Inflammatory and hypoxic stress-induced islet exosomes released during isolation are associated with poor transplant outcomes in islet autotransplantation.

Islets experience enormous stress during the isolation process, leading to suboptimal endocrine function after total pancreatectomy with islet autotransplantation (TPIAT). Our investigation focused on inducing isolation stress in islets ex vivo, where proinflammatory cytokines and hypoxia prompted the release of stress exosomes (exoS) sized between 50 and 200 nm. Mass spectrometry analysis revealed 3 distinct subgroups of immunogenic proteins within these exoS: damage-associated molecular patterns (DAMPs), chaperones, and autoantigens. The involvement of endosomal-sorting complex required for transport proteins including ras-associated binding proteins7A, ras-associated binding protein GGTA, vacuolar protein sorting associated protein 45, vacuolar protein sorting associated protein 26B, and the tetraspanins CD9 and CD63, in exoS biogenesis was confirmed through immunoblotting. Next, we isolated similar exoS from the islet infusion bags of TPIAT recipients (N = 20). The exosomes from infusion bags exhibited higher DAMP (heat shock protein family A [Hsp70] member 1B and histone H2B) levels, particularly in the insulin-dependent TPIAT group. Additionally, elevated DAMP protein levels in islet infusion bag exosomes correlated with increased insulin requirements (P = .010) and higher hemoglobin A1c levels 1-year posttransplant. A deeper exploration into exoS functionality revealed their potential to activate monocytes via the toll-like receptor 3/7: DAMP axis. This stimulation resulted in the induction of inflammatory phenotypes marked by increased levels of CD68, CD80, inducible nitric oxide synthase, and cyclooxygenase-2. This activation mechanism may impact the successful engraftment of transplanted islets.

Islet damage during isolation as assessed by miRNAs and the correlation of miRNA levels with posttransplantation outcome in islet autotransplantation.

High-quality pancreatic islets are essential for better posttransplantation endocrine function in total pancreatectomy with islet autotransplantation (TPIAT), yet stress during the isolation process affects quality and yield. We analyzed islet-enriched microRNAs (miRNAs) -375 and -200c released during isolation to assess damage and correlated the data with posttransplantation endocrine function. The absolute concentration of miR-375, miR-200c, and C-peptide was measured in various islet isolation steps, including digestion, dilution, recombination, purification, and bagging, in 12 cases of TPIAT. Posttransplantation glycemic control was monitored through C-peptide, hemoglobin A1c , insulin requirement, and SUITO index. The amount of miR-375 released was significantly higher during enzymatic digestion followed by the islet bagging (P < .001). Mir-200c mirrored these changes, albeit at lower concentrations. In contrast, the C-peptide amount was significantly higher in the purification and bagging steps (P < .001). Lower amounts of miR-375 were associated with a lower 6-month insulin requirement (P = .01) and lower hemoglobin A1c (P = .04). Measurement of the absolute quantity of miRNA-375 and -200c released during islet isolation is a useful tool to assess islet damage. The quantity of released miRNA is indicative of posttransplantation endocrine function in TPIAT patients.

Risk factors for early readmission after total pancreatectomy and islet auto transplantation.

BACKGROUND: Little published data exist examining causes of hospital readmission following total pancreatectomy with islet autotransplantation (TPIAT). METHODS: A retrospective analysis was performed of a prospectively collected institutional TPIAT database. Primary outcome was unplanned readmission to the hospital within 30 days from discharge. Reasons and risk factors for readmission as well as islet function were evaluated and compared by univariate and multivariate analysis. RESULTS: 83 patients underwent TPIAT from 2006 to 2014. 21 patients (25.3%) were readmitted within 30 days. Gastrointestinal problems (52.4%) and surgical site infection (42.8%) were the most common reasons for readmission. Initial LOS and reoperation were risk factors for early readmission. Patients with delayed gastric emptying (DGE) were three times more likely to get readmitted. In multivariate analysis, patients undergoing pylorus preservation surgery were nine times more likely to be readmitted than the antrectomy group. CONCLUSION: Early readmission after TPIAT is common (one in four patients), underscoring the complexity of this procedure. Early readmission is not detrimental to islet graft function. Patients undergoing pylorus preservation are more likely to get readmitted, perhaps due to increased incidence of delayed gastric emptying. Decision for antrectomy vs. pylorus preservation needs to be individualized.

