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Kidney transplantation can significantly enhance living standards for people suffering from end-stage renal disease. A significant factor that affects graft survival time (the time until the transplant fails and the patient requires another transplant) for kidney transplantation is the compatibility of the Human Leukocyte Antigens (HLAs) between the donor and recipient. In this paper, we propose 4 new biologically-relevant feature representations for incorporating HLA information into machine learning-based survival analysis algorithms. We evaluate our proposed HLA feature representations on a database of over 100,000 transplants and find that they improve prediction accuracy by about 1%, modest at the patient level but potentially significant at a societal level. Accurate prediction of survival times can improve transplant survival outcomes, enabling better allocation of donors to recipients and reducing the number of re-transplants due to graft failure with poorly matched donors., Extended version of AIME 2021 conference paper

A growing body of evidence suggests that postural orthostatic tachycardia syndrome (POTS) may be an autoimmune disorder. We have reported in a previous manuscript that 89% of POTS patients (n = 55) had elevations in G-protein-coupled adrenergic A1 receptor autoantibodies and 53% had elevations in muscarinic acetylcholine M4 receptor autoantibodies, as assessed by ELISA. Patients with autoimmune disorders have been reported with a variety of elevated cytokines and cytokines (such as rheumatoid arthritis), thus, we evaluated a limited number of cytokines/chemokines in POTS patients with elevated adrenergic and muscarinic receptor autoantibodies. We utilized the plasma of 34 patients from a previous study, all of the patients (100%) had autoantibodies against the A1 adrenergic receptor and 55.9% (19/34) had autoantibodies against the M4 muscarinic acetylcholine receptor. In particular, the plasma cytokine/chemokine levels were measured as biomarkers of inflammation by Quantibody® technology (Raybiotech, Peachtree Corners, GA, USA). We also evaluated the platelet dense granule numbers, as these patients frequently complain of symptoms related to platelet dysfunction. Patients were predominantly young females who displayed a multitude of co-morbidities but generally reported viral-like symptoms preceding episodes of syncope. Eighty five percent (29/34) had platelet storage pool deficiency. Patients had elevations in five of ten cytokine/chemokines biomarkers (IL1β, IL21, TNFα, INFγ, and CD30), whereas two biomarkers had decreased levels (CD40L and RANTES). Our observations demonstrate that POTS patients known to have autoantibodies against the G-protein-coupled adrenergic A1 receptor have abnormal plasma concentrations of inflammatory cytokines.

Background Renal artery stenosis is a common cause of renal ischemia, contributing to the development of chronic kidney disease. To investigate the role of local CD 40 expression in renal artery stenosis, Goldblatt 2‐kidney 1‐clip surgery was performed on hypertensive Dahl salt‐sensitive rats (S rats) and genetically modified S rats in which CD 40 function is abolished ( Cd40 mutant ). Methods and Results Four weeks following the 2‐kidney 1‐clip procedure, Cd40 mutant rats demonstrated significantly reduced blood pressure and renal fibrosis in the ischemic kidneys compared with S rat controls. Similarly, disruption of Cd40 resulted in reduced 24‐hour urinary protein excretion in Cd40 mutant rats versus S rat controls (46.2±1.9 versus 118.4±5.3 mg/24 h; P Cd40 mutant rats versus S rat controls ( P Cd40 mutant rats demonstrated a significant decrease in gene expression of the profibrotic mediator, plasminogen activator inhibitor‐1 ( P P P P PCR assay. Reciprocal renal transplantation documented that CD 40 exclusively expressed in the kidney contributes to ischemia‐induced renal fibrosis. Furthermore, human CD 40‐knockout proximal tubule epithelial cells suggested that suppression of CD 40 signaling significantly inhibited expression of proinflammatory and ‐fibrotic genes. Conclusions Taken together, our data suggest that activation of CD 40 induces a significant proinflammatory and ‐fibrotic response and represents an attractive therapeutic target for treatment of ischemic renal disease.

