Department of Pediatrics Works

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1,25-Dihydroxyvitamin D3 enhances glucose-stimulated insulin secretion in mouse and human islets: a role for transcriptional regulation of voltage-gated calcium channels by the vitamin D receptor
(Elsevier, 2018) Kjalarsdottir, Lilja; Tersey, Sarah A.; Vishwanath, Mridula; Chuang, Jen-Chieh; Posner, Bruce A.; Mirmira, Raghavendra G.; Repa, Joyce J.; Pediatrics, School of Medicine
Aim Vitamin D deficiency in rodents negatively affects glucose-stimulated insulin secretion (GSIS) and human epidemiological studies connect poor vitamin D status with type 2 diabetes. Previous studies performed primarily in rat islets have shown that vitamin D can enhance GSIS. However the molecular pathways linking vitamin D and insulin secretion are currently unknown. Therefore, experiments were undertaken to elucidate the transcriptional role(s) of the vitamin D receptor (VDR) in islet function. Methods Human and mouse islets were cultured with vehicle or 1,25-dihydroxyvitamin-D3 (1,25D3) and then subjected to GSIS assays. Insulin expression, insulin content, glucose uptake and glucose-stimulated calcium influx were tested. Microarray analysis was performed. In silico analysis was used to identify VDR response elements (VDRE) within target genes and their activity was tested using reporter assays. Results Vdr mRNA is abundant in islets and Vdr expression is glucose-responsive. Preincubation of mouse and human islets with 1,25D3 enhances GSIS and increases glucose-stimulated calcium influx. Microarray analysis identified the R-type voltage-gated calcium channel (VGCC) gene, Cacna1e, which is highly upregulated by 1,25D3 in human and mouse islets and contains a conserved VDRE in intron 7. Results from GSIS assays suggest that 1,25D3 might upregulate a variant of R-type VGCC that is resistant to chemical inhibition. Conclusion These results suggest that the role of 1,25D3 in regulating calcium influx acts through the R-Type VGCC during GSIS, thereby modulating the capacity of beta cells to secrete insulin.
100 years of insulin: celebrating the past, present and future of diabetes therapy
(Springer Nature, 2021) Sims, Emily K.; Carr, Alice L.J.; Oram, Richard A.; DiMeglio, Linda A.; Evans-Molina, Carmella; Pediatrics, School of Medicine
The year 2021 marks the centennial of Banting and Best's landmark description of the discovery of insulin. This discovery and insulin's rapid clinical deployment effectively transformed type 1 diabetes from a fatal diagnosis into a medically manageable chronic condition. In this Review, we describe key accomplishments leading to and building on this momentous occasion in medical history, including advancements in our understanding of the role of insulin in diabetes pathophysiology, the molecular characterization of insulin and the clinical use of insulin. Achievements are also viewed through the lens of patients impacted by insulin therapy and the evolution of insulin pharmacokinetics and delivery over the past 100 years. Finally, we reflect on the future of insulin therapy and diabetes treatment, as well as challenges to be addressed moving forward, so that the full potential of this transformative discovery may be realized.
12-Lipoxygenase governs the innate immune pathogenesis of islet inflammation and autoimmune diabetes
(The American Society for Clinical Investigation, 2021-07-22) Kulkarni, Abhishek; Pineros, Annie R.; Walsh, Melissa A.; Casimiro, Isabel; Ibrahim, Sara; Hernandez-Perez, Marimar; Orr, Kara S.; Glenn, Lindsey; Nadler, Jerry L.; Morris, Margaret A.; Tersey, Sarah A.; Mirmira, Raghavendra G.; Anderson, Ryan M.; Pediatrics, School of Medicine
Macrophages and related myeloid cells are innate immune cells that participate in the early islet inflammation of type 1 diabetes (T1D). The enzyme 12-lipoxygenase (12-LOX) catalyzes the formation of proinflammatory eicosanoids, but its role and mechanisms in myeloid cells in the pathogenesis of islet inflammation have not been elucidated. Leveraging a model of islet inflammation in zebrafish, we show here that macrophages contribute significantly to the loss of β cells and the subsequent development of hyperglycemia. The depletion or inhibition of 12-LOX in this model resulted in reduced macrophage infiltration into islets and the preservation of β cell mass. In NOD mice, the deletion of the gene encoding 12-LOX in the myeloid lineage resulted in reduced insulitis with reductions in proinflammatory macrophages, a suppressed T cell response, preserved β cell mass, and almost complete protection from the development of T1D. 12-LOX depletion caused a defect in myeloid cell migration, a function required for immune surveillance and tissue injury responses. This effect on migration resulted from the loss of the chemokine receptor CXCR3. Transgenic expression of the gene encoding CXCR3 rescued the migratory defect in zebrafish 12-LOX morphants. Taken together, our results reveal a formative role for innate immune cells in the early pathogenesis of T1D and identify 12-LOX as an enzyme required to promote their prodiabetogenic phenotype in the context of autoimmunity.