Total pancreatectomy with islet autotransplantation: recent updates and outcomes.

PURPOSE OF REVIEW: Total pancreatectomy with islet autotransplantation (TPIAT) is a reliable therapy to retain endocrine function, to alleviate pain and improve quality of life in adult and pediatric patients suffering from refractory chronic pancreatitis and recurrent acute pancreatitis. Recently, an expansion of indications to include benign and malignant pancreatic disease has been suggested. Improved methods for evaluating the functional quality of islets and predicting transplant outcome have been discussed. Furthermore, this review will discuss the recent advances in the procedure, anti-inflammatory strategies and outcomes of TPIAT. RECENT FINDINGS: New assays to measure posttransplant islet damage and improved methods to assess islet quality by monitoring the oxygen consumption rate have shown great promise. Anti-inflammatory strategies such as an interleukin-1 receptor antagonist, α-1 antitrypsin, tumor necrosis factor α inhibitor and an inhibitor of CXCR1/2 have been widely investigated under clinical trials. SUMMARY: The primary objective of TPIAT, which is to relieve pain and reduce narcotic usage, has been shown to have long-term success. However, achieving long-term insulin independence is still a challenge that needs to be addressed. The mechanism that leads to chronic graft failure in TPIAT needs to be delineated.

Essential phospholipids prevent islet damage induced by proinflammatory cytokines and hypoxic conditions.

BACKGROUND: The pancreatic islet damage that occurs through an inflammatory response and hypoxia after infusion is a major hurdle in islet transplantation. Because essential phospholipids (EPL) have been shown to exhibit anti-inflammatory properties in liver disease, we analysed their protective effect on islets in inflammatory or hypoxic conditions. METHODS: We evaluated the viability of mouse and human islets cultured with cytokines or in hypoxic conditions for 48 h and measured cytokine expression in islets by quantitative polymerase chain reaction. We then employed an in vivo mouse assay, transplanting a marginal dose of human islets treated with or without EPL into the subcapsule of the kidney in diabetic nude mice and determining the cure rate. RESULTS: The viability of mouse and human islets damaged by cytokines was significantly improved by supplementation of EPL in the culture (p = 0.003 and <0.001 for mouse and human islets respectively). EPL significantly inhibited intracellular expression of IL-1ß and IL-6 in cytokine-damaged human islets (p < 0.001). The viability of human islets in hypoxic conditions was significantly better when treated with EPL (p < 0.001). In the in vivo mouse assay, the EPL-treated islet group had a higher cure rate than the untreated control, with marginal statistical significance (75 and 17% respectively, p = 0.07). CONCLUSIONS: EPL could be a potent agent to protect islets from inflammatory and hypoxic conditions after isolation procedures. Further studies to clarify the effect of EPL in islet transplantation are warranted.

Aptabinding of tumor necrosis factor-α (TNFα) inhibits its proinflammatory effects and alleviates islet inflammation.

Pancreatic islet cell transplantation (ICT) has emerged as an effective therapy for diabetic patients lacking endogenous insulin production. However, the islet graft function is compromised by a nonspecific inflammatory and thrombotic reaction known as the instant blood-meditated inflammatory reaction (IBMIR). Here, we report the characterization of four single-stranded DNA aptamers that bind specifically to TNFα - a pivotal cytokine that causes proinflammatory signaling during the IBMIR process - using single molecule binding analysis and functional assays as a means to assess the aptamers' ability to block TNFα activity and inhibiting the downstream proinflammatory gene expression in the islets. Our single-molecule fluorescence analyses of mono- and multivalent aptamers showed that they were able to bind effectively to TNFα with monoApt2 exhibiting the strongest binding (Kd  âˆ¼ 0.02 ± 0.01 nM), which is ∼3 orders of magnitude smaller than the Kd of the other aptamers. Furthermore, the in vitro cell viability analysis demonstrated an optimal and safe dosage of 100 µM for monoApt2 compared to 50 µM for monoApt1 and significant protection from proinflammatory cytokine-mediated cell death. More interestingly, monoApt2 reversed the upregulation of IBMIR mediating genes induced by TNFα in the human islets, and this was comparable to established TNFα antagonists. Both monoaptamers showed high specificity and selectivity for TNFα. Collectively, these findings suggest the potential use of aptamers as anti-inflammatory and localized immune-modulating agents for cellular transplant therapy.

