Experimental Organ Transplantation Research Articles

CD26/DPP IV in experimental and clinical organ transplantation.

The T-cell activation-Ag CD26 possesses dipeptidyl peptidase IV (DPP IV) enzymatic activity. Costimulatory efficacy and immunocompetence are associated with the enzymatic activity. In models of experimental cardiac allograft transplantation (HTx), we analyzed the role of CD26/DPP IV during organ rejection. Also, we investigated CD26 enzymatic and cellular expression in human recipients of kidney transplants (Tx). Heterotopic HTx in rats, models of acute and accelerated rejection. Monitoring of DPP IV serum levels and humoral immunity. Propro-diphenyl phosphonate was employed to inhibit DPP IV activity during rejection. In a prospective study, surface expression of CD26, 3, 4, 8, 45, 122 and ADA on PBL and DPP IV serum activity were measured in kidney recipients for 24 months post-transplantation. Acute rejection was . associated with increased serum DPP IV activity (p < 0.005). Specific inhibition abrogated acute (p < 0.0001) and accelerated (p < 0.01) rejection, impairing cytotoxicity and allospecific Ig-synthesis. Kidney recipients displayed a significant drop in CD26 expression on PBL for up to 18 months postoperatively (p < 0.001). CD4, 8, 45, 122 and ADA expression kinetics were only briefly affected. DPP IV enzymic activity stayed depressed for at least 12 months (p < 0.001). CD26/DPP IV is pivotal in T-cell mediated immune responses toward allo-Ag. In clinical transplantation, engraftment/immunosuppression are reflected by CD26 cellular and enzymatic expression posttransplantation and may serve as an indicator for immunomodulation.

Historical Events on Development of Experimental Microsurgical Organ Transplantation

Historical Events on Development of Experimental Microsurgical Organ Transplantation

Behavior of male-specific minor histocompatibility antigen in skin and limb transplantation

Background. Although the role of male-specific minor histocompatibility antigen H-Y has been increasingly understood in both experimental and clinical organ transplantation, little has been investigated on musculoskeletal tissue transplantation. This study was performed to describe the behavior of male-specific minor histocompatibility H-Y antigen in rat skin and whole limb transplantation. Materials and methods. Using three different strains of inbred rats (Lewis, F344, and Dark Agouti), 75 donor hindlimbs and eighteen skin grafts were isogenically transplanted to the sex-mismatched recipients. Recipients were observed up to 48 weeks postoperatively. Rejection was monitored by the appearance of the skin of the grafted limb and histology. Systemic microchimerism was assessed by polymerase chain reaction using Y-chromosome specific primers. Results. Skin rejection didn’t occur in all limb transplant recipients and histology did not show any rejection findings in all components of the limb graft through 48 weeks. Successful functional recovery was expected. Stable and high level of chimerism (>1%) was detected in the lymphoid tissues in nontreated female recipients. Male skin grafts were rejected by Lewis and F344 female recipients within 6 weeks postoperatively. All female skin grafts survived in male recipients. Conclusion. Our results suggest that H-Y antigen can induce graft rejection in rat skin graft but causes no rejection reaction in whole limb transplantation. Systemic chimerism may play an important role for acceptance of sex-mismatched limb graft.

The impact of immune-activating processes following transplantation on chronic allograft nephropathy

The long-term success of organ transplantation is influenced by numerous alloantigen-dependent and -independent risk factors. However, only very little information is presently available on the influence of systemic immune-activating processes following organ engraftment. To simulate the clinical situation of sequential organ transplantation, rat renal allograft recipients received additional immune activating stimuli (secondary donor-specific and third-party skin grafts) after transplantation at serial time intervals (4 and 8 weeks). The overall observation period was 16 weeks. All control animals survived the observation period. In contrast, recipients receiving additional third-party or donor-specific skin grafts were beginning to die 12 weeks after organ engraftment with only few animals surviving 16 weeks. Systemic immune activation by additional third-party and in particular by additional donor-specific skin grafts resulted in significant temporary and long-term functional deterioration. Morphologic changes progressed significantly, particularly after a secondary challenge with donor-specific skin grafts. ED1+ monocytes/macrophages, T-cell infiltrates, and intragraft mRNA expression for CD25 were significantly elevated by 16 weeks, following an additional immune challenge. Analysis of early intragraft events showed strong up-regulation of CD25 transcripts, suggesting fast stimulation of intragraft immune processes. Both alloantigen-specific and -unspecific systemic immune activation processes, following experimental organ transplantation, contribute to chronic graft deterioration. Those results seem relevant for long-term immunosuppressive protocols and clinical situations of sequential organ transplantation.

