DataSheet_6_Optimal temperature for the long-term culture of adult porcine islets for xenotransplantation.pdf

Porcine islet xenotransplantation represents a promising therapy for severe diabetes mellitus. Long-term culture of porcine islets is a crucial challenge to permit the on-demand provision of islets. We aimed to identify the optimal temperature for the long-term culture of adult porcine islets for xenotransplantation. We evaluated the factors potentially influencing successful 28-day culture of islets at 24°C and 37°C, and found that culture at 37°C contributed to the stability of the morphology of the islets, the proliferation of islet cells, and the recovery of endocrine function, indicated by the expression of genes involved in pancreatic development, hormone production, and glucose-stimulated insulin secretion. These advantages may be provided by islet-derived CD146-positive stellate cells. The efficacy of xenotransplantation using islets cultured for a long time at 37°C was similar to that of overnight-cultured islets. In conclusion, 37°C might be a suitable temperature for the long-term culture of porcine islets, but further modifications will be required for successful xenotransplantation in a clinical setting.</p

DataSheet_7_Optimal temperature for the long-term culture of adult porcine islets for xenotransplantation.docx

Porcine islet xenotransplantation represents a promising therapy for severe diabetes mellitus. Long-term culture of porcine islets is a crucial challenge to permit the on-demand provision of islets. We aimed to identify the optimal temperature for the long-term culture of adult porcine islets for xenotransplantation. We evaluated the factors potentially influencing successful 28-day culture of islets at 24°C and 37°C, and found that culture at 37°C contributed to the stability of the morphology of the islets, the proliferation of islet cells, and the recovery of endocrine function, indicated by the expression of genes involved in pancreatic development, hormone production, and glucose-stimulated insulin secretion. These advantages may be provided by islet-derived CD146-positive stellate cells. The efficacy of xenotransplantation using islets cultured for a long time at 37°C was similar to that of overnight-cultured islets. In conclusion, 37°C might be a suitable temperature for the long-term culture of porcine islets, but further modifications will be required for successful xenotransplantation in a clinical setting.</p

DataSheet_3_Optimal temperature for the long-term culture of adult porcine islets for xenotransplantation.pdf

Porcine islet xenotransplantation represents a promising therapy for severe diabetes mellitus. Long-term culture of porcine islets is a crucial challenge to permit the on-demand provision of islets. We aimed to identify the optimal temperature for the long-term culture of adult porcine islets for xenotransplantation. We evaluated the factors potentially influencing successful 28-day culture of islets at 24°C and 37°C, and found that culture at 37°C contributed to the stability of the morphology of the islets, the proliferation of islet cells, and the recovery of endocrine function, indicated by the expression of genes involved in pancreatic development, hormone production, and glucose-stimulated insulin secretion. These advantages may be provided by islet-derived CD146-positive stellate cells. The efficacy of xenotransplantation using islets cultured for a long time at 37°C was similar to that of overnight-cultured islets. In conclusion, 37°C might be a suitable temperature for the long-term culture of porcine islets, but further modifications will be required for successful xenotransplantation in a clinical setting.</p

DataSheet_1_Optimal temperature for the long-term culture of adult porcine islets for xenotransplantation.pdf

Porcine islet xenotransplantation represents a promising therapy for severe diabetes mellitus. Long-term culture of porcine islets is a crucial challenge to permit the on-demand provision of islets. We aimed to identify the optimal temperature for the long-term culture of adult porcine islets for xenotransplantation. We evaluated the factors potentially influencing successful 28-day culture of islets at 24°C and 37°C, and found that culture at 37°C contributed to the stability of the morphology of the islets, the proliferation of islet cells, and the recovery of endocrine function, indicated by the expression of genes involved in pancreatic development, hormone production, and glucose-stimulated insulin secretion. These advantages may be provided by islet-derived CD146-positive stellate cells. The efficacy of xenotransplantation using islets cultured for a long time at 37°C was similar to that of overnight-cultured islets. In conclusion, 37°C might be a suitable temperature for the long-term culture of porcine islets, but further modifications will be required for successful xenotransplantation in a clinical setting.</p

DataSheet_4_Optimal temperature for the long-term culture of adult porcine islets for xenotransplantation.pdf

Porcine islet xenotransplantation represents a promising therapy for severe diabetes mellitus. Long-term culture of porcine islets is a crucial challenge to permit the on-demand provision of islets. We aimed to identify the optimal temperature for the long-term culture of adult porcine islets for xenotransplantation. We evaluated the factors potentially influencing successful 28-day culture of islets at 24°C and 37°C, and found that culture at 37°C contributed to the stability of the morphology of the islets, the proliferation of islet cells, and the recovery of endocrine function, indicated by the expression of genes involved in pancreatic development, hormone production, and glucose-stimulated insulin secretion. These advantages may be provided by islet-derived CD146-positive stellate cells. The efficacy of xenotransplantation using islets cultured for a long time at 37°C was similar to that of overnight-cultured islets. In conclusion, 37°C might be a suitable temperature for the long-term culture of porcine islets, but further modifications will be required for successful xenotransplantation in a clinical setting.</p

DataSheet_5_Optimal temperature for the long-term culture of adult porcine islets for xenotransplantation.pdf

Porcine islet xenotransplantation represents a promising therapy for severe diabetes mellitus. Long-term culture of porcine islets is a crucial challenge to permit the on-demand provision of islets. We aimed to identify the optimal temperature for the long-term culture of adult porcine islets for xenotransplantation. We evaluated the factors potentially influencing successful 28-day culture of islets at 24°C and 37°C, and found that culture at 37°C contributed to the stability of the morphology of the islets, the proliferation of islet cells, and the recovery of endocrine function, indicated by the expression of genes involved in pancreatic development, hormone production, and glucose-stimulated insulin secretion. These advantages may be provided by islet-derived CD146-positive stellate cells. The efficacy of xenotransplantation using islets cultured for a long time at 37°C was similar to that of overnight-cultured islets. In conclusion, 37°C might be a suitable temperature for the long-term culture of porcine islets, but further modifications will be required for successful xenotransplantation in a clinical setting.</p

