University Of Wisconsin Solution Research Articles

Production of high-quality islets from goettingen minipigs: Choice of organ preservation solution, donor pool, and optimal cold ischemia time.

The transplantation of porcine islets into man might soon become reality for patients with type 1 diabetes mellitus. Therefore, porcine islets of high quality and quantity, and a scalable isolation process with strict quality control will be an unconditional prerequisite to enable the best possible transplantation graft. In this study, we provide a comparative study evaluating islet isolation outcome and in vitro survival based upon donor age, organ preservation solution (OPS), and cold ischemia time (CIT). Goettingen minipigs of younger age (1year) and retired breeder animals (3.5years) were studied. Pancreata were harvested according to the standards of human organ retrieval including in situ cold perfusion with either Custodiol® -HTK or Belzer® UW solution. Pancreatic tissue was characterized by quantification of apoptotic cells. Islet isolations were performed according to a modified Ricordi method, and isolation outcome was assessed by determining islet particle numbers (IP), islet equivalents (IEQ), and isolation factor (IF). Isolated islets were cultured for 24 and 48h for the assessment of in vitro survival. Islet viability was significantly higher in Custodiol® -HTK preserved pancreas organs compared to Belzer® UW. Furthermore, organs harvested from retired breeder preserved in Custodiol® -HTK resulted in stable islet isolation yields even after prolonged CIT and showed superior survival rates of islets in vitro compared to the Belzer® UW group. Younger porcine donor organs resulted generally in lower islet yield and survival rates. In summary, Custodiol® -HTK solution should be preferred over Belzer® UW solution for the preservation of pancreata from porcine origin. Custodiol® -HTK allows for maintaining islet viability and promotes reproducible isolation outcome and survival even after longer CIT. The usage of retired breeder animals over young animals for islet isolation is highly advisable to yield high quality and quantity.

Pretreatment Donors after Circulatory Death with Simvastatin Alleviates Liver Ischemia Reperfusion Injury through a KLF2-Dependent Mechanism in Rat.

Objective Severe hepatic ischemia reperfusion injury (IRI) can result in poor short- and long-term graft outcome after transplantation. The way to improve the viability of livers from donors after circulatory death (DCD) is currently limited. The aim of the present study was to explore the protective effect of simvastatin on DCD livers and investigate the underlying mechanism. Methods 24 male rats randomly received simvastatin or its vehicle. 30 min later, rat livers were exposed to warm ischemia in situ for 30 min. Livers were removed and cold-stored in UW solution for 24 h, subsequently reperfused for 60 min with an isolated perfused rat liver system. Liver injury was evaluated during and after warm reperfusion. Results Pretreatment of DCD donors with simvastatin significantly decreased IRI liver enzyme release, increased bile output and ATP, and ameliorated hepatic pathological changes. Simvastatin maintained the expression of KLF2 and its protective target genes (eNOS, TM, and HO-1), reduced oxidative stress, inhibited innate immune responses and inflammation, and increased the expression of Bcl-2/Bax to suppress hepatocyte apoptosis compared to DCD control group. Conclusion Pretreatment of DCD donors with simvastatin improves DCD livers' functional recovery probably through a KLF2-dependent mechanism. These data suggest that simvastatin may provide a potential benefit for clinical DCD liver transplantation.

Hydrogen Sulfide Protects Renal Grafts Against Prolonged Cold Ischemia-Reperfusion Injury via Specific Mitochondrial Actions.

Ischemia-reperfusion injury is unavoidably caused by loss and subsequent restoration of blood flow during organ procurement, and prolonged ischemia-reperfusion injury IRI results in increased rates of delayed graft function and early graft loss. The endogenously produced gasotransmitter, hydrogen sulfide (H2 S), is a novel molecule that mitigates hypoxic tissue injury. The current study investigates the protective mitochondrial effects of H2 S during in vivo cold storage and subsequent renal transplantation (RTx) and in vitro cold hypoxic renal injury. Donor allografts from Brown Norway rats treated with University of Wisconsin (UW) solution + H2 S (150 μM NaSH) during prolonged (24-h) cold (4°C) storage exhibited significantly (p < 0.05) decreased acute necrotic/apoptotic injury and significantly (p < 0.05) improved function and recipient Lewis rat survival compared to UW solution alone. Treatment of rat kidney epithelial cells (NRK-52E) with the mitochondrial-targeted H2 S donor, AP39, during in vitro cold hypoxic injury improved the protective capacity of H2 S >1000-fold compared to similar levels of the nonspecific H2 S donor, GYY4137 and also improved syngraft function and survival following prolonged cold storage compared to UW solution. H2 S treatment mitigates cold IRI-associated renal injury via mitochondrial actions and could represent a novel therapeutic strategy to minimize the detrimental clinical outcomes of prolonged cold IRI during RTx.

