Eugene H. Chang

Cystic fibrosis pigs develop lung disease and exhibit defective bacterial eradication at birth.

The lungs of just-born piglets with cystic fibrosis fail to efficiently eliminate bacteria, suggesting that lung problems in cystic fibrosis patients may be secondary to impaired antibacterial defense mechanisms. A Matter of Life and Breath The CafePress and Zazzle Web sites and most yoga-wear boutiques sport an array of teeshirts, bumper stickers, and water bottles prepared to offer simple advice to those living a harried life: “Just breathe.” Not so simple for a cystic fibrosis (CF) patient. Very early on, physicians recognized that difficulty breathing was the most ominous of the mosaic of symptoms that characterize this syndrome. Indeed, lung disease is the main cause of death in cystic fibrosis patients, but the lack of an animal model that mirrors the CF lung pathology seen in people has slowed translational cystic fibrosis research. Now, Stoltz et al. report findings in cystic fibrosis pigs that survive long enough to develop human-like lung disease. At the heart of this recessive genetic disease is the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride-ion channel. CF-causing mutations in the CFTR gene give rise to an aberrant channel that is defective in its ability to transport ions and water across cell membranes, resulting in a dizzying array of defects in the pancreas, intestines, reproductive system, liver, and lungs. It has been hypothesized that the impaired channel causes cells that line body cavities and passageways to become coated with thick mucus. In such an environment, bacteria thrive, leading to the chronic infections characteristic of this disease. However, the precise mechanisms by which CFTR mutations manifest as the complex phenotypes that constitute CF remain unclear, particularly with respect to the inflamed and infected airways of the CF lung. Despite substantial research efforts, scientists have been unable to achieve two crucial goals,to mold an animal model that mimics human CF lung disease and to pinpoint the trigger of CF lung pathology in pristine airways. Stoltz et al. tackled both of these obstacles by producing genetically modified CF pigs and analyzing their airways from birth to 6 months of age. Their studies revealed a spontaneously arising human-like lung disease that developed over time and had the CF hallmarks: multibacterial infections, inflammation, and mucus buildup. Although the lungs of the newborn CF piglets were not yet inflamed, they were less likely to be sterile and less able to eliminate bacteria that had been introduced into their lungs, relative to wild-type animals. Together, these findings suggest that bacterial infiltration spurs the pattern of lung inflammation and pathogenesis associated with CF. Having a clearer conception of CF lung disease can help clinicians devise preventive treatments that can be initiated early in the lives of CF patients. Such interventions may let CF suffers live and breath more fully. Lung disease causes most of the morbidity and mortality in cystic fibrosis (CF). Understanding the pathogenesis of this disease has been hindered, however, by the lack of an animal model with characteristic features of CF. To overcome this problem, we recently generated pigs with mutated CFTR genes. We now report that, within months of birth, CF pigs spontaneously developed hallmark features of CF lung disease, including airway inflammation, remodeling, mucus accumulation, and infection. Their lungs contained multiple bacterial species, suggesting that the lungs of CF pigs have a host defense defect against a wide spectrum of bacteria. In humans, the temporal and causal relations between inflammation and infection have remained uncertain. To investigate these processes, we studied newborn pigs. Their lungs showed no inflammation but were less often sterile than controls. Moreover, after introduction of bacteria into their lungs, pigs with CF failed to eradicate bacteria as effectively as wild-type pigs. These results suggest that impaired bacterial elimination is the pathogenic event that initiates a cascade of inflammation and pathology in CF lungs. Our finding that pigs with CF have a host defense defect against bacteria within hours of birth provides an opportunity to further investigate CF pathogenesis and to test therapeutic and preventive strategies that could be deployed before secondary consequences develop.

Intestinal CFTR expression alleviates meconium ileus in cystic fibrosis pigs

Cystic fibrosis (CF) pigs develop disease with features remarkably similar to those in people with CF, including exocrine pancreatic destruction, focal biliary cirrhosis, micro-gallbladder, vas deferens loss, airway disease, and meconium ileus. Whereas meconium ileus occurs in 15% of babies with CF, the penetrance is 100% in newborn CF pigs. We hypothesized that transgenic expression of porcine CF transmembrane conductance regulator (pCFTR) cDNA under control of the intestinal fatty acid-binding protein (iFABP) promoter would alleviate the meconium ileus. We produced 5 CFTR-/-;TgFABP>pCFTR lines. In 3 lines, intestinal expression of CFTR at least partially restored CFTR-mediated anion transport and improved the intestinal phenotype. In contrast, these pigs still had pancreatic destruction, liver disease, and reduced weight gain, and within weeks of birth, they developed sinus and lung disease, the severity of which varied over time. These data indicate that expressing CFTR in intestine without pancreatic or hepatic correction is sufficient to rescue meconium ileus. Comparing CFTR expression in different lines revealed that approximately 20% of wild-type CFTR mRNA largely prevented meconium ileus. This model may be of value for understanding CF pathophysiology and testing new preventions and therapies.

