Jeffrey Rawson

Glucose‐Stimulated Increment in Oxygen Consumption Rate as a Standardized Test of Human Islet Quality

Standardized assessment of islet quality is imperative for clinical islet transplantation. We have previously shown that the increment in oxygen consumption rate stimulated by glucose (ΔOCRglc) can predict in vivo efficacy of islet transplantation in mice. To further evaluate the approach, we studied three factors: islet specificity, islet composition and agreement between results obtained by different groups. Equivalent perifusion systems were set up at the City of Hope and the University of Washington and the values of ΔOCRglc obtained at both institutions were compared. Islet specificity was determined by comparing ΔOCRglc in islet and nonislet tissue. The ΔOCRglc ranged from 0.01 to 0.19 nmol/min/100 islets (n = 14), a wide range in islet quality, but the values obtained by the two centers were similar. The contribution from nonislet impurities was negligible (ΔOCRglc was 0.12 nmol/min/100 islets vs. 0.007 nmol/min/100 nonislet clusters). The ΔOCRglc was statistically independent of percent beta cells, demonstrating that ΔOCRglc is governed more by islet quality than by islet composition. The ΔOCRglc, but not the absolute level of OCR, was predictive of reversal of hyperglycemia in diabetic mice. These demonstrations lay the foundation for testing ΔOCRglc as a measurement of islet quality for human 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.

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.

An oral vaccine for type 1 diabetes based on live attenuated Salmonella.

Type 1 diabetes (T1D) is a metabolic disease that is initiated by the autoimmune destruction of pancreatic insulin-producing beta cells that is accompanied by the development of antigen-specific antibodies and cytotoxic T lymphocytes (CTLs). Several studies have shown that vaccination with diabetic autoantigens provides some protection against this process. In this report we describe a new oral vaccine that utilizes live attenuated Salmonella for simultaneous delivery of autoantigens in conjunction with immunomodulatory cytokine genes to immune cells in the gut mucosa. Recent data showed that live attenuated Salmonella is a safe, simple and effective vector for expression of antigens and cytokines by antigen-presenting cells (APCs) of gut-associated lymphatic tissue (GALT). This novel strategy was tested by fusion of the diabetic autoantigen preproinsulin with Salmonella secretory effector protein (SseF) of pathogenicity island-2 (SPI2). In this way the autoantigen is only expressed inside the host immune cells and translocated to the host cell cytosol. In addition Salmonella was used to deliver the gene for the immunomodulatory cytokine transforming growth factor beta (TGFβ) for host cell expression. Oral co-vaccination of 8 week-old non-obese diabetic (NOD) mice with three weekly doses of both the autoantigen and cytokine significantly reduced the development of diabetes, improved the response to glucose challenge, preserved beta cell mass, and reduced the severity of insulitis compared with controls and autoantigen alone. Combination therapy also resulted in increased circulating levels of IL10 four weeks post-vaccination and IL2 for 12 weeks post-vaccination, but without effect on proinflammatory cytokines IL6, IL12(p70), IL17 and IFNγ. However, in non-responders there was a significant rise in IL12 compared with responders. Future studies will examine the mechanism of this vaccination strategy in more detail. In conclusion, Salmonella-based oral vaccines expressing autoantigens combined with imunomodulatory cytokines appears to be a promising therapy for prevention of T1D.

Cells with surface expression of CD133^(high)CD71^(low) are enriched for tripotent colony-forming progenitor cells in the adult murine pancreas

Progenitor cells in the adult pancreas are potential sources of endocrine beta cells for treating type 1 diabetes. Previously, we identified tri-potent progenitor cells in the adult (2-4month-old) murine pancreas that were capable of self-renewal and differentiation into duct, acinar, and endocrine cells in vitro. These progenitor cells were named pancreatic colony-forming units (PCFUs). However, because PCFUs are a minor population in the pancreas (~1%) they are difficult to study. To enrich PCFUs, strategies using cell-surface marker analyses and fluorescence-activated cell sorting were developed. We found that CD133(high)CD71(low) cells, but not other cell populations, enriched PCFUs by up to 30 fold compared to the unsorted cells. CD133(high)CD71(low) cells generated primary, secondary, and subsequent colonies when serially re-plated in Matrigel-containing cultures, suggesting self-renewal abilities. In the presence of a laminin hydrogel, CD133(high)CD71(low) cells gave rise to colonies that contained duct, acinar, and Insulin(+)Glucagon(+) double-hormonal endocrine cells. Colonies from the laminin hydrogel culture were implanted into diabetic mice, and five weeks later duct, acinar, and Insulin(+)Glucagon(-) cells were detected in the grafts, demonstrating tri-lineage differentiation potential of CD133(high)CD71(low) cells. These CD133(high)CD71(low) cells will enable future studies of putative adult pancreas stem cells in vivo.

Determination of islet viability using a zinc-specific fluorescent dye and a semi-automated assessment method.

Islet transplantation is an effective therapy that allows the achievement of insulin independence in patients with type 1 diabetes (T1D). To ensure successful transplantation, islet viability and function are of great importance. Viability assessments most often use fluorescein diacetate (FDA)/propidium iodide (PI) staining. However, results using this method often do not correlate well with graft function. Because FDA nonspecifically penetrates all cells present in the islet preparation, including islets and contaminating acinar cells, its use often complicates viability assessments of the overall cell population. Furthermore, the manual method for determining viability percentages is highly subjective. Shortcomings of the conventional islet viability assay can be potentially improved by staining cells with Newport Green (NG). NG, is a zinc-specific fluorescent dye that specifically reacts with zinc-rich β cells. Two kinds of NG dyes, NG-DCF and NG-PDX, are currently available. We examined the zinc specificity of these NG dyes and compared NG staining with traditional FDA staining to explore the potential of NG dyes to improve islet viability assessment. Of the two NGs tested, NG-DCF showed the higher specificity toward a β-cell line as well as human islets. NG-DCF accurately identified the islet area, even in low-purity islets, while neither FDA nor NG-PDX did. Although NG-DCF staining required a longer incubation time, the addition of poloxamer F127 and incubation at 37°C allowed viability assessment to take place within 30 min. Unlike FDA/PI staining, NG-DCF/PI staining allowed for islet-specific assessment. We also introduced a semiautomated measurement to determine NG-DCF/PI staining results, which enabled us to obtain objective and reproducible results. NG-DCF/PI staining is easy and reliable, and this method permits highly objective islet-specific viability assessments.

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.

Gene expression signature predicts human islet integrity and transplant functionality in diabetic mice

There is growing evidence that transplantation of cadaveric human islets is an effective therapy for type 1 diabetes. However, gauging the suitability of islet samples for clinical use remains a challenge. We hypothesized that islet quality is reflected in the expression of specific genes. Therefore, gene expression in 59 human islet preparations was analyzed and correlated with diabetes reversal after transplantation in diabetic mice. Analysis yielded 262 differentially expressed probesets, which together predict islet quality with 83% accuracy. Pathway analysis revealed that failing islet preparations activated inflammatory pathways, while functional islets showed increased regeneration pathway gene expression. Gene expression associated with apoptosis and oxygen consumption showed little overlap with each other or with the 262 probeset classifier, indicating that the three tests are measuring different aspects of islet cell biology. A subset of 36 probesets surpassed the predictive accuracy of the entire set for reversal of diabetes, and was further reduced by logistic regression to sets of 14 and 5 without losing accuracy. These genes were further validated with an independent cohort of 16 samples. We believe this limited number of gene classifiers in combination with other tests may provide complementary verification of islet quality prior to their clinical use.