Culture of endocrine pancreatic cells in protein-free,chemically defined media

Summary Cell suspensions prepared by collagenase digestion of pancreata obtained from 21.5-d-old rat fetuses were preincubated in RPMI medium containing 10% fetal bovine serum (FBS), to ensure cell adhesion. Twenty hours later, this medium was replaced by a chemically defined medium. Dulbecco's modified Eagle's (DME)-F12 was used alone or supplemented with various combinations of transferrin, sodium selenite, or Ultroser G. The evolution of the culture and the islet ultrastructure were similar in defined and serum-containing media. However, in the defined medium, the neoformed islets seemed less numerous, and the fibroblast layer less dense, when compared to the RPMI+10% FBS control medium. At Day 7, in defined media, the total insulin content per dish was half that of control cultures. None of the tested additives improved the yield of the cultures. The fractional insulin release per day was elevated in defined media. In subsequent incubations, glucose and leucine stimulated insulin release in a way characteristic of these cells of fetal origin. The labeling index of islet cells cultured in DME-F12 reached 10.7%, which is not far from that observed in RPMI+10% FBS. Such a defined medium is useful to study B cell physiology, avoiding the possible interaction of serum components with substances to be tested. The support of the Fonds National de la Recherche Scientifique and the ‘Loterie Nationale’ of Belgium is also acknowledged.     

Fine Needle Aspiration of A Pancreatic Oxyphilic Carcinoma With Pulmonary and Subcutaneous Metastases

The cytological appearances of an oxyphilic pancreatic carcinoma are reported and the histological and clinical features are described. Despite the presence of pulmonary and subcutaneous metastases this tumour did not behave in an aggressive fashion.     

Meal-stimulated and atropine-inhibited secretion of pancreatic polypeptide in healthy subjects, members of MEA I families and patients with malignant endocrine tumours of the gastrointestinal tract

Pancreatic polypeptide has been suggested as a marker for endocrine malignancies of the gastrointestinal tract. However, the secretion of PP shows great intra- and inter-individual variation, causing both false negative and positive results. In order to reduce these risks, we have evaluated a new combined stimulatory and inhibitory test of PP secretion. Six healthy subjects, 23 members of three MEA I families, seven patients with malignant pancreatic endocrine tumours and four patients with carcinoid tumours of the gastrointestinal tract were subjected to a standardized test meal, followed by intravenous atropine 60 min after the start of the meal. Serum PP was monitored during 2 h. In healthy subjects the meal caused a rapid increase in serum PP within 20 min and intravenous atropine caused a significant (P less than 0.05) decrease of serum PP within 15 min. Patients with malignant endocrine pancreatic tumours or carcinoids had a delayed response after the test meal, with maximum levels at 45 min, but still with a significant inhibition by atropine. Even tumour patients with initially normal or slightly increased basal PP levels showed this secretion pattern. Healthy members of MEA I families displayed identical PP curves to healthy subjects, whereas members with elevated basal PP levels who had been previously affected by hyperparathyroidism and/or prolactinomas showed similar secretion patterns to pancreatic tumour patients. We think that a meal stimulatory test is of great value in the diagnosis of gastrointestinal endocrine tumours and also in the identification of subjects with the MEA I trait, who are at high risk of having pancreatic endocrine tumours.     

Value of EUS‐FNA cytological preparations compared with cell block sections in the diagnosis of pancreatic solid tumours

Y. Kopelman, S. Marmor, I. Ashkenazi and Z. Fireman
Value of EUS‐FNA cytological preparations compared with cell block sections in the diagnosis of pancreatic solid tumours Objective: Endoscopic ultrasound‐guided fine needle aspiration (EUS‐FNA) is performed in order to achieve a definite tissue diagnosis of pancreatic lesions. This in turn is a guide to the appropriate treatment for the patient. Tissue samples collected by the same needle for cytological preparations and cell block histological sections (often referred to as FNA‐cytology and FNA–biopsy, respectively) are handled differently. The specific contribution of each of these tests was evaluated. Methods: One hundred and two consecutive patients underwent EUS‐FNA while being investigated for pancreatic solid lesions. Diagnosis was made by cytology, cell block sections or both. The diagnosis was confirmed by clinical outcome. Results: Male/female ratio was 61/41. Mean age was 65 ± 12 years (range, 22–94). Mean lesion size was 3.1 ± 1.8 cm (range, 0.6–10 cm); 68% were >2 cm and 75% were located in the pancreatic head. The average number of needle passes was two (range, 1–4 passes). Final tissue diagnosis was malignant in 66 (65%) patients. Sensitivity, specificity and accuracy were 73%, 94% and 81%, respectively, for cytology alone, and 63%, 100% and 78%, for cell blocks alone. Eighty‐two patients (80%) had cytology and cell blocks, which matched in 64 (78%) patients. EUS‐FNA results that relied on both techniques had 84% sensitivity, 94% specificity and 88% accuracy. Cytology revealed 13 malignancies not diagnosed on cell blocks, while cell blocks revealed five malignancies not diagnosed by cytology. Malignant lesions were more common in men; they were larger in size and located in the pancreatic head. Conclusion: EUS‐FNA cytology was more sensitive than cell blocks but less specific for the diagnosis of solid pancreatic lesions. The two methods are complementary and implementing both improves the diagnostic value of EUS‐FNA.     