Exosomes in transplantation: Role in allograft rejection, diagnostic biomarker, and therapeutic potential.

Exosomes are 50-200 nm-sized extracellular vesicles that are secreted by cells to transfer signals and communicate with other cells. Recent research has revealed that allograft-specific exosomes containing proteins, lipids, and genetic materials are released into circulation post-transplantation which are powerful indicators of graft failure in solid-organ and tissue transplantations. The macromolecular content of exosomes released by the allograft and the immune cells serve as potential biomarkers for assessing the function and the acceptance/rejection status of the transplanted grafts. Identifying these biomarkers could aid in the development of therapeutic strategies to improve graft longevity. Exosomes can be used to deliver therapeutic agonists/antagonists to grafts and prevent rejection. Inducing long-term graft tolerance has been demonstrated in many studies using exosomes from immunomodulatory cells such as immature DCs, T regulatory cells, and MSCs. The use of graft-specific exosomes for targeted drug therapy has the potential to reduce the unwanted side effects of immunosuppressive drugs. Overall, in this review, we have explored the critical role of exosomes in the recognition and cross-presentation of donor organ-specific antigens during allograft rejection. Additionally, we have discussed the potential of exosomes as a biomarker for monitoring graft function and damage, as well as their potential therapeutic applications in mitigating allograft rejection.

Single-Molecule Sensor for High-Confidence Detection of miRNA.

MicroRNAs (miRNAs) play a crucial role in regulating gene expression and have been linked to many diseases. Therefore, sensitive and accurate detection of disease-linked miRNAs is vital to the emerging revolution in early diagnosis of diseases. While the detection of miRNAs is a challenge due to their intrinsic properties such as small size, high sequence similarity among miRNAs and low abundance in biological fluids, the majority of miRNA-detection strategies involve either target/signal amplification or involve complex sensing designs. In this study, we have developed and tested a DNA-based fluorescence resonance energy transfer (FRET) sensor that enables ultrasensitive detection of a miRNA biomarker (miRNA-342-3p) expressed by triple-negative breast cancer (TNBC) cells. The sensor shows a relatively low FRET state in the absence of a target but it undergoes continuous FRET transitions between low- and high-FRET states in the presence of the target. The sensor is highly specific, has a detection limit down to low femtomolar (fM) without having to amplify the target, and has a large dynamic range (3 orders of magnitude) extending to 300 000 fM. Using this strategy, we demonstrated that the sensor allows detection of miRNA-342-3p in the miRNA-extracts from cancer cell lines and TNBC patient-derived xenografts. Given the simple-to-design hybridization-based detection, the sensing platform developed here can be used to detect a wide range of miRNAs enabling early diagnosis and screening of other genetic disorders.

Assessment of Culture/Preservation Conditions of Human Islets for Transplantation.

Islet culture before clinical transplantation has been adopted by various centers, but its effect on the survival and function of islets relative to the culture conditions and media needs further assessment. Human islets were cultured or preserved under four different conditions and three media options. Parameters such as recovery, viability, function, islet damage, and gene expressions for markers of hypoxia, and inflammation were assessed after 48-h culture or preservation. Preservation of islets was performed at 4°C in Connaught's Medical Research Lab (CMRL) and University of Wisconsin (UW) media. Islets were cultured at 22°C, 37°C, and 37°C-22°C in CMRL and PRODO culture media. Islets preserved in UW solution had visually good morphology and exhibited higher recovery with less islet damage compared with the rest of the groups, whereas islets preserved in CMRL at 4°C resulted in poor morphology, recovery, viability, and function compared with the rest of the treatment conditions. Culture at 22°C and 37°C demonstrated an increase in the expression of inflammatory and hypoxia-related genes. In conclusion, islets preserved at 4°C in UW solution showed the best overall outcomes after 48 h compared with islets cultured at 22°C, 37°C, or 37°C-22°C in PRODO. Advancement in islet culture media is warranted to reduce inflammatory gene activation and improve recovery of islets for transplantation.

Differential zinc accumulation and expression of human zinc transporter 1 (hZIP1) in prostate glands.

