Carbachol induces TGF-alpha expression and colonic epithelial cell proliferation in sensory-desensitised rats.

OBJECTIVE/BACKGROUND: Signals for the expression of the peptide growth factors epidermal growth factor and transforming growth factor-alpha (TGFalpha) in the gastrointestinal mucosa are largely unknown. We have shown earlier that extrinsic afferents in the gastrointestinal tract induce TGFalpha expression in colonic mucosa via the deliberation of neurotransmitters substance P and calcitonin gene-related peptide. The aim of our present study was to determine the effects of carbachol on mucosal TGFalpha expression and epithelial cell proliferation in vivo. DESIGN/METHODS: Rats were divided in three groups. Group 1 was treated with vehicle only, group 2 received one single subcutaneous injection of 250 microg/kg of carbachol and animals in group 3 were sensory-desensitised prior to the injection of 250 microg/kg carbachol. TGFalpha expression and epithelial cell proliferation was evaluated by polymerase chain reaction, Western blot analysis and bromodeoxyuridine staining. RESULTS: Carbachol induced a significant increase in mucosal epithelial cell proliferation and TGFalpha expression. Sensory desensitisation did neither abolish the increased TGFalpha expression nor the increase in epithelial cell proliferation. CONCLUSION: Parasympathetic pathways are involved in the control of TGFalpha expression in gastrointestinal mucosa as well as in epithelial cell proliferation.

Bacteribilia after preoperative bile duct stenting: a prospective study.

STUDY DESIGN: A prospective analysis of intraoperative bile duct cultures in patients undergoing surgery for both, malignant or benign periampullary diseases at the Department of Surgery, St Josef Hospital, Bochum, Germany, during a period of 18 months, between January 2004 and June 2005. GOALS: The goals of the presented study were to investigate the effects of preoperative bile duct stenting on intraoperative bile duct cultures and postoperative outcome in patients undergoing pancreatic surgery. BACKGROUND: In pancreatic surgery, bile duct stenting is often aimed at improving postoperative outcome. As implantation of xenograft material in the main bile duct facilitates bacterial contamination and cholangitis, a critical evaluation of stenting is mandatory. STUDY: In all patients with a hepaticojejunostomy (n=80), a bile duct culture was collected during the operation. All patients received antibiotic prophylaxis perioperatively and a retrograde flushing of bile ducts with warm saline after bile duct resection. Fifty-one percent (41/80) patients had biliary drainage before surgery, whereas 49% (39/80) were operated without preoperative draining procedures. RESULTS: After bile duct stenting, 98% of patients had a positive bile culture, whereas only 21% of infected bile was seen in patients without drainage (P<0.001). Despite infected bile, only 2% stented patients developed acute cholangitis postoperatively, versus 13% patients in the group without stent (P=0.231). After stenting, major complications occurred in 12%, versus 8% in patients without stent (P=0.817). CONCLUSIONS: Preoperative biliary drainage leads to an almost 100% bacterial contamination of bile ducts. With hospital-adjusted antibiotic prophylaxis and retrograde flushing of bile ducts, the postoperative rate of acute cholangitis and morbidity is not elevated. A critical evaluation of benefits from preoperative biliary drainage for each patient is necessary.

Histopathological features of patients with chronic pancreatitis due to mutations in the PRSS1 gene: evaluation of BRAF and KRAS2 mutations.

BACKGROUND/AIMS: Hereditary pancreatitis (HP) is a rare cause of chronic pancreatitis (CP; 1%) and more than 25 mutations in the PRSS1 gene have been detected. HP patients with the p.R122H mutation have a 35% lifetime risk of developing pancreatic cancer, but the oncogenetic process remains unknown. We have investigated the histopathological features and frequency of BRAF and KRAS2 mutations in 2 patients with PRSS1 mutations (p.A121T, p.R122H) and patients with CP (n = 11). METHODS: Pancreatic tissue was stained with hematoxylin-eosin and examined by light microscopy. Mutational analysis of the BRAF (exon 5, 11) and KRAS2 (exon 1) genes was performed using PCR and direct DNA sequencing. RESULTS: Histopathological features revealed similar results in both patients, pancreata showed strong fibrosis and ducts with signs of distortion, irregular size and noticeable dilatations. We identified one BRAF mutation (p.V600E) in the p.R122H patient and two KRAS2 (p.G12D; p.G12C) mutations in CP controls. CONCLUSIONS: Our results sustain the knowledge about the clinical phenotype of patients with PRSS1 mutations who have a high risk of pancreatic cancer. Whether the histopathological picture or the BRAF mutation is specific for patients with PRSS1 mutations or plays a specific role in the tumorigenesis of patients with HP needs to be further evaluated.

Increased duodenal expression of transforming growth factor-alpha and epidermal growth factor during experimental colitis in rats.

