How does xenotransplantation occur

Regarding the first question, this area is currently being hotly debated. No one knows whether transplanting animal organs into humans can possibly result in the outbreak of an animal virus in the human species.

Noted authorities in virology are concerned about the possibility, but no one really knows if it is likely or not,and many scientists feel it is very unlikely. The National Institutes of Health is currently studying the subject and should issue guidelines or a report at some point. The chief concern is the use of primate organs, pig organs should pose much less of a threat. Regarding the animal "rights" concern: although small groups of people have annoyed serious researchers in this country and Europe for decades, this opposition is generally taken to be extroidinarily naive.

The fact is that our society values human life and we have customs and laws to protect humans. However, we eat animals for food and use their skins and other products for our shoes and warmth. It seems perfectly consistant with these customs and beliefs to use a pig liver to save a human's life.

The issue with primates is more heatedly debated since the intelligence level of some monkeys rivals that of some humans. However, primates are not ideal as donors anyway for other reasons including the small litter size, the long latency before pregnancy is possible, and the high frequency of viral infection among primates.

Thus, most xenotransplant researchers are concentrating on animals other than primates. Where can I learn more about Xenotransplants?

Kidney Transplantation: Past, Present, and Future. Frequently asked questions about xenotransplants.

What is a xenotransplant? What is a species? What research is being done in the area? Another study, conducted by the Mayo Clinic, will test pork slaughtering and processing workers to determine whether PoERV is transferred to people who have a history of extensive exposure to swine tissues and fluids.

At this time, there is no evidence that PoERV has been transmitted in vivo or that it poses a risk to humans; however, researchers are proceeding with caution to address these outstanding safety concerns. While governments of the United States, the United Kingdom, Spain and elsewhere share similar hopes and concerns regarding xenotransplantation, each is working independently to establish or revise guidelines regarding the regulation of xenotransplantation research.

In the U. The guidelines, shaped in part by public dialogue, include long-term monitoring of recipients and the establishment of a registry to archive patient records and donor samples. The various agencies of the U. Department of Health and Human Services, including the FDA, the Centers for Disease Control and Prevention and the National Institutes of Health, have encouraged open communication about xenotransplantation at public workshops for several years.

In the United States, several scientists who attended the public conference in January , "Developing U. Public Health Policy on Xenotransplantation," urged the FDA to ban cross-species transplantation research until ethical issues and health risks are resolved.

They specifically discussed the potential risk to public health from a viral transfer across species that could result in a new disease epidemic. In April , the FDA released a guidance document stating that any clinical protocols proposing the use of nonhuman primates should include sufficient clinical evidence addressing the risks of such use. At the time of the guidance document, the FDA felt that this evidence did not exist.

The FDA decided it was appropriate to allow well-defined and highly controlled clinical trials, which are proceeding at a cautious pace. Department of Health and Human Services is developing several important mechanisms to facilitate participation by the public, scientists and industry in the progress of xenotransplantation. Public Health Service is also planning to develop a national patient registry and a biological specimen repository for tissues.

The biotechnology industry continues to work closely with the government in the responsible development of regulations and guidelines on the appropriate safeguards for xenotransplantation. Spain and the UK announced their guidelines in June and July , respectively. People who receive organs, as well as their families and friends, will have to sign informed consent documents indicating that they understand and accept the medical risks and inconveniences involved in these early transplant procedures, such as potential for infections, life-long medical surveillance and potential media attention associated with xenotransplantation.

In January , the parliamentary assembly of the Council of Europe called for a worldwide moratorium on xenotransplantation, until technology is evaluated and guidelines are established. A month later, members established a Working Group for the purpose of drafting xenotransplantation guidelines. The guidelines include the following safeguards: the UK Secretaries of State for Health will review each application for a human trial for an organ transplant and make a decision based on advice from UKXIRA; the welfare of animals bred for human transplants will be protected; and the existing Advisory Committee on Dangerous Pathogens will advise on the risks of infection from different types of xenotransplantation therapies proposed.

In addition to the critically important potential public health issues in xenotransplantation, there are a number of ethical issues that should be addressed. These include: deciding upon the fairest way to allocate donor animal organs in a society where thousands of people die while waiting for a transplant; deciding whether or not persons who receive xenografts may be compelled to participate in long term follow-up programs because of the theoretical public health risk from endogenous viruses; developing a carefully constructed ethics concerning the creation and care of those animals that will be created to serve as donors; determining when and under what circumstances children and infants may be considered as recipients of xenografts; and studying the potential emotional impact on people of having had their lives prolonged with donor animal organs.

