A microRNA-based test improves endoscopic ultrasound-guided cytologic diagnosis of pancreatic cancer.

BACKGROUND & AIMS: Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) in combination with cytopathology is the optimal method for diagnosis and staging of pancreatic ductal adenocarcinoma (PDAC) and other pancreatic lesions. Its clinical utility, however, can be limited by high rates of indeterminate or false-negative results. We aimed to develop and validate a microRNA (miRNA)-based test to improve preoperative detection of PDAC. METHODS: Levels of miRNAs were analyzed in a centralized clinical laboratory by relative quantitative polymerase chain reaction in 95 formalin-fixed paraffin-embedded specimens and 228 samples collected by EUS-FNA during routine evaluations of patients with solid pancreatic masses at 4 institutions in the United States, 1 in Canada, and 1 in Poland. RESULTS: We developed a 5-miRNA expression classifier, consisting of MIR24, MIR130B, MIR135B, MIR148A, and MIR196, that could identify PDAC in well-characterized, formalin-fixed, paraffin-embedded specimens. Detection of PDAC in EUS-FNA samples increased from 78.8% by cytology analysis alone (95% confidence interval, 72.2%-84.5%) to 90.8% when combined with miRNA analysis (95% confidence interval, 85.6%-94.5%). The miRNA classifier correctly identified 22 additional true PDAC cases among 39 samples initially classified as benign, indeterminate, or nondiagnostic by cytology. Cytology and miRNA test results each were associated significantly with PDAC (P < .001), with positive predictive values greater than 99% (95% confidence interval, 96%-100%). CONCLUSIONS: We developed and validated a 5-miRNA classifier that can accurately predict which preoperative pancreatic EUS-FNA specimens contain PDAC. This test might aid in the diagnosis of pancreatic cancer by reducing the number of FNAs without a definitive adenocarcinoma diagnosis, thereby reducing the number of repeat EUS-FNA procedures.

Is there adaptation of the exocrine pancreas in wild animal? The case of the Roe deer.

BACKGROUND: Physiology of the exocrine pancreas has been well studied in domestic and in laboratory animals as well as in humans. However, it remains quite unknown in wildlife mammals. Roe deer and cattle (including calf) belong to different families but have a common ancestor. This work aimed to evaluate in the Roe deer, the adaptation to diet of the exocrine pancreatic functions and regulations related to animal evolution and domestication. RESULTS: Forty bovine were distributed into 2 groups of animals either fed exclusively with a milk formula (monogastric) or fed a dry feed which allowed for rumen function to develop, they were slaughtered at 150 days of age. The 35 Roe deer were wild animals living in the temperate broadleaf and mixed forests, shot during the hunting season and classified in two groups adult and young. Immediately after death, the pancreas was removed for tissue sample collection and then analyzed. When expressed in relation to body weight, pancreas, pancreatic protein weights and enzyme activities measured were higher in Roe deer than in calf. The 1st original feature is that in Roe deer, the very high content in pancreatic enzymes seems to be related to specific digestive products observed (proline-rich proteins largely secreted in saliva) which bind tannins, reducing their deleterious effects on protein digestion. The high chymotrypsin and elastase II quantities could allow recycling of proline-rich proteins. In contrast, domestication and rearing cattle resulted in simplified diet with well digestible components. The 2nd feature is that in wild animal, both receptor subtypes of the CCK/gastrin family peptides were present in the pancreas as in calf, although CCK-2 receptor subtype was previously identified in higher mammals. CONCLUSIONS: Bovine species could have lost some digestive capabilities (no ingestion of great amounts of tannin-rich plants, capabilities to secrete high amounts of proline-rich proteins) compared with Roe deer species. CCK and gastrin could play an important role in the regulation of pancreatic secretion in Roe deer as in calf. This work, to the best of our knowledge is the first study which compared the Roe deer adaptation to diet with a domesticated animal largely studied.

Epithelial BMP signaling is required for proper specification of epithelial cell lineages and gastric endocrine cells.

