End Of Preservation Research Articles

The Effect of Continuous Liver Normothermic Machine Perfusion on the Severity of Histological Bile Duct Injury.

Static Cold Storage (SCS) injures the bile duct, while the effect of Normothermic Machine Perfusion (NMP) is unknown. In a sub-study of the COPE trial on liver NMP, we investigated the impact of preservation type on histological bile duct injury score (BDIS). Transplants with at least one bile duct biopsy, either at end of preservation or 1h post-reperfusion, were considered. BDIS was determined by assessing peribiliary glands injury, stromal and mural loss, haemorrhage, and thrombosis. A bivariate linear model compared BDIS (estimate, CI) between groups. Sixty-five transplants and 85 biopsies were analysed. Twenty-three grafts were preserved with SCS and 42 with NMP, with comparable baseline characteristics except for a shorter cold ischemic time in NMP. The BDIS increased over time regardless of preservation type (p = 0.04). The BDIS estimate was higher in NMP [8.02 (7.40-8.65)] than in SCS [5.39 (4.52-6.26), p < 0.0001] regardless of time. One patient in each group developed ischemic cholangiopathy, with a BDIS of 6 for the NMP-preserved liver. In six other NMP grafts, BDIS ranged 7-12 without development of ischemic cholangiopathy. In conclusion, BDIS increases over time, and the higher BDIS in NMP did not increase ischemic cholangiopathy. Thus, BDIS may overestimate this risk after liver NMP.

Liver Ischemia and Reperfusion Induce Periportal Expression of Necroptosis Executor pMLKL Which Is Associated With Early Allograft Dysfunction After Transplantation.

BackgroundEarly allograft dysfunction (EAD) following liver transplantation (LT) remains a major threat to the survival of liver grafts and recipients. In animal models, it is shown that hepatic ischemia-reperfusion injury (IRI) triggers phosphorylation of Mixed Lineage Kinase domain-like protein (pMLKL) inducing necroptotic cell death. However, the clinical implication of pMLKL-mediated cell death in human hepatic IRI remains largely unexplored. In this study, we aimed to investigate the expression of pMLKL in human liver grafts and its association with EAD after LT.MethodsThe expression of pMLKL was determined by immunohistochemistry in liver biopsies obtained from both human and rat LT. Human liver biopsies were obtained at the end of preservation (T0) and ~1 hour after reperfusion (T1). The positivity of pMLKL was quantified electronically and compared in rat and human livers and post-LT outcomes. Multiplex immunofluorescence staining was performed to characterize the pMLKL-expressing cells.ResultsIn the rat LT model, significant pMLKL expression was observed in livers after IRI as compared to livers of sham-operation animals. Similarly, the pMLKL score was highest after IRI in human liver grafts (in T1 biopsies). Both in rats and humans, the pMLKL expression is mostly observed in the portal triads. In grafts who developed EAD after LT (n=24), the pMLKL score at T1 was significantly higher as compared to non-EAD grafts (n=40). ROC curve revealed a high predictive value of pMLKL score at T1 (AUC 0.70) and the ratio of pMLKL score at T1 and T0 (pMLKL-index, AUC 0.82) for EAD. Liver grafts with a high pMLKL index (>1.64) had significantly higher levels of serum ALT, AST, and LDH 24 hours after LT compared to grafts with a low pMLKL index. Multivariate logistical regression analysis identified the pMLKL-index (Odds ratio=1.3, 95% CI 1.1-1.7) as a predictor of EAD development. Immunohistochemistry on serial sections and multiplex staining identified the periportal pMLKL-positive cells as portal fibroblasts, fibrocytes, and a minority of cholangiocytes.ConclusionPeriportal pMLKL expression increased significantly after IRI in both rat and human LT. The histological score of pMLKL is predictive of post-transplant EAD and is associated with early liver injury after LT. Periportal non-parenchymal cells (i.e. fibroblasts) appear most susceptible to pMLKL-mediated cell death during hepatic IRI.

NonFreezable Preservation of Human Red Blood Cells at -8 °C.

