Pancreatic cancer: The microenvironment needs attention too!

The abundant stromal/desmoplastic reaction, a characteristic feature of a majority of pancreatic adenocarcinomas (PDAC), has only recently been receiving some attention regarding its possible role in the pathobiology of pancreatic cancer. It is now well established that the cells predominantly responsible for producing the collagenous stroma are pancreatic stellate cells (PSCs). In addition to extracellular matrix proteins, the stroma also exhibits cellular elements including, immune cells, endothelial cells and neural cells. Evidence is accumulating to indicate the presence of significant interactions between PSCs and cancer cells as well as between PSCs and other cell types in the stroma. The majority of research reports to date, using in vitro and in vivo approaches, suggest that these interactions facilitate local growth as well as distant metastasis of pancreatic cancer, although a recent study using animals depleted of myofibroblasts has raised some questions regarding the central role of myofibroblasts in cancer progression. Nonetheless, novel therapeutic strategies have been assessed, mainly in the pre-clinical setting, in a bid to interrupt stromal-tumour interactions and inhibit disease progression. The next important challenge is for the translation of such pre-clinical strategies to the clinical situation so as to improve the outcome of patients with pancreatic cancer.

Alcohol, signaling, and ECM turnover.

Alcohol is recognized as a direct hepatotoxin, but the precise molecular pathways that are important for the initiation and progression of alcohol-induced tissue injury are not completely understood. The current understanding of alcohol toxicity to organs suggests that alcohol initiates injury by generation of oxidative and nonoxidative ethanol metabolites and via translocation of gut-derived endotoxin. These processes lead to cellular injury and stimulation of the inflammatory responses mediated through a variety of molecules. With continuing alcohol abuse, the injury progresses through impairment of tissue regeneration and extracellular matrix (ECM) turnover, leading to fibrogenesis and cirrhosis. Several cell types are involved in this process, the predominant being stellate cells, macrophages, and parenchymal cells. In response to alcohol, growth factors and cytokines activate many signaling cascades that regulate fibrogenesis. This mini-review brings together research focusing on the underlying mechanisms of alcohol-mediated injury in a number of organs. It highlights the various processes and molecules that are likely involved in inflammation, immune modulation, susceptibility to infection, ECM turnover and fibrogenesis in the liver, pancreas, and lung triggered by alcohol abuse.

Pancreatic MAP kinase pathways and acetaldehyde.

Alcohol abuse is a major cause of pancreatitis, a condition that can manifest as both acute necroinflammation and chronic damage (acinar atrophy and fibrosis). It is generally accepted that alcohol-induced pancreatic injury is a consequence of the metabolism of alcohol by the pancreas (via the oxidative and non-oxidative pathways) producing the toxic metabolites acetaldehyde and fatty acid ethyl esters (FAEEs) respectively. Ethanol oxidation within the pancreas also leads to oxidant stress within the gland. Acetaldehyde, oxidant stress and FAEEs cause numerous molecular changes in pancreatic acinar cells which predispose the gland to autodigestion and necroinflammation. An important recent development relates to the identification of pancreatic stellate cells (PSCs) as the key mediators of alcohol-induced pancreatic fibrosis, when activated by ethanol, acetaldehyde or oxidant stress. Recent studies implicate the mitogen activated protein kinase (MAPK) pathway, a major signalling pathway in mammalian cells, as a critical regulator of the effects of ethanol and acetaldehyde on acinar cells as well as PSCs. Particularly important are the modulatory effects of ethanol and its metabolites on downstream transcription factors NF-kappaB and AP-1 (which regulate inflammatory responses via cytokine production) in acinar cells. In PSCs, additional signalling molecules identified as important to the process of ethanol and acetaldehyde-induced PSC activation include protein kinase C (PKC), phosphatidylinositol-3-kinase (PI3K) and peroxisome proliferator-activated receptor gamma (PPARgamma). Interestingly, cross-talk has been demonstrated between PI3K and MAPK in acetaldehyde-treated PSCs. The above advances in the identification of relevant signalling molecules may enable therapeutic targeting of these pathways so as to prevent/reduce alcohol-induced acute as well as chronic injury of the pancreas.

