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).