Representative islet profiles, sampling of sections, counting Q⁻ islets.

<p>Example of an islet profile of a control (A) and a GIPR^dn transgenic mouse (B) immunohistochemically stained for insulin; (C) Sampling scheme for drawing primary and reference sections; (D) Primary section and (E) reference section for counting Q⁻ islets, one Q⁻ may be counted in the example (arrow in E).</p

Islet-cell replication, isolated beta-cells, and islet-cell apoptosis.

<p>(A) number of BrdU positive islet cells per 10⁵ cells (N_(BrdU)), (B) total volume of isolated beta-cells (V_(Neo,Pan), (C) number of apoptotic islet cells per 10⁵ cells (N_(Apoptosis)). Open bars: female control mice; filled bars: female GIPR^dn transgenic mice; Data represent means and SEM. * p<0.05 vs. age-matched control; +p<0.05 vs. previous time point.</p

Lipid peroxidation.

<p>Serum malondialdehyde (MDA) levels of GIPR^dn transgenic (tg) and control (co) mice at 45, 90 and 180 days of age. Data represent means and SEM;</p><p>*p<0.05 vs. age-matched control;</p><p>+ p<0.05 vs. previous time point.</p

In vivo insulin secretion studies at 10 days of age.

<p>(A) Blood glucose, (B) serum insulin levels, (C) increase of serum insulin levels from basal levels (fold insulin secretion), (D) serum insulin to pancreatic insulin ratio; Open bars: control mice; filled bars: GIPR^dn transgenic mice; Data represent means and SEM. * p<0.05 vs. age-matched control; + p<0.05 vs. basal values.</p

In vivo investigations.

<p>(A) Randomly fed (10 days) and fasting blood glucose (45-180 days), and (B) randomly fed (10 days) and postprandial serum insulin levels (45–90 days), (C) insulin tolerance test, (D), and area under glucose curve (AUC) during insulin tolerance test shown in C; co, control; tg, GIPR^dn transgenic; Data represent means and SEM, * p<0.05 vs. age-matched control.</p

Quantitative-stereological investigations of the endocrine pancreas.

<p>(A) Total islet volume (V_(Islets,Pan), (B) total beta-cell volume (V_{(B-cells,Islets)}, (C) Number of islets (N_{(Islets, Pan)}, (D) mean islet volume (v_(islets), (E) beta-cell number (N_{(B-cells,Islets)}, (F) mean beta-cell volume (v_(B-cells). Open bars: female control mice; filled bars: female GIPR^dn transgenic mice; Data represent means and SEM. * p<0.05 vs. age-matched control; +p<0.05 vs. previous time point.</p

Biological evaluation of transgenic founder animals.

<p>(A) Binding studies of LEA29Y to the CD80/CD86-positive porcine B cell line L23. Cells were incubated with serial dilutions of sera from the three founder animals as well as from wild-type controls. Binding of LEA29Y was assessed by using a goat anti-human IgG-FITC antibody. Labeled cells were analyzed by flow cytometry. The results are expressed as the mean fluorescence intensity. (B) Inhibition of human anti-pig T cell proliferation by serum from LEA29Y-tg pigs. 10⁵ human PBMC were stimulated with 2 x 10³ irradiated L23 cells. Cultivation was performed in the presence of sera taken from transgenic and wild-type control pigs. Proliferation was determined after 5 d by [³H]-TdR incorporation (ccpm, counts/min). The results are expressed as the mean ± SD of triplicate cultures.</p

T cell subpopulations in transgenic pigs.

<p>PBMCs were isolated from LEA pigs, 3-week-old wild-type (WT) piglets and 6-month old WT pigs and analyzed by flow cytometry for the phenotype of CD4⁺ T cells and production of IFN-γ and TNF-α. (A) CD4 and CD8α expression on lymphocytes. Total CD4⁺ T cells were gated (black gates, red population) and analyzed for CD8α expression (green gates). The numbers indicate percent CD8α⁺ cells within CD4⁺ T cells. (B) CD8α and CD27 expression on gated CD4⁺ T cells. (C) CD8α and CD45RC expression on gated CD4⁺ T cells. (D) IFN-γ and TNF-α production in gated CD4⁺ T cells following stimulation with PMA/Ionomycin for four hours as a representative flow cytometry plot (upper panel) and a graph comprising data of all animals as the mean value + standard deviation (lower panel). (E) CD4 and Foxp3 expression of lymphocytes. The numbers indicate the percent of gated CD4⁺Foxp3⁺ regulatory T cells within lymphocytes. The data are representative of two three-week-old WT pigs, two tg pigs and two six-month-old pigs.</p

Lymph node histology in transgenic pigs.

<p>Developmental stage of lymph nodes of a 6-month-old CAG-LEA transgenic pig (B, E) in comparison with WT porcine lymph nodes; the age of the animals was 19 days (A, D) and 6 months (C, F). Immunohistochemistry using the PPT3 anti-CD3 antibody demonstrates nearly T cell-free follicles (Fo) near the trabecules (T) in all samples. Follicles in samples of 6-month-old WT animals were generally larger than in transgenic or young animals (compare C versus A and B). Follicles of 6-month-old WT porcine lymph nodes showed a distinct reaction center (F) with heavily proliferating centroblasts in the dark zone (DZ) and less proliferating cells in the pale zone (PZ)–comparing strong and weak Ki67 immunopositivity (proliferation marker) in DZ and PZ, respectively. In contrast, marked proliferation of lymphocytes could be detected in either the follicles of transgenic (E) nor 19-day-old WT animals (D). The amount of proliferating T cells did not seem to differ between the samples. Visualization of the immunoreaction diaminobenzidine—horseradish peroxidase (positive staining = brown), counterstaining hematoxylin, scale bar = 200 μm.</p

Genetic distance between mammalian CTLA4, B7.1/CD80 and B7.2/CD86.

<p>The percentage of amino acid identities between pairwise comparisons are shown as a matrix for CTLA4 (A), CD80 (B) and CD86 (C). The distribution of pairwise identities was calculated in segments of 4% (D) and illustrates higher amino acid conservation in CTLA4 compared to CD80 and CD86.</p