Geraldine Kent

Characteristic multiorgan pathology of cystic fibrosis in a long-living cystic fibrosis transmembrane regulator knockout murine model

The lack of an appropriate animal model with multiorgan pathology characteristic of the human form of cystic fibrosis has hampered our understanding of the pathobiology of the disease. We evaluated multiple organs of congenic C57BL/6J cystic fibrosis transmembrane regulator (Cftr)(-/-) and Cftr(+/+) mice maintained from weaning on a liquid diet then sacrificed between 1 and 24 months of age. The lungs of the Cftr(-/-) animals showed patchy alveolar overdistention, interstitial thickening, and fibrosis, with progression up to 6 months of age. The proximal and distal airway surface was encased with mucus-like material but lacked overt evidence of chronic bacterial infections or inflammation. All Cftr(-/-) animals showed progressive liver disease, with hepatosteatosis, focal cholangitis, inspissated secretions, and bile duct proliferation; after 1 year of age there was progression to focal biliary cirrhosis. The intercalated, intralobular and interlobular ducts and acinar lumina of the exocrine pancreas, the parotid and submaxillary glands of the Cftr(-/-) animals were dilated and filled with inspissated material, as well as mild inflammation and acinar cell drop out. Quantitative measurements of the pancreas showed significant acinar atrophy and increased acinar volume in comparison with age-matched Cftr(+/+) littermates. The ileal lumen and crypts were filled with adherent fibrillar material. After 3 months of age the vas deferens of the Cftr(-/-) animals could not be identified. None of the aforementioned pathological changes were observed in the Cftr(+/+) littermates fed the same liquid diet. We show, for the first time, that long-lived C578L/6J Cftr(-/-) mice develop manifestations of cystic fibrosis-like disease in all pathologically affected organs in the human form of cystic fibrosis.

Lung disease in mice with cystic fibrosis.

The leading cause of mortality and morbidity in humans with cystic fibrosis is lung disease. Advances in our understanding of the pathogenesis of the lung disease of cystic fibrosis, as well as development of innovative therapeutic interventions, have been compromised by the lack of a natural animal model. The utility of the CFTR-knockout mouse in studying the pathogenesis of cystic fibrosis has been limited because of their failure, despite the presence of severe intestinal disease, to develop lung disease. Herein, we describe the phenotype of an inbred congenic strain of CFTR-knockout mouse that develops spontaneous and progressive lung disease of early onset. The major features of the lung disease include failure of effective mucociliary transport, postbronchiolar over inflation of alveoli and parenchymal interstitial thickening, with evidence of fibrosis and inflammatory cell recruitment. We speculate that the basis for development of lung disease in the congenic CFTR-knockout mice is their observed lack of a non-CFTR chloride channel normally found in CFTR-knockout mice of mixed genetic background.

Phenotypic abnormalities in long-term surviving cystic fibrosis mice.

Mouse models for cystic fibrosis (CF) with no CFTR function(Cftr-/-) have the disadvantage that most animals die of intestinal obstruction shortly after weaning. The objective of this research was to extend the lifespan of CF mice and characterize their phenotype. Weanlings were placed on a nutrient liquid diet, and histologic and functional aspects of organs implicated in the disease were subsequently examined. Approximately 90% of Cftr-/- mice survived to 60 d, the majority beyond 100 d. Cftr-/- mice were underweight and had markedly abnormal intestinal histology. The intestinal epithelia did not respond to challenges with agents that raised intracellular cAMP, consistent with the absence of functional CFTR. No lesions or functional abnormalities were evident in the lungs. Liquid-fed Cftr-/- mice were infertile, although some males weaned to a solid diet were fertile before they died. Thus, we have succeeded in using dietary means to prolong the lives ofCftr-/- mice.

