Transplantation Proceedings

Editorial Board

2010-07-01

Similar Early Complication Rate in Simultaneous Pancreas and Kidney Recipients on Tacrolimus/Mycophenolate Mofetil Versus Tacrolimus/Sirolimus Immunosuppressive Regimens

2010-07-01

Abstract: Introduction: We compared the incidence of severe complications among 123 consecutive simultaneous pancreas and kidney (SPK) recipients randomized for treatment either with tacrolimus plus mycophenolate mofetil (MMF) or tacrolimus plus sirolimus during their initial postoperative hospital stay.Methods: Patients with type 1 diabetes mellitus (T1DM) and renal failure with no age limit who underwent SPK were randomly assigned to tacrolimus/sirolimus or tacrolimus/MMF immunosuppressive protocols. We analyzed the rate of adverse events that led to death, graft loss, operative revision, or prolonged hospital stay.Results: From 2002 to 2009, 62 recipients were included in the MMF and 61 in the Rapamycin (Rapa) groups. More than 2/3 of recipients suffered from at least 1 complication: 74% MMF and 77 % Rapa group (P > .05). No patient died in the MMF and 3 in the Rapa group (P = .11). Pancreas graftectomy was performed in 13% of the MMF group and in 5% of the Rapa group (P = .20). Ten of 62 recipients in the MMF and 13/61 in the Rapa group required operative treatment of wound infections (P = .49). There were no differences in the rates of gastrointestinal bleeding (11% and 8%), kidney lymphocele (6% and 5%), ileus (1.6% both), pancreatic leak (1.6% both), or ureteral leak (0 and 3%) between the groups.Conclusion: We did not observe a difference in the rate of severe postoperative complications between groups. With the use of extraperitoneal placement of the pancreatic graft, fluid collections and wound infections remain the most frequent albeit curable postoperative complications.

Anti-CD25 Antibody (Daclizumab) Maintenance Therapy in Pancreas Transplantation

2010-07-01

Abstract: Background: Calcineurin inhibitors (CNI) are the basis of contemporary immunosuppression in clinical pancreas transplantation (PT). Nevertheless, CNI toxicities, especially nephrotoxicity, have stimulated the search for CNI-sparing protocols. We performed a retrospective analysis of 25 PT patients with progressive CNI toxicities that were switched to a daclizumab (DAC)–based maintenance regimen.Methods: From 2003 to 2007, 25 PT patients with progressive CNI toxicity (predominantly nephrotoxicity) were identified and switched from CNI to monthly DAC maintenance therapy. The DAC group was compared with matched control subjects (1:1) by transplant type and number, age, year of transplant, and duct management.Results and conclusions: Results showed improved graft survival rates and decreased immunologic loss rates at 1, 3, and 5 years in the DAC group compared with the control group. There was no difference in patient survival rate between the 2 groups. Analysis demonstrates that DAC maintenance therapy is safe and effective for PT patients experiencing CNI toxicities. A randomized trial to compare DAC- and CNI-based regimens is needed in CNI-intolerant patients, with particular attention to the impact on renal function and patient morbidity (eg, infection rates).

Alemtuzumab with Rapid Steroid Taper in Simultaneous Kidney and Pancreas Transplantation: Comparison to Induction with Antithymocyte Globulin

2010-07-01

Abstract: We compared our experience with alemtuzumab induction and rapid steroid taper (RST) in simultaneous kidney and pancreas transplantation (SKPT) with a historic control group who received rabbit antithymocyte globulin (r-ATG) induction with RST. 74 SKPTs performed at our center between January 2005 to November 2008 who underwent immunosuppression with RST in combination with r-ATG induction (n = 33; 1.5 mg/kg × 4 for a total dose of 6 mg/kg) or alemtuzumab induction (n = 41; 30 mg single dose). Maintenance immunosuppression consisted of tacrolimus and mycophenolate mofetil. Steroids were discontinued after postoperative day 4. Recipient and transplant characteristics were similar between the 2 groups, with 82% of the r-ATG and 80% of the alemtuzumab group steroid free at 1 year. The rate of clinical acute rejection episodes was 12% in the r-ATG group and 15% in the alemtuzumab group. The rates of cytomegalovirus (CMV) infection, BK nephropathy, and graft survival were similar between the 2 groups. There was no difference in mean serum creatinine, calculated GFR, or fasting blood sugar at 1 year between the 2 groups, whereas glycosylated hemoglobin (HbA1c) was lower at 1 year in the alemtuzumab (5.3 ± 0.4) versus the r-ATG group (5.6 ± 0.4; P = .0021). Induction with r-ATG or alemtuzumab with RST was safe and effective in SKPT. The incidences of acute rejection episodes, CMV infection, and BK nephropathy were similar. Mean HbA1C at 1 year was lower among the alemtuzumab group. Further long-term follow-up is needed to confirm these results.

Simultaneous Pancreas and Kidney Transplantation With Concurrent Allograft Nephrectomy for Recipients With Prior Renal Transplants Lost to BK Virus Nephropathy: Two Case Reports

S. Kubal, J.A. Powelson, T.E. Taber, M.L. Goble, J.A. Fridell — 2010-07-01

Abstract: Candidacy for retransplantation after allograft loss due to BK virus–associated nephropathy (BKVN) with or without allograft nephrectomy is controversial. This report describes 2 renal transplant recipients who lost their grafts to BKVN and subsequently underwent simultaneous kidney and pancreas transplantation with allograft nephrectomy.

Pancreas Oxygen Persufflation Increases ATP Levels as Shown by Nuclear Magnetic Resonance

2010-07-01

Abstract: Background: Islet transplantation is a promising treatment for type 1 diabetes. Due to a shortage of suitable human pancreata, high cost, and the large dose of islets presently required for long-term diabetes reversal; it is important to maximize viable islet yield. Traditional methods of pancreas preservation have been identified as suboptimal due to insufficient oxygenation. Enhanced oxygen delivery is a key area of improvement. In this paper, we explored improved oxygen delivery by persufflation (PSF), ie, vascular gas perfusion.Methods: Human pancreata were obtained from brain-dead donors. Porcine pancreata were procured by en bloc viscerectomy from heparinized donation after cardiac death donors and were either preserved by either two-layer method (TLM) or PSF. Following procurement, organs were transported to a 1.5-T magnetic resonance (MR) system for 31P nuclear magnetic resonance spectroscopy to investigate their bioenergetic status by measuring the ratio of adenosine triphosphate to inorganic phosphate (ATP:Pi) and for assessing PSF homogeneity by MRI.Results: Human and porcine pancreata can be effectively preserved by PSF. MRI showed that pancreatic tissue was homogeneously filled with gas. TLM can effectively raise ATP:Pi levels in rat pancreata but not in larger porcine pancreata. ATP:Pi levels were almost undetectable in porcine organs preserved with TLM. When human or porcine organs were preserved by PSF, ATP:Pi was elevated to levels similar to those observed in rat pancreata.Conclusion: The methods developed for human and porcine pancreas PSF homogeneously deliver oxygen throughout the organ. This elevates ATP levels during preservation and may improve islet isolation outcomes while enabling the use of marginal donors, thus expanding the usable donor pool.

Persufflation Improves Pancreas Preservation When Compared With the Two-Layer Method

2010-07-01

Abstract: Islet transplantation is emerging as a promising treatment for patients with type 1 diabetes. It is important to maximize viable islet yield for each organ due to scarcity of suitable human donor pancreata, high cost, and the large dose of islets required for insulin independence. However, organ transport for 8 hours using the two-layer method (TLM) frequently results in low islet yields. Since efficient oxygenation of the core of larger organs (eg, pig, human) in TLM has recently come under question, we investigated oxygen persufflation as an alternative way to supply the pancreas with oxygen during preservation. Porcine pancreata were procured from donors after cardiac death and preserved by either TLM or persufflation for 24 hours and subsequently fixed. Biopsies collected from several regions of the pancreas were sectioned, stained with hematoxylin and eosin, and evaluated by a histologist. Persufflated tissues exhibited distended capillaries and significantly less autolysis/cell death relative to regions not exposed to persufflation or to tissues preserved with TLM. The histology presented here suggests that after 24 hours of preservation, persufflation dramatically improves tissue health when compared with TLM. These results indicate the potential for persufflation to improve viable islet yields and extend the duration of preservation, allowing more donor organs to be utilized.

Continuous Real-time Viability Assessment of Kidneys Based on Oxygen Consumption

2010-07-01

Abstract: Background: Current ex vivo quality assessment of donor kidneys is limited to vascular resistance measurements and histological analysis. New techniques for the assessment of organ quality before transplantation may further improve clinical outcomes while expanding the depleted deceased-donor pool. We propose the measurement of whole organ oxygen consumption rate (WOOCR) as a method to assess the quality of kidneys in real time before transplantation.Methods: Five porcine kidneys were procured using a donation after cardiac death (DCD) model. The renal artery and renal vein were cannulated and the kidney connected to a custom-made hypothermic machine perfusion (HMP) system equipped with an inline oxygenator and fiber-optic oxygen sensors. Kidneys were perfused at 8°C, and the perfusion parameters and partial oxygen pressures (pO2) were measured to calculate WOOCR.Results: Without an inline oxygenator, the pO2 of the perfusion solution at the arterial inlet and venous outlet diminished to near 0 within minutes. However, once adequate oxygenation was provided, a significant pO2 difference was observed and used to calculate the WOOCR. The WOOCR was consistently measured from presumably healthy kidneys, and results suggest that it can be used to differentiate between healthy and purposely damaged organs.Conclusions: Custom-made HMP systems equipped with an oxygenator and inline oxygen sensors can be applied for WOOCR measurements. We suggest that WOOCR is a promising approach for the real-time quality assessment of kidneys and other organs during preservation before transplantation.

Improved Method of Human Islet Isolation for Young Donors

2010-07-01

Abstract: Background: Although islet transplantation using young donors is more effective than older donors, islet isolation from young donor is notoriously difficult. This may relate to islet ontogeny and collagen composition in the young pancreas. Therefore, we examined whether a high concentration of collagenase could improve the separation of islets from exocrine tissues resulting in an high islet yield.Methods: We used six human pancreata from brain-dead donors of less than 30 years old. Islet isolation was performed based on the Edmonton protocol with modifications. All pancreata were digested with Collagenase NB1 Premium Grade (Serva). The pancreas was expanded by injecting either 200 mL of cold collagenase solution (2.5 mg/mL, standard group, n = 3) or 100 mL of solution (5 mg/mL, new group, n = 3) in a controlled manner under low pressure for 5 minutes. Then the pressure was raised for another 5 minutes. The following procedure and evaluation were performed based on the Edmonton protocol.Results: Phase II time in the new group was significantly shorter than the standard group. The ratio of embedded islets in the new group was significantly lower than the standard group. The postpurification islet equivalents per pancreas weight (IEQ/g) and the recovery rate in the new group were higher than the standard group, but not significantly. There was no significant difference in the postpurification purity, viability, and final tissue volume.Conclusion: Our simple modification with an initially concentrated collagenase preparation using a syringe significantly improved the ratio of embedded islets, resulting in a higher yield from young donors.

Islet Isolation From Human Pancreas With Extended Cold Ischemia Time

W.M. Kühtreiber, L.T. Ho, A. Kamireddy, J.A.W. Yacoub, D.W. Scharp — 2010-07-01

Abstract: The general consensus among transplant centers is that a cold ischemia time (CIT) beyond 8 hours results in reduced yields and quality of human islets. We sought to optimize the isolation process and enzymes for pancreata with extended CIT. We processed 16 extended CIT pancreata (13.2 ± 0.7 hours). Donors averaged 50.8 ± 2.6 (standard error of the mean) years old with a body mass index of 28.6 ± 1.5. Glands were shipped in cold organ preservation solution without oxygenated perfluorocarbon. Isolations were performed under a protocol optimized for digestion with the new cGMP collagenase from Roche. Purification used continuous Euroficoll/University of Wisconsin gradients. Islets were cultured in two types of Prodo cGMP islet culture media and/or in Miami 1A media. Glucose-stimulated insulin secretion assays were performed after 8 to 16 days of culture. Prepurification yield averaged 415 ± 41 KIEQ postpurification, 359 ± 29 KIEQ (purification loss 13.5%); and postculture 317 ± 27 KIEQ (culture loss 11.7%). Our process liberated an average of 4278 IEQ/g of pancreas (97 ± 5 g). Most islets were recovered in the purest fraction (purity 79.7% ± 1.9%). Culture loss in our enhanced culture media was 11.7%. After 2 to 3 days in culture, viability was 92% ± 1%. Islets exhibited compactness and dithizone staining. Glucose-stimulated insulin secretion assays performed after 3 to 23 days in our PIM(R) media resulted in a stimulation index of 6.8 ± 1.7 (G50 to G350). We concluded that our human islet isolation process permitted the recovery of large numbers of high-quality human islets from extended CIT pancreata and that our cGMP islet culture media was superior to the current standard CMRL-based media.

Improved Method of Porcine Pancreas Procurement With Arterial Flush and Ductal Injection Enhances Islet Isolation Outcome

2010-07-01

Abstract: Background: Several pancreas procurement procedures have been used for porcine islet isolation; however, their impact on outcomes has not been extensively studied. We evaluated an advanced procurement technique for porcine islet isolation designed to reduce warm ischemia, to remove blood content, and enhance cooling of the pancreas by implementing a vascular flush and ductal preservation.Method: Pancreata procured from adult Landrace pigs were divided into 3 different surgical protocols: Pancreatectomy utilizing only surface cooling (group 1; n = 24); surface cooling and ductal injection with cold preservation solution before pancreatectomy (group 2; n = 12); or surface cooling, ductal injection, and an approach by selectively flushing through the celiac trunk and the superior mesenteric artery (group 3; n = 14). We assessed the islet isolation results and quality using in vitro and in vivo assays.Results: Significantly higher overall yield and islet yield per gram pancreas were obtained from group 3 pigs compared with the other groups. Measurements of islet viability after 7 days of culture, as assessed by oxygen consumption rate per DNA, showed that group 3 islets displayed the highest values. Sustained normoglycemia was observed in diabetic nude mice transplanted with 2000 islet equivalents from all 3 groups.Discussion: This study demonstrated that an advanced pancreas procurement technique including ductal preservation and selective arterial flush with cold preservation solution provided significant improvements in porcine islet isolation outcomes.

Rapid Quantitative Assessment of the Pig Pancreas Biopsy Predicts Islet Yield

2010-07-01

Abstract: Background: The cost of islet procurement from donor pigs is increased by the use of organs that produce low yields. We developed an assessment system using dithizone-stained pig pancreas biopsies to enable the preselection of donor organs.Methods: Pig pancreas biopsy slices were soaked in dithizone solution. The islets were evaluated before islet isolation by converting the islet counts (IC) to islet equivalents (IE), and then determining the IE/cm2, IE/IC, % islets >150 μm, and % islets >200 μm. These parameters were evaluated in 3 different areas of the pancreas (duodenal, splenic, and connecting lobe; n = 42 each). Stepwise multivariate linear regression analysis was performed to assess for correlations with islet yield and decide which area of the pancreas had the most predictive value. To identify other predictors, including donor and islet isolation variables, we performed binary logistic regression analysis with significant variables from the univariate analysis (n = 67). For this analysis, the pigs were categorized into high (n = 23) and low (n = 44) yield groups.Results: Stepwise multivariate linear regression analysis revealed that IE/cm2 of the splenic lobe significantly predicted islet yield. Binary logistic regression analysis indicated that the IE/mm2 of the splenic lobe was the only parameter that significantly correlated with successful pig islet isolations (P = .01; odds ratio 3.605). Variables associated with donor and islet isolation, such as age, gender, ischemic time, or enzyme lot, were not significantly correlated with islet yield.Conclusion: Our study suggests that the islet distribution of splenic lobe biopsies can be a reliable predictor of islet yield from pig pancreata.

The Impact of Ischemic Stress on the Quality of Isolated Pancreatic Islets

2010-07-01

Abstract: Background: Although the ischemic stress of donated organs has been shown to have strong negative effects on islet recovery, the impact on islet quality remains uncertain. In the present study, therefore, we examined the influence of ischemic stress on the expression of inflammatory mediators among isolated islets.Materials and methods: Islets were isolated from adult porcine pancreata subjected to 16-hour cold ischemia time (CIT) in addition to 40-minute warm ischemia time (WIT). We evaluated the islet yield, islet loss during the first 24 hours in culture, adenosine diphosphate (ADP)/adenosine triphosphate (ATP) ratio, ATP/DNA ratio, glucose-stimulated respiratory activity, in vivo bioassay, and the expression of inflammatory mediators (tissue factor [TF], [MCP-1], macrophage migration inhibitory factor) on the isolated islets. We also analyzed ATP/DNA ratios of the exocrine tissues during isolation procedures.Results: The islet yield, survival rate during culture, and glucose-stimulated respiratory activity were significantly lower in cases of 16-hour CIT plus 40-minute WIT compared with the control group (P < .0001, .0006, and .002, respectively). In contrast, ADP/ATP ratio as well as TF and MCP-1 expressions on the isolated islets were higher among the ischemic group (P = .005, .16, and .005, respectively). During isolation procedures, the ATP/DNA of the exocrine tissues was extremely lower in the ischemic compared to the control group (P < .0001). Notably, however, both ATP/DNA and ADP/ATP ratio of isolated islets were well preserved even in the ischemic group (P = .45 and .40).Discussion: These data suggest that ischemic stress during the preservation period negatively affects the energy status of exocrine tissues. Destruction of the exocrine tissues, in combination with warm ischemic stress during the isolation procedures, subsequently decreases isolated islet activity, inducing the expression of inflammatory mediators.

A New Method to Value Efficiency of Enzyme Blends for Pancreatic Tissue Digestion

2010-07-01

Abstract: Islet transplantation, since the 1990s, has been an example of human cell therapy. Nevertheless, the islet isolation procedure is not completely standardized; in fact, >50% of islet procedures do not eventuate in transplantation due both to the variability of a donor's pancreas and to the unpredictable efficiency of an enzymatic blend. The enzymes used in pancreas isolation to digest several substrates are extracted from Clostridium histolyticum. In particular, they have strong collagenolytic activity compared with vertebrate collagenases. However, several impediments persist in human islet isolation success, probably owing to the variable composition and concentration of collagenases employed during the digestion phase. For islet isolation processes, neutral proteases play important roles. However, they should be considered to be double-edged swords, contributing to tissue dissociation but, sometimes, decreasing islet yield through fragmentation, breakdown, and inactivation. Protease activities cannot be preciously adjusted in a narrow range, there is no approach to determine the optimal dosage and composition of enzymes for extraction of human islets from the pancreas. At this time, available data on commercial enzymatic activity are not sufficient to predict their efficiency for pancreas digestion; consequently, it is difficult to select enzyme batches. For these reasons, we sought to generate an innovative evaluation assay to select enzymes useful for isolation procedures of pancreatic islets.