We examined what happened during a 6-year period to 1121 end-stage renal disease patients who registered with their willing/incompatible living donors for kidney exchanges with the Alliance for Paired Donation (APD). Of all patients, 65% were transplanted: 37% in kidney paired donation (APD-KPD, APD-other-KPD); 10% with compatible live donors (APD-LD); and 18% with deceased donors (APD-DD). The remaining patients were withdrawn (sick/died/others; 15%), or were still waiting (20%). For those patients with a cPRA 0-94%, 72% received a transplant. In contrast, only 49% of very highly sensitized (VHS; cPRA 95-100%) were transplanted. Of the VHS patients, 50% were transplanted by KPD/APD-LD while 50% benefited through prioritization of deceased donors in the modified kidney allocation system (KAS introduced in 2014). All APD transplanted groups had similar death-censored 4-year graft survivals as their relevant Organ Procurement and Transplantation Network (OPTN) groups. It is noteworthy that VHS graft and patient survival results were comparable to less sensitized and nonsensitized patients. All patients should be encouraged to search for compatible donors through different options. Expanding the donor pool through KPD and the new KAS of the OPTN increases the likelihood of transplantation for VHS patients.

T cells play a major role in the pathogenesis of type 1 diabetes, and there is great interest in developing curative immunotherapies targeting these cells. In this study, a monoclonal antibody (mAb) targeting the T cell receptor β-chain (TCRβ) was investigated for its ability to prevent and reverse disease in mouse models of diabetes.RIP-OVA(hi) (C57BL/6-Tg(Ins2-OVA)59Wehi/WehiJ) mice adoptively transferred with ovalbumin-specific T cells (an induced model of diabetes) and NOD mice (a spontaneous model of diabetes) were used to test anti-TCRβ mAb therapy as a means of preventing and reversing type 1 diabetes.A single dose of anti-TCRβ completely prevented disease in RIP-OVA(hi) mice without inducing the release of inflammatory cytokines. Transient anti-TCRβ therapy prevented diabetes in 90% of NOD mice and reversed the disease after its onset in 73% of NOD mice. Long after the remission of type 1 diabetes, the anti-TCRβ treated mice were able to reject BALB/c skin allografts with normal kinetics while maintaining normoglycaemia. Treatment did not cause significant reductions in lymphocyte numbers in the spleen or pancreatic lymph nodes, but did result in a decreased percentage of chemokine receptor 9 (CCR9) positive, CD8(+) T cells. Notably, anti-TCRβ therapy increased the expression of programmed death 1 (PD-1) on the surface of the T cells; PD-1 expression is important for maintaining anti-TCRβ-induced self-tolerance, as type 1 diabetes recurs in mice following a blockade of PD-1 signalling.Anti-TCRβ mAb is a safe and effective immunotherapy that results in reduced numbers of CCR9(+) T cells, an increased expression of PD-1 on T cells and the restoration of self-tolerance in NOD mice.

Memory T cells resist co-stimulatory blockade and present a unique therapeutic challenge in transplantation and autoimmune diseases. Herein, we determined whether memory T cells express less "tolerogenic" genes than naïve T cells to reinforce a proliferative response under the deprivation of co-stimulatory signals. The expression of ∼40 tolerogenic genes in memory and naïve CD4(+) T cells was thus assessed during an in vitro TCR stimulation without co-stimulation. Briefly, upon TCR stimulation with an anti-CD3 mAb alone, memory CD4(+) T cells exhibited more proliferation than naïve CD4(+) T cells. To our surprise, at 24h upon anti-CD3 mAb stimulation, memory CD4(+) T cells expressed more than a 5-fold higher level of the transcription factor Egr2 and a 20-fold higher level of the transmembrane E3 ubiquitin ligase GRAIL than those in naïve T cells. Hence, the high-level expression of tolerogenic genes, Egr2 and GRAIL, in memory CD4(+) T cells does not prevent cell proliferation. Importantly, anti-CD3 mAb-stimulated memory CD4(+) T cells expressed high protein/gene levels of phosphorylated STAT5, Nedd4, Bcl-2, and Bcl-XL. Therefore, co-stimulation-independent proliferation of memory CD4(+) T cells may be due to elevated expression of molecules that support cell proliferation and survival, but not lack of tolerogenic molecules.

