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A.N.A was funded by ARUK Fellowships Non-Clinical Career Development Fellowship Ref No: 18440. I.L. was funded by an ARUK PhD studentship Ref No: 17868. A.N.A and S.J.P were also in part funded by ARUK (grant 21261) Objective Salmonella enterica infections can lead to Reactive Arthritis (ReA), which can exhibit an association with human leucocyte antigen (HLA)-B*27:05, a molecule prone to misfolding and initiation of the unfolded protein response (UPR). This study examined how HLA-B*27:05 expression and the UPR affect the Salmonella life-cycle within epithelial cells. Methods Isogenic epithelial cell lines expressing two copies of either HLA-B*27:05 and a control HLA-B*35:01 heavy chain (HC) were generated to determine the effect on the Salmonella infection life-cycle. A cell line expressing HLA-B*27:05.HC physically linked to the light chain beta-2-microglobulin and a specific peptide (referred to as a single chain trimer, SCT) was also generated to determine the effects of HLA-B27 folding status on S. enterica life-cycle. XBP-1 venus and AMP dependent Transcription Factor (ATF6)-FLAG reporters were used to monitor UPR activation in infected cells. Triacin C was used to inhibit de novo lipid synthesis during UPR, and confocal imaging of ER tracker stained membrane allowed quantification of glibenclamide-associated membrane. Results S. enterica demonstrated enhanced replication with an altered cellular localisation in the presence of HLA-B*27:05.HC but not in the presence of HLA-B*27:05.SCT or HLA-B*35:01. HLA-B*27:05.HC altered the threshold for UPR induction. Salmonella activated the UPR and required XBP-1 for replication, which was associated with endoreticular membrane expansion and lipid metabolism. Conclusions HLA-B27 misfolding and a UPR cellular environment are associated with enhanced Salmonella replication, while Salmonella itself can activate XBP-1 and ATF6. These data provide a potential mechanism linking the life-cycle of Salmonella with the physicochemical properties of HLA-B27 and cellular events that may contribute to ReA pathogenesis. Our observations suggest that the UPR pathway maybe targeted for future therapeutic intervention. Publisher PDF

Improvements in immunosuppression have modified short-term survival of deceased-donor allografts, but not their rate of long-term failure. Mismatches between donor and recipient HLA play an important role in the acute and chronic allogeneic immune response against the graft. Perfect matching at clinically relevant HLA loci does not obviate the need for immunosuppression, suggesting that additional genetic variation plays a critical role in both short- and long-term graft outcomes. By combining patient data and samples from supranational cohorts across the United Kingdom and European Union, we performed the first large-scale genome-wide association study analyzing both donor and recipient DNA in 2094 complete renal transplant-pairs with replication in 5866 complete pairs. We studied deceased-donor grafts allocated on the basis of preferential HLA matching, which provided some control for HLA genetic effects. No strong donor or recipient genetic effects contributing to long- or short-term allograft survival were found outside the HLA region. We discuss the implications for future research and clinical application.

Objective HLA–B27 is associated with the inflammatory spondyloarthritides (SpA), although subtypes HLA–B*27:06 and HLA–B*27:09 are not. These subtypes differ from the HLA–B*27:05 disease‐associated allele primarily at residues 114 and 116 of the heavy chain, part of the F pocket of the antigen‐binding groove. Dimerization of HLA–B27 during assembly has been implicated in disease onset. The purpose of this study was to investigate the factors that influence differences in dimerization between disease‐associated and non–disease‐associated HLA–B27 alleles. Methods HLA–B*27:05 and mutants resembling the HLA–B*27:06 and 09 subtypes were expressed in the rat C58 T cell line, the human CEM T cell line and its calnexin‐deficient variant CEM.NKR. Immunoprecipitation, pulse–chase experiments, flow cytometry, and immunoblotting were performed to study the assembly kinetics, heavy‐chain dimerization, and chaperone associations. Results By expressing HLA–B*27:05, 06‐like, and 09 alleles on a restrictive rat transporter associated with antigen processing background, we demonstrate that a tyrosine expressed at p116, either alone or together with an aspartic acid residue at p114, inhibited HLA–B27 dimerization and increased the assembly rate. F‐pocket residues altered the associations with chaperones of the early major histocompatibility complex class I folding pathway. Calnexin was demonstrated to participate in endoplasmic reticulum (ER) stress–mediated degradation of dimers, whereas the oxidoreductase ERp57 does not appear to influence dimerization. Conclusion Residues within the F pocket of the peptide‐binding groove, which differ between disease‐associated and non–disease‐associated HLA–B27 subtypes, can influence the assembly process and heavy‐chain dimerization, events which have been linked to the initiation of disease pathogenesis.

We demonstrate a miniaturized single beam fiber optical trapping probe based on a high numerical aperture graded index (GRIN) micro-objective lens. This enables optical trapping at a distance of 200μm from the probe tip. The fiber trapping probe is characterized experimentally using power spectral density analysis and an original approach based on principal component analysis for accurate particle tracking. Its use for biomedical microscopy is demonstrated through optically mediated immunological synapse formation.

Exosomes are small microvesicles (40–100 nm) that are formed from the endosome to generate multi-vesicular bodies (MBVs). In mammals, exosomes play a significant role in cellular communication. Exosomes have also been reported for salmon and fruit fly but have not previously been studied in crustaceans. Therefore we undertook to study them in decapods. We chose the hyaline haemocytes of brachyuran crabs, Carcinus maenas and Hyas araneus, because these cells are abundant in haemolymph, are phagocytic and can be cultured on glass or plastic surfaces. Furthermore these cells have been observed to contain MVB-like structures that bear some resemblance to those in mammals that produce exosomes. Preliminary results show that highly enriched hyaline cells of both C. maenas and H. araneus, express actin and haemocyanin during 24 h in vitro. The presence of haemocyanin subunits is interesting because haemocyanin is a multifunctional protein that participates in immunity in crustaceans. As far as we are aware, the present study is the first to show that hyaline cells from a crustacean secrete this biologically important protein. Other proteins were found but we were unable to identify them by mass spectroscopy. Treatment of the hyaline cells in vitro with LPS at sub-lethal concentrations failed to reveal a change in the flow cytometric scatter plots or in the protein profiles on SDS-PAGE.

The majority of patients referred to a breast clinic will have benign disease (BBD) and, after appropriate assessment, most can be reassured and discharged. However, some patients will require excision biopsy to confirm the diagnosis. We have performed a prospective study to determine whether routine outpatient follow-up of these patients can be safely omitted. A series of 100 consecutive patients undergoing breast biopsy for disease, assessed as benign in outpatients, were studied. Before discharge each was given an information sheet outlining their postoperative recovery and advised to see their general practitioner (GP) between the 7th and 10th postoperative day for wound check, suture removal and histology. Of the 100 women in the study group, 94 had benign histology. At the postoperative visit to the GP a full discharge summary including histology was available for 88 patients. In six patients there was a delay in discharge summary generation. Malignant or premalignant disease was found in six patients. All were safely identified and recalled for counselling and further treatment as appropriate. We believe that the routine follow-up of patients undergoing benign breast surgery can safely be avoided if there is a satisfactory protocol which is understood by both the patients and GPs.