Epigenetic Reprogramming Drives Epithelial Disruption in Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) remains a major public health challenge that contributes greatly to mortality and morbidity worldwide. Although it has long been recognized that the epithelium is altered in COPD, there has been little focus on targeting it to modify the disease course. Therefore, mechanisms that disrupt epithelial cell function in patients with COPD are poorly understood. In this study, we sought to determine whether epigenetic reprogramming of the cell-cell adhesion molecule E-cadherin, encoded by the CDH1 gene, disrupts epithelial integrity. By reducing these epigenetic marks, we can restore epithelial integrity and rescue alveolar airspace destruction. We used differentiated normal and COPD-derived primary human airway epithelial cells, genetically manipulated mouse tracheal epithelial cells, and mouse and human precision-cut lung slices to assess the effects of epigenetic reprogramming. We show that the loss of CDH1 in COPD is due to increased DNA methylation site at the CDH1 enhancer D through the downregulation of the ten-eleven translocase methylcytosine dioxygenase (TET) enzyme TET1. Increased DNA methylation at the enhancer D region decreases the enrichment of RNA polymerase II binding. Remarkably, treatment of human precision-cut slices derived from patients with COPD with the DNA demethylation agent 5-aza-2'-deoxycytidine decreased cell damage and reduced air space enlargement in the diseased tissue. Here, we present a novel mechanism that targets epigenetic modifications to reverse the tissue remodeling in human COPD lungs and serves as a proof of concept for developing a disease-modifying target.

Microphysiological Models of Lung Epithelium-Alveolar Macrophage Co-Cultures to Study Chronic Lung Disease.

The interactions between immune cells and epithelial cells influence the progression of many respiratory diseases, such as chronic obstructive pulmonary disease (COPD). In vitro models allow for the examination of cells in controlled environments. However, these models lack the complex 3D architecture and vast multicellular interactions between the lung resident cells and infiltrating immune cells that can mediate cellular response to insults. In this study, three complementary microphysiological systems are presented to delineate the effects of cigarette smoke and respiratory disease on the lung epithelium. First, the Transwell system allows the co-culture of pulmonary immune and epithelial cells to evaluate cellular and monolayer phenotypic changes in response to cigarette smoke exposure. Next, the human and mouse precision-cut lung slices system provides a physiologically relevant model to study the effects of chronic insults like cigarette smoke with the dissection of specific interaction of immune cell subtypes within the structurally complex tissue environment. Finally, the lung-on-a-chip model provides an adaptable system for live imaging of polarized epithelial tissues that mimic the in vivo environment of the airways. Using a combination of these models, a complementary approach is provided to better address the intricate mechanisms of lung disease.

Urinary Catheters Coated with a Novel Biofilm Preventative Agent Inhibit Biofilm Development by Diverse Bacterial Uropathogens.

Despite the implementation of stringent guidelines for the prevention of catheter-associated (CA) urinary tract infection (UTI), CAUTI remains one of the most common health care-related infections. We previously showed that an antimicrobial/antibiofilm agent inhibited biofilm development by Gram-positive and Gram-negative bacterial pathogens isolated from human infections. In this study, we examined the ability of a novel biofilm preventative agent (BPA) coating on silicone urinary catheters to inhibit biofilm formation on the catheters by six different bacterial pathogens isolated from UTIs: three Escherichia coli strains, representative of the most common bacterium isolated from UTI; one Enterobacter cloacae, a multidrug-resistant isolate; one Pseudomonas aeruginosa, common among patients with long-term catheterization; and one isolate of methicillin-resistant Staphylococcus aureus, as both a Gram-positive and a resistant organism. First, we tested the ability of these strains to form biofilms on urinary catheters made of red rubber, polyvinyl chloride (PVC), and silicone using the microtiter plate biofilm assay. When grown in artificial urine medium, which closely mimics human urine, all tested isolates formed considerable biofilms on all three catheter materials. As the biofilm biomass formed on silicone catheters was 0.5 to 1.6 logs less than that formed on rubber or PVC, respectively, we then coated the silicone catheters with BPA (benzalkonium chloride, polyacrylic acid, and glutaraldehyde), and tested the ability of the coated catheters to further inhibit biofilm development by these uropathogens. Compared with the uncoated silicone catheters, BPA-coated catheters completely prevented biofilm development by all the uropathogens, except P. aeruginosa, which showed no reduction in biofilm biomass. To explore the reason for P. aeruginosa resistance to the BPA coating, we utilized two specific lipopolysaccharide (LPS) mutants. In contrast to their parent strain, the two mutants failed to form biofilms on the BPA-coated catheters, which suggests that the composition of P. aeruginosa LPS plays a role in the resistance of wild-type P. aeruginosa to the BPA coating. Together, our results suggest that, except for P. aeruginosa, BPA-coated silicone catheters may prevent biofilm formation by both Gram-negative and Gram-positive uropathogens.

Effect of tympanostomy tube surface on occlusion.

