Cross-tissue immune cell analysis reveals tissue-specific features in humans.

Despite their crucial role in health and disease, our knowledge of immune cells within human tissues remains limited. We surveyed the immune compartment of 16 tissues from 12 adult donors by single-cell RNA sequencing and VDJ sequencing generating a dataset of ~360,000 cells. To systematically resolve immune cell heterogeneity across tissues, we developed CellTypist, a machine learning tool for rapid and precise cell type annotation. Using this approach, combined with detailed curation, we determined the tissue distribution of finely phenotyped immune cell types, revealing hitherto unappreciated tissue-specific features and clonal architecture of T and B cells. Our multitissue approach lays the foundation for identifying highly resolved immune cell types by leveraging a common reference dataset, tissue-integrated expression analysis, and antigen receptor sequencing.

Mitochondria-targeted antioxidant MitoQ ameliorates ischaemia-reperfusion injury in kidney transplantation models.

BACKGROUND: Ischaemia-reperfusion (IR) injury makes a major contribution to graft damage during kidney transplantation. Oxidative damage to mitochondria is an early event in IR injury. Therefore, the uptake, safety, and efficacy of the mitochondria-targeted antioxidant MitoQ were investigated in models of transplant IR injury. METHODS: MitoQ uptake by warm and cooled pairs of pig and declined human kidneys was measured when preserved in cold static storage or by hypothermic machine perfusion. Pairs of pigs' kidneys were exposed to defined periods of warm and cold ischaemia, flushed and stored at 4°C with or without MitoQ (50 nmol/l to 250 µmol/l), followed by reperfusion with oxygenated autologous blood in an ex vivo normothermic perfusion (EVNP). Pairs of declined human kidneys were flushed and stored with or without MitoQ (5-100 µmol/l) at 4°C for 6 h and underwent EVNP with ABO group-matched blood. RESULTS: Stable and concentration-dependent uptake of MitoQ was demonstrated for up to 24 h in pig and human kidneys. Total blood flow and urine output were significantly greater in pig kidneys treated with 50 µmol/l MitoQ compared with controls (P = 0.006 and P = 0.007 respectively). In proof-of-concept experiments, blood flow after 1 h of EVNP was significantly greater in human kidneys treated with 50 µmol/l MitoQ than in controls (P ≤ 0.001). Total urine output was numerically higher in the 50-µmol/l MitoQ group compared with the control, but the difference did not reach statistical significance (P = 0.054). CONCLUSION: Mitochondria-targeted antioxidant MitoQ can be administered to ischaemic kidneys simply and effectively during cold storage, and may improve outcomes after transplantation.

A pilot study evaluating GSK1070806 inhibition of interleukin-18 in renal transplant delayed graft function.

INTRODUCTION: Delayed graft function (DGF) following renal transplantation is a manifestation of acute kidney injury (AKI) leading to poor long-term outcome. Current treatments have limited effectiveness in preventing DGF. Interleukin-18 (IL18), a biomarker of AKI, induces interferon-γ expression and immune activation. GSK1070806, an anti-IL18 monoclonal antibody, neutralizes activated (mature) IL18 released from damaged cells following inflammasome activation. This phase IIa, single-arm trial assessed the effect of a single dose of GSK1070806 on DGF occurrence post donation after circulatory death (DCD) kidney transplantation. METHODS: The 3 mg/kg intravenous dose was selected based on prior studies and physiologically based pharmacokinetic (PBPK) modeling, indicating the high likelihood of a rapid and high level of IL18 target engagement when administered prior to kidney allograft reperfusion. Utilization of a Bayesian sequential design with a background standard-of-care DGF rate of 50% based on literature, and confirmed via extensive registry data analyses, enabled a statistical efficacy assessment with a minimal sample size. The primary endpoint was DGF frequency, defined as dialysis requirement ≤7 days post transplantation (except for hyperkalemia). Secondary endpoints included safety, pharmacokinetics and pharmacodynamic biomarkers. RESULTS: GSK1070806 administration was associated with IL18-GSK1070806 complex detection and increased total serum IL18 levels due to IL18 half-life prolongation induced by GSK1070806 binding. Interferon-γ-induced chemokine levels declined or remained unchanged in most patients. Although the study was concluded prior to the Bayesian-defined stopping point, 4/7 enrolled patients (57%) had DGF, exceeding the 50% standard-of-care rate, and an additional two patients, although not reaching the protocol-defined DGF definition, demonstrated poor graft function. Six of seven patients experienced serious adverse events (SAEs), including two treatment-related SAEs. CONCLUSION: Overall, using a Bayesian design and extensive PBPK dose modeling with only a small sample size, it was deemed unlikely that GSK1070806 would be efficacious in preventing DGF in the enrolled DCD transplant population. TRIAL REGISTRATION: NCT02723786.

