Studies of the transmissibility of the agent of bovine spongiform encephalopathy to the domestic chicken

Background

Escherichia coli O157:H7 is the most common serovar of enterohemorrhagic E. coli associated with serious human disease outbreaks. Cattle are the main reservoir with E. coli O157:H7 inducing hemorrhagic enteritis in persistent shedding beef cattle, however little is known about how this pathogen affects cattle health. Jejunal Hemorrhage Syndrome (JHS) has unclear etiology but the pathology is similar to that described for E. coli O157:H7 challenged beef cattle suggestive that E. coli O157:H7 could be involved. There are no effective treatments for JHS however new approaches to managing pathogen issues in livestock using prebiotics and probiotics are gaining support. The first objective of the current study was to characterize pathogen colonization in hemorrhaged jejunum of dairy cattle during natural JHS outbreaks. The second objective was to confirm the association of mycotoxigenic fungi in feeds with the development of JHS and also to identify the presence of potential mycotoxins. The third objective was to determine the impact of a prebiotic, Celmanax?, or probiotic, Dairyman's Choice? paste, on the cytotoxicity associated with feed extracts in vitro. The fourth objective was to determine the impact of a prebiotic or a probiotic on E. coli O157:H7 colonization of mucosal explants and a bovine colonic cell line in vitro. The final objective was to determine if prebiotic and probiotic feed additives could modify the symptoms that preceded JHS losses and the development of new JHS cases.

Findings

Dairy cattle developed JHS after consuming feed containing several types of mycotoxigenic fungi including Fusarium culmorum, F. poae, F. verticillioides, F. sporotrichioides, Aspergillus flavus, Penicillium roqueforti, P. crustosum, P. paneum and P. citrinum. Mixtures of Shiga toxin - producing Escherichia coli (STEC) colonized the mucosa in the hemorrhaged tissues of the cattle and no other pathogen was identified. The STECs expressed Stx1 and Stx2, but more significantly, Stxs were also present in the blood clot blocking the jejunum. Mycotoxin analysis of the corn crop confirmed the presence of fumonisin, NIV, ZEAR, DON, 15-ADON, 3-ADON, NEO, DAS, HT-2 and T-2. Feed extracts were toxic to enterocytes and 0.1% Celmanax? removed the cytotoxicity in vitro. There was no effect of Dairyman's Choice? paste on feed-extract activity in vitro. Fumonisin, T-2, ZEAR and DON were toxic to bovine cells and 0.1% Celmanax? removed the cytotoxicity in vitro. Celmanax? also directly decreased E. coli O157:H7 colonization of mucosal explants and a colonic cell line in a dose-dependent manner. There was no effect of Dairyman's Choice? paste on E. coli O157:H7 colonization in vitro. The inclusion of the prebiotic and probiotic in the feed was associated with a decline in disease.

Conclusion

The current study confirmed an association between mycotoxigenic fungi in the feed and the development of JHS in cattle. This association was further expanded to include mycotoxins in the feed and mixtures of STECs colonizing the severely hemorrhaged tissues. Future studies should examine the extent of involvement of the different STEC in the infection process. The prebiotic, Celmanax?, acted as an anti-adhesive for STEC colonization and a mycotoxin binder in vitro. Future studies should determine the extent of involvement of the prebiotic in altering disease.     

Polymorphisms in the estrogen receptor 1 and vitamin C and matrix metalloproteinase gene families are associated with susceptibility to lymphoma

Background

Non-Hodgkin lymphoma (NHL) is the fifth most common cancer in the U.S. and few causes have been identified. Genetic association studies may help identify environmental risk factors and enhance our understanding of disease mechanisms.

Methodology/Principal Findings

768 coding and haplotype tagging SNPs in 146 genes were examined using Illumina GoldenGate technology in a large population-based case-control study of NHL in the San Francisco Bay Area (1,292 cases 1,375 controls are included here). Statistical analyses were restricted to HIV- participants of white non-Hispanic origin. Genes involved in steroidogenesis, immune function, cell signaling, sunlight exposure, xenobiotic metabolism/oxidative stress, energy balance, and uptake and metabolism of cholesterol, folate and vitamin C were investigated. Sixteen SNPs in eight pathways and nine haplotypes were associated with NHL after correction for multiple testing at the adjusted q<0.10 level. Eight SNPs were tested in an independent case-control study of lymphoma in Germany (494 NHL cases and 494 matched controls). Novel associations with common variants in estrogen receptor 1 (ESR1) and in the vitamin C receptor and matrix metalloproteinase gene families were observed. Four ESR1 SNPs were associated with follicular lymphoma (FL) in the U.S. study, with rs3020314 remaining associated with reduced risk of FL after multiple testing adjustments [odds ratio (OR) = 0.42, 95% confidence interval (CI) = 0.23–0.77) and replication in the German study (OR = 0.24, 95% CI = 0.06–0.94). Several SNPs and haplotypes in the matrix metalloproteinase-3 (MMP3) and MMP9 genes and in the vitamin C receptor genes, solute carrier family 23 member 1 (SLC23A1) and SLC23A2, showed associations with NHL risk.