Assessment of Human Islet Isolation With Four Different Collagenases

2010-07-01

Abstract: Background: The isolation of islets from the human pancreas critically depends on the efficiency of the digestive enzymes. Liberase HI had been used as a standard preparation until the issues concerning bovine spongiform encephalopathy. Thus, we must now use other collagenases for clinical islet transplantation, four of which we have evaluated herein.Methods: The digestion of each of 17 pancreata from brain-dead donors was performed using the following collagenases: Liberase HI (HI; Roche, n = 9); Liberase MTF C/T (MTF; Roche, n = 4); Collagenase NB1 Premium Grade (NB1; Serva, n = 7); or Clzyme Collagenase HA (CI, VitaCyte, n = 4). Islet isolations were based on the Edmonton protocol for HI, whereas our modified islet isolation method was used for the three new enzymes (MTF, NB1, and CI).Results: There were no significant differences in donor age, body mass index, pancreas size, and cold ischemic time among the four groups. The phase I time in the NB1 group was significantly shorter than in the CI group (P = .0014). The prepurification IEQ/g in the HI group was significantly lower than the others (P = .0003 vs MTF, .0007 vs NB1, and .0009 vs CI, respectively). The postpurification IEQ/g in the MTF group was significantly higher than in the HI group (P = .006). The viability in the NB1 group was significantly greater than the HI group (P = .003).Conclusion: Three new enzymes (MTF, NB1, and CI) may enable us to obtain higher islet yields than with HI.

Tissue Dissociation Enzyme Neutral Protease Assessment

A.G. Breite, F.E. Dwulet, R.C. McCarthy — 2010-07-01

Abstract: Neutral proteases, essential components of purified tissue dissociation enzymes required for successful human islet isolation, show variable activities and effects of substrate on their activities. Initially we used a spectrophotometric endpoint assay with azocasein substrate to measure neutral protease activity. After critical review of the results, we observed these data to be inconsistent and not correlating expected differences in specific activities between thermolysin and Bacillus polymyxa proteases. This observation led to the development of a fluorescent microplate assay using fluorescein isothyocyanate–conjugated bovine serum albumin (FITC-BSA) as the substrate. This simpler, more flexible method offered a homogeneous, kinetic enzyme assay allowing determination of steady state reaction rates of sample replicates at various dilutions. The assay had a linear range of 4- to 8-fold and interassay coefficients of variation for B polymyxa protease and thermolysin of <9% and <15%, respectively, which were lower than those using the spectrophotometric endpoint assay, namely, 54% and 36%, respectively. This format allowed for incorporation of enzyme inhibitors, as illustrated by addition of sulfhydryl protease inhibitors, which, consistent with earlier reports, strongly indicated that the main contaminant in purified collagenase preparations was clostripain. Determination of the specific activities for several purified neutral proteases showed that the B polymyxa and Clostridium histolyticum proteases had approximately 40% and 15% specific activities, respectively, of those obtained with purified thermolysin, indicating the different characteristics of neutral protease enzymes for cell isolation procedures.

Culture of Impure Human Islet Fractions in the Presence of Alpha-1 Antitrypsin Prevents Insulin Cleavage and Improves Islet Recovery

2010-07-01

Abstract: Background: Exocrine tissue is commonly cotransplanted with islets in autografting and allotransplantation of impure preparations. Proteases and insulin are released by acinar cells and islets, respectively, during pretransplantation culture and also systemically after transplantation. We hypothesized that released proteases could cleave insulin molecules and that addition of alpha-1 antitrypsin (A1AT) to impure islet cultures would block this cleavage, improving islet recovery and function.Methods: Trypsin, chymotrypsin, and elastase (TCE) activity and insulin levels were measured in culture supernates of pure (n = 5) and impure (n = 5) islet fractions, which were isolated from deceased donors. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect insulin after incubation with proteases. We assessed the effects of A1AT supplementation (0.5 mg/mL; n = 4] on TCE activity, insulin levels, culture recovery, and islet quality. The ultrastructure of islets exposed to TCE versus control medium was examined using electron microscopy (EM).Results: Protease (TCE) activity in culture supernatants was indirectly proportional to the percentage purity of islets: pure, impure, or highly impure. Increasingly lower levels of insulin were detected in culture supernatants when higher protease activity levels were present. Insulin levels measured from supernatants of impure and highly impure islet preparations were 61 ± 23.7% and 34 ± 33% of that in pure preparations, respectively. Incubation with commercially available proteases (TCE) or exocrine acinar cell supernatant cleaved insulin molecules as assessed using SDS-PAGE. Addition of A1AT to impure islet preparations reduced protease activity and restored normal insulin levels as detected using enzyme-linked immunosorbent assay (ELISA) and SDS-PAGE of culture supernates. A1AT improved insulin levels to 98% ± 1.3% in impure and 78% ± 34.2% in highly impure fractions compared with pure islet fractions. A1AT supplementation improved postculture recovery of islets in impure preparations compared with nontreated controls (72% ± 9% vs 47% ± 15%). Islet viability as measured using membrane integrity assays was similar in both the control (98% ± 2%) and the A1AT-treated groups (99% ± 1%). EM results revealed a reduction or absence of secretory granules after exposure to proteases (TCE).Conclusion: Culture of impure human islet fractions in the presence of A1AT prevented insulin cleavage and improved islet recovery. A1AT supplementation of islet culture media, therefore, may increase the proportion of human islet products that meet release criteria for transplantation.

Inhibition of Inflammatory Cytokine-Induced Response in Human Islet Cells by Withaferin A

H. Peng, G. Olsen, Y. Tamura, H. Noguchi, S. Matsumoto, M.F. Levy, B. Naziruddin — 2010-07-01

Abstract: Background: After islet cell transplantation, a substantial mass of islets are lost owing to nonspecific inflammatory reactions. Cytokine exposure before or after transplantation can upregulate expression of proinflammatory genes via the nuclear factor-κB signaling pathway, eventually resulting in islet loss.Objective: To test the effects of a naturally occurring nuclear factor-κB inhibitor, withaferin A, on regulation of inflammatory genes in human islets.Methods: Human pancreatic islets were isolated using a modified Ricordi protocol. Purified islets were cultured for 2 days. The effect of withaferin A treatment on islet cell viability was examined using the fluorescein diacetate-propidium iodide dye exclusion test, and on function using a static glucose stimulation assay. Islet cells were treated with a cytokine mixture (50 U/mL of interleukin-1β, 1000 U/mL of tumor necrosis factor-α, and 1000 U/mL of interferon-γ) for 48 hours with or without withaferin A, 1 μg/mL. Treated islets were used for real-time polymerase chain reaction (PCR) array analysis for expression of inflammatory genes, and expression of other selected genes was analyzed using real-time PCR with single primers.Results: Glucose stimulation and viability assays demonstrated that withaferin A was not toxic to islet cells. Of 84 inflammation-related genes examined using real-time PCR array analysis, 9 were significantly upregulated by cytokine treatment compared with the control group. However, addition of withaferin A to the culture significantly inhibited expression of all genes.Conclusion: Withaferin A significantly inhibits the inflammatory response of islet cells with cytokine exposure.

Excellence of Suito Index for Assessing Clinical Outcome of Islet Transplantation

2010-07-01

Abstract: Background: Monitoring functional islet mass after transplantation is critical to follow patients. Previously we demonstrated that the average secretory unit of islet transplant objects (SUITO) index within 1 month was an excellent predictor of insulin-free status or reduction in insulin dose. In this study, we analyzed the usefulness of daily SUITO index to assess clinical outcomes.Methods: Five patients underwent islet transplantation, including 3 who received 2 transplantations and 2 who received a single graft. All 5 patients achieved insulin-free status with 3 remaining insulin free at the time of evaluation. We analyzed the daily relative insulin dose and SUITO index. The daily relative insulin dose was calculated as the total daily insulin dose/average pretransplant insulin dose. The SUITO index was calculated as [fasting C-peptide (ng/mL)]/[fasting blood glucose (mg/mL) − 63] × 1,500. The data analyzed based on time after islet transplantation were categorized as within or after 1 month.Results: Within 1 month after islet transplantation, there was no correlation between the daily relative insulin dose and the daily SUITO index (P = .068; R = −0.33). After 1 month, the daily relative insulin dose and the daily SUITO index were strongly correlated (P < .0001; R = −0.70). When the cutoff value of the SUITO index was decided at 26 for insulin-free status, the positive predictive value was 84.1% and negative predictive value 89.4%.Conclusion: SUITO index was an excellent index to assess clinical outcomes beyond 1 month after islet transplantation.

Secretory Unit of Islet Transplant Objects (SUITO) Index Can Predict Outcome of Intravenous Glucose Tolerance Test

2010-07-01

Abstract: Background: Simple monitoring of engrafted islet function is important for follow-up of recipients after islet transplantation. We previously developed a simple assessment tool for islet graft function; the secretory unit of islet transplant objects (SUITO) index. The aim of this study was to clarify the relationship between the SUITO index and the outcomes of intravenous glucose tolerance tests (IVGTT).Methods: Fifteen series of blood samples from 6 islet recipients were collected before 3, 5, 10, 20, and 30 minutes after injection of 0.5 g/kg 50% dextrose. The SUITO index was calculated using plasma C-peptide and glucose level at fasting baseline. Samples were divided into the following 3 groups; low-SUITO (SUITO index <10; n = 3); middle-SUITO (SUITO index ≥10 to <26; n = 4); and high-SUITO (SUITO index ≥26; n = 8).Results: A threshold SUITO index of 26 showed good sensitivity (85.7%) and specificity (75.0%) to predict a blood glucose level of >10 mmol/L at 30 minutes. Blood glucose levels in the low-SUITO group were significantly higher than among the other 2 groups at baseline and 10, 20 and 30 minutes (P < .05). Glucose-level areas under the receiver-operating characteristic curve during IVGTT in the low-SUITO group were also significantly larger than among the other 2 groups (P < .05).Conclusion: The SUITO index, using only a fasting blood sample, predicted IVGTT outcomes.

Pancreata From Pediatric Donors Restore Insulin Independence in Adult Insulin-Dependent Diabetes Mellitus Recipients

2010-07-01

Abstract: Context: The use of pediatric donors can increase the number of donors available for pancreas transplantation.Aim: The aim of this study was to verify if pancreas transplantation from pediatric donors is as effective as transplantation from adult donors to restore metabolic control in type 1 diabetic patients.Materials and Methods: From 2000 to April 2009 we performed 17 pancreas transplantations from pediatric donors: 9 simultaneous kidney-pancreas (SPK), 6 pancreas transplantation alone (PTA), and 2 pancreas after kidney (PAK). All subjects received whole organs with enteric diversion of exocrine secretions; 11 underwent systemic and 6 underwent portal venous graft drainage. The immunosuppressive therapy was as follows: prednisone, mycophenolate mofetil, anti-thymocyte globulin (ATG), and cyclosporine or tacrolimus. The pediatric donor population had a mean age of 15.3 years (range, 12–17), a mean weight of 60.1 kg (range, 42–75), and a mean body mass index (BMI) of 21 (range, 17.9–23.4).Results: After 9 years the overall patient survival rate was 94.12%, whereas the graft survival rate was 63.35%. Normal glucose and insulin levels were maintained either fasting or during oral glucose tolerance test (OGTT). The group of recipients of pediatric organs was compared with patients receiving organs from adult donors (n = 125); the mean glucose values were lower in the pediatric group, whereas insulin production was higher in the adult patients. Early venous thrombosis was 17.6% in the pediatric group and 20% in adult recipients (Fisher exact test, P = not significant [NS]).Conclusion: Pediatric donors restored insulin independence in adult diabetic recipients, representing a valid source of organs for pancreas transplantation.

Neurogenic Differentiation 1 Directs Differentiation of Cytokeratin 19–Positive Human Pancreatic Nonendocrine Cells into Insulin-Producing Cells

M. Shimoda, S. Chen, H. Noguchi, S. Matsumoto, P.A. Grayburn — 2010-07-01

Abstract: Background: It has been reported that the human pancreatic nonendocrine fraction, which remains after islet isolation, can be differentiated toward beta cells. However, the optimal method to accomplish this goal has not been established. In this study, we introduced the human neurogenic differentiation 1 (NeuroD1) gene into human nonendocrine pancreatic epithelial cells (NEPECs) and promoted insulin-producing cells in vitro.Methods: The human pancreatic nonislet fractions were obtained from brain-dead donors and cultured in suspension for 2-3 days followed by culture with G418 for 4 days. These cells (NEPECs) were then plated on dishes. The NEPECs spread into a cell monolayer within 7 days and all of the cells were cytokeratin-19 (CK19) positive. Seven days after plating, plasmids encoding human NeuroD1 gene under human CK19 promoter were transfected 3 times every other day (termed NEPEC+ND). Seven days after starting induction, these cells were characterized.Results: Seven days after starting the induction of human NeuroD1, NEPEC+ND strongly expressed NeuroD1 and insulin mRNA. The ratio of NeuroD1-positive cells in NEPEC+ND was significantly higher than in NEPEC. Human insulin-positive cells in NEPEC+ND were also significantly greater than in NEPEC. Human insulin and C-peptide levels in culture medium in NEPEC+ND were significantly higher than in NEPEC.Conclusions: These findings demonstrated that human NeuroD1 under control of the CK19 promoter can induce the differentiation of CK19-positive NEPECs into insulin-producing cells.

An Acidic pH and Activation of Phosphoinositide 3-Kinase Stimulate Differentiation of Pancreatic Progenitors Into Insulin-Producing Cells

T. Koblas, K. Zacharovova, Z. Berkova, P. Girman, F. Saudek — 2010-07-01

Abstract: Adult pancreatic nonendocrine cells represent a potential alternative source of insulin-producing tissue for the treatment of diabetes. Differentiation of these cells is regulated by various signaling pathways including the phosphoinositide 3-kinase (PI3K) pathway. Therefore, we evaluated the effect of PI3K on this process. Compared with untreated cells the differentiation of human nonendocrine pancreatic cells into insulin-producing elements was increased after treatment with IGF-1, EGF, and Exendin-4, growth factors known to be activators of the PI3K pathway (12.2 ± 3.2% vs 9.1 ± 3.2%). Treatment with PI3K pathway inhibitor wortmannin reduced the number of differentiated beta cells from 9.1 ± 3.2 to 0.7 ± 0.4%. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed that insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), and Exendin-4 significantly increased the expression of the transcription factor neurogenin-3, whereas the expressions of pancreatic and duodenal homeobox 1 (PDX-1), neurogenic differentiation 1 (NeuroD) were increased only among samples treated with ZnCl2 and not significantly affected by treatment with the tested growth factors. Successful differentiation of IGF-1, EGF-, and Exendin-4–treated cells into functional beta cells was confirmed by C-peptide secretion in response to 5 versus 20 mmol glucose stimulation (0.24 vs 0.91 pmol C-peptide/μg DNA). These results showed that activation of the PI3K signaling pathway might be used to stimulate the differentiation of nonendocrine pancreatic cells into insulin-producing elements.

Induction of Insulin-Producing Cells From Human Pancreatic Progenitor Cells

2010-07-01

Abstract: Introduction: We previously established a mouse pancreatic stem cell line without genetic manipulation. In this study, we sought to identify and isolate human pancreatic stem/progenitor cells. We also tested whether growth factors and protein transduction of pancreatic and duodenal homeobox factor-1 (PDX-1) and BETA2/NeuroD into human pancreatic stem/progenitor cells induced insulin or pancreas-related gene expressions.Materials and method: Human pancreata from brain-dead donors were used for islet isolation with the standard Ricordi technique modified by the Edmonton protocol. The cells from a duct-rich population were cultured in several media, based on those designed for mouse pancreatic or for human embryonic stem cells. To induce cell differentiation, cells were cultured for 2 weeks with exendin-4, nicotinamide, keratinocyte growth factor, PDX-1 protein, or BETA2/NeuroD protein.Results: The cells in serum-free media showed morphologies similar to a mouse pancreatic stem cell line, while the cells in the medium for human embryonic stem cells formed fibroblast-like morphologies. The nucleus/cytoplasm ratios of the cells in each culture medium decreased during the culture. The cells stopped dividing after 30 days, suggesting that they had entered senescence. The cells treated with induction medium differentiated into insulin-producing cells, expressing pancreas-related genes.Conclusion: Duplications of cells from a duct-rich population were limited. Induction therapy with several growth factors and transduction proteins might provide a potential new strategy for induction of transplantable insulin-producing cells.

Comparison of Fresh and Cultured Islets From Human and Porcine Pancreata

2010-07-01

Abstract: Introduction: For clinical islet transplantation, many centers have recently introduced of human islet cultures prior to transplantation. They provide flexibility to evaluate isolated islets and pretreat patients. However, isolated islets deteriorate rapidly in culture. In the present study, we compared fresh human and porcine islets with cultured islets for c-Jun NH2-terminal kinase (JNK) activity.Materials and methods: Islet isolations from human and porcine pancreata were performed using the standard Ricordi technique with a modified Edmonton protocol. Isolated islets cultured for 24 hours at 37°C with 5% CO2 in culture medium were evaluated for counts and JNK activity.Results: After 24 hours of culture, the percentages of surviving islets were 86.9% for human and 47.3% for porcine sources. JNK activity in isolated islets declined to a low baseline level after 24-hour culture.Conclusion: Both human and porcine islets deteriorated rapidly in 24-hour cultures, although the in vitro conditions did not induce JNK activation.

Effects of Pancreas Transplantation on Oxidative Stress in Pulmonary Tissue from Alloxan-Induced Diabetic Rats

C.T. Spadella, O.A.X. Suarez, A.N. Lucchesi, S.F.G. Marques, A.J.M. Cataneo — 2010-07-01

Abstract: Purpose: There is considerable evidence that cellular oxidative stress caused by hyperglycemia plays an important role in the genesis and evolution of chronic diabetic lesions. In this study, we determined the effectiveness of pancreas transplantation (PT) in preventing the imbalance caused by excessive production of reactive oxygen species over antioxidant defenses in lungs of rats rendered diabetic by alloxan injection.Methods: Sixty inbred male Lewis rats, weighing 250–280 g, were randomly assigned to 3 experimental groups: NC, 20 nondiabetic control rats; DC, 20 untreated diabetic control rats; and PT, 20 diabetic rats that received syngeneic PT from normal donor Lewis rats. Each group was further divided into 2 subgroups of 10 rats each which were killed after 4 and 12 weeks of follow-up. Plasma glucose, glycosylated hemoglobin, and insulin levels were determined in all rats. Lipid hydroperoxide (LPO) concentrations and enzyme activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) were measured in the pulmonary tissue of all rats.Results: The DC rats showed elevated blood glucose and glycosylated hemoglobin levels, with insulin blood levels significantly lower than the NC rats (P < .001). They also showed significantly increased LPO concentrations in the lungs (P < .01) after 4 and 12 weeks of follow-up. In contrast, SOD, CAT, and GSH-Px antioxidant activities were significantly reduced in these periods (P < .01) 12 weeks after diabetes induction. Successful PT corrected all clinical and metabolic changes in the diabetic rats, with sustained normoglycemia throughout the study. Excessive lung LPO production and low SOD, CAT, and GSH-Px antioxidant activities were already back to normal 4 weeks after PT.Conclusion: PT can control oxidative stress in pulmonary tissue of diabetic rats. It may be the basis for preventing chronic diabetic lesions in lungs.

