Imre Ignáth

CFTR gene transfer to human cystic fibrosis pancreatic duct cells using a Sendai virus vector

Cystic fibrosis (CF) is a fatal inherited disease caused by the absence or dysfunction of the CF transmembrane conductance regulator (CFTR) Cl− channel. About 70% of CF patients are exocrine pancreatic insufficient due to failure of the pancreatic ducts to secrete a HCO  3− ‐rich fluid. Our aim in this study was to investigate the potential of a recombinant Sendai virus (SeV) vector to introduce normal CFTR into human CF pancreatic duct (CFPAC‐1) cells, and to assess the effect of CFTR gene transfer on the key transporters involved in HCO  3− transport. Using polarized cultures of homozygous F508del CFPAC‐1 cells as a model for the human CF pancreatic ductal epithelium we showed that SeV was an efficient gene transfer agent when applied to the apical membrane. The presence of functional CFTR was confirmed using iodide efflux assay. CFTR expression had no effect on cell growth, monolayer integrity, and mRNA levels for key transporters in the duct cell (pNBC, AE2, NHE2, NHE3, DRA, and PAT‐1), but did upregulate the activity of apical Cl−/HCO  3− and Na+/H+ exchangers (NHEs). In CFTR‐corrected cells, apical Cl−/HCO  3− exchange activity was further enhanced by cAMP, a key feature exhibited by normal pancreatic duct cells. The cAMP stimulated Cl−/HCO  3− exchange was inhibited by dihydro‐4,4′‐diisothiocyanostilbene‐2,2′‐disulfonic acid (H2‐DIDS), but not by a specific CFTR inhibitor, CFTRinh‐172. Our data show that SeV vector is a potential CFTR gene transfer agent for human pancreatic duct cells and that expression of CFTR in CF cells is associated with a restoration of Cl− and HCO  3− transport at the apical membrane. J. Cell. Physiol. 214: 442–455, 2008.

CFTR Expression But Not Cl- Transport Is Involved in the Stimulatory Effect of Bile Acids on Apical Cl-/HCO3- Exchange Activity in Human Pancreatic Duct Cells

Objectives: Low doses of chenodeoxycholate (CDC) stimulate apical anion exchange and HCO3− secretion in guinea pig pancreatic duct cells (Gut. 2008;57:1102-1112). We examined the effects of CDC on intracellular pH (pHi), intracellular Ca2+ concentration ([Ca2+]i), and apical Cl−/HCO3− exchange activity in human pancreatic duct cells and determined whether any effects were dependent on cystic fibrosis transmembrane conductance regulator (CFTR) expression and Cl− channel activity. Methods: Polarized CFPAC-1 cells (expressing F508del CFTR) were transduced with Sendai virus constructs containing complementary DNAs for either wild-type CFTR or &bgr;-galactosidase. Microfluorimetry was used to record pHi and [Ca2+]i and apical Cl−/HCO3− exchange activity. Patch clamp experiments were performed on isolated guinea pig duct cells. Results: Chenodeoxycholate induced a dose-dependent intracellular acidification and a marked increase in [Ca2+]i in CFPAC-1 cells. CFTR expression slightly reduced the rate of acidification but did not affect the [Ca2+]i changes. Luminal administration of 0.1 mmol/L of CDC significantly elevated apical Cl−/HCO3− exchange activity but only in cells that expressed CFTR. However, CDC did not activate CFTR Cl− conductance. Conclusions: Bile salts modulate pHi, [Ca2+]i, and apical anion exchange activity in human pancreatic duct cells. The stimulatory effect of CDC on anion exchangers requires CFTR expression but not CFTR channel activity.

Determination of 17α-hydroxylase-C17,20-lyase (P45017α) enzyme activities and their inhibition by selected steroidal picolyl and picolinylidene compounds

17α-hydroxylase-C17,20-lyase (P45017α) is a key regulator enzyme of the steroid hormone biosynthesis in both the adrenals and the testes. Inhibition of this enzyme can block androgen synthesis in an early step, and may thereby be useful in the treatment of several androgen-dependent diseases. We developed radio-substrate in vitro incubation methods for the determination of the distinct 17α-hydroxylase and C17,20-lyase activities of the enzyme using rat testicular homogenate as enzyme source. With this method we have studied the inhibiting activity of selected steroidal picolyl and picolinylidene compounds. Tests revealed a substantial inhibitory action of the 17-picolinyliden-androst-4-en-3-one compound.

X-ray structural analysis and antitumor activity of new salicylic acid derivatives

Tetrakis-, tris-, bis-, and mono salicylic acid derivatives 1–4 were synthesized by reaction of methyl 2-hydroxy benzoate (methyl salicylate) with 2,2-bis (hydroxymethyl) propane-1,3-diol (pentaerythritol) in the presence of sodium. Yields of different salicyloyloxy derivatives were changed by varying the molar ratios of reactants. For compounds 2 and 3, X-ray structure analysis was performed, as well as molecular energy minimization, to define their conformation in terms of their energy minima. Comparison of crystal and energy minimized structures for these two compounds (2 and 3) revealed that the intramolecular hydrogen bonds play an important role, stabilizing conformation of the most part of the molecule. The antioxidant activity and cytotoxicity of the synthesized derivatives were evaluated in a series of in vitro tests, as well as 17β-hydroxysteroid dehydrogenase type 2 inhibition potency. Tetrakis salicyloyloxy derivative 1 expressed the highest antioxidant potency, tris salicyloyloxy derivative 2 was the best inhibitor of 17βHSD2 enzyme, while bis salicyloyloxy derivative 3 showed strong cytotoxicity against prostate and breast cancer cells with no cytotoxicity against healthy cells.

Analysis of research activity in gastroenterology: Pancreatitis is in real danger

Objective Biomedical investment trends in 2015 show a huge decrease of investment in gastroenterology. Since academic research usually provides the basis for industrial research and development (R&D), our aim was to understand research trends in the field of gastroenterology over the last 50 years and identify the most endangered areas. Methods We searched for PubMed hits for gastrointestinal (GI) diseases for the 1965–2015 period. Overall, 1,554,325 articles were analyzed. Since pancreatology was identified as the most endangered field of research within gastroenterology, we carried out a detailed evaluation of research activity in pancreatology. Results In 1965, among the major benign GI disorders, 51.9% of the research was performed on hepatitis, 25.7% on pancreatitis, 21.7% on upper GI diseases and only 0.7% on the lower GI disorders. Half a century later, in 2015, research on hepatitis and upper GI diseases had not changed significantly; however, studies on pancreatitis had dropped to 10.7%, while work on the lower GI disorders had risen to 23.4%. With regard to the malignant disorders (including liver, gastric, colon, pancreatic and oesophageal cancer), no such large-scale changes were observed in the last 50 years. Detailed analyses revealed that besides the drop in research activity in pancreatitis, there are serious problems with the quality of the studies as well. Only 6.8% of clinical trials on pancreatitis were registered and only 5.5% of these registered trials were multicentre and multinational (more than five centres and nations), i.e., the kind that provides the highest level of impact and evidence level. Conclusions There has been a clear drop in research activity in pancreatitis. New international networks and far more academic R&D activities should be established in order to find the first therapy specifically for acute pancreatitis.