A National Registry Analysis of Kidney Allografts Preserved With Marshall's Solution in the United Kingdom.

The preservation fluids most commonly used for renal allograft preservation in the UK are University of Wisconsin Solution (UW, £120 per liter) and Marshall's Solution (hyperosmolar citrate, £10 per liter). The aim of this study was to compare the outcomes of deceased donor renal allografts preserved with these fluids using data from the UK national transplant registry. Data regarding transplants performed between January 1, 2005, and December 31, 2008, was analyzed (n = 5027 kidneys). Kidneys from Donation after Brain Death (DBD) and Donation after Circulatory Death (DCD) were included. After univariate analysis, multivariate logistic and linear regression models were fitted for adult recipients of first grafts (n = 3703 kidneys). Marshall's solution was associated with longer cold ischemic time, older donors, kidney-only donors, donors with hypertension, and DBD (all P < 0.01). After adjusting for confounding, the choice of preservation fluid was not associated with the risk of PNF (OR, 0.82; 95% CI, 0.46-1.46; P = 0.50), DGF (OR, 1.22; 95% CI, 0.96-1.56; P = 0.11), acute rejection (OR, 0.95; 95% CI, 0.76-1.19; P = 0.63), renal function at 1 year (coefficient, 0.97; 95% CI, 0.91-1.04; P = 0.41), or graft survival (DBD HR, 0.71; 95% CI, 0.46-1.10; P = 12; DCD HR, 0.99; 95% CI, 0.58-1.73; P = 1.00). Marshall's solution has been used for the preservation of large numbers of kidneys in the UK. It is associated with transplant outcomes that are equivalent to those with UW solution. Thus, on the basis of this analysis and cost, a strong case can be made for the continued use of Marshall's solution as a preferred fluid for renal allograft preservation.

Blood type conversion in human liver – an experimental study on α-galactosidase

Objective To study blood type B antigen elimination with α-galactosidase in human liver tissue, and discuss the feasibility of blood type conversion in human liver. Methods The liver specimens from patients with blood type B in liver transplantation were collected, and an in vitro liver perfusion model was established. The in vitro livers were perfused with UW solution + /- α-galactosidase. The effect of enzyme in B antigen of human liver were analyzed by immunofluorescence. Results With UW solution containing α-galactosidase to perfuse the in vitro livers, immunohistochemistry showed the level of blood type B antigen in liver was significantly reduced after hypothermic perfusion and preservation. The B antigen level in 1 h perfusion was reduced to approximate 58% of this figure prior to perfusion, in 2 h was 10%, and in 4 h was 4%. Among the different intervals, the blood group antigen levels showed significant differences (P<0.05). In the control group, the blood group antigen levels showed no obvious change on statistical analysis. Conclusions α-galactosidase was effective to clear blood type B antigen in isolated liver tissue. In the experimental group, Although the B antigen did not fall to a undetectable level, liver blood type conversion from B→O remains a promising potential which has been meaningful for related researches on blood type conversion of human organs. Key words: α-galactosidase; Human body; Liver; Blood type conversion

Cross-talk between autophagy and KLF2 determines endothelial cell phenotype and microvascular function in acute liver injury

