Cells were cultured in medium alone, or in the presence of intact functional GiADI (produced, purified and tested as described in Jerlstrom-Hultqvist et al [41]), heat denatured (80°C for 10 min) GiADI (GiADIb), as well

as an equal dilution of BSA 1 μg/mL and PreScission enzyme containing buffer used for elution of GiADI, in combinations with 0.4 mM arginine or citrulline and Selleckchem Danusertib T-cell stimulatory anti-CD3 (mouse IgE moab; CLB-T3/4.E; final concentration 0.3 μg/mL) and anti-CD28 (mouse IgG1moab; CLB-CD28/1; final concentration 0.8 μg/mL) from the Central Laboratory of the Netherlands Red Cross Blood Transfusion Services (Amsterdam, The Netherlands). Cultures were performed in triplicates for 6 days at 37°C in a humidified atmosphere of 5% CO2. PBMC proliferation assay Cellular proliferative responses were measured by the incorporation

of 3H-thymidine into newly synthesized DNA by conventional proliferation assay [42]. After 5 days of culture cells were pulsed with 37 kBq/well of 3H-thymidine (Perkin Elmer, Boston, MA, USA) and harvested 18 h later onto glass-fibre pads. Amounts of DNA-incorporated radioactivity were determined by Epacadostat order liquid scintillation counting. Proliferation was determined as counts per minute (cpm). Data analysis If not mentioned otherwise, ACP-196 nmr all data were analyzed using Microsoft Office Excel 2010. Figures were prepared in Adobe Illustrator CS4. Statistical analyses were performed by two-tailed student’s t-test (p-value <0.5, significant; < 0.01, highly significant). Acknowledgements Steinar Sørnes is thanked for assistance in the lab. Alessandro Giuffre, University of Rome, is acknowledged for sharing of the anti-flavohemoglobin antibody. This study was supported by VR-M and FORMAS (Sweden). Electronic supplementary material Additional file 1: Describes primers used in RT-PCR analyses also (Table S1), expressions of arginine consuming

enzymes in IECs interacting with strain WB (Table S2) , GS (Table S3) and P15 (Table S4). Table S5 describes expression of arginine-consuming enzymes in Giardia WB trophozoites during interaction with IECs. (XLSX 22 KB) References 1. Svard SG, Hagblom P, Palm JE: Giardia lamblia – a model organism for eukaryotic cell differentiation. FEMS Microbiol Lett 2003, 218:3–7.PubMed 2. Ankarklev J, Jerlstrom-Hultqvist J, Ringqvist E, Troell K, Svard SG: Behind the smile: cell biology and disease mechanisms of Giardia species. Nat Rev Microbiol 2010, 8:413–422.PubMed 3. Savioli L, Smith H, Thompson A: Giardia and cryptosporidium join the ‘neglected diseases initiative. Trends Parasitol 2006, 22:203–208.PubMedCrossRef 4. Adam R: Biology of Giardia lamblia. Clin Microbiol Rev 2001, 14:447–475.PubMedCrossRef 5. Ali S, Hill D: Giardia intestinalis. Curr Opin Infect Dis 2003, 16:453–460.PubMedCrossRef 6. Wensaas KA, Langeland N, Hanevik K, Morch K, Eide GE, Rortveit G: Irritable bowel syndrome and chronic fatigue 3 years after acute giardiasis: historic cohort study. Gut 2012, 61:214–219.PubMedCrossRef 7.