Red blood cell (RBC) preservation is very important in human health. The RBCs are usually preserved at 4 ± 2 °C without freezing or at a very low temperature (-80 °C or liquid nitrogen) with deep freezing. Herein, non freezable preservation of RBCs at a subzero temperature is reported to prolong the preservation time compared with that at 4 ± 2 °C. By adding glycerol and poly(ethylene glycol) (PEG) (average number molecular weight 400, PEG-400) into the preservation solution, the freezing point is decreased and the hemolysis is kept low. The cell metabolism of stored RBCs at -8 °C is reduced, and the shelf life of RBCs extends up to at least 70 days. At the end of preservation, the pH decreases a little bit to demonstrate the low metabolic rate of RBCs stored at subzero temperatures. After quick washing, the RBC survival rate is ca. 95%. The adenosine triphosphate, 2,3-diphosphoglycerate, and cell deformation ability of the washed RBCs are maintained at a high level, while the malondialdehyde is relatively low, which verifies the high quality of RBCs stored at this condition.

The Antibacterial and Antioxidant Roles of Buckwheat Honey (BH) in Liquid Preservation of Boar Semen.

In the present study, we hypothesized that buckwheat honey (BH) should be regarded as a potential alternative to antibacterial and antioxidant agent in liquid storage of boar semen. To this end, boar semen was firstly studied for in vitro dose tolerability to BH by measuring sperm progressive motility. The optimum progressive motility of boar spermatozoa was observed in extender with 0.5% and 0.6% BH addition. Afterward, sperm quality parameters, bacterial profile and composition, total antioxidant (T-AOC), catalase (CAT), superoxide dismutase (SOD), and malondialdehyde (MDA) levels of control, BH supplementation, antibiotics supplementation, and incorporated supplementation were compared during liquid storage period, to further investigate antibacterial and antioxidant properties of BH. The results showed that BH supplementation significantly improved sperm motility, acrosome integrity, plasma membrane integrity, inhibited opportunistic bacterial growth, and altered microbial compositions at the end of preservation. Additionally, T-AOC, SOD, and CAT levels were significantly higher in the BH supplementation group than those in the control and antibiotic supplementation group, whereas MDA level exhibited opposite change pattern. Importantly, BH addition to the extender was able to exert a synergistic effect in combination of antibiotic use. Our findings suggested that the appropriate concentrations (0.5% and 0.6%) of BH were added to the extender could act antibacterial and antioxidant roles in liquid preservation of boar semen.

Resolving the graft ischemia-reperfusion injury during liver transplantation at the single cell resolution

Ischemia–reperfusion injury (IRI) remains the major reason for impaired donor graft function and increased mortality post-liver transplantation. The mechanism of IRI involves multiple pathophysiological processes and numerous types of cells. However, a systematic and comprehensive single-cell transcriptional profile of intrahepatic cells during liver transplantation is still unclear. We performed a single-cell transcriptome analysis of 14,313 cells from liver tissues collected from pre-procurement, at the end of preservation and 2 h post-reperfusion. We made detailed annotations of mononuclear phagocyte, endothelial cell, NK/T, B and plasma cell clusters, and we described the dynamic changes of the transcriptome of these clusters during IRI and the interaction between mononuclear phagocyte clusters and other cell clusters. In addition, we found that TNFAIP3 interacting protein 3 (TNIP3), specifically and highly expressed in Kupffer cell clusters post-reperfusion, may have a protective effect on IRI. In summary, our study provides the first dynamic transcriptome map of intrahepatic cell clusters during liver transplantation at single-cell resolution.

Biobanking of Dehydrated Human Donor Corneal Stroma to Increase the Supply of Anterior Lamellar Grafts.

