Joel Shilyansky

Disruption of the CFTR Gene Produces a Model of Cystic Fibrosis in Newborn Pigs

Almost two decades after CFTR was identified as the gene responsible for cystic fibrosis (CF), we still lack answers to many questions about the pathogenesis of the disease, and it remains incurable. Mice with a disrupted CFTR gene have greatly facilitated CF studies, but the mutant mice do not develop the characteristic manifestations of human CF, including abnormalities of the pancreas, lung, intestine, liver, and other organs. Because pigs share many anatomical and physiological features with humans, we generated pigs with a targeted disruption of both CFTR alleles. Newborn pigs lacking CFTR exhibited defective chloride transport and developed meconium ileus, exocrine pancreatic destruction, and focal biliary cirrhosis, replicating abnormalities seen in newborn humans with CF. The pig model may provide opportunities to address persistent questions about CF pathogenesis and accelerate discovery of strategies for prevention and treatment.

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.

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.

Nonoperative management of pancreatic injuries in children

PURPOSE The safety and efficacy of nonoperative management of pancreatic contusions and transections was examined by reviewing the case histories of 35 consecutive children with pancreatic injuries treated over the past 10 years. METHODS/RESULTS Surgical exploration was performed for the management of associated injuries only. The diagnosis of pancreatic trauma was suspected in children with abdominal pain, tenderness, elevated serum amylase levels and findings consistent with pancreatic injury on abdominal ultrasound scan or computerized tomography (CT) examination. After children were diagnosed with pancreatic injury, enteral feedings were withheld and total parenteral nutrition administered until abdominal pain resolved and serum amylase levels and radiographic findings improved. Twenty-three children received diagnosis within 24 hours of injury, and in 12, the diagnosis was delayed 2 to 14 days. Hyperamylasemia was found in 27 of 35 children. Twenty-eight children sustaining pancreatic injuries were treated nonoperatively. Abdominal imaging in these children demonstrated pancreatic contusion in 14, transection in 11, and pseudocyst in three. Enteral feeding resumed an average of 15 days after injury. The average hospital stay was 21 days. Pseudocysts formed in 10 children (2 of 14 with contusion; 5 of 11 with transection; three children presented late, and the type of pancreatic injury could not be determined), whose average hospital stay was 25 days. All pseudocysts were successfully managed nonoperatively, although percutaneous aspiration or drainage was required in six children. Children underwent follow-up for an average of 10 months after injury (range, 1 to 144 months). Abdominal pain and radiological abnormalities resolved in all children before discharge from the clinic. CONCLUSIONS Nonoperative management of pancreatic contusion and transection diagnosed radiologically is effective and safe. Pseudocysts may form after pancreatic injury, and if large or symptomatic, can be managed successfully by percutaneous drainage.

Phosphatidylserine Regulates the Maturation of Human Dendritic Cells

Phosphatidylserine (PS), which is exposed on the surface of apoptotic cells, has been implicated in immune regulation. However, the effects of PS on the maturation and function of dendritic cells (DCs), which play a central role in both immune activation and regulation, have not been described. Large unilamellar liposomes containing PS or phosphatidylcholine were used to model the plasma membrane phospholipid composition of apoptotic and live cells, respectively. PS liposomes inhibited the up-regulation of HLA-ABC, HLA-DR, CD80, CD86, CD40, and CD83, as well as the production of IL-12p70 by human DCs in response to LPS. PS did not affect DC viability directly but predisposed DCs to apoptosis in response to LPS. DCs exposed to PS had diminished capacity to stimulate allogeneic T cell proliferation and to activate IFN-γ-producing CD4+ T cells. Exogenous IL-12 restored IFN-γ production by CD4+ T cells. Furthermore, activated CTLs proliferated poorly to cognate Ag presented by DCs exposed to PS. Our findings suggest that PS exposure provides a sufficient signal to inhibit DC maturation and to modulate adaptive immune responses.

Is 95% Pancreatectomy the Procedure of Choice for Treatment of Persistent Hyperinsulinemic Hypoglycemia of the Neonate?

