Molecular modelling, chemical synthesis and analytical studies
Longer molecular dynamics with explicit solvent and molecular docking were performed as previously described (Barata et al, 2011a, b, c). The anionic G3.5 PAMAM dendrimer (diaminobutane core) was synthesized by Dendritic Nanotechnologies Michigan. PAMAM-(COONa)64 dendrimer: 1H NMR (500 MHz, D2O, 303K): δ 3.55 − 3.50 (br m, 65H), 3.38 − 3.34 (br m, 56H), 3.18 − 3.10 (br m, 120H), 3.05 − 3.00 (br m, 62H), 2.91 − 2.86 (br m, 123H), 2.72 − 2.67 (br m, 64H), 2.57 − 2.47 (br m, 244H), 1.56 (br s, 4H). 13C NMR (125 MHz, D2O, 303K): δ 179.5, 175.2, 174.6, 51.3, 50.3, 48.9, 48.8, 36.7, 35.6, 32.5.
The anionic G3 PETIM dendrimer was synthesized as previously described with minor modifications (Jain et al, 2010; Jayamurugan & Jayaraman, 2006; Krishna and Jayaraman, 2003). PETIM-(COOH)16·14HCl dendrimer: 1H NMR (500 MHz, D2O, 303K): δ 3.71 − 3.64 (m, 52H), 3.59 − 3.55 (m, 32H), 3.43 − 3.39 (m, 16H), 3.35 − 3.30 (m, 36H), 3.03 − 2.99 (m, 32H), 2.16 − 2.04 (m, 52H). 13C NMR (125 MHz, D2O, 303 K): δ 174.1, 68.0, 67.7, 67.5, 52.3, 50.8, 50.7, 49.5, 28.4, 23.6, 23.5, 23.3.
D-(+)-glucosamine (purity 99%) was conjugated to both the PAMAM dendrimer's carboxylic acid groups and the PETIM dendrimer's carboxylic acid groups using EDCI as previously described (Sam et al, 2010; Shaunak et al, 2004). PAMAM-DG: 1H NMR (500 MHz, D2O, 303K): δ 5.25 (d, J = 3.5 Hz, 5H), 3.96 − 2.99 (m, 555H), 2.93 − 2.61 (m, 235H), 2.11 − 2.02 (m, 10H), 1.84 (br s, 4H), 1.24 − 1.19 (m, 15H).
PETIM-DG: 1H NMR (500 MHz, D2O, 303K): δ 5.26 − 5.23 (m, 3H), 3.96 − 3.04 (m, 218H), 2.93 − 2.88 (m, 30H), 2.69 − 2.66 (m, 14H), 2.18 − 1.90 (m, 64H), 1.27 − 1.19 (m, 8H), 1.11 (t, J = 7.4 Hz, 2H). 13C NMR (125 MHz, D2O, 303 K): δ 177.9, 177.7, 177.5, 174.2, 173.4, 172.5, 172.2, 172.0, 94.8, 90.8, 76.0, 73.9, 73.8, 71.7, 70.8, 70.2, 70.0, 67.7, 67.6, 67.4, 60.8, 60.7, 57.0, 56.7, 55.4, 55.0, 54.2, 54.1, 50.9, 50.8, 50.6, 49.7, 49.5, 49.4, 49.3, 42.9, 42.8, 41.7, 40.2, 37.3, 36.5, 35.9, 35.0, 30.7, 30.2, 30.1, 30.0, 29.5, 29.2, 25.0, 24.3, 23.6, 14.5, 13.6, 13.4.
The NMR-based analytical methods used for compound identity included 1D 1H NMR in 100% D2O and 90:10 H2O:D2O, 2D 1H–13C HSQC NMR and 2D 1H–1H ROESY NMR spectra (500/600/800 MHz Bruker). The MS based analytical methods used for compound identity included MALDI-TOF-MS, trimethylsilyl gas chromatography–mass spectrometry (TMS GC–MS), and esterification (methanolic-hydrochloric acid) of the peripheral carboxylic acids. HPLC with CAD and UV detection at 214 nm, as well as CE (pH 8.55–9.51) were used to determine compound purity as previously described (Barata et al, 2011c; Lalwani et al, 2009a, b; Shaunak et al, 2004).
In vivo biology
Animal studies were performed under a French Ministry of Agriculture licence for animal experimentation no 75-305. They took account of the 3Rs of replacement, reduction and refinement, and were performed in accordance with EU laws and regulations.
Ligated ileal loop segments of 5 cm with a Peyer's patch were surgically created in 1 h in New Zealand White rabbits (Oryctolagus cuniculus) weighing 2.4–2.7 kg as previously described (Perdomo et al, 1994; Schnupf & Sansonetti, 2012). Typically, four ligated loops with Peyer's patches could be created in each rabbit. Wild-type S. flexneri (M90T) in exponential phase growth (107 bacteria) were used to infect each loop, endotoxin free (<0.06 EU/ml) DG added, and the abdomen closed. The DG dose range was 0.1–50 mg/loop in 1 ml normal saline. Rabbits were culled at 12 h for the PAMAM-DG experiments and at 18 h for the PETIM-DG experiments. Tissues were harvested for histology [haematoxylin–eosin (H&E) and immuno-histochemistry], and RNA using TriReagent. Loop fluid bacterial titres were determined using Congo red plates.
