Standard Cold Storage Research Articles

Innovations in Liver Preservation Techniques for Transplants from Donors after Circulatory Death: A Special Focus on Transplant Oncology.

Liver transplantation is the preferred treatment for end-stage liver disease. Emerging evidence suggests a potential role for liver transplantation in treating liver tumors such as colorectal liver metastases and cholangiocarcinoma. However, due to a limited donor pool, the use of marginal grafts from donation after circulatory death (DCD) donors is increasing to meet demand. Machine perfusion is crucial in this context for improving graft acceptance rates and reducing ischemia-reperfusion injury. Few studies have evaluated the role of machine perfusion in the context of transplant oncology. Perfusion machines can be utilized in situ (normothermic regional perfusion-NRP) or ex situ (hypothermic and normothermic machine perfusion), either in combination or as a complement to conventional in situ cold flush and static cold storage. The objective of this analysis is to provide an up-to-date overview of perfusion machines and their function in donation after circulatory death with particular attention to their current and likely potential effects on transplant oncology. A literature review comparing standard cold storage to machine perfusion methods showed that, so far, there is no evidence that these devices can reduce the tumor recurrence rate. However, some evidence suggests that these innovative perfusion techniques can improve graft function, reduce ischemia-reperfusion injury, and, based on this mechanism, may lead to future improvements in cancer recurrence.

Ex vivo heart perfusion: an updated systematic review.

Due to the discrepancy between patients awaiting a heart transplant and the availability of donor hearts, strategies to expand the donor pool and improve the transplant's success are crucial. This review aims to summarize current knowledge on the ex vivo heart preservation (EVHP) experience as an alternative to standard cold static storage (CSS). EVHP techniques can improve the preservation of the donor's heart before transplantation and allow for pre-transplant organ evaluation.

Short-term outcomes after heart transplantation using donor hearts preserved with ex vivo perfusion

The standard Conventional Cold Storage (CCS) during heart transplantation procurement is associated with time-dependent ischemic injury to the graft, which is a significant independent risk factor for post-transplant early morbidity and mortality – especially when cold ischemic time exceeds four hours. Since 2018, Rigshospitalet (Copenhagen, Denmark) has been utilising ex vivo perfusion (Organ Care System, OCS) in selected cases. The objective of this study was to compare the short-term clinical outcomes of patients transplanted with OCS compared to CCS. Methods: This retrospective single-centre study was based on consecutive patients undergoing a heart transplant between January 2018 and April 2021. Patients were selected for the OCS group when the cold ischemic time was expected to exceed four hours. The primary outcome measure was six-month event-free survival. Results: In total, 48 patients were included in the study; nine were transplanted with an OCS heart. The two groups had no significant differences in baseline characteristics. Six-month event-free survival was 77.8% [95% CI: 54.9–100%] in the OCS group and 79.5% [95% CI: 67.8–93.2%] in the CCS group (p = 0.91). While the OCS group had a median out-of-body time that was 183 min longer (p < 0.0001), the cold ischemic time was reduced by 51 min (p = 0.007). Conclusion: In a Scandinavian setting, our data confirms that utilising OCS in heart procurement allows for a longer out-of-body time and a reduced cold ischemic time without negatively affecting safety or early post-transplant outcomes.

Characteristics and outcomes of lung transplants performed with ex-situ lung perfusion

Ex-situ lung perfusion (ESLP) can be used to assess and rehabilitate donor lungs, potentially expanding the donor pool. We examined the characteristics and outcomes of lung transplants performed with ESLP in the United States. Retrospective review of the United Network for Organ Sharing registry of primary adult lung transplant recipients from February 28, 2018, to June 30, 2021, was performed, comparing baseline characteristics, in-hospital outcomes, and 1-year survival of ESLP vs no ESLP lung transplants. Of 8204 lung transplants, 426 (5.2%) were performed with ESLP. ESLP donors were older, more donation after circulatory death (DCD), and had lower PaO2:FiO2 (P:F) ratios. Recipients had lower lung allocation scores. ESLP lungs traveled further, had longer preservation times, and were more likely double lung transplants. Reintubation rates, extracorporeal membrane oxygenation at 72hours, and hospital length of stay were greater in the ESLP group. On multivariable analysis, ESLP was not an independent predictor of 1-year survival. However, further analysis showed that DCD lungs managed on ESLP had worse 1-year survival compared to DCD lungs preserved with standard cold storage or with donation after brain death donor lungs. ESLP is used in a small percentage of lung transplants in the US and is not independently associated with 1-year survival. ESLP combined with DCD lungs, however, is associated with worse 1-year survival and warrants further investigation.