Donor-specific alloantibodies (DSA) to HLA-DP may cause antibody-mediated rejection (AMR), especially in re-transplants. We describe the immunization history of a patient who received 3 kidney transplants; the 3rd kidney was completely matched except at DPA1 and DPB1. Prior to the 3rd transplant, single antigen bead analysis (SAB) showed DSA reactivity against DPA1 shared by the 1st and 3rd donors, but B and T flow crossmatch (FXM) results were negative. Within 11 days the 3rd transplant underwent acute C4d+ AMR which coincided with the presence of complement (C1q)-binding IgG1 DSA against donor DPA1 and DPB1. Using HLAMatchmaker and SAB, we provide evidence that eplet (epitope) spreading on DPA1 and eplet sharing on differing DPB1 alleles of the 1st and 3rd transplants was associated with AMR. Since weak DSA to DPA1/DPB1 may induce acute AMR with negative FXM, donor DPA1/DPB1 high resolution typing should be considered in sensitized patients with DP-directed DSA.

IL-21 is the most recently discovered common gamma-chain cytokine that promotes persistent T-cell responses in chronic infections, autoimmunity and cancer. However, the therapeutic potential of inhibiting the IL-21-BATF signaling axis, particularly in transplant rejection, remains unclear. We used heart transplant models to examine the effects of IL-21 blockade in prevention of chronic cardiac allograft vasculopathy (CAV) using genetic knock-out and therapeutic approaches. Both wild-type C57BL/6 and IL-21-/- strains acutely rejected Balb/c skin grafts and once immunized with this skin graft, rejected Balb/c heart allografts in an accelerated fashion. However, when transplanted with heart grafts from the class-II major histocompatibility complex mutant, B6bm12 mice; wild-type recipients developed CAV, while IL-21-/- recipients were protected, even at day 100 post-transplant. Similarly, BATF-/- recipients, lacking the transcription factor BATF responsible for IL-21 production, did not develop CAV in B6-bm12 heart allografts. Strikingly, in a transient treatment protocol, the development of CAV in wild-type recipients of B6-bm12 hearts allografts was blocked by the administration of IL-21 receptor fusion protein (R-Fc). Thus, we demonstrate that CAV is regulated at least in part by IL-21 signaling and its blockade by genetic approaches or therapy with IL-21R-Fc prevents CAV in mice.

Apart from clonal deletion of self-reactive T cells in the thymus, Tregs are the major regulators of immune responses in the periphery and maintain a state of self-tolerance free from autoimmune diseases. Due to their inherent suppressive function, Tregs are being explored for their therapeutic potential in preventing autoimmunity and improving survival of allografts. This review provides recent updates on Treg biology and their use in animal as well as clinical models of transplantation. We discuss potential problems that limit the widespread clinical application of Tregs and provide future perspectives on how to optimize their medical use. Special consideration is given to methods by which Tregs should be isolated and expanded in order to facilitate clinical therapies. We also focus on recent discussions of Treg stability and plasticity, with specific insights into preventing the loss of Treg suppression by allotransplant-mediated inflammation.

The molecular mechanism of the extrathymic generation of adaptive, or inducible, CD4+Foxp3+ regulatory T cells (iTregs) remains incompletely defined. We show that exposure of splenic CD4+CD25+Foxp3− cells to IL-2, but not other common γ-chain cytokines, resulted in Stat5 phosphorylation and induced Foxp3 expression in ∼10% of the cells. Thus, IL-2/Stat5 signaling may be critical for Foxp3 induction in peripheral CD4+CD25+Foxp3– iTreg precursors. In this study, to further define the role of IL-2 in the formation of iTreg precursors as well as their subsequent Foxp3 expression, we designed a two-step iTreg differentiation model. During the initial “conditioning” step, CD4+CD25−Foxp3− naive T cells were activated by TCR stimulation. Inhibition of IL-2 signaling via Jak3–Stat5 was required during this step to generate CD4+CD25+Foxp3− cells containing iTreg precursors. During the subsequent Foxp3-induction step driven by cytokines, IL-2 was the most potent cytokine to induce Foxp3 expression in these iTreg precursors. This two-step method generated a large number of iTregs with relatively stable expression of Foxp3, which were able to prevent CD4+CD45RBhigh cell–mediated colitis in Rag1−/− mice. In consideration of this information, whereas initial inhibition of IL-2 signaling upon T cell priming generates iTreg precursors, subsequent activation of IL-2 signaling in these precursors induces the expression of Foxp3. These findings advance the understanding of iTreg differentiation and may facilitate the therapeutic use of iTregs in immune disorders.