Laryngeal muscle and diaphragmatic activities n conscious dog pups

A new chronic animal preparation (i.e. puppies) for the study of respiratory laryngeal muscle activity is described. Posterior cricoarytenoid (PCA), thyroarytenoid (TA) and diaphragmatic activities were monitored in conscious puppies (13-43 days old) using chronically implanted electrodes. PCA activity was prominent during inspiration in all behavioral states. TA and post-inspiratory diaphragmatic activities were consistently high during expiration in quiet wakefulness and quiet sleep, but expiratory TA activity decreased dramatically and post-inspiratory diaphragmatic activity became highly variable in rapid eye movement sleep. There was also a tendency for these expiratory muscle activities to become less pronounced with increasing postnatal age. When a chronically implanted, low resistance T-tube tracheal cannula was suddenly opened, thus effectively eliminating the effects of upper airway resistance on airflow, respiratory frequency dramatically increased. These results indicate that the puppy exhibits muscle activities consistent with the maintenance of an elevated end-expiratory lung volume achieved by the retardation of expiratory airflow using the larynx and diaphragm.

Protection of Cftr knockout mice from acute lung infection by a helper-dependent adenoviral vector expressing Cftr in airway epithelia

We developed a helper-dependent adenoviral vector for cystic fibrosis lung gene therapy. The vector expresses cystic fibrosis transmembrane conductance regulator (Cftr) using control elements from cytokeratin 18. The vector expressed properly localized CFTR in cultured cells and in the airway epithelia of mice. Cftr RNA and protein were present in whole lung and bronchioles, respectively, for 28 days after a vector dose. Acute inflammation was minimal to moderate. To test the therapeutic potential of the vector, we challenged mice with a clinical strain of Burkholderia cepacia complex (Bcc). Cftr knockout mice (but not Cftr+/+ littermates) challenged with Bcc developed severe lung histopathology and had high lung bacteria counts. Cftr knockout mice receiving gene therapy 7 days before Bcc challenge had less severe histopathology, and the number of lung bacteria was reduced to the level seen in Cftr+/+ littermates. These data suggest that gene therapy could benefit cystic fibrosis patients by reducing susceptibility to opportunistic pathogens.

Enhanced susceptibility to pulmonary infection with Burkholderia cepacia in Cftr(-/-) mice.

ABSTRACT Progressive pulmonary infection is the dominant clinical feature of cystic fibrosis (CF), but the molecular basis for this susceptibility remains incompletely understood. To study this problem, we developed a model of chronic pneumonia by repeated instillation of a clinical isolate of Burkholderia cepacia (genomovar III, ET12 strain), an opportunistic gram-negative bacterium, from a case of CF into the lungs of Cftr m1unc−/−(Cftr−/−) and congenicCftr+/+ controls. Nine days after the last instillation, the CF transmembrane regulator knockout mice showed persistence of viable bacteria with chronic severe bronchopneumonia while wild-type mice remained healthy. The histopathological changes in the lungs of the susceptibleCftr−/− mice were characterized by infiltration of a mixed inflammatory-cell population into the peribronchiolar and perivascular spaces, Clara cell hyperplasia, mucus hypersecretion in airways, and exudation into alveolar airspaces by a mixed population of macrophages and neutrophils. An increased proportion of neutrophils was observed in bronchoalveolar lavage fluid from the Cftr−/− mice, which, despite an increased bacterial load, demonstrated minimal evidence of activation. Alveolar macrophages from Cftr−/− mice also demonstrated suboptimal activation. These observations suggest that the pulmonary host defenses are compromised in lungs from animals with CF, as manifested by increased susceptibility to bacterial infection and lung injury. This murine model of chronic pneumonia thus reflects, in part, the situation in human patients and may help elucidate the mechanisms leading to defective host defense in CF.

The influence of hypothermia on the disposition of fentanyl — Human and animal studies

SummaryThe effect of hypothermia on the disposition of fentanyl was evaluated in 18 children undergoing corrective cardiac surgery. They received a bolus of fentanyl followed by a continuous infusion which was stopped when cardiopulmonary bypass was established and profound hypothermia was achieved (18 °C–25 °C). Fentanyl plasma concentration remained essentially unchanged during hypothermia (6.45 ng/ml 5 min into hypothermia and 5.26 ng/ml 100–140 min later; p>0.1).In subsequent experiments, the effect of hypothermia on the pharmacokinetics of fentanyl was studied in 4 piglets serving as their own controls. Both distribution volume (Vz) and total body clearance (CL) were significantly smaller during hypothermia. Our studies indicate that being a drug with a large distribution volume and a high hepatic extraction ratio, both CL and Vz are significantly reduced by hypothermia-induced hypoperfusion. In addition, TBC is influenced by the temperature-dependent hepatic metabolism of fentanyl.