To investigate the effect of dehydration on human donor corneal stroma for biobanking. Epithelium and endothelium of research-grade human donor corneas (n = 12) were scraped off, leaving a bare stroma with attached sclera. The tissues were placed in a large Petri dish prefilled with silica gel in the periphery and stored at room temperature for 14 days. At the end of preservation, the tissues were rehydrated by being submerged in phosphate-buffered saline for 15 minutes. Transparency (using a custom-built device) and thickness (using optical coherence tomography) measurements were recorded before dehydration, after dehydration, and after rehydration of the tissues. Periodic acid-Schiff and alpha-smooth muscle actin (α-SMA) staining before dehydration and after rehydration were performed to determine the presence of keratocytes and expression of α-SMA. Tensile stress-strain before dehydration and after rehydration was performed to evaluate the biomechanical properties. No difference in corneal transparency before dehydration (69.57 ± 6.41%) and after rehydration (67.37 ± 2.82%), P = 0.36, was observed. The corneas were more compact after dehydration. A significant change in thickness between before dehydration (625.8 ± 75.58 μm) and after rehydration (563.6 ± 15.77 μm) stage, P = 0.03, was noticed. The thickness was reduced to 147.6 ± 3.71 μm when dehydrated. Periodic acid-Schiff staining showed presence of stromal keratocytes and α-SMA protein expressed in control, dehydrated, and rehydrated corneas. There was no significant difference in the stiffness between control (27.86 ± 11.65 MPa) and rehydrated corneas (31.46 ± 11.41 MPa). Human donor corneal stroma can be biobanked for up to 2 weeks in a dehydrated condition without losing their molecular or biomechanical properties after rehydration.

Proteomic Analysis of Perfusate from Machine Cold Perfusion of Transplant Kidneys: Insights Into Protection from Injury.

BackgroundMachine cold perfusion is beneficial to the preservation of kidneys for transplantation. At the end of preservation, the perfusion solution contains many proteins. Using a proteomics approach, we searched for useful biomarkers and potential therapeutic targets in the perfusate. Our program is unique in that all transplant kidneys (even living donor kidneys, LKD) are placed on machine cold perfusion prior to transplantation.Material/MethodsPerfusates from donation after neurological and circulatory determination of death (DNDD and DCDD respectively) and LKD were collected (n=41) and analyzed for LDH, neutrophil gelatinase-associated lipocalin (NGAL), and matrix metalloproteinase-2 (MMP-2) as markers of injury. Perfusate from each kidney was subjected to 2-dimensional gel electrophoresis, then analyzed using software to identify those spots which are significantly different between the 3 groups. Mass spectrometry was used to identify the proteins and their identity was confirmed with Western blot.ResultsThe highest levels of MMP-2, LDH, and NGAL were seen for the DCDD kidneys, followed by the DNDD kidneys and then LDK. Peroxiredoxin-2, NGAL, and alpha-1-antitrypsin were identified as significantly different between the different types of donor kidneys, and their role and possible therapeutic strategies are discussed. Collagen fragments, albumin, and immunoglobulin were also identified as possible byproducts of the injury and may be useful is assessing the degree of injury.ConclusionsComparison of the perfusates from the different types of kidneys has allowed us to identify proteins that will be useful in future research into reducing injury in transplant kidneys.

Machine Perfusion at 20°C Prevents Ischemic Injury and Reduces Hypoxia-Inducible Factor-1α Expression During Rat Liver Preservation.

BACKGROUND Ischemic cholangitis is the main cause of liver failure after transplantation and subnormothermic machine perfusion may represent a better strategy than conventional cold storage, minimizing preservation injury. We compared livers preserved by machine perfusion at 20°C (MP20) or by cold storage at 4°C (CS4) with regard to hypoxia-inducible factor (HIF)-1α mRNA expression and protein stabilization in hypoxic conditions. MATERIAL AND METHODS Livers from male Wistar rats were stored on ice at 4°C in UW solution (CS4) or perfused with oxygenated Krebs-Henseleit buffer at 20°C (MP20) for six hours. After preservation, the livers were reperfused for two hours with oxygenated Krebs-Henseleit buffer at 37°C to simulate reimplantation. We collected bile, perfusate, and tissue samples. Transaminases, lactate dehydrogenase, bilirubin, and lactic acid were assayed in the perfusate and bile. ATP/ADP, glycogen, HIF-1α mRNA, and protein expression were measured in the tissue homogenates. RESULTS At the end of preservation, as well as after reperfusion, HIF-1α mRNA expression was significantly higher in the ischemic CS4 livers. Although the hypoxic conditions found in CS4 preservation stabilized HIF-1α protein was significantly higher in the CS4 livers at the end of preservation, no difference was observed after reperfusion, likely because of the oxygen in the reperfusion medium. After reperfusion, the MP20 livers released less transaminases and LDH. The MP20 livers had higher ATP/ADP, glycogen, and biliary bilirubin after both preservation and reperfusion when compared with the CS4 livers. CONCLUSIONS The data demonstrated that MP20 was associated with a lower HIF-1α expression and organ injury with respect to CS4, suggesting that oxygen provided by this preservation setting might approximate the organ request, thus avoiding the ischemic injury usually observed during organ preservation by cold storage.

