Wen Yuan Chung

Evaluation of Microrna375 as a Novel Biomarker for Graft Damage in Clinical Islet Transplantation

Background Early and sensitive detection of islet graft damage is essential for improving posttransplant outcomes. MicroRNA 375 (miR375) has been reported as a biomarker of pancreatic &bgr;-cell death in small animal models. Methods The miR375 levels were measured in purified human islets, sera from patients with autologous and allogeneic islet transplantation as well as total pancreatectomy alone (nontransplanted group). The miR375 levels were also determined in a miniaturized in vitro tube model comprising human islets and autologous blood. Results The miR375 expression level in islets was dose-dependent (P < 0.001) and significantly elevated after islet damage in plasma in the in vitro model (P = 0.003). Clinical analysis revealed that circulating miR375 levels in both autologous and allogeneic islet recipients were significantly elevated for 7 days after islet infusion, compared with the nontransplanted group (P = 0.005 and <0.001, respectively). Furthermore, miR375 detected the difference in islet graft damage among 3 different anti-inflammatory protocols for clinical autologous transplantation (P < 0.01). Conclusions Circulating miR375 can be a reliable biomarker to detect graft damage in clinical islet transplantation because serum C-peptide and proinsulin levels are difficult to interpret due to the influence of multiple factors, such as &bgr;-cell stress and physiological response.

Addition of a kidney to the normothermic ex vivo perfused porcine liver model does not increase cytokine response.

The addition of a kidney to the ex vivo liver perfused model may facilitate the circuit homeostatic balance of important biochemical parameters (i.e. pH changes, urea and creatinine, or glucose levels) but might also increase the inflammatory reaction produced. In this study, we compared the production of various cytokines between liver–kidney and liver-alone circuits. Seven livers were harvested from female pigs and perfused for 6xa0h. In five additional experiments, a kidney was also harvested and connected in parallel. Blood samples for interleukins (IL) 1, 2, 4, 6, 8, 10, and 12, interferon (IFN)-γ and tumor necrosis factor (TNF)-α were collected before perfusion and at hours 1, 2, 4 and 6 postperfusion. In the combined liver–kidney circuit, a significant increase was present only for IL-6 and IL-8, but this did not differ significantly from those recorded in the liver-alone circuit. All other cytokines were not modified from baseline levels. The addition of a kidney to the perfusion circuit does not stimulate a greater inflammatory reaction than that of the liver alone and therefore further confirms the safety of the experimental setups in view of more delicate experiments requiring strict homeostatic conditions.

Use of the Recanalised Umbilical Vein for Islet Autotransplantation following Total Pancreatectomy

Introduction: Islet autotransplantation requires access to the portal vein or tributaries. We originally infused islets into the liver via the middle or right colic veins, but since 2005 we have used the recanalised umbilical vein. Here, we describe the technique, the advantages and the early results achieved. Materials and Methods: After removal of the pancreas and restoration of the biliary and enteric continuity, the ligamentum teres is transected. The obliterated umbilical vein is identified and recanalised with Bakes dilators giving access to the left portal vein. A Vygon® Nutricath ‘S’ 11-Fr catheter is inserted and used for the islet infusion. If the ligamentum teres is to be exteriorised for postoperative measurements or subsequent access, it is pulled through a 10-mm laparoscopic port in the epigastrium, sutured to the skin and covered with a dressing. Results: We have used this approach in 17 patients and exteriorised the falciform ligament in 4. There have been no intra- or postoperative complications. Conclusions: The recanalised umbilical approach is safe and allows for venous sampling and postoperative measurements of the portal pressure. Under local anaesthetic, the umbilical vein can be approached above the umbilicus and exteriorised if repeated transplants are required for allograft patients.

Current principles and practice in autologous intraportal islet transplantation: a meta-analysis of the technical considerations.

Autologous islet transplantation (IAT) following pancreatectomy is now a recognized, albeit highly specialized procedure carried out in a small number of centers worldwide. Current clinical principles and best practice with emphasis on examining the technical aspects of surgery in centers with significant IAT experience are reviewed.

The “kidney–liver” multiorgan ex vivo perfused model improves the circuit’s biochemical milieu during perfusion compared to the “liver–kidney” counterpart

The multiorgan ex vivo perfused liver–kidney model allows studying the hepatic pathophysiology and purifying waste products. We tested if the addition of the kidney first followed by the liver (KL circuit) produces better results compared to the classic liver-first approach (LK). Intact livers and kidneys were obtained post mortem from ten female domestic white pigs, five experiments were conducted with the KL circuit and five with the LK. Bile, urine production, arterial blood gases, glucose, renal and liver tests were collected hourly during the perfusions. The KL circuit had values more close to physiological ranges, more stable over time and showed less variability compared to the LK circuit for urine production, glucose, PH, anion gap, lactate, urea, sodium, potassium and Alanine Transaminase (ANOVA test for repeated measures pxa0<xa00.05). The KL circuit produced a more physiological and reliable biochemical milieu.

