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Pax6 is reactivated druing pancreatic inflammation
T1866
higher risk of developing pancreatitis or whether this mutation is frequent in idiopathic pancreatitis. Studies in Puerto Rican patients with pancreatnis--especially idiopathic pancreatitis---could be useful, especially since this single mutation is associated with the highest risk of pancreatitis in the CF population.
CFTR Deletion Ameliorates Cerulein Acute Pancreatitis in Mice Matthew Dimagno, Ylbai Hao, Chung Owyang PURPOSE: Idiopathic chronic pancreatitis is strongly associated with compound heterozygous CF gene mutations, or non-cla~acal CF, but subjects with classic CF, specifically' homozygous CF gene mutations and pancreatic sufficiency, rarely (0.5%) develop acute pancreatitis (AP) (Pancreas 22:395, 2001). CFTR (-/-) mice have been shown to have a mild pancreatic phenotype similar to early human CF but susceptibility to AP has been incompletely characterized. We investigated whether CFTR (-/-) mine exhibited an altered susceptibility to AP and how this correlated with in vivo pancreatic secretion. METHODS: A colony of UNC CFTR (+/-) exon 10 knockout mice was bred on a C57BL/6 background and oflspnng were fed Peptamen fulluwnig weaning. In vivo AP was induced by hourly ceralein rejections (50 ug/kg, lP). Mice were sacrificed 30 minutes after 1 injection or i hour after 12 injections. An in vivo CCK model of pancreatic" secretion was performed in anesthetized mice in which the pancreatic duct was cannulated and juice collected every 15 minutes. Proteni output (ug/min), expressed as % basal, was used as a marker of pancreatic secretion, in response to 160 pmol/kg/h CCK. RESULTS: CFTR (4-) mice exhibited a blunted biochemical response to ceruleni AP. Basal pancreatic trypsin activity and serum amylase and lipase values showed no difference between CFTR ( + / + ) and CFTR (-/-) groups. 30 minutes after ceralein treatment, trypsin activity (ng/mg protein) increased by 5 5 (+Al.3) in CFTR ( + / + ) mice and 2.6 ( + / - 0 9 ) in CFTR (-/-) mice, and serum lipase (U/L) nicreased by 225 (+/-28) in CFTR ( + / + ) mice and 128 (+/-16) in CFTR (4-) mice. At 12 hours trypain activity (ng/mg protein) increased by 0.82 (+/'-0.19) in CFTR ( + / + ) mice and 0.42 (4-/0.15) in CFTR (-/-) mice, and serum amylase (KU/I_) increased by- 143 (+/-18) in CFTR ( + / + ) mice and only 52 (+/- 13) in CFTR (-A) mice. Further studies assessed alterations in pancreatm secretion in vivo. In normal C57BL/6 mice, CCK induced a dose dependent response, maximal at 160 pmol~g/h. No difference in basal protein output was observed between CFTR (4-) and ( + / + ) mice, however protein secretion was reduced 51% in CFTR (4-) mice compared to controls in ~vsponse to maximal CCK stimulation, CONCLUSION: CFTR (-A) mice exhibit a blunted biochemical response to in vivo cerulein AP and have z~duced in vivo pancreatic secretion to CCK stimulation. These findnigs may lead to an explanatmn for why humans with classic CF and pancreatic sufficiency rarely develop AP.
