Doctor of Philosophy, Baylor University (2018)
Bachelor of Science, Baylor University (2013)
Reliance on rodents for understanding pancreatic genetics, development and islet function could limit progress in developing interventions for human diseases like diabetes mellitus. Similarities of pancreas morphology and function suggest that porcine and human pancreas developmental biology may have useful homologies. However, little is known about pig pancreas development. To fill this knowledge gap, we investigated fetal and neonatal pig pancreas at multiple, crucial developmental stages using modern experimental approaches. Purification of islet ?-, ?- and ?-cells followed by transcriptome analysis (RNA-Seq) and immunohistology identified cell- and stage-specific regulation, and revealed that pig and human islet cells share characteristic features not observed in mice. Morphometric analysis also revealed endocrine cell allocation and architectural similarities between pig and human islets. Our analysis unveiled scores of signaling pathways linked to native islet ?-cell functional maturation, including evidence of fetal ?-cell GLP-1 production and signaling to ?-cells. Thus, the findings and resources detailed here show how pig pancreatic islet studies complement other systems for understanding the developmental programs that generate functional islet cells, and that are relevant to human pancreatic diseases.
View details for DOI 10.1242/dev.186213
View details for PubMedID 32108026
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.
View details for DOI 10.1111/ajt.14615
View details for Web of Science ID 000428844400024
View details for PubMedID 29210193
The systemic administration of immunosuppressive and anti-inflammatory drugs is routinely employed in organ transplantation to minimize graft rejection and improve graft survival. Localized drug delivery has the potential to improve transplant outcomes by providing sustained exposure to efficacious drug concentrations while avoiding systemic immunosuppression and off-target effects. Here, we describe the synthesis of a novel prodrug and its direct covalent conjugation to pancreatic islets via a cleavable linker. Post-transplant, linker hydrolysis results in the release of a potent anti-inflammatory antagonist of TLR4, localized to the site of implantation. This covalent islet modification significantly reduces the time and the minimal effective dose of islets necessary to achieve normoglycemia in a murine transplantation model. In streptozotocin-induced diabetic C57BL/6 mice a syngeneic transplant of ?100 modified islets achieved a 100% cure rate by the end of a 4-week monitoring period, compared to a 0% cure rate for untreated control islets. Overall, this direct prodrug conjugation to islets is well tolerated and preserves their functionality while affording significantly superior transplant outcomes. The development of drug-eluting tissues that deliver sustained and localized doses of small-molecule therapeutics represents a novel pathway for enhancing success in transplantation.
View details for DOI 10.1016/j.biomaterials.2017.12.020
View details for Web of Science ID 000425070900002
View details for PubMedID 29309990
The efficacy of islet transplantation is compromised by a significant loss of islet mass posttransplant due to an innate inflammatory reaction. Here we report the use of a combination of etanercept and anakinra to block inflammatory islet damage in 100 patients undergoing total pancreatectomy with islet autotransplantation. The patients were divided into three groups: no treatment (CTL), etanercept alone (ETA), or a combination of etanercept and anakinra (ANA+ETA). Peritransplant serum samples were analyzed for protein markers of islet damage and for inflammatory cytokines. Graft function was assessed by fasting blood glucose, basal C-peptide, SUITO index, and hemoglobin A1c. Administration of both antiinflammatory drugs was well tolerated without any major adverse events. Reductions in interleukin-6, interleukin-8, and monocyte chemoattractant protein 1 were observed in patients receiving ANA+ETA compared with the CTL group, while also showing a modest improvement in islet function as assessed by basal C-peptide, glucose, hemoglobin A1c, and SUITO index but without differences in insulin dose. Together these results suggest that double cytokine blockade (ANA+ETA) reduces peritransplant islet damage due to nonspecific inflammation and may represent a promising strategy to improve islet engraftment, leading to better transplant outcomes. This article is protected by copyright. All rights reserved.
View details for DOI 10.1111/ajt.14961
View details for PubMedID 29862647
During the isolation process, pancreatic islets are exposed to an environment of sterile inflammation resulting in an upregulated inflammatory state prior to transplantation. Toll-like receptor 4 (TLR4) has been identified as a major mediator of sterile inflammation. Therefore, we sought to determine whether early TLR4 blockade would be effective in reducing the inflammatory burden in islets pretransplant.Islets from C57BL/6 mice were treated with a TLR4 antagonist during the pancreatic ductal perfusion and digestion steps of the isolation process. Islets were then analyzed for inflammation by RT-PCR and western blot, and for viability and function in vitro. A syngeneic transplant model using a marginal mass of islets transplanted intraportally into mice with streptozotocin-induced diabetes was used to study transplant outcomes after early TLR4 blockade.Diabetic mice receiving 150 islets treated with early TLR4 blockade achieved euglycemia at a higher rate than mice receiving untreated islets (75% vs 29%; p < 0.05) and had improved long-term function (P < 0.05). Serum markers for islet damage and inflammation were significantly reduced posttransplant (P < 0.05). Both the expression of key inflammatory genes and the activation of mitogen-activated protein kinases were reduced by early TLR4 blockade. Islet viability was improved (P < 0.05) while preserving islet insulin secretory capacity postisolation.Early TLR4 blockade protects islets from sterile inflammation-mediated stress sustained during isolation and promotes positive transplant outcomes. Our findings support the use of early TLR4 blockade during clinical islet isolation procedures to reduce pretransplant inflammation and improve transplant outcomes.