Mechanistic Analysis of Nonoxygenated Hypothermic Machine Perfusion’s Protection on Warm Ischemic Kidney Uncovers Greater eNOS Phosphorylation and Vasodilation

Protection of endothelial cell function may explain the benefits of nonoxygenated hypothermic machine perfusion (MP) for marginal kidney preservation. However, this hypothesis remains to be tested with a preclinical model. We postulated that MP protects the nitric oxide (NO) signaling pathway, altered by static cold storage (CS), and improves renal circulation recovery compared to CS. The endothelium releases the vasodilator NO in response to flow via either increased endothelial NO synthase (eNOS) expression (KLF2-dependent) or activation of eNOS by phosphorylation (via Akt, PKA or AMPK). Using a porcine model of kidney transplantation, including 1 h of warm ischemia and preserved 24 h by CS or MP (n=5), we reported that MP did not alter the cortical levels of KLF2 and eNOS at the end of preservation, but significantly increased eNOS activating phosphorylation compared to CS. eNOS phosphorylation appeared AMPK-dependent and was concomitant to an increased NO-dependent vasodilation of renal arteries measured, ex situ, at the end of preservation. In vivo, laser Doppler showed that cortical microcirculation was improved at reperfusion in MP kidneys. In conclusion, we demonstrate for the first time, in a large-animal model, that MP protects the NO signaling pathway, confirming the value of MP for marginal kidney preservation.

NaOH treatment reduces capsaicin content and pungency of ‘Makedoniko’ pepper (Capsicum annuum L.)

SummaryPickled peppers are preferred by certain consumer groups at reduced pungency. To investigate the reduction in pepper pungency and capsaicin content, fresh ‘Makedoniko’ hot peppers were treated with 0, 0.5 or 1% NaOH before preserved in acid brine for 60 days. Pepper physicochemical traits were monitored following NaOH treatment and during preservation. Capsaicin content was measured in the beginning and at the end of preservation, and the peppers were also tested organoleptically. Preservation in acid brine induced the typical green–yellow product colour, increased firmness and decreased the dry mass in all treatments. NaOH‐treated pepper increased phenolics, chlorophyll and antioxidant content in dry mass during preservation faster than the control. Capsaicin content was reduced by 25 or 50% following 0.5 or 1% NaOH treatment, respectively. Pepper organoleptic pungency was also decreased following 1% NaOH treatment. NaOH reduces capsaicin content and pungency of ‘Makedoniko’ pepper, with minor effects on its processed product quality.

Determination and Extension of the Limits to Static Cold Storage using Subnormothermic Machine Perfusion

Static cold storage (SCS) of the liver for transplantation is limited by time. Continuation of metabolic activity leads to depletion of energy stores and loss of cellular function, which results in poor post-transplant function. Machine perfusion (MP) applied at the end of preservation may improve the viability of marginal liver grafts and provides information on the quality of the organ. We attempt to define the limits to SCS in terms of easily measurable perfusion parameters and investigate whether MP can improve liver viability. Rat livers were cold-stored for 0, 24, 48, 72, and 120 h, after which they were treated with subnormothermic machine perfusion (SNMP). Livers cold-stored for 48 and 72 h were transplanted orthotopically with or without SNMP. During SNMP easily measurable parameters were monitored and adenosine triphosphate (ATP) content was measured following preservation and SNMP. ATP increased significantly during SNMP, but the recovered ATP content deteriorated with increased duration of SCS, with minimal improvement after 72 h of SCS. Vascular resistance during SNMP increased with extended preservation. After 48 h of SCS, orthotopic transplantation survival increased significantly from 50% to 100% with SNMP, but did not improve after 72 h. Vascular resistance and ATP recovery suggest a decrease in viability after 48 h of SCS. Survival data confirms the loss of post-transplant graft function and supports the use of ATP and vascular resistance as useful indicators. Further, we show that the recoverability of a liver using SNMP is limited to 48 h of SCS.