Determination of 5-chloro-7-iodo-8-quinolinol (clioquinol) in plasma and tissues of hamsters by high-performance liquid chromatography and electrochemical detection.

This paper describes a method of determining clioquinol levels in hamster plasma and tissue by means of HPLC and electrochemical detection. Clioquinol was separated on a Nucleosil C18 300 mm x 3.9 mm i.d. 7 microm column at 1 ml/min using a phosphate/citrate buffer 0.1M (400 ml) with 600 ml of a methanol:acetonitrile (1:1, v/v) mobile phase. The retention times of clioquinol and the IS were, respectively, 11.6 and 8.1 min; the quantitation limit (CV>8%) was 5 ng/ml in plasma and 10 ng/ml in tissues. The intra- and inter-assay accuracies of the method were more than 95%, with coefficients of variation between 3.0 and 7.7%, and plasma and tissue recovery rates of 72-77%. There was a linear response to clioquinol 5-2000 ng/ml in plasma, and 10-1000 ng/g in tissues. The method is highly sensitive and selective, makes it possible to study the pharmacokinetics of plasma clioquinol after oral administration and the distribution of clioquinol in tissues, and could be used to monitor plasma clioquinol levels in humans.

Vitamin A inhibits pancreatic stellate cell activation: implications for treatment of pancreatic fibrosis.

BACKGROUND AND AIMS: Activated pancreatic stellate cells (PSCs) are implicated in the production of alcohol induced pancreatic fibrosis. PSC activation is invariably associated with loss of cytoplasmic vitamin A (retinol) stores. Furthermore, retinol and ethanol are known to be metabolised by similar pathways. Our group and others have demonstrated that ethanol induced PSC activation is mediated by the mitogen activated protein kinase (MAPK) pathway but the specific role of retinol and its metabolites all-trans retinoic acid (ATRA) and 9-cis retinoic acid (9-RA) in PSC quiescence/activation, or its influence on ethanol induced PSC activation is not known. Therefore, the aims of this study were to (i) examine the effects of retinol, ATRA, and 9-RA on PSC activation; (ii) determine whether retinol, ATRA, and 9-RA influence MAPK signalling in PSCs; and (iii) assess the effect of retinol supplementation on PSCs activated by ethanol. METHODS: Cultured rat PSCs were incubated with retinol, ATRA, or 9-RA for varying time periods and assessed for: (i) proliferation; (ii) expression of alpha smooth muscle actin (alpha-SMA), collagen I, fibronectin, and laminin; and (iii) activation of MAPKs (extracellular regulated kinases 1 and 2, p38 kinase, and c-Jun N terminal kinase). The effect of retinol on PSCs treated with ethanol was also examined by incubating cells with ethanol in the presence or absence of retinol for five days, followed by assessment of alpha-SMA, collagen I, fibronectin, and laminin expression. RESULTS: Retinol, ATRA, and 9-RA significantly inhibited: (i) cell proliferation, (ii) expression of alpha-SMA, collagen I, fibronectin, and laminin, and (iii) activation of all three classes of MAPKs. Furthermore, retinol prevented ethanol induced PSC activation, as indicated by inhibition of the ethanol induced increase in alpha-SMA, collagen I, fibronectin, and laminin expression. CONCLUSIONS: Retinol and its metabolites ATRA and 9-RA induce quiescence in culture activated PSCs associated with a significant decrease in the activation of all three classes of MAPKs in PSCs. Ethanol induced PSC activation is prevented by retinol supplementation.

Molecular mechanisms of alcoholic pancreatitis.

Alcoholic pancreatitis is a major complication of alcohol abuse. Since only a minority of alcoholics develop pancreatitis, there has been a keen interest in identifying the factors that may confer individual susceptibility to the disease. Numerous possibilities have been evaluated including diet, drinking patterns and a range of inherited factors. However, at the present time, no susceptibility factor has been unequivocally identified. In contrast, considerable progress has been made with respect to the constant effects of alcohol on the pancreas. The molecular mechanisms of alcohol-induced pancreatic injury are being increasingly defined with an emphasis, in recent years, on the acinar cell itself as the principal site on ethanol-related damage. It has now been established that the acinar cell is capable of metabolizing alcohol and that the direct toxic effects of alcohol and/or its metabolites on acinar cells may predispose the gland to autodigestive injury in the presence of an appropriate triggering factor. A significant recent development relates to the characterization of pancreatic stellate cells, increasingly implicated in alcoholic pancreatic fibrosis. Here the current concepts regarding the mechanisms/pathways mediating alcohol-induced pancreatic injury are outlined.

