Hans Weidenbach

Identification, culture, and characterization of pancreatic stellate cells in rats and humans☆☆☆

BACKGROUND & AIMS Until now, the basic matrix-producing cell type responsible for pancreas fibrosis has not been identified. In this report, retinoid-containing pancreatic stellate cells (PSCs) in rat and human pancreas are described, and morphological and biochemical similarities to hepatic stellate cells are shown. METHODS Electron and immunofluorescence microscopy (collagen types I and III, fibronectin, laminin, alpha-actin, and desmin) was performed using pancreatic tissue and cultured PSCs. Extracellular matrix synthesis was shown using quantitative immunoassay and Northern blot analysis. RESULTS PSCs are located in interlobular areas and in interacinar regions. Early primary cultured PSCs contain retinol and fatty acid retinyl-esters. Addition of retinol to passaged cells resulted in retinol uptake and esterification. During primary culture, the cells changed from a quiescent fat-storing phenotype to a highly synthetic myofibroblast-like cell expressing iso-alpha-smooth muscle actin (>90%) and desmin (20%-40%) and showing strong positive staining with antibodies to collagen types I and III, fibronectin, and laminin. As determined on protein and messenger RNA level, serum growth factors stimulated the synthesis of collagen type I and fibronectin. CONCLUSIONS The identification of PSCs, particularly in fibrotic areas, and the similarities of these cells to hepatic stellate cells suggest that PSCs participate in the development of pancreas fibrosis.

NF-κB/Rel activation in cerulein pancreatitis

Abstract Background & Aims: Recent evidence suggests that a number of rapid signaling cascades are initiated during secretagogue-induced pancreatitis. However, little is known about the nuclear events. The aim of this study was to explore activation of the transcription factor NF-κB/Rel after supramaximal stimulation with the cholecystokinin analogue cerulein in the pancreas. Methods & Results: Nuclear appearance of NF-κB/Rel–binding activity was detectable 15 minutes after cerulein injection. The DNA-binding activity consisted of NF-κB1 p50, NF-κB2 p52, and RelA p65 as judged by supershift assays and Western blot analysis. The onset and termination of NF-κB/Rel activation correlated with the degradation and reappearance of IκBα. Cerulein in supramaximal but not in physiological doses activated NF-κB/Rel in vitro. After blocking of NF-κB/Rel activation with pyrrolidine dithiocarbamate, the degree of morphological alterations was more pronounced than in controls, serum amylase and lactate dehydrogenase levels were significantly increased, and messenger RNA levels of pancreatitis-associated protein were more strongly induced, reflecting a more severe degree of pancreatitis. Similar results were obtained when N -acetyl-L-cysteine was used as an inhibitor of NF-κB activation. Conclusions: These data show that NF-κB/Rel is rapidly activated during cerulein pancreatitis. This activation may induce a self-defending genetic program before the onset of cellular injury, which might prevent higher degrees of damage of pancreatic acinar cells after secretagogue hyperstimulation. GASTROENTEROLOGY 1999;116:420-430

Acute Experimental Pancreatitis and NF-κB/Rel Activation

Acute pancreatitis is a serious disease with a high morbidity and an overall mortality rate of about 10%. However, in its most severe form, which is characterized by pancreatic necrosis, 20–30% of the patients die. Death is often the result of multiorgan dysfunction, including acute respiratory, kidney, and hepatic failure as well as generalized diffuse capillary leak water retention, hypoxia, and acid/base disturbance. The mechanisms by which distant organ systems are involved still remain obscure, but several lines of evidence suggest the participation of cytokines (IL-1, IL-6, and TNF-α) as a response to local tissue damage. A series of studies have now shed new light on the pivotal pathogenic role of the transcription factor NF-ĸB/Rel that binds to the promoter regions of many proinflammatory genes and regulates their transcription.

