Ludwig Jonas

Pancreatic fibrosis in experimental pancreatitis induced by dibutyltin dichloride

BACKGROUND & AIMS 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. METHODS 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. RESULTS 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. CONCLUSIONS 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.

Multi-walled carbon nanotubes induce oxidative stress and apoptosis in human lung cancer cell line-A549

Abstract Multi-walled carbon-nanotubes (MWCNTs)-induced apoptotic changes were studied in human lung epithelium cell line-A549. Non-cytotoxic doses of MWCNTs were identified using tetrazolium bromide salt (MTT) and lactate dehydrogenase (LDH) release assays. Cells were exposed to MWCNTs (0.5–100 μg/ml) for 6–72 h. Internalization and characterization of CNTs was performed by electron microscopy. Apoptotic changes were estimated by nuclear condensation, DNA laddering, and confirmed by expression of associated markers: p53, p21WAF1/CIP1, Bax, Bcl2 and activated caspase-3. MWCNTs induced the production of reactive oxygen species and malondialdehyde along with significant decrease in the activity of catalase and glutathione. MWCNTs-induced ROS generation was found not to be associated with the mitochondrial activity. In general, the changes were significant at 10 and 50 μg/ml only. Results indicate the involvement of oxidative stress and apoptosis in A549 cells exposed to MWCNTs. Our studies provide insights of the mechanisms involved in MWCNTs-induced apoptosis at cellular level.

Nanoparticles Induce Changes of the Electrical Activity of Neuronal Networks on Microelectrode Array Neurochips

Background Nanomaterials are extensively used in industry and daily life, but little is known about possible health effects. An intensified research regarding toxicity of nanomaterials is urgently needed. Several studies have demonstrated that nanoparticles (NPs; diameter < 100 nm) can be transported to the central nervous system; however, interference of NPs with the electrical activity of neurons has not yet been shown. Objectives/methods We investigated the acute electrophysiological effects of carbon black (CB), hematite (Fe2O3), and titanium dioxide (TiO2) NPs in primary murine cortical networks on microelectrode array (MEA) neurochips. Uptake of NPs was studied by transmission electron microscopy (TEM), and intracellular formation of reactive oxygen species (ROS) was studied by flow cytometry. Results The multiparametric assessment of electrical activity changes caused by the NPs revealed an NP-specific and concentration-dependent inhibition of the firing patterns. The number of action potentials and the frequency of their patterns (spike and burst rates) showed a significant particle-dependent decrease and significant differences in potency. Further, we detected the uptake of CB, Fe2O3, and TiO2 into glial cells and neurons by TEM. Additionally, 24 hr exposure to TiO2 NPs caused intracellular formation of ROS in neuronal and glial cells, whereas exposure to CB and Fe2O3 NPs up to a concentration of 10 μg/cm2 did not induce significant changes in free radical levels. Conclusion NPs at low particle concentrations are able to exhibit a neurotoxic effect by disturbing the electrical activity of neuronal networks, but the underlying mechanisms depend on the particle type.

Acute interstitial pancreatitis in rats induced by dibutyltin dichloride (DBTC): pathogenesis and natural course of lesions.

Dibutyltin dichloride (DBTC; 6 mg/kg body weight, i.v.) induced acute interstitial pancreatitis in rats. The course of the pancreatitis was examined within 28 days by light and electron microscopy as well as by pathobiochemistry (amylase, lipase, alkaline phosphatase, and bilirubin in serum; tin concentration in biliopancreatic juice, tissue, and concretions). The pathogenesis of the DBTC-induced pancreatitis in rats was studied by different experimental designs (in intact animals, after bile duct ligation, after surgical bypass of the bile duct). DBTC caused toxic necrosis of the biliopancreatic duct epithelium, which is then shed into the duct and forms obstructing plugs in the distal common bile duct. Interstitial pancreatitis occurred during the first 4 days, accompanied by significantly increased activities of serum α-amylase and lipase. After 7 days extensive infiltration of the pancreatic interstitium with mononuclear cells was observed. Twenty-eight days after administration of DBTC one-third of the rats showed periductal and interstitial fibrosis as well as an active inflammatory process in the pancreas. The findings suggest a twofold pathogenesis of the DBTC-induced pancreatitis: first, the cytotoxic effects on the biliopancreatic duct epithelium lead to epithelial necrosis with obstruction of the duct, subsequent cholestasis, and interstitial pancreatitis; and second, the hematogenic DBTC effects cause direct injury of pancreatic cells (mitochondrial damage, autophagy, cell necrosis) followed by interstitial edema and inflammation. Both processes lead to this special type of DBTC-induced acute pancreatitis with a tendency to a chronic course, when the obstruction of the duct and cholestasis persist.

