Chronic pancreatitis: the perspective of pain generation by neuroimmune interaction.

Chronic pancreatitis (CP) is an inflammatory, often painful, disease of the exocrine pancreas which leads to exocrine insufficiency. The pathophysiology of pain in CP is incompletely understood. Several hypotheses have been advanced, including pancreatic and extrapancreatic causes. Here, the different pain hypotheses are discussed and evidence is presented that neuroimmune interactions are significant in the pathogenesis of pain generation and inflammation in CP. A better understanding of the complex cellular and molecular mechanisms of neuroimmune interactions should offer possibilities for innovative therapy and long term disease prevention.

Ventral neural tube cells differentiate into hepatocytes in the chick embryo.

A population of ventral neural tube cells has recently been shown to migrate out of the hindbrain neural tube via the vagus nerve and contribute to the developing gastrointestinal tract. Since liver is also innervated by the vagus nerve, we sought to determine if these cells also migrate into the liver. Ventral neural tube cells in the caudal hindbrain of chick embryos were tagged with a replication-deficient retroviral vector containing the LacZ gene on embryonic day 2. Embryos were processed for detection of labeled cells on embryonic day 5 and 11. Labeled cells were seen in the liver on both days and identified as hepatocytes. Previously, it was believed that all hepatocytes develop from the gut endoderm. Results of the present study show an additional source for the formation of liver cells.

A new source of cells contributing to the developing gastrointestinal tract demonstrated in chick embryos.

BACKGROUND & AIMS: Smooth muscle cells in the walls of the gastrointestinal tract are thought to derive solely from mesoderm surrounding the primitive gut. A population of neuroepithelial cells has recently been shown to migrate from the ventral part of the neural tube in the region joined by the vagus nerve. We sought to determine if these cells contributed to the development of the stomach and intestine. METHODS: Cells of the ventral hindbrain of chick embryos were tagged by replication-deficient retroviral vectors containing the lacZ gene, providing a permanent label that is transmitted without dilution as the cells divide. Embryos were processed for detection of labeled cells. Specific markers were used to determine differentiation of progeny in the gastrointestinal tract. RESULTS: Cells labeled in the ventral neural tube migrate in association with the vagus nerve. Labeled cells are found in the intestine and stomach after time for further migration and differentiation. Using a specific marker, they were clearly identified as smooth muscle cells. CONCLUSIONS: Some of the smooth muscle cells of the gastrointestinal tract are derived from precursor cells that originate in the ventral part of the hindbrain neural tube. Their developmental importance and functional significance remain to be determined.

[Membrane injuries of pancreatic acinar cells are the first changes in early stage acute experimental pancreatitis]. / Membranverletzungen von Pankreasazinuszellen stellen eine der ersten Veränderungen im Frühstadium der akuten experimentellen Pankreatitis dar.

We tested the hypothesis that membrane wounding of acinar cells is one of the earliest changes during the induction of acute pancreatitis. Wounding of cell membranes was detected by the penetration of the animals own albumin into cells. The pancreatitis was induced by the intraperitoneal injection of supramaximal doses of caerulein. The controls received saline. Fifteen to 180 min. after the injection the animals were perfused with buffer followed by fixative. Frozen sections of pancreas were processed identically for immunocytological localization of albumin. The intensity of staining was quantified by image analysis. Animals receiving caerulein consistently display significantly greater (p < 0.001) anti-albumin immunostaining in the cytoplasm of acinar cells than controls. The penetration of albumin into acinar cells indicates that wounding of their plasma membrane occurs during the onset of acute pancreatitis. Wounding of membranes may allow the exit of molecules such as enzymes from the acinar cells during this period.

Development of the thymus.

Proper development of the thymus is critical for an individual to acquire full immune capability. A full complement of the components that participate in thymic development, interacting with each other at the correct time, is required for maturation. In order to establish the microenvironment necessary for T-cell differentiation, the epithelial primordium of the thymus must expand from pharyngeal endoderm with the aid of contributions from the ectoderm. Experimental studies have established the importance of mesenchymal derivatives from the neural crest in functional development of the epithelial primordium. Interfering with this process inhibits thymic development in a manner similar to that observed in congenital conditions such as the DiGeorge syndrome and the fetal alcohol syndrome. These observations provide clues to understanding the origin of defects in thymus-dependent immunity, and point the way to studies that will expand our understanding of the controls that are involved in genetic and environmental factors impacting on this process.

