Person:

Fishman, Jay

Five patients with end-stage renal disease received combined bone marrow and kidney transplants from HLA single-haplotype mismatched living related donors, with the use of a nonmyeloablative preparative regimen. Transient chimerism and reversible capillary leak syndrome developed in all recipients. Irreversible humoral rejection occurred in one patient. In the other four recipients, it was possible to discontinue all immunosuppressive therapy 9 to 14 months after the transplantation, and renal function has remained stable for 2.0 to 5.3 years since transplantation. The T cells from these four recipients, tested in vitro, showed donor-specific unresponsiveness and in specimens from allograft biopsies, obtained after withdrawal of immunosuppressive therapy, there were high levels of P3 (FOXP3) messenger RNA (mRNA) but not granzyme B mRNA.

Background In December 2003 and April 2005, signs and symptoms suggestive of infection devel- oped in two groups of recipients of solid-organ transplants. Each cluster was inves- tigated because diagnostic evaluations were unrevealing, and in each a common do- nor was recognized. Methods We examined clinical specimens from the two donors and eight recipients, using viral culture, electron microscopy, serologic testing, molecular analysis, and histo- pathological examination with immunohistochemical staining to identify a cause. Epidemiologic investigations, including interviews, environmental assessments, and medical-record reviews, were performed to characterize clinical courses and to determine the cause of the illnesses. Results Laboratory testing revealed lymphocytic choriomeningitis virus (LCMV) in all the recipients, with a single, unique strain of LCMV identified in each cluster. In both investigations, LCMV could not be detected in the organ donor. In the 2005 cluster, the donor had had contact in her home with a pet hamster infected with an LCMV strain identical to that detected in the organ recipients; no source of LCMV infection was found in the 2003 cluster. The transplant recipients had abdominal pain, altered mental status, thrombocytopenia, elevated aminotransferase levels, coagulopathy, graft dysfunction, and either fever or leukocytosis within three weeks after transplan- tation. Diarrhea, peri-incisional rash, renal failure, and seizures were variably present. Seven of the eight recipients died, 9 to 76 days after transplantation. One recipient, who received ribavirin and reduced levels of immunosuppressive therapy, survived. Conclusions We document two clusters of LCMV infection transmitted through organ trans- plantation.

Porcine endogenous retrovirus (PERV), porcine cytomegalovirus (PCMV), and porcine lymphotropic her- pesvirus (PLHV) are common porcine viruses that may be activated with immunosuppression for xenotrans- plantation. Studies of viral replication or transmission are possible due to prolonged survival of xenografts in baboon recipients from human decay-accelerating factor transgenic or -1,3-galactosyltransferase gene knock- out miniature swine. Ten baboons underwent xenotransplantation with transgenic pig organs. Graft survival was 32 to 179 days. Recipient serial samples of peripheral blood mononuclear cells (PBMC) and plasma were analyzed for PCMV, PERV, and PLHV-1 nucleic acids and viral replication using quantitative PCR assays. The PBMC contained PERV proviral DNA in 10 animals, PLHV-1 DNA in 6, and PCMV in 2. PERV RNA was not detected in any PBMC or serum samples. Plasma PLHV-1 DNA was detected in one animal. Pig cell micro- chimerism (pig major histocompatibility complex class I and pig mitochondrial cytochrome c oxidase subunit II sequences) was present in all recipients with detectable PERV or PLHV-1 (85.5%). Productive infection of PERV or PLHV-1 could not be demonstrated. The PLHV-1 viral load did not increase in serum over time, despite prolonged graft survival and pig cell microchimerism. There was no association of viral loads with the nature of exogenous immune suppression. In conclusion, PERV provirus and PLHV-1 DNA were detected in baboons following porcine xenotransplantation. Viral detection appeared to be due to persistent pig cell microchimerism. There was no evidence of productive infection in recipient baboons for up to 6 months of xenograft function.

The replication of porcine endogenous retrovirus (PERV) in human cell lines suggests a potential infectious risk in xenotransplantation. PERV isolated from human cells following cocultivation with porcine peripheral blood mononuclear cells is a recombinant of PERV-A and PERV-C. We describe two different recombinant PERV-AC sequences in the cellular DNA of some transmitting miniature swine. This is the first evidence of PERV-AC recombinant virus in porcine genomic DNA that may have resulted from autoinfection following exogenous viral recombination. Infectious risk in xenotransplantation will be defined by the activity of PERV loci in vivo.

Clinical transplantation for the treatment of end-stage organ disease is limited by a shortage of donor organs. Successful xenotransplantation could immediately overcome this limitation. The development of homozygous α1,3-galactosyltransferase knockout (GalT-KO) pigs removed hyperacute rejection as the major immunologic hurdle to xenotransplantation. Nevertheless, GalT- KO organs stimulate robust immunologic responses that are not prevented by immunosuppressive drugs. Murine studies show that recipient thymopoiesis in thymic xenografts induces xenotolerance. We transplanted life-supporting composite thymokidneys prepared in GalT-KO miniature swine to baboons in an attempt to induce tolerance in a pre-clinical xenotransplant model. Here, we report the results of 7 xenogenic thymokidney transplants using a steroid-free immunosuppressive regimen that eliminated whole body irradiation in all but 1 recipient. The regimen resulted in average recipient survival of over 50 days. This was associated with donor- specific unresponsiveness in vitro and early baboon thymopoiesis in the porcine thymus tissue of these grafts, suggesting the development of T cell tolerance. The kidney grafts had no signs of cellular infiltration or deposition of IgG, and no grafts were lost due to rejection. These results show that xenogeneic thymus transplantation can support early human thymopoiesis, which in turn may induce T cell tolerance to solid organ xenografts.