OBJECTIVE/BACKGROUND: Epidermal growth factor (EGF) and transforming growth factor alpha (TGFalpha) protect gastrointestinal mucosa against injury. Having shown earlier, that TGFalpha but not EGF is locally increasingly expressed after mucosal injury in the colon, we now wanted to explore the pattern of expression of EGF and TGFalpha in the remaining gastrointestinal tract and to infer from the pattern of expression, to possible signals for the induction of the growth factor expression and further mechanisms for mucosal protection. DESIGN/METHODS: The trinitrobenzene sulfonic acid/ethanol-induced model of colitis in rats was used. TGFalpha-mRNA and EGF-mRNA expression was evaluated in inflamed and noninflamed colon, in the ileum, jejunum, duodenum, stomach, and in the submandibulary glands. RESULTS: A significant increase of TGFalpha-mRNA and EGF-mRNA expressions was detected in the duodenal mucosa and a significant increase in TGFalpha-mRNA expression was observed in the inflamed colonic mucosa after mucosal injury in the colon within the first hours of colitis. CONCLUSION: The increased expression of EGF and TGFalpha in the duodenum may lead to neutralization of gastric acid and proteolytic enzymes in the upper gastrointestinal tract during the course of colitis. Possible signals for the increased expression of EGF and TGFalpha presumably are fasting, parasympathetic, or adrenergic parts of the enteric nervous system or yet unknown mechanisms.

Tailored resective pancreatic surgery for pediatric patients with chronic pancreatitis.

BACKGROUND: Surgical treatment for chronic pancreatitis (CP) in children comprises predominantly nonresective draining procedures. The purpose of this study was to identify indications, techniques, and results of organ-preserving resective pancreatic procedures for pediatric CP at our institution. PATIENTS AND METHODS: A retrospective chart review was performed of all children undergoing pancreatic surgery for CP over a period of 4 years. RESULTS: Overall, 6 pediatric patients (3 male, 3 female, ages 7-18 years) underwent a duodenum-preserving pancreatic head resection (3), a middle segmental pancreatic resection (2), or a distal pancreatectomy (1) for CP of different etiologies (idiopathic 2, posttraumatic 2, pancreas divisum 1, situs inversus 1). No mortality or major surgical complication occurred. Mean operative time was 294 min (207-412 min) and intraoperative blood loss was 541 mL (100-1300 mL). Postoperative hospital stay was 13 days (10-18 days). No endocrine or exocrine insufficiency occurred during follow up of 46 months (25-50 m), and pain control was improved in 5 of 6 patients. CONCLUSIONS: Tailored organ-preserving resective pancreatic surgery can be performed with low morbidity and mortality in pediatric patients with CP and not responding to conservative treatment.

Sensory neuropeptides and epithelial cell restitution: the relevance of SP- and CGRP-stimulated mast cells.

BACKGROUND: Calcitonin-gene-related peptide (CGRP) and substance P (SP) are neurotransmitters of extrinsic primary afferent neurons located within the dorsal root ganglia. In experimental models of colitis in rats and rabbits, a protective role of SP and CGRP on intestinal mucosa was presumed. The mucosal protection partly depends on a CGRP-mediated modulation of mucosal blood flow. Limited data are available regarding CGRP- or SP-mediated effects on epithelial cell restitution. Having shown earlier that SP-stimulated fibroblasts but not CGRP-stimulated fibroblasts induce epithelial cell migration in vitro, the aim of this study was to explore whether mast cells mediate effects of SP and CGRP on epithelial cell restitution in vitro. METHODS: Mast cells (C57) were exposed to SP [10(-12)-10(-6 M)] and CGRP [10(-12)-10(-7 M)]. After a 24-h incubation period, the cell supernatants (conditioned media, CDM) were taken from mast cell cultures and directly applied to rat intestinal epithelial cell lines-18 or Caco-2 monolayers, which had been wounded with a razor blade 24 h prior to the experiments. Epithelial cell migration was assessed by counting cells across the wound edge and epithelial cell proliferation was measured using 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide test. RESULTS: CGRP significantly induced epithelial cell migration and proliferation via mast cells when supernatants were directly applied to epithelial cells in vitro. The effects on epithelial cell migration were abolished after neutralizing anti-transforming growth factor-beta (TGF-beta) had been added to the cell cultures. SP had no effects on epithelial cells following stimulation of mast cells. CONCLUSION: CGRP modulates epithelial cell restitution in vitro mediated by mast cells. The CGRP- and mast-cell-induced epithelial cell migration is TGF-beta dependent. This observation underlines an important role for extrinsic primary afferent neurons in mucosal defence and repair and in keeping the mucosal homeostasis. This knowledge leads to a better understanding of the interaction of the enteric nervous system and wound healing and may, in the future, lead to new therapeutic approaches to inflammatory diseases of the intestine.

