Development of transplantable B-cell lymphomas in the MHC-defined miniature swine model.

BACKGROUND: Establishment of transplantable tumors in clinically relevant large animals allows translational studies of novel cancer therapeutics. METHODS: Here we describe the establishment, characterization, and serial transplantation of a naturally occurring B-cell lymphoma derived from a unique, highly inbred sub-line of Massachusetts General Hospital (MGH) major histocompatibility complex (MHC)-defined miniature swine. RESULTS: The lymphoblastic cell line (LCL) originated from peripheral blood of a 2.5 year old female swine leukocyte antigen (SLA)dd-inbred miniature swine breeder demonstrating clinical signs of malignancy. Flow cytometric phenotypic analysis of subclones derived from the original cell line revealed surface markers commonly expressed in a B-cell lineage neoplasm. A subclone of the original LCL was transplanted into mildly-conditioned histocompatible miniature swine and immunocompromised NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ (NSG) mice. Tissue and blood samples harvested 2 weeks following subcutaneous and intravenous injection in a highly inbred SLAdd pig were cultured for tumor growth and phenotypic analysis before serial transfer into NSG mice. Evidence of tumor growth in vivo was found in all tumor cell recipients. In vitro growth characteristics and surface phenotype were comparable between the original and serially transplanted tumor cell lines. CONCLUSIONS: These results indicate the feasibility of developing a large-animal transplantable tumor model using cells derived from spontaneously occurring hematologic malignancies within the highly inbred miniature swine herd.

Absence of interaction between porcine endogenous retrovirus and porcine cytomegalovirus in pig-to-baboon renal xenotransplantation in vivo.

BACKGROUND: Studies of xenotransplantation from swine have identified porcine viruses as potential barriers to clinical trials. The biology of these viruses has not been extensively investigated in the in vivo xeno-environment. Enhancement of viral gene expression by viral and cellular factors acting in trans has been demonstrated for certain viruses, including bidirectional interactions between human herpesviruses and endogenous (HERV) and exogenous (HIV) retroviruses. Both porcine cytomegalovirus (PCMV) and porcine endogenous retrovirus (PERV) infections have been identified in xenografts from swine. PERV receptors exist on human cells with productive infection in vitro in permissive human target cell lines. PCMV is largely species-specific with infection restricted to the xenograft in pig-to-baboon transplants. It is unknown whether coinfection by PCMV affects the replication of PERV within xenograft tissues which might have implications for the risk of retroviral infection in the human host. METHODS: A series of 11 functioning, life-supporting pig-to-baboon kidney xenografts from PERV-positive miniature swine were studied with and without PCMV co-infection. Frozen biopsy samples were analyzed using quantitative, real-time PCR with internal controls. RESULTS: PERV replication was not altered in the presence of PCMV coinfection (P = .70). The absence of variation with coinfection was confirmed when PERV quantitation was expressed relative to simultaneous cellular GAPDH levels with or without PCMV coinfection (P = .59). CONCLUSIONS: PCMV coinfection does not alter the replication of PERV in life-supporting renal xenotransplantation in vivo in baboons.

Discrepant serological assays for Pneumococcus in renal transplant recipients - a prospective study.

Vaccine immunoprotection for Streptococcus pneumoniae is mediated by opsonizing antibodies targeting serotype-specific capsular polysaccharides. Quantitative antibody levels enzyme-linked immunosorbent assay (ELISA) and antibody-mediated opsonophagocytic assays (OPA) measure vaccine-induced protection; correlation of these assays in transplantation requires investigation. This study examines the laboratory assessment of antibody titers in vaccinated renal recipients. Streptococcus pneumoniae 19A is common in immunocompromised hosts and is represented in protein-conjugate vaccines (PCV) and polysaccharide vaccines (PSV). Antibodies to 19A in serial sera from 30 vaccinated renal transplant recipients were compared using ELISA and OPA assays. Subject titers were classified as protected or not by ELISA (>0.35 µg/ml) and OPA titer (>1:8). Antibody titers analyzed using McNemar's test indicate that protection measured by the two assays are not the same (P = 0.0078); simple linear regression of within-subject geometric means of 19A enzyme-linked immunosorbent assay (ELISA) antibody levels versus 19A opsonophagocytic assays (OPA) titers demonstrates significant correlation between the two assays (P < 0.001). Vaccination is increasingly important given increasing antimicrobial resistance worldwide. OPA and ELISA antibody assays do not correlate well using current values for protective immunity against the Pneumococcus in immunosuppressed transplant recipients. Future studies of vaccination in transplant recipients should evaluate protective antibody levels using both functional antibody assays and standard ELISA antibody titers. (ClinicalTrials.gov: NCT00307125).

