Cystic Fibrosis Research and Translation Center
Cystic Fibrosis Research and Translation Center
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Current Pilot Projects


Pilot 3: Biochemical Mechanisms Mediating Impaired Insulin Secretion in Mouse Models of Cystic Fibrosis


P.I.: Ian Sweet, PhD

Research Associate Professor

Metabolism, Endocrinology and Nutrition

Director, Islet Core, UW DERC

Affiliate Investigator, Benaroya Research Institute


Cystic fibrosis (CF) is a congenital disease arising from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) and affects about 30,000 people nationwide. Mutations of the CFTR gene affect functioning of the chloride ion channels in epithelial cell membranes, leading to the many symptoms of CF. As CF patients age, there is an increased incidence of diabetes mellitus, occurring in almost half the patients with the disease. This form of diabetes has features of both type 1 and type 2 diabetes and is called Cystic Fibrosis Related Diabetes (CFRD). The most conceptually attractive factor responsible for the increased incidence of diabetes is diminished insulin secretion due to impaired beta cell function. We have obtained preliminary data showing that insulin secretion is significantly decreased in islets from CFTR knockout mice. Based on these data, we hypothesize that the loss of CFTR function is related to a loss in cAMP-stimulated insulin secretion. The PI of this application has been Director of the Islet Cell Functional Analysis Core, part of an NIDDK-funded Diabetes Research Center, for the last 10 years, and has established and validated a wide array of assays specifically to characterize biochemical mechanisms regulating islet secretory function involving metabolic, electrogenic and signaling factors. We propose to use these assays, as well as assays available through the Inflammation Core of the CF Research and Translation Center, to characterize and study the properties of islets from mouse models of CF that indicate the role of CFTR mutations in CFRD. To accomplish this, we will carry out 2 specific aims, one that will focus on in vitro experiments designed to determine the intracellular mechanisms mediating the impaired secretory function due to the CFTR mutation. The second aim will focus on in vivo conditions where it will be determined whether conditions resulting from the development of CF (as simulated by infecting mice with Pseudomonas aeruginosa) further decrease secretory function. The results of these studies will provide data that has both fundamental and clinical implications and will support a future R01 application to be submitted by the PI.



Pilot 5: Role of Islet Amyloid and Il-1β Signaling in β-cell Loss in Cystic Fibrosis-Related Diabetes


P.I.: Rebecca Hull, PhD

Research Assistant Professor of Medicine

Metabolism, Endocrinology and Nutrition


Srinath Sanda, MD

Associate Professor, Pediatrics

University of California at San Francisco


This proposal seeks to determine the role of ILβ in the pathogenesis of cystic fibrosis related diabetes (CFRD). CFRD is a unique form of diabetes and new treatments are needed to improve the morbidity and mortality of cystic fibrosis patients. Therefore the goals of this grant proposal are directly relevant to the mission of the National Institute of Diabetes, Digestive, and Kidney Diseases. The grant proposal consists of 2 broad aims. First we will compare the degree of amyloid deposition between autopsy pancreas sections from CFRD patients, CF patients without diabetes and age-matched controls. Subjects with type 2 diabetes, and appropriately-matched subjects without type 2 diabetes will serve as positive and negative controls for the measurements of interest. We anticipate that CFRD patients will show extensive deposition of islet amyloid, together with decreased β-cell area, reduced insulin expression, and increased β-cell apoptosis. Second we will analyze the same samples for evidence that the IL-1β pathway is active. In particular we will stain for IL-1β, IL-1Ra, NF-κB, MyD88, and CCL2. Aim 2: Do pancreatic sections from patients with CFRD show evidence of IL-1β activation? We will also determine whether correlations exist between these markers of the IL-1β pathway and measured obtained in Aim1, namely islet amyloid deposition, β-cell area and measures of β-cell apoptosis and replication. We anticipate that IL-1β activation will be a major feature of the islet in CFRD, suggesting that this pathway may play a role in the pathogenesis of islet dysfunction in this patient population.



Pilot 6: Investigating the effects of CFTR Correction on Myeloid Cell Function


P.I.: Lev Becker, PhD

Assistant Professor

Department of Pediatrics

University of Chicago


We propose to more directly test the hypothesis that CFTR dysfunction alters myeloid cell gene expression and function. To reduce confounding factors associated with comparison of healthy controls to CF patients, and to overcome the limitations of using mouse models, we propose to study patients with the CFTR-G551D mutation before and Ivacaftor treatment.


Aim 1: Determine the effect of Ivacaftor therapy on global gene and cell surface protein expression patterns in peripheral blood monocytes. Peripheral blood monocytes were isolated from 12 patients the day before initiation of Ivacaftor therapy, and on days 2 and 7 of treatment. Sweat chloride levels had dropped significantly in all patients at the day 2 time point, demonstrating efficacy of Ivacafor therapy. Total RNA and plasma membrane protein extracts from peripheral blood monocytes were collected at each time point, and shipped to University of Chicago for analysis by the Becker laboratory. We propose to perform microarray and cell surface proteomics analysis of peripheral blood monocytes in CF patients undergoing Ivacaftor therapy. The overall goal of these studies is to determine the direct effect of CFTR correction on myeloid cell gene/protein expression networks and function.



