper reports the results of the investigations on the role of outer membrane exopolymers (LPS) of the chemolithotroph, Acidithiobacillus ferrooxidans, in adsorption of the cells onto pyrite and chalcopyrite. Optimization of EDTA treatment for removal of LPS from cell surface and the surface characterization of EDTA-treated cells are outlined. There was no change in cell morphology or loss in cell motility upon treatment with upto 0.04 mM EDTA for 1 h. Partial removal of LPS by EDTA treatment resulted in reduced adsorption of the cells on both pyrite and chalcopyrite. The protein profile of the EDTA-extractable fraction showed presence of certain outer membrane proteins indicating that EDTA treatment results in temporary gaps in the outer membrane. Also, specificity towards pyrite compared to chalcopyrite that was exhibited by untreated cells was lost when their exopolymer layers were stripped off, which could be attributed to the role of outer membrane proteins in the mineral-specificity exhibited by the bacteria. (C) 2013 Elsevier B.V. All rights reserved.

Toxic blue-green algae: The "problem" in perspective

The incidence of blue-green algal blooms and surface scum-formation are certainly not new phenomena. Many British and European authors have been faithfully describing the unmistakable symptoms of blue-green algal scums for over 800 years. There is no disputing that blue-green algal toxins are extremely harmful. Three quite separate categories of compound have been separated: neurotoxins; hepatotoxins and lipopolysaccharides. There is a popular association between blue-green algae and eutrophication. Certainly the main nuisance species - of Microcystis, Anabaena and Aphanizomenon are rare in oligotrophic lakes and reservoirs. Several approaches have been proposed for the control of blue-green algae. Distinction is made between methods for discharging algae already present (eg algicides; straw bales; viruses; parasitic fungi and herbivorous ciliates), and methods for averting an anticipated abundance in the future (phosphorous control, artificial circulation etc).

A novel C1q-domain-containing protein from Zhikong scallop Chlamys farreri with lipopolysaccharide binding activity

The C1q-domain-containing (C1qDC) proteins are a family of proteins characterized by a globular C1q (gC1q) domain in their C-terminus. They are involved in various processes of vertebrates and supposed to be an important pattern recognition receptor in innate immunity of invertebrates. In this study, a novel member of C1q-domain-containing protein family was identified from Zhikong scallop Chlamys farreri (designated as CfC1qDC) by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of CfC1qDC was of 777 bp, consisting of a T-terminal untranslated region (UTR) of 62 bp and a 3' UTR of 178 bp with a polyadenylation signal sequence AATAAA and a poly (A) tail. The CfC1qDC cDNA encoded a polypeptide of 178 amino acids, including a signal peptide and a C1q-domain of 158 amino acids with the theoretical isoelectric point of 5.19 and the predicted molecular weight of 17.2 kDa. The C1q-domain in CfC1qDC exhibited homology with those in sialic acid binding lectin from mollusks and C1qDC proteins from higher vertebrates. The typical 10 beta-strand jelly-roll folding topology structure of C1q-domain and the residues essential for effective packing of the hydrophobic core were well conserved in CfC1qDC. By fluorescent quantitative real-time PCR, mRNA transcripts of CfC1qDC were mainly detected in kidney, mantle, adductor muscle and gill, and also marginally detectable in hemocytes. In the bacterial challenge experiment, after the scallops were challenged by Listonella anguillarum, there was a significant up-regulation in the relative expression level of CfC1qDC and at 6 h post-injection, the mRNA expression reached the maximum level and was 4.55-fold higher than that of control scallops. Similarly, the expression of CfC1qDC mRNA in mixed primary cultures of hemocytes stimulated by lipopolysaccharides (LPS) was up-regulated and reached the maximum level at 6 h post-stimulation, and then dropped back to the original level gradually. In order to investigate its function, the cDNA fragment encoding the mature peptide of CfC1qDC was recombined and expressed in Escherichia coli BL21 (DE3). The recombinant CfC1qDC protein displayed a significantly strong activity to bind LIDS from E. coli, although no obvious antibacterial or agglutinating activity toward Gram-negative bacteria E. coli JM109, L. anguillarum and Gram-positive bacteria Micrococcus luteus was observed. These results suggested that CfC1qDC was absolutely a novel member of the C1qDC protein family and was involved in the recognition of invading microorganisms probably as a pattern recognition molecule in mollusk. (c) 2008 Elsevier Ltd. All rights reserved.

