Béla Ózsvári

Chymotrypsin C (CTRC) variants that diminish activity or secretion are associated with chronic pancreatitis.

Chronic pancreatitis is a persistent inflammatory disease of the pancreas, in which the digestive protease trypsin has a fundamental pathogenetic role. Here we have analyzed the gene encoding the trypsin-degrading enzyme chymotrypsin C (CTRC) in German subjects with idiopathic or hereditary chronic pancreatitis. Two alterations in this gene, p.R254W and p.K247_R254del, were significantly overrepresented in the pancreatitis group, being present in 30 of 901 (3.3%) affected individuals but only 21 of 2,804 (0.7%) controls (odds ratio (OR) = 4.6; confidence interval (CI) = 2.6–8.0; P = 1.3 × 10−7). A replication study identified these two variants in 10 of 348 (2.9%) individuals with alcoholic chronic pancreatitis but only 3 of 432 (0.7%) subjects with alcoholic liver disease (OR = 4.2; CI = 1.2–15.5; P = 0.02). CTRC variants were also found in 10 of 71 (14.1%) Indian subjects with tropical pancreatitis but only 1 of 84 (1.2%) healthy controls (OR = 13.6; CI = 1.7–109.2; P = 0.0028). Functional analysis of the CTRC variants showed impaired activity and/or reduced secretion. The results indicate that loss-of-function alterations in CTRC predispose to pancreatitis by diminishing its protective trypsin-degrading activity.

Effects of bile acids on pancreatic ductal bicarbonate secretion in guinea pig

Background and aims: Acute pancreatitis is associated with significant morbidity and mortality. Bile reflux into the pancreas is a common cause of acute pancreatitis and, although the bile can reach both acinar and ductal cells, most research to date has focused on the acinar cells. The aim of the present study was to investigate the effects of bile acids on HCO3− secretion from the ductal epithelium. Methods: Isolated guinea pig intralobular/interlobular pancreatic ducts were microperfused and the effects of unconjugated chenodeoxycholate (CDC) and conjugated glycochenodeoxycholate (GCDC) on intracellular calcium concentration ([Ca2+]i) and pH (pHi) were measured using fluorescent dyes. Changes of pHi were used to calculate the rates of acid/base transport across the duct cell membranes. Results: Luminal administration of a low dose of CDC (0.1 mM) stimulated ductal HCO3− secretion, which was blocked by luminal H2DIDS (dihydro-4,4′-diisothiocyanostilbene-2,2′-disulfonic acid). In contrast, both luminal and basolateral administration of a high dose of CDC (1 mM) strongly inhibited HCO3− secretion. Both CDC and GCDC elevated [Ca2+]i, and this effect was blocked by BAPTA-AM (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid), caffeine, xestospongin C and the phospholipase C inhibitor U73122. BAPTA-AM also inhibited the stimulatory effect of low doses of CDC on HCO3− secretion, but did not modulate the inhibitory effect of high doses of CDC. Conclusions: It is concluded that the HCO3− secretion stimulated by low concentrations of bile acids acts to protect the pancreas against toxic bile, whereas inhibition of HCO3− secretion by high concentrations of bile acids may contribute to the progression of acute pancreatitis.

Neutrophils are required for both the sensitization and elicitation phase of contact hypersensitivity

Weber et al. report that neutrophils are required for both the sensitization and elicitation phase of contact hypersensitivity. Their results identify a novel role for neutrophils in shaping the adaptive immune response.

Trypsin Reduces Pancreatic Ductal Bicarbonate Secretion by Inhibiting CFTR Cl− Channels and Luminal Anion Exchangers

BACKGROUND & AIMS The effects of trypsin on pancreatic ductal epithelial cells (PDECs) vary among species and depend on the localization of proteinase-activated receptor 2 (PAR-2). We compared PAR-2 localization in human and guinea-pig PDECs, and used isolated guinea pig ducts to study the effects of trypsin and a PAR-2 agonist on bicarbonate secretion. METHODS PAR-2 localization was analyzed by immunohistochemistry in guinea pig and human pancreatic tissue samples (from 15 patients with chronic pancreatitis and 15 without pancreatic disease). Functionally, guinea pig PDECs were studied by microperfusion of isolated ducts, measurements of intracellular pH and intracellular Ca(2+) concentration, and patch clamp analysis. The effect of pH on trypsinogen autoactivation was assessed using recombinant human cationic trypsinogen. RESULTS PAR-2 localized to the apical membrane of human and guinea pig PDECs. Trypsin increased intracellular Ca(2+) concentration and intracellular pH and inhibited secretion of bicarbonate by the luminal anion exchanger and the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. Autoactivation of human cationic trypsinogen accelerated when the pH was reduced from 8.5 to 6.0. PAR-2 expression was strongly down-regulated, at transcriptional and protein levels, in the ducts of patients with chronic pancreatitis, consistent with increased activity of intraductal trypsin. Importantly, in PAR-2 knockout mice, the effects of trypsin were markedly reduced. CONCLUSIONS Trypsin reduces pancreatic ductal bicarbonate secretion via PAR-2-dependent inhibition of the apical anion exchanger and the CFTR Cl(-) channel. This could contribute to the development of chronic pancreatitis by decreasing luminal pH and promoting premature activation of trypsinogen in the pancreatic ducts.