Effect of 3D-scaffold formation on differentiation and survival in human neural progenitor cells

Background3D-scaffolds have been shown to direct cell growth and differentiation in many different cell types, with the formation and functionalisation of the 3D-microenvironment being important in determining the fate of the embedded cells. Here we used a hydrogel-based scaffold to investigate the influences of matrix concentration and functionalisation with laminin on the formation of the scaffolds, and the effect of these scaffolds on human neural progenitor cells cultured within them.MethodsIn this study we used different concentrations of the hydrogel-based matrix PuraMatrix. In some experiments we functionalised the matrix with laminin I. The impact of concentration and treatment with laminin on the formation of the scaffold was examined with atomic force microscopy. Cells from a human fetal neural progenitor cell line were cultured in the different matrices, as well as in a 2D culture system, and were subsequently analysed with antibody stainings against neuronal markers. In parallel, the survival rate of the cells was determined by a live/dead assay.ResultsAtomic force microscopy measurements demonstrated that the matrices are formed by networks of isolated PuraMatrix fibres and aggregates of fibres. An increase of the hydrogel concentration led to a decrease in the mesh size of the scaffolds and functionalisation with laminin promoted aggregation of the fibres (bundle formation), which further reduces the density of isolated fibres. We showed that laminin-functionalisation is essential for human neural progenitor cells to build up 3D-growth patterns, and that proliferation of the cells is also affected by the concentration of matrix. In addition we found that 3D-cultures enhanced neuronal differentiation and the survival rate of the cells compared to 2D-cultures.ConclusionsTaken together, we have demonstrated a direct influence of the 3D-scaffold formation on the survival and neuronal differentiation of human neural progenitor cells. These findings emphasize the importance of optimizing 3D-scaffolds protocols prior to in vivo engraftment of stem and progenitor cells in the context of regenerative medicine.

Increased cytosolic Ca2+ amplifies oxygen radical-induced alterations of the ultrastructure and the energy metabolism of isolated rat pancreatic acinar cells

Background: Oxygen radicals have been implicated as important mediators in the early pathogenesis of acute pancreatitis, but the mechanism by which they produce pancreatic tissue injury remains unclear. We have, therefore, investigated the effects of oxygen radicals on isolated rat pancreatic acinar cells as to the ultrastructure, cytosolic Ca2+ concentration and energy metabolism. Methods: Acinar cells were exposed to an oxygen radical-generating system consisting of xanthine oxidase, hypoxanthine and chelated iron ions. Cell injury was assessed by LDH release and electron microscopy. Cytosolic Ca2+ levels and mitochondrial membrane potential were determined by flow cytometry; adenine nucleotide concentrations by HPLC. Mitochondrial dehydrogenase activity was measured by spectrophotometric assay. Results: Oxygen radicals damaged the plasma membrane as shown by a 6-fold LDH increase in the incubation medium within 180 min. At the ultrastructural level, mitochondria were the most susceptible to oxidative stress. In correlation to the pronounced mitochondrial damage, the mitochondrial dehydrogenase activity declined by 70%, whereas the mitochondrial membrane potential was enhanced by 27% after 120 min. Together this may cause the 85% decrease in the ATP concentration and the corresponding increase in ADP/AMP observed in parallel. In addition, an immediate 26% increase in cytosolic Ca2+ was found, a change which could be inhibited by BAPTA, reducing cellular damage. Conclusion: Cytosolic Ca2+ synergizes with oxygen radicals causing alterations of the ultrastructure and energy metabolism of acinar cells which might contribute to the cellular changes found in early stages of acute pancreatitis.

Caerulein pancreatitis increases mRNA but reduces protein levels of rat pancreatic heat shock proteins

We have recently reported that preconditioning through hyperthermia induces expression of pancreatic heat shock proteins (HSPs) and protects against caerulein pancreatitis. Here, we investigate caerulein-mediated effects on pancreatic HSPs without prior hyperthermia. Caerulein time and dose dependently increased pancreatic mRNA levels of the constitutive isoform of HSP70 (HSC70). However, pancreatic HSC70 protein levels were decreased, as were HSP60 protein levels. Also, in contrast to hyperthermia preconditioning, caerulein did not induce measurable levels of mRNA or protein of the inducible isoform of HSP70. Thus the pancreas reacts to different kinds of stress (hyperthermia vs. hyperstimulation) with differential induction of HSP mRNAs. Clearly, hyperthermia leads to induction of HSP protein expression, whereas caerulein treatment does not. Therefore, our current study further supports the idea that hyperthermia-induced protection against caerulein pancreatitis may be mediated through increased protein levels of pancreatic HSPs. It is further tempting to hypothesize that failure to appropriately increase HSP protein levels in response to high doses of caerulein might be a factor in the development of pancreatitis.We have recently reported that preconditioning through hyperthermia induces expression of pancreatic heat shock proteins (HSPs) and protects against caerulein pancreatitis. Here, we investigate caerulein-mediated effects on pancreatic HSPs without prior hyperthermia. Caerulein time and dose dependently increased pancreatic mRNA levels of the constitutive isoform of HSP70 (HSC70). However, pancreatic HSC70 protein levels were decreased, as were HSP60 protein levels. Also, in contrast to hyperthermia preconditioning, caerulein did not induce measurable levels of mRNA or protein of the inducible isoform of HSP70. Thus the pancreas reacts to different kinds of stress (hyperthermia vs. hyperstimulation) with differential induction of HSP mRNAs. Clearly, hyperthermia leads to induction of HSP protein expression, whereas caerulein treatment does not. Therefore, our current study further supports the idea that hyperthermia-induced protection against caerulein pancreatitis may be mediated through increased protein levels of pancreatic HSPs. It is further tempting to hypothesize that failure to appropriately increase HSP protein levels in response to high doses of caerulein might be a factor in the development of pancreatitis.

