Basic Research Open Access
Copyright ©The Author(s) 2004. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Mar 15, 2004; 10(6): 885-888
Published online Mar 15, 2004. doi: 10.3748/wjg.v10.i6.885
Effect of local CTLA4Ig gene transfection on acute rejection of small bowel allografts in rats
Yi-Fang Wang, Ai-Gang Xu, Yi-Bing Hua, Wen-Xi Wu, Department of Gastrointestinal Surgery, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, Jiangsu Province, China
Author contributions: All authors contributed equally to the work.
Supported by the Innovative Foundation of Nanjing Medical University, N0.200106
Correspondence to: Wen-Xi Wu, Department of Gastrointestinal Surgery, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, Jiangsu Province, China. wuwenxi@yahoo.com
Telephone: +86-25-3718836-6828
Received: October 8, 2003
Revised: October 23, 2003
Accepted: December 16, 2003
Published online: March 15, 2004

Abstract

AIM: To evaluate the local expression of CTLA4Ig gene in small bowels and its effect on preventing acute rejection of the small bowel allografts.

METHODS: Groups of Wistar rats underwent heterotopic small bowel transplantation from SD rats. The recipients were randomly divided into experimental group (allografts were transfected with CTLA4Ig gene) and control group (non CTLA4Ig gene transfected). In the experimental group, the donor small bowels were perfused ex vivo with CTLA4Ig cDNA packaged with lipofectin vector via intra-superior mesenteric artery before transplantation, and the CTLA4Ig expression in the small bowel grafts post-transplantation was assessed by immunohistology. On d 3, 7 and 10 post-transplantation, the allografts in each group were harvested for the examination of histology and detection of apoptosis.

RESULTS: Small bowel allografts treated with CTLA4Ig cDNA showed abundant CTLA4Ig expression after transplantation. Acute rejection of grade I on d 7 and grade II on d 10 after transplantation was noticed in the control allografts, and a dramatically increased number of apoptotic enterocytes in parallel to the progressive rejection could be recognized. In contrast, the allografts treated with CTLA4Ig cDNA showed nonspecific histological changes and only a few apoptotic enterocytes were found after transplantation.

CONCLUSION: Local CTLA4Ig gene transfection of small bowel allograft is feasible, and the local CTLA4Ig expression in the allograft can prevent acute rejection after transplantation.




INTRODUCTION

CTLA4Ig is a soluble recombinant fusion protein constructed with an extracellular domain of mouse CTLA4 and Fc portion of human IgG. This protein binds to the mouse and rat B7-1/2 molecules, and blocks the co-stimulatory signals from antigen processing cell (APC) to antigen specific T cell. Treatment with CTLA4Ig gene transfection has been shown to prolong graft survival in mouse and rat heart, liver, pancreatic islet, kidney and lung transplantations and to induce donor-specific tolerance in some of these cases[1-7].

There are two methods of gene transduction: systemic administration (ie. intravenous injection) and local transfection of allograft. Gene transfer of sequences coding for soluble immunosuppressive molecules into transplanted organs aims to create a local microenvironment directly modulating the activation state of immune cells responsible for graft rejection[8]. Therefore, when compared with systemic administration, local and continuous production of biologically active compounds might increase their bioavailability and allow a more effective treatment. Furthermore, cells not involved in the rejection process could be spared, and side effects or generalized immunosuppression may thus be avoided.

Intragraft expression of CTLA4Ig by gene transfection at the time of transplantation can successfully prolong survival of several grafts[9-12]. Nevertheless, study of CTLA4Ig expression within the small bowel allograft has not been reported. In the present study, we transfected gene by ex vivo intra-superior mesenteric artery infusion of mCTLA4Ig cDNA packaged with lipofectin vector before transplantation, evaluated the local expression of CTLA4Ig gene and its effect on preventing acute rejection of small bowel allografts in rats.

MATERIALS AND METHODS
Animals and transplantation

Inbred male SD and Wister rats weighing 250 to 300 g were used as donors and recipients, respectively. All rats were obtained from the Animal Center of Nanjing Medical University.

