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The Consortium for Organ Preservation in Europe (COPE) – an EU funded surgical research project on organ preservation – is running three clinical trials in five European countries. As of December 2014, all COPE trials are now live.
Bariatric surgery improves access to renal transplantation and is safe in renal failure as well as after transplantation: A systematic review and meta-analysis.
INTRODUCTION: Effective workup and listing of end-stage renal disease (ESRD) patients for renal transplantation, often with multiple co-morbidities, poses a challenge for transplant teams. Obesity is a common co-morbidity associated with adverse outcomes in ESRD and kidney transplant (KT) recipients. Bariatric and metabolic surgery (BMS) has long been established as a safe and effective treatment for morbid obesity. In this study, the authors aimed to evaluate the strength of evidence for both the efficacy and safety of bariatric surgery in patients with ESRD or kidney transplantation. METHODS: A literature search was performed using key terms including "transplantation", "kidney", "renal", "obesity", and "bariatric". Databases searched include MEDLINE, EMBASE and Web of Science from inception to date (April 2021). Methodological quality was assessed using the Newcastle-Ottawa tool. Selected articles were then categorised into patients awaiting waiting list acceptance, patients awaiting transplantation, patients undergoing simultaneous BMS + KT and patients undergoing BMS following a previous renal transplant. Summary effects are presented with a level of statistical significance and 95% Confidence Intervals. RESULTS: A total of 28 articles were selected following the literature search. Fourteen studies on patients awaiting listing (n = 1903), nine on patients on the KT waiting list (n = 196), a single study on simultaneous BMS and KT and ten studies on patients undergoing BMS following KT (n = 198). Mean change in BMI for patients awaiting listing was -11.3 kg/m2 (95%CI: -15.3 to -7.3, p
Abstract 1369: Predicting clinical pharmacokinetics and toxicity of current and emerging oncology therapeutics by normothermic perfusion of isolated human-sized organs
Abstract Novel cancer therapeutics have less than a 12% probability of translating from bench to bedside. Unwarranted toxicity and inadequate therapeutic delivery due to uptake by clearance organs, not predicted by current preclinical methods, have contributed towards this high rate of attrition. In the present work, we propose normothermic machine perfusion of human or human-sized organs as a more predictive, closer-to-human model to investigate drug pharmacokinetics and toxicity. Over the past decade, developments in the field of organ preservation for transplantation have enabled prolonged (>12 hours) normothermic machine perfusion (NMP) of isolated porcine or human organs ex vivo, maintaining quasi-physiological haemodynamic, synthetic and metabolic function using a packed red cell perfusate with physiological oxygenation and nutrient levels at normal body temperature. This preserves physiological processes such as metabolism and drug elimination, and enables easy access to tissue, blood and excreted biological fluids, with NMP livers producing bile and NMP kidneys producing urine. We hypothesise that this will provide a physiologically relevant platform to investigate drug pharmacokinetics and toxicity. We selected a widely used small-molecule chemotherapeutic (Irinotecan hydrochloride 2mg/ml, Medac, UK) which has seen decades of clinical use and benefits from extensive clinical pharmacokinetic data. The small-molecule drug was infused into isolated porcine and human livers and kidneys, with quantification of concentration time profiles of the prodrug and its main metabolites in plasma, bile and urine over 16-24 hours of NMP. In addition to irinotecan (CPT11), three of its metabolites (APC, SN38G, SN38) were successfully detected and quantified, demonstrating peak plasma concentrations (Cmax ~ 10,000 ng/mL, 1000 ng/mL, 100 ng/g, 30 ng/g), plasma decay rates and percentages of injected dose in bile (%ID ~20%, 8%, 25%, 1%) and urine (%ID ~20%, 0.06%, 0.5%,0.1%) that are comparable to clinical data. Drug-tissue toxicity could also be adequately replicated in the NMP model. In conclusion, we have demonstrated that human-sized isolated, normothermically perfused livers and kidneys accurately represent the clinically observed pharmacokinetic and toxicity profiles of an established small-molecule therapeutic. Further model validation is ongoing for biologics and other nanomedicines which are susceptible to clearance by the mononuclear phagocytic system or are hepato- or nephrotoxic. If this proves successful, normothermic machine perfusion of isolated porcine and human organs could greatly aid the early screening of candidate therapeutics and significantly enhance the pace and success rate with which they are translated into patients. Citation Format: Tamsyn Clark, Luca Bau, Fungai Dengu, Daniel Voyce, Robert Carlisle, Peter Friend, Constantin Coussios. Predicting clinical pharmacokinetics and toxicity of current and emerging oncology therapeutics by normothermic perfusion of isolated human-sized organs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1369.