Purification and characterisation of a natural lectin from the serum of the shrimp Litopenaeus vannamei

A natural lectin from the serum of the shrimp Litopenaeus vannamei was purified to homogeneity by a single-step affinity chromatography using fetuin-coupled agarose. The purified serum lectin (named LVL) showed a strong affinity for human A/B/O erythrocytes (RBC), mouse RBC, chicken RBC and its haemagglutinating (HA) activity was specifically dependent on Ca2+ and reversibly sensitive to EDTA. LVL inactive form had a molecular mass estimate of 172 kDa and was composed of two non-identical subunits (32 and 38 kDa) cross-linked by interchain disulphide bonds. Significant LVL activity was observed between pH 7 and 11. In HA-inhibition assays performed with several carbohydrates and glycoproteins, LVL showed a distinct and unique specificity for GalNAc/GluNAc/NeuAc which had an acetyl group, while glycoproteins fetuin and bovine submaxillary mucin (BSM) had sialic acid. Moreover, this agglutinin appeared to recognise the terminal N- and O-acetyl groups in the oligosaccharide chain of glycoconjugates. The HA activity of L. vannamei lectin was also susceptible to inhibition by lipopolysaccharides from diverse Gram-negative bacteria, which might indicate a significant in vivo role of this humoral agglutinin in the host immune response against bacterial infections. (C) 2006 Elsevier Ltd. All rights reserved.

The role of presenilin 1 and presenilin 2 in IL-1β-, LPS- and TNFα- mediated signalling events

The importance of γ-secretase protease activities in development, neurogenesis and the immune system are highlighted by the diversity of its substrates and phenotypic characterization of Presenilin (PS)-deficient transgenic animals. Since the discovery of Amyloid precursor protein (APP) and it’s cleavage by γ-secretase complexes, over 90 other type I membrane proteins have been identified as γ-secretase substrates. We have identified interleukin-1 (IL-1) receptor type I (IL-1R1), toll-like receptor 4 (TLR4) and tumour necrosis factor-α (TNFα) receptor-1 (TNFR1) as novel substrates for - secretase cleavage, which play an important role in innate immunity. In this study, using PS-deficient cells and PS-knockout animal models we examined the role of PS proteins, PS1 and PS2, in IL-1R1-, TLR4- and TNFR1- mediated inflammatory responses. Data presented show that in response to IL- 1β, lipopolysaccharide (LPS) or TNFα, immortalised fibroblasts from PS2- deficient animals have diminished production of specific cytokines and chemokine, with differential reduction in nuclear factor-κB (NF-κB) and (mitogen activated protein kinase) MAPK activities. In contrast, no defect in the response to IL-1β, LPS or TNFα was observed in PS1-deficient immortalised fibroblasts. These observations were confirmed using bone marrow-derived macrophages from PS2-null mice, which also display impaired responsiveness to IL-1β- and LPS, with decreased production of inflammatory cytokines. Furthermore, in whole animal in vivo responses, we show that PS2-deficient animals display ligand (IL-1β, LPS and TNFα)-dependent alterations in the production of specific pro-inflammatory cytokines or chemokines. Importantly, this reduced responsiveness to IL-1β, LPS or TNFα is independent of γ- secretase protease activity and γ-secretase cleavage of TNFR1, IL-1R1 or TLR4. These observations suggest a novel γ-secretase-independent role of PS2 in the regulation of innate immune responsiveness and challenge current concepts regarding the regulation of IL-1β-, LPS- and TNFα-mediated immune signalling.

Differential inhibition of human immunodeficiency virus type 1 in peripheral blood mononuclear cells and TZM-bl cells by endotoxin-mediated chemokine and gamma interferon production.

