Human AAT inhibited pDC maturation and cytokine secretion.

<p>BM pDCs from B6 mice were differentiated with or without hAAT using Flt3L for 8 days then stimulated with 10μg/ml CpG for an additional 24 hr prior to FACS analysis. (A) CD80, (B) CD40, and (C) CCR9 expression. (D) TNF-α and (E) IL-6 levels in pDC culture media. P values of One-Way-ANOVA using Tukey’s post-hoc test are indicated as * P<0.05; ** P<0.01; *** P<0.001, n = 3.</p

Human AAT inhibited cDC maturation and cytokine secretion.

<p>BM cDCs from B6 mice were generated in vitro in the presence of GM-CSF and IL-4 for 4 days with or without hAAT and then stimulated with 0.5 μg/ml LPS or 10μg/ml CpG for an additional 24 hr prior to FACS analysis. (A) CD80, CD86 and I-A^b expression (measured as mean fluorescence intensity, MFI) in B6 DCs stimulated with LPS. (B) TNF-α, IFNI, and IL-1β secretions in supernatants of B6 DCs stimulated with LPS. (C) CD80, CD86 and I-Ab expression in B6 DCs stimulated with CpG. (D) Secretion of TNF-α, IFN-I, IL-1β, and IL-6 by B6 DCs stimulated with CpG. P values of One-Way-ANOVA using Tukey’s post-hoc test are indicated as * P<0.05; ** P<0.01; *** P<0.001, n = 3.</p

<i>In vivo</i> hAAT treatment attenuated BMDCs differentiation and maturation in MRL/lpr mice.

<p>BMDCs from MRL/lpr mice treated with hAAT or PBS for 11 weeks were stimulated with LPS for 24 hrs. (A) Percentages of CD11c⁺ and (B) CD11b⁺CD11c⁺. (C-D) Percentages of CD80⁺ (C) and I-A (D) expressing BMDCs. P values of Student’s <i>t</i>-test are indicated as * P<0.05; ** P<0.01; *** P<0.001.</p

Detection of endogenous mouse AAT, exogenous human AAT, and anti-human AAT neutralizing antibody in MRL/lpr mice (N = 10) by ELISA.

<p>(A) Mouse AAT levels (relative unit to C57BL/6) in PBS-treated MRL mice. (B) hAAT levels detected in hAAT injected group. Note: at week 2 and 8, animals were bled at 2 days after hAAT injection; at week 4, animals were bled at 1 day after the injection; at week 11, animals were bled 3 days after the injection. Dashed line is the lower limit of quantification (LLOQ). The serum concentration of human AAT from the PBS-treated group was below LLOQ. (C) Relative levels of anti-human AAT neutralizing antibody in MRL/lpr mice following multiple-dose human AAT administrations in MRL/lpr mice.</p

Human AAT treatment attenuated lupus nephritis in MRL/lpr mice.

<p>(A) Proteinuria scores after 9 weeks after hAAT treatment (Mann-Whitney test). (B) Albuminuria levels after 11 weeks of hAAT treatment. *P = 0.0727. (C) Average area of glomeruli (um²). (D) Average number of nuclei per glomerulus. **P<0.01 and***P<0.001 by student’s <i>t</i>-test. (F) Representative PAS-stained kidney sections from control MRL/lpr mice and hAAT treated MRL/lpr mice. All imaged were photographed at the same magnification (20X).</p

Human AAT inhibited autoantibodies production in MRL/lpr mice.

<p>Disease development was evaluated in MRL/lpr mice after 11 weeks of treatment with hAAT or PBS (n = 10 per group). (A) Interscapular lesion surface (cm²). (B) Lymph node weight (cervical, brachial, and inguinal). (C) Terminal serum IFN-I, (D) BAFF and (E) TNF-α. (F) Terminal serum anti-dsDNA IgG. (G) Serum ANA staining. Representative images are shown on the left, and FITC relative intensities are graphed on the right. * P<0.05, and*** P<0.001 by Student’s <i>t</i>-test.</p

The survival of bioencapsulated ADSCs in the liver 2 weeks after <i>in situ</i> transplantation.

<p>(A) Detection of intact alginate microspheres 2 hours after transplantation; (B) The enlarged picture of picture A; (C) The hAAT positive donor cells (brown) are detected surrounding the degraded alginate microsphere (DA) 2 weeks after transplantation; (D) The hAAT positive donor cells are detected surrounding the residual alginate microsphere (RA) 2 weeks after transplantation. (E-F) Validation of the specificity of anti-hAAT antibody: (E) Human liver stained by anti-hAAT antibody as a positive control, whereas (F) Mouse liver stained by anti-hAAT antibody as a negative control (N = 2).</p

The distribution of free ADSCs in different organs 3 weeks after intrasplenic. injection.

<p>(A) Pancreas; (B) Brain; (C) Kidney; (D) Muscle; (E) Testes; (F) Intestine; (G) Spleen; (H) Lung; and (I) Bone Marrow. The arrows indicated hAAT positive cells (N = 4).</p

The distribution of bioencapsulated ADSCs in different organs 3 weeks after in situ transplantation.

<p>(A) Pancreas; (B) Brain; (C) Kidney; (D) Muscle; (E) Testes; (F) Intestine; (G) Spleen; (H) Lung; and (I) Bone Marrow. The arrows indicated hAAT positive cells (N = 4).</p

Characterization of adipose tissue derived stem cells (ADSCs) isolated from M-hAAT transgenic mice.

<p>(A) Representative pictures of FACS analysis showing isolated mouse ADSC expressed mesenchymal stem cell markers CD90 and CD105, but not hematopoietic lineage markers, such as CD45, or endothelial cell markers, such as CD31; (B) Detection of human AAT protein in cell culture medium by ELISA; (C) Detection of mouse albumin in cell culture medium by ELISA. Mouse albumin was used as a positive control (PC) and cell culture medium from RAW264.7 was used as a negative control.</p