It would be naive to think that all these and other ethical issues will be resolved in advance of the technological readiness to attempt animal to human xenografts. However, it is crucial that those in the biotechnology industry who are working in this area help to initiate and sustain an ongoing public dialogue on these and related issues.

BIO is committed to assisting in this process. BIO supports a full and open debate about xenotransplantation to further understanding and broaden knowledge which will hopefully , offer innovative treatments for previously untreated conditions. Understandably, this subject raises concerns among many people.

BIO encourages a full and open discussion of this issue and welcomes the opportunity to address any concerns people have. BIO is committed to a responsible research program that is consistent with regulatory guidelines as well as with the recommendations of ethics advisory boards within both industry and government. Our colleagues at EuropaBIO have made the same pledge helping to ensure international consistency.

BIO also is working with the FDA to ensure that appropriate safeguards exist for the proper use of this promising new medical treatment. A number of independent bodies across the world have considered the ethics of xenotransplantation and found it to be ethically acceptable.

When HAR, AHXR, and T-cell response are prevented, coagulation dysregulation becomes more obvious following xenograft transplantation and is considered another major barrier to prolonged xenograft survival in NHPs Coagulation dysregulation results in the development of thrombotic microangiopathy in the graft.

Features of thrombotic microangiopathy include fibrin deposition and platelet aggregation resulting in thrombosis within the vessels of the graft and eventual ischemic injury 77 , With the development of coagulation dysregulation, systemic consumptive coagulopathy may be observed in the recipient and lead to the recipient's death, but this phenotype does not occur in all xenograft organs Coagulation is a complex pathway that involves interactions with inflammation and innate immunity Normally, coagulation occurs continuously within the bloodstream but is restrained by anticoagulants, thus maintaining coagulation balance When endothelial cells are injured, tissue factor TF is liberated into circulation, triggering the extrinsic coagulation pathway.

Figure 3. The coagulation cascade related to xenotransplantation. A Coagulation cascade in primates. Black arrows designate cascade amplification steps. The coagulation cascade is initiated by tissue factor TF extrinsic pathway or negatively charged surface contact intrinsic pathway. TF is expressed by vascular subendothelial cells.

Factor Xa converts prothrombin to thrombin. Thrombin then cleaves fibrinogen into fibrin monomers and activates factor XIII, which cross-links fibrin monomers into an insoluble clot. In response to shear stress, von Willebrand Factor vWF binds to glycoprotein 1b GPIb on platelets leading to platelet activation and adhesion Activated platelet bind to fibrinogen to mediate platelet aggregation and endothelial adherence.

Red lines show the natural inhibitors of coagulation. These processes consequently prevent the formation of thrombin B Dysregulated coagulation in pig-to-primate xenotransplantation. Red and black arrows designate incompatibility between pig and primates.

When pig endothelium is activated, pig TF is expressed and released into the circulation. After interaction with the pig endothelium, recipient platelets and peripheral blood mononuclear cells PBMCs express primate tissue factor hTF. The porcine TF pTF pathway inhibitor is an ineffective inhibitor of the human Xa factor and may ineffectively shut down the activation of the major TF.

Pig TBM pTBM binds only weakly to primate thrombin, leading to levels of activated PC that are insufficient to inhibit coagulation, resulting in thrombotic microangiopathy in pig grafts within a matter of weeks Porcine vWF spontaneously could aggregate primate platelets through GPIb receptors even in the absence of shear stress Small vessels in the graft become occluded by fibrin and platelet aggregation. In the context of xenotransplantation, the assault by antibodies and complement-activated pig endothelial cells converts endothelial cells from an anticoagulant phenotype to a procoagulant state, leading to vascular destruction, and infiltration by various immune cells Both recipient- and donor-derived TF contribute to activation of the extrinsic coagulation cascade 85 , The molecular incompatibilities between primate and pig coagulation—anticoagulation systems exaggerate this process Figure 3B.

