Hypothermic Storage of Periportal and Perivenous Rat Hepatocytes

High yields of intact parenchymal cells are produced by the two-step Digitonin-collagenase perfusion of whole liver, and it has gained wide acceptance for biochemical and cellular analyses of zonal hepatocytes. The development reached by this methodology is in contrast to the time-limited use of the isolated cells unless those other methods, such as primary cultures, are employed. An alternative option to have cells ready to be used for several days, is the cold storage in University of Wisconsin solution as a preservation solution. This procedure is easy, not too expensive, and does not require specialized equipment. We study the competence of this system to maintain liver cells: mixed or total cells and cell-enriched fractions. We affirm viability of hepatocytes during hypothermic storage (UW-96h-4°C) by Trypan Blue exclusion, the capacity to retain cytoplasmic enzymes, metabolic competence to maintain total Glutathione content, and immunocytochemistry (gene detection). After 96 h of cold storage, mixed cells and cell-enriched fractions, were submitted to normothermic incubation (120 min, 37°C) and we check Trypan Blue exclusion, cytoplasmic enzyme release, and the capacity of cell populations to synthesize urea. The results show that it is possible to use, after several days of storage, mixed liver cells and cell-enriched fractions in metabolic and gene expression studies. This procedure allows us to reduce the number of experimental animals needed, to save experimental time and costs, and to facilitate further studies in vitro about the basis and consequences of metabolic heterogeneity of the liver cell plate.     

Effects of Atrial Natriuretic Peptide After Prolonged Hypothermic Storage of the Isolated Rat Heart

Primary graft failure (PGF) caused by ischemia‐reperfusion injury (IRI) is the strongest determinant of perioperative mortality after heart transplantation. Atrial natriuretic peptide (ANP) has been found to reduce the IRI of cardiomyocytes and may be beneficial in alleviating PGF after heart transplantation, although there is a lack of evidence to support this issue. The purpose of this study was to investigate the cardioprotective effects of ANP after prolonged hypothermic storage. For this purpose, an isolated working‐heart rat model was used. After the preparation, the hearts were arrested with and stored in an extracellular‐based cardioplegic solution at 3–4°C for 6 h and followed by 25 min of reperfusion. The hearts were divided into four groups (n = 7 in each group) according to the timing of ANP administration: Group 1 (in perfusate before storage), Group 2 (in cardioplegia), Group 3 (in reperfusate), and control (no administration of ANP). Left ventricular functional recovery and the incidence of ventricular fibrillation (VF) were compared. ANP administration at the time of reperfusion improved the percent recovery of left ventricular developed pressure (control, 45.5 ± 10.2; Group 1, 47.4 ± 8.8; Group 2, 45.3 ± 12 vs. Group 3, 76.3 ± 7; P < 0.05) and maximum first derivative of the left ventricular pressure (control, 47.9 ± 8.7; Group 1, 46.7 ± 8.8; Group 2, 49.6 ± 10.8 vs. Group 3, 76.6 ± 7.5; P < 0.05). The incidence of VF after reperfusion did not differ significantly among these four groups (71.4, 85.7, 57.1, and 85.7% in Groups 1, 2, 3, and control, respectively). This result suggests that the administration of ANP at the time of reperfusion may have the potential to decrease the incidence of PGF after heart transplantation.     

Impact of Allograft Injury Time of Onset on the Development of Chronic Lung Allograft Dysfunction After Lung Transplantation

The impact of allograft injury time of onset on the risk of chronic lung allograft dysfunction (CLAD) remains unknown. We hypothesized that episodes of late‐onset (≥6 months) allograft injury would produce an augmented CXCR3/ligand immune response, leading to increased CLAD. In a retrospective single‐center study, 1894 transbronchial biopsy samples from 441 lung transplant recipients were reviewed for the presence of acute rejection (AR), lymphocytic bronchiolitis (LB), diffuse alveolar damage (DAD), and organizing pneumonia (OP). The association between the time of onset of each injury pattern and CLAD was assessed by using multivariable Cox models with time‐dependent covariates. Bronchoalveolar lavage (BAL) CXCR3 ligand concentrations were compared between early‐ and late‐onset injury patterns using linear mixed‐effects models. Late‐onset DAD and OP were strongly associated with CLAD: adjusted hazard ratio 2.8 (95% confidence interval 1.5–5.3) and 2.0 (1.1–3.4), respectively. The early‐onset form of these injury patterns did not increase CLAD risk. Late‐onset LB and acute rejection (AR) predicted CLAD in univariable models but lost significance after multivariable adjustment for late DAD and OP. AR was the only early‐onset injury pattern associated with CLAD development. Elevated BAL CXCR3 ligand concentrations during late‐onset allograft injury parallel the increase in CLAD risk and support our hypothesis that late allograft injuries result in a more profound CXCR3/ligand immune response.     