Pancreatitis risk in primary hyperparathyroidism: relation to mutations in the SPINK1 trypsin inhibitor (N34S) and the cystic fibrosis gene.

OBJECTIVE: Primary hyperparathyroidism (pHPT)-related hypercalcemia is considered to represent a risk factor for the development of pancreatitis. We therefore explored whether mutations in genes that were previously identified to increase the risk for pancreatitis coexist in a cohort of 826 patients with pHPT prospectively studied between 1987 and 2002. METHODS: Among 826 patients with pHPT, 38 patients were identified with pancreatitis (4.6%). DNA was available from 25 patients (13 women/12 men, 16 acute pancreatitis/9 chronic pancreatitis). These individuals and 50 patients with pHPT without pancreatitis were analyzed for mutations in the serine protease inhibitor Kazal type I (SPINK1) gene (N34S) and the cationic trypsinogen gene (PRSS1) (N29I, R122H) by melting curve analysis and DNA sequencing. Sequence analysis of the cystic fibrosis transmembrane conductance regulator (CFTR) gene was carried out for the detection of 36 mutations and the Tn polymorphism. RESULTS: Four of 25 patients with pHPT and pancreatitis carried the N34S missense mutation in the SPINK1 gene (16%), while all 50 controls (pHPT without pancreatitis) showed no mutation in SPINK1 or PRSS1 genes (P < 0.05 vs controls, P < 0.001 vs general population). CF-causing CFTR mutations were present in four patients (P < 0.05 vs general population), while one patient carried a 5T allele. One patient was transheterozygous (SPINK1: N34S/CFTR: R553X). Mean serum calcium levels in pancreatitis patients (3.1 mmol/L) did not differ significantly from the mean of the entire cohort (3.0 mmol/L) or pHPT patients without pancreatitis (3.1 mmol/L). CONCLUSION: Pancreatitis risk is approximately 10-fold elevated in pHPT, but pancreatitis occurs infrequently. This indicates an existing but minor impact of pHPT-related hypercalcemia. If pancreatitis occurs, it seems associated with genetic risk factors such as mutations in the SPINK1 and CFTR genes. In contrast, a combination of both hypercalcemia and genetic variants in SPINK1 or CFTR increases the risk to develop pancreatitis in patients with pHPT.

Glucagon like peptide-2 induces intestinal restitution through VEGF release from subepithelial myofibroblasts.

Glucagon like peptide-2 (GLP-2) exerts intestinotrophic actions, but the underlying mechanisms are still a matter of debate. Recent studies demonstrated the expression of the GLP-2 receptor on fibroblasts located in the subepithelial tissue, where it might induce the release of growth factors such as keratinocyte growth factor (KGF) or vascular endothelial growth factor (VEGF). Therefore, in the present studies we sought to elucidate the downstream mechanisms involved in improved intestinal adaptation by GLP-2. Human colonic fibroblasts (CCD-18Co), human colonic cancer cells (Caco-2 cells) and rat ileum IEC-18 cells were used. GLP-2 receptor mRNA expression was determined using real time RT-PCR. Conditioned media from CCD-18Co cells were obtained following incubation with GLP-2 (50-250 nM) for 24 h. Cell viability was assessed by a 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide (MTT)-assay, and wound healing was determined with an established migration-assay. Transforming Growth Factor beta (TGF-beta), VEGF and KGF mRNA levels were determined by RT-PCR. Protein levels of VEGF and TGF-beta in CCD-18Co cells following GLP-2 stimulation were determined using ELISA. Neutralizing TGF-beta and VEGF-A antibodies were utilized to assess the role of TGF-beta and VEGF-A in the process of wound healing. GLP-2 receptor expression was detected in CCD-18Co cells. Conditioned media from CCD-18Co cells dose-dependently induced proliferation in Caco-2 cells, but not in IEC-18 cells. Conditioned media also enhanced cell migration in IEC-18 cells (P<0.01), while migration was even inhibited in Caco-2 cells (P<0.0012). GLP-2 significantly stimulated mRNA expression of VEGF and TGF-beta, but not of KGF in CCD-18Co. The migratory effects of GLP-2 were completely abolished in the presence of TGF-beta and VEGF-A antibodies. GLP-2 exerts differential effects on the epithelium of the small intestine and the colon. Thus, in small intestinal cells GLP-2 stimulates wound repair, whereas no such effects were observed in colonic cells. The mechanisms underlying GLP-2 induced intestinal wound repair seem to involve the secretion of VEGF and, subsequently, TGF-beta from subepithelial fibroblasts, whereas KGF appeared to be less important.

Substance P induces intestinal wound healing via fibroblasts--evidence for a TGF-beta-dependent effect.