SARS-CoV-2-Specific T Cell Responses Are Not Associated with Protection against Reinfection in Hemodialysis Patients.
Patients on hemodialysis (HD) are vulnerable to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and mount poor neutralizing antibody responses after two-dose vaccination. Although serological responses have been associated with reduced rates of reinfection, the relationship between cellular immunogenicity and protection has not been established. We report, for the first time, high incidence of reinfection in patients on HD who are vaccine naive (25%), which identifies that T cell responses do not predict protection against reinfection. Instead, patients on HD who went on to become reinfected had mounted highly variable and sometimes robust proliferative T cell responses to a broad array of SARS-CoV-2 peptide pools during the primary infection. The understanding that SARS-CoV-2–specific T cell responses are not predictive of protection against future infection will be a critical issue when measuring clinical efficacy of vaccination in these vulnerable cohorts, particularly when facing rapidly emerging variants of concern.
Development of ex situ normothermic reperfusion as an innovative method to assess pancreases after preservation.
Static cold storage (SCS) is the standard method for pancreas preservation prior to transplantation; however, it does not permit organ assessment. Normothermic reperfusion (NR) is utilized clinically for other organs to assess viability. Our aim was to develop NR using normothermic machine perfusion technique to simulate reperfusion at the time of transplantation, enabling evaluation of oxygenated hypothermic machine perfusion (HMPO2) as a newer strategy to optimize pancreas preservation. 13 porcine pancreases procured after circulatory death were divided into 3 groups: 4 pancreases preserved using SCS, and 2 groups preserved by HMPO2 (n = 4 and n = 5, differing by type of preservation solution). Duration of perfusion or cold storage was 6 hours before the 1-hour assessment using NR. Outcome measures were perfusion characteristics, biochemistry and change in tissue water mass as oedema assessment. During NR, the HMPO2 groups demonstrated better perfusion characteristics, normal macroscopic appearances, decreased water mass and one HMPO2 group demonstrated a response to glucose stimulation. Conversely, the SCS group showed an increased water mass and developed early macroscopic appearances of oedema, interstitial haemorrhage and minimal portal outflow. This study suggests that ex situ assessment of pancreases by NR is promising, and that HMPO2 may be better than SCS.
Ischemia-Reperfusion Injuries Assessment during Pancreas Preservation.
Maintaining organ viability between donation and transplantation is of critical importance for optimal graft function and survival. To date in pancreas transplantation, static cold storage (SCS) is the most widely practiced method of organ preservation. The first experiments in ex vivo perfusion of the pancreas were performed at the beginning of the 20th century. These perfusions led to organ oedema, hemorrhage, and venous congestion after revascularization. Despite these early hurdles, a number of factors now favor the use of perfusion during preservation: the encouraging results of HMP in kidney transplantation, the development of new perfusion solutions, and the development of organ perfusion machines for the lung, heart, kidneys and liver. This has led to a resurgence of research in machine perfusion for whole organ pancreas preservation. This review highlights the ischemia-reperfusion injuries assessment during ex vivo pancreas perfusion, both for assessment in pre-clinical experimental models as well for future use in the clinic. We evaluated perfusion dynamics, oedema assessment, especially by impedance analysis and MRI, whole organ oxygen consumption, tissue oxygen tension, metabolite concentrations in tissue and perfusate, mitochondrial respiration, cell death, especially by histology, total cell free DNA, caspase activation, and exocrine and endocrine assessment.
Assessment of Mitochondrial Function and Oxygen Consumption Measured During Ex Vivo Normothermic Machine Perfusion of Injured Pig Kidneys Helps to Monitor Organ Viability.
