Transient-mixed Chimerism With Nonmyeloablative Conditioning Does Not Induce Liver Allograft Tolerance in Nonhuman Primates.

BACKGROUND: Although short-term outcomes for liver transplantation have improved, patient and graft survival are limited by infection, cancer, and other complications of immunosuppression. Rapid induction of tolerance after liver transplantation would decrease these complications, improving survival and quality of life. Tolerance to kidneys, but not thoracic organs or islets, has been achieved in nonhuman primates and humans through the induction of transient donor chimerism. Since the liver is considered to be tolerogenic, we tested the hypothesis that the renal transplant transient chimerism protocol would induce liver tolerance. METHODS: Seven cynomolgus macaques received immune conditioning followed by simultaneous donor bone marrow and liver transplantation. The more extensive liver surgery required minor adaptations of the kidney protocol to decrease complications. All immunosuppression was discontinued on postoperative day (POD) 28. Peripheral blood chimerism, recipient immune reconstitution, liver function tests, and graft survival were determined. RESULTS: The level and duration of chimerism in liver recipients were comparable to those previously reported in renal transplant recipients. However, unlike in the kidney model, the liver was rejected soon after immunosuppression withdrawal. Rejection was associated with proliferation of recipient CD8 T effector cells in the periphery and liver, increased serum interleukin (IL)-6 and IL-2, but peripheral regulatory T cell (Treg) numbers did not increase. Antidonor antibody was also detected. CONCLUSIONS: These data show the transient chimerism protocol does not induce tolerance to livers, likely due to greater CD8 T cell responses than in the kidney model. Successful tolerance induction may depend on greater control or deletion of CD8 T cells in this model.

Impact of CMV Reactivation, Treatment Approaches, and Immune Reconstitution in a Nonmyeloablative Tolerance Induction Protocol in Cynomolgus Macaques.

BACKGROUND: Cytomegalovirus (CMV) infection is a serious complication in immunosuppressed patients, specifically transplant recipients. Here, we describe the development and use of an assay to monitor the incidence and treatment of CMV viremia in a Cynomolgus macaque model of bone marrow transplantation (BMT) for tolerance induction. We address the correlation between the course of viremia and immune reconstitution. METHODS: Twenty-one animals received a nonmyeloablative conditioning regimen. Seven received cyclosporine A for 28 days and 14 received rapamycin. A CMV polymerase chain reaction assay was developed and run twice per week to monitor viremia. Nineteen recipients were CMV seropositive before BMT. Immune reconstitution was monitored through flow cytometry and CMV viremia was tracked via quantitative polymerase chain reaction. RESULTS: Recipients developed CMV viremia during the first month post-BMT. Two animals developed uncontrollable CMV disease. CMV reactivation occurred earlier in cyclosporine A-treated animals compared with those receiving rapamycin. Post-BMT, T-cell counts remained significantly lower compared with pretransplant levels until CMV reactivation, at which point they increased during the viremic phase and approached pretransplant levels 3 months post-BMT. Management of CMV required treatment before viremia reached 10 000 copies/mL; otherwise clinical symptoms were observed. High doses of ganciclovir resolved the viremia, which could subsequently be controlled with valganciclovir. CONCLUSIONS: We developed an assay to monitor CMV in Cynomolgus macaques. CMV reactivation occurred in 100% of seropositive animals in this model. Rapamycin delayed CMV reactivation and ganciclovir treatment was effective at high doses. As in humans, CD8 T cells proliferated during CMV viremia.

Characterization, biology, and expansion of regulatory T cells in the Cynomolgus macaque for preclinical studies.

Reliable in vitro expansion protocols of regulatory T cells (Tregs) are needed for clinical use. We studied the biology of Mauritian Cynomolgus macaque (MCM) Tregs and developed four in vitro Treg expansion protocols for translational studies. Tregs expanded 3000-fold when artificial antigen presenting cells (aAPCs) expressing human CD80, CD58 and CD32 were used throughout the culture. When donor peripheral blood mononuclear cells (PBMCs) were used as the single source of APCs followed by aAPCs, Tregs expanded 2000-fold. Tregs from all protocols suppressed the proliferation of anti-CD2CD3CD28 bead-stimulated autologous PBMCs albeit with different potencies, varying from 1:2-1:4 Treg:PBMC ratios, up to >1:32. Reculture of cryopreserved Tregs permitted reexpansion with improved suppressive activity. Occasionally, CD8 contamination was observed and resolved by resorting. Specificity studies showed greater suppression of stimulation by anti-CD2CD3CD28 beads of PBMCs from the same donor used for stimulation during the Treg cultures and of autologous cells than of third-party PBMC responders. Similar to humans, the Treg-specific demethylated region (TSDR) within the Foxp3 locus correlated with suppressive activity and expression of Foxp3. Contrary to humans, FoxP3 expression did not correlate with CD45RA or CD127 expression. In summary, we have characterized MCM Tregs and developed four Treg expansion protocols that can be used for preclinical applications.

