Transplantation Proceedings

Editorial Board

2012-05-01

Evolution of Registry and Tracking System for Organ Transplantation in Japan

2012-05-01

Abstract: Previously, the renal and liver transplantation registry in Japan was enforced yearly using registration and tracking papers only on recipients. The input of all patient data and announcement of statistical analysis to the public required a long time. Following The Declaration of Istanbul 2008, the committees planned to establish new registry and tracking systems for renal and liver transplantations on both recipients and donors. As the first step, for renal transplantation, we established a new registry and tracking system, JARTRE (JApan Renal Transplantation REgistry), using flash (USB) memory in 2009. The recipient and donor data were inputted into the USB memory in the transplantation centers. The memory was collected once a year by the committees with performed at 3 months at 1 year and every year after, the operation. As the second step, for liver transplantation, we established an online registry and tracking system, LITRE-J (LIver Transplantation REgistry in Japan), using the Internet in 2011. The recipient and donor data are inputted online in the centers just after transplantation. The tracking is performed at 3 months, at 1 year and every year after the operation. In 2012, we will convert the JARTRE system to an online registration and tracking system using the Internet like LITRE-J. The advantages of these system are the ease of input, scope of the data, and rapidly for statistical processing. Herein we have reported the details of JARTRE and LITRE-J, as well as the evaluation of the registry and tracking systems for renal and liver transplantation in Japan.

Difficulties in Organ Procurement and Transplantation in the Aboriginal Minority People in Taiwan

2012-05-01

Abstract: Background: Aboriginal people (AP) are a minority group in Taiwan. Little information on their perspectives on organ transplantation (OT) is available. Their rights for organ donation (OD) and as OT recipients (OTR) are constrained as a vulnerable population in society. This research sought to explore various Highland Aborigine Tribes beliefs systems and concepts related to OT. Methods: We employed a qualitative design on a purposive sample including seven categories of Taiwanese AP. Data collected by face-to-face interviews were evaluated by content analysis. Results: Seventy-five informants (45 female and 30 males) of 18 to 82 years from seven tribes completed interviews: Bunun (n = 20), Shao (n = 18), Tsou (n = 15), Amis (n = 12), Truku (n = 4), Rukai (n = 3), and Puyuma (n = 3). Of there, 33% had no idea of OT. All informants reported lack of knowledge of OD, organ procurement, and OTR. Eighty percent (45–82 years) had no willingness for OD or OTR; others might consult family members and health professionals (HP) to learn about OT. Seven hindering factors were identified: (1) having no background of OT; (2) limited impressions obtained from television news reports; (3) negative concepts of donating one's organs to others; (4) OT concepts contrast with cultural meanings of death; (5) possibility of being stigmatized; (6) fear of being rejected by others; and (7) HP had never mentioned OT. Conclusions: Taiwan APs' perspectives of OT concepts showed the majority to be unfamiliar with the concept and benefits of OT. Future research is necessary to explore the possible avenues to facilitate communications between HP and AP leaders, as well as elders in each AP category in Taiwan.

The Dilemma of “To Be or Not To Be”: Developing Electronically e-Health & Cloud Computing Documents for Overseas Transplant Patients from Taiwan Organ Transplant Health Professionals' Perspective

F.-J. Shih, Y.-W. Fan, C.-M. Chiu, F.-Ji. Shih, S.-S. Wang — 2012-05-01

Abstract: Aims: The development of mutually accessible e-health documents (ehD) and cloud computing (CC) for overseas organ transplant health professionals (OTHP) in two medical parties (domestic and overseas) would ensure better quality of care. This project attempted to compare pro and con arguments from the perspective of Taiwan's OTHP. Methods: A sample was obtained from three leading medical centers in Taiwan. Results: Eighty subjects including transplant surgeons (n = 20), registered nurses (RN; n = 30), coordinating nurses (OTCN; n = 15), and e-health information and communication technologies experts (ehICTs; n = 15) participated in this research. The pros of developing ehD were: (1) better and continuous care through communication and cooperation in two parties (78%); (2) better collaborative efforts between health professionals, information technology experts in two medical parties is (74%); (3) easier retrieval and communication of personal health documents with the trustworthy OTHP in the different countries (71%); and (4) CC may help develop transplant patients medical cloud based on the collaboration between medical systems in political parties of Taiwan and mainland China (69%). The cons of developing ehD and CC included: (1) inadequate knowledge of benefits and manuals of developing ehD and CC (75%); (2) no reliable communication avenues in developing ehD and CC (73%); (3) increased workload in direct care and documentation in developing new ehD and CC (70%); (4) lack of coaching and accreditation systems in medical, electronic, and law aspects to settle discrepancies in medical diagnosis and treatment protocols between two parties (68%); and (5) lacking systematic ehD and CC plans developed by interdisciplinary teams in two parties (60%). Conclusion: In this initial phase, the establishment of an interdisciplinary team including transplant leaders, transplant surgeon, RN, OTCN, ehICTs, and law experts from two parties might be helpful in working out developing plans with careful monitoring mechanisms.

National Survey of Filipinos on Acceptance of Incentivized Organ Donation

2012-05-01

Abstract: Background: The increasing number of patients requiring transplantation has brought about a shortage of donor kidneys. Incentives can potentially improve organ donation. There is a need to know if the public can accept incentivized organ donation. Objectives: To evaluate knowledge and opinions on organ donation and compensating the donor/donor family and to determine factors affecting consent. Methods: The third survey in 2009 covered 15 regions, 29 provinces, and 14 cities in the National Capital Region. There were 1500 respondents interviewed using a structured questionnaire. Analysis used Statistical Package for Social Science and chi-square. Results: Of the respondents, 63% were females and 74% were married. Nearly half were between 26 and 45 years old. Fewer than 5% were unschooled. Monthly household income was less than USD$222.00 in 70% of respondents. A majority knew about donation from 2001 to 2009. Fewer than 20% knew about deceased donors. Those who wanted to become donors decreased. Sixty-five percent were willing to donate a brain-dead relative's organs. Respondents felt that kidney donors deserve a token of gratitude. Options included livelihood (32%), cash (31%), and educational assistance (26%). Sixty percent wanted the donor assistance termed a “token of gratitude.” Consent for donation was positively correlated (P < .05) with higher education and monthly income. Conclusion: Awareness on organ transplantation and donation increased. Factors that promote organ donation are higher education and monthly income. A majority of Filipinos felt that the donor deserves a token of gratitude. Public acceptance of incentivized organ donation may be pursued. Strategies to improve the national advocacy campaign for deceased donation are needed.

Outcomes of Management for Potential Deceased Donors

2012-05-01

Abstract: Backgrounds: Potential deceased donor management optimization is important for organ recovery maximization. Before optimization, the current state of donor management and predictors for organ recovery require analysis. Methods: We retrospectively analyzed organ procurement activity and medical management for 2005 to 2010 potential brain death donors at Seoul National University Hospital. Results: Of 316 contacts for potential brain-dead donors, 129 (39.7%) patients were transferred to the donor management team. Among the causes of transfer failure, issues related to proper donor management affected 33%. Expanded criteria donors were 17.9% of transferred donors. Organ recovery was successful in 111 (90.2%) donors. A total of 360 organs were recovered, corresponding to a mean of 2.92 ± 1.37 organs per donor. The absence of organ demand was an important cause of recovery failure among less transplanted organs. Brain death-related complications were identified as follows: acute kidney injury (AKI), defined by AKI network criteria, occurred in 19 (15.4%); cardiopulmonary resuscitation in 5 (3.1%); bacteremia in 12 (9.7%); thrombocytopenia in 24 (19.5%); and diabetes insipidus in 42 (34.1%). AKI was a significant independent risk factor for organ recovery failure in both the liver and kidney (odds ratio [OR] 0.147, 95% confidence interval [0.045, 0.473], P = .001; OR 0.096, 95% confidence interval [0.023, 0.392], P = .001, for kidney and liver, respectively). Conclusions: Both the transfer success rate and rate of organs transplanted per donor of potential deceased donors remained low in Korea. AKI during potential donor management was a risk factor for kidney and liver recovery failure.

Professional Education and Hospital Development for Organ Donation

N. Fukushima, S. Konaka, O. Kato, J. Ashikari — 2012-05-01

Abstract: Because of the strict Organ Transplantation Act, only 81 brain dead (BD) organ donations had been performed in Japan for 13 years since 1997. The Act was revised on July 17, 2010, allowing, organs to be donated after BD with consent from the family, if the subject had not denied organ donation previously. This act has lead to an expectation of a 6–7-fold increase in BD donation. The 82 organ procurement coordinators (OPC) in Japan include 32 belonging to the Japanese Organ Network (JOT) and the others to each administrative division. JOT has guideline manuals of standard roles and procedures of OPC during organ procurement from BD and cardiac death donors. To manage the increased organ donations after the revision of the act, we have modified the education system. First, we modified the guideline manuals for OPC to correspond to the revised Transplant Act and governmental guidelines. Second, all OPC gathered in a meeting room to learn the new organ procurement system to deal with the revised Transplant Act and guidelines. Third, a special education program for 2 months was provided for the 10 newcomers. Last, the practical training in each donor case for newcomers was performed by older OPC. Topics of the education program were the revised transplant act and guidelines, family approach to organ donation, BD diagnosis, donor evaluation and management, organ procurement and preservation, allocation system, hospital development and family care. In the future, each OPC will be divided into special categories, such as the donor family OPC, the donor management OPC, and the operating room OPC. Therefore, we need to construct separate special education programs for each category.

Modification of the Education System for Organ Procurement Coordinators in Japan After the Revision of the Japanese Organ Transplantation Act

S. Konaka, O. Kato, J. Ashikari, N. Fukushima — 2012-05-01

Abstract: Background: From October 1997 to July 2010, only 86 brain-dead (BD) organ donations were obtained and no organs were retrieved from children under 15 years of age because of the strict Japan Organ Transplantation Act. The Act was revised on July 17, 2010, allowing organs to be donated after BD with family consent. Objective: To manage the increased donations after the revision, the Japan Organ Transplant Network (JOT) employed 10 organ procurement coordinators (OPCs) and modified its education systems. We retrospectively reviewed the modified education programs to evaluate whether they were effective and whether the processes of organ donation were promptly performed after the revision of the Act. Methods: The modifications of education program were: changing OPC to guideline manuals to correspond to the revised Transplant Act; OPCs were taught the new organ procurement system; and a special education program was provided for the 10 newcomers for 2 months. Results: After 12 months of the revision, 58 BD organ donations were accomplished, whereas they had averaged 6.6 in a year before the revision. Two pediatric BD organ donations were accomplished without problem. One priority organ donation to a relative was performed uneventfully. After applying the modified education program, skilled JOT OPCs and leader JOT OPCs increased. Conclusions: To manage increased organ donations after the revision of the Act, the educational system was modified and 58 brain dead organ donations were performed safely.

A Newly Developed Container for Safe, Easy, and Cost-effective Overnight Transportation of Tissues and Organs by Electrically Keeping Tissue or Organ Temperature at 3 to 6°C

H. Ohkawara, T. Kitagawa, N. Fukushima, T. Ito, Y. Sawa, T. Yoshimine — 2012-05-01

Abstract: Background: As there is only one skin procurement organization in Japan the Japan Skin Bank Network (JSBN), all skin grafts procured in Japan are sent by a commercialized delivery system. Preliminarily, bottles containing saline were transported in a cardboard box using a so-called “cooled home delivery service” using a truck with a refrigerated cargo container. During transportation the temperature in the cardboard box increased to 18°C in summer and decreased to −5°C in winter. For these reasons, we investigated whether a newly developed container “Medi Cube” would be useful to transport skin grafts. Objectives: Four bottles with a capacity of 300 mL containing 150 mL of saline in a Medi Cube container were transported from Osaka to the JSBN in Tokyo between 4 PM and 10 AM using a commercialized cooled home delivery service. Two bottles were transported in a Medi Cube container without phase change materials (PCM) in winter and summer, respectively. Another two bottles were transported in the Medi Cube with PCMs in winter. The temperatures inside saline, inside a transportation container, and outside the container, and air temperature were monitored continuously with a recordable thermometer. Results: The temperatures inside saline and inside a Medi Cube container were maintained between 3 and 6°C, even when the temperature outside the container increased during parking. The temperature inside a Medi Cube container without PCM decreased to −3°C when the inside of the cargo container was overcooled in winter. However, the temperatures inside saline and inside a Medi Cube container with PCM were between 3 and 6°C, even when the temperature outside the container decreased to below 0°C in winter. Conclusion: A Medi Cube container with PCM provided a safe, easy, and cost-effective method for overnight transportation of skin grafts.

A Study on Knowledge and Attitude toward Brain Death and Organ Retrieval among Health Care Professionals in Korea

K.O. Jeon, B.N. Kim, H.S. Kim, N.-I. Byeon, J.J. Hong, S.H. Bae, S.Y. Son — 2012-05-01

Abstract: Purpose: The practice of retrieving vital organs from brain-dead donors is legally and medically accepted in Korea, but health care professionals' beliefs and opinions regarding these matters have not been sufficiently explored. The purpose of this study was to evaluate the knowledge and attitudes of health care professionals to the concepts of brain death and organ retrieval. Methods: Data were collected using a 41-item questionnaire during a week in June 2011. Sixty-one doctors and 109 nurses from five hospitals with more than 2000 beds in Seoul, Korea, participated in the survey. The data was analyzed using SPSS version 17.0 (SPSS Inc. Chicago, Illinois, USA). Results: There were statistically significant differences in the scores on knowledge according to marital status (P = .001) education level (P = .019), whether the participants were informed about organ donation from a brain-dead donor (P = .002), and the participant's experience managing potential brain-dead patients (P = .037). There were statistically significant differences in the scores on the attitude according to gender (P < .001), age (P < .001), marital status (P < .001), education level (P = .003), job position (P < .001), and the participant's experience referring brain-dead patients to the hospital-based organ procurement organization (P = .001). Significantly, attitude's positively correlated with knowledge about brain-dead organ donation (P < .001). Conclusion: Compared with previous studies, the knowledge and attitudes of health care professionals' regarding brain death and organ retrieval were not improved. There are passive attitudes to brain death and organ retrieval. More research must be performed to promote knowledge and understanding toward brain death and organ retrieval among health care professionals.

Kidney Transplantation From Donation After Cardiac Death Donors in China—A Single-Center Experience

2012-05-01

Abstract: Objective: To report clinical outcomes of kidney transplantation from cardiac death donors (DCD) in China, and to investigate its feasibility to expand the organ donor pool. Patients and methods: We retrospectively studied clinical data of 46 DCD kidneys from 31 donors from February 2007 to August 2011. Recipients were followed for patient and graft survival. Results: We discarded the organs from 3 of 29 (10.3%) DCD donors and 7 of 42 (16.7%) kidneys that displayed renal thrombosis. Of the 39 recipients engrafted with DCD kidneys successfully, the mean follow-up was 16 months, (range = 50 days to 43 months). Delayed graft function (DGF) occurred in 15 (38.5%) recipients, who except one recovered within 3 months. Three biopsy-proven acute rejection episodes were observed in two recipients (5.1%). All patients survived through the follow-up. The graft survival rate was 97.4% at 12 months and 94.9% at 24 months. A 45-year-old male recipient who received a pair of grafts from a 6-year-old child survived with good renal function. Conclusion: Although kidney transplantations from DCD donors showed a higher rate of DGF with a longer duration of graft recovery, we achieved favorable short-term clinical outcome in terms of graft survival and function. Donation after cardiac death can expand the organ donor pool in China.

The Fate of Patients on the Waiting List for Lung Transplantation in Korea

2012-05-01

Abstract: Lung transplantation for end-stage lung disease results in prolonged actuarial survival and improved pulmonary function. However, the shortage of donor lungs has been a major limiting factor in transplantation. The purpose of this study was to analyze the waiting time and mortality rate for each disease entity. The medical records of all patients listed in The Korean Network for Organ Sharing (KONOS) from May 1996 to May 2011 were analyzed to identify waiting times and causes of death. During the study period, 146 patients (86 males and 60 females) of mean age of 46.6 years (range; 5 to 73 years) showed idiopathic pulmonary fibrosis (IPF; n = 61), chronic obstructive pulmonary disease (COPD; n = 19) or bronchiectasis (n = 15). Sixty-five patients (44.5%) underwent lung or heart-lung transplantation. Sixty-two patients (42.5%) expired during the waiting period, and 19 patients are still on the waiting list. The mortality rate while waiting was highest among patients with primary pulmonary hypertension (62.5%) followed by IPF (57.4%), and acute respiratory distress syndrome (ARDS) (55.6%). The mean time from diagnosis to registration in KONOS was 15.5 months among the expired and 13.2 months in the transplanted group (P = .455). The mean time on waiting list was 8.2 months in the expired group and 3.7 months in the transplanted group (P = .012). In the expired group, the mean survival time was significantly shorter among patients with ARDS (2.2 months, P = .004) compared to IPF (7.9 months), COPD (10.7 months), and primary pulmonary hypertension (PPH) (30.0 months). The high mortality rate (42.5%) during the waiting period in Korea may result from the lack of donors and the delay in registration.

Donor Evaluation for Lung Transplantation in Korea

2012-05-01

Abstract: Lung transplantation for end-stage lung disease results in prolonged survival and improved pulmonary function. However, the shortage of donor lungs has been a major limiting factor in Korea. We sought to investigate the number and utilization of donor lungs by the five institutions performing LTx in Korea, retrospectively reviewing outcomes of organs registered in the Korean Network for Organ Sharing from January to December, 2010. Lungs were offered from 270 brain-dead patients (189 males and 81 females) of mean age of 45.2 ± 14.2 years (range, 12 to 77 years). The most common cause of brain death was hemorrhage (n = 219, 81%). Only 18 (6.7%) donor lungs were used, which was low compared with kidney (93.3%), liver (86.3%), heart (26.7%), and pancreas (11.1%) use. The mean age of donors of transplanted lungs was 35.7 years (range, 14 to 51 years) compared with 45.9 years for other organs (P = .003). The characteristics of utilized donor lungs were: mean partial pressure of oxygen (PaO2), 300.9 mm Hg; mean smoking history, as 2.7 pack-years; and mean body mass index, 21.2 kg/m2. The causes of refusal were medical ineligibility (n = 129) including poor PaO2, abnormal chest x-ray, long smoking history, older age (n = 46), no properly matched recipient (n = 46), unknown (n = 17), and family withdrawal (n = 14). Only 8 (33.3%) were transplanted from standard criteria and 10 from the lungs that did not satisfy these criteria. An efficient utilization system is needed to improve lung transplantations.

Relationship Between Postoperative Lung Atelectasis and Position of the Endotracheal Tube in Pediatric Living-Donor Liver Transplantation

2012-05-01

Abstract: Objective: The aims of current study were: 1) to evaluate the incidence of lung atelectasis; and 2) to investigate whether or not the position of the endotracheal (ET) tube is associated with this complication. Methods: The medical records and chest roentgenograms of 183 pediatric patients who underwent living-donor liver transplantation were retrospectively reviewed and analyzed. Patients without atelectasis were grouped in group I (GI) and those with atelectasis in group II (GII). The patients' characteristics and ET tube level between groups were compared with unpaired Student's t test. Multiple binary logistic regressions were also performed to identify the important risk factors associated with lung atelectasis. Results: Right upper lung (RUL) atelectsis could be found in ET tube at any level from T1 to T5, with incidence rates of 12.7%, 15.2%, 26.3%, 6.7%, and 100% for T1, T2, T3, T4, and T5, respectively. The incidence of atelectasis is 16.6%, and all of the atelectasis occurred in the RUL. No significant difference between groups was observed in the patients' characteristics, except for the amount of preoperative ascites. The likelihood of this risk factor could not be confirmed by multivariate binary logistic regression analysis. Conclusions: The incidence of lung atelectasis in our study was 16.6%, which all occurred in the RUL. No predictive risk factor from the patients' characteristics could be found, and no correlation between the level of the ET tube and the occurrence of RUL atelectasis could be observed.

Extracorporeal Membrane Oxygenation and Thoratec Pneumatic Ventricular Assist Devices as Double Bridge to Heart Transplantation

2012-05-01

Abstract: Introduction: Ventricular assist devices have benefited patients with end-stage heart failure as a bridge to heart transplantation (HTx). We present our experiment of HTx using extracorporeal membrane oxygenation (ECMO) with Thoratec pneumatic ventricular assist device (TpVAD). Methods: From May 1996 to June 2011, among 410 patients who underwent HTx 23 required mechanical circulatory support (MCS) with implantation of the TpVAD and 15 (65%) of them received grafts. Results: The 23 patients included 4 female and 19 male patients of age range 10 to 80 years. Eighteen (78%) of them needed ECMO before TpVAD implantation. Twelve (67%) were implanted with a TpVAD double bridge to HTx. The demand for MCS among patients with acute hemodynamic collapse has led to major improvements in the existing systems such as ECMO with double bridge to TpVAD. Conclusion: We used ECMO as a rescue procedure for acute hemodynamic deterioration. However, during ECMO support, left ventricular afterload increased. If prolonged support is required, TpVAD might be required: 15 (65%) of patients supported by ECMO with TpVAD needed to a wait a suitable donor. We recommend the application of ECMO for short-term support (within 1 week), and TpVAD as a bridge for medium- or long-term support.

