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.
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.
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.
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.
We report the successful transplantation of a pair of human kidneys that were declined for transplantation due to inadequate in situ perfusion but subsequently transplanted after perfusion and ...assessment using ex vivo normothermic perfusion (EVNP). The kidneys were from a 35‐year‐old man, a donation after circulatory death donor. Both kidneys were declined by all UK transplant centers. On arrival, the kidneys had significant areas of incomplete clearance of blood from the microcirculation that did not clear after a further attempt to flush them. Kidneys underwent 60 min of EVNP with an oxygenated packed red blood cell–based solution warmed to 35.2°C. During EVNP, the patchy areas cleared in both kidneys. The mean renal blood flow and total urine output were 68.0 mL/min/100 g and 560 mL in the left kidney and 59.9 mL/min/100 g, 430 mL in the right, respectively. Based on the EVNP perfusion parameters, both kidneys were deemed suitable for transplantation. They were transplanted without any complications, and both recipients had initial graft function. The serum creatinine levels at 3 months were 1.2 mg/dl in the recipient of the left kidney and 1.62 mg/dl in the recipient of the right kidney. EVNP technology can be used to assess and rescue kidneys previously deemed unsuitable for transplantation.
Ex vivo normothermic perfusion allows the transplantation of two kidneys deemed untransplantable by all UK centers due to inadequate in situ perfusion.
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.
The Cambridge Pancreatic Allograft Thrombosis grading system may be used to standardize reports of thrombosis, identify patients who would benefit from therapeutic anticoagulation, provide prognostic information, and allow better comparison of reports from different centers for improved understanding and management of this common condition.
Human epithelial tissues accumulate cancer-driver mutations with age
, yet tumour formation remains rare. The positive selection of these mutations suggests that they alter the behaviour and fitness ...of proliferating cells
. Thus, normal adult tissues become a patchwork of mutant clones competing for space and survival, with the fittest clones expanding by eliminating their less competitive neighbours
. However, little is known about how such dynamic competition in normal epithelia influences early tumorigenesis. Here we show that the majority of newly formed oesophageal tumours are eliminated through competition with mutant clones in the adjacent normal epithelium. We followed the fate of nascent, microscopic, pre-malignant tumours in a mouse model of oesophageal carcinogenesis and found that most were rapidly lost with no indication of tumour cell death, decreased proliferation or an anti-tumour immune response. However, deep sequencing of ten-day-old and one-year-old tumours showed evidence of selection on the surviving neoplasms. Induction of highly competitive clones in transgenic mice increased early tumour removal, whereas pharmacological inhibition of clonal competition reduced tumour loss. These results support a model in which survival of early neoplasms depends on their competitive fitness relative to that of mutant clones in the surrounding normal tissue. Mutant clones in normal epithelium have an unexpected anti-tumorigenic role in purging early tumours through cell competition, thereby preserving tissue integrity.
A significant number of pancreases procured for transplantation are deemed unsuitable due to concerns about graft quality and the associated risk of complications. However, this decision is ...subjective and some declined grafts may be suitable for transplantation. Ex vivo normothermic perfusion (EVNP) prior to transplantation may allow a more objective assessment of graft quality and reduce discard rates. We report ex vivo normothermic perfusion of human pancreases procured but declined for transplantation, with ABO‐compatible warm oxygenated packed red blood cells for 1–2 h. Five declined human pancreases were assessed using this technique after a median cold ischemia time of 13 h 19 min. One pancreas, with cold ischemia over 30 h, did not appear viable and was excluded. In the remaining pancreases, blood flow and pH were maintained throughout perfusion. Insulin secretion was observed in all four pancreases, but was lowest in an older donation after cardiac death pancreas. Amylase levels were highest in a gland with significant fat infiltration. This is the first study to assess the perfusion, injury, as measured by amylase, and exocrine function of human pancreases using EVNP and demonstrates the feasibility of the approach, although further refinements are required.
This study reports the first use of a short period of ex vivo normothermic perfusion to assess the quality of human donor pancreases deemed unsuitable for transplantation, reporting injury markers, endocrine function, and histopathology.
