A rat model reproducing key pathological responses of alcoholic chronic pancreatitis.

Although alcohol abuse is the major cause of chronic pancreatitis, the pathogenesis of alcoholic chronic pancreatitis (ACP) remains obscure. A critical obstacle to understanding the mechanism of ACP is lack of animal models. Our objective was to develop one such model. Rats were pair-fed for 8 wk ethanol or control Lieber-DeCarli liquid diet. For the last 2 wk, they received cyclosporin A (CsA; 20 mg/kg once daily) or vehicle. After 1 wk on CsA, one episode of acute pancreatitis was induced by four 20 microg/kg injections of cerulein (Cer); controls received saline. Pancreas was analyzed 1 wk after the acute pancreatitis. CsA or Cer treatments alone did not result in pancreatic injury in either control (C)- or ethanol (E)-fed rats. We found, however, that alcohol dramatically aggravated pathological effect of the combined CsA+Cer treatment on pancreas, resulting in massive loss of acinar cells, persistent inflammatory infiltration, and fibrosis. Macrophages were prominent in the inflammatory infiltrate. Compared with control-fed C+CsA+Cer rats, their ethanol-fed E+CsA+Cer counterparts showed marked increases in pancreatic NF-kappaB activation and cytokine/chemokine mRNA expression, collagen and fibronectin, the expression and activities of matrix metalloproteinase-2 and -9, and activation of pancreatic stellate cells. Thus we have developed a model of alcohol-mediated postacute pancreatitis that reproduces three key responses of human ACP: loss of parenchyma, sustained inflammation, and fibrosis. The results indicate that alcohol impairs recovery from acute pancreatitis, suggesting a mechanism by which alcohol sensitizes pancreas to chronic injury.

Related papers:

title: Alcohol-induced pancreatic injury.
abstract: Alcoholic pancreatitis is a major complication of alcohol abuse. Until recently, it was generally accepted that alcoholic pancreatitis was a chronic disease from the outset. However, evidence is now emerging in support of the 'necrosis-fibrosis' hypothesis that alcoholic pancreatitis begins as an acute process and that repeated episodes of acute injury lead to the changes of chronic pancreatitis (acinar atrophy and fibrosis) resulting in exocrine and endocrine dysfunction. The treatment of acute pancreatitis follows the regimen of bed rest, nasogastric suction, analgesia and intravenous support. The role of additional therapeutic measures such as prophylactic antibiotics, antioxidants and enteral nutrition in severe cases has not yet been precisely defined. The treatment of chronic pancreatitis involves attention to its three cardinal features: pain, maldigestion and diabetes. With respect to the pathogenesis of alcoholic pancreatitis, the focus of research over the past 30 years has shifted from the sphincter of Oddi and ductular abnormalities to the acinar cell itself. It has now been established that the acinar cell is capable of metabolizing alcohol and that direct toxic effects of alcohol and/or its metabolites on acinar cells may predispose the gland to injury in the presence of an appropriate trigger factor. A significant recent development relates to the characterization of pancreatic stellate cells, increasingly implicated in alcoholic pancreatic fibrosis. This chapter summarizes the natural history, clinical features, current trends in treatment as well as recent advances in our understanding of the pathogenesis of alcoholic pancreatitis.

title: [Alcohol and the pancreas].
abstract: In the last decenniums the importance of alcohol for the pathology of the pancreas has increased in Middle Europe. The inhibition of the pancreas secretion which was to be observed at an occasional alcohol intake is when a regular ethanol supply is present changed by a hyperfunction with secretion of a pancreatic juice rich in protein with high enzyme and calcium content. This predisposes to the protein precipitation in the small and medium ducts with resulting intrapancreatic obstruction, which on its part gives the impact to the changes of chronic pancreatitis. In 80-90% of the cases the ethanol-induced lesion of the pancreas is manifest under the picture of an acute pancreatitis. Nevertheless, in most cases the first shift of a chronic relapsing pancreatitis is concerned, the peculiarity of which is the frequent occurrence of calcifications. Clinical picture, complications and sequelae are described.

title: Potentiation of ethanol-induced pancreatic injury by dietary fat. Induction of chronic pancreatitis by alcohol in rats.
abstract: Effects of sustained ethanol intoxication and dietary fat content on pancreatic morphology were investigated in the rat model implanted with gastrostomy catheters, which permitted continuous intragastric infusion of ethanol plus liquid diet containing one of three levels of corn oil: 5% (low-fat), 25% (high-fat), and 35% (extra-high-fat) of total calories. After various durations of infusion ranging from 30 to 160 days, the pancreatic histology was examined. Mean blood alcohol levels achieved in the low, high, and extra-high fat diet groups were similarly high: 210 +/- 120, 224 +/- 122, and 289 +/- 110 mg/dl. The average weight gain of these ethanol-fed groups during the first 8 weeks of experiments was 15.4 +/- 1.9, 19.6 +/- 8.0, and 14.9 +/- 5.2 g/wk, respectively, and was not statistically different from that of pair-fed controls infused with isocaloric amount of dextrose and respective diet, nor from that of age-matched animals given the regular chow. None of control animals showed abnormal pancreatic morphologic features except occasional mild steatosis in those fed the extra-high-fat diet. With the low dietary intake of unsaturated fat, chronic ethanol intoxication produced only mild pancreatic pathology such as steatosis and interstitial edema. Administration of ethanol and the high-fat and extra-high-fat diets caused hypogranulation and apoptosis of acinar cells. Focal lesions of chronic pancreatitis were also observed in 20% or 30% of ethanol-fed animals given the high-fat or extra-high-fat diet. These lesions were characterized by fat necrosis, mononuclear cell infiltration, fibrosis, acinar atrophy, ductal dilatation, and intraductal mucious or proteinacious plugs. The incidence of focal acute pancreatitis was less (7-20%) but appeared increased with higher dietary fat content. Induction of either acute or chronic pancreatitis was not correlated with plasma levels of triglycerides or cholesterol. These results demonstrate potentiation by dietary unsaturated fat of ethanol-induced pancreatic injury. This model possesses many features analogous to those seen in alcoholic pancreatic injury in man. The hyperlipidemia does not appear to be an important pathogenetic factor for ethanol-induced pancreatitis produced in this model.

title: Development of an animal model of chronic alcohol-induced pancreatitis in the rat.
abstract: This study was designed to develop an animal model of alcoholic pancreatitis and to test the hypothesis that the dose of ethanol and the type of dietary fat affect free radical formation and pancreatic pathology. Female Wistar rats were fed liquid diets rich in corn oil (unsaturated fat), with or without a standard or high dose of ethanol, and medium-chain triglycerides (saturated fat) with a high dose of ethanol for 8 wk enterally. The dose of ethanol was increased as tolerance developed, which allowed approximately twice as much alcohol to be delivered in the high-dose group. Serum pancreatic enzymes and histology were normal after 4 wk of diets rich in unsaturated fat, with or without the standard dose of ethanol. In contrast, enzyme levels were elevated significantly by the high ethanol dose. Increases were blunted significantly by dietary saturated fat. Fibrosis and collagen alpha1(I) expression in the pancreas were not detectable after 4 wk of enteral ethanol feeding; however, they were enhanced significantly by the high dose after 8 wk. Furthermore, radical adducts detected by electron spin resonance were minimal with the standard dose; however, the high dose increased carbon-centered radical adducts as well as 4-hydroxynonenal, an index of lipid peroxidation, significantly. Radical adducts were also blunted by approximately 70% by dietary saturated fat. The animal model presented here is the first to demonstrate chronic alcohol-induced pancreatitis in a reproducible manner. The key factors responsible for pathology are the amount of ethanol administered and the type of dietary fat.

title: Animal Models in Alcoholic Pancreatitis – What Can We Learn?
abstract: Although the majority of patients with chronic pancreatitis present a history of excessive alcohol consumption, the pathophysiology underlying chronic alcoholic pancreatitis remains poorly defined. Since experimental animal models represent helpful tools in understanding human disease, numerous laboratory studies have been designed to study the effects of alcohol on the pancreas. In the present article we summarize the existing animal models that have been used to investigate the effects of acute and chronic alcohol application on the development of morphological alterations and pancreatic injury. Despite considerable experimental effort, acute or chronic ethanol feeding alone failed to cause acute or chronic pancreatitis in animals. However, ethanol-feeding and the combination with other procedures has demonstrated several mechanisms that play a role in ethanol-induced pancreatic injury. Among these ethanol-induced alterations and mechanisms are the reduction of pancreatic blood-flow and microcirculation, damaging effects of ethanol metabolites, increased pancreatic acinar cell expression of digestive and lysosomal enzymes, increased glandular enzyme content, additional nutritional factors, pancreatic duct obstruction, and limitations of pancreatic regeneration. Although no satisfactory animal model for alcoholic pancreatitis has been developed, these animal models have provided insights in several factors that predispose the pancreas to development of pancreatic injury and contribute to alcoholic pancreatitis.

title: Theories, mechanisms, and models of alcoholic chronic pancreatitis.
abstract: Alcoholic chronic pancreatitis is a severe, disabling, chronic inflammatory condition of the pancreas that is seen in fewer than 5% of alcoholics. The severity and unpredictability of this condition has lead to several theories on the mechanism causing chronic pancreatitis based on careful clinical observation. Hypothetical mechanisms were applied to various animal models. Finally, following multiple lines of evidence, there is a convergence of thought and development of some new models that are quite instructive. Taken together, chronic alcohol consumption by rats results in multiple effects on the pancreas that increase the risk of acute pancreatitis, including ongoing acinar cell injury that lowers the threshold for hyperstimulation-induced acute pancreatitis, neurohormonal injury, and adaptation that results in acinar cell hyperstimulation, increased susceptibility to viral mediated acute pancreatitis, and possibly other factors. After acute pancreatitis initiates the inflammatory process, the chronic inflammation and fibrosis of alcoholic chronic pancreatitis are driven by diet, the acinar cell stress response to continued alcohol that may be potentiated by toxic alcohol metabolites, hypoxia, hyperstimulation, and partial duct obstruction; plus the effects of proinflammatory immunocytes and cytokines; and by stellate cell-mediated fibrosis driven by anti-inflammatory cytokines, alcohol, and alcohol metabolites. The factors determining which alcoholic will develop alcoholic chronic pancreatitis likely involve genetic factors, dietary factors, and susceptibility to pancreatic injury through several mechanisms ranging from trauma to gallstones to viruses.

title: Hereditary pancreatitis: new insights into acute and chronic pancreatitis
abstract: Hereditary pancreatitis has recently emerged as an important pancreatic disease. Although relatively rare, this genetic disorder has provided major breakthroughs in our understanding of acute and chronic pancreatitis and pancreatic cancer. Furthermore, study of this disorder promises additional insights into the pathophysiology of human pancreatitis, new strategies for developing animal models of disease, and new therapies to treat or prevent acute and chronic pancreatitis. A review of recent findings and mechanisms of disease should benefit scientists who study the pancreas and physicians who care for individuals with pancreatitis. This report will focus on acute and chronic pancreatitis.