The Influence of the Organ Allocation Policy on a Patient's Chances of Undergoing Heart Transplantation and the Posttransplantation Survival Rate

2012-05-01

Abstract: The Taiwan Organ Registry and Sharing Center (TORSC) was established by the Department of Health on June 6, 2002. According to the organ allocation policy, the computer-based organ-matching program began on April 1, 2005. In order to encourage organ donations, “donor hospitals” were given the highest priority. On October 1, 2010, the TORSC implemented a new allocation policy allowing highest priority to the most critically ill patients listed as 1A status. The aim of this study was to investigate the influence of the allocation policy on the likelihood of undergoing a heart transplantation (HTx) as well as the survival after the procedure. Based on the timeline of changes in the organ allocation policy, the patients were divided into three groups: “individual decision,” “donor hospital first,” and “urgency status first.” We observed the waiting time of status 1A patients to decrease and their chance to receive a donor heart increase but their survival rate after HTx to decrease. Further research is needed to define the optimal organ allocation policy.

Ventricular Assist Device Application as a Bridge to Pediatric Heart Transplantation: A Single Center's Experience

2012-05-01

Abstract: Objectives: There are limited options for mechanical circulatory support to treat end-stage heart failure in pediatric patients. Although extracorporeal membrane oxygenation is commonly used in infants and children, ventricular assist devices (VAD) provide a longer duration of support with fewer complications before recovery or as a bridge to heart transplantation (HTx), as described herein. Methods: This retrospective chart review of eight patients transplanted from April 2008 to December 2011, after left ventricular assist device (LVAD) implantation due to end-stage heart failure. Their mean age was 12 years (9–15 y) and mean body weight, 48 kg (20–78). All were New York Heart Association functional class IV with mean left ventricular ejection fractions less than 15%. Results: The six patients (75%) received HTx after a mean LVAD support duration of 43.2 days; 2 (25%) died before a suitable heart became available. Their mean duration of LVAD support was 30 days. There were 4 (50%) who experienced clinically evident thromboembolic events: 3 (37.5%) cerebrovascular with 1 mortality and 1 (12.5%) as acute limb ischemia. Transient hemodialysis was performed in 4 (50%). Bloodstream infection identified in 6 (75%) was controlled with intravenous antibiotics. Driveline infection identified in 4 (50%) was treated successfully with local wound dressing changes and intravenous antibiotics. One 9-year-old boy died of rejection at 16 months after transplantation. Conclusions: Because of the organ shortage, pediatric patients have a low chance to undergo HTx. VAD provides long-term support for children with end-stage heart failure before a suitable heart becomes available. A thromboembolic event remains a major complication influencing their survival.

Combined St. Thomas and Histidine-Tryptophan-Ketoglutarat Solutions for Myocardial Preservation in Heart Transplantation Patients

2012-05-01

Abstract: Background: To establish quicker cardiac arrest and less myocardial distension injury during heart procurement, we combined St. Thomas and histidine-tryptophan-ketoglutarate (HTK) solutions for donor heart preservation since June 2008. Methods: From June 2008 to March 2010, we enrolled 31 heart transplantation (HT) patients in this study. During heart procurement we initially infused 1,000 mL cold St Thomas cardioplegic solution to achieve cardiac arrest. After procurement, a further 2,000 mL of cold HTK solution was infused at low perfusion pressure. Another 1,000 mL cold HTK solution was perfused before donor heart implantation. We examined donor age, recipient preoperative characteristics, ischemia time, hospital stay, postoperative graft function, major cardiac events, and transplant vasculopathy (TCAD). Results: Twenty-two patients (71.0%) presented with dilated cardiomyopathy and 7 (23.3%) with ischemia cardiomyopathy. There were 23 (76.7%) male donors, and the mean donor age was 38.4 ± 13.8 years. Six patients underwent a redo sternotomy, 1 patient needed a third-do sternotomy, and 1 a seventh sternotomy (third HT) for repeated endocarditis and graft failure. The average ischemia time was 224.9 ± 71.0 minutes and the postoperative hospital stay was 57.7 ± 47.7 days. The surgical mortality (3.2%) was not accompanied by hospital or follow-up mortality. Patient left ventricular ejection fraction postoperative was 59.6 ± 2.3% with good functional status. Major cardiac events occurred in 8 patients (26.7%) without major complications. There were two subjects with TCAD but normal graft function. The correlation between ischemia time and hospital stay was insignificant (r = 0.21; P = .26). Conclusions: Donor heart preservation combining St Thomas cardioplegic arest and low-pressure perfusion with HTK solution seemed to be safe with. short-term survival similar to other approaches.

The Outcome of Heart Transplantation in Hepatitis C-Positive Recipients

2012-05-01

Abstract: Background: Clinical outcomes of heart transplantation (HTx) among recipients with chronic hepatitis C virus (HCV) infection are poorly understood especially in Asia. Therefore, this study evaluated these clinical outcomes. Methods: Using retrospective chart review we collected data on 385 patients including 20 HCV-positive recipients at the time of transplantation. We obtained information on demographics features, serial transaminases, graft function, patient survival as well as the incidences of acute hepatitis and transplant coronary artery disease. Results: Between 1987 and 2010, the 20 HCV-positive patients had a median age at transplantation of 52 years (range, 30–63). Seventeen were men and three women. All the patients were classified as Child-Pugh class A; two had cirrhosis prior to HTx. Over a mean follow-up of 63 months (range, 2 days to 187 months), there were 11 deaths, including two hospital mortalities and nine subsequent deaths. Only one mortality (5%) was related to Child-Pugh class C cirrhosis, despite liver transplantation. Among the other 19 deceased or surviving recipients, there was no evidence of hepatic dysfunction or hepatocellular carcinoma. Transplant coronary artery disease was detected in six patients (30%). There was no significant difference in Kaplan-Meier actuarial survival between the HCV-positive and HCV-negative recipients (P = .59). Conclusions: There was no significant difference in patient survival or graft function between HCV-positive and HCV-negative HTx recipients. Additionally, HCV-positive recipients were not at an increased risk of hepatic failure or accelerated transplant coronary artery disease.

Endomyocardial Biopsy in Heart Transplantation: Schedule or Event?

2012-05-01

Abstract: Background: Endomyocardial biopsy is the gold standard to identify rejection after heart transplantation. Due to its invasiveness, discomfort, and difficult vascular access, some patients are not willing to accept routine scheduled biopsies years after heart transplantation. The purpose of this study was to identify whether there was a difference in outcomes among the scheduled versus event biopsy groups. Methods: We studied 411 patients who underwent heart transplantation from 1987 to 2011, reviewing biopsy results and pathology reports. There were 363 patients who followed the scheduled biopsy protocol, and 48 patients who were assigned to the event biopsy group. We extracted data on biopsy results, rejection episodes, rejection types, and survival time. Results: The 2481 reviewed biopsies over 24 years, showed most rejection episodes (86.4%) to occur within 2 years after heart transplantation. The rejection incidence was low (2.1%) at 3 years after transplantation. The major reason for an event biopsy was poor vascular access, such as tiny central vein or congenital disease without a suitable central vein. Event biopsy group patients were younger than schedule biopsy patients (19.7 years old vs 47.6 years old; P < .05). The 10-year survival rates were 64% among the event versus 53% among the scheduled biopsy group (P = .029). The 10-year rates of freedom from rejection were similar. Conclusions: The rejection rate was low after 3 years; episodes occurred within 2 years. Although the long-term survival in the event group was better, they had a younger man age. The rejection and freedom from rejection rates were similar. As the rejection rate was low at 3 years after transplantation, we suggest that the event principle could be applied for biopsy at 3 years after heart transplantation.

Cardiac Allograft Vasculopathy Compared by Intravascular Ultrasound Sonography: Everolimus to Mycophenolate Mofetil—One Single-Center Experience

2012-05-01

Abstract: Cardiac allograft vasculopathy (CAV) remains one of the leading causes of late graft failure and death. Cyclosporine microemulsion Neoral (CsA) had been used in heart transplantation (HTx) recipients. Meanwhile, Everolimus (EVL; Certican, Norvatis Pharmaceuticals; Basel, Switzerland) or mycophenolate mofetil (MMF) have been combined with CsA for maintenance treatment. We compared atherosclerosis in HTx patients showing CAV by intravascular ultrasound (IVUS) in two groups: the CE who received CsA, EVL, and steroid versus the CM group, who received CsA, MMF, and steroid. Materials and Methods: We explored IVUS parameters such as plaque thickness (PT), lumen circumference (LC), media adventitial circumference, lumen diameter (LD), and media adventitial diameter to characterize the atherosclerosis among CE versus CM groups. Results: In this study, both the CE and CM groups showed increased plaque thickening in the first year posttransplantation (P < .05). However, MMF significantly reduced LC and LD (P < .05) Upon multivariate linear regression analysis, the CE group seemed to show less effect on the maximal difference in PT between 2 and 12 months after adjusting for age at transplantation and gender (P < .05). There was no acute clinical adverse event of CAV reported in either both group during the follow-up. The atherosclerosis of CAV revealed by LC, LDmax, and LDmin was significantly less among patients treated with CE than CM. Conclusion: These results suggested that everolimus-treated patients showed benefits compared with MMF-treated subjects as extrapolated from these IVUS data.

Steroid Pulse Therapy Combined with Plasmapheresis for Clinically Compromised Patients after Heart Transplantation

2012-05-01

Abstract: Background: The most serious complication after heart transplantation is allograft dysfunction. Patients presenting with compromised hemodynamics show a high incidence of mortality. The most common reason for allograft dysfunction is rejection. We have employed steroid pulse therapy combined with plasmapheresis for hemodynamically compromised patients after heart transplantation. Methods and Results: Steroid pulse therapy and plasmapheresis were performed on 35 patients who underwent orthotopic heart transplantation for graft dysfunction. Thus treatment rescued ventricular function and improved the ejection fraction in 77% of patients, among who ever 71.4% showed improved New York Heart Association (NYHA) functional class. Conclusions: Steroid pulse therapy combined with plasmapheresis improved the cardiac contractility and NYHA functional class of most heart transplant recipients with graft dysfunction.

Isolated Cardiac Sarcoidosis in Heart Transplantation

T.-I. Chang, N.-H. Chi, N.-K. Chou, C.-I. Tsao, H.-Y. Yu, Y.-S. Chen, S.-S. Wang — 2012-05-01

Abstract: Background: Heart transplantation is the ultimate treatment for end-stage heart failure. Cardiac sarcoidosis has rarely been reported in heart transplantation worldwide. Their long-term prognosis after heart transplantation is unknown. Herein we have presented clinical and pathological observations among heart transplantation patients with isolated cardiac sarcoidosis. Methods: From 1987 to 2011, we performed 411 heart transplantations including five patients retrospectively reviewed due to the presence of sarcoidosis and giant-cell cardiomyopathy in the recipient heart. Results: Among the heart transplantations from 2003 to 2011, the four male and one female patients were ages 31 to 40 years. None of them had extra-cardiac sarcoidosis. All five subjects presented with dilated cardiomyopathy with patent coronary arteries. The commonest clinical presentations were atrioventricular block, ventricular arrhythmia, electrocardiographic findings of ST elevations, and poor left ventricular ejection fractions (17%–23%). All patients survived without allograft heart failure to date with the longest survivor at 8 years postoperatively. No recurrence of sarcoidosis has been observed clinically or among the post–heart transplantation endomyocardial biopsies. Conclusion: Heart transplantation is a useful treatment for isolated cardiac sarcoidosis patients suffering end-stage heart failure. Often the diagnosis is difficult to establish before heart transplantation despite endomyocardial biopsy. No recurrence of sarcoidosis was observed among the allografted hearts.

Clinical Experience of Tacrolimus With Everolimus in Heart Transplantation

2012-05-01

Abstract: Background: Tacrolimus (Tac) in combination with mycophenolate mofetil is widely used after heart transplantation (HT). Everolimus (EVR), a new potent proliferation signal inhibitor can be used with a carcineurin inhibitor to reduce the occurrence of rejection. The purpose of this study was to evaluate the efficacy and safety of Tac combined with EVR in de novo HT. Materials and methods: From January 2009 to April 2011, 33/62 patients who underwent HT were prescribed Tac and EVR as de novo immunosuppression. The main exclusion criteria were poor kidney function (serum creatinine > 2.8 mg/dL), panel-reactive antibodies > 25%, donors > 60 years old, or cold ischemia time > 6 hours. All patients received Tac (C0 blood level 5–10 ng/mL during the first 6 months, then 3–5 ng/mL), EVR (C0 target 3–8 ng/mL), and corticosteroids. After transplantation, routine examinations included echocardiogram and protocol endomyocardial biopsy. Results: There was no operative mortality. The 1- and 3-year actuarial survivals were 95.74% ± 3.49%. One patient who had undergone coronary artery bypass grafting previously and received intra-aortic balloon pumping and extracorporeal membrane oxygenator-assisted cardiopulmonary resuscitation before HT died of Aspergillus septicemia 58 days after HT. No biopsy-proven acute rejection > grade 2R or acute rejection associated with hemodynamic compromise was observed. Hyperlipemia was noted in 16 cases (48.5%), hypertension in 11 (33.3% 5%), and diabetes mellitus in 12 (36.4%). No other severe adverse events were noted. Conclusions: Concentration-controlled EVR (C0 target 3–8 ng/mL) in combination with Tac achieved good efficacy and safety. The 1- and 3-year actuarial survivals were 95.74% ± 3.49%.

Twenty-four Year Single-Center Experience of Hepatitis B Virus Infection in Heart Transplantation

2012-05-01

Abstract: Objective: Hepatitis B virus (HBV) infection is hyperendemic in Taiwan. We have reported the outcome of (1) recipients with hepatitis B surface antigen (HBsAg)-positive; HBsAg-negative recipients who receive donor hearts from HBsAg-positive donors; and treatment with lamivudine of hepatitis B flare-ups after heart transplantation, using case numbers that range from 100 to 200. Methods: From July 1987 to May 2011, all 412 orthotopic heart transplant recipients and donors underwent routine preoperative screening for hepatitis B virus markers and liver function parameters. Lamivudine was prescribed prophylactically for recipients with elevated serum enzyme levels or an HBV DNA virus load before transplantation, or when there was evidence of hepatitis B flare-up after transplantation. Postoperative HBV markers and liver function parameters were collected over a mean follow-up time of 7.8 years. Results: Thirty-four recipients were HBsAg-positive before heart transplantation, and 23 experiencing HBV reactivation upon follow-up requiring lamivudine treatment. Clinical responses were achieved in all of them: 15 were complete and two, slow partial responses. Twenty-six recipients with an HBV naïve status at the time of heart transplantation, and three patients received donor hearts from an HBsAg-positive donor under perioperative hepatitis B immunoglobulin prophylaxis. HBV infection was successfully prevented in two patients, but the other one contracted HBV hepatitis, which was successfully treated with lamivudine. Conclusions: HBV reactivation after the heart transplantation was common but usually well controlled with lamivudine treatment. Although posttransplantation liver function deteriorated for a period, there was no HBV infection-related morbidity or mortality. Perioperative hepatitis B immunoglobulin prophylaxis can successfully prevent HBV naïve recipients from infection in some cases, but HBsAg-positive donors should only be considered in high risk situations.

Heart Retransplantation for Pediatric Primary Allograft Failure

2012-05-01

Abstract: Purpose: Heart transplantation is indicated for children with end-stage heart failure or complex inoperable congenital defects. When the transplanted heart fails, retransplantation is suggested and herein we have presented the prognosis of these pediatric cases. Materials and methods: From March 1987 to March 2011, we performed 404 heart transplantations including 45 pediatric patients, 6 (13.3%) of whom experienced graft failure requiring retransplantation. Only four of the six patients (66.7%) had a chance for retransplantation. Results: Six of 45 pediatric heart transplant patients (13.3%) experienced graft failure requiring retransplantation. Four of them (66.7%) underwent retransplantation. Only one of the four died due to severe postoperative sepsis with acute respiratory distress. The other three patients recovered well and remain alive with no neurological sequelae; all are in New York Heart Association functional classification I at present. Conclusion: Pediatric post–heart graft failure require expectations retransplantation, which shows a good prognosis.

Changes in Health-Related Quality of Life Across Three Post–Heart Transplantation Stages: Preoperative Extracorporeal Membrane Versus Non–Extracorporeal Membrane Group/Clinical Trial Plan Group Versus Non–Clinical Trial Plan Group in Taiwan

P.-H. Tseng, S.-S. Wang, F.-J. Shih — 2012-05-01

Abstract: Aims: The aims of this research were to compare changes in overall health-related quality of life (HRQoL), working competence (WC), physical functions (PF), and quality of sleep across 3 crucial post–heart transplantation (HT) stages (1 month, 6 months, and 1 year post-HT) between the following: (1) preoperative extracorporeal membrane (preop-ECMO) versus non-ECMO group and (2) postoperative Clinical Trial Plan (CTP) group versus non-CTP group in Taiwan. Patients and Methods: A between-method triangulation design was used. Subjects who had undergone HT in the last 1–4 years were recruited from a leading medical center in Taipei. Quantitative data were collected using Visual Analog scale (VAS) and Taiwan's version of the World Health Organization Quality of Life (WHOQOL) questionnaire. Semistructured qualitative questions were added to explore the factors influencing the changes in social domains of HRQoL. Results: A total of 62 heart transplant recipients (HTRs) participated in this study. Their ages ranged from 20 to 70 (mean, 47.16 ± 12.09) years; 80.6% were male. Compared with the subjects with preop-ECMO, HRQoL, WC, and PF of the subjects without preop-ECMO were less at 1 month post-HT; the difference reached statistical significance for HRQoL and PF for 1 month post-HT, but they recovered at the 6 months post-HT stage. HTRs who had participated in the CTP had higher HRQoL and perceived WC in the period of 1 month post-HT, 6 months post-HT, and 1 year post-HT as compared with the group not in CTP; meanwhile, the difference was statistically significant for HRQoL at 1 month post-HT and 6 months post-HT and for PF at 1 month post-HT. Conclusions: The efficacy of postop-CTP including HRQoL, WC, and PF was promising across the 3 post-HT stages. Postop-CTP was suggested both clinically and was shown to be statistically significant to HTR's recovery of their health status.

Role of Killer Immunoglobulin-like Receptor–Ligand Interactions in Human Leukocyte Antigen–Matched Sibling Hematopoietic Stem Cell Transplantation

2012-05-01

Abstract: Introduction: Killer immunoglobulin-like receptor (KIR)-ligand mismatches lead to natural killer cell alloreactivity after hematopoietic stem cell transplantation (HSCT). However, their clinical impact on HSCT outcomes is controversial due to complexity of KIR haplotypes, genotypes, and phenotypes as well as their diversity among patient populations. The present study investigated the role of KIR-ligand interactions in human leukocyte antigen (HLA)-matched sibling transplants. Methods: The recipient cohort, which included patients diagnosed with aplastic anemia, acute leukemia, and myelodysplastic syndrome, received granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood stem cells. HLA typing was performed using polymerase chain reaction - sequence specific oligo probes (PCR-SSO). The KIR genotype of the donors and the ligands C1 (Asparagine 80), C2 (Lysine 80), and Bw4 recipient typings were performed using polymerase chain reaction - sequence specific primers (PCR-SSP). We assessed acute and chronic graft-versus-host disease (GVHD), relapse, and overall survival. Results: While 84.5% of donors carried a Bx KIR, 15.5% carried the AA haplotype. The effect of a recipient's lack of ligands among 88.5% of cases was associated with 39% of subjects developing GvHD. Lack of C1 may lead to manifestations of acute GvHD and lack of C2 to manifestation of chronic GvHD. The presence of both C1 and C2 seemed to be protective against both forms of GvHD. The role of two Bw4 alleles, threonine (T) or isoleucine (I) at position 80, was evaluated. 73% of recipients who carried Bw4 80(I) versus 27% with the Bw4 80(T) allele. The presence of Bw4-80(T) allele appeared to reduce the risk of GvHD, indicating its stronger inhibitory effect than its 80(I) counterpart. Conclusion: KIR-ligand interactions influenced HSCT outcomes.