Morphology of the exocrine pancreas related to pancreatitis.

It has been assumed in the past that pancreatic acinar cells represent an irreversible end stage in development. Consequently, when there was an increase in structures that had the morphology of ductules, the interpretation was that they were derived from the proliferation of stem cells and/or pre-existing ductular cells. Pancreatitis, however, is regressive in nature [Bockman (1984) In: Pancreatitis: Concepts and Classification. Gyr, K.E., Singer, M.V., Sarles, H., eds. Elsevier, Amsterdam, pp. 11-15]. That is, it is characterized by parenchymal destruction and loss, rather than by expansion of parenchyma. Furthermore, it was assumed that the organization of the pancreatic parenchyma is like bunches of grapes, with spheroidal acini representing the grapes, and the ductules representing the stems. Given this organization, it would be difficult to understand how regressive changes could lead to clusters of ductular structures. Investigations using three-dimensional reconstruction and retrograde injections have altered our idea of pancreatic organization. In addition to spheroidal acini, there also are other shapes, including tubular acini. Moreover, ductules do not necessarily stop when they encounter an acinus. They may emerge on the other side. Combined ductular and acinar lumina may anastomose with each other. It is now clear that pancreatic acini may undergo redifferentiation, taking on the morphology of ductules and forming tubular complexes during pancreatitis, as well as in response to pancreatic cancer, cystic fibrosis, or blockage of the ductal system. With this understanding of pancreatic architecture and morphological plasticity, it is easier to understand the changes one sees with pancreatic diseases.

Pathological changes in pancreatic ducts from patients with chronic pancreatitis.

CONCLUSION: Chronic pancreatitis and restricted pancreatic outflow are accompanied by pathological changes in the ducts, including inflammation and alterations in the microvasculature. These changes and loss of epithelium provide a likely explanation for increased release of serum proteins, immunoglobulins, and lactoferrin into the juice, and the possibility of luminal contents entering the extracellular space and bloodstream. BACKGROUND: Enlargement of pancreatic ducts is a well-known phenomenon accompanying chronic pancreatitis and conditions restricting outflow of pancreatic juice. However, the relationship between ductal pathology and concomitant changes in the pancreatic juice is incompletely understood. METHODS: Segments of pancreatic ducts removed at surgery from patients with chronic pancreatitis and conditions restricting outflow were studied by light and electron microscopy to assess the pathological changes. RESULTS: Pathological changes in ducts from patients with chronic pancreatitis include chronic inflammation in the wall, enlarged and numerous capillaries packed with erythrocytes and leukocytes close to the lumen, and loss of epithelium and sometimes basement membrane. Plasma cells provide a source for increased immunoglobulins. Ducts from patients with diseases restricting outflow show significant pathology.

Localization of peptide transporter in nuclei and lysosomes of the pancreas.

CONCLUSIONS: These studies show for the first time the localization of a H+/peptide cotrasporter in nuclei of vascular smooth muscle cells and Schwann cells and its localization in lysosomes of the exocrine pancreas. It is likely that the transporter functions to move small peptides from the lysosome to the cytoplasm following intralysosomal protein degradation. The nature of the transporter function in the nucleus remains to be determined, including the possibility that peptide signaling molecules may be transmitted between nucleus and cytoplasm. BACKGROUND: PEPT1 transports di- and tripeptides through plasma membranes. Peptides are cotransported with H+, thus deriving the energy for the active transport process from an electrochemical H+ gradient. The main regions in which PEPT1 has been thought to function are the plasma membranes of the small intestinal epithelial cells for absorption of protein digestion products and in the kidney tubules for recovery of small peptides from the glomerular filtrate. METHODS: Pancreas was removed from rats and quick frozen with liquid nitrogen. Frozen sections were fixed in cold acetone. Sections were incubated with primary antibody against PEPT1, followed by a secondary antibody conjugated with fluorescein, then examined with a fluorescence microscope. RESULTS: Three major structures were immunopositive with the antibody to PEPT1: the nuclei of smooth muscle cells in the wall of arterioles, the nuclei of Schwann cells in unmyelinated pancreatic nerves, and lysosomes in acinar cells.

DiI labeling and homeobox gene islet-1 expression reveal the contribution of ventral neural tube cells to the formation of the avian trigeminal ganglion.

