CMV-specific T cells in HLA-A*02:01-positive/CMV-seropositive donors were identified directly by HLA-A02/CMVpp65 (A02pp65) multimer staining and, after short in vitro stimulation with HLA-A*02:01-restricted pp65 peptide, by ELISPOT, ELISA, ICS and CSA. A peptide-specific T-cell response was detected in only 4 FILA-A*02:01-positive donors (50%). Despite A02pp65 peptide negativity, T-cell responses to CMVpp65 protein and/or overlapping peptide pool were detected. Comparing the specific immune response against EBV antigens in healthy donors overall, BZLF1-specific T cells ( smaller than 92.9% peptides, smaller than 56.3% peptide pool) were more frequent than EBNA-specific

T cells ( smaller than 64.3% peptides, smaller than 46.9% peptide AZD2014 pool) with higher percentage of positive findings for single HLA-restricted EBV peptides. T-cell response against HLA-B*08 peptide

epitopes was predominant (multimer staining: EBNA3A: MCC950 mechanism of action 9/14 and BZLF1: 7/14, IFN-’y ELISPOT: EBNA3A: 13/14 and BZLF1: 11/14). The fact that responses to EBV-specific antigens were not detected in every single EBV-seropositive donor as well as that the T-cell frequencies in response to the investigated EBV antigens differed strongly in the donor cohort indicates that these epitopes are less immunodominant than CMVpp65. Taken together, precise monitoring of T-cell immunity against infectious agents in potential T-cell donors and post-transplant recipients requires individual selection of antigens and immunoassays for the efficient detection and generation of clinically relevant T cells. Due to its lower detection limit and direct visualization

of each IFN-y-secreting cell we identified ELISPOT analysis to be preferable for high-throughput pre-screening. CSA was found to be advantageous for a more detailed analysis Evofosfamide manufacturer of antigen-specific T-cell subsets. (C) 2014 Elsevier B.V. All rights reserved.”
“Orexins are produced from neurons which are restricted to a few regions of the lateral hypothalamus (LH), where they are important in pain modulation. The orexin receptors and orexinergic projections are localized in regions previously shown to play a role in pain modulation such as rostral ventromedial medulla (RVM). The effect of orexin-A (ORXA) microinjection into the RVM on nociceptive behaviors was examined using the formalin test. Microinjection of ORXA into the RVM, but not adjacent reticularis gigantocellularis (Gi) nucleus, decreased formalin induced nociceptive behaviors. Pretreatment with a selective OX1R antagonist, SB-334867 inhibited the antinociception produced by ORXA, while the administration of SB-334867 alone had no effect. These data demonstrate that ORXA-induced antinociception in the formalin test is mediated in part through orexin1 receptors in the RVM. (C) 2014 Published by Elsevier Inc.