Paola Neri

Localized Surface Plasmon Resonance Detection of Biological Toxins Using Cell Surface Oligosaccharides on Glyco Chips

We have detected biological toxins using localized surface plasmon resonance (LSPR) and synthetic glycosyl ceramides (β-lactoside, globosyl trisaccharide (Gb3), or GM1 pentasaccharide) attached to gold (Au) nanoparticles. The particle diameters ranged from 5-100 nm. The detection sensitivity for three toxins (ricin, Shiga toxin, and cholera toxin) was found to depend not only on the attached glycoside but also on the diameter of the Au nanoparticles. For the detection of ricin, the 20-nm β-lactoside-coated Au nanoparticle exhibited the highest LSPR response, whereas 40-nm Gb3- and GM1-coated Au nanoparticles gave the best results for Shiga toxin and cholera toxin, respectively. In addition, a blocking process on the nanoparticle surface greatly improved the detection sensitivity for cholera toxin. The LSPR system enabled us to detect ricin at 30 ng/mL, Shiga toxin at 10 ng/mL, and the cholera toxin at 20 ng/mL.

Glycochips from polyanionic glycopolymers as tools for detecting shiga toxins

An alternating layer‐by‐layer adsorption methodology was applied to the assembly of glycochips by using synthetic polyanionic glycopolymers. Three glycochips carrying globobioside (Gb2), β‐lactoside (β‐Lac), or α‐D‐mannoside (α‐Man) residues were prepared, and used for the detection of Shiga toxins, Stx‐1 and Stx‐2, by using surface plasmon resonance (SPR). Using this method, we could confirm that both Stx‐1 and Stx‐2 show binding specificity for the Gb2 glycochip as well as a weak affinity for the β‐Lac glycochip. The affinity constants of these toxins depended strongly on the sugar content of the Gb2 polymer used to prepare the glycochip. Greater affinity was observed for chips with a higher sugar content (up to 43 %) in the Gb2 glycopolymer. The maximal affinity constants of Stx‐1 and Stx‐2 (Ka=108–109 M−1) enabled highly sensitive and facile analysis (10 ng mL−1, 30 min). When Gb2 glycopolymers were used as competitors, Stx‐1 and Stx‐2 behaved differently from one another in terms of their SPR response; this allowed us to perform discriminative analysis between the two toxins.

A nasal vaccine comprising B-subunit derivative of Shiga toxin 2 for cross-protection against Shiga toxin types 1 and 2

Enterohemorrhagic Escherichia coli (EHEC) produces Stx1 and Stx2 causing severe diseases. Their B-subunits (StxBs) exhibit low immunogenicity and the anti-StxB antibodies neutralizing both Stxs has not been prepared yet. By intranasal vaccination with His-tagged-StxB (Stx1B-His or Stx2B-His) plus a mutant heat-labile enterotoxin (mLT) in mice, their serum and lung fluid reacted with appropriate StxB. Mice vaccinated with Stx2B-His plus mLT had antibodies reacting Stx1B and showed the resistance to toxemia of Stx1 and Stx2. This is the first demonstration to get anti-Stx2B serum neutralizing both Stxs. These suggest that the nasal vaccination with Stx2B-His and mLT is effective for preventing toxemias by EHEC.

Neutralizing Activity of Polyvalent Gb3, Gb2 and Galacto‐Trehalose Models against Shiga Toxins

Shiga toxin (Stx) is one of the most critical factors in the development of hemolytic uremic syndrome and other systemic complications following enterohemorrhagic Escherichia coli (EHEC) infection. Substances neutralizing Stx by interfering with toxin‐receptor binding have been explored as therapeutic candidates for EHEC infection. In this study, we examined globotriaosyl (Gb3), galabiosyl (Gb2) and galacto‐trehalose, each of which was synthetically conjugated with a polyacrylamide backbone, for Stx‐neutralizing activity. Galacto‐trehalose was designed as a Gb2 mimicking, unnatural Stx‐ligand that was expected to show tolerance to enzymatic degradation in vivo. Galacto‐trehalose copolymer showed neutralizing activity against Stx‐1 but not Stx‐2 in a HeLa cell cytotoxicity assay. It was thought that galacto‐trehalose copolymer could be a lead compound for the treatment of Stx‐mediated diseases, although it requires modification to show neutralizing activity to Stx‐2. The Gb3 copolymer with high sugar unit density showed stronger neutralizing activity against Stx‐2 than those with lower density. However, the density‐dependency of the neutralizing activity was less obvious against Stx‐1. Intravenous administration of the Gb3 copolymer prevented death in mice lethally infected with Stx‐1‐ and Stx‐2‐producing E. coli O157:H7. Thus, we demonstrated that the artificial Gb3 copolymer could neutralize Stx‐1 and the more clinically relevant Stx‐2 in vitro and effectively inhibit Stx toxicity in vivo.