Cells of the neural tube are thought to be committed to form only the central nervous system, whereas the peripheral nervous system is believed to be derived from neural crest cells and from placodes, which are specialized regions of the surface ectoderm. Neural crest cells arise early from the dorsal part of the neural tube. The possibility that after emigration of the neural crest cells, another population of cells arising from the ventral part of the neural tube also emigrates via a different route was examined. Here we report that, after labeling cells of the ventral neural tube in the rostral hindbrain of E3 duck embryos with DiI, they were later found in the trigeminal ganglion of the fifth cranial nerve. A trail of labeled cells could be traced from the ventral part of the neural tube to the peripheral ganglion. Further, expression of the homeobox gene Islet-1 in cells of the neural tube and the ganglion also indicated that some ventral neural tube cells may normally emigrate to the trigeminal ganglion. It is concluded that not all neural tube cells are committed to form the central nervous system; the ventral part of the neural tube also provides cells for the formation of the trigeminal ganglion. These results raise the possibility that the ventral neural tube may serve as an additional source of cells for the formation of various other components of the peripheral nervous system.

Scatter factor/hepatocyte growth factor as a regulator of skeletal muscle and neural crest development.

Factors that regulate cellular migration during embryonic development are essential for tissue and organ morphogenesis. Scatter factor/hepatocyte growth factor (SF/HGF) can stimulate motogenic and morphogenetic activities in cultured epithelial cells expressing the Met tyrosine kinase receptor and is essential for development; however, the precise physiological role of SF/HGF is incompletely understood. Here we provide functional evidence that inappropriate expression of SF/HGF in transgenic mice influences the development of two distinct migratory cell lineages, resulting in ectopic skeletal muscle formation and melanosis in the central nervous system, and patterned hyperpigmentation of the skin. Committed TRP-2 positive melanoblasts were found to be situated aberrantly within defined regions of the transgenic embryo, including the neural tube, which overproduced SF/RGF. Our data strongly suggest that SF/HGF possesses physiologically relevant scatter activity, and functions as a true morphogenetic factor by regulating migration and/or differentiation of select populations of premyogenic and neural crest cells during normal mammalian embryogenesis.

Toward understanding pancreatic disease: from architecture to cell signaling.

Each year the American Pancreatic Association sponsors the Frank Brooks Memorial State of the Art Lecture. The following Frank Brooks Memorial Lecture was delivered at the combined meeting of the American Pancreatic Association and the International Association of Pancreatology, which was held in Chicago, Illinois, November 2-4, 1994. The presentation emphasizes the necessity for understanding the normal architecture of the pancreas, and the nature of the changes that occur, as important steps in understanding the origins and progression of pancreatic disease. A central theme is the plasticity of the cells that comprise the pancreas. Encouraging new insights are being derived from studies that determine the signaling molecules that regulate proliferation and differentiation, including those studies using transgenes.

Regulation of terminal differentiation of zymogenic cells by transforming growth factor alpha in transgenic mice.

BACKGROUND/AIMS: Transforming growth factor (TGF) alpha affects the growth of gastric mucosa. Its overexpression alters the mucosa. The aim of this study was to test the possibility that it regulates differentiation of gland cells. METHODS: Transgenic mice that overexpress TGF-alpha were used to detect its effect on zymogenic (chief) cells in the stomach. To test for a general regulatory role of TGF-alpha in differentiation of zymogen-producing cells, salivary glands from transgenic mice were studied. RESULTS: In these mice, messenger RNA for pepsinogen C is present in the stomach at normal levels during the neonatal period and then decreases markedly. Zymogenic cells are present in the stomach during the neonatal period but are missing in transgenic adults. The bases of gastric glands, normally rich in zymogenic cells, are occupied by undifferentiated cells and mucous neck cells, the precursors of zymogenic cells. Zymogen granules in submandibular glands of transgenic female mice are reduced in number. Zymogen granule-containing cells in the parotid gland undergo redifferentiation to form tubular complexes, collections of ductularlike structures like those formed in the transgenic pancreas. CONCLUSIONS: TGF-alpha is a major participant in the regulation of terminal differentiation of zymogenic cells in the stomach and salivary glands.

Transforming growth factor alpha disrupts the normal program of cellular differentiation in the gastric mucosa of transgenic mice.

