Establishment of a novel extracorporeal bowel model to study luminal approaches to treat inflammatory bowel disease.

We have established an extracorporeal bowel model system for the analysis of early events in inflammatory bowel disease (IBD) and therapeutic applications. This model consists of an intestinal segment that is cannulated and perfused in situ, allowing the investigation of cellular responses of apical mucosa cells on luminal applied substances. Short-term treatment with iodoacetamide mimicked experimental intestinal inflammation in IBD, as indicated by histological alterations such as hemorrhage, hyperemia and loss of regular crypt architecture, as well as enhanced expression of cytokines (e.g. IL-6, IL-10 and MCP-1) compared with control segments perfused with media. Perfusion of therapeutic agents (e.g. dexamethasone or Mutaflor) in the small intestine segment significantly reduced the features of early inflammation that are induced by iodoacetamide. Moreover, similar data were obtained for Resormin(®), a montmorillonite-illite mixed-layer mineral (smectite), indicating that smectites might be a newly identified therapeutic option for IBD. In summary, this model could provide novel insights into epithelial injury as well as genesis of IBD and, therefore, be useful in testing the therapeutic potential of compounds for IBD therapy.

First human application of a novel adsorptive-type cytapheresis module in patients with active ulcerative colitis: a pilot study.

The aim of this study was to evaluate the safety, tolerability, technical performance and clinical efficacy of a novel adsorptive-type cytapheresis module in patients with active ulcerative colitis. Ten patients with ulcerative colitis (clinical activity index 6-10) were recruited. The new adsorber (Nikkiso, Tokyo, Japan) was specifically designed to remove platelets, granulocytes and monocytes from peripheral blood using an extracorporeal circulation. Cytapheresis treatments were performed weekly for five consecutive weeks (each with a 60-min duration). Safety and tolerability were evaluated by investigating vital parameters, routine laboratory tests, adverse event reporting and a questionnaire. Disease activity was evaluated by assessing the clinical activity index as well as the endoscopic index, according to Rachmilewitz. Technical performance and biocompatibility were investigated by repeated measurements of cellular blood count, complement factor C3a and cell surface markers before, during and after the apheresis treatments. The cytapheresis treatments were "well" to "very well" tolerated by the patients. All measured safety parameters remained essentially unchanged. Performance data showed that platelets, monocytes and neutrophil granulocytes were effectively reduced during the cytapheresis treatments. Apheresis treatment was associated with high remission rates (80% at week 10). Clinical remission was accompanied by the reduction of the endoscopic index in four out of the nine eligible patients. Levels of C3a did not significantly increase during cytapheresis treatments. The novel device has been shown to be safe, well tolerated and clinically efficient. It offered a very good biocompatibility and platelet elimination capacity.

Bone marrow-derived pancreatic stellate cells in rats.

Origin and fate of pancreatic stellate cells (PSCs) before, during and after pancreatic injury are a matter of debate. The crucial role of PSCs in the pathogenesis of pancreatic fibrosis is generally accepted. However, the turnover of the cells remains obscure. The present study addressed the issue of a potential bone marrow (BM) origin of PSCs. We used a model of stable hematopoietic chimerism by grafting enhanced green fluorescence protein (eGFP)-expressing BM cells after irradiation of acceptor rats. Chimerism was detected by FACS analysis of eGFP-positive cells in the peripheral blood. Dibutyltin dichloride (DBTC) was used to induce acute pancreatic inflammation with subsequent recovery over 4 weeks. Investigations have been focused on isolated cells to detect the resting PSC population. The incidence of eGFP-positive PSC obtained from the pancreas of chimeric rats was approximately 7% in healthy pancreatic tissue and increased significantly to a mean of 18% in the restored pancreas 4 weeks after DBTC-induced acute inflammation. Our results suggest that BM-derived progenitor cells represent a source of renewable stellate cells in the pancreas. Increased numbers of resting PSCs after regeneration point toward enhanced recruitment of BM-derived cells to the pancreas and/or re-acquisition of a quiescent state after inflammation-induced activation.

