Paola G. Blanco

Acute regulation of the SLC26A3 congenital chloride diarrhoea anion exchanger (DRA) expressed in Xenopus oocytes

Mutations in the human SLC26A3 gene, also known as down‐regulated in adenoma (hDRA), cause autosomal recessive congenital chloride‐losing diarrhoea (CLD). hDRA expressed in Xenopus oocytes mediated bidirectional Cl−‐Cl− and Cl−‐HCO3− exchange. In contrast, transport of oxalate was low, and transport of sulfate and of butyrate was undetectable. Two CLD missense disease mutants of hDRA were nonfunctional in oocytes. Truncation of up to 44 C‐terminal amino acids from the putatively cytoplasmic C‐terminal hydrophilic domain left transport function unimpaired, but deletion of the adjacent STAS (sulfate transporter anti‐sigma factor antagonist) domain abolished function. hDRA‐mediated Cl− transport was insensitive to changing extracellular pH, but was inhibited by intracellular acidification and activated by NH4+ at acidifying concentrations. These regulatory responses did not require the presence of either hDRAs N‐terminal cytoplasmic tail or its 44 C‐terminal amino acids, but they did require more proximate residues of the C‐terminal cytoplasmic domain. Although only weakly sensitive to inhibition by stilbenes, hDRA was inhibited with two orders of magnitude greater potency by the anti‐inflammatory drugs niflumate and tenidap. cAMP‐insensitive Cl−‐HCO3− exchange mediated by hDRA gained modest cAMP sensitivity when co‐expressed with cystic fibrosis transmembrane conductance regulator (CFTR). Despite the absence of hDRA transcripts in human cell lines derived from CFTR patients, DRA mRNA was present at wild‐type levels in proximal colon and nearly so in the distal ileum of CFTR(‐/‐) mice. Thus, pharmacological modulation of DRA might be a useful adjunct treatment of cystic fibrosis.

Decreased expression of peroxisome proliferator activated receptor γ in CFTR-/- mice

Some of the pathological manifestations of cystic fibrosis are in accordance with an impaired expression and/or activity of PPARγ. We hypothesized that PPARγ expression is altered in tissues lacking the normal cystic fibrosis transmembrane regulator protein (CFTR). PPARγ mRNA levels were measured in colonic mucosa, ileal mucosa, adipose tissue, lung, and liver from wild‐type and cftr−/− mice by quantitative RT‐PCR. PPARγ expression was decreased twofold in CFTR‐regulated tissues (colon, ileum, and lung) from cftr−/− mice compared to wild‐type littermates. In contrast, no differences were found in fat and liver. Immunohistochemical analysis of PPARγ in ileum and colon revealed a predominantly nuclear localization in wild‐type mucosal epithelial cells while tissues from cftr−/− mice showed a more diffuse, lower intensity labeling. A significant decrease in PPARγ expression was confirmed in nuclear extracts of colon mucosa by Western blot analysis. In addition, binding of the PPARγ/RXR heterodimer to an oligonucletotide containing a peroxisome proliferator responsive element (PPRE) was also decreased in colonic mucosa extracts from cftr−/− mice. Treatment of cftr−/− mice with the PPARγ ligand rosiglitazone restored both the nuclear localization and binding to DNA, but did not increase RNA levels. We conclude that PPARγ expression in cftr−/− mice is downregulated at the RNA and protein levels and its function diminished. These changes may be related to the loss of function of CFTR and may be relevant to the pathogenesis of metabolic abnormalities associated with cystic fibrosis in humans.

Decreased expression of peroxisome proliferator activated receptor gamma in cftr-/- mice.

Some of the pathological manifestations of cystic fibrosis are in accordance with an impaired expression and/or activity of PPARγ. We hypothesized that PPARγ expression is altered in tissues lacking the normal cystic fibrosis transmembrane regulator protein (CFTR). PPARγ mRNA levels were measured in colonic mucosa, ileal mucosa, adipose tissue, lung, and liver from wild‐type and cftr−/− mice by quantitative RT‐PCR. PPARγ expression was decreased twofold in CFTR‐regulated tissues (colon, ileum, and lung) from cftr−/− mice compared to wild‐type littermates. In contrast, no differences were found in fat and liver. Immunohistochemical analysis of PPARγ in ileum and colon revealed a predominantly nuclear localization in wild‐type mucosal epithelial cells while tissues from cftr−/− mice showed a more diffuse, lower intensity labeling. A significant decrease in PPARγ expression was confirmed in nuclear extracts of colon mucosa by Western blot analysis. In addition, binding of the PPARγ/RXR heterodimer to an oligonucletotide containing a peroxisome proliferator responsive element (PPRE) was also decreased in colonic mucosa extracts from cftr−/− mice. Treatment of cftr−/− mice with the PPARγ ligand rosiglitazone restored both the nuclear localization and binding to DNA, but did not increase RNA levels. We conclude that PPARγ expression in cftr−/− mice is downregulated at the RNA and protein levels and its function diminished. These changes may be related to the loss of function of CFTR and may be relevant to the pathogenesis of metabolic abnormalities associated with cystic fibrosis in humans.