Desmoplastic reaction in pancreatic cancer: role of pancreatic stellate cells.

OBJECTIVES: Pancreatic cancer has a very poor prognosis, largely due to its propensity for early local and distant spread. Histopathologically, most pancreatic cancers are characterized by a prominent stromal/fibrous reaction in and around tumor tissue. The aims of this study were to determine whether (1) the cells responsible for the formation of the stromal reaction in human pancreatic cancers are activated pancreatic stellate cells (PSCs) and (2) an interaction exists between pancreatic cancer cells and PSCs that may facilitate local and distant invasion of tumor. METHODS: Serial sections of human pancreatic cancer tissue were stained for desmin and glial fibrillary acidic protein (stellate cell selective markers) and alpha-smooth muscle actin (alphaSMA), a marker of activated PSC activation, by immunohistochemistry, and for collagen using Sirius Red. Correlation between the extent of positive staining for collagen and alphaSMA was assessed by morphometry. The cellular source of collagen in stromal areas was identified using dual staining methodology, ie, immunostaining for alphaSMA and in situ hybridization for procollagen alpha1I mRNA. The possible interaction between pancreatic cancer cells and PSCs was assessed in vitro by exposing cultured rat PSCs to control medium or conditioned medium from 2 pancreatic cancer cell lines (PANC-1 and MiaPaCa-2) for 24 hours. PSC activation was assessed by cell proliferation and alphaSMA expression. RESULTS: Stromal areas of human pancreatic cancer stained strongly positive for the stellate cell selective markers desmin and GFAP (indicating the presence of PSCs), for alphaSMA (suggesting that the PSCs were in their activated state) and for collagen. Morphometric analysis demonstrated a close correlation (r = 0.77; P < 0.04; 8 paired sections) between the extent of PSC activation and collagen deposition. Procollagen mRNA expression was localized to alphaSMA-positive cells in stromal areas indicating that activated PSCs were the predominant source of collagen in stromal areas. Exposure of PSCs to pancreatic cancer cell secretions in vitro resulted in PSC activation as indicated by significantly increased cell proliferation and alphaSMA expression. CONCLUSIONS: Activated PSCs are present in the stromal reaction in pancreatic cancers and are responsible for the production of stromal collagen. PSC function is influenced by pancreatic cancer cells. Interactions between tumor cells and stromal cells (PSCs) may play an important role in the pathobiology of pancreatic cancer.

Pharmacokinetics and distribution of sodium 3,4-diaminonaphthalene-1-sulfonate, a Congo Red derivative active in inhibiting PrP(res) replication.

Sodium 3,4-diaminonaphthalene-1-sulfonate (CRA) is a compound, synthesised by our group from Congo Red (CR), that is active in preventing the pathological conversion of normal prion protein (PrP). As the precise mechanisms controlling the ways in which prions are distributed and infect the brain and other organs are not fully understood, studying the pharmacokinetics of drugs that are active against prions may clarify their targets and their means of inhibiting prion infection. This paper describes the pharmacokinetics of CRA in plasma, spleen and brain after single or repeated intraperitoneal or subcutaneous administration, as determined by means of specific and sensitive fluorimetric HPLC. A single intraperitoneal administration led to peak plasma CRA concentrations after 15 min, followed by biphasic decay with an apparent half-life of 4.3 h. After subcutaneous administration, T(max) was reached after 30 min, and was followed by a similar process of decay: Cmax and the AUC0-last were 25% those recorded after intraperitoneal administration. The mean peak concentrations and AUCs of CRA after a single intraperitoneal or subcutaneous administration in peripheral tissue (spleen) were similar to those observed in blood, whereas brain concentrations were about 2% those in plasma. After repeated intraperitoneal or subcutaneous doses, the Cmax values in plasma, brain and spleen were similar to those observed at the same times after a single dose. After repeated intraperitoneal doses, CRA was also found in the ventricular cerebrospinal fluid at concentrations of 1.8 +/- 0.2 microg(-1) mL, which is similar to, or slightly higher than, those found in brain. Brain concentrations may be sufficient to explain the activity of CRA on PrP reproduction in the CNS. However, peripheral involvement cannot be excluded because the effects of CRA are more pronounced after intraperitoneal than after intracerebral infection.

