Helicobacter ISSN 1523-5378 © 2009 Blackwell Publishing Ltd, Helicobacter 14: 177–179 177 Blackwell Publishing Ltd Oxford, UK HEL Helicobacter 1083-4389 1523-5378 © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd, Helicobacter XX: xx–xx XXX Original Article Neutrophil Activation by C-terminal HPNAP Region Kottakis et al. Letter to the Editor The C-terminal Region of HPNAP Activates Neutrophils and Promotes Their Adhesion to Endothelial Cells Filippos Kottakis,* Christina Befani,* Antonios Asiminas,* Maria Kontou,† Georgios Koliakos‡ and Theodora Choli-Papadopoulou* *Laboratory of Biochemistry, School of Chemistry, Aristotle University of Thessaloniki, 54124, Greece, †Department of Biochemistry and Biotechnology, University of Thessaly, Ploutonos 26,TK 41221 Larissa, Greece, ‡Department of Biological Chemistry, Medical School, Aristotle University of Thessaloniki, 54124, Greece Abstract Entire Helicobacter Pylori Neutrophil Activated Protein (HPNAP) and its truncated forms NH 2 -terminal region HPNAP 1–57 and C-terminal region HPNAP 58–144 after cloning into pET29c vector, purification and removal of LPS traces were subjected to human neutrophil activation. Our results revealed that the C-terminal region of HPNAP is indispensable for human neutrophil stimulation and their further adhesion to endothelial cells – a step necessary to H. pylori inflammation – in a ratio equal to that exhibited by the entire protein. In addition, experiments concerning the implication of Arabino-Galactan- Proteins (AGPs) derived from Chios Mastic Gum (CMG), the natural resin of the plant Pistacia lentiscus var. Chia revealed the inhibition of neutrophil activation and therefore their adhesion to endothelial cells, in vitro. Both, the involvement of HPNAP C-terminal region in stimulation-adhesion of neutrophils to endothelial cells as well as the inhibition of this process by AGPs have to be further investigated and may be exploited in a future anti- inflammatory therapy for H. pylori patients. Keywords HPNAP, Helicobacter, AGPs, anti-inflammatory therapy. Reprint request to: Theodora Choli-Papadopoulou, Laboratory of Biochemistry, School of Chemistry, Aristotle University of Thessaloniki, TK 54124, Thessaloniki, Greece. E-mail: tcholi@chem.auth.gr Helicobacter pylori infection is among the most common human infections and the major risk factor for peptic ulcer disease and gastric cancer. The H. pylori virulence factors are three conserved antigens, namely the vacillating cytotoxin A (VacA), the cytotoxin-associated antigen (GagA), and Helicobacter pylori – neutrophil-activating protein (HPNAP). HPNAP attracts and activates neutrophils, monocytes, and mast cells, resulting in the release of proinflammatory mediators [1]. The same molecule promotes Th1-type immune responses, likely acting as a Toll-like receptor-2 agonist [2]. It has been also reported that the broad C- terminal region of HPNAP stimulates neutrophil activation indicated by the production of reactive oxygen inter- mediates (ROIs) after nicotinamide adenine dinucleotide phosphate oxidase activation [3]. We present here evidence that the C-terminal region of HPNAP is indispensable for neutrophil adhesion to endo- thelial cells, a step necessary to H. pylori inflammation. In addition we show that arabino galactan proteins (AGPs) derived from Chios mastic gum (CMG), the natural resin of the plant Pistacia lentiscus var. Chia [4], inhibit neutrophil activation in vitro. Materials and Methods Cloning and Purification of the Entire HPNAP and its Truncation Forms The entire protein HPNAP as well as its truncated forms HPNAP 1–57 and HPNAP 58–144 were cloned into pET 29c vector and purified as previously described [3]. Endothelial Cell Preparation and Culture Endothelial cells were isolated from the umbilical cords of healthy newborns by the collagenase perfusion method as previously described by Jaffe et al. [5] with minor modifica- tions. In brief, the umbilical cord veins were carefully washed with sterile phosphate buffered saline (PBS) (0.9% NaCl, pH 7.4) and were then filled and incubated with Earle 199 medium that contained 0.5 μg/mL collagenase, for 10 minutes at room temperature. The content was then emptied in a clean, sterile falcon and the umbilical cord vein was washed with Earle 199 medium (without collagenase). The total content was then centrifuged for 10 minutes at 800 g and