Pancreatic L-Glutamine Administration Protects Pig Islets From Cold Ischemic Injury and Increases Resistance Toward Inflammatory Mediators.

The isolation and transplantation of porcine islets represent a future option for the treatment of type 1 diabetic patients. Stringent product release criteria and limited availability of transgenic and specific pathogen-free pigs will essentially require processing of explanted pig pancreata in specialized, possibly remote isolation facilities, whereby pancreata are exposed to cold ischemia due to prolonged tissue transit time. In the present study we investigated whether pancreas oxygenation can be efficiently combined with an antioxidant strategy utilizing intraductal L-glutamine administration. Pig pancreata were intraductally perfused after retrieval and after cold storage in oxygen-precharged perfluorohexyloctane utilizing University of Wisconsin solution supplemented with (n = 16) or without (n = 14) 5 mmol/L L-glutamine. After isolation purified islets were subjected to extensive quality assessment. Islet recovery postpurification was significantly higher in glutamine-treated pancreata (77.0 ± 3.3% vs. 60.3 ± 6.0%, p < 0.05). Glutamine administration increased intraislet content of reduced glutathione (117.8 ± 16.5 vs. 15.9 ± 2.8 ng/ng protein, p < 0.001) associated with increased islet recovery after culture (65.8 ± 12.1% vs. 40.3 ± 11.7%, p < 0.05), enhanced glucose stimulation index (1.82 ± 0.16 vs. 1.38 ± 0.10, p < 0.05), and improved posttransplant function in diabetic nude mice (p < 0.05). Furthermore, intraductally administered glutamine increased pig islet resistance toward reactive oxygen species, nitric oxide, and high-dose proinflammatory cytokines. The present study demonstrates that quality and function of pig islets exposed to warm and cold ischemia can significantly be improved using intraductal l-glutamine administration. As the efficiency of the intraductal route may be inferior compared to intravascular administration further studies should aim on assessment of l-glutamine as supplement for pancreas perfusion during organ procurement.

Normothermic perfusion of donor lungs for preservation and assessment with the Organ Care System Lung before bilateral transplantation: a pilot study of 12 patients.

BACKGROUND: Cold flush and static cold storage is the standard preservation technique for donor lungs before transplantations. Several research groups have assessed normothermic perfusion of donor lungs but all devices investigated were non-portable. We report first-in-man experience of the portable Organ Care System (OCS) Lung device for concomitant preservation, assessment, and transport of donor lungs. METHODS: Between Feb 18, and July 1, 2011, 12 patients were transplanted at two academic lung transplantation centres in Hanover, Germany and Madrid, Spain. Lungs were perfused with low-potassium dextran solution, explanted, immediately connected to the OCS Lung, perfused with Steen's solution supplemented with two red-cell concentrates. We assessed donor and recipient characteristics and monitored extended criteria donor lung scores; primary graft dysfunction scores at 0, 24, 48, and 72 h; time on mechanical ventilation after surgery; length of stays in hospital and the intensive-care unit after surgery; blood gases; and survival of grafts and patients. FINDINGS: Eight donors were female and four were male (mean age 44·5 years, range 14-72). Seven recipients were female and five were male (mean age 50·0 years, range 31-59). The preharvest donor ratio of partial pressure of oxyen (PaO(2)) to fractional concentration of oxygen in inspired air (F(I)O(2)) was 463·9 (SD 91·4). The final ratio of PaO(2) to F(I)O(2) measured with the OCS Lung was 471·58 (127·9). The difference between these ratios was not significant (p=0·72). All grafts and patients survived to 30 days; all recipients recovered and were discharged from hospital. INTERPRETATION: Lungs can be safely preserved with the OCS Lung, resulting in complete organ use and successful transplantation in our series of high-risk recipients. In November, 2011, we began recruitment for a prospective, randomised, multicentre trial (INSPIRE) to compare preservation with OCS Lung with standard cold storage. FUNDING: TransMedics and German Federal Ministry of Education and Research.

Long-term preservation of human saphenous vein by green tea polyphenol under physiological conditions.

Polyphenolic compounds are well known as a functional food with various bioactivities. However, less attention has been paid to the effect of phenolic antioxidants on the preservation of blood vessels. In this study, the possible effects of green tea polyphenolic compounds (GTPCs) on the longterm preservation of the human saphenous vein (HSV) were investigated under physiological conditions. HSV segments were pretreated with GTPCs (0.5 or 1.0 mg/mL) for 1 day and then incubated for 1, 2, 4, or 8 weeks. After incubation, cellular viability, endothelial nitric oxide synthase (eNOS) expression level, biomechanical properties, and vein histology were evaluated. When HSV segments were incubated without GTPC treatment, endothelial cell viability was significantly (p < 0.05) reduced with incubation time, and none of the endothelial cells expressed eNOS after 2 weeks. Furthermore, nontreated veins demonstrated appreciable inferiority in such mechanical properties as failure strength, elastic modulus, and compliance, compared with fresh veins. These results were confirmed by histological observations, which showed severe structural changes in nontreated veins. On the other hand, these phenomena were markedly prevented by preincubating veins with GTPCs (1.0 mg/mL) at 37 degrees C in a CO(2) incubator for 1 day. GTPC-pretreated veins could be preserved for at least 2 weeks under physiological conditions, retaining cellular viability and eNOS expression level and maintaining both biomechanical properties and vascular structures without any morphological alterations. These results demonstrate that GTPC treatment may be a useful method for preserving the HSV and could be exploited to craft strategies for the long-term preservation of other tissues under physiological conditions.