Pancreatic stellate cell activation by ethanol and acetaldehyde: is it mediated by the mitogen-activated protein kinase signaling pathway?

BACKGROUND: Pancreatic fibrosis is a characteristic feature of alcoholic chronic pancreatitis. Recent studies suggest that activated pancreatic stellate cells (PSCs) are the major cell-type involved in pancreatic fibrogenesis. Cultured PSCs become activated when exposed to ethanol or its metabolite acetaldehyde (as indicated by increased alpha-smooth muscle actin [alpha-SMA] expression and increased collagen synthesis). However the intracellular signaling mechanisms responsible for ethanol- or acetaldehyde-induced PSC activation remain to be fully elucidated. One of the major signaling pathways known to regulate protein synthesis in mammalian cells is the mitogen-activated protein kinase (MARK) pathway. AIMS: To examine the effects of ethanol and acetaldehyde on the MAPK pathway (by assessing the activities of the 3 major subfamilies (extracellular-regulated kinases 1 and 2 [ERK 1/2], JNK and p38 kinase) in PSCs and to examine the role of p38 kinase in mediating the ethanol- and acetaldehyde-induced increase in alpha-SMA expression in activated rat PSCs. METHODS: Rat PSCs were incubated with ethanol (50 mM) or acetaldehyde (200 microM) for 15 min, 30 min, 60 min, and 24 h; and activities of ERK 1/2, JNK, and p38 kinase were assessed in cell lysates using kinase assays and Western blot. In addition, rat PSCs were treated with the specific p38 MAPK inhibitor SB203580 in the presence or absence of ethanol or acetaldehyde for 24h, and activation of the downstream protein kinase MAPKAP kinase-2 (an indicator of p38 MAPK activity) was assessed by Western blot. Specific inhibitors were also used to inhibit the activity of ERK 1/2 and JNK. Following inhibition of the above signaling pathways, alpha-SMA expression by PSCs was assessed by Western blot. RESULTS: Ethanol and acetaldehyde increased the activation of all 3 subfamilies (ERK 1/2, JNK and p38 kinase) of the MAPK pathway in PSCs. Treatment of PSCs with SB203580 abolished the ethanol- and acetaldehyde-induced increase in p38 MAPK activity and also prevented the induction of alpha-SMA expression in PSCs. However, inhibition of ERK 1/2 and JNK had no effect on ethanoland acetaldehyde-induced alpha-SMA expression in PSCs. CONCLUSIONS: (1) The MAP kinase pathway is induced in PSCs after exposure to ethanol or acetaldehyde and this induction is sustained for at least 24h. (2) The p38 MAPK pathway mediates the activation (as indicated by increased alpha-SMA expression) of PSCs by ethanol or acetaldehyde.

Cell migration: a novel aspect of pancreatic stellate cell biology.