HCV and HGV in B-cell non-Hodgkin's lymphoma

BACKGROUND/AIMS A causative role of hepatitis C virus infection (HCV) has been discussed in the pathogenesis of mixed cryoglobulinaemia and in B-cell non-Hodgkins lymphoma. No data are available concerning the newly discovered hepatitis G virus (HGV) and extrahepatic manifestations such as haematological malignancies. But, HCV and HGV most probably belong to the same family of Flavivirus. Consequently, we looked for the prevalence of HCV, HGV and cryoglobulins in patients with B-cell non-Hodgkins lymphoma. METHODS Serum samples from 69 patients with non-Hodgkins lymphoma were studied. Diagnosis of non-Hodgkins lymphoma was established according to the Kiel classification. Active HCV- and HGV infections were investigated using polymerase chain reaction for detection of viral RNA. Cryoglobulins were detected from serum and monoclonal immunoglobulin components were analysed with immunofixation electrophoresis. In addition, we assessed the clinical course of HCV- and HGV-infected patients under chemotherapy. RESULTS Three of 69 (4.3%) patients with B-cell non-Hodgkins lymphoma were HCV-infected and nine non-Hodgkins lymphoma patients (13.0%) were positive for hepatitis G virus RNA. All HGV infected patients were suffering from low-grade non-Hodgkins lymphoma. No HGV-infected patient was co-infected by HCV and neither HCV- nor HGV-infected patients showed clinical signs of chronic liver disease before, during or after chemotherapy. Serum samples from all patients were devoid of cryoglobulins. CONCLUSIONS HCV seems to have no significance for the pathogenesis of non-Hodgkins lymphoma in Germany. The increased prevalence of hepatitis G (16.3%) in patients with low-grade non-Hodgkins lymphoma could suggest a pathological consequence of HGV infection outside of the liver. Evidence of clinically relevant hepatic disease in HGV infected patients was not obtained. Further, chemotherapy does not seem to affect the subsequent clinical course of HGV infection.

Enhancement of transforming growth factor β1 expression in the rat pancreas during regeneration from caerulein-induced pancreatitis

Abstract Synthesis of extracellular matrix components is enhanced in the rat pancreas during regeneration from caerulein‐induced pancreatitis. To study the involvement of transforming growth factor β1 (TGFβ1), one of the most potent modulators of the extracellular matrix, in the process of pancreatic regeneration we examined the expression of this gene on the transcript and protein level. Pancreatic RNA was extracted from rats killed 0h, 12h, 24h, 2, 3 and 7 days after induction of caerulein pancreatitis. Transcript levels for TGFβ1 were measured by slot–blot analysis and mRNA in situ hybridization. Total amount of TGFβ1‐protein was measured using a radioligand binding assay. TGFβ1 protein was increased twofold after 24h and 48h and returned to control values 7 days after induction of pancreatitis, TGFβ1‐mRNA reached maximal values (3‐fold over controls) after 2 days. The largest amount of TGFβ1‐mRNA was found in pancreatic acinar cells and in stromal cells. In summary, expression of TGFβ1 in acinar and stromal cells of the rat pancreas is enhanced during regeneration from caerulein‐induced pancreatitis, which may indicate an involvement of TGFβ1 in the regulation of extracellular matrix regeneration in the rat pancreas after caerulein‐induced pancreatitis.

Lipopolysaccharide-Activated Macrophages Stimulate the Synthesis of Collagen Type I and C-Fibronectin in Cultured Pancreatic Stellate Cells

