Classic Transplantation Research Articles

Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an attractive alternative to classic organ transplantation. This technology utilizes an organ's natural biological extracellular matrix (ECM) as a scaffold onto which autologous or stem/progenitor cells may be seeded and cultured in such a way that facilitates regeneration of the original tissue. The natural ECM is isolated by a process called decellularization. Decellularization is accomplished by treating tissues with a series of detergents, salts, and enzymes to achieve effective removal of cellular material while leaving the ECM intact. Studies conducted utilizing decellularization and subsequent recellularization of rodent lungs demonstrated marginal success in generating pulmonary-like tissue which is capable of gas exchange in vivo. While offering essential proof-of-concept, rodent models are not directly translatable to human use. Nonhuman primates (NHP) offer a more suitable model in which to investigate the use of bioartificial organ production for eventual clinical use. The protocols for achieving complete decellularization of lungs acquired from the NHP rhesus macaque are presented. The resulting acellular lungs can be seeded with a variety of cells including mesenchymal stem cells and endothelial cells. The manuscript also describes the development of a bioreactor system in which cell-seeded macaque lungs can be cultured under conditions of mechanical stretch and strain provided by negative pressure ventilation as well as pulsatile perfusion through the vasculature; these forces are known to direct differentiation along pulmonary and endothelial lineages, respectively. Representative results of decellularization and cell seeding are provided.

Modified Multivisceral Transplantation in the Rat

Modified multivisceral transplantation (MMT) is a current therapy consisting of multiple abdominal organs implantation: mostly stomach, duodenum, pancreas, and intestine. It is indicated for gastrointestinal disorders without liver involvement as pseudo-obstruction syndrome, extensive inflammatory bowel diseases, and diffuse abdominal tumors (1–3). Evisceration techniques may vary depending on the underlying abdominal disease. However, different from the classic multivisceral transplantation, the recipient’s liver is always maintained (1–3). The result of MMT is improving; nevertheless, models to study the immunologic and physiologic aspects of this innovation are lacking. Here, we portray an original model of MMT in the rat. Donor surgery starts with a cross incision and isolation of portal triad components near the liver (Fig. 1A). The abdominal aorta is isolated from above celiac trunk to below superior mesenteric artery. After systemic heparinization, the aorta is sectioned above celiac trunk, tied below superior mesenteric artery, and sectioned below the tie, creating a long conduit containing the superior mesenteric artery and celiac trunk (Fig. 1B). The portal vein, biliary duct, and hepatic artery are divided close to the liver (Fig. 1C). The distal esophagus and ascending colon are sectioned and the graft containing esophagus, stomach, duodenum, small bowel, pancreas, spleen, cecum, and ascending column is removed, flushed by the aorta with cold lactate Ringer (Fig. 1B), and stored in the same solution. A polyurethane cuff is positioned in the portal vein as described previously (4, 5). In the recipient, the infrarenal aorta is cross-clamped, and an end-to-side microanastomosis is performed between donor’s aortic conduit and recipient’s aorta (Fig. 1D). Recipient’s portal vein and biliary duct are sectioned, the hepatic artery is preserved, and the cuff anastomosis is performed between donor’s portal vein and recipient’s superior mesenteric vein below the inferior mesenteric vein drainage (Fig. 1E) as described previously (4, 5). The graft was reperfused after clamps removal. Recipient’s stomach, duodenum, pancreas, spleen, small bowel, cecum, and ascending colon were removed after division of their vasculature near the organs to avoid lesions in recipient’s hepatic artery and inferior mesenteric vein. The biliary reconstruction was performed using an internal stent and gastrointestinal continuity was established by anastomosis between recipients’ and donors’ esophagus and ascending colon (Fig. 1F). We performed seven procedures using Lewis rat as donor and recipient and observed the recipients for 2 hr. Donor and recipient operation time ranged from 42 to 51 min (median, 46) and 53 to 101 min (median, 84), respectively. During the experiment, all recipients were kept under artificial ventilatory assistance and received intensive care including 3 to 4 mL of warm lactate Ringer solution plus 3 to 4 mL of isograft peripheral blood transfusion by the penile vein. One case died 30 min after reperfusion due to bleeding. The remaining six cases survived up to the experimental end point. Autopsy of the graft revealed mild reperfusion injury in two grafts and normal appearance in four grafts. This model is viable but need intraoperative and postoperative intensive care for good result. It may allow studies of graft rejection and physiology in this challenging procedure.FIGURE 1: A, recipient surgery with identification of PV, HA, and BD and abdominal esophagus (ES). B, allograft containing the stomach (ST), duodenum (D), pancreas (P), spleen (SP), and intestine (I), with special attention to the aortic conduit (Ao), including CT and SMA. C, PV and BD were transected near the hilum for cold solution perfusion drainage. D, final aspect of arterial anastomosis between recipient and donor aorta. E, dissection of recipient’s vascular pedicles showing the HA, SMA, and SMV, IMV, CT, LGV, and cuff anastomosis site at SMV. F, composition of the final aspect of the transplantation showing the graft (G) in gray and the PVA, AA, and BDA by stent. AA, arterial anastomosis; BD, bile duct; BDA, bile duct anastomosis; CT, celiac trunk; HA, hepatic artery; IMV, inferior mesenteric vein; LGV, left gastric vein; PV, portal vein; PVA, portal cuff anastomosis; SMA, superior mesenteric artery; SMV, superior mesenteric vein. Flavio Henrique Ferreira Galvão Daniel Reis Waisberg Ruy Jorge Cruz Jr Eleazar Chaib Luiz Augusto Carneiro D’Albuquerque Laboratory of Medical Investigation (LIM37) Division of Transplantation Department of Gastroenterology University of São Paulo School of Medicine São Paulo, Brazil ACKNOWLEDGMENT The authors thank Marcos Retzer for his help in the illustration of Figure 1.

