E toxate

Monolayers of Spodoptera frugiperda Sf9 insect cells (Invitrogen, catalogue number B82501) grown in Excell medium culture (Sigma) at 27°C were infected with budded virions of AcMNPV obtained from BaculoGold (Becton Dickinson Argentina S.R.L.). To purify BVs, we infected the cells at a multiplicity of infection (moi) of 0.05. At 5 dpi, the supernatants containing BVs were harvested and cell debris were removed by centrifugation (4,000× g, 15 min, 8°C). Viral stocks were stored at 4°C until use. The moi used for polyhedron purification was 0.5. At 5 dpi, cells were harvested, lysed with SDS 0.5%, and sedimented by centrifugation (5,000× g, 10 min, 4°C). After two washes, one with NaCl 0.5 M and another with distilled water, the stock of polyhedra was stored at -80°C until use. ODVs were released from the polyhedra by dissolving them in 0.1 M sodium carbonate solution. Partially and non-dissolved polyhedra were removed by centrifugation (5,000× g, 10 min, 4°C).
All the material used in the purification procedures was treated with E-TOXATE (Sigma) to eliminate traces of endotoxin.

Specific pathogen-free, male, 6- to 8-week-old BALB/c mice were purchased from the National Laboratory Animal Center, Republic of China. Der p extract (1 g lyophilized whole body extract in ether; Allergon, Engelholm, Sweden) was dissolved in pyrogen-free isotonic saline, filtered through a 0.22 mm filter, and stored at −80°C until further use. LPS levels in the Der p preparations were <0.96 EU/mg of Der p (limulus amebocyte lysate test, E-Toxate; Sigma-Aldrich, St. Louis, MO). FUT (Futhen; Torii Pharmaceuticals Co, Chiba, Japan), FOY (Ono Pharmaceutical Co, Osaka, Japan), and UTI (Mochida Pharmaceuticals Co, Tokyo, Japan), which have been used clinically, were dissolved in sterile PBS before use.

To prepare T. congolense whole cell extract (WCE), isolated parasites were resuspended in TSG at a final concentration of 10⁸/ml and then subjected to 3–5 sonication cycles (5 min per cycle). Thereafter, the sonicate was further subjected to freeze/thawing (at −80°C) up to about 8 cycles (30 min/cycle), aliquoted and stored at −80°C until used. Endotoxin level in WCE preparations was determined by the LAL kit (E-TOXATE, Sigma) according to the manufacturer's suggested protocol. Endotoxin level was <0.05 EU/ml.

Human recombinant Hsp70 was purified from Escherichia coli cells transformed with pMSHsp70 plasmid and detoxified with the use of polymyxin B–agarose gel (Sigma, St.Loise, MO, USA) as described elsewhere [56 (link)] The concentration of bacterial lipopolysaccharide in the final preparation was lower than 0.1 MU/mL, according to LAL test (E-toxate, Sigma, St. Louis, USA); this value is much lower than one that can have any endotoxic effect.

Antibiotics (gentamicin [Gm], ciprofloxacin [Cip], and meropenem [Mero]) were purchased from Sigma-Aldrich (United States Pharmacopeia [USP]) (Reference Standard grade). All compounds were dissolved in endotoxin-free water (E-Toxate; Sigma-Aldrich). For in vivo experiments, compounds were diluted into 0.9% NaCl (Sigma-Aldrich).

Peptides HHC-10 (KRWWKWIRW-NH₂) [37 (link)], 1002 (VQRWLIVWRIRK-NH₂) [38 (link)], 1018 (VRLIVAVRIWRR-NH₂) [39 (link)] and the D-enantiomer DJK-5 (VQWRAIRVRVIR-NH₂) [25 (link)] were synthesized by CPC Scientific using solid-phase 9-flurenylmethoxy carbonyl (Fmoc) chemistry and purified to >95% purity using reverse-phase high-performance liquid chromatography (HPLC). The lyophilized peptides were resuspended in endotoxin-free water. The antibiotics gentamicin, ciprofloxacin, meropenem, erythromycin, clindamycin, vancomycin, azithromycin, and colistin were purchased from Sigma-Aldrich at a United States Pharmacopeia (USP) Reference Standard grade. erythromycin and azithromycin were initially dissolved in 70% ethanol, while all other antibiotics were dissolved in endotoxin-free water (E-Toxate, Sigma-Aldrich). Antibiotics and peptides were further diluted into saline (Sigma-Aldrich) for in vivo application.