After fasting for 24 h, donors and recipients were anesthetized with an intraperitoneal injection of pentobarbital (50 mg/kg). The vasculature of the donor small bowel was perfused with 10 mL heparinized saline solution at 4 °C. A segment of 20 cm small bowel with portal vein and superior mesenteric artery attached to a cuff of aorta were removed. The lumen of the donor small bowel perfused with 20 mL pure saline solution at 4 °C. The small bowel graft was transplanted with an end - to - side anastomosis of the cuff of aorta and portal vein of the graft to the infrarenal aorta and infrarenal vena cava (Figure 1). After revascularization of the graft, the oral end and the anal end of the small bowel graft were constructed as a stoma respectively through the right abdominal wall of the recipient. All animals had free access to water within 24 h after transplantation. Starting from postoperative d 1, they received standard rat chow.

Figure 1
Figure 1 Heterotopic small bowel transplantation in the rat. The vasculature of the graft has been anastomosed. RSB: recipient’s small bowel. TSB: transplanted small bowel.
Experimental groups

Animals were placed into two groups: One group of recipients did not receive treatment (control group, n = 21), and the other group of recipients received CTLA4Ig gene transfection (experimental group, n = 21).

Delivery of mCTLA4Ig to small intestine

The plasmid of AAVmCTLA4Ig was a kind gift of Professor I. Anegon (INSERM U437, Nants, France). DOTAP:Chole (in vivo GenSHUTTLE, Qbiogene) was used as the vector.

The AAVmCTLA4Ig was mixed with DOTAP:Chole at room temperature for 15 min to create the DNA-lipid complex. The final concentration of DNA in the complex was 0.5 μg/μL. Before cold preservation, the small bowel graft was irrigated with cold saline and then 50 μL lipid (control group) or 50 μL DNA-lipid complex (experimental group) was delivered into the superior mesenteric artery by slow infusion over 5-10 min. After 1.5 h of cold preservation, the superior mesenteric artery of small bowel was reperfused with 5 mL cold saline for 10 min before transplantation.

Graft histology, apoptosis detection and immunohistology

Three, seven and ten days after transplantation, 7 rats were killed in each group. Samples of the small bowel allografts were obtained. One half was fixed in paraformaldehyde for histology examination and cell apoptosis detection. Another half was preserved in nitrogen liquid for immunohistology.

For histology, sections from paraffin embedded blocks were stained with hematoxylin-eosin (H&E). Kuusanmaki’s protocol[13] served as basis for the grading of acute graft rejection and determination of diagnostic categories.

Apoptosis was detected on sections from paraffin-embedded blocks by the terminal deoxynucleotidyl transferase (TdTase) mediated d-UTP-biotin nick end labeling (TUNEL) technique[14-17]. Apoptosis assay with a detection kit from Bochinger (Mannheim, Germany), conformed to the manufacturer’s protocol strictly except that the sections were finally counterstained with methylgreen (Vector Laboratories). The nuclei of apoptotic cells were stained brown as detected under light microscope, and the number of apoptotic cells was determined by counting labeled enterocytes in 10 randomly chosen high-power fields[18].

Immunohistology was performed in cryostat sections. To detect CTLA4Ig in tissues, sections were subsequently incubated (60 min) with hamster anti-murine CTLA4 mAb (UC-4F10-11, BD Biosciences). Tissues probed with the mAb were then incubated with a biotin-conjugated mouse IgG-absorbed anti-hamster IgG Ab (60 min; G70-204 & G94-56, BD Biosciences), followed by HRP-conjugated streptavidin (45 min; Woburn MA) and DAB substrate, and sections were counterstained with hematoxylin.

Statistical analysis

Data were expressed as mean ± SD and Student’s t test was used. Significant difference was assumed when P < 0.05.

RESULTS
Immunohistology

The CTLA4Ig expression of implanted small bowels was detected by immunohistology, and the small bowel grafts transduced with CTLA4Ig showed the presence of abundant labeling in mesenteric vascular walls, muscularis, submucosa and villus. Higher densities of CTLA4Ig were detected in grafts sampled at early time points, and persistent expression of CTLA4Ig was confirmed within 10 d after transplantation (Figure 2: A, B, C). As anticipated, there was no detectable expression of CTLA4Ig in the control small intestines (Figure 2: D).