The transcription factor Krüppel-like factor 2 (KLF2), inducible by simvastatin, confers endothelial vasoprotection. Considering recent data suggesting activation of autophagy by statins, we aimed to: 1) characterize the relationship between autophagy and KLF2 in the endothelium, 2) assess this relationship in acute liver injury (cold ischemia/reperfusion) and 3) study the effects of modulating KLF2-autophagy in vitro and in vivo. Autophagic flux, the vasoprotective KLF2 pathway, cell viability and microvascular function were assessed in endothelial cells and in various pre-clinical models of acute liver injury (cold storage and warm reperfusion). Positive feedback between autophagy and KLF2 was observed in the endothelium: KLF2 inducers, pharmacological (statins, resveratrol, GGTI-298), biomechanical (shear stress) or genetic (adenovirus containing KLF2), caused endothelial KLF2 overexpression through a Rac1-rab7-autophagy dependent mechanism, both in the specialized liver sinusoidal endothelial cells (LSEC) and in human umbilical vein endothelial cells. In turn, KLF2 induction promoted further activation of autophagy. Cold ischemia blunted autophagic flux. Upon reperfusion, LSEC stored in University of Wisconsin solution did not reactivate autophagy, which resulted in autophagosome accumulation probably due to impairment in autophagosome-lysosome fusion, ultimately leading to increased cell death and microvascular dysfunction. Simvastatin pretreatment maintained autophagy (through the upregulation of rab7), resulting in increased KLF2, improved cell viability, and ameliorated hepatic damage and microvascular function. We herein describe for the first time the complex autophagy-KLF2 relationship, modulating the phenotype and survival of the endothelium. These results help understanding the mechanisms of protection conferred by KLF2-inducers, such as simvastatin, in hepatic vascular disorders. Autophagy and the transcription factor KLF2 share a common activation pathway in the endothelium, being able to regulate each other. Statins maintain microvascular function through the inhibition of Rac1, which consequently liberates Rab7, activates autophagy and increments the expression of KLF2.

Inhibition of coagulation proteases Xa and IIa decreases ischemia–reperfusion injuries in a preclinical renal transplantation model

Coagulation is an important pathway in the pathophysiology of ischemia-reperfusion injuries. In particular, deceased after circulatory death (DCD) donors undergo a no-flow period, a strong activator of coagulation. Hence, therapies influencing the coagulation cascade must be developed. We evaluated the effect of a new highly specific and effective anti-Xa/IIa molecule, with an integrated innovative antidote site (EP217609), in a porcine preclinical model mimicking injuries observed in DCD donor kidney transplantation. Kidneys were clamped for 60minutes (warm ischemia), then flushed and preserved for 24 hours at 4°C in University of Wisconsin (UW) solution (supplemented or not). EP217609-supplemented UW solution (UW-EP), compared with unfractionated heparin-supplemented UW solution (UW-UFH) or UW alone (UW). A mechanistic investigation was conducted invitro: addition of EP217609 to endothelial cells during hypoxia at 4°C in the UW solution inhibited thrombin generation during reoxygenation at 37°C in human plasma and reduced tumor necrosis factor alpha, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 messenger RNA cell expressions. Invivo, function recovery was markedly improved in the UW-EP group. Interestingly, levels of thrombin-antithrombin complexes (reflecting thrombin generation) were reduced 60minutes after reperfusion in the UW-EP group. In addition, 3 months after transplantation, lower fibrosis, epithelial-mesenchymal transition, inflammation, and leukocyte infiltration were observed. Using this new dual anticoagulant, anti-Xa/IIa activity during kidney flush and preservation is protected by reducing thrombin generation at revascularization, improving early function recovery, and decreasing chronic lesions. Such an easy-to-deploy clinical strategy could improve marginal graft outcome.