12-Lipoxygenase Inhibitor Improves Functions of Cytokine-Treated Human Islets and Type 2 Diabetic Islets
(Oxford University Press, 2017-08-01) Ma, Kaiwen; Xiao, An; Park, So Hyun; Glenn, Lindsey; Jackson, Laura; Barot, Tatvam; Weaver, Jessica R.; Taylor-Fishwick, David A.; Luci, Diane K.; Maloney, David J.; Mirmira, Raghavendra G.; Imai, Yumi; Nadler, Jerry L.; Pediatrics, School of Medicine
Context: The 12-lipoxygenase (12-LO) pathway produces proinflammatory metabolites, and its activation is implicated in islet inflammation associated with type 1 and type 2 diabetes (T2D). Objectives: We aimed to test the efficacy of ML355, a highly selective, small molecule inhibitor of 12-LO, for the preservation of islet function. Design: Human islets from nondiabetic donors were incubated with a mixture of tumor necrosis factor α , interluekin-1β, and interferon-γ to model islet inflammation. Cytokine-treated islets and human islets from T2D donors were incubated in the presence and absence of ML355. Setting: In vitro study. Participants: Human islets from organ donors aged >20 years of both sexes and any race were used. T2D status was defined from either medical history or most recent hemoglobin A1c value >6.5%. Intervention: Glucose stimulation. Main Outcome Measures: Static and dynamic insulin secretion and oxygen consumption rate (OCR). Results: ML355 prevented the reduction of insulin secretion and OCR in cytokine-treated human islets and improved both parameters in human islets from T2D donors. Conclusions: ML355 was efficacious in improving human islet function after cytokine treatment and in T2D islets in vitro. The study suggests that the blockade of the 12-LO pathway may serve as a target for both form of diabetes and provides the basis for further study of this small molecule inhibitor in vivo.
12-Lipoxygenase Promotes Obesity-Induced Oxidative Stress in Pancreatic Islets
(American Society for Microbiology (ASM), 2014-10) Tersey, Sarah A.; Maier, Bernhard; Nishiki, Yurika; Maganti, Aarthi V.; Nadler, Jerry L.; Mirmira, Raghavendra G.; Department of Pediatrics, IU School of Medicine
High-fat diets lead to obesity, inflammation, and dysglycemia. 12-Lipoxygenase (12-LO) is activated by high-fat diets and catalyzes the oxygenation of cellular arachidonic acid to form proinflammatory intermediates. We hypothesized that 12-LO in the pancreatic islet is sufficient to cause dysglycemia in the setting of high-fat feeding. To test this, we generated pancreas-specific 12-LO knockout mice and studied their metabolic and molecular adaptations to high-fat diets. Whereas knockout mice and control littermates displayed identical weight gain, body fat distribution, and macrophage infiltration into fat, knockout mice exhibited greater adaptive islet hyperplasia, improved insulin secretion, and complete protection from dysglycemia. At the molecular level, 12-LO deletion resulted in increases in islet antioxidant enzymes Sod1 and Gpx1 in response to high-fat feeding. The absence or inhibition of 12-LO led to increases in nuclear Nrf2, a transcription factor responsible for activation of genes encoding antioxidant enzymes. Our data reveal a novel pathway in which islet 12-LO suppresses antioxidant enzymes and prevents the adaptive islet responses in the setting of high-fat diets.
