TBG-driven transgene expression in tissues of rats injected with AAV2/8.

<p>A) Hepatocyte-restricted eGFP expression in the liver of rats injected with AAV2/8-TBG-eGFP. Wild-type rats were injected at P4 (upper pictures), or at P30 (lower pictures) with 4×10e13 gc/kg of AAV2/8-TBG-eGFP vectors. Animals were sacrificed at P15 (those injected at P4) or at P90 (those injected at P30). The identity of eGFP expressing cells in the liver from injected rats was confirmed by confocal microscopy analysis on sections after immuno-fluorescence with anti-albumin (left panels), anti-CD163 (middle panels, white arrows) or anti CD-31 (right panels, white arrows) antibodies. Representative pictures from rats in each group are shown. Magnification: 63×. B, C) Analysis of vector bio-distribution and eGFP expression in tissues from rats injected with AAV2/8-TBG-eGFP. B) AAV vector genome copies/molecule of diploid genome (gc/mdg) were measured in tissues (reported under each bar) from rats injected at P4 (left, for spleen, kidney, muscle, heart and gonads data are pooled from tissues harvested at P15, P30 and P90) or at P30 (right, all tissues harvested at P90) by Real-time PCR. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033286#s2" target="_blank">Results</a> are reported as mean ± SE. The number of animals analyzed in each group is reported in the corresponding bar. C) eGFP expression was assessed by Western blot analysis on the spleen (70 µg of proteins) , kidney (70 µg of proteins) and heart (150 µg of proteins). Representative animals from each group are shown. −: tissues from uninjected rats; +: liver lysate from a rat injected at P4 with AAV2/8-TBG-eGFP and collected at P15 used as eGFP-positive control (10 ug of proteins).</p

Inclusion of target sequences for miR142-3p in the AAV vector genome does not reduce immune responses to ARSB in MPS VI rats injected with AAV2/8-TBG-hARSB.

<p>MPS VI rats were injected either at P4 or at P30 with AAV2/8-TBG-hARSB or AAV2/8-TBG-hARSB-miR142-Tx4 vectors. Animals were followed up for 6 months after vector delivery when they were sacrificed for analysis of tissue ARSB expression. A) Liver ARSB activity was measured at the time of sacrifice in rats injected at P4 with AAV2/8-TBG-hARSB or AAV2/8-TBG-hARSB-miR142-Tx4. The number of animals analyzed in each group is reported in each bar. NR: control rats AF: uninjected MPS VI rats. * = p<0.05. The p-value between the TBG-hARSB and TBG-hARSB-miR142-Tx4 groups is 0.12. B, C) Serum ARSB activity was measured each month in NR and AF rats (light grey horizontal grids) and in rats injected with either AAV2/8-TBG-hARSB or AAV2/8-TBG-hARSB-miR142-Tx4 vectors at P4 (B) or at P30 (C). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033286#s2" target="_blank">Results</a> are reported as mean ± SE for each group. D, E) Serum anti-ARSB antibodies were measured by ELISA each month in AF rats injected with either AAV2/8-TBG-hARSB or AAV2/8-TBG-hARSB-miR142-Tx4 vectors at P4 (D) or at P30 (E). As negative control anti-ARSB antibodies were measured in sera from NR and AF rats that did not receive AAV vectors (uninjected controls). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033286#s2" target="_blank">Results</a> are reported as mean ± SE for each group.</p

Lysosomal storage does not impact on AAV2/8-mediated liver transduction.

<p>A) Alcyan blue staining performed on liver sections from wild-type (NR) and MPS VI (AF) rats injected with AAV2/8-TBG-eGFP shows GAG storage (blue staining) in AF but not NR rats. Representative pictures from animals in each group are shown. Magnification: 20×. B) NR and AF rats were injected at P30 with 4×10e13 gc/kg of AAV2/8-TBG-eGFP vectors. Animals were sacrificed at P90 and eGFP expression levels were assessed under a fluorescence microscope on sections from transduced livers. Representative pictures from animals in each group are shown. Magnification: 20×. C) eGFP expression levels were assessed by Western blot analysis of liver lysates from the same animals as in panel B. The intensity of eGFP bands was measured and normalized to that of the corresponding tubulin band. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033286#s2" target="_blank">Results</a> are reported as mean ± SE. The number of animals analyzed in each group is reported in each bar. D) AAV vector genome copies/molecule of diploid genome (gc/mdg) were measured by Real-time PCR in livers of rats injected with AAV2/8. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033286#s2" target="_blank">Results</a> are reported as mean ± SE. The number of animals analyzed is reported as before.</p

Inclusion of the WPRE-m variant is associated with decreased hepatocyte transduction levels in rats.

<p>A) Rats were injected at postnatal day (P)30 with 4×10e13 gc/kg of AAV2/8-TBG-eGFP or AAV2/8-TBG-eGFP-WPRE-m vectors. Animals were sacrificed at P90 and eGFP-positive cells were visualized on liver sections under a fluorescence microscope. Representative pictures from animals in each group are shown. Magnification 20×. B) eGFP expression levels were assessed by Western blot analysis on liver lysates from the same animals as in panel A. The intensity of eGFP bands was measured and normalized on that of the corresponding tubulin band. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033286#s2" target="_blank">Results</a> are reported as mean ± SE of three independent experiments. The number of animals analyzed in each group is reported inside each bar. *: p<0.05. C) AAV vector genome copies/molecule of diploid genome (gc/mdg) were measured by Real-time PCR in livers of rats injected with AAV. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033286#s2" target="_blank">Results</a> are reported as mean ± SE of three independent experiments. The number of animals analyzed in each group is reported inside each corresponding bar. The p-value between the TBG-eGFP and TBG-eGFP-WPRE-m groups is 0.44.</p

Effect of p31–43 and p57–68 on [Ca²⁺]_i in Caco-2 cells.