RGDfK-Peptide Modified Alginate Scaffold for Cell Transplantation and Cardiac Neovascularization.

Cell implantation for tissue repair is a promising new therapeutic strategy. Although direct injection of cells into tissue is appealing, cell viability and retention are not very good. Cell engraftment and survival following implantation are dependent on a sufficient supply of oxygen and nutrients through functional microcirculation as well as a suitable local microenvironment for implanted cells. In this study, we describe the development of a porous, biocompatible, three-dimensional (3D) alginate scaffold covalently modified with the synthetic cyclic RGDfK (Arg-Gly-Asp-D-Phe-Lys) peptide. Cyclic RGDfK peptide is protease resistant, highly stable in aqueous solutions, and has high affinity for cellular integrins. Cyclic RGDfK-modified alginate scaffolds were generated using a novel silicone sheet sandwich technique in combination with freeze-gelation, resulting in highly porous nonimmunogenic scaffolds that promoted both human and rodent cell survival in vitro, and neoangiogenesis in vivo. Two months following implantation in abdominal rectus muscles in rats, cyclic RGDfK-modified scaffolds were fully populated by host cells, especially microvasculature without an overt immune response or fibrosis, whereas unmodified control scaffolds did not show cell ingrowth. Importantly, modified scaffolds that were seeded with human mesenchymal precursor cells and were patched to the epicardial surface of infarcted myocardium induced myocardial neoangiogenesis and significantly improved cardiac function. In summary, purified cyclic RGDfK peptide-modified 3D alginate scaffolds are biocompatible and nonimmunogenic, enhance cell viability, promote angiogenesis, and may be used as a means to deliver cells to myocardial infarct areas to improve neovascularization and cardiac function.

Novel H-shunt Venovenous Bypass for Liver Transplantation in Cynomolgus Macaques.

Cynomolgus monkeys are often used in preclinical transplantation research. Performing liver transplantation in cynomolgus monkeys is challenging because they poorly tolerate portal vein clamping during the anhepatic phase. Finding an alternative to portal vein clamping is necessary before preclinical liver transplant models can be performed with reliable outcomes. We used 3 different techniques to perform 5 liver transplants in male cynomolgus macaques (weight, 7.4-10.8 kg; mismatched for MHC I and II; matched for ABO). In procedure A, we clamped the portal vein briefly, as in human transplants, as well as the superior mesentery artery to minimize congestion at the expense of temporary ischemia (n = 2). In procedure B, we performed a temporary portocaval shunt with extracorporeal venovenous bypass (n = 1). For procedure C, we developed an H-shunt system (modified portocaval shunt) with extracorporeal bypass (n = 2). Postoperative immunosuppression comprised cyclosporine A, mycophenolate mofetil, and steroids. Recipients in procedure A developed hemodynamic instability and were euthanized within 2 d. The recipient that underwent procedure B was euthanized within 11 d due to inferior vena caval thrombosis. The H-shunt in procedure C led to minimal PV congestion during the anhepatic phase, and both recipients reached the 21-d survival endpoint with good graft function. Our novel H-shunt bypass system resulted in successful liver transplantation in cynomolgus macaques, with long-term posttransplant survival possible. This technical innovation makes possible the use of cynomolgus monkeys for preclinical liver transplant tolerance models.

Effect of Ex Vivo-Expanded Recipient Regulatory T Cells on Hematopoietic Chimerism and Kidney Allograft Tolerance Across MHC Barriers in Cynomolgus Macaques.