Donor kidney assessment may improve organ utilisation. Normothermic Machine Perfusion (NMP) has the potential to facilitate this advance. The mechanism of action is not yet determined and we aimed to assess mitochondrial function during NMP. Anaesthetised pigs (n = 6) had one kidney clamped for 60 min. The healthy contralateral kidney was removed and underwent NMP for 8 h (healthy control (HC), n = 6). Following 60 min warm ischaemia the injured kidney underwent HMP for 24 h, followed by NMP for 8 h (n = 6). Mitochondria were extracted from fresh tissue for analysis. Injured kidneys were analysed as two separate groups (IMa, n = 3 and IMb, n = 3). Renal resistance was higher (0.39ï, ± 0.29 vs. 1.65ï, ± 0.85; p = 0.01) and flow was lower (55ï, ± 28 vs. 7ï, ± 4; p = 0.03) during HMP in IMb than IMa. NMP blood flow was higher in IMa versus IMb (2-way ANOVA; p < 0.001) After 60 min NMP, O2 consumption was significantly lower in IMb versus IMa (p ≤ 0.002). State-3 respiration was significantly different between the groups (37ï, ± 19 vs. 24ï, ± 14 vs. 10ï, ± 8; nmolO2/min/mg; p = 0.049). Lactate levels were significantly lower in IMa versus IMb (p = 0.028). Mitochondrial respiration levels during NMP may be suggestive of kidney viability. Oxygen consumption, renal blood flow and lactate can differentiate severity of kidney injury during NMP.
Abdominal multiorgan procurement from slaughterhouse pigs: a bespoke model in organ donation after circulatory death for ex vivo organ perfusion compliant with the 3 Rs (Reduction, Replacement & Refinement).
BACKGROUND: Advances in organ preservation, reconditioning and assessment have been driven by the increasing necessity to utilise organs from extended criteria donors, particularly donors after circulatory death. Research efforts in this area have aided translation of machine perfusion technology into clinical practice. Pigs are anatomically and physiologically similar to humans and are an excellent model. However, conducting large animal experimental research is challenging and typically limited by ethical and economic constraints. Here we describe a reproducible, cost-effective multi-organ abdominal procurement model of porcine organs from the slaughterhouse. METHODS: Domestic pigs are electrically stunned and exsanguinated following the standard abattoir process. Via a longitudinal midline incision, the thoracoabdominal viscera are removed en bloc by incising along the anterior vertebral plane. The abdominal organs are isolated, perfused and separated preserving their respective vasculature, allowing individual organ use for specific experiments. RESULTS: The warm ischaemic time is kept between 15-30 minutes. Using this highly protocolized procurement technique we have procured 12 livers, 162 kidneys and 12 pancreata for research, the majority of which have been utilized for ex situ perfusion experiments. CONCLUSIONS: We have described a reliable and reproducible procedure for abdominal multi-organ procurement from slaughterhouse pigs.
Auditory cues modulate the short timescale dynamics of STN activity during stepping in Parkinson's disease.
BACKGROUND: Gait impairment has a major impact on quality of life in patients with Parkinson's disease (PD). It is believed that basal ganglia oscillatory activity at β frequencies (15-30 Hz) may contribute to gait impairment, but the precise dynamics of this oscillatory activity during gait remain unclear. Additionally, auditory cues are known to lead to improvements in gait kinematics in PD. If the neurophysiological mechanisms of this cueing effect were better understood they could be leveraged to treat gait impairments using adaptive Deep Brain Stimulation (aDBS) technologies. OBJECTIVE: We aimed to characterize the dynamics of subthalamic nucleus (STN) oscillatory activity during stepping movements in PD and to establish the neurophysiological mechanisms by which auditory cues modulate gait. METHODS: We studied STN local field potentials (LFPs) in eight PD patients while they performed stepping movements. Hidden Markov Models (HMMs) were used to discover transient states of spectral activity that occurred during stepping with and without auditory cues. RESULTS: The occurrence of low and high β bursts was suppressed during and after auditory cues. This manifested as a decrease in their fractional occupancy and state lifetimes. Interestingly, α transients showed the opposite effect, with fractional occupancy and state lifetimes increasing during and after auditory cues. CONCLUSIONS: We show that STN oscillatory activity in the α and β frequency bands are differentially modulated by gait-promoting oscillatory cues. These findings suggest that the enhancement of α rhythms may be an approach for ameliorating gait impairments in PD.
