Xenotransplantation literature update, November–December 2014 (2015)

Advances in pig production and immunosuppression have brought pig islet xenografts to a previously unrealized level of success. However, Wright et al. suggest that the cost and availability of pig islet xenografts may be a barrier to their application. These authors present a review of the application of islet-like Brockman bodies of boney fish, namely Tilapia 1. When transplanted into diabetic mice, piscine islets restore normoglycemia and evade the recipient immune response when encapsulated. The authors highlight benefits of piscine islets such as a natural resistance to hypoxia, lack of aGal, and significantly less expensive isolation costs. The application of transgenic tilapia that express a humanized insulin may be an alternative source of abundant and cheap xeno-islets. Salvatori et al. 2 review a different approach to overcome the human immune response to pig xenografts: semi-xenotransplantation (SX). The author's description of SX entails decellularizing a xenograft leaving a scaffold of the extracellular matrix upon which recipient cells can grow. Citing several recent descriptions of reduced antigenicity among decellularized grafts, Salvatori et al. suggest that SX may be the quickest route to the use of porcine products in the clinic. Those who publish in and read Xenotransplantation recognize the importance of using genetically modified animals as a solution to the shortage of human organs for transplantation. In fact, one could argue that we become so embedded in our goals that we assume the ethical debate regarding the suitability of pig organs is over. The publication Cell Journal formerly the Yakteh Medical Journal is the English printed quarterly journal of the Royan institute in Tehran, Iran. Prior to reading this article, I had no knowledge of xeno research in Iran and it was therefore refreshing to consider the perspective of another culture. Manesh et al. 3 define their ethical review with two groups, the deontological and the consequentialist critiques. According to the authors, the groups by definition reject the use of pigs organs as first morally and second biologically unethical. In spite of this, the authors reflect largely on earlier publications that defined the appropriateness of xenotransplantation and go further to highlight several perspectives in support of genetically modified pig research. Their conclusion suggests that use of these organs in humans and perhaps their use in general is unethical. While many of these issues have been debated elsewhere, it is interesting to read a current interpretation from the authors perspective. Inclusion of recent studies by Wynyard et al. and Fishman et al. 2012 would have provided a more scientifically balanced debate 4, 5. Definitely worth reading if only to enhance our worldview of xenotransplantation. It is fitting that Sobbrio and Jorqui have provided an ethical review of how we have historically and currently consider public opinion and consultation 6. The authors suggest that xenotransplantation as an emerging biotechnology may be an example of how engaging the public has evolved from a passive discussion to assessing the shared responsibility of risk acceptance and possible regulatory issues. The manuscript spends considerable time defining who the public is and in conclusion reflects the authors personal opinion that an international regulatory framework engaged with public representatives from all cultures will be ethically necessary to move forward. In the end, those “public” individuals, if relevant to making policies, will have to rely on the scientific interpretation of our research and the risks if any. The commentary by Dr. Fishman published earlier this year summarizes a modern view of risk and gives a reasonable position with which to advance xenotransplantation to the clinic 7. In a concise letter to the editor of Xenotransplantation, Dr. Cooper has described the importance of circulating triiodothyronine (fT3) in nonhuman primates as an early indicator of heart and liver xenografts 8. The author first describes the normal levels of fT3 in monkeys and then in detail describes the concentrations and timing of fT3 in circulation before and after transplantation. The author emphasizes that a decrease of fT3 shortly after heart or liver xenotransplantation may correlate to xenograft and recipient survival. It should be evaluated if T3 replacement therapy would improve graft function and extend xenograft function. The PD-1/PD-L1 interaction regulates T-cell activation in mice and humans alike. Plege-Fleck et al. 9 define the effect of PD-L1 expression in the pig-to-rat model of xenotransplantation as a proof-of-concept study. The authors transplanted a B-cell line expressing PD-L1 (or a mock control) under the kidney capsule and looked for infiltrating T cells and the production of anti-pig antibodies. As predicted, expressing a functional PD-L1 reduced T-cell activation and infiltration as well as blunting the production of antibody against the L23 B-cell line. Applying these cells and methods to evaluation in nonhuman primates may produce similar results and justify the production of transgenic pigs. Porcine and bovine xenografts are currently being used in the clinic as heart valve replacements. The durability of the valve cusps is limited by the recipient immune recognition of Gal alpha 1,3 gal epitopes that remain after gluteraldehyde treatment. Barone et al. 10 describe the immunoreactivity of the acidic and non-acidic glycosphingolipids