DataSheet_2_Optimal temperature for the long-term culture of adult porcine islets for xenotransplantation.pdf

Porcine islet xenotransplantation represents a promising therapy for severe diabetes mellitus. Long-term culture of porcine islets is a crucial challenge to permit the on-demand provision of islets. We aimed to identify the optimal temperature for the long-term culture of adult porcine islets for xenotransplantation. We evaluated the factors potentially influencing successful 28-day culture of islets at 24°C and 37°C, and found that culture at 37°C contributed to the stability of the morphology of the islets, the proliferation of islet cells, and the recovery of endocrine function, indicated by the expression of genes involved in pancreatic development, hormone production, and glucose-stimulated insulin secretion. These advantages may be provided by islet-derived CD146-positive stellate cells. The efficacy of xenotransplantation using islets cultured for a long time at 37°C was similar to that of overnight-cultured islets. In conclusion, 37°C might be a suitable temperature for the long-term culture of porcine islets, but further modifications will be required for successful xenotransplantation in a clinical setting.</p

Development of vaccine for dyslipidemia targeted to a proprotein convertase subtilisin/kexin type 9 (PCSK9) epitope in mice

<div><p>Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates expression of low-density lipoprotein (LDL) receptors via receptor internalization and subsequent lysosomal degradation. Thus, an anti-PCSK9 antibody is well known as an anti-hyperlipidemia drug. Here, we aimed to develop vaccine for a long-term treatment of dyslipidemia targeted to PCSK9. In This study, we designed a peptide vaccine for mouse PCSK-9, which consisted of short peptides conjugated to keyhole limpet hemocyanin (KLH) as a carrier protein. Vaccines were administered to male <i>apolipoprotein E (ApoE) deficient mice</i> with adjuvants and significantly elicited an antibody response against PCSK9. The PCSK9 vaccines were administered to mice three times in 2-week intervals, and antibody titers and lipoprotein levels were evaluated up to 24 weeks after the first immunization to determine the therapeutic effect. Anti-PCSK9 antibody titers reached peak levels 6 weeks after the first immunization, and theses titers were maintained for up to 24 weeks. Decreased plasma levels of total cholesterol, very low-density lipoprotein (VLDL), and chylomicron (CM) were maintained for up to 24 weeks. Immunized mice exhibited a significant increase in cell-surface LDL receptor expression. Stimulation with KLH, but not PCSK9, induced the production of INF-gamma and interleukin-4 (IL-4), as determined with ELISPOT assays, thus indicating that PCSK9 vaccine did not elicit T-cell activation in our vaccine system. The present anti-PCSK9 vaccine induced long-lasting anti-PCSK9 antibody production and improved lipoprotein profiles. Thus, anti-PCSK9 vaccine could become a new option for the treatment of dyslipidemia as a long-acting therapy in future.</p></div

Evaluation of the PCSK9 vaccine (V2 vaccine) in male <i>ApoE-deficient mice</i> (N = 5–7).

<p>PCSK9 vaccine (V2 vaccine) or control (Saline) was injected at different doses (Low; 5 μg and High; 50 μg peptides per mouse) three times in biweekly intervals (0, 2, and 4 weeks). (A) Anti-PCSK9 antibody titers (OD50%) were evaluated at pre-immunization (pre) and post-immunization time points (2, 4, 6, 8, 12, 16, 20, and 24 weeks). Data are presented as the average of each groups; error bars indicate the SEM. **P<0.01 and ****P<0.0001 show significant changes between low dose group and saline group. ††††P<0.0001 shows significant changes between high dose group and saline group. (B) PCSK9 levels in plasma samples from pre-immunized (pre) and post-immunized (6 weeks) mice. Bars represent mean levels of detected mouse PCSK9 levels in plasma samples, and error bars represent ± SEM. Significance values relative to saline group (*P<0.05, ***P<0.001) were obtained using two-way ANOVA with subsequent Tukey's multiple comparisons tests. (C) Cell-surface LDLR levels in liver hepatocytes were measured in immunized mice at 6 weeks post-immunization via ELISA. The results are presented as a fold-increase relative to saline-treated groups. Significance values relative to saline (****P<0.0001) were obtained with one-way ANOVA with subsequent Tukey’s tests for multiple comparisons. All data in this Figure are expressed as the means ± SEM.</p

Screening of PCSK9 peptide vaccines in male <i>ApoE-deficient mice</i>.

<p>Two candidate vaccines (V1 and V2 vaccines) or control (KLH) was injected (5 μg peptide per mouse) (N = 4 per group). (A) The antibody titers against candidate PCSK9 peptides conjugated with bovine serum albumin were evaluated pre-immunization (pre) and post-immunization (4 or 8 weeks), and the results are expressed as half-maximal binding (optical density: OD50%). Significance values were obtained with a 2-factor repeated-measure ANOVA with subsequent Tukey’s multiple comparisons tests. (B) Mouse plasma PCSK9 levels were measured at pre-immunization (pre) and post-immunization (4 weeks) time points. Significance values were obtained using two-way ANOVA with subsequent Tukey’s multiple comparisons test. (C and D) Mean values of TC and TG levels (mg/dL) were measured post-immunization (4 weeks). Significance values relative to KLH (*P<0.05) were obtained with one-way ANOVA with subsequent Tukey’s multiple comparisons tests. All data in this Figure are expressed as the means ± SEM. *P<0.05, **P<0.01, and ****P<0.0001.</p