View details for DOI 10.1097/TP.0000000000002287
View details for PubMedID 29787520
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-?-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 ?-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 ?-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 ?-cells and reveal IP-10 as a primary therapeutic target to prevent ?-cell-induced inflammatory loss of graft function after islet cell transplantation.
View details for DOI 10.2337/db17-0578
View details for Web of Science ID 000413559100015
View details for PubMedID 28855240
View details for PubMedCentralID PMC5652609
Transplantation of allogenic pancreatic islets is a minimally invasive treatment option to control severe hypoglycemia and dependence on exogenous insulin among type 1 diabetes (T1D) patients. This overview summarizes the current issues and progress in islet transplantation outcomes and research.Several clinical trials from North America and other countries have documented the safety and efficacy of clinical islet transplantation for T1D patients with impaired hypoglycemia awareness. A recently completed phase 3 clinical trial allows centres in the United States to apply for a Food and Drug Administration Biologics License for the procedure. Introduction of anti-inflammatory drugs along with T-cell depleting induction therapy has significantly improved long-term function of transplanted islets. Research into islet biomarkers, immunosuppression, extrahepatic transplant sites and potential alternative beta cell sources is driving further progress.Allogeneic islet transplantation has vastly improved over the past two decades. Success in restoration of glycemic control and hypoglycemic awareness after islet transplantation has been further highlighted by clinical trials. However, lack of effective strategies to maintain long-term islet function and insufficient sources of donor tissue still impose limitations to the widespread use of islet transplantation. In the United States, wide adoption of this technology still awaits regulatory approval and, importantly, a financial mechanism to support the use of this technology.
View details for PubMedID 28692442
Fifteen new substituted adenines were synthesized as potential TLR7 agonists. These compounds, along with 9 previously reported compounds, were analyzed for TLR7 activity and for the selective stimulation of B cell proliferation. Several functionalized derivatives exhibit significant activity, suggesting their potential for use as vaccine adjuvants.
View details for DOI 10.1016/j.bmcl.2016.07.049
View details for Web of Science ID 000381959900016
View details for PubMedID 27476423
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.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.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).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.
View details for PubMedID 27429015
View details for Web of Science ID 000391783500367
View details for Web of Science ID 000391783500366
Pancreatic islet transplantation is a promising beta cell replacement treatment for patients with "brittle" type 1 diabetes (T1D) or intractable chronic pancreatitis to restore or preserve pancreatic endocrine function. Early after transplant, a significant islet mass is lost due to an innate inflammatory response, and further loss of the islet graft occurs over time due to immune response, drug toxicity, or metabolic exhaustion. Thus, clinically feasible techniques are essential to monitor islet graft function and survival to maintain appropriate therapy. Currently, islet graft function is monitored using blood glucose levels, insulin and C-peptide levels, and islet imaging. However, these tests are influenced by physiological changes, including beta cell stimulation. Biomarkers that are independent of metabolic stimuli would be more accurate and reliable in detecting islet damage. Antibodies against islet autoantigens are useful but not reliable markers of islet injury due to their presence during the pretransplant period. Several islet-specific proteins such as Glutamate decarboxylase-65, doublecortin, protein phosphatase 1, regulatory (inhibitor) subunit 1A, ubiquitin C-terminal hydrolase-L1, and the high-mobility group box-1 protein have been proposed as candidates to monitor islet damage, but these biomarkers have short half-lives and unreliable detection. Unmethylated insulin DNA has been studied in T1D patients and has been documented as a highly correlative and selective biomarker for beta cell death. More recently, microRNAs (miRNAs) that are selectively expressed in islets have been shown to provide sensitive and accurate quantification of islet damage. Analysis of plasma samples from autologous and allogeneic islet transplant patients has demonstrated the value of miRNA-375 as a specific biomarker to accurately assess islet damage. Use of selective, sensitive, and measurably reproducible biomarkers of islets will lead to effective monitoring of beta cell replacement therapy and may also lead to development of preventative and interventional treatment strategies to improve outcomes.
View details for Web of Science ID 000374620200007
View details for PubMedID 26763742
Protein conjugates of toll-like receptor 7 agonists have been shown to elicit powerful immune responses. In order to facilitate our studies in this area our group has developed efficient syntheses for a number of functionalized derivatives that retain immune stimulatory activity.
View details for DOI 10.1016/j.tetlet.2014.11.126
View details for Web of Science ID 000348265100040
View details for PubMedCentralID PMC4296738