Long cytoplasmic processes in pancreatic polypeptide cells

Pancreatic polypeptide (PP) cells were studied in human endocrine pancreatic tumours and in normal human pancreata by immunohistochemical techniques and electron miscroscopy. The existence of long cytoplasmic processes was demonstrated both in tumours and normal tissue. These processes are in close contact with other endocrine cells or with acinar cells. This particular morphological aspect suggests that PP cells may control the function of other cells via paracrine secretion.     

Zebrafish model of KRAS-initiated pancreatic endocrine tumor

Pancreatic cancer constitutes a genetic disease in which somatic mutations in the KRAS proto-oncogene are detected in 95% of cases. Activation of the KRAS proto-oncogene represents an initiating event in pancreatic tumorigenesis. Here, we established a zebrafish pancreatic neoplasia model that recapitulates human pancreatic tumors. Toward this end, we generated a stable CRE/Lox-based zebrafish model system to express oncogenic KRAS^G12D  in the elastase3I domain of the zebrafish pancreas. Lineage tracing experiments showed that early KRAS^G12D -responsive pancreatic progenitors contribute to endocrine in addition to exocrine cells. In this system, 10% and 40% of zebrafish developed pancreatic tumors by 6 and 12 months, respectively. The histological profiles of these experimental tumors bore a striking resemblance to those of pancreatic endocrine tumors. Immunohistochemical analysis including the endocrine cell-specific marker confirmed the pancreatic tumor region as a characteristic endocrine tumor. Taken together, our zebrafish model data revealed that pancreatic endocrine tumors originate from early KRAS^G12D -responsive pancreatic progenitor cells. These findings demonstrated that this zebrafish model may be suitable as an experimental and preclinical system to evaluate different strategies for targeting pancreatic endocrine tumors and ultimately improve the outcome for patients with pancreatic endocrine tumors.     

Endoscopic ultrasound-guided fine needle aspiration cytology of pancreatic neuroendocrine tumours: cytomorphological and immunocytochemical evaluation

OBJECTIVES: Endoscopic ultrasound (EUS)-guided fine needle aspiration (FNA) is increasingly used in preoperative localization and diagnosis of pancreatic neoplasms including neuroendocrine tumours (NETs). The objective of the present study was to identify the cytological features of pancreatic NETs obtained by EUS-FNA. METHODS: The study group consisted of nine cases of pancreatic tumours correctly diagnosed or strongly suggestive of NETs based on EUS-FNA. Cytological smears were retrospectively reviewed. The clinical data and immunocytochemical stains applied to the cell block preparations were also reviewed and examined. RESULTS: All cases except one showed characteristic cytomorphological features sufficient for their recognition and separation from pancreatic adenocarcinoma and other lesions. The most helpful cytological features that facilitated the cytological diagnosis of NET were a richly cellular aspirate with a monotonous, poorly cohesive population of small cells with a speckled or dusty chromatin pattern and plasmacytoid morphology. The neuroendocrine differentiation of these tumours was further confirmed by immunocytochemistry. CONCLUSION: EUS-FNA is a valuable method in the recognition of pancreatic NETs. By adherence to the characteristic cytomorphological criteria of pancreatic NET together with collection of suitable material for ancillary immunocytochemical stains, cytopathologists could reach a correct diagnosis in most instances.     