Effect of semi-invasive cardiovascular monitoring on the perioperative outcome of kidney or lung xenotransplantation in cynomolgus monkeys

Summary >Background: Cynomolgus monkeys (Macaca fascicularis) are one of the predominant primate models in experimental transplantation. Particularly in xenotransplantation experiments, they can be subjected to profound cardiovascular instability by surgical trauma, volume shifts and inflammatory mediator release. Meticulous perioperative management including deliberate intraoperative monitoring is therefore required. Methods: 26 porcine kidneys (group A) and 3 single lung lobes (group B), obtained from unmodified pigs or from pigs transgenic for human complement regulators, were transplanted into 29 cynomolgus monkeys. In group A 1 (n = 11) the monkeys were monitored intraoperatively by ECG, non-invasive arterial, and central venous blood pressure (standard cardiovascular monitoring, SCVM). In group A 2 (n = 15) measurements of cardiac output (CO), intrathoracic blood volume and extravascular lung water (extended cardiovascular monitoring, XCVM) were added to the intraoperative monitoring regime. XCVM plus continuous pulse contour CO was used in the 3 monkeys undergoing porcine single lung xenotransplantation. Results: In group A 2 significantly more animals had initial graft function if compared to group A 1 . 40% of the monkeys of group A 2 but 73% of group A 1 died within the first postoperative week. Group A 2 displayed a trend towards greater overall survival rates, but this difference did not reach statistical significance (p = 0,07). In group B stable lung xenograft reperfusion was achieved in all 3 recipients despite recurrent phases of severe cardiovascular instability. Two lung recipients could be weaned from the ventilator, and 1 monkey survived more than 24 hours postoperatively. Conclusions: The application of XCVM in small primates undergoing kidney xenotransplantation led to significantly better early postoperative results and longer survival times. In single lung xenotransplantation XCVM plus continuous pulse contour CO showed to be indispensable for the therapeutic management of the profound cardiovascular instabilities observed. Our data indicate that comprehensive anaesthesiologic monitoring can contribute substantially to the advancement of experimental organ transplantation in small monkeys.

Novel agents or strategies for immunosuppression after renal transplantation

There have been several recent advances in the use of immunosuppression after organ transplantation. The main goal is to induce transplantation tolerance without immunosuppressive drug toxicity. We reviewed both experimental and clinical organ transplantation studies such as costimulatory blockade agents, sphingosine 1-phosphate receptor analogs, lymphocyte depletion strategies, and chemokine blockade agents in the context of renal transplantation. The newer and safer immunosuppression protocols might provide significant benefit to organ transplant recipients as alternatives to conventional immunosuppressive therapy.

A novel leflunomide derivative, FK778, for immunosuppression after kidney transplantation in dogs

Background. Leflunomide and its metabolite, A77 1726, interfere with pyrimidine metabolism and exert potent immunosuppression in experimental organ transplantation. However, clinical use of the agents has been restrained because of an extended half-life. FK778, one synthetic malononitrilamide derived from A77 1726, is synthesized to overcome this problem while maintaining similar therapeutic efficacy. Methods. Immunosuppressive effect, pharmacokinetics, and adverse events of FK778, as a single drug treatment or combination with tacrolimus or cyclosporine, were determined in a canine kidney transplantation model. The agents were daily administered orally to the animals for 90 days after surgery. Animal survival, pharmacokinetics, biochemistry, hematology, and histopathology were evaluated. Results. FK778 at 4 mg/kg prolonged median survival of the control animals from 10 days to 30.5 days. Administration of 4 mg/kg FK778 with 0.3 mg/kg tacrolimus or 10 mg/kg cyclosporine increased median survival to 75.5 days and 50.5 days, respectively. In combined treatments, trough levels of FK778 at 4 mg/kg ranged between 40 μg/mL and 100 μg/mL after 1 month. Vomiting and diarrhea were common in animals given FK778. Bone marrow suppression was seen at higher doses. Conclusions. FK778 is a promising new immunosuppressant that may be used in combination with current standard drugs in organ transplantation. (Surgery 2002;132:72-9.)