Figure 2
Figure 2 Presence of CTLA4Ig in the small bowel allografts. CTLA4Ig was stained as the brown granules. A, B, C represent the cryostat sections of CTLA4Ig gene transfected grafts on d 3, 7, 10 after transplantation, respectively. D represent the cryostat sections of non-CTLA4Ig gene transfected grafts, expression of CTLA4Ig was not detected. [original magnification ×200].
Morphological findings

In the control group, on d 3 after transplantation, only nonspecific changes were noted . Focal mesenteric inflammation, mild endothelial vacuolization, and minimal swelling and desquamation of enterocytes were observed in allografts, and a relatively normal villiform mucosal structure was retained. On d 7, a widespread inflammatory infiltrate in the mesentery, with moderate invasion of the intestinal wall, villous blunting and part of the crypt destruction, was noticed in allografts. In addition, endothelial swelling and proliferation with intimal thickening were found in small mesenteric arteries and arterioles. On d 10, a pronounced mesenteric infiltrate with severe invasion of the intestinal wall was found in allografts. Endothelial proliferation with intimal thickening resulted in luminal obliteration. Furthermore, moderate to extreme enterocystic necrosis could be recognized as erosions and focal ulcerations. However, the allografts treated with CTLA4Ig gene transfection showed nearly normal mucosal structure, slight cell infiltration and minimal swelling and desquamation of enterocytes on d 3, 7 and 10 post-transplantation.

Detection of apoptotic enterocytes by TUNEL

Apoptotic cells were detected mainly in the crypts. On d 3 after transplantation, a small number of labeled enterocytes were observed both in control (5.3 ± 1.5, n = 7) and experimental (5.8 ± 1.8, n = 7) groups. There was no significant difference in the number of apoptotic enterocytes between the two groups. On d 7, the number of labeled enterocytes increased dramatically (61.7 ± 2.8, n = 7) and reached a higher level on d 10 (101 ± 6.1, n = 7) in control group after transplantation (Figure 3: A1, B1, C1), whereas an increasing number of labeled nuclei could not be recognized in experimental group on d 7 (3.4 ± 1.0, n = 7) and on d 10 (3.6 ± 1.3, n = 7) after transplantation (Figure 3: A2, B2, C2). The difference in the number of apoptotic enterocytes in allografts between control and experimental groups on d 7 and 10 was extremely significant (bP < 0.0005, t = 41.2876 on day 7 and dP < 0.005, t = 39.2437 on d 10).

Figure 3
Figure 3 Apoptotic crypt cells in the small bowel allografts. A1, B1, C1 represent the tissue sections of non-CTLA4Ig gene transfected grafts obtained on d 3, 7, 10 after transplantation, respectively. A2, B2, C2 represent the tissue sections of CTLA4Ig gene transfected grafts obtained on d 3, 7, 10 after transplantation, respectively. [original magnification ×400].
DISCUSSION

Small bowel transplantation has emerged as a life-saving therapy for patients with irreversible intestinal failure and is now a routine therapeutic tool at some transplant centers[19,20]. Nevertheless, graft rejection is still the major obstacle to the transplantation[21-23]. Blockade of the B7/CD28 co-stimulation signal by systemic transfer of CTLA4Ig gene through intravenous infusion of AdCTLA4Ig has been shown to prolong small intestinal graft survival[24]. However, different from heart, liver, kidney and other organs, the small bowel is unique among vascularized organ grafts with rich lymphoid components and large amounts of bacteria, severe infection as well as rejection occurs easier after transplantation. Local CTLA4Ig gene transfection of small bowel allograft could be the ideal therapeutic strategy of anti-rejection. It allows inhibition of the recipient’s rejection response, while maintaining the recipient’s efficient peripheral immune function (not suppressing the antibacterial response). The local transfection of CTLA4Ig gene has been successfully achieved in rat heart, liver, kidney, islet and lung allografts[9-12], but it has not been studied in the small bowel transplantation. In the present study, we perfused CTLA4Ig cDNA packaged with lipofectin vector via intra-superior mesenteric artery into the small bowels. By immunohistology, there were a large amount of CTLA4Ig expression in the small bowel allografts on days 3, 7 and 10 after transplantation. The result suggests that local CTLA4Ig gene transfection of small bowel allograft is feasible.