A Multicenter Study on Long‐Term Outcomes After Lung Transplantation Comparing Donation After Circulatory Death and Donation After Brain Death

The implementation of donation after circulatory death category 3 (DCD3) was one of the attempts to reduce the gap between supply and demand of donor lungs. In the Netherlands, the total number of potential lung donors was greatly increased by the availability of DCD3 lungs in addition to the initial standard use of donation after brain death (DBD) lungs. From the three lung transplant centers in the Netherlands, 130 DCD3 recipients were one‐to‐one nearest neighbor propensity score matched with 130 DBD recipients. The primary end points were primary graft dysfunction (PGD), posttransplant lung function, freedom from chronic lung allograft dysfunction (CLAD), and overall survival. PGD did not differ between the groups. Posttransplant lung function was comparable after bilateral lung transplantation, but seemed worse after DCD3 single lung transplantation. The incidence of CLAD (p = 0.17) nor the freedom from CLAD (p = 0.36) nor the overall survival (p = 0.40) were significantly different between both groups. The presented multicenter results are derived from a national context where one third of the lung transplantations are performed with DCD3 lungs. We conclude that the long‐term outcome after lung transplantation with DCD3 donors is similar to that of DBD donors and that DCD3 donation can substantially enlarge the donor pool.     

Effect of Immunological Differences on Rat Aortic Valve Allograft Calcification

Calcification may be a cause of allograft valve degeneration. To determine whether immunological differences between donor and recipient affect the degree of calcification that occurs, adult Lewis rats received aortic valve allografts transplanted heterotopically into the abdominal aorta. All valves were transplanted immediately after harvest. The valves were not exposed to antibiotics or albumin before insertion. Valve donors were of the Lewis (syngeneic), F344 (weakly allogeneic, RT1 compatible, non-RT1 incompatible), LBN F1 (moderately allogeneic, one haplotype identical, one haplotype incompatible at the RT1 and non-RT1 loci), and Brown Norway (strongly allogeneic, RT1 and non-RT1 incompatible) strains. Valves were harvested 3-12 weeks following transplantation. Scanning electron microscopy and energy dispersion x-ray microanalysis were performed on one leaflet of each valve to evaluate calcium content. Calcium content expressed in counts (mean +/- standard error) according to donor strain were: Lewis, 1642 +/- 233; F344, 4853 +/- 1412; LBN F1, 4714 +/- 823; and Brown Norway, 4358 +/- 835. Significant differences (p less than 0.05) existed between valves from Lewis donors and those from each other strain. No differences among the other strains were statistically significant. It is concluded that syngeneic valve allografts calcify less than allogeneic grafts. However, the degree of allogenicity did not influence the magnitude of calcification.     

Prevention of Acute Lung Allograft Rejection in Rat by CTLA4Ig

CTLA4 immunoglobulin (CTLA4Ig), which binds with a high affinity to B7-1 and B7-2, interrupts T-cell activation by inhibiting costimulatory signal. CTLA4Ig has been used in hopes of achieving antigen-specific tolerance induction in several solid organ transplants. In lung allograft rejection, however, its use has been controversial in terms of its effect on prevention of rejection. In the present study, the effect of murine CTLA4Ig on rat-lung allograft rejection was investigated. Rat left-lung transplantation was performed in an RT1 incompatible donor (Brown Norway; BN)-recipient (F344) combination. All allografts (n = 12) without any treatment were rejected within 7 days after transplantation. A single injection of murine form CTLA41g at a dose of 100 microg intraperitoneally (ip) or intravenously (iv) on day 1 post-transplantation achieved long-term graft survival (>90days) in 2/5 (40%) and 3/8 (38%), respectively. Moreover, 6/7 (86%) allografts in rats that received iv injection of 500 microg CTLA4Ig survived more than 90days. Allograft survival in the CTLA4Ig 500 microg iv recipient group was significantly longer than that in the no-treatment control or control immunoglobulin group (p <0.01). Four out of seven recipients bearing functional allografts for more than 90 days with the CTLA4Ig treatment accepted donor-specific skin grafts, whereas all third-party skin grafts (n=3) were rejected. Prevention of rat-lung allograft rejection could be achieved by intravenous administration of CTLA4Ig, resulting in long-term allograft survival with acceptance of donor-specific skin grafts.     