BACKGROUND: Pancreatic stellate cells (PSCs), implicated as key mediators of pancreatic fibrogenesis, are found in increased numbers in areas of pancreatic injury. This increase in PSC number may be due to increased local proliferation and/or migration of these cells from adjacent areas. The ability of PSCs to proliferate has been well established but their potential for migration has not been examined. AIMS: Therefore, the aims of this study were to determine whether cultured rat PSCs have the capacity to migrate and, if so, to characterise this migratory capacity with respect to the influence of basement membrane components and the effect of platelet derived growth factor (PDGF, a known stimulant for migration of other cell types). METHODS: Migration of freshly isolated (quiescent) and culture activated (passaged) rat PSCs was assessed across uncoated or Matrigel (a basement membrane-like substance) coated porous membranes (pore size 8 micro m) in the presence or absence of PDGF (10 and 20 ng/ml) in the culture medium. A checkerboard assay was performed to assess whether the effect of PDGF on PSC migration was chemotactic or chemokinetic. RESULTS: Cell migration was observed with both freshly isolated and passaged PSCs. However, compared with passaged (culture activated) cells, migration of freshly isolated cells was delayed, occurring only at or after 48 hours of incubation when the cells displayed an activated phenotype. PSC migration through Matrigel coated membranes was delayed but not prevented by basement membrane components. PSC migration was increased by PDGF and this effect was predominantly chemotactic (that is, in the direction of a positive concentration gradient). CONCLUSIONS: (i) PSCs have the capacity to migrate. (ii) Activation of PSCs appears to be a prerequisite for migration. (iii) PDGF stimulates PSC migration and this effect is predominantly chemotactic. IMPLICATION: Chemotactic factors released during pancreatic injury may stimulate the migration of PSCs through surrounding basement membrane towards affected areas of the gland.

Rat pancreatic stellate cells secrete matrix metalloproteinases: implications for extracellular matrix turnover.

BACKGROUND: Pancreatic fibrosis is a characteristic feature of chronic pancreatic injury and is thought to result from a change in the balance between synthesis and degradation of extracellular matrix (ECM) proteins. Recent studies suggest that activated pancreatic stellate cells (PSCs) play a central role in pancreatic fibrogenesis via increased synthesis of ECM proteins. However, the role of these cells in ECM protein degradation has not been fully elucidated. AIMS: To determine: (i) whether PSCs secrete matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) and, if so (ii) whether MMP and TIMP secretion by PSCs is altered in response to known PSC activating factors such as tumour necrosis factor alpha (TNF-alpha), transforming growth factor beta1 (TGF-beta1), interleukin 6 (IL-6), ethanol, and acetaldehyde. METHODS: Cultured rat PSCs (n=3-5 separate cell preparations) were incubated at 37 degrees C for 24 hours with serum free culture medium containing TNF-alpha (5-25 U/ml), TGF-beta1 (0.5-1 ng/ml), IL-6 (0.001-10 ng/ml), ethanol (10-50 mM), or acetaldehyde (150-200 micro M), or no additions (controls). Medium from control cells was examined for the presence of MMPs by zymography using a 10% polyacrylamide-0.1% gelatin gel. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to examine gene expression of MMP9 and the tissue inhibitors of metalloproteinases TIMP1 and TIMP2. Western blotting was used to identify a specific MMP, MMP2 (a gelatinase that digests basement membrane collagen and the dominant MMP observed on zymography) and a specific TIMP, TIMP2. Reverse zymography was used to examine functional TIMPs in PSC secretions. The effect of TNF-alpha, TGF-beta1, and IL-6 on MMP2 secretion was assessed by densitometry of western blots. The effect of ethanol and acetaldehyde on MMP2 and TIMP2 secretion was also assessed by this method. RESULTS: Zymography revealed that PSCs secrete a number of MMPs including proteinases with molecular weights consistent with MMP2, MMP9, and MMP13. RT-PCR demonstrated the presence of mRNA for metalloproteinase inhibitors TIMP1 and TIMP2 in PSCs while reverse zymography revealed the presence of functional TIMP2 in PSC secretions. MMP2 secretion by PSCs was significantly increased by TGF-beta1 and IL-6, but was not affected by TNF-alpha. Ethanol and acetaldehyde induced secretion of both MMP2 and TIMP2 by PSCs. CONCLUSIONS: Pancreatic stellate cells have the capacity to synthesise a number of matrix metalloproteinases, including MMP2, MMP9, and MMP13 and their inhibitors TIMP1 and TIMP2. MMP2 secretion by PSCs is significantly increased on exposure to the proinflammatory cytokines TGF-beta1 and IL-6. Both ethanol and its metabolite acetaldehyde increase MMP2 as well as TIMP2 secretion by PSCs. IMPLICATION: The role of pancreatic stellate cells in extracellular matrix formation and fibrogenesis may be related to their capacity to regulate the degradation as well as the synthesis of extracellular matrix proteins.