MECHANISMS TO EXPLAIN PANCREATIC DYSFUNCTION IN CYSTIC FIBROSIS

This article focuses on three potential mechanisms by which pancreatic dysfunction occurs in cystic fibrosis. These include (1) obstruction of pancreatic ducts by inspissated plugs, (2) inhibition of endocytosis in acinar cells, and (3) imbalance in membrane lipids in cystic fibrosis regulated cells. Any of these abnormalities alone or in combination may explain the development of pancreatic exocrine insufficiency.

Fatty acids in cystic fibrosis.

Cystic fibrosis (CF) is associated with deficiencies in certain essential fatty acids. These deficiencies have been studied in plasma, red blood cells, and mucus and were previously thought to be a result of malnutrition or malabsorption. More recent studies have indicated that these deficiencies are independent of nutritional status. However, these studies examined fatty acids in plasma but not in CF–regulated tissues. In the pancreas, lungs, and ileum of CF knock-out mice, membrane-bound arachidonic acid levels have been shown to be increased while docosahexaenoic acid levels are decreased. This lipid abnormality is reversed following oral administration of docosahexaenoic acid (DHA). In addition, DHA therapy reverses the increased neutrophil infiltration in the lungs of CF knock-out mice. Further studies are required to determine the mechanism by which CF gene mutations lead to this lipid abnormality.

Revitalizing pathology laboratories in a gastrointestinal pathophysiology course using multimedia and team-based learning techniques

In 2008, we changed the gastrointestinal pathology laboratories in a gastrointestinal pathophysiology course to a more interactive format using modified team-based learning techniques and multimedia presentations. The results were remarkably positive and can be used as a model for pathology laboratory improvement in any organ system. Over a two-year period, engaging and interactive pathology laboratories were designed. The initial restructuring of the laboratories included new case material, Digital Atlas of Video Education Project videos, animations and overlays. Subsequent changes included USMLE board-style quizzes at the beginning of each laboratory, with individual readiness assessment testing and group readiness assessment testing, incorporation of a clinician as a co-teacher and role playing for the student groups. Student responses for pathology laboratory contribution to learning improved significantly compared to baseline. Increased voluntary attendance at pathology laboratories was observed. Spontaneous student comments noted the positive impact of the laboratories on their learning. Pathology laboratory innovations, including modified team-based learning techniques with individual and group self-assessment quizzes, multimedia presentations, and paired teaching by a pathologist and clinical gastroenterologist led to improvement in student perceptions of pathology laboratory contributions to their learning and better pathology faculty evaluations. These changes can be universally applied to other pathology laboratories to improve student satisfaction.

Liver and skeletal muscle lipids have differing fatty acid profiles in short-gut rats fed via parenteral nutrition.

BACKGROUND In short-gut rats, we showed marked abnormalities in plasma lipid fatty acids using parenteral nutrition (PN) with lipid vs sham surgery rats. This suggests that either sensing or metabolism of parenteral lipid is abnormal in malabsorption. The goal of this study was to determine fatty acid profiles in skeletal muscle and liver in short-gut rats treated with PN compared with sham rats. METHODS Sprague-Dawley rats underwent laparotomy and massive small bowel resection (or sham surgery). Rats (n = 32, 16 sham, 16 short gut) were randomly assigned to PN with lipid or fat-free PN. After 5 days, weight loss was similar in all groups, and mixed hindlimb skeletal muscle and liver were biopsied. RESULTS We found marked differences between liver and skeletal muscle. In livers of short-gut animals, 22:4omega6, 22:5omega6, and 22:6omega3 were higher (all p < .05) than in sham. In skeletal muscle, short gut had no effect on fatty acid profiles. In liver, fat-free PN led to significant increases in 20:3omega6, 22:4omega6, 22:5omega6, 20:3omega9, 20:5omega3, 22:6omega3, and triene/tetraene ratio (all p < .05) compared with feeding PN with lipid, irrespective of short gut. In muscle, levels of the distal long-chain fatty acid metabolites and triene/tetraene ratio were minimally affected by nutrition. Serum glucose and insulin concentrations were similar in all 4 groups. CONCLUSIONS Both the presence of short gut and type of PN led to increases in distal metabolites of fatty acids on omega:3 and omega:6 pathway in liver phospholipids but not in skeletal muscle during short-term PN feeding in rats.