Conclusions/Significance

Our findings suggest a role for estrogen, vitamin C and matrix metalloproteinases in the pathogenesis of NHL that will require further validation.     

A novel mimetic antigen eliciting protective antibody to Neisseria meningitidis.

Molecular mimetic Ags are of considerable interest as vaccine candidates. Yet there are few examples of mimetic Ags that elicit protective Ab against a pathogen, and the functional activity of anti-mimetic Abs has not been studied in detail. As part of the Neisseria meningitidis serogroup B genome sequencing project, a large number of novel proteins were identified. Herein, we provide evidence that genome-derived Ag 33 (GNA33), a lipoprotein with homology to Escherichia coli murein transglycosylase, elicits protective Ab to meningococci as a result of mimicking an epitope on loop 4 of porin A (PorA) in strains with serosubtype P1.2. Epitope mapping of a bactericidal anti-GNA33 mAb using overlapping peptides shows that the mAb recognizes peptides from GNA33 and PorA that share a QTP sequence that is necessary but not sufficient for binding. By flow cytometry, mouse antisera prepared against rGNA33 and the anti-GNA33 mAb bind as well as an anti-PorA P1.2 mAb to the surface of eight of nine N. meningitidis serogroup B strains tested with the P1.2 serosubtype. Anti-GNA33 Abs also are bactericidal for most P1.2 strains and, for susceptible strains, the activity of an anti-GNA33 mAb is similar to that of an anticapsular mAb but less active than an anti-P1.2 mAb. Anti-GNA Abs also confer passive protection against bacteremia in infant rats challenged with P1.2 strains. Thus, GNA33 represents one of the most effective immunogenic mimetics yet described. These results demonstrate that molecular mimetics have potential as meningococcal vaccine candidates.     

Mimicking the nicotinic receptor binding site by a single chain Fv selected by competitive panning from a synthetic phage library

We have developed a novel competitive method to select from a phage display library a single chain Fv which is able to mimic the alpha-bungarotoxin binding site of the muscle nicotinic receptor. The single chain Fv was selected from a large synthetic library using alpha-bungarotoxin-coated magnetic beads. Toxin-bound phages were then eluted by competition with affinity purified nicotinic receptor. Recognition of the toxin by the anti-alpha-bungarotoxin single chain Fv was very similar to that of the receptor, such as indicated by the epitope mapping of alpha-bungarotoxin through overlapping synthetic peptides. Moreover, several positively charged residues located in the toxin second loop and in the C-terminal region were found to be critical, to a similar extent, for toxin recognition by the single chain Fv and the receptor. However, although the anti-alpha-bungarotoxin single chain Fv seems to mimic the toxin binding site of the nicotinic receptor, it does not bind other nicotinic agonists or antagonists. Our results suggest that competitive selection of anti-ligand antibody phages can allow the production of receptor-mimicking molecules directly and exclusively targeted at one specific ligand. Since physiologically and pharmacologically different ligands can produce opposite effects on receptor functions, such selective ligand decoys can have important therapeutic applications.     

Iron release in erythrocytes and plasma non protein-bound iron in hypoxic and non hypoxic newborns

Iron is released in a desferrioxamine (DFO)-chelatable form (DCI) when erythrocytes are challenged by an oxidative stress. In β-thalassemic erythrocytes, both DCI content and release (after aerobic incubation for 24 h) are increased and correlated with the fetal hemoglobin (HbF) levels. Since erythrocytes from newborns have an extremely high content of HbF and are exposed to conditions of oxidative stress, the release of iron in these erythrocytes was investigated. The erythrocyte DCI content was increased in preterm but not in term newborns as compared to adults, while the release was increased in both preterm and term erythrocytes. The level of plasma non protein-bound iron (NPBI), which was not detectable in adults, was much higher in preterm than in term newborns. When term plus preterm newborns were divided in two groups, normoxic and hypoxic, according to cord blood pH, it was found that both iron release and NBPI were markedly higher in the hypoxic newborns compared to normoxic ones. Similar results were also obtained when the preterm and term infants were considered separately on the basis of cord blood pH. Therefore, iron release and NPBI are higher when conditions of hypoxia occur. In fact, when the values for iron release and NPBI were separately plotted against cord blood pH values, significant negative correlations were seen in both cases. NPBI was correlated with iron release seen in all the newborns and a significant part of the released iron could be recovered into the incubation medium at the end of the incubation.     

