Limulus amoebocyte lysate assay

PMMA particles with mean diameter 6 μm (range 0.1–10 μm) were purchased from a commercial resource (Polysciences, Warrington, PA). Endotoxin was removed from the particles according to the protocol described by Ragab et al. [15 (link)]. The particles were rinsed in ethanol four times and sterilized in 70% ethanol with shaking overnight. The particles were then rinsed four times with PBS and resuspended in serum-free α-MEM. The removal of endotoxin was confirmed by a Limulus amoebocyte lysate assay (Cat number QCL-1000; BioWhittaker, Walkersville, MD) with sensitivity of 0.005 endotoxin unit (EU)/mL.

Plasma endotoxin content was determined by using the limulus amoebocyte lysate assay (Lonza). Briefly, defrosted plasma was diluted 1:10 in pyrogen-free water and then heated at 70 °C for 10 min, to inactivate potential endotoxin-neutralizing agents. Fifty microlitres of heat-inactivated plasma was combined with 50 μl limulus amoebocyte lysate reagent for 10 min at 37 °C and 100 μl of substrate solution were added before reading the absorbance at 405 nm. Plasma endotoxin concentrations were estimated by using a standard curve prepared from kit-supplied Escherichia coli reference standard endotoxin in the same plate.

A Tat expression vector was prepared as described in the previous study.^{43 (link)} To construct a Tat-PDIA3 protein, PDIA3 cDNA was amplified by polymerase chain reaction (PCR) with the use of the following primers: sense primer 5′-CTCGAGATGCGCCTCCGC-3′ and antisense primer 5′-GGATCCTTAGAGATCCTCCTGTGCC-3′. After subcloning the PCR product into a TA cloning vector, it was ligated into the Tat expression vector. A PDIA3 expression vector without the Tat-protein transduction domain was also constructed to be used as a control (Control-PDIA3).
The Tat-PDIA3 and control-PDIA3 plasmids were expressed in Escherichia coli BL21 cells, which were treated with 0.1 mM isopropyl-β-d-thiogalactoside (IPTG; Duchefa, Haarlem, Netherlands) at 18 °C for 8 h, and purified using a Ni²⁺-nitrilotriacetic acid Sepharose affinity column and PD-10 column chromatography (Amersham, Braunschweig, Germany) according to the manufacturer's instructions. The purified proteins were treated using Detoxi-Gel™ endotoxin removing gel (Pierce, Rockford, IL, USA) to remove endotoxins. Endotoxin levels in the proteins were below the detection limit (<0.1 EU/ml) as tested using a Limulus amoebocyte lysate assay (BioWhittaker, Walkersville, MD, USA). The Bradford assay^{44 (link)} was used to estimate the quantity of purified Tat-PDIA3 and control-PDIA3 protein.

Intestinal permeability was determined at 11 and 15 weeks. Plasma samples taken 2 h after oral administration of FITC-dextran were analyzed for amount of fluorescence and plotted on a FITC-dextran standard curve to calculate FITC-dextran concentrations in the plasma. Endotoxin levels in cecum were assessed at 16 weeks using the Limulus Amoebocyte Lysate assay (Lonza) according to the manufacturer’s instructions.

The pCMV-βgal ( = pCMV-LacZ) plasmid (Clontech, St Germain en Laye, France) encoding β-galactosidase, and the pVAX-Der f1 plasmid encoding Der f1, under the control of the human cytomegalovirus immediate promoter was used. The pVAX-Der f1 plasmid was obtained by cloning the Der f1 gene sequence into pVAX (Invitrogen, Courtaboeuf, France) with HindIII and Xho1 restriction enzymes. All plasmids were purified using EndoFree plasmid purification columns (Qiagen, Courtaboeuf, France). They were confirmed to be free of endotoxin contamination (endotoxin <0.1 EU/µg plasmid DNA) by the Limulus amoebocyte lysate assay (Lonza, Clermont-Ferrand, France). Then purified plasmids were subjected to quality control by loading 1.5 µg of plasmid DNA with or without restriction enzyme according manufacturer’s protocol (New England Biolab, Ipswich, UK) of an agarose gel 1% containing ethidium bromide (Sigma, Saint-Quentin Fallavier, France). Samples were left to migrate for 30 min at 100 V in Tris–EDTA buffer. The obtained enzymatic restrictions profiles were conform for each plasmid used. The tetrafunctional block copolymer 704 was kindly supplied by In-Cell-Art (Nantes, France) (more Information in Pitard B et al. [8] (link)). Plasmid DNA was formulated immediately prior to IM injection. Two different doses of each plasmid were used (5 and 10 µg).