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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.

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.

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.

The Effects of Digestion Enzymes on Islet Viability and Cellular Composition

The choice of enzyme blend is critical for successful islet isolation. Islet yield, viability, integrity, and function are important factors that influence the outcome of islet transplantation. Liberase HI has been used as a standard enzyme for pancreas digestion and has successfully produced islets that reversed diabetes. However, the replacement of Liberase HI with collagenase NB1 has significantly influenced the process outcome, both in quality and quantity of the isolated islets. The assessment of islet cells by Flow Cytometry (FC) has been reported to be useful for evaluating islet quality. The aim of this study was to assess the isolation outcomes and islet quality when comparing human islet cell processed with Liberase HI and NB1. A total of 66 islet isolations, 46 processed using Liberase HI and 20 using Serva NB1, were retrospectively analyzed. Islet yield, function in vitro, islet cell viability by FC, as well as isolation-related factors were compared. There was no significant difference in donor characteristics such as age and height; however, body mass index (BMI) in the Liberase HI group was significantly higher. There was also no significant difference in prepurification, postisolation, or postculture IEQ or percent recovery between the two groups. Flow data showed Liberase HI preparations had a significantly higher percent of live cells (DAPI-) and NG+/ TMRE+ when compared to NB1. Stimulation Indices (SI) for Liberase HI (n = 45) showed 3.17 and NB1 (n = 18) 2.71 (p = NS). The results of Annexin V/DAPI staining for live, apoptotic, and necrotic cells were 50.7 ± 2.24%, 14.4 ± 1.02%, and 27.8 ± 1.92% for Liberase HI versus 48.1 ± 1.93%, 12.3 ± 0.92%, and 33.9 ± 2.28% for NB1. Islets isolated using Liberase HI showed higher viable β cells by NG/TMRE staining and decreased necrosis by Annexin V/DAPI staining. FC assessment may be useful for determining the choice of digestion enzyme to maximize viable islets.

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.

Testing Combinations of Protease Inhibitor and Preservation Solution to Improve Islet Quality and Yield

UNLABELLED Pancreas preservation using an oxygenated two-layer method (TLM) has been reported to improve islet yields, as has supplementation of Liberase with Pefabloc. We hypothesized that using both TLM and Pefabloc could enhance islet yield as compared with preservation in University of Wisconsin (UW) or Histidine-Tryptophan Ketoglutarate (HTK) solution. METHODS Ninety-eight pancreata with no significant differences of age, body mass index, or cold ischemia time preserved randomly with UW (n = 40), TLM (n = 48), or HTK (n = 10) were processed with (n = 36) or without (n = 66) Pefabloc. RESULTS The total islet equivalent (IEQ) from TLM-preserved pancreata processed with Pefabloc (n = 12) showed lower yields versus those processed without Pefabloc (n = 36): 216,120 +/- 27,906 vs. 301,427 +/- 21,447 IEQ (P < .05). Islets from 1 of 12 (8.33%) pancreata processed with Pefabloc in TLM were transplanted, in contrast with 15/36 TLM (41.67%) pancreata processed without it. Islet yields were not significantly different among pancreata preserved in UW and processed with Pefabloc (n = 17) versus without Pefabloc (n = 23): 342,693 +/- 45,588 versus 266,609 +/- 29,006 IEQ (P = .149). The number of transplants from UW-preserved pancreata was 3/17 (17.65%) when processed with Pefabloc and 4/23 (17.39%) without. Among the HTK group, there was no significant difference in islet yields between pancreata processed with (n = 7) versus without Pefabloc (n = 3): 248,227 +/- 65,294 versus 483,555 +/- 144,070 IEQ (P = .118). CONCLUSIONS Pefabloc showed no benefit to improve islet yields. Pancreata preserved in TLM provided better transplant quality islets when processed in the absence of Pefabloc.

Evaluation of collagenase gold plus BP protease in isolating islets from human pancreata

ABSTRACT Selection of enzymes for optimal pancreas digestion is essential for successful human islet isolations. The aim of this study was to evaluate the efficacy and outcome of using Collagenase Gold plus BP protease (VitaCyte) (n = 8) by comparing it to two commercially available enzymes, Liberase MTF C/T (Roche) (n = 48) and Collagenase NB1/NP (Serva) (n = 15). The isolation outcomes were assessed by islet counting, viability, glucose-stimulated oxygen consumption rate (OCR), and successful graft-rate following transplantation in diabetic NOD scid mice. The pancreas donor characteristics were not significantly different between the tested enzyme groups regarding their BMI, pancreas weight, cold ischemia time (CIT) and HbA1c. The results show that digested tissue volume was not statistically significant between the VitaCyte enzyme (34.25 ± 5.4 mL) and the Roche enzyme (55.25 ± 3.42 mL, p = 0.073), however, this was significant with Serva enzyme (64.07 ± 7.95 mL, p = 0.020). Interestingly, the islet yields were not statistically different between all enzyme groups. Moreover, when islets were transplanted into NOD scid mice, the reversal rate of diabetes for the VitaCyte enzyme group was similar to all enzyme groups. In conclusion, the effectiveness of Collagenase Gold plus BP protease is comparable to the MTF C/T and the Collagenase NB1/NP enzymes; the low cost could facilitate the use of more pancreata for islet isolations.

Prophylactically Decontaminating Human Islet Product for Safe Clinical Application: Effective and Potent Method.

Background Transplanting pancreatic islets into recipients must be safe and effective to treat type 1 diabetes. Islet quality and quantity are important; however, the final product must also be free from microbial contamination and low endotoxin levels. Methods This study explored a method to eliminate contamination in manufacturing islets for transplantation. A simple (single antibiotic n = 164) and refined (triple antimicrobial agents, n = 279) pancreas decontaminating methods were used to test their effects on reducing the contamination rates in the islet final product. A total of 443 pancreata were processed for islet isolations. Three samples for microbial tests (Gram stain, aerobic, and anaerobic culture) were taken at preprocess (pancreas preservation), postisolation, and postculture. Endotoxin levels were measured only for islets considered for transplantation. Results Of 443 pancreata used for islet isolation, 79 (17.8%) showed signs of contamination in preprocess samples; 10 (2.3%) were contaminated in both preprocess and in the final product (postisolation and postculture) samples. Contamination rates in which preprocess and final product samples were positive for contamination was significantly lower using the refined method (refined vs simple method: 5% vs 20.5%, P = 0.045). Identical microbial species were present in both preprocess and in the final product. Conclusions This study demonstrated that the refined method reduces the rate of contamination of the islet final product and is safe for clinical application. Moreover, it may be used as a standard method during human islet manufacturing facilitating the application of a biological license agreement from United States Food and Drug Administration.