Synergistic growth inhibitory effects of the dual endothelin-1 receptor antagonist bosentan on pancreatic stellate and cancer cells.

Pancreatic stellate cells (PSC) play a key role in pancreatic fibrosis. Activation of PSC occurs in response to pro-fibrogenic stimuli and is maintained by autocrine loops of mediators, such as endothelin (ET)-1. Here, we have evaluated effects of the dual ET receptor antagonist bosentan in models of pancreatic fibrogenesis and cancer. Cell culture studies revealed that PSC and DSL6A pancreatic cancer cells expressed both ET-1 and ET receptors. Bosentan efficiently inhibited proliferation of both cell types and collagen synthesis in PSC. Expression of the myofibroblastic marker alpha-smooth muscle actin, connective tissue growth factor, and ET-1 itself in PSC was reduced, while expression of matrix metalloproteinase-9 was enhanced. Like PSC, DSL6A cells secrete less ET-1 when cultured with bosentan. In a rat model of pancreatic fibrosis, chronic pancreatitis induced by dibutyltin dichloride, a tendency towards a diminished disease progression was observed in a subgroup of rats with less severe disease. Together, our results indicate that bosentan exerts antifibrotic and antitumor effects in vitro. Its efficiency in vivo warrants further investigation.

Molecular insights into connective tissue growth factor action in rat pancreatic stellate cells.

Pancreatic fibrosis, a key feature of chronic pancreatitis and pancreatic cancer, is mediated by activated pancreatic stellate cells (PSC). Connective tissue growth factor (CTGF) has been suggested to play a major role in fibrogenesis by enhancing PSC activation after binding to alpha5beta1 integrin. Here, we have focussed on molecular determinants of CTGF action. Inhibition of CTGF expression in PSC by siRNA was associated with decreased proliferation, while application of exogenous CTGF stimulated both cell growth and collagen synthesis. Real-time PCR studies revealed that CTGF target genes in PSC not only include mediators of matrix remodelling but also the proinflammatory cytokines interleukin (IL)-1beta and IL-6. CTGF stimulated binding of NF-kappaB to the IL-6 promoter, and siRNA targeting the NF-kappaB subunit RelA interfered with CTGF-induced IL-6 expression, implicating the NF-kappaB pathway in the mediation of the CTGF effect. In further studies, we have analyzed regulation of CTGF expression in PSC. Transforming growth factor-beta1, activin A and tumor necrosis factor-alpha enhanced expression of the CTGF gene, while interferon-gamma displayed the opposite effect. The region from -74 to -125 of the CTGF promoter was revealed to be critical for its activity in PSC as well as for the inhibitory effect of interferon-gamma. Taken together, our results indicate a tight control of CTGF expression in PSC at the transcriptional level. CTGF promotes fibrogenesis both directly by enhancing PSC proliferation and matrix protein synthesis, and indirectly through the release of proinflammatory cytokines that may accelerate the process of chronic inflammation.

Antifibrogenic effects of histone deacetylase inhibitors on pancreatic stellate cells.

Pancreatic stellate cells (PSCs) are essentially involved in pancreatic fibrogenesis and considered as a target for antifibrotic therapies. Here, we have analyzed the effects of three histone deacetylase inhibitors (HDACIs), sodium butyrate, sodium valproate (VPA) and trichostatin A (TSA), on profibrogenic activities of PSC and elucidated molecular targets of HDACI action. Therefore, cultured PSCs were exposed to HDACI. Cell proliferation and viability were assessed by 5-bromo-2'-deoxyuridine (BrdU) incorporation and trypan blue staining assays. Exhibition of the myofibroblastic PSC phenotype was monitored by immunofluorescence analysis of alpha-smooth muscle actin (alpha-SMA) expression. [(3)H]-proline incorporation into acetic acid-soluble proteins was measured to quantify collagen synthesis. Levels of mRNA were determined by quantitative reverse transcriptase real-time PCR. Protein expression, phosphorylation and acetylation were analyzed by immunoblotting, and gel shift assays were performed to study DNA binding of nuclear proteins. HDACI enhanced histone H3 acetylation in a dose-dependent manner. In the same dose range, they strongly inhibited cell proliferation, alpha-SMA expression and collagen synthesis. A significantly increased rate of cell death was observed in response to TSA at 1 microM. While all three HDACI inhibited mRNA expression of endothelin-1, only VPA significantly reduced expression of transforming growth factor-beta1. Both mediators exert autocrine profibrogenic effects on PSC. Furthermore, HDACI-treated PSC displayed a diminished DNA binding of AP-1, a key transcription factor in profibrogenic signaling. Together, the results suggest that HDACI exert antifibrogenic effects on PSC. Interruption of AP-1 signaling and autocrine loops enhancing PSC activation might be key mechanisms of HDACI action.