Ex Vivo Porcine Organ Perfusion Models as a Suitable Platform for Translational Transplant Research

In transplantation surgery, extending the criteria for organ donation to include organs that may have otherwise been previously discarded has provided the impetus to improve organ preservation. The traditional method of cold static storage (CS) has been tried and tested and is suitable for organs meeting standard criteria donation. Ex vivo machine perfusion is, however, associated with evidence suggesting that it may be better than CS alone and may allow for organ donation criteria to be extended. Much of our knowledge of organ preservation is derived from animal studies. We review ex vivo porcine organ perfusion models and discuss the relevance to the field of transplantation surgery. Following a systematic literature search, only articles that reported on experimental studies with focus on any aspect(s) of ex vivo and porcine perfusion of organs yet limited to the context of organ transplantation surgery were included. The database search and inclusion/exclusion criteria identified 22 journal articles. All 22 articles discussed ex vivo porcine organ perfusion within the context of transplant preservation surgery: 8 liver, 3 kidney, 3 lung, 2 pancreas/islet, 4 discussed a combined liver-kidney multiorgan model, 1 small bowel, and 1 cardiac perfusion model systems. The ex vivo porcine perfusion model is a suitable, reliable, and safe translational research model. It has advantages to investigate organ preservation techniques in a reproducible fashion in order to improve our understanding and has implications to extend the criteria for organ donation.

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an extended perfusion period is challenging. We have added the kidney to our previous ex vivo perfused liver experiment model to reproduce a more accurate physiological state for prolonged experiments without using live animals. Five intact livers and kidneys were retrieved post-mortem from sacrificed pigs on different days and perfused for a minimum of 6 hr. Hourly arterial blood gases were obtained to analyze pH, lactate, glucose and renal parameters. The primary endpoint was to investigate the effect of adding one kidney to the model on the acid base balance, glucose, and electrolyte levels. The result of this liver-kidney experiment was compared to the results of five previous liver only perfusion models. In summary, with the addition of one kidney to the ex vivo liver circuit, hyperglycemia and metabolic acidosis were improved. In addition this model reproduces the physiological and metabolic responses of the liver sufficiently accurately to obviate the need for the use of live animals. The ex vivo liver-kidney perfusion model can be used as an alternative method in organ specific studies. It provides a disconnection from numerous systemic influences and allows specific and accurate adjustments of arterial and venous pressures and flow.

Ex vivo normothermic porcine pancreas: A physiological model for preservation and transplant study

INTRODUCTIONnAn ex vivo normothermic porcine pancreas perfusion (ENPPP) model was established to investigate effects of machine perfusion pressures on graft preservation.nnnMETHODOLOGYnNine porcine pancreata were perfused with autologous blood at 50u202fmmHg (control) pressure. Graft viability was compared against four ex-vivo porcine pancreata perfused at 20u202fmmHg (low) pressure. Arterio-venous oxygen gas differentials, biochemistry, and graft insulin responses to glucose stimulation were compared. Immunohistochemistry stains compared the cellular viability.nnnRESULTSnControl pancreata were perfused for a median of 3u202fh (range 2-4u202fh) with a mean pressure 50u202fmmHg and graft flow 141u202fmLu202fmin-1. In comparison, all of the low pressure models were perfused for 4u202fh, with mean perfusion pressure 20u202fmmHg and graft flow 40u202fmL.min-1. All pancreata demonstrated cellular viability with evidence of oxygen consumption with preserved endocrine and exocrine function. However, following statistical analysis, the low pressure perfusion of porcine pancreata compared favourably in important biochemical and immunohistochemistry cellular profiles; potentially arguing for an improved method for graft preservation.nnnCONCLUSIONnENPPP will facilitate whole organ preservation to be studied in further detail and avoids use of expensive live animals. ENPPP is reproducible and mimics a donation after circulatory death scenario.

Case Report of Mannose-Binding Lectin (MBL) Deficiency and Postoperative Sepsis and Coagulopathy in a Patient Following Total Pancreatectomy for Chronic Pancreatitis

CONTEXTnComplement plays a central role against infection and coordinates the activity of coagulation and fibrinolysis. In this report we present a patient that underwent total pancreatectomy experienced sepsis, coagulopathy and bleeding that endangered the postoperative course.nnnCASE REPORTnA sixty-five-year-old woman underwent total pancreatectomy for intractable pain without islet transplant, this patient was diagnosed as AP and MBL deficient from a blood test performed preoperatively. On the postoperative course she experienced severe haemorrhages and sepsis for 3 weeks postoperatively. An analysis of serial perioperative serum samples conducted which showed further depletion of the alternate and MBL complement pathway without restoration to baseline levels.nnnCONCLUSIONnThis is the first reported case of alternative and mannose-binding lectin pathways depletion associated with major postoperative bleeding and sepsis following pancreatic surgery. Future research should examine the relationship between complement pathways activity and postoperative complications in order to possibly introduce it as a preoperative screening and possible replacement therapy prior to any major surgical intervention.