We have recently identified and characterized pancreatic stellate cells (PSC) in rats and humans (Gastroenterology 1998, 15:421-435). PSC are suggested to represent the main cellular source of extracellular matrix in chronic pancreatitis. Now we describe a paracrine stimulatory loop between human macrophages and PSC (rat and human) that results in an increased extracellular matrix synthesis. Native and transiently acidified supernatants of cultured macrophages were added to cultured PSC in the presence of 0.1% fetal calf serum. Native supernatants of lipopolysaccharide-activated macrophages stimulated the synthesis of collagen type I 1.38 +/- 0.09-fold of control and c-fibronectin 1.89 +/- 0.18-fold of control. Transiently acidified supernatants stimulated collagen type I and c-fibronectin 2.10 +/- 0.2-fold and 2.80 +/- 0.05-fold of control, respectively. Northern blot demonstrated an increased expression of the collagen-I-(alpha-1)-mRNA and fibronectin-mRNA in PSC 10 hours after addition of the acidified macrophage supernatants. Cell proliferation measured by bromodeoxyuridine incorporation was not influenced by the macrophage supernatants. Unstimulated macrophages released 1.97 pg TGFbeta1/microgram of DNA over 24 hours and lipopolysaccharide-activated macrophages released 6.61pg TGFbeta1/microgram of DNA over 24 hours. These data together with the results that, in particular, transiently acidified macrophage supernatants increased matrix synthesis, identify TGFbeta as the responsible mediator. In conclusion, our data demonstrate a paracrine stimulation of matrix synthesis of pancreatic stellate cells via TGFbeta1 released by activated macrophages. We suggest that macrophages might play a pivotal role in the development of pancreas fibrosis.

Caerulein-induced NF-κB/Rel activation requires both Ca2+ and protein kinase C as messengers

The eukaryotic transcription factor NF-κB/Rel is activated by a large variety of stimuli. We have recently shown that NF-κB/Rel is induced during the course of caerulein pancreatitis. Here, we show that activation of NF-κB/Rel by caerulein, a CCK analog, requires increasing intracellular Ca2+ levels and protein kinase C activation. Caerulein induces a dose-dependent increase of nuclear NF-κB/Rel binding activity in pancreatic lobules, which is paralleled by degradation of IκBα. IκBβ was only slightly affected by caerulein treatment. Consistent with an involvement of Ca2+, the endoplasmic reticulum-resident Ca2+-ATPase inhibitor thapsigargin activated NF-κB/Rel in pancreatic lobules. The intracellular Ca2+ chelator TMB-8 prevented IκBα degradation and subsequent nuclear translocation of NF-κB/Rel induced by caerulein. BAPTA-AM was less effective. Cyclosporin A, a Ca2+/calmodulin-dependent protein phosphatase (PP2B) inhibitor, decreased caerulein-induced NF-κB/Rel activation and IκBα degradation. The inhibitory effect of bisindolylmaleimide suggests that protein kinase C activity is also required for caerulein-induced NF-κB/Rel activation. These data suggest that Ca2+- as well as protein kinase C-dependent mechanisms are required for caerulein-induced NF-κB/Rel activation.

Vasoactive mediators and the progression from oedematous to necrotising experimental acute pancreatitis.

Little is known about the pathophysiological factors that determine the clinical severity of acute pancreatitis. Because impairment of pancreatic circulation and oxygenation is associated with greater disease severity and morphological damage in experimental pancreatitis it has been suggested that various vasoactive mediators might participate in the progression from the oedematous to the necrotising variety of the disease. This study used an animal model of acute pancreatitis induced by intravenous caeruleint (10 micrograms/kg/h for up to six hours), which does not entail either haemorrhage or significant necrosis of the pancreas. This study considered whether the administration or the inhibition of either nitric oxide, bradykinin, or adrenergic mediators can convert this mild variety into haemorrhagic and necrotising pancreatitis. Neither nitric oxide nor catecholamines were involved in the progression from oedematous to haemorrhagic pancreatitis. Their substitution, activation, and inhibition all failed to change the severity of the disease process. Bradykinin alone seemed to be critically involved in the pathogenesis of pancreatic haemorrhage and necrosis. However, the inhibition of bradykinin and not its activation or substitution increased the severity of the disease.

