Keiko Omori

Inhibition of p38 Pathway Suppresses Human Islet Production of Pro-Inflammatory Cytokines and Improves Islet Graft Function

Nonspecific inflammation is associated with primary graft nonfunction (PNF). Inflammatory islet damage is mediated at least partially by pro‐inflammatory cytokines, such as interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α) produced by resident islet macrophages. The p38 pathway is known to be involved in cytokine production in the cells of the monocyte–macrophage lineage. Therefore, inhibition of the p38 pathway may prevent pro‐inflammatory cytokine production by resident islet macrophages and possibly reduce the incidence of PNF. Our present study has demonstrated that inhibition of the p38 pathway by a chemical p38 inhibitor, SB203580, suppresses IL‐1β and TNF‐α production in human islets exposed to lipopolysaccharide (LPS) and/or inflammatory cytokines. Although IL‐1β is predominantly produced by resident macrophages, ductal cells and islet vascular endothelial cells were found to be another cellular source of IL‐1β in isolated human islets. SB203580 also inhibited the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2) in the treated islets. Furthermore, human islets treated with SB203580 for 1 h prior to transplantation showed significantly improved graft function. These results suggest that inhibition of the p38 pathway may become a new therapeutic strategy to improve graft survival in clinical islet transplantation.

Generation of human islets through expansion and differentiation of non-islet pancreatic cells discarded (pancreatic discard) after islet isolation.

Objectives: Islet transplantation is hampered by the shortage of donor tissues. Our objective was to generate islet-like cell clusters (ICCs) from cultures of non-islet pancreatic cells. Methods: The starting cultured cells came from the non-islet fractions of human pancreases after enzymatic digestion and purification for the purpose of islet isolation. Initially, these cells expanded in monolayer cultures and became confluent on collagen-coated flasks. After trypsination and suspension of these cells in a defined islet differentiation medium, the cells aggregated to form ICCs. Results: The initial cell population consisted of less than 1% of insulin-positive cells, 44% amylase-positive cells, and 41% cytokeratin (CK) 7-positive, or CK19+ cells, but PDX-1+ cells were absent. Cells from later stages of the monolayer cultures showed signs of dedifferentiation/transdifferentiation. At the time of harvesting, more than 90% of the cells were positive for CK 7/19 and PDX-1, but less than 1% of the cells were insulin-positive. After aggregation, the ICCs appeared redifferentiated, and contained glucose-responsive, insulin-secreting cells with an insulin content measuring 20% of that found in freshly isolated islets isolated from the same pancreas. ICCs transplanted into athymic mice and removed after 4 months did acquire the morphology of mature islets, indicating further maturation of the ICCs in vivo after transplantation. Human C-peptide was detected in recipient animal sera. Conclusion: Using the specified culture methods, non-islet pancreas cells can generate cell clusters resembling islets. These ICCs, obtained from fractions of the pancreas that are otherwise discarded, continue to differentiate after transplantation to become mature islets.

64Cu labeled sarcophagine exendin-4 for microPET imaging of glucagon like peptide-1 receptor expression

The Glucagon-like peptide 1 receptor (GLP-1R) has become an important target for imaging due to its elevated expression profile in pancreatic islets, insulinoma, and the cardiovascular system. Because native GLP-1 is degraded rapidly by dipeptidyl peptidase-IV (DPP-IV), several studies have conjugated different chelators to a more stable analog of GLP-1 (such as exendin-4) as PET or SPECT imaging agents with various advantages and disadvantages. Based on the recently developed Sarcophagin chelator, here, we describe the construction of GLP-1R targeted PET probes containing monomeric and dimeric exendin-4 subunit. The in vitro binding affinity of BarMalSar-exendin-4 and Mal2Sar-(exendin-4)2 was evaluated in INS-1 cells, which over-express GLP-1R. Mal2Sar-(exendin-4)2 demonstrated around 3 times higher binding affinity compared with BaMalSar-exendin-4. After 64Cu labeling, microPET imaging of 64Cu-BaMalSar-exendin-4 and 64Cu-Mal2Sar-(exendin-4)2 were performed on subcutaneous INS-1 tumors, which were clearly visualized with both probes. The tumor uptake of 64Cu-Mal2Sar-(exendin-4)2 was significantly higher than that of 64Cu-BaMaSarl-exendin-4, which could be caused by polyvalency effect. The receptor specificity of these probes was confirmed by effective blocking of the uptake in both tumor and normal positive organs with 20-fold excess of unlabeled exendin-4. In conclusion, sarcophagine cage conjugated exendin-4 demonstrated persistent and specific uptake in INS-1 insulinoma model. Dimerization of exendin-4 could successfully lead to increased tumor uptake in vivo. Both 64Cu-BaMalSar-exendin-4 and 64Cu-Mal2Sar-(exendin-4)2 hold a great potential for GLP-1R targeted imaging.