A new intratracheal stent for tracheobronchial reconstruction: Experimental and clinical studies

An intraluminal tracheal stent (ITS) was used experimentally in rabbits and piglets, as well as clinically in infants with tracheal stenosis, to facilitate airway reconstruction. The ITSs were constructed of stainless-steel springs covered with silicone rubber. They were implanted in seven piglets (6 to 8 kg), five rabbits (3 to 5 kg) and three infants. No animals developed severe respiratory distress and all appeared to tolerate the ITS. Postmortem examinations 1 to 8 weeks after surgery showed (1) loss of stent fixation (one pig), (2) increased tracheal secretions, (3) pneumonia (one pig, two rabbits), and (4) focal squamous metaplasia of tracheal mucosa. Stents used to treat three infants (2 to 5 months of age) with complex tracheobronchial stenosis were placed at the time of periosteal tracheoplasty in two. Recurrent stenosis necessitated a second tracheoplasty and stenting in one, and a long tracheostomy tube and balloon dilatations in the other. The third child had endoscopic stent insertion to alleviate severe airway collapse after esophageal tracheoplasty. The child died from progressive respiratory failure after stent dislodgment. Although the stents were well tolerated in animals and they enhanced critical ventilation of all pulmonary lobes in infants after tracheal reconstruction, certain modifications such as alternative methods of fixation, accommodation for tracheal growth, and reduction in tissue reactivity are necessary before further use of the ITS can be advocated.

The successful reconstruction of thoracic tracheal defects with free periosteal grafts

A defect was created in the thoracic trachea in nine piglets (10 kg) by excising a segment from the entire thoracic trachea proximal to the right upper lobe bronchus (3 to 3.5 cm in length) and 30% of the circumference in width (1 to 1.5 cm). The defect was repaired with a free tibial periosteal graft (FPG) and in four piglets an omental pedicle graft (OPG) was applied to the surface of the FPG. An internal tracheal splint was inserted for 12 to 16 days to support the repair and prevent air leak. The splints were removed bronchoscopically. None of the pigs developed signs of airway obstruction at any stage following the repair. Eight were killed 3 months postoperatively when four to six times their original weight. One pig died on the 18th postoperative day of unknown cause. The tracheas were removed, x-rayed, and examined grossly and histologically. The results demonstrated normal tracheal growth with absence of either stenosis, collapse, or granulation tissue. All grafts were viable, bone formation was present, and the lumenal surfaces were lined with columnar and respiratory epithelium. The OPG did not improve vascularization of the FPG. This model suggests that tracheal stenosis in children may be surgically corrected by simply incising the stenotic segment longitudinally and enlarging the tracheal diameter by inserting a FPG into the defect.

Exocrine Pancreatic Alterations in Long-Lived Surviving Cystic Fibrosis Mice

We investigated the development of the exocrine pancreas inCftr-/- mice in comparison with age-matched littermates(Cftr+/+, Cftr +/- ) up to 100 d postnatally. Controls were weaned either to mouse chow or a liquid diet;Cftr-/- mice were weaned solely to a liquid diet. Solid-fed control mice gained weight and showed a progressive increase in pancreatic protein, DNA, amylase, lipase, trypsin, and chymotrypsin activities. Liquid-fed control mice showed similar postnatal somatic and pancreatic growth, except that amylase and lipase activities were lower than in the solid-fed controls. Cftr-/- mice exhibited significantly lower body and pancreatic weights than did controls. Pancreatic protein content and enzyme activities (notably amylase and lipase) were consistently lower than in the age-matched littermates fed either diet. The reduction in lipase activity in Cftr-/- mice was noted before weaning. We concluded that the liquid diet influenced postnatal exocrine pancreatic development in mice. However, a further reduction in postnatal pancreatic growth and enzymatic activities in the Cftr-/- mice was noted. These alterations could be due to the primary cystic fibrosis defect, although secondary factors, such as malnutrition induced by decreased dietary intake or abnormal absorptive capacity, may be responsible.