Transforming growth factor alpha (TGF alpha) evokes diverse responses in transgenic mouse tissues in which it is over-expressed, including the gastric mucosa, which experiences aberrant growth and a coincident repression of hydrochloric acid production. Here we show that ectopically expressed TGF alpha induces an age-dependent cellular reorganization of the transgenic stomach, in which the surface mucous cell population in the gastric pit is greatly expanded at the expense of cells in the glandular base. Immunohistochemical analysis of BrdU incorporation into DNA demonstrated that although mature surface mucous cells were not proliferating, DNA synthesis was enhanced by approximately 67% in the glandular base and isthmus, where progenitor cells reside. RNA blot and in situ hybridization were employed to determine temporal and spatial expression patterns of specific markers representing a variety of exocrine and endocrine gastric cell types. Mature parietal and chief cells were specifically depleted from the glandular mucosa, as judged by a 6- to 7-fold decrease in the expression of genes encoding H+,K(+)-ATPase, which is required for acid secretion, and pepsinogen C, respectively. The reduction of these markers coincided in time with the activation of TGF alpha transgene expression in the neonatal stomach. The rate of cell death in the glandular region was not overtly different. Significantly, the loss of parietal and chief cells occurred without a concomitant loss of their respective cellular precursors. In contrast to exocrine cells, D and G endocrine cells were much less severely affected, based on analysis of somatostatin and gastrin expression. Analysis of these dynamic changes indicates that TGF alpha can induce selective alterations in terminal differentiation and proliferation in the gastric mucosa, and suggests that TGF alpha plays an important physiological role in the normal regulation of epithelial cell renewal.

Interaction of pancreatic ductal carcinoma with nerves leads to nerve damage.

BACKGROUND/AIMS: Perineural extension of pancreatic adenocarcinoma has been explained as a mechanical extension along planes of least resistance. This study tests whether the cancer is limited to following these planes and if substances involved in cell signaling are involved in the interaction of cancer cells with nerves. METHODS: Samples of tissue from patients undergoing resection of pancreatic cancer were studied by electron microscopy and light microscopy. Transforming growth factor alpha (TGF-alpha) and epidermal growth factor receptor (EGFR) were localized in sections. RESULTS: The adenocarcinoma is not confined to the periphery of nerves. It penetrates the perineurium and becomes intimately associated with Schwann cells and axons in the endoneurium. Neural elements are damaged. Neural invasion likely is a factor in associated pain. TGF-alpha is abundant in nerves in the pancreas. EGFR is prominent in the cells of the adenocarcinoma. CONCLUSIONS: The interaction of pancreatic cancer with nerves involves more than the cancer following a perineural space. Interaction of TGF-alpha in nerves with EGFR on cancer cells constitutes a possible paracrine mechanism that provides a growth advantage for pancreatic adenocarcinoma and serves as an example of potential interactions that might be active in biological interaction of cancer with nerves.

Nesidioblastosis as a mechanism to prevent fibrosis-induced diabetes after pancreatic duct obstruction.

Does obstruction of the main pancreatic duct that results in exocrine atrophy also result in diabetes? We followed 11 mongrel dogs for 3 years after approximately three-fourths of the pancreas had been removed and the main pancreatic duct obstructed in the pancreatic tail remnant. At 3 years after surgery, all dogs showed total exocrine atrophy (remnants weighed 0.2-1.2 g). None of the animals became diabetic despite only 6% of the original pancreatic tail remaining. During the 3-year period, fasting blood insulin increased during the last half of the study, and this was associated with islets that were 1,300% larger than in control animals (nesidioblastosis). An in situ pancreatic remnant with an obstructed duct and total exocrine atrophy can maintain a nondiabetic state for > 3 years. Perhaps the mechanism is associated with the observed nesidioblastosis.

Cytological changes in the pancreas of transgenic mice overexpressing transforming growth factor alpha.

Transgenic mice overexpressing human transforming growth factor alpha (TGF-alpha) predictably develop an enlarged, firm pancreas. The present study investigated the changes that occur in the different components of the pancreas in these animals. The increase in size of the pancreas may be accounted for by increased connective tissue. The added collagen is mainly type I. Thin, elongate fibroblasts are frequently bordered by a basal lamina, a relationship that is normally restricted to the perineurium. Collagen is intimately associated with epithelial cells. Fingers of connective tissue extend close to acinar lumina. Redifferentiation of acinar cells produces tubular complexes. In some cases, acinar cells take on the appearance of ductular cells. In some, there is a transition to mucin-producing cells. Intermediate forms between acinar and mucin-producing cells are present. The growth factor is localized in acinar cells and decreases with redifferentiation. The pancreas of these animals routinely displays characteristics that also are observed in diseases of the exocrine pancreas in humans, including fibrosis and redifferentiation. It is likely that the changes are the result of both direct and indirect effects of TGF-alpha, some of which may parallel altered control mechanisms in human pancreatic disease. Study of this model may provide clues to understanding the initiation of fibrosis and redifferentiation in human pancreas.

