Iranian J Parasitol: Vol. 7, No.1, 2012, pp. 18-25 isolated from the culture of each patient’s lesion were centrifuged at 700 × g, 4ºC for 10min, and then were washed 3 times with PBS. DNA was extracted using High Pure PCR Template Preparation Kit (Roche, Ger- many). Purified DNA was eluted in 200μl of elution buffer and stored in -20ºC until use. DNA from L. major (MRHO/IR/75/ER) was used as a control. PCR amplification Specific oligonucleotide primer was designed based on GenBank accession no. AJ300485, (Forward: CAA CAC GCC GCC TCC TCT CT, Reverse: CCT CTC TTT TTT CGC TGT GC) (Bioneer). Amplifications were performed in 25 μl containing, 2.5μl PCR buffer(10xbuffer), 0.75mM MgCl2 (50mM), 0.5 mM dNTP (10mM), 5pmol of each primer, 0.25unit of Taq polymerase (5u/ μl) and 1μl of DNA template. DNA was amplified using thermal cycler (Cor- bett) under the following conditions: at 94ºC for 5min followed by 25 repetitive cycles of 30s at 94ºC, 30s at 54ºC, 30s at 72 ºC and a final elon- gation at 72ºC for 10min. PCR products were run on 1.5% agarose gel in TBE buffer. The bands were visualized under UV illumination and the size of the products was determined. Isolation of mononuclear cells Peripheral Blood Mononuclear Cells (PBMCs) were isolated using Ficoll-hypaque density gra- dient (Lymphodex, Germany). PBMCs were washed 3 times with PBS and the pellet was resuspended in 4 ml of RPMI 1640 medium (Gibco, Germany), supplemented with 2mM L- glutamine, penicillin (100u/ml), gentamicin (100μg/ml) and 10% heat-inactivated Fetal Bo- vine Serum (Gibco). The cell number was esti- mated using light microscopy and the viability of the cells was checked by trypan blue (0.4%). PBMCs were distributed in two 3.5 cm tissue culture plates with a concentration of 5x106 cell/ml in each plate and incubated at 37ºC with 5% CO2 for 2 hours and then non adherent cells 20 were removed by washing with PBS. Adherent cells were incubated at 37ºC with 5% CO2 for additional 6 days in order to differentiate to macrophages (24) and the medium was changed every 72h. The size of the cells was morphologically increased and numerous short pseudopods were formed. The cellular pheno- type was analyzed by flowcytometry using anti CD14 monoclonal antibody (Abcam, United Kingdom) (data not shown). Parasite L. major (MRHO/IR/75/ER), was used for leishmanization and preparation of experi- mental Leishmania vaccine and leishmanin was used in this experiment (25). Parasite was grown on biphasic NNN medium and sub passaged in RPMI 1640 supplemented with 10% FBS. Promastigotes were harvest- ed at stationary phase. Macrophage and parasite interaction After 6 days monocyte derived macrophages were infected with L. major promastigotes harvested at stationary phase at a ratio of 5:1 parasite/macrophage. After 6h, non-interna- lized free promastigotes were removed by washing. Then, the infected cells were incu- bated in complete RPMI 1640 for additional 18h. The culture supernatants were collected and stored at -70ºC for IL-23 and IL-27 titra- tion using ELISA assay. Cytokines measurement The frozen culture supernatants were thawed and cytokine levels of IL-23 and IL-27 were determined using commercial kits (Abcam, United Kingdom) according to the manufac- turer's instructions. Briefly during the first incubation IL-23or IL-27 antigen was added to the wells. After washing, biotinylated monoclonal antibody specific for IL-23or IL-27 was incubated. Then the enzyme (streptavidin-peroxydase) was added. After incubation and washing to remove all un- bounded enzyme, a substrate solution which Available at: http://ijpa.tums.ac.ir