Bone morphogenetic protein (BMP) signaling within the gastrointestinal tract is complex. BMP ligands and their receptors are expressed in both epithelial and mesenchymal compartments, suggesting bidirectional signaling between these two entities. Despite an increasing interest in BMP signaling in gut physiology and pathologies, the distinct contribution of BMP signaling in the epithelium vs. the mesenchyme in gastrointestinal homeostasis remains to be established. We aimed to investigate the role of epithelial BMP signaling in gastric organogenesis, gland morphogenesis, and maintenance of epithelial cell functions. Using the Cre/loxP system, we generated a mouse model with an early deletion during development of BMP receptor 1A (Bmpr1a) exclusively in the foregut endoderm. Bmpr1a(ΔGEC) mice showed no severe abnormalities in gastric organogenesis, gland epithelial proliferation, or morphogenesis, suggesting only a minor role for epithelial BMP signaling in these processes. However, early loss of BMP signaling in foregut endoderm did impact on gastric patterning, leading to an anteriorization of the stomach. In addition, numbers of parietal cells were reduced in Bmpr1a(ΔGEC) mice. Epithelial BMP deletion significantly increased the numbers of chromogranin A-, ghrelin-, somatostatin-, gastrin-, and serotonin-expressing gastric endocrine cells. Cancer never developed in young adult (<100 days) Bmpr1a-inactivated mice although a marker of spasmolytic polypeptide-expressing metaplasia was upregulated. Using this model, we have uncovered that BMP signaling negatively regulates the proliferation and commitment of endocrine precursor cells. Our data also indicate that loss of BMP signaling in epithelial gastric cells alone is not sufficient to induce gastric neoplasia.

Life with the pancreas: A personal experience.

This review article has primary objective to summarize pancreatic research which has been done in our laboratory since 1965, the first year of the author's registration in the Ph.D. program at the University of Sherbrooke (Canada). It covers the following major topics of pancreatic physiology: controls of pancreatic adaptation to diet, control of pancreatic enzyme secretion, control of pancreatic enzyme synthesis, control of pancreatic growth, intracellular events stimulated during pancreatic growth, pancreas regeneration after pancreatitis and pancreatectomy, the pancreatic cholecystokinin receptor types 1 and 2, growth control and cell signaling in pancreatic cancer cells and finally, cystic fibrosis.

'The most important duty of a mentor remains his availability'. An interview with Dr. Jean Morisset. Interview by Martin E Fernandez-Zapico.

We interview Dr. Jean Morisset, an internationally recognized leader in the field of pancreatic physiology. His work on the regulation of pancreatic secretion by hormones and the parasympathetic nervous system has been fundamental to the understanding of pancreatic adaptation to diet in normal as well as pathological conditions. Here he provides advices for young investigators starting in the field of pancreatic research and emphasizes the key role of the relationship mentor-mentee during the career development. and IAP.

Digestive enzyme concentrations and activities in healthy pancreatic tissue of horses.

OBJECTIVE: To measure concentrations and activities of major digestive enzymes in healthy equine pancreatic tissue. ANIMALS: 7 adult horses with normal pancreatic tissues. PROCEDURES: Small pieces of pancreatic tissue were collected immediately after euthanasia, immersed in liquid nitrogen, and maintained at -80 degrees C until analyzed. Concentrations and activities of amylase, lipase, chymotrypsin, trypsin, and elastase were determined by use of a microtiter technique. Relative pancreatic protein concentrations were determined by use of bovine serum albumin as the standard. Pancreatic DNA was extracted and con-centrations determined by use of the diphenylamine method with calf thymus DNA as the standard. RESULTS: The pancreatic cellular concentration of each enzyme, expressed as units per milligram of DNA, was consistent among horses. Cellular concentration of lipase (1,090.8 +/- 285.3 U/mg of DNA) was highest, followed by amylase (59.5 +/- 9.8 U/mg of DNA). Elastase, trypsin, and chymotrypsin were detected in small concentrations (1.9 +/- 0.6, 3.5 +/- 1.5, and 9.6 +/- 2.9 U/mg of DNA, respectively). Similar results were obtained for specific activities of the enzymes. CONCLUSIONS AND CLINICAL RELEVANCE: Results were unexpected because, under natural conditions, the predominant energy source for horses is carbohydrate. These results may indicate, in part, the reason horses seem to tolerate large amounts of fat added to their diet.