The first family with hereditary pancreatitis was described by Comfort and Steinberg in 1952.1 Careful evaluation of four definite and two probably affected family members revealed an autosomal dominant inheritance pattern, onset of symptoms between five and 23 years, but no other “. . .earmarks which distinguish it from non-hereditary chronic relapsing pancreatitis”. These observations remain accurate with the caveat that penetrance, the fraction of patients with the mutation that eventually express the disease, is 80%.2-6 Attacks of acute pancreatitis usually begin in childhood,7 but age of onset can range from infancy to the fifth or sixth decade of life. The acute attacks may vary from mild abdominal discomfort to severe life-threatening episodes with pancreatic necrosis, splenic vein thrombosis,8 pseudocysts,9 and death.3 10 Chronic pancreatitis follows recurrent attacks of acute pancreatitis with all of its common complications—unrelenting pain, parenchymal and ductal calcifications, duct distortion, fibrosis, maldigestion and diabetes mellitus.3-6 10 11 These features make hereditary pancreatitis indistinguishable from other causes of acute and chronic pancreatitis, save the relatively early age of onset, the autosomal dominant inheritance pattern and lack of other identifiable aetiologies.

Many investigators pursued the aetiology of hereditary pancreatitis via access to hereditary pancreatitis …

title: Course of alcoholic chronic pancreatitis: a prospective clinicomorphological long-term study.
abstract: The pathogenesis of alcoholic chronic pancreatitis and its relationship to alcoholic acute pancreatitis are debated. According to our recent clinical long-term study, alcoholic chronic pancreatitis seems to evolve from severe acute pancreatits. The aim of this study was to correlate clinical findings to the pancreatic histopathology at early and advanced stages of the disease.



Morphological changes (pseudocysts, autodigestive necrosis, calcification, and perilobular and intralobular fibrosis) were recorded in 37 surgical and 46 postmortem pancreas specimens of 73 patients from our long-term series, who progressed from clinically acute to chronic pancreatitis (mean follow-up, 12 years). Pancreatic function was monitored at yearly intervals.



Surgical interventions were performed at a mean of 4.1 years from onset. Histologically, focal necrosis (49%) and mild perilobular fibrosis (54%) predominated, Pseudocysts (n = 41, mostly postnecrotic) occurred in 88% within 6 years from onset. Autopsy specimens were obtained at a mean of 12 years. These pancreata often showed severe perilobular and intralobular fibrosis (85%) and calcifications (74%), but rarely necrosis (4%). Fibrosis correlated with progressive pancreatic dysfunction (P < 0.001), particularly in the 10 patients with two histological assessments (mean interval between biopsy and autopsy, 8 years).



The data support an evolution from severe alcoholic acute pancreatitis to chronic pancreatitis.

title: Effects of ethanol and protein deficiency on pancreatic digestive and lysosomal enzymes.
abstract: The pathogenesis of alcoholic pancreatitis is not fully understood. An increase in pancreatic digestive and lysosomal enzyme synthesis because of ethanol consumption could contribute to the development of pancreatic injury in alcoholics. This study aimed, firstly, to determine the effect of ethanol on the content and messenger RNA levels of pancreatic digestive enzymes and on the messenger RNA level of the lysosomal enzyme cathepsin B, and secondly, to examine the influence of concomitant protein deficiency (a known association of alcoholism and pancreatic injury) on these effects. A rat model of chronic ethanol administration was used in which rats were fed in groups of four, and for four weeks, protein sufficient and protein deficient diets with or without ethanol. Ethanol increased the pancreatic content of lipase but did not influence chymotrypsinogen or trypsinogen values. mRNA levels for lipase, trypsinogen, and chymotrypsinogen were raised in rats fed ethanol. Protein deficiency resulted in reduced tissue levels of lipase, chymotrypsinogen, and amylase but did not influence trypsinogen values. mRNA levels for proteases were increased in protein deficient rats, while those for lipase remained unaltered. Both ethanol and protein deficiency increased mRNA levels for cathepsin B. It is concluded that chronic ethanol consumption, in both protein sufficient and protein deficient states, increases the capacity of the pancreatic acinar cell to synthesise digestive and lysosomal enzymes.

title: Ethanol metabolism and transcription factor activation in pancreatic acinar cells in rats.
abstract: Ethanol metabolism by pancreatic acinar cells and the role of its metabolites in ethanol toxicity to the pancreas remain largely unknown. Here, we characterize ethanol metabolism in pancreatic acinar cells and determine the effects of ethanol metabolites on nuclear factor kappa B (NF-kappa B) and activator protein (AP)-1, transcription factors that are activated in pancreatitis and mediate expression of inflammatory molecules critical for this disease.



We measured activities of fatty acid ethyl ester (FAEE) synthase and alcohol dehydrogenase (ADH), as well as accumulation of ethanol metabolites. We measured the effects of ethanol and its metabolites on NF-kappa B and AP-1 activation by using a gel shift assay.



Pancreas metabolizes ethanol via both oxidative and nonoxidative pathways. Acinar cells are the main source of ethanol metabolism in the pancreas. Compared with the liver, FAEE synthase activity in the pancreas is greater, whereas that of ADH is much less. FAEEs activated NF-kappa B and AP-1, whereas acetaldehyde inhibited NF-kappa B activation. Ethanol decreased NF-kappa B binding activity in acinar cells, which was potentiated by cyanamide.



Oxidative and nonoxidative ethanol metabolites regulate transcription factors differently in pancreatic acinar cells. Ethanol may regulate NF-kappa B and AP-1 positively or negatively, depending on which metabolic pathway's effect predominates. These regulatory mechanisms may play a role in ethanol toxicity to the pancreas.

title: Non-Oxidative Metabolism of Ethanol by Rat Pancreatic Acini
abstract: The pathogenesis of alcoholic pancreatitis may involve the metabolism of ethanol (via oxidative and non-oxidative pathways) within the pancreas. The aims of this study were to determine the rate of non-oxidative metabolism in isolated rat pancreatic acini and to compare this to the rate of ethanol oxidation.  Pancreatic acini were isolated from male Sprague-Dawley rats and incubated with 14C-ethanol. Radiolabelled fatty acid ethyl esters (non-oxidative metabolites) were isolated from lipid extracts by thin-layer chromatography. Radiolabelled acetate (oxidative metabolite) was isolated from the incubation medium by ion-exchange chromatography.  Non-oxidative metabolism by isolated pancreatic acini was demonstrated. At 50 and 100 mmol/l ethanol, fatty acid ethyl ester concentrations were 49.6 ± 13.3 and 199 ± 93 µmol/l, respectively. These levels have previously been shown to result in tissue injury. Non-oxidative metabolism was increased 9-fold by addition of oleic acid and inhibited by the lipase inhibitor, tetrahydrolipstatin, by 91.05 ± 1.99%. The rate of oxidative metabolism was 21-fold higher than that of non-oxidative metabolism.  Intact pancreatic cells metabolize ethanol by the non-oxidative pathway, generating fatty acid ethyl esters at a rate sufficient to cause pancreatic damage. Oxidative metabolism of ethanol occurs at a much higher rate and may also play a role in pancreatitis.

title: Chronic ethanol administration causes oxidative stress in the rat pancreas.
abstract: There is increasing evidence implicating oxidative stress in the pathogenesis of both acute and chronic pancreatitis. Because ethanol is a major cause of pancreatitis in Western society, the aim of this study was to determine whether chronic ethanol administration results in oxidative stress in the pancreas. Twelve pairs of rats were fed a diet containing ethanol as 36% of calories or an isocaloric control diet for 4 weeks. Ethanol feeding resulted in a 46% increase in pancreatic malondialdehyde (p=0.006). In addition, total pancreatic glutathione was increased by 22% (p=0.005). These biochemical changes occurred in the absence of histologic evidence of inflammation or necrosis, implying that the observed oxidative stress is a primary phenomenon rather than part of an inflammatory response.

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



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.



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.



