TLR3 but not TLR7/8 ligand induces allergic sensitization to inhaled allergen

Epidemiological studies suggest that viral infections during childhood are a risk factor for the development of asthma. However, the role of virus-specific pattern recognition receptors in this process is not well defined. In the current study, we compare the effects of the inhaled viral TLR ligands polyinosinic-polycytidylic acid (TLR3) and resiquimod (TLR7/8) on sensitization to a model allergen (OVA) in a murine model. Both compounds enhance the migration, activation, and Ag-processing of myeloid dendritic cells from the lung to the draining lymph nodes comparable to the effects of LPS. Application of polyinosinic-polycytidylic acid [poly(I:C)] or LPS induces production of allergen-specific IgE and IgG1, whereas resiquimod (R848) had no effect. In addition, rechallenge of mice with OVA resulted in airway inflammation and mucus production in animals that received either poly(I:C) or LPS but not after application of R848. In summary, these results show that activation of TLR3 in combination with inhaled allergen results in induction of dendritic cell activation and migration similar to the effects of LPS. This leads to the development of allergic airway disease after allergen rechallenge, whereas mice treated with R848 did not develop allergic airway disease. These findings give further insight into the effects of stimulation of different TLRs on the development of asthma.     

Self-assembly and structural characterization of Echinococcus granulosus antigen B recombinant subunit oligomers

Echinococcus granulosus antigen B is an oligomeric protein of 120-160 kDa composed by 8-kDa (AgB8) subunits. Here, we demonstrated that the AgB8 recombinant subunits AgB8/1, AgB8/2 and AgB8/3 are able to self-associate into high order homo-oligomers, showing similar properties to that of parasite-produced AgB, making them valuable tools to study AgB structure. Dynamic light scattering, size exclusion chromatography and cross-linking assays revealed approximately 120- to 160-kDa recombinant oligomers, with a tendency to form populations with different aggregation states. Recombinant oligomers showed helical circular dichroism spectra and thermostability similar to those of purified AgB. Cross-linking and limited proteolysis experiments indicated different degrees of stability and compactness between the recombinant oligomers, with the AgB8/3 one showing a more stable and compact structure. We have also built AgB8 subunit structural models in order to predict the surfaces possibly involved in electrostatic and hydrophobic interactions during oligomerization.     

Attitudes of Dutch Citizens toward Sow Husbandry with Regard to Animals,Humans, and the Environment

The pig industry is struggling with negative attitudes of people toward sow husbandry. To be able to respond to these attitudes, the pig industry first has to understand people’s attitudes. The first objective of this study was to determine the attitudes of Dutch people toward sow husbandry with regard to animals, humans, and the environment. The second objective was to group people based on their attitudes toward sow husbandry and determine and compare the socio-demographic characteristics of these groups. An online survey was conducted in the Netherlands and there were 1,607 respondents. On average, respondents had negative attitudes toward all issues defined in this study. The most negative attitudes were toward the effect on both animals and consumers of the use of antibiotics, the number of animals kept per square meter, the possibility for animals to go outside, food safety risks, public health risks, and environmental waste. The findings indicate the importance of considering all the issues identified in this study during the process of developing measures to improve people’s attitudes toward sow husbandry. Respondents could be divided into four clusters; each cluster represented different attitudes toward sow husbandry and had different socio-demographic characteristics. This makes it possible for the pig industry to assign people with specific socio-demographic characteristics to one of the clusters and predict their attitudes toward sow husbandry. Knowledge of these attitudes enables the pig industry to predict how different groups of people will respond to different animal welfare measures. Results of this study are therefore useful for the pig industry to help improve people’s attitudes toward sow husbandry.     

Structural basis for inhibition promiscuity of dual specific thrombin and factor Xa blood coagulation inhibitors

BACKGROUND: A major current focus of pharmaceutical research is the development of selective inhibitors of the blood coagulation enzymes thrombin or factor Xa to be used as orally bioavailable anticoagulant drugs in thromboembolic disorders and in the prevention of venous and arterial thrombosis. Simultaneous direct inhibition of thrombin and factor Xa by synthetic proteinase inhibitors as a novel approach to antithrombotic therapy could result in potent anticoagulants with improved pharmacological properties. RESULTS: The binding mode of such dual specific inhibitors of thrombin and factor Xa was determined for the first time by comparative crystallography using human alpha-thrombin, human des-Gla (1--44) factor Xa and bovine trypsin as the ligand receptors. The benzamidine-based inhibitors utilize two different conformations for the interaction with thrombin and factor Xa/trypsin, which are evoked by the steric requirements of the topologically different S2 subsites of the enzymes. Compared to the unliganded forms of the proteinases, ligand binding induces conformational adjustments of thrombin and factor Xa active site residues indicative of a pronounced induced fit mechanism. CONCLUSION: The structural data reveal the molecular basis for a desired unselective inhibition of the two key components of the blood coagulation cascade. The 4-(1-methyl-benzimidazole-2-yl)-methylamino-benzamidine moieties of the inhibitors are able to fill both the small solvent accessible as well as the larger hydrophobic S2 pockets of factor Xa and thrombin, respectively. Distal fragments of the inhibitors are identified which fit into both the cation hole/aromatic box of factor Xa and the hydrophobic aryl binding site of thrombin. Thus, binding constants in the medium-to-low nanomolar range are obtained against both enzymes.