Somatostatin: One of the Rare Multifunctional Inhibitors of Mammalian Species.

This review article has for major main objectives to give an overlook of the major physiological effects of somatostatin on different organs. It will cover first the general aspect of the hormone, its cDNA and its protein maturation process, as well as its characterization in various organs. This aspect will be followed by the factors involved in the control of its secretion, its intracellular mode of action, and its general action on physiological processes. Secondly, the review will focus on the pancreas, looking at its in vivo and in vitro actions with special attention on its effects on normal pancreas growth and pancreatic tumors.

Regulation of rat pancreatic CCKB receptor and somatostatin expression by insulin.

The cholecystokinin B receptor (CCK(B)R) is localized on pancreatic endocrine somatostatin delta-cells. Pancreatic somatostatin content was increased in diabetic rats. The mechanisms involved in this phenomenon are unknown, and we believe insulin is involved. In this study, four groups of rats were used: controls, streptozotocin-induced diabetic, streptozotocin-induced diabetic with insulin, and streptozotocin-induced diabetic with insulin and its cessation. Rats were killed after 7-28 days of treatment for diabetes, and somatostatin mRNA expression and pancreatic somatostatin content, CCK(B)R mRNA and protein expression evaluation in total pancreas and purified islets, and the cellular localization of somatostatin and CCK(B)R in islets was measured. Data indicate that diabetes is established after 7 days, is controlled by insulin, and reappears after treatment cessation. Pancreatic somatostatin mRNA expression and somatostatin content were increased during diabetes, normalized during insulin treatment, and reaugmented after treatment cessation. Gland and islet CCK(B)R mRNA and protein almost disappeared during diabetes; CCK(B) mRNA reappeared in response to insulin, but the protein did not. Confocal microscopy confirmed data obtained on somatostatin and CCK(B)R as established biochemically in the course of the treatments. In conclusion, these data strongly suggest that insulin can negatively control pancreatic somatostatin mRNA and hormone content and positively control CCK(B)R mRNA; the CCK(B)R protein appears to be delayed.

Convergent alteration of granulopoiesis, chemotactic activity, and neutrophil apoptosis during mouse selection for high acute inflammatory response.

Neutrophil homeostasis was investigated in two mouse lines, AIRmax and AIRmin, genetically selected for high or low acute inflammatory response (AIR) and compared with unselected BALB/c mice. Mature neutrophil phenotype and functions appeared similar in the three mouse lines. However, an unprecedented phenotype was revealed in AIRmax animals characterized by a high neutrophil production in bone marrow (BM), a high number of neutrophils in blood, a high concentration of chemotactic agents in acrylamide-induced inflammatory exudates, and an increased resistance of locally infiltrated neutrophils to spontaneous apoptosis. In vitro, BM production of neutrophils and eosinophils was accompanied by an unusual high up-regulation of cytokine receptors as assessed by antibodies to CD131, which bind the common beta chain of receptors to interleukin (IL)-3, IL-5, and granulocyte macrophage-colony stimulating factor. An accelerated neutrophil maturation was also observed in response to all-trans retinoic acid. Several candidate genes can be proposed to explain this phenotype. Yet, more importantly, the results underline that genetic selection, based on the degree of AIR and starting from a founding population resulting from the intercross of eight inbred mouse lines, which display a continuous range of inflammatory responses, can lead to the convergent selection of alleles affecting neutrophil homeostasis. Similar gene combinations may occur in the human with important consequences in the susceptibility to inflammatory or infectious diseases and cancer.