Genome-wide inactivation of porcine endogenous retroviruses (PERVs).

The shortage of organs for transplantation is a major barrier to the treatment of organ failure. Although porcine organs are considered promising, their use has been checked by concerns about the transmission of porcine endogenous retroviruses (PERVs) to humans. Here we describe the eradication of all PERVs in a porcine kidney epithelial cell line (PK15). We first determined the PK15 PERV copy number to be 62. Using CRISPR-Cas9, we disrupted all copies of the PERV pol gene and demonstrated a >1000-fold reduction in PERV transmission to human cells, using our engineered cells. Our study shows that CRISPR-Cas9 multiplexability can be as high as 62 and demonstrates the possibility that PERVs can be inactivated for clinical application of porcine-to-human xenotransplantation.

Porcine cytomegalovirus infection is associated with early rejection of kidney grafts in a pig to baboon xenotransplantation model.

BACKGROUND: Recent survivals of our pig-to-baboon kidney xenotransplants have been markedly shorter than the graft survivals we previously reported. The discovery of high levels of porcine cytomegalovirus (pCMV) in one of the rejected xenografts led us to evaluate whether this reduction in graft survival might be because of the inadvertent introduction of pCMV into our α1,3-galactosyltransferase gene knockout swine herd. METHODS: Archived frozen sections of xeno-kidney grafts over the past 10 years were analyzed for the presence of pCMV, using real-time polymerase chain reaction. Three prospective pig-to-baboon renal transplants using kidneys from swine delivered by cesarean section (C-section) and raised in isolation were likewise analyzed. RESULTS: Kidney grafts, from which 8 of the 18 archived samples were derived were found to be pCMV-negative, showed a mean graft survival of 48.3 days and were from transplants performed before 2008. None showed signs of disseminated intravascular coagulopathy and were lost because of proteinuria or infectious complications. In contrast, 10 of the archived samples were pCMV positive, were from kidney transplants with a mean graft survival of 14.1 days, had been performed after 2008, and demonstrated early vascular changes and decreased platelet counts. Three prospective xenografts from swine delivered by C-section were pCMV negative and survived an average of 53.0 days. CONCLUSIONS: Decreased survivals of α1,3-galactosyltransferase gene knockout renal xenografts in this laboratory correlate temporally with latent pCMV in the donor animals and pCMV in the rejected xeno-kidneys. Transmission of pCMV to swine offspring may be avoided by C-section delivery and scrupulous isolation of donor animals.

Results of life-supporting galactosyltransferase knockout kidneys in cynomolgus monkeys using two different sources of galactosyltransferase knockout Swine.

BACKGROUND: Various durations of survival have been observed in the xenotransplantation of life-supporting α-1,3-galactosyltransferase knockout (GalT-KO) porcine kidneys into nonhuman primates. Although others have demonstrated loss of GalT-KO-transplanted kidneys within 2 weeks, we have reported an average survival of 51 days with the cotransplantation of the kidney and vascularized thymus and an average of 29 days with the kidney alone. To determine the factors responsible for this difference in survival time, we performed xenogeneic kidney transplantations into cynomolgus monkeys with an anti-CD40L-based regimen using two different strains of GalT-KO swine, one derived from MGH miniature swine and the other obtained from Meji University. MATERIALS AND METHODS: Eight cynomolgus moneys received GalT-KO kidneys. Three kidney grafts were from Massachusetts General Hospital (MGH)-Nippon Institute for Biological Science (NIBS) GalT-KO pigs and five GalT-KO grafts were from MEIJI GalT-KO swine. All cynomolgus recipients were treated identically. RESULTS: Recipients of kidneys from the MGH GalT-KO kidneys swine, produced by nuclear transfer in Japan, survived an average of 28.7 days, whereas recipients of MEIJI GalT-KO kidneys swine survived an average of 9.2 days. Among the differences between these two groups, one potentially revealing disparity was that the MEIJI swine were positive for porcine cytomegalovirus, whereas the MGH-derived swine were negative. CONCLUSION: This is the first study comparing renal xenotransplantation from two different sources of GalT-KO swine into nonhuman primates at a single center. The results demonstrate that porcine cytomegalovirus may be responsible for early loss of GalT-KO swine kidney xenografts.