Zinc (Zn) is essential for a very large number and variety of cellular functions but is also potentially toxic. Zn homeostasis is therefore dynamically maintained by a variety of transporters and other proteins distributed in distinct cellular and subcellular compartments. Zn transport is mediated by two major protein families: the Zip family, which mediates Zn influx, and the ZnTs which are primarily linked to Zn sequestration into intracellular compartments and are, thereby, involved in lowering cytoplasmic Zn free ion concentrations. In the prostate epithelial cell, the accumulation of high cellular zinc is a specialized function that is necessary for these cells to carry out the major physiological functions of production and secretion of prostatic fluids. The loss of Zn accumulation is the most consistent and persistent characteristic of prostate malignancy. Currently, there are no direct methods to determine the relative Zn levels in various cell types of prostate gland (i.e. stroma, glandular epithelia, acini, and muscular) and no reliable ways to compare the Zn in normal versus malignant areas of the gland. Here we report a new method to show a differential Zn staining method that correlates with various stages of prostate cancer development in situ and expression of a human Zn transporter1-hZIP1 -in situ by in situ reverse transcriptase-polymerase chain reaction hybridization (ISRTPCR) that correlate with the relative Zn levels determined by the differential Zn staining method. By utilizing these methods, we show for the first time that: (1) the relative Zn levels are very low to absent in the malignant glands, (2) normal glands show high Zn levels in both glandular epithelia as well as in stromal tissues, (3) the Zn levels begin to decrease in pre-malignant glands and precedes the development of malignancy, and (4) the expression of human Zn transporter1 (hZIP1) appears to correlate with the Zn levels in the prostate glands and may be the major Zn regulator in this organ.

Withaferin A inhibits lymphocyte proliferation, dendritic cell maturation in vitro and prolongs islet allograft survival.

The immunosuppressive regimen for clinical allogeneic islet transplantation uses beta cell-toxic compounds such as tacrolimus that cause islet graft loss. Previously we reported that the plant-derived steroidal lactone Withaferin A (WA) can protect islet grafts by inhibiting nuclear factor-kappa B (NF-κB). Since the NF-κB signaling pathway is essential for T-cell activation, we hypothesized that long-term WA administration may also provide an immunosuppressive effect. Treatment of BALB/c donor islets and C57BL/6N recipients with WA alone resulted in 80% islet graft long-term survival vs. 40% in low-dose FK506-treated mice. In vitro, WA significantly blocked mouse and human T-cell proliferation by CD3/CD28 bead stimulation and in mixed lymphocyte reaction assay. Treatment of immature dendritic cells with WA prevented their maturation in response to inflammatory stimuli, as seen by decreased expression of CD83 and human leukocyte antigen-DR isotype. Exosomes released by islets treated with WA contained significantly fewer proinflammatory molecules interleukin-6, interleukin-8, monocyte chemoattractant protein-1, interferon-gamma-induced protein-10, inducible nitric oxide synthase, and cyclooxygenase-2. In conclusion, WA treatment not only reduced inflammation but also prolonged allograft survival, possibly through suppression of dendritic cell maturation and T-cell proliferation. WA has the potential to inhibit both the innate and adaptive immune response to prolong allograft survival.

Outcomes of Islet Autotransplantation in Chronic Pancreatitis Patients with Complete Acinar Atrophy.

Total pancreatectomy with islet autotransplantation (TPIAT) is a promising treatment for refractory chronic pancreatitis (CP). Pathological features of CP include progressive fibrosis in pancreas parenchyma, atrophy, and/or ductal occlusion. Complete acinar atrophy (CAA) caused by chronic fibrosis and necroinflammation results in exocrine sufficiency and may influence islet isolation characteristics during TPIAT. In this analysis of patients who underwent TPIAT at our center, we compared transplant outcomes among those with CAA (n = 5) vs non-acinar atrophy (NAA; matching controls, n = 36). Data were analyzed using one-way analysis of variance with Bonferroni post hoc test or Student's t test. Pancreas digestion was longer in CAA than in NAA cases (18.6 vs 14.6 min) despite a lower pancreas weight (55.2 vs 91.2 g). Obtained tissue volume was 1.0 ml in the CAA group and 12.1 ml in the NAA group. Both groups had similar islet viability (96%) and islet dose (CAA, 3,391 IEQ/kg; NAA, 4141.1 IEQ/kg). During islet infusion, serum cytokine (IL-6, IL-8, and MCP-1) levels and plasma hsa-miR-375 levels were lower in the CAA group than in the NAA group, but not significantly. Serum tumor necrosis factor α levels at 3 h after infusion were significantly higher in CAA group than in NAA group. After TPIAT, the metabolic outcomes of the CAA group were comparable with that of the NAA group. Narcotics usage decreased significantly over 24 months in both groups, with the CAA group reporting being pain free at 12 months. Complete atrophy of acinar cells of pancreas did not significantly impact islet yield or endocrine function after TPIAT.