integral to porcine heart valves as they are used for valve replacements. Liquid chromatography and mass spectrometry were applied to detect fucosylated and galactosylated glycosphingolipids, while structures were elucidated using accepted nomenclature. The authors deduced several structures that correspond to potentially antigen-bearing lipids and create a foundation to ask additional questions about immunoreactivity. The authors provide immunoblots to non-acidic lipids using human anti-aGal antibody and conclusively illustrate the reactivity of porcine valve glycolipids. The immune work presented supports the glycol structures suggested. It is interesting that Neu5Gc was not detected in the lipids tested and suggests that pigs engineered to be heart valve donors may only require removal of aGal expression but not deletion of the CMAH gene. The expression of other carbohydrates on proteins and lipids may yet still contribute to a significant immune response. Byrne et al. 11 have previously described the β 1,4 N-acetylgalactosaminyl transferase gene product as a target of baboon after xenotransplantation. Their most recent work characterized the gene and presented immune data to support their findings. Stably expressed B4GalNT2 gene in human embryonic kidney cells lead to increased human antibody and complement binding when measured by flow cytometry. The B4GalNT2 enzyme product expressed on these cells was also recognized by the DBA lectin and anti-Sda blood group antibody, known to contain the galactose β 1,4 N-acetylgalactosamine epitope. It will be compelling to see the impact of the B4GalNT2 gene knockout in pigs with and without the GGTA1 and CMAH genes deleted on the ability to bind naïve and seroconverted human and baboon antibodies. In a brief communication, Zhao et al. 12 overcome the limitations of immune-deficient mouse models of type 1 diabetes by inducing transplant tolerance to human islets in immune sufficient mice treated with anti-CD45RB (the B isoform of the CD45 receptor). Expressed on peripheral B cells, naïve T cells, thymocytes, and other antigen-presenting cells, CD45RB is critically involved in TCR and BCR signaling. Twenty-six percent of mice that received anti-CD45RB therapy prior to injection of human islets under the kidney capsule had no signs of rejection for the duration of the experiment. The addition of anti-CD20 antibody therapy removed all incidence of rejection. The authors suggest that early administration of the described antibody therapy may allow the long-term survival of xeno-islets likely due to the expansion and recruitment of FoxP3-positive cells to the site of transplantation. Weegman et al. 13 build on the development of pig islet production protocols with a critical analysis of the warm ischemia time (WIT) during isolation. From the time of cardiac death to pancreas cooling, the length of WIT is associated with reduced islet yields and poor function. The authors compared surface cooling with crushed ice versus an intravascular flush with cold solution. A statistically significant decrease in WIT was achieved when the intravascular flush included an intraductal infusion reducing the pancreas core temperature by 15–20 °C within 2 min. An approach incorporating genetically engineering pigs to extend islet xenograft function, blockade of the remaining recipient immune responses in addition to optimization of islet isolation techniques as described in this manuscript will undoubtedly be necessary for successful xenotransplantation. As researchers delete and add genes to the pig genome to modify the expression of xenoantigens, the most common method for assessing early success is antibody binding. The assays used vary from ELISAs to flow cytometry but have no standardization for quantifying bound antibody. Azimzadeh et al. 14 have developed a consensus protocol based on the common flow cytometric techniques of four research laboratories and defined commercially available antibodies. Application of this protocol will allow the research community to compare data and hopefully accelerate the field's assessment of pig organ donors to be used clinically. Despite advances in immune suppression, the presence of anti-HLA antibodies in transplant recipients is a potent indicator of antibody-mediated irreversible graft damage and cross-react with pig MHC class I molecules. Deletion of the pig MHC class I molecule will reduce human antibody binding and perhaps block CD8 T-cell activation. Reyes et al. 15 have targeted the MHC class I genes and pseudogenes resulting in undetectable MHC class I expression. In spite of reduced CD8 T cells, piglets appeared healthy. The authors point out the advantages of the CRISPR/cas9 technology and emphasize the usefulness of these pigs for the study of viral diseases and undoubtedly xenotransplantation. Similarly, Yao et al. 16 have used TALEN gene targeting tools to delete the GGTA1, Parkin, and DJ-1 genes while simultaneously inserting single-strand DNA into the cut site of the DJ-1 gene to develop a model to study Parkinson's disease. The authors confirm the frequency of gene deletions and allelic targeting with undetectable off target effects. It is likely that the deletion of these Parkinson's-related genes will create a model more similar to human disease progression. Reviewing the work of Reyes et al. and Yao et al. side-by-side suggests that both CRISPR and TALEN technologies result in similar efficiency of gene deletion, yet the authors suggest that TALENs may provide a significantly reduced likelihood of off target effects.