Notch signaling in pancreatic endocrine cell and diabetes

Recent studies have improved our understanding of the physiological function of Notch signaling pathway and now there is compelling evidence demonstrating that Notch is a key regulator of embryonic development and tissue homeostasis. Although further extensive studies are necessary to illustrate the molecular mechanisms, new insights into the role of Notch signaling in pancreas development and diabetes have been achieved. Importantly, the ability to regulate Notch signaling intensity both positively and negatively may have therapeutic relevance for diabetes. Thus, this paper reviews the current knowledge of the roles of Notch signaling in the pancreatic endocrine cell system.     

Spontaneous pathology of the baboon endocrine system

Background  Study of endocrine pathology in animal models is critical to understanding endocrine pathology in humans.
Methods  We evaluated 434 endocrine-related diagnoses from 4619 baboon necropsies, established the incidence of spontaneous endocrine pathology, and analyzed the clinical and biochemical data associated with the individual cases.
Results  The most common diagnoses in descending order, were pancreatic islet cell amyloidosis (n = 259), ovarian cysts (n = 50), pituitary adenoma (n = 37), pancreatic islet cell adenoma (n = 20), granulosa cell tumor (n = 15), thyroid adenoma (n = 11), adrenal hyperplasia (n = 10), thyroid carcinoma (n = 8), and pheochromocytoma (n = 6). The incidence of pancreatic islet cell amyloidosis progressively increased with age. Pheochromocytomas were associated with renal and heart failure. The incidence of pancreatic islet cell amyloidosis and adrenal pathology was similar to humans; the incidence of pituitary adenoma and thyroid pathology was lower than in humans.
Conclusions  Endocrine disease in baboons is common and shares clinical and biochemical characteristics with endocrine disease in humans.     

The endocrine pancreas in the adult gray short-tailed opossum, Monodelphis domestica (Marsupialia)

Monodelphis domestica, a South American marsupial proposed as a laboratory animal, presents some characteristics that are favourable for research on the development of pancreatic tissue. Therefore, it is important to know if Monodelphis domestica is similar to other mammals concerning the cellular composition of the pancreas and if the different regions of the organ share similar morphologic features. In the present study the pancreas of ten adult male or female Monodelphis domestica were examined: serial paraffin sections were stained immunohistochemically using polyclonal antibodies against insulin or glucagon. The size of the immunoreactive area was measured with a computer-based image-analysing system differentiating lobus dexter, corpus pancreatis, and lobus sinister.

The islets' profiles varied in size and shape. The insulin-positive cells were mainly located in the centre of the islets, while the glucagon-positive cells were arranged at the islets' periphery. However, some glucagon-positive cells also occurred in the centre of most islets. On average, 0.66% of the entire pancreatic section area was insulin-positive, while 0.23% was glucagon-positive. Thus, the ratio between glucagon-positive and insulin-positive area was 1:2.9. Differences between the three pancreatic parts, lobus dexter, corpus pancreatis, and lobus sinister, were revealed. In general, the findings compared well with those of species that have a history as laboratory animals, i.e. mice, rats, cats, and dogs.     

Quantitative energy dispersive X-ray microanalysis of eight elements in pancreatic endocrine and exocrine cells after cryo-fixation

Quantitative X-ray microanalysis of 8 elements was performed on ultrathin, freeze-dried sections of islets and pancreas pieces from non-inbredob/ob-mice. Diffusion of elements was reduced to a minimum by rapidly freezing the tissue samples between nitrogen-cooled polished copper surfaces and avoiding the use of chemical fixatives and stains. The ultrastructural morphology was adequately maintained to allow measurements on secretory granules, mitochondria, cell nuclei, and cytoplasm free of these organelles. The distribution of the various elements between cellular compartments was similar in islet -cells and exocrine pancreas cells. However, the insulin secretory granules were outstanding in exhibiting the highest concentrations of zinc and calcium. In comparison with cytoplasm in the -cells, the insulin granules accumulated calcium 2-fold and zinc as much as 40-fold. As no correlation could be made for endoplasmic reticulum in the cytoplasmic measurements areas, the true accumulations above cytosol are likely to be even higher.     