Ex Vivo Adenoviral Vector Gene Delivery Results in Decreased Vector-associated Inflammation Pre- and Post–lung Transplantation in the Pig

Acellular normothermic ex vivo lung perfusion (EVLP) is a novel method of donor lung preservation for transplantation. As cellular metabolism is preserved during perfusion, it represents a potential platform for effective gene transduction in donor lungs. We hypothesized that vector-associated inflammation would be reduced during ex vivo delivery due to isolation from the host immune system response. We compared ex vivo with in vivo intratracheal delivery of an E1-, E3-deleted adenoviral vector encoding either green fluorescent protein (GFP) or interleukin-10 (IL-10) to porcine lungs. Twelve hours after delivery, the lung was transplanted and the post-transplant function assessed. We identified significant transgene expression by 12 hours in both in vivo and ex vivo delivered groups. Lung function remained excellent in all ex vivo groups after viral vector delivery; however, as expected, lung function decreased in the in vivo delivered adenovirus vector encoding GFP (AdGFP) group with corresponding increases in IL-1β levels. Transplanted lung function was excellent in the ex vivo transduced lungs and inferior lung function was seen in the in vivo group after transplantation. In summary, ex vivo delivery of adenoviral gene therapy to the donor lung is superior to in vivo delivery in that it leads to less vector-associated inflammation and provides superior post-transplant lung function.

Use of Diffusion Tensor Imaging to Predict Myocardial Viability After Warm Global Ischemia: Possible Avenue for Use of Non-beating Donor Hearts

The assessment of myocardial viability after global warm ischemia (WI) but before reperfusion is challenging. We hypothesized that fractional anisotropy (FA), a magnetic resonance imaging (MRI) parameter of water diffusion that characterizes cellular integrity within tissues, provides a rapid and useful method for evaluating the viability of hearts after WI. Dog hearts were exposed to 60 minutes of WI after exanguination, explanted and preserved in a cold, non-beating state for 6 hours, using continuous perfusion (CP) or static cold storage (CS). Toward the end of preservation, a global FA assessment, acquired using MRI, was compared with analyses obtained from myocardial biopsies that included adenosine triphosphate (ATP), endothelin-1 (ET-1) and caspase-3 levels, light microscopy and tetrazolium staining. Functional recovery was analyzed after restoration of blood flow on a non-working Langendorff preparation. FA measured at the end of CP showed strong correlations with all parameters of functional recovery (developed pressure, R = 0.60; dP/dt, R = 0.96; -dP/dt, R = 0.96). Although FA also correlated with tissue levels of ATP, ET-1 and caspase-3 (R = 0.77, -0.84, -0.64), recovery of myocardial function did not correlate with these markers or any other conventional analyses of myocardial injury (troponin I, changes on light microscopy or tetrazolium staining). FA, an MRI-based parameter that indicates cellular integrity, was found to reflect better myocardial ATP stores, less induction of ET-1 and caspase-3 and improved functional recovery of hearts after global WI. As a clinically applicable tool capable of rapidly differentiating reversible from lethal injury, diffusion tensor imaging may prove useful in the eventual adoption of non-beating donor hearts for transplantation.

Effect of hyperbaric oxygen on cold storage of the liver in rats

The depletion of biochemical energy stores during prolonged cold storage is one of the most critical events of cold ischemia-reperfusion (CI/R) injury. The aim of this study was to evaluate the effect of hyperbaric oxygen (HBO) treatment on CI/R injury. Livers were harvested from male Wistar rats and stored for 24 h at 4 degrees C in University of Wisconsin solution (Group 1). Others were additionally treated with HBO during the preservation period (Group 2). At the end of the 24 h cold preservation, the concentrations of hepatic enzymes and lipid peroxidation (LPO) in the effluent and the hepatic adenosine triphosphate (ATP) levels were measured. After preservation, the livers were reperfused for 90 min with an oxygenated Krebs-Henseleit bicarbonate buffer. Perfusate samples were obtained serially, and portal flow rates were also recorded. In group 2, aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and LPO into the effluent at the end of preservation were decreased and the depletion of ATP was prevented (P<0.05). After reperfusion, the portal flow was significantly improved in group 2 (P<0.05). The time-dependent increase of alanine aminotransferase levels (ALT) observed in group 1 was suppressed significantly in group 2, and total bile production during 90 min of reperfusion was significantly greater in group 2 (P<0.05). The structure of the livers in group 2 was significantly well maintained, and the liver weight change ratio was significantly greater in group 1 (P<0.05). HBO treatment during cold storage seems to prevent hepatic ischemic injury and have protective effects against CI/R injury by attenuating the depletion of energy stores.