BACKGROUND: Infusion of recipient regulatory T (Treg) cells promotes durable mixed hematopoietic chimerism and allograft tolerance in mice receiving allogeneic bone marrow transplant (BMT) with minimal conditioning. We applied this strategy in a Cynomolgus macaque model. METHODS: CD4 CD25 Treg cells that were polyclonally expanded in culture were highly suppressive in vitro and maintained high expression of FoxP3. Eight monkeys underwent nonmyeloablative conditioning and major histocompatibility complex mismatched BMT with or without Treg cell infusion. Renal transplantation (from the same BMT donor) was performed 4 months post-BMT without immunosuppression to assess for robust donor-specific tolerance. RESULTS: Transient mixed chimerism, without significant T cell chimerism, was achieved in the animals that received BMT without Treg cells (N = 3). In contrast, 2 of 5 recipients of Treg cell BMT that were evaluable displayed chimerism in all lineages, including T cells, for up to 335 days post-BMT. Importantly, in the animal that survived long-term, greater than 90% of donor T cells were CD45RA CD31, suggesting they were new thymic emigrants. In this animal, the delayed (to 4 months) donor kidney graft was accepted more than 294 days without immunosuppression, whereas non-Treg cell BMT recipients rejected delayed donor kidneys within 3 to 4 weeks. Early CMV reactivation and treatment was associated with early failure of chimerism, regardless of Treg cell administration. CONCLUSIONS: Our studies provide proof-of-principle that, in the absence of early CMV reactivation (and BM-toxic antiviral therapy), cotransplantation of host Treg cell can promote prolonged and high levels of multilineage allogeneic chimerism and robust tolerance to the donor.

Optimization of alginate purification using polyvinylidene difluoride membrane filtration: Effects on immunogenicity and biocompatibility of three-dimensional alginate scaffolds.

Sodium alginate is an effective biomaterial for tissue engineering applications. Non-purified alginate is contaminated with protein, lipopolysaccharide, DNA, and RNA, which could elicit adverse immunological reactions. We developed a purification protocol to generate biocompatible alginate based on (a) activated charcoal treatment, (b) use of hydrophobic membrane filtration (we used hydrophobic polyvinylidene difluoride membranes to remove organic contaminants), (c) dialysis, and finally (d) ethanol precipitation. Using this approach, we could omit pre-treatment with chloroform and significantly reduce the quantities of reagents used. Purification resulted in reduction of residual protein by 70% down to 0.315 mg/g, DNA by 62% down to 1.28 µg/g, and RNA by 61% down to less than 10 µg/g, respectively. Lipopolysaccharide levels were reduced by >90% to less than 125 EU/g. Purified alginate did not induce splenocyte proliferation in vitro. Three-dimensional scaffolds generated from purified alginate did not elicit a significant foreign body reaction, fibrotic overgrowth, or macrophage infiltration 4 weeks after implantation. This study describes a simplified and economical alginate purification method that results in alginate purity, which meets clinically useful criteria.

Restimulation After Cryopreservation and Thawing Preserves the Phenotype and Function of Expanded Baboon Regulatory T Cells.

BACKGROUND: Regulatory T cells (Treg) are being explored for their tolerance-inducing capabilities. Freezing and banking Treg for future use makes this strategy more clinically applicable. We aimed to devise an improved method of expanding and cryopreserving Treg to maximize yield, purity, and function for use in xenotransplantation. METHODS: Baboon peripheral blood mononuclear cells (PBMC) were isolated from whole blood. CD4+/CD25hi cells were isolated by flow cytometric sorting and expanded for 26 days in culture with IL-2, anti-CD3 antibody, artificial APCs transfected with human CD58, CD32, and CD80, and rapamycin with weekly restimulations. Expanded Treg were frozen for 2 months then thawed and cultured for 48 hours in medium plus 1) no additives, 2) IL-2, 3) anti-CD3 antibody, 4) IL-2 + anti-CD3 antibody, and 5) IL-2 + anti-CD3 antibody + L cells. Phenotype and suppression were assessed after expansion, immediately after thawing, and after culturing. RESULTS: We expanded purified baboon Treg more than 10,000-fold. Expanded Treg exhibited excellent suppression in functional assays. Cryopreservation decreased suppressive function without changing phenotype, but increasing amounts of reactivation after thawing produced significantly better viability and suppressive function with a trend towards greater Treg purity. CONCLUSIONS: We produced numbers of expanded Tregs consistent with clinical use. In contrast to some previous reports, both Treg phenotype and suppressive function were preserved or even enhanced by increasing amounts of restimulation after thawing. Thus, banking of expanded recipient Tregs for in vivo infusion should be possible.