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.

title: Identification of mediators stimulating proliferation and matrix synthesis of rat pancreatic stellate cells.
abstract: The aim of this study was to identify fibrogenic mediators stimulating activation, proliferation, and/or matrix synthesis of rat pancreatic stellate cells (PSC). PSC were isolated from the pancreas of normal Wistar rats and from rats with cerulein pancreatitis. Cell activation was demonstrated by immunofluorescence microscopy of smooth muscle alpha-actin (SMA) and real-time quantitative RT-PCR of SMA, fibronectin, and transforming growth factor (TGF)-beta(1). Proliferation was measured by bromodeoxyuridine incorporation. Matrix synthesis was demonstrated on the protein and mRNA level. Within a few days in primary culture, PSC changed their phenotype from fat-storing to SMA-positive myofibroblast-like cells expressing platelet-derived growth factor (PDGF) alpha- and PDGF beta-receptors. TGF-beta(1) and tumor necrosis factor (TNF)-alpha accelerated the change in the cells' phenotype. Addition of 50 ng/ml PDGF and 5 ng/ml basic fibroblast growth factor (bFGF) to cultured PSC significantly stimulated cell proliferation (4.37 +/- 0.49- and 2.96 +/- 0.39-fold of control). Fibronectin synthesis calculated on the basis of DNA was stimulated by 5 ng/ml bFGF (3.44 +/- 1.13-fold), 5 ng/ml TGF-beta(1) (2.46 +/- 0.89-fold), 20 ng/ml PDGF (2.27 +/- 0.68-fold), and 50 ng/ml TGF-alpha (1.87 +/- 0.19-fold). As shown by RT-PCR, PSC express predominantly the splice variant EIII-A of fibronectin. Immunofluorescence microscopy and Northern blot confirmed that in particular bFGF and TGF-beta(1) stimulated the synthesis of fibronectin and collagens type I and III. In conclusion, our data demonstrate that 1) TGF-beta(1) and TNF-alpha accelerate the change in the cell phenotype, 2) PDGF represents the most effective mitogen, and 3) bFGF, TGF-beta(1), PDGF, and, to a lesser extent, TGF-alpha stimulate extracellular matrix synthesis of cultured rat PSC.

title: Experimental animal models of acute pancreatitis.
abstract: Experimental animal models have been employed to study pancreatitis for more than a century. Although not always related to the disease in humans, they have contributed greatly to our current understanding of the pathophysiology and cell biology of this disease. They have also become a standard tool for testing new and promising treatments against pancreatitis. The following article will review the most commonly used experimental models of pancreatitis and some of the lessons we have learned from them.

title: Inflammatory mediators in acute pancreatitis
abstract: Inflammatory mediators play a key role in acute pancreatitis and the resultant multiple organ dysfunction syndrome, which is the primary cause of death in this condition. Recent studies have confirmed the critical role played by inflammatory mediators such as TNF‐α, IL‐1β, IL‐6, IL‐8, PAF, IL‐10, C5a, ICAM‐1, and substance P. The systemic effects of acute pancreatitis have many similarities to those of other conditions such as septicaemia, severe burns, and trauma. The delay between the onset of inflammation in the pancreas and the development of the systemic response makes acute pancreatitis an ideal experimental and clinical model with which to study the role of inflammatory mediators and to test novel therapies. Elucidation of the key mediators involved in the pathogenesis of acute pancreatitis will facilitate the development of clinically effective anti‐inflammatory therapy.

title: NF-kappaB activation in pancreas induces pancreatic and systemic inflammatory response.
abstract: The role of nuclear factor kappaB (NF-kappaB) activation in acute pancreatitis is uncertain. The transcription factor NF-kappaB is activated early in acute pancreatitis, and NF-kappaB is widely considered a key element in inflammatory responses based on its ability to regulate the expression of inflammatory mediators in vitro. However, its role in vivo in specific diseases remains unclear, and the current data on the role of NF-kappaB in acute pancreatitis is primarily correlative.



In this study, NF-kappaB was directly activated within the pancreas using adenoviral-mediated transfer of an active subunit, RelA/p65 (Adp65), delivered by intraductal injection.



Administration of Adp65 led to the infection of a population of acinar cells within the pancreas, the activation of NF-kappaB, the expression of NF-kappaB target genes, and an inflammatory response. Administration of Adp65 increased the infiltration of neutrophils to the pancreas and lung and caused widespread damage to pancreatic acinar cells. In contrast, at the same titer, control adenovirus (AdGFP) had no effect on these parameters. The level of NF-kappaB activation and the severity of inflammation were reduced when an adenovirus bearing the inhibitory subunit IkappaB-alpha was coadministered with Adp65.



Thus, activation of NF-kappaB within the pancreas was sufficient for the initiation of an inflammatory response in this model. These results help define the specific role of NF-kappaB activation in acute pancreatitis.

title: Neutrophils and NADPH oxidase mediate intrapancreatic trypsin activation in murine experimental acute pancreatitis.
abstract: Intrapancreatic activation of digestive enzymes is a key event in the parenchymal cell injury of pancreatitis. We hypothesized that neutrophils recruited to the pancreas during pancreatitis may contribute to such activation.



To cause experimental pancreatitis, rats and mice were treated with high doses of cerulein. Activation of the digestive enzyme, trypsin, was measured in pancreatic homogenates using a fluorogenic assay and localized immunocytochemically with antibody to trypsin-activation peptide (TAP).



Compared with controls, rats depleted of neutrophils with antineutrophil serum exhibited a marked attenuation in intrapancreatic trypsin activation and acinar cell TAP labeling induced by high-dose cerulein. To examine the mechanism, mice deficient in either nicontinamide adenine dinucleotide phosphate (NADPH) oxidase, or myeloperoxidase (MPO) were studied for trypsin activation. Mice deficient in NADPH oxidase exhibited attenuation of the cerulein-induced trypsin activation, but those deficient in MPO did not. Using measurements of Western blot analysis, generation of reactive oxygen species, and immunocytochemistry, we demonstrated the NADPH oxidase activity is in neutrophils and not pancreatic acinar tissue.



The results demonstrate a novel role for neutrophils infiltrating the pancreas in pathologic activation of digestive enzymes in acute pancreatitis and indicate that this effect is mediated by products of NADPH oxidase.

title: Early NF-kappaB activation is associated with hormone-induced pancreatitis.
abstract: Inflammation and cell death are critical to pathogenesis of acute pancreatitis. Here we show that transcription factor nuclear factor-kappaB (NF-kappaB), which regulates these processes, is activated and plays a role in rat cerulein pancreatitis. NF-kappaB was strongly activated in the pancreas within 30 min of cerulein infusion; a second phase of NF-kappaB activation was prominent at 3-6 h. This biphasic kinetics could result from observed transient degradation of the inhibitory protein IkappaBalpha and slower but sustained degradation of IkappaBbeta. The hormone also caused NF-kappaB translocation and IkappaB degradation in vitro in dispersed pancreatic acini. Both p65/p50 and p50/p50, but not c-Rel, NF-kappaB complexes were manifest in pancreatitis and in isolated acini. Coinfusion of CCK JMV-180, which abolishes pancreatitis, prevented cerulein-induced NF-kappaB activation. The second but not early phase of NF-kappaB activation was inhibited by a neutralizing tumor necrosis factor-alpha antibody. Antioxidant N-acetylcysteine (NAC) blocked NF-kappaB activation and significantly improved parameters of pancreatitis. In particular, NAC inhibited intrapancreatic trypsin activation and mRNA expression of cytokines interleukin-6 and KC, which were dramatically induced by cerulein. The results suggest that NF-kappaB activation is an important early event that may contribute to inflammatory and cell death responses in acute pancreatitis.

title: Cholecystokinin induction of mob-1 chemokine expression in pancreatic acinar cells requires NF-κB activation.
abstract: Inflammatory mediators are involved in the early phase of acute pancreatitis, but the cellular mechanisms responsible for their generation within pancreatic cells are unknown. We examined the role of nuclear factor-κB (NF-κB) in cholecystokinin octapeptide (CCK-8)-induced mob-1 chemokine expression in pancreatic acinar cells in vitro. Supraphysiological, but not physiological, concentrations of CCK-8 increased inhibitory κB (IκB-α) degradation, NF-κB activation, and mob-1 gene expression in isolated pancreatic acinar cells. CCK-8-induced IκB-α degradation was maximal within 1 h. Expression of mob-1 was maximal within 2 h. Neither bombesin nor carbachol significantly increased mob-1 mRNA or induced IκB-α degradation. Thus the concentration, time, and secretagogue dependence of mob-1 gene expression and IκB-α degradation were similar. Inhibition of NF-κB with pharmacological agents or by adenovirus-mediated expression of the inhibitory protein IκB-α also inhibited mob-1 gene expression. These data indicate that the NF-κB signaling pathway is required for CCK-8-mediated induction of mob-1 chemokine expression in pancreatic acinar cells. This supports the hypothesis that NF-κB signaling is of central importance in the initiation of acute pancreatitis.

title: Cholecystokinin Induces Caspase Activation and Mitochondrial Dysfunction in Pancreatic Acinar Cells
abstract: Apoptosis and necrosis are critical parameters of pancreatitis, the mechanisms of which remain unknown. Many characteristics of pancreatitis can be studied in vitro in pancreatic acini treated with high doses of cholecystokinin (CCK). We show here that CCK stimulates apoptosis and death signaling pathways in rat pancreatic acinar cells, including caspase activation, cytochromec release, and mitochondrial depolarization. The mitochondrial dysfunction is mediated by upstream caspases (possibly caspase-8) and, in turn, leads to activation of caspase-3. CCK causes mitochondrial alterations through both permeability transition pore-dependent (cytochrome c release) and permeability transition pore-independent (mitochondrial depolarization) mechanisms. Caspase activation and mitochondrial alterations also occur in untreated pancreatic acinar cells; however, the underlying mechanisms are different. In particular, caspases protect untreated acinar cells from mitochondrial damage. We found that caspases not only mediate apoptosis but also regulate other parameters of CCK-induced acinar cell injury that are characteristic of pancreatitis; in particular, caspases negatively regulate necrosis and trypsin activation in acinar cells. The results suggest that the observed signaling pathways regulate parenchymal cell injury and death in CCK-induced pancreatitis. Protection against necrosis and trypsin activation by caspases can explain why the severity of pancreatitis in experimental models correlates inversely with the extent of apoptosis.

title: Cell Death in Pancreatitis
abstract: Mechanisms of cell death in pancreatitis remain unknown. Parenchymal necrosis is a major complication of pancreatitis; also, the severity of experimental pancreatitis correlates directly with necrosis and inversely with apoptosis. Thus, shifting death responses from necrosis to apoptosis may have a therapeutic value. To determine cell death pathways in pancreatitis and the possibility of necrosis/apoptosis switch, we utilized the differences between the rat model of cerulein pancreatitis, with relatively high apoptosis and low necrosis, and the mouse model, with little apoptosis and high necrosis. We found that caspases were greatly activated during cerulein pancreatitis in the rat but not mouse. Endogenous caspase inhibitor X-linked inhibitor of apoptosis protein (XIAP) underwent complete degradation in the rat but remained intact in the mouse model. Furthermore, XIAP inhibition with embelin triggered caspase activation in the mouse model, implicating XIAP in caspase blockade in pancreatitis. Caspase inhibitors decreased apoptosis and markedly stimulated necrosis in the rat model, worsening pancreatitis parameters. Conversely, caspase induction with embelin stimulated apoptosis and decreased necrosis in mouse model. Thus, caspases not only mediate apoptosis but also protect from necrosis in pancreatitis. One protective mechanism is through degradation of receptor-interacting protein (RIP), a key mediator of “programmed” necrosis. We found that RIP was cleaved (i.e. inactivated) in the rat but not the mouse model. Caspase inhibition restored RIP levels; conversely, caspase induction with embelin triggered RIP cleavage. Our results indicate key roles for caspases, XIAP, and RIP in the regulation of cell death in pancreatitis. Manipulating these signals to change the pattern of death responses presents a therapeutic strategy for treatment of pancreatitis.

