Abstract

Bratko Filipič40808*, Sandor Toth40809, Lidija Gradišnik40810, Adriana Pereyra40811 and Hrvoje Mazija40812

As a type I Interferon’s, Human leukocyte Interferonalfa (HuIFN-αN3) is a complex pleiotropic molecule composed from several natural subtypes, showing: Antiviral, proor anti-inflammatory and antiproliferative activity in vitro. The present work was aimed to detect, analyze and compare the natural subtype’s composition of HuIFN-αN3 from EGIS (Budapest, Hungary) or IMZ (Zagreb, Croatia) and to measure theirs proor anti-inflammatory, antiproliferative and cytocidal activity in vitro. HuIFN-αN3 from EGIS, (Budapest, Hungary) or IMZ, (Zagreb, Croatia) contains seven “minor” (a-g) and eight “major” (1-8) subtypes with the isoelectric points: 8.12, 7.40, 7.02, 6.90, 6.45, 6.06, 4.45, and 5.80, 5.65, 5.50, 5.30, 5.05, 4.88, 4.72, 4.53. Subtypes show different pro and anti-inflammatory effects. Those separated in the range of pI 8.0 to 6.0 show pro-inflammatory activity. Those separated in the range 5.80 to 3.50 show anti-inflammatory activity. The subtypes isolated from HuIFN-αN3 (IMZ, Zagreb, Croatia) show very weak pro and strong anti-inflammatory activity. The strongest antiinflammatory activity has subtype 3. The antiproliferative assays on Human Embryonic Fibroblasts (HEF) or Human amnion cells line (FL) cells shows, that the total antiproliferative activity of HuIFN-αN3 is bigger than this from different subtypes, with the exception of subtype a. The cytocidal activity measured as IU/ml needed to get the 50% cytocidal effect on the Adult pig kidney cell line (PLA) cells shows: In the subtype a with 78.1, 1 with 312.5, 2 with 625, 3 with 0, 4 with 1.250, 5 with 0, 6 with 2.500, 7 with 625 and 8 with 0. When different HuIFN-αN3’s were tested, this from IMZ (Zagreb, Croatia) and SAV (Bratislava, Slovakia) has 156.2. HuIFN-αN3 from EGIS, (Budapest, Hungary) has 312.5, HuIFN-γ has 156.2, rHuIFN-α1 (recombinant Interferon's) has 2500 and rHuIFN-α2 has 5000. The results show, that all of the subtypes (a-g, 1-8) can be neutralized with the polyclonal anti-IFN-αN3. The values of NI were in the range from: -1.15 till -2.21. It can be concluded that this is the picture of different natural subtypes’ content in both preparations from EGIS or IMZ because of different technology. EGIS use concentrated purified preparation, while IMZ use concentrated non purified one.