Effect of EPIT on systemic response induced in mice after oral sensitization.

<p>(A) Quantity of specific IgE expressed in µg.ml⁻¹ for each group. (B) Quantity of specific IgG2a expressed in µg.ml⁻¹ for each group. (C) Determination of the IgG1/IgG2a ratio expressed for each group. D44 (week 6) concords with the end of sensitization and from D44 to D99 (weeks 7 to 14) with the immunotherapy. Data are expressed as means ± SD for each group of 10 mice. (D–H) Measurement of Th2 cytokine levels (IL-4, IL-5, IL-10, IL-13) and IFN-γ secretion by splenocytes collected from each group of mice (EPIT, Sham, and naïve) immediately after sacrifice. Splenocytes were prepared and stimulated with PPE for 72 h. Cytokines were measured by ELISA. Data are presented as means ± SD for each group of 10 mice. ns: non significant, * p<0.05, ** p<0.01 and *** p<0.001.</p

Establisment of the model of peanut-induced allergic esophagus-gastro-enteropathy by histological and RT-qPCR analyses.

<p>Microscopic analysis of eosinophils at 100× in the esophagus of naive (A) and sensitized (B) mice. Most eosinophils (arrows) are located in the lamina propia, submucosa, and epithelial layer of the sensitized group. (C) Measurement of eosinophil infiltration in the esophagus at 40×; the results are expressed as number of eosinophils per mm² (means ± SD). Cytokine mRNA from esophagus segments was assayed by RT-qPCR. The relative levels of gene expression were calculated by reference to the mRNA levels of SDHA and β-actin in each sample. (D) IL-5, (E) eotaxin, (F) IL-13. Jejunum segments collected and analyzed by microscopy after HES coloration (×40); inflammatory infiltration, particularly of eosinophils is shown in (G). Measurement of the ratio of villous height to crypt depth for each group of 10 mice at 10×. Results are expressed as means ± SD (H). N: naïve mice, S: sensitized mice.* p<0.05, ** p<0.01, *** p<0.001.</p

Effect of EPIT on the induction of peanut-induced allergic esophagus inflammation established by histological and RT-qPCR analyses Microscopic analysis of eosinophils in the esophagus at 40× (A–C) and 100× high-powered fields (D–F).

<p>Most eosinophils (arrows) are located in the lamina propia, submucosa, and epithelial layer of the sham group and to a lesser extent of the EPIT group. A difference in the thickness of epithelium is observed between naive/EPIT and sham. Analysis of (G) chorion inflammation, (H) acanthosis, (I) measurement of eosinophil infiltration in the esophagus and in 40× high-powered fields. Grading for chorion inflammation is as follows: 0 = nothing, 1 = slight diffuse infiltration, 2 = perivascular area. Grading for acanthosis is as follows: 0 = nothing, 1 = layer with more than 4 cells, 2 = layer with more than 6 cells. The epithelium thickness was expressed as mean (µm) ±SD. For eosinophils, the results are expressed as number of eosinophils per mm² (means ± SD). Cytokine mRNA from esophagus segments collected 24 h after stopping peanut diet was assayed by RT-qPCR. Results are presented as mRNA expression of naive, Sham or EPIT animals. The relative levels of gene expression were calculated by reference to the mRNA levels of SDHA and β-actin in each sample. (J) eotaxin, (K) IL-5, (L) IL-13, (M) Foxp3, (N)GATA-3, (O) Tbet. * p<0.05, ** p<0.01.</p

Effect of EPIT on the induction of peanut-induced allergic enteropathy.

<p>(A–F) Jejunum segments collected and analyzed by microscopy after HES coloration (×40). Upper layer: villous atrophia could be observed in the sham group. Lower layer: inflammatory infiltration, particularly of eosinophils is shown. (G) Measurement of the ratio of villous height by crypt depth for each group of 10 mice under 10× high-powered fields. Results are expressed as means ± SD. ns: non significant, ** p<0.01, *** p<0.001.</p

Study design for induction of eosinophilic esophagatis and enteropathy and for the effect of EPIT on the induction of digestive lesions.

<p>(A) Fourty mice were sensitized to peanut proteins in the first phase. Then a resting period with no treatment and no peanut administration was applied. After that, a peanut regimen for 10 days was given to sensitized and naïve mice (n = 40). Mice were then sacrificed to analyze esophagus and jejunum samples by histology and RT-qPCR. (B) Twenty mice were sensitized to peanut proteins in the first phase. Epicutaneous immunotherapy was conducted for 8 weeks in 1à sensitized mice (EPIT) and 10 other sensitized mice received a Sham treatment (Sham). After a sustained oral challenge, mice were sacrificed to analyze esophagus and jejunum samples by histology and RT-qPCR. Blood samples were taken every 2 weeks to measure specific immunoglobulins (IgE, IgG1, IgG2a).</p

Systemic response induced in mice after oral sensitization analyzed in plasma and spleens.