title: Acute pancreatitis and bile duct lesions in rat induced by dibutyltin dichloride.
abstract: After i.v. or i.p. administration of a single dose of dibutyltin dichloride (DBTC) the following changes were observed in pancreas and bile ducts of male rats: acute interstitial pancreatitis, enlargement of diameter and destruction of epithelial cells of the bile ducts, increase of serum alkaline phosphatase activity. The effects were dependent on the dose of DBTC (1, 4 or 6 mg/kg body weight), the route of administration and the time after treatment. The pancreatitis induced by DBTC is under further investigation as a model of experimental pancreatitis.

title: Repetitive Self-Limited Acute Pancreatitis Induces Pancreatic Fibrogenesis in the Mouse
abstract: The role of repetitive acute injury in the pathogenesis of chronic pancreatitis remains unknown. To determine if repetitive injury induced by pancreatic hyperstimulation would reproduce the characteristic features of human chronic pancreatitis, acute reversible pancreatic injury was induced in mice by twice weekly cerulein treatment, 50 μg/kg/hr × 6 hr, for 10 weeks. Procollagen α1(I) mRNA was markedly increased by week 2. Sirius red staining of interstitial collagen demonstrated progressive accumulation of extracellular matrix surrounding acinar units and in interlobular spaces. Atrophy, transdifferentiation of acinar units to ductlike tubular complexes, and dilatation of intraacinar lumina also developed. Electron microscopy demonstrated the presence of stromal cells in areas of fibrosis with morphologic characteristics of pancreatic stellate cells. These findings demonstrate that, in a murine model, repetitive acute injury to the pancreas by hyperstimulation can reproduce the major morphological characteristics of human chronic pancreatitis.

title: Induction of chronic pancreatic disease by trinitrobenzene sulfonic acid infusion into rat pancreatic ducts.
abstract: Despite being a common disease in humans, little is known about the etiopathogenesis of and effective therapeutic approaches to chronic pancreatitis, due mainly to the fact that few simple animal models suitable to study inflammatory and fibrogenetic processes have been described in the pancreas. Trinitrobenzene sulfonic acid (TNBS) induces chronic colitis and cholangitis in the rat. We hypothesized that TNBS instillation into the pancreatic ducts could also result in the development of a chronic pancreatic disease. The biliopancreatic duct of rats was cannulated and tied close to the liver. TNBS [0.4 ml of 2% TNBS in phosphate-buffered saline (PBS)-10% ethanol, pH 8] was infused into the pancreas under a continuous controlled-pressure system. Control rats underwent the same procedure using vehicle only. Pathology assessment of TNBS-treated rats examined at 48 h was consistent with severe acute necrotizing pancreatitis, having a morality rate of 31% and serum amylase activity of 37.4 +/- 8.8 U/ml at 24 h and 13.3 +/- 1.7 U/ml at 48 h (p < 0.01 for both time points compared to PBS/ethanol-treated rats). Groups of 10 rats each were killed at 3, 4, and 6 week after the surgical procedure. Morphological examination revealed changes mimicking features of chronic pancreatitis in humans in 80% (32 of 40) of TNBS-treated rats, consisting in various degrees of periductal and lobular fibrosis, duct stenosis, patchy acute and chronic inflammatory cell infiltrates, and signs of gland atrophy. Animals developing chronic disease had a weight gain rate significantly lower than that of control rats. Serum amylase, fasting glucose, and a glucose tolerance test were not different in diseased or control rats. In conclusion, we were able to induce chronic fibrogenetic inflammatory disease in the pancreas after a single pulse instillation of TNBS into the pancreatic ducts. This might be a useful animal model to study the pathophysiology of inflammatory, fibrogenetic, and reparative processes in pancreatic tissue.

title: Pancreatic fibrosis in experimental pancreatitis induced by dibutyltin dichloride.
abstract: Regulatory mechanisms in chronic pancreatitis finally resulting in pancreatic fibrosis cannot be studied sufficiently in human pancreas. Results of a new pancreatitis model in rats suitable for investigation of the processes leading to pancreatic fibrosis are presented.



Experimental pancreatitis was induced by intravenous application of 8 mg/kg body wt dibutyltin dichloride. Pancreatitis was characterized by histology, serum parameters, and immunohistochemistry, detecting inflammatory cells. Gene expression of collagen type I and transforming growth factor beta1 was shown by Northern blot analysis.



Dibutyltin dichloride induced an acute edematous pancreatitis within 24 hours. Extensive infiltration with mononuclear cells could be observed after day 7 followed by the development of fibrosis. Parallel to the cell infiltration, an upregulation of messenger RNA-encoding collagen type I and transforming growth factor beta1 could be shown. An active inflammatory process could be shown until the end of the observation period, i.e., 2 months.



The findings suggest that dibutyltin dichloride-induced pancreatitis in rats is suitable to study cellular interactions and mediators involved in the development of pancreatic fibrosis.

title: Myofibroblast proliferation, fibrosis, and defective pancreatic repair induced by cyclosporin in rats
abstract: Full recovery is always achieved after caerulein induced pancreatitis. Cyclosporin stimulates transforming growth factor β (TGF-β) and may interfere with pancreatic regeneration.  To investigate the effects of cyclosporin after caerulein induced pancreatitis or after caerulein injury.  Protocol A: rats received cyclosporin daily (20 mg/kg) and caerulein pancreatitis was induced on days 2 and 8. Protocol B: six courses of caerulein pancreatitis were induced at weekly intervals. Cyclosporin was administered on induction and the day before. Rats recovered for two weeks before being killed. Control groups received saline, cyclosporin, or caerulein alone.  Protocol A: plasma TGF-β1 and tissue collagenase rose after pancreatitis but decreased towards baseline values on day 15, matching a low collagen content. Morphology disclosed minimal inflammatory infiltration and some interstitial cells immunoreactive for smooth muscle α-actin (SMA). TGF-β1 increased, and remained high in cyclosporin treated groups (cyclosporin alone and cyclosporin plus caerulein). Rats treated with cyclosporin and caerulein showed severe pancreatic weight reduction, abundant inflammatory infiltrates, increased SMA immunoreactive interstitial cells, high collagen content, and delayed collagenase response. No SMA immunoreactive cells were detected in normal rats. Cyclosporin alone also increased SMA immunoreactive cells, despite the absence of inflammatory infiltration and fairly conserved pancreatic structure. Protocol B: the combined pulse treatment induced appreciable collagen deposition and resulted in a smaller pancreas than controls. Morphological examination showed atrophy, fibrosis, fibroblast proliferation, and mononuclear infiltrates.  Cyclosporin greatly distorts pancreatic repair, transforming caerulein induced pancreatitis into a fibrotic chronic-like disease. The mechanism involves TGF-β, myofibroblasts, and defective collagenase activation.

title: Endogenous interleukin-10 modulates fibrosis and regeneration in experimental chronic pancreatitis.
abstract: Interleukin (IL)-10, a potent anti-inflammatory cytokine, limits the severity of acute pancreatitis and downregulates transforming growth factor (TGF)-beta release by inflammatory cells on stimulation. Proinflammatory mediators, reactive oxygen species, and TGF-beta can activate pancreatic stellate cells and their synthesis of collagen I and III. This study evaluates the role of endogenous IL-10 in the modulation of the regeneration phase following acute pancreatitis and in the development of pancreatic fibrosis. IL-10 knockout (KO) mice and their C57BL/6 controls were submitted to repeated courses (3/wk, during 6 wk, followed by 1 wk of recovery) of cerulein-induced acute pancreatitis. TGF-beta(1) release was measured on plasma, and its pancreatic expression was assessed by quantitative RT-PCR and immunohistochemistry. Intrapancreatic IL-10 gene expression was assessed by semiquantitative RT-PCR, and intrapancreatic collagen content was assessed by picrosirius staining. Activated stellate cells were detected by immunohistochemistry. S phase intrapancreatic cells were marked using tritiated thymidine labeling. After repeated acute pancreatitis, IL-10 KO mice had more severe histological lesions and fibrosis (intrapancreatic collagen content) than controls. TGF-beta(1) plasma levels, intrapancreatic transcription, and expression by ductal and interstitial cells, as well as the number of activated stellate cells, were significantly higher. IL-10 KO mice disclosed significantly fewer acinar cells in S phase, whereas the opposite was observed for pseudotubular cells. Endogenous IL-10 controls the regeneration phase and limits the severity of fibrosis and glandular atrophy induced by repeated episodes of acute pancreatitis in mice.

title: Chronic alcohol consumption accelerates fibrosis in response to cerulein-induced pancreatitis in rats.
abstract: Alcohol consumption is a risk factor for chronic pancreatitis (CP), but the mechanism in humans remains obscure because prolonged alcohol consumption in most humans and animal models fails to produce alcoholic chronic pancreatitis (ACP). We hypothesize that the process leading to ACP is triggered by a sentinel acute pancreatitis (AP) event; this event causes recruitment of inflammatory cells, which initiates fibrosis driven by the anti-inflammatory response to recurrent AP and/or chronic oxidative stress. The aim was to determine whether chronic alcohol consumption accelerates fibrosis in response to cerulein-induced pancreatitis in the rat. Wistar male rats were pair-fed control (C) or 5% ethanol (E) Lieber-DeCarli liquid diets. Animals were studied without pancreatitis (P0), with cerulein pancreatitis induced once (P1), or with cerulein-induced pancreatitis weekly for 3 weeks (P3). AP markers, inflammation, and fibrosis were measured histologically, by gene expression profiling and protein expression. Macrophage infiltration was reduced in EP0 versus CP0 rats, but the pattern was reversed after AP. Microabscess, severe necrosis, and early calcification were only induced in the EP3 rats. Fibrosis was significantly induced in the EP3 rats versus EP1, CP1, and CP3 by histology, hydroxyproline content, and mRNA expression for collagen alpha1(1) and procollagen alpha2(1). Proinflammatory cytokine mRNAs were up-regulated shortly after induction of AP, while the anti-inflammatory cytokines (interleukin-10 and transforming growth factor-beta) were strongly up-regulated later and in parallel with fibrogenesis, especially in the EP3 rats. Pancreatic fibrosis develops after repeated episodes of AP and is potentiated by alcohol. Expression of fibrosis-associated genes was associated with expression of anti-inflammatory cytokines in alcohol-fed rats.