In non-CTLA4Ig gene transfected small bowel allografts, a histological change of progressive acute allograft rejection could be recognized. Although the allografts did not show any specific morphological changes on d 3, acute allograft rejection of grade I on d 7 and grade II on d 10 after transplantation was noticed. In contrast, no evidence of acute rejection was observed in CTLA4Ig gene transfected allografts on d 3, 7 and 10 after transplantation. These data indicate that local CTLA4Ig expression in small bowel allografts can prevent acute rejection after transplantation. The protection of local CTLA4Ig expression in small bowel allografts from acute rejection may be because of the inhibition of expansion of alloantigen-induced T cell priming. The local transfection of CTLA4Ig can block B7 ligand expression on the surface of intragraft APCs, and thus blocking B7/CD28 co-stimulatory pathway. In the absence of B7/CD28 co-stimulatory signal, the interaction of T cell with the alloantigen commonly produces T cell anergy[25-29].

Apoptosis is a DNA-dependent cell death mechanism, which occurs under physiological and pathological conditions. In organ transplantation, apoptosis is an important biochemical indicator of allograft rejection[14-17]. In the process of allograft rejection, the target cells damage is mainly mediated by cytotoxic T lymphocytes through the perforin-dependent granule-exocytosis pathway and the Fas/Fas ligand (FasL) pathway[30-32]. Both perforin- and Fas-mediated pathways of cytotoxicity can result in target cell apoptosis. Numerous reports have demonstrated up-regulation of transcripts for perforin, granzyme B, and FasL during allograft rejection[33-36]. In the present study, TUNEL illustrated a few apoptotic enterocytes on d 3 and showed a dramatically increased number of apoptotic enterocytes on d 7 and 10 after transplantation in non-CTLA4Ig gene transfected small bowel allografts. This result demonstrates the existence of acute rejection after transplantation. Nevertheless, in CTLA4Ig gene transfected small bowel allografts, a few apoptotic crypt cells was observed on days 3, but the number of apoptotic enterocytes did not increase on d 7 and 10 after transplantation. It indicates that the apoptosis in small bowel allografts can be prevented by local CTLA4Ig gene transfection, and the cytotoxic roles of infiltrated T lymphocytes and the acute rejection after transplantation can be prevented by CTLA4Ig expression in the allografts. The few apoptosis in allografts on d 3 was likely caused by cold preservasion damage and postischemic reperfusion damage[37-39].

In conclusion, local CTLA4Ig gene transfection of small bowel allograft can be achieved by perfusing CTLA4Ig cDNA packaged with lipofectin vector via intra-superior mesenteric artery into the small bowels, and the local CTLA4Ig expression in the allograft can prevent acute rejection after transplantation.

ACKNOWLEDGMENT

The authors are grateful to Professor I. Anegon (INSERM U437, Nantes, France) for the kind gift of the AAVmCTLA4IgG plasmid, and thank Professor Xirong Guo (Experimental Research Center of Nanjing Medical University) for the experimental help.