Audit of Peripheral Stem Cell Transplantation for Aplastic Anemia in Multitransfused Infected Patients

T. Seth, U. Kanga, P. Sood, V. Sharma, P. Mishra, M. Mahapatra — 2012-05-01

Abstract: Introduction: Allogeneic hematopoietic stem cell transplantation is a curative modality for aplastic anemia; the preferred stem cell source is bone marrow. However, allogeneic peripheral blood stem cell transplantation (PBSCT) used in high-risk patients is associated with higher risk of chronic graft-versus-host disease (GVHD). Our center receives multitransfused, alloimmunized, infected, late referrals for transplant. Methods: Forty-one patients of median age 22 years (range 8–37) received allogeneic-PBSCT from human leukocyte antigen (HLA)-matched sibling donors. The median time since diagnosis was 12 months (range 4–65) and median pretransplant transfusions were 37 (range 6–160). Six patients were platelet refractory and one alloimmunized for pan-red blood cell (RBC) antigens. Several patients had pretransplant icterus or renal dysfunction and 26 (63.4%) had unresponsive bacterial/fungal infections. Our conditioning regimen included fludarabine 30 mg/m2 for 6 days (days –10 to –5), cyclophosphamide 60 mg/kg/d for 2 days (days –6 to –5), and antithymocyte globulin (ATGAM) 30 mg/kg/d for 4 days (day –4 to –1), which was reduced to 2 days in 2 patients. We used standard GVHD prophylaxis with cyclosporine and methotrexate on days 1, 3, 6, 11. Results: The median follow-up period was 29 months (range 6–78) and median engraftment time 10 days (range 8–17). Thirty-one patients (75.6%) were treated for infections, with 20 of these on antifungals for preexisting infections. There were two graft rejections and 10 (24.4%) deaths, with three intracranial hemorrhages, two rejections with infection, three cases of refractory GVHD (acute/overlap syndrome) with cytomegalovirus reactivation, and two invasive fungal infections. Overall incidence of acute GVHD was 39% with 2 grade IV cases. Ten (25%) cases developed chronic GVHD, with extensive GVHD in four. Conclusion: With more experience using shortened course of ATGAM, HLA-matched donor transfusions, and availability of newer antifungals, we have been able to decrease PBSCT-related mortality. Further improvement will be possible with early referrals.

Single-Center Experience with Pancreas Transplantation

2012-05-01

Abstract: Background: Recently improved patient and graft survivals, as well as decreased of postoperative morbidity have ushered in pancreas transplantation (PT) due to technical refinements as well as better immunosuppression and postoperative management. Herein we analyzed the outcomes of PT over a 19-year experiences at a single center. Methods: All recipients who underwent deceased donor or living donor PT from July 1992 to July 2011 were enrolled in this study. We reviewed their medical records, including operative records, as well as clinical and laboratory findings. We analyzed graft and patient survival rates using the Kaplan-Meier method. Results: One hundred fifty-three cases were performed between July 1992 and July 2011. The indication for PT was type I diabetes in 125 (81.7%), and type II diabetes in 28 (18.3%) patients. The pancreas donor was deceased in 139 (90.8%) and living in 14 cases (9.2%). The type of PT was simultaneous pancreas-kidney transplantation (n = 91, 59.5%), pancreas alone (n = 49; 32.0%), or pancreas after kidney (n = 13, 8.5%). Median follow-up was 43.0 months (range 0–228). At 1, 5, and 10 years overall patient survivals were 93.8%, 88.1%, and 85.1%, and graft survivals, 82.3%, 70.6%, and 64.6%, respectively. When we divided the deceased donor PT recipients into two groups according to when they underwent PT (up to 2005 [n = 54]) vs 2006 and later [n = 85]), the recent group showed significantly improved patient and graft survival rates (P < .001). With no difference between type I (n = 65) and type II (n = 20) patients (P = .159). Conclusion: Considering the improved quality of life and long-term patient survival, PT can be an effective treatment strategy in diabetic patients requiring insulin regardless of type of disorder.

Inducible Cyclooxygenase Expression Mediating Hypoxia/Reoxygenation–Induced Pulmonary Vasoconstriction is Attenuated by a Cyclooxygenase Inhibitor in Rats

C.L. Su, D.W. Yuan, L.L. Chiang, H.L. Lee, K.H. Chen, D. Wang — 2012-05-01

Abstract: Objective: Hypoxic pulmonary vasoconstriction (HPV) is a well known phenomenon to temporarily offset a ventilation-perfusion mismatch. Sustained HPV may lead to pulmonary hypertension. In this protocol, we studied the relationships between the HPV response and inducible cyclooxygenase II (COX II) activation after hypoxia-reoxygenation (H-R) challenge in an isolated perfused lung model. Methods: An in situ isolated perfused rat lung model underwent inaction of hypoxia by ventilation with 5% CO2–95% N2 for 10 minutes instead of 5% CO2–95% air; they were then reoxygenated with 5% CO2–95% air. We measured pulmonary arterial pressure (PAP) changes before, during, and after H-R challenge. We also estimated changes in blood concentrations of hydroxyl radicals, nitric oxide (NO) and thromboxane B2 (TxB2) before and after H-R as well as mRNA expressions of COX II in lung tissue thereafter. A COX II inhibitor, celecoxib (10 mg/kg), was administered between 2 consecutive challenges. Results: Hypoxia induced pulmonary vasoconstriction by increasing PAP (4.1 ± 0.8 mm Hg). Consecutive hypoxic challenges did not show tachyphylaxis (P > .05). H-R of lung tissues induced significant increases in blood concentrations of hydroxyl radicals (48.5 ± 7.6 vs 75.8 ± 11.5 mmol/L; P < .01), NO (54.3 ± 12.3 vs 77.7 ± 15.7 pmol; P < .05), and TxB2 (42.3 ± 6.9 vs 58.7 ± 8.6 pg/mL; P < .05). Lung tissue H-R also significantly increased COX II mRNA expression compared with sham tissues (1 ± 0 vs 4.0 ± 2.8; P < .001). The COX II inhibitor celecoxib significantly attenuated HPV responses (P < .05) and attenuated the elevated blood concentrations of TxB2 (P < .05), hydroxyl radicals (P < .01), nitric oxide (P < .05), and COX II mRNA expression (P < .05) after H-R challenge. Conclusions: Lung tissue H-R induced significant increases blood concentrations of inflammatory mediators and tissue mRNA expression of COX related to elevation of HPV responses. COX II inhibitor celecoxib attenuated the HPV responses by reducing TxB2 release.

L-Ascorbic Acid and Alpha-tocopherol Attenuates Liver Ischemia-Reperfusion Induced of Cardiac Function Impairment

C.-C. Hsu, J.-J. Wang — 2012-05-01

Abstract: Objectives: The Pringle maneuver is a surgical procedure to minimize hemorrhage during hepatectomy, which however, can induce production of reactive oxygen species causing remote organ injury. We sought to study the impact of the Pringle maneuver on cardiac function as well as the protective effects of L-ascorbic acid and α-tocopherol pretreatments. Methods: Rats were divided into four study groups: L-ascorbic acid (60 mg/kg/d) or α-tocopherol (200 mg/kg/d), and surgical interventions (Sham-operated or liver ischemia-reperfusion [I/R]). Liver ischemia was performed by clamping the hepatic artery and portal vein for 30 minutes, followed by reperfusion by releasing the clamps for 2 hours. Cardiac function was evaluated by a high-fidelity pressure-volume catheter positioned in the left ventricle. Myocardial injury was assessed through plasma creatine kinase-MB (CKMB) and troponin I (cTnI). Cardiac lipid peroxidation and systemic hydroxyl radical levels were assessed by cardiac tissue malondialdehyde and plasma methylguanidine, respectively. Results: Cardiac function was significantly depressed in the I/R group, where plasma CKMB and cTnI were markedly increased (P < .05). L-ascorbic acid and α-tocopherol pretreatments improved cardiac function and significantly reduced cardiac injury (P < .05). L-ascorbic acid pretreatment demonstrated better heart protection than α-tocopherol, in terms of cTnI and CKMB (P < .05), but no significant difference in terms of cardiac functional improvement. Conclusions: L-ascorbic acid and α-tocopherol pretreatment 3 days prior to the Pringle maneuver attenuated myocardial injury and protected cardiac function by scavenging hydroxyl radical and reducing lipid peroxidation. L-ascorbic acid demonstrated better protection than α-tocopherol.

The Role of CD14 and Toll-Like Receptor 4 of Kupffer Cells in Hepatic Ischemia-Reperfusion Injury in Rats

X. Luan, Y. Liu, M. Li — 2012-05-01

Abstract: Objective: The objective of this study was to study the role of CD14 and Toll-like receptor 4 (TLR4) in Kupffer cells (KCs) on ischemia reperfusion injury (IRI) in rat liver grafts. Methods: Isolated KCs were obtained from control, IRI, and IRI plus anti-CD14 antibody groups. We measured messenger RNA (mRNA) and protein expression of the lipopolysaccharide receptor CD14 and TLR4, nuclear factor kappa B (NF-κβ) activity, and TNF-α levels. Results: mRNA and protein expressions of CD14 and TLR4 were significantly higher in the IRI than in the control group, as were protein expressions of CD14 and TLR4 by flow cytometry and by Western blots. NF-κβ activity and tumor necrosis factor-α level in the IRI group were significantly higher than in the control group (3.17 ± 0.21 and 0.28 ± 0.03 vs 654.2 ± 3.6 pg/mL and 147.4 ± 1.1 pg/mL; t value = 4.11 and 4.29 for each; P < .01). Compared with the IRI group they were greatly decreased after anti-CD14 antibody treatment: 2.14 ± 0.17 vs 3.17 ± 0.21, 298.7 ± 1.8 pg/mL vs 654.2 ± 3.6 pg/mL (t value = 2.52 and 2.92 for each; P < .05). They were still significantly higher than the control group (t values of 3.01 and 3.27 for each; P < .01). Conclusion: IRI up-regulated CD14 and TLR4 gene expression in KCs, and subsequently activated NF-κβ to produce cytokines.

A Basic Consideration for Porcine Liver Preservation Using a Novel Continuous Machine Perfusion Device

2012-05-01

Abstract: Introduction: The aims of this study were to compare extracellular and intracellular-type University of Wisconsin (UW) solutions for liver grafts and to assess oxygenation in this perfusion system. Materials and Methods: The organ preservation system consisted of 3 circulating systems for the portal vein, hepatic artery, and maintenance of the perfusion solution. The portal vein or hepatic artery system had a roller pump, a flow meter, and a pressure sensor. In this study, we perfused livers with UW or extracellular type UW-gluconate at 4°C–6°C for 4 hours. The flow rates at the entrance were 0.5 mL/min/g liver in the portal vein and 0.2 mL/min/liver in the hepatic artery. Orthotopic liver transplantation was performed in pigs: group 1-a, grafts procured after acute hemorrhagic shock were preserved by a solution without O2; group 1-b, grafts were preserved with O2; group 2-a, grafts were perfused using intracellular type solution (UW); and group 2-b, grafts were perfused using extracellular-type solution (UW-gluconate). Results: Effluent aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) levels in group 1-b were lower than those in group 1-a. Survival rates in group 2-a and group 2-b were 1/4 and 3/3, respectively. Effluent AST and LDH levels in the perfusate of group 2-b were lower than group 2-a. Histological study revealed necrosis of hepatocytes and sinusoidal congestion in group 2-a. Conclusion: A beneficial effect of extracellular-type solution with oxygenation in a novel continuous machine preservation system yielded well-preserved liver graft function.

Functional Recovery of Donation After Cardiac Death Liver Graft by Continuous Machine Perfusion Preservation in Pigs

2012-05-01

Abstract: Introduction: Grafts from donation after cardiac death (DCD) will greatly contribute to the expand the donor pool. However, these grafts may require the development of the preservation methods because of primary nonfunction and severe ischemic bile duct injury. Methods: Porcine livers were perfused with a newly developed machine perfusion (MP) system. Each system for the portal vein or the hepatic artery had a roller pump, a flow meter, and a pressure sensor. The livers were perfused with University of Wisconsin (UW)-gluconate at 4°C–6°C for 3 hours after 2 hours simple cold storage (CS). The portal vein flow rate was 0.5 mL/min/g liver (pressure, 10 mm Hg) and the hepatic artery flow rate was 0.2 mL/min/g liver (pressure, 30 mm Hg). Orthotopic liver transplantation was performed in pigs comparing Group 1 (n = 4) procured after acute hemorrhagic shock preserved by MP, Group 2 (n = 3) procured after warm ischemia time (WIT) of 30 minutes with CS preservation, and Group 3 (n = 4) procured with 30 minutes of WIT and MP preservation. Results: Collected effluent aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) levels in the perfusion solution and serum AST and LDH were significantly lower in Group 1. AST and LDH results were lower in Group 3 than Group 2. Survival rates in Groups 1 and 3 were 3/4, but 0/3 in Group 2. Conclusion: MP preservation was a useful promising preservation mode for DCD liver grafts.

Protective Role of Nitric Oxide Induced by Ischemic Preconditioning on Cold Ischemic-Reperfusion Injury of Rat Liver Graft

Q. Xue, Z. Yuan, Z. Chen, R. Hao, C. Liu, B. Tu — 2012-05-01

Abstract: Aims: To investigate the protective role of nitric oxide (NO) induced by ischemic preconditioning (IP) on cold ischemic-reperfusion (IR) injury of rat liver grafts. Methods: One hundred twenty-eight male Sprague Dawley rats used for orthotopic liver transplantation were randomly divided into four groups (n = 32): administering heparin before ischemic reperfusion (control group); IP with 10-minute ischemia and 10-minute reperfusion before IR (IP group); adenosine before IR (Ade group); and L-NAME (NG-nitro-L-arginine methyl ester) + IP before IR (NAME group). Half of each group were used to investigate 1-week recipient survival rate, and another to obtain blood and hepatic tissue samples after 2-hour reperfusion. Results: One-week survival bile production, serum NO, and antioxidase activity were higher but serum alanine aminotransferase, tumor necrosis factor-α, and superoxide levels in hepatic tissue were lower in the IP group and Ade group versus the control group or NAME group. Liver sinusoidal endothelial cells in the IP and Ade groups showed less injury than the other groups. Conclusion: NO induced by IP can improve 1-week survival and rat liver function as well as protect liver sinusoidal endothelial cells.

The Effect of Perfusion Speed on Graft Reperfusion Injury after Rat Orthotopic Liver Transplantation

S. Zhou, P. Li, T. Chen, M. Li, Y. Zhang, Z. Yu — 2012-05-01

Abstract: Objective: The objective of this study was to investigate the effects of various perfusion speeds on reperfusion injury of grafts after liver transplantation. Methods: Liver transplantation was performed from Sprague-Dawley (SD) to SD rats. Recipients were divided into 4 groups according to perfusion speed: 50 mL/h for group A, 100 mL/h for group B, 150 mL/h for group C, and 200 mL/h for group D. Peripheral blood was collected from the caudal vein. All survivors were humanely killed at 24 hours posttransplantation. The morphological changes in grafts were evaluated using light microscopy, serum tumor necrosis factor-α (TNF-α), and endothelial nitric oxide synthase (eNOS) proteins as well as their messenger RNA (mRNA)-intragraft levels. Results: The pathohistological damage of grafts in the 150 mL/h and 200 mL/h groups were compared with the 50 mL/h and 100 mL/h groups. TNF-α in serum was increased at 6 hours, reaching a peak at 12 hours posttransplantation. The TNF-α levels in the 50 mL/h and 100 mL/h groups were significantly lower than the 150 mL/h and 200 mL/h groups; the 50 mL/h group was greater than the 100 mL/h group as well as the 200 mL/h group was greater than the 150 mL/h group. The expressions of eNOS protein and mRNA intragraft in the 150 mL/h and 200 mL/h groups were significantly reduced compared with the 50 mL/h and 100 mL/h groups, and the 50 mL/h group was decreased when compared with the 100 mL/h group. There was no significant difference between the 150 mL/h group and the 200 mL/h group. Conclusion: The optimal perfusion speed for harvesting rat liver graft is 100 mL/h. High-speed perfusion may impair sinusoid endothelial cells and low-speed perfusion may extend the warm ischemia time and increase microthrombus formation.

Quantification of Macrovesicular and Microvesicular Hepatic Steatosis in Rats Using 3.0-T 1H-Magnetic Resonance Spectroscopy

2012-05-01

Abstract: Background and objective: Hepatic steatosis (HE), which is common among the general population, is present in donor organs, potentially affecting their graft survival as well as the recovery of the donor. Our goal was to develop an experimentally and clinically reliable, noninvasive method to quantify macrovesicular and microvesicular hepatic steatosis using 3-T 1H-magnetic resonance spectroscopy (MRS). Materials and methods: Macrovesicular and microvescular steatosis were induced in rats using methylcholine deficiency and choline deficiency diets. A MayoBC10 coil was used for radiofrequency transmission and signal recept. Measurements of hepatic fat content were performed using 1H spectroscopy on a 3.0-T whole-body GE Signa system. The ratio of the areas under the curve of fat (0.8–1.3 ppm) and water (4.7 ppm) was used to determine hepatic fat content, which was compared with the degree of histopathologic and biochemical steatosis. Results: Twenty rats were divided into two groups based on the percentage of microvesicular liver steatosis. Group A (n = 13) was the lower percentage group (microvesicular < 10%) while group B (n = 7), the higher group (microvesicular ≥ 10%). The mean total fatty change in the liver was 58.4% ± 47.2% and 67.6% ± 39.1% in groups A and B, respectively. A highly significant linear correlation between 1H-MRS and total fatty change was observed in group A (r = .986, P < .001) while there was a relatively poor correlation in group B (r = .764, P = .05). The power to predict fatty change in the liver in groups A and B was significantly different (P = .004). Conclusions: The degree of hepatic steatosis with a small amount of microvesicular steatosis (<10%) can be precisely predicted using 3-T 1H-MRS.

Pretransplant Screening and Evaluation of Liver Graft Viability Using Machine Perfusion Preservation in Porcine Transplantation

2012-05-01

Abstract: A novel method using machine perfusion for pretransplant screening and evaluation of the viability of liver grafts has been proposed, seeking to prevent severe ischemia-reperfusion injury and to reduce the risk of primary graft nonfunction. This study sought to evaluate the viability of critical grafts, which were obtained from expanded criteria donors or donation after cardiac death donors during preservation with a new machine preservation perfusion system (NES-01). The normalized pressure transition in the hepatic artery was employed as an evaluation index for liver viability. As a result, the normalized pressure (p/p0) in the hepatic artery showed a distinctive transition under each experimental conditions controlled by warm ischemic time (WIT). The high viability graft, obtained under the condition of WIT as 0 minutes (WIT0), showed a quick response to hepatic artery pressure after initiating perfusion, whereas the normalized pressure showed a sudden decrease. In contrast, the normalized pressure among WIT60, which may cause the graft to lose viability, showed a poor hepatic artery response. These findings corresponded to the cumulative release of enzymes. The findings of our study suggest that monitoring of the pressure drop rate in the hepatic artery during machine perfusion can be used to evaluate liver graft viability.

Protective Effect of Melatonin on Liver Ischemia-Reperfusion Induced Pulmonary Microvascular Injury in Rats

M.-H. Chiu, C.-L. Su, C.-F. Chen, K.-H. Chen, D. Wang, J.-J. Wang — 2012-05-01

Abstract: Objective: Reactive oxygen species generated during liver reperfusion have been implicated in remote lung injury. In this study, we evaluate the protective effects of melatonin pretreatment against the increased pulmonary microvascular permeability. Methods: Male Sprague-Dawley rats were divided into three groups: shame-operated, liver ischemia-reperfusion (I/R), and melatonin pretreated (15 mg/kg, intraperitoneally) 15 minutes prior to the liver I/R). The duration of ischemia was 30 minutes, followed by 2 hours of reperfusion. Lungs were isolated in situ and parameters of the capillary filtration coefficient (Kfc), lung wet-to-dry weight ratio (W/D), lung weight-to-body weight (LW/BW), and protein concentration in bronchial lavage fluid (PCBAL), the percentage of macrophages and neutrophils in bronchial lavage fluid (BALF), and lung tissue malonedealdehyde were used to assess the lung injury. Results: Liver I/R-induced lung injury was noted by the markedly increased Kfc, W/D, LW/BW, PCBAL, and the presence of neutrophils and macrophages in BALF. Lipid peroxidation was also increased (P < .05). All indicators were markedly decreased in melatonin-pretreated rats (P < .05), suggesting that lung injury was attenuated. Conclusions: Melatonin pretreatment prior to liver I/R can effectively reduce the pulmonary microvascular permeability and attenuate lipid peroxidation in the lungs.