title: A mouse model of ethanol dependent pancreatic fibrosis
abstract: The majority of patients with chronic pancreatitis are alcoholics. Our goal was to develop a mouse model of alcohol dependent chronic pancreatitis.  Mice were fed either the non-alcohol containing Lieber-DeCarli diet or the Lieber-DeCarli diet containing 24% of calories as ethanol. After eight weeks and while on their respective diets, mice were subjected to repeated episodes of acute pancreatitis elicited by administration of caerulein. They were sacrificed 1, 3, and 5 weeks after the last dose of caerulein. Pancreatic morphology and collagen deposition were evaluated in samples stained with haematoxylin-eosin and Sirius red. Collagen content was quantitated by measuring OH-proline. Gene expression was determined by quantitative polymerase chain reaction.  Both groups of mice gained weight at the same rate. Those receiving the alcohol containing diet had serum alcohol levels of approximately 100 mM. No histological or gene expression differences were found in mice that were not subjected to acute pancreatitis, regardless of their diet. Necrosis, Sirius red staining, OH-proline content, and expression of α-1 collagen I, α-smooth muscle actin, transforming growth factor β1, and tissue inhibitor of metalloproteinase 1 were all increased in mice fed the alcohol containing diet and given caerulein compared with those fed the control diet and given caerulein. Matrix metalloproteinase 9 expression was transiently decreased in mice fed ethanol and given caerulein compared with the group given caerulein but not fed ethanol.  We have developed a mouse model of alcohol dependent chronic pancreatic fibrosis. This mouse model may be useful in studies examining the effects of genetic manipulation on chronic pancreatitis.

title: The effect of chronic alcohol administration on cerulein-induced pancreatitis
abstract: SummaryAlcohol consumption is associated with pancreatitis, but the mechanism underlying this injury remains unclear. Alcohol consumption has recently been shown to increase the fragility of both rat pancreatic lysosomes and zymogen granules in vitro, which may predispose to autodigestion via the intracellular activation of digestive enzymes by lysosomal enzymes. Cerulein-induced pancreatitis is also associated with lysosomal fragility. To determine the effect of alcohol consumption on this form of pancreatic injury, the severity of pancreatitis was compared in three groups of rats following iv cerulein infusion: rats fed alcohol in a liquid diet, pair-fed dextrose controls, and chow-fed controls. The histological severity of pancreatitis induced by supramaximal cerulein infusion was not found to be increased by prior alcohol consumption. Since alcohol did not appear to increase the severity of pancreatic injury induced by cerulein, we sought to define biochemical parameters that might precede obvious injury. The subcellular distribution of cathepsin B activity and markers of lysosomal fragility were compared in the same groups of experimental animals. Cerulein infusion led to a marked redistribution of cathepsin B activity from the lysosomal to the zymogen-granule-enriched fractions. For animals killed in the fed state, a redistribution of cathepsin B activity toward the zymogen-granule-enriched fraction was also demonstrated in alcohol-fed animals compared to their pair-fed controls. However, chronic alcohol administration did not influence the effect of cerulein on subcellular cathepsin B distribution or lysosomal fragility. In this rat study, administration of alcohol did not increase the effects of supramaximal doses of cerulein on the pancreas.

title: Curcumin ameliorates ethanol and nonethanol experimental pancreatitis.
abstract: Treatments for pancreatitis are limited. Activation of transcription factor NF-kappaB, a key regulator of inflammatory molecule expression, is an early event in experimental pancreatitis and correlates with the inflammatory response. We report here that curcumin, a natural phytochemical known to inhibit NF-kappaB and activator protein (AP)-1, another important proinflammatory transcription factor, ameliorates pancreatitis in two rat models. In both cerulein pancreatitis and pancreatitis induced by a combination of ethanol diet and low-dose CCK, curcumin improved the severity of the disease as measured by a number of parameters (histology, serum amylase, pancreatic trypsin, and neutrophil infiltration). Curcumin markedly inhibited NF-kappaB and AP-1 activation, assessed by DNA binding and degradation of inhibitory IkappaB proteins, and the induction of mRNAs for cytokines IL-6 and TNF-alpha, the chemokine KC, and inducible nitric oxide synthase in pancreas. Curcumin also blocked CCK-induced NF-kappaB and AP-1 activation in isolated pancreatic acini. Our findings indicate that blocking key signals of the inflammatory response ameliorates pancreatitis in both ethanol and nonethanol models. They suggest that curcumin, which is currently in clinical trials for cancer prevention, may be useful for treatment of pancreatitis.

title: Nonoxidative ethanol metabolites alter extracellular matrix protein content in rat pancreas.
abstract: The mechanisms involved in ethanol-induced pancreas fibrosis are poorly understood. Here we show that fatty acid ethyl esters (FAEEs), nonoxidative ethanol metabolites, increase extracellular matrix (ECM) protein levels in pancreas.



Rat pancreatic acini were incubated for 1-4 hours with FAEEs or acetaldehyde. In another set of experiments, rats received an intravenous infusion of FAEEs for 6 hours. Collagens were assessed by a hydroxyproline assay. Laminin and fibronectin were analyzed by Western blotting. Gene expression of ECM proteins was measured by conventional and real-time reverse-transcription polymerase chain reaction (RT-PCR). Matrix metalloproteinase (MMP), plasmin, and urokinase-type plasminogen activator (uPA) activities were determined by zymography and fluorogenic assays.



FAEEs increased collagen, laminin, and fibronectin levels in pancreatic acini without affecting messenger RNA (mRNA) expression for these proteins. Actinomycin D, a transcriptional inhibitor, did not block the increase in ECM proteins induced by FAEEs. FAEEs reduced the activity of the serine protease, plasmin, and that of the uPA. Consistent with these results, the serine protease inhibitor aprotinin reproduced the effects of FAEEs and prevented the further increase in ECM proteins induced by FAEEs. In vivo administration of FAEEs reduced plasmin and uPA activities and increased ECM protein levels in pancreas. Acetaldehyde had minor effects on ECM protein levels and did not affect plasmin activity.



FAEEs increase ECM protein levels in pancreas. The results suggest that this effect is caused primarily by an inhibition in ECM degradation via serine proteases including the plasminogen system.

title: Ethanol diet increases the sensitivity of rats to pancreatitis induced by cholecystokinin octapeptide.
abstract: Although alcoholism is a major cause of pancreatitis, the pathogenesis of this disorder remains obscure. Failure to produce experimental alcoholic pancreatitis suggests that ethanol may only increase predisposition to pancreatitis. This study sought to develop a model of ethanol pancreatitis by determining if an ethanol diet sensitizes rats to pancreatitis caused by cholecystokinin octapeptide (CCK-8).



Rats were fed intragastrically either control or ethanol diet for 2 or 6 weeks. The animals were then infused for 6 hours with either saline or CCK-8 at a dose of 3000 pmol. kg(-1). h(-1), which by itself did not induce pancreatitis. The following parameters were measured: serum amylase and lipase levels, pancreatic weight, inflammatory infiltration, number of apoptotic acinar cells, pancreatic messenger RNA (mRNA) expression of cytokines and chemokines, and nuclear factor (NF)-kappaB activity.



All measures of pancreatitis, as well as NF-kappaB activity and mRNA expression for tumor necrosis factor alpha, interleukin 6, monocyte chemotactic protein 1, macrophage inflammatory protein 2, and inducible nitric oxide synthase, were significantly increased only in rats treated with ethanol plus CCK-8.



An ethanol diet sensitizes rats to pancreatitis caused by CCK-8. The combined action of ethanol and CCK-8 results in NF-kappaB activation and up-regulation of proinflammatory cytokines and chemokines in the pancreas. These mechanisms may contribute to the development of alcoholic pancreatitis.

title: Course and spontaneous regression of acute pancreatitis in the rat
abstract: Rat exocrine pancreatic function was studied structurally and biochemically after the in vivo production of acute interstitial pancreatitis by supramaximal stimulation with caerulein. Two major phases in the reaction of the gland were observed: During the first two days after cessation of the supramaximal stimulation a progressive infiltration of the interstitium and the pancreatic tissue with polymorphonuclear leucocytes, lymphocytes and macrophages occurred which led to further destruction of the gland and to decreased functional response. From two days after the cessation of the treatment, hypertrophy of centro-acinar cells and an increased rate of mitotic activity indicated regeneration of the pancreas. This was combined with an accelerated in vitro discharge of newly synthesized proteins over a period of four days. Between days three and six after the initial treatment mitotic activity was also observed in fully differentiated exocrine cells. Total structural and functional recovery of the pancreas was achieved nine to twelve days after the cessation of the supramaximal stimulation.