Summary Kidney transplantation is the treatment of choice for patients with end-stage renal disease. While living donors provide anywhere from a small to a large fraction of kidneys for transplantation in different countries, at least one-third of these donors are incompatible with their potential recipients. To overcome these challenges, kidney paired donation (KPD) programs have been established that organize donor exchanges to find matches among the pool of incompatible pairs. Each program has developed its own features to accommodate local needs. Reasons for participating in KPD include blood group incompatibility, sensitization of the recipient against the donor, and the potential for improvement in transplant quality (e.g., age difference or graft size), and tissue compatibility. KPD programs use sophisticated algorithms to find matches among the pool of donor-recipient pairs to create simultaneous 2-way, 3-way, or 4-way exchanges or more complex non-simultaneous chains of transplants. These KPD allocation systems should be medically sound and ethically acceptable according to the principles of equity, utility, and justice. The variety of possible exchanges provided by these algorithms allows for maximizing the number of transplants, increasing the quality of transplants, and accommodating patients who are difficult to match. In this review, we describe several examples of successful KPD programs with diverse organizational approaches. By highlighting the strategies used by these programs to meet the needs of their patient populations, we aim to inspire improvements in existing programs and to provide a framework for expanding KPD to better serve international transplant communities.

The human immune system is a myriad of diverse cellular populations, each contributing to maintaining an effective and optimal immune response against infectious agents. It is important to maintain a "self-check" in the immune system so that responses do not go haywire, leading to the development of autoimmune diseases. Regulatory/suppressor T (Treg) cells are a specialized subpopulation of T cells that suppress the activation, expansion, and function of other T cells, thereby maintaining homeostasis through a fine balance between reactivity to foreign and self antigens. Tregs are characterized by surface expression of interleukin (IL)-2 receptor alpha chain (CD25) and intracellular expression of forkhead box protein P3 (FoxP3). There are at least two important functional populations of Treg cells, namely natural Treg (nTreg), which are continuously derived from the thymus, and induced Treg (iTreg), which are converted from naive T cells. The development and function of both nTreg and iTreg cells are regulated by several factors, such as antigen T-cell receptor, co-stimulatory receptors (i.e., cytotoxic T lymphocyte-associated antigen, or CTLA-4), and cytokines (IL-2, IL-10, and tumor growth factor-beta, or TGF-beta). In addition, the TGF-beta inhibitor ALK5, retinoid acid, and rapamycin influence the expansion of nTreg cells and the conversion of iTreg cells in vitro and in vivo. The heightening of Treg expansion may be harnessed to therapeutic methods for the treatment of autoimmune diseases and the induction of transplantation tolerance.

Agonists of the type 1 sphingosine-1-phosphate receptor (S1P1) render lymphocytes unable to migrate along an S1P gradient, thereby causing sequestration in lymphoid organs. While the S1P agonist FTY720 prolongs the survival of organ allografts and blocks T-cell mediated autoimmune diseases in experimental models, it is a non-selective agonist of four out of five S1P receptors. A derivative compound, KRP-203, is a novel more selective agonist of the S1P1 receptor. In the present study, MRL/lpr mice were treated orally with KRP-203 or vehicle alone starting at 12 (prevention) or 16 (treatment) wks of age. Eighty percent of mice in the prevention group survived to 24 wks, compared to 50% of controls. There was less tubulointerstitial disease and 4- to 8- fold fewer infiltrating CD4+ and CD8+ T-cells (p

Maintaining T cell homeostasis is critical for normal immune response. Three sequential signals activate T cells, with signal 3 delivered by multiple cytokines that regulate cell proliferation, differentiation, and survival/death. Cytokines binding to their receptors engages two key molecular families, namely, Janus tyrosine kinases (Jaks) and signal transducers and activators of transcription (Stats). Among Stats, Stat3 is involved in the generation of T helper 17 (Th17) cells, regulation of dendritic cells, and acute inflammatory response. These aspects of Stat3 function are important for transplantation. We discuss Stat3's role in innate and adaptive immunity as well as its potential for therapeutic intervention.