Donor heart ischemic time can be extended beyond 9 hours using hypothermic machine perfusion in sheep

The global shortage of donor hearts available for transplantation is a major problem for the treatment of end-stage heart failure. The ischemic time for donor hearts using traditional preservation by standard static cold storage (SCS) is limited to approximately 4 hours, beyond which the risk for primary graft dysfunction (PGD) significantly increases. Hypothermic machine perfusion (HMP) of donor hearts has been proposed to safely extend ischemic time without increasing the risk of PGD. Using our sheep model of 24 hours brain death (BD) followed by orthotopic heart transplantation (HTx), we examined post-transplant outcomes in recipients following donor heart preservation by HMP for 8 hours, compared to donor heart preservation for 2 hours by either SCS or HMP. Following HTx, all HMP recipients (both 2 hours and 8 hours groups) survived to the end of the study (6 hours after transplantation and successful weaning from cardiopulmonary bypass), required less vasoactive support for hemodynamic stability, and exhibited superior metabolic, fluid status and inflammatory profiles compared to SCS recipients. Contractile function and cardiac damage (troponin I release and histological assessment) was comparable between groups. Overall, compared to current clinical SCS, recipient outcomes following transplantation are not adversely impacted by extending HMP to 8 hours. These results have important implications for clinical transplantation where longer ischemic times may be required (e.g., complex surgical cases, transport across long distances). Additionally, HMP may allow safe preservation of "marginal" donor hearts that are more susceptible to myocardial injury and facilitate increased utilization of these hearts for transplantation.

Effect of long-term storage on the change in the expression of selected Mal d 1 gene isoforms in the apple cultivar Opal&lt;sup&gt;®&lt;/sup&gt;

Apples are available all year round since they can be stored for long time. However, the expression of the major apple allergen Mal d 1 can increase in the fruit during storage and incease so the risk of allergies in sensitive consumers. Therefore, we studied the change in the expression of the <i>Mal d 1</i> gene during storage in the modern cultivar UEB 32642 (known under the brand name Opal^®) and the cultivar Gala Brookfield (cv. Gala) as a reference. The cultivars were stored under two different conditions, ultra-low oxygen (ULO) and standard cold conditions. The gene expression was evaluated both in fresh fruits and in fruits stored for four, six, and nine months. Nine isoforms of the gene <i>Mal d 1</i> with the highest published expression were quantified using real-time PCR. The most expressed isoforms <i>Mal d 1.01</i>, <i>Mal d 1.02</i> and <i>Mal d 1.06A</i> were found in the fresh and also in the stored fruits. The expression of the <i>Mal d 1.03G</i> and <i>Mal d 1.06D</i> isoforms was higher the sored fruits. Our study confirmed that (<i>i</i>) Opal^® had a lower overall expression of the <i>Mal d 1</i> gene than cv. Gala, both in the fresh and stored fruits; (<i>ii</i>) standard cold storage is superior in preserving lower <i>Mal d 1</i> levels in Opal^®apples compared to the ULO conditions; and (<i>iii</i>) less expressed isoforms may be responsible for the general increase in the <i>Mal d 1</i> gene expression during storage.

Machine Perfusion for Extended Criteria Donor Livers: What Challenges Remain?

Based on the renaissance of dynamic preservation techniques, extended criteria donor (ECD) livers reclaimed a valuable eligibility in the transplantable organ pool. Being more vulnerable to ischemia, ECD livers carry an increased risk of early allograft dysfunction, primary non-function and biliary complications and, hence, unveiled the limitations of static cold storage (SCS). There is growing evidence that dynamic preservation techniques—dissimilar to SCS—mitigate reperfusion injury by reconditioning organs prior transplantation and therefore represent a useful platform to assess viability. Yet, a debate is ongoing about the advantages and disadvantages of different perfusion strategies and their best possible applications for specific categories of marginal livers, including organs from donors after circulatory death (DCD) and brain death (DBD) with extended criteria, split livers and steatotic grafts. This review critically discusses the current clinical spectrum of livers from ECD donors together with the various challenges and posttransplant outcomes in the context of standard cold storage preservation. Based on this, the potential role of machine perfusion techniques is highlighted next. Finally, future perspectives focusing on how to achieve higher utilization rates of the available donor pool are highlighted.