Simultaneous determination of clomipramine and its desmethyl and hydroxy metabolites in plasma of patients by high-performance liquid chromatography after solid-phase extraction.

Clomipramine (CMI) is a typical tricyclic antidepressant with a wide clinical spectrum, being used in major depressive, panic and obsessive-compulsive disorders. The relationship between clinical response and plasma levels of clomipramine and its N-desmethylated (N-desmethylclomipramine, DMCMI) and hydroxy-metabolites remains unclear. In particular, limited information is available on the correlation with clinical response in patients with obsessive-compulsive disorder (OCD). This study describes a new sensitive method to simultaneously determine CMI and its major N-desmethylated and hydroxy-metabolites present in human plasma by HPLC with a UV detector. After a solid-phase extraction from plasma (Isolute C2 columns) the separation of the compounds was performed on a Lichrospher CN column (250 x 4 mm, 5 microm with a 2-cm pre-column) by an eluent consisting of 10 mM K(2)HPO(4)-acetonitrile-methanol (35:25:40 v/v/v) at a flow of 1.5 ml/min. UV detector was set at 214 nm. The lower limit of quantification for all the analytes was at least 5 ng/ml. The coefficients of variation ranged between 2.0 and 4.9% with recovery rates between 97.0 and 100.3%. Linear regression analyses showed correlation coefficients between 0.98 and 0.99. This method is simple, fast and reliable with good specificity and sensitivity. Solid phase extraction is efficient and rapid, allowing the extraction of several plasma samples on the same day and may therefore be usefully and realistically applied in the clinical context. We thus investigated the relevance of plasma levels of CMI and its metabolites as a predictor of clinical outcome in a group of 15 patients with OCD.

Determination of sodium 3,4-diaminonaphthalene-1-sulfonate, a Congo Red derivative, in plasma and brain of hamsters by high-performance liquid chromatography after solid-phase extraction and ultraviolet absorbance.

In the search for compounds with similar or greater activity than Congo Red (CR) in protecting normal prion protein from being converted into the pathological form, we have synthesized various compounds which derive from CR. One of these is the sodium 3,4-diaminonaphthalene-1-sulfonate (RCA) which has an activity similar to CR in preliminary experiments. This study describes a method to determine RCA in plasma and in brain tissue by high-performance liquid chromatography (HPLC), using a solid-phase extraction and UV detection. RCA is an amphoteric molecule difficult to separate from biological matrices. Extraction was achieved by solid-phase extraction (ENV+ columns) together with the use of a counter ion. The resulting solid-phase extraction is efficient and rapid. RCA was separated on a Symmetry C18 250 x 4.6 mm I.D. 5 lm column at 1 ml/min using a 50 mM NaSO4 in 5 mM tetra-n-butylammoniumiodide (TEBA) in water-methanol (82:18, v/v) mobile phase. Retention times of RCA and I.S. were 21 and 24 min. The UV detector was set at 210 nm. The limit of quantitation was 0.5 microg/ml. The method has intra-assay and inter-assay accuracies higher than 95%, coefficients of variation ranging between 2.8 and 8.6%, and recovery rates between 74.3 and 80.1% in plasma and in brain tissue. A linear response to quantities of RCA from 0.5 to 100 microg/ml or 10 microg/g in plasma or brain was obtained. The present method allows the study of the pharmacokinetics of RCA in plasma after i.p. administration, and the distribution of the compound into the brain at the peak time.

Pancreatic stellate cells respond to inflammatory cytokines: potential role in chronic pancreatitis.