<p>(<b>A</b>) and (<b>B</b>) Single-cell traces representative of the effect of 20 µg/ml p31–43 and p57–68, respectively, on [Ca²⁺]_i. Starting time of perfusion is indicated by the arrows. Ionomycin (1µM) was added as control (grey arrows) at the end of the experiment. (<b>C</b>) and (<b>D</b>) Dose-dependent effect of p31–43 and p57–68, respectively, on [Ca²⁺]_i increase. PCTR (20 µg/ml) served as control. From 40–65 cells were monitored in each experiment. Each bar represents the mean ± SEM of data obtained in three independent experimental sessions. *p<0.05 versus its respective control (basal values); **p<0.05 versus previous concentrations and control.</p

Quantification of tTG activity by the microplate assay.

<p>(<b>A</b>) The microplate assay was performed on 25 µg of cell lysates obtained after treatment. Values are the means ± SD of at least 3 independent experiments in triplicate. *p<0.05 <i>versus</i> tTG basal activity. (<b>F</b>) Inhibition by cystamin of tTG activity induced by ionomycin and p31–43. Values are the means ± SD of three independent experiments in triplicate. *p<0.05 <i>versus</i> the respective activity in the absence of the inhibitor.</p

Microscopic visualization of tTG transamidating activity in Caco-2 cells.

<p>(<b>A</b>) Microscopic visualization of pentylamine-biotin incorporation <i>in situ</i>, ×40, in the presence of a complete medium. Peptides were used at 20 µg/ml, ionomycin at 10 µM, and THP at 1µM. Where indicated, the tTG inhibitor cystamin (200 µM) was added 5 min before treatment. (<b>B</b>) Microscopic visualization of p31–43-biotin and p57–68-biotin incorporation <i>in situ</i>, x40, in the presence of ionomycin. (<b>C</b>) Confocal images of pentylamine-biotin incorporation <i>in situ</i>, x63 (LSM 510 Zeiss microscope), in the absence or presence of p31–43 20 µg/ml. (<b>D</b>) tTG localization (red) in ionomycin-treated cells and superimposition (merge) with intracellular tTG activity (green). Nuclei are in blue (Hoescht staining). Arrows indicate nuclei in which tTG is increased (<b>E</b>) Microscopic visualization of pentylamine-biotin incorporation <i>in situ</i>, ×40, in the presence of a Ca²⁺-free medium.</p

Effect of FCCP on [Ca²⁺]_i increase induced by gliadin peptides in Caco-2 cells.

<p>(<b>A</b>) and (<b>B</b>) Superimposed single-cell traces representative of the effect of 20 µg/ml p31–43 and p57–68, respectively, in a Ca²⁺-free buffer, and in a Ca²⁺-free buffer plus 1µM THP and 300 nM FCCP, on [Ca²⁺]_i. Before peptide perfusion (black arrows), cells were preincubated with FCCP and THP for 10 min (grey arrows). (<b>C</b>) Quantification of the effect of the treatments reported in (A) and (B) on [Ca²⁺]_i. Each bar represents the mean ± SEM of data obtained in three independent experimental sessions. *p<0.05 versus peptide alone; **p<0.05 versus peptide plus THP.</p

Analysis of CHOP expression in Caco-2 cells.

<p>(<b>A</b>) Quantification of CHOP mRNA by real-time RT-PCR after 24 h of treatment with 1 µM THP, or 20 µg/ml p31–43 and p57–68. The amount of CHOP mRNA is normalized to that of GAPDH. Values are the means ± SD of three independent experiments. *p<0.05 versus untreated. (<b>B</b>) Western blot analysis of CHOP protein level after 24 h of treatments. The blot shown is representative of three independent experiments. (<b>C</b>) Densitometric analysis (means of three independent western blot experiments). The amount of CHOP is normalized to that of tubulin. Values are the means ± SD *p<0.05 <i>versus</i> untreated.</p

Analysis of p31–43 induced tTG expression in Caco-2 cells.

<p>(<b>A</b>) Quantification of tTG mRNA by real-time RT-PCR after 24 and 48 h of treatment with 20 µg/ml p31–43 and p57–68. The amount of mRNA of tTG is normalized to that of GAPDH. Values are the means ± SD of three independent experiments. *p<0.05 versus untreated. (<b>B</b>) Western blot analysis of tTG protein level after 48 h of treatment with 20 µg/ml p31–43 and p57–68. The blot shown is representative of three independent experiments. (<b>C</b>) Densitometric analysis (means of three independent western blot experiments) of protein levels after 24 and 48 h of treatments. The amount of tTG is normalized to that of tubulin. Values are the means ± SD *p<0.05 <i>versus</i> untreated.</p