Unloading of the rodent heart activates the fetal gene program, decreases peroxisome proliferator-activated receptor alpha (PPARalpha) and PPARalpha-regulated gene expression (MCAD), and induces cardiomyocyte atrophy. NF-kappaB regulates the fetal gene program and PPARalpha-regulated gene expression during cardiac hypertrophy and induces atrophy in skeletal muscle. Our objective was to test the hypothesis that NF-kappaB is the regulator for activation of the fetal gene program, for downregulation of PPARalpha and PPARalpha-regulated gene expression, and for cardiomyocyte atrophy in the heart subjected to mechanical unloading.Activation of the inhibitory kappa B kinase beta (IKKbeta)/NF-kappaB pathways were measured in the heterotopically transplanted rat heart using Western blotting of total and phospho-IKKbeta and using transcription factor ELISA's for the five members of the NF-kappaB family (p65 (Rel A), p105/p50, c-Rel, RelB, and p100/p52). In loss of function experiments, we transplanted hearts of p105/p50 knockout mice into wildtype mice and compared changes in gene expression and cardiomyocyte size with wildtype hearts transplanted into wildtype mice.Total and phospho-IKKbeta levels significantly increased in the transplanted heart seven days after surgery. The activation of IKKbeta was paralleled by increased DNA binding activity of p65 and p105/p50. Mechanical unloading induced myosin heavy chain beta expression and decreased cardiomyocyte size in hearts of both wildtype and p105/p050 knockout animals. In contrast, the downregulation of PPARalpha and MCAD was significantly attenuated or prevented in the hearts of p105/p50 knockout mice.The IKKbeta/p65/p50 pathway is activated in the unloaded rodent heart and a likely regulator for the downregulation of PPARalpha and PPARalpha-regulated gene expression.

High blood pressure is a common cause of chronic kidney disease. Because CD40, a member of the tumor necrosis factor receptor family, has been linked to the progression of kidney disease in ischemic nephropathy, we studied the role of Cd40 in the development of hypertensive renal disease. The Cd40 gene was mutated in the Dahl S genetically hypertensive rat with renal disease by targeted-gene disruption using zinc-finger nuclease technology. These rats were then given low (0.3%) and high (2%) salt diets and compared. The resultant Cd40 mutants had significantly reduced levels of both urinary protein excretion (41.8 ± 3.1 mg/24 h vs. 103.7 ± 4.3 mg/24 h) and plasma creatinine (0.36 ± 0.05 mg/dl vs. 1.15 ± 0.19 mg/dl), with significantly higher creatinine clearance compared with the control S rats (3.04 ± 0.48 ml/min vs. 0.93 ± 0.15 ml/min), indicating renoprotection was conferred by mutation of the Cd40 locus. Furthermore, the Cd40 mutants had a significant attenuation in renal fibrosis, which persisted on the high salt diet. However, there was no difference in systolic blood pressure between the control and Cd40 mutant rats. Thus, these data serve as the first evidence for a direct link between Cd40 and hypertensive nephropathy. Hence, renal fibrosis is one of the underlying mechanisms by which Cd40 plays a crucial role in the development of hypertensive renal disease.

Failure to convert computer-identified possible kidney paired donation (KPD) exchanges into transplants has prohibited KPD from reaching its full potential. This study analyzes the progress of exchanges in moving from "offers" to completed transplants. Offers were divided into individual segments called 1-way transplants in order to calculate success rates. From 2007 to 2014, the Alliance for Paired Donation performed 243 transplants, 31 in collaboration with other KPD registries and 194 independently. Sixty-one of 194 independent transplants (31.4%) occurred via cycles, while the remaining 133 (68.6%) resulted from nonsimultaneous extended altruistic donor (NEAD) chains. Thirteen of 35 (37.1%) NEAD chains with at least three NEAD segments accounted for 68% of chain transplants (8.6 tx/chain). The "offer" and 1-way success rates were 21.9 and 15.5%, respectively. Three reasons for failure were found that could be prospectively prevented by changes in protocol or software: positive laboratory crossmatch (28%), transplant center declined donor (17%) and pair transplanted outside APD (14%). Performing a root cause analysis on failures in moving from offer to transplant has allowed the APD to improve protocols and software. These changes have improved the success rate and the number of transplants performed per year.