Pancreatltis in OF patients with "mild" CFTR alleles G ~ 3849r I any RI17H / any 2789*SG..',A I any G85E I any R347P / any P-a34WI any A455E / any
Number of CF patients 256 249 IM ~00 95 77 ........ 80
Patients wi~ panereatitls N % 23 9% 29 12% 20 15% 8 8% 1t 12% 15 ~% 5 8%
T1869 Keratin 8 Mutations in Patients with Chronic Pancreatitis Martina Cavestro, kuca Frulloni, A. Nom/erme Barbara Calore, T. Neri, Laios Okolicsanyi, Francesco Di Mario, Gmrgio Cavallini Background: The pathogenesis of chronic pancreatitis (CP) is still unknown Genetic mutations in the SPINK1, CFTR and PRSS1 gene has been associated with CP. Keratin 8 (K8) and 18 (K18) are the major components of the intermediate filament cytoskeleton of pancreatic acinar cells and play a relevant role in pancreatic exocrine omeostasis. In transgenic mice, disruption of the normally regulate keratin expression in vivo could be related to inflammatory pancreatic disorders. We therefore investigated mutations in the Keratin 8 gene as a genetic background of CP. Methods: The substitution of cysteine for glycine at position 61 (G61C mutation), and the substitution of hlstidine for tyrosnie at position 53 (Y53H mutation) in the keratin 8 gene, were determined by PCR~estriction Fragment Length Polymorphism (RFLP) in 67 CP patients (55 male, 12 female) and 100 healthy normal controls (70 male, 30 female) of the same ethnical group. Sequence analysis, was performed when the digestion of PCR products resulted in two different-sized fragments suggesting the presence of mutation. All patients and controls gave their informed consent. Results: The G61C heterozygous mutation of the keratin 8 gene was found in six patients with chronic pancreatitis (8.9% vs 0%, pc<0.003, OR= 21.24, CI=2.74-164.42); none of the 100 normal controls presented the mutation. No Y53H mutation was detected in any subiect, Conclusion: Mutations in the Keratin 8 gene, together with other environmental factors (alcohol intake, smoking habits) and/or genetic factors, could predispose to chronic pancreatitis by interfering with the normal organization of keratin filaments.
T1867 CFTR Gone Analysis in Patients with Alcohol Induced Pancreatitis Michele ]7). Bishop, Steven D. Freedman, Sheelagh Martin, juhan Zielenski, Lynda Ellis, Peter R. Durie Cystic fibrosis (CFTR) gene mutations have been found m a significant number of patients with idiopathic chronic pancreatitis ti'om multiple centers. The mutation rate in patients with alcollol-induced pancreatitis is unclear. In addition, complete genetic sequencing of the cystic fibrosis gene has never been periormed on a group of control subjects m estimate the true incidence in the general population. METHODS: Complete DNA analysis of the CE gene was performed using PCR-based multiplex, beteroduplex analysis on MDE gel matrix, Mlowed by DNA sequencing. Patmnts were enrolled with alcohol induced chronic pancreatitis defined as at least 15 drinks per week tbr over 3 years, who had characteristic findings by histology,, ERCP, calcifications of the pancreas on x-ray, or abnomral secretin or CCK pancreatic function test. Identical genetic testing was pertormed on a group of 50 random,anonyanous blood donors as a control group. RESUI.TS: To date, all patients enrolled are male. 9/19 pancreatitis patients (47%) have CFTR mutations or variants. Of these, one patient was found to have two mutations (AF508/R75Q), five have one mutation (V754M,1716Cr~A,-799deIT,R75Q,S1235R), and three have the 5T variant. Average age of disease onset in patients with abnormal CFTR was 42 years (range 32-63). In the control group, 11/50 (22%) were fouod to have CFTR mutations(p= 0.072). None of the control subjects had two mutations CONCLUSIONS: Abnormalities of the cystic fibrosis gene seem to occur more frequently in patients with alcohol-induced chronic pancreatitis than in the geneial population, although the limited number of patients with completed genotype data so lar dues not allow statistical stgnit~cance The rate of mild CFTR mutations detected in the general population with complete analysis is also somewhat high. Historical data based on classic cystic fibrosis disease rates in a Caucasian population, or from limited gene screens of the most common 15-20 mutations (of the >900 known) suggest a carrier rate of approximately 3%. This represents the corner rate of severe mutations, but clearly, larger studies are warranted to estimate the true carrier rate of mild mutations