Pancreas Transplantation Prevents Morphologic and Ultrastructural Changes in Pulmonary Parenchyma of Alloxan-Induced Diabetic Rats

C.T. Spadella, O.A.X. Suarez, A.N. Lucchesi, A.J.M. Cataneo — 2010-07-01

Abstract: Purpose: The aim of this study was to evaluate whether pancreas transplantation (PT) is a suitable method for controlling histopathologic changes in lungs of alloxan-induced diabetic rats.Methods: Sixty inbred male Lewis rats were randomly assigned to 3 experimental groups: NC, 20 nondiabetic control rats; DC, 20 untreated diabetic control rats; and PT, 20 diabetic rats that received syngeneic PT from normal donor Lewis rats. Each group was further divided into 2 subgroups of 10 rats each, which were killed after 4 and 12 weeks of follow-up. Clinical and laboratory parameters, fresh and fixed lung weights, and fixed lung volumes were recorded for all rats. Total number of alveoli, alveolar perimeter, alveolar surface area, and alveolar epithelial (AE) and endothelial capillary (EC) basal laminae thickening were randomly measured in 5 rats from each subgroup by using an image analyzer. For light microscopy, 250 alveoli were analyzed in each subgroup. For electron microscopy, 50 electron micrographs were examined for each subgroup.Results: The DC rats showed elevated blood glucose and glycosylated hemoglobin levels, with insulin blood levels significantly lower than the NC rats (P < .001). Fresh and fixed lung weights and fixed volumes were significantly reduced in these rats, although their proportions to body weight were increased at 12 weeks (P < .01). The total number of alveoli in diabetic rats was higher than in control rats, whereas alveolar perimeter and surface area were significantly diminished (P < .01). AE and EC basal laminae were significantly thicker in DC than in NC (P < .01). Successful PT corrected all clinical and metabolic changes in diabetic rats, with sustained normoglycemia throughout the study. Morphologic and morphometric changes observed in diabetic lungs were completely prevented in PT rats from 4 weeks after transplant.Conclusion: We conclude that PT can control morphologic and ultrastructural changes in pulmonary parenchyma, suggesting a promising perspective for preventing other chronic diabetic lesions.

Vascularization of Artificial Beds for Pancreatic Islet Transplantation in a Rat Model

J. Kriz, D. Jirak, G.J. Vilk, P. Girman, D.J. White, M. Hajek, F. Saudek — 2010-07-01

Abstract: An alternative prevascularized bed with a subcutaneously located entrance would substantially improve islet engraftment, requiring much less invasive surgery. Studies have described times necessary for the creation of an artificial pouch suitable for subsequent islet transplantation. Polymeric mesh shaped in rounded scaffolds were implanted both subcutaneously and into the major omentum of Brown Norway female rats (n = 7). The connective tissue together with vessels were embedded into scaffolds at 1 week without regard to site. In contrast to the major omentum, vessels within the subcutaneous connective tissue surrounding the devices started to decline in 2 weeks and almost disappeared 1 week later. Magnetic resonance imaging (MRI) detected changes in fibrous tissue surrounding the wall, but only large veins located beside the devices were visible using basic MRI. The blood supply to the internal surface of the created beds was important for islet engraftment, but information could be obtained only by using dynamic contrast-enhanced MRI.

C5a-Inhibitory Peptide Combined with Gabexate Mesilate Prevents the Instant Blood-Mediated Inflammatory Reaction in a Rat Model of Islet Transplantation

K. Tokodai, M. Goto, A. Inagaki, W. Nakanishi, N. Okada, H. Okada, S. Satomi — 2010-07-01

Abstract: Background: The instant blood-mediated inflammatory reaction (IBMIR), in which the activation of both the coagulation and the complement cascades plays a key role, is one of the main obstacles to successful islet transplantation. At present, however, no useful protocol is clinically available. Therefore the aim of this study was to examine whether complementary peptides against an active region of C5a were safe to suppress IBMIR, owing to their extremely low molecular mass, when combined with a clinically available anticoagulant.Methods: Complement receptors on pancreatic tissues and isolated islets were analyzed by immunohistochemical staining and flow cytometry. Two-and-a-half islet equivalents per gram of syngeneic rat islet grafts were transplanted intraportally into 4 groups of 10–13 animals each after streptozotocin induction of diabetes: control, gabexate (Gab), C5a-inhibitory peptide (C5aINH), and C5aINH plus Gab. Recipients injected with equivalent amounts of saline solution served as control subjects. Plasma samples were collected at 0, 0.5, 1, 3, 6, and 24 h after transplantation for analysis. We also evaluated the curative rate, intravenous glucose tolerance test, and insulin amounts in the liver of the recipients.Results: C3a receptor (C3aR) was scarcely expressed on the isolated islets with relatively strong expression of C5a receptor (C5aR): C3aR: 0.44 ± 0.38%; C5aR: 7.91 ± 2.83%). However, C5aR was not expressed on pancreatic tissues before the isolation procedures. Thrombin-antithrombin complex was significantly suppressed in the 3 treated groups (P = .0015). The curative rate was also significantly improved (0% vs 33% vs 67% vs 100%, respectively; P = .03). Glucose tolerance was significantly improved among the 3 treated groups (P < .0005). Insulin amounts in the liver were considerably higher among treated versus control hosts. Notably, the treatment did not affect the increased body weight of the recipient.Conclusions: This study suggested that C5a-inhibitory peptide combined with gabexate mesilate may be a useful approach to control the IBMIR induced in clinical islet transplantation and one that is free of side effects.

Magnetic Resonance Imaging of Transplanted Mouse Islets Labeled With Chitosan-Coated Superparamagnetic Iron Oxide Nanoparticles

2010-07-01

Abstract: Although only 10% of islet recipients maintain insulin independence, 80% of them are C-peptide positive at 5 years after transplantation. To better understand the fate of transplanted islets, a magnetic resonance imaging (MRI) technique has been used to detect Feridex-labeled islet grafts in rodents. In this study, we used a novel MRI contrast agent, chitosan-coated superparamagnetic iron oxide (CSPIO) nanoparticles, to monitor mouse islet grafts. Male inbred C57BL/6 mice were used as donors and recipients of islet transplantation. The islet cytotoxicity was evaluated by fluorescein diacetate and propidium iodide staining for RAW cells incubated with CSPIO. After being incubated overnight with and without CSPIO (10 mg/mL), 300 islets were transplanted under the left kidney capsule of each mouse. After transplantation, 3.0-Tesla MRI of the recipients was performed biweekly until 19 weeks. At the end of study, the islet graft was removed for insulin and Prussian blue staining. The cell death rates in RAW cells did not increase with increasing CSPIO concentrations or incubation time. The grafts of CSPIO-labeled islets were visualized on MRI scans as distinct hypointense spots homogeneously located at the upper pole of left kidney. Their MRI signal was 30%–50% that of control islets and was maintained throughout the follow-up period. At 18 weeks, the histology of CSPIO-labeled islet graft revealed the insulin- and iron-stained areas to be almost identical. Our results indicate that isolated mouse islets labeled with CSPIO nanoparticles can be effectively and safely imaged by using MRI as long as 18 weeks after transplantation.

Effect of Triple Costimulation Blockade on Islet Allograft Survival in Sensitized Mice

2010-07-01

Abstract: Background: Islet allograft rejection in sensitized recipients is difficult to control by costimulation blockade using anti-CD154 and cytotoxic T-lymphocyte antigen-4 immunoglobulin (CTLA4Ig). Because leukocyte function antigen (LFA) 1 is highly expressed on memory T cells, adding an LFA-1 blockade may inhibit memory T-cell activities. We examined the effects on islet allograft survival of triple costimulation blockade in presensitized recipient mice.Methods: C57BL/6 mice were sensitized by transplantation under the kidney capsule or intraperitoneal injection of Balb/c islets. Four weeks after transplantation, sensitization was confirmed by flow-cytometric detection of alloreactive antibodies. Diabetes was induced by a single intravenous injection of streptozotocin. Recipients were transplanted with 200 Balb/c islets under the right kidney capsule. Graft function was assessed by daily blood glucose and body weight records. Transplanted animals were divided into 3 treatment groups: group 1, control antibody; group 2, anti-CD154 and CTLA-4 Ig double therapy; group 3, anti-CD154, CTLA4Ig, and anti–LFA-1 triple therapy. Injections were administered every second day from day −2 to day 8.Results: Naïve mice rejected islet allografts between days 7 and 29 (mean 16 ± 6 d; n = 5), sensitized mice in group 1 between days 0 and 14 (mean 7 ± 5 d; n = 8), in group 2 between days 4 and 16 (mean 8 ± 4 d; n = 7), and in group 3 between days 4 and 26 (mean 11 ± 7 d; n = 10).Conclusion: Triple costimulation blockade with anti-CD154, CTLA4Ig, and anti–LFA-1 was not sufficient to improve islet allograft survival in sensitized recipients.

Imaging of Human Islet Vascularization Using a Dorsal Window Model

O. Sabek, M.W. Gaber, C.M. Wilson, J.A. Zawaski, D.W. Fraga, O. Gaber — 2010-07-01

Abstract: Background: The islets of Langerhans are micro-organs rich in blood vessels. The process of islet isolation and culture disrupts the vasculature of the islets. The reestablishment of an appropriate microvascular supply is an essential prerequisite for long-term survival and function of islet grafts. In this study, we examined the effects on the process of neovascularization of coating the islets with fibrin.Methods: Isolated human islets were stained using the dioctadecylindocarbocyanine (DII) dye. An aliquot of the human islets were embedded in 3-dimensional fibrin. Human islets (100 islets-equivalents) were transplanted into a mouse dorsal window model to evaluate angiogenesis over 17 days. Transplanted islets were divided into 2 groups: either free islets or islets coated with fibrin gel. Animals were imaged using intravital microscopy immediately and at 3, 4, 8, 11, and 17 days after surgery. The DII dye caused the islets to be fluorescent and visible using a rhodamine filter. Fluorescein isothiocyanate dextran was used to visualize vasculature structures surrounding the islets.Results: Human islets coated with fibrin demonstrated an early appearance of a network of immature blood vessels that produced a significantly higher density/unit area for neovascularization by day 8 after transplantation.Conclusion: Our preliminary data showed that fibrin played a role in early neovascularization and support to sustain development of new blood vessels. Fibrin formed a matrix that helped to maintain the 3-dimensional structure of, and therefore reducing the environmental stress on islets.

A Survey of the Attitudes of Scientists Toward Xenotransplantation in Taiwan

S.-Y. Yen, S.-M. Lee, C.-F. Tu, S.-M. Tang, J.d.D. Tapsoba — 2010-07-01

Abstract: This study examined the attitudes of scientists in Taiwan's leading animal research institution toward xenotransplantation. The aim was primarily to evaluate the opinions of professionals in the biomedical field on key issues including ethical moral, legal, and regulatory issues raised by the biotechnology. A secondary objective was to identify potential factors that influenced opinions. A questionnaire-based survey was used to evaluate opinions. A test for internal consistency of the questionnaires to sample of 91 scientists was performed as well as a principal component analysis. We evaluated associations between variables using the nonparametric Kruskal-Wallis test. Among the subjects 85.2% thought that xenotransplantation can be more beneficial than harmful to human society and 94.3% believed that it is important to develop xenotransplantation. Also, 97.8% of participants believed that legislative guidelines should be adopted to regulate research in biotechnology. Gender was an influencing factor, whereas, variables such as religion, marital status, and age did not have obvious effects. Further studies on the general public are needed to detect other factors and to examine the attitude of nonprofessionals toward xenotransplantation.

Some Ethical Aspects of Xenotransplantation in Light of the Proposed European Directive on the Protection of Animals Used for Scientific Purposes

M. Jorqui-Azofra, C.M. Romeo-Casabona — 2010-07-01

Abstract: Unlike what has happened in other times, society in general and especially the scientific community has become aware that animals share our sensitivity to pain and the capacity to suffer. In this regard, it is generally accepted that animals must be protected from all types of abuse. In fact, it is unavoidable today that animals used in scientific experiments enjoy the maximum degree of protection and well-being. This view is based on an ecocentric notion of living matter as opposed to the traditional anthropocentric approach because it has become evident that ethics should not be limited to those belonging to the same species. Likewise, there is a broad consensus—with the exception of members of certain animal protection groups—regarding the need to experiment with animals, when no alternative methods (AM) are available, given that the current state of scientific knowledge still does not allow for this type of experimentation to be entirely abolished. Nevertheless, we must keep in mind that not every scientific procedure in which animals are used is legitimate. On one side of the scale that symbolizes the legislation in this field, we find the weight of science and safety, and on the other side, the weight of ethics. In this article we have reviewed some of the main ethical criteria that serve as a basis to balance the scale, in other words, to guide and legalize animal experimentation in the field of xenotransplantation (XT). To that end, we take into account the current revisions made to the European Directive regarding the welfare of animals used in scientific procedures (86/609/EEC), in order to reflect, in turn, on the following issue: where is European institutional ethics headed on this issue?

The Attitude of Scottish Citizens to Xenotransplantation in the South East of Spain: An Emerging Population Subgroup

2010-07-01

Abstract: Introduction: It is important to discover whether xenotransplantation would be accepted in society. In populations where there are preclinical projects there is the possibility of xenotransplantation to humans. In the South East of Spain in recent years there has been a significant social change, due to the migratory influx, which is making it necessary to reconsider the level of acceptance of xenotransplantation. The objective of this study was to analyze the attitudes of and to determine relevant variables among the population from southeastern Spain who were born in Scotland.Method: A random sample of the population from the South East of Spain that was born in Scotland. (n = 350) answered a questionnaire with categories validated for our geographical area. The self-administered survey was completed anonymously between November 2005 and March 2006. The statistical analysis employed Student's t-test, the χ2 test, and Fisher's exact test.Results: The questionnaire completion rate was 93% (n = 325). Assuming the results were comparable with those obtained using human organs, most respondents (67%) favored xenotransplantation with 8% against and 25% undecided. With regard to xenotransplantation, if the results were worse than those achieved using human organs, 26% (n = 83) would be in favor, 55% (n = 178) undecided, and the remaining 19% (n = 62) against these procedures.The attitude was related to belonging to the male gender (P < .001), marital status (P = .008), level of education (P = .041), a partner's favorable attitude toward transplantation (P < .001), and a favorable attitude toward organ donation, either from a deceased (P = .001) or a living (P < .001) donor.Conclusion: The attitudes of Scottish residents in southeastern Spain were similar to those of the native Spanish population and determined by many psychosocial factors, mainly related to previous attitudes toward various types of human organ donation.

Are Veterinary Students in Favor of Xenotransplantation? An Opinion Study in a Spanish University With a Xenotransplantation Program

2010-07-01

Abstract: Background: The shortage of available transplant organs has made it necessary to search for new alternatives, one of which is xenotransplantation. However, the use of animal organs and the personnel involved in its implementation could face opposition. Our objective was to analyze the attitudes of veterinary degree students in a Spanish university toward xenotransplantation and to determine the factors that affect its acceptance.Methods: Of the 515 students registered in a veterinary degree course (2007–2008), we surveyed 482 94% of whom completed the questionnaire. Attitudes toward organ xenotransplantation were evaluated using a validated, self-administered questionnaire concerning organ donation, which was completed anonymously. We applied Student's t-tests and χ2 tests.Results: If xenotransplantation was confirmed as a clinical reality, 91% (n = 436) of those students surveyed would accept a xenotransplanted organ, whereas 9% (n = 46) would not. Furthermore, 95% (n = 457) would accept tissue xenotransplantation and 97% (n = 467) cell xenotransplantation. Attitudes toward xenotransplantation were not affected by the academic year in which a student was studying, even when this was the year in which it was taught as a subject. Attitudes were not associated with any pyschosocial variable or attitude toward deceased organ donation; (P = .779). The students who believed that the demand for organs is not covered had a more favorable attitude toward xenotransplantation than those who think that there is no shortage (91% vs 70%; P = .027).Conclusion: Veterinary students had favorable attitudes toward xenotransplantation, assuming that the animal organs functioned as well as human organs. Therefore, these students could play important roles in the future promotion of this technique.

Infection of Porcine Cells with Human Herpesviruses

2010-07-01

Abstract: Porcine organs are valuable candidate materials for xenotransplantation to humans. Long-term maintenance of well functioning transplants is a prerequisite for success. Transplanted organs may be damaged by immune reactions or by infectious agents in hosts. Human herpesviruses (HHVs) establish life-long latency in humans after a primary infection. They can be reactivated with various stimuli, including immunosuppression. This study was performed to verify the infectivity of some HHVs toward porcine cells. PK-15 cells infected with HHV-1 and HHV-2 showed cytopathology from 1 day after infection. Immunofluorescent (IF) staining of HHV-1– and HHV-2–infected PK-15 cells with respective antibodies demonstrated the expression of the respective viral antigens. Permissiveness of PK-15 to HHV-1 and -2 was confirmed by an infection test on Vero cells. Islet cells infected with HHV-5 showed no gross morphologic changes during the experimental course. A limited portion of islet cells reacted only to anti-IE1 and anti-IE2, but not to anti-UL44 or anti-gB antibody by IF staining, whereas a small portion of endothelial cells reacted to anti-IEs and anti-UL44, but not to anti-gB antibody. HHV-1 and -2 can permissively infect porcine cells, but HHV-5 infects a small proportion of cells with limited viral protein expression. HHV-4 could not transform peripheral blood mononuclear cells from miniature pigs. Collectively, because some HHVs can infect and damage porcine cells or impair their functions, HHVs should be cautiously monitored and controlled in humans when porcine cells or organs are transplanted to human beings.

The In Vitro Protection of Human Decay Accelerating Factor and hDAF/Heme Oxygenase-1 Transgenes in Porcine Aortic Endothelial Cells Against Sera of Formosan Macaques

2010-07-01

Abstract: To mitigate hyperacute rejection, pigs have been generated with α-Gal transferase gene knockout and transgenic expression of human decay accelerating factor (hDAF), MCP, and CD59. Additionally, heme-oxygenase-1 (HO-1) has been suggested to defend endothelial cells. Sera (MS) (0%, 1%, 5%, 10%, and 15%) from Formosan macaques (Macaca cyclopis, MC), an Old World monkey wildly populated in Taiwan, was used to test the protective in vitro, effects of hDAF or hDAF/hHO-1 on porcine aortic endothelial cells (pAEC) derived from hDAF+, hDAF+/hHO-1+, and hDAF+/hHO-1− and 1 nontransgenic pAEC. Ten percent human serum (HS) served as a positive control. When MS addition increased to 10% or 15%, all transgenic pAEC exhibited a greater survival than nontransgenic pAEC. Noticeably, 15% MS reduced survived to <10% versus >40% in nontransgenic and transgenic pAEC, respectively. These results revealed that hDAF exerted protective effects against MC complement activation. However, comparing with 10% MS and HS in pAEC of nontransgenic pigs, the survivability was higher in HS, suggesting that complement activation by MS was more toxic than that by HS. Furthermore, hDAF+/hHO-1+ showed no further protection against effects of MS on transgenic pAEC.

