Menno Verhave

Combined use of cyclosporine and azathioprine or 6-mercaptopurine in pediatric inflammatory bowel disease.

The aim of this study was to assess whether in steroid-resistant patients with pediatric inflammatory bowel disease (IBD) a combination of cyclosporine and azathioprine (or 6-mercaptopurine) could induce remission and subsequently permit maintenance on azathioprine/6-mercaptopurine as the sole immunosuppressive agent. Two boys and six girls (six with ulcerative colitis and two with Crohns disease; ages 3-17 years) received 100-200 micrograms/kg/day cyclosporine intravenously and then 4-10 mg/kg/day orally. Doses were adjusted to achieve trough serum cyclosporine levels of 100-200 mu/L (Abbotts TDX assay). Seven of the eight patients received azathioprine/6-mercaptopurine, and all were given a 5-aminosalicylate preparation and corticosteroids. The latter drugs were continued and then tapered off as clinical status allowed. Cyclosporine was continued for 3-10 months in those who responded. In seven of eight patients, there was a rapid clinical response; one patient showed a transient response, but recurrent bleeding led to total colectomy 9 days after starting cyclosporine. Of the seven responders, three were able to discontinue prednisone and cyclosporine and are doing well on azathioprine at long-term follow-up (2-5 years). One who did not receive azathioprine/6-mercaptopurine maintained remission for 2 years after cyclosporine was stopped, one experienced a disease flare-up 5 months after start of cyclosporine treatment and required colectomy, one who did not tolerate 6-mercaptopurine had a flare-up during cyclosporine tapering and underwent surgery at 6 months, and one started to flare up with cyclosporine tapering at 6 months and was scheduled for surgery. No significant complications of treatment were observed. Seven patients had an initial response and four of them have so far not required surgery. These preliminary findings suggest that azathioprine/6-mercaptopurine can be used safely to maintain cyclosporine-induced remission in children with IBD.

Identification of nonsteroidal antiinflammatory drug-induced gastroduodenal injury in children with juvenile rheumatoid arthritis

In a cohort of children with juvenile rheumatoid arthritis treated with nonsteroidal antiinflammatory drugs and referred for gastrointestinal complaints, more than 75% had gastritis, antral erosions, or ulcers. Epigastric pain strongly correlated with documented gastroduodenal injury. Therapy with ranitidine or misoprostol led to clinical improvement. Nonsteroidal antiinflammatory drugs are associated with significant gastrointestinal abnormalities in children.

Thrombosis and inflammatory bowel disease: A call for improved awareness and prevention

&NA; Thrombotic complications in patients with inflammatory bowel disease (IBD) are common and require improved awareness and prevention. In this review the interface between IBD and thrombosis is discussed, with emphasis on risk assessment and data to aid clinical decision making. Thromboembolic complications are 3‐fold more likely in IBD patients than controls and the relative risk exceeds 15 during disease flares. Improved assessment of thrombosis risk for an individual patient includes thorough personal and family history and awareness of prothrombotic medications and lifestyle choices. Patients with the highest risk of thrombosis are those with active colonic disease, personal or strong family history of thrombosis, and those with significant acquired risk factors. Combined risk factors or hospitalization should prompt mechanical thromboprophylaxis. Indications for prophylactic anticoagulation are not defined currently by clinical studies, especially in pediatric patients, although some groups now advocate prophylactic anticoagulation for all hospitalized IBD patients and even some outpatients with disease flares. Thrombosis management requires a multidisciplinary therapeutic approach to balance anticoagulation and bleeding risk. While bleeding may occur with anticoagulation in IBD, data and experience indicate that therapeutic heparin is safe and bleeding manifestations can be managed supportively in most patients. Until prospective trials of prophylactic anticoagulation are published, management of thrombotic risk and prophylaxis in IBD will remain a clinical challenge. (Inflamm Bowel Dis 2011;)

Rat lactase-phlorizin hydrolase/human growth hormone transgene is expressed on small intestinal villi in transgenic mice

