Paula S. Ludwig

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.

Sevoflurane and halothane reduce focal ischemic brain damage in the rat : possible influence on thermoregulation

BackgroundThere has been little systematic examination concerning the comparative effects of the anesthetized versus the awake state on outcome from cerebral ischemia. This experiment evaluated infart volume and neurologic function in rats subjected to temporary focal ishemia while anesthetized with either sevoflurane or halothane. Outcome in these animals was compared to that observed in rats maintained unanesthetized during a similar ischemic insult. MethodsAll rats were anesthetized with halothane and surgically prepared for filament occlusion of the middle cerebral artery. After preparation, one group (Halothane) remained anesthetized with approximately 1.4 MAC halothane. In another group (Sevoflurane), halothane was discontinued and substituted with sevoflurane, which was administered until electroencephalographic burst suppression was evident (approximately 1.4 MAC). The final group (A wake) was allowed to awaken immediately after the onset of ischemia. Middle cerebral artery occlusion persisted for 90 min all groups. The middle cerebral artery filament then was removed, and a 96-h survival interval was allowed. Neurologic function and infarct volume were determined. Recent evidence indicates that transient mild hyperthermia occurs in awake rats undergoing filament occlusion of the middle cerebral artery. To examine the potential role of mild hyperthermia in this experiment, a second experiment was performed in which rats anesthetized with halothane underwent 90-min focal ischemia, with pericranial temperatures held at either 38.0° C or 39.2° C. ResultsIntraischemic mean arterial pressure was 20–25 mmHg lower in the two anesthetized groups compared with awake animals. Despite this finding, cortical infarct volumes (mean ± SD; Halothane, 17 ± 32 mm3; Sevoflurane, 36 ± 57 mm3; Awake, 115 ± 104 mm3) and subcortical infarct volumes (mean ±:SD; Halothane, 39 ± 57 mm3; Sevoflurane, 50 ± 29 mm3; Awake, 88 ± 46 mm3) were reduced in both groups of anesthetized rats. This reduction correlated with improved neurologic function. The rats in whom the pericranial temperature was maintained at 39.2° C had a larger total infarct volume (218 ± 81 mm3) and increased neurologic deficits when compared to those in whom the pericranial temperature was maintained at 38.0° C (total infarct volume, 75 ± 77 mm3). ConclusionsBoth halothane and sevoflurane substantially reduced damage in this focal ischemia model when compared to outcome resulting from the same insult induced in awake rats. The reduction in intraischemic mean arterial pressure caused by the anesthetics did not seem contributory to out-come. Brain temperature differences among the groups were not defined. Because small differences in pericranial temperature were shown to have major effects on outcome, further work is required to determine if differences in brain temperature explain the observed protective effects of these anesthetics.

The ΔF508 Mutation Causes CFTR Misprocessing and Cystic Fibrosis–Like Disease in Pigs

A common mutation in human cystic fibrosis, CFTR-ΔF508, results in misprocessed CFTR and a cystic fibrosis–like clinical phenotype in pigs. Four Legs Good, Two Legs Bad In Animal Farm, George Orwell describes a pasture in which the pigs lead an animal revolt, resulting eventually in the porcine dwellers becoming indistinguishable from the human ones against whom they revolted. Scientists similarly wish for pigs to model humans, although as large animal models of human disease, not despotic rulers. Ostedgaard et al. extended this idea to cystic fibrosis (CF), generating pigs that carry the most common human CF mutation, Δ508. CF is a devastating genetic disease characterized by difficulty breathing, progressive disability, persistent infections, and, often, early death. CF is caused by a mutation in the gene that encodes the CF transmembrane conductance regulator (CFTR), which is an anion channel that modulates the components of sweat, digestive juices, and mucus. The most common mutation in CF patients results in an altered version of CFTR, CFTR-Δ508, which is found in 1 of 25 people of Caucasian descent. CF is difficult to study in human patients, and mouse models do not accurately reflect the human disease. Pigs may provide a better model of CF because they have more similar anatomy, biochemistry, physiology, size, and genetics to humans than mice. Thus, the authors generated a pig model of CF with the CFTR-Δ508 mutation. Similar to pigs that completely lack expression of CFTR, the CFTR-Δ508 pigs developed CF symptoms that mimicked those in human patients. In these animals, much of the CFTR-Δ508 protein was misprocessed; specifically, it was retained in the endoplasmic reticulum and rapidly degraded. However, pigs with CFTR-Δ508 retained small amounts of CFTR conductance (~6%), although this level of function was not sufficient to prevent disease. This new model may help to determine which levels of CFTR are sufficient for function and, therefore, guide future therapeutic strategies. After all, all animal models are equal, but some are more equal than others. Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. The most common CF-associated mutation is ΔF508, which deletes a phenylalanine in position 508. In vitro studies indicate that the resultant protein, CFTR-ΔF508, is misprocessed, although the in vivo consequences of this mutation remain uncertain. To better understand the effects of the ΔF508 mutation in vivo, we produced CFTRΔF508/ΔF508 pigs. Our biochemical, immunocytochemical, and electrophysiological data on CFTR-ΔF508 in newborn pigs paralleled in vitro predictions. They also indicated that CFTRΔF508/ΔF508 airway epithelia retain a small residual CFTR conductance, with maximal stimulation producing ~6% of wild-type function. Cyclic adenosine 3′,5′-monophosphate (cAMP) agonists were less potent at stimulating current in CFTRΔF508/ΔF508 epithelia, suggesting that quantitative tests of maximal anion current may overestimate transport under physiological conditions. Despite residual CFTR function, four older CFTRΔF508/ΔF508 pigs developed lung disease similar to human CF. These results suggest that this limited CFTR activity is insufficient to prevent lung or gastrointestinal disease in CF pigs. These data also suggest that studies of recombinant CFTR-ΔF508 misprocessing predict in vivo behavior, which validates its use in biochemical and drug discovery experiments. These findings help elucidate the molecular pathogenesis of the common CF mutation and will guide strategies for developing new therapeutics.