Downregulation of the CXC chemokine receptor 4/stromal cell-derived factor 1 pathway enhances myocardial neovascularization, cardiomyocyte survival, and functional recovery after myocardial infarction.

OBJECTIVES: Although adequate numbers of hematopoietic progenitor cells reside in the human bone marrow, the extent of endogenous neovascularization after myocardial infarction remains insufficient. The aim of this study was to identify the role of the CXC chemokine receptor 4/stromal cell-derived factor 1 axis in the mobilization and homing of hematopoietic progenitor cells in the ischemic heart. METHODS: Human bone marrow-derived hematopoietic progenitor cells or saline were injected systemically into athymic nude rats 48 hours after myocardial infarction. Myocardial and bone marrow expression of stromal cell-derived factor 1 and chemotaxis of hematopoietic progenitor cells were measured in vitro in the presence or absence of stromal cell-derived factor 1. The role of the CXC chemokine receptor 4/stromal cell-derived factor 1 axis was investigated by means of antibody blockade or systemic administration of granulocyte colony-stimulating factor. Morphologic analysis included measurement of the infarct area, capillary density, and apoptosis, whereas left ventricular function was measured by means of echocardiographic analysis. RESULTS: Expression of postinfarct stromal cell-derived factor 1 was increased by 67% in the bone marrow and decreased by 43% in myocardium. Disruption of bone marrow stromal cell-derived factor 1/CXC chemokine receptor 4 interactions by antibody blockade resulted in a redirection of human hematopoietic progenitor cells from the bone marrow to the ischemic heart and augmented neovascularization and cardiomyocyte survival. Similarly, systemic administration of granulocyte colony-stimulating factor to block CXC chemokine receptor 4/stromal cell-derived factor 1 interaction resulted in increased mobilization and homing of hematopoietic progenitor cells to the ischemic heart, which translated to augmented myocardial neovascularization, prevention of apoptosis, and improved cardiac function. CONCLUSIONS: Bone marrow stromal cell-derived factor 1 upregulation after myocardial ischemia prevents mobilization of endogenous hematopoietic progenitor cells. We provide evidence that disruption of stromal cell-derived factor 1/CXC chemokine receptor 4 interactions allows redirection of hematopoietic progenitor cells to ischemic myocardium and enhances recovery of left ventricular function.

Therapeutic effects of human STRO-3-selected mesenchymal precursor cells and their soluble factors in experimental myocardial ischemia.

Stromal precursor antigen (STRO)-3 has previously been shown to identify a subset of adult human bone marrow (BM)-derived mesenchymal lineage precursors, which may have cardioprotective potential. We sought to characterize STRO-3-immunoselected and culture-expanded mesenchymal precursor cells (MPCs) with respect to their biology and therapeutic potential in myocardial ischemia. Immunoselection of STRO-3(+) MPCs enriched for fibroblastic colony forming units from unfractionated BM mononuclear cells (MNCs). Compared to mesenchymal stem cells conventionally isolated by plastic adherence, MPCs demonstrated increased proliferative capacity during culture expansion, expressed higher levels of early 'stem cell' markers and various pro-angiogenic and cardioprotective cytokines, and exhibited greater trilineage developmental efficiency. Intramyocardial injection of MPCs into a rat model of myocardial infarction (MI) promoted left ventricular recovery and inhibited left ventricular dilatation. These beneficial effects were associated with cardioprotective and pro-angiogenic effects at the tissue level, despite poor engraftment of cells. Treatment of MI rats with MPC-conditioned medium (CM) preserved left ventricular function and dimensions, reduced myocyte apoptosis and fibrosis, and augmented neovascularization, involving both resident vascular cells and circulating endothelial progenitor cells (EPCs). Profiling of CM revealed various cardioprotective and pro-angiogenic factors, which had biological activity in cultures of myocytes, tissue-resident vascular cells and EPCs. Prospective immunoselection of STRO-3(+) MPCs from BM MNCs conferred advantage in maintaining a population of immature MPCs during ex vivo expansion. Transplantation of culture-expanded MPCs into the post-MI heart resulted in therapeutic benefit, attributable at least in part to paracrine mechanisms of action. Thus, MPCs represent a promising therapy for myocardial ischemia.