Negative regulation of CD95 ligand gene expression by vitamin D3 in T lymphocytes.

Fas (APO-1/CD95) and its ligand (FasL/CD95L) are cell surface proteins whose interaction activates apoptosis of Fas-expressing targets. In T lymphocytes, the Fas/FasL system regulates activation-induced cell death, a fundamental mechanism for negative selection of immature T cells in the thymus and for maintenance of peripheral tolerance. Aberrant expression of Fas and FasL has also been implicated in diseases in which the lymphocyte homeostasis is compromised, and several studies have described the pathogenic functions of Fas and FasL in vivo, particularly in the induction/regulation of organ-specific autoimmune diseases. The 1,25(OH)(2)D(3) is a secosteroid hormone that activates the nuclear receptor vitamin D(3) receptor (VDR), whose immunosuppressive activities have been well studied in different models of autoimmune disease and in experimental organ transplantation. We and others have recently described the molecular mechanisms responsible for the negative regulation of the IFN-gamma and IL-12 genes by 1,25(OH)(2)D(3) in activated T lymphocytes and macrophages/dendritic cells. In this study, we describe the effect of 1,25(OH)(2)D(3) on the activation of the fasL gene in T lymphocytes. We show that 1,25(OH)(2)D(3) inhibits activation-induced cell death, fasL mRNA expression, and that 1,25(OH)(2)D(3)-activated VDR represses fasL promoter activity by a mechanism dependent on the presence of a functional VDR DNA-binding domain and ligand-dependent transcriptional activation domain (AF-2). Moreover, we identified a minimal region of the promoter containing the transcription start site and a noncanonical c-Myc-binding element, which mediates this repression. These results place FasL as a novel target for the immunoregulatory activities of 1,25(OH)(2)D(3), and confirm the interest for a possible pharmacological use of this molecule and its derivatives.

Why do we reject grafts?

One of the puzzles in clinical and experimental organ and tissue transplantation has been why it is that, with the exception of transplantation between genetically identical twin pairs, transplantation results in a rapidly progressing immune response to the point of tissue necrosis in a matter of a few days, even though the recipient has not been immunized to the donor. Indeed, the numbers of T lymphocytes predetermined to react against grafts are relatively abundant compared to those that recognize microbial antigens. Cellular, molecular, and genetic studies are now revealing the nature of T-cell recognition, and why it is that we cannot freely exchange tissue grafts.

MICROCHIMERIC CELLS FROM THE PERIPHERAL BLOOD ASSOCIATED WITH CARDIAC GRAFTS ARE BONE MARROW DERIVED, LONG-LIVED AND MAINTAIN ACQUIRED TOLERANCE TO MINOR HISTOCOMPATIBILITY ANTIGEN H-Y 1

Although it has been well established that the microchimerism occurs in the peripheral blood of the recipients after various settings in both clinical and experimental organ transplantation, nevertheless, their roles in inducing and maintaining acquired transplantation tolerance are controversial. Furthermore, regarding the cell lineages, kinetics, and functions of the cells that constitute the microchimerism after organ transplantation, solid information is not available. Using rat heterotopic heart isografts from bone marrow chimeras between cross-sex and applying polymerase chain reaction with specific primers to rat sex determining region of Y chromosome, a relationship between a state of microchimerism and induction as well as maintenance of acquired tolerance to H-Y antigen were examined. Microchimeric cells of the peripheral blood (MCPB) after cardiac grafting contain bone marrow-derived and radiation-sensitive cells. Furthermore, removal of the primary cardiac grafts revealed that microchimeric cells in the peripheral blood are long-lived cells, i.e., more than 6 months. When the female rats that had contained long-lasting MCPB, were innoculated with syngeneic male dendritic cells, failure to sensitize female toward male specific antigen H-Y was found to occur. Thus it was suggested that radiation-sensitive, bone marrow derived, long-lived MCPB play a significant role in maintaining acquired transplantation tolerance to minor histocompatibility antigen H-Y.