Localization of cholecystokinin receptor subtypes in the endocine pancreas.

This study was undertaken to clarify the controversy in the literature about pancreatic localization of the cholecystokinin (CCK) CCK(A) and CCK(B) receptors. With antibodies used by other investigators, we first established their specificity by Western blotting, indirect immunofluorescence, and confocal microscopy with each antibody's peptide antigen. Co-localization assays between the CCK receptors and the pancreatic hormones insulin, glucagon, and somatostatin revealed that the CCK(A) RAbs 1122 and R1-2 recognized insulin and glucagon cells in rat, pig, and human pancreas but not in the somatostatin cells. Conversely, the three CCK(B) RAbs tested, 9262, 9491, and GR4, identified the somatostatin cells. Abs 9491 and GR4 occasionally co-localized with glucagon, a feature that never occurred with Ab 9262. Finally, the specificity of Ab 9262 for the pancreatic CCK(B) R was confirmed in six different species. It co-localized with somatostatin but never with glucagon in these species. Our data suggest the use of Abs 1122 and 9262 to specifically identify and localize pancreatic CCK(A) and CCK(B) receptors, respectively. Confusion in the literature may result from the lack of specificity of most antibodies used, as established in this study.

The rat pancreatic islets: a reliable tool to study islet responses to cholecystokinin receptor occupation.

This study was undertaken to show that rat purified islets can be used as a reliable tool to study some aspects of human islet's physiology related to CCKR occupation. Therefore, isolated foetal, adult human and rat islets were compared for (1) CCKR subtypes mRNA and protein expression and somatostatin (SS) mRNA and (2) co-localization of these receptors with insulin, glucagon and SS. Finally, rat islets were tested for their responsiveness to stimulation. Purified human and rat islets were used for CCKR subtypes and SS mRNA estimation by RT-PCR and protein by Western blots. Receptors and hormones co-localizations were evaluated by confocal microscopy. Hormones secretion served to determine rat islets responsiveness. Islets of both species express CCKA and CCKBR mRNA and proteins and SS mRNA. The CCKAR co-localizes with insulin and glucagon and the CCKBR with SS. Insulin release was increased 5-fold in response to 16 mM glucose and SS secretion reached 1.3- and 1.7-fold increments above basal in response to forskolin and IBMX. In conclusions, human and rat islets have comparable CCKR subtypes localized on the same cells; they also express SS mRNA. The rat islets are functional as they secrete but their response to hormonal stimulation remains to be clarified. These rat islets can thus serve as tools to study islets physiology.

Presence and localization of CCK receptor subtypes in calf pancreas.

This study was undertaken to confirm the presence of CCK receptor subtypes in calf pancreas and establish their cellular localization. Using specific antibodies against CCKA and CCKB receptors, somatostatin, glucagon and insulin, we were able to confirm by Western blot the presence of both CCK receptor protein subtypes in the calf pancreas as a 80-85-kDa CCKA receptor and 40-45-kDa CCKB receptor. By immunofluorescence, the CCKB receptor colocalizes with the islets' somatostatin delta cells, confirming what was previously shown in other species, as well as on ductal cells. We could not reproduce in the calf its colocalization with glucagon alpha cells as observed in human and rat. Any specific localization of CCKA receptors with our multiple antibodies failed. Our observation that the CCKB receptor subtype is specifically localized on pancreatic delta cells as well as on ductal cells lets us support the hypothesis that in this species, CCK could be involved in somatostatin metabolism as well as hydrelatic secretion; its effect on enzyme secretion would be indirect.

Bovine pancreatic secretion in the first week of life: potential involvement of intestinal CCK receptors.