Liver Reperfusion-Induced Decrease in Dynamic Compliance and Increase in Airway Resistance Are Ameliorated by Preischemic Treatment with Melatonin through Scavenging Hydroxyl Radicals in Rat Lungs

J.-H. Yeh, C.-L. Su, C.F. Chen, D. Wang, J.-J. Wang — 2012-05-01

Abstract: Objectives: Acute lung injury is frequently observed in patients subsequent to liver ischemia and reperfusion (I/R) injury. However, the changes in pulmonary function, eg, lung dynamic compliance (Cdyn) and airway resistance (RI), are not well understood. We sought to study the alternations in pulmonary function during liver I/R and the protective effects of preischemic treatment with melatonin. Methods: Animals were divided into 3 groups: sham-operated, liver I/R, and intraperitoneal (i.p.) pretreatment with melatonin (15 mg/kg). Liver I/R was performed by clamping the hepatic artery and portal vein for 30 minutes followed by releasing for 2 hours. The Cdyn and RI were studied at baseline and at 2 hours of reperfusion. We assessed the level of pulmonary hydroxyl radicals by methylguanidine (MG) content in the bronchoalveolar lavage fluid (BALF) as well as the liver damage using plasma levels of lactate dehydrogenase (LDH), glutamic oxaloacetic transaminase (GOT), and glutamic pyruvic transaminase (GPT). Results: After 2 hours of liver reperfusion, Cdyn was reduced by ∼25%, while RI increased by ∼16% (P < .05). The decreased Cdyn and increased RI were markedly attenuated by melatonin pretreatment (P < .05). Melatonin pretreatment also protected the liver against I/R injury (P < .05), as seen by reduced LDH, GOT and GPT along with markedly reduced hydroxyl radicals (P < .05). Conclusions: Preischemic treatment with melatonin protected lung function against damage by liver I/R. The improvement in lung function was strongly associated with decreased hydroxyl radicals in the lungs.

Preischemic Treatment With Melatonin Attenuates Liver Reperfusion-Induced Impairment of Cardiac Function

T.-H. Chen, K.-H. Chen, J.-J. Wang — 2012-05-01

Abstract: Objectives: Cardiac functional impairment is frequently observed in patients with end-stage liver disease and after reperfusion of an ischemic liver. Excessive production of reactive oxygen species (ROS) through activation of Kupffer cells and leukocytes during reperfusion may play important roles. We evaluated the cardiac protective effects of preischemic treatment with melatonin. Methods: Studies were performed on 3 groups of male Sprague-Dawley rats; shame-operated controls, liver ischemia and reperfusion (I/R), and melatonin pretreatment prior to I/R. Liver I/R was performed by clamping the hepatic artery and portal vein for 30 minutes, followed by releasing the clamps for 2 hours. The cardiac function was assessed using a high-fidelity dual pressure-volume catheter positioned in the left ventricle (LV). We also evaluated heart injury using plasma creatine kinase-MB (CKMB), and Troponin I (cTnI). The level of hydroxyl radical production was evaluated using plasma methylguanidine (MG). Results: LV function was severely impaired after 2 hours of reperfusion; stroke volume and LV contractility were significantly reduced (P < .05). Markedly increased CKMB and cTnI indicated serious myocardial injury. Preischemic treatment with melatonin protected the heart as seen by the reduced plasma CKMB and cTnI (P < .05), and decreased systemic hydroxyl radical production. Conclusions: Liver I/R severely impaired cardiac functions by production of hydroxyl radicals. Melatonin pretreatment effectively scavenged oxidants and hydroxyl radicals, protecting cardiac function against liver I/R-induced injury.

The Protective Effect of Curcumin on Ischemia-Reperfusion–Induced Liver Injury

C.M. Lin, J.F. Lee, L.L. Chiang, C.F. Chen, D. Wang, C.L. Su — 2012-05-01

Abstract: Objective: Reperfusion of the ischemic liver results in the generation of oxidative and nitrosative stresses and reaction product of peroxynitrite, which induce rapid cytotoxicity and liver injury. In this study we demonstrated that curcumin, an antioxidant, attenuated ischemia/reperfusion (I/R)-induced liver injury. Materials and Methods: Ischemia was induced by clamping the common hepatic artery and portal vein of rats for 30 minutes. Thereafter, flow was restored and the liver was reperfused for 80 minutes. Blood samples collected prior to ischemia and after reperfusion were analyzed for methyl guanidine (MG), nitric oxide (NO), tumor necrosis factor-alpha (TNF-α), and adenosphate triphosphate (ATP). Blood levels of serum glutamic oxaloacetic transaminase (sGOT), serum glutamate pyruvate transaminase (sGPT), and lactic dehydrogenase (LDH), which served as indexes of liver injury, were measured. Results: The protocol resulted in elevation of blood NO (P < .001), TNF-α (P < .001), and MG (P < .001). sGOT, sGPT, and LDH were elevated significantly (P < .001), whereas ATP was significantly diminished (P < .001). Pretreatment with curcumin (25 mg/kg) significantly attenuated the reperfusion liver injury, while the ATP content reversed. In addition, MG, TNF-α, and NO release were attenuated. Conclusions: These results indicated that curcumin exerted potent anti-inflammatory effects in I/R-induced liver injury due to its antioxidant effects.

Protective Role of Shenfu on Ischemia-Reperfusion Injury of Rat Liver Grafts

T. Chen, M. Cheng, Z. Yuan, S. Zhou, Z. Yu — 2012-05-01

Abstract: Aims: We sought to investigate the protective role of Shenfu (SF),a traditional Chinese formulation comprising Radix Ginseng and Radix Aconitum Carmichaeli on ischemia-reperfusion injury in rat liver grafts. Methods: Ninety-six male Sprague Dawley rats were used as donors (n = 48) and recipients (n = 48) of orthotopic liver transplantation. They were randomly divided into a control group with donor livers injected with saline through the portal vein immediately after recovery versus the SF group, with livers injected with SF. Each group was further divided into 3 subgroups equally to obtain bood and hepatic tissues samples at 2, 4, and 6 hours reperfusion. Results: At each phase, the SF group, showed significantly higher bile production (P < .05) with lower serum levels of alanine aminotransferase and tumor necrosis factor-α, and nuclear factor-κB expression in the hepatic tissues. (P < .05). SF group hepatic tissues showed less injury compared with controls. Conclusion: SF injection seemed to protect hepatocytes from injury during the early reperfusion phase and to improve subsequent rat liver graft function.

Apoptosis in Endothelial Cells by Cyclosporine

E.A. Hwang, H.S. Kim, E. Ha, K.C. Mun — 2012-05-01

Abstract: Objectives: The immunosuppressive drug cyclosporine (CsA) is a potent agent widely used after organ transplantations and to treat various autoimmune disorders. After using CsA, some patients suffer severe complications including renal and vascular toxicity, which are influenced by the degree of the endothelial damage. Several studies have demonstrated CsA treatment to directly induce apoptosis in several cell types. Thus, CsA may induce endothelial damage via activation of proapoptotic proteins. The present study was undertaken to investigate the effects of CsA on apoptosis of endothelial cells using human umbilical vein endothelial cells. Methods: Proliferation was measured by using the Cell Counting Assay Kit after cells were exposed to CsA (0 L, 10 L, 30 L, 50 L or 100 μg/mL). Apoptotic cells were identified by fluorescence microscopy of 4′, 6-diamidino-2-phenylidole-stained nuclei. Western blot analysis was done for poly(ADP-ribose) polymerase (PARP), p27, p53 and caspase. Results: Cell viability decreased dependent on the CsA concentration. CsA treatment group showed chromatin condensation and nuclear fragmentation. CsA produced a dose-dependent induction of p27 and reduction of procasapase-3. CsA treatment induced the degradation of 116-kDa PARP into an 89-kDa fragment. Conclusions: CsA induced apoptosis of endothelial cells.

Effect of Cyclosporine on Apoptosis in Bronchial Epithelial Cells

E.Y. Ha, K.C. Mun — 2012-05-01

Abstract: Objectives: Among airway complications, posttransplantation infections are related to impaired mucociliary clearance, which may represent a toxicity of cyclosporine (CsA), a potent, widely used immunosuppressive drug after organ transplantations. Since several recent studies have demonstrated CsA treatment to directly induce apoptosis in several cell types, we investigated its effects on airway cells using the human bronchial epithelial cell line BEAS-2B. Methods: Proliferation was measured by using a Cell Counting Assay Kit by exposing cells to CsA (0, 10, 30, 50, or 100 μg/mL). Apoptotic cells were identified using fluorescence microscopy after 4′, 6-diamidino-2-phenylidole (DAPI) staining. Western blot analysis was performed to evaluate the contents of poly(adenosine diphosphate-ribose) polymerase (PARP), p27, Bcl-2, and caspase-3. Results: Cell viability decreased dependent on the CsA concentration: 100.00 ± 0.01% with 0 μg CsA as control; 98.65 ± 0.02% with 10 μg (P < .05 vs control); 95.41 ± 0.05% with 30 μg (P < .05 vs control); 38.84 ± 0.04% (P < .001 vs control) with 50 μg; and 15.28 ± 0.05% with 100 μg (P < .001 vs control). Apoptotic cells detected with DAPI showed chromatin condensation and nuclear fragmentation. CsA induced p27 and p53, as well as degradation of 116-kd PARP into an 89-kd fragment. Conclusion: CsA induced apoptosis in human bronchial epithelial cells.

Effects of Cyclosporine on Oxidative Stress in Human Bronchial Epithelial Cells

D.S. Jeon, E.Y. Ha, K.C. Mun — 2012-05-01

Abstract: Objectives: Some of the airway complications relate to the use of cyclosporine (CsA), a potent agent widely used after organ transplantations. Several recent studies have demonstrated CsA treatment to induce reactive oxygen species (ROS). The present study was undertaken to investigate effects of CsA on production of ROS and antoxidant defense of airway cells using the human bronchial epithelial cell line BEAS-2B. Methods: We measured biological antioxidant potential (BAP), as well as ROS and malondialdehyde levels in BEAS-2B cells after CsA treatment, using Free Radical Analytical System 4 kits (Diacron, Grosseto, Italy). ROS production was expressed as Carr Units as established by the manufacturer and BAP as μmol/2 × 105 cells; malondialdehyde, by the thiobarbituric acid assay. Results: ROS production was increased in the BEAS-2B cells after CsA treatment: 73.5 at 0 (controls); 82.5 at 10; 84.0 at 30; 86.0 at 50; and 93.0 Carr Unit/2 × 105 cells at 100 μg/mL of CsA. The levels of BAP were 1821 at 0 (controls), 1698 at 10; 1653 at 30; 1366 at 50 μg/mL; and 1391 at 100 μg/mL. The levels of malondialdehyde were increased: 3.8 at 0 (controls); 3.4 at 10; 4.4 at 30; 4.2 at 50: and 5.0 nmol/106 cells at 100 μg/mL. Conclusions: Increased production of ROS and decreased BAP by CsA in BEAS-2B cells may increase malondialdehyde levels by radical-induced damage.

Effects of Cyclosporine on Metalloproteinase in Endothelial Cells

E. Ha, K.C. Mun — 2012-05-01

Abstract: Objectives: Cyclosporine (CsA) is a potent agent widely used after organ transplantations and in treatment of various autoimmune disorders. Some patients suffer severe complications including renal and vascular toxicity that are influenced by the degree of endothelial damage. Dysregulation of metalloproteinase (MMP) activity is known to contribute to renal and vascular diseases. To investigate the possible mechanisms of posttransplantation complications in the kidney and vessels by CsA, we examined its effects on metalloproteinases in endothelial cells using human umbilical vein endothelial cells (HUVECs). Methods: HUVECs isolated from umbilical cords by collagenase digestion were seeded in 6-well plates at a density of 1 × 105 cells/well before treatment with 2–250 μmol/L CsA and a 24-hour incubation. Thereafter we performed gelatin zymography of MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, and MMp-13 to evaluate band density using a luminescent image analyzer system with controls calculated as 100%. Results: MMP-1, MMP-3, MMP-8, MMP-9, and MMP-13 activities were increased after CsA treatment; MMP-1 = 121; MMP-3 = 164; MMP-8 = 133; MMP-9 = 124; and MMP-13 = 121. In contrast, MMP-2 activity was decreased after CsA treatment; MMP-2 = 79. Conclusions: This study showed CsA to activate most MMPs (except MMP-2) in endothelial cells. This result suggests that CsA may dysregulate MMPs in endothelial cells.

Tumor Necrosis Factor-α Pathway Plays a Critical Role in Regulating Interferon-γ Induced Protein-10 Production in Initial Allogeneic Human Monocyte–Endothelial Cell Interactions

Y.S. Fang, L.M. Zhu, Z.G. Sun, L.Z. Yu, H. Xu — 2012-05-01

Abstract: T-cell infiltration of allografts is a major pathologic component defining acute rejection episodes (ARE). We have shown that monocytes interact with allogeneic endothelial cells (ECs) for costimulation to achieve T-cell allorecognition. However, the production of T-cell interferon-γ induced protein-10 (IP-10) and regulation of this chemokine during the initial monocyte–EC interaction are unclear. We hypothesized that the tumor necrosis factor (TNF)-α pathway plays a key role to regulate IP-10 production during the initial monocyte–EC interaction. Cytokine-activated ECs were analyzed for IP-10 production and adhesion molecule expression. Established, monocyte–EC cocultures were analyzed using real-time polymerase chain reaction and a chemokine assay for IP-10 and activation factors. Anti–TNF-α antibody was used to neutralize TNF-α release during monocyte–EC interactions. TNF-α–activated ECs upregulated CD62E and CD54 as determined by flow cytometry, releasing high levels of IP-10 and interleukin (IL)-6. Interferon-γ–stimulated ECs also produced high levels of IP-10 and IL-6. Monocyte–EC interactions demonstrated upregulation of gene transcripts for TNF-α, IL-6, and IP-10. The cytokine/chemokine assay detected high levels of TNF-α, IL-6, and IP-10 in coculture supernates in a time-dependent manner. Anti–TNF-α antibody dramatically reduced IP-10 production by monocyte–ECs interactions. However, anti–TNF-α antibody did not prevent the release of IL-6 by monocytes in EC cocultures. Our results showed that ECs activated by TNF-α are an important source of IP-10. The monocyte–EC interaction produces high levels of IP-10. The TNF-α pathway plays a key role to regulate IP-10 production during monocyte-EC interactions. We thus proposed that the initial monocyte–EC interaction with increased expression of IP-10 may play a critical role to initiate and augment T-cell–mediated ARE.

Monocyte-Derived CD40 Expression Is Regulated by Interferon-γ/Interferon-γ Receptor-1 Pathway when Acting as a Bridge During Their Interaction with T Cells and Allogeneic Endothelial Cells

L.M. Zhu, Y.S. Fang, Z.G. Sun, L.Z. Yu, H. Xu — 2012-05-01

Abstract: Previous studies have showed the lack of CD40 expression on monocytes during monocyte and endothelial cell (EC) interaction in the absence of T cells indicating that the interaction between T cells, monocytes, and ECs is required for monocyte-derived CD40 expression. We investigated the role of monocytes acting as a bridge between ECs and T cells and the possible mechanisms for monocyte-derived CD40 up-regulation in allogeneic immune responses. A coculture system with tanswell was established between purified monocytes, T cells, and ECs, and the cells were analyzed by flow cytometry to detect monocyte-derived CD40 expression. Purified monocytes stimulated by ECs did not show up-regulation of CD40 expression. Ec-stimulated monocytes up-regulated interferon (IFN)-γ receptor-1 expression. Monocytes, stimulated by ECs, up-regulated CD40 expression in the presence of T cells. However, when T cells were separated from monocyte-EC interaction, these monocytes did not show CD40 up-regulation. Furthermore, IFN-γ receptor-1 blockade but not IFN-γ receptor-2 blockade inhibited monocyte-derived CD40 expression during monocyte-EC-T cell interaction. Neutralizing antibody directed to IFN-γ inhibited up-regulation of monocyte-derived CD40. We showed here that the interaction between T cells and EC-stimulated monocytes and up-regulation of monocyte-derived CD40 expression are contact-dependent, suggesting that monocytes act as bridge between ECs and T cells. The IFN-γ receptor-1 blockade inhibited the monocyte-derived CD40 up-regulation. These data suggest that the Th1 lymphocytes provide help for monocytes via IFN-γ and IFN-γ receptor-1 pathway following their interaction.

A Novel Cannulation Technique for Isolation of Human Hepatocytes from Explanted Diseased Whole Livers

D.C. Kehr, N. Raschzok, I.M. Sauer — 2012-05-01

Abstract: Diseased human organs explanted during liver transplantation can be used as a cell source for basic research and future therapeutic applications in regenerative medicine. Enzymatic digestion using the perfusion technique has become the gold standard in liver cell isolation. Usually the portal vein is used as a vascular access for liver cell isolation from explanted livers, that were rejected from whole organ transplantation. No special techniques are required for cannulation; the cannulas are simply introduced into the vessels and a ligature is then thrown around the vessel to secure the cannulation. This method is not applicable to organs explanted during liver transplant surgery, because as much of the vessels as possible has to be kept in situ, to facilitate anastomosis of the new organ. Therefore, when perfusing the explanted organ, normal perfusion catheters are easily displaced and a more complex “vascular reconstruction” must be performed to secure hold of the catheters. We established a novel cannulation technique using commercially available Foley catheters for liver cell isolation from diseased whole organs explanted during transplant surgery. We evaluated this technique in 15 diseased organs. 5 were isolated in the conventional setting and 10 were cannulated using Foley catheters. The average cannulation time was significantly shortened using Foley catheters compared with the conventional approach (12 ± 5.2 min vs 40 ± 14.1 min; P = .0001). Foley catheter cannulation is fast, simple, and efficient. It appears to be favorable for hepatocyte isolation from diseased whole livers or from explanted organs with technically difficult vascular access.

Slow Cooling Rate with a Shock Cooling Program Can Effectively Cryopreserve Pig Hepatocytes

J.-H. Lee, D.-H. Jung, D.-H. Lee, J.-K. Park, S.-K. Lee — 2012-05-01

Abstract: Hepatocyte transplantation is a potential alternative to whole organ liver transplantation. To realize this procedure, a hepatocyte bank system capable of supplying large numbers of hepatocytes must be established. The aim of this study was to develop a cryopreservation protocol using controlled rate freezers (CRF) for the application of a bioartificial system of porcine hepatocytes. Hepatocytes were harvested from 3- to 4-week-old male pigs weighing 11–14 kg. Liver cell preparations were prepared and the spheroid hepatocytes were cryopreserved using University of Wisconsin (UW) solution in controlled freezing. After thawing, viability, plating efficiency, urea synthesis, and ammonia removal were measured to assess the effects of freezing methods. Freezing methods had effects on the viability and specific functions of hepatocytes after thawing. About 80% of the cell viability could be obtained with an optimal computer programming method (−1°C slow cooling rate with shock cooling, using UW solution with 15% dimethyl sulfoxide [DMSO]). The cryopreservation method for hepatocytes was significantly improved by using the above cryopreservation conditions. However, research on application to large-scale cryopreservation is needed for practical use.

Cryopreservation of Immobilized Rat Hepatocytes for the Development of a Bioartificial Liver System

J.-H. Lee, J.-H. Park, H.-J. Park, T. General, M.-G. Cho, S.-K. Lee — 2012-05-01

Abstract: Liver transplantation, the only effective treatment for end-stage disease, is limited by donor availability. Cell transplantation offers the possibility to restore liver mass and function. Cryopreserved primary hepatocytes that can be thawed as needed might address this problem. Hepatocytes were harvested from a male Sprague Dawley rat, weighing approximately 250 g, using a 2-step in situ collagenase perfusion technique modified from the method described by Seglen. Hepatocytes were immobilized using a 100-mmol/L calcium with 1.5% alginate solution. Primary, immobilized hepatocytes transferred to various cryopreservation solutions containing 15% dimethyl sulfoxide were immediately placed into an isopropanol progressive freezing container at −80°C. We analyzed 4 cryopreservation solutions: Hormonally defined medium, histidine-tryptophan-ketoglutarate (HTK), fetal bovine serum (FBS), and HTK-modified cryopreservation solution (JH). After thawing, we measured viability, plating efficiency, urea synthesis, and albumin secretion to assess the effects of cryopreservation. Primary hepatocytes in HTK solution showed the better results in hepatocytes viability and urea synthesis after thawing. Cryopreserved immobilized hepatocytes in FBS maintained their viability and urea synthesis function. However, JH seemed to be the most effective medium for albumin secretion by both cyropreserved primary and immobilized hepatocytes.Our study suggested that HTK and JH cryopreservation solutions without FBS can be used to develop a bioartificial liver system.