Hydrogen peroxide-induced activation of defense mechanisms against oxidative stress in rat pancreatic acinar AR42J cells.

Oxidative stress has been implicated in the pathogenesis of acute pancreatitis. Generally, cells respond to oxidative stress with adaptive changes in gene expression aimed at preventing cellular damage and increasing their survival. However, the overall extent of these genetic changes remains poorly defined. This issue was, therefore, examined in the current study. Following exposure of rat pancreatic AR42J cells to 0.08 mM hydrogen peroxide (H(2)O(2)), a concentration failing to induce necrotic cell death, the expression of 96 stress-related genes was monitored by cDNA microarray analysis. H(2)O(2) provoked a time-dependent reorientation of 54 genes. In particular, at 6 and 24 h, 27 and 11 genes were induced, whereas 10 and 6 genes were suppressed, respectively, showing that the degree of change was stronger at the early time point, and that the number of up-regulated genes was obviously larger than the number of down-regulated genes. Reverse transcription-PCR for selected genes confirmed the gene expression pattern. Many of the differentially up-regulated genes can be related to the antioxidant enzymatic defense system, to cell cycle arrest, to repair and/or replacement of damaged DNA, to repair of damaged protein, and to activation of the NF-kappaB pathway. The results suggest that AR42J cells respond to sublethal oxidative stress with transient transcriptional activation of multiple defense mechanisms that may be an indication for a complex adaptation process. An understanding of the cellular stress responses may lead to new insights into the pathogenesis of oxidative stress-related diseases including acute pancreatitis.

Inhibitory effects of interferon-gamma on activation of rat pancreatic stellate cells are mediated by STAT1 and involve down-regulation of CTGF expression.

Pancreatic stellate cells (PSCs) are the main source of extracellular matrix proteins in pancreatic fibrosis, a pathological feature of chronic pancreatitis and pancreatic cancer. Interferon-gamma (IFN-gamma) is an antifibrotic cytokine, but how precisely it exerts its effects on PSCs is largely unknown. Here, we have focussed on the role of STAT1 as well as target genes of IFN-gamma signalling. Our data indicate that IFN-gamma regulates the expression of two autocrine mediators of PSC activation, connective tissue growth factor and endothelin-1, in a transforming growth factor-beta1-antagonistic manner. STAT1 overexpression under the control of a tetracycline-dependent promoter revealed a close correlation between STAT1 expression and activation, the biological effects of IFN-gamma (growth inhibition, induction of apoptosis), and target gene expression. Our data further support the hypothesis that IFN-gamma interferes with stellate cell activation in the pancreas and suggest activated STAT1 as an inductor of a quiescent PSC phenotype.

Adenovirus-mediated gene transfer of interleukin-4 into pancreatic stellate cells promotes interleukin-10 expression.

Pancreatic stellate cells (PSC) are crucially involved in the development of fibrosis, a hallmark of chronic pancreatitis. Therefore, PSC represent an attractive target for the modulation of cellular functions providing the prerequisite for the establishment of novel therapeutic strategies like transfer of genetic material to the cells. Based on recent studies suggesting that the chronic course of pancreatitis is associated with immune deviation towards a Th1 cytokine profile, we have investigated the applicability of primary PSC to an adenovirus-mediated transfer of the cDNA encoding the Th2 cytokine interleukin (IL) 4 and the autocrine-acting effects of IL 4 on the cells in vitro. The transduction of primary PSC with a replication-incompetent adenovirus type 5 vector carrying the cDNA encoding rat IL- 4 resulted in a distinct expression of the cytokine on mRNA and protein level for two weeks. Similar to recombinant IL 4, effects of the endogenously synthesized cytokine were mediated by the signal transducer and activator of transcription (STAT)6. Interestingly, beside the increase of PSC proliferation, IL 4 transduction was accompanied by an up-regulation in the endogenous expression of the anti-inflammatory cytokine IL 10. In summary, our data suggest that PSC are suitable targets for gene therapy modulating cellular interactions in the pancreas.