Specific egg yolk immunoglobulin as a new preventive approach for Shiga-toxin-mediated diseases.

Shiga toxins (Stxs) are involved in the development of severe systemic complications associated with enterohemorrhagic Escherichia coli (EHEC) infection. Various neutralizing agents against Stxs are under investigation for management of EHEC infection. In this study, we immunized chickens with formalin-inactivated Stx-1 or Stx-2, and obtained immunoglobulin Y (IgY) from the egg yolk. Anti-Stx-1 IgY and anti-Stx-2 IgY recognized the corresponding Stx A subunit and polymeric but not monomeric B subunit. Anti-Stx-1 IgY and anti-Stx-2 IgY suppressed the cytotoxicity of Stx-1 and Stx-2 to HeLa 229 cells, without cross-suppressive activity. The suppressive activity of these IgY was abrogated by pre-incubation with the corresponding recombinant B subunit, which suggests that the antibodies directed to the polymeric B subunits were predominantly involved in the suppression. In vivo, the intraperitoneal or intravenous administration of these IgY rescued mice from death caused by intraperitoneal injection of the corresponding toxin at a lethal dose. Moreover, oral administration of anti-Stx-2 IgY reduced the mortality of mice infected intestinally with EHEC O157:H7. Our results therefore suggest that anti-Stx IgY antibodies may be considered as preventive agents for Stx-mediated diseases in EHEC infection.

Inhibitory effect of 10-hydroxy-trans-2-decenoic acid on LPS-induced IL-6 production via reducing IκB-ζ expression

The effect of 10-hydroxy-trans-2-decenoic acid (10H2DA), a major fatty acid component of royal jelly, was investigated on LPS-induced cytokine production in murine macrophage cell line, RAW264 cells. 10H2DA inhibited LPS-induced IL-6 production dose-dependently, but did not inhibit TNF-α production. 10H2DA inhibited LPS-induced NF-κB activation in a dose-dependent fashion. In addition, NF-κB activation induced by over-expression of either MyD88 or Toll/IL-1 receptor domain-containing adaptor inducing IFN-β (TRIF) was also inhibited by 10H2DA. Degradation of IκB-α and phosphorylation of IκB kinase-α were not inhibited by 10H2DA. On the other hand, reduction of LPS-induced IκB-ζ expression was discovered. Production of lipocalin-2 and granulocyte colony-stimulating factor (G-CSF), which is dependent on IκB-ζ, was also inhibited by 10H2DA, whereas that of IκB-ζ–independent cytokines/chemokines, such as IFN-β, murine monocyte chemotactic protein-1 (JE), macrophage inflammatory protein (MIP)-1α and MIP-2, was not. Together, 10H2DA specifically inhibited LPS-induced IκB-ζ expression, followed by inhibition of IκB-ζ-dependent gene production. These results suggest that 10H2DA is one of the components of royal jelly to show anti-inflammatory effects and could be a therapeutic drug candidate for inflammatory and autoimmune diseases associated with IκB-ζ and IL-6 production.

Single chain variable fragment antibodies against Shiga toxins isolated from a human antibody phage display library.

Shiga toxins (Stxs) are involved in the pathogenesis of hemolytic-uremic syndrome and other severe systemic complications following enterohemorrhagic Escherichia coli infection in humans. Passive immunotherapies using monoclonal antibodies have been shown to be effective for neutralizing the toxic effects of Stxs. However, animal-derived monoclonal antibodies are sometimes immunogenic and their production is both laborious and expensive. We here report the isolation of single-chain variable fragment antibodies against Stxs by screening a phage display library constructed from a naïve human repertoire. An antibody among the selected clones designated B22 bound to the binding subunits of both Stx-1 and Stx-2, and strongly neutralized the cytotoxicity of Stx-1. This is the first example of a monovalent antibody showing Stx-neutralizing activity. The B22 antibody is also completely naturally occurring in human, which reduces the possibility of adverse immunological effects, and can be easily produced using bacterial protein synthesis systems.

Inhibitory mechanism of 10-hydroxy-trans-2-decenoic acid (royal jelly acid) against lipopolysaccharide- and interferon-β-induced nitric oxide production.