Long-term outcomes after heart transplantation using ex vivo allograft perfusion in standard risk donors: A single-center experience.

The Organ Care System (OCS) is an ex vivo perfusion platform for donor heart preservation. Short/mid-term post-transplant outcomes after its use are comparable to standard cold storage (CS). We evaluated long-term outcomes following its use. Between 2011 and 2013, 38 patients from a single center were randomized as a part of the PROCEED II trial to receive allografts preserved with CS (n=19) or OCS (n=19). Endpoints included 8-year survival, survival free from graft-related deaths, freedom from cardiac allograft vasculopathy (CAV), non-fatal major adverse cardiac events (NF-MACE), and rejections. Eight-year survival was 57.9% in the OCS group and 73.7% in the CS group (p=.24). Freedom from CAV was 89.5% in the OCS group and 67.8% in the CS group (p=.13). Freedom from NF-MACE was 89.5% in the OCS group and 67.5% in the CS group (p=.14). Eight-year survival free from graft-related death was equivalent between the two groups (84.2%vs. 84.2%, p=.93). No differences in rejection episodes were observed (all p>.5). In select patients receiving OCS preserved allografts, late post-transplant survival trended lower than those transplanted with an allograft preserved with CS. This is based on a small single-center series, and larger numbers are needed to confirm these findings.

Heart transplantation of patients with ventricular assist devices: impact of normothermic ex-vivo preservation using organ care system compared with cold storage

BackgroundOrgan Care System (OCS) minimizes the cold ischemic time and allows for optimization of logistics and meticulous recipient preparation. Impact of normothermic ex-vivo preservation using OCS compared with cold storage (CS) for prolonged heart preservation especially beneficial for high-risk recipients bridged to transplantation with Mechanical Circulatory Support (MCS).MethodsBetween 2012 and 2018, we performed a retrospective single-center review of prospectively collected data. All patients who underwent heart transplantation with MCS using the OCS Heart (n = 25) versus standard cold storage (n = 10) were included in this study.ResultsDuring this period, 353 patients were implanted with left ventricular assisted device (LVAD) and 35 (10%) were bridged to heart transplantation. There was no significant difference in donor and recipient characteristics and risk factors. The Index for Mortality Prediction after Cardiac Transplantation (IMPACT) score was a trend towards higher estimated risk of death at 1y in the OCS group (14.2 vs. 10.8% p = 0.083). Mean total ischemic time during preservation was statistically significantly longer in CS vs OCS group (210 (23) Vs 74.6 (13) min p = 0.001). Median ex vivo normothermic heart perfusion time in OCS was 348.4(132; 955) min. There was significant difference in total out of body time between OCS group 423(67) Vs CS group 210(23) min p = 0.002). All patients were alive on the 30th days post implant in CS groups and 96% in OCS group (p = 0.5).ConclusionNormothermic ex-vivo preservation of the allograft during transportation with the organ care system might be beneficial for long-time out of body organ preservation in comparison of cold storage especially for recipients on mechanical circulatory support.

The Condition of the Rat Myocardium and Isolated Sheep Heart after Prolonged 24-Hour Hypothermic Preservation in a Pressurized Carbon Monoxide–Oxygen Gas Mixture

High organoprotective properties of a carbon monoxide (CO)–oxygen (O2) gas mixture were confirmed after prolonged (24-h) preservation of the papillary muscle and an isolated rat heart at 4°C. Hypothermic preservation in the high-pressure gas mixture (6 atm) provided efficient restoration of the contractile activity of the isolated rat heart after 24-h storage at 4°C. The isolated retrograde-perfused Langendorff heart performed physically relevant mechanical work, which was similar in duration to that of an intact control heart. Staining with triphenyltetrazolium chloride did not detect infarcted regions in the myocardium. After preservation, the heart tissue was highly capable of performing its function in a test for electrically stimulated contractile activity of papillary muscles. In the test group, The frequency–intensity relationship, the potentiation effect induced by a pause, and the response to stimulation with isoproterenol of test hearts generally corresponded to the parameters of a normal rat myocardium. A sheep heart, which is comparable in size and weight to a human heart, was for the first time successfully preserved using the gas mixture. Normal heartbeat was spontaneously restored after the start of perfusion in all experiments. Histology did not detect a significant difference between test and control sheep hearts. The normal tissue structure of the myocardium was preserved in the test hearts. The 24-h preservation achieved in the study was four times longer than the maximum allowable preservation time of standard static cold storage. The results obtained with the large laboratory animal heart model showed that the hypothermic preservation protocol is promising for prolonged storage of human hearts.