Evaluation of the right ventricular ejection fraction during classic orthotopic liver transplantation without venovenous bypass

Right ventricular (RV) function is sensitive to changes in cardiac loading conditions, and RV dysfunction may contribute to hemodynamic instability during orthotopic liver transplantation (OLT). Thus, we evaluated RV function and its role in hemodynamic instability during classic OLT without venovenous bypass (VVB). Thirty patients undergoing classic OLT without VVB were studied. Right ventricular ejection fraction (RVEF) was measured using a modified pulmonary artery catheter. Hemodynamic data were recorded at pre-determined time points: T0, baseline; T1-T3: 5, 15, and 30 min after clamping; T4-T7: 5, 15, 30, and 120 min after reperfusion; T8 and T9: 24 and 48 h after surgery. The baseline RVEF was lower than normal value. RVEF decreased significantly from T1 to T4 and returned to baseline beginning at T5. At 24 and 48 h after surgery, RVEF was higher than baseline value. RVEF was correlated with stroke volume index and post-reperfusion syndrome during OLT. Compared to the low MELDs group, RVEF in the high MELDs group was lower at T1, T2, and T4. Right ventricular function was compromised during the anhepatic and early reperfusion stages in patients undergoing classic OLT without VVB, particularly in the high MELD score patients. Close monitoring of RV function in these patients should be considered.

Clinical analysis of liver transplant from a child of brain death to an adult

Objective To explore clinical feasibility of liver transplant from child of brain death to adult, to summarize the clinical experiences that a child of brain death transplants liver to an adult. Methods The recipient was a 39-year-old woman patient with primary hepatic carcinoma and posthepatitis cirrhosis (decompensation stage); while the donor was a 8-old-year child of brain death because of brain neoplasms. Donated liver was gained by the method of en bloc multivisceral procurement in a short time; the operative method was classic orthotopic liver transplantation. The postoperative managements included immunosuppression, prevention of infection, hepatic protection, and other relevant supports etc. Results The transplantation operative duration was 6 hours, after which not only did the recipient survive but also her body functioned well including the liver part, with no severe postoperative complications. Conclusions The technology of transplanting livers from children to adults is feasible. The key to ensure the success of transplant operation is systematic preoperative evaluation, excellent operative technique, and perfect postoperative treatment. Key words: Liver transplantation; Brain death; The donate liver from child

Selection of four surgical techniques in orthotopic liver transplantation

To compare the advantages and disadvantages of four surgical techniques in orthotopic liver transplantation. A total of 135 adult recipients receiving cadaveric whole liver grafts were divided into four groups according to the surgeries they received: group A (n=22) underwent classic orthotopic liver transplantation, group B (n=79) underwent modified piggyback liver transplantation, group C (n=18) underwent classical piggyback liver transplantation, and group D (n=16) underwent modified classic orthotopic liver transplantation. The clinical data of these recipients were retrospectively analyzed. The operation time, anhepatic time, and intra-operation bleeding volume among these four groups were significantly different (P < 0.05). The incidence of transient renal damage in group C was significantly lower than that in other groups (P < 0.05), while the complication rates and survival rates were not significantly different in the early stage after the operation. Surgery techniques should be carefully selected based on the individual patients's pre-operative condition. The modified classic orthotopic liver transplantation is a preferred technique for tumor patients or patients with surgical history of upper abdomen.