Evaluation of 3D C-Arm Fluoroscopy versus Diagnostic CT for Deep Brain Stimulation Stereotactic Registration and Post-Operative Lead Localization.
INTRODUCTION: DBS efficacy depends on accuracy. CT-MRI fusion is established for both stereotactic registration and electrode placement verification. The desire to streamline DBS workflows, reduce operative time, and minimize patient transfers has increased interest in portable imaging modalities such as the Medtronic O-arm® and mobile CT. However, these remain expensive and bulky. 3D C-arm fluoroscopy (3DXT) units are a smaller and less costly alternative, albeit incompatible with traditional frame-based localization and without useful soft tissue resolution. We aimed to compare fusion of 3DXT and CT with pre-operative MRI to evaluate if 3DXT-MRI fusion alone is sufficient for accurate registration and reliable targeting verification. We further assess DBS targeting accuracy using a 3DXT workflow and compare radiation dosimetry between modalities. METHODS: Patients underwent robot-assisted DBS implantation using a workflow incorporating 3DXT which we describe. Two intra-operative 3DXT spins were performed for registration and accuracy verification followed by conventional CT post-operatively. Post-operative 3DXT and CT images were independently fused to the same pre-operative MRI sequence and co-ordinates generated for comparison. Registration accuracy was compared to 15 consecutive controls who underwent CT-based registration. Radial targeting accuracy was calculated and radiation dosimetry recorded. RESULTS: Data were obtained from 29 leads in 15 consecutive patients. 3DXT registration accuracy was significantly superior to CT with mean error 0.22 ± 0.03 mm (p < 0.0001). Mean Euclidean electrode tip position variation for CT to MRI versus 3DXT to MRI fusion was 0.62 ± 0.40 mm (range 0.0 mm-1.7 mm). In comparison, direct CT to 3DXT fusion showed electrode tip Euclidean variance of 0.23 ± 0.09 mm. Mean radial targeting accuracy assessed on 3DXT was 0.97 ± 0.54 mm versus 1.15 ± 0.55 mm on CT with differences insignificant (p = 0.30). Mean patient radiation doses were around 80% lower with 3DXT versus CT (p < 0.0001). DISCUSSION: Mobile 3D C-arm fluoroscopy can be safely incorporated into DBS workflows for both registration and lead verification. For registration, the limited field of view requires the use of frameless transient fiducials and is highly accurate. For lead position verification based on MRI co-registration, we estimate there is around a 0.4 mm discrepancy between lead position seen on 3DXT versus CT when corrected for brain shift. This is similar to that described in O-arm® or mobile CT series. For units where logistical or financial considerations preclude the acquisition of a cone beam CT or mobile CT scanner, our data support portable 3D C-arm fluoroscopy as an acceptable alternative with significantly lower radiation exposure.
LDHB contributes to the regulation of lactate levels and basal insulin secretion in human pancreatic β cells.
Using 13C6 glucose labeling coupled to gas chromatography-mass spectrometry and 2D 1H-13C heteronuclear single quantum coherence NMR spectroscopy, we have obtained a comparative high-resolution map of glucose fate underpinning β cell function. In both mouse and human islets, the contribution of glucose to the tricarboxylic acid (TCA) cycle is similar. Pyruvate fueling of the TCA cycle is primarily mediated by the activity of pyruvate dehydrogenase, with lower flux through pyruvate carboxylase. While the conversion of pyruvate to lactate by lactate dehydrogenase (LDH) can be detected in islets of both species, lactate accumulation is 6-fold higher in human islets. Human islets express LDH, with low-moderate LDHA expression and β cell-specific LDHB expression. LDHB inhibition amplifies LDHA-dependent lactate generation in mouse and human β cells and increases basal insulin release. Lastly, cis-instrument Mendelian randomization shows that low LDHB expression levels correlate with elevated fasting insulin in humans. Thus, LDHB limits lactate generation in β cells to maintain appropriate insulin release.
