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Analysis of anti-human CD22 human mAbs affinity and specificity on human PBMCs. a) Affinity determination of anti-human CD22 mAbs using SPR by flowing various concentration of CD22 antibody over CD22 chip-bound. b) Flow cytometry analysis of CD22 mAbs on human PBMC. Human PBMC were labeled with anti human CD19 (APC) and purified CD22 mAbs (clone γ1λ1, γ3λ3-5, γ23κ5-2 or γ27λ26) coupled with Alexa Fluor 568 fluorochrome or with a commercial mouse mAb anti-human CD22 (Ms anti-human CD22). (PPT 666 kb

A: CAR expression in the liver and the adrenal glands.

<p>Total RNAs were extracted from liver biopsies or total adrenal glands and subjected to reverse transcription and PCR amplification. The PCR products were separated by electrophoresis. B and C: Mice were injected systemically with 10⁹ PFU of either AdHwt (n = 7) or AdH[GA]24 (n = 7) or PBS (n = 4). Forty-eight hours later, the animals were culled and the livers and adrenal glands were collected. B: Whole liver were stained for β-galactosidase expression. C: Total RNA were extracted from the adrenal glands (two glands from the same animal were pooled), reversed-transcribed and subjected to quantitative PCR to detect LacZ expression or 18S RNA. The ratio LacZ/18S of adrenal glands collected from animals injected with AdHwt was used as 100%. The data presented are means+SEM. Statistical test: ANOVA (Prism, Graph-Pad softwares).</p

Localization of the kidneys using ^99mTc-DMSA.

<p>Mice were injected intra-peritoneally with 95 MBq of ^99mTc-DMSA. Five hours later, the animals were anaesthetized and SPECT/CT scans performed. A) Transverse, coronal and sagittal sections centered on the kidneys. B) Volume rendering of the whole animal in which the kidneys appear in yellow. Legend: K: kidney, L: left, post: posterior side of the animal.</p

Influence of Factor X on adenovirus transduction in warfarin-treated mice.

<p>A replication-deficient recombinant adenovirus encoding the Lac-Z gene (6×10⁸ PFU) was injected in warfarin-pretreated animals (n = 7) or warfarin-pre-treated, factor X-complemented animals (n = 5). Forty-eight hours later, liver biopsies or adrenal glands were collected. A) Whole liver were stained for β-galactosidase expression. B: Total RNA were extracted from the adrenal glands (two glands from the same animal were pooled), reversed-transcribed and subjected to quantitative PCR to detect LacZ expression or 18S RNA. The ratio LacZ/18S of adrenal glands collected from warfarin-pre-treated, factor X-complemented animals injected with adenovirus was set at 100%. The data presented are means+SEM. Statistical analysis: Student t test (Prism, Graph-Pad softwares).</p

Figure 2

<p><b>Effect of warfarin pre-treatment on adenoviral transgene expression.</b> A replication-deficient recombinant adenovirus encoding the Lac-Z gene (6×10⁸ PFU) was injected in control- (Adenovirus) or warfarin-pretreated animals (Warfarin and Adenovirus). Twenty-four hours later, liver biopsies or adrenal glands were collected. A: Measurement of the β-galactosidase protein in the samples was performed. The data presented are percentages of mean +/− SEM from untreated animals (n = 2), adenovirus-injected animals (n = 10) and adenovirus-injected animals, pre-treated with warfarin (n = 10). 100% represents the average of β-galactosidase activity in the adrenal glands (100% = 7584 β-gal units/mg of protein) or liver (100% = 24805 β-gal units/mg of protein) of adenovirus-injected animals. Statistical test: ANOVA (Prism, Graph-Pad softwares). B) Total RNA were collected from individual adrenal glands or liver biopsies, reversed-transcribed and subjected to quantitative PCR to detect LacZ or 18S RNA. The ratio LacZ/18S of liver or adrenal glands collected from animals injected with adenovirus was set at 100%. Data presented are duplicate determinations from a single adrenal gland and liver biopsy and is representative of 4 independent experiments.</p

Visualization of gene transfer in the liver upon intra-renal injection: Effect of warfarin.

<p>Direct injection of 5×10⁸ PFU Ad-CMV-rNIS in the left kidney was performed on control (A) or warfarin-treated mice (B). Forty-eight hours later, the mice were anaesthetized and SPECT/CT scans performed. The transverse, coronal and sagittal sections presented are centered on the liver. Legend: L: liver, S: Stomach.</p

Effect of warfarin pre-treatment on adenoviral transgene expression following intra-renal administration.

<p>Ad-CMV-rNIS, a replication-deficient recombinant adenovirus encoding the NIS gene (5×10⁸ PFU) was injected into the left kidney of control- (Adenovirus) or warfarin-pretreated animals (Warfarin and Adenovirus). Fourty-eight hours later, liver biopsies and adrenal glands were collected. Total RNA were collected from individual adrenal glands (on the left kidney) or liver biopsies, reversed-transcribed and subjected to quantitative PCR to detect rNIS expression or GAPDH RNA. The ratio rNIS/GAPDH of liver or adrenal glands collected from animals injected with adenovirus was set at 100%. Data presented are triplicate determinations from three adrenal gland and liver biopsies. The data presented are means+SEM. Statistical analysis: Student t test (Prizm, Graph-Pad softwares). **p≤0,01.</p

Visualization of gene transfer in the adrenal gland upon intra-renal injection: Effect of warfarin.

<p>Direct injection of 5×10⁸ PFU Ad-CMV-rNIS in the left kidney was performed on control (A) or warfarin-treated mice (B). Forty-eight hours later, the mice were anaesthetized and SPECT/CT scans performed. The transverse, coronal and sagittal sections presented are centered on the adrenal glands. Legend: AG: adrenal glands, L: liver, K: kidneys, S: stomach, post: posterior side of the animal.</p