Royal jelly acid, 10-hydroxy-trans-2-decenoic acid (10H2DA), is a major lipid component of royal jelly, which is the exclusive diet of queen honeybees. Previously, we showed partial inhibition of lipopolysaccharide (LPS)-induced NF-κB activation by 10H2DA. In this study, the ability of 10H2DA to inhibit LPS-induced nitric oxide (NO) production was investigated. LPS-induced NO production and inducible NO synthase (iNOS) gene transcription were inhibited by 10H2DA. LPS-stimulated interferon (IFN)-β production, IFN regulatory factor-1 induction and IFN-stimulated response element activation, which are required for iNOS induction, were unaffected by 10H2DA. IFN-β-induced NO production, however, was significantly inhibited by 10H2DA. Furthermore, IFN-β-induced nuclear factor (NF)-κB activation and tumour necrosis factor (TNF)-α production were significantly inhibited by 10H2DA, and TNF-α-induced NF-κB activation was also inhibited by 10H2DA. These results and our previous study suggest that 10H2DA inhibits LPS- and IFN-β-induced NO production via inhibition of NF-κB activation induced by LPS or IFN-β.

Mechanism of inhibition of lipopolysaccharide-induced interferon-β production by 2-aminopurine

2-Aminopurine (2-AP) is widely used as an inhibitor for double stranded RNA-dependent protein kinase (PKR). Previously, we reported that 2-AP inhibits Toll-like receptor (TLR) ligand-induced nitric oxide production through the prevention of interferon (IFN)-β production. In this study, we investigated the mechanisms for 2-AP inhibition of lipopolysaccharide (LPS)-induced IFN-β production. A reporter gene assay showed that LPS-induced IFN-β promoter, but not nuclear factor (NF)-κB, activation was significantly inhibited by 2-AP. IFN-β promoter activation induced by the overexpression of Toll/interleukin-1 receptor domain-containing adaptor inducing IFN-β (TRIF) was significantly inhibited by 2-AP in a dose-dependent manner, while TRIF- or myeloid differentiation primary response gene 88-dependent NF-κB activation was not inhibited. IFN-β promoter activation induced by expression of the downstream signaling molecules, tumor necrosis factor receptor-associated factor family member-associated NF-κB activator-binding kinase 1, inhibitor of NF-κB kinase i and a constitutively active mutant of interferon regulatory factor (IRF)-3, was also inhibited by 2-AP. Another PKR inhibitor harboring the imidazolo-oxindole structure, however, did not affect TRIF signaling molecules-induced IFN-β promoter activation, suggesting that the inhibition of IFN-β transcription by 2-AP is independent of PKR inhibition. Further, we examined the effect of 2-AP on LPS-induced IRF-3 activation by immunoblotting. While 2-AP did not affect LPS-induced phosphorylation of IRF-3, nuclear translocation of IRF-3 was inhibited. Moreover, we revealed that LPS-induced phosphorylation of Akt, another key molecule involved in IRF-3 activation, was inhibited by 2-AP. These results suggest that 2-AP inhibits nuclear translocation of phosphorylated-IRF-3 by inhibiting Akt activation.

Inhibition of interferon-γ-induced nitric oxide production by 10-hydroxy-trans-2-decenoic acid through inhibition of interferon regulatory factor-8 induction

10-Hydroxy-trans-2-decenoic acid (10H2DA) is a major lipid component of royal jelly, a honey bee secretion used to nourish the queen bee and young larvae. In this study, we examined the effect of 10H2DA on interferon (IFN)-γ-induced nitric oxide (NO) production. IFN-γ-induced NO production and activation of the inducible NO synthase promoter were significantly inhibited by 10H2DA. IFN-γ-induced phosphorylation of signal transducer and activator of transcription-1 was not affected by 10H2DA. In contrast, IFN-γ-induced tumor necrosis factor (TNF)-α production and nuclear factor (NF)-κB activation were inhibited by 10H2DA. IFN-γ-mediated induction of interferon regulatory factor (IRF)-8, but not IRF-1, was also inhibited by 10H2DA. IFN-γ-induced TNF-α production followed by activation of NF-κB is known to be essential for NO production. Together, 10H2DA inhibited IFN-γ-induced NO production by inhibiting IRF-8 induction and TNF-α production. 10H2DA might modulate IFN-γ-mediated cellular responses by inhibiting the induction of IRF-8 and IRF-8-dependent genes.