Delayed response toward activation stimuli in pancreatic stellate cells.

OBJECTIVES: Pancreatic stellate cells (PSCs) are known to be crucially involved in the development of pancreatic fibrosis, a characteristic feature of chronic pancreatitis and pancreatic cancer. The key event in the pathogenesis of fibrosis represents a transition process of quiescent PSCs into a myofibroblastlike phenotype associated with cell activation in terms of proliferation and synthesis of profibrogenic substances. There is little information available regarding the dynamics of the complex processes initiated by an activating stimulus in quiescent stellate cells. METHODS: Using microarray analysis, we characterized the expression profiles during PSC activation caused by in vitro cultivation on days 2, 4, 7, and 14 after cell isolation. Activation status has been identified by the expression of the activation marker alpha-smooth muscle actin. Genes of interest were subjected to reverse transcription-polymerase chain reaction. To test biologic functions, the responsiveness of stellate cells toward the activators activin A and transforming growth factor-beta1 was investigated in dependence on the cell transition status. RESULTS: Our results revealed that freshly isolated (=quiescent) PSCs were refractory to stimuli for several days, a phenomenon that we referred to as delay phase. CONCLUSION: The retarded response could be considered a protection mechanism to prevent inappropriate stellate cell activation.

Cis-hydroxyproline-induced inhibition of pancreatic cancer cell growth is mediated by endoplasmic reticulum stress.

AIM: To investigate the biological effects of cis-hydroxyproline (CHP) on the rat pancreatic carcinoma cell line DSL6A, and to examine the underlying molecular mechanisms. METHODS: The effect of CHP on DSL6A cell proliferation was assessed by using BrdU incorporation. The expression of focal adhesion kinase (FAK) was characterized by Western blotting and immunofluorescence. Induction of endoplasmic reticulum (ER) stress was investigated by using RT-PCR and Western blotting for the glucose-related protein-78 (GRP78) and growth arrest and DNA inducible gene (GADD153). Cell viability was determined through measuring the metabolic activity based on the reduction potential of DSL6A cells. Apoptosis was analyzed by detection of caspase-3 activation and cleavage of poly(ADP-ribose) polymerase (PARP) as well as DNA laddering. RESULTS: In addition to inhibition of proliferation, incubation with CHP induced proteolytic cleavage of FAK and a delocalisation of the enzyme from focal adhesions, followed by a loss of cell adherence. Simultaneously, we could show an increased expression of GRP78 and GADD153, indicating a CHP-mediated activation of the ER stress cascade in the DSL6A cell line. Prolonged incubation of DSL6A cells with CHP finally resulted in apoptotic cell death. Beside L-proline, the inhibition of intracellular proteolysis by addition of a broad spectrum protease inhibitor could abolish the effects of CHP on cellular functions and the molecular processes. In contrast, impeding the activity of apoptosis-executing caspases had no influence on CHP-mediated cell damage. CONCLUSION: Our data suggest that the initiation of ER stress machinery by CHP leads to an activation of intracellular proteolytic processes, including caspase-independent FAK degradation, resulting in damaging pancreatic carcinoma cells.

Inhibitory effects of interferons on pancreatic stellate cell activation.