A new storage solution for the hypothermic preservation of corneal grafts: an experimental study.

In this experimental study we used for the first time Tiprotec® as a solution for corneal preservation and cold storage. We compared the resultant endothelial cell morphology and viability with this obtained after preservation of the ex-vivo corneas with both usual standard techniques: conventional cold storage (using Eusol-C) and organ culture. This prospective, in vitro, 3-armed parallel study was performed with the use of 90 porcine corneas (examined for their endothelial quality and transparency) randomly selected for preservation in three storage methods (each 30 corneas): organ culture, standard cold storage (Eusol-C) and experimental cold storage (Tiprotec®) Endothelium cell quantity and quality as well as corneal opacification were assessed. The degree of endothelial transparency was significantly reduced over time with all preservation media, without any significant difference among the three groups at any point of time. A reduction in endothelial cell density was also observed with all three preservation media after 30days of storage without statistically significant differences between groups. The number of hexagonal and pentagonal endothelium cells was significantly reduced overtime in all media with significantly more hexagonal and pentagonal in the organ culture group compared to the cold storage groups. We could show that the cryopreservation medium Tiprotec®, used until now for the preservation of vascular grafts, was of similar quality compared to the medium Eusol-C for the hypothermic storage of corneal tissue for an extended period of time up to 30days. In comparison to organic culture with culture medium KII, both Tiprotec® and Eusol-C were found less effective in preserving endothelial cell quality, as assessed by the morphometric analysis, and viability, as assessed by the degree of vacuolization at least up to the 30th day of storage. However, both, Tiprotec®- and Eusol-C-preserved corneas demonstrated a certain capacity to recover after their submission in organ culture.

Heart Preservation with the Organ Care System in Extended Criteria Donor Hearts: A Three-Center Experience

The Organ Care System (OCS) Heart® is non-inferior to standard cold storage for preservation of donor hearts. We hypothesized that its properties for prolonged heart preservation might be especially beneficial for extended criteria donor (ECD) hearts. Patients transplanted with ECD hearts preserved using the OCS Heart® at two German and one Italian centers between 11/2011 and 10/2019 were prospectively followed and included into the study. ECD were defined as fulfilling at least one of the following criteria: age between 45 and 55 years without coronary angiography; age >55 years; echocardiographic evidence of left ventricular hypertrophy (LVH); need for cardiopulmonary resuscitation (CPR) longer than 20 minutes; left ventricular ejection fraction (LVEF)<40%; evidence of coronary artery disease (CAD); carbon monoxide intoxication with preserved LVEF; moderate-severe alcohol consumption; diabetes mellitus without CAD. During the study period, the OCS Heart® was employed in 122 patients transplanted at the three institutions. Among these 122 patients, 82 (67%) patients were transplanted with an ECD heart, as defined by the aforementioned criteria (median donor age, 48 years; LVH, n=17; CPR, n=28; mild CAD, n=5; LVEF<40%, n=2; alcohol consumption, n=36; diabetes mellitus, n=1), and thus considered in the study. Five further ECD hearts were considered non-transplantable while on OCS and were discarded. Before transplantation, 64 (78%) recipients had undergone at least one cardiac operation (LVAD/BVAD, n=47, 57%), 12 (15%) were on ECLS, and 65 (79%) were on the high-urgency waiting list. Median OCS perfusion and out-of-body times were 4.5 and 6.2 hours, respectively. Postoperatively, 20 (24%) and 38 (46%) patients required re-thoracotomy for bleeding and dialysis (temporary, n=32), respectively. Sixteen (19%) patients required postoperative ECLS or intraaortic balloon pump (IABP), due to PGD in 11 cases. Eleven (13%) patients died in-hospital. At discharge, all surviving patients showed a LVEF>60% in echocardiography. At 2-year follow-up, survival and freedom from biopsy-confirmed rejection (ISHLT grade >1R) were 78% and 86%, respectively. OCS Heart® allowed safe transplantation of extended criteria donor hearts. Despite out-of-body time was longer than 6 hours, patient and graft outcomes were favorable.