Immunohistochemical expression of rabphilin-3A-like (Noc2) in normal and tumor tissues of human endocrine pancreas

Involvement of rabphilin-3A-like (RPH3AL), or Noc2, the potential effector of Ras-associated binding proteins Rab3A and Rab27A in the regulation of exocytotic processes in the endocrine pancreas has been demonstrated in experimental models. Noc2 expression together with other regulatory molecules of the exocytotic machinery in human tissues, however, has not been studied. We evaluated immunohistochemical expression of the key molecules of the exocytotic machinery, Noc2, Rab3A, Rab27A, and RIM2, together with the characteristic islet cell hormones, insulin and glucagon in normal and endocrine tumor tissues of human pancreas. Normal pancreatic islets were stained for all of these proteins and showed strong cytoplasmic localization. A similar pattern of strong cytoplasmic expression of these proteins was observed in the majority of endocrine tumors. By contrast, the exocrine portions of normal appearing pancreas completely lacked Rab27A staining and showed decreased expression of the proteins, Noc2, Rab3A, and RIM2. The staining pattern of Noc2 and Rab27A was similar to the staining pattern of glucagon-producing cells within the islets. The concomitant expression of Noc2 with these molecules suggests that Noc2 may serve as an effector for Rab3A and Rab27A and that it is involved in the regulation of exocytosis of the endocrine pancreas in humans.     

Fine Needle Aspiration Cytology and Immunocytochemistry of Metastatic Melanoma

The cytological and immunological findings of 81 metastatic melanomas are described. Fine needle aspiration was performed from secondary deposits in lymph nodes (38), subcutaneous and soft tissue (36), abdomen (5), lung (1) from 67 patients with histologically verified malignant melanoma. One patient had disease which had spread into the cerebrospinal fluid. Cytomorphologically the cases were classified as classical (47%), carcinoma-like (22%), spindle cell type (14%), lymphoma like (6%), undifferentiated (6%), myxoid type (3%), and clear cell type (2%). All cases were immunologically characterized using antibodies to S-100, vimentin and cytokeratin. All cases were S-100 positive and the majority (96%) reacted with antibodies to vimentin. A weak heterogenous reactivity to cytokeratin antibody was detected in only eight cases. The HMB45 antibody was applied to 20 cases and 16 (80%) of these tumours were positive. In summary, we found that an immunological characterization was necessary to conclusively diagnose over 50% of metastatic melanomas which presented with an equivocal cytological picture.     

Natural purified porcine gastric inhibitory polypeptide (GIP) stimulates exocrine pancreas secretion due to contamination with a cholecystokinin-like substance

The effects of purified natural gastric inhibitory polypeptide-enterogastrone III (GIP-EG III) and a fraction which is further purified by high pressure liquid chromatography (GIP-HPLC) were investigated on the endocrine and exocrine isolated perfused pancreas of rats. At the dose of 5 ng/ml used for both GIP preparations, only GIP-EG III significantly stimulated volume and amylase secretion of the exocrine pancreas. The response of insulin release to stimulation by GIP-EG III or GIP-HPLC was not significantly different. In the presence of cholecystokinin-octapeptide (CCK-8) at a concentration which gave half-maximal stimulation of amylase secretion, GIP-EG III almost doubled the response of the exocrine pancreas, whereas GIP-HPLC had no additional effect. CCK-8 alone significantly increased total insulin output under hyperglycemic conditions. We conclude that porcine GIP purified by gel chromatography contains a CCK-like substance which can be removed by further purification on high pressure liquid chromatography without affecting the insulinotropic activity. Some of the reported effects of GIP could be due to contamination.     

Evidence for cholecystokinin receptor subtype in endocrine pancreas

Cholecystokinin (CCK) is a gut hormone that regulates pancreatic endocrine functions via CCK_A receptors. CCK₄ (Trp-Met-Asp-Phe-NH₂) has an insulinotropic effect, but is 1000-fold less potent than CCK₈. The in vitro potencies and selectivity of newly synthesized CCK₄ analogs were investigated. Exchanging various a amino acids, for example Met by Nle and modifying Phe and/or Trp, led to compounds that were much more effective than CCK₄ itself and show insulinotropic effects comparable with those of CCK₈. Compounds that possess electron withdrawing groups on the C-terminal phenylalanine were especially effective; compounds with electron-donating groups had no effect. In contrast to CCK₈ the synthetic CCK₄ compounds were selective for the endocrine pancreas: they had no agonistic or antagonistic effect on the contraction of the guinea pig ileum, amylase release from isolated acini, and no major effect on the feeding behavior of mice being supplied with either compound by an implantable Alzet^R pump for 8 days. The data indicate that some of the synthetic tetrapeptides exhibit a high affinity for the CCK receptor of the endocrine pancreas and that they are highly selective for this (peripheral) CCK_A receptor subtype. The β-cell CCK_A receptors are different from those in exocrine pancreas, smooth muscle, and those for regulating appetite; these peripheral receptor subtypes can be discriminated for the first time.