"1"1870 Pax6 is Reactivated During Pancreatic Inflammation Saleem Desai, David Q Wang, Richard L Maas Introduction: Pancreatitis is characterized by inflammation of tire exocrine pancreas including the pancreatic ducts. Pancreatitis can also result in diabetes secondary to destruction of the islets. It is believed that stem cells reside within the pancreatic ducts and it has been proposed that pancreatic inflammation induces budding of pancreatic stem cells or their progeny f~'om the ducts, and their migration through the parenchy~la and cytodift~rentiation to forul new islet cells. In fact, it has been shown that islet cell neogenesis can be stimulated by inducing pancreatitis by ligation of the pancreatic duct. A fundamental understanding of the developmemal pathways leading to formation of difteremiated cells in the endocrine pancreas would have significant clinical implications. Members of the Pax gene family of transcription factors plays a crucial role in the development of the pancreas. In adults, Pax6 is a key regulator of pancreatic islet hormone gene transcription and is not only expressed in the islets but it is required for normal islet development. In humans, heterozygons Pax6 nmtations have been associated with glucose intolerance. Methods: Pancreatic inflammation was induced by ligating panei~atic ducts of mice Pancreata were harvested on POD4. H6~E, LacZ, & immunostaining (insnfin&glucagon) was pertbrmed. Staining patterns in acini, islets, and ducts from inflamed pancreata were compared to non-inflamed pancreata. Results: A distinct Pax6 regulatory enhancer region was identified that is required for Pax6 expression in the developing and adult pancreas and this region was narrowed down to a 450 bp upstream of the Pax6 P0 promoter. Pax6-LacZ transgene containing this regulatory region is expressed in the embryonic pancreatic rudiment and also in the adult islet in a way that suggests a role in the ontogeny of those cell populations. During pancreatic inflammation, budding of the ductal epithelium occurs, and Pax6-LacZ transgene appears to be expressed in the budding cells that migrate from the duct through the parench~nna to generate new islet ceils ConcJusions: Data suggest that Pax6 expressing cells may represent muhipotent pancreatic cells that arise from the duct and then migrate through the parenchyma to form new islet ceils in the setting of pancreatic inflammation. The Pax6-LacZ transgenic line with the 450bp pancreatic regulatory, element offers a powerful and potentially unique tool to track these cells. These experiments have the potential to permit further investigation of the functional role of Pax6.
T1868 High Rate of Pancreatitis in Hispanic CF Patients Carrying The R334W Mutation Patrick Maisonneuve, Preston Campbell tIl, Peter R Durie, Albert B Lowenfels INTRODUCTION: Pancreatitis, while uncommon in the majority of patients with cystic fibrosis (CF), is relatively common among the subgroup with preserved pancreatic function. AIM: To investigate tim mutation-specific t~requency of attacks of pancreatins in pancreatic sufficient CF patients. METHODS: We studied all ptvvalem and new CF patients collected between 1990 and 1999 by the Cysnc Fibrosis Foundation and recorded the number of patients who reported at least one episode of pancreatitis durmg the study period, according to their genotype. RESULTS: Patients carrying one R334W mutation had the highest frequency of pancreatins (table). Overall, the R334 mutation is rare and has been identified in only 77 (0.43%) of all genotyped patients in the CFF registry'; 38 (49%) were classified as Hispanics, a much higher proportion than in the global CE population (5.6%) (p<0.001). Fourteen lived in Puerto Rico, where this mutation is over-represented, occurring in 31% of all CE patients. DISCUSSION: Several study groups have idemihed the RI 17H and other todd CFTR mutations in patients with idiopathic pancreatitis but failed to detect the R334W mutation. This is understandable because none of the populations studied were likely to include Puerto Ricans or other populations in which this mutation is common. There is no current data to determine whether as)anptonxatic carriers of the R334W mutation are at
T1871 A Novel Mutation of the Calcium Sensing Receptor Gene Is Associated with Chronic Pancreatitis in Individuals with Heterozygons SPINK1 Mutations Peter Felderbauer, Nikolans Ansorge, Kerem Bulut, Henrik Einwaechter, Henning Schrader, Frank Scbmitz, Wolfgang E. Schmidt, Peter Hofth~ann Introduction: The role of mutations in the serine protease inhibitor type Kazal 1 (SPINK1) gene in chronic pancreatitis (CP) is still a matter of debate. Active SPINK1 is thought to be potent enough to antagonize approximately 20% of the activated trypsin and hence a
A-583
AGA
Abstracts
higher risk of developing pancreatitis or whether this mutation is frequent in idiopathic pancreatitis. Studies in Puerto Rican patients with pancreatnis--especially idiopathic pancreatitis---could be useful, especially since this single mutation is associated with the highest risk of pancreatitis in the CF population.
