Lipopolysaccharide Contamination Research Articles

In Silico Analysis and Development of the Secretory Expression of D-Psicose-3-Epimerase in Escherichia coli.

D-psicose-3-epimerase (DPEase), a key enzyme for D-psicose production, has been successfully expressed in Escherichia coli with high yield. However, intracellular expression results in high downstream processing costs and greater risk of lipopolysaccharide (LPS) contamination during cell disruption. The secretory expression of DPEase could minimize the number of purification steps and prevent LPS contamination, but achieving the secretion expression of DPEase in E. coli is challenging and has not been reported due to certain limitations. This study addresses these challenges by enhancing the secretion of DPEase in E. coli through computational predictions and structural analyses. Signal peptide prediction identified PelB as the most effective signal peptide for DPEase localization and enhanced solubility. Supplementary strategies included the addition of 0.1% (v/v) Triton X-100 to promote protein secretion, resulting in higher extracellular DPEase (0.5 unit/mL). Low-temperature expression (20 °C) mitigated the formation of inclusion bodies, thus enhancing DPEase solubility. Our findings highlight the pivotal role of signal peptide selection in modulating DPEase solubility and activity, offering valuable insights for protein expression and secretion studies, especially for rare sugar production. Ongoing exploration of alternative signal peptides and refinement of secretion strategies promise further enhancement in enzyme secretion efficiency and process safety, paving the way for broader applications in biotechnology.

Generation of Endotoxin-Specific Monoclonal Antibodies by Phage and Yeast Display for Capturing Endotoxin.

Endotoxin, a synonym for lipopolysaccharide (LPS), is anchored in the outer membranes of Gram-negative bacteria. Even minute amounts of LPS entering the circulatory system can have a lethal immunoactivating effect. Since LPS is omnipresent in the environment, it poses a great risk of contaminating any surface or solution, including research products and pharmaceuticals. Therefore, monitoring LPS contamination and taking preventive or decontamination measures to ensure human safety is of the utmost importance. Nevertheless, molecules used for endotoxin detection or inhibition often suffer from interferences, low specificity, and low affinity. For this reason, the selection of new binders that are biocompatible, easy to produce, and that can be used for biopharmaceutical applications, such as endotoxin removal, is of high interest. Powerful techniques for selecting LPS-binding molecules in vitro are display technologies. In this study, we established and compared the selection and production of LPS-specific, monoclonal, human single-chain variable fragments (scFvs) through two display methods: yeast and phage display. After selection, scFvs were fused to a human constant fragment crystallizable (Fc). To evaluate the applicability of the constructs, they were conjugated to polystyrene microbeads. Here, we focused on comparing the functionalized beads and their LPS removal capacity to a polyclonal anti-lipid A bead. Summarized, five different scFvs were selected through phage and yeast display, with binding properties comparable to a commercial polyclonal antibody. Two of the conjugated scFv-Fcs outperformed the polyclonal antibody in terms of the removal of LPS in aqueous solution, resulting in 265 times less residual LPS in solution, demonstrating the potential of display methods to generate LPS-specific binding molecules.

Direct Electrochemical Sensing of Lipopolysaccharide by Graphene Nanoplatlets@Cytochrome c Hydrogel Using a Microfluidic Chip