scRNA-seq assessment of the human lung, spleen, and esophagus tissue stability after cold preservation.

BACKGROUND: The Human Cell Atlas is a large international collaborative effort to map all cell types of the human body. Single-cell RNA sequencing can generate high-quality data for the delivery of such an atlas. However, delays between fresh sample collection and processing may lead to poor data and difficulties in experimental design. RESULTS: This study assesses the effect of cold storage on fresh healthy spleen, esophagus, and lung from ≥ 5 donors over 72 h. We collect 240,000 high-quality single-cell transcriptomes with detailed cell type annotations and whole genome sequences of donors, enabling future eQTL studies. Our data provide a valuable resource for the study of these 3 organs and will allow cross-organ comparison of cell types. We see little effect of cold ischemic time on cell yield, total number of reads per cell, and other quality control metrics in any of the tissues within the first 24 h. However, we observe a decrease in the proportions of lung T cells at 72 h, higher percentage of mitochondrial reads, and increased contamination by background ambient RNA reads in the 72-h samples in the spleen, which is cell type specific. CONCLUSIONS: In conclusion, we present robust protocols for tissue preservation for up to 24 h prior to scRNA-seq analysis. This greatly facilitates the logistics of sample collection for Human Cell Atlas or clinical studies since it increases the time frames for sample processing.

A randomized prospective study comparing mobile-bearing against fixed-bearing PFC Sigma cruciate-retaining total knee arthroplasties with ten-year minimum follow-up.

AIMS: This study compares the PFC total knee arthroplasty (TKA) system in a prospective randomized control trial (RCT) of the mobile-bearing rotating-platform (RP) TKA against the fixed-bearing (FB) TKA. This is the largest RCT with the longest follow-up where cruciate-retaining PFC total knee arthroplasties are compared in a non-bilateral TKA study. PATIENTS AND METHODS: A total of 167 patients (190 knees with 23 bilateral cases), were recruited prospectively and randomly assigned, with 91 knees receiving the RP and 99 knees receiving FB. The mean age was 65.5 years (48 to 82), the mean body mass index (BMI) was 29.7 kg/m2 (20 to 52) and 73 patients were female. The Knee Society Score (KSS), Knee Society Functional Score (KSFS), Oxford Knee Score (OKS), Western Ontario and McMaster Universities Arthritis Index (WOMAC), and 12-Item Short-Form Health Survey Physical and Mental Component Scores (SF-12 PCS, SF-12 MCS) were gathered and recorded preoperatively, at five-years' follow-up, and at ten years' follow-up. Additionally, Knee Injury and Osteoarthritis Outcome Scores (KOOS) were collected at five- and ten-year follow-ups. The prevalence of radiolucent lines (RL) on radiographs and implant survival were recorded at five- and ten-year follow-ups. RESULTS: At the ten-year follow-up, the RP group (n = 39) had a statistically significant superior score in the OKS (p = 0.001), WOMAC (p = 0.023), SF-12 PCS (p = 0.019), KOOS Activities of Daily Living (ADL) (p = 0.010), and KOOS Sport and Recreation (Sport/Rec) (p = 0.006) compared with the FB group (n = 46). The OKS, SF-12 PCS, and KOOS Sport/Rec at ten years had mean scores above the minimal clinically important difference (MCID) threshold. There was no significant difference in prevalence of radiolucency between groups at five-years' follow-up (p = 0.449), nor at ten-years' follow-up (p = 0.08). Implant survival rate at 14 years postoperative was 95.2 (95% CI 90.7 to 99.8) and 94.7 (95% CI 86.8 to 100.0) for the RP and FB TKAs, respectively. CONCLUSION: At ten-year follow-up, the mobile-bearing knee joint arthroplasty had statistically and clinically relevant superior OKS, SF-12 PCS, and KOOS (Sport/Rec) than the fixed-bearing platform. No difference was seen in prevalence of radiolucent lines. There was a greater than 94% implant survival rate for both cohorts at 14 years. Cite this article: Bone Joint J 2018;100-B:1336-44.