Porcine TBM also fails to regulate primate thrombin. Even in the absence of shear stress, pvWF spontaneously aggregates primate platelets through GPIb receptors Activated platelets develop thrombosis after being recruited to the place of the endothelial cells' injury, which leads to widespread activation of the coagulation system Above all, recent advances in the field of xenotransplantation have enabled a better understanding of the immune mechanisms underlying the failure of porcine xenografts.

It is vital for xenotransplantation be introduced into clinic. However, many molecular mechanisms underlying xenograft rejection needed further elucidation, especially in pig-to-NHP models. As a consequence, diverse strategies are required to overcome the various immunological barriers involved in the rejection of various forms of xenotransplantation procedures. According to studies on immunological rejection and coagulation dysregulation, plenty of genetically modified pigs were generated to bridge cross-species molecular incompatibilities.

Since , most of the advances that have been made in the field of xenotransplantation because of the production of genetically engineered pigs. In this section, we summarize current genetically modified pigs available for xenotransplantation Table 1. Table 1. Genetically modified pigs currently available for xenotransplantation research. Complement activation is a clearly detriment factor in contributing to xenograft failure.

One approach is administering an agent to inhibit complement, but such treatment only had a temporary effect and enhanced the risk of infection 15 , Another approach is engineering genetically modified pigs to overcome immunological rejection. Pigs possess complement regulatory proteins CRPs that are similar to those of humans, but pig CRPs are not sufficient to protect pig epithelium cells from human complement-mediated injury.

In the s, two independent research groups first proposed the suggestion that production of transgenic pigs expressing the human CRPs CD59 and CD55 to protect from hyperacute xenograft rejection.

These advances introduced the possibility of genetic modification of the organ-source pig for xenotransplantation. Today, many pigs expressing hCPRs have been produced [reviewed in ]. Researches have also demonstrated that expression of hCRPs can inhibit complement-mediated graft injury and prolong xenograft survival time , Furthermore, studies have also demonstrated that a combination of hCRPs offers greater protection than the expression of just one hCRP , Rejection of anti-Gal antibodies can be prevented through plasmapheresis or using immunoaffinity columns However, these approaches have demonstrated only partial success because the graft is lost when antibody levels recover.

The production of GTKO donor pigs is a milestone of xenotransplantation field. These results suggest that the deletion of Neu5Gc epitope in pigs is crucial for increasing xenograft survival time.

In vitro evidence has also suggested that inactivation of the B4GalNT2 gene reduce human antibody binding 34 , Therefore, this animal is the preferred candidate model for evaluating the effect of the Neu5Gc deletion xenograft in NHP models. Although expression of hCRP alone does not enable make graft long-term survival, even in the GTKO pigs, the complement system is still activated by ischemia—reperfusion injury.

Herein, the deletion of identified xenoantigens with expression of one or more hCRPs in pigs would form the foundation for future clinical trial. In vitro , porcine cells expressing hCD47 can reduce their phagocytosis by human macrophages In vivo , hCD47 expression increased xenogeneic hematopoietic engraftment chimerism in the murine model and prolonged the survival porcine skin grafts in baboons These findings collectively suggest the beneficial role of hCD47 expression in xenografts.

However, hCD47 expression did not completely prevent phagocytosis from primate macrophages; therefore, the pathway of xenoantigen-activated macrophages may also need to be suppressed. Martin et al. The beneficial effect of hCTL4-Ig expression extended xenograft survival time in a rat skin transplantation model and a NHP neuronal transplantation model These in vivo evidence also suggested that the expression of hCTLA4-Ig alone could not prevent xenograft rejection, which is consistent with the result blocking costimulatory pathway against B7-CD28 only.

However, these pigs were susceptible to infection because of high levels of pCTLA4-Ig expression in the blood. Therefore, the expression of this agent only in specific target cells of the pig is favorable. However, the role of these modified genes in protecting xenograft from rejection response requires further evaluation in NHPs.

However, both thrombotic microangiopathy and systemic consumptive coagulopathy are increasingly recognized in xenograft and NHP recipients. Therefore, coagulation dysregulation becomes a non-negligible barrier to successful xenotransplantation. The graft vascular endothelial cells enter into a procoagulant state, which cannot be successfully controlled by the pig's anticoagulant factors, resulting in coagulation dysregulation and graft failure.

