Xenotransplantation 2014: 21: 301–305 doi: 10.1111/xen.12112
© 2014 John Wiley & Sons A/S XENOTRANSPLANTATION
Xenotransplantation literature update, March–April 2014 Burlak C, Taylor RT. Xenotransplantation literature update, March– April 2014. Xenotransplantation 2014: 21: 301–305. © 2014 John Wiley & Sons A/S.
Christopher Burlak1 and R. Travis Taylor2 1
Department of Surgery, Indiana University School of Medicine, Indianapolis, IN, 2Department of Medical Microbiology and Immunology, University of Toledo Medical Center, Toledo, OH, USA Key words: coagulation disorder – mesenchymal stem cells – pig – xenotransplantation Abbreviations: GTKO, a-galactosyl transferase knockout; aGal, galactose a-1,3 galactose; Neu5Gc, N-glycolylneuraminic acid; STZ, streptozotocin; NOD, non-obese diabetic; NOD-SCID, non-obese diabetic severe combined immunodeficiency; hPBMC, human peripheral blood monocytes; tDCs, tolerogenic-like dendritic cells; PERV, porcine endogenous retrovirus. Address reprint requests to Christopher Burlak, Department of Surgery, Indiana University School of Medicine, 635 Barnhill Dr., MS B-008, Indianapolis, IN, USA (E-mail: [email protected]
) Received 5 January 2014; Accepted 5 April 2014
During the period of March–April 2014, two reviews on xenotransplantation were published. The developed world has historically focused on pig islets, livers, and kidneys as a solution for the shortage of human organs. The developing countries, however, have a unique burden with 90% of the world’s blind (roughly 35.1 million people). Lamm et al.  outline the causes for corneal diseases that lead to blindness and describe the potential for pig corneal transplantation to help an estimated 4 million people. Readers are given an education in corneal structure as it relates to disease. Importantly, disease state alters the cornea and may indicate the appropriate surgical procedure; penetrating keratoplasty or endothelial keratoplasty. While prevention with anti-fungal, viral or bacterial agents early in disease is suggested, the existing population of the blind would beneﬁt from current research strategies using human embryonic stem cells, synthetic and biosynthetic corneas, amnionic membrane transplantation, and lastly xenotransplantation. The authors remind us that
corneal xenografts are non-vascularized and therefore do not suﬀer an acute anti-body mediated rejection as observed in vascularized grafts. Instead, the corneal endothelial cells appear to be the target of a delayed immune response in non-human primates. In conclusion, the application of genetically modiﬁed pig corneas with a decellularization protocol may provide a graft resistant to immune attack and require reduced immunosuppression. The last decade of research has revealed much of the immune mechanisms of xenograft rejection. Vadori and Cozzi discuss the advances in both humoral and adaptive immunity in a balanced and concise review . The authors skillfully build this review on the basics that every xenograft researcher should know about the immunology of xenotransplantation. The summary of the recent history of xenoantigens and immune cells important to xenotransplantation prepares the reader for a discussion of strategies to extend xenograft survival. After thought provoking summaries of complement regulation, neutralizing recipient antibodies and B- cell and T-cell tolerance induction, immunosuppression, and encapsulation barriers 301
Burlak and Taylor the authors conclude that the development of a genetically modiﬁed pig that addresses some of these issues in combination with optimized immunosuppression may allow xenotransplantation to proceed clinically in the near future. Immunology
Intraportal injection of islets could be a therapy for diabetics if we could better understand why 50–70% are immediately removed from the liver by the instant blood-mediated inﬂammatory reaction (IBMIR). Models using non-human primates suggest that once injected the pig islets induce complement activation that in some way leads to islet loss. Kang et al.  isolated adult wild-type pig islets and challenged them with human serum including various complement inhibitors. The authors conﬁrmed that islets induce complement ﬁxation in both classical and alternative pathways. However, the alternative pathway likely leads to islet cell damage. The authors then intraportally injected the adult pig islets into non-human primates and assessed the components of the alternative complement pathway in the presence and absence of complement inhibitors. As predicted from their in vitro data, intraportal islets were damaged by the alternative complement pathway and when blocked by cobra venom factor or factor H, saw a decrease in complement split products. Further