Effect of Fulminant Hepatic Failure Porcine Plasma Supplemented with Essential Components on Encapsulated Rat Hepatocyte Spheroids

J.-H. Lee, D.-H. Lee, J.-K. Park, S.-K. Kim, C.H.D. Kwon, S.-K. Lee — 2012-05-01

Abstract: The development of bioartificial liver (BAL) systems has required detailed information about the functional capabilities of cultured hepatocytes during blood or plasma passage. In this study we investigated the effects of porcine plasma and various supplements on the viability and function of adult rat hepatocytes in vitro. Primary rat hepatocytes cultured in porcine plasma supplemented with various substances showed albumin synthesis rates and viability equal to or higher than those of controls. Supplementation with calcium chloride, magnesium sulfate, trace elements, amino acids, insulin, and epidermal growth factor were essential to maintain viability and high albumin synthesis. Especially, trace elements showed significantly higher and longer albumin secretion. Isolated rat hepatocytes were cultured in Spinner flasks for 24 hours to form spheroids that were harvested and encapsulated with chitosan-alginate solution before transfer to the bioreactor in the BAL system. Encapsulated rat hepatocyte spheroids cultured with porcine plasma including trace elements showed higher viability (57%) than controls (40%) after 24 hours, with ammonia removal values of 30.92 μg/106 cells versus the control 9.04 μg/106 cells. After 24 hours of operation the urea secretion value of encapsulated rat hepatocyte spheroids cultured in porcine plasma in the presence versus absence of trace elements was 76.73 μg/106 cells and 18.80 μg/106 cells, respectively. We concluded that encapsulated hepatocyte spheroids in a packed-bed bioreactor operated with human plasma including trace elements enhanced cell viability and liver function as a bases for an in vivo clinical trial of the BAL system.

Potentiality of Immobilized Pig Hepatocyte Spheroids in Bioartificial Liver System

J.-H. Lee, D.-H. Lee, J.-K. Park, S.-K. Kim, C.H.D. Kwon, S.-K. Lee — 2012-05-01

Abstract: Various extracorporeal bioartificial liver (BAL) systems have been developed. To treat fulminant hepatic failure (FHF) patients. Direct cell-cell interaction is one of the major factors influencing the functions of cultured hepatocytes, which increase with progressing of cell aggregation in this study, we investigated the effects of plasma viability and function single and spheroid pig hepatocytes in vitro. Hepatocytes were cultured as spheroids by suspension culture in spinner flasks. We obtained pig plasma from animals in hepatic failure. Immobilized single pig hepatocytes exposed to the toxic pig plasma lost viability and liver function. However, immobilized pig hepatocyte spheroids showed stable ammonia removal functions and urea synthesis and lower lactate dehydrogenase, glutamine oxaloacetate transaminase, and glutamine pyruvate transminase levels during BAL operation. At 5 hours, the ammonia concentration in plasma decreased to 370 and 150 μg/dL by immobilized single and spheroid hepatocytes, respectively, the concentrations at which they were maintained thereafter. The urea concentrations in plasma were 44 versus 72 μg/dL in immobilized single versus spheroid hepatocytes respectively, at 5 hours of operation. Spheroid hepatocytes not only showed in vivo structure, but also maintained high levels of liver-specific functions. The spheroid-based BAL system may be a good candidate to treat FHF patients.

Effect of Spheroid Aggregation on Susceptibility of Primary Pig Hepatocytes to Cryopreservation

J.-H. Lee, D.-H. Jung, D.-H. Lee, J.-K. Park, S.-K. Lee — 2012-05-01

Abstract: Bioartificial liver support systems, which use freshly isolated primary hepatocytes (PH), present severe logistical difficulties. Stored frozen PH that are thawed as required could answer this problem. The aim of this study was to develop a cryopreservation protocol for large-scale preparation of porcine PH. We cryopreserved single and spheroid hepatocytes. Harvested hepatocytes were cryopreserved in various concentrations of dimethyl sulfoxide (DMSO) using hormonally defined medium (HDM) and various presentation solutions, such as University of Wisconsin (UW) and fetal bovine serum. After thawing the hepatocytes, we measured the viability, plating efficiency, ammonia removal, urea synthesis, and albumin secretion. UW solution was most effective for cryopreservation, as evidenced by the viability and liver-specific functions of the thawed hepatocytes. The optimal DMSO concentration for porcine hepatocyte cryopreservation was 15%. After cryopreservation, spheroid hepatocytes maintained greater viability and functional activity compared with single hepatocytes. Moreover, spheroid hepatocytes showed native cell structures and maintained high levels of liver-specific functions. In conclusion, cryopreserved spheroid hepatocytes were superior to cryopreserved single hepatocytes in terms of viability and liver-specific functions.

Self-Organization of Human Hepatic Organoid by Recapitulating Organogenesis In Vitro

2012-05-01

Abstract: Background: Careful orchestration among endodermal epithelial, endothelial, and mesenchymal cells initiate liver organogenesis prior to vascular function. Nonparenchymal endothelial or mesenchymal cells not only form passive conduits, but also establish an organogenic stimulus. Herein, we have evaluated the potential roles of primitive endothelial and mesenchymal cells toward hepatic organization in vitro. Methods: To track the cellular movements and localization, we retrovirally transduced enhanced green fluorescence protein and kusabira orange into human fetal liver cells (GFP-hFLCs) and human umbilical vein endothelial cells (KO-HUVECs), respectively. GFP-hFLCs were cocultivated with KO-HUVECs and human mesenchymal stem cells (hMSCs) under conventional two-dimensional (2D) conditions. Results: Even under 2D culture, fetal liver, endothelial, and mesenchymal cells self-organized into a macroscopically visible three-dimensional (3D) organoid. Time-lapse confocal imaging showed dynamic cellular organizations of GFP-hFLCs and KO-HUVECs. Endothelial cells organized into patterned clusters wrapping fetal liver cells, forming vessel-like lumens inside. Mesenchymal cells supported the generated organoid from outside. Conclusion: Generation of whole organ architecture remains a great challenge so far. Our preliminary results showed that recapitulation of primitive cellular interactions during organogenesis elicit the intrinsic self-organizing capacity to form hepatic organoids. Future studies to define precise conditions mimicking organogenesis may ultimately lead to the generation of a functional liver for transplantation and for other applications such as drug development.

High Glucose Increases Mesangial Lipid Accumulation via Impaired Cholesterol Transporters

K.H. Song, J. Park, H. Ha — 2012-05-01

Abstract: Diabetes, whether it occurs before or after transplantation, plays an important role to decrease graft function and survival. In addition renal lipid accumulation has been suggested to play a role in the development and progression of chronic renal allograft rejection. Intracellular lipid accumulation is governed by a balance between the influx and efflux of lipid. Cholesterol transporters, such as scavenger receptor (SR)-A1, CD36, and ATP binding cassette (ABC) A1 and G1 (ABCG1), coordinate to regulate cellular lipid status. Therefore, in the present study, we examined whether high glucose caused lipid accumulation in mesangial cells as a result of altered cholesterol transporters. Mouse mesangial cells were stimulated with 30 mmol/L D-glucose (high glucose); 100 μmol/L oleic acid (OA) used as a positive control. Cellular lipid accumulation was measured by Oil Red O staining. Protein and mRNA expression of cholesterol influx (SR-A1 and CD36) and efflux (ABCA1 and ABCG1) transporters were evaluated using Western blot analysis and real-time quantitative polymerase chain reaction, respectively. High glucose was shown to significantly increase lipid accumulation in mesangial cells at 24 hours as was observed for OA. SR-A1 and CD36 mRNA expression levels were 1.5-fold and 3.5-fold higher, respectively, in high glucose-stimulated than control mesangial cell, whereas ABCG1 mRNA expression decreased to 60% of controls; however, there was no decrease in ABCA1 mRNA. Altered protein expression of each transporter in mesangial cells cultured under conditions of high glucose concentrations was consistent with mRNA expression. Osmotic control using mannitol did not significantly affect any of the measured parameters in the present study. These results demonstrated that high glucose, in itself, can induce mesangial lipid accumulation; this effect may be associated with an impaired balance between the influx and efflux of cholesterol.

Fractalkine Increases Mesangial Cell Proliferation Through Reactive Oxygen Species and Mitogen-Activated Protein Kinases

J. Park, K.H. Song, H. Ha — 2012-05-01

Abstract: Mesangial cell proliferation is one of the main features of chronic renal allograft rejection. One unique feature of fractalkine (CX3CL1) is its existence as both a membrane-tethered and a soluble form. Fractalkine expression is increased in acute and chronic allograft rejection. However, its role in mesangial cell proliferation has not yet been clearly explored. Thus, the present study examined whether fractalkine induced mesangial cell proliferation through production of reactive oxygen species (ROS) and activation of mitogen-activated protein kinase (MAPK), two known mediators of mesangial cell proliferation. Growth-arrested and synchronized mouse mesangial cells were stimulated with fractalkine in the presence versus absence of inhibitors against ROS, extracellular signal-regulated protein kinase (ERK), and p38 MAPK. Cell proliferation was assessed by methylthiazoletetrazolium assay, dichlorofluorescein (DCF)-sensitive cellular ROS production by a fluorometer, and MAPK activation by Western blot analysis. Fractalkine (10–50 ng/mL) significantly increased mesangial cell proliferation at 24 hours in a dose-dependent manner, an effect that was abrogated by the ROS and MAPK inhibitors. Fractalkine (50 ng/mL) also induced cellular ROS production and activation of ERK1/2 and p38 MAPK in mesangial cells. These results demonstrated that fractalkine can induce mesangial cell proliferation through production of cellular ROS and activation of MAPK.

Lipopolysaccharide Increases Monocyte Binding to Mesangial Cells Through Fractalkine and Its Receptor

J. Park, K.H. Song, H. Ha — 2012-05-01

Abstract: Fractalkine (CX3CL1) is a unique chemokine that functions not only as a chemokine but also as an adhesion molecule. Fractalkine plays an important role in the recruitment of macrophages into the kidneys by binding to its specific receptor CX3CR1, and renal fractalkine expression was shown to be increased in chronic renal allograft rejection. Considering that microcapillary inflammation is a key feature of chronic renal allograft rejection, the present study examined whether monocytes bind to mesangial cells cultured in the presence of lipopolysaccharide (LPS) through fractalkine/CX3CR1 in order to understand their regulation with respect to inflammation-induced renal allograft dysfunction. Mouse mesangial cells were stimulated with LPS in the presence or absence of fractalkine or CX3CR1 siRNA. Calcein-AM-labeled monocytes were used to evaluate monocyte binding. Fractalkine and CX3CR1 mRNA and protein expression were measured by real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. LPS at 100 ng/mL significantly increased monocyte binding to mesangial cells. Each siRNA against fractalkine or CX3CR1 effectively inhibited LPS-induced monocyte-mesangial cell binding. Fractalkine and CX3CR1 mRNA expression were enhanced in mesangial cells stimulated with LPS. Fractalkine protein synthesis in media and lysate of mesangial cells were also induced by LPS. These results demonstrated that LPS induces monocyte-mesangial cell binding through the fractalkine/CX3CR1 system and suggested that fractalkine/CX3CR1 system may contribute to renal inflammation leading to chronic renal allograft rejection.

Selectively Expanding Subsets of T Cells in Mice by Injection of Interleukin-2/Antibody Complexes: Implications for Transplantation Tolerance

O. Boyman, C. Krieg, S. Letourneau, K. Webster, C.D. Surh, J. Sprent — 2012-05-01

Abstract: The biological activity of interleukin (IL)-2 and other cytokines in vivo can be augmented by binding to certain anti-cytokine monoclonal antibodies (mAb). Here, we review evidence on how IL-2/anti–IL-2 mAb complexes can be used to cause selective stimulation and expansion of certain T-cell subsets. With some anti–IL-2 mAbs, injection of IL-2/mAb complexes leads to expansion of CD8 T effector cells but not CD4 T regulatory cells (Tregs); these complexes exert less adverse side effects than soluble IL-2 and display powerful antitumor activity. Other IL-2/mAb complexes have minimal effects on CD8 T cells but cause marked expansion of Tregs. Preconditioning mice with these complexes leads to permanent acceptance of MHC-disparate pancreatic islets in the absence of immunosuppression.

Immunology Mini-review: The Basics of TH17 and Interleukin-6 in Transplantation

T. Nakagiri, M. Inoue, M. Minami, Y. Shintani, M. Okumura — 2012-05-01

Abstract: The outcomes of organ transplantation are determined by graft rejection, the mechanisms of which are some of the most important areas of study in the transplantation field. The main cause of rejection is the immunologic response of the recipient toward the transplanted organ. The immunologic responses are regulated by T-cell subsets, especially helper T-cells, which have been characterized as TH1 or TH2 cells according to their profiles of cytokines production. A unique subset of recently identified lymphocytes, the regulatory T cells (Tregs), seem to play a role in tolerance. The recently identified TH17 cells are a subset of effector-helper lymphocytes that specifically secrete interleukin (IL) 17. Interestingly, TH17 and Treg both develop from naïve T cells on stimulation by transforming growth factor β. The difference is only the existence of IL-6, a proinflammatory cytokine. TH17 clears pathogens that are not adequately handled by TH1 and TH2 elements, as well as contributing to autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, and inflammatory diseases. Autoimmune diseases are caused by reactions to self-antigens. TH17 (or IL-17) and IL-6 are also thought to be involved in rejection after organ transplantation. We examined the contributions of TH17 and IL-6 in bronchiolitis obliterans (BO), the histologic finding in chronic rejection of lung transplantations. Earlier studies have reported that TH17 and IL-6 contribute not only to chronic rejection of lung transplantations, but also to the rejection of other solid organs, e.g., heart, liver, and kidney. In addition, prospective avenues of research on TH17 and IL-6 in transplantation have emerged from the perspectives of recent studies.

The Role of Invariant NKT Cells in Liver Transplant Tolerance in Rats

Y. Liu, X. Luan, J. Li, Y. He, M. Li — 2012-05-01

Abstract: Objective: The aim of this study was to investigate the role of invariant natural killer T (iNKT) cells in liver transplant tolerance in rats. Methods: Animals were randomly divided into 3 groups. The α-galactosylceramide (α-GalCer) group underwent injection through the caudal vein; the saline group received the same dose of saline and the control group received no treatment. Ten rats in each group were examined for survival the others were humanely killed on the seventh day posttransplantation. Liver tissues were used to assess histopathologic changes. Real-time polymerase chain reaction (PCR) was performed to determine the relative expressions of messanger RNAs of Th1/Th2-related cytokine (mRNAs) in the liver allograft. The serum levels of related cytokines were determined using enzyme-linked immunosorbent assay (ELISA). Results: Allograft survival was significantly higher among the α-galactosylceramide α-GalCer group than the saline or control groups. The histopathology showed mild changes in the α-galactosylceramide α-GalCer compared with the other 2 groups. Real-time PCR showed the relative expression of Th1-related cytokine interferen (IFN)-γ mRNA to be significantly lower in the α-galactosylceramide α-GalCer compared with the other 2 groups, while the Th2-related cytokine interleukin (IL)-10 mRNA was much higher. The ELISA results confirmed these differential expressions. Conclusion: The iNKT cells may play a pivotal role in liver transplant tolerance due to their regulatory functions on the Th1/Th2 imbalance. iNKT cells should be considered to be significant targets because of their attractive specificity and induction of liver allograft tolerance.

Dynamic Changes of Indoleamine 2,3-Dioxygenase of Kupffer Cells in Rat Liver Transplant Rejection and Tolerance

X. Luan, W. Liao, X. Lai, Y. He, Y. Liu, J. Gong, J. Li — 2012-05-01

Abstract: Aims: To study the dynamic changes and the immunologic role of indoleamine 2, 3-dioxygenase (IDO) in Kupffer cells (KCs) after rat liver transplantation. Methods: Animals were randomly divided into two groups: a rejection group (REJ; LEW to BN) and a tolerance group (TOL; BN to LEW). Liver morphological changes were observed optically with hematoxylin/eosin staining. KCs were isolated from recipients. mRNA and protein expressions of IDO were detected by real-time polymerase chain reaction and Western blotting at 1, 3, 5, and 7 days after transplantation. Results: The levels of IDO mRNA and protein in KCs of TOL groups were similar to those in REJ groups at day 1 posttransplantation. However, the expression of IDO mRNA and protein time-dependently increased to much higher levels in the TOL than the in REJ groups at 3, 5, and 7 days posttransplantation (P < .05). The peak was observed at 7 days. Conclusions: The IDO level of KCs was closely associated with immune tolerance induction. IDO-mediated immune modulation appears to be an attractive means to assess transplant tolerance induction.

Can Immune Function Assay Predict Infection or Recovery?

2012-05-01

Abstract: Background: Recently, the ImmuKnow assay (Cylex Inc., Columbia, Md) has been reported to be a global immune monitoring tool for organ transplants recipients. We assessed whether immunKnow ATP values predicted infectious syndromes. Methods: We prospectively enrolled 71 kidney transplant patients between September 2008 and May 2011. lmmuKnow assay monitoring was performed at one dav before as well as 4, 8, 12, 16, 20, 24, 36, and 52 weeks after the operation. ImmuKnow assay values were compared as well as BK viral infection pre-infection(PI), at first detection of infectious syndrome (DI), 4 weeks there after (4W), 8 weeks there after (8W) and 12 weeks there after (12W) and pre-recovery (PR), recovery (R) times. Results: Serial ImmuKnow assays showed significant differences over time and BK viral infectious state (P = .026). Interestingly, PI was significantly lower than DI and PR but PR significant greater than PI, 8W and 12W. However, we did not observe an adequate or absolute cutoff value of ImmuKnow by ROC curve: 377 ng/mL ImmuKnow showed 0.471 of AUC and 57.1% and 56.2%, of sensitivity and specificity. Conclusion: Longitudinal evaluation and adjustment of the value of ImmuKnow assay seemed to be a favorable modality to monitor infectious syndromes especially those involving BK virus.

Immunomodulation of Human CD8+ T Cells by Thymoglobulin In Vitro

L.-Z. Yu, Y. Fang, L. Zhu, Z.-G. Sun, H. Xu — 2012-05-01

Abstract: The major mechanism of Thymoglobulin in inhibiting allograft rejection involves lymphocyte depletion. However, its role in modulating CD8+ cells has remained unclear. This study evaluated the immunologic effects of Thymoglobulin on human CD8+ cells. Purified CD8+ cells were pretreated with Thymoglobulin followed by incubation for 72 hours. The culture supernates and cells were analyzed using real-time quantitative polymerase chain reaction (RT-PCR), multiplex cytokine detection assay, and flow cytometry. RT-PCR showed that Thymoglobulin-treated CD8+ cells up-regulated transcripts for CD25, CTLA-4, OX40, GITR, Foxp3, IFN-γ, interleukin (IL)-10, and IL-2 when compared with isotype control immunoglobulin (Ig). The expression for GITR and IL-2 transcripts was down-regulated at 72 hours after incubation. The release of IFN-γ, IL-10, and IL-2 from Thymoglobulin-pretreated CD8+ cells was detected using multiplex assay after 24-hour incubation, and these cytokine levels were decreased after incubation for 48 and 72 hours. Flow cytometry demonstrated up-regulation of CD69 and CD25 expression after treatment. The surface CTLA-4 and intracellular Foxp3 expression was not increased in Thymoglobulin-treated cells. Our results demonstrated that Thymoglobulin-treated CD8+ cells up-regulate CD25, and multiple costimulatory molecules at the transcriptional level. The up-regulation of transcripts of immune regulatory cytokines accompanies the release of these cytokines. The unique effects of Thymoglobulin on CD8+ cells may be an important mechanism for its action in inducing immunosuppression and immunomodulation.