Transgenic expression of hTBM in donor pig is one of most important approaches to overcoming coagulopathy currently. Pig aortic endothelial cells expressing hTBM were reported to substantially suppress prothrombinase activity, delay human plasma clotting time, and exhibit less activity in inducing human platelet aggregation , In the pig-to-baboon model, hTBM expression on cardiac xenografts confers an independent protective effect for prolonging graft survival time , Another key player in the anticoagulation system is EPCR, which also mediates anti-inflammatory and cytoprotective signaling Therefore, it is speculated that overexpression of hEPCR in donor pigs is a potential solution to overcoming related barriers, providing potent local anti-inflammatory, anticoagulant, and cytoprotective cell signaling.

CD39 plays a key role in the regulation of coagulation. In addition, vWF-deficient donor pigs exhibited prolonged lung graft survival time in NHP models and caused a less substantial platelet decrease in receipts , Graft coagulation varies among different xenograft organs after transplantation, perhaps because of differences in vascular structure and protein expression pattern. Recently, considerable progress has been made in cardiac and renal xenotransplantation.

However, improvements have been limited in liver and lung xenotransplantation. After pig liver xenotransplantation, severe thrombocytopenia can occur within minutes to hours, which exacerbates coagulation dysfunction, resulting in lethal hemorrhage PvWF is a glycoprotein that plays a key role in the pathogenesis of xenograft failure, especially in pulmonary xenotransplantation, because the lung releases more vWF than the heart or kidneys Moreover, the transcription of genes involved in coagulation, fibrinolysis, and platelet function differs in heart and kidney xenografts, which may account for the different courses of coagulation dysregulation in the recipients of these organs Pulmonary xenografts release larger quantities of vWF than do heart and kidney xenografts These data collectively suggest that successful control of coagulation dysregulation in xenotransplantation may require different genetic and pharmacological strategies for different organs.

An increasing amount of evidence suggests that inflammatory response plays a considerable role in graft failure in cases of a condition called systemic inflammatory response in xenograft recipients Therefore, the engineering of donor pigs that express one or more human anti-inflammatory or antiapoptotic genes may be an approach to xenograft protection.

Transgenic pigs that express human hemeoxygenase-1 and human A20 are available , The expression of human hemeoxygenase-1 reportedly protected porcine kidneys from xenograft rejection in the case of ex vivo perfusion with human blood and transgenic porcine aortic endothelial cells However, several human transgenes, including hHO1, hCD47, and human A20, have been introduced in pigs with multiple genetic manipulations As discussed above, numbers of genes have been found to be involved in xenograft rejection.

Because of the immune response to a pig xenograft cannot be considered in isolation, successful control of immunological rejection in xenotransplantation requires the altering of multiple genes in donor pigs. Genetically modified pigs with multiple genes, with up to seven manipulations, have been produced. The in vivo evaluation of their individual specific benefits will be difficult, and it remains unknown whether the manipulation of so many genes in donor pigs has adverse effects.

Therefore, the optimization of combinations of modified genes in donor pigs and evaluation of these xenografts in NHP models are important in further studies. Xenotransplantation has a long history with a number of animal models, including mouse, rat, and NHP, and has been used to reveal the mechanisms of rejection responses , Old World NHPs are the preferred surrogate for humans in exploring the response to pig xenograft transplantation because of their immunological similarities to humans 6.

Today, the pig-to-NHP model is the standard model for testing the primate immune response to organs or tissue from genetically modified pigs and the effect of novel immunosuppressive regimens. It is considered the optimal testing ground for predicting human responses as the final step before a human clinical trial Two comprehensive reviews explored pig solid organ graft survival in an NHP until , More recently, several studies reported key advances in NHP models. In this section, we summarize the studies of solid organs in preclinical models in recent years Table 2.

Table 2. Best survival time of solid organ xenotransplantation from pigs to non-human primates. Most studies on pig heart transplantation in NHPs have been heterotopic. The survival time of the graft was only 4—6 h following transplant with a wild-type pig heart In , Mohiuddin et al.

They extended the longest survival for heterotopic cardiac xenografts to days. However, thrombotic microangiopathy was observed in the xenograft, and coagulation dysregulation is likely to be the major obstacle in achieving longer survival rates.