supporting their hypothesis, cobra venom factor enabled longer islet survival time and C peptide production. The authors paint a clear picture of what pathways can be investigated further to maximize islet survival after intraportal injection. An immune privilege site is known to contain cells expressing the FAS ligand (FASL). It is thought the FASL interacts with incoming cellular immune responses and essentially “turns oﬀ” activation. Lee et al.  have used these observations combined with a tolerogenic-like dendritic cells (tDCs) to prevent destruction of pig ﬁbroblasts by human peripheral blood monocytes (hPBMCs) in vitro. Interestingly, FASL expressing cells in coculture for 3 days induced apoptosis in numerous cell types but to the greatest extent in CD4+ T cells. The authors explored the tDCs for expression changes of activation and immune receptors CD80, CD86, and MHC class I and II, as well as the production of several cytokines. tDCs in coculture suppressed the immune destruction of nonFASL expressing pig cells. However, when tDCs, FASL expressing cells and hPBMC were in coculture, no synergistic beneﬁt was observed. Unfortunately, cells bearing FASL also induced apoptosis in the tDCs negating a possible synergistic eﬀect. 302
The authors provide a thought provoking discussion of advantages and pitfalls of this potential therapy. The expression of immune regulatory molecules in the background of current genetically modiﬁed pigs may be necessary to enable longterm xenograft survival. Studies such as this are necessary to better understand the impact of immune receptor expression in pigs or pig cells. Activated platelets produce soluble CD154, which had been show to activate T cells and induce B cells to produce anti-body. Ezzelerab et al.  hypothesized that therapy with an anti-CD154 anti-body would reduce xeno-anti-body production. The authors used an arterial patch xenograft in non-human primates to tested the anti-CD154 therapy along side CTLA4-Ig and non-immunosuppressed animals. As predicted non-immunosuppressed subjects had abundant T- and B-cell responses and an increase in IgG and IgM production. The authors previous experience with CTLA4-Ig immune suppression was further supported when CTLA4-Ig therapy reduced T-cell responses but had little eﬀect on anti-pig IgG. Blocking CD154 successfully reduced anti-pig IgG production and reduced the development of germinal centers. The authors summarize their ﬁndings in an easy to follow ﬁgure that highlights the signiﬁcance of their ﬁndings. This work is important because, if accurate in humans, suggests that blocking sCD154 from perhaps activated platelets with concomitant costimulation blockade may have the beneﬁcial eﬀect of suppressing the production of graft-speciﬁc anti-bodies and preventing a robust T-cell response. Preclinical
As we push closer to testing xenografts clinically, several groups have begun to survey the attitude of populations that may take part in these trials. Rios et al.  have identiﬁed a growing population of Latin–American residents who have relocated to Spain. Using an anonymous and self-administered questionnaire, 1314 individuals responded with an impressive 89% completion rate. The authors summarize the ﬁndings in tables that separate the responders and their attitudes by many factors such as country of origin, age, and marital status. Not surprisingly those individuals who were not in favor of xenotransplantation were also against allotransplantation. In general, Spaniards have a more favorable opinion of xenografts than their new Latin–American neighbors. Choi et al.  have published a brief communication on their observations when using decellularized porcine corneal lamellae as a bridge to corneal
Xenotransplantation literature update allotransplantation in non-human primates. The authors ask whether decellularized pig corneas will sensitize patients to allografts through cross-reactivity to allo-antigens. Using established techniques to measure the immune response from ﬁve primates; the authors found no increase in panelreactive IgG or T-cell activation. This work continues to strengthen the case for corneal xenografts and therefore outcomes for long-term studies with select genetically modiﬁed pig corneas seem promising. As a natural extension of this work, Cohen et al.  examine the distribution of non-gal antigens in pig cornea. The authors validate their techniques by illustrating a lack of aGal in GTKO corneas and highlight the relative signiﬁcance of Neu5Gc. It appears that the epithelium and collagen in the stromal layer are the source of non-aGal antigens. The authors suggest that decellularization of the cornea from pigs deﬁcient in aGal and Neu5Gc will be necessary to create long lasting functional xenografts. Zoonosis
