Sequence analysis and identification of new isoform of EP4 receptors in different atlantic salmon tissues (Salmo salar L.) and its role in PGE2 induced immunomodulation in vitro.

PGE2 plays an important role in a broad spectrum of physiological and pathological processes mediated through a membrane-bound G protein-coupled receptor (GPCR) called EP receptor. In mammals, four subtypes of EP receptor (EP 1-4) are identified and each of them functions through different signal transduction pathways. Orthologous EP receptors have also been identified in other non-mammalian species, such as chicken and zebrafish. EP4 is the only identified PGE2 receptor to date in Atlantic salmon but its tissue distribution and function have not been studied in any detail. In this study, we first sequenced EP4 receptor in different tissues and found that the presence of the 3nt deletion in the 5' untranslated region was accompanied by silent mutation at nt 668. While attempting to amplify the same sequence in TO cells (an Atlantic salmon macrophage-like cell line), we failed to obtain the full-length product. Further investigation revealed different isoform of EP4 receptor in TO cells and we subsequently documented its presence in different Atlantic salmon tissues. These two isoforms of EP4 receptor share high homology in their first half of sequence but differ in the second half part with several deletion segments though the final length of coding sequence is the same for two isoforms. We further studied the immunomodulation effect of PGE2 in TO cells and found that PGE2 inhibited the induction of CXCL-10, CCL-4, IL-8 and IL-1ß genes expression in a time dependent manner and without cAMP upregulation.

IL-2-driven regulation of NK cell receptors with regard to the distribution of CD16+ and CD16- subpopulations and in vivo influence after haploidentical NK cell infusion.

To characterize natural killer (NK) cell subpopulations during activation, we analyzed the NK cell receptor repertoire and functionality of purified clinical scale CD56CD3 donor NK cells during stimulation with 1000 U/mL interleukin (IL)-2 for up to 14 days. In a phase I/II trial, we investigated the efficacy and feasibility of nonidentical NK cell infusion in patients with neuroblastoma after haploidentical stem cell transplantation. After IL-2 stimulation, large differences in the distribution of CD16 and CD16 subpopulations were found in 12 donors. Thereby, surface expression for all natural cytotoxicity receptors (NCRs) and NKG2D increased. In addition, killer cell immunoglobulin-like receptor (KIR) NK cells were overgrown by KIR proportion and the homing receptor CD62L was lost during stimulation. NK cell cytotoxicity against K562 and neuroblastoma cells increased and significantly higher cytokine secretion (eg, interferon-gamma, tumor necrosis factor-beta, macrophage inflammatory protein-1alpha, macrophage inflammatory protein-1beta) was observed after IL-2 stimulation compared with freshly isolated NK cells. However, NK cells of donors showing an initially enhanced cytotoxicity combined with NCR and CD69 expression, seemed to be exhausted and did not favor a stimulation period over 9 days. When IL-2-stimulated NK cells were given to transplant recipients, they induced a decrease of peripheral blood NK, in particular of CD56-NK cells. Our data indicate that IL-2 stimulation increases the expression of activating receptors and emphasizes mechanisms beside KIR/human leukocyte antigen. Furthermore, the results suggest that the expansion period of purified NK cells has to be individualized to optimize NK cell immunotherapy.

Impaired pneumococcal immunity in children after treatment for acute lymphoblastic leukaemia.

Although the substantial risk for invasive pneumococcal disease is well recognized in children after allogeneic stem cell transplantation, little is known about the specific immunity against pneumococci in children after cytotoxic therapy for acute lymphoblastic leukaemia (ALL). We therefore assessed the spontaneous reconstitution of humoral immunity against pneumococcal antigens, of total IgG and the IgG2 subclass, and of lymphocyte subsets in a total of 53 children treated for ALL. None of the patients had received pneumococcal vaccination prior to or after therapy for ALL. At 3 and 9 months after completion of chemotherapy, most patients had levels of specific antibodies to pneumococcal antigens below the presumed threshold of protection and significantly lower than those of age-matched unvaccinated healthy controls. In contrast, at 9 months after completion of therapy, only a minority of patients had immunoglobulin concentrations or lymphocyte subset counts below the age-matched reference value. Our data indicate that patients with ALL who are unvaccinated against pneumococci have a selective immunodeficiency with an impaired antibody protection against pneumococci for up to 9 months after completion of therapy. Therefore, effective prevention, including chemoprophylaxis and active immunization, has to be considered in this patient population.