CFTR Deletion Ameliorates Cerulein Acute Pancreatitis in Mice Matthew Dimagno, Ylbai Hao, Chung Owyang PURPOSE: Idiopathic chronic pancreatitis is strongly associated with compound heterozygous CF gene mutations, or non-cla~acal CF, but subjects with classic CF, specifically' homozygous CF gene mutations and pancreatic sufficiency, rarely (0.5%) develop acute pancreatitis (AP) (Pancreas 22:395, 2001). CFTR (-/-) mice have been shown to have a mild pancreatic phenotype similar to early human CF but susceptibility to AP has been incompletely characterized. We investigated whether CFTR (-/-) mine exhibited an altered susceptibility to AP and how this correlated with in vivo pancreatic secretion. METHODS: A colony of UNC CFTR (+/-) exon 10 knockout mice was bred on a C57BL/6 background and oflspnng were fed Peptamen fulluwnig weaning. In vivo AP was induced by hourly ceralein rejections (50 ug/kg, lP). Mice were sacrificed 30 minutes after 1 injection or i hour after 12 injections. An in vivo CCK model of pancreatic" secretion was performed in anesthetized mice in which the pancreatic duct was cannulated and juice collected every 15 minutes. Proteni output (ug/min), expressed as % basal, was used as a marker of pancreatic secretion, in response to 160 pmol/kg/h CCK. RESULTS: CFTR (4-) mice exhibited a blunted biochemical response to ceruleni AP. Basal pancreatic trypsin activity and serum amylase and lipase values showed no difference between CFTR ( + / + ) and CFTR (-/-) groups. 30 minutes after ceralein treatment, trypsin activity (ng/mg protein) increased by 5 5 (+Al.3) in CFTR ( + / + ) mice and 2.6 ( + / - 0 9 ) in CFTR (-/-) mice, and serum lipase (U/L) nicreased by 225 (+/-28) in CFTR ( + / + ) mice and 128 (+/-16) in CFTR (4-) mice. At 12 hours trypain activity (ng/mg protein) increased by 0.82 (+/'-0.19) in CFTR ( + / + ) mice and 0.42 (4-/0.15) in CFTR (-/-) mice, and serum amylase (KU/I_) increased by- 143 (+/-18) in CFTR ( + / + ) mice and only 52 (+/- 13) in CFTR (-A) mice. Further studies assessed alterations in pancreatm secretion in vivo. In normal C57BL/6 mice, CCK induced a dose dependent response, maximal at 160 pmol~g/h. No difference in basal protein output was observed between CFTR (4-) and ( + / + ) mice, however protein secretion was reduced 51% in CFTR (4-) mice compared to controls in ~vsponse to maximal CCK stimulation, CONCLUSION: CFTR (-A) mice exhibit a blunted biochemical response to in vivo cerulein AP and have z~duced in vivo pancreatic secretion to CCK stimulation. These findnigs may lead to an explanatmn for why humans with classic CF and pancreatic sufficiency rarely develop AP.