In their experiments, the longest survival time was extended to days with a median survival of days. Furthermore, none of the subjects experienced consumptive coagulopathy or thrombocytopenia. This study demonstrated the efficacy and safety of a CD40 antibody-based immunomodulatory regimen 2C10R4 in recipients and suggested the important role of hTBM in donor pigs. Although considerable progress has been made in non-life supporting heart xenotransplantation, the life-supporting heart xenotransplantation is still difficult in NHPs; moreover, it is also vital to justify the potential clinical application of heart xenotransplantation.

Until , the longest survival time of life-supporting heart transplantation in pig-to-NHP cases was only 57 days On the bases of previous studies, Langin et al.

In their protocol of heart xenotransplantation, two steps were crucial to prolong the survival of functional xenografts in baboons. First, non-ischemic porcine heart preservation was performed instead of cold static storage. Second, detrimental xenograft overgrowth was restricted by a drug called temsirolimus Table 2. The immunosuppression protocol used in this study seems to have been tolerated by the baboons because of no major immunosuppression-related infection observed.

Their encouraging data suggest that their method might be safe for use in humans, and their research constitutes vital progress toward making clinical heart xenotransplantation a reality. Although the kidney is transplanted as a vital organ, progress in the use of kidneys in pig-to-NHP models has been slower than that for the use of the heart. Before , life-sustaining pig kidney xenotransplantation was limited to only a few weeks on average, with the longest reported survival in pig-to-NHP models being 90 days [review in ].

Recipients with lower titers of antipig antibodies exhibited prolonged kidney xenograft survival more than days Compared with previous reports, features of consumptive coagulopathy and proteinuria were delayed for many months. Iwase et al. This study suggested that the anti-CD40mAb-based regimen was likely to be of equal benefit to anti-CD mAb; it also noted the potential beneficial effects of anti-inflammatory agent.

Two baboons died from infection rather than from immune rejection, and no features of consumptive coagulopathy were observed They suggested that the expression of EPCR is critical to prevent kidney xenograft from coagulation dysregulation.

The longest survival achieved in these recipients with functioning transplants was days However, analysis of xenografted kidneys revealed that antibody-meditated rejection and coagulation dysregulation are still the causes of graft failure. Additional deletion of xenoantigen and introduction of human anticoagulation gene would be required in kidney xenografts. More recently, Kim et al.

This is the first translation model of life-sustaining kidney xenotransplantation, achieving the longest survival time for pig kidney xenografts in NHP models to date. Whether additional modification such as SLA class II knockout is necessary for donor pigs requires further investigation. Another question in kidney xenograft is hypoalbuminemia, which developed from proteinuria and uniformly documented in the early studies.

However, only modest proteinuria without accompanying hypoalbuminemia has been observed in NHPs with pig kidney grafts recently.

More effective control of immunological rejection by genetically modified pig and immunosuppressive agents might be beneficial for this problem.

Pig liver xenotransplantation seems to be more difficult to perform compared with heart and kidney xenotransplantation. The relevant molecular mechanisms of xenogeneic rejection involved in liver xenografts are more complex. After liver xenotransplantation, thrombotic microangiopathy in the graft and systemic consumptive coagulopathy appear to be more severe after pig liver xenotransplantation The first report of pig liver orthotopic xenotransplantation to NHPs dates back to , at which time immunosuppression was limited and donor pigs were wild type, resulting in a maximum survival of only 3.

Livers from genetically modified pigs were transplanted into baboons, extending liver graft survival time up to 9 days. The limited survival time of liver xenograft was predominantly due to the development of a lethal coagulopathy.

Recently, the survival time for life-supporting orthotopic GTKO pig liver xenografts was extended to 25 and 29 days with hepatic function in baboons, which represents the longest survival time following pig-to-primate liver xenotransplantation to date , In their modified experimental protocol, the addition of a costimulation blockade agent, anti-CD40 mAb, was ostensibly critical to prolonging liver survival.

Moreover, baboons were treated using a continuous infusion of human prothrombin concentrate complex, an exogenous human coagulation factor, to prevent coagulation dysregulation and allow spontaneous platelet count recovery. The pig lung is the organ most severely damaged by rapid coagulation dysfunction Most research on lung transplantation has employed ex vivo pig lung xenoperfusion with human donor blood models , but this model is limited to only short-term effects, usually those occurring within 4 h Lung xenotransplantation research has begun to use pig-to-NHP models.