AbstractContamination by lipopolysaccharide (LPS), an endotoxin which is present in the outer membrane of Gram‐negative bacteria causes adverse toxic effects on human health. Herein, we report a facile graphene nanoplatelets@cytochrome c (GNP@Cyt c) hydrogel based voltammetric electrochemical sensor for LPS. Firstly, the resultant hydrogel sensor facilitated a large electroactive surface coverage for electron transfer, and secondly provided a 3D porous microenvironment for interaction of abundant electrocatalytic sites in Cyt c with LPS. Under optimal incubation conditions, the heme iron of Cyt c in its reduced form (Fe2+) can directly bind to highly anionic carbohydrate groups of LPS, thereby giving rise to specific interaction. Moreover, the binding of Cyt c (Fe2+) with LPS served as a electrocatalytic unit, generating a strong oxidation peak current at 0.019 V. Importantly, no separate redox mediator was required and the electrochemical signal obtained by LPS‐Cyt c (Fe2+) complex sufficed for detection purposes. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were employed to investigate the electrochemical aspects of GNP@Cyt c hydrogel modified screen‐printed carbon electrode (SPCE). Further, the GNP@Cyt c hydrogel/SPCE demonstrated LPS dose‐dependent changes in oxidation current which enabled selective quantitative detection. This method exhibited a detection limit as low as 8.4×10−4 ng mL−1 with a wide linear range of 0.01–2.0 ng mL−1. For continuous on‐chip monitoring of LPS, a customized microfluidic electrochemical device approach was established which endorses the practical utility of the developed sensor. Further, the integrated microfluidic electrochemical device showed excellent recovery upon spiking LPS in lemon juice and tap water samples. Overall, the fabricated sensor holds great promise for point‐of‐care screening of trace LPS contamination, especially for food safety.

Recombinant human TIM-3 ectodomain expressed in bacteria and recovered from inclusion bodies as a stable and active molecule.

Introduction: Microbial systems, such as Escherichia coli, as host recombinant expression is the most versatile and the cheapest system for protein production, however, several obstacles still remain, such as recovery of soluble and functional proteins from inclusion bodies, elimination of lipopolysaccharides (LPS) contamination, incomplete synthesis, degradation by proteases, and the lack of post-translational modifications, which becomes even more complex when comes to membrane proteins, because they are difficult not only to produce but also to keep in solution in its active state. T-cell Immunoglobulin and Mucin domain 3 (TIM-3) is a type I transmembrane protein that is predominantly expressed on the surface of T lymphocytes, natural killer (NK) cells, dendritic cells, and macrophages, playing a role as a negative immune checkpoint receptor. TIM-3 comprises a single ectodomain for interaction with immune system soluble and cellular components, a transmembrane domain, and a cytoplasmic tail, responsible for the binding of signaling and scaffolding molecules. TIM-3 pathway holds potential as a therapeutic target for immunotherapy against tumors, autoimmunity, chronic virus infections, and various malignancies, however, many aspects of the biology of this receptor are still incompletely understood, especially regarding its ligands. Methods: Here we overcome, for the first time, the challenge of the production of active immune checkpoint protein recovered from bacterial cytoplasmic inclusion bodies, being able to obtain an active, and non-glycosylated TIM-3 ectodomain (TIM-3-ECD), which can be used as a tool to better understand the interactions and roles of this immune checkpoint. The TIM-3 refolding was obtained by the association of high pressure and alkaline pH. Results: The purified TIM-3-ECD showed the correct secondary structure and was recognized from anti-TIM-3 structural-dependent antibodies likewise commercial TIM-3-ECD was produced by a mammal cells system. Furthermore, immunofluorescence showed the ability of TIM-3-ECD to bind to the surface of lung cancer A549 cells and to provide an additional boost for the expression of the lymphocyte activation marker CD69 in anti-CD3/CD28 activated human PBMC. Discussion: Taken together these results validated a methodology able to obtain active checkpoint proteins from bacterial inclusion bodies, which will be helpful to further investigate the interactions of this and others not yet explored immune checkpoints.

Effect of Odanacatib on the release of NTX (Amino Terminal Telopeptide) from LPS contaminated type I dentin collagen.

To evaluated the Odanacatib inhibitor treatment on lipopolysaccharide (LPS) contamination effect on cathepsin-K mediated dentin degradation by analysis of type I collagen C- and N-termini telopeptides. Pulverized and disks of human dentin were demineralized and LPS contaminated, or stored in deionized water (DW) for 12h. Samples were challenged with lactic acid (LA). Aliquots of dentin powder were treated with 1mL Odanacatib or stored in DW for 30min. Dentin collagen degradation was determined by sub-product release of C-terminal (ICTP and CTX) and N-terminal (NTX) telopeptides, normalized to total protein (tp) concentration (n=3). Dentin matrix was evaluated for gravimetric (n=8) and ultrastructural changes. Data were analyzed by Student t-test, one-way ANOVA and Tukey's test (α=5 %). LA incubation significantly increased telopeptide release compared with DW (p<0.05). In untreated groups, significantly higher CTXtp, NTXtp telopeptide rates were observed for LA+LPS samples compared with DW (p<0.01). Odanacatib significantly reduced ICTPtp, CTXtp, and NTXtp telopeptide release for LPS, LA, and LA+LPS conditions. In untreated groups, LPS and LA+LPS challenge significantly increased dentin weight loss (p=0.02). Within each storage condition, Odanacatib treatment did not affect weight change (p>0.05) of dentin disks. This study showed that LPS contamination resulted in significantly higher rates of NTX than CTX from dentin matrix. Odanacatib significantly reduced telopeptide release rates of LPS contaminated dentin matrix.