BACKGROUND: It is now generally accepted that chronic pancreatic injury and fibrosis may result from repeated episodes of acute pancreatic necroinflammation (the necrosis-fibrosis sequence). Recent studies suggest that pancreatic stellate cells (PSCs), when activated, may play an important role in the development of pancreatic fibrosis. Factors that may influence PSC activation during pancreatic necroinflammation include cytokines known to be important in the pathogenesis of acute pancreatitis, such as tumour necrosis factor alpha (TNF-alpha), and the interleukins 1, 6, and 10 (IL-1, IL-6, and IL-10). AIM: To determine the effects of these cytokines on PSC activation, as assessed by cell proliferation, alpha smooth muscle actin (alpha-SMA) expression, and collagen synthesis. METHODS: Cultured rat PSCs were incubated with cytokines for 24 hours. Cell proliferation was assessed by measuring (3)H thymidine incorporation into cellular DNA, alpha-SMA expression by western blotting, and collagen synthesis by incorporation of (14)C proline into collagenase sensitive protein. mRNA levels for procollagen alpha(1)(1) in PSCs were determined by northern and dot blotting methods. RESULTS: Expression of alpha-SMA by PSCs was increased on exposure to each of the cytokines used in the study. Stellate cell proliferation was stimulated by TNF-alpha but inhibited by IL-6, while IL-1 and IL-10 had no effect on PSC proliferation. Collagen synthesis by PSCs was stimulated by TNF-alpha and IL-10, inhibited in response to IL-6, and unaltered by IL-1. Changes in collagen protein synthesis in response to TNF-alpha, IL-10, and IL-6 were not regulated at the mRNA level in the cells. CONCLUSION: This study has demonstrated that PSCs have the capacity to respond to cytokines known to be upregulated during acute pancreatitis. Persistent activation of PSCs by cytokines during acute pancreatitis may be a factor involved in the progression from acute pancreatitis to chronic pancreatic injury and fibrosis.

Alcohol and the pancreas.

This article represents the proceedings of a workshop at the 2000 ISBRA Meeting in Yokohama, Japan. The presentations were (1) Phenotypic alteration of myofibroblast during ethanol-induced pancreatic injury: its relation to bFGF, by Masahiko Nakamura, Kanji Tsuchimoto, and Hiromasa Ishii; (2) Activation of pancreatic stellate cells in pancreatic fibrosis, by Paul S. Haber, Gregory W. Keogh, Minoti V. Apte, Corey S. Moran, Nancy L. Stewart, Darrell H.G. Crawford, Romano C. Pirola, Geoffrey W. McCaughan, Grant A. Ramm, and Jeremy S. Wilson; (3) Pancreatic blood flow and pancreatic enzyme secretion on acute ethanol infusion in anesthetized RAT, by H. Nishino, M. Kohno, R. Aizawa, and N. Tajima; (4) Genotype difference of alcohol-metabolizing enzymes in relation to chronic alcoholic pancreatitis between the alcoholic in the National Institute on Alcoholism and patients in other general hospitals in Japan, by K. Maruyama, H. Takahashi, S. Matsushita, K. Okuyama, A. Yokoyama, Y. Nakamura, K. Shirakura, and H. Ishii; and (5) Alcohol consumption and incidence of type 2 diabetes, by Katherine M. Conigrave, B. Frank Hu, Carlos A. Camargo Jr, Meir J. Stampfer, Walter C. Willett, and Eric B. Rimm.

Does alcohol directly stimulate pancreatic fibrogenesis? Studies with rat pancreatic stellate cells.

BACKGROUND & AIMS: Activated pancreatic stellate cells have recently been implicated in pancreatic fibrogenesis. This study examined the role of pancreatic stellate cells in alcoholic pancreatic fibrosis by determining whether these cells are activated by ethanol itself and, if so, whether such activation is caused by the metabolism of ethanol to acetaldehyde and/or the generation of oxidant stress within the cells. METHODS: Cultured rat pancreatic stellate cells were incubated with ethanol or acetaldehyde. Activation was assessed by cell proliferation, alpha-smooth muscle actin expression, and collagen synthesis. Alcohol dehydrogenase (ADH) activity in stellate cells and the influence of the ADH inhibitor 4-methylpyrazole (4MP) on the response of these cells to ethanol was assessed. Malondialdehyde levels were determined as an indicator of lipid peroxidation. The effect of the antioxidant vitamin E on the response of stellate cells to ethanol or acetaldehyde was also examined. RESULTS: Exposure to ethanol or acetaldehyde led to cell activation and intracellular lipid peroxidation. These changes were prevented by the antioxidant vitamin E. Stellate cells exhibited ethanol-inducible ADH activity. Inhibition of ADH by 4MP prevented ethanol-induced cell activation. CONCLUSIONS: Pancreatic stellate cells are activated on exposure to ethanol. This effect of ethanol is most likely mediated by its metabolism (via ADH) to acetaldehyde and the generation of oxidant stress within the cells.