Viral infections including those due to cytomegalovirus (CMV) have been associated with accelerated cardiac allograft vasculopathy (CAV) in clinical trials and some animal models. Evidence demonstrating a direct causal relationship between such infections and de novo formation of coronary vascular lesions is lacking. Heterotopic murine cardiac transplants were performed in a parental to F1 combination in animals lacking both T- and B-lymphocytes (RAG−/−). Coronary vasculopathy developed almost exclusively in the presence of recipient infection with lymphocytic choriomeningitis virus (LCMV) but not in uninfected controls. This process was also dependent upon the presence of NK cells as depletion of NK cells abrogated the process. These data show that a viral infection in its native host, and not previously implicated in the production of CAV, can contribute to the development of advanced coronary vascular lesions in cardiac allotransplants in mice. These data also suggest that virus-induced CAV can develop via an NK-cell dependent pathway in the absence of T- and B-lymphocytes.

Xenotransplantation is one promising approach to bridge the gap between available human cells, tissues, and organs and the needs of patients with diabetes or end-stage organ failure. Based on recent progress using genetically-modified source pigs, improving results with conventional and experimental immunosuppression, and expanded understanding of residual physiologic hurdles, xenotransplantation appears likely to be evaluated in clinical trials in the near future for some select applications. This review offers a comprehensive overview of known mechanisms of xenograft injury, a contemporary assessment of preclinical progress and residual barriers, and our opinions regarding where breakthroughs are likely to occur.

Xenotransplantation of porcine organs carries the risk of reactivation of latent virus in donor and recipient tissues as well as transmission of viruses between species. We have investigated the activation of baboon cytomegalovirus (BCMV) and porcine CMV (PCMV) in a pig-to-primate model of xenotransplantation. Tissues originating from a series of six swine-to-baboon composite thymokidney xenotransplants were inves- tigated. Four immunosuppressed baboons died (survival range, 7 to 27 days) with the graft in situ. Increases in BCMV DNA copy numbers occurred in three (75%) of these baboons and was thought to be responsible for pneumonitis and the death of one animal. In two baboons, disseminated intravascular coagulation was successfully treated by graftectomy and discontinuation of immunosuppression. PCMV was upregulated in five of six xenografts (83%). PCMV infection was associated with ureteric necrosis in one xenograft. Although significantly increased in native tissues, low levels of BCMV and PCMV were also detected in tissues other than that of the native viral host species. The cross-species presence of CMV did not appear to cause clinical or histological signs of invasive disease. Thus, viral infections with clinical disease were restricted to tissues of the native species of each virus. Intensive immune suppression currently required for xenotransplantation results in a significant risk of reactivation of latent infections by BCMV and PCMV. It is not yet known whether viral DNA detected across species lines represents cellular microchimerism, ongoing viral infection, or uptake of free virus. The observation of graft injury by PCMV demonstrates that CMV will be an important pathogen in immunosuppressed xenograft recipients. Strategies must be developed to exclude CMV from porcine organ donors.

A quantitative-competitive PCR for the quantification of porcine cytomegalovirus (PCMV) was developed. The virus was detected in a variety of pig organs (including potential xenotransplant donations), with viral loads ranging from <10 to 97 genome copies/ g of DNA. This assay will have significant utility for studying the activation and replication of PCMV and in swine models for allo- and xenotransplantation.

Caspase-11 (Cas11) is a cysteine protease involved in programmed cell death and cytokine maturation. Through activation of Cas1 (interleukin-1 [IL-1 ]-converting enzyme), Cas11 is directly involved in the maturation of IL-1 and IL-18. Apoptosis is mediated through Cas3. Given the role of apoptosis and cytokine signaling during the innate immune response in intracellular infection, we examined Cas11-deficient (Cas11 / ) mice during infection with Listeria monocytogenes. Cas11 / and wild-type C57BL/6 mice were equally susceptible to intravenous infection with L. monocytogenes, resulting in similar bacterial burdens in tissue and similar survival rates. By contrast, enhanced susceptibility was observed in control mice on a mixed genetic 129/C57BL/DBA2 background. Cas11 / and wild-type mice infected with Listeria had similar hepatic microabscess formation in terms of histologic appearance, size, and number. Apoptosis of L. monocytogenes- infected hepatocytes in vivo and in vitro in primary culture was not altered by the absence of Cas11. Serum IL-18 and IL-1 levels were similar in Cas11 / mice and controls. Endotoxin (lipopolysaccharide [LPS])- challenged Cas11 / mice were deficient in the production of gamma interferon. IL-1 responses in Cas11 / were normal with intravenous administration of LPS but decreased with intraperitoneal administration. Our findings suggest that Cas11 deficiency does not impair the immune response to infection with L. monocytogenes. Apoptosis and maturation of IL-18 and IL-1 were normal despite Cas11 deficiency. LPS-induced proinflam- matory pathways are altered by the absence of Cas11. While Cas11-mediated Cas1 and Cas3 activation is crucial for cytokine maturation and apoptosis during inflammation, alternative pathways allow normal in- flammatory and apoptotic responses during infection with L. monocytogenes.