Pancreatltis in OF patients with "mild" CFTR alleles G ~ 3849r I any RI17H / any 2789*SG..',A I any G85E I any R347P / any P-a34WI any A455E / any
Number of CF patients 256 249 IM ~00 95 77 ........ 80
Patients wi~ panereatitls N % 23 9% 29 12% 20 15% 8 8% 1t 12% 15 ~% 5 8%
T1869 Keratin 8 Mutations in Patients with Chronic Pancreatitis Martina Cavestro, kuca Frulloni, A. Nom/erme Barbara Calore, T. Neri, Laios Okolicsanyi, Francesco Di Mario, Gmrgio Cavallini Background: The pathogenesis of chronic pancreatitis (CP) is still unknown Genetic mutations in the SPINK1, CFTR and PRSS1 gene has been associated with CP. Keratin 8 (K8) and 18 (K18) are the major components of the intermediate filament cytoskeleton of pancreatic acinar cells and play a relevant role in pancreatic exocrine omeostasis. In transgenic mice, disruption of the normally regulate keratin expression in vivo could be related to inflammatory pancreatic disorders. We therefore investigated mutations in the Keratin 8 gene as a genetic background of CP. Methods: The substitution of cysteine for glycine at position 61 (G61C mutation), and the substitution of hlstidine for tyrosnie at position 53 (Y53H mutation) in the keratin 8 gene, were determined by PCR~estriction Fragment Length Polymorphism (RFLP) in 67 CP patients (55 male, 12 female) and 100 healthy normal controls (70 male, 30 female) of the same ethnical group. Sequence analysis, was performed when the digestion of PCR products resulted in two different-sized fragments suggesting the presence of mutation. All patients and controls gave their informed consent. Results: The G61C heterozygous mutation of the keratin 8 gene was found in six patients with chronic pancreatitis (8.9% vs 0%, pc<0.003, OR= 21.24, CI=2.74-164.42); none of the 100 normal controls presented the mutation. No Y53H mutation was detected in any subiect, Conclusion: Mutations in the Keratin 8 gene, together with other environmental factors (alcohol intake, smoking habits) and/or genetic factors, could predispose to chronic pancreatitis by interfering with the normal organization of keratin filaments.
T1867 CFTR Gone Analysis in Patients with Alcohol Induced Pancreatitis Michele ]7). Bishop, Steven D. Freedman, Sheelagh Martin, juhan Zielenski, Lynda Ellis, Peter R. Durie Cystic fibrosis (CFTR) gene mutations have been found m a significant number of patients with idiopathic chronic pancreatitis ti'om multiple centers. The mutation rate in patients with alcollol-induced pancreatitis is unclear. In addition, complete genetic sequencing of the cystic fibrosis gene has never been periormed on a group of control subjects m estimate the true incidence in the general population. METHODS: Complete DNA analysis of the CE gene was performed using PCR-based multiplex, beteroduplex analysis on MDE gel matrix, Mlowed by DNA sequencing. Patmnts were enrolled with alcohol induced chronic pancreatitis defined as at least 15 drinks per week tbr over 3 years, who had characteristic findings by histology,, ERCP, calcifications of the pancreas on x-ray, or abnomral secretin or CCK pancreatic function test. Identical genetic testing was pertormed on a group of 50 random,anonyanous blood donors as a control group. RESUI.TS: To date, all patients enrolled are male. 9/19 pancreatitis patients (47%) have CFTR mutations or variants. Of these, one patient was found to have two mutations (AF508/R75Q), five have one mutation (V754M,1716Cr~A,-799deIT,R75Q,S1235R), and three have the 5T variant. Average age of disease onset in patients with abnormal CFTR was 42 years (range 32-63). In the control group, 11/50 (22%) were fouod to have CFTR mutations(p= 0.072). None of the control subjects had two mutations CONCLUSIONS: Abnormalities of the cystic fibrosis gene seem to occur more frequently in patients with alcohol-induced chronic pancreatitis than in the geneial population, although the limited number of patients with completed genotype data so lar dues not allow statistical stgnit~cance The rate of mild CFTR mutations detected in the general population with complete analysis is also somewhat high. Historical data based on classic cystic fibrosis disease rates in a Caucasian population, or from limited gene screens of the most common 15-20 mutations (of the >900 known) suggest a carrier rate of approximately 3%. This represents the corner rate of severe mutations, but clearly, larger studies are warranted to estimate the true carrier rate of mild mutations