A 12-lipoxygenase-Gpr31 signaling axis is required for pancreatic organogenesis in the zebrafish
(Wiley, 2020-11) Hernandez-Perez, Marimar; Kulkarni, Abhishek; Samala, Niharika; Sorrell, Cody; El, Kimberly; Haider, Isra; Aleem, Ansari Mukhtar; Holman, Theodore R.; Rai, Ganesha; Tersey, Sarah A.; Mirmira, Raghavendra G.; Anderson, Ryan M.; Pediatrics, School of Medicine
12-Lipoxygenase (12-LOX) is a key enzyme in arachidonic acid metabolism, and alongside its major product, 12-HETE, plays a key role in promoting inflammatory signaling during diabetes pathogenesis. Although 12-LOX is a proposed therapeutic target to protect pancreatic islets in the setting of diabetes, little is known about the consequences of blocking its enzymatic activity during embryonic development. Here, we have leveraged the strengths of the zebrafish-genetic manipulation and pharmacologic inhibition-to interrogate the role of 12-LOX in pancreatic development. Lipidomics analysis during zebrafish development demonstrated that 12-LOX-generated metabolites of arachidonic acid increase sharply during organogenesis stages, and that this increase is blocked by morpholino-directed depletion of 12-LOX. Furthermore, we found that either depletion or inhibition of 12-LOX impairs both exocrine pancreas growth and unexpectedly, the generation of insulin-producing β cells. We demonstrate that morpholino-mediated knockdown of GPR31, a purported G-protein-coupled receptor for 12-HETE, largely phenocopies both the depletion and the inhibition of 12-LOX. Moreover, we show that loss of GPR31 impairs pancreatic bud fusion and pancreatic duct morphogenesis. Together, these data provide new insight into the requirement of 12-LOX in pancreatic organogenesis and islet formation, and additionally provide evidence that its effects are mediated via a signaling axis that includes the 12-HETE receptor GPR31.
143 Training & Sustaining: Training and learning collaborative outcomes across a statewide network for early diagnosis of children with autism
(Cambridge University Press, 2024-04-03) McNally Keehn, Rebecca; Paxton, Angela; Delaney, Mary; Ciccarelli, Mary; Pediatrics, School of Medicine
OBJECTIVES/GOALS: Community-based primary care autism diagnostic models are one promising solution to delays in autism diagnosis. Our objective is to describe the development and report on outcomes related to primary care professional (PCP) training and sustained engagement in a longitudinal learning collaborative across a statewide network for autism diagnosis. METHODS/STUDY POPULATION: We developed ADAPT (i.e., Accelerating the Diagnosis of Autism with Primary care Training), a training program to prepare PCPs to develop independent competency in evaluation of autism in children ages 14-48 months. ADAPT includes didactic and case-based modules and expert practice-based coaching delivered by a diagnostic specialist; following training PCPs participate in a longitudinal learning collaborative. Aligned with competency-based medical education standards, measures of autism evaluation knowledge and diagnostic competency are collected. RESULTS/ANTICIPATED RESULTS: To date, 13 PCPs have completed ADAPT didactic and practicum training reaching competency in independent autism evaluation. Clinicians demonstrated significant improvement in total autism knowledge following didactic training (p=.02). There was an overall trend toward increased scoring agreement on an autism observational assessment over case observations and practicum evaluations. Similarly, PCPs demonstrated improved evaluation competence, moving on average from Advanced Beginner to Competent Performer as rated by expert trainers. Following training, PCPs attended 57% of monthly learning collaborative sessions. DISCUSSION/SIGNIFICANCE: Training PCPs to deliver autism evaluations as part of community-based models of care is a promising solution to address access and waitlist challenges. ADAPT is an intensive, standard PCP training model which results in achievement of independent competency and sustained engagement in in autism evaluation.