落新妇甙对肺移植术后急性排斥反应的作用

目的探讨落新妇甙对大鼠肺移植后机体急性排斥反应的影响和机制，以明确落新妇甙对大鼠肺移植急性排斥反应的作用。方法建立大鼠原位肺移植模型，术后将60只受体大鼠随机分为两组，对照组：术后用生理盐水1ml／d灌胃，实验组：术后用落新妇甙1ml／kg·d灌胃。观察肺移植后大鼠的存活时间、大鼠脾细胞T淋巴细胞转化率、脾淋巴细胞白细胞介素2（IL-2）的活性以及外周血中活化T淋巴细胞凋亡情况。在电子显微镜下观察肺血管超微结构变化。结果实验组大鼠肺移植后存活时间较对照组明显延长（25．4±2．1d vs．13．4±1．2d；t=2．042，P〈0．05）。实验组脾细胞T淋巴细胞转化率较对照组明显降低（23465．8±8783．4 cpm vs．74567．3±12874．6cpm；t=2．284，P〈0．05）；实验组移植大鼠脾淋巴细胞IL-2活性较对照组明显降低（4．25±2．65U／ml vs．23．46±1．82 U／ml；t=3．165，P〈0．01）。实验组能有效地诱导急性排斥反应中活化T淋巴细胞凋亡。实验组肺组织超微结构损伤较对照组减轻。结论落新妇甙通过下调IL-2产生，诱导活化T淋巴细胞凋亡，抑制T淋巴细胞增殖分化，广泛抑制了以T淋巴细胞为主的肺移植术后急性排斥反廊，从而延长肺移槽大鼠的存活时间．     

Effect of Circulatory Assistance on Premature Death and Long-term Prognosis

Background

Patients undergoing urgent heart transplantation (HT) have a poorer prognosis and more long-term complications. The objective of this study was to compare the preoperative course in patients undergoing urgent HT according to the need for preoperative intra-aortic balloon counterpulsation (IABP).

Materials and Methods

We studied 102 consecutive patients including 23 patients with IABP who underwent urgent HT between January 2000 and September 2006. We excluded patients who received combination transplants, those who underwent repeat HT, and pediatric patients who underwent HT. The statistical methods used were the t test for quantitative variables and the χ² test for qualitative variables. A logistic regression model was constructed to assess the possible relationship between IABP and other variables on premature death within 30 days after HT.

Results

Mean (SD) patient-age was 50 (10) years. No significant differences were observed in baseline characteristics between the IABP and the non-IAPB groups. The IABP patient group had higher rates of acute graft failure (45.5% vs 35.4%; P = .46) and premature death (18.8% vs 14.8%; P = .67) and shorter long-term survival (40.6 [34.9] vs 54.5 [43.7] mo; P = .30). Multivariate analysis demonstrated no association between the need for IABP and increased frequency of premature death.

Conclusions

Use of IABP is not associated with premature or late death. We recommend use of IABP in patients with acute decompensated heart failure to stabilize them before HT.     

前列腺素E1改善离体鼠心保存效果的研究

目的　探讨在冷缺血期供者心脏保存液中前列腺素 Ｅ１（ｐｒｏｓｔａｇｌａｎｄｉｎ Ｅ１）对改善鼠心的保存效果。　方法　将１４ 只大白鼠随机分为对照组和实验组，每组７ 只。对照组用 Ｓｔ Ｔｈｏｍ ａｓ停搏液停搏并保存大鼠心脏６ 小时；实验组在 Ｓｔ Ｔｈｏｍ ａｓ液中加入前列腺素 Ｅ１（５ μｇ／ Ｌ）停搏并冷保存６ 小时，利用离体鼠心非循环式 Ｌａｎｇｅｎｄｏｒｆｆ 灌流功能测定模型，测定左心室舒张期末压（ Ｌ Ｖ Ｅ Ｄ Ｐ）、左心室发展压（ Ｌ Ｖ Ｄ Ｐ）和左心室压力变化率（＋ ｄｐ／ｄｔ），并检测心肌细胞线粒体三磷酸腺苷（ Ａ Ｔ Ｐ）、腺嘌呤核苷酸总量（ Ｔ Ａ Ｎ）。　结果　实验组保存后的左心功能恢复明显优于对照组（ Ｐ＜ ０．０５），实验组保存后心肌细胞三磷酸腺苷和腺嘌呤核苷酸总量的浓度明显高于对照组（ Ｐ＜ ０．０５）。　结论　供者心脏停搏液和保存液中的前列腺素 Ｅ１ 能改善供心的保护效果。     