OBJECTIVE: Premature tympanostomy tube (TT) occlusion frequently leads to TT replacement surgery. TT surface preparations have been suggested as a means of reducing TT occlusion. The purpose of this study is to determine if commercial TT compositions or surface preparations impact the rate of TT occlusion using an in vitro model. METHODS: Commercial TTs composed of titanium, fluoroplastic, and silicone, as well as human serum albumin coated titanium, phosphorylcholine coated fluoroplastic, and polyvinylpyrrolidone coated silicone TTs, were tested for occlusion development in a previously validated in vitro model that simulates middle ear air and mucus flow. RESULTS: Time to occlusion was longer with all coated TTs relative to all uncoated TTs (p=0.038). Polyvinylpyrrolidone coated silicone TTs had the lowest rate of occlusion and improvement relative to silicone (36% vs. 70%). Time to occlusion was longer in all coated TTs, but individually, none reached statistical significance. CONCLUSION: TT composition and surface preparations do not dramatically impact the development of TT occlusion. All tested surface coatings seem to delay TT occlusion in this in vitro model. In vivo testing will be necessary to validate these findings.

Analysis of flap tension in surgical rejuvenation of the midface: a cadaver study.

OBJECTIVE: To determine the flap tension present for 2 different surgical approaches for midface lifts and whether a gingivobuccal incision for release of the midface periosteum will diminish the resultant load on the midface flap. STUDY DESIGN: Cadaver study. SETTING: Anatomy laboratory at the University of Florida. SUBJECTS AND METHODS: Twelve fresh-frozen cadaver heads are subjected to a transblepharoplasty (TB) approach on one side of the cadaver head while a transtemporal (TT) approach is performed on the contralateral side. The tension (grams) generated by moving the midface flap a distance of 1.03 cm is recorded by a digital load cell. A gingivobuccal incision is made on each side to allow transoral periosteal release (TOPR) of the midface flap, and the experiment is repeated. Three trials are performed for each operation, and the average load is calculated. A paired t test is applied. RESULTS: The tension resulting from the TT approach (172 g) was not found to be significantly different from that of the TB approach (179 g; P = .75). Significant differences in the resultant tension were not appreciated when comparing standard techniques to TT with TOPR (141.5 g; P = .27) or for TB with TOPR (164.1 g; P = .45). DISCUSSION: An experimental method is described for determining flap tension in facial rejuvenation surgery. No significant difference is found between flap tensions generated in the TB and TT approaches to the midface.

Development of a calcium alginate tympanostomy tube.

OBJECTIVES/HYPOTHESIS: Tympanostomy tubes (TTs) are prone to complications resulting in part from the unpredictable duration that the TT remains in the tympanic membrane. General anesthesia may be necessary to remove TTs that fail to extrude. The purpose of this study was to develop a TT that could be dissolved on demand but remain functional with exposure to common otologic exposures. STUDY DESIGN: Prospective in vitro analysis. METHODS: Dissolvable TTs were developed from calcium alginate. Mechanical properties and occlusion susceptibility were optimized by varying ingredient concentrations and compared to commonly used commercial TTs using in vitro measures. RESULTS: Alginate TTs had a greater compressive strength than commercial silicone tubes. TTs composed of 0.5 M CaCl were stronger than high molarity CaCl concentrations. Uncoated alginate TTs showed a 20% reduction in occlusion propensity. Exposure of alginate TTs to otological solutions for 24 hours resulted in degradation of their mechanical properties, but they remained superior to commercial silicone TTs. CONCLUSIONS: Alginate TTs appear to be a good alternative to commercial tubes based on high mechanical strength and low occlusion propensity. Furthermore, unlike commercial TTs, alginate TTs have the potential to be dissolved in vivo if retained.

In vitro testing of tympanostomy tube occlusion.

OBJECTIVE: Tympanostomy tubes (TTs) are commonly rendered nonfunctional by mucus plug formation. The purpose of this study was to determine whether an in vitro model could be developed to assess TT plug formation with results consistent with human trials. STUDY DESIGN: An ear chamber was designed to mimic middle ear air and mucus flow conditions in post-TT otorrhea. TT occlusion was tested and correlated to published in vivo results. METHODS: TTs that had previously been studied in vivo (Goode "T" and Reuter Bobbin collar buttons) were placed in the model chamber. Pooled, homogenized human middle ear mucus and an analog, egg white, were delivered at 80 muL per hour through the TTs. An air bolus was delivered every two minutes to simulate swallowing. Chamber pressure was monitored over 2.5 hours. Occlusion was determined by a pressure peak and visual confirmation. RESULTS: Obstruction was found in 60 percent of the Reuter Bobbin and 40 percent of the Goode TTs using the mucus analog. These results are similar to those reported from previous in vivo studies. No plugging was reported for either TT using homogenized human ear mucus. CONCLUSIONS: The in vitro TT chamber simulates the in vivo environment and yields results consistent with in vivo observations. This model system may allow for rapid prototyping and evaluation of new TTs that may be less vulnerable to occlusion.