Immunosuppressant FTY720 induces apoptosis by direct induction of permeability transition and release of cytochrome c from mitochondria.

FTY720 has immunosuppressive activity in experimental organ transplantation and shows a prompt and protracted decrease of blood T lymphocytes upon oral administration. The blood lymphocyte decrease in vivo was mainly a result of FTY720-induced apoptosis. However, this apoptotic mechanism is not well understood. We examined the mechanism of FTY720-induced apoptosis in lymphoma. Western blotting and fluorescent caspase-specific substrate revealed that caspase-3 is involved in FTY720-induced apoptosis, whereas caspase-1 is not. Apoptotic cell death was inhibited by the pan-caspase inhibitor, Z-VAD-FMK, suggesting that caspase activation is essential for FTY720-induced apoptosis. FTY720 reduced mitochondrial transmembrane potential and released cytochrome c from the mitochondria of intact cells as well as in a cell-free system even in the presence of Z-VAD-FMK. As these mitochondrial reactions occurred before caspase activation, we concluded that FTY720 directly influences mitochondrial functions. The inhibition of mitochondrial permeability transition by Bcl-2 overexpression or by chemical inhibitors prevented all apoptotic events occurring in intact cells and in a cell-free system. Moreover, using a cell-free system, FTY720 did not directly affect isolated nuclei or cytosol. These results indicate that FTY720 directly affects mitochondria and triggers permeability transition to induce further apoptotic events.

Heparin interference with reverse transcriptase polymerase chain reaction of RNA extracted from lungs after ischemia-reperfusion.

The reverse transcriptase polymerase chain reaction (RT-PCR) is a rapid and sensitive method for detecting gene expression. However, when we used this technique to study gene expression of cytokines in ischemic and ex-vivo-reperfused rat lungs as a model for transplantation, significant inhibition of RT-PCR reaction was observed. To optimize RT-PCR conditions, RNA was extracted from rat lungs after flushing, preservation, and reperfusion. RNA was further purified and PCR conditions were modified with various strategies. We found that heparinase I pretreatment completely overcame the inhibitory effects of RT-PCR using RNA extracted from lung tissues after ischemia-reperfusion. With this treatment, a dramatic increase in tumor necrosis factor-a (TNF-a) mRNA was revealed from lung tissues after ischemia-reperfusion. This result suggests that residual heparin in lung tissue interferes with RT-PCR. Because heparinization is routinely used during clinical and experimental organ transplantation, we recommend the treatment of RNA samples with heparinase prior to RT-PCR.

Oxygen free radicals in experimental organ transplantation

Oxygen free radicals in experimental organ transplantation

Immunohistochemical characterization of transgenic mice highly expressing human lysosomal α-galactosidase

Human lysosomal α-galactosidase predominantly hydrolyzes ceramide trihexoside. A transgenic mouse line, C57BL/6CrSIc–TgN(GLA) 1951 Rin, highly expressing human α-galactosidase, has been established and investigated biochemically and immunohistochemically in order to clarify the distribution of the expressed enzyme proteins and to evaluate it as a donor model of organ transplantation therapy for Fabry disease caused by a genetic defect of α-galactosidase. In these transgenic mice, about five copies of the transgene were integrated, and α-galactosidase activity was expressed in liver, kidney, heart, spleen, small intestine, submaxillary gland, skeletal muscle, cerebrum, cerebellum, bone marrow cells and serum. The enzyme activity was about 22 to 11,080-fold higher than that in non-transgenic mice. In liver, heart and kidney tissues, which are important organs for transplantation studies, sufficient amounts of α-galactosidase mRNAs were transcribed, and the expressed enzymes, with molecular weights of 54–60 kDa, are abundant in the liver (enzyme activity: 53,965 nmol h −1 mg −1 protein) and heart (39,906 nmol h −1 mg −1 protein), followed by in the kidney tissue (9177 nmol h −1 mg −1 protein), respectively. An immunohistochemical microscopic study clearly demonstrated the distribution of the expressed enzyme proteins in kidney and liver tissues. Highly expressed α-galactosidase was detected in glomerular cells, tubular cells and hepatocytes. These transgenic mice will be useful as a donor model for experimental organ transplantation, and also it will enable recurrent biopsies and long-term observation. The organ transplantation data on mice will provide us with important information.

