Materials and Methods
LL particles. Aqueous suspensions of LL particles in the tens to hundreds of µm (largest dimension) range were prepared from hydatid walls obtained from cattle naturally infected with E. granulosus sensu stricto from Uruguay. Parasite samples were genotyped by amplification and sequencing of mitochondrial cytochrome c oxidase according to (23); all samples in the study corresponded to genotype G1. Particles were prepared as in (16), using freeze-drying for the dehydration step and applying the modification described next. CaCl2 (0.5 mM) was included in the initial washes and in the rehydration step (omitting EDTA) in order to preserve the calcium InsP 6 deposits (9, 24). This yielded a preparation that contains all the components found in the native LL, which we called “wpLL” (from “w hole p articulatel aminated l ayer”). A fraction of wpLL was treated with EDTA-containing buffer so as to obtain the particulate preparation that contains the mucin meshwork but lacks calcium InsP 6, which we previously termed pLL (16). A fraction of pLL was treated with sodium periodate in order to destroy the terminal monosaccharide residues of the mucin glycans followed by sodium borohydride to reduce to alcohols the aldehyde groups formed (“pLL periodate”), or treated with sodium borohydride only (“pLL mock”) (16). All final materials were kept as suspensions in sterile PBS (with 0.5 mM CaCl2 in the case of wpLL) containing antibiotic/antimycotic, at 4°C for 6 months at most (16). The dry mass content of the final preparations was determined as in (16). Precautions to exclude endotoxin contamination were applied as in (16). The final materials were assumed not to contain biologically significant levels of endotoxins on the basis of extensive experience with previous batches in terms of negative LAL tests and lack of elicitation of inflammatory cytokines from bone marrow-derived dendritic cells (16, 18, 25).