Peptide-protein interactions studied by surface plasmon and nuclear magnetic resonances

The structural features of the complexes that alpha-bungarotoxin forms with three different synthetic peptides, mimotopes of the nicotinic acetylcholine receptor binding site, have been compared to the corresponding nuclear magnetic resonance (NMR) and surface plasmon resonance (SPR) data. For the considered peptides, the observed different affinities towards the toxin could not be accounted simply by static structural considerations. A combined analysis of the SPR- and NMR-derived dynamic parameters shows new correlations between complex formation and dissociation and the overall pattern of intramolecular and intermolecular nuclear Overhauser effects. These features could be crucial for a rational design of protein ligands.     

A branched peptide mimotope of the nicotinic receptor binding site is a potent synthetic antidote against the snake neurotoxin alpha-bungarotoxin

We previously produced synthetic peptides mimicking the snake neurotoxin binding site of the nicotinic receptor. These peptide mimotopes bind the snake neurotoxin alpha-bungarotoxin with higher affinity than peptides reproducing native receptor sequences and inhibit toxin binding to nicotinic receptors in vitro; yet their efficiency in vivo is low. Here we synthesized one of the peptide mimotopes in a tetrabranched MAP form. The MAP peptide binds alpha-bungarotoxin in solution and inhibits its binding to the receptor with a K(A) and an IC(50) similar to the monomeric peptide. Nonetheless, it is at least 100 times more active in vivo. The MAP completely neutralizes toxin lethality when injected in mice at a dose compatible with its use as a synthetic antidote in humans. The in vivo efficacy of the tetrameric peptide cannot be ascribed to a kinetic and thermodynamic effect and is probably related to different pharmacokinetic behavior of the tetrameric molecule, with respect to the monomer. Our findings bring new perspectives to the therapeutic use of multimeric peptides.     

NMR structure of alpha-bungarotoxin free and bound to a mimotope of the nicotinic acetylcholine receptor

A combinatorial library approach was used to produce synthetic peptides mimicking the snake neurotoxin binding site of nicotinic receptors. Among the sequences, which inhibited binding of alpha-bungarotoxin to muscle and neuronal nicotinic receptors, HRYYESSLPWYPD, a 14-amino acid peptide with considerably higher toxin-binding affinity than the other synthesized peptides, was selected, and the structure of its complex with the toxin was analyzed by NMR. Comparison of the solution structure of the free toxin and its complex with this peptide indicated that complex formation induced extensive conformational rearrangements mainly at finger II and the carboxy terminus of the protein. The peptidyl residues P10 and Y4 seemed to be critical for peptide folding and complex stability, respectively. The latter residue of the peptide strongly interacted with the protein by entering a small pocket delimited by D30, C33, S34, R36, and V39 toxin side chains.     

Proteomic approach to the identification of voltage-dependent anion channel protein isoforms in guinea pig brain synaptosomes

Voltage-dependent anion channel (VDAC) proteins are small, abundant, pore-forming proteins belonging to the eukaryotic mitochondrial porins. At least three different VDAC genes have been identified in vertebrates. VDAC proteins are known to play an essential role in cellular metabolism and in the early stages of apoptosis. A proteomic approach, consisting of two-dimensional gel electrophoresis followed by two-dimensional immunoblotting with anti-VDAC and anti-phosphotyrosine antibodies and by matrix-assisted laser desorption/ionization-time of flight mass spectrometry, was exploited to define the expression pattern of VDAC isoforms in guinea pig brain synaptosomes, both in normoxic and hypoxic conditions. In this way a total of five different VDAC isoforms were identified, as both VDAC1 and VDAC2 were detected in more than one electrophoretic spot. Moreover, VDAC isoforms selectively undergo hypoxia-induced tyrosine phosphorylation, suggesting that tyrosine phosphorylation may contribute to the modulation of VDAC protein function/conformation or interaction with other proteins in hypoxic conditions.