Role of Inflammasomes in the Development of Gastrointestinal Diseases.

Many diseases of the gastrointestinal tract have been attributed to chronic inflammation, and a few have identified the role of inflammasomes in their pathogenesis. Inflammasomes are a group of protein complexes comprising of several intracellular proteins that link the sensing of microbial products and metabolic stress to the proteolytic activation of the proinflammatory cytokines. Recent studies have implicated activation of several families of NOD-like receptors (NLRs) which are major components of inflammasomes in the development and exacerbation of many diseases of human systems. In this chapter, we discuss the role of inflammasomes in some of the most prevalent diseases of the gastrointestinal tract and highlight potential targets for treatment.

A small molecule inhibitor of NFκB blocks ER stress and the NLRP3 inflammasome and prevents progression of pancreatitis.

BACKGROUND: The underlying molecular mechanism that leads to development of chronic pancreatitis remains elusive. The aim of this study is to understand the downstream inflammatory signaling involved in progression of chronic pancreatitis, and to use withaferin A (WA), a small molecule inhibitor of nuclear factor κB (NFκB), to prevent progression of chronic pancreatitis. METHODS: Two different protocols were used to induce pancreatitis in mice: standard and stringent administration of cerulein. The severity of pancreatitis was assessed by means of pancreatic histology and serum amylase levels. Immunohistochemistry and flow-cytometric analysis was performed to visualize immune cell infiltration into the pancreas. Real-time PCR and Western blot were used to analyze the downstream signaling mechanism involved in the development of chronic pancreatitis. RESULTS: The severity of cerulein-induced pancreatitis was reduced significantly by WA, used as either preventive or curative treatment. Immune cell infiltration into the pancreas and acinar cell death were efficiently reduced by WA treatment. Expression of proinflammatory and proapoptotic genes regulated by NFκB activation was increased by cerulein treatment, and WA suppressed these genes significantly. Sustained endoplasmic reticulum stress activation by cerulein administration was reduced. NLRP3 inflammasome activation in cerulein-induced pancreatitis was identified, and this was also potently blocked by WA. The human pancreatitis tissue gene signature correlated with the mouse model. CONCLUSIONS: Our data provide evidence for the role of NFκB in the pathogenesis of chronic pancreatitis, and strongly suggest that WA could be used as a potential therapeutic drug to alleviate some forms of chronic pancreatitis.

Pancreatic ß-Cell-Derived IP-10/CXCL10 Isletokine Mediates Early Loss of Graft Function in Islet Cell Transplantation.

Pancreatic islets produce and secrete cytokines and chemokines in response to inflammatory and metabolic stress. The physiological role of these "isletokines" in health and disease is largely unknown. We observed that islets release multiple inflammatory mediators in patients undergoing islet transplants within hours of infusion. The proinflammatory cytokine interferon-γ-induced protein 10 (IP-10/CXCL10) was among the highest released, and high levels correlated with poor islet transplant outcomes. Transgenic mouse studies confirmed that donor islet-specific expression of IP-10 contributed to islet inflammation and loss of ß-cell function in islet grafts. The effects of islet-derived IP-10 could be blocked by treatment of donor islets and recipient mice with anti-IP-10 neutralizing monoclonal antibody. In vitro studies showed induction of the IP-10 gene was mediated by calcineurin-dependent NFAT signaling in pancreatic ß-cells in response to oxidative or inflammatory stress. Sustained association of NFAT and p300 histone acetyltransferase with the IP-10 gene required p38 and c-Jun N-terminal kinase mitogen-activated protein kinase (MAPK) activity, which differentially regulated IP-10 expression and subsequent protein release. Overall, these findings elucidate an NFAT-MAPK signaling paradigm for induction of isletokine expression in ß-cells and reveal IP-10 as a primary therapeutic target to prevent ß-cell-induced inflammatory loss of graft function after islet cell transplantation.