In Vitro Production of Multigene Transgenic Blastocysts via Sperm-Mediated Gene Transfer Allows Rapid Screening of Constructs to Be Used in Xenotransplantation Experiments

2010-07-01

Abstract: Multigene transgenic pigs would be of benefit for large animal models and in particular for xenotransplantation, where extensive genetic manipulation of donor pigs is required to make them suitable for organ grafting to humans. We have previously produced multitransgenic pigs via sperm-mediated gene transfer (SMGT) using integrative constructs expressing 3 different reporter genes. The aim of the present work was to evaluate the efficacy and safety of using 3 integrative constructs carrying 3 different human genes involved in the modulation of inflammatory responses. We developed an in vitro fertilization system to demonstrate that SMGT can be used to efficiently produce multigene transgenic embryos through a 1-step genetic modification using multiple integrative constructs each carrying a different human gene involved in the modulation of inflammatory processes (hHO1, hCD39, and hCD73). The results suggest that this system allowed an effective preliminary test of transgenesis optimization, greatly reducing the number of animals used in the experiments and fulfilling important ethical issues. We performed 5 in vitro fertilization experiments using sperm cells preincubated with all 3 integrative constructs. A total of 1,498 oocytes were fertilized to obtain 775 embryos, among which 340 further developed into blastocysts. We did not observe any toxicity related to the transgenesis procedure that affected normal embryo development. We observed 68.5% transgenesis efficiency. Blastocysts were 48% single, 31% double, and 21% triple transgenic.

ABO and RH1 Blood Group Phenotyping in Pigs (Sus scrofa) Using Microtyping Cards

2010-07-01

Abstract: Transplantation or transfusion with ABO disparity is a cause for rejection or for severe hemodynamic alterations. ABO groups in pigs are commonly an unknown variable, which has been previously assessed by means of hemagglutination tests or immunohistochemical procedures on tissues. Herein, we have reported a simple method using commercial microcards for human ABO typing. However, the reagents directly derived from human sera included in these cards can result in false determinations due to α-gal interference. The ABO groups of 19 wild-type pigs (Landrace × Large White) were assessed using 2 commercial cards: Human sera-based and monoclonal antibody-based cards. The human sera cards determined that 8 pigs belonged to the AB group and 11 to the B group. The monoclonal antibody cards determined that 8 pigs belonged to the A group and 11 to the O group. None of the pigs showed reactions to Rh1 antibodies. Because the B group has not been described in pigs, the reaction in human sera cards represented an interference with α-gal antigen, a molecule structurally similar to the B blood antigen. Thus, microtyping cards based on monoclonal antibodies provided simple, quick way to assess ABO groups in pigs used for xenotransplantation. ABO concordance should always be investigated for these types of procedures.

Surgical and Nonsurgical Complications of a Pig to Baboon Heterotopic Heart Transplantation Model

2010-07-01

Abstract: A modified immunosuppressive regimen, developed at the National Institutes of Health, has been employed in a large animal model of heterotopic cardiac xenotransplantation. Graft survival has been prolonged, but despite this, our recipients have succumbed to various surgical or nonsurgical complications. Herein, we have described different complications and management strategies. The most common complication was hypercoagulability (HC) after transplantation, causing thrombosis of both small and large vasculature, ultimately leading to graft loss. While managing this complication we discovered that there was a delicate balance between HC and consumptive coagulopathy (CC). CC encountered in some recipient baboons was not able to be reversed by stopping anticoagulation and administering multiple blood transfusions.Some complications had iatrogenic components. To monitor the animals, a solid state left ventricular telemetry probe was placed directly into the transplanted heart via the apex. Induction of hypocoagulable states by continuous heparin infusion led to uncontrollable intra-abdominal bleeding in 1 baboon from this apical site. This occurrence necessitated securing the probe more tightly with multiple purse strings and 4-quadrant pledgeted stay sutures. One instance of cardiac rupture originated from a lateral wall infarction site. Earlier studies have shown infections to be uniformly fatal in this transplant model. However, owing to the telemetry placement, infections were identified early by temperature spikes that were treated promptly with antibiotics.We had several cases of wound dehiscence due to recipients disrupting the suture line. These complications were promptly resolved by either re-approximating the wound or finding distractions for the baboon. A few of the most common problems we faced in our earlier experiments were related to the jacket, tether, and infusion pumps. It was difficult to keep the jackets on some baboons and the tether had to be modified several times before we assured long-term success. Infusion catheter replacement resulted in transplant heart venous obstruction and thrombosis from a right common femoral venous line. Homeostatic perturbations such as HC and CC and baboon-induced wound complications comprised most complications. Major bleeding and death due to telemetry implantation and infarct rupture occurred in 2 baboons. Despite the variety of complications, we achieved significant graft prolongation in this model.

Left Ventricular Pressure Measurement by Telemetry Is an Effective Means to Evaluate Transplanted Heart Function in Experimental Heterotopic Cardiac Xenotransplantation

2010-07-01

Abstract: Evaluation of the function of heterotopic cardiac transplants has traditionally been accomplished by either manual palpation or serial biopsies. Both methods have drawbacks. Palpation can be difficult to differentiate a pulse from the graft versus a transmitted pulse from the native aorta. Serial biopsies, though accurate, require multiple laparotomies, leading to increased morbidity and possibly mortality rates. In this study we used an advanced telemetry system, consisting of an intra-abdominal implant, that was capable of continuously monitoring simultaneously several parameters of the transplanted heart and the status of the recipient. In a large animal model of heterotopic cardiac xenotransplantation (pig donor to baboon recipient), we implanted the device in 12 animals: 8 with and 4 without immunosuppression. We monitored and continuously recorded the left ventricular pressure (both peak-systolic and end-diastolic [LVEDP]), heart rate, and the electrocardiogram pattern of the transplanted heart as well as the temperature of the recipient. The left ventricular pressure proved to be the most valuable parameter to assess graft heart function. In the 4 nonimmunosuppressed cases, grafts were rejected acutely. In these cases, the end-diastolic pressure increased sharply and the heart stopped contracting when the difference between the systolic and the diastolic pressure decreased to <10 mm Hg. The earliest reproducible sign of rejection was an increased LVEDP. Among long-term survivors, the increase in diastolic pressure was gradual, indicating progressive thickening of the myocardium and decreased compliance of the ventricle. Six of 8 immunosuppressed animals died of other complications before rejecting the transplanted heart. The telemetry was also helpful to indicate early onset of fever in the recipients, thus allowing us to intervene early and prevent potentially lethal septic complications. Continuous monitoring of several parameters via telemetry allowed detection of changes associated with rejection as well as other complications at an early stage, allowing prompt intervention, treatment, and possibly reversal of rejection.

Super-High-Dose Islet Transplantation Is Associated With High SUITO Index and Prolonged Insulin Independence: A Case Report

2010-07-01

Abstract: Introduction: One of the current issues of clinical islet transplantation is the difficulty to achieve a prolonged insulin-free status. Functional islet mass gradually decreased after transplantation. We developed the SUITO index, which reflects engrafted islet mass. The SUITO (Secretory Unit of Islet Transplant Objects) index more than 26.0 is associated with an insulin-free status. In this study, we have experienced that super-high-dose islet transplantation maintained insulin-free status and a high SUITO index for a prolonged period.Materials and methods: Two islet isolations were performed in February 2007 and January 2008. Ductal injections were performed at the procurement site using the ET-Kyoto solution and pancreata preserved by a two-layer method. Islets were isolated using the modified Ricordi method. Both isolated islets were transplanted into a type 1 diabetic patient. Efficacy of islet transplantation was assessed by the amount of insulin requirements and SUITO index.Results: Islet yields were 514,467 islet equivalents (IE) and 872,174 IE, with purities of 49% and 85% for the first and second islet transplantations, respectively. The patient received a total of 24,327 IE/kg body weight. The immunosuppression was based on the Edmonton protocol. After the second islet transplantation, the average SUITO index for the following 1 month was 48.5, and the patient became insulin-free. At postoperative day 1006, the SUITO index was 44.6 and the patient maintained an insulin-free status with excellent glycemic control.Conclusion: Super-high-dose islet transplantation was associated with an high SUITO index and prolonged insulin independence.

ET-Kyoto Ductal Injection and Density-Adjusted Purification Combined With Potent Anti-Inflammatory Strategy Facilitated Single-Donor Islet Transplantation: Case Reports

2010-07-01

Abstract: Background: The necessity to use multiple donors for achieving insulin independence in clinical islet transplantation is still a major issue. We have developed a modified islet isolation method for non–heart-beating donors (Kyoto method) to significantly increase islet yield. In this study, we further modified the method for brain-dead donors and in addition, introduced a potent anti-inflammatory strategy aiming for single-donor islet transplantation.Materials and methods: Two islet isolations used pancreatic ductal preservation with the modified Kyoto solution and a density-adjusted purification method. Anti-interleukin-1-beta antibody (Anakinra) and anti-tumor necrosis factor-alpha (Eternacept) were administered during and after transplantation. The efficacy of the islet transplantation was assessed by the insulin requirement and SUITO (Secretory Unit of Islet Transplant Objects) index, wherein a value of more than 26.0 seems to be associated with insulin independence.Results: Both isolated islet preparations met the criteria for transplantation. They were transplanted into two type 1 diabetic patients, both of whom became insulin independent with stable glycemic control. The average SUITO index within 1 month was 29.2 and 45.3.Conclusion: The islet isolation method combined with a potent anti-inflammation strategy made it possible to achieve single-donor islet transplantation achieving a high SUITO index.

Percutaneous Embolization of Periduodenal Varix Due to Portal Hypertension in a Patient With Kidney–Pancreas Transplantation: A Case Report

2010-07-01

Abstract: Kidney–pancreas transplantation is a valid therapeutic option for patients with insulin-dependent diabetes mellitus. However, vascular complications associated with pancreas transplantation are not uncommon. Herein we have reported a 32-year-old woman with a history of insulin-dependent diabetes mellitus and celiac disease. She underwent liver transplantation for acute hepatitis. After 7 years, the patient developed end-stage kidney disease beginning hemodialysis and being listed for a kidney–pancreas transplantation, which was successfully performed when she was 29 years old with enteric diversion (Roux intestinal loop reconstruction). Five years after kidney–pancreas transplantation, she was admitted to our hospital with serious intestinal bleeding and poor liver function. The ultrasound showed a pattern like a arteriovenous fistula near the head of the pancreas. Computed Tomography was not diagnostic; an arteriogram showed the presence of a mesenteric varix and a mesenteric-caval shunt through the duodenum of the pancreatic graft. The liver biopsy and portal pressure gradient showed portal hypertension and liver cirrhosis. To obtain time a waiting a new liver, the patient underwent percutaneous embolization of the mesenteric varix through jugular access. The procedure was uneventful. The patient was successfully transplanted 2 months later. Pancreas function was always satisfactory.

Preface

Franco Filipponi — 2010-07-01

The 1st Congress of the Italian Society for Safety and Quality in Transplantation (Società Italiana per la Sicurezza e la Qualità nei Trapianti, SISQT) took place in Florence from November 12 to 14, 2009. It was the first time that transplant care professionals gathered to discuss about the issues of safety and quality of current donation and transplant procedures together with patients and stakeholders. The SISQT Board of Directors planned a multi-stakeholder congress with the aim of integrating the different points of view that make up the complexity of the transplant community. The aims of the SISQT is to involve all health care professionals across the continuum of donation and transplantation, championing a collaborative, inclusive, inter-disciplinary, inter-professional, and multi-stakeholder approach, in order to ease translation of the results of research into clinical practice.

The Italian Society for Safety and Quality in Transplantation (SISQT)

F. Filipponi — 2010-07-01

Abstract: The Italian Society for Safety and Quality in Transplantation (La Societâ Italiana per Ia Sicurezza e la Qualità nei Trapianti, SISQT) was founded in 2008 to bring quality and safety issues at the center of donation and transplantation practice. In doing so, the SISQT seeks to involve, all health care professionals across the continuum of donation and transplantation, championing a collaborative, inclusive, inter-disciplinary, inter-professional, and multi-stakeholder approach, in order to ease translation of the results of research into clinical practice. The program of the SISQT aims to (1) set a patient safety agenda with all professionals and stakeholders; (2) design and implement patient-centered care processes and procedures; 3) help professionals harmonize and integrate operational practice with policy and regulatory mandates of the European union; (3) lay the scientific evidence on management of complex care across the continuum of donation and transplantation; (4) promote behaviors and cultural attributes in light of quality and safety. Accomplishment of these results requires cooperation of each care provider at all levels, from hospital to home to achieve integration of patient expectations within the scope of current transplant practice.

Electrical Safety During Transplantation

2010-07-01

Abstract: Technologic innovations enable management of medical equipment and power supply systems, with improvements that can affect the technical aspects, economics, and quality of medical service. Herein are outlined some technical guidelines, proposed by Istituto Superiore per la Prevenzione e la Sicurezza del Lavoro, for increasing the effectiveness of the power supply system and the safety of patients and surgeons in the operating room, with particular focus on transplantation. The dependence of diagnoses and therapies on operation of the electrical equipment can potentially cause great risk to patients. Moreover, it is possible that faulty electrical equipment could produce current that may flow through the patient. Because patients are particularly vulnerable when their natural protection is considerably decreased, as during transplantation or other surgery, power supply systems must operate with a high degree of reliability and quality to prevent risk, and must be designed to reduce hazards from direct and indirect contact. Reliability of the power supply system is closely related to the quality of the project, choice of materials, and management of the system (eg, quality and frequency of servicing). Among the proposed guidelines, other than normal referencing, are (1) adoption of a monitoring system to improve the quality of the electrical parameters in the operating room, (2) institution of emergency procedures for management of electrical faults, (3) a procedure for management of fires in the operating room, (4) and maintenance interventions and inspections of medical devices to maintain minimal requirements of safety and performance.

National Policies for Risk Management in Italy

2010-07-01

Abstract: The Ministry of Health in Italy considers risk management (RM) to be one of the specific objectives to be developed by its national policies, as suggested by the European Union recommendations and by several international organizations, such as the World Health Organization (WHO), the Council of Europe, and the Organization for Economic Cooperation and Development. The National Health Plan stated the need to guarantee and monitor safety of health care and biomedical technology, with the development of a standardized computerized method to collect and analyze data on adverse events and with specific actions for education and training of all stakeholders, which is to be conducted at different levels of the health system governance, national, regional, and local. Over a 4-year period, the National Observatory for the Monitoring of Sentinel Events has collected data on 385 sentinel events, with a mortality rate of 54.8%. Compared with earlier reports, we have observed a reduction from 41% to 17% of sentinel events classified in the “nonspecified sentinel event” and an increase from 20% to 40% of cases in which an action plan has been developed. A manual for root cause analysis has been released with the aim of offering health operators an instrument to analyze the occurrence of an adverse event. Ten recommendations and a manual for safety in the operating room, which includes a checklist for safe surgery adapted on the basis of WHO suggestions, have been published. To date, eight guidelines for safety have been released to improve stakeholders' accountability. The Ministry of Health has also elaborated a program of specific actions to be developed over the next 2 years in several areas of RM. These initiatives confirm the strategic role of policies for RM in our country, allowing for a dynamic and proactive process, ensuring continuity of action and promoting a deep understanding of patient safety issues.

Unintended Transplantation of Three Organs from an HIV-Positive Donor: Report of the Analysis of an Adverse Event in a Regional Health Care Service in Italy

T. Bellandi, S. Albolino, R. Tartaglia, F. Filipponi — 2010-07-01

Abstract: In February 2007, three organs from an human immunodeficiency virus (HIV)–positive donor were transplanted at two hospitals in the Tuscany Regional Health Care Service, owing to a chain of errors during the donation process. The heart-beating donor was a 41-year-old woman who died as a result of head trauma. The patient's history did not highlight any risky behavior. The available data on previous hospital admissions reported a negative result on HIV testing. During the donation process, the result of the lab test performed for evaluation of organ suitability was mistakenly transcribed from positive to negative. This wrong negative result was then included in the donation record without any cross-check. Therefore, the Regional Transplant Center allocated the liver and both kidneys. The patient also donated tissues, and a second laboratory conducted an evaluation of suitability for the tissue banks. During this process, only 5 days after the successful transplantation procedures, the positive HIV result was fed back to the Regional Transplant Center and the previous error discovered. Transplanted patients were immediately assessed and then treated with antiretroviral medications. A national commission soon performed a systems analysis of the adverse event. Besides the active error committed during the manual transcription for the HIV lab test result, the commission also identified technological factors, such as the lack of integration between the lab machine, the laboratory information system (LIS), and the donor record, as well as organizational factors, such as the distribution to two different labs of the suitability evaluation for organs and tissues. Recommendations included: automatic transmission of lab test results from the lab machine to the LIS and to the donor record, centralization of lab tests for suitability evaluation of organs and tissues, a training program to develop a proactive quality and safety culture in the regional network of donation and transplantations.

Do “Silent” Brain Deaths Affect Potential Organ Donation?

F. Procaccio, L. Rizzato, A. Ricci, S. Venettoni, A. Nanni Costa — 2010-07-01

Abstract: Brain death (BD) is not a stable, objective condition; in fact, it strongly depends on early intensive treatment before death, brain stem reflex testing, and intensive care unit (ICU) physician attitudes. Consequently, unpredictable “silent” BDs due to inadequate treatment or omitted declaration may affect potential organ donations. Several lines of evidence suggest that 55% to 65% of all deaths among patients with acute cerebral lesions (DACL) in the ICU may become brain deaths. Since DACL are easily measurable, deviations from the expected ratio of declarations will disclose “silent” BDs. Results from the National Registry of DACL in ICU settings have confirmed that BD declarations are consistently fewer than the number expected in Italy, particularly in regions where organ donation rates are low. Only 43% of the 10,304 referred DACL were potential donors in a 2-year period. Thus, around 1000 BDs per year are missing in Italy. Significant clinical factors for lost BDs may be older age and timing of death. As DACL represent the global donation potentiality (possible donors), we suggest the use of a new indicator—DACL in ICU per million population—and careful analyses of differences in DACL per million people among regions. In conclusion, since striking deviations from the expected ratio between BD declarations and deaths with an acute cerebral lesion exist in some regions, targeted training and support to ICUs should be planned. As adequate neurointensive treatment can improve outcomes and reduce “silent” BDs, more organ donors may exist where patients with acute cerebral lesion are better treated.