Pilot 8: Investigating the effects of CFTR Correction on Myeloid Cell Function


P.I.: Anne Manicone, MD

Assistant Professor of Medicine

Center for Lung Biology

Pulmonary and Critical Care Medicine


Pulmonary macrophages have dichotomous roles in promoting and resolving inflammatory responses in the lung; and they do this in part by their ability to adopt different activation or polarized states. In an "M1" polarized state, they participate in clearance of bacteria and secretion of pro-inflammatory cytokines/chemokines. They can also adopt an "M2" polarized state, and this activation state is important in promoting wound resolution, including clearance of debris and apoptotic neutrophils. A balance of M1 and M2 polarization is likely necessary for a normal inflammatory response to infection; and an imbalance in these activation states may contribute to lung disease. The functional role of M1 and M2 cells in regulating chronic inflammatory diseases is far from clear, and potentially an imbalance of M1/M2 function may contribute to lung disease. In fact, an increase in M2 cells has been reported in CF airways and is associated with worse lung function.1 Particularly in CF, macrophage polarization may be altered by chronic exposure to apoptotic neutrophils (a stimulus for M2 polarization) and to bacteria (a stimulus for M1 polarization). In addition, other factors specific to or associated with CF, such as a role of CFTR or hyperglycemia in macrophage plasticity and M2 repolarization remain unexplored.


My laboratory is interested in the functional roles of M2 cells in acute and chronic lung inflammation and the signaling pathways involved in promoting M2 programming. Our murine studies reveal the following: (1) alveolar macrophage repolarize from M1 to M2 in vivo during the resolution of P. aeruginosa pneumonia, (2) repolarized alveolar macrophages contribute to lung injury resolution in an acute model (not shown), and (3) greater M2 responses is associated with excessive wound repair in a chronic injury model.2,3 We hypothesize that in an acute infection, M2 repolarization is important to resolve inflammation and clear debris. However, persistence of these cells in the context of ongoing inflammatory stimuli may contribute to disease pathogenesis. Our preliminary studies demonstrate that

M2 repolarization promotes immunoparalysis to further challenge with endotoxin. Although this may be a favorable response, it may also contribute to chronic bacterial colonization, such as that seen in CF airways. In this pilot grant application, we seek to uncover the functional consequences of macrophage plasticity and the signaling pathways involved to expand upon a growing interest in macrophage biology in human lung disease. Expanding upon our knowledge will lead to novel therapeutics to modify the immune response in CF lung disease.4 This proposal will address these basic yet unknown areas in macrophage biology as outlined below. Results of these pilot studies will be the basis of larger NIH funded initiatives.



Pilot 9: Vitamin D metabolism in cystic fibrosis


P.I.: Ian de boer, MD

Associate Professor of Medicine

Nephrology


Bryan Kestenbaum, MD

Associate Professor of Medicine

Nephrology


Vitamin D deficiency is one of the most common nutritional deficits in cystic fibrosis (CF) patients, is resistant to treatment, and may contribute to bone disease and infections. Possible reasons for vitamin D deficiency in CF include intestinal malabsorption, altered liver metabolism, and loss of vital carrier proteins in the urine; however, empiric evidence to support these mechanisms is lacking.


In this application we propose a series of experiments designed to comprehensively define the vitamin D metabolic axis in CF. First, we will characterize the circulating profile of vitamin D metabolites, vitamin D carrier proteins, and downstream hormonal responses in 100 adult CF patients and 50 control subjects. Next we will conduct formal pharmacokinetic studies of radiolabeled tracer to probe the fate of substrate vitamin D in CF patients. We will then measure transcription of key vitamin D metabolism genes in circulating monocytes. Identifying the underlying causes of vitamin D deficiency in CF patients could suggest novel treatments that target vitamin D deficiency as a means to improve clinical outcomes in this disorder.



Pilot 10: Mechanisms of Late Hypoglycemia in Cystic Fibrosis


P.I.: Steven Kahn, MB, ChB

Professor of Medicine

Metabolism, Endocrinology and Nutrition


Kristina Utzschneider, MD

Associate Professor of Medicine

Metabolism, Endocrinology and Nutrition


With the increased life expectancy of patients with cystic fibrosis (CF), other co-morbidities have become apparent in these patients. One of these is abnormal glucose metabolism, where CF-related diabetes (CFRD) is common. More recently, another abnormality of glucose metabolism has been recognized; namely late hypoglycemia following oral glucose loading. In this study, we propose to test the hypothesis that the post-glucose load hypoglycemia observed in patients with CF results from a deficient counterregulatory hormone response and/or an insulin response that is exaggerated and delayed. This increased insulin response could be the result of an exaggerated incretin hormone response or altered gastric emptying. To address this hypothesis, we will perform a 3-hour oral glucose tolerance test during which we will measure counterregulatory, islet and incretin hormone responses and determine the rate of gastric emptying using acetaminophen. To determine whether patients with CF and late hypoglycemia also have episodes of hypoglycemia during daily living that includes mixed meals, we will use a continuous glucose monitoring system (CGMS) to examine 24-hour glucose profiles for 3 days. All these measures will be compared between patients with CF who develop late hypoglycemia, CF patients who do not develop hypoglycemia, and age and body mass index-matched healthy controls. The findings from this study will provide important new information regarding the mechanism(s) responsible for the late hypoglycemia observed in patients with CF and the data could be used as the basis for future grant applications. The ultimate goal is to gain insight into the condition of late hypoglycemia in order to better manage patients with CF.





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