Requirements for Ion and Solute Transport, and pH Regulation During Enamel Maturation

Transcellular bicarbonate transport is suspected to be an important pathway used by ameloblasts to regulate extracellular pH and support crystal growth during enamel maturation. Proteins that play a role in amelogenesis include members of the ABC transporters (SLC gene family and CFTR). A number of carbonic anhydrases (CAs) have also been identified. The defined functions of these genes are likely interlinked during enamel mineralization. The purpose of this study is to quantify relative mRNA levels of individual SLC, Cftr, and CAs in enamel cells obtained from secretory and maturation stages on rat incisors. We also present novel data on the enamel phenotypes for two animal models, a mutant porcine (CFTR‐ΔF508) and the NBCe1‐null mouse. Our data show that two SLCs (AE2 and NBCe1), Cftr, and Car2, Car3, Car6, and Car12 are all significantly up‐regulated at the onset of the maturation stage of amelogenesis when compared to the secretory stage. The remaining SLCs and CA gene transcripts showed negligible expression or no significant change in expression from secretory to maturation stages. The enamel of CFTR‐ΔF508 adult pigs was hypomineralized and showed abnormal crystal growth. NBCe1‐null mice enamel was structurally defective and had a marked decrease in mineral content relative to wild‐type. These data demonstrate the importance of many non‐matrix proteins to amelogenesis and that the expression levels of multiple genes regulating extracellular pH are modulated during enamel maturation in response to an increased need for pH buffering during hydroxyapatite crystal growth. J. Cell. Physiol. 227: 1776–1785, 2012.

The role of connexins in human disease.

Connexins are the building blocks of gap junctions. In forming a gap junction, six connexins oligomerize to form a hexameric torus called a connexon. The number of gap junctions in a cell ranges from a few to over 105 and imparts to interconnected cells a uniform phenotype. The crucial role that gap junctions play in normal physiology is reflected by the diverse spectrum of human diseases in which allele variants of different gap junction genes are implicated. In particular, mutations in GJB2 are a major cause of autosomal recessive non-syndromic deafness. This discovery has impacted medical practice and makes it incumbent on clinicians to familiarize themselves with the genetic advances that are rapidly occurring in our field.

Sinus hypoplasia precedes sinus infection in a porcine model of cystic fibrosis.

Chronic sinusitis is nearly universal in humans with cystic fibrosis (CF) and is accompanied by sinus hypoplasia (small sinuses). However, whether impaired sinus development is a primary feature of loss of the cystic fibrosis transmembrane conductance regulator (CFTR) or a secondary consequence of chronic infection remains unknown. Our objective was to study the early pathogenesis of sinus disease in CF.

Elevated body mass index and risk of postoperative CSF leak following transsphenoidal surgery

OBJECT Postoperative CSF leakage can be a serious complication after a transsphenoidal surgical approach. An elevated body mass index (BMI) is a significant risk factor for spontaneous CSF leaks. However, there is no evidence correlating BMI with postoperative CSF leak after transsphenoidal surgery. The authors hypothesized that patients with elevated BMI would have a higher incidence of CSF leakage complications following transsphenoidal surgery. METHODS The authors conducted a retrospective review of 121 patients who, between August 2005 and March 2010, underwent endoscopic endonasal transsphenoidal surgeries for resection of primarily sellar masses. Patients requiring extended transsphenoidal approaches were excluded. A multivariate statistical analysis was performed to investigate the association of BMI and other risk factors with postoperative CSF leakage. RESULTS In 92 patients, 96 endonasal endoscopic transsphenoidal surgeries were performed that met inclusion criteria. Thirteen postoperative leaks occurred and required subsequent treatment, including lumbar drainage and/or reoperation. The average BMI of patients with a postoperative CSF leak was significantly greater than that in patients with no postoperative CSF leak (39.2 vs 32.9 kg/m(2), p = 0.006). Multivariate analyses indicate that for every 5-kg/m(2) increase in BMI, patients undergoing a transsphenoidal approach for a primarily sellar mass have 1.61 times the odds (95% CI 1.10-2.29, p = 0.016, by multivariate logistic regression) of having a postoperative CSF leak. CONCLUSIONS Elevated BMI is an independent predictor of postoperative CSF leak after an endonasal endoscopic transsphenoidal approach. The authors recommend that patients with BMI greater than 30 kg/m(2) have meticulous sellar reconstruction at surgery and close monitoring postoperatively.