Manual of Experimental Muscle Flap and Organ Transplantation in the Rat

Manual of Experimental Muscle Flap and Organ Transplantation in the Rat

Use of allogeneic bone marrow labeled with neomycin resistance gene to examine bone marrow-derived chimerism in experimental organ transplantation

Posttransplant infusion of viable donor bone marrow cells (DBMC) has been shown in our previous studies to promote acceptance of incompatible kidney allografts in rhesus monkeys after treatment with polyclonal antithymocyte globulin to deplete peripheral T-lymphocytes. In this nonhuman primate model, the infusion of the DBMC is requisite for the induction of functional graft tolerance and specific MLR and CTLp unresponsiveness, although the relevant role and fate of bone marrow-derived chimeric cells is uncertain. Standard immunological and molecular techniques applied to this monkey model are unable to differentiate between chimeric cells derived from the infused DBMC and those derived from allograft-borne passenger leukocyte emigrants. To distinguish chimerism due to infused DBMC, we transduced DBMC with a functional neomycin resistance gene (Neo r) using the retroviral vector pHSG-Neo. Neo r-transduced BMC were infused into recipients approximately 2 wk after kidney transplantation and treatment with rabbit antithymocyte globulin. No maintenance immunosuppressive drugs were given. Genomic DNA isolated from peripheral blood leukocytes was used to monitor the presence of Neo r-positive cells. Tissue samples obtained at necropsy also were assessed for Neo r-positive chimeric cells. The presence of DBMC-derived chimerism was assessed by polymerase chain reaction using Neo r sequence-specific primers (PCR-SSP). Chimerism was detectable in recipient tissues at various times for up to 6 mo after DBMC infusion. These studies using gene transduction methodology indicate that a stable genetic marker can provide capability to examine DBMC-derived chimerism for prolonged periods in a non-human primate model. This approach should facilitate future studies in preclinical models to study the role and type of chimeric cell lineages in relation to functional allograft tolerance.

Use of Allogeneic Bone Marrow Labeled with Neomycin Resistance Gene to Examine Bone Marrow-Derived Chimerism in Experimental Organ Transplantation

Posttransplant infusion of viable donor bone marrow cells (DBMC) has been shown in our previous studies to promote acceptance of incompatible kidney allografts in rhesus monkeys after treatment with polyclonal antithymocyte globulin to deplete peripheral T-lymphocytes. In this nonhuman primate model, the infusion of the DBMC is requisite for the induction of functional graft tolerance and specific MLR and CTLp unresponsiveness, although the relevant role and fate of bone marrow-derived chimeric cells is uncertain. Standard immunological and molecular techniques applied to this monkey model are unable to differentiate between chimeric cells derived from the infused DBMC and those derived from allograft-borne passenger leukocyte emigrants. To distinguish chimerism due to infused DBMC, we transduced DBMC with a functional neomycin resistance gene (Neo(r)) using the retroviral vector pHSG-Neo.Neo(r)-transduced BMC were infused into recipients approximately 2 wk after kidney transplantation and treatment with rabbit antithymocyte globulin. No maintenance immunosuppressive drugs were given. Genomic DNA isolated from peripheral blood leukocytes was used to monitor the presence ofNeo(r)-positive cells. Tissue samples obtained at necropsy also were assessed forNeo(r)-positive chimeric cells. The presence of DBMC-derived chimerism was assessed by polymerase chain reaction usingNeo(r) sequence-specific primers (PCR-SSP). Chimerism was detectable in recipient tissues at various times for up to 6 mo after DBMC infusion. These studies using gene transduction methodology indicate that a stable genetic marker can provide capability to examine DBMC-derived chimerism for prolonged periods in a nonhuman primate model. This approach should facilitate future studies in preclinical models to study the role and type of chimeric cell lineages in relation to functional allograft tolerance.