Establishment of vein bypass assisted porcine liver auto transplantation model

Objective To establish the vein bypass assisted porcine liver auto transplantation model and study the methods of liver cold storage during the operation, in order to provide experience for the clinical application of liver auto transplantation and experimental evidence for liver machine perfusion. Methods A total of 5 pigs were operated to establish the portal-vein bypass by inserting cannula into vena jugularis interna, femoral vein and splenic vein, with liver being hypothermic perfused and stored in situ by 4℃ UW solution through hepatic artery and portal vein. Vital signs and clinical outcomes were monitored during the operation, and the changes of liver function indexes such as aminotransferase, total bilirubin and albumin were detected at different time points. Results During the operation, the vital signs kept steady and liver were perfused well, achieving fully blood supply after reperfusion. All animals survived over 120 h. Both alanine aminotransferase and aspartate transaminase peaked at 24 h after blood reperfusion (69.8±16.2 U/L and 457.7±50.1 U/L), and then dropped within normal range at 96 ho after reperfusion. Total bilirubin peaked at 48 h after blood reperfusion (7.1±3.3 μmol/L), and then decreased rapidly. The plasma albumin kept falling after blood reperfusion, reaching the minimum of 11.1±2.7 g/L at 4 h after reperfusion, and then rose to normal level steadily at 16 h. Conclusions The vein bypass assisted liver auto transplantation could guarantee the steady vital signs and survival rate, which may supply an experimental basis for developing new efficient liver preservation and restoration methods. Key words: Liver auto transplantation; Bypass; Animal model

Contrastive study on cryopreservation of rat amputated limb with UW solution and HTK solution

Objective To compare the effect on preserving rat amputated extremities during the cold preservation between the UW solution and HTK solution. Methods Thirty healthy adult Wistar rats were randomly divided into 3 groups (10 rats in each group) for producing the models of amputated limbs of rats. The three groups were separately irrigated the amputated limbs with UW solution, HTK solution and saline from femoral artery. After irrigation, the extremities were preserved in the refrigerator at 4 ℃. Samples of skeleton muscle tissue were taken for pathological and biochemical examination every 6 hours since the amputation. Results After perfusion, HE: changes of organizational structures of skeletal muscle were lighter in experimental groups than in control group (saline-perfusion group) at the same time. While samples in UW solution-perfusion group has lighter changes of organizational structure than those in HTK solution-perfusion group. TEM: at 12 h, all the three groups have shown the mitochondria edema. At 24 h, the myofibril in saline-perfusion group become fragments. Biochemical examination: at 24 h, indexes in saline-perfusion group, HTK solution group and UW solution group were: ALP (U/L) 3.62 ± 1.32, 3.37 ± 0.84 and 2.68 ± 1.59, respectively; ALT (U/L) 542.25 ± 129.36, 521.82 ± 97.32 and 462.53 ± 74.18, respectively; AST (U/L) 2200.12 ± 687.61, 2002.20 ± 632.27 and 1742.87 ± 396.09, respectively; CK(U/L) 313190.83 ± 42041.32, 283173.50 ± 31488.87 and 271319.67 ± 41147.52, respectively; LDH(U/L) 32409.50 ± 4253.20, 30382.50 ± 2337.18 and 30047.83 ± 4628.78, respectively; GLU(mmol/L) 0.242 ± 0.041、0.240 ± 0.044 and 0.252 ± 0.049, respectively; LAC (mmol/L) 1.790 ± 0.160, 1.792 ± 0.196 and 1.993 ± 0.366, respectively; SOD(U/mL) 80.82 ± 9.46, 91.62 ± 14.97 and 73.71 ± 10.60, respectively. There were statistical difference among the UW solution-perfusion group and centrol group in ALP and GLU (P < 0.05). UW solution-perfusion group has better results than HTK solution-perfusion group in SOD (P < 0.05). Conclusion Amputated limbs irrigated with organ preservation solution as a way to protect skeletal muscle had great effects on lightening tissue damage and keeping tissue active. Between the two usual organ preservation solution, UW solution had a stronger protective effects on the amputated limbs than HTK solution. Key words: Amputated limbs; Organ preservation solution; Skeletal muscle; Cryopreservation; Rat

Protective role of normothermic machine perfusion during reduced-size liver transplantation in pigs.

