One third of deceased donor kidneys for transplantation in the UK are donated following cardiac death (DCD). Such kidneys have a high rate of delayed graft function (DGF) following transplantation. We conducted a multicenter, randomized controlled trial to determine whether kidney preservation using cold, pulsatile machine perfusion (MP) was superior to simple cold storage (CS) for DCD kidneys. One kidney from each DCD donor was randomly allocated to CS, the other to MP. A sequential trial design was used with the primary endpoint being DGF, defined as the necessity for dialysis within the first 7 days following transplant. The trial was stopped when data were available for 45 pairs of kidneys. There was no difference in the incidence of DGF between kidneys assigned to MP or CS (58% vs. 56%, respectively), in the context of an asystolic period of 15 min and median cold ischemic times of 13.9 h for MP and 14.3 h for CS kidneys. Renal function at 3 and 12 months was similar between groups, as was graft and patient survival. For kidneys from controlled DCD donors (with mean cold ischemic times around 14 h), MP offers no advantage over CS, which is cheaper and more straightforward.

The implementation of bacterial screening of PLT components with the NHSBT BacT/ALERT protocol was an effective risk reduction measure and increased the safety of the blood supply.

Summary The refusal rate for organ donation in the UK is 42%, among the highest in Europe. We extracted data on every family approach for donation in UK ICUs or Emergency Departments between 1st April 2012 and 30th September 2013, and performed multiple logistic regression to identify modifiable factors associated with consent. Complete data were available for 4703 of 4899 approaches during the study period. Consent for donation after brain death was 68.9%, and for donation after circulatory death 56.5% (p < 0.0001). Patient ethnicity, knowledge of a patient's wishes and involvement of a specialist nurse in organ donation in the approach were strongly associated with consent (p < 0.0001). The impact of the specialist nurse was stronger for donation after circulatory death than for donation after brain death, even after accounting for the impact of prior knowledge of patients' wishes. Involvement of the specialist nurse in the approach, encouraging family discussions about donation wishes and promotion of the organ donor register are key strategies to increase UK consent rates, and are supported by this study.

SUMMARYThe activation of dendritic cells (DCs) by microbes is mediated by pattern recognition receptors including the Toll-like receptors (TLR). Bacterial lipopolysaccharide acts via TLR4 whereas peptidoglycan and lipoprotein responses are mediated by TLR2. It is generally accepted that TLR binding to microbes occurs at the cell surface but this has not been directly demonstrated for human DCs. We show here that TLR2 and TLR4 are expressed inside DCs in an abundant tubulovesicular pattern with a focus of intense staining adjacent to the nucleus. In contrast, there was no detectable expression on the cell surface. TLR2 and TLR4 were readily found both intracellularly and on the surface of monocytes. They were shown to be closely associated with the Golgi complex and colocalized with a-tubulin, displaying a high focal concentration at the microtubule organizing centre. Alignment of TLR2 and TLR4 with microtubules was observed, suggesting that microtubules serve as transport tracks for TLR vesicles. Depolymerization of the microtubule network disrupted the intracellular expression of TLR2 and TLR4 and profoundly inhibited interleukin-12 (IL-12) production in response to Neisseria meningitidis but did not prevent phagocytosis. These data are consistent with the bacterial signalling through TLR2 and TLR4 required for IL-12 production occurring inside DCs after phagocytosis.

We have constructed a physical map of the human genome by using a panel of 90 whole-genome radiation hybrids (the TNG panel) in conjunction with 40,322 sequence-tagged sites (STSs) derived from random genomic sequences as well as expressed sequences. Of 36,678 STSs on the TNG radiation hybrid map, only 3604 (9.8%) were absent from the unassembled draft sequence of the human genome. Of 20,030 STSs ordered on the TNG map as well as the assembled human genome draft sequence and the Celera assembled human genome sequence, 36% of the STSs had a discrepant order between the working draft sequence and the Celera sequence. The TNG map order was identical to one of the two sequence orders in 60% of these discrepant cases.

The feasibility of large-scale genome-wide association studies of complex human disorders depends on the availability of accurate and efficient genotyping methods for single nucleotide polymorphisms (SNPs). We describe a new platform of the invader assay, a biplex assay, where both alleles are interrogated in a single reaction tube. The assay was evaluated on over 50 different SNPs, with over 20 SNPs genotyped in study cohorts of over 1500 individuals. We assessed the usefulness of the new platform in high-throughput genotyping and compared its accuracy to genotyping results obtained by the traditional monoplex invader assay, TaqMan genotyping and sequencing data. We present representative data for two SNPs in different genes (CD36 and protein tyrosine phosphatase 1beta) from a study cohort comprising over 1500 individuals with high or low-normal blood pressure. In this high-throughput application, the biplex invader assay is very accurate, with an error rate of <0.3% and a failure rate of 1.64%. The set-up of the assay is highly automated, facilitating the processing of large numbers of samples simultaneously. We present new analysis tools for the assignment of genotypes that further improve genotyping success. The biplex invader assay with its automated set-up and analysis offers a new efficient high-throughput genotyping platform that is suitable for association studies in large study cohorts.

SummaryGroup B Neisseria meningitidis is a human pathogen, for which a universally effective vaccine is still not available. Immune responses to bacteria are initiated by dendritic cells (DC), which internalize and process bacterial antigens for presentation to T cells. We show here that optimal IL-12 and TNF-a a a a production by human monocyte derived DC in response to killed serogroup B N. meningitidis depends on physical contact and internalization of the bacteria by DC. The majority of DC producing cytokines had internalized N. meningitidis while inhibition of bacterial internalization markedly impaired IL-12 and TNF-a a a a , but not IL-6 production. Internalization of N. meningitidis was shown to depend on lipooligosaccharide (LOS) expressed by the bacteria with poor internalization of LOS deficient bacteria compared to wild-type bacteria. Restoration of LOS biosynthesis in a LOS regulatory strain also restored both internalization and cytokine production and was enhanced in the presence of LPS binding protein (LBP). These results suggest that DC phagocytosis depends on expression of LOS within the bacteria and that optimal cytokine production, particularly IL-12, requires internalization of the bacteria. These findings have important implications for designing vaccines that will induce protective immune responses to group B N. meningitidis.

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