The aim of this study was to evaluate pancreatic juice secretion of calves in the first postnatal days, and determine a potential involvement of cholecystokinin (CCK) and intestinal CCK receptor in its regulation. Nine neonatal Friesian calves (five controls and four treated intraduodenally with FK480, a CCK-A receptor antagonist) were surgically fitted with a pancreatic duct catheter and a duodenal cannula before the first colostrum feeding. Collections of pancreatic juice and duodenal luminal pressure recordings were started early after recovery from anaesthesia and continued for 6 days. From day 2 or 3 of life, periodic fluctuations in pancreatic secretions were observed in concert with duodenal myoelectric motor complex (MMC) and variations in plasma pancreatic polypeptide (PP) concentrations. Intraduodenal administration of FK480 reduced pancreatic juice secretion while intravenous infusion of CCK had no effect. Immunocytochemistry indicated an association of mucosal CCK-A and -B receptors with neural components of the small intestine. In conclusion, periodic activity of the exocrine pancreas exists in neonatal calves soon after birth and local neural intestinal CCK-A receptors could be partly responsible for the modulation of neonatal calf pancreatic secretion.

Seventy years of pancreatic physiology: take a look back.

This review article has 4 major objectives to follow pancreatic physiology development more than close to 70 years of intensive and productive basic research. At first, the review will focus on secretion of the pancreatic enzymes with (1) the controls involved, (2) the interrelations existing between secretion and synthesis of these enzymes, (3) the enzymes' adaptation to the constituents of the diet, and (4) whether secretion of the different enzymes is parallel or nonparallel. Second, growth and regeneration of the pancreatic gland will be looked at in relation to the factors involved and the target cells implicated.

Control of somatostatin (SS) secretion by CCK-1 and CCK-2 receptors' occupation in RIN-14B cells, a rat pancreatic islet cell line.

OBJECTIVES: This study evaluated the role played by cholecystokinin (CCK) receptors' occupation in the control of somatostatin (SS) secretion in RIN-14B cells. METHODS: The presence of the CCK receptors 1 and 2 was confirmed by immunofluorescence, and SS secretion was evaluated by enzyme-linked immunosorbent assay. RESULTS: By immunofluorescence, 95% of the cell population was composed of SS cells bearing both CCK-R subtypes with 5% of beta cells (data not shown). Cerulein (Cae), a CCK-1R agonist, and pentagastrin, a CCK-2R agonist, dose-dependently increased SS release, 3-fold at 1 mumol/L Cae, 2.5-fold at 10 mumol/L pentagastrin, with occupation of both CCKRs confirmed by L-364,178 and L-365,260 inhibition of CCK receptors 1 and 2. The occupation of high-affinity CCK-1R by Cae was confirmed on SS release with JMV-180, a high-affinity CCK-1R agonist, and absence of SS release inhibition at high Cae concentration occupying the low-affinity CCK-1R. These cells release more than 60% of their SS content by constitutive secretion, confirmed by cycloheximide and brefeldin inhibiting SS synthesis and intracellular trafficking, respectively. CONCLUSIONS: Both CCKR subtypes occupy RIN-14B cells and initiate SS secretion through constitutive secretion controlled at SS synthesis level. Somatostatin secretion via the CCK-1R occupation mobilizes its high-affinity sites.

Cholecystokinin and Somatostatin Negatively Affect Growth of the Somatostatin-RIN-14B Cells.

With the exclusive presence of the pancreatic CCK-2 receptors on the pancreatic delta cells of six different species, this study was undertaken to determine the role of cholecystokinin and gastrin on growth of these somatostatin (SS) cells. For this study, the SS-RIN-14B cells were used in culture and their growth was evaluated by cell counting. Results. To our surprise, we established by Western blot that these RIN cells possess the two CCK receptor subtypes, CCK-1 and CCK-2. Occupation of the CCK-1 receptors by caerulein, a CCK analog, led to inhibition of cell proliferation, an effect prevented by a specific CCK-1 receptor antagonist. Occupation of the CCK-2 receptors by the gastrin agonist pentagastrin had no effect on cell growth. Proliferation was not affected by SS released from these cells but was inhibited by exogenous SS. Conclusions. Growth of the SS-RIN-14B cells can be negatively affected by occupation of their CCK-1 receptors and by exogenous somatostatin.