HEPATOCYTE GROWTH FACTOR IMPROVES SURVIVAL OF STEATOTIC LIVER TRANSPLANTATION IN RATS

P71 Background/Aims; Transplantation of liver with severe steatosis may result in primary nonfunction (PNF) of the graft. As described previously, human recombinant hepatocyte growth factor (hrHGF) protected fatty grafts against cold preservation injury in rats. In the present study, we examined the effects of hrHGF on survival and graft function in rat fatty liver transplantation model. Methods; Severe steatosis was induced by a 15-day choline-deficient diet in male Lewis rats. The fatty graft was orthotopically transplanted in normal male Lewis rat after 12 hr cold preservation in UW solution. In the HGF group, donor rats were pretreated with a single bolus of systemic injection of hrHGF (10μg/rat), and 30μg of hrHGF was also added to the perfusion/preservation solution. Animal survival, graft function and morphological changes were compared with those of the untreated group. Liver cell apoptosis was detected by TUNEL stain and liver regeneration was evaluated by BrdU stain at 2 hrs after reperfusion of the liver graft. Results; One week survival was significantly improved in the HGF group compared with the untreated group (83% vs. 33%, p<0.01, n=18/group). AST, ALT and LDH levels in the rinse effluent at the end of cold preservation and serum ALT and LDH at 2 hrs after reperfusion were significantly lower in the HGF group. Although there was no significant difference in hyaluronic acid (HA) level in the rinse effluent at the end of preservation between the two groups, exogenous HA uptake rate (HAUR) at 2 hrs after reperfusion was improved in the HGF group (0.62±0.05 vs. 0.79±0.11, p<0.05). hrHGF pretreatment also significantly reduced the sinusoidal endothelial cell (SEC) apoptotic index (1.65±0.60% vs. 4.90±1.25%, p<0.01) and increased BrdU incorporation index (2.07±0.62% vs. 0.06±0.04%, p<0.01) at 2 hrs after reperfusion. More severe sinusoidal SEC damage was observed by transmission electron microscope in the untreated group than that in HGF group. Conclusions; hrHGF prevented the death of PNF in steatotic liver transplantation by attenuating preservation–reperfusion injury and facilitating recovery of the graft, probably through its antiapoptotic properties and acceleration of liver regeneration at early stage after transplantation.

The effects of preconditioning on the oxidative stress in small-bowel autotransplantation

Background. One determining factor in intestinal transplantation is the extreme sensitivity of the small bowel to ischemia-reperfusion injury. This study investigated the effect of ischemic preconditioning prior to autotransplantation. Methods. Total orthotopic intestinal autotransplantation was performed in 40 mongrel dogs. In 4 groups (GI-GIV), grafts were stored for 3 hours in cold Euro Collins (GI,GIII) and University of Wisconsin (GII,GIV) solutions. In GIII and GIV, before preservation, preconditioning was induced by 4 cycles (5-min ischemia + 10-min reperfusion). Bowel samples were collected after laparotomy (control), at the end of preservation and reperfusion periods. We determined oxidative stress markers (reduced glutathione [GSH], superoxide dismutase [SOD]), production of oxygen free radicals, activity of nuclear factor-κB (NF-κB), and DNA damage. Results. In the non-preconditioned groups, GSH concentration increased slightly, while SOD activity decreased significantly during reperfusion. In the preconditioned groups, GSH increased markedly, and better preservation of SOD was observed. The number of oxygen free radicals increased during reperfusion mainly in non-preconditioned groups. Activation of NF-κB peaked by 1 hour, and decreased 3 hours after preconditioning. We observed DNA-damaged cells in all groups. Conclusions. Our findings confirm that preconditioning prior to preservation can moderate the severity of oxidative stress and activate the endogenous cellular adaptation in bowel tissue. (Surgery 2002;132:877-84.)