15-Lipoxygenase worsens renal fibrosis, inflammation, and metabolism in a murine model of ureteral obstruction
(American Physiological Society, 2022) Montford, John R.; Bauer, Colin; Rahkola, Jeremy; Reisz, Julie A.; Floyd, Deanna; Hopp, Katharina; Soranno, Danielle E.; Klawitter, Jelena; Weiser-Evans, Mary C. M.; Nemenoff, Raphael; Faubel, Sarah; Furgeson, Seth B.; Pediatrics, School of Medicine
15-Lipoxygenase (15-LO) is a nonheme iron-containing dioxygenase that has both pro- and anti-inflammatory roles in many tissues and disease states. 15-LO is thought to influence macrophage phenotype, and silencing 15-LO reduces fibrosis after acute inflammatory triggers. The goal of the present study was to determine whether altering 15-LO expression influences inflammation and fibrogenesis in a murine model of unilateral ureteral obstruction (UUO). C57BL/6J mice, 15-LO knockout (Alox15-/-) mice, and 15-LO transgenic overexpressing (15LOTG) mice were subjected UUO, and kidneys were analyzed at 3, 10, and 14 days postinjury. Histology for fibrosis, inflammation, cytokine quantification, flow cytometry, and metabolomics were performed on injured tissues and controls. PD146176, a specific 15-LO inhibitor, was used to complement experiments involving knockout animals. Compared with wild-type animals undergoing UUO, Alox15-/- mouse kidneys had less proinflammatory, profibrotic message along with less fibrosis and macrophage infiltration. PD146176 inhibited 15-LO and resulted in reduced fibrosis and macrophage infiltration similar to Alox15-/- mice. Flow cytometry revealed that Alox15-/- UUO-injured kidneys had a dynamic change in macrophage phenotype, with an early blunting of CD11bHiLy6CHi "M1" macrophages and an increase in anti-inflammatory CD11bHiLy6CInt "M2c" macrophages and reduced expression of the fractalkine receptor chemokine (C-X3-C motif) receptor 1. Many of these findings were reversed when UUO was performed on 15LOTG mice. Metabolomics analysis revealed that wild-type kidneys developed a glycolytic shift postinjury, while Alox15-/- kidneys exhibited increased oxidative phosphorylation. In conclusion, 15-LO manipulation by genetic or pharmacological means induces dynamic changes in the inflammatory microenvironment in the UUO model and appears to be critical in the progression of UUO-induced fibrosis. NEW & NOTEWORTHY: 15-Lipoxygenase (15-LO) has both pro- and anti-inflammatory functions in leukocytes, and its role in kidney injury and repair is unexplored. Our study showed that 15-LO worsens inflammation and fibrosis in a rodent model of chronic kidney disease using genetic and pharmacological manipulation. Silencing 15-LO promotes an increase in M2c-like wound-healing macrophages in the kidney and alters kidney metabolism globally, protecting against anaerobic glycolysis after injury.
156. Daily Association of Drug Use Cravings and Physical and Emotional Well-Being among Students Attending a Recovery High School during COVID-19: Results from an EMA Study
(Elsevier, 2023) Hensel, Devon J.; Wilburn, Victoria Garcia; Pediatrics, School of Medicine
Purpose: Ongoing COVID-19 restrictions are now well-known to increase youth substance use. Little research has addressed this vulnerability among adolescents in substance use recovery (AIR), who may be at heightened risk for relapse within ongoing pandemic management. We used ecological momentary assessment-focused (EMA) to characterize daily shifts in recovery management among adolescents attending a recovery high school. We engage the first wave of these EMA-data collected during third wave of the pandemic (January-February 2022) to: 1) document day-to-day changes in drug craving context (e.g., frequency, temporality, duration, and intensity) and; 2) examine its association with daily shifts in physical and emotional well-being. Methods: Data were from an EMA-study (ongoing through 2022-2023 school-year) intended to understand the social/emotional context of drug use cravings among a cohort of AIR attending a recovery high school in Indianapolis, IN. Our analytic-sample includes six-students (N=40 total) who were enrolled during the second pandemic wave (57.3% female, 42.5% heterosexual, 71.0% White, 52.3% 12th-grade, 57.4% had weekly urges use). Drug use context measures: any drug-use urge (no/yes), urge frequency (5-point item: once-6+ times), urge temporality/duration (6-point item: AM, aft or PM only, more than half the day, all day) and urge intensity (single item: 0-10). Physical and emotional variables: emotional pain (5-point single item: not at all-extremely), self-perceived health (5-point single item: poor to excellent), positive and negative mood (PANAS) and somatic symptoms (summed 12-point: all no/yes). Descriptive statistics and intraclass correlation coefficients (ICC) examined the prevalence and day-to-day variability of each outcome; random intercept mixed effects binary or ordinal regression assessed the impact of physical and emotional predictors on each outcome (Stata v. 18). Results: Participants contributed 81.2% (147/180) of expected EMAs, one-third of which were associated with an urge to use drugs. 