Normothermic Ex-Vivo Perfusion for Donor Heart Preservation in Transplantation of Patients Bridged with Ventricular Assist Devices

Recently, it has been shown that survival after heart transplantation is impaired in patients bridged with left ventricular assist devices (LVAD), mostly due to increased rates of primary graft dysfunction (PGD). Ex vivo perfusion (EVP) is a graft preservation technique non-inferior to standard cold storage (CS). It allows to maintain the heart in a beating and perfused status during challenging surgical dissection in LVAD patients undergoing heart transplantation, and to reduce ischemic time potentially lowering the risk of PGD. Patients bridged on LVAD and transplanted in two European centers (Udine, Italy and Hanover, Germany) since 2016 were included in the study and prospectively followed. Grafts were transported with EVP using the Organ Care System (Transmedics, Andover, MA) in 44 cases and 21 with CS. The underlying demographics were not different, except for urgency status (86% vs. 57%, p=0.01) and chronic renal failure (59% vs. 29%, p=0.02) in EVP and CS respectively. Median age was 56(21-71) vs. 54(25-65), p=0.73; male sex was 77% vs. 81%, p=0.50 and most frequent ethiology was dilated cardiomyopathy in 73% vs. 52%, p=0.11. No differences were found in donors characteristics, with the employment of extended criteria donors in many cases (41% vs. 43%, p=0.55).Five extended criteria donor hearts were assessed as non-transplantable while on EVP.The remaining grafts were transported for a mean time of 296±64 min in EVP.Ischemic times were significantly shorter in the EVP group (132±29 vs. 223±48 min, p<0.01). The early outcome showed no significant statistical differences between the two groups, but a favourable trend in 30-day mortality after HTx in the EVP group (5% vs. 14%, p=0.19). Severe PGD (11% vs. 19% p=0.32), need of mechanical ventilation >96h (25% vs. 33%, p=0.48), of dialysis (66% vs. 52%, p=0.29), revision for bleeding (16% vs. 19%, p=0.50) and ICU stay (6.1-123 vs. 5.1-195 days, p=0.48) were reported. Furthermore, a trend to better survival at mid-term was found (p=0.25) with 88% vs. 76% survival at 3 years. VAD patients have a high risk of morbidity during HTx. EVP warrants a trend towards improved outcomes in this difficult recipient cohort. The longer available time to perform surgery and the reduced ischemia permit a safer procedure including cases of extended criteria donor employment.

Ex-Vivo Normothermic Limb Perfusion With a Hemoglobin-Based Oxygen Carrier Perfusate.

Ex-vivo normothermic limb perfusion (EVNLP) has been proven to preserve limb viability better than standard cold storage. Perfusates containing packed red blood cells (pRBC) improve outcomes when compared to acellular perfusates. Limitations of pRBC-based perfusion include limited availability, need for cross match, mechanical hemolysis, and activation of pro-inflammatory proteins. Hemoglobin-based oxygen carrier (HBOC)-201 (Hemopure) is a solution of polymerized bovine hemoglobin, characterized by low immunogenicity, no risk of hemolytic reaction, and enhanced convective and diffusive oxygen delivery. This is a preliminary study on the feasibility of EVNLP using HBOC-201 as an oxygen carrier. Three porcine forelimb perfusions were performed using an established EVNLP model and an HBOC-201-based perfusate. The perfusion circuit included a roller pump, oxygenator, heat exchanger, and reservoir. Electrolytes, limb temperature, weight, compartment pressure, nerve conduction, and perfusion indicated by indocyanine green angiography and infra-red thermography were monitored. Histological evaluation was performed with hematoxylin and eosin and electron microscopy. Three limbs were perfused for 21.3±2.1hours. Muscle contractility was preserved for 10.6±2.4hours. Better preservation of the mitochondrial ultrastructure was evident at 12hours in contrast to crystallization and destruction features in the cold-storage controls. An HBOC-201-EVNLP produced outcomes similar to RBC-EVNLP with preservation of muscle contractility and mitochondrial structure.