Quantitative assessment of β-cell apoptosis and cell composition of isolated, undisrupted human islets by laser scanning cytometry.

Background. Assays for assessing human islet cell quality, which provide results before transplantation, would be beneficial to improve the outcomes for islet transplantation therapy. Parameters such as percent &bgr;-cell apoptosis and cell composition are found to vary markedly between different islet preparations and may serve as markers of islet quality. We have developed fluorescence-based assays using laser scanning cytometry for assessing &bgr;-cell apoptosis and islet cell composition on serial sections of intact isolated islets. Methods. Isolated human islets were fixed in formalin and embedded in paraffin. Serial sections were immunostained for the pancreatic hormones and acinar and ductal cell markers. DNA fragmentation was used to label apoptotic cells. Stained cells were quantified using an iCys laser scanning cytometer. Results. Islet preparations from 102 human pancreatic islet isolations were analyzed. For the whole set of islet preparations, we found a mean islet cell composition of 54.5%±1.2% insulin-positive, 33.9%±1.2% glucagon, 12.1%±0.7% somatostatin, and 1.5%±0.2% pancreatic polypeptide-positive cells. The apoptotic &bgr; cells were 2.85%±0.4% with a range of 0.27% to 18.3%. The percentage of apoptotic &bgr; cells correlated well (P<0.0001, n=59) with results obtained in vivo by transplantation of the corresponding islets in diabetic NODscid mice. Conclusions. The analysis of whole, nondissociated islets for cell composition and &bgr;-cell apoptosis using laser scanning cytometry gives reliable and reproducible results and could be performed both before islet transplantation and on preserved cell blocks at any time in future. Thus, they can be a powerful tool for islet quality assessment.

Liver natural killer cells play a role in the destruction of islets after intraportal transplantation.

Background. A significant loss of islet function observed in type 1 diabetic recipients after intraportal islet transplantation raises a question about the suitability of the liver as a transplant site. We hypothesize that natural killer (NK) cells in the liver play a role in the islet graft destruction. Methods. Phenotypical and functional differences between liver and splenic NK cells isolated from mice were examined by flow cytometry and in vitro cytotoxicity assays. In vivo, the role of liver NK cells was determined by examining the function of intraportally administered islet iso- and allografts by treating recipients with anti-asialo GM1 to deplete NK cells. Results. NK cell-depleted diabetic C57BL/6 mice receiving 400 syngeneic islets into the liver rapidly ameliorated hyperglycemia, whereas control recipients did not. The same number of BALB/c islets grafted in the liver of diabetic nonimmunosuppressed C57BL/6 mice failed to function, whereas NK cell-depleted recipients reversed hyperglycemia for up to 10 days. NK cells from the liver of naive C57BL/6 mice showed significantly higher cytotoxicity than splenic NK cells as tested with a &bgr;-cell line and allogeneic islets. The cell proportion and the expression level of activation markers on liver NK cells significantly increased after intraportal islet transplantation as compared with the control. Liver NK cells also increased anti-islet cytotoxicity, but not splenic NK cells, after islet transplantation. Conclusions. Our results clearly show the destructive activity of liver NK cells toward islets, suggesting that NK cells play a role in early islet graft loss after intraportal islet transplantation.

Improvement of human islet cryopreservation by a p38 MAPK inhibitor.