Absence of replication of porcine endogenous retrovirus and porcine lymphotropic herpesvirus type 1 with prolonged pig cell microchimerism after pig-to-baboon xenotransplantation.

Porcine endogenous retrovirus (PERV), porcine cytomegalovirus (PCMV), and porcine lymphotropic herpesvirus (PLHV) are common porcine viruses that may be activated with immunosuppression for xenotransplantation. Studies of viral replication or transmission are possible due to prolonged survival of xenografts in baboon recipients from human decay-accelerating factor transgenic or alpha-1,3-galactosyltransferase gene knockout 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 microchimerism (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.

Pre-screening of miniature swine may reduce the risk of transmitting human tropic recombinant porcine endogenous retroviruses.

BACKGROUND: It has been reported that peripheral blood mononuclear cells from miniature swine are capable of transmitting human tropic porcine endogenous retrovirus (PERV) recombinants to both human and pig cells. It has been suggested that these recombinants are exogenous and/or driven by one or more critical loci present in the pig genome. METHODS AND RESULTS: Genomic analysis of a miniature swine capable of transmitting human tropic replication competent (HTRC) recombinant PERV-A/C identified a PERV-C provirus in a region with homology to sequences located on chromosome 7. In "null" swine, incapable of in vitro transmission of PERV to human or pig cells, amplification using specific primers revealed that only two of five animals retained this locus in comparison to a total of five out of five transmitters (recombinant PERV-A/C transmission to both human and pig cells) and seven out of seven non-transmitters (replication of non-recombinant PERV in pig cells only). CONCLUSION: These data suggest that further analysis of these loci may provide a genetic basis for identifying pigs that are less likely to transmit human tropic PERV and would, therefore, be more suitable as source animals for human xenotransplantation.

Gene expression of porcine lymphotrophic herpesvirus-1 in miniature Swine with posttransplant lymphoproliferative disorder.

Porcine lymphotropic herpesvirus-1 (PLHV-1) is a gamma-herpesvirus related to Epstein-Barr virus (EBV) and associated with development of posttransplant lymphoproliferative disorder (PTLD) following allogeneic stem cell or spleen transplantation in miniature swine. Oligonucleotide microarrays were designed based on known open reading frames (ORFs) of PLHV-1. Expression was compared by cohybridization of cDNA from lymph nodes of PLHV-1+ swine after allogeneic spleen transplantation between either: 1) PTLD-affected and PTLD-unaffected swine; or 2) PTLD-affected swine vs. samples from the same animal prior to diagnosis. In PTLD-affected animals, consistent upregulation (nine ORFs) and downregulation (four ORFs) of PLHV-1 mRNA was observed in comparison to those without PTLD. No differences in gene expression were discovered at the time of clinical PTLD diagnosis compared to six to nine days prior to diagnosis in the same animals. This model provides insights into the pathogenesis of PTLD and, by extension, potential diagnostic and therapeutic tools for human EBV-associated PTLD.

Early weaning of piglets fails to exclude porcine lymphotropic herpesvirus.