Safety of Bone Marrow Stem Cell Donation: A Review

A. Bosi, B. Bartolozzi — 2010-07-01

Abstract: Allogeneic hematopoietic stem cell transplantation (HSCT) represents the first choice of treatment or an important therapeutic option for several diseases, but it is still marked by morbidity and mortality. In contrast, the donation of hematopoietic stem cells (HSCs) is considered to be a safe procedure. The invaluable ethical source of donation and its central role in transplantation implies that the greatest attention be due to the donor and to the donation process through a serious monitoring protocol for donor safety. Both the Joint Accreditation Committee and the European Committee pay particular attention to the notification of adverse events and adverse reactions. Bone marrow donation is a well established procedure, that has now been performed for >30 years. Although it does not require drug administration, there is hospital admission for 1–3 days with 7–10 days off work. The main risk is related to the anesthesia. Pain in the aspiration area, together with astenia are considered to be the most frequent side effects, as shown by the USA National Marrow Donor Program experience in 1,193 donations. In the European Group for Blood and Marrow Transplantation analysis performed between 1993 and 2005 on 27,770 first HSCTs from bone marrow, only 1 fatal event (pulmonary embolism) and 12 serious adverse events were observed. The most frequent adverse events were cardiac. The incidence of adverse events was significantly lower (P < .05) compared with peripheral blood HSC donors, which confirms the necessity of accurate attention to donor selection and evaluation in bone marrow donation.

Efficiency of the Tissue Procurement Process: What Evaluation Tools?

A. Saviozzi, G. Bozzi, P. De Simone, F. Filipponi — 2010-07-01

Abstract: Evaluation of the efficiency of tissue procurement (TP) requires appropriate indicators. We report the results of a survey on all in-hospital deaths in Tuscany performed to identify potential indicators of efficiency with regard to donor identification and evaluation. In January 2004, we established a regional, compulsory, prospective program to monitor all in-hospital deaths in Tuscany. Currently, in Tuscany TP is performed if donors are ≤75 years without evidence of infectious or malignant diseases. Between January 2004 and December 2008, we analyzed data on 75,921 in-hospital deaths, including 50,001 (66.9%) in subjects older than 75 years, while 25,920 (33.1%) were ≤75 years and thus considered for TP. Among the latter cohort, 11,657 (15.3%) presented with clinical contraindication(s), while 14,263 (18.8%) were fit for tissue donation. Of the latter population, tissue donation occurred in 3083 cases (ie, 4% of in-hospital deaths or 11.8% of potential tissue donors). Contraindications to tissue donation were identified in 9861 cases (12.9%) based on clinical files, and in 1796 (2.4%) after interview with the family. There was a great variability by regional hospital in the percent of contraindications identified after the family interview, from a low of 4% to a high of 45%. Based on our experience, implementation of efficiency parameters and improvement of the efficiency of the entire TP process requires compulsory reporting of in-hospital deaths by local transplant coordinators.

Ensuring Safety and Quality of Tissues in Italy: Application of European Directives

E. Porta, D. Fehily, F. Bariani, A. Nanni Costa — 2010-07-01

Abstract: Three European Directives published in 2004 and 2006 require Member States to take a series of regulatory actions to ensure appropriate quality and safety of tissues and cells. These directives define responsibilities to inspect and certify centers, to put vigilance systems in place and to publish information on certified centers and their activities. A European-funded project led by the Italian National Transplant Centre has supported Member States in the development of common guidelines for inspection, tools for vigilance, and training for inspectors. In Italy, inspections are conducted every 2 years at each tissue bank, and a vigilance system has been launched. Information on Italian centers and their activity is published in the Eurocet Registry.

The Role of Nurses in the Risk Management of Organ and Tissue Donation

A. Saviozzi — 2010-07-01

Abstract: In the setting of organ and tissue procurement, lack of transplantation and the resulting missed opportunity for wait-listed patients might be considered as an untoward effect, since it results in longer wait-list times, higher mortality rates for potential candidates, and harm to the entire society. Beyond the classical definition holding that an adverse event is the inadvertent transmission of disease from a donor to a recipient, we advocate it should also include nonreporting of potential deceased donors; unsuccessful donor management; failure in organ/tissue procurement as the result of impossibility to assess brain death, lack of clinical data, or technical problems during surgical procedures. Based on their education, experience, and competencies, nurses share the responsibility to participate in the evaluation of risks and in the implementation of appropriate strategies for error prevention during the entire procurement process.

Calcineurin Inhibitors in Renal Transplantation Still Needed but in Reduced Doses: A Review

C. Ponticelli, M.P. Scolari — 2010-07-01

Abstract: Despite their contribution in the success of organ transplantation, calcineurin inhibitors (CNIs) may be responsible for frequent and severe side effects that can affect graft survival and life expectancy. In this article, we have reviewed registry studies and randomized controlled trials (RCTs) that seek to avoid, withdraw, or minimize CNIs in renal transplant recipients. Attempts to completely avoid CNIs by administering mycophenolate mofetil (MMF) and/or sirolimus (SRL) have resulted in increased risks of rejection and side effects, with small advantage to improve renal graft function. Early withdrawal of CNIs after transplantation using administration of MMF can improve graft function but may be associated with a greater risk of acute or chronic rejection and graft failure. RCTs in which CNIs were replaced a few months after transplantation by SRL reported improved graft function among SRL-treated patients, but such a treatment was complicated by iatrogenic toxicity. Late replacement of CNIs with SRL did not produce a particular advantage and again was complicated by more frequent side effects. On the basis of these trials, it seems that CNI elimination can trigger rejection or side effects. Recent RCTs showed that minimization of CNI doses in association with everolimus does not increase the risk of rejection, allows one to obtain good graft function, and is well tolerated. Such an approach seems therefore preferable to complete elimination of CNIs with substitution of the current immunosuppressive drugs.

Analysis of Four Scoring Systems and Monocentric Experience to Optimize Criteria for Marginal Kidney Transplantation

M. Messina, F. Fop, E. Gallo, M. Tamagnone, G.P. Segoloni — 2010-07-01

Abstract: There is a strong need among the transplantation community to identify common criteria to utilize the pool of expanded criteria donors (ECD), considering the disparity between organ demand and supply as well as the benefits of transplantation on long-term mortality compared with survival on dialysis, also in patients transplanted with these organs. The purpose of this article was to analyze scoring systems proposed in literature by Nyberg, Anglicheau, Rao (Kidney Donor Risk Index), and Schold, seeking to verify whether our clinical and histological allocation strategy matched the Nyberg score. Herein we have reported the results of a preliminary retrospective study on the 5-year outcomes of organs from 60 marginal donors, who were older than 50 years and histologically evaluated before implantation. The donors matched Nyberg class C and D, that is, marginal donors. We noted a tendency toward an association between global and vascular scores with class D (odds ratio 2.2 and 4.3, respectively). Kaplan–Meier graft survival curves were similar to Nyberg data: 83% for class C versus 73% for class D at 5 years. Without any comparison to the Nyberg score, the only feature that was predictive of renal function at 5 years in our population was hypertension in the donor. Further studies are required to identify which of the scoring systems—clinical and/or histological—is more suitable to allocate ECD kidneys and to predict recipient outcomes.

C4d-Positive Renal Allograft Rejection Biopsies in Cyclosporine-Treated Patients: Single-Center Incidence and Outcome

2010-07-01

Abstract: T cell–mediated acute rejection (ATCMR) in renal transplant patients can have an antibody-mediated component. The aim of this study was to evaluate the incidence of renal biopsies showing ATCMR with C4d immunoreactivity and the correlation between C4d-positive ATCMRs and graft outcomes. We studied 216 renal transplant patients receiving cyclosporine-based immunosuppression (mean follow-up = 203.5 ± 42.5 months). Of these, 79 experienced biopsy-proven ATCMR (group 1), whereas 137 did not show clinical or laboratory evidence of ATCMR (group 2). Mean serum creatinine levels were evaluated at 6 months, as well as 2 and 5 years after transplantation. The number of graft losses due to interstitial fibrosis and tubular atrophy (IF/TA) was greater in group 1 than in group 2 (P < .001 and P < .02, respectively), while graft survival was lower (P < .03). Staining with anti-C4d antibody was performed in 61/77 type I ATCMR biopsies: seven cases showed diffuse C4d positivity with CD68+ monocytes in peritubular capillaries observed in all cases. Three cases showed focal C4d positivity. Two ATCMRs were steroid, resistant. Graft loss due to IF/TA occurred in 4/7 patients (57.1%) who had previously experienced ATCMRs with diffuse C4d positivity; whereas it occurred in 5/51 patients (9.8%) with previous C4d negative ATCMRs (P < .001). Patients with focal C4d positivity did not undergo graft loss due to IF/TA. In conclusion, at our center the diffuse C4d positivity that occurred in 11.4% of type I ATCMRs was associated with a poor prognosis.

Kidney Graft Survival Rates Do Not Improve by Era: The Impact of the Age Factor

2010-07-01

Abstract: We performed this study to evaluate whether older ages of donors and recipients negatively affected long-term graft survival. We compared 5-year graft survival rates of 89 recipients transplanted between 1991 and 1995 (period A) versus 221 recipients transplanted between 1996 and 2000 (period B). Acute rejection rates and the number of donors and recipients >50 years of age were compared in the two periods. The 5-year graft survival rate in period B was 76.3% versus 82% in period A. In period B, the acute rejection incidence was 18% versus 40% in period A (P < .001). The overall 5-year graft survival was 86.2% for donors aged 21–50 years and 65.7% for donor's aged >50 years (P < .0001) in period A versus 84.1% and 68%, respectively, in period B (P = .0023). In period A, 23.6% of donors and 35.9% of recipients were >50 years old, versus 50.2% and 42.9%, respectively, in period B. The graft survival rate in period B was worse than in period A, although the acute rejection rate was lower. The older age of both donors and recipients in period B seemed to be an important cause of worse outcomes.

Fibrosis Progression and the Pros and Cons of Antiviral Therapy for Hepatitis C Virus Recurrence After Liver Transplantation: A Review

2010-07-01

Abstract: The progression of fibrosis due to hepatitis C virus (HCV) recurrence after liver transplantation (OLT) is faster than in the pretransplant setting, leading to histologically documented cirrhosis within 5 years in 25% to 30% of cases. Whether it is associated with biliary complications or previous alcohol abuse, recurrent HCV is the main cause of graft failure and death after OLT. The most important donor risk factor for HCV recurrence is advanced donor age. The disease's course is even more aggressive if it is associated with anti-HCV positivity or graft steatosis. The type of calcineurin inhibitor does not seem to influence HCV recurrence. Avoiding or slowly tapering steroids has been associated with less disease recurrence, while steroid pulses to treat acute rejection episodes have been associated with a worse progression of fibrosis. Antiviral therapy (AT) is not always recommended in OLT patients, but is of some benefit. Fibrosis has been shown to ameliorate in sustained virological responders to AT and to progress significantly more in nonresponders. Using long-term maintenance, AT has recently been shown to increase the probability of biochemical and histological responses, regardless of the timing of the HCV recurrence. In conclusion, the donor- recipient match should be assessed to limit HCV recurrences and their severity; AT is recommended to reduce or reverse the progression of fibrosis.

Frozen-Section Diagnosis in Donor Livers: Error Rate Estimation of Steatosis Degree

E. D'Alessandro, F. Calabrese, E. Gringeri, M. Valente — 2010-07-01

Abstract: A high degree of macrovesicular steatosis is associated with a significant risk of graft dysfunction. Most centers, including ours, consider 60% the limit value for transplantability, while others have adopted 30% as a cutoff. Pretransplant frozen-section (PFS) evaluation is used for reliable quantification of steatosis. However, the accuracy of PFS analysis for the degree of steatosis has largely been debated due to its high grade of variability and subjectivity. The aim of our study was to evaluate the accuracy of PFS diagnosis compared with subsequent paraffin histology samples. We retrospectively analyzed PFS from 52 consecutive liver donors. All PFS were blindly reviewed by two pathologists. The results were graded according to two classifications: (A) lower or higher than 60% steatosis, or (B) mild (0%–30%), moderate (30%–60%), or severe (>60%) steatosis. The rate of error for A (two-grade classification score) was 1.9% with the erroneous discharge of a viable organ. The error rate for B (three-grade classification score) was 7.7% with four discrepancies. In three cases, the discrepancy was related to the mild to moderate cutoff value, leading to a clinical error rate of 5.8% (discharging organs with statosis >30%, when we used more strict criteria). Our study validated PFS analysis as a reliable technique when the maximum value for organ transplantation was 60% steatosis. There was a higher error rate when a cutoff value of 30% was used. This finding suggests the usefulness of another technique to support a more precise steatosis evaluation.

Liver Transplant Quality and Safety Plan in Anesthesia and Intensive Care Medicine

2010-07-01

Abstract: Patients scheduled for orthotopic liver transplantation (OLT) may have coexisting diseases and more likely receive grafts of poorer quality than in the past. Perioperative mortality and morbidity are usually due to a combination of factors related to the patient, graft, surgery, anesthesia, and intensive care management. Anesthesia and intensive care are the areas with the highest frequency and severity of errors. Error and accident risks are always present in this context where a human component is unavoidable. The matter of medical errors is becoming noteworthy worldwide. Nevertheless, data concerning medical errors during OLT are not available in Italy. There are only hypothetical evaluations. The number of adverse events may be high, but so far no specific programs have been developed to increase patient safety. To improve patient safety, anesthesia and intensive care units must use a proactive approach dedicated to an OLT program. We have presented herein a prevention policy to detect errors before they happen through incident reporting, anonymous and voluntary reports of adverse events or near misses, operating room checklists (patient, drugs, devices, equipment), improved training, safer facilities, equipment function, and adequate drug supplies for an OLT program.

Patient Satisfaction Among Liver Transplant Recipients: Single-Center Survey

2010-07-01

Abstract: A single-center survey using a semistructured questionnaire was conducted in liver transplantation recipients at discharge after the primary surgery. The objectives of the study were to assess patient satisfaction and to identify critical points that negatively affected their perception of the quality of care received, and to derive information to enable improvement in current standards of care. The questionnaire included 5 sections about quality and 1 section for suggestions. Patients were asked to provide answers on a 5-item Likert scale. Areas assessed included quality of staff, organization, boarding, privacy, and transfer of care. Among 51 recipients, satisfaction was high (>50%) in all areas. Lower satisfaction scores were given for room services, diet, and background music. The most frequently reported area of dissatisfaction (12%) was lack of availability of in-hospital physical rehabilitation programs. Despite overall satisfaction with quality of care, recipients reported lack of appropriate physical rehabilitation programs in the early posttransplantation period.

Transplantation of Hematopoietic Stem Cells: Role of National Transplantation Center

L. Lombardini, A. Nanni Costa — 2010-07-01

Transplantation of hematopoietic stem cells (HSCs) has been, for many years, a therapy choice for the treatment of many hematologic and nonhematologic diseases: leukemias, lymphomas, myeloma, congenital diseases of metabolism, immunodeficiencies, autoimmune disease, and solid tumor. The biologic basis of transplantation was first known in the 1950s with the identification of hematopoietic stem cells in bone marrow and elucidation of the major histocompatibility system. The first pioneering transplantation attempts were performed in the 1950s and 1960's, and the first successful procedure was described by Gatti et al in November 1968. In 1975, the Seattle group published the results of the first 110 implanted patients, providing the basis for the clinical application of bone marrow transplantation on a large scale. Until the early 1990s, the only source of stem cells was bone marrow (BM); thereafter, HSCs were also derived from peripheral blood and then cord blood.

Human De-epidermized Dermis as a Stem Cell Carrier

E. Pianigiani, F. Ierardi, B. Mazzanti, R. Saccardi, C. Cuciti, M. Fimiani — 2010-07-01

Abstract: Recently several types of skin equivalents have been developed, consisting of differentiated keratinocytes cultured on various dermal substitutes. Different models of reconstructed human skin have been proposed, such as human and animal de-epidermized dermis, inert filters, collagen matrices, lyophilized collagen membranes populated with fibroblasts, and other models populated with melanocytes and/or Langerhans cells. These skin equivalents mimic native skin in vivo. They have provided information about dermal-epidermal interactions, cell-cell, and cell-matrix interactions; responses of dermal and epithelial cells to biological signals and pharmacological agents; as well as effects of drugs and growth factors on wound healing. Human allodermis from tissue banks has been used for clinical purposes, namely, as support for autologous keratinocyte cultures and as a potentially ideal scaffold for dermal replacement. This bioproduct is considered to be the most suitable clinical carrier for autologous fibroblasts and keratinocytes, as well as an useful experimental model to study angiogenesis and to stimulate vascularization in reconstructed human skin. Because it is human-derived, it is in our opinion the safest of all available types of skin equivalent. Having epidermal and dermal structures, it can be used in one-stage grafting procedures for wound closure.

A Common Polymorphism in the Monocyte Chemoattractant Protein-1 (MCP-1) Gene Regulatory Region Influences MCP-1 Expression and Function of Isolated Human Pancreatic Islets

2010-07-01

Abstract: Background and aims: Islet transplantation is an attractive approach to treat type 1 diabetic patients. However, suboptimal islet engraftment still represents an unsolved problem. It has been shown that human islets release monocyte chemoattractant protein-1 (MCP-1), one of the most powerful macrophage chemokines, which may impair the fate of the transplant. The aim of this study was to evaluate the presence and role of MCP-1 in isolated human islets, including genotyping for a common polymorphism.Methods: Pancreatic islets were isolated by enzymatic digestion and gradient purification from 41 nondiabetic multiorgan donors. We measured MCP-1 mRNA expression by quantitative real- time reverse-transcriptase polymerization chain reaction, analyzed the MCP-1 single nucleotide polymorphism, −2518 G/A (SNP, rs 1024611) and evaluated glucose-stimulated insulin release (IR; μU/islet/min).Results: MCP-1 mRNA expression was found in all studied batches of islets. Overall, IR was significantly higher at 16.7 mmol/L than 3.3 mmol/L glucose. We observed a significant negative correlation between MCP-1 mRNA expression and stimulation index (SI). We found that MCP-1 mRNA expression was significantly higher in CC and CT compared with TT genotype groups. Finally, SI was significant lower in the CC with respect to the TT genotype group.Conclusions: These data show that MCP-1 gene expression regulated by the −2518 G/A polymorphism, is correlated with glucose-stimulated insulin release. The study of MCP-1 expression and genotype on isolated islets before transplantation may be useful to understand the inflammatory response after infusion of human islets into patients with type 1 diabetes mellitus.