BACKGROUND: Substance P (SP) and calcitonin gene-related peptide (CGRP) are neurotransmitters of the afferent sensory nervous system. In experimental models of colitis in rats and rabbits, a protective role of SP and CGRP on the intestinal mucosa was presumed. In part, mucosal protection depends on a SP-mediated and CGRP-mediated modulation of mucosal blood flow after injury. We thought to explore whether there is a fibroblast-mediated effect of SP and CGRP on epithelial cell restitution in vitro. MATERIALS AND METHODS: Rat kidney fibroblast (NRK-49F) cell lines were exposed to CGRP or SP in various concentrations. After incubation, the cell culture supernatants were taken from the fibroblast cultures and were directly applied to IEC-18 or Caco-2 monolayers, which had been wounded with a razor blade 24 h before the experiments. Epithelial cell migration was assessed by counting cells across the wound edge. Epithelial cell proliferation was assessed using the 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide (MTT) test. RESULTS: SP significantly induced epithelial cell migration and inhibited epithelial cell proliferation via stimulation of fibroblasts when supernatants were directly applied to epithelial cells in vitro. The effects on epithelial cell migration were abolished after neutralising anti-transforming growth factor-beta (TGF-beta) was added to the cell cultures. CGRP had no effect on epithelial cells via stimulation of fibroblasts. Neither CGRP nor SP had any effect on epithelial cell migration or proliferation when directly applied to epithelial cells. CONCLUSION: SP modulates epithelial cell restitution in vitro mediated by fibroblasts. The epithelial cell migration depends on a TGF-beta release from SP-stimulated fibroblasts. This observation underlines an important role for the sensory nervous system in mucosal defence and repair and in keeping mucosal homeostasis. Modulation of SP may be potentially useful for the treatment of various intestinal disorders characterised by injury and ineffective repair of the intestinal mucosa.

Pathophysiology and treatment of acute pancreatitis: new therapeutic targets--a ray of hope?

Acute pancreatitis is a life-threatening disease with putatively high mortality rates, particularly in the setting of systemic inflammatory response and multiple organ failure when superinfection of necrosis occurs. Although the APACHE II and Ranson score are widely accepted as clinical scores to predict the prognosis, current medical treatment is still based upon state of the art intensive care treatment largely unrelated to the pathogenesis of the disease. The mechanisms by which premature enzyme activation and autodigestion of the pancreatic gland is triggered and maintained are still ill-defined. It is well known that activation of chemokines, cytokines and pancreatic enzymes characterize the cause of the disease, but disease-phase specific treatment attempts have thus far not resulted in successful molecular based medical treatments. The current summary describes the novel understanding in the pathophysiology of acute pancreatitis with special emphasis on specific disease phases. It outlines promising and novel experimental and medical therapeutic approaches which might become clinical targets and successful strategies to significantly reduce pancreatitis-associated mortality rates.

A novel mutation of the calcium sensing receptor gene is associated with chronic pancreatitis in a family with heterozygous SPINK1 mutations.

BACKGROUND: The role of mutations in the serine protease inhibitor Kazal type 1 (SPINK1) gene in chronic pancreatitis is still a matter of debate. Active SPINK1 is thought to antagonize activated trypsin. Cases of SPINK1 mutations, especially N34S, have been reported in a subset of patients with idiopathic chronic pancreatitis. However, the inheritance pattern is still unknown. Some cases with N34S heterozygosity have been reported with and without evidence for CP indicating neither an autosomal recessive nor dominant trait. Therefore SPINK1 mutations have been postulated to act as a disease modifier requiring additional mutations in a more complex genetic model. Familial hypocalciuric hypercalcemia (FHH) caused by heterozygous inactivating mutations in the calcium sensing receptor (CASR) gene is considered a benign disorder with elevated plasma calcium levels. Although hypercalcemia represents a risk factor for pancreatitis, increased rates of pancreatitis in patients with FHH have not been reported thus far. METHODS: We studied a family with a FHH-related hypercalcemia and chronic pancreatitis. DNA samples were analysed for mutations within the cationic trypsinogen (N29I, R122H) and SPINK1 (N34S) gene using melting curve analysis. Mutations within CASR gene were identified by DNA sequencing. RESULTS: A N34S SPINK1 mutation was found in all screened family members. However, only two family members developed chronic pancreatitis. These patients also had FHH caused by a novel, sporadic mutation in the CASR gene (518T>C) leading to an amino acid exchange (leucine->proline) in the extracellular domain of the CASR protein. CONCLUSION: Mutations in the calcium sensing receptor gene might represent a novel as yet unidentified predisposing factor which may lead to an increased susceptibility for chronic pancreatitis. Moreover, this family analysis supports the hypothesis that SPINK1 mutations act as disease modifier and suggests an even more complex genetic model in SPINK1 related chronic pancreatitis.