INTRODUCTION AND OBJECTIVES: Non-simultaneous Extended Altruistic Donor (NEAD) chains and bridge donors (BD) are increasingly utilized in the field of Kidney Paired Donation (KPD), but remain controversial due to the possibility that such donors could renege on their promise to donate a kidney. The donor at the end of a NEAD chain is referred to as a BD, who is re-entered into the pool and trusted to donate after their incompatible recipient has received a transplant. This study evaluates how long bridge donors should wait. METHODS: Data from a single United States KPD matching program was analyzed. To understand the impact of a BD subsequently failing to donate, a theoretical model was created in which a wait time limit (WTL) was placed on BDs. If the WTL was reached, the bridge donor was directed to the deceased donor waitlist and the chain was ended. If the WTL was not reached before a donor reneged, the chain ended with no additional transplant to the deceased donor waitlist. RESULTS: A total of 90 BDs were used in 26 NEAD chains to complete 151 transplants. Nine BDs (10%) are currently suspended or withdrawn from the KPD pool. Though some of these donors may eventually donate, for the purpose of this study they were considered to have reneged. Of these 9 BD who appear to have reneged most were subsequently denied as donors for medical reasons; only 2 of 90 (2.2%) simply chose not to donate. No BD in a planned NEAD chain has failed to donate, so only BDs without initially identified potential recipients were included in the model (n1⁄461). The maximum number of transplants was achieved with a WTL of 365 days (Figure 1), which would have resulted in 152 transplants/6 reneged BDs. By comparison, an unlimited WTL resulted in 148 transplants/9 reneges, while not allowing BDs to wait (WTL1⁄40) would have achieved 89 transplants/0 reneges. The most significant gains in the number of transplants were seen with a WTL between 120 and 210 days (97 to 144 transplants). Four BDs reneged at 210 days, with an additional 2 reneging between 210 and 365 days. CONCLUSIONS: This data suggests that a reasonable BD WTL should be at least 7 months, but not more than 1 year. To date, most BD reneging is actually medical disqualification, rather than a choice by the donor not to donate.

Background In skeletal muscle, transcript levels of proteins regulating the ubiquitin proteasome system (UPS) increase with atrophy and decrease with hypertrophy. Whether the same is true for heart muscle is not known. Aim of the study We set out to characterize the transcriptional profile of regulators of the UPS during atrophy-, hypertrophy-, and hypoxia-induced remodeling of the heart. Methods and results Cardiac atrophy was induced by heterotopic transplantation of the rat heart. Left ventricular hypertrophy was induced by banding of the ascending aorta in rats. To study the effects of hypoxemia on the left ventricle, rats were exposed to hypobaric hypoxia. Transcript levels of six known regulators of the UPS, ubiquitin B (UbB), the ubiquitin conjugating enzymes UbcH2 and E2-14 kDa, the ubiquitin ligases Mafbx/Atrogin-1 and MuRF-1, and the proteasomal subunit PSMB4 were measured using quantitative RT-PCR. Unloading-induced atrophy increased mRNA levels of UbB and decreased levels of both ubiquitin ligases. Transcript levels of all UPS genes investigated increased in the hypertrophied and hypoxic heart (with the exception of E2-14 kDa). Conclusions Cardiac atrophy, hypertrophy, and hypoxemia all increase myocardial UbB expression, suggesting that UbB is a transcriptional marker for load-induced and hypoxia-mediated cardiac remodeling.