BACKGROUND & AIMS Lactase-phlorizin hydrolase (LPH) is an absorptive enterocyte-specific gene that is expressed in a well-characterized pattern along the cryptvillus (vertical), proximal-distal (horizontal), and developmental (temporal) gradients. The aim of this study was to characterize the capacity of regulatory elements within the rat LPH gene to direct appropriate cell lineage and topographical patterns of expression in vivo in transgenic mice. METHODS Transgenic mouse lines were established using a construction containing bases -2038 to +15 of the rat LPH gene fused to a human growth hormone reporter gene. RESULTS In one line, the transgene was expressed only in small intestine and was localized to absorptive enterocytes on villi. The transgene was not expressed in goblet or enteroendocrine cells or in crypts. Transgene expression along horizontal and developmental gradients was different from that of the native mouse LPH gene. CONCLUSIONS The results suggest that the region from -2038 to +15 of the rat LPH gene contains regulatory elements that direct correct tissue, cell, and vertical expression but may not contain all the elements necessary for appropriate horizontal and temporal control. This investigation provides further insight into the complexities of the molecular control of intestinal gene expression.

Risk factors, morbidity, and treatment of thrombosis in children and young adults with active inflammatory bowel disease.

Objectives: Pediatric inpatients with inflammatory bowel disease (IBD) are rarely considered for thromboprophylaxis because of concerns about safety and underappreciation of thrombotic risk. We characterized thromboembolism (TE) in children and young adults with inflammatory bowel disease (IBD) at a single tertiary care hospital. Methods: We performed a retrospective review of an inpatient billing database for all IBD admissions with colonic involvement and an anticoagulation database for thrombotic complications from 2006 to 2011. Results: Of 532 patients admitted with IBD with colonic involvement, 10 (1.9%) had TE (9 venous, 1 arterial), 2 of whom had recurrent thrombosis. Many of the events resulted in considerable morbidity, including 4 cerebrovascular events and 2 pulmonary emboli. Established risk factors in IBD colitis inpatients with TE included: indwelling catheter (4/10), first-degree family member with TE (2/10), hereditary thrombophilia (3/10), smoking (1/10), oral contraceptive (1/5 females), and thalidomide (1/10). Additionally, most (8/10) patients had acquired thrombophilia, mostly elevation of factor VIII and anticardiolipin antibodies. Patients with IBD and TE received therapeutic anticoagulation without significantly increased bleeding. Thrombus resolution was documented in 7 cases, persistence in 2 cases and recurrence in 2 cases. Conclusions: Pediatric inpatients hospitalized with IBD with colonic involvement have increased risk of TE, including complications of pulmonary embolism, recurrence, persistence, and indefinite long-term anticoagulation. Therapeutic anticoagulation in patients with IBD with active colitis appears safe. We identified both inherited thrombophilias and acquired risk factors in patients with IBD and TE. We presently use risk stratification and recommend prophylactic anticoagulation in high-risk patients.

Lactase; Origin, gene expression, localization, and function

Abstract Lactase-phlorizin hydrolase is a small intestinal enzyme responsible for the hydrolysis of the carbohydrate lactose in mammalian milk. During the neonatal period the enzyme is crucial for the nutrition of humans and most other mammals. Subsequently, the specific activity of lactase decreases to low adult levels. In most adult humans and other mammals, large amounts of lactose are no longer tolerated, and lactose ingestion may lead to gastrointestinal symptoms. People of Caucasian extraction and a few isolated other groups form a clear exception with regard to this pattern; high levels of lactase activity are maintained, enabling these people to consume diary products during adult life. This review will describe the role of lactase in the digestion of lactose in mammalian milk. The function and origin of the enzyme will be outlined, and the review will examine relevant issues regarding the consumption of lactose and the clinical syndrome of lactose intolerance. Furthermore, insight provided by molecular and cell biology into gene structure, promoter function, gene transcription, localization of expression in the small intestine, biosynthesis of lactase protein, and enzyme specificities will be outlined. Current thinking with respect to the regulation of expression of lactase during development, and the differences in expression between species and different human populations will also be discussed.