Microvasculature of the pancreas. Relation to pancreatitis.

Local or generalized alteration of microcirculation may be expected in diseases of the pancreas. Changes may range from increased permeability of capillaries to hemorrhage. Tissue necrosis may result from prolonged ischemia owing to intravascular coagulation and severely impaired blood flow. It is possible to observe early microvascular changes by intravital microscopy. Klar and coworkers have demonstrated by this method that isovolemic hemodilution improves blood flow under conditions that would otherwise lead to tissue damage. This paper presents the basic microcirculation of the pancreas and the changes that accompany pancreatic disease. It emphasizes that concentration on the changes in microcirculation that accompany the early manifestations of pancreatic diseases, particularly pancreatitis, may reveal important clues to their pathogenesis.

Inhibition of epibranchial placode-derived ganglia in the developing rat by bisdiamine.

Although bisdiamine has been shown to affect the development of mammals, its effect on the nervous system has gone largely unrecognized. In the present study, rats were given bisdiamine by gavage on days 9 and 10 of pregnancy. They were sacrificed at intervals and the fetuses were prepared for study of serial sections stained with hematoxylin and eosin, or by immunohistochemical reaction with HNK-1 monoclonal antibody. HNK-1 reacted strongly with the nervous system, allowing precise analysis of the components and their relationships. Controls receiving no bisdiamine were prepared and studied in parallel with the experimental fetuses. Administration of bisdiamine inhibited development of the petrosal and nodose ganglia, altered associations of the glossopharyngeal, vagus, and hypoglossal nerves, and inhibited contributions of vagal nerve fibers to the developing enteric system. The proximal ganglia of the glossopharyngeal and vagus nerves developed normally. It is concluded that bisdiamine affects, directly or indirectly, the differentiation of nervous components derived from the epibranchial placodes. It seems likely that these placode-derived components serve as pioneer neurons in establishing the pathway for the posteriorly extending trunks of the glossopharyngeal and vagus nerves. The early changes in congenital conditions such as the DiGeorge syndrome may not be limited to alterations in neural crest derivatives. It may be worthwhile to investigate more closely whether there are alterations in the nervous system associated with these syndromes.

Capacity of neural crest cells from various axial levels to participate in thymic development.

In order to assess the capacity of neural crest from different sources to participate in thymic development, neural crest from selected axial levels was transplanted unilaterally from quail donors to the region in chick hosts from which neural crest cells normally migrate to interact with the primordial thymus. The greatest representation of donor cells was observed after isotopic transplantation and when donor tissue was taken from the hyoid and mesencephalic regions of the neural crest. The capacity for transplants to contribute cells decreased both anteriorly and posteriorly, so that neural crest close to the usual origin of mesenchyme-producing cells contributed a large number of donor cells around the developing thymus than neural crest from anterior and posterior regions. Cells from the transplant were inserted as an addition to the host chick cells. Thus, a special relationship and capacity for interaction in thymic development is expressed by neural crest at usual levels over a limited span of axial regions, but to some extent by all regions. This study has established that the capacity for neural crest cells from different axial levels to interact with developing organs is not uniform, but may vary, depending upon the nature of the interaction with a particular organ.

Impaired development of the thymic primordium after neural crest ablation.

Impaired thymic development as a result of ablation of neural crest has been observed in embryos late in development. The present study was initiated to determine what changes are effected early in thymic development by neural crest ablation. The epithelial primordia of the thymus were studied in chick embryos on the sixth day of incubation. Embryos with neural crest ablations were compared with sham-operated and untreated controls. Neural crest ablation inhibited formation of epithelial thymic primordia. Primordia in experimental embryos were fewer in number and were smaller than in shams and untreated controls. When primordia from shams and controls were transplanted to the chorioallantoic membrane of chick hosts, they were able to develop into organs with the typical features of embryonic thymus. Similar transplantation from neural crest-ablated animals, on the other hand, led to small, predominantly epithelial structures with meager lymphoid development. These findings are consistent with the hypothesis that mesenchyme derived from cranial neural crest is critical in initiating and sustaining the development from pharyngeal pouches of epithelial structures competent to attract and support the proliferation and differentiation of lymphoid stem cells.