The pH of Exhaled Breath Condensate of Patients with Allograft Rejection After Lung Transplantation

Endogenous airway acidification, as assessed by the condensate pH, has been implicated in the pathophysiology of inflammatory airway diseases such as cystic fibrosis and asthma. The aim of this study was to investigate the pH of condensate in patients after lung transplantation (LTX). From the cohort of transplanted patients at our center, 83 patients (9 heart-lung transplantation, 48 double-lung transplantation, 26 single-lung transplantation) were recruited and analyzed in a cross-sectional manner: 26 patients were diagnosed with chronic rejection or bronchiolitis obliterans syndrome (BOS), 7 patients were diagnosed with acute rejection (AR) while 50 patients had no evidence of rejection according to the International Society for Heart and Lung Transplantation criteria. The condensate pH was significantly reduced in patients with BOS and AR when compared to patients without rejection and control subjects (5.8 +/- 0.5 and 6.2 +/- 0.4 versus 6.6 +/- 0.4 and 6.5 +/- 0 .4, respectively; p < 0.05). Moreover, there was a significant correlation between condensate pH levels and the BOS grade (r =-0.62; p < 0.01), the FEV(1) (r = 0.39; p < 0.01) and the total cell and neutrophil count in bronchoalveolar lavage fluid (r =-0.39 and r =-0.56, respectively; p < 0.01). Airway acidification occurs in BOS and may directly or indirectly reflect airway inflammation in patients with allograft rejection after LTX. Measuring condensate pH might thus be a new tool for the evaluation of rejection in lung transplant patients.     

Effect of Transient BK Viremia and Viruria on Long-term Renal Allograft Survival and Function

IntroductionThe purpose of this study is to present the five-year survival and function of the renal allograft of recipients who were diagnosed with BK viremia and viruria during the first year after renal transplantation.Patients and MethodsBK virus was studied in 32 new renal allograft recipients, from the first postoperative day until 18 months after the transplantation. Real-time polymerase chain reaction was used to detect and quantitate BK viral load in serum and urine samples.ResultsQualitative analysis with PCR for the DNA of BK virus showed 31 (31/228, 14%) positive serum samples originating from 20 (20/32, 62%) renal allograft recipients and 57 (57/228, 25%) positive urine samples originating from 23 (23/32, 72%) recipients. During the follow up period of 5 years, renal allograft function remained stable (eGFR 18^th month: 53.9 ± 23.9 mL/min/1.73 m² and eGFR 5^th year: 52.6 ± 20.6 mL/min/1.73 m²). Comparison of recipients that presented with either BK viremia or viruria with a group that did not present viral reactivation did not reveal a statistically significant difference in eGFR. Furthermore, recipients with significantly high viral load in serum or urine did not present renal allograft dysfunction.ConclusionBK virus is potentially pathogenic in renal allograft recipients. It is certain that there is a reactivation of the virus in a high percentage of transplanted patients mostly in the first year after the surgery, without however a negative effect of the transient viremia and viruria in renal allograft function.     

Efficacy of Total Lymphoid Irradiation in Azithromycin Nonresponsive Chronic Allograft Rejection After Lung Transplantation

Bronchiolitis obliterans syndrome (BOS) remains a major problem after lung transplantation. Azithromycin seems to be beneficial in some patients with established BOS. We investigated the efficacy of total lymphoid irradiation (TLI) in 6 BOS patients with a continuous decline in FEV₁, despite treatment with azithromycin for a mean of 12 ± 13 (range, 1-35) months. A historical control group consisted of 5 patients with declining FEV₁, also nonresponders to azithromycin and those not treated with TLI. All 6 TLI patients received the total dose of 8 Gy in 10 sessions. There was a significant change in the decline of the FEV₁ after TLI treatment (from 221 ± 107 to 94 ± 79 mL/mo; P = .041). Three patients died, due to BOS progression, overwhelming pneumonia, and sudden cardiac arrest, respectively, 3.5, 11, and 26 months after TLI; two patients underwent retransplantation at 6 and 19 months after TLI, respectively. The sixth patient remains stable in BOS stage 3 after a follow-up period of 24 months. In the control group, there was no significant change in FEV₁ decline (209 ± 97 mL/mo before versus 193 ± 81 mL/mo after starting azithromycin; P = not significant). Two patients remain stable in BOS stage 3, 1 died of BOS progression, and the 5th patient is scheduled for retransplantation. We conclude that patients who do not or no longer respond to azithromycin may benefit from TLI, as suggested by a decreased rate in decline of the FEV₁.     