title: Time Course and Cellular Source of Pancreatic Regeneration Following Acute Pancreatitis in the Rat
abstract: The regenerative capacity of the different cell types in the rat exocrine pancreas has been studied in a model of hormone-induced acute pancreatitis in which pancreatic edema, inflammation, and acinar cell destruction were induced within 12 h of infusion of supramaximal concentrations of cerulein (5 μg/kg/h). A sequential biochemical and structural analysis of the pancreas in daily intervals was combined with the autoradiographic quantitation of labeling indices of five cell populations following 3H-thymidine injection at days 1–7 after induction of pancreatitis. Desquamation of acinar cell apical cytoplasm and release of cytoplasmic segments into the acinar lumen on the first day following induction of pancreatitis led to formation of duct-like tubular complexes. Enzyme content in the pancreas decreased progressively following the formation of the edema to levels 15–20% of controls and remained reduced during the initial 5 days. Thymidine incorporation into total DNA showed a biphasic pattern with a distinct peak at day 1 and a second broader peak between days 4 and 7. Autoradiographic quantitation of labeling indices demonstrated the exclusive incorporation into intercalated duct cells and interstitial cells during the initial 24 h, while the second peak was predominantly due to labeling of acinar cells. Larger interlobular ducts and islets did not show changes in labeling index. In vivo labeling with 3H-thymidine during the first day and analysis of labeling indices 14 days later showed the persistence of label in intercalated duct cells and interstitial cells and argued against the stem cell hypothesis and against transformation of duct cells into acinar cells. It was concluded that regeneration following acute pancreatitis is mainly due to mitotic activity of remaining acinar cells. The initial and transient increase in replication of intercalated duct cells and interstitial cells could be due to the inflammatory reaction.

title: Repetitive cerulein-induced pancreatitis and pancreatic fibrosis in the rat.
abstract: The effect of repetitive inductions of pancreatitis by supramaximal doses of cerulein on pancreatic morphology and collagen content was studied in the rat. Pancreatitis was induced nine times at intervals of about 20 days; 3 days after the last injection of cerulein, pancreatitis was still observed, as indicated by pancreatic weight loss, increase of protein-bound hydroxyproline content, acinar-cell destruction, cellular infiltration, and deposition of collagen fibers. However, 6 weeks later, no differences in the parameters mentioned above were observed between control and cerulein-treated animals. Thus, repetitive induction of pancreatitis in the rat, according to the experimental protocol we used, did not result in pancreatic fibrosis.

title: Involvement of Tubular Complexes in Pancreatic Regeneration After Acute Necrohemorrhagic Pancreatitis
abstract: Localized acute necrohemorrhagic pancreatitis was induced in rats by multiple trypsin injections. Morphological alterations were monitored by light and electron microscopy until complete recovery. In the acute phase, typical pictures of focal acute necrohemorrhagic pancreatitis were observed. In the postacute phase, fibrosis and tubular complexes are characteristic of damaged areas. Tubular complexes appear from the dedifferentiation of acinar cells. They are characterized by duct-like cells bordering wide, empty luminae. In the recovery phase, cellular proliferation was accompanied by differentiation, with progressive acquisition of the morphological characteristics of acinar cells at the periphery of the tubular complexes. In that instance, cellular proliferation was concomitant with the development of collagen septa in tubular complexes. In these structures both duct-like and acinar-like cells presented mitoses. Cell division persisted in the dedifferentiated cells until tubular complexes disappeared. A very similar process was observed in the embryonic pancreas, where organized parenchyma originated from proliferation and differentiation of protodifferentiated cells. We concluded that pancreatic repair following necrohemorrhagic pancreatitis involves proliferation of cells from intact acini and from tubular complexes, at variance with edematous pancreatitis, where regeneration is exclusively due to acinar cell proliferation.

title: Tubular Complexes in Cerulein‐and Oleic Acid‐Induced Pancreatitis in Rats: Glycoconjugate Pattern, Immunocytochemical, and Ultrastructural Findings
abstract: Ductlike tubular complexes in cerulein-induced pancreatitis and oleic acid-induced pancreatic insufficiency were studied to analyze further their origin and development. Irnmunocytochemistry for pancreatic enzymes, lectin-binding studies, and ultrastructural investigations were combined with autoradiographic quantitation of labeling indices of ductlike cells in tubular complexes. In one group of rats, pancreatitis was induced by infusion of cerulein (10 pg kg−1 h−1). In a second group, pancreatic insufficiency was induced by intraductal injection of oleic acid (50 μ1). The investigations were carried out at distinct intervals following induction of pancreatic injury. In both groups of animals, after 3 days, a significant widening of acinar lumina was paralleled by a decreasing height of acinar cells, which showed pronounced retrogressive changes. At this time, acinar cells bound all of the lectins used and retained their imrnunoreactivity for amylase, trypsinogen, chymotrypsinogen, and lipase. At further intervals, acinar structures formed typical ductlike complexes, with a progressive loss of immunoreactivity for pancreatic enzymes and a reduced lectinbinding for L-fucose and N-acetylgalactosamine. Autoradiographic quantitation demonstrated no significant labeling of acinar cells undergoing tubular dedifferentiation. In both models, tubular complexes were removed by macrophages. It is concluded that lining cells in tubular complexes represent degenerating acinar cells that have no regenerative potency and have lost their secretory and membrane characteristics.

title: Effect of Chronic Ethanol Feeding on Digestive Enzyme Synthesis and mRNA Content in Rat Pancreas
abstract: Chronic ethanol ingestion is a primary factor in the development of pancreatitis in humans. Alterations in both enzyme secretion and protein synthesis have been implicated as causative factors. We determined the effect of chronic ethanol feeding on the content and synthesis rates of digestive enzymes in dispersed acini from rats that were pair-fed isocaloric diets with or without ethanol for 3–6 months. Total protein content and synthesis were unchanged. The relative synthetic rates of individual digestive enzymes were analyzed using scanning laser den-sitometry of 1-D sodium dodecylsulfate (SDS) gels. The content of all measurable digestive enzymes except amylase increased in acini from ethanol-fed rats. Relative synthetic rates were examined in pancreatic acini labeled in vitro with [35S]methionine. Liquid scintillation counting of radiolabeled digestive enzymes extracted from gelslices revealed that amylase synthesis in ethanol-fed rats decreased 2.8-fold compared with controls whereas the synthetic rates of proelastase 1 and 2, chymotrypsinogen, and trypsinogen increased by 1.5-, 1.4-, 1.8-, and 1.6-fold, respectively. Total cellular RNA was extracted from control and ethanol-fed rats and subjected to Northern and dot blot analysis. Amylase mRNA decreased in ethanol-fed rats whereas chymotrypsinogen and trypsinogen mRNA content increased, indicating that the effect of ethanol on expression of these genes was regulated at a step prior to translation. Elastase mRNA content was not altered, suggesting that the increased synthesis of proelastase may be regulated posttranscriptionally.

title: Growth of rat pancreatic acinar cells quantitated with a monoclonal antibody against the proliferating cell nuclear antigen
abstract: The monoclonal antibody PC10 raised against the proliferating cell nuclear antigen (PCNA) was used to study acinar cell replication in the pancreas of rats under different functional conditions. In Western blots, the antibody recognized a single band of 37 kDa in pancreatic homogenates indicating its specificity in this particular species and organ. Three conditions of growth were chosen for immunohistochemical analysis: pancreatic preand postnatal development, pancreatic regeneration after injury, and cholecystokinin-stimulated acinar cell proliferation. The time course of acinar cell replication under each condition was the same as that obtained after tritiated thymidine incorporation with subsequent autoradiography, indicating that the percentage of PCNA-positive cells reflects the pool of cycling cells in the models investigated. However, the absolute number of PCNA-positive cells was two to ten times higher than comparable labeling indices from 3H-thymidine autoradiography. This finding might reflect the half life of PCNA, which exceeds the duration of the S-phase. Thus, PCNA-positive cells not only represent S-phase cells, but also cells that have recently completed the cell cycle.

title: Rat macrophages: membrane glycoproteins in differentiation and function
abstract: Macrophages (mφ) play a crucial role in the immune system. The rat offers unique advantages for studying the biology of mφ. Firstly, monoclonal antibodies (mAb) against many rat mφ surface glycoproteins have become available. These have not only demonstrated a considerable heterogeneity among mφ, but have also allowed the characterization of various mφ surface molecules in terms of structure, expression regulation and function. Furthermore, substantial numbers of rat mφ can be isolated from various sites (e.g. blood, peritoneal cavity, lung and other tissues), enabling proper molecular and functional analysis of these mφ populations. Finally, a number of (unique) experimental models for human diseases have been developed in the rat, making possible the evaluation of the involvement of mφ in pathogenesis. For this, a method for the selective elimination of mφ from various tissues in vivo has proven very useful. Here, we will review the contribution that the rat has made to understanding the immunobiology of mφ. In particular, we will discuss the surface (glyco)proteins on rat mφ in differentiation and function, and the involvement of mφ in rat models of disease.

title: Distinct Macrophage Subpopulations in Pancreas of Prediabetic BB/E Rats: Possible Role for Macrophages in Pathogenesis of IDDM
abstract: Use of monoclonal antibodies directed against rat macrophages and serial pancreatic biopsy in the prediabetic period have enabled us to document the involvement of macrophages in the pancreatic events leading to onset of diabetes in the spontaneously diabetic BB/E rat. A few weeks before onset of disease, there is marked recruitment and accumulation of ED1+ macrophages at periductal and perivascular locations adjacent to noninfiltrated islets. These recruited cells, distinct from the resident ED2+ tissue macrophages, then infiltrate the islets. Infiltration of the pancreas by ED1 + macrophages is therefore a very early event in the prediabetic period and suggests a possible role for macrophages in the pathogenesis of insulin-dependent diabetes mellitus (IDDM) in this animal model.

title: OX-40: life beyond the effector T cell stage.
abstract: The OX-40 receptor (OX-40R) is a transmembrane protein found on the surface of activated CD4(+) T cells. When engaged by an agonist such as anti-OX-40 antibody or the OX-40 ligand (OX-40L) during antigen presentation to T cell lines, the OX-40R generates a costimulatory signal that is as potent as CD28 costimulation. Engagement of OX-40R enhances effector and memory-effector T cell function by up-regulating IL-2 production and increasing the life-span of effector T cells. We hypothesize that the signal generated by the OX-40R inhibits activation-induced T cell death (AICD) and thereby increases the number of cells differentiating from the effector to memory T cell stage. In experimental autoimmune encephalomyelitis (EAE) OX-40R+ T cells are found only within the inflammatory site [central nervous system (CNS)]. Sorting OX-40R+ T cells from the CNS of animals with EAE revealed that they are autoantigen-specific T cells. Therefore, OX-40R-specific therapies were devised to eliminate or inhibit autoreactive T cells, while sparing the remainder of the T cell repertoire. In contrast, in vivo costimulation through the OX-40R in animals with cancer generated enhanced tumor-specific immunity leading to improved tumor-free survival. Thus, manipulation of the OX-40R during inflammatory responses can alter effector CD4(+) T cell function by enhancing or limiting T cell activation and survival.

title: Macrophage migration inhibitory factor is a critical mediator of severe acute pancreatitis.
abstract: Macrophage migration inhibitory factor (MIF), originally described as an inhibitor of the random migration of macrophages, has been shown recently to be involved in the pathogenesis of several inflammatory diseases such as sepsis. The aim of this study was to clarify the role of MIF in acute pancreatitis (AP).