AIM: To analyze and to compare the effects of interferon (IFN)-alpha, IFN-beta, and IFN-gamma on pancreatic stellate cell (PSC) activation in vitro and to elucidate the molecular basis of IFN action. METHODS: PSCs were isolated from rat's pancreatic tissue, cultured and stimulated with recombinant rat IFNs. Cell proliferation and collagen synthesis were assessed by measuring the incorporation of 5-bromo-2'-deoxyuridine (BrdU) into DNA and (3H)-proline into acetic acid-soluble proteins, respectively. Apoptotic cells were determined by FACS analysis (sub-G1 peak method). Exhibition of the myofibroblastic PSC phenotype was monitored by immunoblot analysis of alpha-smooth muscle actin (alpha- SMA) expression. To assess the activation of signal transducer and activator of transcription (STAT), Western blots using phospho-STAT-specific antibodies were performed. In studies on STAT1 function, expression of the protein was inhibited by siRNA. RESULTS: IFN-beta and IFN-gamma but not IFN-alpha significantly diminished PSC proliferation and collagen synthesis. IFN-gamma was the only IFN that clearly inhibited alpha-SMA expression. Under the experimental conditions used, no enhanced rate of apoptotic cell death was observed in response to any IFN treatment. IFN-beta and IFN-gamma induced a strong increase of STAT1 and STAT3 tyrosine phosphorylation, while the effect of IFN-alpha was much weaker. Inhibition of STAT1 expression with siRNA was associated with a significantly reduced growth-inhibitory effect of IFN-gamma. CONCLUSION: IFN-beta and particularly IFN-gamma display inhibitory effects on PSC activation in vitro and should be tested regarding their in vitro efficiency. Growth inhibition by IFN-gamma action requires STAT1.

Peroxisome proliferator-activated receptor gamma overexpression inhibits pro-fibrogenic activities of immortalised rat pancreatic stellate cells.

Pancreatic stellate cells (PSCs) play a key role in the development of pancreatic fibrosis, a constant feature of chronic pancreatitis and pancreatic cancer. In response to pro-fibrogenic mediators, PSCs undergo an activation process that involves proliferation, enhanced production of extracellular matrix proteins and a phenotypic transition towards myofibroblasts. Ligands of the peroxisome proliferator-activated receptor gamma (PPARgamma), such as thiazolidinediones, are potent inhibitors of stellate cell activation and fibrogenesis in pancreas and liver. The effects of PPARgamma ligands, however, are at least in part mediated through PPARgamma-independent pathways. Here, we have chosen a different approach to study regulatory functions of PPARgamma in PSCs. Using immortalised rat PSCs, we have established a model of tetracycline (tet)-regulated PPARgamma overexpression. Induction of PPARgamma expression strongly inhibited proliferation and enhanced the rate of apoptotic cell death. Furthermore, PPARgamma-overexpressing cells synthesised less collagen than controls. To monitor effects of PPARgamma on PSC gene expression, we employed Affymetrix microarray technology. Using stringent selection criteria, we identified 21 up- and 19 down-regulated genes in PPARgamma-overexpressing cells. Most of the corresponding gene products are either involved in lipid metabolism, play a role in signal transduction, or are secreted molecules that regulate cell growth and differentiation. In conclusion, our data suggest an active role of PPARgamma in the induction of a quiescent PSC phenotype. PPARgamma-regulated genes in PSCs may serve as novel targets for the development of antifibrotic therapies.

Interleukin-4 gene transfer into rat pancreas by recombinant adenovirus.

OBJECTIVE: Adenovirus-mediated gene transfer technology may provide a novel approach in the treatment of pancreatic diseases. In the rat model of chronic pancreatitis induced by dibutyltin dichloride (DBTC), Th1 lymphocytes are known to be involved in the mediation of inflammation. We therefore investigated whether local expression of the Th2 cytokine interleukin (IL)-4 might modulate the inflammatory response. To address this question, we have established a protocol of efficient gene transfer into rat pancreas. MATERIAL AND METHODS: Recombinant adenovirus constructs carrying the Escherichia coli beta-galactosidase gene (Adbeta-gal) or the rat IL-4 gene (AdrIL-4) were injected into the left gastric artery of healthy LEW.1W rats. Expression of beta-Gal and IL-4 in pancreatic cells was analyzed by X-Gal staining and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. After optimization of the transduction protocol, effects of the IL-4 gene transfer on pancreatic inflammation and fibrosis were studied in DBTC-treated rats. RESULTS: Seven days after Adbeta-gal injection, beta-gal-positive cells were detectable in the rat pancreas. RT-PCR analysis using RNA from pancreata of AdrIL-4-treated rats indicated that IL-4 was expressed for at least 14 days after adenovirus application. Expression of the IL-4 transgene was accompanied by a transient increase of the IL-10 mRNA level in the pancreas. In DBTC-treated rats, adenovirus-mediated transfer of the IL-4 gene modified the pattern of infiltrating inflammatory cells in the pancreas. Importantly, a decrease of CD4+ helper cells was observed. CONCLUSIONS: Our data suggest that the injection of recombinant adenoviruses into the left gastric artery is a promising approach to achieving expression of therapeutic transgenes in the pancreas.