A 95% pancreatectomy became the treatment of choice for persistent hyperinsulinemic hypoglycemia of the neonate (PHHN, Nesidioblastosis) at the authors institution, when lesser resections failed to prevent hypoglycemia in 25% to 50% of cases. With few outcome data available in the literature, the authors reviewed their 25-year experience to assess the efficacy and the long-term consequences of this procedure. Since 1971, 27 infants underwent a 95% pancreatectomy for the treatment of PHHN. None had responded to medical treatment (glucose infusion, glucagon, octreotide, diazoxide), and two had 85% pancreatectomy that failed. The procedure consisted of resecting the pancreas including the uncinate process, leaving only the gland lying between the common bile duct (CBD) and the duodenum and a small rim of pancreas along the duodenal sweep. Hyperinsulinemia and hypoglycemia recurred in nine children (33%), all within 2 to 5 days. Seven of them were subsequently cured with near-total pancreatic resection. Partial pancreatic regrowth was evident at reoperation. In two cases hypoglycemia was controlled with diazoxide and frequent feedings because reoperation was refused. The gross anatomic findings and the histopathology were not predictive of treatment failure. Perioperative complications occurred in four of 27 children (15%) after 95% pancreatectomy and in four of seven children (57%) after near-total pancreatectomy. Clinical follow-up ranged from 0.5 to 18 years (mean, 8 years; median, 8 years). To date, diabetes has developed in 15 children (56%), nine of 20 (45%) after 95% pancreatectomy (mean age, 9.7 years) and six of seven (86%) after a near-total pancreatectomy (mean age, 1.7 years). After 95% pancreatectomy, the incidence of diabetes increased with age, developing in nine of the 13 (69%) children followed up for more than 4 years. The failure of 95% pancreatectomy to prevent hypoglycemia in one third of children with PHHN and the ultimate development of diabetes in a minimum of two-thirds, indicates that an alternative treatment strategy is needed for this disease.

T-Cell Recognition of Human Melanoma Antigens

The adoptive transfer of tumor-infiltrating lymphocytes (TILs) with interleukin-2 (IL-2) has antitumor activity in some patients with metastatic melanoma. We have analyzed molecular mechanisms of TIL recognition of human melanoma. Some cultured TILs specifically lysed autologous and some allogeneic melanomas sharing a variety of class I major histocompatibility complex (MHC) molecules. HLA-A2-restricted melanoma-specific TILs lysed many HLA-A2+ melanoma cell lines from different patients but failed to lyse HLA-A2- melanoma and HLA-A2+ nonmelanoma cell lines. However, these TILs were capable of lysing many naturally HLA-A2- melanomas after introduction of the HLA-A2.1 gene by vaccinia virus. These results indicate that shared melanoma antigens (Ag) are expressed in melanomas regardless of their human leukocyte antigen types. In order to identify these shared melanoma Ags, we have tested some known proteins expressed in melanoma. Expression of tyrosinase or HMB45 Ag correlated with lysis of TILs. We are also attempting to isolate antigenic peptides by high performance liquid chromatography separation and genes encoding melanoma Ag by cDNA expression cloning. The T-cell component of the antimelanoma response was also analyzed by determining the genetic structure of the T-cell receptor (TCR) used by melanoma TILs. However, we did not observe common TCR variable region usage by different melanoma TILs. We could establish melanoma cell clones and lines resistant to TIL lysis due to the absence of or defects in the expression of Ag, MHC, or beta 2-microglobulin molecules. These data indicate multiple mechanisms for melanoma escape from T-cell immunosurveillance.(ABSTRACT TRUNCATED AT 250 WORDS)

Diagnosis and management of duodenal injuries in children

Traumatic duodenal perforations in children pose a diagnostic and therapeutic challenge. To identify specific diagnostic criteria and define an optimal therapeutic protocol, we reviewed all duodenal injuries treated at our institution in the past 10 years. There were 14 hematomas and 13 perforations. The diagnosis was confirmed by computed tomography (CT), ultrasound scan (US), upper gastrointestinal contrast studies (UGI), or at laparotomy. The clinical findings and CT findings of the two groups were compared. Children with suspected duodenal hematomas were treated expectantly, and children with duodenal perforations were treated surgically. Twenty-five associated injuries (10 pancreatic) occurred in 19 children. Children with perforations had higher injury severity scores (ISS) (25 v 9), but the two groups could not be differentiated based on presenting signs, symptoms, or laboratory findings. CT findings of retroperitoneal air or contrast were seen in 9 of 9 perforations and in 0 of 10 hematomas. CT findings of intraabdominal or retroperitoneal fluid, mesenteric enhancement, and thickened duodenal wall did not differentiate the two groups. Duodenojejunostomy was performed in one patient, and primary repair was performed in 11 children who had perforation. In five children, duodenostomy tube drainage with feeding jejunostomy or gastrojejunostomy were added. Complications occurred in three of four children in the first 5 years of the study and in two of nine children in the last 5 years. The decreased morbidity rate correlated with reduced time to definitive therapy (28 v 7.8 hours). Duodenal fistulae resulted in three of seven children treated without duodenostomy tube drainage and zero of five treated with drainage. Enteral feeds resumed faster (average, 12 v 27 days) if repair of perforation was combined with feeding jejunostomy or pyloric exclusion and gastrojejunostomy. Children with duodenal hematoma resumed eating an average of 16 days after injury. Only one child required surgery for persistent obstruction. The findings of retroperitoneal air and contrast extravasation on CT accurately distinguish duodenal perforation from hematoma. Conservative management of hematoma is safe and effective. Primary repair of perforation with duodenal drainage results in fewer postoperative complications, and gastrojejunostomy or feeding jejunostomy shorten the time to resumption of feeds.

