Adult Cystic Fibrosis and Host Defense

The long-term and central goal of the Cystic Fibrosis Research Center at the University of Pittsburgh is to provide improved treatments, and ultimately a cure, for cystic fibrosis (CF).  A collaborative effort by investigators in the Departments of Cell Biology and Physiology, Medicine, and Pediatrics, our strategy is to implement this goal using our strengths in the basic science of CFTR and epithelial ion transport and to translate our increasingly applied basic science knowledge to clinical investigations.

In order to achieve these goals we have established centers of excellence in three broad categories: Cell and Molecular Biology of CF, Infection and Inflammation in CF, and Clinical Studies in CF.   These centers are summarized below:

Cell and Molecular Biology:  The bulk of our research is conducted using primary human airway cell cultures established from CF and non-CF tissues that are made available by the more than 100 lung transplants that are performed annually at the University of Pittsburgh Medical Center.   State of the art biochemistry, molecular biology, cell biology, and electrophysiology are used to study CFTR and ENaC functions in epithelial membranes, how these pathways contribute to normal airway function, how they are trafficked between various compartments of the protein secretory and recycling pathways, and how therapeutic approaches can be derived from this understanding of channel function.  The overall concept guiding many of these studies is that airway ion and water transport determine the properties of the airway surface liquid, which in turn affects the efficiency of mucociliary clearance mechanisms.  Studies of the pharmacological manipulation of channel biogenesis (correction) or channel activity (potentiation) fall into this category.  Most of this activity is currently supported by a an NIH P30 grant, a Research Development Program  grant from The Cystic Fibrosis Foundation, and individual NIH R01 or Cystic Fibrosis Foundation (CFF) mechanisms of support.

Because the airway surface liquid is critical to the regulation of mucus clearance from the lung, numerous novel techniques are employed at our center to measure the properties of this thin 10 micrometer layer of fluid, including live cell fluorescence and phase microscopy, as shown in the accompanying figures.

ASL absorption in CF and non-CF HBE. Cells stained with calcein-AM are layered with PBS containing Texas Red-dextran and subjected to confocal x-z scanning to measure the time-course of ASL height. Using this method we can differentiate CF from non-CF airway epithelium and have demonstrated that AMPK agonists, such as metformin, have a beneficial effect on the ASL volume and inflammation in CF lung disease. From Myerburg et al., Am J Respir Cell Mol Biol 2010)

Comparison of the ASL volume of HBE cultures with various CFTR genotypes. The ASL volume of several CF and non-CF HBE cultures was measured using a novel method which relies on the refracted light properties induced by a liquid meniscus.

Movie of primary HBE cultures demonstrating coordinated ciliary beat and mucus movement.  When the ASL is properly hydrated, ciliary function is optimized and the accumulated mucus overlying HBE cultures is transported in a circular direction.

Infection and Inflammation:  The course of CF lung disease changes qualitatively when patients become colonized by opportunistic infections.  This area of research is designed to determine the inflammatory response to chronic Pseudomonas aeruginosa and Aspergillus fumigatus infections, the cytokines that mediate this response, the host defense mechanisms that attempt to clear bacteria from the lung and the disease and bacterial mechanisms responsible for clearance failure.  The response to pro-inflammatory cytokines may contribute to lung injury during sustained infection.  Thus, it is of critical importance to understand the interrelationship between the CFTR defect and host immunity to: 1) improve our understanding of the pathogenesis of the diseases, 2) define bio-markers which may be better surrogates of disease progression as well as novel outcome measures for future clinical trials, and 3) identify potential CF-specific targets for anti-inflammatory therapy.  This work is currently supported primarily by a NIH SCOR, R01 and CFF grants.

Clinical Studies:  A critical issue in CF research has been translation of laboratory findings to new therapies, particularly identification of the most appropriate and efficient endpoints for clinical trials.  The need for new outcome measurements for assessing CF therapies has been long recognized, and although progress has been made recently towards validation of induced sputum markers and the use of improved airway clearance methods, further refinement is needed.Investigators in the CF Center have focused efforts for several years on proof of principle nasal epithelial ion transport studies for new ion channel modulators, and more recently, on identifying surrogate endpoints for disease activity and short-term responses to new therapies aimed at overcoming defects in mucociliary clearance.  Collaborative investigations between Center investigators and scientists at Carnegie Mellon University have focused on improvements and airway clearance techniques and the use of surfactants as airway delivery reagents.  Whole-lung mucociliary clearance measurements have been used successfully as a short-term outcome measure in previous studies, but new isotope methods are providing the promise for assessing transepithelial airway liquid movements as a means to test therapeutics that target the basic defect in CFTR.  These studies are supported by the NIH, NSF, and the CF Foundation.

In vivo method of measuring ASL absorption and mucus clearance as a biomarker in CF.

We have developed a novel method to simultaneously measureASL absorption and mucus clearance in patients.  The difference in the clearance between an absorbable radiolabeled particle (Indium 111 -DTPA) and a non- absorbable radiolabeled particle (Technetium 99m- SC) represents the rate of ASL absorption.  To the right is a movie of representative clearance scan.   Corcoran, T.E., Thomas, K.M., Myerburg, M.M., Muthukrishnan, A., Weber, L., Frizzell, R.,Pilewski, J.M. (2010) Eur Resp J, 35(4):781-6

The CF research group at the University of Pittsburgh is composed of faculty, graduate students, and post graduate students, from The Division of Pulmonary, Allergy, and Critical Care MedicineThe Pediatric Cystic Fibrosis CenterThe Department  of Cell Biology and Physiology, and The Division of Renal-Electrolyte Medicine.

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