Escitalopram for the Prevention of Peginterferon-α2a–Associated Depression in Hepatitis C Virus–Infected Patients Without Previous Psychiatric Disease: A Randomized Trial

BACKGROUND Depression is a major complication during treatment of chronic hepatitis C virus (HCV) infection with interferon-α (IFN-α). It is unclear whether antidepressants can prevent IFN-induced depression in patients without psychiatric risk factors. OBJECTIVE To examine whether preemptive antidepressant treatment with escitalopram can decrease the incidence or severity of depression associated with pegylated IFN-α in HCV-infected patients without a history of psychiatric disorders. DESIGN Randomized, multicenter, double-blind, prospective, placebo-controlled, parallel-group trial. (ClinicalTrials.gov registration number: NCT00136318) SETTING 10 university and 11 academic hospitals in Germany. PATIENTS 181 HCV-infected patients with no history of psychiatric disorders enrolled between August 2004 and December 2008. INTERVENTION Escitalopram, 10 mg/d (n = 90), or placebo (n = 91) administered 2 weeks before and for 24 to 48 weeks during antiviral therapy. MEASUREMENTS The primary end point was the incidence of depression, defined as a Montgomery-Asberg Depression Rating Scale (MADRS) score of 13 or higher. Secondary end points were time to depression, incidence of major depression according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, quality of life, sustained virologic response, tolerability, and safety. RESULTS 32% (95% CI, 21% to 43%) of the patients in the escitalopram group developed a MADRS score of 13 or higher compared with 59% (CI, 48% to 69%) in the placebo group (absolute difference, 27 percentage points [CI, 12 to 42 percentage points]; P < 0.001). Major depression was diagnosed in 8% of the patients in the escitalopram group and 19% in the placebo group (absolute risk difference, 11 percentage points [CI, 5 to 15 percentage points]; P = 0.031). Tolerability and safety parameters did not differ between the groups. In the escitalopram group, 56% (CI, 46% to 66%) of patients achieved a sustained virologic response compared with 46% (CI, 37% to 57%) in the placebo group (P = 0.21). LIMITATIONS Results might not be generalizable to patients with previous psychiatric disease. Some patients withdrew or developed temporary elevated MADRS scores after randomization but before the study medication was started. CONCLUSION Prophylactic antidepressant treatment with escitalopram was effective in reducing the incidence and severity of IFN-associated depression in HCV-infected patients without previous psychiatric disease. PRIMARY FUNDING SOURCE Roche Pharma and Lundbeck.

Role of Extracellular Matrix in Pancreatic Diseases

Accessible online at: http://BioMedNet.com/karger The extracellular matrix (ECM) has been traditionally thought of as the structurally stable material that provides support for cells and tissues. However, a number of discoveries over the past decades have changed this view. First, it has been shown that four major classes of macromolecules – the collagens, proteoglycans, structural glycoproteins, and elastin – collectively comprise the ECM of animal cells [1–6]. Furthermore, with the exception of elastin, each class of matrix macromolecules has been found to contain families of related proteins with each member being a unique gene product. Second, individual members of each class and family of ECM molecules were found to exhibit a degree of tissue-specific distribution implicating the matrix in development and tissue function [7, 8]. Third, specific cell-surface receptors for ECM components were identified, which provided a rational basis for linking the ECM with the cell [9–11]. From these discoveries it is now evident that the extracellular matrix is composed of a number of different macromolecules whose structural integrity and functional composition are important in maintaining normal tissue architecture, in development and in tissue-specific function [2, 4, 6, 8, 12]. Finally, it has been recognized that dysfunctional matrix components and abnormalities in ECM biosynthesis and catabolism are of importance in both inherited and acquired diseases and in normal wound healing [2, 3, 5, 6]. In particular pancreatic diseases as, e.g., pancreatic cancer and chronic pancreatitis are characterized by profound alterations of ECM formation and composition. The following review summarizes some of the major aspects that have emerged in the recent years concerning composition, formation and regulation of the ECM in human pancreatic diseases and in experimental models of pancreatic fibrosis.