Mokhtari Amirmajdi et al. : Apoptosis of Human Lymphocytes after Exposure …. Introduction Hdisease caused by the larval stage of ydatidosis is a zoonotic helminthic the platyhelminth parasite Echinococcus granulosus, which is highly prevalent in the Middle East, and especially in north of Iran (1, 2). Oncospheres hatching from eggs migrate to liver and several or- gans of the intermediate hosts (including man), and developed into hydatid cysts in a few weeks. In a fertile cyst, the germinal layer expresses many antigens that are important stimulators of the host immune re- sponse (3). The main antigens, Ag B and Ag 5, which are used for both diagnosis and fol- low-up (4), are released into the hydatid fluid (HF) (5, 6). Apoptosis is one of the main mechanism by which host defends against this parasite. The fertile hydatid cyst containing protoscolex is unfertilized through induction of an apop- totic cascade by the host (7, 8). On the other hand, E. granulosus is capable of modulat- ing immune responses of the host by several mechanisms (9-13). Some of these mecha- nisms are the interference with modulation of dendritic cells maturation (14), and induc- tion of a non-protective Th2 cell response by AgB (9). Apoptosis is another proposed mechanism (15) that provides suitable situa- tion for survival of the cyst by inducing apoptosis in the host immune cells. Several factors play role in apoptosis, but the cas- pase enzymes and Bcl-2 family are the two main families in this process. The first is a cascade of enzyme, of which caspase 3 is the most important member affecting the lymphocyte apoptosis. The second is Bcl-2 family, a set of cytoplasmic proteins mem- bers that regulate apoptosis (16). In this fam- ily, the most considerable components are Bcl-2 and Bax proteins. While Bcl-2 pro- teins inhibit apoptosis, Bax counteracts this (17). Furthermore, Macintyre et al. studied 10 the effect of E. granulosus hydatid fluid on the lymphocytes in 3-day cultures of the T- cell line, and suggested that the cytotoxicity of hydatid fluid could be resulted from cell- cycle arrest (18). This study aims to assess the in vitro apop- totic features of hydatid fluid on human lym- phocytes treated with fertile and infertile hy- datid fluid by evaluating the expression lev- els of Bcl-2, Bax mRNA as well as the Cas- pase-3 activity, which is the central enzyme in the apoptosis cascade (19). Materials and Methods In our study in Medical Mycoparasitology laboratory and Bu Ali Research Institute of Mashhad University of Medical Science 2008, four human hydatid cysts (two fertile and two infertile cysts) excised from the lower lobe of the lungs and the spleen were used to assess apoptotic effects of hydatid fluid on human lymphocytes. Fertile cysts were determined by the presence of free protoscoleces in the hydatid fluid and the growing protoscoleces attached to the germi- nal layer. The infertile cysts do not have these protoscoleces. After diagnosis by clini- cal examination and imaging techniques, hy- datid cysts were surgically removed un-rup- tured under sterile condition and were aspi- rated by syringe and stored at -70 °C for fur- ther analysis (Fig. 1). For later studies on Fas-L and TRAIL (as in- duced apoptotic molecules), the germinal and fibrous layers and adjacent normal tissue were dissected by scalpels and stored at -70 °C in sterile vials. Lymphocytes were sepa- rated from 20 ml blood sample of a healthy control by Ficoll-Hypaque gradient cen- trifug-ation method (20). Monocytes were removed by adhesion to plastic flasks in a RPMI-1640 medium in pH 7.4 at 37°C, hu- midified atmosphere of 5% CO2 for 2 hours.