Pharmacological testing of therapeutics using normothermic machine perfusion: A pilot study of 2,4-dinitrophenol delivery to steatotic human livers.

INTRODUCTION: Normothermic machine perfusion (NMP) provides a platform for drug-delivery. However, pharmacological considerations for therapeutics delivered during NMP are scarcely reported. We aimed to demonstrate the ability of NMP as a platform for pharmacological testing, using a drug which increases metabolism (2,4-dinitrophenol; DNP) as an example therapeutic. METHODS: We performed 25 h of NMP on human livers which had been declined for transplant due to steatosis (n = 7). Three livers received a DNP bolus, three were controls, and one received a DNP infusion. RESULTS: Toxicity studies revealed DNP delivery was safe, without hepatotoxic effects. The liver surface temperature was increased in the DNP group (p = 0.046), but no livers suffered hyperthermia-the mechanism of DNP toxicity in vivo. Pharmacokinetic studies revealed DNP elimination with first-order kinetics and 7.7 h half-life (95% CI = 5.1-15.9 hrs). The clearance of DNP in bile was negligible. As expected, DNP significantly increased oxygen consumption (p = 0.023); this increase was closely correlated with perfusate DNP concentration (r2  = 0.975; p = 0.002) and the effect was lost as DNP was eliminated by the liver. A DNP infusion rate, calculated using our pharmacokinetic data, successfully maintained perfusate DNP concentration. DISCUSSION: Detailed pharmacological testing can be performed during NMP. Our therapeutic (DNP) is rapidly eliminated by the ex vivo liver, meaning the drug effect of increased metabolism is only transient. This demonstrates the importance of assessing pharmacokinetics when delivering therapeutics during NMP, especially for prolonged perfusion of organs with established roles in drug elimination. Rigorous pharmacological testing is needed to unlock the potential of NMP as a clinical drug-delivery platform.

Novel delivery of cellular therapy to reduce ischemia reperfusion injury in kidney transplantation.

Ex vivo normothermic machine perfusion (NMP) of donor kidneys prior to transplantation provides a platform for direct delivery of cellular therapeutics to optimize organ quality prior to transplantation. Multipotent Adult Progenitor Cells (MAPC® ) possess potent immunomodulatory properties that could minimize ischemia reperfusion injury. We investigated the potential capability of MAPC cells in kidney NMP. Pairs (5) of human kidneys, from the same donor, were simultaneously perfused for 7 hours. Kidneys were randomly allocated to receive MAPC treatment or control. Serial samples of perfusate, urine, and tissue biopsies were taken for comparison. MAPC-treated kidneys demonstrated improved urine output (P = .009), decreased expression of injury biomarker NGAL (P = .012), improved microvascular perfusion on contrast-enhanced ultrasound (cortex P = .019, medulla P = .001), downregulation of interleukin (IL)-1ß (P = .050), and upregulation of IL-10 (P < .047) and Indolamine-2, 3-dioxygenase (P = .050). A chemotaxis model demonstrated decreased neutrophil recruitment when stimulated with perfusate from MAPC-treated kidneys (P < .001). Immunofluorescence revealed prelabeled MAPC cells in the perivascular space of kidneys during NMP. We report the first successful delivery of cellular therapy to a human kidney during NMP. Kidneys treated with MAPC cells demonstrate improvement in clinically relevant parameters and injury biomarkers. This novel method of cell therapy delivery provides an exciting opportunity to recondition organs prior to transplantation.

Hypothermic machine perfusion is superior to static cold storage in deceased donor kidney transplantation: A meta-analysis.