Negative control of human pancreatic secretion: physiological mechanisms and factors.

The negative control of pancreatic exocrine secretion in man occurs during the interdigestive and postprandial periods of the digestive cycle. The physiological mechanisms involved include negative feedback mechanisms, well described and accepted in animals, and controlled by the cholecystokinin- and secretin-releasing factors of pancreatic and duodenal origin, along with the active pancreatic proteases present in the upper gut. The presence of these factors and their efficacy in humans, however, have their supporters and detractors, with a possibility for reconciliation among opponents. Besides these releasing factors, hormones, mostly from the intestine, are also involved in this inhibitory process of pancreatic secretion. Somatostatin, peptide YY, pancreatic polypeptide, glucagon, ghrelin, and leptin were described as potentially involved from studies mostly performed on animals. Finally, bile and bile salts have mixed responses on this inhibition, and their effects seem to be at the intestine level with gastrointestinal hormones involved. Future studies will have to be performed in humans to determine the presence of cholecystokinin- and secretin-releasing factors and their role. Finally, the demonstrated modulatory action of hormones and bile acids in other species needs to be confirmed in humans.

What are the pancreatic target cells for gastrin and its CCKB receptor? Is this a couple for cancerous cells?

The objectives of this review are to summarize, analyse and discuss the roles played by the CCK receptor subtypes and their agonists on pancreatic enzyme secretion, pancreas growth and regeneration, define the receptors specific target cells and evaluate the role of gastrin in pancreatic pathologies including cancer. In rodents, it is clear that the CCKARs present on pancreatic acinar cells play a major role in enzyme secretion. In large mammals, CCK does not seem to be the final mediator of enzyme release. In rat, gastrin and its CCKBR seem responsible for foetal pancreas growth while after birth, CCK was shown to be the most potent trophic factor via occupation of its CCKAR. In pig and human, no one has yet established a direct link between CCK, gastrin and pancreas growth. In rodent's pancreas, the CCKAR were observed on acinar cells as well as on islet's alpha and beta cells; in six other species, the CCKAR were present only on alpha and beta cells with the CCKBR always present on delta cells. The CCKBRs were overexpressed in acute pancreatitis and in metaplastic pancreas following duct ligation. In pancreatic cancer cells, a gastrin autocrine loop involving the CCKBR was suggested. The presence of both CCKR-subtypes and gastrin was observed in many pancreatic tumors; however, their role in cancer growth remains controversial.

Differential allele-specific accumulation of bovine kappa-casein mRNA throughout lactation.

A differential allele-specific accumulation of kappa-casein mRNA that is not linked to the kappa-casein protein variants is described in Holstein cows. Actually, cows genotyped kappa-casein AB were a mixed population. For the first group of kappa-casein AB cows, allele A-specific kappa-casein mRNA contents within mammary epithelial cells were lower than the allele B-specific ones (cows LH), suggesting that the allele A-specific kappa-casein gene was expressed with lower efficiency in mRNA. For the other group of kappa-casein AB cows, allele A- and B-specific kappa-casein mRNA accumulated to a similar level within mammary epithelial cells (cows HH). The objective of this study was to determine whether the accumulation of allele-specific kappa-casein mRNA remained constant throughout lactation for the two groups of cows. Quantitative RT-PCR was used to monitor Holstein cows kappa-casein AB genotyped HH and LH throughout lactation for the proportion of allele B-specific mRNA accumulation relative to the total kappa-casein encoded mRNA within mammary epithelial cells: RNA was extracted from milk somatic cells known to contain a small proportion of mammary epithelial cells. Mean values of allele B-specific mRNA content were 50.6+/-0.5 and 54.0+/-0.9%, for cows HH and cows LH, respectively, and did not vary during lactation (P> 0.10). This suggests that the phenotypic expression of the genetic mutation that causes the differential allele-specific accumulation of kappa-casein mRNA was not affected by physiological and environmental factors, which tend to vary considerably throughout lactation.