Sensitive detection of lipopolysaccharides by monitoring of interleukin-10 secretion from human PBMCs.

Lipopolysaccharide (LPS) contaminations may falsify immunological experiments and are crucial for pharmaceutical products because they cause life-threatening immune reactions. Here, we present interleukin-10 (IL-10) as a reliable marker to measure LPS contents when the readout of pro-inflammatory cytokines is not favored. This animal free source assay is able to detect LPS with a limit of detection (LOD) of 0.024 EU/ml by monitoring IL-10 secretions from isolated human peripheral blood mononuclear cells (PBMCs).

Plant produced endotoxin binding recombinant proteins effectively remove endotoxins from protein samples

Lipopolysaccharides (LPS) are highly toxic compounds, even at a trace amount. When recombinant proteins are produced in E. coli, it is inevitable that LPS contaminates. However, LPS removal is still technically challenging and costly due to the high degree of solubility in a wide range of solvents. In this study, we explored the possibility of using the N-terminal region containing cysteine-rich, EGF-like, and sushi1–3 domains (CES3) of Factor C from the horseshoe crab Carcinoscorpius rotundicauda to develop a platform to remove LPS from recombinant proteins. We expressed CES3 as part of a recombinant protein, BiP:NT:CBM3:SUMO:CES3:His:HDEL, in Nicotiana benthamiana and found that purified or microcrystalline cellulose (MCC) bead-immobilised CES3 showed strong binding to LPS-containing E. coli. To produce CES3:CBM3 in an LPS-free environment, we generated Arabidopsis transgenic plants harbouring a recombinant gene, BiP:NT:SUMO:CES3:CBM3:HDEL, and found that transgenic plants mainly produce CES3:CBM3:His:HDEL, a truncated version of BiP:NT:SUMO:CES3:CBM3:HDEL via endogenous protease-mediated proteolytic processing in vivo. CES3:CBM3:HDEL purified from Arabidopsis plant extracts and immobilised onto MCC beads removed LPS contamination from protein samples. We propose that the CES3:CBM3 fusion protein produced in plants and immobilised on MCC beads can be a robust and easy platform for LPS removal from recombinant proteins.

Neutrophil‐Derived Microvesicles Enhance Pulmonary Vascular Inflammation via a Toll‐like Receptor 4 Signaling‐Dependent Mechanism