A modified model of hindlimb osteomyocutaneous flap for the study of tolerance to composite tissue allografts

Composite tissue allotransplantation (CTA) is the new frontier in transplantation. More than 25 hand allograft transplants have been performed worldwide, and the feasibility has been well established. The classical experimental model of CTA involves rat orthotopic hindlimb transplantation, a time-consuming procedure associated with high mortality and morbidity. We describe a rat heterotopic osteomyocutaneous flap that serves as a nonfunctional CTA, allowing the study of tolerance induction to a highly antigenic vascularized allograft of bone, muscle, and skin while minimizing the morbidity and mortality of full hind limb transplantation. In the present studies, we explored whether establishing chimerism by nonmyeloablative conditioning would induce tolerance to CTA. When compared with the classic hind limb transplantation model, these results demonstrate that our heterotopic hind limb flap is less morbid and as an effective experimental model for the study of CTA tolerance.

Causes for early postoperative renal dysfunction in classic orthotopic liver transplantation without venoveno bypass

目的 观察经典非转流原位肝移植(orthotopic liver transplantation,OLT)术后早期肾功能障碍(renal dysfunction,RD)发生的原因并提供临床参考.方法前瞻性研究了连续48例经典非转流OLT病例,根据术后早期(术后第1周)血清肌酐水平进行分组.对单因素分析后有显著性差异的资料进行多因素回归分析.结果经典非转流OLT术后早期RD的发生率为35.4%.Binary Logistic多元回归分析显示:术前RD、Child-Pugh评分、无肝期门静脉开放后1 h尿量是该术后早期RD的危险因素.结论对于术前明显肾功能障碍或Child-Pugh评分较高的病人,应避免使用经典非转流术式.对于接受经典非转流手术的病人,术前应纠正肾功能异常;术中应避免血流动力学的剧烈波动,保持稳定有效的肾血流灌注,以减少术后早期RD的发生。

Retransplantation for liver transplanter with poor graft function

To summarize the experience of liver retransplantation for patients with poor graft function. The clinical data of 9 patients undergone liver retransplantation at our center from April 1993 to April 2005 were retrospectively analyzed. The main indications for liver retransplantation at our center were early hepatic artery thrombosis (2/9), early portal vein thrombosis (1/9), and biliary tract complication (6/9). Of the 9 patients received liver retransplantation with cadaveric allografts, 3 received classic orthotopic liver transplantation, and 6 piggyback liver transplantation. Roux-en-Y biliary tract reconstruction was performed in 6 patients, Donor spleen vein was used as a conduit between donor portal vein and recipient portal vein in 1, and donor spleen artery as a conduit between donor hepatic artery and recipient aorta in 1. No perioperative mortality occurred. Of them, 5 had no complications after the operation, 1 had stricture in anastomotic stoma of portal vein, and 3 died in 6 months after the operation. Poor graft function due to biliary tract complications and vessel complications after primary liver transplantation are the chief indications of liver retransplantation. Liver retransplantation is the only suitable treatment of poor graft function.

Classic orthotopic liver transplantation without venovenous bypass: a report of 45 cases

THE CLASSIC orthotopic liver transplantation with venovenous bypass has both advantages and disadvantages. Advantages include maintaining the balance of the internal environment, relieving gastrointestinal congestion, decreasing blood loss, and protecting of bypass renal function during the anhepatic period. Disadvantages include the amount of time consumed, expensive cost, blood cell destruction, and complications to the blood vessels used for bypass. This article reviews the experience in 45 cases of classic orthotopic liver transplantation without venovenous bypass.

Liver repopulation for the treatment of metabolic diseases

Orthotopic liver transplantation is the treatment of choice for several inborn errors of metabolism. Unfortunately, the supply of donor organs is limiting and therefore many patients cannot benefit from this therapy. In contrast, hepatocyte transplantation could potentially overcome the shortage in donor livers by use of cells from a single donor for multiple recipients. In classic hepatocyte transplantation, however, only 1% of the liver mass or less can be replaced by donor cells. Recently, though, it has been shown in animal models that >90% of host hepatocytes can be replaced by a small number of transplanted donor cells in a process we term 'therapeutic liver repopulation'. This phenomenon is analogous to repopulation of the haematopoietic system after bone marrow transplantation. Liver repopulation occurs when transplanted cells have a growth advantage in the setting of damage to recipient liver cells. It has been discovered that transplanted cells from extrahepatic sources such as the adult pancreas or bone marrow can also be used for liver repopulation. Because bone marrow donors are widely available, this finding raises the hope of therapeutic application of these cells in the future. Here, the current knowledge regarding therapeutic liver repopulation and the hopeful implications for treatment of liver diseases will be discussed.