MUCOSAL DAMAGE AND RECOVERY OF THE INTESTINE AFTER PROLONGED PRESERVATION AND TRANSPLANTATION IN DOGS1

Although much is known about the mucosal damage that occurs after intestinal warm ischemia and reperfusion and its recovery, little is known about the effect of cold preservation and transplantation on the mucosa. We studied the electrophysiological, biochemical, and histological changes of the intestinal mucosa after preservation for 24 hr and subsequent transplantation. The small intestines from adult mongrel dogs were harvested. The intestines were orthotopically autotransplanted immediately (control group) or after preservation for 24 hr (preservation group). Jejunal and ileal tissues were taken before harvesting, at the end of preservation, 1 hr after reperfusion, and on postoperative days 3, 7, 14, and 28. The Ussing chamber method was used to study the electrophysiologic changes. Tissue maltase, diamine oxidase, and ornithine decarboxylase were measured. A histological analysis was also performed. Control group grafts showed no evident deterioration in electrophysiology, biochemistry, or morphology. In contrast, preservation group grafts exhibited electrophysiological and biochemical degradation, complete denudation of the villi, and crypt injury (especially in the ileum) after reperfusion. Electrophysiologic function and the mucosa biochemical marker recovered within 3 days in the jejunum and within 7-14 days in the ileum; however, histological recovery of mucosal injury required 28 days in the jejunum and more than 28 days in the ileum. Our study showed that despite severe destruction of mucosal integrity by prolonged preservation and transplantation, the intestinal mucosa has an enormous regenerative capacity. Our study also showed that regeneration was more pronounced in the jejunum than in the ileum.

Fludarabine and cyclophosphamide with filgrastim support in patients with previously untreated indolent lymphoid malignancies

To evaluate the response rate and potential toxicities, a phase II trial was conducted of fludarabine and cyclophosphamide with filgrastim support in patients with previously untreated low-grade and select intermediate-grade lymphoid malignancies. Symptomatic patients with preserved end organ function received cyclophosphamide 600 mg/m(2) intravenous (iv) day 1 and fludarabine 20 mg/m(2) iv days1 through 5, followed by filgrastim 5 microg/kg subcutaneous starting approximately day 8. Treatment was repeated every 28 days until maximum response or a maximum of 6 cycles. Sixty patients, median age 53.5 years, were enrolled. Thirty-seven patients with non-Hodgkin lymphoma (NHL) were stage IV and 6 were stage III. Eleven of 17 patients with chronic lymphocytic leukemia (CLL) were Rai intermediate risk and 6 were high risk. The overall complete response (CR) rate was 51% and the partial response (PR) rate was 41%. Of patients with CLL, 47% achieved a CR and the remaining 53% achieved a PR. Of patients with follicular lymphoma, 60% achieved CR and 32% achieved a PR. Although the toxicity of this regimen was mainly hematologic, significant nonhematologic toxicities, including infections, were seen. Twenty-four patients subsequently received an autologous or allogeneic stem cell transplant. Engraftment was rapid, and there were no noticeable procedure toxicities in the immediate posttransplant period attributable to the fludarabine and cyclophosphamide regimen. Fludarabine, cyclophosphamide, and filgrastim make up a highly active and well-tolerated regimen in CLL and NHL.

Alteration in the control of mitochondrial respiration by outer mitochondrial membrane and creatine during heart preservation.