BackgroundCirculating neutrophil‐derived microvesicles (NMVs) are markedly elevated during sepsis and therefore could have a role in the development of indirect acute lung injury (ALI). We recently found that NMV‐enriched CD11b+ MVs, immunoaffinity isolated from lipopolysaccharide (LPS)‐stimulated healthy volunteer blood, have potent pro‐inflammatory activity in a human peripheral blood mononuclear cell (PBMC) and lung microvascular endothelial cell (HLMEC) coculture model of pulmonary vascular inflammation (1). By contrast, immunoaffinity isolated platelet‐MVs (CD61+) produced negligible responses in these assays, suggesting specificity of the NMV‐enriched CD11b+ MV, activity. Here, we investigated the signaling mechanisms responsible for NMV‐mediated activation of monocytes and HLMECs in this model.MethodsHeparinized blood from healthy donors was treated with LPS (100 ng/ml, 3 h) and NMVs (CD66b+) were isolated by positive immunoaffinity selection. NMVs were then incubated in PBMC‐HLMEC cocultures for 4 h. NMV‐induced responses were determined by flow cytometric quantification of cell surface activation markers (ICAM‐1 for HLMECs, ICAM‐1 and tissue factor for monocytes) and pro‐inflammatory cytokine release by ELISA (TNFα, IL‐8, IL‐6, MCP‐1).ResultsNMV‐induced HLMEC activation required the presence of monocytes and was completely attenuated by anti‐TNFα neutralizing antibody (ICAM‐1: isotype control 302±103 vs anti‐TNFα 66±29; p&lt;0.01). Pharmacological inhibition of mitogen‐activated protein kinases p38, MEK1‐MEK2 and phosphoinositide‐3 kinase ablated monocyte and HLMEC activation. Monocyte activation was prevented in the presence of the Toll‐like receptor 4 (TLR4) inhibitor TAK‐242 (e.g., TNFα release: vehicle 1108±257 vs TAK‐242 16±12; p&lt;0.001) and partially inhibited by an anti‐TLR4 neutralizing antibody. However, the LPS inhibitor polymyxin‐B had no effect on these responses, indicating that LPS contamination was unlikely. Furthermore, NMV activity was found to be sensitive to proteinase K and heat‐treatment (55 °C, 15 min) (e.g., TNFα release: untreated 474±267 vs proteinase K 12±15; p&lt;0.0001 vs heat‐treatment 200±84; p&lt;0.01).ConclusionsWe have demonstrated that NMVs produced within LPS‐stimulated blood are potent mediators of lung microvascular endothelial cells via monocyte‐derived TNFα signaling. NMV‐induced monocyte activation involves TLR4 signaling and is protease sensitive, suggesting that MVs serve as vehicles of damage‐associated molecular pattern (DAMP) activity, mediated by surface proteins. Together these findings support a major role for NMVs as novel long‐range mediators for systemic propagation of inflammation, contributing to the pathogenesis of sepsis‐induced indirect acute lung injury.(1) Tsiridou et al. European Respiratory Journal. 2020;56:4468

Production of Single-Domain Antibodies in Pichia pastoris.

Single-domain antibodies (sdAbs) are binders that consist of a single immunoglobulin domain. SdAbs have gained importance as therapeutics, diagnostic reagents, and research tools. Functional sdAbs are commonly produced in Escherichia coli, which is a simple and widely used host for production of recombinant proteins. However, there are drawbacks of the E. coli expression system, including the potential for misfolded recombinant proteins and pyrogeniccontamination with toxic lipopolysaccharides. Pichia pastoris is an alternative host for the production of heterologous proteins because of its high recombinant protein yields and the ability to produce soluble, properly folded proteins without lipopolysaccharide contamination. Here, we describe a method to produce sdAbs in P. pastoris. We present methods for the cloning of sdAb-encoding genes into a P. pastoris expression vector, production and purification of sdAbs, and measurement of sdAb-binding kinetics. Functional sdAbs are easily and routinely obtained using these methods.

SARS-CoV-2 N Protein Induces Acute Lung Injury in Mice via NF-ĸB Activation.

BackgroundInfection of SARS-CoV-2 may cause acute respiratory syndrome. It has been reported that SARS-CoV-2 nucleocapsid protein (N-protein) presents early in body fluids during infection. The direct involvement of N-protein in lung injury is poorly understood.MethodsRecombinant N-protein was pretreated with polymyxin B, a lipopolysaccharide (LPS)-neutralizing agent. C57BL/6, C3H/HeJ (resistant to LPS), and C3H/HeN (control for C3H/HeJ) mice were exposed to N-protein via intratracheal administration to examine acute lung injury. In vitro, bone marrow–derived macrophages (BMDMs) were cultured with N-protein to study phosphorylation of nuclear factor kappa B (NF-ĸB) p65, macrophage polarization, and expression of proinflammatory cytokines.ResultsN-protein produced acute lung injury in C57BL/6 mice, with elevated protein permeability, total cell count, neutrophil infiltration, and proinflammatory cytokines in the bronchioalveolar lavage. N-protein also induced lung injury in both C3H/HeJ and C3H/HeN mice, indicating that the effect could not be attributed to the LPS contamination. N-protein triggered phosphorylation of NF-ĸB p65 in vitro, which was abolished by both N-protein denaturation and treatment with an antibody for N-protein, demonstrating that the effect is N-protein specific. In addition, N-protein promoted M1 macrophage polarization and the expression of proinflammatory cytokines, which was also blocked by N-protein denaturation and antibody for N-protein. Furthermore, N-protein induced NF-ĸB p65 phosphorylation in the lung, while pyrrolidine dithiocarbamate, an NF-ĸB inhibitor, alleviated the effect of N-protein on acute lung injury. ConclusionsSARS-CoV-2 N-protein itself is toxic and induces acute lung injury in mice. Both N-protein and NF-ĸB pathway may be therapeutic targets for treating multi-organ injuries in Coronavirus disease 2019 (COVID-19).