Islet grafting and imaging in a bioengineered intramuscular space.

BACKGROUND: Because the hepatic portal system may not be the optimal site for islet transplantation, several extrahepatic sites have been studied. Here, we examine an intramuscular transplantation site, bioengineered to better support islet neovascularization, engraftment, and survival, and we demonstrate that at this novel site, grafted beta cell mass may be quantitated in a real-time noninvasive manner by positron emission tomography (PET) imaging. METHODS: Streptozotocin-induced rats were pretreated intramuscularly with a biocompatible angiogenic scaffold received syngeneic islet transplants 2 weeks later. The recipients were monitored serially by blood glucose and glucose tolerance measurements and by PET imaging of the transplant site with [11C] dihydrotetrabenazine. Parallel histopathologic evaluation of the grafts was performed using insulin staining and evaluation of microvasularity. RESULTS: Reversal of hyperglycemia by islet transplantation was most successful in recipients pretreated with bioscaffolds containing angiogenic factors when compared with those who received no bioscaffolds or bioscaffolds not treated with angiogenic factors. PET imaging with [11C] dihydrotetrabenazine, insulin staining, and microvascular density patterns were consistent with islet survival, increased levels of angiogenesis, and with reversal of hyperglycemia. CONCLUSIONS: Induction of increased neovascularization at an intramuscular site significantly improves islet transplant engraftment and survival compared with controls. The use of a nonhepatic transplant site may avoid intrahepatic complications and permit the use of PET imaging to measure and follow transplanted beta cell mass in real time. These findings have important implications for effective islet implantation outside of the liver and offer promising possibilities for improving islet survival, monitoring, and even prevention of islet loss.

Suppression of xenogeneic graft-versus-host disease by treatment with immunoglobulin-like transcript 3-Fc.

Allogeneic hematopoietic cell transplantation represents an important therapy for certain malignant and nonmalignant diseases. However, graft-versus-host disease (GVHD) is a major cause of mortality and morbidity. The search for agents that can efficiently suppress GVHD has been going on for more than half a century. GVHD is particularly strong in xenogeneic donor-recipient combinations, given the unlimited number of potentially immunogenic antigens donor lymphocytes encounter in the host. Using a hu-nonobese diabetic/severe combined immunodeficiency (hu-NOD/SCID) gamma-null model of xenogeneic GVHD, we have demonstrated that treatment with recombinant immunoglobulin-like transcript 3-Fc protein induces the differentiation of CD8(+) T suppressor cells and blocks the cellular and humoral arm of the GVH reaction.

Alloantibodies in heart transplantation.

The presence of complement fixing anti-human leukocyte antigen (HLA) antibodies in the circulation of organ transplant recipients may result in heart allograft rejection. Here, we assessed the clinical impact of pre- and post-transplantation allosensitization on long-term survival of heart allografts. Sequential samples of sera from heart allograft recipients were screened pretransplantation for panel reactive antibodies using the complement-dependent cytotoxicity test. Patients were monitored post-transplantation for donor specific anti-HLA class I and class II antibodies. Kaplan-Meier graft survival plots were generated to analyze the effect of anti-HLA antibodies on transplantation outcomes. Statistical analysis showed that the post-transplantation development of alloantibodies was a significant risk factor that was associated with low long-term survival rates; in contrast, recipients' gender, age, previous transplantations, and degree of HLA matching with the donor had no effect on long-term survival. The presence in pretransplantation sera of antibodies against more than 10% of the HLA reference panel (PRA >10%) was associated with AMR and with a relatively lower rate of graft survival after 1 year but did not affect 10-year survival. The present data underline the importance of monitoring the development of anti-HLA antibodies as a tool for early diagnosis and treatment of AMR.

A DNA enzyme against plasminogen activator inhibitor- type 1 (PAI-1) limits neointima formation after angioplasty in an obese diabetic rodent model.