The Conversional Efficacy of Ex Vivo Lung Perfusion and Clinical Outcomes in Patients Undergoing Transplantation of Donor Lungs by Ex Vivo Lung Perfusion: A Meta-Analysis.

BackgroundEx vivo lung perfusion (EVLP) is a relatively new technique that can be used to assess and repair the donor lungs, increasing the utilization of high-risk lungs. However, its effect on outcomes of lung transplantation patients is uncertainty. This meta-analysis is conducted to assess the impact of EVLP on donor lungs and outcomes of recipients compared with the standard lung transplantation.Material/MethodsWe systematically searched for studies comparatively analyzing the efficacy of EVLP and standard cold storage in lung transplantation. The hazard ratio (HR), relative risk (RR), and weighted mean difference (WMD) were used as the effect size (ES) to evaluate the survival outcomes, categorical variables, and continuous variables respectively.ResultsA total of 20 published articles (including 2574 donors and 2567 recipients) were eligible. The chest x-ray manifestations and PaO2/FiO2 100% were more deficient in the EVLP group than the standard group. EVLP improved the function of high-risk donor lungs with the conversion rate ranging from 34% to 100%. The EVLP group had a lower incidence of primary graft dysfunction 3, but longer intensive care unit stay. Other clinical outcomes between the 2 groups were similar.ConclusionsThe pooled results indicated that EVLP could be used to assess and improve high-risk donor lungs and had non-inferior postoperative outcomes compared with the standard cold storage. EVLP not only increased the utilization of marginal donors, but also could extend preservation time and reduce the total ischemia time of donors.

Evaluation of the Functional Effects of an African American Glucose-6-Phosphate Dehydrogenase (G6PD) Polymorphism (Val68Met) on RBC Hemolytic Propensity and Post-Transfusion Recovery in a Humanized Mouse Model

Background: Growing evidence suggest that donor genetic variation is associated with RBCs storage integrity and post-transfusion recovery. In collaboration with the REDS III program, we performed a large-scale genome-wide association study (GWAS) study in ~13,000 healthy human blood donors, which demonstrated that RBCs with an African American G6PD-deficient A- variant (rs1050828, Val68Met) was associated with higher oxidative hemolysis after cold storage in normal volunteers. Despite a high prevalence of X-linked G6PD mutation in African American population (&amp;gt; 10%), blood donors are not routinely checked for G6PD deficiency and its importance in transfusion medicine is relatively understudied. We hypothesize that G6PD V68M SNP is associated with G6PD deficiency and modulates RBCs in vitro hemolytic propensity and in vivo post-transfusion recovery. Methods and Results: CRISPR-Cas9 technology was used to create non-synonymous human G6PD X-linked SNP (Val68Met) in C57B6 mice, and the desired genotypes were isolated by strategic back-crossing and sequential genotyping to ensure passage of SNP. G6PD enzymatic activity in erythrocytes was measured in fresh collected blood using a colorimetric assay kit. The predisposition of fresh and stored RBCs (after 11 days cold storage) to hemolysis was evaluated by subjecting washed mouse RBCs to selected stress assays, including osmotic fragility, mechanical fragility, and oxidative hemolysis using AAPH, diamide or H2O2. Hemolysis was measured by detection of supernatant cell-free hemoglobin using Drabkin's assay. Hematological values were measured using a Hemavet® 950 Hematology Analyzer System. Reticulocyte count was obtained using thiazole orange staining and analyzed by flow cytometry. We found severe disruption of G6PD enzymatic activity in erythrocytes from G6PD V68M SNP mice compared to WT mice (~5% residual activity in hemizygous male and ~60% in heterozygous female mice). Significant increased oxidative hemolysis was observed in both fresh and stored mouse RBCs with G6PD SNP, consistent with the GWAS study in human. G6PD V68M SNP hemizygous male mice had higher mean corpuscular volume (MCV) and lower mean corpuscular hemoglobin concentration (MCHC) compared to WT mice. However, no difference was observed in storage hemolysis, osmotic fragility, mechanical fragility and reticulocyte counts. Transfusion experiments with stored red blood cells from G6PD hemizygote males into GFP positive recipients will evaluate red blood cell recovery and half-life after standard cold storage and transfusion. Conclusions: We successfully generated a novel mouse strain carrying a "humanized" African American G6PD V68M variant which resembles the phenotype of humans with G6PD deficiency and increased oxidative hemolysis. Studies are undertaken to further investigate the effects of G6PD V68M SNP on RBCs structure, functions and in vivo post-transfusion recovery. Disclosures Gladwin: Bayer Pharmaceuticals: Other: Co-investigator; United Therapeutics: Patents &amp; Royalties: Co-inventor on an NIH government patent for the use of nitrite salts in cardiovascular diseases ; Globin Solutions, Inc: Patents &amp; Royalties: Provisional patents for the use of recombinant neuroglobin and heme-based molecules as antidotes for CO poisoning.