In vivo models of cerebral ischemia : effects of parenterally administered NMDA receptor glycine site antagonists

Both in vitro and in vivo experiments have implicated extracellular glycine in the pathogenesis of ischemic brain damage. Recently, halogenated derivatives of quinoxaline-2,3-dione have been synthesized that possess bioavailability when parenterally administered and minimal psychotomimetic properties. Such compounds have allowed investigation into the efficacy of glycine receptor antagonism as a strategy for protection against cerebral ischemic insults. Rats underwent either 90 min of middle cerebral artery filament occlusion or 10 min of forebrain ischemia with recovery while receiving intraperitoneal injections of either a glycine receptor antagonist (ACEA-1021, ACEA-1031, or ACEA-1011) or vehicle (dimethyl sulfoxide). Both ACEA-1021 and ACEA-1031 reduced cerebral infarct volumes and were associated with a reduced incidence of hemiparesis resulting from MCA occlusion. ACEA-1011, administered in a smaller dose had no effect. In the forebrain ischemia model, glycine receptor antagonism had no effect on delayed neuronal necrosis in the hippocampal CA1 sector, neocortex, or caudoputamen. We conclude that pharmacologic antagonism of glycine at the strychnine-insensitive glycine receptor presents a neuroprotective profile similar to that previously observed for antagonists of glutamate at the N-methyl-d-aspartate complex with a potential for fewer side effects.

Halothane reduces focal ischemic injury in the rat when brain temperature is controlled.

Background Previous work has demonstrated that rats anesthetized with halothane during focal cerebral ischemia have better histologic and neurologic outcome than do rats undergoing the same insult when awake. The purpose of this experiment was to determine whether this difference persists when brain temperature is held similar in halothane‐ anesthetized and awake experimental groups. Methods Two ischemia experiments were performed. In both, the middle cerebral artery was occluded for 90 min. Temperature was monitored from a radiotelemetered thermistor implanted in the cerebral cortex. Four days after ischemia, infarct volume and neurologic function were assessed. In experiment 1, brain temperature was not controlled in awake rats. Temperature in rats anesthetized with halothane, approximately 1 minimum alveolar concentration, was regulated by servomechanism by surface heating or cooling to replicate the temperature profiles generated by awake animals. To address methodologic issues regarding infarct volume analysis, a subset of nine rats was examined for the effect of the histologic staining technique and the mathematical modeling algorithms used for computation of infarct volume values. In experiment 2, the brain temperature of awake and halothane‐ anesthetized rats was maintained normothermic (38.0 degrees Celsius) throughout ischemia and early recirculation. Results In experiment 1 no difference between groups was observed for cortical (halothane 146 plus/minus 95 mm3 and awake 126 plus/minus 108 mm3; P = 0.64) or subcortical (halothane 110 plus/minus 48 mm3 and awake 100 plus/minus 66 mm3; P 0.66) infarct volume. Neurologic function was also similar between groups. Total infarct volume was approximately 11% greater when histologic sections were stained with hematoxylin and eosin than when they were stained with nitro blue tetrazolium, although volumes correlated closely between the two techniques (r2 0.996). Analysis by orthogonal or frustum projection from two‐dimensional planimetric areas to three‐ dimensional volumes resulted in nearly identical values (r2 0.999). In experiment 2, halothane‐anesthetized rats experienced a 46% reduction in cortical infarct volume (halothane 106 plus/minus 97 mm3 and awake 197 plus/minus 103 mm3; P = 0.03). The incidence of hemiparesis was reduced in the anesthetized group (P = 0.03). Conclusions When brain temperature was maintained normothermic throughout the focal ischemic insult, a neurologic and histologic protective effect for halothane anesthesia was observed. This effect of halothane was not sufficient to persist when large variations in brain temperature were allowed. Regulation of brain temperature is a critical factor in the determination of the effects of anesthetics on focal ischemic brain damage.