The effect of sucrose esters on a culture model of the nasal barrier

Sucrose esters are effective solubilizers and there is an interest to use them as pharmaceutical excipients for nasal drug delivery. We have determined for the first time the non-toxic doses of laurate and myristate sucrose esters by four independent methods, and their effects on epithelial permeability using RPMI 2650 human nasal epithelial cell line. Based on real-time cell electronic sensing, MTT dye conversion and lactate dehydrogenase release methods reference surfactant Cremophor RH40 proved to be the least toxic excipient, and could be used at 5mg/mL concentration for 1h in epithelial cells without cellular damage. The non-toxic dose of Tween 80 was 1 mg/mL, while the dose of laurate and myristate sucrose esters that could be safely used on cells for 1 h was 0.1 mg/mL. Both the reference surfactants and the sucrose esters significantly enhanced the permeability of epithelial cell layers for the paracellular marker FITC-labelled 4.4 kDa dextran at 0.1 mg/mL concentration. The effects of sucrose esters on epithelial permeability were dose-dependent. These data indicate that laurate and myristate sucrose esters can be potentially used as permeability enhancers in nasal formulations to augment drug delivery to the systemic circulation.

A cell-microelectronic sensing technique for the screening of cytoprotective compounds.

In recent years, a new cell-based high throughput paradigm has emerged, which seeks to identify novel, pharmacologically active cytoprotective compounds. The essence of this approach is to create experimental models of cell injury relevant for a particular disease by establishing in vitro cell-based models, followed by high-throughput testing of compounds that affect the cellular response in a desired manner. Prior approaches typically used simple end-point analyses. To assess the cytoprotective effects of novel drug candidates in real-time, we have applied a cell-microelectronic sensing technique (RT-CES), which measures changes in the impedance of individual microelectronic wells that correlates linearly with cell index (reflecting cell number, adherence and cell growth), thereby allowing the continuous determination of cell viability during oxidative stress. In vitro cytotoxicity was elicited by hydrogen peroxide in myocytes (H9c2) and hepatocytes (Hep3B). Cells were post-treated at 30 min with various reference molecules and novel cytoprotective compounds. Cytoprotection detected in the RT-CES system correlated well with the results of two classical end-point-based methods (improvement in MTT and reduction of LDH release). The RT-CES method, when used as described in the current report, is suitable for the screening of molecular libraries to identify molecules or molecule combinations that attenuate oxidative stress-induced cell damage.

Polyunsaturated fatty acids synergize with lipid droplet binding thalidomide analogs to induce oxidative stress in cancer cells.

BackgroundCytoplasmic lipid-droplets are common inclusions of eukaryotic cells. Lipid-droplet binding thalidomide analogs (2,6-dialkylphenyl-4/5-amino-substituted-5,6,7-trifluorophthalimides) with potent anticancer activities were synthesized.ResultsCytotoxicity was detected in different cell lines including melanoma, leukemia, hepatocellular carcinoma, glioblastoma at micromolar concentrations. The synthesized analogs are non-toxic to adult animals up to 1 g/kg but are teratogenic to zebrafish embryos at micromolar concentrations with defects in the developing muscle. Treatment of tumor cells resulted in calcium release from the endoplasmic reticulum (ER), induction of reactive oxygen species (ROS), ER stress and cell death. Antioxidants could partially, while an intracellular calcium chelator almost completely diminish ROS production. Exogenous docosahexaenoic acid or eicosapentaenoic acid induced calcium release and ROS generation, and synergized with the analogs in vitro, while oleic acid had no such an effect. Gene expression analysis confirmed the induction of ER stress-mediated apoptosis pathway components, such as GADD153, ATF3, Luman/CREB3 and the ER-associated degradation-related HERPUD1 genes. Tumor suppressors, P53, LATS2 and ING3 were also up-regulated in various cell lines after drug treatment. Amino-phthalimides down-regulated the expression of CCL2, which is implicated in tumor metastasis and angiogenesis.ConclusionsBecause of the anticancer, anti-angiogenic action and the wide range of applicability of the immunomodulatory drugs, including thalidomide analogs, lipid droplet-binding members of this family could represent a new class of agents by affecting ER-membrane integrity and perturbations of ER homeostasis.