BACKGROUND: Xenotransplantation using pigs as source species carries a risk for the activation of latent herpesviruses from the porcine donor and potential transmission to the recipient. In pig-to-baboon xenotransplantation, activation of porcine cytomegalovirus (PCMV) has been associated with xenograft injury and an increased incidence of consumptive coagulopathy and graft loss. Activation of porcine lymphotropic herpesvirus (PLHV)-1 was not observed in pig-to-baboon solid organ xenotransplantation, but was associated with a syndrome of post-transplantation lymphoproliferative disorder (PTLD) after allogeneic stem cell transplantation in pigs. MATERIAL AND METHODS: Early weaning of piglets was used to try to reduce the viral burden of xenograft donors. This consisted of separating the piglets of a litter from the sow within the first 2 weeks after birth and raising them in isolation from the remaining herd. RESULTS: We have previously demonstrated that PCMV could be excluded from source animals by early weaning of piglets. However, early weaning failed to exclude PLHV-1 from source pigs. CONCLUSIONS: This disparity between PCMV and PLHV-1 reflects differing pathogenesis of infection of these herpesviruses. New approaches will be needed to exclude PLHV-1 from pig colonies.

Reduction of consumptive coagulopathy using porcine cytomegalovirus-free cardiac porcine grafts in pig-to-primate xenotransplantation.

BACKGROUND: Xenotransplantation using pigs as the source species for organs carries a potential risk for transmission and activation of porcine herpesviruses. Activation of porcine cytomegalovirus (PCMV) in pig-to-baboon xenotransplantation is associated with xenograft injury and possibly an increased incidence of consumptive coagulopathy (CC). METHODS: To further investigate the role of PCMV activation in the occurrence of CC, a strategy to exclude PCMV from the donor was developed. To exclude PCMV, piglets were early-weaned and raised separated from other swine. These piglets were used as donors in an experimental protocol of pig-to-baboon heart xenotransplantation. RESULTS: Early weaning of piglets was successful in excluding PCMV. Use of PCMV-free cardiac porcine xenografts in baboons resulted in prolonged graft survival and prevented consumptive coagulopathy in all recipients. CONCLUSIONS: The use of PCMV-free cardiac grafts is beneficial in reducing the direct effects of PCMV activation in the graft (tissue damage) and the indirect effects of PCMV activation in the recipient (consumptive coagulopathy).

Mouse retrovirus mediates porcine endogenous retrovirus transmission into human cells in long-term human-porcine chimeric mice.

Porcine endogenous retrovirus (PERV) is a potential pathogen in clinical xenotransplantation; transmission of PERV in vivo has been suggested in murine xenotransplantation models. We analyzed the transmission of PERV to human cells in vivo using a model in which immunodeficient NOD/SCID transgenic mice were transplanted with porcine and human lymphohematopoietic tissues. Our results demonstrate, we believe for the first time, that human and pig cells can coexist long-term (up to 25 weeks) without direct PERV infection of human cells. Despite the transplantation of porcine cells that did not produce human-tropic PERV, human cells from the chimeric mice were frequently found to contain PERV sequences. However, this transmission was due to the pseudotyping of PERV-C (a virus without human tropism) by xenotropic murine leukemia virus, rather than to de novo generation of human-tropic PERV. Thus, pseudotyping might account for the PERV transmission previously observed in mice. The absence of direct human cell infection following long-term in vivo coexistence with large numbers of porcine cells provides encouragement regarding the potential safety of using pigs that do not produce human-tropic PERV as source animals for transplantation to humans.

Posttransplant lymphoproliferative disease after allogeneic transplantation of the spleen in miniature swine.

Spleen transplantation (SpTx) was performed in miniature swine across full major histocompatibility complex barriers to study the tolerogenic effect of the spleen. This study describes the development of posttransplant lymphoproliferative disease (PTLD) after allogeneic SpTx. Recipient pigs underwent whole body irradiation (100 cGy), thymic irradiation (700 cGy), and native splenectomy (day 0), and received a 45-day course of intravenous cyclosporine (trough level 400-800 ng/ml). After SpTx, two of seven pigs developed PTLD (1 donor-type, 1 host-type). These two pigs had greater T cell depletion and higher trough levels of cyclosporine. Early changes that occurred prior to the development of clinical features of PTLD were increased porcine lymphotropic herpesvirus-1 viral loads in blood and tissues, and increased numbers of leukocytes, B cells, and total serum IgM. PTLD can occur after allogeneic SpTx in swine. This model may be useful in studies of the pathogenesis of PTLD.