This study aimed to evaluate the nature and extent of mitochondrial alterations during heart preservation. Rat hearts, isolated after cardioplegia in situ, were preserved for 6 or 15 h at 4 degrees C either by immersion in cardioplegic solution or by low-flow perfusion (0.3 ml/min) with cardioplegic solution. The energy state of hearts at the end of preservation was determined by 31P-NMR spectroscopy and functional recovery was evaluated on reperfusion. Variables of mitochondrial respiration (maximal rate of respiration in the presence of ADP = delta Vmax, apparent Km for ADP, effect of creatine) were evaluated on skinned fibers and compared with those determined in controls and in hearts subjected to 1 hour of ischemia at 37 degrees C. Serious mitochondrial alterations were detected in fibers from 15 h immersed hearts: decrease of delta Vmax and of apparent Km for ADP, loss of the stimulatory effect of creatine, and disruption of the outer mitochondrial membrane. The extent of alternations was more accentuated in fibers from normothermic ischemic hearts, in which some damage of the inner mitochondrial membrane also occurred. In fibers from hearts preserved for 6 h, no significant changes in mitochondrial variables could be detected. When the hearts were preserved under low-flow perfusion for 15 h, only the stimulatory effect of creatine on respiration was significantly decreased. The extent of the loss of the stimulatory effect of creatine paralleled the accumulation of inorganic phosphate (Pi) during preservation and the decrease in left ventricular function on reperfusion. Alterations related to the outer membrane and the intermembrane space are among the earliest signs of damage to mitochondrial function during heart preservation. These alterations could be attributed to the swelling of mitochondria under the effect of Pi. The determination of mitochondrial parameters in biopsy samples could allow a simple and rapid evaluation of energy-producing and transfer capacities of the myocardium.

Extending the margin of safety of preservation period for resuscitation of ischemically damaged pancreas during preservation using the two-layer (University of Wisconsin solution/perfluorochemical) method at 20 degrees C with thromboxane A2 synthesis inhibitor OKY046.

We have shown that 5-hr preservation using the two-layer (University of Wisconsin solution/perfluorochemical) method at 20 degrees C allows ATP synthesis and makes it possible to resuscitate a canine pancreas subjected to 90 min of warm ischemia. However, 8 hr of preservation using this method caused a disturbance of vascular microcirculation and did not resuscitate the grafts. The aim of this study was to examine the effect of thromboxane A2 synthesis inhibitor OKY046 on vascular endothelial cells and ATP tissue levels of canine pancreas during preservation using the two-layer (University of Wisconsin solution/perfluorochemical) method at 20 degrees C, and vascular microcirculation and pancreas viability after transplantation. Graft viability was judged by graft survival following autotransplantation. ATP tissue levels were measured by high-performance liquid chromatography at the end of preservation. Viability of the vascular endothelial cells was judged using nuclear trypan blue uptake of the graft after preservation. Pancreatic tissue perfusion was measured using an H2 clearance technique after reperfusion. Pancreas grafts subjected to 90 min of warm ischemia were not viable (0/5). However, 5-hr preservation made it possible to recover the pancreas (5/5); 8-hr preservation was not successful (0/3). ATP tissue levels after 5-hr and 8-hr preservation were 9.40+/-2.09 and 7.37+/-1.06 micromol/g dry weight, respectively, and OKY046 did not affect ATP synthesis during 8-hr preservation (8.44+/-0.92 micromol/g dry weight). The percentage of nuclear trypan blue uptake of endothelial cells in 8-hr-preserved grafts was 37.6+/-11.6% and was significantly higher than the value in 5-hr-preserved grafts (5.0+/-3.0%; P<0.01). However, OKY046 significantly reduced trypan blue uptake in 8-hr-preserved grafts (8.2+/-3.6%; P<0.01). Pancreatic tissue perfusion in 8-hr-preserved grafts after 2 hr of reperfusion was 28.5+/-7.5 ml/min/100 g, and was significantly lower than the value in 5-hr-preserved grafts (57.1+/-4.4 ml/ min/100 g; P<0.01), but OKY046 dramatically improved pancreatic tissue perfusion (97.1+/-14.6 ml/min/100 g; P<0.01). As a consequence, 8-hr-preserved grafts were resuscitated (4/5). We conclude that OKY046 protects the vascular endothelium during preservation by the two-layer method at 20 degrees C and consequently improves vascular microcirculation on reperfusion. Together with ATP synthesis, which is essential for repairing damaged cells, the canine pancreas graft subjected to 90 min of warm ischemia is resuscitated during 8-hr preservation by the two-layer method at 20 degrees C. This method holds promise for pancreas-kidney transplantation from cardiac arrest donors.