Nguyen et al. However, the xenografts were functional for only 3. Recently, Watanabe et al. These studies have demonstrated that the introduction of hCD47 can mitigate acute vascular rejection of lung xenografts and prolong porcine lung transplant survival time in NHP models. However, the limited survival time suggests the necessity of additional strategies in lung xenotransplantation. Considering the above-mentioned achievements, heart and the kidney may be the first two solid organs to be used for clinical xenotransplantation.

In addition, a new problem of rapid and detrimental growth of xenografts after transplantation has emerged.

This problem has been observed in both heart and kidney xenotransplantation , The combination approaches of therapeutic reduction in blood pressure, reduction in corticosteroid dose, and administration of the mTOR inhibitor appeared to successfully prevent this problem.

Moreover, the mechanisms of appropriate xenograft size require further study. The miniature pig as the donor animal may be necessary for the solid xenograft. Although some progress has been made in lung and liver xenotransplantation, survival time is limited, and preclinical results suggest that new genetic engineering and immunosuppression strategies must be developed before considering a transition to clinical trials.

A major concern in the field of xenotransplantation is the transmission of porcine pathogens to human recipients. Most porcine viruses, bacteria, and fungi can be eliminated by the selection of negative donor animals, breeding in sterile and isolated conditions, early weaning, and embryo transfer , However, such strategies are impossible in the case of porcine endogenous retroviruses PERVs because PERVs are integrated into the porcine genome with multiple copies and the number of PERV proviruses varies among pig breeds and organs, ranging from 1 to more than No consensus has yet been reached regarding whether it is necessary to guarantee PERV inactivation in donor pigs by genetic manipulation , PERV transmission of pig-to-human and human-to-human cells was detected in several in vitro studies , However, the infection was only observed in certain types of cells, as PERVs are unable to infect certain cell types because of the absence of a functional receptor on most cell surfaces To the best of our knowledge, PERV transmission has not yet been reported in preclinical pig-to-NHP models or in clinical cells transplantations to humans If necessary, PERV inactivation can also be accomplished by genetically engineering pig donors.

In , Yang et al. The engineered cells reduced PERV transmission to human cells in vitro In , the same group inactivated all PERVs in a porcine primary cell line and generated healthy PERV-inactivated pigs through somatic cell nuclear transfer These pigs provide a new strategy that eliminates the potential risk of PERVs in xenotransplantation.

However, the susceptibility of these pigs to reinfection by PERV remains unclear. Godehardt et al. However, these results were obtained through a monitoring period only up to 55 days, the reinfectivity remains a concern, and the persistent information and observation in PERV-inactivated pigs are necessary. With recent achievements and the accumulation of experience with xenotransplantation in preclinical research, the first-in-human clinical trial may be possible in the near future.

It is an inevitable trend that pigs modified with multiple genes are to be used as donor animals for xenotransplantation. New gene-editing technologies enable the production of multiple genetically engineered pigs in shorter periods of time and with greater efficiency. Various types of gene-modified pigs already exist, most of which are being tested in preclinical pig-to-NHP xenotransplantation models. In addition, new xenoreactive antigens are continually being discovered , , from which new knockout and transgenic pigs may be generated.

Although assessment of current genetically modified pigs combined with immunosuppressive therapy in NHP models is complex and expensive, we agree with the opinion that substantial results should be obtain in NHP models before clinic application.

Data and experience based on studies with NHP models suggest that combining various genetically modified pigs with different immunosuppressive therapies is necessary for the effective transplantation of different organs. Therefore, determining the optimal genetically engineered organ-source pig and immunosuppressive regimen strategy in pig-to-NHP models is a key step toward further clinical study. Questions regarding the regulatory challenges and ethical concerns regarding clinical xenotransplantation are being asked worldwide.

Scientists suggest that national regulatory authorities worldwide should reconsider guidelines and regulations regarding xenotransplantation so as to better enable design and safe conduction of informative clinical trials of xenotransplantation when supported by preclinical data The current research makes some progress in meeting the criteria outlined by the recommendations of the International Society for Heart and Lung Transplantation published in However, it is unclear which regulatory agencies consider current evidence to be sufficient for moving forward with clinical xenotransplantation.

An alternative potential approach that could alleviate the current shortage of human organs for transplantation is to create human—animal chimeras through various techniques, including stem cell biotechnology, regenerative medicine, and blastocyst complementation ,