Pancreatic allograft thrombosis: Suggestion for a CT grading system and management algorithm.

Pancreatic allograft thrombosis (PAT) remains the leading cause of nonimmunologic graft failure. Here, we propose a new computed tomography (CT) grading system of PAT to identify risk factors for allograft loss and outline a management algorithm by retrospective review of consecutive pancreatic transplantations between 2009 and 2014. Triple-phase CT scans were graded independently by 2 radiologists as grade 0, no thrombosis; grade 1, peripheral thrombosis; grade 2, intermediate non-occlusive thrombosis; and grade 3, central occlusive thrombosis. Twenty-four (23.3%) of 103 recipients were diagnosed with PAT (including grade 1). Three (2.9%) grafts were lost due to portal vein thrombosis. On multivariate analysis, pancreas after simultaneous pancreas-kidney transplantation/solitary pancreatic transplantation, acute rejection, and CT findings of peripancreatic edema and/or inflammatory change were significant risk factors for PAT. Retrospective review of CT scans revealed more grade 1 and 2 thromboses than were initially reported. There was no significant difference in graft or patient survival, postoperative stay, or morbidity of recipients with grade 1 or 2 thrombosis who were or were not anticoagulated. Our data suggest that therapeutic anticoagulation is not necessary for grade 1 and 2 arterial and grade 1 venous thrombosis. The proposed grading system can assist clinicians in decision-making and provide standardized reporting for future studies.

Local Expansion of Donation After Circulatory Death Kidney Transplant Activity Improves Waitlisted Outcomes and Addresses Inequities of Access to Transplantation.

In the United Kingdom, donation after circulatory death (DCD) kidney transplant activity has increased rapidly, but marked regional variation persists. We report how increased DCD kidney transplant activity influenced waitlisted outcomes for a single center. Between 2002-2003 and 2011-2012, 430 (54%) DCD and 361 (46%) donation after brain death (DBD) kidney-only transplants were performed at the Cambridge Transplant Centre, with a higher proportion of DCD donors fulfilling expanded criteria status (41% DCD vs. 32% DBD; p = 0.01). Compared with U.K. outcomes, for which the proportion of DCD:DBD kidney transplants performed is lower (25%; p < 0.0001), listed patients at our center waited less time for transplantation (645 vs. 1045 days; p < 0.0001), and our center had higher transplantation rates and lower numbers of waiting list deaths. This was most apparent for older patients (aged >65 years; waiting time 730 vs. 1357 days nationally; p < 0.001), who received predominantly DCD kidneys from older donors (mean donor age 64 years), whereas younger recipients received equal proportions of living donor, DBD and DCD kidney transplants. Death-censored kidney graft survival was nevertheless comparable for younger and older recipients, although transplantation conferred a survival benefit from listing for only younger recipients. Local expansion in DCD kidney transplant activity improves survival outcomes for younger patients and addresses inequity of access to transplantation for older recipients.

Donors With Immune Thrombocytopenia: Do They Pose a Risk to Transplant Recipients?