Oxygen Radical Generation and Acute Pancreatitis : Effects of Dibutyltin Dichloride/Ethanol and Ethanol on Rat Pancreas

Recent studies suggest that enhanced release of free oxygen radicals plays an important role in the pathogenesis of acute pancreatitis. Therefore, we studied the activity of the oxygen radical generating xanthine ox-idase (XOD) in pancreatic tissue from rats treated with either dibutyltin dichloride/ethanol (DBTCiEtOH: 6 mg kg−1/13.7 mg kg−I, i.v.), ethanol alone (EtOH: 13.7 mmol kg−1, i.v.), or isotonic saline (NaCl) as control. We also investigated activities of the oxygen radical scavengers superoxide dismutase (SOD) and glutathione peroxidase (GPX). In addition, levels of the lipid peroxidation marker malondialdehyde (MDA) were determined. Enhanced activity of XOD was not detected. While SOD activity 1 and 6 h after treatment was significantly more reduced by DBTC/EtOH than by EtOH alone, no difference was found thereafter. Correspondingly, both regimens diminished GPX activity. Moreover, DBTC/EtOH and EtOH rapidly increased MDA levels within 1 h, in dicating release of oxygen radicals early on after administration. After 16 h the MDA concentration was still elevated only in the DBTCiEtOH group. Although similar metabolic alterations were observed in both groups, only DBTCiEtOH induced acute interstitial pancreatitis within 24 h. We conclude that (a) a tissue imbalance between oxidants and antioxidants might be of importance in the pathogenesis of DBTCiEtOH-induced acute interstitial pancreatitis; (b) although EtOH increases oxygen radical levels, additional damage is required for development of acute pancreatitis; (c) XOD does not seem to be responsible for significant oxygen radical generation; and (d) the DBTC/EtOH model is a useful tool to study acute interstitial pancreatitis in rats.

Heat shock response is associated with protection against acute interstitial pancreatitis in rats.

We recently reported that hyperthermia induces pancreatic expression of heat shock proteins (HSPs), particularly HSP70 isoforms, and protects against cerulein pancreatitis. We have now studied whether a double hyperthermia amplifies these effects and whether hyperthermia also protects against dibutyltin dichloride (DBTC)-induced pancreatitis. A further aim was to examine whether hyperthermia induces changes in transforming growth factor-β1 (TGF-β1). Following pretreatment without or with a single or double hyperthermia, pancreatitis was induced by application of cerulein or DBTC. Pancreatic HSP and TGF-β1 expression were studied by immunoblotting. Pancreas injury was assessed by light microscopy and serum pancreatic enzyme activity. Hyperthermia as well as DBTC induced HSP72, whereas cerulein did not. A double hyperthermia led to a further increase in HSP72 compared to a single heat stress. In both models, hyperthermia significantly reduced pancreatic injury. Although a double hyperthermia slightly decreased the severity of cerulein pancreatitis compared to a single heat treatment, an improved pancreas protection against DBTC cytotoxicity was not achieved. We also found that hyperthermia induces the expression of TGF-β1. In conclusion, hyperthermia preconditioning exerts protective effects against two pathophysiologically different types of pancreatitis by a mechanism that involves the up-regulation of HSP70 isoforms as well as TGF-β1.

The mtDNA nt7778 G/T polymorphism affects autoimmune diseases and reproductive performance in the mouse

Mitochondria are organelles of all nucleated cells, and variations in mtDNA sequence affect a wide spectrum of human diseases. However, animal models for mtDNA-associated diseases are rare, making it challenging to explore mechanisms underlying the contribution of mitochondria. Here, we identify a polymorphism in the mitochondrial genome, G-to-T at position 7778, which results in an aspartic acid-to-tyrosine (D-Y) substitution in the fifth amino acid of the highly conserved N-terminus of ATP synthase 8 (ATP8). Using a series of conplastic strains we show that this polymorphism increases susceptibility to multiple autoimmune diseases, including collagen-induced arthritis, autoimmune diabetes, nephritis and autoimmune pancreatitis. In addition, it impairs reproductive performance in females, but only in the MRL/MpJ strain. We also demonstrate that the mtAtp8 polymorphism alters mitochondrial performance, increasing H(2)O(2) production and affecting mitochondrial structure. Functional analysis reveals that the polymorphism increase the CD4 T cell adaptive potential to an oxidative phosphorylation impaired condition. Our findings provide direct experimental evidence for the role of mitochondria in autoimmunity and reproduction.