Functional and Survival Analysis of Isolated Human Islets

2010-07-01

Abstract: Background and Aims: Pancreatic islet transplantation has become one of the potential treatments for type 1 diabetes. We evaluated functional and viability parameters of isolated islets in relation to donors clinical characteristics and preparation variables.Methods: Islets were isolated from 70 nondiabetic multiorgan donors of overall age of 62.5 ± 15.9 years. There were 41 men and 29 women. Their mean body mass index (BMI) was 25.62 ± 3.09 kg/m2. We evaluated the islet number (IEQ/g pancreatic tissue) insulin release (IR; μU/islet/min) in response to 3.3 (g) or 16.7 (G) mmol/L glucose; calcium flux concentration (CFC); and islet cell viability.Results: IEQ was 5249 ± 1505, with 73.7 ± 14.96% viable islet cells. IR was 0.03 ± 0.01 at g and 0.11 ± 0.06 at G (stimulation index [S] = 3.24 ± 1.96). CFC was 1.95 ± 1.03 ΔRFU. We observed positive correlations between viable cells and IR at g (R2 = 0.260; P = .013), IR at G (R2 = 0.165; P = .013), and CFC (R2 = 0.175; P = .047). A positive correlation was documented between BMI and g (R2 = 0.245; P = .016) and negative correlations between age with SI (R2 = 0.188; P = .052) and cold ischemia time with IEQ (R2 = 0.865; P = .0061).Conclusions: These results showed that quality control of isolated human pancreatic islets allowed assessment of beta-cell function and survival before transplantation, revealing several important variables.

Risk Analysis of Hematopoietic Stem Cell Transplant Process: Failure Mode, Effect, and Criticality Analysis and Hazard Analysis Critical Control Point Methods Integration Based on Guidelines to Good Manufacturing Practice for Medicinal Product ANNEX 20 (February 2008)

S. Gianassi, S. Bisin, B. Bindi, I. Spitaleri, F. Bambi — 2010-07-01

Abstract: The collection and handling of hematopoietic stem cells (HSCs) must meet high quality requirements. An integrated Quality Risk Management can help to identify and contain potential risks related to HSC production. Risk analysis techniques allow one to “weigh” identified hazards, considering the seriousness of their effects, frequency, and detectability, seeking to prevent the most harmful hazards. The Hazard Analysis Critical Point, recognized as the most appropriate technique to identify risks associated with physical, chemical, and biological hazards for cellular products, consists of classifying finished product specifications and limits of acceptability, identifying all off-specifications, defining activities that can cause them, and finally establishing both a monitoring system for each Critical Control Point and corrective actions for deviations. The severity of possible effects on patients, as well as the occurrence and detectability of critical parameters, are measured on quantitative scales (Risk Priority Number [RPN]). Risk analysis was performed with this technique on manipulation process of HPC performed at our blood center. The data analysis showed that hazards with higher values of RPN with greater impact on the process are loss of dose and tracking; technical skills of operators and manual transcription of data were the most critical parameters. Problems related to operator skills are handled by defining targeted training programs, while other critical parameters can be mitigated with the use of continuous control systems. The blood center management software was completed by a labeling system with forms designed to be in compliance with standards in force and by starting implementation of a cryopreservation management module.

Key Role of Staff Competencies for Patient and Donor Safety in a Bone Marrow Transplantation Unit: Design and Implementation of an Accredited Training and Self-Assessment Program

C. Lamanna, M. Baroni, S. Bisin, S. Gianassi, F. Bambi, D. Caselli, M. Aricò — 2010-07-01

Abstract: Human resources represent at the moment the most critical factor in an hospital setting characterized by a high rate of staff turnover. It is important to ensure a consistent level of expertise and knowledge of professionals who work in health care facilities to provide quality services and simultaneously support the implementation of strategies for patient safety. Unfortunately, the development of effective interventions for training newly added staff and self-evaluation of skills possessed by trained staff are closely related to understanding critical aspects of the organization. At the new Center for Bone Marrow Transplantation and Blood Transfusion Service in Meyer Hospital, during the last year, a group of professional nurses and technicians completed a specific plan to train new staff and, at the same time, a program of self-assessment of skills for experienced staff. The main purpose of this project was to promote skills development by newly added as well as experienced staff, to identify areas of weaknesses, and to correct them with training (organized by the hospital, departmental, or individual) designed to improve performance.

Patient Satisfaction With Nursing Staff in Bone Marrow Transplantation and Hematology Units

A. Piras, M. Poddigue, E. Angelucci — 2010-07-01

Abstract: Several validated questionnaires for assessment of hospitalized patient satisfaction have been reported in the literature. Many have been designed specifically for patients with cancer. User satisfaction is one indicator of service quality and benefits. Thus, we conducted a small qualitative survey managed by nursing staff in our Bone Marrow Transplantation Unit and Acute Leukemia Unit, with the objectives of assessing patient satisfaction, determining critical existing problems, and developing required interventions. The sample was not probabilistic. A questionnaire was developed using the Delphi method in a pilot study with 30 patients. Analysis of the data suggested a good level of patient satisfaction with medical and nursing staffs (100%), but poor satisfaction with food (48%), services (38%), and amenities (31%). Limitations of the study were that the questionnaire was unvalidated and the sample was small. However, for the first time, patient satisfaction was directly measured at our hospital. Another qualitative study will be conducted after correction of the critical points that emerged during this initial study, in a larger sample of patients.

Case Report: HIV Infection From a Kidney Transplant

B. Borchi, S. Ambu, S. Bresci, M. Zanazzi, M. Salvadori, F. Leoncini — 2010-07-01

Abstract: The transmission of human immunodeficiency virus (HIV) through transplantation of human tissues and organs is rare but not impossible. A 27-year-old Bulgarian woman received a kidney transplant from a cadaveric donor owing to chronic renal failure due to glomerulonephritis of unknown etiology. Five days after the donation, the tissues showed HIV-1 infection, so she was immediately initiated on highly active antiretroviral therapy (HAART) with lopinavir/ritonavir, zidovudine, enfuvirtide, and lamivudine. Subsequently, according to the genotypic test which revealed a complex resistance pattern of the HIV-1, we changed the regimen to darunavir/ritonavir, etravirine, lamivudine, and enfuvirtide. The HIV-1 genome (550 UI/mL), which was detected at 5 days after transplantation, rapidly declined to undetectable levels at 3 weeks after HAART. The CD4+ T-cell nadir was 432 cells/μL (40%) to 1,400 cells/μL after 2 years. The posttransplantation course was complicated by cytomegalovirus pneumonia. At 32 months after transplantation, the patient had experienced hypertension with secondary retinopathy, bilateral cataracts, diabetes, hypothyroidism, osteoporosis with multiple vertebral fractures, a hip prosthesis, and a bone infarction of the femur. Major management problems had been related to steroid and HAART treatment side effects. Therapeutic interactions between the immunosuppressants and the antiretroviral drugs were complex for management, requiring frequent checks of drug levels and dose-adjustments. We finally obtained a stable clinical and viroimmunologic condition. The transmission of multiresistant strains of HIV from unknown patients requires complex multidisciplinary management.

Case Report: Cystic Fibrosis, Lung Transplantation, and the Novel H1N1 Flu

S. Bresci, B. Borchi, S. Ambu, G. Taccetti, C. Braggion, F. Leoncini — 2010-07-01

Abstract: The H1N1 pandemic flu is a significant risk factor for both patients with chronic disease who need organ transplantation and transplant recipients. This population needs special care regarding comorbidities and related complications. MB, a 38-year-old Italian cystic fibrosis male patient with lung and pancreatic involvement, was referred to our division in July 2009 for fever-associated arthromyalgia, headache, and rhinitis. Lung transplantation had been performed in September 2005, and he was subsequently treated with immunosuppressive therapy: tacrolimus, everolimus, and prednisolone. In the past, chronic respiratory colonization with Pseudomonas aeruginosa and intermittent infection with Aspergillus flavus, chronic renal failure, hypertension, and diabetes mellitus complicated his clinical history. He started antiviral treatment with oseltamivir despite no travel history and no respiratory symptoms. H1N1 swab was positive. Three days later, the patient was admitted to the hospital for the persistence of fever and the onset of cough. Chest x-ray showed a left lower pneumonia, which was confirmed by computerized tomography. Broad-spectrum antibiotic therapy led to an improvement of the clinical condition. The patient was discharged 8 days later; a control swab was negative. This case report suggests some general considerations regarding solid organ recipients: 1) Flu-related complications require early treatment (both antiviral and antibiotic); 2) active microbiologic surveillance is important to prevent lethal infections (ie, invasive aspergillosis); 3) evaluation of immunosuppressant blood levels is necessary for drug-drug interactions. Active prevention is the best option for decreasing morbidity and mortality in the transplanted patient.

Transplantation in Hungary—Preface on the Occasion of Transplantation Proceedings Becoming the Official Journal of the Hungarian Transplantation Society

Robert M. Langer, Ferenc Perner — 2010-07-01

Abstract: The first, long-term successful kidney transplantation happened 37 years ago in Hungary. At the same time an organized renal program was initiated followed by transplantations of other solid organs. The authors remember previous milestone operations and the preceding events. In 1982, Hungary was the first country in the Eastern block to introduce cyclosporine. After the Iron Curtain fell new circumstances and possibilities opened for the transplant community also. Young transplant surgeons were sent to Western countries returning with new experiences. In 1992 the heart transplantation program started in Budapest. The Universities of Debrecen and Pécs joined Budapest and Szeged with renal transplant programs. In 1994, a new Department was initiated at Semmelweis University with an immediate increase of 50%. The next year a liver transplantation program was launched. Pancreas transplants were performed in 1998 in Pécs, followed by Budapest. In 2003, a collaboration was initiated between Geneva and Budapest for islet transplantation and another with Vienna for lung transplantation. This article provides an overview of Hungarian transplant activities.

Changes in Pituitary Adenylate Cyclase-Activating Polypeptide Following Renal Ischemia-Reperfusion in Rats

2010-07-01

Abstract: PACAP (pituitary adenylate cyclase-activating polypeptide) occurs in two biologically active forms, with 38 and 37 amino acid residues (PACAP38 and PACAP27). In mammalian tissues, PACAP38 is the dominant form. Diverse effects have been described in the cardiovascular, respiratory, gastrointestinal, and urogenital systems. PACAP is known for its strong cytoprotective effects, which are present endogenously as well, as proven by knockout studies and results showing that PACAP is up-regulated following diverse injuries. Little is known about such effects in the kidney. We have previously shown that PACAP is protective in renal ischemia-reperfusion injury. Therefore, the aim of the present study was to investigate the changes of endogenous PACAP following 60-minute renal ischemia using radioimmunoassay. Changes were observed within 24 hours following renal vessel clamping. In the cortex, an acute decrease was followed by an increase on the intact side, and levels returned to original ones on the operated side. In the medulla, changes were only observed on the clamped side: a marked up-regulation was detected in PACAP38-like immunoreactivity within the first 24 hours. The present study showed that PACAP38- and PACAP27-like immunoreactivities sensitively react to renal ischemia-reperfusion, the physiological importance of which awaits further investigation.

Examination of Protective Effect of Ischemic Postconditioning After Small Bowel Autotransplantation

2010-07-01

Abstract: Ischemia/reperfusion (I/R) injury is a serious condition that results from some surgical procedures, including intestinal transplantation. Ischemic postconditioning is defined as brief periods of reperfusion alternating with reocclusion applied during the early minutes after reperfusion. The objective of this study was to investigate the effect of ischemic postconditioning before small bowel autotransplantation. Total orthotopic intestinal autotransplantation was performed in 30 white domestic pigs. Grafts were stored in cold University of Wisconsin solution for 1, 3, or 6 hours. Duration of reperfusion was 3 hours in all grafts. Before reperfusion, the intestine was postconditioned via 3 cycles of ischemia for 30 seconds and reperfusion for 30 seconds (ischemic postconditioning protocol). Tissue from the small intestine was obtained after laparotomy (control group) and at the end of reperfusion periods. To monitor oxidative stress, tissue concentrations of malondialdehyde and reduced glutathione, and activity of superoxide dismutase were determined at spectrophotometry. Tissue damage on sections stained with hematoxylin- eosin was evaluated using a quantitative method (Scion Image software; Scion Corp, Frederick, Maryland). Our results demonstrated that ischemic postconditioning significantly decreased the reperfusion-ended lipid peroxidation value (mean ± SEM, 142.0 ± 7.1 μmol/g vs 125.0 ± 2.1 μmol/g; P < .05). Moreover, the capacity and activity of endogenous antioxidant protective systems (glutathione 789±8.0 μmol/g vs 934 ± 5.7 μmol/g, and superoxide dismutase 110 ± 9 IU/g vs 126 ± 4 IU/g; P < .05) remained higher in the ischemic postconditioning groups compared with tissues without ischemic postconditioning. At quantitative analysis, tissue injury was increased by the duration of cold preservation. The greatest injury was observed in the mucosal and submucosal layers and in the depth of crypts after 6 hours of preservation. Ischemic postconditioning significantly decreased intestinal wall injury in each group (P < .05). It was concluded that ischemic postconditioning before reperfusion mitigated oxidative stress and histologic damage during small bowel autotransplantation.

Comparison of Intestinal Cold Preservation Injury on Pituitary Adenylate Cyclase–Activating Polypeptide in Knockout and Wild-Type Mice

2010-07-01

Abstract: Tissue injury caused by cold preservation remains a problem in small intestinal transplantation. Pituitary adenylate cyclase-activating polypeptide (PACAP) has a central role in intestinal physiology. The objective of the present study was to compare the effects of cold ischemia injury in PACAP-38 knockout and wild-type mice after cold storage of small bowel. Cold ischemia was produced using small bowel preservation in University of Wisconsin solution at 4°C in 20 PACAP-38 wild-type mice for 1, 3, and 6 hours (groups 1, 2, and 3, respectively) and 20 PACAP-38 knockout mice for 1, 3, and 6 hours (groups 4, 5, and 6, respectively). Biopsy samples of small bowel were obtained after laparotomy (control) and at the end of preservation periods. To determine oxidative stress, malondialdehyde, reduced glutathione, and superoxide dismutase concentrations were measured. Tissue damage was assessed using a quantitative method on sections stained with hematoxylin-eosin. After 6 hours, tissue lipid peroxidation was increased significantly in PACAP-38 knockout mice (mean ± SD, 153.04 ± 7.2 μmol/g) compared with sham-operated mice (110.44 ± 5.5 μmol/g) and wild-type mice (120.0 ± 1.1 μmol/g) (P < .05). The capacity and activity of the endogenous antioxidant system decreased significantly after 3 and 6 hours of preservation (reduced glutathione, 808.7 ± 5.2 μmol/g and 720.4 ± 8.7 μmol/g; and superoxide dismutase, 125.1 ± 1.4 IU/g and 103.3 ± 1.9 IU/g vs 212.11 ± 5.8 IU/g; P < .05). Quantitative histologic analysis demonstrated destruction of mucosal and submucosal layers and crypts in knockout mice compared with wild-type mice. These processes depended on duration of cold preservation. These findings demonstrate that PACAP-38 has a key role in protection against intestinal cold preservation injury.

Mice Deficient in Neuropeptide PACAP Demonstrate Increased Sensitivity to In Vitro Kidney Hypoxia

2010-07-01

Abstract: One of the well-known effects of pituitary adenylate cyclase activating polypeptide (PACAP) is its neuroprotective and cytoprotective actions including renoprotective effects. Mice deficient in endogenous PACAP exhibit several behavioral, metabolic, and developmental alterations. Furthermore, PACAP-deficient mice have larger infarct volume in a model of cerebral ischemia, delayed axonal regeneration, and increased cell death in cerebellar oxidative stress. We have previously demonstrated that PACAP-deficient mice have increased susceptibility to in vitro oxidative stress, which can be counteracted by exogenous PACAP treatment. These results demonstrate that endogenous PACAP has a protective role against various stressors. The objective of the present study was to investigate whether endogenous PACAP has a protective effect in the kidney against in vitro hypoxia. Kidney cell cultures were isolated from wild-type and PACAP-deficient mice, and cell viability was assessed after in vitro hypoxia induced using CoCl2. The sensitivity of cells from PACAP-deficient mice was increased to hypoxia: both after 24 and 48 hours of exposure, cell viability was significantly reduced compared with that in control wild-type mice. These results show that endogenous PACAP protects against noxious stimuli in the kidney and that PACAP may act as a stress sensor in renal cells.

Arterial Stiffness in Chronic Renal Failure and After Renal Transplantation

2010-07-01

Abstract: Arterial stiffness is an independent cardiovascular risk factor, along with aging, hypertension, and cardiovascular disease. The augmentation index (AIx) and pulse wave velocity (PWV) are early markers of atherosclerotic vascular changes. Arteriography was used to determine systolic and diastolic blood pressure, pulse pressure (PP), AIx, and PWV in 82 male and 64 female renal transplant recipients (mean [SD] age, 45.3 [11.2] years). Cardiovascular risk was assessed using echocardiography and ultrasonography of the carotid arteries. The left ventricular wall thickness, ejection fraction, and stenosis of the carotid arteries were also measured. Fasting serum creatinine, cystatin C, homocysteine, C-reactive protein, immunoreactive parathyroid hormone, lipid, and calcium-phosphorus concentrations were determined. The serum cystatin concentration was 2.1 (0.2) mg/L, and the homocysteine concentration, 15.2 (2.6) μmol/L. After transplantation, body mass index, fat mass, and visceral fat area increased significantly (P < .01). The AIx was increased (AIx ≥10%) in 20% of men and 37% of women, PWV was increased (>10 m/s) in 43% of men and 34% of women, and PP was pathologically high (>12 m/s) in 10% of men and 12% of women. The PWV was significantly related to age (r = 0.52) and ventricular wall thickness (r = 0.46). Pulse pressure, BMI, and systolic and diastolic blood pressure correlated positively but modestly with PWV. There was a significant relationship between AIx80 and systolic (r = 0.42) and diastolic (r = 0.39) blood pressure and PP (r = 0.33). The ejection fraction correlated negatively with PWV and AIx. There was a strong association between carotid artery stenosis, PWV, and AIx80. All patients with PWV greater than 10 m/s demonstrated carotid artery stenosis. In conclusion, arteriography is an objective, noninvasive, and convenient method for early diagnosis and follow-up of atherosclerosis.

Insertion/Deletion Polymorphism of Angiotensin-Converting Enzyme as a Risk Factor for Chronic Allograft Nephropathy

2010-07-01

Abstract: Angiotensin-converting enzyme (ACE) inhibitor therapy is widely used to treat chronic allograft nephropathy (CAN), which suggests a possible role of the renin-angiotensin system in the pathologic mechanism of the disease. The objective of this study was to investigate the possible link between CAN and ACE. The ACE insertion/deletion polymorphism and the amount and activity of ACE were determined in cadaver kidney recipients with CAN (n = 38) or normal renal function (n = 34). The DD genotype was observed significantly more frequently in the CAN group compared with the group with normal renal function. Moreover, the DD genotype was associated with a higher serum ACE concentration and greater serum ACE activity, compared with II genotype homozygotes. The insertion/deletion polymorphism of ACE affects ACE expression and activity in serum, and, therefore, may have an important role in the pathogenesis of CAN. These findings suggest that determination of the ACE genotype may be useful in identifying patients at high risk. In particular, the DD genotype may be considered an indication for ACE inhibitor therapy.