Histological assessment of baseline chronic kidney injury may discriminate kidneys that are suitable for transplantation, but has not been validated for appraisal of donation after circulatory death ...(DCD) kidneys. ‘Time‐zero' biopsies for 371 consecutive, solitary, deceased‐donor kidneys transplanted at our center between 2006 and 2010 (65.5% DCD, 34.5% donation after brain death DBD) were reviewed and baseline chronic degenerative injury scored using Remuzzi's classification. High scores correlated with donor age and extended criteria donors (42% of donors), but the spectrum of scores was similar for DCD and DBD kidneys. Transplant outcomes for kidneys scoring from 0 to 4 were comparable (1 and 3 year graft survival 95% and 92%), but were much poorer for kidneys scoring ≥5, with 1 year graft survival only 73%, and 12.5% suffering primary nonfunction. Critically, high Remuzzi scores conferred the same survival disadvantage for DCD and DBD kidneys. On multi‐variable regression analysis, time‐zero biopsy score was the only independent predictor for graft survival, whereas one‐year graft estimated glomerular filtration rate (eGFR) correlated with donor age and biopsy score. In conclusion, the relationship between severity of chronic kidney injury and transplant outcome is similar for DCD and DBD kidneys. Kidneys with Remuzzi scores of ≤4 can be implanted singly with acceptable results.
This study investigating the role of “time‐zero” histological assessment of baseline donor chronic kidney injury shows that high Remuzzi biopsy scores confer the same survival disadvantage for DCD as DBD kidney transplants, and suggests that a Remuzzi biopsy score of 5 represents the cut‐off below which deceased‐donor kidneys can be implanted singly.
To evaluate the effect of pre-biopsy magnetic resonance imaging (MRI) on cancer diagnostic times, and to report MRI-directed pathology outcomes.
In total, 1483 patients were referred with prostate ...cancer suspicion during a 30-month period. Upfront MRI was performed in 745 patients: 332 MRIs in the 15 months prior to dedicated scanning slots (group 1), and 413 in the 15 months post-introduction (group 2). A further 88 patients had initial MRI following clinical assessment. Biopsy via the transrectal (TR) or transperineal (TP) approach was performed, with MRI/ultrasound fusion for MRI targets. Clinically significant cancer (csPCa) was defined as Gleason ≥3+4. Negative MRIs were defined as Likert 1–2. Per-case clinical decisions were taken to biopsy or not.
44.4% of patients avoided biopsy. 484/833 (58.1%) MRIs were negative; 37.4% of these patients had biopsy with a negative predictive value (NPV) of 92.8% for Gleason ≥3+4 and 98.3% for ≥4+3. Overall prostate cancer prevalence was 34.3% (24.6% csPCa). In 323 MRI-positive cases, any cancer was present in 78.9% (csPCa 60.4%). Of the 1483 patients, 1232 (83.1%) completed all diagnostic tests within 28 days. Upfront MRI patients met this standard in 621/833 (74.5%), improving from 66.9% to 81.1% with reserved slots (group 2) with a reduced diagnostic time from median 25.5 to 20.9 days. Biopsy scheduling delayed the pathway in 69.7%, with MRI responsible in 22.3%, reducing to 10.3% in group 2. TP biopsies met the 28-day standard in significantly less cases (29.7%), compared to TR (67.4%, p<0.0001).
Reserved MRI slots reduces time-to-diagnosis, and upfront MRI safely avoids biopsy in a significant proportion of men, whilst maintaining expected csPCa detection rates.
•Overall biopsy avoidance rate in the upfront MRI cohort was 44% in a 3 year period.•Use of reserved MRI slots reduced diagnostic time from median 25.5 to 20.9 days.•NPV in men with negative MR undergoing biopsy for high clinical risk was high at 92%.•Cancer detection matched literature reported rates for clinically significant cancer.
This article documents a single-centre experience with the use of the Isiris
TM
single-use flexible cystoscope with integrated grasper system for the removal of ureteric stents from kidney transplant ...recipients in the operating theatre or in clinic. The data demonstrated that the Isiris
TM
cystoscope can be used safely for ureteric stent removal in transplant recipients, and that an outpatient ureteric stent removal service is feasible, cost-effective, and safe in experienced hands.
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.
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.
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).
Mitochondria-targeted antioxidant MitoQ can be administered to ischaemic kidneys simply and effectively during cold storage, and may improve outcomes after transplantation.
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