Sucrose Esters Increase Drug Penetration, But Do Not Inhibit P-Glycoprotein in Caco-2 Intestinal Epithelial Cells

Sucrose fatty acid esters are increasingly used as excipients in pharmaceutical products, but few data are available on their toxicity profile, mode of action, and efficacy on intestinal epithelial models. Three water-soluble sucrose esters, palmitate (P-1695), myristate (M-1695), laurate (D-1216), and two reference absorption enhancers, Tween 80 and Cremophor RH40, were tested on Caco-2 cells. Caco-2 monolayers formed a good barrier as reflected by high transepithelial resistance and positive immunostaining for junctional proteins claudin-1, ZO-1, and β-catenin. Sucrose esters in nontoxic concentrations significantly reduced resistance and impedance, and increased permeability for atenolol, fluorescein, vinblastine, and rhodamine 123 in Caco-2 monolayers. No visible opening of the tight junctions was induced by sucrose esters assessed by immunohistochemistry and electron microscopy, but some alterations were seen in the structure of filamentous actin microfilaments. Sucrose esters fluidized the plasma membrane and enhanced the accumulation of efflux transporter ligands rhodamine 123 and calcein AM in epithelial cells, but did not inhibit the P-glycoprotein (P-gp)-mediated calcein AM accumulation in MES-SA/Dx5 cell line. These data indicate that in addition to their dissolution-increasing properties sucrose esters can enhance drug permeability through both the transcellular and paracellular routes without inhibiting P-gp.

Curcumin and Its Analogue Induce Apoptosis in Leukemia Cells and Have Additive Effects with Bortezomib in Cellular and Xenograft Models

Combination therapy of bortezomib with other chemotherapeutics is an emerging treatment strategy. Since both curcumin and bortezomib inhibit NF-κB, we tested the effects of their combination on leukemia cells. To improve potency, a novel Mannich-type curcumin derivative, C-150, was synthesized. Curcumin and its analogue showed potent antiproliferative and apoptotic effects on the human leukemia cell line, HL60, with different potency but similar additive properties with bortezomib. Additive antiproliferative effects were correlated well with LPS-induced NF-κB inhibition results. Gene expression data on cell cycle and apoptosis related genes, obtained by high-throughput QPCR, showed that curcumin and its analogue act through similar signaling pathways. In correlation with in vitro results similar additive effect could be obsereved in SCID mice inoculated systemically with HL60 cells. C-150 in a liposomal formulation given intravenously in combination with bortezomib was more efficient than either of the drugs alone. As our novel curcumin analogue exerted anticancer effects in leukemic cells at submicromolar concentration in vitro and at 3 mg/kg dose in vivo, which was potentiated by bortezomib, it holds a great promise as a future therapeutic agent in the treatment of leukemia alone or in combination.

Extracellular deposition of matrilin-2 controls the timing of the myogenic program during muscle regeneration.

ABSTRACT Here, we identify a role for the matrilin-2 (Matn2) extracellular matrix protein in controlling the early stages of myogenic differentiation. We observed Matn2 deposition around proliferating, differentiating and fusing myoblasts in culture and during muscle regeneration in vivo. Silencing of Matn2 delayed the expression of the Cdk inhibitor p21 and of the myogenic genes Nfix, MyoD and Myog, explaining the retarded cell cycle exit and myoblast differentiation. Rescue of Matn2 expression restored differentiation and the expression of p21 and of the myogenic genes. TGF-&bgr;1 inhibited myogenic differentiation at least in part by repressing Matn2 expression, which inhibited the onset of a positive-feedback loop whereby Matn2 and Nfix activate the expression of one another and activate myoblast differentiation. In vivo, myoblast cell cycle arrest and muscle regeneration was delayed in Matn2−/− relative to wild-type mice. The expression levels of Trf3 and myogenic genes were robustly reduced in Matn2−/− fetal limbs and in differentiating primary myoblast cultures, establishing Matn2 as a key modulator of the regulatory cascade that initiates terminal myogenic differentiation. Our data thus identify Matn2 as a crucial component of a genetic switch that modulates the onset of tissue repair.