The purpose of this study is to explore whether normothermic machine perfusion (NMP) preservation is superior to cold preservation during reduced-size liver transplantation (RSLT) in pigs. Twenty-four healthy Ba-Ma mini pigs were used (aged >13 months; weight 25-35 kg; regardless of sex). The animals were randomized into 2 groups. In group A (NMP), donor livers were harvested without warm ischemia time and heartbeats and then were connected to the NMP system to reduce the livers' size under the normothermic condition. In group B (University of Wisconsin [UW] solution), donor livers were harvested without warm ischemia time and heartbeats after being perfused by UW solution and were then preserved in 0°C-4°C UW solution to reduce the livers' size under cold conditions. After that, liver transplantation without venovenous bypass was performed. General RSLT information of the pigs from the 2 groups was recorded; the serological indices were measured; and routine pathological examination of liver tissue was observed. A significant difference was observed in the intraoperative bleeding between the 2 groups (P < 0.05), whereas no significant difference was found in the other indices (all P > 0.05). Significant differences of alanine aminotransferase levels, aspartate aminotransferase levels, and lactate dehydrogenase levels between the 2 groups were observed between postoperative days 3 and 5 (P < 0.05). Significant differences of lactic acid levels between the 2 groups were observed between postoperative days 2 and 5 (P < 0.05). Compared with the cold preservation group, the liver tissues of the NMP preservation group only rarely experienced liver cell necrosis and maintained integrities in the hepatic sinusoid spaces and endothelial cells. In conclusion, NMP preservation is superior to cold preservation during RSLT in pigs. Liver Transplantation 22 968-978 2016 AASLD.

Advantages of replacing hydroxyethyl starch in University of Wisconsin solution with hyperbranched polyglycerol for cold kidney perfusion

BackgroundEfficient and effective perfusion during organ procurement is required for the best prevention of donor organ injury preceding transplantation. However, current organ preservation solutions, including hydroxyethyl starch (HES)–based University of Wisconsin (UW) solution, do not always yield the best outcomes. Our previous study demonstrated that replacing HES with hyperbranched polyglycerol (HPG) reduced donor heart injury during cold storage. The current research was designed to examine the advantages of HPG-based solution for cold kidney perfusion. MethodsPerfusion efficiency of HPG versus UW solution was tested using mouse kidneys at 4°C. The blood washout was evaluated by using a semiquantitative scoring system and tissue damage by histologic analysis. The interaction of HPG or UW solution with human red blood cells (RBCs) was examined by measuring RBC sedimentation and aggregation. ResultsThe lower viscosity of HPG solution was correlated with faster and more efficient perfusion through donor kidneys as compared with UW. HPG solution was also more effective than UW in removing RBCs from the kidney and was associated with less tissue damage to donor kidneys. In vitro UW solution caused significant RBC sedimentation and hyperaggregation, whereas HPG showed minimal impact on RBC sedimentation and prevented RBC aggregation. ConclusionsThis experimental study demonstrated that compared with UW, HPG solution was more efficient and effective in the removal of the blood from donor kidneys and offered better protection from donor tissue damage, suggesting that the HPG solution is a promising candidate to supplant standard UW solution for donor kidney perfusion in transplantation.

Hydrogen Gas Ameliorates Hepatic Reperfusion Injury After Prolonged Cold Preservation in Isolated Perfused Rat Liver.

Hydrogen gas reduces ischemia and reperfusion injury (IRI) in the liver and other organs. However, the precise mechanism remains elusive. We investigated whether hydrogen gas ameliorated hepatic I/R injury after cold preservation. Rat liver was subjected to 48-h cold storage in University of Wisconsin solution. The graft was reperfused with oxygenated buffer with or without hydrogen at 37° for 90 min on an isolated perfusion apparatus, comprising the H2 (+) and H2 (-) groups, respectively. In the control group (CT), grafts were reperfused immediately without preservation. Graft function, injury, and circulatory status were assessed throughout the perfusion. Tissue samples at the end of perfusion were collected to determine histopathology, oxidative stress, and apoptosis. In the H2 (-) group, IRI was indicated by a higher aspartate aminotransferase (AST), alanine aminotransferase (ALT) leakage, portal resistance, 8-hydroxy-2-deoxyguanosine-positive cell rate, apoptotic index, and endothelial endothelin-1 expression, together with reduced bile production, oxygen consumption, and GSH/GSSG ratio (vs. CT). In the H2 (+) group, these harmful changes were significantly suppressed [vs. H2 (-)]. Hydrogen gas reduced hepatic reperfusion injury after prolonged cold preservation via the maintenance of portal flow, by protecting mitochondrial function during the early phase of reperfusion, and via the suppression of oxidative stress and inflammatory cascades thereafter.

