Alicia Smith

Writer’s comment: I was keeping an eye out for an intriguing topic to research for the literature review paper in my English 102 class (Writing in the Biological Sciences) when I came across a section on xenotransplantation in my physiology text. The possibilities mentioned in the article amazed me since I had no idea that science had made so much progress toward successfully putting animal organs into people. I also recognized that the field seemed relatively new, so writing a review on some of the original research would be productive since so little general information on it is currently available. Obtaining and reading the original research papers was an education in itself, since I had not read this type of paper before. Fortunately, I was motivated to fully understand the papers because the details I discovered while wading through all of the technical words allowed me to make interesting connections between the papers for the review.
- Alicia Smith

Instructor’s comment: Alicia wrote this literature review for my Advanced Composition course, English 102: Writing in the Biological Sciences. The task was to take from six to ten recent, original research articles that focus on an issue and to synthesize them in order to bring readers up to speed on that topic. Alicia did a great job of organizing the material in a logical way and of explaining it coherently. Her introduction quickly establishes the importance of xenotransplantation, then sets the reader up for not only the topics that will be addressed but also the relationship between those topics. After that, it’s smooth sailing through the complexities of tissue rejection to a conclusion that points out a further avenue for research. It isn’t easy to write one of these, but Alicia makes it look that way.
- Jared Haynes, English



Introduction

     In the last few years, progress has been made toward successfully using animal organs in humans who need transplants, an operation called xenotransplantation. The biggest obstacle has been preventing the body from destroying the transplant as a foreign body. The speed of rejection depends on the species and tissue involved. In transplants between discordant species, such as pig to human, the recipient has natural antibodies against the donor organ. In untreated discordant vascularized xenografts, hyperacute rejection (HAR) occurs within minutes or hours after transplantation.
     Recently, HAR has been successfully inhibited, and a second stage of rejection, termed delayed xenograft rejection (DXR), has surfaced. DXR takes place three to four days after transplantation and results from a cell-mediated response. Such a response involves a massive invasion of macrophages, which engulf the xenograft cells. Successful suppression of DXR is currently the most researched area of xenotransplantation because this stage of rejection must be inhibited before even later types can be researched.

Hyperacute Rejection (HAR)

The Immune Response that Causes HAR

     Several researchers have evaluated the specific antibody response that is responsible for HAR. An in vitro kinetic experiment combined rat endothelial cells with primate serum and then measured bound human and monkey antibodies, number of lysed cells, and C complement activity (Azimzadeh et al., 1996). The results showed that IgM antibodies were produced rapidly in the earliest stage, after which a large number of IgG antibodies were produced. Components of the C cascade were present on the endothelial cells. These results agree with the accepted theory that IgM antibodies activate IgG and the C cascade.
     To conclude with more certainty that the IgM class of antibodies is most responsible for HAR, Kroshus, Bolman, and Dalmasso (1996) removed IgM from human blood prior to perfusing a pig heart with it. While one heart perfused with normal blood survived for only 25 ± 5.6 minutes, the heart perfused with IgM-depleted blood survived for 228.5 ± 45.2 minutes. Furthermore, there were lower levels of complement in the IgM-depleted model. The experiment revealed that the elimination of IgM from the blood reduced C activation ability by 80%. An understanding of the exact mechanism of hyperacute rejection has allowed for more effective inhibition of HAR since there are several stages which can be blocked.

Prevention of HAR with CVF

     Cobra Venom Factor (CVF) is known to deplete the C complement cascade. In one experiment, Azimzadeh et al. (1996) transplanted rat hearts into monkeys without removing the original heart. Untreated hearts stopped beating after 5.5 ± 1.4 minutes, while CVF-treated hearts survived for 18 or 94 hours. Because of this success, investigators in a more recent study administered CVF to all subjects to prevent HAR and to study other drugs for the suppression of DXR (Hancock et al., 1997). However, prevention of HAR with high doses of CVF came at a cost for the monkeys treated with CVF by Azimzadeh et al. (1996); CVF apparently caused the monkeys to die within days of the operation. This observation suggests a need for alternatives to countering HAR with high doses of immunosuppressive drugs.

Inhibition of HAR

     One alternative to immunosuppressive drugs is decay accelerating factor (DAF), which is one of the body’s natural C regulators. Every species has its own DAF, so the endothelial cells of a pig heart must express human DAF for successful inhibition of the human immune system. An in vitro study evaluated how well human DAF would incorporate into pig endothelial cells and then how well it protects them against C activation by human serum (Dalmasso, Vercellotti, Platt, and Bach, 1991). The endothelial cells were shown to take up and hold DAF. Furthermore, the DAF inhibited C activation, and resulting endothelial cell lysis, by up to 80%.
     The next step in the use of DAF to prevent HAR was to genetically engineer pigs that express human DAF in their endothelial cells. Rosengard et al. (1995) confirmed successful genetic engineering by staining engineered pig endothelial tissue with immunoperoxidase. They found that human DAF was expressed in 10 out of 13 pigs, in 7 of which it was widespread. In yet another test of DAF, human blood was perfused through engineered pig hearts and normal pig hearts (Schmoeckel et al., 1996). While tissue from normal hearts demonstrated the complete symptoms of tissue rejection, including cell rupture and severe damage in the mitochondria, tissue from transgenic hearts showed only minor vasculitis and myocyte damage. Thus, engineering pig hearts with human DAF is a successful approach to inhibiting the C cascade before it begins its attack on the membranes of pig heart cells.