The Lipopolysaccharide Mutant Re-LPS Is a Useful Tool for Detecting LPS Contamination in Rheumatoid Synovial Cell Cultures

Introduction: Lipopolysaccharide (LPS) contamination of commercially available proteins has seriously impeded research on citrullinated fibrinogen (cit-Fb) in rheumatoid synovial cells (RSCs). Methods: RSCs obtained from 4 rheumatoid arthritis patients who underwent full knee arthroplasty were cultured, stimulated with cit-Fb, and cytokine expression levels were measured. We then evaluated polymyxin-B (PMB), heat inactivation, and rough (R)-type LPS mutants for rapid detection of LPS contamination. Results: cit-Fb induced expression of CXCL10 and IFNB in RSCs via the toll-like receptor. PMB inhibited cit-Fb-mediated CXCL10 gene expression but not protein expression induced by 20 μg/mL cit-Fb. Heat inactivation did not affect LPS-mediated CXCL10 or IL-6 induction; however, cit-Fb-mediated CXCL10expression was inhibited. Wild-type LPS from Escherichia coli (WT-LPS) strongly induces CXCL10 expression, but induction by Ra-LPS was weak, and induction by Rc- and Re-LPS was minimal. Re-LPS suppression of WT-LPS-mediated CXCL10 induction in RSCs and peripheral blood monocytes (PBMs) was dose dependent. Furthermore, Re-LPS completely suppressed cit-Fb-mediated CXCL10 induction in RSCs and PBMs. Conclusion: To easily identify LPS contamination during routine experiments, our results suggest that Re-LPS is a better tool for rapid detection of LPS contamination compared to PMB and heat treatment.

Efficacy of bioactive nanoparticles on tissue-endotoxin induced suppression of stem cell viability, migration and differentiation.

To characterize a lipopolysaccharide (LPS)-treated dentine tissue model (LPS dentine) to analyse the efficacy of polycationic chitosan nanoparticles (CSnp) and/or dexamethasone conjugate chitosan nanoparticles (Dex-CSnp) on the viability/differentiation potential of stem cells from apical papilla (SCAP) when exposed to LPS dentine. A further aim was to understand the effect of macrophage-dependent inflammation on SCAP migration in the presence of LPS dentine. A total of 88 dentine slabs were used. TOF-SIMS analysis was performed amongst the LPS-treated and untreated dentine groups (n=2/group). The study was conducted using four dentine groups: no treatment (control); LPS treatment only; LPS treatment followed by CSnp conditioning; and LPS treatment followed by Dex-CSnp conditioning groups. SCAP adherence, viability, differentiation and biomineralization potential on dentine from different groups were studied using fluorescent and scanning electron microscopy. Inflammation by macrophages in response to LPS dentine was quantified, and effect on SCAP migration was analysed. Statistical analysis was performed using Student's t-test with a significance level of P<0.05. TOF-SIMS analysis confirmed LPS contamination. LPS dentine affected SCAP viability but not adherence to dentine (P<0.001). Conditioning of LPS dentine with either nanoparticles improved SCAP viability (P<0.01) and rescued other LPS related adverse effects on SCAPs, such as F-actin disruption, decrease in differentiation/biomineralization potential. IL-6 produced by macrophages in response to LPS-treated dentine impeded SCAP migration (P<0.001), diminished on CSnp and Dex-CSnp conditioning groups (P<0.01). This study developed an LPS-dentine model and highlighted the ability of CSnp and Dex-CSnp to promote stem cell viability, migration, differentiation potential and reduce inflammation, providing an environment conducive for tissue regeneration/repair.