Apoptosis inhibition during preservation by fructose-1,6-diphosphate and theophylline in rat intestinal transplantation.

OBJECTIVE: This study evaluated the effect of fructose-1,6-diphosphate (FDP), theophylline, or the addition of both together to the preservation solution (University of Wisconsin [UW]) on apoptosis during preservation and the effect of apoptosis minimization on the early reperfusion period after transplantation. DESIGN: Prospective, randomized, and controlled animal study. SETTING: Laboratory of a research institute. SUBJECT: Male Wistar rats. INTERVENTIONS: The jejunum was isolated and preserved for 6 hrs in UW solution. FDP and theophylline were added to the UW solution to evaluate their effects on apoptosis both alone and together. The role of adenosine with respect to FDP was examined by increasing endogenous adenosine. In addition, rats were subjected to intestinal transplantation for the evaluation of the effect of apoptosis on bacterial translocation, histology, and neutrophil infiltration after reperfusion. MEASUREMENTS AND MAIN RESULTS: Caspase-3 activity, assayed both in vitro or by cleaved caspase-3 levels in Western blots or immunohistochemically, and the number of terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling (TUNEL)-positive cells decreased with FDP and with theophylline addition to UW solution. Increase of endogenous adenosine reversed the antiapoptotic effect of FDP. FDP and theophylline together demonstrated a more pronounced antiapoptotic effect and prevented bacterial translocation after transplantation. CONCLUSION: Supplementary FDP to UW solution decreased apoptosis through an adenosine-independent mechanism. Addition of theophylline to UW solution decreased both apoptosis and bacterial translocation. Concomitant theophylline and FDP addition to preservation solution is recommended to maintain low levels of apoptosis during intestinal hypothermic preservation and to decrease bacterial translocation.

Functional recovery of human mesenteric and coronary arteries after cryopreservation at -196 degrees C in a serum-free medium.

PURPOSE: Long-term patency of cryopreserved vascular grafts is determined by maintained cellular and tissue viability, which implies preservation of various biochemical, smooth muscle, and endothelial functions. Therefore, it was investigated whether the presence of fetal calf serum (FCS) in the cryomedium improves the postthaw contractile and endothelial function of human arteries. METHODS: Rings from human mesenteric (HMA) and left circumflex coronary arteries (HCA) obtained from organ donors were randomized into three groups and studied either unfrozen or after storage for 3 to 6 weeks at -196 degrees C while suspended in Krebs-Henseleit solution without or with 20% FCS as the vehicles and 1.8 mol/L dimethyl sulfoxide and 0.1 mol/L sucrose as cryoprotecting agents. The samples were slowly frozen to -70 degrees C and then stored in liquid nitrogen. Before use, the tissues were thawed within 3 minutes in a 40 degrees C water bath. RESULTS: After thawing the sensitivity to various agonists and maximal responses to the endothelium-independent relaxing agent sodium nitroprusside were unchanged. However, after cryopreservation of HMA was performed without and with FCS, maximal contractile responses to noradrenaline were significantly reduced to 10.1 +/- 0.7 gm and 9.9 +/- 0.9 gm compared with 13.3 +/- 0.6 gm in unfrozen HMA (mean +/- SEM, n = 15). After cryopreservation of HCA was performed without and with FCS, maximal contractile responses to prostaglandin F2 alpha (6.9 +/- 0.4 gm in unfrozen HCA) were significantly reduced to 4.3 +/- 0.3 gm and 3.8 +/- 0.2 gm (mean +/- SEM, n = 6). In both types of arteries cryopreservation also attenuated significantly the endothelium-dependent relaxant responses to bradykinin during U46619 (10 nmol/L)-induced tone. In HMA the maximal bradykinin-induced relaxation (85% +/- 4%) was significantly diminished to 29% +/- 7% and 38% +/- 9% after cryopreservation without and with FCS (mean +/- SEM, n = 6). In HCA maximal bradykinin-induced relaxation (88% +/- 4%) was significantly diminished to 26% +/- 10% and 36% +/- 11% after cryopreservation without and with FCS (mean +/- SEM, n = 6). This result was reflected by a marked endothelial denudation in all groups of cryopreserved arteries. Neither functional nor morphologic preservation of the endothelial cell lining was significantly improved by FCS supplementation of the cryomedium. CONCLUSIONS: Cryopreservation diminished contractile and endothelium-dependent relaxant responses of human arteries. The presence of FCS in the cryomedium did not modify these changes.