1% BSA and incubated with equal volume of 1.25 μM CFSE (Molecular Probes Europe, Leiden, The Netherlands) for 10 min at room temperature. Unbound dye was quenched by the addition of equal volume of RPMI+10% FBS and 15 min incubation at 37°C. CFSE-labeled CbT cells were washed twice in RPMI+10% FBS and plated at 5×104 cells/well in 96-well round-bottom plate (Corning, Corning, NY, USA). mDC incubated for 24 h with isotype-matched control mAb (MOPC-21), anti-CD300e (UP-H2) mAb or stimulated with LPS at 100 ng/mL were collected and plated over the CbT at ratios

(CbT:mDC) 10:1; 20:1; 40:1; 80:1 and 160:1. After 4 days samples were examined by flow cytometry for sequential dilution of CFSE fluorescence and analyzed using FlowJo software GSI-IX datasheet (Three Star). FlowJo Proliferation Platform was used to analyzed CbT-cell proliferation expressing BAY 80-6946 the results as “% divided” that is defined as the percentage of CbT cells in the starting population that divided (assuming that no cells died in culture). Statistical analysis was performed using either the Student’s t-test or the non-parametric Kolmogorov–Smirnov test. This work was supported

by a grant from Plan Nacional de I+D (SAF2007-61814) and Red Heracles, Ministerio de Ciencia e Innovación (MICINN). TB is supported by a fellowship from MICINN. BPC is supported by grant FI 07/00054 and FEB by contract CES 07/015 both from Instituto de Salud Carlos III. The authors thank Marta Donini (University of Verona, Verona, Italy) for technical advice in monocyte manipulation and Dr. Oscar Fornas (University Pompeu Fabra, Barcelona, Spain) for advice in flow cytometry analysis. They are very grateful to Marco A. Fernández (Germans Trias and Pujol Health Sciences Research Institute, Badalona, Spain) for the support in mDC isolation.

They thank Gemma Heredia (University Pompeu Fabra, Barcelona, Spain) for technical support in checking the specificity of UP-H mAb on CD300 transfectants. They also thank blood donors for their contribution. Conflict of interest: The authors declare no financial or commercial conflict of interest. “
“The obligate intracellular bacterium PRKACG Parachlamydia acanthamoebae is a potential human pathogen, but the host range of the bacteria remains unknown. Hence, the growth of P. acanthamoebae Bn9 in protozoa (Tetrahymena, Acanthamoeba, Dictyostelium) and mammalian cells (HEp-2, Vero, THP-1, PMA-stimulated THP-1, Jurkat) was assessed using an AIU assay which had been previously established by the current authors. P. acanthamoebae grew in Acanthamoeba but not in the other cell types. The growth was also confirmed using DAPI staining, FISH and TEM. These results indicate that the host range of P. acanthamoebae is limited. Parachlamydia acanthamoebae is an environmental chlamydia of the order Chlamydiales. It is an obligate intracellular bacterium that is widely distributed in the natural environment, including in rivers and soil (1).