Inhibition of Allogenic T-Cell Cytotoxicity by Hepatic Stellate Cell via CD4+ CD25+ Foxp3+ Regulatory T Cells In Vitro

T.-J. Wu, Y.-C. Wang, T.-H. Wu, C.-F. Lee, K.-M. Chan, W.-C. Lee — 2012-05-01

Abstract: Background: The liver is considered to be an immune-privileged organ. Several types of liver cells have been implicated in the induction of immunologic tolerance. Hepatic stellate cells (HSCs) seem to participate in hepatic fibrosis and to display immunological properties. Materials and results: In this study, HSCs isolated from C3H mice were highly positive for GFAP (98.4%) and α-SMA (95.4%). After stimulation by interferon-γ (IFN-γ), HSCs were more active in morphology with enhanced expression of H2-KK, I-AK, CD80, and CD54, similar to mature myelogenic dendritic cells (MDCs). Through allogeneic stimulation, C3H HSCs induced proliferation of both CD8+ and CD4+ T cells in B6 mice. However, the T cells activated by allogeneic HSCs produced less INF-γ, interleukin (IL)-4, IL-10, and IL-17, but large amount of transforming growth factor-β. These T cells expressed immunoregulatory rather than effector functions. Naïve T cells stimulated by allogeneic HSCs expressed Foxp3 compared with MDCs (8.67% vs 2.14%, P < .05). CD8+ T cells activated by HSCs lost their allogeneic cytotoxicity, and CD4+ T cells activated in this fashion suppressed the allogeneic cytotoxicity of CD8+ T cells activated by MDCs. Conclusion: HSCs seem to act as liver-resident antigen-presenting cells instructing the generation of Foxp3+ regulatory T cells, a property suggestion of induction of immunologic tolerance.

CD28 Superagonist Antibody Treatment Attenuated Obliterative Bronchiolitis in Rat Allo-Orthotopic Tracheal Transplantation by Preferentially Expanding Foxp3-Expressing Regulatory T Cells

Q. Shi, Y. Niu, H. Cao, X. Zhou, S. Jiang, Z. Liu, H. Fan — 2012-05-01

Abstract: Obliterative airway disease (OAD) due to chronic alloantigen rejection remains a major challenge for long-term graft survival in lung transplantation. It is known that superagonistic CD28-specific monoclonal antibody JJ316 (supCD28 MAb) has the ability to induce regulatory T cells (Tregs) efficiently. Here we used a rat orthotopic tracheal transplantation model to investigate the effects of supCD28 MAb on expanding Tregs in vivo and its application in suppression of acute and chronic airway allograft rejection. SupCD28 MAb administration revealed a significant increase in the CD4+CD25+ T cells, CD4+FoxP3+ T cells, and CD4+CD25+ FoxP3+ T cells population among CD4+ T cells in spleen, peripheral blood, as well as cervical lymph nodes. The allografts from animals treated with supCD28 MAb showed significantly less airway obliteration and rejection of the respiratory epithelium compared with allografts of the mouse immunoglobulin G-treated group on the 5th day and the 60th day after transplantation. Overall, our data demonstrated that an intraperitoneally administrated low dose of supCD28 MAb was sufficient to induce Treg cell expansion in vivo and was effective in protecting the airway graft from early rejection and chronic OAD development. These findings provide the basis for new therapies to prevent OAD and perhaps rejection of allografts in other human transplantations.

Induction of Regulatory CD4+ Cells and Prolongation of Survival of Fully Allogeneic Murine Cardiac Grafts by Danazol

M. Uchiyama, X. Jin, Q. Zhang, A. Amano, T. Watanabe, M. Niimi — 2012-05-01

Abstract: Danazol, a modified testosterone, has been used to treat endometriosis and pretreatment before in vitro fertilization and embryo transfer, although its reproductive mechanisms remain unclear. We investigated the effect of danazol on alloimmune responses in murine heart transplantation. CBA male mice (H2k) that underwent transplantation of C57BL/6 (B6, H2b) hearts received danazol (0.4 and 4 mg/kg/d) by intraperitoneal injection from the day of transplantation to days 6. We performed an adoptive transfer study to determine regulatory cells as well as cell proliferation, cytokine, and flow cytometry assessments. Danazol-treated (4 mg/kg/d) CBA mice showed prolonged allograft survival (median survival time [MST], 63 days). Moreover, secondary CBA recipients of whole splenocytes and CD4+ cells from primary danazol-treated (4 mg/kg/d) CBA recipients at 30 days after transplantation displayed prolonged allograft survival (MSTs, 29 and 60 days, respectively). Cell proliferation, interleukin (IL)-2, and interferon-γ were suppressed in danazol-treated mice, whereas IL-4 and IL-10 were up-regulated. Moreover, danazol directly suppressed alloproliferation in mixed leukocyte cultures. Flow cytometry studies showed an increased CD4+CD25+Foxp3+ cell population among splenocytes from danazol-treated mice. In conclusion, danazol induced prolonged cardiac allograft survival and generation of regulatory CD4+ cells.

The Smell of Tokishakuyaku-san (TJ-23) Induces Generation of Regulatory T Cells and Prolongation of Survival of Fully Allogeneic Cardiac Grafts in Mice

X. Jin, M. Uchiyama, Q. Zhang, T. Watanabe, M. Niimi — 2012-05-01

Abstract: Oral administration of Tokishakuyaku-san (TJ-23), a Japanese herbal medicine, induces prolongation of cardiac allograft survival and generates regulatory cells in mice. Because herbal medicines usually have unique odor, and because smell is supposed to modulate the immune system, we examined whether the odor of TJ-23 induced prolonged allograft survival and regulatory cell generation. Naïve CBA mice (H2k) and olfactory-dysfunctional CBA mice after a stereotaxic operation underwent transplantation of C57BL/6 (B6, H2b) hearts, receiving fumigated water only or TJ-23 until rejection. Untreated or treated with water fumigation CBA mice rejected B6 cardiac grafts acutely (median survival times [MSTs], 7 and 8.5 days). When CBA mice were treated with fumigation of TJ-23, allograft survival was significantly prolonged (MST, 48 days). Olfactory-dysfunctional CBA mice treated with fumigation of TJ-23 rejected grafts acutely (MST, 7 days). Treatment with fumigation of TJ-23 also suppressed splenocytes proliferation and interferon-γ production. Secondary CBA recipients of whole splenocytes or CD4+ cells from primary TJ-23–treated CBA recipients of B6 cardiac allografts at 30 days after grafting showed prolonged survival of B6 hearts (MST, >60 days). Flow cytometry studies showed increased CD4+CD25+Foxp3+ regulatory cells in recipients given fumigation of TJ-23. In conclusion naïve but not olfactory-dysfunctional CBA mice treated with fumigation of TJ-23 displayed prolonged survival of fully allogeneic cardiac allografts and generation of regulatory cells.

Artemisiae capillaris herba Induces Prolonged Survival of Fully Cardiac Allografts and Generates Regulatory Cells in Mice

X. Jin, M. Uchiyama, Q. Zhang, T. Watanabe, M. Niimi — 2012-05-01

Abstract: Inchingorei-san (TJ-117), a 6-component herbal medicine, is used in Japan for the treatment of vomiting, urticaria, and liver and kidney disorders with few side effects. In this study we investigated the effect of TJ-117 on alloimmune responses in murine cardiac allograft transplantation. CBA (H2k) mice underwent transplantation of a C57BL/6 (B6, H2b) heart with oral administration of TJ-117 (or 1 component of TJ-117) from the day of transplantation for 7 days. CBA recipients given 1 g/kg/d of TJ-117 showed prolonged B6 allograft survival (median survival time [MST], 23 days). Naive CBA mice rejected B6 cardiac grafts acutely (MST, 7 days). Moreover, Artemisiae capillaris herba (ACH; 1g/kg/d) 1 component of TJ-117, significantly prolonged B6 allograft survival (MST, > 100 days). However, the other 5 components of TJ-117 were individually less effective than TJ-117 or ACH. ACH also suppressed splenocyte proliferation and interferon-γ production. Secondary CBA recipient showed prolonged survival of B6 hearts after treatments with whole splenocytes from primary ACH-treated CBA recipients carrying B6 cardiac allografts for 30 days (MST, >50 days). Flow cytometry studies showed increased CD4+CD25+Foxp3+ regulatory cells in transplant recipients given ACH. In conclusion, ACH, 1 component of TJ-117, as well as TJ-117 induced hyporesponsiveness to fully allogeneic cardiac allografts with generation of CD4+CD25+Foxp3+ regulatory cells.

Music Exposure Induced Prolongation of Cardiac Allograft Survival and Generated Regulatory CD4+ Cells in Mice

M. Uchiyama, X. Jin, Q. Zhang, A. Amano, T. Watanabe, M. Niimi — 2012-05-01

Abstract: In clinical practice, music has been used to decrease stress, heart rate, and blood pressure and to provide a distraction from disease symptoms. We investigated sound effects on alloimmune responses in murine heart transplantation. Naïve and eardrum-ruptured CBA/N (CBA, H2K) underwent transplantation of a C57BL/6 (B6, H2b) heart and were exposed to 1 of 3 types of music-opera (La Traviata), classical (Mozart), and New Age (Enya)-or 1 of 6 different single sound frequencies for 7 days. An adoptive transfer study was performed to determine whether regulatory cells were generated in allograft recipients. Cell-proliferation, cytokine, and flow cytometry assessments were also performed. CBA recipients of a B6 graft exposed to opera and classical music had significantly prolonged allograft survival (median survival times [MSTs], 26.5 and 20 days, respectively), whereas those exposed to 6 single sound frequencies and New Age did not (MSTs, 7, 8, 9, 8, 8, 8, and 11 days, respectively). Untreated and eardrum-ruptured CBA rejected B6 grafts acutely (MSTs, 7 and 8.5 days, respectively). Adoptive transfer of whole splenocytes, CD4+ cells, and CD4+CD25+ cells from opera-exposed primary recipients resulted in significantly prolonged allograft survival in naive secondary recipients (MSTs, 36, 68, and >50 days, respectively). Cell-proliferation, interleukin (IL)-2 and interferon-γ were suppressed in opera-exposed mice, whereas IL-4 and IL-10 from opera-exposed recipients were up-regulated. Flow cytometry studies showed an increased CD4+CD25+Foxp3+ cell population in splenocytes from opera-exposed mice. In conclusion, exposure to some types of music may induce prolonged survival of fully allogeneic cardiac allografts and generate CD4+CD25+Foxp3+ regulatory cells.

The Antiproteinuric Effects of Green Tea Extract on Acute Cyclosporine-Induced Nephrotoxicity in Rats

B.C. Shin, Y.E. Kwon, J.H. Chung, H.L. Kim — 2012-05-01

Abstract: Background: It has been reported that the proteinuria is an early useful marker to detect cyclosporine (CsA) nephrotoxicity. The aim of this study was to investigate the antiproteinuric effects of green tea extract (GTE) on CsA-induced acute renal injury in rats. Methods: The rats (n = 28) were divided into four groups (n = 7/group); controls intraperitoneally (IP) injected with 0.9% saline; CsA group IP injected CsA (50 mg/kg); inducible nitric oxide synthase (iNOS) inhibitor group administered in addition NG-nitro-L-arginine-methyl ester (12 mmol/L) subcutaneously and CsA-GTE group of CsA IP plus GTE (100 mg/kg) subcutaneously. Results: The 24-hour urine proteins were significantly increased among the CsA (22.6 ± 3.1 mg/d) compared with the control (7.1 ± 1.5 mg/d) and significantly decreased in the CsA-GTE group (8.2 ± 1.8 mg/d, P < .01). Nitric oxide production induces by CsA treatment was significantly suppressed by GTE and iNOS inhibitor. Renal tissue malondialdehyde level was significantly increased in the CsA compared with controls and significantly decreased in the CsA-GTE group. The antioxidant enzyme activities of superoxide dysmutase and catalase, which were significantly suppressed in the CsA compared with the control group, were restored in the CsA-GTE cohort. Conclusion: GTE treatment of rats showed meaningful antiproteinuric effects through antioxidative activity in kidneys from CsA-induced acute renal injury.

Cellular Function of RhoGDI-α Mediates the Cycling of Rac1 and the Regulation of Pancreatic Beta Cell Death

Y. Cho, K.H. Huh, Y.-J. Park, J.H. Do, D.J. Joo, M.S. Kim, Y.S. Kim — 2012-05-01

Abstract: Mycophenolic acid (MPA) is an immunosuppressive agent that is widely used in clinical therapy, including pancreas and islet transplantation. Previously, we showed that MPA induces significant apoptosis of insulin-secreting cells by downregulating RhoGDI-α and increasing JNK expression. In this study, we investigated Rac1 directly associated with RhoGDI-α during MPA-induced apoptosis in INS-1E cells (an insulin-secreting cell line). Cells were treated with MPA for 24 and 36 hours. Immunoprecipitation was used to examine physical interactions between RhoGDI-α and Rac1. Activation and immunoprecipitation assays showed expressions of Rac1 and RhoGDI-α to be directly correlated. Rac1 binding to RhoGDI-α decreased after MPA treatment, and Rac1 was induced and subsequently activated by MPA. We concluded that this novel RhoGDI-α/Rac1/JNK pathway induced apoptosis of transplanted islet cells after MPA treatment.

Alpha-Melanocyte Stimulating Hormone Preserves Islet Graft Survival Through Down-Regulation of Toll-Like Receptors

E.-J. Jung, S.-C. Kim, S.-H. Jeong, J.-Y. Lee, D.-J. Han — 2012-05-01

Abstract: The induction of Toll-like receptors (TLRs) in β cells is involved in β-cell death and graft rejection after transplantation. This study investigated the ability of alpha-melanocyte stimulating hormone (α-MSH) to protect pancreatic islets and improve graft survival through regulation of TLRs. To test the effect of α-MSH on TLR regulation, we first isolated pancreatic islets from rats pretreated with/without α-MSH and assayed inflammatory cytokines and insulin release, and measured the expression of TLRs. Pancreatic islets were transplanted into the kidney capsule of a diabetes mellitus (DM) mouse with and without prior injection of α-MSH. The blood glucose levels were measured and TLR4 expression in transplanted kidney tissue was assessed. Islet morphology, including size and total mass, was improved in the α-MSH group compared to the control group. The expression of TLRs as well as nitric oxide and monocyte chemoattractant protein 1 production were decreased in islets isolated from α-MSH–treated rats. In DM mice, the normoglycemic ratio was higher in the α-MSH–treated group than in the sham group. Moreover, the high levels of TLR4 expression observed in DM kidney tissue were significantly decreased in islet-transplanted tissue with α-MSH. This study showed that α-MSH protects pancreatic islets from cell death and dysfunction through downregulation of TLRs. In conclusion, α-MSH could contribute to improved islet graft survival and function in pancreatic islet transplantation.

Microencapsulation of Pancreatic Islets With Canine Ear Cartilage for Immunoisolation

2012-05-01

Abstract: Improving human islet transplantation is often limited by the shortage of donors and the side effects of immunosuppressive agents. If immunoisolation is properly used, it can overcome these obstacles. Because artificial materials are adopted in this technique, however, there are still multiple issues with biocompatibility and foreign body reactions. We developed a chondrocyte microencapsulated immunoisolated islet (CMI-islet) that allows living cells to act as the immunoisolating material. To manufacture CMI-islets for xenotransplantation, isolated rat pancreatic islets were placed on low cell-binding culture dishes. Subsequently, expanded canine auricular cartiage primary cells were seeded on these dishes at a high density and maintained in a suspended state via a shaking culture system. Morphological evaluations showed good islet viability and a clear progression of the islet- encapsulation events. When the cells were challenged with glucose, they were able to secrete sufficient insulin according to glucose concentrations. The CMI-islets responded better to the glucose challenge than did nude pancreatic islets and created better glucose-insulin feedback regulation. Moreover, insulin secretion into the culture medium was confirmed over a period of 100 days, showing the survival and secretory capacity of the CMI-islet cells. By microencapsulating pancreatic islets with recipient ear cartilage cells, long-term insulin secretion can be maintained and the response to glucose challenges improved. This new immunodelusion technology differs from other immunoisolation techniques in that the donor tissue is enclosed with the recipient's tissue, thus allowing the transplanted cells to be recognized as recipient cells. This microencapsulation method may lead to developing viable xenotransplantation techniques that do not use immunosuppressive drugs.

Hybrid Cellular Spheroids From Hepatocellular Carcinoma and Insulin-Secreting Cell Lines

2012-05-01

Abstract: During islet transplantation into the portal vein of the liver, the islet cells are expected to have complex interactions with hepatocytes. However, the mechanism underlying this interaction is not yet understood. Hence, we developed cellular complexes containing a mixture of human hepatocellular carcinoma cell line (Hep-G2) and rat insulin-secreting cell line (RIN-5F) by using a co-culture model and studied the function and morphology of the resultant hybrid cellular spheroids (HCSs). The RIN-5F and Hep-G2 cells were suspension cultured and, within 5 days of culture, the two types of cells aggregated to yield spheroids. The functionality of the thus formed HCSs was evaluated by measuring the levels of insulin and albumin in the culture supernatant. The HCSs retained their insulin- and albumin-secreting ability and their morphology, as revealed by immunohistological staining. The insulin and albumin levels secreted by the HCSs were considerably higher than those secreted by spheroids of single-cell origin. Generally, obtaining complexes from more than two types of cells is difficult. However, we were able to generate HCSs. We believe that this culture method could have various applications such as studying the in vitro cell-cell interactions and developing new cell transplantation models.

Impact of Coculture with Ischemic Preconditioned Hepatocellular Carcinoma Cell Line (Hep-G2) Cells on Insulin Secreting Function of Rat Insulin-secreting Cell Line (RIN-5F) Cells

2012-05-01

Abstract: Introduction: Although Islet cell isolation and culture have been well developed, there has been little progress to prolong transplanted islet sruvival. Hepatic ischemia and insufficient neovascularization of islets are considered to be the barriers to long-term survival, Hepatocytes that survive ischemic injury have been reported to protect themeslves and regenerate using the IL-6 interleukin 6 and STAT3 pathways. Materials and Methods: The hepatocellular carcinoma (Hep-G2) cell line preconditioned for 0, 2, 4, 6, and 24 hours in a hypoxic chamber, was cocultured with rat insulin-secreting celline (RIN-5F) cells. We measured cell viabilities, insulin secretion, and p-STAT3, IL-6, and NF-κB levels. Results: Cocultured Hep-G2 and RIN-5F cells aggregated to form spheroids. Viabilities of Hep-G2 cells were no different after various ischemic preconditioning times, but insulin secretion increased in a time-dependent fashion with preconditioning. Western blotting showed p-STAT3, NF-κB, and IL-6 levels to increase with preconditioning time. Conclusion: The IL-6/STAT3 pathway of Hep-G2 cells after ischemic injury showed beneficial effects on insulin secretion of RIN-5f cells cocultured with themselves.

Regenerative Medicine Approach as an Alternative Treatment to Islet Transplantation

K. Sekine, T. Takebe, M. Enomura, C. Matsui, H. Tanaka, H. Taniguchi — 2012-05-01

Abstract: Islet transplantation is considered to be one of the most promising treatment for type I diabetes mellitus (TID). Development of the Edmonton protocol opened the possibility of insulin independence for the patients with TID. However, there is the problem of the donor shortage. Herein we have discussed recent approaches to overcome the problem. It is neccessary to develop a new cellular source for donor islets and to achieve a high engraftment rate. One advantage in TID therapy is that allogeneic islet transplantation is allowed to avoid autoimmunity. That opens broad candidates for the beta-cell source. To achieve a high engraftment rate, is several attempts have sought to develop an appropriate site for transplantation and to modify beta-cells for long-term survival. It is also important to achieve early onset of blood perfusion after transplantation by prevascularization of the islets in vitro. These multiple approaches will bring a milestone in diabetes therapy.

Stimulation of Liver Regeneration After Hepatectomy in Mice by Injection of Bone Marrow Mesenchymal Stem Cells via the Portal Vein

2012-05-01

Abstract: Aim: To investigate whether mouse bone marrow mesenchymal stem cells (BMC) stimulate liver regeneration after partial hepatectomy. Methods: Isolated BMCs were purified by density gradient centrifugation. We performed a 70% hepatectomy in male BALB/c mice followed by injection of BMCs into the portal vein (PV-BMC group), or the tail vein (IV-BMC group), or of saline into the portal vein (control group). Results: The wet weight of the liver remnant increased significantly in the PV-BMC group at 3 and 5 days after hepatectomy compared with the IV-BMC and control groups. The Ki-67 labeling index revealed that the increase to result from stimulation of DNA synthesis. The constitutive interleukin-6 and hepatocyte growth factor mRNAs in the remnant liver tended to increase in the PV-BMC group at 3 days after hepatectomy. Conclusions: These results demonstrated that BMC injection into the portal vein enhanced liver growth after partial hepatectomy in mice.