14-3-3ζ-Mediated Stimulation of Oxidative Phosphorylation Exacerbates Oxidative Damage Under Hypothermic Oxygenated Conditions in Human Renal Tubular Cells (HK-2)

Cellular survival and death are at least partially regulated by the phosphorylation of proteins. A chaperon protein, 14-3-3ζ, regulates the activity of many proteins by covering the phosphorylation site within a 14-3-3 binding motif. Therefore, regulation of 14-3-3ζ activity may affect the fate of cells subjected to cold preservation and/or hypothermic oxygenated conditions. The present study assessed whether 14-3-3ζ protects cells from hypothermic oxygenation-induced injury and clarified its role in mitochondrial functions. Human renal tubular cell line HK-2 or 14-3-3ζ-overexpressed HK-2 (ζHK-2) cells were subjected to 72 hours of normoxic cold preservation in UW solution with or without antioxidants and hydroperoxides. Cellular death, adenosine triphosphate (ATP) content, and MTT catabolism were evaluated. Deferoxamine treatment reduced cellular death and augmented ATP content in both cell types. These indices were higher in ζHK-2, regardless of deferoxamine treatment. Exposure to hydroperoxides did not affect cellular death in either cell type, whereas hydroperoxide supplementation significantly reduced ATP content, except for low-dose hydrogen peroxide in HK-2 cells. MTT assay at normal state showed higher values in ζHK-2 cells, whereas it was impaired by hydroperoxides in both cell types. These results suggest that accumulation of hydroperoxides as a byproduct of the augmented oxidative phosphorylation by 14-3-3ζ overexpression causes mitochondrial dysfunction. In conclusion, despite possessing many potentially protective functions, 14-3-3ζ exacerbates cellular injury under hypothermic oxygenated conditions. 14-3-3ζ accelerates mitochondrial functions together with iron-dependent oxidative damage. Although further investigations are necessary, upregulation of 14-3-3ζ could be a method to maintain mitochondrial function under hypothermic oxygenated conditions, as shown in hypothermic machine preservation of renal grafts, when appropriate antioxidant treatment is administered.

Graft preservation solutions in cardiovascular surgery.

Vein grafts are still the most commonly used graft material in cardiovascular surgery and much effort has been spent in recent years on investigating the optimal harvesting technique. One other related topic of similar importance remained more or less an incidental one. The storage solutions of vein grafts following procurement and prior to implantation are, despite their assumed impact, a relatively neglected theme. There is no doubt that the endothelium plays a key role in long-term patency of vein grafts, but the effects of the different storage solutions on the endothelium remain unclear : In a review of the literature, we could find 20 specific papers that addressed the question, of which the currently available preservation solutions are superior, harmless, damaging or ineffective. The focus lies on saline and autologous whole blood. Besides these two storage media, novel or alternative solutions have been investigated with surprising findings. In addition, a few words will be spent on potential alternatives and novel solutions on the market. As there is currently no randomized clinical trial regarding saline versus autologous whole blood available, this review compares all previous studies and methods of analysis to provide a certain level of evidence on this topic. In summary, saline has negative effects on the endothelial layers and therefore may compromise graft patency. Related factors, such as distension pressure, may outbalance the initial benefit of autologous whole blood or storage solutions and intensify the harmful effects of warm saline. In addition, there is no uniform consent on the superiority of autologous whole blood for vein graft storage. This may open the door to alternatives such as the University of Wisconsin solution or one of the specific designed storage solutions like TiProtec™ or Somaluthion™. Whether these preservation solutions are superior or advantageous remains the subject of further studies.