Hemorrhagic necrotizing pancreatitis and edematous pancreatitis were induced by the injection of taurocholic acid (TCA pancreatitis) and cerulein (cerulein pancreatitis), respectively, on male Wistar rats. MIF levels in ascitic fluids, serum, and the organs were determined. The effects of anti-MIF antibody were examined on the prognosis of rats with TCA pancreatitis and of female CD-1 mice with choline-deficient, ethionine-supplemented, diet-induced model of severe AP. In addition, serum MIF levels in AP patients and in healthy controls were measured.



Serum and ascitic MIF levels in TCA pancreatitis were increased rapidly and decreased gradually thereafter. Ascitic MIF levels were also increased in cerulein pancreatitis, but to a lesser degree. MIF level was increased in the lung in TCA pancreatitis, but not in the pancreas and the liver. Prophylactic (1 hour before and immediately after induction) administration of anti-MIF antibody significantly improved the survival rate of rats with TCA pancreatitis. The survival rate of mice with severe AP was also improved significantly by the antibody treatment. Serum MIF levels were higher in severe AP patients than mild AP patients and healthy controls.



These results suggest a role of MIF in the pathogenesis of severe AP.

title: Directing Transition from Innate to Acquired Immunity: Defining a Role for IL-61
abstract: Appropriate control of leukocyte recruitment and activation is a fundamental requirement for competent host defense and resolving inflammation. A pivotal event that defines the successful outcome of any inflammatory event is the transition from innate to acquired immunity. In IL-6 deficiency, this process appears defective, and a series of in vivo studies have documented important roles for IL-6 in both the resolution of innate immunity and the development of acquired immune responses. Within this review, particular attention will be given to the regulatory properties of the soluble IL-6 receptor and how its activity may affect chronic disease progression.

title: Apoptosis of neutrophils
abstract: Neutrophils are the most abundant leukocytes and serve as a first line of defense against infectious microorganisms. For this purpose, neutrophils contain granules filled with proteolytic and other cytotoxic enzymes. Neutrophils have the shortest lifespan of all leukocytes. To prevent senescent neutrophils from releasing their toxic contents into the surrounding tissues, these cells become apoptotic and are then internalized by tissue macrophages. Recent studies have revealed more details about effects of cytokines on neutrophil apoptosis and on the uptake of apoptotic neutrophils by macrophages. In addition, the intracellular events leading to apoptosis are slowly being unraveled.

title: The many roles of chemokines and chemokine receptors in inflammation.
abstract: In this review, the authors discuss the properties of chemokines and their receptors and highlight the roles of these chemoattractants in selected clinical disorders.

title: Monocyte Chemoattractant Protein‐1 (CCL2) in Inflammatory Disease and Adaptive Immunity: Therapeutic Opportunities and Controversies
abstract: Monocyte chemoattractant protein (MCP)‐1 (CCL2) specifically attracts monocytes and memory T cells. Its expression occurs in a variety of diseases characterized by mononuclear cell infiltration, and there is substantial biological and genetic evidence for its essential role in atherosclerosis and multiple sclerosis. Despite intensive screening, there are as yet no small‐molecule antagonists of the receptor of MCP‐1/CCL2, CCR2. However, biological agents, including antibodies and inhibitory peptides, have been developed and may be useful for these indications. Recent evidence from genetically modified mice indicates that MCP‐1 and CCR2 have unanticipated effects on T helper (Th) cell development. However, unlike the identical phenotypes of MCP‐1/CCL2−/− and CCR2−/− mice in inflammatory diseases, the phenotypes of these mice are disparate in adaptive immunity: MCP‐1 stimulates Th2 polarization, whereas CCR2 activation stimulates Th1 polarization. This presents both a challenge and an opportunity for targeting the MCP‐1/CCL2/CCR2 axis in disease.

title: STRUCTURAL BIOLOGY OF THE FIBRES OF THE COLLAGENOUS AND ELASTIC SYSTEMS
abstract: The different types of fibres of the collagenous and elastic systems can be demonstrated specifically in tissue sections by comparing the typical ultrastructural picture of each of the fibre types with studies using selective staining techniques for light microscopy. A practicalmodus operandi, which includes the recommended staining procedures and interpretation of the results, is presented. Micrographs and tables are provided to summarize the differential procedures. Reticulin fibres display a distinct argyrophilia when studied by means of silver impregnation techniques, and show up as a thin meshwork of weakly birefringent, greenish fibres when examined with the aid of the Picrosirius‐polarization method. In addition, electron‐microscopic studies showed that reticulin fibres are composed of a small number of thin collagen fibrils, contrasting with the very many thicker fibrils that could be localized ultrastructurally to the sites where non‐argyrophilic, coarse collagen fibres had been characterized by the histochemical methods used. The three different fibre types of the elastic system belong to a continuous series: oxytalan—elaunin—elastic (all of the fibre types comprising collections of microfibrils with, in the given sequence, increasing amounts of elastin). The three distinct types of elastic system fibres have different staining characteristics and ultrastructural patterns. Ultrastructurally, a characteristic elastic fibre consists of two morphologically different components: a centrally located solid cylinder of amorphous and homogeneous elastin surrounded by tubular microfibrils. An oxytalan fibre is composed of a bundle of microfibrils, identical to the elastic fibre microfibrils, without amorphous material. In elaunin fibres, dispersed amorphous material (elastin) is intermingled among the microfibrils.

title: Cyclosporin and chronic pancreatitis: a supermodel?
abstract: See article on page 269 

Most patients with acute pancreatitis develop their disease in association with either biliary tract stones or abuse of ethanol, whereas chronic pancreatitis is most commonly the result of prolonged ethanol abuse. Attempts to study the pathogenesis and pathophysiology of these inflammatory conditions have been hampered by the relative inaccessibility of the pancreas to study in humans, as well as the difficulty in identifying appropriate patients during the earliest stages of their disease.

Over the past two decades, several models of acute pancreatitis induced in laboratory animals have been developed.1-3 Use of these models has permitted the performance of studies that have advanced our understanding of the early cellular events that underlie the development of acute pancreatitis.4 Unfortunately, similar progress in the area of chronic pancreatitis has not been made, mainly because no good models of that disease have been developed.

The hallmarks of chronic pancreatitis are the combined presence …

title: Treatment with bindarit, a blocker of MCP-1 synthesis, protects mice against acute pancreatitis.
abstract: Chemokines are believed to play a key role in the pathogenesis of acute pancreatitis. We have earlier shown that pancreatic acinar cells produce the chemokine monocyte chemotactic protein (MCP)-1 in response to caerulein hyperstimulation, demonstrating that acinar-derived MCP-1 is an early mediator of inflammation in acute pancreatitis. Blocking chemokine production or action is a major target for pharmacological intervention in a variety of inflammatory diseases, such as acute pancreatitis. 2-Methyl-2-[[1-(phenylmethyl)-1H-indazol-3yl]methoxy]propanoic acid (bindarit) has been shown to preferentially inhibit MCP-1 production in vitro in monocytes and in vivo without affecting the production of the cytokines IL-1, IL-6, or the chemokines IL-8, protein macrophage inflammatory-1alpha, and RANTES. The present study aimed to define the role of MCP-1 in acute pancreatitis with the use of bindarit. In a model of acute pancreatitis induced by caerulein hyperstimulation, prophylactic as well as therapeutic treatment with bindarit significantly reduced MCP-1 levels in the pancreas. Also, this treatment significantly protected mice against acute pancreatitis as evident by attenuated hyperamylasemia neutrophil sequestration in the pancreas (pancreatic MPO activity), and pancreatic acinar cell injury/necrosis on histological examination of pancreas sections.

title: Activation of pancreatic acinar cells on isolation from tissue: cytokine upregulation via p38 MAP kinase.
abstract: Cytokines produced by pancreatic acinar cells may mediate cell death and recruitment of inflammatory cells into pancreas in pancreatitis and other disorders. Here, we demonstrate mRNA expression for a number of cytokines in acini isolated from rat pancreas. Using RNA from microscopically selected individual cells, we confirmed the acinar cell as a source for cytokine expression. Competitive RT-PCR, Western blot analysis, and immunocytochemistry showed large amounts of monocyte chemotactic protein-1 and interleukin-6 compared with other cytokines. Cytokine expression was inhibited by either inhibitors of p38 mitogen-activated protein kinase (MAPK), SB-202190 and SB-203580, or (less strongly) by the transcription factor nuclear factor (NF)-kappaB inhibitor MG-132. A combination of SB-203580 and MG-132 inhibited mRNA expression of all cytokines by >90%. The results suggest a major role for p38 MAPK and involvement of NF-kappaB in cytokine expression in pancreatic acinar cells. In contrast to isolated acini, we detected no or very low cytokine expression in normal rat pancreas. Our results indicate that activation of p38 MAPK, transcription factors, and cytokines occurs during removal of the pancreas from the animal and isolation of acini.