Footnotes

Edited by Zhu LH and Xu FM

References
1.  Laumonier T, Potiron N, Boeffard F, Chagneau C, Brouard S, Guillot C, Soulillou JP, Anegon I, Le Mauff B. CTLA4Ig adenoviral gene transfer induces long-term islet rat allograft survival, without tolerance, after systemic but not local intragraft expression. Hum Gene Ther. 2003;14:561-575.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 18]  [Cited by in F6Publishing: 20]  [Article Influence: 1.0]  [Reference Citation Analysis (0)]
2.  Kosuge H, Suzuki J, Gotoh R, Koga N, Ito H, Isobe M, Inobe M, Uede T. Induction of immunologic tolerance to cardiac allograft by simultaneous blockade of inducible co-stimulator and cytotoxic T-lymphocyte antigen 4 pathway. Transplantation. 2003;75:1374-1379.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 65]  [Cited by in F6Publishing: 66]  [Article Influence: 3.1]  [Reference Citation Analysis (0)]
3.  Shiraishi T, Yasunami Y, Takehara M, Uede T, Kawahara K, Shirakusa T. Prevention of acute lung allograft rejection in rat by CTLA4Ig. Am J Transplant. 2002;2:223-228.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 21]  [Cited by in F6Publishing: 22]  [Article Influence: 1.0]  [Reference Citation Analysis (0)]
4.  Kita Y, Nogimura H, Ida M, Kageyama Y, Ohi S, Ito Y, Matsushita K, Takahashi T, Suzuki K, Kazui T. Effects of adenoviral vectors containing CTLA4Ig-gene in rat heterotopic lung implants. Transplant Proc. 2002;34:1434-1436.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 1]  [Cited by in F6Publishing: 2]  [Article Influence: 0.1]  [Reference Citation Analysis (0)]
5.  Yanagida N, Nomura M, Yamashita K, Takehara M, Murakami M, Echizenya H, Konishi K, Kitagawa N, Furukawa H, Uede T. Tolerance induction by a single donor pretreatment with the adenovirus vector encoding CTLA4Ig gene in rat orthotopic liver transplantation. Transplant Proc. 2001;33:573-574.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 11]  [Cited by in F6Publishing: 12]  [Article Influence: 0.5]  [Reference Citation Analysis (0)]
6.  Shindo J. [Effect of CTLA4Ig gene transfer with adenovirus vector on allogeneic renal graft survival in the rat]. Hokkaido Igaku Zasshi. 2001;76:251-261.  [PubMed]  [DOI]  [Cited in This Article: ]
7.  Iwasaki N, Gohda T, Yoshioka C, Murakami M, Inobe M, Minami A, Uede T. Feasibility of immunosuppression in composite tissue allografts by systemic administration of CTLA4Ig. Transplantation. 2002;73:334-340.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 17]  [Cited by in F6Publishing: 18]  [Article Influence: 0.8]  [Reference Citation Analysis (0)]
8.  Guillot C, Ménoret S, Guillonneau C, Braudeau C, Castro MG, Lowenstein P, Anegon I. Active suppression of allogeneic proliferative responses by dendritic cells after induction of long-term allograft survival by CTLA4Ig. Blood. 2003;101:3325-3333.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 32]  [Cited by in F6Publishing: 33]  [Article Influence: 1.6]  [Reference Citation Analysis (0)]
9.  Kita Y, Li XK, Nogimura H, Ida M, Kageyama Y, Ohi S, Suzuki K, Kazui T, Suzuki S. Prolonged graft survival induced by CTLA4IG gene transfection in rat lung allografting. Transplant Proc. 2003;35:456-457.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 4]  [Cited by in F6Publishing: 5]  [Article Influence: 0.2]  [Reference Citation Analysis (0)]
10.  Umeda Y, Iwata H, Yoshikawa S, Matsuno Y, Marui T, Nitta T, Idia Y, Takagi H, Mori Y, Miyazaki J. Gene gun-mediated CTLA4Ig-gene transfer for modification of allogeneic cardiac grafts. Transplant Proc. 2002;34:2622-2623.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 6]  [Cited by in F6Publishing: 7]  [Article Influence: 0.3]  [Reference Citation Analysis (0)]
11.  Cheung ST, Tsui TY, Wang WL, Yang ZF, Wong SY, Ip YC, Luk J, Fan ST. Liver as an ideal target for gene therapy: expression of CTLA4Ig by retroviral gene transfer. J Gastroenterol Hepatol. 2002;17:1008-1014.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 23]  [Cited by in F6Publishing: 23]  [Article Influence: 1.0]  [Reference Citation Analysis (0)]