Bacterial lipopolysaccharide (endotoxin) is a frequent contaminant of biological specimens and is also known to be a potent inducer of beta-chemokines and other soluble factors that inhibit HIV-1 infection in vitro. Though lipopolysaccharide (LPS) has been shown to stimulate the production of soluble HIV-1 inhibitors in cultures of monocyte-derived macrophages, the ability of LPS to induce similar inhibitors in other cell types is poorly characterized. Here we show that LPS exhibits potent anti-HIV activity in phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMCs) but has no detectable anti-HIV-1 activity in TZM-bl cells. The anti-HIV-1 activity of LPS in PBMCs was strongly associated with the production of beta-chemokines from CD14-positive monocytes. Culture supernatants from LPS-stimulated PBMCs exhibited potent anti-HIV-1 activity when added to TZM-bl cells but, in this case, the antiviral activity appeared to be related to IFN-gamma rather than to beta-chemokines. These observations indicate that LPS stimulates PBMCs to produce a complex array of soluble HIV-1 inhibitors, including beta-chemokines and IFN-gamma, that differentially inhibit HIV-1 depending on the target cell type. The results also highlight the need to use endotoxin-free specimens to avoid artifacts when assessing HIV-1-specific neutralizing antibodies in PBMC-based assays.

Co-regulation of nuclear respiratory factor-1 by NFkappaB and CREB links LPS-induced inflammation to mitochondrial biogenesis.

The nuclear respiratory factor-1 (NRF1) gene is activated by lipopolysaccharide (LPS), which might reflect TLR4-mediated mitigation of cellular inflammatory damage via initiation of mitochondrial biogenesis. To test this hypothesis, we examined NRF1 promoter regulation by NFκB, and identified interspecies-conserved κB-responsive promoter and intronic elements in the NRF1 locus. In mice, activation of Nrf1 and its downstream target, Tfam, by Escherichia coli was contingent on NFκB, and in LPS-treated hepatocytes, NFκB served as an NRF1 enhancer element in conjunction with NFκB promoter binding. Unexpectedly, optimal NRF1 promoter activity after LPS also required binding by the energy-state-dependent transcription factor CREB. EMSA and ChIP assays confirmed p65 and CREB binding to the NRF1 promoter and p65 binding to intron 1. Functionality for both transcription factors was validated by gene-knockdown studies. LPS regulation of NRF1 led to mtDNA-encoded gene expression and expansion of mtDNA copy number. In cells expressing plasmid constructs containing the NRF-1 promoter and GFP, LPS-dependent reporter activity was abolished by cis-acting κB-element mutations, and nuclear accumulation of NFκB and CREB demonstrated dependence on mitochondrial H(2)O(2). These findings indicate that TLR4-dependent NFκB and CREB activation co-regulate the NRF1 promoter with NFκB intronic enhancement and redox-regulated nuclear translocation, leading to downstream target-gene expression, and identify NRF-1 as an early-phase component of the host antibacterial defenses.

Gene expression signatures of radiation response are specific, durable and accurate in mice and humans.

BACKGROUND: Previous work has demonstrated the potential for peripheral blood (PB) gene expression profiling for the detection of disease or environmental exposures. METHODS AND FINDINGS: We have sought to determine the impact of several variables on the PB gene expression profile of an environmental exposure, ionizing radiation, and to determine the specificity of the PB signature of radiation versus other genotoxic stresses. Neither genotype differences nor the time of PB sampling caused any lessening of the accuracy of PB signatures to predict radiation exposure, but sex difference did influence the accuracy of the prediction of radiation exposure at the lowest level (50 cGy). A PB signature of sepsis was also generated and both the PB signature of radiation and the PB signature of sepsis were found to be 100% specific at distinguishing irradiated from septic animals. We also identified human PB signatures of radiation exposure and chemotherapy treatment which distinguished irradiated patients and chemotherapy-treated individuals within a heterogeneous population with accuracies of 90% and 81%, respectively. CONCLUSIONS: We conclude that PB gene expression profiles can be identified in mice and humans that are accurate in predicting medical conditions, are specific to each condition and remain highly accurate over time.