Reduced efficacy of ganciclovir against porcine and baboon cytomegalovirus in pig-to-baboon xenotransplantation.

In pig-to-baboon xenotransplantation, porcine cytomegalovirus (PCMV) causes viremia, consumptive coagulopathy, and tissue-invasive disease. Baboon cytomegalovirus (BCMV) is associated with invasive disease in xenograft recipients. The efficacy of prophylaxis with intravenous ganciclovir (GCV) was studied for prevention of PCMV and BCMV infections in pig-to baboon xenotransplantation. GCV prophylaxis did not alter the incidence of BCMV activation in recipients, but reduced the amount of virus in tissues (mean 8.38 x 10(2) vs. 3.24 x 10(5) copies/ micro g DNA without treatment) and prevented tissue-invasive infections. PCMV viral loads were unaltered by GCV prophylaxis (8.36 x 10(8) copies/ micro g DNA without prophylaxis vs. 1.20 x 10(9) copies/ micro g DNA with prophylaxis). In vitro, PCMV was relatively resistant to GCV [90% inhibitory concentration (IC90) of 10 micro m, IC50 = 3 micro m], acyclovir (100 micro m), and leflunomide (not achievable). Only cidofovir (IC90 1 micro m) and foscarnet (IC90 100 micro m) might have therapeutic efficacy for PCMV in vivo in achievable concentrations, although these agents often carry significant toxicity in transplant recipients. GCV has limited activity against BCMV and no therapeutic efficacy against PCMV at standard doses in vivo. GCV and other antiviral agents have limited activities against PCMV in vitro. Breeding of PCMV-free xenograft donors may be necessary to prevent PCMV infections in clinical trials.

Listeria monocytogenes infection in caspase-11-deficient mice.

Caspase-11 (Cas11) is a cysteine protease involved in programmed cell death and cytokine maturation. Through activation of Cas1 (interleukin-1beta [IL-1beta]-converting enzyme), Cas11 is directly involved in the maturation of IL-1beta 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-1beta levels were similar in Cas11(-/-) mice and controls. Endotoxin (lipopolysaccharide [LPS])-challenged Cas11(-/-) mice were deficient in the production of gamma interferon. IL-1beta 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-1beta were normal despite Cas11 deficiency. LPS-induced proinflammatory 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 inflammatory and apoptotic responses during infection with L. monocytogenes.

Pharmacokinetics of 18F-labeled trovafloxacin in normal and Escherichia coli-infected rats and rabbits studied with positron emission tomography.

OBJECTIVE: To measure tissue pharmacokinetics of trovafloxacin (CP 99,219) in normal and infected animals by both direct tissue radioactivity measurements and positron emission tomography (PET). METHODS: Concentrations of [18F]trovafloxacin were measured in normal and infected rats (n=6/group), at 10, 30, 60, and 120 min after injection, by radioactivity measurements. In normal rabbits (n=4) and rabbits with Escherichia coli thigh infection (n=4), tissue concentrations of drug were measured over 2 h with PET. After acquiring the final images, the rabbits were killed and tissue concentrations measured with PET were compared to the results of direct tissue radioactivity measurements. RESULTS: In both species, there was rapid distribution of [18F] trovafloxacin in most peripheral organs. Peak concentrations of more than five times the MIC90 of most Enterobacteriaceae and anaerobes (>100-fold for most organisms) were achieved in all tissues and remained above this level for >2 h. Particularly high peak concentrations were achieved in the kidney (>75 micro g/g), liver (>100 micro g/g), blood (>40 micro g/g), and lung (>10 micro g/g). Even though the concentration of trovafloxacin in infected muscle was reduced (p<0.01), the peak concentration was still >4 micro g/g and tissue levels remained above 2 micro g/g for more than 2 h. Due to the lower concentrations that were achieved in the brain (peak approximately 5 micro g/g), it is expected that trovafloxacin will have limited central nervous system toxicity. CONCLUSION: PET with [18F]trovafloxacin is a useful technique for non-invasive measurements of tissue pharmacokinetics.