The Phenotypic Characteristic of Liver-Derived Stem Cells From Adult Human Deceased Donor Liver

2012-05-01

Abstract: Liver transplantation is the only effective treatment for end-stage liver disease. Because of the limited donor availability, attention has been focused on the possibility to restore liver mass and function through cell transplantation. Stem cells are a promising source for liver repopulation after cell transplantation, but whether or not the adult liver contains hepatic stem cells is highly controversial. Several studies have suggested the presence of stem cells in the adult normal human liver. However, a population with stem cell properties has not yet been isolated. The purpose of this study was to identify and characterize progenitor cells in normal adult human liver. We isolated and expanded human liver stem cells (HLSCs) from a donated liver not suitable for liver transplantation or characterizing them by fluorescence-activated cell sorter, polymerase chain reaction, and immunofluorescence assay. HLSCs expressed the mesenchymal stem cell markers CD29, CD73, CD44, CD90, CD105, and CD166 but not the hematopoietic stem cell markers CD34, CD45, and CD117. HLSCs were also positive for vimentin and nestin, a stem cell marker. The absence of staining for cytokeratin-19, CD117, and CD34 indicated that HLSCs were not oval stem cells. In addition, HLSCs expressed CD26, and in a small percentage of cells, cytokeratin-8 and cytokeratin-18, indicating a partial commitment to hepatic cells. We concluded that HLSCs expressed several mesenchymal but not hematopoietic stem cell markers as well as CD26 and CK18, indicating a partial commitment to hepatic cells.

Differentiation and Major Histocompatibility Complex Antigen Expression in Human Liver–Derived Stem Cells

2012-05-01

Abstract: Stem cells are a promising source for liver repopulation after cell transplantation, but whether the adult liver contains hepatic stem cells is controversial. The purpose of this study was to characterize the properties and expression profile of major histocompatibility complex (MHC) antigens on the surface of human-derived stem cells. Human liver–derived stem cells (HLSC7) were isolated from the nontumorous tissue of a patient who underwent a resection of an hepatic hemangioendothelioma. We characterized HLSC7 using a fluorescence-activated cell sorter, polymerase chain reactions, and immunofluorescence assays. HLSC7 expressed mesenchymal but not hematopoietic stem cell markers. HLSC7 underwent osteogenic, chondrogenic, and hepatogenic differentiation when cultured in appropriate differentiation media. However, HLSC7 did not differentiate into adipocytes. In addition, HLSC7 did not express MHC class II (HLA-DP, -DQ, and -DR) antigens. However, they did express MHC class I antigens. These results suggest that human liver–derived stem cells express MHC class I antigens and thus may be rejected on transplantation. Therefore, in addition to studies on stem cell differentiation, one must overcome immunologic barriers for successful clinical application of this therapy.

Live Cell–Imaging Perfusion Culture System of Liver Sinusoidal Endothelial Cells to Mimic Stem Cell Engraftment in Liver

2012-05-01

Abstract: Hepatocyte and various hepatic stem cell transplantations have been studied as alternative therapies to orthotopic liver transplantation for liver injury. The engraftment of transplanted cells into the parenchyma requires transmigration through sinusoidal endothelial cells (SECs), the only cellular barrier. In this study, we constructed a SEC-imaging perfusion culture system that mimics sinusoids with respect to hemorheologic properties. SECs were successfully maintained for 24 hours. Human liver stem cells (HLSCs) were used as a model of transplanted cells for in vitro engraftment to SECs under perfusion culture conditions. Conditions of high shear stress perfusion with 0.34 dyne/cm2 significantly reduced cell adhesion in contrast to lower shear stress conditions of 0.1 and 0.03 dyne/cm2. Among the biologic perfusion fluids, namely, fetal bovine serum (FBS), pig plasma, and 5% human albumin solution, HLSCs showed significantly greater attachment to SECs when perfused with FBS, which is well known to contain abundant amounts of adhesion molecules. This biomimetic SEC perfusion culture system may provide a useful tool to study engraftment mechanisms and to evaluate the effects of various enhancers as an alternative to animal models.

In Vitro Evaluation of Migratory Capacity of Human Liver Stem Cells Influenced by Soluble Factors

2012-05-01

Abstract: Although several studies have addressed the engraftment of stem cells into the liver, the exact mechanisms in vivo remain unclear. In this study, we investigated the effects of soluble factors on cell migration using purified, expanded human liver stem cells (HLSCs) obtained from a pediatric liver resection. Using a in vitro transwell migration assay, we evaluated the migratory capacity of HLSCs under the influence of the cytokines tumor necross factor- [TNF]-α, interleukin [IL]-6, and interferon (IFN)-γ or the growth factors vascular endothelial growth factor [VEGF], basic fibroblast growth factor [bFGF], and hepatocyte growth factor [HGF], which are known to be highly secreted during liver injury. We also evaluated the migratory capacity indirectly influenced by cryopreserved human hepatocytes. The migration across the transwell membrane was promoted by VEGF, bFGF, TNF-α, IFN-γ, or hepatocytes. The cryopreserved human hepatocytes especially induced significant migration. These results suggested the presence of unidentified soluble factors from hepatocytes. This experiment described a reliable system for quantitative migration studies to broaden our understanding of the directional nature of cell migration.

Delayed Treatment With Human Umbilical Cord Blood-Derived Stem Cells Attenuates Diabetic Renal Injury

J.H. Park, J. Park, S.H. Hwang, H. Han, H. Ha — 2012-05-01

Abstract: Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease worldwide. Excess accumulation of extracellular matrix and the epithelial-to-mesenchymal transition contribute to renal fibrosis, which is associated with DKD. The present study examined whether delayed treatment with human umbilical cord blood–derived stem cells (hUCB-SC) showed a therapeutic effect on DKD progression. Experimental diabetes was induced by intraperitoneal injection of streptozotocin (STZ; 50 mg/kg) into 6-week-old male Sprague-Dawley rats. Age-matched control rats received an equivalent volume of sodium citrate buffer alone. At 4 weeks after the STZ injection when diabetic renal injury had developed, hUCB-SC were administered (1 × 106 cells/rat) through the tail vein. Four weeks after administering the hUCB-SC, rats were sacrificed and we measured indices of DKD, including urinary protein excretion as well as fibronectin, α-smooth muscle actin (α-SMA), and E-cadherin mRNA, and protein expression. Diabetic rats developed significantly increased urinary protein excretion and renal hypertrophy compared to those in control rats. Renal expression of fibronectin and α-SMA mRNA, and protein were increased significantly in diabetic rats compared to those in the controls. E-cadherin protein expression in diabetic kidneys decreased significantly. Intravenously administered hUCB-SC effectively reduced proteinuria, renal fibronectin, and α-SMA up-regulation, as well as renal E-cadherin down-regulation in diabetic rats without a significant effect on blood glucose. Engrafted hUCB-SC in diabetic kidneys were confirmed by human DNA PKcs. The results demonstrated that delayed treatment with hUCB-SC attenuated the progression of diabetic renal injury.

Highly Efficient Generation of Definitive Endoderm Lineage from Human Induced Pluripotent Stem Cells

K. Sekine, T. Takebe, Y. Suzuki, A. Kamiya, H. Nakauchi, H. Taniguchi — 2012-05-01

Abstract: Background: Although hepatocytes can be an option for liver transplantation, the shortage of donor organs continues to worsen. Since the development of induced pluripotent stem (iPS) cell technology, it is eagerly anticipated to produce functional elements from pluripotent stem cells. These functional cells differentiated from iPS cells could be used for transplantation, drug screening, and in vitro toxicology. Methods: Human iPS cells are maintained on Mitomycin C–treated mouse embryonic fibroblast layers in DMEM–Ham F12–based medium supplemented with Knockout Serum Replacement, nonessential amino acids, 2-mercaptoethanol, and Glutamax. Differentiation of human iPS cells into a definitive endodermal lineage was induced with PRMI 1640 medium supplemented with B27 and 100 ng/mL human activin A. Two B27 supplements were examined with and without insulin. Furthermore, the PI3 kinase inhibitor LY294002 was used to examine the effect of inhibiting insulin signaling. Results and Discussion: We established efficient induction of definitive endodermal differentiation from iPS cells. Quantitative analysis revealed efficient (93.03 ± 2.74%) differentiation of human iPS cells into definitive endoderm cells using B27 minus insulin. This protocol may contribute as a fundamental technique to promote human iPS studies to develop cellular sources for transplantation.

Generation of Functional Human Vascular Network

2012-05-01

Abstract: Background: One of the major obstacles in regenerating thick, complex tissues such as the liver is their need for vascularization, which is essential to maintain cell viability during tissue growth and to induce structural organization. Herein, we have described a method to engineer a functional human vascular network. Methods: Enhanced green fluorescence protein-labeled human umbilical vein endothelial cells (GFP-HUVECs) were cocultivated with kusabira orange-labeled human mesenchymal stem cells (KO-hMSCs) inside a collagen/fibronectin matrix. Premature vascular network formation was visualized by fluorescence microscopy imaging. Furthermore, constructs prevascularized in vitro were implanted into a transparency window in immunodeficient mice. Results: Following several days of cultivation, GFP-HUVECs formed vessel-like structures that were stabilized by pericytes differentiated from KO-hMSCs. After implantation in vivo, the patency of human vascular structures was proved by rhodamine dextran infusion. These functional vascular structures remained for over 2 months. Discussion: Vascularization is the key challenge to organ generation. We successfully generated human vascular networks inside a matrix. Integration of parenchymal cells using our engineering technique should facilitate future efforts to reconstitute vascularized human organ systems in vitro.

A Study of the Glycoantigens of Neonatal Porcine Islet-Like Cell Clusters Using a Lectin Microarray

2012-05-01

Abstract: Background: The pig pancreas is considered to be the most suitable source of islets for clinical xenotransplantation. Two types of islet transplantation are: adult pig islets and neonatal porcine islet-like cell clusters (NPCC). However, besides a-Gal expression, differences in glycosylation and xenoantigenicity between both types were not clear so fat to date. In this study, we performed lectin microarray analyses of NPCCs cultured for 1, 5, or 9 days. Methods: We studied differences in gycoantigens among several kinds of wild-type NPCCs isolated from 1- to 3-day-old neonatal wild-type pigs (Large White/Landrace × Duroc) and cultured for 1, 5 and 9 days in Ham's 10 in the presence of nicotinamide, using a previously published technique. After sonication and centrifugation, supernatant proteins from each islet were labeled with Cy3, applied to a lectin array and scanned with an SC-Profiler for evaluation using an Array Pro Analyzer. Results: The overall signals of NPCC at days 5 and 9, showed almost the same values to most lectins, whereas those on day 1 showed differences, suggesting that the NPCC on day 1 contain immature cells that gradually turn to mature NPCCs in culture.

A Cloning of Cytidine Monophospho-N-Acetylneuraminic Acid Hydroxylase From Porcine Endothelial Cells

2012-05-01

Abstract: Introduction: The Hanganutziu-Deicher (H-D) antigen with terminal N-glycolyl neuraminic acid-(NeuGc) is widely distributed in mammalian species including monkeys and apes, but is not found in humans and birds. After the knock out of α1, 3galactosyltransfease, the H-D antigen became a major antigen of the “non-Gal antigen.” The expression of NeuGc is controlled by the activity of cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH). In this study, molecular cloning of pig CMAH was performed, as the first step in producing H-D knockout pigs. Methods: A pig endothelial cell line, MYP30, was used. The DNA sequence of pig CMAH was queried in dbEST (NCBI) using the BLAST program to search for cDNA fragments of pig CMAH, based on an alignment analysis of the mouse CMAH sequence. A polymerase chain reaction experiment was performed and candidate cDNA clones were isolated. To obtain the 5′-end and 3′-end of the open reading frame sequence, a 5′-full RACE Core Set and 3′-full RACE Core Set were used. Results: We cloned and characterized the pig CMAH gene. The ATG is located in exon 4, which corresponds to the mouse gene, and the stop codon is in exon 17. In the case of the 5′ site of the gene, exon 3 was identified but exons 1 and 2 are still being investigated. On the other hand, exon 18 was newly identified in the 3′ site of the gene. Conclusion: The results represent useful information for future clinical xenotransplantation studies.

The Interleukin-2 Receptor α Chain (CD25) Plays an Important Role in Regulating Monocyte-Derived CD40 Expression During Anti-Porcine Cellular Responses

Z.-G. Sun, Z. Wang, L.-M. Zhu, Y.-S. Fang, L.-Z. Yu, H. Xu — 2012-05-01

Abstract: Long-term xenograft survival is limited by delayed xenograft rejection, and monocytes are thought to play an important role in this process. Although typically considered a T cell surface marker, interleukin 2 the receptor chain CD25 is also functional on monocytes. We hypothesized that CD25 expression on monocytes functions to augment monocyte activation in xeno-specific cellular responses. Xenogeneic mixed lymphocyte-endothelial cell reactions were used to study the role of CD25 in facilitating xenogeneic cell-mediated immune responses an in vitro. We also tested the effect of the anti-CD25 antibody daclizumab on monocyte-mediated T cell activation during xeno-specific cellular responses. Co-culture with porcine endothelial cells (PEC) elicited a pronounced proliferative response by human peripheral blood mononuclear cells (PBMC) that was accompanied by upregulation of CD25 and CD40 on CD14+ monocytes. CD4+ cells proliferated in response to PEC-conditioned monocytes, while blockade of CD25 with daclizumab reduced CD4+ cell proliferation in the presence of PEC-conditioned monocytes. In addition, daclizumab inhibited proliferation of PBMC in responses to PEC. Analysis of monocytes from PBMC-PEC cocultures by flow cytometry indicated that daclizumab inhibited CD40 upregulation on PEC-activated monocytes. These data demonstrate that CD25 blockade prevents xenogeneic cellular responses by directly blocking CD25 expression on both activated T cells and monocytes. CD25 blockade on T cells or monocytes may indirectly affect upregulation of CD40 on xenoreactive monocytes. Our data strengthen the rationale for incorporating CD25 directed therapy in discordant xenotransplantation.

Efficacy of Double Filtration Plasmapheresis in Removing Xenoantibodies and Prolonging Xenograft Survival in an Ex Vivo Swine Perfusion Model

Y.-T. Lee, C.-H. Chu, S.-H. Sue, I.-C. Chen, J. Wei — 2012-05-01

Abstract: Introduction: In xenotransplantation, antibodies mediate humoral rejection, resulting in organ dysfunction. Removal of xenoantibodies is likely a first step for successful transplantation. Double filtration plasmapheresis (DFPP) selectively removes large molecular weight pathogenic substances, such as immunoglobulins (Ig), without other plasma proteins. The antibodies fractions removed and the changes in blood biochemistry are unanswered questions after DFPP in addressed this ex vivo swine heart perfusion model. Materials and Methods: Twelve swine hearts were perfused with human blood in a modified Langendorff's apparatus. The perfusate containing human blood was divided into 2 groups: controls (N = 6) and DFPP-treated group (N = 4). Blood counts, biochemistry data, and immunological profiles were compared at 3 time points: before and after DFPP and after heart perfusion. Result: Perfusion times of control and DFPP groups were 5.43 ± 1.81 vs 9.25 ± 3.00 hours, respectively. Only the values of albumin and total protein showed difference. The immunologic profile revealved complete removal of IgM and most IgG, IgA, C3, and C4, namely, 79.95%, 88.58%, 83.15%, and 87.97%, respectively. Conclusion: DFPP showed excellent efficacy to remove xenoantiboidies and prolong xenograft survival in an ex vivo perfusion model.

Porcine Bioengineered Scaffolds as New Frontiers in Regenerative Medicine

K.M. Park, H.M. Woo — 2012-05-01

Abstract: Porcine organs are attractive for xenotransplantation, if severe immunologic concerns can be overcome. Recently, reengineered organs, with heterologous cellular materials removed but preserved organ architecture and vasculature have been created using small rodents in an effort to produce customized bioengineered organs. However, few studies have been performed to generate bioengineered organs from porcine sources. The aim of this work was to produce 3-D bioengineered scaffolds from major porcine organs, preserving the native morphology and vascular structures with complete removal of cellular and nuclear materials. We decellularized porcine heart, liver, and kidney using a peristaltic pump system with 1% sodium dodecyl sulfate. The preservation of major architecture and vasculature was confirmed by gross findings, ultrasonography, and angiography. Hematoxylin and eosin staining revealed no evidence of nuclear or cytoplasmic residues. Quantitative DNA analysis demonstrated a substantial reduction (0%–8%) of porcine DNA in the scaffolds. These results suggested that 3-D bioengineered scaffolds of porcine organs may have tremendous potential to produce non-immunogenic transplantable organs as well as beneficial tools for biomedical studies on organ re-engineering and repair.

Systemic Decellularization for Multi-organ Scaffolds in Rats

K.M. Park, H.M. Woo — 2012-05-01

Abstract: Introduction: Bioscaffolds derived from animal organs are promising materials for xenotransplantation and regenerative medicine. For effective generation of biological scaffolds from diverse organs, there have been many technical challenges. In this study, we introduced a novel approach to create multiorgan bioscaffolds through systemic decellularization. Methods and materials: To obtain acellular bioscaffolds, the healthy adult rats were systemically perfused with ionic detergent through the carotid artery. Additional liver perfusion was set up to prevent potential obstruction from the influx of the decellularized debris via the portal vein. The perfusion system was controlled to maintain a constant physiological cardiac output of approximately 50 mL/min and was designed to minimize air entrapment. After decellularization, every organ designated for bioscaffold was harvested for evaluation of vascular structure and histology. Results: The perfusion times were different for each organ. In our histological analysis, the decellularized bioscaffolds harvested from most organs including major solid organs (ie, heart, liver, and kidney) as well as the others (such as stomach, intestines, spleen, etc) represented no evidence of residual cellular materials. Furthermore, the well-preserved collagen materials and intact vascular structures were also confirmed. Conclusion: The results from this study suggested that this systemic decellularization has the advantages to obtain a variety of bioscaffolds from single donor, and we can even decellularize organs with complex influx vascular structures. This method may also be used to study organ bioengineering for patients who need simultaneous combined organ transplantation.

A Potent Anti-angiogenic Factor, Vasohibin-1, Ameliorates Experimental Bronchiolitis Obliterans

2012-05-01

Abstract: Background: Bronchiolitis obliterans (BO) is a major cause of morbidity and mortality after lung transplantation. BO is pathologically characterized by neovascularized fibro-obliteration of the allograft airway. A recent study has shown that aberrant angiogenesis during fibro-obliteration contributes to the pathogenesis of BO. Vasohibin-1 (VASH1) has been isolated as a vascular endothelial growth factor–inducible gene in endothelial cells (ECs) that inhibits migration and proliferation of ECs and exhibits anti-angiogenic activity in vivo. Purpose: This study examines whether VASH1 inhibits fibro-obliteration of the allograft in a murine intrapulmonary tracheal transplantation model. Method: Tracheal allografts of BALB/c mouse were transplanted into the left lung of recipient C57BL/6J mouse. We performed gene transfer to the recipient lungs using an adenovirus vector encoding human VASH1 (Ad-VASH1) or beta- garactosidase (Ad-LacZ) as the control. Tracheal allografts were harvested and pathological on days 21 and 28. Result: Ad-VASH1 treatment reduced the vascular area on day 21 (4.6% versus 13.0%, P = .037) and day 28 (5.4% versus 13.4%, P = .022) compared with the control group. This was accompanied by significantly inhibited luminal obliteration of the tracheal allografts in the animals transferred with Ad-VASH1 compared with the control (69% versus 93%, P = .028) on day 21. We were not able to observe this effect on day 28 (92% versus 97%, P = .48). Conclusion: Transgene expression of VASH1 in the recipient lung significantly attenuated luminal obliteration of the tracheal allograft; this was associated with significantly reduced aberrant angiogenesis in the fibro-obliterative tissue in a murine model intrapulmonary tracheal transplantation.

Human Elastic Cartilage Engineering from Cartilage Progenitor Cells Using Rotating Wall Vessel Bioreactor

2012-05-01

Abstract: Transplantation of bioengineered elastic cartilage is considered to be a promising approach for patients with craniofacial defects. We have previously shown that human ear perichondrium harbors a population of cartilage progenitor cells (CPCs). The aim of this study was to examine the use of a rotating wall vessel (RWV) bioreactor for CPCs to engineer 3-D elastic cartilage in vitro. Human CPCs isolated from ear perichondrium were expanded and differentiated into chondrocytes under 2-D culture conditions. Fully differentiated CPCs were seeded into recently developed pC-HAp/ChS (porous material consisted of collagen, hydroxyapatite, and chondroitinsulfate) scaffolds and 3-D cultivated utilizing a RWV bioreactor. 3-D engineered constructs appeared shiny with a yellowish, cartilage-like morphology. The shape of the molded scaffold was maintained after RWV cultivation. Hematoxylin and eosin staining showed engraftment of CPCs inside pC-HAp/ChS. Alcian blue and Elastica Van Gieson staining showed of proteoglycan and elastic fibers, which are unique extracellular matrices of elastic cartilage. Thus, human CPCs formed elastic cartilage-like tissue after 3-D cultivation in a RWV bioreactor. These techniques may assist future efforts to reconstruct complicate structures composed of elastic cartilage in vitro.