Choledochal cysts in western adults: complexities compared to children.

Choledochal cysts occur most frequently in East Asian children and rarely in Western adults. Over the past two decades, pediatric treatment has been standardized, but relatively little information is available on the management of Western adults with choledochal cysts. Therefore the aims of this analysis were to compare the presentation, management, and late results of Western adults and children with choledochal cysts. Records were reviewed of patients with choledochal cysts at three academic institutions in Wisconsin. Fifty-seven patients were identified, and 51 of these patients (89%) were managed surgically. Thirty-one patients (54%) were adults, and the adults were more likely to be male (29% vs. 4%, P<0.02). Pain (81% vs. 42%, P<0.01) and cholangitis (35% vs. 15%) were more common in adults. Forty-one patients (71 %) had type I cysts, but type IVa or V cysts with dilated intrahepatic ducts were more common in adults (39% vs. 15%, P=0.05). Seventeen adults had undergone biliary surgery prior to referral compared to only four children (59% vs. 15%, P<0.01). Preoperative endoscopic or percutaneous stents were employed more commonly in adults (42% vs. 15%, P<0.01). Hospital mortality was 0%, and morbidity was low in both adults and children (25% vs. 8%). An associated biliary malignancy correlated with age (P<0.05): 0 to 30 years (0%), 31 to 50 years (19%), and 51 to 70 years (50%). In addition, adults were more likely to have late problems with cholangitis (19% vs. 4%, P<0.07) and secondary biliary cirrhosis (13% vs. 4%). This analysis suggests that compared to children, Western adults with choledochal cysts are more likely to have (1) type IVA or V cysts, (2) undergone prior surgery, (3) preoperative biliary stents, (4) an associated biliary malignancy, and (5) late hepatobiliary problems. We conclude that surgery in Western adults with choledochal cysts is frequently complicated and should be performed by specialists in complex biliary surgery.

Distal splenorenal shunts for the treatment of severe thrombocytopenia from portal hypertension in children

Profound thrombocytopenia resulting from portal hypertension may exacerbate gastrointestinal bleeding, precipitate spontaneous bleeding, preclude surgical intervention for associated disorders, and severely limit life-style because of the danger of splenic injury. Although splenectomy can reverse the thrombocytopenia, the procedure should be avoided in children. We reviewed our experience with distal splenorenal shunting (DSRS) in children, particularly when performed for the sole purpose of reversing severe thrombocytopenia resulting from portal hypertension. DSRS was performed in 11 children between the ages of 7 and 15 years: five for severe thrombocytopenia (group 1), four for advanced hypersplenism and congenital hepatic fibrosis prior to renal transplantation (group 2), and two for esophageal bleeding (group 3). One child in group 1 with severe heart disease and Child’s class C cirrhosis due to hepatitis C died of progressive cardiac failure and was excluded from further analysis. Of the eight remaining patients in groups 1 and 2, four children had congenital hepatic fibrosis, two had portal vein thrombosis, one had hepatitis B, and one had Wilson’s disease. After DSRS, the mean platelet count increased from 37,000 ±18,000 to 137,600 ±81,000 (P = 0.01). The platelet count improved significantly in all seven children with presinusoidal portal hypertension or stable cirrhosis but did not increase in the child with hepatitis Band Child’s class B cirrhosis. The white blood cell count increased from an average of 3.3 ±1.1 to 5.4 ± 2.6 (P= 0.02). There were no postoperative complications in this group. The improved platelet count allowed the four children with congenital hepatic fibrosis and renal failure to undergo renal transplantation with full posttransplant immunosuppression including azathioprine. Postoperative Doppler ultrasound examination demonstrated shunt patency at 6 months in all cases. Spleen size decreased appreciably in all children in groups 1 and 2. All children were able to resume full activity including contact sports. In summary, DSRS effectively controls profound thrombocytopenia resulting from presinusoidal portal hypertension or stable cirrhosis without sacrificing the spleen and should be the treatment of choice for this condition.