It is unclear if porcine endogenous retroviruses (PERV) are a barrier to xenotransplantation in humans using pig-derived organs. All domestic pigs are infected with PERV, and thus may represent a risk for human infections following transplantation. As a retrovirus, PERV genomic RNA is reverse transcribed into DNA and is subsequently integrated into host chromosomes in the form of proviruses. Any tissues coming from pigs therefore expose the organ recipient to a zoonotic virus. Three subtypes of PERV have been identiﬁed (A, B, and C). Of these, A and B are found in all pigs and can eﬃciently infect human cells in culture. PERV-C, while not found in all pigs, is a signiﬁcant concern as recombination with PERV-A can lead to PERV-A/C variants with increased infectivity over the individual wild-type viruses. Current research focuses on (i) improving screening techniques for all subtypes of PERV, (ii) understanding the general risk, tissue/organ distribution and cellular targets of PERV in humans, and (iii) identiﬁcation of host restriction factors and other potential inhibitors of PERV. Two articles in the current review period have addressed PERV screening. Gola and Mazurek reviewed the various methods currently employed to screen donor pigs and herds for PERV . As all pigs carry PERV-A/B, the ﬁrst priority is to identify donors with the lowest levels of integration. To prevent false negatives of PERV-C, the authors recommend using PCR and reverse trans-
criptase (RT)-PCR methods to examine both DNA and RNA genomic forms, respectively. In addition to PCR, infectivity assays can be employed using a coculture system of recipient cells and a highly susceptible cell line actively producing PERV. Rodrigues Costa et al.  employed this system to address whether human peripheral blood mononuclear cells (PBMC) can be infected with PERV. Despite previous reports of infection, the authors found that highly infectious PERV-A/C was unable to productively infect PBMC from supernatants. With the coculture system, however, the authors detected proviral DNA in PBMCs, suggesting a non-productive infection. They conclude that the extended exposure that occurs when coculturing infected/uninfected cells allow virus entry, but virus in cell-free supernatants is not capable of infecting PBMCs. To explain discrepancies with other studies in the literature, the authors suggest that high excess in input virus may lead to positive results. They support their observation with similar data from labs using virus-containing supernatant from pig cell lines. Regardless of whether cells are productively infected, the authors point out that PERV genetic material can be found in PBMCs and thus there is perhaps inherent risk of mutagenesis and chromosomal rearrangements following xenotransplantation with PERV-containing tissues. Natural host virus restriction may be employed to prevent virus infections. As with other retroviruses, PERV is sensitive to human restriction factors APOBEC3G and tetherin . During the current review period, Bae and Jung used molecular virology techniques to compare various tetherin proteins from a diﬀerent species . They conclude that all mammalian tetherins to varying degrees can block the release of PERV from infected cells. Canine and feline proteins were more eﬀective than murine and rhesus homologous. Importantly, human tetherin is induced by interferon treatment and eﬀectively prevented PERV release from a 293T cell line producing PERV. Transgenic expression of these factors may therefore be employed to prevent transmission of PERV during xenotransplantation. Source Animals
There is great potential for insulin-producing islets to treat type 1 diabetes if the problems of loss to immune assault and a suitable donor can be overcome. To that end, encapsulation of islets has proven an eﬀective barrier in both allo- and xenotransplantation studies. Safely et al.  seek to expand our pool of donors for insulin-producing 303
Burlak and Taylor islets to tilapia ﬁsh and examine their survival and function in the STZ-diabetic nude mouse model. The authors demonstrate an impressive 210 days of normalized blood glucose in immune-incompetent NOD-SCID mice. Diabetic NOD mice with an intact immune system functioned for approximately 11 days before rejection due to an apparent inﬁltration of innate and adaptive immune cells and corruption of the capsule with mouse IgG against tilapia islets. The authors then take full advantage of the beneﬁts of working in a mouse model by testing numerous immunosuppressive combinations with the greatest beneﬁt from CTL4Ig+ and anti-CD154 therapy containing therapies. Saﬂey et al. successfully demonstrate that encapsulated tilapia islets show tremendous