Heat Shock Protein Polymorphism Predisposes to Urinary Tract Malformations and Renal Transplantation in Children

2010-07-01

Abstract: Background: Anatomical malformations of the kidney and urinary tract account for 17% of pediatric renal transplantation procedures. Heat shock proteins (HSPs) are molecular chaperones with a protective function that promotes cell survival. HSP72 is an endogenous ligand for toll-like receptor TLR4, thereby stimulating innate immunity. Both in adults and children, decreased expression of HSP70s is associated with a number of kidney diseases.Objective: To assess the prevalence of HSPA1A G(190)C, HSPA1B A(1267)G, and TLR4 A(896)G polymorphisms in children who had undergone kidney transplantation.Patients and Methods: Genotypes were analyzed using allele-specific polymerase chain reaction in 41 pediatric recipients. Allelic prevalence was related to reference values in 65 age- and sex-matched healthy children.Results: Clinical data did not reveal a difference between any of the groups. HSPA1B (1267)GG genotype and HSPA1B (1267)G allele were observed more frequently in the transplant recipients compared with the control group: AA vs AG: odds ratio [OR], 12.6; 95% confidence interval [CI], 1.58–100.0; P = .004; AA vs GG: OR, 20.80; 95% CI, 2.32–187.00; P = .01; and A vs G: OR, 2.10; 95% CI, 1.19–3.07; P = .01. Furthermore, the prevalence of the HSPA1B (1267)GG genotype was greater in transplant recipients with vs without urinary tract malformations: AG vs GG: OR, 0.10; 95% CI, 0.09–0.48; P = .007. No differences were observed in the other studied polymorphisms.Conclusion: Our findings suggest an association between the carrier status of HSPA1B (1267)G with urinary tract malformations, leading to end-stage renal disease requiring kidney transplantation. This observation raises further questions about the clinical and therapeutic relevance of this polymorphism to pediatric nephrology.

Results and Complications after Living Related Kidney Transplantation in Hungary: 30 Years' Experience

G. Dallos, M. Hídvégi, G. Kósa, L. Kóbori, F. Alföldy, F. Perner, J. Járay — 2010-07-01

Abstract: The number of patients suffering from kidney disorders is increasing the need for kidney transplantation. Kidneys originating from living donors (LD) show substantially better results than those originating from cadaveric donors (CD). We performed 3000 kidney transplantations between November 1973 and December 2007, including 154 from LD (5.13%). The early kidney function as measured by the delta creatinine clearance was significantly better among the LD group (P < .001). There was no significant difference in the immunologic data between the LD and the CD groups (P = .047). Four years after transplantation the glomerular filtration rate (GFR) and the serum creatinine level treated to be better among the LD group with tacrolimus versus cyclosporine immunosuppression (P = .089). In the LD group, the acute rejection rate was lower with tacrolimus- versus cyclosporine based immunosuppression (P = .014).

Small Difference in International Normalized Ratio May Yield a Significant Impact on Prioritizing Patients Listed for Liver Transplantation

2010-07-01

Abstract: Priority for liver transplantation is currently based on the Model for End-stage Liver Disease (MELD) score. The aim of our study was to assess in detail the contribution of international normalized ratio (INR) differences for MELD scores because of interlaboratory variability. The samples from 92 cirrhotic patients were measured on different systems combining three coagulometers and three thromboplastin products to determine variations in INR and MELD score. The INR differences among the first four systems varied between 0 and 0.2, resulting in MELD differences of 0 to 2. The MELD scores of 92 patients changed only among 10 possible integers so that normally 2 to 10 patients shared the same MELD value. In some cases, one MELD score difference resulted in a 10 superpositioning on the waiting list. Including one more system (mechanical vs optical) into our investigations achieved a five MELD difference. Supposing an extreme situation where one patient competes with his or her lowest, all the other with their highest possible score (and visa versa), the difference may be even 20 positions, overturning the complete waiting list. In conclusion substantial interlaboratory differences in MELD score have profound clinical consequences.

Can a Cutoff Value for Cystatin C in the Operative Setting Be Determined to Predict Kidney Function After Liver Transplantation?

B. Nemes, G. Zádori, F. Gelley, G. Gámán, D. Görög, A. Doros, E. Sárváry — 2010-07-01

Abstract: Correct assessment and follow-up of kidney function is essential in liver transplant recipients. Glomerular filtration rate (GFR) represents the functional capacity of the kidney. The GFR is generally determined on the basis of creatinine clearance using several methods. It has been suggested that cystatin C be used rather than GFR. Production of cystatin C is not dependent on the same factors as creatinine. It is filtered and completely metabolized in the glomeruli, and is not secreted by the kidney tubules. The objective of this study was to determine a preoperative cutoff value for cystatin C based on kidney function estimated after liver transplantation. At prefixed times before and after orthotopic liver transplantation (OLT), serum cystatin C and creatinine concentrations were measured, and GFR was calculated using the Cockroft-Gault equation. Patients were divided into 2 groups according to GFR on postoperative days 1 to 5. Group 1 (healthy recipients) included patients with post-OLT GFR greater than 70 mL/min; and group 2 (kidney-impaired recipients), post-OLT GFR less than 70 mL/min. Group 2 demonstrated greater risk of postoperative complications, abnormal postoperative creatinine concentrations and GFR values, and worse patient and graft survival. Based on the preoperative cystatin C concentration, postoperative kidney function can be assessed. The cutoff value for preoperative cystatin was determined using receiver operating characteristics analysis. When the preoperative cystatin C concentration exceeded 1.28 mg/L, the postoperative GFR was less than 70 mL/min in the first 5 days after OLT. These findings suggest that if the cystatin C concentration exceeds the cutoff point preoperatively, there will be deterioration of kidney function after OLT. Along with other researchers, we suggest that cystatin C is a sensitive marker of post-OLT kidney function.

Outcome of Liver Transplantation Based on Donor Graft Quality and Recipient Status

2010-07-01

Abstract: Background: Availability of suitable donor organs has always limited the number of liver transplantations performed. Use of marginal donor organs is an alternative to overcome organ shortage.Objective: To analyze the effect of various combinations of donor organ quality and recipient status on the outcome of liver transplantation.Materials and Methods: Data from 260 whole-liver transplantations performed between January 2003 and September 2009 were analyzed retrospectively. Study groups were established according to donor organ quality (marginal score 0–1 vs 2–5) and recipient status (Model for End-Stage Liver Disease [MELD] score <17 or >17). In patients at low risk, 102 received optimal grafts (good-to-good group [G/G], and 75 received marginal grafts (bad-to-good group [B/G]. In patients at high risk, 46 received optimal grafts (good-to-bad group [G/B], and 37 received marginal grafts (bad-to-bad group [B/B].Results: No differences were observed in cumulative patient and graft survival rates; however, total survival differed in the early period after transplantation, that is, within 1 year. There was a higher rate of overall postoperative complications including initial poor graft function, bleeding, infection, and kidney failure in group B/B compared with group G/B (25 of 37 patients [67.5%] vs 27 of 46 patients [59.0%]), group B/G (25 of 37 patients [68%] vs 39 of 75 patients [52%], and group G/G (25 of 37 patients [68%] vs 43 of 102 patients [42%]) (P = .04). Patients with a high MELD score (G/B and B/B) demonstrated increased risk of postoperative complications. Use of donor organs with marginal score of 2 or higher in patients with high MELD scores increased early patient mortality.Conclusion: In summary, patients with a high MELD score (G/B and B/B) are at an increased risk of post-OLT complications. In contrast, use of marginal grafts (B/G and B/B) increased the rate of hepatitis C virus recurrence and decreased the response rate to antiviral therapy. The combination of impaired donor grafts and recipients at high risk should be avoided.

Incidence of Nonmelanoma Skin Cancer After Human Organ Transplantation: Single-Center Experience in Hungary

2010-07-01

Abstract: There is increasing evidence that nonmelanoma skin cancers (NMSCs) are the most frequently observed tumors in transplant recipients. The incidence of posttransplantation NMSC was determined using our dermatologic screening program. Included in the study were 116 white adults (70 men and 46 women; median age, 49.3 years) who had undergone kidney or combined kidney-pancreas transplantation, with follow-up from September 2008 to December 2009. All patients underwent a full skin examination for NMSC, and completed a standardized questionnaire. Screening resulted in detection of 16 NMSCs in 11 patients out of 116 (9.5%). Lesions were equally distributed by sex, and were detected at a median of 4.1 years posttransplantation. Histologic analysis verified 13 basal cell carcinomas and 3 squamous cell carcinomas (ratio, 4:1). The incidence of NMSC was significantly greater in patients who received cyclosporine immunosuppression therapy (16 vs 1; P < .05), had experienced 2 or more painful sunburns before transplantation (10 vs 11), or worked outdoors (10 vs 11). These data indicate the relevance of skin cancer surveillance in transplant recipients. Our results correspond to international statistics except for the ratio of basal cell carcinoma to squamous cell carcinoma. Further studies are needed to elucidate the reasons for this difference.

Changes in Oxidative Stress in Patients Screened for Skin Cancer After Solid-Organ Transplantation

2010-07-01

Abstract: Transplant recipients are at high risk of nonmelanoma skin cancer (NMSC). Ultraviolet radiation can generate oxygen free radicals (OFRs), leading to oxidative stress and carcinogenesis, primarily during immunosuppression therapy. In the present study, changes in oxidative stress were examined in transplant recipients with and without NMSC. The study included 116 white adults who had undergone kidney or combined kidney-pancreas transplantation. Dermatologic follow-up revealed 16 NMSCs (13.8%). To monitor oxidative stress, peripheral blood samples were used to measure malondialdehyde (MDA), reduced glutathione, sulfhydryl (−SH) groups, OFRs, and activity of myeloperoxidase, superoxide dismutase, and catalase. The mean (SD) plasma MDA concentration was significantly greater in patients without NMSC compared with healthy control individuals (0.48±0.05 nmol/mL; P < .05), whereas MDA concentration in hemolysate was slightly increased. In peripheral blood samples, the MDA concentration in both plasma (0.71±0.03 nmol/mL) and hemolysate (87.74±1.25 nmol/mL) was significantly increased in the NMSC group compared with the healthy control group (0.24±0.05 nmol/mL vs 75.87±2.8 nmol/mL; P < .05) or patients without NMSC (0.48±0.04 nmol/mL vs 79.62±2.77 nmol/mL; P < .05). The reduced glutathione concentration was significantly decreased in the −SH groups compared with the healthy control group (P < .05). Antioxidant activity of myeloperoxidase (0.78±0.05 IU/mL) and catalase (1855.8±45.41 IU/mL) was significantly increased in the group without NMSC compared with the healthy control group (0.41±0.1 IU/mL vs 1642.07±82.96 IU/mL) and the NMSC group (0.93±0.03 IU/mL vs 2180.5±15.03 IU/mL). The superoxide dismutase activity was decreased slightly but not significantly. Total production of OFRs was significantly greater in the NMSC group compared with the non-NMSC group or the healthy control group (P < .05). These findings suggest that an imbalance exists between pro-oxidant and antioxidant status in transplant recipients, with a significant difference in patients with vs without NMSC.

Effect of Tacrolimus on the Excitatory Synaptic Transmission Between the Parallel Fibers and Pyramidal Cells in the Rat Dorsal Cochlear Nucleus

L. Szabó, Z. Rusznák, G. Sz″ucs, L. Asztalos, B. Pál — 2010-07-01

Abstract: Aim: The immunosuppressive drug tacrolimus has several effects on the central nervous system. Besides its protective effect in hearing deficiencies, it is also considered to be able to cause tinnitus. In the present work, we attempted to describe its effects on a characteristic synapse of the auditory system that may be involved in the pathogenesis of tinnitus.Methods/materials: Slices of the dorsal cochlear nucleus (200 μm thick) were prepared from 9- to 14-day-old Wistar rats. In response to stimulation targeting the superficial layer of the nucleus, we recorded excitatory postsynaptic currents (EPSCs) developing in the cell bodies of the pyramidal neurons using whole-cell voltage clamps. Inhibitory synaptic activity was inhibited by the application of bicuculline and strychnine. Short-term plasticity was investigated using high-frequency stimulation (50 Hz). Unambiguous identification of the investigated neurons was ensured by employing biocytin in the pipette solution, which allowed the confocal reconstruction of the cells after the functional measurements. A concentration of 1 μmol/L tacrolimus was applied extracellularly.Results: Tacrolimus effectively and reversibly inhibited glutamatergic neurotransmission in the investigated synapse from −145 ± 26 pA to −55 ± 15 pA (n = 7; P = .00928). In contrast, EPSC amplitudes without failures were not significantly reduced (from −153 ± 26 pA to −131 ± 23 pA) in the presence of tacrolimus, but there were increased failure numbers of synaptic transmission. These data suggested that application of tacrolimus produced a combined pre- and postsynaptic inhibition.Conclusion: Tacrolimus affected short-term synaptic plasticity in the rat dorsal cochlear nucleus. It was also capable of inhibiting the glutamatergic neurotransmission. These effects suggested that tacrolimus may have neuroprotective effects in this structure.

Significance and Imaging of Lumbar Veins and Early-Branching Arteries in Planning Living-Donor Laparoscopic Nephrectomy: Two Case Reports From 21 Months' Experience

2010-07-01

Abstract: A key aspect in planning laparoscopic living-donor nephrectomy is mapping of vascular variations. Lumbar veins and early-branching renal arteries are of utmost importance. To date, 43 candidates including 18 men and 25 women aged 25 to 67 years have been examined at our clinic using 16-section multidetector-row computed tomography angiography. Each examination was double-checked by an experienced radiologist. Of the 43 patients, 31 underwent surgery. In 29 of 31 patients (93.5%), the anatomy observed during surgery was identical to that demonstrated on the preoperative computed tomography scan. In 1 of 2 patients, 2 separate arteries were found at surgery, rather than the prognosticated early-branching arteries. In this patient, conversion to open surgery was necessary. In the other patient, a lumbar vein running into a retroaortic renal vein was discovered. In this patient, a 6-mm length of the joint stem contained the wall of the aorta and the periaortic tissue; thus, technically they were of separate origins. Careful mapping of the anatomy helps to prevent unexpected operative complications that are difficult to manage. Correct interpretation of the data must always be based on agreement between the radiologist and the surgeon.

Spontaneous Pneumomediastinum After Kidney Transplantation: Case Report

2010-07-01

Abstract: Spontaneous pneumomediastinum is a rare condition with nonspecific signs and symptoms. A 39-year-old underwent cadaver kidney transplantation. After an uncomplicated operation, progressive dyspnea of unknown origin developed. Findings at chest radiography suggested pneumomediastinum, which was confirmed at computed tomography. Esophageal or tracheal injury was ruled out. The rapidly developing atelectasis of the left lung necessitated urgent bronchoscopy, which revealed occlusion of the left main bronchus. After removal of the occluding mucus plug, the clinical symptoms immediately improved, and the spontaneous pneumomediastinum resolved within 3 days. Asymptomatic increase in airway secretions in patients receiving peritoneal dialysis may result in mucus plug formation during general anesthesia, which can cause spontaneous pneumomediastinum.

Mitigation of Cytokine Storm by Intraoperative Use of Renal Replacement Therapy During Combined Liver-Kidney Transplantation

I. Fehervari, J. Fazakas, Z. Gerlei, B. Nemes, L. Kobori — 2010-07-01

Abstract: Background: Combined liver-kidney transplantation (CLKT) is a widely used multiorgan transplantation with good graft survival rates. Previous studies have shown beneficial effects of renal replacement therapy in critically ill patients. This observation led us to use intraoperative continuous veno-venous hemofiltration (CVVH) during multiorgan transplantations.Methods: We analyzed (CRP) inflammatory response parameters of tumor necrosis factor (TNF)α, interleukin(IL)-6, procalcitonin (PCT) and C-reactive protein (CRP) at various stages of the combined transplantations.Results: All patients survived with well-functioning grafts. Mean ± SD follow-up was 32.8 ± 14.2 months. During the whole operation we used intraoperative CVVH starting at the beginning and continuing in the intensive care unit (ICU) afterward (mean ± SD, 11.2 ± 8.4 hours). Intraoperative TNFα, IL-6, CRP, and PCT were measured before surgery, during hepatectomy in the anhepatic phase, before and after liver reperfusion, exactly before kidney reperfusion, after kidney reperfusion, and upon arrival in the ICU. The wash-out of cytokines together with hemodynamic stability gave optimal circumstances for recovery of the transplanted organs.Conclusions: CVVH-based therapy offered stable intraoperative parameters, prevention of fluid overload, correction of metabolic disturbances, and wash-out of cytokines, which gave optimal circumstances for recovery of transplanted organs.

Smoking Following Renal Transplantation in Hungary and Its Possible Deleterious Effect on Renal Graft Function

P. Gombos, R.M. Langer, R. Korbely, M. Varga, A. Kaposi, E. Dinya, V. Müller — 2010-07-01

Abstract: Smoking is a known risk factor for kidney damage and also influences graft function following renal transplantation. Because smoking habits following kidney transplantation are not systematically evaluated, we analyzed them in a single center in Hungary. The survey was conducted among 402 randomly selected kidney graft recipients. We assessed smoking-related questions as well as clinical kidney disease and transplantation data. Posttransplantation renal function was analyzed based on serum creatinine values at 1 month and at 3 years after transplantation. In our study 25% (n = 102) of patients continued to smoke after transplantation. Smokers who received grafts displayed a significantly younger age compared with nonsmokers (40.1 ± 13.4 vs 47.1 ± 12.7 years; P < .001) independent of underlying kidney disease. Posttransplantation kidney function in smokers did not differ at 1 month after engraftment, but was significantly impaired at 3 years as assessed based on serum creatinine levels: 138.9 ± 42.4 versus 128.4 ± 48.5 μmol/L (P < .05). Decrease of renal function correlated with smoking intensity defined in pack-years (r2 = 0.102; P < .05). Smoking is common following kidney transplantation in Hungary and might represent a risk factor for kidney damage following renal transplantation. Therefore, effective tobacco-dependence treatment is necessary in this patient population.

Should a Complex Uropathy Be a Contraindication for Renal Transplantation in Children?