Case Report: Spleen-preserving Multivisceral Transplant for Peutz–Jeghers Syndrome

Case ReportA 24-year-old man diagnosed with Peutz–Jeghers syndrome as a child underwent multiple surgeries owing to intussusception. Pretransplant workup showed >150 polyps along the gastrointestinal (GI) tract, some of them with high-grade dysplasia. Despite having intestinal sufficiency, a modified multivisceral transplantation was offered. ProcedureAn 18-year-old donor was procured using University of Wisconsin solution. The recipient's surgery started with a midline incision. Mobilization of the right colon and the root of the mesentery was done to isolate the superior mesenteric artery. The same maneuver was done with the left and sigmoid colon. The common bile duct was then isolated and transected at the cystic duct level. The abdominal portion of the esophagus and the proximal stomach were isolated and divided at the gastroesophageal junction. After that, the pancreas was mobilized, preserving the spleen with the splenic vessels. The distal GI tract was transacted at the level of the proximal rectum. For engraftment, an arterial conduit was placed in the infrarenal aorta and anastomosed to the graft's aortic patch. End-to-side portal reconstruction was made at the level of the portal vein, allowing performing a duct-to-duct biliary reconstruction over a 5-Fr T-tube. A hand-sewn gastrogastric anastomosis and piloroplasty were performed; the distal anastomosis was done with circular staplers. A gastrojejunostomy and a loop ileostomy were the final steps of the procedure. ResultsThe patient stayed in intensive care for 2 days and enteral feeds were started on day 7. Currently, 23 months after transplant he is alive with an excellent quality of life.

Donor pretreatment with carbon monoxide prevents ischemia/reperfusion injury following heart transplantation in rats.

Because inhaled carbon monoxide (CO) provides potent anti-inflammatory and antioxidant effects against ischemia reperfusion injury, we hypothesized that treatment of organ donors with inhaled CO would decrease graft injury after heart transplantation. Hearts were heterotopically transplanted into syngeneic Lewis rats after 8 hours of cold preservation in University of Wisconsin solution. Donor rats were exposed to CO at a concentration of 250 parts per million for 24 hours via a gas-exposure chamber. Severity of myocardial injury was determined by total serum creatine phosphokinase and troponin I levels at three hours after reperfusion. In addition, Affymetrix gene array analysis of mRNA transcripts was performed on the heart graft tissue prior to implantation. Recipients of grafts from CO-exposed donors had lower levels of serum troponin I and creatine phosphokinase; less upregulation of mRNA for interleukin-6, intercellular adhesion molecule-1, and tumor necrosis factor-α; and fewer infiltrating cells. Although donor pretreatment with CO altered the expression of 49 genes expressly represented on the array, we could not obtain meaningful data to explain the mechanisms by which CO potentiated the protective effects. Pretreatment with CO gas before organ procurement effectively protected cardiac grafts from ischemia reperfusion-induced injury in a rat heterotopic cardiac transplant model. A clinical report review indicated that CO-poisoned organ donors may be comparable to non-poisoned donors.

Lifor Solution: An Alternative Preservation Solution in Small Bowel Transplantation.

Background and Objectives. The intestinal mucosa is extremely sensitive to ischemia. Better intestinal preservation is the first step to improve the results of intestinal transplantation. The aim of the study is to investigate the effect of cold Lifor solution on preservation of swine small bowel. Methods. Swine ileum segments (200 cm) were allotransplanted heterotopically after 9-hour cold storage with UW solution (group 1, n = 6), with Lifor solution (group 2, n = 6), or without storage (group 3, n = 6), respectively. After cold storage, mucosal adenosine triphosphate (ATP) concentrations and histopathologic analysis after preservation were performed. At day 7 after the transplantation, intestinal absorptive function was also observed. Results. After 9 h cold preservation, pathological changes, the content of ATP in the intestinal mucosa, and the intestinal absorptive function after transplantation in group 2 were similar to those of group 1. Conclusion. The effect of cold storage of swine small bowel with Lifor solution is similar to that of UW solution. It may provide additional rationale for further exploration of Lifor as an alternative preservation solution in small bowel transplantation.

Contribution of alloantigens to hepatic ischemia/reperfusion injury: Roles of natural killer cells and innate immune recognition of nonself.