Effect of humic acid, seaweed extract and essential oils as antioxidants on pre-and post-harvest quality of red radish plants

This study was carried out to assess the effect of certain biomodulators, namely seaweed extract, humic acid, peppermint and thyme oils, on quality-related metabolites during both pre- and post-harvest of Red radish. For this purpose, plants were sprayed with tested biomodulators twice, 20 and 30 days after sowing, and chosen biochemical constituents affecting quality were estimated before harvest, and again in roots of harvested plants after storage in standard cold storage. In addition, to evaluate the involvement of each tested parameter in post-harvest quality of stored roots, a correlation matrix between biochemical constituents after storage and quality parameters of stored roots represented by weight loss and post-harvest decay percentage as well as stored root‘s dry matter percentage. Results indicated that the adopted biomodulators affected the metabolism of treated plants in a way that persisted after storage, hence positively affected roots post-storage quality attributes. Invariably, stored roots of biomodulators-treated plants contained higher concentrations from total anthocyanins, ascorbic acid, total phenols, nutrients concentration as well as flavonoids compared with those of control plants. Moreover, activity of both peroxidase and polyphenol oxidase was higher in stored roots of biomodulators-treated plants. In addition, all applied biomodulators decreased weight loss and post-harvest decay percentage, thus roots of biomodulators-treated plants contained higher dry matter compared with roots of untreated plants. The metabolic and physiological bases of biomodulators-induced quality parameters of roots after storage were discussed.

Hypothermic Oxygenated Perfusion: A Simple and Effective Method to Modulate the Immune Response in Kidney Transplantation.

Hypothermic oxygenated perfusion (HOPE) has been shown to protect liver recipients from acute rejection in an allogeneic model of liver transplantation in rats. Here we investigate the impact of HOPE on the T cell-mediated immune response following kidney transplantation. Kidneys from Lewis rats were transplanted into Brown Norway recipients to trigger acute rejection (allogeneic untreated group). Next, Brown Norway recipients were treated either with tacrolimus,= or donor kidneys underwent 1h-HOPE-treatment before implantation without additional immunosuppression in recipients. Syngeneic kidney transplants (Brown Norway to Brown Norway) served as controls. In a second set of experiments, the immune response was assessed in a donation after circulatory death model of kidney transplantation comparing standard cold storage with subsequent HOPE treatment and hypothermic nitrogenated perfusion, where oxygen was replaced during cold perfusion. Allogeneic kidney transplantation led to death in all untreated recipients within 10 days due to severe acute rejection. In contrast, immune activation was prevented by tacrolimus with significantly improved recipient survival. Similarly, HOPE treatment, without any immunosuppression, protected recipients from acute immune response, as measured by less cytokine release, T-cell, and macrophage activation. Additionally, HOPE-treated kidneys showed better function and less early fibrosis leading to a significantly improved recipient survival, compared with untreated allogeneic controls. Similarly, HOPE treatment protected recipients of extended donation after circulatory death kidneys from immune activation. This effect was lost when deoxygenated perfusate was used. In summary, this is the first study demonstrating the beneficial effects of HOPE on the immune response following kidney transplantation in an allogeneic rodent model.

Hearts Donated After Circulatory Death and Reconditioned Using Normothermic Regional Perfusion Can Be Successfully Transplanted Following an Extended Period of Static Storage.