12.  Benigni A, Tomasoni S, Remuzzi G. Impediments to successful gene transfer to the kidney in the context of transplantation and how to overcome them. Kidney Int. 2002;61:S115-S119.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 5]  [Cited by in F6Publishing: 6]  [Article Influence: 0.3]  [Reference Citation Analysis (0)]
13.  Kuusanmäki P, Halttunen J, Paavonen T, Pakarinen M, Luukkonen P, Häyry P. Acute rejection of porcine small bowel allograft. An extended histological scoring system. Transplantation. 1994;58:757-763.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 24]  [Cited by in F6Publishing: 24]  [Article Influence: 0.8]  [Reference Citation Analysis (0)]
14.  Wu MY, Liang YR, Wu XY, Zhuang CX. Relationship between Egr-1 gene expression and apoptosis in esophageal carcinoma and precancerous lesions. World J Gastroenterol. 2002;8:971-975.  [PubMed]  [DOI]  [Cited in This Article: ]
15.  Sun P, Ren XD, Zhang HW, Li XH, Cai SH, Ye KH, Li XK. Serum from rabbit orally administered cobra venom inhibits growth of implanted hepatocellular carcinoma cells in mice. World J Gastroenterol. 2003;9:2441-2444.  [PubMed]  [DOI]  [Cited in This Article: ]
16.  Huang ZH, Fan YF, Xia H, Feng HM, Tang FX. Effects of TNP-470 on proliferation and apoptosis in human colon cancer xenografts in nude mice. World J Gastroenterol. 2003;9:281-283.  [PubMed]  [DOI]  [Cited in This Article: ]
17.  Zhao AG, Zhao HL, Jin XJ, Yang JK, Tang LD. Effects of Chinese Jianpi herbs on cell apoptosis and related gene expression in human gastric cancer grafted onto nude mice. World J Gastroenterol. 2002;8:792-796.  [PubMed]  [DOI]  [Cited in This Article: ]
18.  Fayyazi A, Schlemminger R, Gieseler R, Peters JH, Radzun HJ. Apoptosis in the small intestinal allograft of the rat. Transplantation. 1997;63:947-951.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 22]  [Cited by in F6Publishing: 24]  [Article Influence: 0.9]  [Reference Citation Analysis (0)]
19.  Platell CF, Coster J, McCauley RD, Hall JC. The management of patients with the short bowel syndrome. World J Gastroenterol. 2002;8:13-20.  [PubMed]  [DOI]  [Cited in This Article: ]
20.  Kato T, Ruiz P, Thompson JF, Eskind LB, Weppler D, Khan FA, Pinna AD, Nery JR, Tzakis AG. Intestinal and multivisceral transplantation. World J Surg. 2002;26:226-237.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 100]  [Cited by in F6Publishing: 102]  [Article Influence: 4.6]  [Reference Citation Analysis (0)]
21.  Ding J, Guo CC, Li CN, Sun AH, Guo XG, Miao JY, Pan BR. Postoperative endoscopic surveillance of human living-donor small bowel transplantations. World J Gastroenterol. 2003;9:595-598.  [PubMed]  [DOI]  [Cited in This Article: ]
22.  Zhang WJ, Liu DG, Ye QF, Sha B, Zhen FJ, Guo H, Xia SS. Combined small bowel and reduced auxiliary liver transplantation: case report. World J Gastroenterol. 2002;8:956-960.  [PubMed]  [DOI]  [Cited in This Article: ]
23.  Ghanekar A, Grant D. Small bowel transplantation. Curr Opin Crit Care. 2001;7:133-137.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 27]  [Cited by in F6Publishing: 28]  [Article Influence: 1.2]  [Reference Citation Analysis (0)]
24.  Echizenya H, Yamashita K, Takehara M, Konishi K, Nomura M, Yanagida N, Kitagawa N, Kobayashi T, Furukawa H, Inobe M. Adenovirus-mediated CTLA4-IgG gene therapy in orthotopic small intestinal transplantation in rats. Transplant Proc. 2001;33:183-184.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 4]  [Cited by in F6Publishing: 5]  [Article Influence: 0.2]  [Reference Citation Analysis (0)]
25.  Vasilevko V, Ghochikyan A, Sadzikava N, Petrushina I, Tran M, Cohen EP, Kesslak PJ, Cribbs DH, Nicolson GL, Agadjanyan MG. Immunization with a vaccine that combines the expression of MUC1 and B7 co-stimulatory molecules prolongs the survival of mice and delays the appearance of mouse mammary tumors. Clin Exp Metastasis. 2003;20:489-498.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 12]  [Cited by in F6Publishing: 13]  [Article Influence: 0.6]  [Reference Citation Analysis (0)]