MiR-375 and miR-200c as predictive biomarkers of islet isolation and transplantation in total pancreatectomy with islet autotransplantation.

BACKGROUND: Total pancreatectomy with islet autotransplantation (TPIAT) is a promising treatment for refractory chronic pancreatitis. Predictable biomarkers for the endocrine function after transplantation would be helpful in selecting patients for TPIAT. This study aims to identify novel biomarkers for predicting the outcome of islet isolation and transplantation in TPIAT patients. METHODS: This paper studied microRNA of 31 TPIAT patients and 11 deceased donors from plasma samples before TPIAT. MiR-7, miR-200a, miR-200c, miR-320, and miR-375 were analyzed along with patient characteristics and the outcomes of islet isolation and transplantation via univariate and multivariate regression analysis. RESULTS: MiR-375 before TPIAT showed a significant correlation with ∆C-peptide (r = -0.396, P = 0.03) and post-digestion islet count (r = -0.372, P = 0.04). And also miR-200c was significantly correlated with insulin requirement, C-peptide, and SUITO index at 1 year after transplantation. Moreover it was confirmed that miR-200c was a predictable factor of endocrine outcome in multi regression analysis (coefficient = -7.081, P = 0.001). CONCLUSIONS: We concluded that miR-375 and miR-200c could potentially serve as novel biomarkers in predicting the islet yield in islet isolation and the metabolic function after transplantation for chronic pancreatitis patients.

Use of Flaviviral genetic fragments as a potential prevention strategy for HIV-1 Silencing.

INTRODUCTION: Coinfection with certain members of the Flaviviridae, such as Dengue Virus (DV), West Nile Virus (WNV) Yellow Fever Virus (YFV) and most importantly, GBV-C have been documented to reduce HIV-1 viral load in vivo. Numerous studies strongly support the notion that persistent coinfection with non-pathogenic virus prolongs survival in HIV-1 infected individuals. Coinfected individuals show higher CD4+ cell counts, lower HIV-1 RNA viral loads and live three times longer than clinically matched HIV-1 monoinfected patients. We have previously shown that one of the major anti-HIV defenses conferred by GBV-C coinfection is the upregulation of intracellular miRNAs in CD4+ cells that share significant mutual homologies with GBV-C and HIV-1 (>80%) genomes. METHODOLOGY: Genome-wide bioinformatics analyses were carried out to search for miRNA binding sites in mutual homologies between HIV and several members of the Flaviviridae. RESULTS: Several miRNAs shared significant mutual homology with HIV-1 genetic sequences and GBV-A, B, C, DV, WNV and YFV. These may be responsible for beneficial effects in HIV-1 infected individuals. Three highly mutual homologous miRNAs (i.e. miR-627-5, miR-369-5 and miR-548f), expressed in CD4+ cell lines, reduce HIV-1 replication by up to 90% whereas miRNAs with low mutual homologies (i.e. miR-34-1 and miR-508) impart only slight inhibition of HIV-1. CONCLUSION: We hypothesize that a recombinant GBV-C-based vector can be constructed which expresses several beneficial genetic motifs of the Flaviviridae without causing any side effects while stimulating a wide array of beneficial miRNAs that can more efficiently prevent HIV-1 infection.

Comparison of surface modification chemistries in mouse, porcine, and human islets.

Beta cell replacement therapy, the transplantation of isolated pancreatic islets by intraportal infusion, offers patients with brittle type 1 diabetes blood glucose regulation with a minimally invasive technique. Chemical modification of islets prior to transplantation, providing a nanothin barrier that potentially includes active protective compounds, has been proposed as a strategy to minimize the inflammatory and immune reactions that often significantly limit graft function and duration. Chemical modification also has the potential to allow the use of alternative sources of islets, such as porcine islets, for transplantation. This investigation compared three orthogonal covalent islet modification techniques across three species (human, porcine, and murine), using multiple measures to determine biocompatibility and effectiveness. All three conjugation chemistries were well tolerated, and the overall efficiency, gross uniformity, and stability of the surface modifications were dependent upon the conjugation chemistry as well as the islet source (human, porcine, or murine). Notably, the reductive modification of surface disulfides was shown to afford intense and long-lasting modification of human islets. This study demonstrates that murine, human, and porcine islets tolerate a variety of covalent modifications, that these modifications are relatively stable, and that the murine islet model may not be predictive for some chemical contexts.