The activation of p38 mitogen‐activated protein kinase (MAPK) has been shown to cause ischemia/reperfusion injury of several organs used for transplantation and also to play a significant role in primary islet graft nonfunction. Activation of p38 MAPK may also occur during islet cryopreservation and thawing. In this study, a p38 MAPK inhibitor (p38IH) was applied to human islet cryopreservation to improve islet yield and quality after thawing. Under serum‐free conditions, human islets were cryopreserved, thawed and cultured using our standard procedures. Three types of solutions were tested: conventional RPMI1640 medium (RPMI), a newly developed islet cryopreservation solution (ICS), and ICS supplemented with a p38IH, SD‐282 (ICS‐p38IH). Activation or inhibition of p38 MAPK was demonstrated by the diminished phosphorylation of HSP27 substrate. Islet recovery on day 2 after thawing was highest with ICS‐p38IH and islet viability was not significantly different in the three groups. β Cell numbers and function were the highest in islets cryopreserved with ICS‐p38IH. Glucose‐stimulated human C‐peptide levels were 86% of that of the nonfrozen islets when measured 4 weeks after transplantation into NODscid mice. This improvement may provide an opportunity to establish islet banks and allow the use of cryopreserved islets for clinical transplantation.

Improvement of canine islet yield by donor pancreas infusion with a p38MAPK inhibitor.

Background. The activation of p38 mitogen-activated protein kinases (MAPK) is implicated in cold ischemia-reperfusion injury of donor organs. The islet isolation process, from pancreas procurement through islet collection, may activate p38MAPK leading to cytokine release and islet damage. This damage may be prevented by treating pancreata with a p38MAPK inhibitor (p38IH) before cold preservation. Methods. Pancreata removed from Beagle dogs were infused with University of Wisconsin solution containing the p38IH, SB203580, and Pefabloc (n=6) or vehicle (dimethyl sulfoxide and Pefabloc) alone (n=7), through the pancreatic duct and preserved using the two-layer method. After 20 to 22 hr, islets were isolated and 3000 IEQ/kg were autotransplanted into the corresponding dog to monitor glucose metabolism. Results. p38IH-treated pancreata yielded significantly more islets than control pancreata (IEQ/g: 2134±297 vs. 1477±145 IEQ/g or 65,012±9385 vs. 45,700±5103 IEQ/pancreas; P<0.05). Apoptotic &bgr;-cell percentages assessed by laser scanning cytometry were lower in p38IH-treated than the controls (44%±9.4% vs. 61.6%±4.8%, P<0.05). Tumor necrosis factor-&agr; expression assessed by real-time reverse transcription polymerase chain reaction was significantly lower in the p38IH-treated group than controls. All dogs (3000 IEQ/kg) transplanted with p38IH-treated islets (n=5) became euglycemic versus four of five dogs that received untreated islets. Plasma C-peptide levels after glucagon challenge were higher in animals receiving p38IH-treated islets (n=5) versus untreated islets (n=4) (0.40±0.78 vs. 0.21±0.05 ng/mL, P<0.05). Conclusions. Infusion of pancreata with University of Wisconsin solution containing p38IH through the duct before preservation suppresses cytokine release, prevents &bgr;-cell apoptosis, and improves islet yield significantly with no adverse effect on islet function after transplantation. p38IH treatment of human pancreata may improve islet yield for use in clinical transplantation.

Microassay for glucose-induced preproinsulin mRNA expression to assess islet functional potency for islet transplantation.

Background. The capacity for insulin synthesis in islets is important for islet transplantation to succeed. We developed a microassay that evaluates the potency of human islets by measuring changes in glucose-induced human insulin gene (INS) expression using a single islet in octuplicate samples. Methods. Poly (A)+ messenger RNA (mRNA) was purified from a set of single handpicked human islets. Glucose-induced mature (postspliced) and premature (prespliced) insulin mRNA were quantified by reverse-transcriptase polymerase chain reaction using several insulin mRNA primers designed at different locations including, intron, exon, and an exon-intron junction. Results. The synthesis of premature INS mRNA was significantly increased in islets exposed to high glucose for 16 vs. 4 hr (P<0.01), whereas mature INS mRNA showed no difference. Glucose-induced premature INS mRNA synthesis was attenuated in heat-damaged islets. Stimulation index (SI) calculated by normalizing premature by mature INS mRNA (SI_INS mRNA) positively correlated with SI of insulin release (SI_16h insulin) from the same set of islets during 16-hr incubation in high or low glucose media, and SI of glucose-mediated insulin release obtained from the same islet lot in a perifusion system (n=12). Furthermore, linear multiple regression analysis using SI_INS mRNA and SI_16h insulin predicted islet transplantation outcome in nonobese diabetic (NOD) scid mice (n=8). Conclusion. The measurement of glucose-induced premature INS mRNA normalized by mature INS mRNA can be used to assess the functional quality of human islets and may predict islet function after transplantation in type 1 diabetic patients.