2010-07-01

Abstract: Background: Anatomic and functional disorders of the lower urinary tract represent up to 40% of the causes of renal failure in children. Several centers avoid renal transplantation in these patients because of the high risk of complications and lower graft survival. The aim of this work was to determine the frequency of urinary tract abnormalities (UTAs) among our pediatric series, and to compare the frequency of complications, function, and long-term graft survival among patients without versus with UTA.Methods: This single-center, retrospective study compared outcomes between pediatric recipients with versus without UTA. We analyzed demographic features, etiology, pretransplant protocol, urinary tract rehabilitation, incidence of complications, rejection events, as well as graft function and survival.Results: Among 328 pediatric cases performed between 1998 and 2008, we excluded nine patients due to incomplete medical records, analyzing 319 procedures in 312 patients. Sixty-seven patients (21%) had UTA. The average age, weight, and height at the time of grafting were significantly lower in the urologic group: 11.1 versus 12.6 years, 28.8 versus 34.4 kg; 125.4 versus 138.4 cm, respectively. There were significantly higher frequencies of a transperitoneal approach and vena cavae and aortic anastomoses among patients with UTA (P < .001), posing a greater technical challenge in this population. No differences in creatinine levels were observed at 0.5, 1, 2, 5, and 10 years: 1.3 versus 1.6 at 5 years, and 1.4 versus 1.5 at 8 years. Urologic complications, including urinary tract infections (UTIs), occurred among 80.6% of patients with UTA versus 42.1% in the non-UTA group (P < .001). UTIs appeared predominantly in patients with UTA (62.7% vs 35.3%, P < .001), representing a 2.7-fold risk compared with those children transplanted for other reasons. Rejection incidence was similar in both groups (49.8%). There was no significant difference in 5-y (89.8% vs 85%) or 10-year (83% vs 67%) graft survivals between the groups (P = .162).Conclusion: Our results demonstrated that with proper interdisciplinary care, graft and patient survivals of pediatric recipients with UTAs were not affected; therefore, these patients should not be rejected for transplantation.

Renal Grafts With Multiple Arteries: A Relative Contraindication for a Renal Transplant?

2010-07-01

Abstract: Introduction: Advances in surgical techniques had achieved good outcomes in renal transplantation. There has been controversy with respect to the impact of multiple arteries on the outcome of the renal transplantations.Objectives: The objectives of this study were to examine the renal function and incidence of complications among grafts with one versus two or more arteries.Materials and methods: We evaluated 86 patients with renal transplantations between January 2006 and January 2008 as a retrospective comparative study. The patients were stratified according to the number of renal graft arteries: group 1 had one artery (n = 66); group 2, two or more arteries (n = 16).Results: The warm ischemia time was shorter among group 1 compared with group 2 (P < .03). There were significant differences between the groups with respect to mean blood pressure at 1 year (P < .04). The kidney biopsies after 1-year follow-up did not show any difference.Conclusion: We considered that the presence of anatomic variations was not a contraindication for renal transplantation, but that it is necessary to continue our follow-up to determine the real impact of these variations on graft and patient survivals.

Role of Major Histocompatibility Complex and Ethnicity in Acute Renal Graft Rejection

2010-07-01

Abstract: Background: The major histocompatibility complex (MHC) plays a main role in antigen presentation. Class I, II, and III genes form defined “blocks” of conserved DNA sequences (conserved extended haplotypes) that are useful to follow the ancestry of a population. Each variant encodes a specific peptide that determines a particular individual's immune response. In addition, differential expression of HLA antigens in certain physiological and pathological conditions may participate in the pathogenesis of allograft rejection versus tolerance.Objectives: The aim of this study was to determine whether the specific HLA ancestry was associated with acute renal graft rejection among the Mexican mestizo population.Materials and Methods: We studied 544 Mexican mestizo renal donors and their respective recipients for their serologically determined HLA and based on antigens haplotype assignments. The acute rejection group was compared with the nonrejection group among donors and recipients, correspondingly.Results: Frequent Mexican alleles were observed in this study. Moreover, HLA-B*61/-DR*04, HLA-A*35/-DR*06 (Amerindian ancestry), HLA-A*68/-DR*01, HLA-A*28/-B*65/-DR*01 (African ancestry), and HLA-A*33/-B*65 (Caucasian ancestry) in donors were associated with acute renal graft rejection episodes.Conclusion: Knowing the ancestry of a donor's HLA molecules may help to individualize immunosuppressive therapy for postransplant surveillance, because they may be more membrane-exposed in parenchymal cells, making them more susceptible of being recognized by the recipient's immune system.

Risk Factors for Banff Borderline Acute Rejection in Protocol Biopsies and Effect on Renal Graft Function

2010-07-01

Abstract: Introduction: The interpretation and handling of Banff borderline acute rejection observed in protocol biopsies from patients with stable renal function continues to be controversial. Our objective was to identify the risk factors for borderline acute rejection on 1-year protocol biopsies and to evaluate their effect on renal graft function after 2 years' follow-up.Methods: We included 82 kidney transplant recipients (KTR), who underwent 1-year protocol biopsies with normal or stable graft function. All KTR had follow-up of at least 2 years posttransplantation. We formed three groups: (1) KTR with a normal biopsy, (2) KTR with borderline changes, and (3) KTR with interstitial fibrosis/tubular atrophy (IF/TA). We searched for risk factors related to borderline injury. The main outcome to evaluate was renal function at 1 month, at protocol biopsy, and 2 years posttransplant.Results: The 82 patients included in this study showed no differences in immunosuppression, gender, etiology of renal failure, or percentage of panel-reactive antibodies. The risk factors associated with borderline lesions were: at least one biopsy due to allograft dysfunction and acute rejection events during the first year posttransplant (P = .011 and P = .021, respectively). Increased serum creatinine and estimated glomerular filtration rate decline were greater among the borderline lesion than the normal group, but similar to patients with IF/TA.Conclusion: Renal function decline was greater among borderline and IF/TA groups. However, the sum of insults, and not only the borderline injury itself, produces greater declines in renal function with greater risk for graft loss.

Long-Term Complications and Survival of Patients After Orthotopic Liver Transplantation

2010-07-01

Abstract: Introduction: The survival rates among patients after orthotopic liver transplantation (OLT) has increased to 83% and 75% at 1 and 5 years, respectively. However, these patients are at increased risk of long-term complications.Objective: To identify long-term complications and survivals among patients after OLT.Methods: From September 1999 to July 2009 we evaluated long-term complications among 78 consecutive patients after OLT including 46 males.Results: Complications de novo after OLT were metabolic (n = 38; 67%), infections (n = 13; 23%), recurrent liver disease (n = 12; 21%), osteopenia/osteoporosis (n = 10; 18%), acute/chronic rejection (n = 8; 14%), renal failure (n = 2; 4%) and Kaposi's sarcoma (n = 1). Their overall survival at 118 months was 55%.Conclusions: The most common long-term complications after OLT were metabolic, infections, and disease recurrence.

Anesthetic Risk Factors Associated With Early Mortality in Pediatric Liver Transplantation

2010-07-01

Abstract: Introduction: Early mortality in pediatric patients after liver transplantation (30 days) may be due to surgical and anesthetic perioperative factors.Objective: To identify anesthetic risk factors associated with early mortality in pediatric patients who undergo liver transplantation (OLT).Materials and Methods: This retrospective study of all patients who underwent a deceased or living donor liver transplantation evaluated demographic variables of age, weight, gender, degree of malnutrition, and etiology, as well as qualitative variables of anesthesia time, bleeding, massive transfusion, acid–base balance, electrolyte and metabolic disorders, as well as graft prereperfusion postreperfusion characteristics. Chi-square tests with corresponding odds ratio (OR) and 95% confidence intervals as well as Interactions were tested among significant variables using multivariate logistic regression models. P ≤.05 was considered significant.Results: We performed 64 OLT among whom early death occurred in 20.3% (n = 13). There were deaths associated with malnutrition (84.6% vs 43.6%) in the control group (P < .01); massive bleeding, 76.9% (n = 10) versus 25.8% in the control group (P < .05) including transfusions in 84.6% (n = 11) versus 43.6% in the control group (P < .03); preperfusion metabolic acidosis in 84.6% (n = 11) versus 72.5% (n = 37; P < .05); posttransplant hyperglycemia in 69.2% (n = 9) versus 23.5% (n = 12; P < .01); and postreperfusion hyperlactatemia in 92.3% (n = 12) versus 68.6% (n = 35; P < .045).Conclusion: Prereperfusion metabolic acidosis, postreperfusion hyperlactatemia, and hyperglycemia were significantly more prevalent among patients who died early. However, these factors were exacerbated by malnutrition, bleeding, and massive transfusions. Postreperfusion hypokalemia and hypernatremia showed high but not significant frequencies in both groups.

Surgical Technique in Cadaveric Donors for Partial Hand Allotransplant in Patients With Rheumatoid Arthritis

2010-07-01

Abstract: For patients with severe hand deformities due to rheumatoid arthritis, we propose an allotransplantation of an osteomyotendinose structure (OMTS), preserving the recipient's skin and sensory nerves. Our objective was to develop the surgical technique in a 10 cadavers, five as donors and five as recipients. The donor's hand was 10% to 15% smaller than the recipient's. Dissections were performed by two surgical teams under magnification. In the donor, the OMTS was procured at the distal third of the forearm, maintaining the integrity of the arterial system, with its concomitant veins and motor branches of the median and ulnar nerves, leaving the skin envelope. In the recipient, the OMTS was removed, taking care to preserve the cutaneous cover with the digital arteries in continuity with the superficial palmar arch and radial and ulnar arteries. Also, the digital nerves were maintained in the skin flap, in continuity with the median and ulnar nerves. Their motor branches were divided after emergence from the main nerves. The superficial dorsal veins and radial nerve were kept adhered to the cutaneous cover. Then, the donor OMTS was placed within the recipient cutaneous flap; all the anatomic structures were repaired. The average surgical time was 780 minutes. Methylene blue was present in the digital arteries. There were no difficulties in the anatomic repair. The surgical technique is quite laborious, especially the dissection of the recipient interdigital spaces. Due to the requirement for arterial system integrity, the cutaneous flap must be viable. Also, the allotransplanted OMTS has all necessary conditions to obtain good tissue perfusion for subsequent function. Procurement without skin permits a greater opportunity to find donors, and greater social and personal acceptance by the recipient.

Successful Renal Transplantation From a Deceased Donor With Pesticide Intoxication: A Case Report

2010-07-01

Abstract: The number of individuals awaiting organ transplantation exceeds the number of organs. Patients who die from intoxication are rarely accepted as potential organ donors. Herein we have presented the results of kidney transplantations performed from a deceased 20-year-old female donor with suicidal ingestion of a pesticide (carbamate). The procured kidneys were successfully transplanted. Patients and grafts are doing well at 4 months following transplantation. There are few reports of successful transplantation of organs obtained from patients who die from various intoxications. Poisoned patients represent another pool of organ donors for transplantation services.

Author Index

2010-07-01

Transplantation Proceedings

Editorial Board

Contents

Similar Early Complication Rate in Simultaneous Pancreas and Kidney Recipients on Tacrolimus/Mycophenolate Mofetil Versus Tacrolimus/Sirolimus Immunosuppressive Regimens

Anti-CD25 Antibody (Daclizumab) Maintenance Therapy in Pancreas Transplantation

Alemtuzumab with Rapid Steroid Taper in Simultaneous Kidney and Pancreas Transplantation: Comparison to Induction with Antithymocyte Globulin

Simultaneous Pancreas and Kidney Transplantation With Concurrent Allograft Nephrectomy for Recipients With Prior Renal Transplants Lost to BK Virus Nephropathy: Two Case Reports

Pancreas Oxygen Persufflation Increases ATP Levels as Shown by Nuclear Magnetic Resonance

Persufflation Improves Pancreas Preservation When Compared With the Two-Layer Method

Continuous Real-time Viability Assessment of Kidneys Based on Oxygen Consumption

Improved Method of Human Islet Isolation for Young Donors

Islet Isolation From Human Pancreas With Extended Cold Ischemia Time

Improved Method of Porcine Pancreas Procurement With Arterial Flush and Ductal Injection Enhances Islet Isolation Outcome

Rapid Quantitative Assessment of the Pig Pancreas Biopsy Predicts Islet Yield

The Impact of Ischemic Stress on the Quality of Isolated Pancreatic Islets

A New Method to Value Efficiency of Enzyme Blends for Pancreatic Tissue Digestion

Assessment of Human Islet Isolation With Four Different Collagenases

Tissue Dissociation Enzyme Neutral Protease Assessment

Culture of Impure Human Islet Fractions in the Presence of Alpha-1 Antitrypsin Prevents Insulin Cleavage and Improves Islet Recovery

Inhibition of Inflammatory Cytokine-Induced Response in Human Islet Cells by Withaferin A

Excellence of Suito Index for Assessing Clinical Outcome of Islet Transplantation

Secretory Unit of Islet Transplant Objects (SUITO) Index Can Predict Outcome of Intravenous Glucose Tolerance Test

Pancreata From Pediatric Donors Restore Insulin Independence in Adult Insulin-Dependent Diabetes Mellitus Recipients

Neurogenic Differentiation 1 Directs Differentiation of Cytokeratin 19–Positive Human Pancreatic Nonendocrine Cells into Insulin-Producing Cells

An Acidic pH and Activation of Phosphoinositide 3-Kinase Stimulate Differentiation of Pancreatic Progenitors Into Insulin-Producing Cells

Induction of Insulin-Producing Cells From Human Pancreatic Progenitor Cells

Comparison of Fresh and Cultured Islets From Human and Porcine Pancreata

Effects of Pancreas Transplantation on Oxidative Stress in Pulmonary Tissue from Alloxan-Induced Diabetic Rats

Pancreas Transplantation Prevents Morphologic and Ultrastructural Changes in Pulmonary Parenchyma of Alloxan-Induced Diabetic Rats

Vascularization of Artificial Beds for Pancreatic Islet Transplantation in a Rat Model

C5a-Inhibitory Peptide Combined with Gabexate Mesilate Prevents the Instant Blood-Mediated Inflammatory Reaction in a Rat Model of Islet Transplantation

Magnetic Resonance Imaging of Transplanted Mouse Islets Labeled With Chitosan-Coated Superparamagnetic Iron Oxide Nanoparticles

Effect of Triple Costimulation Blockade on Islet Allograft Survival in Sensitized Mice

Imaging of Human Islet Vascularization Using a Dorsal Window Model

A Survey of the Attitudes of Scientists Toward Xenotransplantation in Taiwan

Some Ethical Aspects of Xenotransplantation in Light of the Proposed European Directive on the Protection of Animals Used for Scientific Purposes

The Attitude of Scottish Citizens to Xenotransplantation in the South East of Spain: An Emerging Population Subgroup

Are Veterinary Students in Favor of Xenotransplantation? An Opinion Study in a Spanish University With a Xenotransplantation Program

Infection of Porcine Cells with Human Herpesviruses

The In Vitro Protection of Human Decay Accelerating Factor and hDAF/Heme Oxygenase-1 Transgenes in Porcine Aortic Endothelial Cells Against Sera of Formosan Macaques

In Vitro Production of Multigene Transgenic Blastocysts via Sperm-Mediated Gene Transfer Allows Rapid Screening of Constructs to Be Used in Xenotransplantation Experiments

ABO and RH1 Blood Group Phenotyping in Pigs (Sus scrofa) Using Microtyping Cards

Surgical and Nonsurgical Complications of a Pig to Baboon Heterotopic Heart Transplantation Model

Left Ventricular Pressure Measurement by Telemetry Is an Effective Means to Evaluate Transplanted Heart Function in Experimental Heterotopic Cardiac Xenotransplantation

Super-High-Dose Islet Transplantation Is Associated With High SUITO Index and Prolonged Insulin Independence: A Case Report

ET-Kyoto Ductal Injection and Density-Adjusted Purification Combined With Potent Anti-Inflammatory Strategy Facilitated Single-Donor Islet Transplantation: Case Reports

Percutaneous Embolization of Periduodenal Varix Due to Portal Hypertension in a Patient With Kidney–Pancreas Transplantation: A Case Report

Preface

The Italian Society for Safety and Quality in Transplantation (SISQT)

Electrical Safety During Transplantation

National Policies for Risk Management in Italy

Unintended Transplantation of Three Organs from an HIV-Positive Donor: Report of the Analysis of an Adverse Event in a Regional Health Care Service in Italy

Do “Silent” Brain Deaths Affect Potential Organ Donation?

Safety of Bone Marrow Stem Cell Donation: A Review

Efficiency of the Tissue Procurement Process: What Evaluation Tools?

Ensuring Safety and Quality of Tissues in Italy: Application of European Directives

The Role of Nurses in the Risk Management of Organ and Tissue Donation

Calcineurin Inhibitors in Renal Transplantation Still Needed but in Reduced Doses: A Review

Analysis of Four Scoring Systems and Monocentric Experience to Optimize Criteria for Marginal Kidney Transplantation

C4d-Positive Renal Allograft Rejection Biopsies in Cyclosporine-Treated Patients: Single-Center Incidence and Outcome

Kidney Graft Survival Rates Do Not Improve by Era: The Impact of the Age Factor

Fibrosis Progression and the Pros and Cons of Antiviral Therapy for Hepatitis C Virus Recurrence After Liver Transplantation: A Review

Frozen-Section Diagnosis in Donor Livers: Error Rate Estimation of Steatosis Degree

Liver Transplant Quality and Safety Plan in Anesthesia and Intensive Care Medicine

Patient Satisfaction Among Liver Transplant Recipients: Single-Center Survey

Transplantation of Hematopoietic Stem Cells: Role of National Transplantation Center

Human De-epidermized Dermis as a Stem Cell Carrier

A Common Polymorphism in the Monocyte Chemoattractant Protein-1 (MCP-1) Gene Regulatory Region Influences MCP-1 Expression and Function of Isolated Human Pancreatic Islets

Functional and Survival Analysis of Isolated Human Islets

Risk Analysis of Hematopoietic Stem Cell Transplant Process: Failure Mode, Effect, and Criticality Analysis and Hazard Analysis Critical Control Point Methods Integration Based on Guidelines to Good Manufacturing Practice for Medicinal Product ANNEX 20 (February 2008)

Key Role of Staff Competencies for Patient and Donor Safety in a Bone Marrow Transplantation Unit: Design and Implementation of an Accredited Training and Self-Assessment Program

Patient Satisfaction With Nursing Staff in Bone Marrow Transplantation and Hematology Units

Case Report: HIV Infection From a Kidney Transplant

Case Report: Cystic Fibrosis, Lung Transplantation, and the Novel H1N1 Flu

Transplantation in Hungary—Preface on the Occasion of Transplantation Proceedings Becoming the Official Journal of the Hungarian Transplantation Society

Changes in Pituitary Adenylate Cyclase-Activating Polypeptide Following Renal Ischemia-Reperfusion in Rats

Examination of Protective Effect of Ischemic Postconditioning After Small Bowel Autotransplantation

Comparison of Intestinal Cold Preservation Injury on Pituitary Adenylate Cyclase–Activating Polypeptide in Knockout and Wild-Type Mice

Mice Deficient in Neuropeptide PACAP Demonstrate Increased Sensitivity to In Vitro Kidney Hypoxia

Arterial Stiffness in Chronic Renal Failure and After Renal Transplantation