Activation of pancreatic stellate cells in human and experimental pancreatic fibrosis.

The mechanisms of pancreatic fibrosis are poorly understood. In the liver, stellate cells play an important role in fibrogenesis. Similar cells have recently been isolated from the pancreas and are termed pancreatic stellate cells. The aim of this study was to determine whether pancreatic stellate cell activation occurs during experimental and human pancreatic fibrosis. Pancreatic fibrosis was induced in rats (n = 24) by infusion of trinitrobenzene sulfonic acid (TNBS) into the pancreatic duct. Surgical specimens were obtained from patients with chronic pancreatitis (n = 6). Pancreatic fibrosis was assessed using the Sirius Red stain and immunohistochemistry for collagen type I. Pancreatic stellate cell activation was assessed by staining for alpha-smooth muscle actin (alphaSMA), desmin, and platelet-derived growth factor receptor type beta (PDGFRbeta). The relationship of fibrosis to stellate cell activation was studied by staining of serial sections for alphaSMA, desmin, PDGFRbeta, and collagen, and by dual-staining for alphaSMA plus either Sirius Red or in situ hybridization for procollagen alpha(1) (I) mRNA. The cellular source of TGFbeta was examined by immunohistochemistry. The histological appearances in the TNBS model resembled those found in human chronic pancreatitis. Areas of pancreatic fibrosis stained positively for Sirius Red and collagen type I. Sirius Red staining was associated with alphaSMA-positive cells. alphaSMA staining colocalized with procollagen alpha(1) (I) mRNA expression. In the rat model, desmin staining was associated with PDGFRbeta in areas of fibrosis. TGFbeta was maximal in acinar cells adjacent to areas of fibrosis and spindle cells within fibrotic bands. Pancreatic stellate cell activation is associated with fibrosis in both human pancreas and in an animal model. These cells appear to play an important role in pancreatic fibrogenesis.

Alcoholic pancreatitis and polymorphisms of the variable length polythymidine tract in the cystic fibrosis gene.

BACKGROUND: The observation that only a minority of alcoholics develops clinical pancreatic disease has led to a search for a predisposing factor to the disease. One possible predisposing factor is mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene as cystic fibrosis leads to pancreatic injury. We have recently demonstrated that 15 common CFTR mutations are not found in patients with alcoholic pancreatitis. Another common polymorphism of the CFTR gene has recently been implicated in the pathogenesis of idiopathic chronic pancreatitis, the 5T variant of the variable length polythymidine tract in intron 8 (the normal genotypes are 7T and 9T). The 5T variant inhibits transcription of exon 9 resulting in a CFTR protein lacking chloride channel activity. The aim of this study was to determine whether the 5T variant is associated with alcoholic pancreatitis. METHODS: Fifty-two patients with alcoholic pancreatitis were identified using standardized diagnostic criteria. Fifty alcoholics without pancreatitis were also studied as controls. Genomic DNA was extracted from peripheral blood leukocytes and the polythymidine tract of intron 8 was amplified by nested polymerase chain reaction using established primers. The polymerase chain reaction products were digested with MseI, separated by electrophoresis on 15% polyacrylamide gels and genotypes assigned by comparison with known positive controls. RESULTS: The 5T allele we found in only two patients with alcoholic pancreatitis (3.9% of th index group; 95% confidence intervals 0-10%) and in seven alco holic controls. Allele frequencies for 5T, 7T, and 9T in patients with alcoholic pancreatitis were 1.9%, 85.6%, and 12.5%, respectively These did not differ from the allele frequencies in alcoholic controls (7%, 79%, and 14% for 5T, 7T, and 9T, respectively). CONCLUSION: The 5T allele was not associated with alcoholic pancreatitis. Individual susceptibility to this disease remains unexplained.