Inhibition of Delayed Xenograft Rejection (DXR) with Drugs

     The most current experimentation in xenotransplantation is testing the effectiveness of immunosuppressive drugs in preventing DXR. Leflunomide (LEF) is the most popular drug, followed by cyclosporine (CsA), mycophenolate mofetil (MMF), and deoxyspergualin (DSG). Since some of the effects of many of these drugs on the immune system are already known, an understanding of the mechanisms of DXR can be derived from the impact of different drugs on rejection.
     Cyclosporine was used in combination with leflunomide in the following two experiments. One study of pig islets transplanted into rats showed a great reduction of invading macrophages when CsA and LEF were administered (Wennberg et al., 1997). In a hamster to rat heart xenograft where CsA was used in all subjects, HAR was not a problem, but DXR did occur unless LEF was also given for two weeks after transplantation (Lin, Goebels, Vandeputte, and Waer, 1997). CsA is not a significant inhibitor alone, but it probably serves some function in initial inhibition of HAR.
     Leflunomide inhibits multiple B, macrophage, and T cell functions. In Lin et al.’s (1997) hamster to rat heart xenograft, treatment with LEF prevented DXR. A second heart was transplanted, six weeks after the first, and maintained on LEF and CsA therapy. Following the second transplantation, some rats were treated with CsA only, while others received another two-week course of LEF. The second hearts untreated with LEF were rejected, while the ones treated with LEF were not. Surprisingly, none of the originally transplanted hearts underwent rejection, even when the second hearts were being rejected. A guinea pig to rat heart transplant experiment also tested the effectiveness of LEF, finding that it decreased macrophage infiltration of the graft but did not entirely prevent it (Hancock et al., 1997).
     Several experiments have been designed to test other drugs, including MMF and DSG. One experiment investigated the effectiveness of MMF in further preventing rejection of porcine islets in rats (Wennberg et al., 1997). When MMF was given along with LEF and CsA, rejection was prevented more completely than when just LEF and CsA were administered. In the guinea pig to rat heart xenograft, Hancock et al. (1997) experimented with DSG, which blocks T and B cell differentiation and antigen processing by a macrophage. Rats treated with DSG or LEF generally showed similar decreases, with a greater decrease in natural killer cells, macrophage invasion, and T cells when both were given. However, the drugs did not entirely prevent macrophages from invading the xenograft, and eventually enough macrophages accumulated to destroy it.

Conclusion

     The consensus from these drug-related experiments is that macrophages cause DXR, but the process of recruitment is still uncertain because inhibition of the known mechanisms does not entirely prevent rejection. However, Hancock et al. (1997) found that macrophage lectin, which is a macrophage-activating chemical not present in resting macrophages and not activated by the usual pathways, was present in 80% of the macrophages infiltrating the xenograft. This discovery may be the key to complete inhibition of DXR in the future without total destruction of the responsible macrophages because inhibition of the lectin pathway does not affect the other ways in which macrophages are normally activated. Possible ways of blocking macrophage lectin activation are treatments with neutralizing antibodies or peptide inhibitors.


Literature Cited

Azimzadeh A., P. Wolf, A.P. Dalmasso, M. Odeh, J. Beller, M. Fabre, B. Charreau, K. Thimbaudeau, J. Cinqualbre, J. Soulillou, and I. Anegon. 1996. Assessment of Hyperacute Rejection in a Rat to Primate Cardiac Xenograft model. Transplantation61: 1305–1313.

Dalmasso, A.P., G.M. Vercellotti, J.L. Platt, and F.H. Bach. 1991. Inhibition of Complement-Mediated Endothelial Cell Cytotoxicity by Decay-Accelerating Factor. Transplantation52: 530–533.

Hancock, W.W., T. Miyatake, N. Koyamada, J.P. Kut, M. Soares, M.E. Russell, F.H. Bach, and M.H. Sayegh. 1997. Effects of Leflunomide and Deoxyspergualin in the Guinea Pig to Rat Cardiac Model of Delayed Xenograft Rejection. Transplantation64: 696–704.

Kroshus, T., R. M. Bolman, and A. P. Dalmasso. 1996. Selective IgM Depletion Prolongs Organ Survival in an Ex Vivo Model of Pig to Human Xenotransplantation. Transplantation62: 5–12.

Lin, Y., J. Goebels, M. Vandeputte, and M. Waer. 1997. Long-Term Survival of Hamster to Rat Heart Xenografts Based on Mechanisms of Accommodation and Tolerance of CD5 B Cells. Transplantation Proceedings29: 2355.

Rosengard, A.M., N. Cary, J. Horsley, G. Langford, E. Cozzi, J. Wallwork, and D.J.G White. 1995. Endothelial Expression of Human Decay Accelerating Factor in Transgenic Pig Tissue: A Potential Approach for Human Complement Inactivation in Discordant Xenografts. Transplantation Proceedings27: 326–327.

Schmoeckel M., G. Nollert, M. Shahmohammadi, V.K. Young, G. Chavez, W. Ksper-Konig, D.J.G. White, J. Muller-Hocker, R.M. Arendt, U. Wilbert-Lampen, C. Hammer, and B. Reichart. 1996. Prevention of Hyperacute Rejection by Human Decay Accelerating Factor in Xenogeneic Perfused Working Hearts. Transplantation62: 729–734.

Wennberg, L., C.G. Groth, A. Tibell, S. Zhu, J. Liu, E. Rafael, J. Soderlund, P. Biberfeld, R.E. Morris, A. Karlsson-Parra, and O. Korsgren. 1997. Triple Drug Treatment with Cyclosporine, Leflunomide and Mycophenolate Mofetil Prevents Rejection of Pig Islets Transplanted into Rats and Primates. Transplantation Proceedings29: 2498.