Lentiviral vector gene transfer to porcine airways.

In this study, we investigated lentiviral vector development and transduction efficiencies in well-differentiated primary cultures of pig airway epithelia (PAE) and wild-type pigs in vivo. We noted gene transfer efficiencies similar to that observed for human airway epithelia (HAE). Interestingly, feline immunodeficiency virus (FIV)-based vectors transduced immortalized pig cells as well as pig primary cells more efficiently than HIV-1–based vectors. PAE express TRIM5α, a well-characterized species-specific lentiviral restriction factor. We contrasted the restrictive properties of porcine TRIM5α against FIV- and HIV-based vectors using gain and loss of function approaches. We observed no effect on HIV-1 or FIV conferred transgene expression in response to porcine TRIM5α overexpression or knockdown. To evaluate the ability of GP64-FIV to transduce porcine airways in vivo, we delivered vector expressing mCherry to the tracheal lobe of the lung and the ethmoid sinus of 4-week-old pigs. One week later, epithelial cells expressing mCherry were readily detected. Our findings indicate that pseudotyped FIV vectors confer similar tropisms in porcine epithelia as observed in human HAE and provide further support for the selection of GP64 as an appropriate envelope pseudotype for future preclinical gene therapy studies in the porcine model of cystic fibrosis (CF).

New insights into the pathogenesis of cystic fibrosis sinusitis

People with cystic fibrosis (CF) sinus disease have developmental sinus abnormalities with airway bacterial infection, inflammation, impaired mucociliary clearance and thick obstructive mucus. The pathophysiology of airway disease in CF is not completely understood, and current treatments in CF sinus disease ameliorate symptoms but do not provide a cure.

Adenoviral gene transfer corrects the ion transport defect in the sinus epithelia of a porcine CF model.

Cystic fibrosis (CF) pigs spontaneously develop sinus and lung disease resembling human CF. The CF pig presents a unique opportunity to use gene transfer to test hypotheses to further understand the pathogenesis of CF sinus disease. In this study, we investigated the ion transport defect in the CF sinus and found that CF porcine sinus epithelia lack cyclic AMP (cAMP)-stimulated anion transport. We asked whether we could restore CF transmembrane conductance regulator gene (CFTR) current in the porcine CF sinus epithelia by gene transfer. We quantified CFTR transduction using an adenovirus expressing CFTR and green fluorescent protein (GFP). We found that as little as 7% of transduced cells restored 6% of CFTR current with 17-28% of transduced cells increasing CFTR current to 50% of non-CF levels. We also found that we could overcorrect cAMP-mediated current in non-CF epithelia. Our findings indicate that CF porcine sinus epithelia lack anion transport, and a relatively small number of cells expressing CFTR are required to rescue the ion transport phenotype. These studies support the use of the CF pig as a preclinical model for future gene therapy trials in CF sinusitis.

Our initial experience of the transaxillary totally endoscopic approach for hemithyroidectomy.

OBJECTIVE: To report our initial experience with the transaxillary totally endoscopic (TATE) approach to the thyroid gland. STUDY DESIGN: A historic cohort study of patients undergoing TATE procedures compared with open procedures for hemi-thyroidectomy with isthmusectomy. SETTING: Private-practice otolaryngology group. SUBJECT AND METHODS: Patients selected for benign thyroid disease confirmed by fine-needle aspiration and requiring hemithyroidectomy with isthmusectomy. A historic cohort study of 24 patients who underwent TATE procedures for hemithyroidectomy with isthmusectomy. Comparison of the first 10 TATE approaches to a control group of 10 consecutive open approaches by the senior authors group. RESULTS: All 24 TATE patients were successful without the need to convert to an open procedure. The TATE approach had longer operative times than the open group (142 vs 105), but these operative times decreased as the number of procedures increased (first five TATE = 170, last five TATE = 114, n = 24, average = 114). No patients had peri- or postoperative complications. CONCLUSIONS: The TATE approach to the thyroid gland is safe and effective. Operative time is longer but decreases with experience. The TATE approach is one option to treat young patients with unilateral benign thyroid disease who are seeking to avoid visible scars and limit morbidity.