Kidney Tissue Proteome Profiles in Short Versus Long Duration of Delayed Graft Function - A Pilot Study in Donation After Circulatory Death Donors
Introduction: Delayed graft function (DGF) is often defined as the need for dialysis treatment in the first week after a kidney transplantation. This definition, though readily applicable, is generic and unable to distinguish between “types” of DGF or time needed to recover function that may also significantly affect longer-term outcomes. We aimed to profile biological pathways in donation after circulatory death (DCD) kidney donors that correlate with DGF and different DGF durations. Methods: A total of N ¼ 30 DCD kidney biopsies were selected from the UK Quality in Organ Donation (QUOD) biobank and stratified according to DGF duration (immediate function, IF n ¼ 10; “short-DGF” (1–6 days), SDGF n ¼ 10; “long-DGF” (7–22 days), LDGF n ¼ 10). Samples were matched for donor and recipient demographics and analyzed by label-free quantitative (LFQ) proteomics, yielding identification of N ¼ 3378 proteins. Results: Ingenuity pathway analysis (IPA) on differentially abundant proteins showed that SDGF kidneys presented upregulation of stress response pathways, whereas LDGF presented impaired response to stress, compared to IF. LDGF showed extensive metabolic deficits compared to IF and SDGF. Conclusion: DCD kidneys requiring dialysis only in the first week posttransplant present acute cellular injury at donation, alongside repair pathways upregulation. In contrast, DCD kidneys requiring prolonged dialysis beyond 7 days present minimal metabolic and antioxidant responses, suggesting that current DGF definitions might not be adequate in distinguishing different patterns of injury in donor kidneys contrib uting to DGF.
Adaptation to chronic hypoxia in triple-negative breast cancer
Low levels of oxygen (i.e. hypoxia) are often observed in solid tumours. Hypoxia is able to drive several of the cancer hallmarks and promotes resistance to both radio and chemotherapy, resulting in its association with poor prognosis. Triple-negative breast cancer has the worst prognosis of all breast cancer subtypes with a 5 year survival rate of only 76.7%, and only 10.8% if the cancer has already metastasised. In line with this, triple-negative tumours often have higher levels of hypoxia than their hormone receptor expressing counterparts. The length and severity of hypoxia is heterogeneous both within and between tumours, meaning cells within the same tumour may experience both acute and chronic hypoxia. Acute hypoxia (⩽ 24 hours) is relatively well studied, whereas research into chronic hypoxia (⩾ 7 days) is lacking. This thesis therefore aimed to characterise the transcriptional response to chronic hypoxia using RNA sequencing. It also aimed to identify proteins or microRNAs which are necessary for adaptation to chronic hypoxia using genome-wide CRISPR screening. This method enables specific vulnerabilities in hypoxic cells to be identified, meaning drugs can be designed to target these “Achilles heels”. The major pathways altered under chronic hypoxia were related to epithelial-mesenchymal transition (EMT) and cell adhesion, with triple-negative breast cancer cells undergoing an atypical form of EMT. However, none of the genes which were upregulated in response to chronic hypoxia were found to be essential for their survival. Instead, hypoxic cells had a greater dependence on general control nonderepressible 2 (GCN2), an instigator of the integrated stress response usually activated under amino acid starvation. GCN2 was found to be active under chronic hypoxia, and chemical inhibition of GCN2 reduced cell growth in vitro. Previous work has shown that GCN2 knockout mice are healthy unless fed a diet restricted in essential amino acids, meaning targeting GCN2 is unlikely to result in severe toxicities. Together, this means GCN2 constitutes a promising therapeutic target for hypoxic tumours. No microRNAs essential for survival under hypoxia were identified in this study.