Transplant-mediated alloimmune thrombocytopenia (TMAT) from donors with immune thrombocytopenia (ITP) can result in significant bleeding complications in the recipient. The risk to a recipient of TMAT if they receive an organ from a donor with ITP is unknown. The outcomes of recipients of organs from deceased donors with ITP recorded in the UK Transplant Registry between 2000 and 2015 were reviewed. Twenty-one deceased organ donors had a predonation diagnosis of ITP. These donors were significantly more likely to have died from intracranial hemorrhage than were all other deceased organ donors (85% vs. 57%, p < 0.001). Organs from donors with ITP resulted in 49 organ transplants (31 kidney, 14 liver, four heart), with only one case of TMAT, which occurred in a liver transplant recipient and resulted in death from bleeding complications 18 days posttransplantation. The recipient of a kidney from the same organ donor was not affected. Unadjusted 5-year patient and graft survival was significantly worse for liver transplant recipients from donors with ITP compared with liver transplant recipients from donors without ITP (64% vs. 85%, p = 0.012). Organs from donors with ITP may be considered for transplantation, but livers should be used with caution.

B-cell biomarkers in transplantation--from genes to therapy.

An increased understanding of the mechanisms by which the immune system mounts a response to transplanted organs has allowed the development of immunosuppressive regimens that limit acute T-cell-mediated rejection (TCMR). However, the treatment of acute and chronic antibody-mediated rejection (ABMR) in kidney transplants remains sub-optimal. The occurrence and severity of antibody-mediated graft pathology are variable, and genetic polymorphisms that affect the magnitude and nature of the B-cell response, as well as effector functions of antibody, are likely to contribute to such phenotypic variation. Here we review current efforts to understand and quantify the contribution of B cells to renal transplant pathology by studying variation in DNA, mRNA and proteins. Large genetic studies with information on B-cell-specific genetic variants are scarce. At a transcriptomic level, there is evidence that B cells are essential contributors to transplant tolerance and may protect against TCMR and ABMR. In contrast, at the protein level, the detection of donor-specific human leukocyte antigen (HLA) antibodies and an assessment of their capacity to bind complement allow patients of high immunological risk to be identified. Other biomarkers, such as serum B-cell-activating factor (BAFF) or interleukin (IL)-10-producing B cells, may allow this risk stratification to be refined. An increased understanding of the significance of these biomarkers should allow a more accurate assessment of how an individual patient's B cells will impact allograft responses and thereby allow clinicians to adjust therapeutic strategies appropriately.

Preformed donor HLA-DP-specific antibodies mediate acute and chronic antibody-mediated rejection following renal transplantation.

Donor-specific HLA alloantibodies may cause acute and chronic antibody-mediated rejection (AMR) and significantly compromise allograft survival. The clinical relevance of antibodies directed against some HLA class II antigens, particularly HLA-DP, is less clear with conflicting reports on their pathogenicity. We report two patients with high levels of pretransplant donor-specific HLA-DP antibodies who subsequently developed recurrent acute AMR and graft failure. In both cases, there were no other donor-specific HLA alloantibodies, suggesting that the HLA-DP-specific antibodies may be directly pathogenic.

Guided imagery to improve functional outcomes post-anterior cruciate ligament repair: randomized-controlled pilot trial.

Imagery can improve functional outcomes post-anterior cruciate ligament repair (ACLR). Research is needed to investigate potential mechanisms for this effect. The aim of this study was to (a) evaluate the effectiveness of an imagery intervention to improve functional outcomes post-ACLR, and (b) explore potential mechanisms. A randomized-controlled pilot trial was conducted. Participants were randomized to guided imagery and standard rehabilitation or standard rehabilitation alone (control). The primary outcome was knee strength 6-month post-operatively. Secondary outcomes were knee laxity at 6-months, and change in psychological (self-efficacy) and neurohormonal (adrenaline, noradrenaline, dopamine) variables. Participants (n=21; 62% male) were 34.86 (SD 8.84) years. Following the intervention, no statistical differences between groups for knee strength extension at 180°/s (t=-0.43, P=0.67), or at 60°/s (t=-0.72, P=0.48) were found. A statistically significant effect was found for knee laxity, F=4.67, P<0.05, mean difference of -3.02 (95% CI -4.44 to -1.60), favoring the intervention. No differences were found for self-efficacy; however, an overall effect was found for noradrenaline, F(1, 19) 19.65, P<0.001, η(2) =0.52, and dopamine, F(1, 19) 6.23, P=0.02, η(2) =0.29, favoring the intervention. This imagery intervention improved knee laxity and healing-related neurobiological factors.