Influence of poly(I:C) variability on thermoregulation, immune responses and pregnancy outcomes in mouse models of maternal immune activation

Maternal immune activation (MIA) models that are based on administration of the viral mimetic, poly(I:C), are widely used as experimental tools to study neuronal and behavioral dysfunctions in relation to immune-mediated neurodevelopmental disorders and mental illnesses. Evidence from investigations in non-pregnant rodents suggests that different poly(I:C) products can vary in terms of their immunogenicity, even if they are obtained from the same vendor. The present study aimed at extending these findings to pregnant mice, while also controlling various poly(I:C) products for potential contamination with lipopolysaccharide (LPS). We found significant variability between different batches of poly(I:C) potassium salt obtained from the same vendor (Sigma-Aldrich) in terms of the relative amount of dsRNA fragments in the high molecular weight range (1000–6000 nucleotides long) and with regards to their effects on maternal thermoregulation and immune responses in maternal plasma, placenta and fetal brain. Batches of poly(I:C) potassium salt containing larger amounts of high molecular weight fragments induced more extensive effects on thermoregulation and immune responses compared to batches with minimal amounts of high molecular weight fragments. Consistent with these findings, poly(I:C) enriched for high molecular weight dsRNA (HMW) caused larger maternal and placental immune responses compared to low molecular weight (LMW) poly(I:C). These variable effects were unrelated to possible LPS contamination. Finally, we found marked variability between different batches of the poly(I:C) potassium salt in terms of their effects on spontaneous abortion rates. This batch-to-batch variability was confirmed by three independent research groups using distinct poly(I:C) administration protocols in mice. Taken together, the present data confirm that different poly(I:C) products can induce varying immune responses and can differentially affect maternal physiology and pregnancy outcomes. It is therefore pivotal that researchers working with poly(I:C)-based MIA models ascertain and consider the precise molecular composition and immunogenicity of the product in use. We recommend the establishment of reference databases that combine phenotype data with empirically acquired quality information, which can aid the design, implementation and interpretation of poly(I:C)-based MIA models.

Synergistic effect of Dermatophagoides pteronyssinus allergen and Escherichia coli lipopolysaccharide on human blood cells.

PurposeHouse dust mites Dermatophagoides pteronyssinus are the main source of major inhalatory allergens inducing inflammatory response. Mite extract contain both allergenic proteins and lipopolysaccharides (LPS). The main allergenic protein, Der p 2, is a functional homolog of sMD-2, a protein providing blood cell response on LPS. Der p 2 may restore the response to LPS in absence of MD-2, but its interaction with LPS in whole blood is unknown. We studied the effect of Der p 2 on LPS-mediated activation of human whole blood cells.MethodsInteraction of Der p 2 and LPS was studied on eight healthy donors. The whole blood was incubated with extract of house dust mite Dermatophagoides pteronyssinus (DP-e), recombinant antigenic protein Der p 2 variant 5 (rDep 2), Escherichia coli lipopolysaccharide and their combination. Supernatants were collected for ELISA analysis of protein content. Activation degree was determined by change in concentration of TNF-α, IL-8, IL-1Ra cytokines and sMD-2 protein.Resultsextract of mite Dermatophagoides pteronyssinus (DP-e) possessed weak inherent activity and did not cause significant increase of cytokine production. Simultaneous activation of blood cells by LPS and DP-e led to considerable increase of pro-inflammatory cytokine production. We have shown the intrinsic inducing activity of Der p 2 allergen on sMD-2 protein and TNF-α cytokine expression.ConclusionsDer p 2 allergen enhances the response of human whole blood cells to external LPS by inducing additional expression of LPS-transporting protein sMD-2. The obtained data show an important role of LPS contamination of allegrens in the progress of allergic inflammatory response.