Galectin-1 is an inductor of pancreatic stellate cell activation.

Pancreatic stellate cells (PSCs) play a key role in the development of pancreatic fibrosis, a pathological feature of chronic pancreatitis and pancreatic cancer. Here, we show that activation of rat PSCs in vitro is associated with increased expression of galectin-1 (gal-1) and that gal-1 modulates PSC function. Expression of the lectin was stimulated by fetal calf serum and platelet-derived growth factor. PSCs exposed to exogenous gal-1 proliferated at a higher rate and synthesised more collagen than controls. Gal-1-dependent collagen synthesis was blocked by lactose but not by cellobiose, suggesting that gal-1 acts on PSCs through targeting beta-galactoside-containing glycoconjugates. Analysis of gal-1 signalling in PSCs revealed an activation of the extracellular signal-regulated kinases 1 and 2 and enhanced DNA binding of AP-1 transcription factors. Together, our data implicate gal-1 in PSC activation and suggest further studies to analyse the role of endogenous lectins in the development of pancreatic fibrosis in vivo.

Inhibition of lymphocyte apoptosis by pancreatic stellate cells: impact of interleukin-15.

There is growing evidence that pancreatic stellate cells (PSCs) produce cytokines and take part in the regulation of inflammatory processes in the pancreas. IL-15 inhibits apoptosis of various cell populations. This study was performed to investigate whether PSCs produce IL-15 and thereby can affect lymphocytes. Primary PSCs were isolated from the rat pancreas using density gradient centrifugation. mRNA expression of IL-15 was demonstrated by RT-PCR, and IL-15 protein was analyzed by immunoblotting. Lymphocytes obtained from rat mesenterial lymph nodes were cocultured with in vitro activated PSCs. Apoptosis has been quantified by the binding of annexin V-FITC with a flow cytometer. Proliferation was monitored using [3H]thymidine incorporation. PSCs express two splice variants of IL-15. The protein was detectable only in cell lysates but not in the cell culture supernatant. Cocultivation of lymphocytes with PSCs and IL-15 inhibited spontaneous lymphocyte apoptosis, and this effect was reduced by an anti-IL-15 antibody. Lymphocytes induced vice versa the proliferation and collagen production of PSCs. The inhibition of spontaneous lymphocyte apoptosis in cocultures with PSCs was at least partially mediated by cell-bound IL-15. This effect and the stimulation of PSCs by lymphocytes may lead to a circulus vitiosus, resulting in the persistence of inflammatory processes and the development of fibrosis during chronic pancreatitis.

In vitro and in vivo evaluation of Adacolumn cytapheresis in healthy subjects.

