Local immune responses to surgery lead to systemic pro-inflammatory and immunosuppressive phases that are temporally related and proportionate in magnitude. Improved understanding of these mechanisms has implications for clinical study design and has led to the emergence of novel biomarkers such as Toll-like receptor expression. These can be used to stratify patient care pathways to maximize the benefit from current therapies or to select the right target at the right phase of illness for future drug development.

SUMMARYErrors in chromosome segregation in mammalian oocytes lead to aneuploid eggs that are developmentally compromised. In mitotic cells, mitotic centromere associated kinesin (MCAK; KIF2C) prevents chromosome segregation errors by detaching incorrect microtubule-kinetochore interactions. Here, we examine whether MCAK is involved in spindle function in mouse oocyte meiosis I, and whether MCAK is necessary to prevent chromosome segregation errors. We find that MCAK is recruited to centromeres, kinetochores and chromosome arms in mid-meiosis I, and that MCAK depletion, or inhibition using a dominantnegative construct, causes chromosome misalignment. However, the majority of oocytes complete meiosis I and the resulting eggs retain the correct number of chromosomes. Moreover, MCAK-depleted oocytes can recover from mono-orientation of homologous kinetochores in mid-meiosis I to segregate chromosomes correctly. Thus, MCAK contributes to chromosome alignment in meiosis I, but is not necessary for preventing chromosome segregation errors. Although other correction mechanisms may function in mammalian meiosis I, we speculate that late establishment of kinetochore microtubules in oocytes reduces the likelihood of incorrect microtubule-kinetochore interactions, bypassing the requirement for error correction. ] was obtained in pEGFP-N1 and subcloned into pBluescript II KS(+) (Stratagene). Plasmids were amplified, linearised and mRNA generated for microinjection using Ambion mMessage Machine T7 Ultra, as described previously (FitzHarris, 2009). Morpholinos (Gene Tools) were microinjected at an estimated final concentration of 50-100 M. MCAK-MO, 5Ј-CATGGACTCAGGAACAACAACAGGC-3Ј; Control-MO, 5Ј-CCTCTTACCTCATTACAATTTATA-3Ј; Mad2-MO, 5Ј-GCTCTCGGGCGAGCTGCTGTGCCAT-3Ј. ImagingOocytes were fixed and permeablised in PHEM buffer containing 4% paraformaldehyde and 0.5% Triton X-100. Primary antibodies used: rabbit anti-MCAK and anti-KIF2A (1/1000; gifts from Duane Compton, Dartmouth, NH, USA), CREST serum (1/300; a gift from William Earnshaw, Edinburgh, UK) and YL1/2 anti--tubulin (1/1000; Abcam). Chromatin was labelled with 5 g/ml Hoechst 33343 for 5 minutes.Confocal imaging was performed on a Zeiss 510 microscope. Chromosome counts were performed as described (Duncan et al., 2009), with minor modifications. Spindles were collapsed using a 90-minute pulse of monastrol (200 M). Oocytes were labelled with CREST antibodies and Hoechst, and mounted on slides. Serial z-sections were acquired at 0.5 m intervals. All counting was performed blind. For chromosome alignment experiments, fixed oocytes were examined using a Leica inverted microscope fitted with fluorescence optics. Oocytes were rolled using a glass pipette to orientate the M-phase plate, and the number of chromosomes that were separated from the plate was counted. RESULTS AND DISCUSSION MCAK depletion or inhibition causes chromosome misalignment in meiosis IFollowing release from the ovary or from agents that increase cAMP concentration, such as the phosphodiesterase inhi...

PurposeThe graft site microenvironment has a profound effect on alloimmunity and graft survival. We aimed to study the kinetics and phenotype of trafficking antigen-presenting cells (APC) to the draining lymph nodes (DLNs) in a mouse model of corneal transplantation, and to evaluate the homing mechanisms through which graft site inflammation controls APC trafficking.MethodsAllogeneic donor corneas were transplanted onto inflamed or quiescent graft beds. Host- (YAe+) and donor (CD45.1+ or eGFP+)-derived APCs were analyzed by flow cytometry. Protein and mRNA expression of the CC chemokine receptor (CCR)7 ligands CCL19 and CCL21 were assessed using ELISA and Real-Time qPCR, respectively. Transwell migration assay was performed to assess the effect of DLNs isolated from hosts with inflamed graft beds on mature bone marrow–derived dendritic cells (BMDCs).ResultsWe found that inflamed graft sites greatly promote the trafficking of both recipient- and graft-derived APCs, in particular mature CCR7+ CD11c+ dendritic cells (DC). CCL19 and CCL21 were expressed at significantly higher levels in the DLNs of recipients with inflamed graft beds. The supernatant of DLNs from recipients with inflamed graft beds induced a marked increase in mature DC migration compared with supernatant from recipients with quiescent graft beds in a transwell assay. This effect was abolished by neutralizing CCL19 or CCL21. These data suggest that graft site inflammation increases the expression of CCR7 ligands in the DLNs, which promote mature DC homing and allorejection.ConclusionsWe conclude that the graft site microenvironment plays a critical role in alloimmunity by determining DC trafficking through the CCR7-CCL19/21 axis.