6% of urge days also involved a report of drug-use (4/48; ns sample size for additional-analysis). Median within-day urge frequency was 3-5x/day (35.3% of all urge events). We observed significant day-to-day variability in reports of drug urges and the intensity of urges (ICC: 0.209-0.601), but not in temporality and duration of urges. Greater daily emotional pain nearly quadrupled the odds of reporting having drug urges (OR=3.75) and was associated with three-fold higher within-day urge frequency (OR=2.42) and urge intensity (OR=2.92). Higher positive mood was positively associated with urge to use (OR=1.36), urge frequency (OR=1.17) and urge intensity (OR=1.18). More somatic symptoms were associated with greater odds of having drug urges (OR=1.26) and urge frequency during the day (OR=1.10). Conclusions: Our research demonstrates that daily emotional valence and greater somatic symptoms are associated with daily drug urge occurrence, daily urge frequency and daily drug urge intensity among AIR. These EMA data have important clinical implications for “just in time” mental and behavioral health interventions that could focus on mood stability and physical wellness as scaffolds in adolescent recovery management.
158. Diagnostic Yield and Clinical Utility of Broad Range PCR Testing at a Tertiary Children’s Hospital
(Oxford University Press, 2023-11-27) Schneider, Jack; Prabhudas-Strycker, Kirsten; Samaro, Matthew; Goings, Michael; Mellencamp, Kagan A.; Khan, Haseeba; Boyd, LaKeisha; Pediatrics, School of Medicine
Background: Broad range PCR testing (BR-PCR) targets highly conserved DNA sequences of bacteria, fungi, or mycobacteria to detect a broad range of organisms in various clinical samples. Given its potential impact in providing timely diagnoses that cannot always be made through conventional testing (CT), we evaluated the diagnostic yield and clinical impact of BR-PCR at our institution. Methods: We retrospectively evaluated all clinical specimen types obtained for BR-PCR at Riley Hospital for Children from October 2019 to May 2022. Percent positivity (PP) was determined by specific PCR test type (Bacterial/Fungal/NTM/TB), along with median turn-around times (in days) from sample collection. Medical charts were reviewed, and clinical impact of results was determined. Results: We identified 956 BR-PCR tests sent from 271 specimens collected from 178 patients. Only 14.5% yielded a positive result with a median days-to-result being the longest for fungal PCR at 8.1 days (7.0, 10.1) and TB PCR being the fastest at 7.8 days (6.8, 9.9). Bacterial BR-PCR yielded an overall PP of 42.6% while Fungal BR-PCR was 10.7%. Positivity rates for NTM and TB were 0% and 0.5%, respectively. Bronchial lavage was the most common specimen type (35.5%) with an overall PP of 19.9%. Of the 271 specimens, 245 returned conclusive results from both BR-PCR and clinical testing (CT) for comparison. A clinically significant organism based on CT was identified in 68 specimens (27.8%), 45 of which were confirmed by BR-PCR. 23 (33.8%) were detected by CT but not BR-PCR. 21 clinically significant organisms not detected by CT were identified by BR-PCR, which led to a change in clinical management in 12 instances: new diagnosis (91.7%); or appropriate initiation (91.7%), escalation (25.0%), or de-escalation (25.0%) of antimicrobial therapy. Conclusion: BR-PCR overall had low diagnostic yield at our institution but was influenced by specimen type. The clinical utility was predominantly seen in immunocompromised patients in which conventional testing was negative. Further data is needed to determine which specimen types and diagnoses will increase the yield and clinical value of BR-PCR and thus, aid in enhancing diagnostic stewardship.

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