Adacolumn is a medical device for adsorptive cytapheresis. It has been developed for selective adsorption of granulocytes and monocytes from peripheral blood of patients with immune disorders, such as autoimmune diseases and chronic inflammatory diseases. A double blind sham-controlled crossover study design was used in order to evaluate in vivo biological responses of leukocytes as well as biocompatibility during and after Adacolumn cytapheresis in healthy volunteers. In addition, experiments were undertaken to further evaluate leukocyte reactions to Adacolumn carrier (G-1: cellulose diacetate) beads in vitro. Six healthy volunteers, 4 males and 2 females, with a mean age of 26.7 years were randomly assigned to one of the two treatment arms in a crossover fashion. Three subjects received a single Adacolumn treatment, followed by a single sham treatment at an interval of 7 days. The other three subjects received the two treatments in reverse order. All subjects were followed up 7 days after the last treatment. Additionally, in vitro investigations were carried out using blood from the healthy donors to examine the effect of G-1 beads on granulocyte functions. In vitro exposure of human peripheral blood to G-1 beads caused downregulation of L-selectin expression and upregulation of Mac-1 expression on granulocytes, leading to a marked reduction of adhesive capacity of granulocytes to endothelial cells. The exposure also led to decreased granulocyte chemotactic activity to IL-8. The number of granulocytes and monocytes clearly decreased during Adacolumn cytapheresis. Granulocytes showed marked phenotypic changes of L-selectin(Low) and Mac-1(Hi) after passing through Adacolumn in vivo. Expression of TNF-alpha and chemokine receptors was downregulated. In addition, TNF-alpha and IL-1beta producing capacity of peripheral blood leukocytes was decreased after Adacolumn cytapheresis and these changes lasted even one week after the cytapheresis. The level of complement fragments, C3a and C5a, increased, while bradykinin concentration did not change during Adacolumn cytapheresis. Exposure of human peripheral blood to G-1 beads, both in vitro and in vivo, caused a significant reduction of adhesive capacity and proinflammatory cytokine producing capacity of peripheral blood leukocytes. Such changes were not observed after sham apheresis. Despite complement activation, tolerability of Adacolum cytapheresis was not influenced. These findings may at least partly explain the beneficial effect of Adacolumn cytapheresis in the treatment of autoimmune diseases.

In vivo characterization of developing chronic pancreatitis in rats.

Despite numerous experimental and clinical investigations, there is no unifying concept on pathophysiology and pathogenesis of chronic pancreatitis. Defining the interplay between pancreatic microcirculation and parenchymal tissue, we will provide a basis for the better understanding of pancreatic fibrogenesis using in vivo high-resolution multifluorescence microscopy in dibutyltin chloride (DBTC)-exposed rats. Pancreatic microcirculation at days 3 and 7 after DBTC revealed leukocyte activation with a two-fold higher fraction of rolling cells and a nine- to 10-fold increase of cells firmly adherent to the endothelial lining, followed by subsequent transendothelial migration into tissue, as given by chloracetate esterase histology. In vivo staining of acinar tissue with bisbenzimide presented single cells exhibiting nuclear chromatin condensation and fragmentation. Apoptotic cell death was confirmed by immunohistochemical staining for active caspase-3 as well as by TUNEL analysis. Necrotic cells were found dispersed throughout the exocrine tissue under observation. Both modes of cell death were found highest in extent at days 3 and 7 with 15-20 cells/mm2, but progressively decreased below 10 cells/mm2 up to 28 days after DBTC. By means of in vivo microscopy yellow-green autofluorescent collagen deposits were found at day 7 and progressively increased up to approximately 12% at day 28 after DBTC. Concomitantly, density of capillaries progressively decreased and capillaries failing to conduct blood flow became apparent. Present on-line analysis indicates an early inflammatory response with acinar cell death, most probably triggering progression of disease with collagen deposition, capillary rarefication and manifestation of perfusion failure. These temporal and spatial multiparameter measurements of the in vivo microenvironment provide new insights into the pathological processes of pancreatic fibrogenesis.

Generation and characterization of immortalized rat pancreatic stellate cells.

Pancreatic stellate cells (PSCs) are involved in, among other things, the pathogenesis of pancreatic fibrosis. Here, we present the generation of immortalized PSCs 7 and 14 days after isolation by retroviral gene transfer of the SV40 large T antigen encoding region. Propagated cell lines [large T immortalized cells (LTC)-7, LTC-14] retained characteristics of primary cells in terms of morphology, responsiveness to mediators regulating cellular functions such as proliferation, and expression profile of a number of investigated genes. Whereas LTC-14 kept the morphological features of the differentiation status of the primary cells they were made of, LTC-7 appeared similar to an earlier stage. Thus the established cell lines represent a versatile tool to investigate various aspects of PSC biology.