We investigated whether targeted cleavage of PAI-1 mRNA might prevent post-angioplasty neointima formation in diabetic JCR:LA-cp/cp rats with naturally elevated PAI-1 levels. Catalytic DNA enzymes targeting rat PAI-1 mRNA (PAI-1 DNA enzyme, n = 12) or a random sequence as control (scrambled DNA enzyme, n = 12) were infused at the site of arterial damage. Control animals demonstrated prominent PAI-1 protein expression in the arterial endothelium at 48 hours, and robust neointimal proliferation by two weeks, with 60 +/- 10% mean occlusion of the artery lumen. The neointimal lesion consisted of dense fibrin deposition and numerous proliferating smooth muscle cells, as determined by dual alpha-smooth muscle actin/Ki67 expression. Treatment with PAI-1 DNA enzyme resulted in marked early (48 hour) reduction of endothelial PAI-1 protein expression, which persisted for the next two weeks as well as a two fold reduction of expression of PAI-1 mRNA by RT-PCR at the same time point, (P < 0.05). By two weeks, PAI-1 DNA enzyme treated animals demonstrated significantly reduced levels of fibrin deposition and 5-fold lower levels of proliferating smooth muscle cells at the site of arterial injury compared to controls (P < 0.01), and a 2-fold lower neointima/media ratio (0.67 +/- 0.11 vs 1.39 +/- 0.12) (P < 0.05). Treatment with a catalytic PAI-1 DNA enzyme successfully prevents neointimal proliferation after balloon injury in diabetic animals.

Bartonella quintana prosthetic valve endocarditis detected by blood culture incubation beyond 10 days.

We describe the case of a 45-year-old man with mitral and aortic prosthetic valve replacement who presented with symptoms of subacute bacterial endocarditis. Bartonella quintana was grown from blood after prolonged culture. The course of the disease was complicated by splenic infarction, glomerulonephritis resulting in progressive renal insufficiency, and cerebroventricular hemorrhage. Notably, cardiac ultrasonography showed no extensive vegetations but a strand-like lesion. Culture-positive B. quintana prosthetic valve endocarditis in a formerly healthy subject represents a newly observed entity. It should be added to the differential diagnosis of prosthetic valve endocarditis, especially when it presents with features suggesting subacute bacterial endocarditis.

Mesenchymal lineage precursor cells induce vascular network formation in ischemic myocardium.

Mesenchymal lineage precursors can be reproducibly isolated from adult mammalian bone marrow and grown in culture. Immunoselection with monoclonal antibodies against STRO-1 and vascular-cell-adhesion molecule 1 (VCAM1/CD106) prior to expansion results in a 1,000-fold enrichment of mesenchymal precursors compared to standard isolation techniques. Intramyocardial injection of human STRO-1-selected precursors in an athymic rat model of acute myocardial infarction results in induction of vascular network formation and arteriogenesis coupled with global functional cardiac recovery.

Catalytic degradation of vitamin D up-regulated protein 1 mRNA enhances cardiomyocyte survival and prevents left ventricular remodeling after myocardial ischemia.

Vitamin D3 up-regulated protein 1 (VDUP1) is a key mediator of oxidative stress on various cellular processes via downstream effects on apoptosis signaling kinase 1 (ASK1) and p38 mitogen-activated protein kinase (MAPK). Here, we report that VDUP1 expression is significantly increased in rat hearts following acute myocardial ischemia, suggesting it may have important regulatory effects on cardiac physiological processes during periods of oxidative stress. Transfection of H9C2 cardiomyoblasts with a sequence-specific VDUP1 DNA enzyme to down-regulate VDUP1 mRNA expression significantly reduced apoptosis and enhanced cell survival under conditions of H(2)O(2) stress, and these effects involved inhibition of ASK1 activity. Direct intracardiac injection of the DNA enzyme at the time of acute myocardial infarction reduced myocardial VDUP1 mRNA expression and resulted in prolonged reduction in cardiomyocyte apoptosis and ASK1 activity. Moreover, down-regulation of VDUP1 was accompanied by significant reduction in cardiac expression of pro-collagen type I alpha2 mRNA level, as well as marked reduction in myocardial scar formation. These features were accompanied by significant improvement in cardiac function. Together, these results suggest a direct role for VDUP1 in the adverse effects of ischemia and oxidative stress on cardiomyocyte survival, left ventricular collagen deposition, and cardiac function. Strategies to inhibit VDUP1 expression and/or function during acute ischemic events may be beneficial to cardiac functional recovery and prevention of left ventricular remodeling.