The Choice of Enzyme for Human Pancreas Digestion Is a Critical Factor for Increasing the Success of Islet Isolation

Background We evaluated 3 commercially available enzymes for pancreatic digestion by comparing key parameters during the islet isolation process, as well as islet quality after isolation. Methods Retrospectively compared and analyzed islet isolations from pancreata using 3 different enzyme groups: liberase HI (n = 63), collagenase NB1/neutral protease (NP) (n = 43), and liberase mammalian tissue-free collagenase/thermolysin (MTF C/T) (n = 115). A standardized islet isolation and purification method was used. Islet quality assessment was carried out using islet count, viability, in vitro glucose-stimulated insulin secretion (GSIS), glucose-stimulated oxygen consumption rate, and in vivo transplantation model in mice. Results Donor characteristics were not significantly different among the 3 enzyme groups used in terms of age, sex, hospital stay duration, cause of death, body mass index, hemoglobin A1c, cold ischemia time, and pancreas weight. Digestion efficacy (percentage of digested tissue by weight) was significantly higher in the liberase MTF C/T group (73.5 ± 1.5 %) when compared to the liberase HI group (63.6 ± 2.3 %) (P < 0.001) and the collagenase NB1/NP group (61.7 ± 2.9%) (P < 0.001). The stimulation index for GSIS was significantly higher in the liberase MTF C/T group (5.3 ± 0.5) as compared to the liberase HI (2.9 ± 0.2) (P < 0.0001) and the collagenase NB1/NP (3.6 ± 2.9) (P = 0.012) groups. Furthermore, the liberase MTF C/T enzymes showed the highest success rate of transplantation in diabetic non-obese diabetic severe combined immunodeficiency mice (65%), which was significantly higher than the liberase HI (42%, P = 0.001) and the collagenase NB1/NP enzymes (41%, P < 0.001). Conclusions Liberase MTF C/T is superior to liberase HI and collagenase NB1/NP in terms of digestion efficacy and GSIS in vitro. Moreover, liberase MTF C/T had a significantly higher success rate of transplantation in diabetic NOD Scid mice compared to liberase HI and collagenase NB1/NP enzymes.

Human Pancreatic Islets Isolated From Donors With Elevated HbA1c Levels: Islet Yield and Graft Efficacy.

The aim of this study was to investigate the effects of elevated donor HbA1c levels (type 2 diabetes, T2D) on the islet yield and functionality postisolation. In this retrospective analysis, donors for islet isolations were classified into two groups: T2D group (HbA1c ≥ 6.5%, n = 18) and normal group (HbA1c < 6.5%, n = 308). Optimum pancreas digestion time (switch time) was significantly higher in the T2D group compared to the normal group (13.7 ± 1.2 vs. 11.7 ± 0.1 min, respectively, p = 0.005). Islet yields were significantly lower in the T2D group compared to the control (T2D vs. control): islet equivalent (IEQ)/g (prepurification 2,318 ± 195 vs. 3,713 ± 114, p = 0.003; postpurification 1,735 ± 175 vs. 2,663 ± 89, p = 0.013) and islet particle number (IPN)/g (prepurification, 2,519 ± 336 vs. 4,433 ± 143, p = 0.001; postpurification, 1,760 ± 229 vs. 2,715 ± 85, p = 0.007). Islets from T2D pancreata had significantly lower viability (T2D vs. control: 91.9 ± 1.6 vs. 94.4 ± 0.3%, p = 0.004) and decreased oxygen consumption rate (DOCR) (T2D vs. control: 0.09 ± 0.01 and 0.21 ± 0.03 nmol O2 100 islets−1 min−1, p = 0.049). The islets isolated from T2D donor pancreata reversed diabetes in NOD-SCID mice in 9% (2/22) compared to islets from control donor pancreata, which reversed diabetes in 67% (175/260, p < 0.001). In conclusion, this study demonstrates that elevated HbA1c (≥6.5%) is associated with impairment of islet function and lower islet yield; however, these islets could not be suitable for clinical applications.