活血化瘀注射液Ⅰ号对大鼠肾脏低温保存时超微结构及移植肾功能的影响

目的观察活血化瘀注射液Ⅰ号(HHI-1)是否具有延长肾脏低温保存时间、减轻肾脏损伤的作用.方法将HHI-I加入大鼠肾脏的保存液(乳酸钠林格液)中作为HHI-I组,单纯乳酸钠林格液作为对照组,分别低温保存大鼠肾脏.观察保存的各个时点肾脏超微结构的变化;将在两种不同保存液中保存达3 h后进行大鼠肾脏移植,观察移植后24 h肾功能的变化.结果大鼠肾脏低温保存24 h HHI-I组未出现明显的器质性损伤;对照组于保存8h出现明显的器质性损伤.分别于保存液中保存3 h后进行大鼠肾脏移植,HHI-I组术后24 h血肌酐明显低于对照组(P<0.01).结论 HHI-I可以减轻单纯低温保存时肾脏超微结构的改变、延长移植物低温保存的时间、改善大鼠肾脏移植效果、促进移植肾功能尽快恢复.     

供体抗原特异性T细胞疫苗诱导大鼠移植心存活延长

采用大鼠异位(腹腔)心脏移植模型,使T细胞疫苗的提供者和接受者成为同一个体,探讨供体抗原致敏的T细胞疫苗接种对移植物存活期的影响,为其应用于临床防治排斥反应提供可行性依据。结果显示,T细胞疫苗可明显延长同种移植物的存活。注射T细胞疫苗后,虽然也使第三供体的移植心存活期明显延长(21.57±8.02天),但与特异供体移植心的存活期(40.75±25.15天)相比,有明显差异,证明其作用具有供体抗原特异性。本实验结果也表明使疫苗的提供者和接受者成为同一个体是可行的。     

活血化瘀注射液I号对大鼠肾脏低温保存时超微结构及移植肾功能的影响

目的 观察活血化瘀注射液I号（HHI－I）是否具有延长肾脏低温保存时间、减轻肾脏损伤的作用。方法 将HHI－I加入大鼠肾脏的保存液（乳酸钠林格液）中作为HHI－I组，单纯乳酸钠林格液作为对照组，分别低温保存大鼠肾脏。观察保存的各个时点肾脏超微结构的变化；将在两种不同保存液中保存达3h后进行大鼠肾脏移植，观察移植后24h肾功能的变化。结果 大鼠肾脏低温保存24h HHI-I组示出现明显的器质性损伤；对照组于保存8h出现明显的器质性损伤。分别于保存液中保存3h后进行大鼠肾脏移植，HHI－I组术后24h血肌酐明显低于对照组（P＜0．01）。结论 HHI－I可以减轻单纯低温保存时肾脏超微结构的改变、延长移植物低温保存的时间、改善大鼠肾脏移植效果、促进移植肾功能尽快恢复。     

Effect of Hypothermic Machine Perfusion on the Preservation of Kidneys Donated After Cardiac Death: A Single‐Center,Randomized, Controlled Trial

To assess the application of a hypothermic machine perfusion device (LifePort) in kidney transplantation from donation after cardiac death (DCD) donors, 24 pairs of DCD kidneys were randomly divided into two groups: one of the paired kidneys from the same donor was perfused with the LifePort machine (hypothermic machine perfusion [HMP]), and the contralateral kidney was prepared using common static cold preservation (CCP). The two groups were compared with respect to the incidence of delayed graft function (DGF), level of graft function, and pathological changes in time‐zero biopsy specimens. The incidence of DGF was 16.7 and 37.5% in the HMP and CCP groups, respectively; the difference between the two groups was statistically significant (P < 0.05). The incidence of acute rejection was 4.1 (1/24) and 8.3% (2/24) in the HMP and CCP groups, respectively; this difference was not statistically significant (P > 0.05). Forty‐eight kidney patients were followed up for 6 months, and the two groups of recipients all survived, yielding a survival rate of 100%. The mean 6‐month serum creatinine levels were 98.7 ± 23.6 µmol/L in the HMP group and 105.3 ± 35.1 µmol/L in the CCP group; there was no significant difference between the two groups. HMP can reduce the incidence of DGF in DCD kidneys, and this effect is greater for expanded criteria donors kidneys. HMP can also improve early renal function.