The history of intestinal transplantation

In all likelihood it was not the two great pioneers of experimental organ transplantation at the beginning of this century, Carrel or Ullmann, as mistakenly frequently cited, but instead Lillehei and Starzl who pioneered experimental intestinal transplantation. Although the first attempts of clinical bowel transplantation began in Boston in 1964, these cases were never published in a scientific manner. The first formally published case was performed by Lillehei 17 in Minneapolis in 1967. Using conventional immuno-suppression, a total of seven such transplants were carried out until 1970 with a longest survival of 76 days. Between 1985 and 1990, 15 isolated bowel transplants were performed using cyclosporine as the basic immunosuppressant. The patients transplanted in Kiel in 1988 and in Paris in 1989 became the first long-term survivors with sufficient graft function. The first recipient of a combined liver-intestinal graft, performed in London, Ontario in 1988, lived for several years. From 1983 until 1991, nine multivisceral transplants were undertaken, the first by Starzl 34 in Pittsburgh. One patient who received a transplant in Innsbruck in 1989 and two who received transplants in London, Ontario in 1990 and 1991 were discharged with functioning grafts but died between 210 and 320 days later from tumor recurrence or lymphoma. In 1990 a clinical intestinal transplant program was started by the Pittsburgh group using FK-506 as primary immunosuppression. Until August 1993, 59 patients received various forms of intestinal transplants with one-year survival rates exceeding 60%. It is clearly to their credit that bowel transplantation is becoming a clinical reality.

Manual of Experimental Muscle Flap and Organ Transplantation Models in the Rat

The Manual of Experimental Muscle Flap and Organ Transplantation Models in the Rat by Zhang and co-authors represent an impressive compilation of detailed and valuable anatomical and surgical information for laboratory investigators employing microsurgical rat models. Certainly, this well-organized and well-written manual will be a valuable addition to the reference shelf in all laboratories employing rats as surgical models of human disease. The manual is organized into three sections. The first of these concerns basic information about rat handling, anesthesia, surgery, as well as general information about the utility of animal models in basic research. This section will serve as a good introduction for the novice rat surgeon. The second section is entitled, “Muscle and musculocutaneous flaps,” but is in fact the description of virtually every free tissue transfer (except organ transplants) that has been described in the rat. There are separate chapters on the serratus anterior, latissimus dorsi, rectus abdominus, gracilis, gastrocnemius, cutaneous maximus, and pectoralis major muscle and, if applicable, myocutaneous flaps. In addition, the groin free flap, the temporoparietal fascial flap, and the free toe transfer are covered. In each chapter, a rational well-organized discussion of the general utility of the flap in experimental surgery, of the relevant anatomy for each flap, and of the surgical technique, is provided. The detailed, step by step descriptions of the surgical procedures for elevation and transfer of these flaps are especially useful. The author’s vast experience with these techniques is evident in the numerous, important subtle technical points which are emphasized. In section three, the use of microsurgical models of whole organ transplantation is covered. An introductory chapter points out some of the history of organ transplantation in animal models and discusses some important aspects of experimental transplantation surgery, including immunosuppressants. After a chapter on hindlimb transplantation, the remaining chapters cover bowel, kidney, liver, pancreaticoduodenal, heart, testicular, and ovarian transplantation. These chapters provide the same organized and detailed description of the operations as in section two. In all of the chapters in sections two and three, welldrawn, accurate illustrations provide an invaluable resource for the relevant anatomy and the surgical execution of each of the procedures described. These illustrations alone would make this manual a worthwhile addition to any microsurgical laboratory. Like all publications, this manual does have several deficiencies, which the authors could perhaps address in a subsequent edition. Although the description of animal handling is quite useful, no mention is made of the importance of strict adherence to the National Institutes of Health Guide for the Use and Care of Laboratory Animals (NIH Publication 86-23). Secondly, this multi-author manual has chapters by a number of people who are not listed in the list of authors on the cover. It would probably be most appropriate to indicate that Zhang, Lineaweaver, Kao, Walker, and Tonken have in fact edited this multi-author manual, and then to acknowledge each of the authors in a separate location. In addition, this manual has been obviously published with the aid of commercial sponsorship. Although the name of the company appears prominently on the front and

Manual of Experimental Muscle Flap and Organ Transplantation Models in the Rat

Manual of Experimental Muscle Flap and Organ Transplantation Models in the Rat