BACKGROUND: There remains a lack of consensus on the optimal storage method for deceased donor kidneys. This meta-analysis compares storage with hypothermic machine perfusion (HMP) vs traditional static cold storage (SCS). METHODS: The Cochrane Kidney and Transplant Specialised Register was searched to identify (quasi-) randomized controlled trials (RCTs) to include in our meta-analysis. PRISMA guidelines were used to perform and write this review. RESULTS: There is high-certainty evidence that HMP reduces the risk of delayed graft function (DGF) when compared to SCS (2138 participants from 14 studies, RR = 0.77; 0.67-0.90, P = .0006). This benefit is significant in both donation following circulatory death (DCD; 772 patients from seven studies, RR = 0.75; 0.64-0.87, P = .0002) and donation following brainstem death (DBD) grafts (971 patients from four studies, RR = 0.78; 0.65-0.93, P = .006). The number of perfusions required to prevent one episode of DGF was 7.26 and 13.60 in DCD and DBD grafts, respectively. There is strong evidence that HMP also improves graft survival in both DBD and DCD grafts, at both 1 and 3 years. Economic analyses suggest HMP is cost-saving at 1 year compared with SCS. CONCLUSION: Hypothermic machine perfusion is superior to SCS in deceased donor renal transplantation. Direct comparisons with normothermic machine perfusion in RCTs are essential to identify optimal preservation methods in kidney transplantation.

UK registry analysis of donor substance misuse and outcomes following pancreas transplantation.

Substance abuse is unfortunately common in organ donors. Often, these organs are declined for transplant, not only because of concerns around blood-borne virus transmission but also because of perceived poor outcomes. In kidney transplantation, previous studies have demonstrated donor smoking status significantly impacts transplant outcome, but intravenous drug use or alcohol dependence does not. This study aims to clarify these issues in pancreas transplantation. Retrospective data on all UK solid organ pancreas transplants from 1994 to 2015 were obtained from the NHSBT UK Transplant Registry. The impact of illicit drug misuse, alcohol abuse, and smoking on graft and patient survival were analyzed using Kaplan-Meier plots and a Cox regression model. A total of 1175 of the 2317 (49.5%) donors were categorized as substance misusers. Univariate survival analysis revealed no significant impact of substance misuse on 10-year graft or patient survival. Multivariate analysis confirmed substance misuse was not associated with impaired graft or patient survival. A history of donor substance misuse does not negatively impact 10-year graft or patient survival following pancreas transplantation. This is a large national registry analysis with long-term follow-up data and should therefore provide clinicians with reassurance when considering pancreas grafts from substance misuse donors.

Risk factors associated with the reported scores on the symptoms severity and functional limitations scales of the Boston Carpal Tunnel Questionnaire: a cross-sectional study.

Carpal tunnel syndrome (CTS) is the most common entrapment neuropathy. It presents with numbness, paresthesias, and pain. Multiple risk factors are associated with CTS, such as pregnancy, oral contraceptive use, rheumatoid arthritis (RA), and diabetes mellitus (DM). The Boston Carpal Tunnel Questionnaire (BCTQ) is a self-administered questionnaire for assessing the severity of symptoms and functional status of those previously diagnosed with CTS. We aim to identify risk factors associated with higher scores of CTS symptoms severity and functional limitations scales on the BCTQ. Materials and methods: This cross-sectional study was conducted among 366 female participants. The data was mainly collected using the BCTQ. Demographics and risk factors of CTS were added to the study's complete questionnaire; risk factors included RA, DM, hypothyroidism, number of pregnancies, usage of oral contraceptive pills (OCPs), use of smartphones and keyboards. A P value of less than 0.05 was considered statistically significant. Results: Most participants were in their 30s (44%) and housewives. RA, DM, hypothyroidism, and pregnancy were associated with reporting symptoms and functional limitations on BCTQ. OCPs and smartphone use were associated with functional limitations only. Conclusion: Different risk factors are associated with reporting symptoms and functional limitations of CTS on the BCTQ. For example, RA, DM, hypothyroidism, pregnancy, OCPs, and smartphone use have all been found to statistically affect the outcome of the BCTQ in this study. Therefore, clinical confirmation of the CTS diagnosis is required in future studies to ensure that these symptoms and functional limitations are associated with the CTS pathology rather than other risk factors and pathologies for proper targeted treatment plans and outcomes.