title: Anti-monocyte chemoattractant protein 1 gene therapy attenuates experimental chronic pancreatitis induced by dibutyltin dichloride in rats
abstract: Monocyte chemoattractant protein 1 (MCP-1) is a member of the C-C chemokine family and exerts strong chemoattractant activity in monocytes, macrophages, and lymphocytes. Rat pancreatic fibrosis induced by dibutyltin dichloride (DBTC) is considered to be an appropriate chronic pancreatitis model histologically and enzymatically, as has demonstrated in a previous study.  We examined the effect of human dominant negative inhibitor of MCP-1 (mutant MCP-1) on progression of chronic pancreatitis induced by DBTC in a rat model.  We used the experimental model of chronic pancreatitis induced by DBTC in rats. Mutant MCP-1 or empty plasmid at a dose of 50 µg/body weight was administrated into rat thigh muscles on days 4, 11, and 18 after administration of DBTC. On days 14 and 28, we evaluated the effect of mutant MCP-1 morphologically and biochemically.  The mutant MCP-1 treated group inhibited early pancreatic inflammation and later pancreatic fibrosis histologically, and showed a decrease in serum MCP-1 concentration, intrapancreatic hydroxyproline, α-smooth muscle actin, and an increase in intrapancreatic amylase and protein content compared with the empty plasmid treated group. The mutant MCP-1 group also inhibited intrapancreatic mRNA expression of cytokines and chemokines.  Our findings suggest that monocyte/macrophage recruitment and the systemic MCP-1 signal pathway contribute to progression of chronic pancreatitis, and that blockade of MCP-1 may suppress the development of pancreatic fibrosis.

title: Matrix metalloproteinases as modulators of inflammation and innate immunity
abstract: As their name implies, matrix metalloproteinases are thought to be responsible for the turnover and degradation of the extracellular matrix. However, matrix degradation is neither the sole nor the main function of these proteinases. Indeed, as we discuss here, recent findings indicate that matrix metalloproteinases act on pro-inflammatory cytokines, chemokines and other proteins to regulate varied aspects of inflammation and immunity.

title: Inhibitors of the calcineurin/NFAT pathway.
abstract: The well known calcium-sensitive phosphatase calcineurin is implicated in many eukaryotic activation and developmental programmes, including lymphocyte activation, heart-valve morphogenesis, angiogenesis, and neural and muscle development. The importance of this phosphatase is graphically illustrated by the observation that the immunosuppressive actions of the microbial drugs Cyclosporin A (CsA) and FK506 arise from their inhibition of calcineurin. As substrates of calcineurin, transcription factors of the NFAT family play an essential role in lymphocyte activation, and it follows that their function is also inhibited by CsA and FK506. Although the use of these drugs has been crucial for the success of organ transplantation, their therapeutic use is associated with severe side effects. There is, therefore a need to develop better, less toxic immunosuppressive agents. In recent years, a number of endogenous calcineurin inhibitor proteins have been identified that bind calcineurin and block its phosphatase activity. In some cases the calcineurin interaction domains of these proteins, or their corresponding docking sites on calcineurin, have been described. However, their mode of action and regulatory mechanisms are not completely known. In a more recent development, specific amino acidic sequences implicated in the interaction between calcineurin and NFAT have been identified. It is of special interest that specific disruption of this pathway has been obtained through the expression of peptides based on some of these sequences. A more profound analysis of these issues could open up new perspectives in immunosuppressive therapy; promising compounds with features of endogenous calcineurin inhibitors (and thus likely to have fewer toxic effects than CsA and FK506), or selective blockers of calcineurin-NFAT interactions that would not alter the functioning of other calcineurin substrates.

title: Fibrogenic Effects of Cyclosporin A on the Tubulointerstitium: Role of Cytokines and Growth Factors
abstract: The clinical utility of cyclosporin A (CyA) as an immunosuppressive agent has been significantly limited by the frequent occurrence of chronic nephrotoxicity, characterised by tubular atrophy, interstitial fibrosis and progressive renal impairment. The pathogenesis of this condition remains poorly understood, but has been postulated to be due to either direct cytotoxicity or indirect injury secondary to chronic renal vasoconstriction. Using primary cultures of human proximal tubule cells (PTCs) and renal cortical fibroblasts (CFs) as an in vitro model of the tubulointerstitium, we have been able to demonstrate that clinically relevant concentrations of CyA are directly toxic to these cells and promote fibrogenesis by a combination of suppressed matrix metalloproteinase activity and augmented fibroblast collagen synthesis. The latter effect occurs secondary to the ability of CyA to stimulate autocrine secretion of insulin-like growth factor-I by CFs and paracrine secretion of transforming growth factor-β1 by PTCs. Many of these pro-fibrotic mechanisms are completely reversed by concurrent administration of the angiotensin-converting enzyme inhibitor, enalaprilat, which has proven efficacy in preventing chronic CyA nephropathy in vivo. These studies highlight the unique potential that human renal cell cultures offer for studying the role of local cytokine networks in tubulointerstitial disease and for developing more effective treatment strategies which specifically target fibrogenic growth factor activity following nephrotoxic injuries.

title: Exploring cyclosporine A-side effects and the protective role-played by antioxidants: the morphological and immunohistochemical studies.
abstract: Cyclosporine A (CsA) is the immunosuppressor most frequently used in transplant surgery and in the treatment of autoimmune diseases, because of its specific inhibiting effect on the signal transduction pathways of cell T receptor. It has been shown that CsA is able to generate reactive oxygen species and lipid peroxidation, which are directly involved in the CsA nephrotoxicity, hepatotoxicity and cardiotoxicity. So, the use of antioxidants seems to be a useful tool in attempting to reduce CsA adverse effects. The aim of this review is to summarise the general aspect of CsA, the classification of antioxidants, their mechanism of action and their administration for improving CsA side effects. The protective role of different antioxidants has been evaluated on CsA-induced nephrotoxicity. It has been shown that the antioxidants, improved the morphological renal cytoarchitecture, increased the antioxidant enzyme content, decreased lipid peroxidation and reactive species oxygen (ROS). The protective role of antioxidants was also found in CsA hepatotoxicity and was related to the increase in antioxidant capacity of hepatic tissue, which was responsible for ameliorating hepatic morphology. Recently, it has been demonstrated that CsA induces side effects on the heart but the data to this purpose are very few and also the number of results on the protective role played by antioxidants it is very limited. In conclusion, not only do these observations provide insight into the intricate mechanism of CsA adverse effects, but they also present novel opportunities for the design and development of more effective therapeutic strategies against negative effects.

title: Cyclosporine A-induced oxidative stress in rat hepatocytes.
abstract: In man the immunosuppressive drug Cyclosporine A (CsA) has been used successfully in organ transplantation and in the treatment of autoimmune disorders. The drug, however, causes side effects which occur mainly in the kidney but also in the liver. The mechanisms leading to the hepatic side effects are not yet fully understood. Because reactive oxygen production is a common mechanism of drug toxicity, the goal of this study was to evaluate whether CsA induces oxidative stress in rat liver cells. In primary rat hepatocyte 20-h cultures, CsA caused a concentration-dependent increase of free reactive oxygen species, thiobarbituric acid reactive substances, loss of protein thiols and decrease of molar ratios of glutathione and glutathione disulfide in the range of 0 to 50 microM CsA. The weakening or enforcement of the cellular glutathione state by the glutathione synthesis inhibitor buthionine sulfoximine or the glutathione disulfide -reducing agent dithiothreitol either increased or inhibited the CsA cytotoxicity, as determined by lactate dehydrogenase release. CsA also decreased the level of endogenous antioxidant ascorbic acid and increased its oxidation product dehydroascorbic acid. Supplementation of the cell cultures with ascorbic acid significantly reduced the CsA toxicity. The antioxidant DL-alpha-tocopherol-polyethylene-glycol-1000-succinate partly decreased CsA-mediated reactive oxygen species formation, totally decreased thiobarbituric acid reactive substances formation, prevented the loss of protein-bound sulfhydryl groups and in addition totally inhibited the CsA cytotoxicity. The present data provide good evidence that oxidative stress is part of the mechanism by which CsA causes toxicity in rat liver cells.

title: Immunodeficiency associated with ethanol abuse.
abstract: A number of study findings have shown that ETOH has a profound effect on the immune system. The work from my laboratory has established in animal models that the effect of ETOH is complex. It is well established that ingestion of ETOH-containing diets results in a loss of lymphoid cells from the peripheral blood, spleen, and thymus. Some of the cell loss from the thymus is the result of corticosteroid release as a result of the withdrawal from ETOH, but the loss from the spleen and some of the thymocyte loss is independent of corticosteroids, as demonstrated by studies using ADX mice and rats. We have also established that ETOH ingestion is associated with a loss of lymphocyte function, especially T-cell-dependent immune responses. One aspect of the T-cell defect is an inability to use IL-2, an important growth factor for T cells. Similar changes in lymphocyte function have been demonstrated in animals exposed to ETOH only in utero. The inability of a person to respond immunologically in an appropriate fashion to foreign antigens has a profound effect on the survival of the person. It would be predicted that ETOH-associated immunosuppression would result in increased incidences of infections. From the data generated from my laboratory it could also be predicted that these infections would be primarily opportunistic infections that are associated with defects in T-cell function. The available literature would support these predictions. It is also likely that changes in T-cell function would alter immunosurveillance mechanisms with the end result being an increased incidence of tumors. Again, the available literature would support this prediction.(ABSTRACT TRUNCATED AT 250 WORDS)

title: Consequences of alcohol consumption on host defence.
abstract: This communication reviews recent literature and summarizes current views on the immunomodulatory effects of acute and chronic alcohol consumption. Chronic and even acute, moderate alcohol use can increase host susceptibility to infections caused by bacterial and viral pathogens. Impaired host defence after alcohol exposure appears to be linked to a combination of decreased inflammatory response, altered cytokine production, and abnormal reactive oxygen intermediate generation. Furthermore, cellular immunity, particularly antigen-specific immune response, is impaired by both acute and chronic alcohol use. Although T lymphocyte functions can be directly affected by ethanol, decreased antigen presenting cell function appears to be a key element in the ethanol-induced decrease in cell-mediated immunity. In addition, a preferential induction of Th2 vs Th1 immune response has been suggested, based on the increased immunoglobulin levels seen in chronic alcoholics. The effects of chronic and acute alcohol consumption in humans, animal models and in vitro systems on host defence and immunity are discussed in the context of the functional abnormalities of T and B lymphocytes, natural killer cells and monocytes/macrophages resulting in the altered immune response seen after alcohol use.