The molecular basis of allorecognition assessment of the involvement of peptide

The immune system complicates replacement of damaged organs by transplantation because exposure to foreign tissue evokes a vigorous immune response that often results in graft rejection. Not all transplants fail, however, and much has been learned by studying the conditions that satisfy the requirements for graft acceptance. In general, transplanted tissue that is genetically identical with the host exhibits the capacity for prolonged survival. Exceptions to this rule exist, however, as is evident from the observation that immunosuppressive regimens are often required for engraftment of syngeneic bone marrow transplants used for treatment of severe aplastic anemia [1, 2]. The understanding that transplantation success is genetically controlled has stimulated a vast amount of research into the identification and characterization of genes that pose a barrier to successful transplantation. This research has revealed the primary importance of a single set of closely linked genes in the control of transplant success. This group of genes is called the major histocompatibility complex (MHC) or, in humans, the human leukocyte antigen (HLA) complex. The actual physiologic role of the classic transplantation antigens encoded in the MHC is to control the ability of the immune system to recognize foreign antigens. In particular, MHC molecules restrict the recognition of foreign antigen by T cells which recognize

The evolution of single lung transplantation for emphysema

Classic transplantation dogma mandated bilateral lung replacement for lung transplant candidates with end-stage emphysema to avoid air trapping in the native lung and subsequent crowding of the newly transplanted lung. During a recent 12-month period 11 patients with emphysema received a single lung transplant. There was no hospital mortality and only one patient had any notable degree of air trapping in the native lung. Substantial improvement in pulmonary function was seen as early as 2 weeks after transplantation, with significant functional improvement seen by 6 weeks, despite some residual ventilation-perfusion mismatch. We have demonstrated the utility and safety of single lung transplantation for patients with end-stage emphysema, and it is our operation of choice in recipients more than 50 years of age.

Generation of autoreactive cytotoxic T lymphocytes under limiting dilution conditions.

The ability of F1 (H-2k/d) responder cells to generate cytotoxic effector cells against parental and F1 targets was investigated under limiting dilution conditions. One hit limiting dilution curves, consistent with the generated cytolytic cells being the progeny of a single precursor cell, were reproducibly obtained if a culture was scored as "responding" on the basis of its ability to lyse one or more of a panel of Con A blast targets consisting of self F1 and both parents. The calculated frequency of the precursor cell in a nylon wool-nonadherent splenic responder cell population ranged from about 1/500 to 1/1600 in (B10.BR X B10.D2)F1 and (RNC X BALB/c)F1 mice. The patterns of lysis expressed by individual microcultures against the panel of F1 and both parental targets were unusual; besides patterns that were consistent with predictions of classic transplantation rules (i.e., lysis of F1 targets plus one/both parental targets), "anomalous" patterns (i.e., lysis of F1 targets or one/both parental targets exclusively) were also seen. The precursor cells that gave rise to cytolytic cells were devoid of T cell markers, whereas the cytolytic effector cells were Thy-1+, Lyt-2+, as defined by sensitivity to treatment with monoclonal antibody plus complement. The data are discussed in the context of early in vitro development of cytolytic T lymphocytes, with particular emphasis on a possible manifestation of an early T cell repertoire.

The Classic Transplantation of a Portion of the Tibia into the Spine for Pott??s Disease A Preliminary Report

The Classic Transplantation of a Portion of the Tibia into the Spine for Pott??s Disease A Preliminary Report

NEW MODIFICATION OF THE McREYNOLDS TRANSPLANTATION FOR PTERYGIUM

DURING the past three years an increasing number of recurring pterygiums have been observed in the Army following the classic McReynolds transplantation.1These failures have also been noted in civilian life. It is the purpose of this paper to offer an explanation for these recurrences and to present a new modification of the McReynolds procedure for their prevention. This method has been employed in 300 cases, with satisfying results. A brief review of the anatomic framework of the ocular structures involved in the operation for pterygium will prove helpful in the discussion pertaining to the factors which are probably responsible for recurrences. The subconjunctiva is divided into the superficial and the deep fibrous layer. The superficial layer consists of adenoid tissue, which is loosely arranged. The deep fibrous layer is the one more properly considered the subconjunctival connective tissue layer, rather than a constituent of the conjunctiva itself. It