There has been an increased interest in donation after circulatory death (DCD) to expand donor pool for cardiac transplantation. Normothermic regional perfusion (NRP) allows in situ assessment of DCD hearts, allowing only acceptable organs to be procured. We sought to determine if extended cold storage was possible for DCD hearts following NRP and to compare hearts stored using standard cold storage with a novel cardioprotective solution designed for room temperature storage. Donor pigs underwent hypoxic cardiac arrest (DCD) followed by 15 minutes of warm ischemia and resuscitation on NRP. They were then randomly assigned to static storage with histidine-tryptophan-ketoglutarate (HTK) at 4°C (HTK group, n=5) or SOM-TRN-001 at 21°C (SOM group, n=5). Conventional beating-heart donations were used as controls (n=4). Fourteen transplants were successfully performed. HTK hearts showed initial dysfunction following reperfusion; however, they demonstrated significant recovery up to 3 hours post-transplant. No significant differences were seen between HTK and control hearts post-transplantation (cardiac index: control 49.5±6% and HTK 48.5±5% of baseline). SOM improved myocardial preservation; hearts showed stable contractility after transplantation (cardiac index: 113.0±43% of NRP function) and improved diastolic function compared with HTK. Preservation in SOM also significantly reduced proinflammatory cytokine production and release following transplantation and partially prevented endothelial dysfunction. DCD hearts stored using a standard preservation solution demonstrated comparable post-transplantation myocardial function to standard controls. Thus, short periods of cold storage following successful NRP and documented adequate function is an acceptable strategy for DCD hearts. Preservation in SOM at room temperature is feasible and can improve cardiac recovery by minimizing endothelial dysfunction and tissue injury.

Effect of post-harvest interventions on surficial carrot bacterial community dynamics, pathogen survival, and antibiotic resistance

Strategies to mitigate antibiotic-resistant bacteria (ARB), including human pathogens, on raw vegetables are needed to reduce incidence of resistant infections. The objective of this research was to determine the effectiveness of standard post-harvest interventions, sanitizer washing and cold storage, to reduce ARB, including antibiotic resistant strains of the human pathogen E. coli O15:H7 and a common spoilage bacterium Pseudomonas, on raw carrots. To provide a background inoculum representing potential pre-harvest carryover of ARB, carrots were dip-inoculated in dairy cow manure compost slurry and further inoculated with known ARB. Inoculated carrots were washed with one of three treatments: sodium hypochlorite (50 ppm free chlorine), peroxyacetic acid (40 ppm peroxyacetic acid; 11.2% hydrogen peroxide), tap water (no sanitizer), or no washing (control). Washed carrots were air dried, packaged and stored at 10 °C for 7d or 2 °C for up to 60 d. Enumeration was performed using total heterotrophic plate counts (HPC), HPCs on antibiotic-containing media (“ARBs”), E. coli O157:H7, and Pseudomonas sp. immediately after washing (0 d) and after 7 d of storage. In addition to the cultured bacteria, changes to the surficial carrot microbiota were profiled by sequencing bacterial 16S rRNA gene amplicons to determine the effect of sanitizer wash, storage temperature, and time of storage (0, 1, 7, 14 and 60 d). Storage temperature, addition of a sanitizer during wash, and duration of storage significantly affected the bacterial microbiota (Wilcoxon, p < 0.05). Inclusion of either sanitizer in the wash water significantly reduced the log CFU/g of E. coli O157:H7 and Pseudomonas sp., as well as HPCs enumerated on cefotaxime- (10 μg/ml), sulfamethoxazole- (100 μg/ml), or tetracycline (3 μg/ml) supplemented media compared to the unwashed control (ANOVA, p < 0.05). However, no significant reductions to bacteria resistant to vancomycin or clindamycin occurred after washing and storage. Members of the Proteobactetria, Firmicutes, Actinobacteria, Planctomycetes, and Acidobacteria comprised the bacterial carrot microbiota. The diversity of the carrot microbiota was significantly affected by the temperature of storage and by extended storage (60 d), when spoilage began to occur. There were no significant differences to the relative abundance of bacterial groups associated with the type of sanitizer used for washing. Results of this study indicate that inclusion of a sanitizer in wash water, followed by storage at 2 °C, might be an effective strategy to prevent re-growth of pathogenic E. coli O157:H7 and reduce levels of bacteria resistant to certain antibiotics on carrots.