Hepatic ischemia/reperfusion injury (IRI) remains a major clinical problem and involves the innate immune system's recognition of "nonself." Considering the efficient nonself recognition by natural killer (NK) cells, we hypothesize in this study that hepatic IRI associated with liver transplantation (LT) could be augmented in allogeneic rather than in syngeneic (Syn) grafts due to alloantigen recognition by innate immune cells, especially by NK cells. Using green fluorescent protein (GFP)/Sprague-Dawley rats, we tested our hypothesis in a rat LT model with 18 hours of cold storage in University of Wisconsin solution. Hepatic IRI was significantly augmented in allografts with higher alanine transaminase levels, increased necrosis, and vigorous proinflammatory mediator up-regulation compared to Syn grafts. Injury increased in allografts associated with augmented GFP+ host leukocyte infiltration due to significantly increased host CD11b/c+ and RP-1(+) neutrophil recruitment. A large number of liver-resident (donor) mature CD11b/c+ NK cells quickly diminished from allografts, but not from Syn grafts. Depletion of mature NK cells from liver grafts with anti-asialo monosialotetrahexosylganglioside significantly improved hepatic IRI and reduced neutrophil infiltration and proinflammatory mediators. In conclusion, early innate immune responses were more significantly enhanced in allografts than in Syn grafts during hepatic IRI, in part through NK cell recognition of "missing self."

Эффективность сахарозо-содержащего раствора и раствора UW для гипотермического хранения мезенхимальных стромальных клеток человека в суспензии или в составе альгинатных микросфер

The effect of hypothermic storage (HS) on viability and functional activity of human mesenchymal stromal cells (MSCs) in suspension and within alginate microspheres (AMSs) was studied. Cells were stored in sealed cryovials at 4°C in culture medium (CM), sucrose-based solution (SBS) and that of the University of Wisconsin (UW). After 3 and 7 days of storage the viability, metabolic activity and morphology of MSCs were assessed by MTT-assay, Alamar Blue test, adhesive activity, morphology and ability of multilineage differentiation at monolayer culture conditions. Hypothermic storage of MSCs suspension in CM for 3 days was established to result in a 3–4-fold decrease in their viability and adhesive ability. The encapsulation into AMSs partially prevented cell death. Hypothermic storage for 7 days caused death of almost all MSCs both in suspension and AMSs. The substitution of CM for UW and SBS preservation solutions maintained the MSCs viability and metabolic activity following HS within 3 days both in suspension and within AMSs. After 7 days of HS in UW and SBS solutions the MSCs in suspensions and within AMSs preserved their viability on the level of 60–80%, adhesive properties, metabolic activity, as well as the ability for induced adipogenic and osteogenic differentiation. The use of UW and SBS preservation solutions enabled to prolong the hypothermic storage terms for MSCs. Probl Cryobiol Cryomed 2015; 25(4):329-339.

Evaluation of hepatic vascular endothelial injury during liver storage by molecular detection and targeted contrast-enhanced ultrasound imaging.

We hypothesized that lack of the high-energy phosphates during liver storage may potentially cause persistent injury to the vascular endothelium. Biopsies were obtained from livers obtained from beating heart human donors, stored either in the standard storage solution, that is, University of Wisconsin solution (UWS) or Celsior, and examined for various markers related to progressive endothelial injury. The expression of P2Y1 receptor, the major signal transduction machinery for adenosine triphosphate/adenosine diphosphate, decreased in hepatic vascular endothelial cells over time. Despite unaltered endothelial nitric oxide synthase (eNOS) levels, serine1177-phosphorylated eNOS, the active form of eNOS, progressively decreased with time. The production of nitric oxide enzyme decreased with time when liver tissues were examined in vitro. This also coincided with decreased interaction of eNOS with actin nucleating proteins like myristoylated alanine-rich C kinase substrate and Rac1, which plays a role in modulating the cytoskeleton and helps position eNOS in a favorable cytosolic position for active enzymatic activity. Conversely, the interaction of eNOS with caveolin1 was significantly increased 6 H after ex vivo storage. Finally, we demonstrated by targeted contrast-enhanced ultrasound that membrane-bound vascular cell adhesion molecule-1 in the hepatic vascular endothelial cell increased after 6 H of ex vivo storage. Overall, the results of this study provide evidence of a progressive hepatic vascular endothelial injury during the ex vivo storage. This may be a causative factor for ischemic cholangiopathy and delayed graft function post liver transplantation. © 2015 IUBMB Life, 68(1):51-57, 2015.