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.

Glycaemic regulation and insulin secretion are abnormal in cystic fibrosis pigs despite sparing of islet cell mass

Diabetes is a common and significant co-morbidity in cystic fibrosis (CF). The pathogenesis of cystic fibrosis related diabetes (CFRD) is incompletely understood. Because exocrine pancreatic disease is similar between humans and pigs with CF, the CF pig model has the potential to contribute significantly to the understanding of CFRD pathogenesis. We determined the structure of the endocrine pancreas in fetal, newborn and older CF and non-CF pigs and assessed endocrine pancreas function by intravenous glucose tolerance test (IV-GTT). In fetal pigs, pancreatic insulin and glucagon density was similar between CF and non-CF. In newborn and older pigs, the insulin and glucagon density was unchanged between CF and non-CF per total pancreatic area, but increased per remnant lobular tissue in CF reflecting exocrine pancreatic loss. Although fasting glucose levels were not different between CF and non-CF newborns, CF newborns demonstrated impaired glucose tolerance and increased glucose area under the curve during IV-GTT. Second phase insulin secretion responsiveness was impaired in CF newborn pigs and significantly lower than that observed in non-CF newborns. Older CF pigs had elevated random blood glucose levels compared with non-CF. In summary, glycaemic abnormalities and insulin secretion defects were present in newborn CF pigs and spontaneous hyperglycaemia developed over time. Functional changes in CF pig pancreas were not associated with a decline in islet cell mass. Our results suggest that functional islet abnormalities, independent of structural islet loss, contribute to the early pathogenesis of CFRD.

Pancreatic and biliary secretion are both altered in cystic fibrosis pigs

The pancreas, liver, and gallbladder are commonly involved in cystic fibrosis (CF), and acidic, dehydrated, and protein-rich secretions are characteristic findings. Pancreatic function studies in humans have been done by sampling the jejunal fluid. However, it has been difficult to separately study the function of pancreatic and biliary systems in humans with CF, because jejunal fluid contains a mixture of bile and pancreatic fluids. In contrast, pancreatic and biliary ducts open separately into the porcine intestine; therefore, biliary and pancreatic fluid can be individually analyzed in CF pigs. We studied newborn wild-type (WT) and CF pigs and found that CFTR was localized to the pancreatic ducts. We collected bile and pancreatic fluid and analyzed pancreatic enzymes with activity assays and immunoblot. Pancreatic enzyme expression was significantly decreased in CF compared with WT pigs. The volume and pH of pancreatic fluid were significantly lower and protein concentration was >5-fold higher in CF pigs. Secretin stimulation increased pancreatic fluid volume and pH in WT, but not CF, pigs. Baseline bile volume did not differ between WT and CF pigs, but volume did not increase in response to secretin in CF pigs. Bile pH was lower and protein concentration was twofold higher in CF pigs. These results indicate that pancreatic and biliary secretions are altered in CF pigs. Abnormal pancreatic and biliary secretion in CF may have important implications in disease pathogenesis.

Pancreatic and Biliary Secretion Differ in Cystic Fibrosis and Wild-Type Pigs

270* Neuroendocrine characterization of the intestine of F508del CFTR mice L. Hjelte1, M. Flodstrom-Tullberg2, A. Nilsson3, N. Wierup4, F. Sundler4. 1Karolinska Institutet, Karolinska Univ Hosp Huddinge, Stockholm CF Center, Stockholm, Sweden; 2Karolinska Institutet, Center for Infectious Medicine, Dept of Medicine, HS, Stockholm, Sweden; 3Lund Univ Hosp, Dept of Clinical Sciences, Gastroenterology and Nutrition, Lund, Sweden; 4Lund University, Dept of Experimental Medical Science, Division of Neuroendocrine Cell Biology, Lund, Sweden