A Report on the Safety of Acitretin Use in Renal Failure Patients on Hemodialysis
Abstract Acitretin, commonly used for severe psoriasis and keratinocyte carcinoma chemoprevention in high-risk patients, is contraindicated in patients with end stage renal disease on hemodialysis. However, these patients often lack medication choices and in certain clinical scenarios, the benefits of acitretin may outweigh the potential risks. We identified 24 end stage renal disease patients on HD taking acitretin from Duke and Vanderbilt University Medical Centers. While adverse effects were common, patients did not frequently discontinue the medication due to them. We also found no association between acitretin with hospital admissions or mortality. We lastly found statistically significant increases in ALP and total bilirubin when on acitretin and dialysis compared to baseline. However, there was no dose-dependency or temporal association with acitretin or hemodialysis initiation. Based off these preliminary findings, we find that acitretin may safely be used in patients receiving HD with close monitoring of ALP and bilirubin.
Long-term Outcomes With Islet-Alone and Islet-After-Kidney Transplantation for Type 1 Diabetes in the Clinical Islet Transplantation Consortium: The CIT-08 Study.
OBJECTIVE: To determine long-term outcomes for islet-alone and islet-after-kidney transplantation in adults with type 1 diabetes complicated by impaired awareness of hypoglycemia. RESEARCH DESIGN AND METHODS: This was a prospective interventional and observational cohort study of islet-alone (n = 48) and islet-after-kidney (n = 24) transplant recipients followed for up to 8 years after intraportal infusion of one or more purified human pancreatic islet products under standardized immunosuppression. Outcomes included duration of islet graft survival (stimulated C-peptide ≥0.3 ng/mL), on-target glycemic control (HbA1c <7.0%), freedom from severe hypoglycemia, and insulin independence. RESULTS: Of the 48 islet-alone and 24 islet-after-kidney transplantation recipients, 26 and 8 completed long-term follow-up with islet graft function, 15 and 7 withdrew from follow-up with islet graft function, and 7 and 9 experienced islet graft failure, respectively. Actuarial islet graft survival at median and final follow-up was 84% and 56% for islet-alone and 69% and 49% for islet-after-kidney (P = 0.007) with 77% and 49% of islet-alone and 57% and 35% of islet-after-kidney transplantation recipients maintaining posttransplant HbA1c <7.0% (P = 0.0017); freedom from severe hypoglycemia was maintained at >90% in both cohorts. Insulin independence was achieved by 74% of islet-alone and islet-after-kidney transplantation recipients, with more than one-half maintaining insulin independence during long-term follow-up. Kidney function remained stable during long-term follow-up in both cohorts, and rates of sensitization against HLA were low. Severe adverse events occurred at 0.31 per patient-year for islet-alone and 0.43 per patient-year for islet-after-kidney transplantation. CONCLUSIONS: Islet transplantation results in durable islet graft survival permitting achievement of glycemic targets in the absence of severe hypoglycemia for most appropriately indicated recipients having impaired awareness of hypoglycemia, with acceptable safety of added immunosuppression for both islet-alone and islet-after-kidney transplantation.
Neutrophilia, lymphopenia and myeloid dysfunction: a living review of the quantitative changes to innate and adaptive immune cells which define COVID-19 pathology.
Destabilization of balanced immune cell numbers and frequencies is a common feature of viral infections. This occurs due to, and further enhances, viral immune evasion and survival. Since the discovery of the Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), which manifests in coronavirus disease 2019 (COVID-19), a great number of studies have described the association between this virus and pathologically increased or decreased immune cell counts. In this review, we consider the absolute and relative changes to innate and adaptive immune cell numbers, in COVID-19. In severe disease particularly, neutrophils are increased, which can lead to inflammation and tissue damage. Dysregulation of other granulocytes, basophils and eosinophils represents an unusual COVID-19 phenomenon. Contrastingly, the impact on the different types of monocytes leans more strongly to an altered phenotype, e.g. HLA-DR expression, rather than numerical changes. However, it is the adaptive immune response that bears the most profound impact of SARS-CoV-2 infection. T cell lymphopenia correlates with increased risk of intensive care unit admission and death; therefore, this parameter is particularly important for clinical decision-making. Mild and severe diseases differ in the rate of immune cell counts returning to normal levels post disease. Tracking the recovery trajectories of various immune cell counts may also have implications for long-term COVID-19 monitoring. This review represents a snapshot of our current knowledge, showing that much has been achieved in a short period of time. Alterations in counts of distinct immune cells represent an accessible metric to inform patient care decisions or predict disease outcomes.