The specialized epithelial cells overlying the lymphoid follicles called Peyer's patches are the first to be damaged in an infectious diarrhoea. Mild gut wall damage was histologically defined as an intact epithelial lining, minimal goblet cell depletion and a slight increase in the number of neutrophils and lymphocytes in the surface epithelium and lamina propria. Severe gut wall damage was histologically defined as a destroyed surface epithelium, severe goblet cell depletion and a marked increase in the number of neutrophils and lymphocytes in the crypt epithelium and lamina propria (Islam et al, 1994; Islam & Christensson, 2000; Islam et al, 2001; Raqib et al, 1994; Schnupf & Sansonetti, 2012).
Design and construction of human and rabbit multi-gene quantification plasmids for RT-PCR
messenger RNA quantification was performed by real-time PCR using custom made human and rabbit quantification multi-gene plasmid standards based upon methods and primer pairs as previously described (Corware et al, 2011; Schnupf & Sansonetti, 2012; Shaunak et al, 2004). Using this approach to quantitative RT-PCR, it was possible to reliably and reproducibly identify twofold changes in absolute mRNA copy number.
For the human multi-gene quantification plasmid, cDNA was made from human monocytes treated with LPS (25 ng/ml) for 3 h. This induced expression of otherwise low copy number chemokine and cytokine mRNA transcripts. The cDNA was amplified by PCR using the human primer pairs shown in Supporting Information Table S1. A single human multi-gene plasmid was made by sequential ligation and cloning of the authenticated (by sequencing) PCR amplified products of β-actin, HPRT, interferon-1β, interferon-γ, IL-2, IL-6, IL-8, MIF, MIP-2α, MIP-1α (CCL3), MIP-1β (CCL4), chemokine (C-C motif) ligand 5 (CCL5) and TNF-α into a pGEM-TA (Promega) vector. The plasmid was sequenced to confirm its identity and linearized with SacII.
For the rabbit multi-gene quantification plasmids, cDNA was made from the Peyer's patches of Shigella infected rabbits. This induced expression of otherwise low copy number chemokine and cytokine mRNA transcripts. The cDNA was amplified by PCR using the rabbit primer pairs shown in Supporting Information Table S1. Two multi-gene plasmid standards were constructed by ligation of the amplified and authenticated (by sequencing) PCR products as described above. The first plasmid contained GAPDH, IL-6, IL-8, MIP-1β (CCL4) and TNF-α cloned into a TOPO4 (Invitrogen) vector that was linearized with NotI. The second multi-gene plasmid contained HPRT, interferon-1β, interferon-γ, IL-12 p35, IL-12 p40 and IL-10 cloned into a pGEM-TA vector and linearized with NotI. All other genes studied were cloned as individual plasmids into cloning vectors pGEM TA, TOPO4 or pDrive (Qiagen) and the amplified target gene number normalized using the ‘housekeeping’ HPRT reference gene.
Sample extraction and quantitative RT-PCR for mRNA expression
All samples were lysed with Tri-Reagent (Sigma) for RNA extraction. For the rabbit tissue based samples, 1 ml aliquots of Tri-Reagent lysates were centrifuged in 2 ml Max Tract tubes (Qiagen) at 12,000g for 10 min, the aqueous phase above the wax barrier removed, and the RNA precipitated as per the manufacturer's instructions. For tissue culture derived samples, the Max Tract tube step was omitted. Eight hundred ng of purified RNA was then reverse transcribed with a QuantiTect RT kit (Qiagen) which incorporated a DNAse digestion step. The cDNAs were then diluted 1:4 with RNAse free water.
Standard curves were generated by serial 10-fold dilutions of 10 to 107 copies of the relevant plasmid in water containing 5 ng/µl Lambda DNA. Triplicate 2 µl aliquots of sample cDNA and 2 µl aliquots of each of the standard curve dilutions were amplified with each gene specific primer pair in Jumpstart Sybr Green mix (Sigma) in the same PCR run in a Corbett Research Rotor-Gene 3000 machine. The fluorescence acquisition temperature was set at 5–7°C below the melting temperature of each amplified product in order to eliminate any signal from primer-dimers. The authenticity of each amplified product was confirmed by performing a melt curve analysis at the end of each PCR run.
The absolute target copy number for each gene studied was then normalized using the HPRT gene as the reference ‘housekeeping’ gene, and the result expressed as the absolute copy number of target gene mRNA per 105 absolute HPRT copy number. When the result was expressed as a percentage change against control, the absolute target gene number for both genes was first normalized using the HPRT reference gene copy number and the percentage change then calculated.