Long-Term Viability of Transplanted Hybrid Cellular Spheroids within Chondrocyte Sheets

2012-05-01

Abstract: Encapsulation of transplanted cells within an immunoisolating membrane may provide a new strategy for protecting these cells from recipient immune responses without the use of immunosuppressive drugs. We have previously reported a novel concept of immunoisolation and immunodelusion using recipient cells instead of traditional artificial materials. We developed a chondrocyte sheeting immunodelusive immunoisolated bioartificial pancreas (CSI-BAP) that would enable transplantation of cells across allogeneic and xenogeneic barriers without the cells being recognized as donor cells and without the need for immunosuppression. Recently, we have constructed hybrid cellular spheroids (HCSs) containing cells from two different cell lines (RIN-5F, an insulin-secreting cell line, and Hep-G2, a hepatocellular carcinoma cell line) to enhance the function and biocompatibility of the HCSs. These HCSs were then encapsulated with multiple layers of chondrocyte sheets obtained from the auricular cartilage of Sprague-Dawley (SD) rats. The in vitro ability of the CSI-BAP to secrete insulin was tested before transplantation. Histological evaluation of CSI-BAP chondrocyte microencapsulated immunoisolated islet morphology and viability of allogeneic or xenogeneic cell lines was performed 100 days after the CSI-BAP was transplanted into SD rats. Morphological evaluations revealed good viability of the islets and progression of islet encapsulation. In vitro insulin secretion from the CSI-BAP was well maintained. Additionally, insulin and albumin secretion from the CSI-BAP was confirmed by in vivo immunohistochemical examination. Moreover, the cell lines transplanted into the subcutaneous space in the form of HCSs within the chondrocyte sheets showed good viability of more than 100 days and sustained insulin and albumin secreting ability.

Adenosine Receptors Are Up-Regulated in Unilateral Ureteral Obstructed Rat Kidneys

J. Lee, l. Hwang, H. Ha — 2012-05-01

Abstract: Despite recent improvements in immunosuppressive regimens, chronic renal allograft rejection remains a major problem. Tubulointerstitial fibrosis is one of the major histological features of chronic renal allograft rejection, but its exact pathogenic mechanisms are not fully understood. Adenosine present in the normal kidney is significantly elevated in response to cellular damage. The cellular effect of adenosine occurs through 4 known adenosine receptor (AR) subtypes; A1AR, A2AAR, A2BAR, and A3AR. All AR subtypes are expressed in the kidney, but the expression of each AR subtype has not been defined under fibrotic conditions. In the present study, we examined AR subtype expression in kidneys that underwent unilateral ureteral obstruction (UUO), a well-characterized model for tubulointerstitial fibrosis. At 5 days after the induction of UUO, we observed α-smooth muscle actin (α-SMA), fibronectin, and collagen I messenger RNA (mRNA) and protein expressions to be significantly up-regulated in the obstructed compared with the sham kidneys, confirming that fibrosis had occurred in the former organs. A1AR mRNA expression in the obstructed kidney cortex was 3.2-fold higher than an the sham kidney cortex. Relative to the sham kidney A2AAR and A2BAR mRNA expressions were also 2.6- and 2.0-fold increased, respectively. A3AR mRNA expression in the obstructed kidney cortex was also up-regulated by 3.3-fold. These data demonstrated that all 4 subtypes of AR were increased in the obstructed kidney, which was accompanied by tubulointerstitial fibrosis. Further studies are needed to determine which subtypes of AR play a protective or pathogenic role in tubulointerstitial fibrosis.

Mucosal Protective Effect of PGI2 on Canine Small Bowel Auto-transplantation

Y.I. Kim, K.-Y. Chung, B.J. So, J.J. Park — 2012-05-01

Abstract: Purpose: We designed this experimental study to assess the mucosal protective effects of continuous prostaglandin I2 (PGI2) infusion after canine small bowel autotransplantation. Materials and Methods: Six Mongrel dogs were randomly divided two groups: PGI2 (n = 3) and control (n = 3). The small bowel from jejunum to ileum was obtained, including the mesenteric vascular pedicle. After cold flushing ex vivo, the harvested segment was preserved in an icebox for 3 hours. Thereafter we reimplanted the harvested intestinal segment. While completing the anastomosis, PGI2 (50 μg) was slowly infused through the mesenteric artery in the PGI2 group versus the same volume of saline in the control group. At 1, 3 and 6 days after autotransplantation, we obtained blood samples, and at 6 days, small bowel segments. Endotoxin and interleukin 6 (IL-6) levels were measured and all histologic specimens stained with hematoxylin-eosin H-E were reviewed by a pathologist to grade mucosal damage as: mild (1 point), moderate (2 points), or severe (3 points) change. Results: Mean basal serum endotoxin levels were similar in both groups the PGI2 groups versus control group were 0.216 ± 0.018 versus 0.223 ± 0.040 EU/mL, respectively. However, on day 3 after the operation, the PGI2 group showed much decreased levels of serum endotoxin compared to control levels: 0.349 ± 0.196 versus 0.842 ± 0.446 EU/mL. The mean concentration of serum IL-6 on day 1 after operation among the PGI2 versus control group were 32.13 ± 7.13 pg/mL versus 36.96 ± 3.65 pg/mL. The histologic scores at 6 days after the operation were PGI2 group versus control group: 1.33 versus 1.66 (P = NS). Conclusion: Continuous infusions of PGI2 through the mesenteric artery after the canine small bowel autotransplantation may protect the small bowel mucosal barrier.

Prosthetic Endocarditis Treated by Repeated Heart Transplantation: Report of a Successful Case

H.-H. Huang, Y.-C. Chuang, K.-C. Lee, S.-H. Sue, C.-Y. Chang, J. Wei — 2012-05-01

Abstract: The treatment of recurrent prosthetic valve endocarditis is extremely difficult. Heart transplantation (HT) may save the patient's life. Recurrent endocarditis, however, can occur after HT. This report described a patient who had under gone four conventional valve surgeries and three HTs successfully. In May 2000, a 14-year-old boy suffered from endocarditis with severe aortic valve regurgitation. He underwent aortic valve replacement (AVR) at another hospital. Due to prosthetic valve endocarditis, he displayed a severe paravalvular leakage and was transferred to our hospital where he underwent Bentall's operation in October 2000. Despite a full antibiotic course, he experienced a relapse of the prosthetic endocarditis with significant deterioration of the heart function and a progressively more severe paravalvular leak. Considering the difficulties of repair and the poor heart function, he underwent an HT in June 2003 and recovered well. Unfortunately, endocarditis with aortic valve regurgitation attacked him again after 3 years. Remarkably, all blood cultures were negative. A second AVR was performed in October 2006 with a Second Bentall's procedure 1 year later in 2007. In November 2009, the patient suddenly displayed cardiogenic shock with collapse. He was transferred to our hospital and needed extracorporcal membrane oxygenation (ECMO) support. Two days later, he underwent a second HT. However, the donor heart was nonfunctional due to the prolonged ischemia time. ECMO support was continuously needed after the HT. A third HT was performed successfully 10 days later. Due to previous reported experiences of culture-negative endocarditis, minocycline was prescribed twice daily continuously after the third HT/seventh cardiac surgery. The patient was discharged 2 months later. To date he takes minocycline every day and lives a healthy life.

Combined Heart Transplantation and Total Replacement of Thoracic Aorta in Marfan's Syndrome With Recurrent Aortic Dissection: A Case Report

J. Wei, S.H. Sue, Y.T. Lee, C.Y. Chang — 2012-05-01

Abstract: It is extremely rare for a patient to need simultaneous heart transplantation (HTx) and replacement of the thoracic aorta. A 23-year-old woman with Marfan's syndrome underwent Bentall's operation and replacement of the ascending aorta (AsA) due to a type A aortic dissection (AD) in August 2001. In March 2005, she began to experience dyspnea on exertion and was found to have a huge pseudoaneurysm at the aortic root, which had caused dehiscence of the aortic conduit. In July 2009, she suffered acute chest pain followed by hypotension and cold sweating. The computed tomography (CT) scan showed a recurrent dissection with a long intimal tear extending from the arch to the mid-portion of the descending thoracic aorta (DTA). Due to technical difficulties in the repair of the aortic root, she was placed on the HTx waiting list. The next day, she received a donor heart and underwent combined HTx and total replacement of the thoracic aorta. Explantation of the heart improved the exposure of the DTA. With the use of a vascular ring connector (Vasoring), the operation was successfully performed without need for a blood transfusion. The patient was still well at 2 years after the operation. Simultaneous replacement of the heart and the whole segment of the thoracic aorta is technically possible in Marfan patients who are complicated with aortic dissection.

Total Pancreatectomy Combined with Partial Pancreas Autotransplantation for Recurrent Pancreatic Cancer: A Case Report

2012-05-01

Abstract: We describe a patient presenting with a resectable carcinoma of the remnant pancreas at 3 years after undergoing a pylorus-preserving pancreaticoduodenectomy for invasive ductal carcinoma of the pancreatic head. We also performed a distal pancreas autotransplantation using a part of the resected pancreas to preserve endocrine function. Final histologic findings showed the second tumor to be an invasive ductal carcinoma consisting of a well-differentiated tubular adenocarcinoma with similar histopathologic findings as the first tumor. There were no microscopic lymph node metastases and no evidence of microvascular invasion (pStage IA [pT1, pN0, M0] and R0 according to the International Union Against Cancer TNM classification). The patient was discharged at 20 days after surgery without any trouble and followed by adjuvant chemotherapy with S-1. The carbohydrate antigen 19-9 value was again normalized after the second surgery. Twenty months after the second operation, the patient is alive without cancer recurrence. The pancreas graft is functioning with a blood glucose of 108 mg/dL, HbA1C of 6.2%, and serum C-peptide of 1.4 ng/mL.

Chronic Rejection in a Small Bowel Transplant with Successful Revision of the Allograft by Segmental Resection: Case Report

2012-05-01

Abstract: An 8-year-old girl was admitted for severe electrolyte imbalance and for hyponatremic seizure. In July 2005, at 3 years of age, she underwent isolated small-bowel transplantation of 100 cm ileum from her father. Her own bowel was only 50 cm of proximal jejunum which had been directly connected to the anus due to extended total aganglionosis. The graft was placed into the middle of her remaining bowel, using the splenic artery and vein as feeding vessels with saving of the spleen. Daclizumab induction and tacrolimus monotherapy were applied for immunosuppression. Two acute cellular rejection episodes, E on day 10 and 4 years after transplantation, were successfully treated with OKT-3 and recombinant antithymocyte globulin, respectively. However, because of intermittent bowel dysfunction, she was hospitalized several times for hydration and metabolic care. On admission, her abdomen was moderately distended, and a simple abdominal film showed a fixed dilated loop. Colonoscopy could not pass the narrowed lumen, with stiffness at the anastomosis between the graft and the distal bowel. Endoscopic biopsy at the entrance to the stricture showed a nonspecific inflammatory reaction with fibrosis. Similar findings on a gastrograffin enema suggested chronic rejection (CR). On laparotomy, an irregularly narrowed fibrotic loop was noticed at the distal part of the graft, proximal to the anastomosis. We performed a 20-cm segmental resection with an end-to-end anastomosis. Histopathologic findings showed CR with fibrosis and hyalinization of the entire bowel wall and vessel walls with mild cellular infiltrations. She recovered in 10 days. The graft may have been saved, but intermittent requirement of hydration over the following months suggested progressive graft dysfunction. A case of segmental involvement of CR with subsequent successful graft salvage by partial resection is rare in the literature.

Author Index

2012-05-01

Transplantation Proceedings

Editorial Board

Contents

Evolution of Registry and Tracking System for Organ Transplantation in Japan

Difficulties in Organ Procurement and Transplantation in the Aboriginal Minority People in Taiwan

The Dilemma of “To Be or Not To Be”: Developing Electronically e-Health & Cloud Computing Documents for Overseas Transplant Patients from Taiwan Organ Transplant Health Professionals' Perspective

National Survey of Filipinos on Acceptance of Incentivized Organ Donation

Outcomes of Management for Potential Deceased Donors

Professional Education and Hospital Development for Organ Donation

Modification of the Education System for Organ Procurement Coordinators in Japan After the Revision of the Japanese Organ Transplantation Act

A Newly Developed Container for Safe, Easy, and Cost-effective Overnight Transportation of Tissues and Organs by Electrically Keeping Tissue or Organ Temperature at 3 to 6°C

A Study on Knowledge and Attitude toward Brain Death and Organ Retrieval among Health Care Professionals in Korea

Kidney Transplantation From Donation After Cardiac Death Donors in China—A Single-Center Experience

The Fate of Patients on the Waiting List for Lung Transplantation in Korea

Donor Evaluation for Lung Transplantation in Korea

Relationship Between Postoperative Lung Atelectasis and Position of the Endotracheal Tube in Pediatric Living-Donor Liver Transplantation

Extracorporeal Membrane Oxygenation and Thoratec Pneumatic Ventricular Assist Devices as Double Bridge to Heart Transplantation

The Influence of the Organ Allocation Policy on a Patient's Chances of Undergoing Heart Transplantation and the Posttransplantation Survival Rate

Ventricular Assist Device Application as a Bridge to Pediatric Heart Transplantation: A Single Center's Experience

Combined St. Thomas and Histidine-Tryptophan-Ketoglutarat Solutions for Myocardial Preservation in Heart Transplantation Patients

The Outcome of Heart Transplantation in Hepatitis C-Positive Recipients

Endomyocardial Biopsy in Heart Transplantation: Schedule or Event?

Cardiac Allograft Vasculopathy Compared by Intravascular Ultrasound Sonography: Everolimus to Mycophenolate Mofetil—One Single-Center Experience

Steroid Pulse Therapy Combined with Plasmapheresis for Clinically Compromised Patients after Heart Transplantation

Isolated Cardiac Sarcoidosis in Heart Transplantation

Clinical Experience of Tacrolimus With Everolimus in Heart Transplantation

Twenty-four Year Single-Center Experience of Hepatitis B Virus Infection in Heart Transplantation

Heart Retransplantation for Pediatric Primary Allograft Failure

Changes in Health-Related Quality of Life Across Three Post–Heart Transplantation Stages: Preoperative Extracorporeal Membrane Versus Non–Extracorporeal Membrane Group/Clinical Trial Plan Group Versus Non–Clinical Trial Plan Group in Taiwan

Role of Killer Immunoglobulin-like Receptor–Ligand Interactions in Human Leukocyte Antigen–Matched Sibling Hematopoietic Stem Cell Transplantation

Audit of Peripheral Stem Cell Transplantation for Aplastic Anemia in Multitransfused Infected Patients

Single-Center Experience with Pancreas Transplantation

Inducible Cyclooxygenase Expression Mediating Hypoxia/Reoxygenation–Induced Pulmonary Vasoconstriction is Attenuated by a Cyclooxygenase Inhibitor in Rats

L-Ascorbic Acid and Alpha-tocopherol Attenuates Liver Ischemia-Reperfusion Induced of Cardiac Function Impairment

The Role of CD14 and Toll-Like Receptor 4 of Kupffer Cells in Hepatic Ischemia-Reperfusion Injury in Rats

A Basic Consideration for Porcine Liver Preservation Using a Novel Continuous Machine Perfusion Device

Functional Recovery of Donation After Cardiac Death Liver Graft by Continuous Machine Perfusion Preservation in Pigs

Protective Role of Nitric Oxide Induced by Ischemic Preconditioning on Cold Ischemic-Reperfusion Injury of Rat Liver Graft

The Effect of Perfusion Speed on Graft Reperfusion Injury after Rat Orthotopic Liver Transplantation

Quantification of Macrovesicular and Microvesicular Hepatic Steatosis in Rats Using 3.0-T 1H-Magnetic Resonance Spectroscopy

Pretransplant Screening and Evaluation of Liver Graft Viability Using Machine Perfusion Preservation in Porcine Transplantation

Protective Effect of Melatonin on Liver Ischemia-Reperfusion Induced Pulmonary Microvascular Injury in Rats

Liver Reperfusion-Induced Decrease in Dynamic Compliance and Increase in Airway Resistance Are Ameliorated by Preischemic Treatment with Melatonin through Scavenging Hydroxyl Radicals in Rat Lungs

Preischemic Treatment With Melatonin Attenuates Liver Reperfusion-Induced Impairment of Cardiac Function

The Protective Effect of Curcumin on Ischemia-Reperfusion–Induced Liver Injury

Protective Role of Shenfu on Ischemia-Reperfusion Injury of Rat Liver Grafts

Apoptosis in Endothelial Cells by Cyclosporine

Effect of Cyclosporine on Apoptosis in Bronchial Epithelial Cells

Effects of Cyclosporine on Oxidative Stress in Human Bronchial Epithelial Cells

Effects of Cyclosporine on Metalloproteinase in Endothelial Cells

Tumor Necrosis Factor-α Pathway Plays a Critical Role in Regulating Interferon-γ Induced Protein-10 Production in Initial Allogeneic Human Monocyte–Endothelial Cell Interactions

Monocyte-Derived CD40 Expression Is Regulated by Interferon-γ/Interferon-γ Receptor-1 Pathway when Acting as a Bridge During Their Interaction with T Cells and Allogeneic Endothelial Cells

A Novel Cannulation Technique for Isolation of Human Hepatocytes from Explanted Diseased Whole Livers

Slow Cooling Rate with a Shock Cooling Program Can Effectively Cryopreserve Pig Hepatocytes

Cryopreservation of Immobilized Rat Hepatocytes for the Development of a Bioartificial Liver System

Effect of Fulminant Hepatic Failure Porcine Plasma Supplemented with Essential Components on Encapsulated Rat Hepatocyte Spheroids

Potentiality of Immobilized Pig Hepatocyte Spheroids in Bioartificial Liver System

Effect of Spheroid Aggregation on Susceptibility of Primary Pig Hepatocytes to Cryopreservation

Self-Organization of Human Hepatic Organoid by Recapitulating Organogenesis In Vitro

High Glucose Increases Mesangial Lipid Accumulation via Impaired Cholesterol Transporters

Fractalkine Increases Mesangial Cell Proliferation Through Reactive Oxygen Species and Mitogen-Activated Protein Kinases

Lipopolysaccharide Increases Monocyte Binding to Mesangial Cells Through Fractalkine and Its Receptor

Selectively Expanding Subsets of T Cells in Mice by Injection of Interleukin-2/Antibody Complexes: Implications for Transplantation Tolerance

Immunology Mini-review: The Basics of TH17 and Interleukin-6 in Transplantation

The Role of Invariant NKT Cells in Liver Transplant Tolerance in Rats

Dynamic Changes of Indoleamine 2,3-Dioxygenase of Kupffer Cells in Rat Liver Transplant Rejection and Tolerance

Can Immune Function Assay Predict Infection or Recovery?

Immunomodulation of Human CD8+ T Cells by Thymoglobulin In Vitro

Inhibition of Allogenic T-Cell Cytotoxicity by Hepatic Stellate Cell via CD4+ CD25+ Foxp3+ Regulatory T Cells In Vitro

CD28 Superagonist Antibody Treatment Attenuated Obliterative Bronchiolitis in Rat Allo-Orthotopic Tracheal Transplantation by Preferentially Expanding Foxp3-Expressing Regulatory T Cells

Induction of Regulatory CD4+ Cells and Prolongation of Survival of Fully Allogeneic Murine Cardiac Grafts by Danazol

The Smell of Tokishakuyaku-san (TJ-23) Induces Generation of Regulatory T Cells and Prolongation of Survival of Fully Allogeneic Cardiac Grafts in Mice

Artemisiae capillaris herba Induces Prolonged Survival of Fully Cardiac Allografts and Generates Regulatory Cells in Mice

Music Exposure Induced Prolongation of Cardiac Allograft Survival and Generated Regulatory CD4+ Cells in Mice

The Antiproteinuric Effects of Green Tea Extract on Acute Cyclosporine-Induced Nephrotoxicity in Rats

Cellular Function of RhoGDI-α Mediates the Cycling of Rac1 and the Regulation of Pancreatic Beta Cell Death

Alpha-Melanocyte Stimulating Hormone Preserves Islet Graft Survival Through Down-Regulation of Toll-Like Receptors

Microencapsulation of Pancreatic Islets With Canine Ear Cartilage for Immunoisolation

Hybrid Cellular Spheroids From Hepatocellular Carcinoma and Insulin-Secreting Cell Lines

Impact of Coculture with Ischemic Preconditioned Hepatocellular Carcinoma Cell Line (Hep-G2) Cells on Insulin Secreting Function of Rat Insulin-secreting Cell Line (RIN-5F) Cells