Bovine Lactoferrin Modulates Dendritic Cell Differentiation and Function

Lactoferrin is an abundant glycoprotein in bovine milk that has immunomodulatory effects on human cells. Bovine lactoferrin (LF) binds lipopolysaccharides (LPS) with high affinity and is postulated to act via TLR4-dependent and -independent mechanisms. It has been shown that LF modulates differentiation of human monocytes into tolerogenic dendritic cells. However, in a previous study, we showed that LPS also mediates differentiation into tolerogenic dendritic cells (DC). Since LF binds LPS with high affinity, it remains to be investigated whether LF or LPS is mediating these effects. We, therefore, further investigated the LPS-independent effect of LF on differentiation of human monocytes into dendritic cells (DC). Human monocytes were isolated by magnetic cell sorting from freshly isolated PBMCs and cultured for six days in the presence of IL-4 and GM-CSF with or without LF or proteinase K treated LF to generate DC. These immature DC were stimulated for 48 h with LPS or Poly I:C + R848. Cell surface marker expression and cytokine production were measured by flow cytometry. DC differentiated in the presence of LF produced higher IL-6 and IL-8 levels during differentiation and showed a lower expression of CD1a and HLA-DR. These LFDCs showed to be hyporesponsive towards TLR ligands as shown by their semi-mature phenotype and reduced cytokine production. The effect of LF was abrogated by proteinase K treatment, showing that the functional effects of LF were not mediated by LPS contamination. Thus, LF alters DC differentiation and dampens responsiveness towards TLR ligands. This study indicates that LF can play a role in immune homeostasis in the human GI tract.

Role of C-Reactive Protein at Sites of Inflammation and Infection.

C-reactive protein (CRP) is an acute inflammatory protein that increases up to 1,000-fold at sites of infection or inflammation. CRP is produced as a homopentameric protein, termed native CRP (nCRP), which can irreversibly dissociate at sites of inflammation and infection into five separate monomers, termed monomeric CRP (mCRP). CRP is synthesized primarily in liver hepatocytes but also by smooth muscle cells, macrophages, endothelial cells, lymphocytes, and adipocytes. Evidence suggests that estrogen in the form of hormone replacement therapy influences CRP levels in the elderly. Having been traditionally utilized as a marker of infection and cardiovascular events, there is now growing evidence that CRP plays important roles in inflammatory processes and host responses to infection including the complement pathway, apoptosis, phagocytosis, nitric oxide (NO) release, and the production of cytokines, particularly interleukin-6 and tumor necrosis factor-α. Unlike more recent publications, the findings of early work on CRP can seem somewhat unclear and at times conflicting since it was often not specified which particular CRP isoform was measured or utilized in experiments and whether responses attributed to nCRP were in fact possibly due to dissociation into mCRP or lipopolysaccharide contamination. In addition, since antibodies for mCRP are not commercially available, few laboratories are able to conduct studies investigating the mCRP isoform. Despite these issues and the fact that most CRP research to date has focused on vascular disorders, there is mounting evidence that CRP isoforms have distinct biological properties, with nCRP often exhibiting more anti-inflammatory activities compared to mCRP. The nCRP isoform activates the classical complement pathway, induces phagocytosis, and promotes apoptosis. On the other hand, mCRP promotes the chemotaxis and recruitment of circulating leukocytes to areas of inflammation and can delay apoptosis. The nCRP and mCRP isoforms work in opposing directions to inhibit and induce NO production, respectively. In terms of pro-inflammatory cytokine production, mCRP increases interleukin-8 and monocyte chemoattractant protein-1 production, whereas nCRP has no detectable effect on their levels. Further studies are needed to expand on these emerging findings and to fully characterize the differential roles that each CRP isoform plays at sites of local inflammation and infection.

Quality control of commercial bovine lactoferrin.