Association of TNF-α rs1800629 with Adult Acute B-Cell Lymphoblastic Leukemia.

TNF−α influences lymphomagenesis by upregulating proinflammatory and antiapoptotic pathways. In this study, we evaluated the frequency of TNF−α rs1800629 (−308 G>A) polymorphism in newly diagnosed adult patients with acute lymphoblastic leukemia (ALL) and its correlation with age at diagnosis, gender and subtype of ALL. In this case control study, a total of 330 individuals were recruited, including 165 newly diagnosed adult patients with ALL, from the Radiation and Isotope Center in Khartoum (RICK) and 165 healthy normal controls. TNF−α rs1800629 polymorphism was tested through allele-specific polymerase chain reaction (PCR) assay. The frequency of the rs1800629 GA genotype was high (70.9% vs. 60%, OR = 1.84) in the patient group as compared to healthy controls, whereas GG and AA genotypes did not exhibit any statistically significant difference between controls and patients. Based on subtype, GG and GA rs1800629 genotypes showed increased risk of B-ALL (OR 0.46 and 2.12, respectively), whereas rs1800629 GG, GA and AA genotypes did not show any disease association with T-ALL (p > 0.05). Age at diagnosis and gender did not exhibit any association of rs1800629 with ALL in the patient group. In conclusion, rs1800629 is associated with high risk of adult B-ALL, with an insignificant effect of age at diagnosis and gender.

Too Fat for Transplant? The Impact of Recipient BMI on Pancreas Transplant Outcomes.

BACKGROUND: In many transplant centers, a recipient body mass index (BMI) >30 kg/m2 would be considered a contraindication for pancreas transplantation. This study aims to investigate the impact of recipient BMI on graft outcomes after pancreas transplantation. METHODS: Retrospective data on all UK solid organ pancreas transplants from 1994 to 2016 were obtained from the National Health Service Blood and Transplant UK Transplant Registry, n = 2618. Cases missing BMI data were excluded, resulting in a final cohort of n = 1452. Graft and patient survival analysis were conducted using Kaplan-Meier plots and Cox regression models. RESULTS: The mean recipient BMI was 24.8 kg/m2 (±2.4). There were 507 overweight (BMI 25-29.9) and 146 obese (>30) recipients receiving pancreas transplants. Univariate analysis showed no statistically significant difference between overweight BMI categories compared with normal BMI (18.5-24.9 kg/m2). Multivariate analysis revealed increasing recipient BMI had a significant impact on graft survival (P = 0.03, hazard ratio 1.04, 95% confidence interval, 1.00-1.08). Receiver operating characteristic curve analyses revealed no value of BMI that provided both specific and sensitive discrimination between death and survival of both grafts or patients. Recipients on dialysis with a BMI >30 kg/m2 had a statistically significant decrease in both graft (P = 0.002) and patient survival (P = 0.015). CONCLUSIONS: Analysis of available UK Pancreas data has shown recipient BMI is an independent risk factor for patient survival after transplantation. However, we have been unable to define a specific cutoff value above which patients have poorer outcomes. Obese patients on hemodialysis had the poorest graft survival, and preemptive transplantation may be beneficial in this cohort.

Dual MicroRNA Blockade Increases Expression of Antioxidant Protective Proteins: Implications for Ischemia-Reperfusion Injury.