Targeting B cells and antibody in transplantation.

There has been increasing interest in the role played by B cells, plasma cells and their associated antibody in the immune response to an allograft, driven by the need to undertake antibody-incompatible transplantation and evidence suggesting that B cells play a role in acute cellular rejection and in acute and chronic antibody-mediated rejection. A number of immunosuppressive agents have emerged which target B cells, plasma cells and/or antibody, for example, the B cell-depleting CD20 antibody rituximab. This review describes recent developments in the use of such agents, our understanding of the role of B cells in alloimmunity and the application of this knowledge toward novel therapies in transplantation. It also considers the evidence to date suggesting that B cells may act as regulators of an alloimmune response. Thus, future attempts to target B cells will need to address the problem of how to inhibit effector B cells, while enhancing those with regulatory capacity.

Fabry disease: results of the first UK hemodialysis screening study.

BACKGROUND: Fabry disease is an X-linked lysosomal storage disorder in which deficiency of α-Galactosidase A (α-Gal A), leads to accumulation of glycosphingolipids in the vascular endothelium, kidneys and heart. Males with classical disease present in childhood, however some individuals with low levels of α-Gal A activity present atypically with adult onset renal impairment. Screening studies in patients with established end-stage renal failure (ESRF) suggest that up to 1.5% of patients have sub-normal α-Gal A levels. We used the dried blood spot (DBS) enzyme activity test to screen for undiagnosed Fabry disease in patients with ESRF. METHODS: Male hemodialysis patients treated at a single UK center (n = 155) were screened using the DBS assay. In patients with low enzyme activity on DBS, α-Gal A activity was assessed in plasma and leucocytes. RESULTS: 8 of the 155 (5%) patients screened showed low enzyme activity on the DBS assay. Confirmatory testing of plasma and leucocyte α-Gal A activity showed normal activity in all cases tested, indicating a false positive DBS result. CONCLUSIONS: This study is the first screening program in UK hemodialysis patients using the DBS test and did not identify any new cases of Fabry disease. In this cohort, the DBS enzyme assay had a false positive rate of 2.6%, emphasizing the need for validation with alternative techniques.

The generation and maintenance of serum alloantibody.

Donor-specific alloantibodies (DSA) mediate hyperacute and acute antibody-mediated rejection (AMR), which can lead to early graft damage and loss, and are also associated with chronic AMR and reduced long-term graft survival. Such alloantibodies can be generated by previous exposure to major histocompatibility (MHC) antigens (usually via blood transfusions, previous allografts or pregnancy) or can occur de novo after transplantation. Recent studies also suggest that non-MHC antibodies, including those recognising major histocompatibility complex class I-related chain A (MICA), MICB, vimentin, angiotensin II type I receptor may also have an adverse impact on allograft outcomes. In this review, we consider how the dose, route and context of antigen exposure influences DSA induction and describe factors which control the generation, maintenance and survival of alloantibody-producing plasma cells. Finally, we discuss the implications of these variables on therapeutic approaches to DSA.

Daystay hamstring ACL reconstruction performed under regional anaesthesia.

Daycase ACL reconstruction is commonly performed under general anaesthesia with a patella tendon graft. We report our experience with hamstring reconstruction under regional anaesthesia. Over a 14-month period, 104 daycase arthroscopic ACL reconstructions were performed by one surgeon and one anaesthetist. All operations were performed under spinal anaesthesia with a femoral nerve block. Patients were discharged with oral analgesia, brace and a cryocuff. One hundred and two patients were prospectively evaluated with a visual analogue pain score (0-10) and a patient satisfaction questionnaire. Of these 102 patients, 101 (99%) were happy to be discharged on the same day. One patient was admitted from the daycase unit, and one patient was re-admitted. Patients were very satisfied with the pain relief provided. The mean visual analogue pain score was 1.0 at discharge, 1.8 in the middle of the first night, and 2.1 on the first post-op day. Patients experienced significantly more pain the day after surgery than the evening of surgery (p=0.04). We conclude that hamstring ACL reconstruction under regional anaesthesia is well tolerated by patients as a daycase procedure.