26.  Chung JB, Wells AD, Adler S, Jacob A, Turka LA, Monroe JG. Incomplete activation of CD4 T cells by antigen-presenting transitional immature B cells: implications for peripheral B and T cell responsiveness. J Immunol. 2003;171:1758-1767.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 37]  [Cited by in F6Publishing: 38]  [Article Influence: 1.8]  [Reference Citation Analysis (0)]
27.  Elhalel MD, Huang JH, Schmidt W, Rachmilewitz J, Tykocinski ML. CTLA-4. FasL induces alloantigen-specific hyporesponsiveness. J Immunol. 2003;170:5842-5850.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 26]  [Cited by in F6Publishing: 27]  [Article Influence: 1.3]  [Reference Citation Analysis (0)]
28.  Arpinati M, Terragna C, Chirumbolo G, Rizzi S, Urbini B, Re F, Tura S, Baccarani M, Rondelli D. Human CD34(+) blood cells induce T-cell unresponsiveness to specific alloantigens only under costimulatory blockade. Exp Hematol. 2003;31:31-38.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 14]  [Cited by in F6Publishing: 15]  [Article Influence: 0.7]  [Reference Citation Analysis (0)]
29.  Appleman LJ, Boussiotis VA. T cell anergy and costimulation. Immunol Rev. 2003;192:161-180.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 208]  [Cited by in F6Publishing: 221]  [Article Influence: 10.5]  [Reference Citation Analysis (0)]
30.  Mandrup-Poulsen T. Beta cell death and protection. Ann N Y Acad Sci. 2003;1005:32-42.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 61]  [Cited by in F6Publishing: 62]  [Article Influence: 3.1]  [Reference Citation Analysis (0)]
31.  Abrahams VM, Straszewski-Chavez SL, Guller S, Mor G. First trimester trophoblast cells secrete Fas ligand which induces immune cell apoptosis. Mol Hum Reprod. 2004;10:55-63.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 188]  [Cited by in F6Publishing: 187]  [Article Influence: 23.4]  [Reference Citation Analysis (0)]
32.  Catalfamo M, Henkart PA. Perforin and the granule exocytosis cytotoxicity pathway. Curr Opin Immunol. 2003;15:522-527.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 109]  [Cited by in F6Publishing: 106]  [Article Influence: 5.3]  [Reference Citation Analysis (0)]
33.  D'Errico A, Corti B, Pinna AD, Altimari A, Gruppioni E, Gabusi E, Fiorentino M, Bagni A, Grigioni WF. Granzyme B and perforin as predictive markers for acute rejection in human intestinal transplantation. Transplant Proc. 2003;35:3061-3065.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 20]  [Cited by in F6Publishing: 22]  [Article Influence: 1.1]  [Reference Citation Analysis (0)]
34.  Veale JL, Liang LW, Zhang Q, Gjertson DW, Du Z, Bloomquist EW, Jia J, Qian L, Wilkinson AH, Danovitch GM. Noninvasive diagnosis of cellular and antibody-mediated rejection by perforin and granzyme B in renal allografts. Hum Immunol. 2006;67:777-786.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 1]  [Cited by in F6Publishing: 2]  [Article Influence: 0.1]  [Reference Citation Analysis (0)]
35.  Simon T, Opelz G, Wiesel M, Ott RC, Süsal C. Serial peripheral blood perforin and granzyme B gene expression measurements for prediction of acute rejection in kidney graft recipients. Am J Transplant. 2003;3:1121-1127.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 87]  [Cited by in F6Publishing: 85]  [Article Influence: 4.0]  [Reference Citation Analysis (0)]
36.  Zhang SG, Wu MC, Tan JW, Chen H, Yang JM, Qian QJ. Expression of perforin and granzyme B mRNA in judgement of immunosuppressive effect in rat liver transplantation. World J Gastroenterol. 1999;5:217-220.  [PubMed]  [DOI]  [Cited in This Article: ]
37.  Wang SF, Li GW. Early protective effect of ischemic preconditioning on small intestinal graft in rats. World J Gastroenterol. 2003;9:1866-1870.  [PubMed]  [DOI]  [Cited in This Article: ]
38.  Ma K, Yu Y, Bu XM, Li YJ, Dai XW, Wang L, Dai Y, Zhao HY, Yang XH. Prevention of grafted liver from reperfusive injury. World J Gastroenterol. 2001;7:572-574.  [PubMed]  [DOI]  [Cited in This Article: ]
39.  Zhu XH, Qiu YD, Shen H, Shi MK, Ding YT. Effect of matrine on Kupffer cell activation in cold ischemia reperfusion injury of rat liver. World J Gastroenterol. 2002;8:1112-1116.  [PubMed]  [DOI]  [Cited in This Article: ]