Open ArchivePublished:July 03, 2014DOI:
        Dr Joren Madsen (Boston, Mass). I congratulate the speaker and the Society for recognizing the importance of this work. Enthusiasm in xenotransplantation has waxed and waned over the last 20 years. Dr Shumway used to say, half jokingly but half seriously, that xenotransplantation was the future of heart transplantation and always would be. However, these compelling new findings suggest that the future of xenotransplantation may be closer than we all thought. Indeed, achieving a greater than 500-day survival of a pig heart in a discordant baboon recipient sets a new standard for the field and allows us to begin thinking about human application again.
        My major concern in reading your article was your use of the heterotopic heart transplant model in which the heart graft is not life sustaining and graft survival times can be easily overestimated. However, your use of the innovative telemetry techniques to continuously monitor intracardiac pressures has tempered those concerns. I have 3 questions that relate to your recipients in group D.
        What was the specific immune or nonimmune mechanism that led to the loss of 2 of the 5 recipients in group D? Will additional immunotherapeutics or genetic modifications be required to achieve 100% survival?
        Dr Mohiuddin. The 2 animals that rejected in group D were explanted. One of them had long-term fever with no cause to be found, and because of strict animal care rules, we had to euthanize this animal. This animal showed no significant necropsy finding but had some inclusion CMV bodies in its testes. There was no evidence of any active CMV disease in this animal. The other animal that we explanted had repeated episodes of ventricular fibrillation, and we took the heart out to satisfy our Animal Care and Use Committee concerns.
        In both these hearts, we did see a lot of fibrosis and myocyte damage on histology, but there were areas preserved that had normal myocardium. It is hard to pinpoint the exact cause of rejection, and all these animals had high levels of antibodies against non-GAL antigens. It is possible that both the antibody and the TM played significant roles in the delayed rejection of these hearts. Additional expression of human genes to avert TM and careful adjustment of anti-CD40 dose may yield an even better survival.
        Dr Madsen. Many investigators believe that successful xenotransplantation will require the induction of immune tolerance. Do you believe or have any evidence that your protocol resulted in immune tolerance, and if so, would you consider weaning all drugs at a certain time point to limit the morbidity and mortality related to chronic immunosuppression?
        Dr Mohiuddin. In one of the animals, which has survived for approximately 600 days now, we have done some preliminary studies. We have done mixed lymphocyte reaction assays from the lymphocytes and saved cells from the donor pig, and we have seen that this animal was hyporesponsive to the donor-specific antigen. But when we tested responses against cells from a third-party pig, these cells were responsive. So there is an element of “tolerance” in there, but we need to do a lot more studies to evaluate that. We have already started to taper the immunosuppressive drugs in 2 of 3 surviving animals. In explanted grafts, we have found no evidence of chronic vasculopathy.
        Dr Madsen. The last question concerns infections. Other have found that reactivation of pig and even baboon CMV seems to have serious adverse effects in pig-to-baboon xenotransplantation. Given your exclusive use of SPF animals, are you concerned that the same excellent results may not be achieved in non-SPF animals or, for that matter, in humans?
        Dr Mohiuddin. We tested the levels of the non-Gal antibodies in SPF baboons, and they were found to be comparable to levels found in humans. As far as the antibody levels, we are satisfied with what we have, but I fully acknowledge your concern for CMV. Although we have not seen active CMV disease in any of our animals, the literature shows that this is a big concern, and we will have to keep a close eye on that.
        Dr Madsen. Thank you and congratulations.
        Dr R. Duane Davis (Durham, NC). This is an interesting series of experiments. Let me follow up a little bit on what Joren was asking in terms of the antibody-mediated rejection assessment on the organs that are still working. Have you been following the anti-swine antibody titers? You mentioned you started with low-titer baboons as recipients. How low were they? What has happened over the period of time in terms of the development of anti-pig, non-GAL antibodies?
        Dr Mohiuddin. As I mentioned earlier, the non-Gal antibody levels in SPF baboons are lower than in the conventional baboons and comparable to the levels in humans. The levels vary from baboon to baboon. We give 4 doses of rituximab that completely takes care of B cells, and these B cells do not return until day 60. After that period, we do see somewhat higher titers of antibodies than their baseline levels. In the earlier groups, we did find antibody deposition in the heart, but what role they are playing is not evident.
        Dr Davis. When you say by using that dose you are clearing all of the B cells, are you looking at the B1 cells in the peritoneal cavity, and along those lines are you looking at the central compartment? Obviously, you have plasma cells and some of the other long lines that antiCD20 does not actually address.
        Dr Mohiuddin. Exactly, that is our concern also, because CD20 does not take care of plasma cells, people have shown the role of bortezomib in eliminating plasma cells, which we have not tried. A single dose of rituximab can eliminate the peripheral circulating B cells. But what we have shown earlier, and Chris McGregor's group, is that you need at least 4 doses to completely wipe out B cells in all compartments.
        Dr Y. Joseph Woo (Philadelphia, Pa). Do the standard xenotransplantation models entail continuous heparin anticoagulation?
        Dr Mohiuddin. Yes, and we have seen a beneficial effect of it. We keep these animals on tether and continuous heparin infusion.
        Dr Woo. Among the prior models that yielded survival only up to 500 days, did these involve anticoagulation? How much is the anticoagulation benefiting your model?
        Dr Mohiuddin. We monitor their ACT and try to keep the level twice the level of the baseline number, and we maintain it throughout. Others have tried various forms of anticoagulation for variable periods of time; some of them have tried for a few months and others for only a few weeks. We are the only ones, I think, who have used it on a continuous basis.
        Dr Woo. So it may be contributing significantly?
        Dr Mohiuddin. Definitely.
        Dr Woo. In this heterotopic model, can the grafts undergo an endovascular endomyocardial biopsy?
        Dr Mohuddin. Yes, the histology results I showed in my presentation are the 1-year biopsy results from our long-term graft.
        Dr Woo. They weren't the final pathology?
        Dr Mohiuddin. The long-term graft results were biopsy results, but for the other groups, we have explanted the heart, and the results shown are from the final pathology.
        Dr Woo. Did you ever modify the anti-rejection regimen on the basis of biopsy findings?
        Dr Mohiuddin. Not really, because the major problem is the antibodies in xenotransplantation. Our main goal is to first tackle the antibody and the molecules that are incompatible with this model. We do initiate rescue therapy with steroids when we see onset of rejection, but this therapy has not helped us so far.