"1"1870 Pax6 is Reactivated During Pancreatic Inflammation Saleem Desai, David Q Wang, Richard L Maas Introduction: Pancreatitis is characterized by inflammation of tire exocrine pancreas including the pancreatic ducts. Pancreatitis can also result in diabetes secondary to destruction of the islets. It is believed that stem cells reside within the pancreatic ducts and it has been proposed that pancreatic inflammation induces budding of pancreatic stem cells or their progeny f~'om the ducts, and their migration through the parenchy~la and cytodift~rentiation to forul new islet cells. In fact, it has been shown that islet cell neogenesis can be stimulated by inducing pancreatitis by ligation of the pancreatic duct. A fundamental understanding of the developmemal pathways leading to formation of difteremiated cells in the endocrine pancreas would have significant clinical implications. Members of the Pax gene family of transcription factors plays a crucial role in the development of the pancreas. In adults, Pax6 is a key regulator of pancreatic islet hormone gene transcription and is not only expressed in the islets but it is required for normal islet development. In humans, heterozygons Pax6 nmtations have been associated with glucose intolerance. Methods: Pancreatic inflammation was induced by ligating panei~atic ducts of mice Pancreata were harvested on POD4. H6~E, LacZ, & immunostaining (insnfin&glucagon) was pertbrmed. Staining patterns in acini, islets, and ducts from inflamed pancreata were compared to non-inflamed pancreata. Results: A distinct Pax6 regulatory enhancer region was identified that is required for Pax6 expression in the developing and adult pancreas and this region was narrowed down to a 450 bp upstream of the Pax6 P0 promoter. Pax6-LacZ transgene containing this regulatory region is expressed in the embryonic pancreatic rudiment and also in the adult islet in a way that suggests a role in the ontogeny of those cell populations. During pancreatic inflammation, budding of the ductal epithelium occurs, and Pax6-LacZ transgene appears to be expressed in the budding cells that migrate from the duct through the parench~nna to generate new islet ceils ConcJusions: Data suggest that Pax6 expressing cells may represent muhipotent pancreatic cells that arise from the duct and then migrate through the parenchyma to form new islet ceils in the setting of pancreatic inflammation. The Pax6-LacZ transgenic line with the 450bp pancreatic regulatory, element offers a powerful and potentially unique tool to track these cells. These experiments have the potential to permit further investigation of the functional role of Pax6.
T1868 High Rate of Pancreatitis in Hispanic CF Patients Carrying The R334W Mutation Patrick Maisonneuve, Preston Campbell tIl, Peter R Durie, Albert B Lowenfels INTRODUCTION: Pancreatitis, while uncommon in the majority of patients with cystic fibrosis (CF), is relatively common among the subgroup with preserved pancreatic function. AIM: To investigate tim mutation-specific t~requency of attacks of pancreatins in pancreatic sufficient CF patients. METHODS: We studied all ptvvalem and new CF patients collected between 1990 and 1999 by the Cysnc Fibrosis Foundation and recorded the number of patients who reported at least one episode of pancreatitis durmg the study period, according to their genotype. RESULTS: Patients carrying one R334W mutation had the highest frequency of pancreatins (table). Overall, the R334 mutation is rare and has been identified in only 77 (0.43%) of all genotyped patients in the CFF registry'; 38 (49%) were classified as Hispanics, a much higher proportion than in the global CE population (5.6%) (p<0.001). Fourteen lived in Puerto Rico, where this mutation is over-represented, occurring in 31% of all CE patients. DISCUSSION: Several study groups have idemihed the RI 17H and other todd CFTR mutations in patients with idiopathic pancreatitis but failed to detect the R334W mutation. This is understandable because none of the populations studied were likely to include Puerto Ricans or other populations in which this mutation is common. There is no current data to determine whether as)anptonxatic carriers of the R334W mutation are at
T1871 A Novel Mutation of the Calcium Sensing Receptor Gene Is Associated with Chronic Pancreatitis in Individuals with Heterozygons SPINK1 Mutations Peter Felderbauer, Nikolans Ansorge, Kerem Bulut, Henrik Einwaechter, Henning Schrader, Frank Scbmitz, Wolfgang E. Schmidt, Peter Hofth~ann Introduction: The role of mutations in the serine protease inhibitor type Kazal 1 (SPINK1) gene in chronic pancreatitis (CP) is still a matter of debate. Active SPINK1 is thought to be potent enough to antagonize approximately 20% of the activated trypsin and hence a
A-583
AGA
Abstracts