potential as diabetes therapy if capsule integrity can be improved. Many of those who tout the beneﬁts of using pigs for their large litter size, social acceptance, and metabolic similarity to humans may see transgenic ﬁsh islets as a reasonable alternative. In addition to tilapia, Hani et al.  put forth evidence that goats (caprine) may serve as an additional source of islets. The authors immunosuppressed diabetic mice with thalidomide and transplanted islets under the kidney capsule. Impressively, blood glucose levels were normalized for up to 30 days by detectable production of goat insulin. The authors weren’t clear about the percentage of cells rejected after 30 days, but state that viable cells remained, of which a small percentage was insulin producing. This work creates a solid foundation upon which further studies into immunosuppression and immune responses to goat islets can be explored. Yi et al.  have used state-of-the-art techniques to deduce the pancreas proteome of the miniature pig. These authors have accomplished a very diﬃcult task as proteins and glycoproteins are exposed to the numerous degradative enzymes released during analysis. Another unique aspect of this analysis was the authors chose to stager the samples by development; 4 days, 19 days, and 14 months old pigs. Yi et al. are providing important information of pancreas development which may enhance our understanding or islet development or an as yet unknown temporal expression of xenoantigen. The quality of the two-dimensional analysis is excellent and only limited by existing databases of homologous proteins. The authors found 13 proteins that changed signiﬁcantly throughout development of which nine could be identiﬁed. This article is a proof of concept for Yi et al., and as they suggest, will be useful in further analysis of the miniature pig pancreas as a xenograft. 304
To compensate for the ethical and social dilemma of using human stem cells in research Dong et al.  have evaluated the inbred Wuzhishan miniature pigs as stem cell donors. Creating genetically modiﬁed pigs require cells that can be maintained in culture for long periods to allow for multiple genetic modiﬁcations and selection strategies. Dong et al. isolated two embryonic germ cell lines that were positive for four stem cell markers; Oct-4, SSEA-1, SSEA-3, SSEA-4 and production of alkaline phosphatase. The germ cells were stimulated in vitro with fetal serum and retinoic acid to form neural precursor, ﬁbroblast, and endoderm cells. The authors additionally showed the utility of the germ cells by microinjection and creation of chimeric pigs identiﬁed in part by skin pigmentation. Taken together, these authors have provided anyone interested in pig genetics with a very useful tool and is a very much appreciated leap forward in technology. References 1. LAMM V, HARA H, MAMMEN A, DHALIWAL D, COOPER DKC. Corneal blindness and xenotransplantation. Xenotransplantation 2014; 21: 99–114. 2. VADORI M, COZZI E. Immunological challenges and therapies in xenotransplantation. Cold Spring Harb Perspect Med Cold Spring Harbor Laboratory Press 2014; 4: a015578. 3. KANG HJ, LEE H, HA J-M et al. The role of the alternative complement pathway in early graft loss after intraportal porcine islet xenotransplantation. Transplantation 2014; 97: 999–1008. 4. LEE IK, SON YM, JU YJ et al. Survival of porcine ﬁbroblasts enhanced by human FasL and dexamethasone-treated human dendritic cells in vitro. Transpl Immunol 2014; 30: 99–106. 5. EZZELARAB MB, EKSER B, ISSE K et al. Increased soluble CD154 (CD40 ligand) levels in xenograft recipients correlate with the development of de novo anti-pig IgG antibodies. Transplantation 2014; 97: 502–508. -NAVAS AI, MARTINEZ-ALARCON L et al. A 6. RIOS A, LOPEZ study of the attitude of Latin-American residents in Spain toward organ xenotransplantation. Xenotransplantation 2013; 21: 149–161. 7. CHOI HJ, LEE JJ, KIM MK et al. Cross-reactivity between decellularized porcine corneal lamellae for corneal xenobridging and subsequent corneal allotransplants. Xenotransplantation 2013; 21: 115–123. 8. COHEN D, MIYAGAWA Y, MEHRA R et al. Distribution of non-gal antigens in pig cornea: relevance to corneal xenotransplantation. Cornea 2014; 33: 390–397. 9. GOLA J, MAZUREK U. Detection of porcine endogenous retrovirus in xenotransplantation. Reprod Biol 2014; 14: 68–73. € RR. 10. RODRIGUES COSTA M, FISCHER N, GULICH B, TONJES Comparison of porcine endogenous retroviruses infectious potential in supernatants of producer cells and in cocultures. Xenotransplantation 2014; 21: 162–173. 11. BAE EH, JUNG Y-T. Comparison of the eﬀects of retroviral restriction factors involved in resistance to porcine
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