<p>(A) Quantity of specific IgE and IgG2a expressed in µg.ml⁻¹ for each group. Data are expressed as means ± SD for each group mice, D44 after oral sensitization, D89 after the 8-week resting period of peanut free diet. (B–E) Measurement of Th2 cytokine levels (IL-4, IL-5, IL-13) and IFN-γ secretion by splenocytes collected from each group of mice (EPIT, Sham, and naïve) immediately after sacrifice. Splenocytes were prepared and stimulated with PPE for 72 h. Cytokines were measured by ELISA. Data are presented as means ± SD for each group of mice. N: naïve mice, S: sensitized mice. ** p<0.01, *** p<0.001.</p

rAAV.TetR-KRAB/mEpo in the mouse muscle results in Dox-mediated transgene regulation.

<p>(<b>A</b>) Vector design: the vector encoded the murine <i>Epo</i> (<i>mEpo</i>) under the control of a <i>TetO</i>.lCMVlg, <i>TetO</i>.CMVsh, or <i>TetO</i>.PGK promoter and the trans-inhibitor TetR-KRAB under the control of the CAG promoter. pA (<i>mEpo</i>), SV40 polyadenylation signal; pA (TetR-KRAB), BGH polyadenylation signal; ITR, inverted terminal repeat of AAV2. Arrows indicate the transcription start sites. All expression cassettes were cloned between AAV2 ITRs in the same orientation (<i>forward</i>). (<b>B</b>) Follow-up of mEpo levels in animal groups before and after Dox induction. Six to 8 mice were injected IM in the <i>tibialis</i> anterior muscle with a rAAV1 vector harboring either <i>TetO</i>-CMVlg (n = 8), <i>TetO</i>-CMVsh (n = 6) or <i>TetO</i>-PGK (n = 8) promoters at a dose of 3×10⁹ total viral genomes (vg) in a volume of 30 µL. A rAAV1 vector carrying an rtTA-<i>mEpo</i> cassette (<i>forward</i> orientation) was injected at the same dose and volume in the control group (n = 8). Epo levels in the absence and in the presence of Dox are presented as the mean + SD <i>per</i> group in pg/ml. Murine Epo baseline (dotted line) was determined as the mean Epo level (115 pg/ml) in non-injected mice (n = 83). Mann-Whitney statistical test was performed between (-Dox) and (+Dox) conditions for each experimental group. ***: p<0.001, **: p<0.01, *p<0.05.</p

Regulation of d2GFP expression in the rat retina using the TetR-KRAB system over a time period of 48 weeks.

<p>Expression of d2GFP was induced several times with Dox for over 48 weeks to evaluate the long-term regulation and functionality of the regulatory system after subretinal injection of (<b>A</b>) rAAV5.d2GFP.KRAB <i>forward</i> and (<b>B</b>) rAAV5.d2GFP.KRAB <i>opposite</i> vectors. Images of fundus fluorescence were taken 14 days after starting the administration of Dox (+Dox) or the withdrawal of Dox (-Dox) at 15, 20 and 38 weeks post-injection.</p

Regulation of mEpo expression using the TetR-KRAB system in mice over 42 weeks.

<p>Long-term follow up of the mouse group injected with the PGK-TetR-KRAB construct (n = 8) consisted of the monitoring of hematocrit and Epo levels under alternate Dox cycles for 42 weeks. Two subgroups of mice (n = 4 for each group, black and dark gray lines) were monitored with alternated induction kinetics. The hematocrit was measured weekly by collecting ≈40 µL of blood <i>via</i> retro-orbital puncture and is represented as mean values (%) for each group. Discontinous lines correspond to Dox administration periods, and continuous lines to periods during which Dox was withdrawn. The dotted light gray line represents hematocrit mean values in non-injected mice (n = 10 mice <i>per</i> measure). For injected mice, Epo concentration values (pg/ml) are indicated on the hematocrit graphs at the time points they were measured by ELISA, in the presence of Dox (top numbers) and its absence (bottom numbers).</p

rAAV.TetR-KRAB/d2GFP in the rat retina results in Dox-mediated transgene regulation.

<p>(<b>A</b>) Vector design: Vectors encode the destabilized GFP (d2GFP) under the control of the <i>TetO</i>.CMVlg promoter and the trans-inhibitor TetR-KRAB under the control of the CAG promoter. pA (d2GFP), SV40 polyadenylation signal; pA (TetR-KRAB), bovine growth hormone (BGH) polyadenylation signal; pA (bidirectional), SV40 polyadenylation signal; ITR, inverted terminal repeat of AAV2. Both expression cassettes <i>TetO</i>.CMVlg-d2GFP-pA and CAG-TetR-KRAB-pA were cloned between AAV2 ITRs either in the same orientation (<i>forward</i>) using two pA signals or in opposite orientations (<i>opposite</i>) using a bidirectional pA. Arrows indicate the transcription start sites, and the distance between the two promoters is indicated above both constructions. (<b>B and C</b>) Fluorescence retinal images showing induction and de-induction kinetics of d2GFP expression in the retina of representative rats subretinally injected with (<b>B</b>) rAAV5.d2GFP.KRAB <i>forward</i> and (<b>C</b>) rAAV5.d2GFP.KRAB <i>opposite</i>. A volume of 2.5 µL of vector solution was injected subretinally in 12 rats (n = 6 for each vector). Dox at a dose of 10 mg/kg/day was added to (+Dox) or removed from (-Dox) the drinking water, as indicated by the arrows. d2GFP expression was monitored in representative rats by direct retinal imaging after vector delivery (before induction) and following Dox administration or withdrawal during 14 to 21 days at the indicated time points.</p