Somatostatin gene regulation: An overview

The somatostatinergic system has proven to be one of the best models of neuropeptide biology. Originally characterized as a hypothalamic regulator of growth hormone secretion, somatostatin also regulates the secretion of several other pituitary, pancreatic, and gastrointestinal (GI) hormones including thyrotropin-stimulating hormone, insulin, glucagon, and gastrin. Disorders in somatostatin metabolism have been proposed to contribute to the pathogenesis of Alzheimers disease, epilepsy, GI motility disorders, and diabetes. On a more basic level, studies of somatostatin action have integrated divergent concepts of intracellular signal transduction. Advances in the understanding of somatostatin biosynthesis have had an impact on areas outside the field of endocrinology by providing new concepts of eukaryotic gene regulation. This report focuses on the transcriptional regulation of somatostatin gene expression. Two aspects of somatostatin gene transcription will be considered--regulated expression by second messengers and tissue-specific basal expression.

Asymmetrical Localization of mRNAs in Enterocytes of Human Jejunum

Intracellular localization of specific mRNAs is known to be a mechanism for targeting proteins to specific sites within the cell. Previous studies from this laboratory have demonstrated co-localization of mRNAs and proteins for a number of genes in absorptive enterocytes of fetal rat intestine. The present study was undertaken to examine in human enterocytes the intracellular localization patterns of mRNAs for the microvillous membrane proteins lactase–phlorizin hydrolase (LPH), sucrase-isomaltase (SI), and intestinal alkaline phosphatase (IAP), and the cytoskeletal protein β-actin. In sections of human jejunum, mRNAs were localized by in situ hybridization using digoxigenin-labeled anti-sense RNA probes. Both LPH and SI mRNAs were localized to the apical region of villous enterocytes, whereas IAP and β-actin mRNAs were detected both apically and basally relative to the nucleus. Therefore, in contrast to LPH, SI, and β-actin mRNAs, which co-localize with their encoded proteins, that of IAP is present in the basal region of the cell where IAP protein has not directly been demonstrated to be present. Absorptive enterocytes from humans possess the mechanisms for intracellular mRNA localization, but not all mRNAs co-localize with their encoded proteins.

Three distinct messenger RNA distribution patterns in human jejunal enterocytes

BACKGROUND & AIMS The importance of messenger RNA (mRNA) localization in human enterocytes is poorly understood. Previous studies from our laboratory have indicated that mRNAs are asymmetrically distributed in human intestinal epithelial cells, but in general colocalized with their encoded proteins. The aim of this study was to characterize, in human enterocytes, mRNA localization patterns of three genes with distinctly different functions. METHODS mRNA distribution was determined by in situ hybridization with digoxigenin-labeled RNA probes in tissue sections of human jejunum. RESULTS The mRNA for villin, a well-characterized microvillus cytoskeletal protein, was sorted to the basal region of the enterocyte. The mRNA for human sodium glucose cotransporter 1 was localized to the apical region, and the mRNA for human liver fatty acid-binding protein was distributed diffusely in the cytoplasm. CONCLUSIONS The three distinct mRNA distribution patterns suggest that active mRNA sorting mechanisms exist in human enterocytes. This study also reveals for the first time that dichotomies may occur between the distribution patterns of sorted mRNAs and their encoded proteins.

Verification of the lactase site of rat lactase-phlorizin hydrolase by site-directed mutagenesis☆

BACKGROUND & AIMS Lactase-phlorizin hydrolase (LPH) is an intestinal microvillus membrane glycoprotein that hydrolyzes lactose and phlorizin. These enzymatic activities have been assigned to glutamic acid (E) residues 1271 and 1747 in rabbit LPH. The aim of this study was to determine directly if this assignment was correct and if these two amino acids are the only nucleophiles required for LPH enzyme activity. METHODS Site-directed mutagenesis of a full-length rat LPH complementary DNA was used to convert the rat homologues E1274 and E1750 to aspartic acid or glycine. Mutants were analyzed by enzyme activity assays. RESULTS All tested activities of E1274D and E1274G were virtually unaffected. In contrast, mutations E1750D and E1750G resulted in total loss of lactase and cellobiose activities, leaving only low ONP-glc and ONP-gal hydrolase activities detectable. A double mutant containing both E1274G and E1750G had no activity. CONCLUSIONS These studies directly confirm that the two previously identified glutamic acids are essential to the enzymatic activity of rat LPH. Rat lactase activity is not associated with the E1274 site. This study provides the first evidence that rat LPH has its major catalytic site at E1750, representing all of the lactase and the majority of the phlorizin hydrolase activity.