BACKGROUND: MicroRNAs (miRNAs) are short noncoding RNAs which each cause repression of many target genes. Previous work has demonstrated that therapeutic blockade of single miRNAs is possible. miR-24-3p and miR-145-5p are reported to have a detrimental role in ischemia-reperfusion injury. As the action of miRNAs is inhibitory, we hypothesized that dual blockade of both miRNAs could synergistically upregulate shared target genes. METHODS: Quantification of miRNA expression in donated kidneys was performed using polymerase chain reaction (PCR) panels. Ischemia-reperfusion injury was modeled in vitro by placing human umbilical vein endothelial cells into a hypoxic incubator (1% O2) for 24 hours, with reoxygenation for 6 hours. RNA expression was quantified with reverse transcription quantitative polymerase chain reaction and protein expression assessed with Western blot. Antisense oligonucleotides were used to inhibit miRNAs. RESULTS: miR-24-3p and miR-145-5p were highly expressed in human kidneys following extended cold ischemia. In vitro, hypoxia caused significant upregulation of miR-24-3p (P ≤ 0.001) and miR-145-5p (P ≤ 0.001) and significant downregulation in messenger RNA of shared targets superoxide dismutase 2 (P ≤ 0.001) and heme oxygenase 1 (P ≤ 0.001). These changes were mirrored at the protein level. Dual inhibition of both miR-24-3p and miR-145-5p caused significant upregulation of superoxide dismutase 2 and heme oxygenase 1 protein following hypoxia-reoxygenation; fold change of 3.17 (P ≤ 0.05) and 6.97 (P ≤ 0.05) respectively. Dual inhibition resulted in reduced cellular reactive oxygen species production compared with negative control (P ≤ 0.05) and single blockade of miR-24-3p (P ≤ 0.01) or miR-145-5p (P ≤ 0.05). CONCLUSIONS: Dual blockade of 2 miRNAs can act synergistically to increase the expression of shared gene targets. Dual blockade of miR-24-3p and miR-145-5p represents a novel therapeutic option worthy of further research.

Comparative study of NMP-22, telomerase, and BTA in the detection of bladder cancer.

PURPOSE: The diagnostic efficacy of Nuclear Matrix Protein-22 (NMP-22), bladder tumor antigen (BTA TRAK), and telomerase activity was evaluated in urine in a trial to assess their value in the detection of bladder cancer and to compare it to that of routine urine cytology. SUBJECTS AND METHODS: The study included 46 newly diagnosed bladder cancer patients, diagnosed by cystoscopy and histopathological typing, in addition to 20 patients with benign bladder lesions and 20 healthy age and sex matched volunteers as a control group. Fifty percent of the cancer patients (23/46) had proven bilharzial history. Most patients (27/46) had transitional cell carcinoma (TCC), 17/46 had squamous cell carcinoma (SCC), while only 2 patients had adenocarcinoma. A single freshly voided urine sample (approximately 100ml) was collected from each patient and control subject and aliquoted for each test. All assays were conducted according to the manufacturer's guidelines and the results were compared to those of urine cytology. RESULTS: The optimal cutoffs for NMP-22, BTA and telomerase activity as calculated by ROC curves were 12.1 U/ml, 78 U/ml, 0.48 (Ratio) respectively. The levels of the three parameters were significantly higher in the malignant group compared to either the benign group or normal controls, (p<0.001) and the positive rates were also higher in the malignant group for all 3 parameters. The overall sensitivity of urine cytology, NMP-22, BTA and telomerase was 54.3%, 91.3%, 100% and 80.4% respectively. For bilharzial cancer bladder respective sensitivities were 69.6%, 95.6%, 100% and 73.9%, while for nonbilharzial cancer bladder the respective sensitivities were 39.1%, 87%, 100% and 87%. The overall specificities with urine cytology, NMP-22, BTA and telomerase was 100%, 87.5%, 92.5% and 95.0%, respectively. Combined sensitivity of voided urine cytology with one or more of the 3 biomarkers, or the use of these biomarkers in double or triple combinations gave higher positivity than each biomarker alone. CONCLUSION: BTA showed the highest sensitivity in all the studied parameters in the bladder cancer group, bilharzial bladder cancer subgroup, and non bilharzial bladder subgroup, (100%), while the highest specificity was recorded with urine cytology (100%), followed by telomerase (95%), then BTA (92.5%), and lastly NMP- 22 (87.5%). Use of markers in combination with cytology, or in a panel, improved the sensitivity, and specificity.