Decreased expression of peroxisome proliferator activated receptor ? in CFTR?/? mice

Some of the pathological manifestations of cystic fibrosis are in accordance with an impaired expression and/or activity of PPARγ. We hypothesized that PPARγ expression is altered in tissues lacking the normal cystic fibrosis transmembrane regulator protein (CFTR). PPARγ mRNA levels were measured in colonic mucosa, ileal mucosa, adipose tissue, lung, and liver from wild‐type and cftr−/− mice by quantitative RT‐PCR. PPARγ expression was decreased twofold in CFTR‐regulated tissues (colon, ileum, and lung) from cftr−/− mice compared to wild‐type littermates. In contrast, no differences were found in fat and liver. Immunohistochemical analysis of PPARγ in ileum and colon revealed a predominantly nuclear localization in wild‐type mucosal epithelial cells while tissues from cftr−/− mice showed a more diffuse, lower intensity labeling. A significant decrease in PPARγ expression was confirmed in nuclear extracts of colon mucosa by Western blot analysis. In addition, binding of the PPARγ/RXR heterodimer to an oligonucletotide containing a peroxisome proliferator responsive element (PPRE) was also decreased in colonic mucosa extracts from cftr−/− mice. Treatment of cftr−/− mice with the PPARγ ligand rosiglitazone restored both the nuclear localization and binding to DNA, but did not increase RNA levels. We conclude that PPARγ expression in cftr−/− mice is downregulated at the RNA and protein levels and its function diminished. These changes may be related to the loss of function of CFTR and may be relevant to the pathogenesis of metabolic abnormalities associated with cystic fibrosis in humans.

How to Create an Unfunded Teaching Fellowship During the Gastroenterology Fellowship That Positively Impacts Subsequent Teaching Activities and Career Path

The gastrointestinal (GI) pathophysiology course is a 2.5-week required preclinical course at the end of medical school’s second-year pathophysiology organ system blocks. In 1993, I was appointed co-course director of this course. teaching using the Case-Based Method. In October 2002, I went for the first of numerous times to Harvard Business School to observe and learn from Professor Garvin. Here the professors consistently use the Case Method with question, listen, and respond as the prime teaching strategy. In addition, each professor summarizes with high-yield, takehome points at the end of class. Learning these 3 key teaching strategies made all the difference in my individual ratings, overall course ratings, and how I taught the current teaching and academic fellows to teach.

Mechanism of altered cytokine secretion from monocytes of patients heterozygous and homozygous for CFTR mutations: Potential role in chronic inflammatory disorders

Introduction Intragastru balloon systems have been used tot weight loss m obese patients. Because of a high complication rate this method was abandoned in the 1980s. Recently, a new generation of intragastric balloon system has been introduced in clinical use. We report the eNciency and tolerance of the Bioenterics lntragastric Balloon System (BIB). Methods Obese patients willing to reduce weight wetv oticred the possibility of weight reduction assistance by the BIB Initially a questionnaire was completed by all patients, inquiring about medical history, comorbidiv, dietary" habits and previous treatment t0r obesity. BIB placement was pertormed during upper endoscopy on an outpatient basis under intravenous sedation. The system was tilled with 500ml saline, coloured with methyienblue to identity, any leakage, A dieticians consultation was pertomled every month. lhe BIB was removed endoscopically aher 6 months Weight loss was a s se~d each month and alter removal ot the BIB. Results From July 2001 to November 2002, 60 Patients (19 male, 49 iemale, mean age 44.2 years (range 26 67)) underwent BIB iruplacement. Mean initial BMi was 38.6 kg/ru 2 (range 2952) witil a mean imtial excess weight of 41.8 kg (range 13-80) and nman initial %excess wright ot 3 5 8 (133-549) Iniual upper endoscopy was normal in 43 and abnormal in 17 patients (9 esophagitis, 3 small hiatal hernia, 4 gastritis, 3 duodenal ulcers, 2 patienl:s with double pattmlognes). In all 60 patients the insertion procedure was uneventful, in 2 patients the BIB had to be removed within the first week due to intolerance, in the retraining 58 patients the mean intmgastric residing time of tbe BIB was 6 6 months (range 4.5-9.0). There was no spomaneons loss ot the device. The mean weight loss was l l .2kg (range 035~ corresponding to a %excess weight loss of 30% (0-116.7). In the first week of treatment 48 patients (82%) suffered t~rom vomiting over a mean time of 3 7 days, heartburn occurred in 13 patients (22%) and hospitalisation was necessary m 2 patients Vomiting and heartburn requinng PPi-therapy lasted longer than one week in 8 (13%) and 14 (24%) patients, respectively No major complications like pertoration or obstruction were observed. Conclusion [he insertion of BIB offers a highly efhcient method tot rapid weight reduction in obese patients Vomiting and retlux s}~nptoms are common within the tirst 4 days alter placement, but can be controlled by drug therapy Overall, the therapy is well tolerated, sate and associated with a low complication rate.