Herein we review commercial bovine lactoferrin quality issues by describing an example of industrial production, the current status of global quality standardization, and quality-activity concerns for further discussion. Morinaga Milk Industry has been industrially producing bovine lactoferrin in Milei GmbH, Germany, since 1989. We delineate its production and quality as an example of safe and high-quality manufacturing. Currently, global standardization in the quality of bovine lactoferrin is progressing through Novel Food and GRAS in the EU and USA, respectively. Novel Food was applied or notified to seven lactoferrin manufacturers and GRAS was notified to three manufacturers, two of which are for infant use and one is for adult use, by the end of 2017. The specifications of these regulations are relatively high, including more than 95% lactoferrin purity in protein, which means that such companies can supply relatively high-grade lactoferrin. There appear to be several concerns regarding lactoferrin quality affecting activities, including contamination of lipopolysaccharide (LPS) and angiogenin, purity, and degradation of lactoferrin sample. Although LPS is immunologically toxic when invading the body, it is distributed normally in foods and the gut. However, an industrial lactoferrin sample may contain LPS at a maximum LPS/lactoferrin molecule ratio = 1/1724, which means 99.9% of the lactoferrin molecule is LPS-free. It is difficult to speculate that LPS contained in a lactoferrin sample affects its activities. Finally in order to achieve good and reproducible results, we make proposals to researchers a use of high-grade lactoferrin, careful storage, and indication the manufacturers' names and specifications in the paper.

Lipopolysaccharide interferes with the use of the human Cell Line Activation Test to determine the allergic potential of proteins

It was believed that high molecular weight molecules including proteins cannot penetrate the skin. However, protein penetration through disrupted/ruptured skin has been reported recently, thus carrying the potential for inducing an allergic response. We used the human Cell Line Activation Test (h-CLAT), an in vitro skin sensitization test, to assess the allergic potential of proteins by measuring levels of CD86 and CD54 in the human monocytic leukemia cell line THP-1. Six allergens including ovalbumin (OVA) and human serum albumin (HSA; negative control) upregulated CD86 and/or CD54; a false-positive result was obtained using HSA. This was caused by lipopolysaccharide (LPS) contamination. Naturally derived materials often include LPS at various concentrations and may influence protein induction of CD86 and CD54. Additionally, polymyxin B, an LPS inhibitor, could not completely overcome the effect of LPS. Therefore, if test proteins contain ≥0.1 EU/mL LPS, their allergenic potency will not be assessed accurately using h-CLAT. These data show that naturally occurring materials or those derived from living organisms should be evaluated for their LPS content. It is important to confirm the applicability of in vitro methods such as h-CLAT for assessing the allergenic potency of naturally occurring proteins; our findings can be a foundation for future studies.

The hidden toxin. Is bacterial lipopolysaccharide contamination responsible for nanomaterial toxicity?

The hidden toxin. Is bacterial lipopolysaccharide contamination responsible for nanomaterial toxicity?

Plasmodium falciparum Heat Shock Protein 70 Lacks Immune Modulatory Activity.

Heat shock protein 70 (Hsp70) family are conserved molecules that constitute a major part of the cell's protein folding machinery. The role of Hsp70s of parasitic origin in host cell immune modulation has remained contentious. This is largely due to the fact that several studies implicating Hsp70 in immune modulation rely on the use of recombinant protein derived from bacteria which is often fraught with lipopolysaccharide (LPS) contamination. For this reason, there is need to clarify the role of parasite Hsp70 in modulating host immune cells. The current study sought to investigate the role of Plasmodium falciparum Hsp70 (PfHsp70) in immune modulation. We expressed recombinant PfHsp70 using three bacterial expression hosts: E. coli XL1 Blue, E. coli ClearColi BL21 and Brevibacillus choshinensis, respectively. We further investigated the immunostimulatory capability of PfHsp70 by monitoring cytokine production by murine immune cells cultured in the presence of the protein. Recombinant PfHsp70 produced using E. coli XL1 Blue expression host induced IL6 and IL8 cytokines. On the other hand, PfHsp70 produced in E. coli ClearColi and B. choshinensis expression systems was associated with no detectable traces of LPS and exhibited no immunomodulatory activity. Our findings demonstrate that PfHsp70 does not possess immunomodulatory function. Furthermore, our study further confirm E. coli ClearColi and B. choshinensis as appropriate expression systems for the production of LPS-free recombinant protein.