Background The addition of phosphorothioate (PS) groups to natural phosphodiester (PD) antisense oligodeoxynucleotides (oligo) prevents their in vivo hydrolysis by nucleases allowing an RNase-dependent elimination of targeted mRNA. To further improve oligo function 2'-methoxyethyl (ME) groups were attached to selected nucleotides at the 3'-end because ME groups block RNase activity. Methods/results ME modification of PS- or PD/PS-oligo targeting human intracellular adhesion molecule (ICAM)-1 mRNA significantly increased the degree and duration of the in vitro inhibitory effects without compromising selectivity and specificity. A 7-day intravenous or oral therapy with rat ME/PS-modified ICAM-1 antisense oligo extended the survivals of kidney allografts. In addition, ME/PS-modified ICAM-1 antisense oligo reduced ischemic-reperfusion injury in kidneys, as measured by glomerular filtration rate, creatinine levels, and infiltration with leukocytes. Finally, a 14-day treatment with cyclosporine (CsA)-induced nephrotoxicity in syngeneic kidney transplants correlated with both increased ICAM-1 protein expression and infiltration with leukocytes. Graft perfusion and treatment of recipients with ICAM-1 antisense ME/PS-oligo alleviated the nephrotoxic effect and decreased ICAM-1 expression and leukocyte infiltration. Conclusions ME/PS-modified ICAM-1 antisense oligo is very effective in inhibiting the ICAM-1-dependent mechanism of graft infiltration and tissue damage involved in allograft rejection, ischemic-reperfusion injury, and CsA-induced nephrotoxicity.

Binding of the P-, L-, and E-selectins to sialyl Lewis(x) (sLe(x)) retards circulating leukocytes, thereby facilitating their attachment to the blood vessels of allografts. Whether the selectin inhibitor bimosiamose (BIMO; C(46)H(54)O(16) . 0.25 H(2)O [867.4 molecular weight]) inhibits the rejection process of kidney allografts in a rat model was examined. Rat recipients acutely rejected kidney allografts at a mean survival time of 8.8 +/- 0.75 d. An intravenous 7-d infusion by osmotic pump of 2.5, 5, 10, or 20 mg/kg BIMO extended kidney allograft survival to 11.5 +/- 2.2 d (P0.03), 25.4 +/- 11.4 d (P0.006), 37.4 +/- 13.6 d (P0.001), and 39.8 +/- 34.5 d (P0.01), respectively. Combination of BIMO with cyclosporine produced synergistic interactions, as documented by the combination index (CI) values of 0.34 to 0.43 (CI1 is synergistic; CI = 1 is additive; and CI1 is antagonistic). Similarly, BIMO interacted synergistically with sirolimus (CI = 0.64) and FTY720 (CI = 0.22). While the mechanism of immunosuppression was being analyzed, decreased infiltration of CD4(+), CD8(+), and macrophages on day 7 after grafting was observed. Multiple cytokines were also expressed, including IL-1alpha, IL-1beta, IL-2, IL-4, IL-6, IL-10, IL-12, IL-18, TNF-alpha, and IFN-gamma in kidney allografts on days 3, 5, and 7 after grafting, as measured by a ribonuclease protection assay. Furthermore, at similar time points, BIMO treatment reduced intragraft expression of P-selectin glycoprotein ligand-1, CX(3)CL1, CCL19, CCL20, and CCL2. Thus, BIMO blocks allograft rejection by reduction of intragraft expression of cytokines and chemokines.

Background. ISIS 2302, an antisense oligonucleotide that inhibits the expression of human intercellular adhesion molecule (ICAM)-1, was evaluated in combination with a cyclosporine (CsA)-prednisone (Pred) regimen first in a phase I safety and pharmacokinetic study and then in a phase II assessment of prophylaxis of acute rejection episodes in deceased donor renal allografts. Methods. Both phase I and phase II trials were double-blinded and placebo-controlled, including 17 stable and 39 de novo patients, respectively, in time-lagged, ascending-dose regimens. Each study compared the outcomes of 8 alternate-day intravenous infusions of four ISIS 2302 dose levels (0.05, 0.5, 1.0, or 2.0 mg/kg) versus placebo (3:1 ratio). Patients were followed for 34 days (phase I) or 6 months (phase II). All transplant patients were followed for 3 years. Results. ISIS 2302 produced no evident toxicity; a significant, dose-related increase in activated partial thromboplastin time was accompanied by a trend toward a decreased platelet count. ISIS 2302 did not alter the pharmacokinetic behavior of CsA. At 6 months, the rates of acute rejection episodes were 38.1% in the ISIS 2302 group versus 20.0% in the placebo group. Three-year graft survivals were similar. The mean creatinine values at 1, 2, and 3 years for all ISIS dose groups combined versus placebo over 3 years showed no significant differences. Conclusions. ISIS 2302 did not evoke side-effects and produced slightly improved renal function. However, in this pilot study, it did not further reduce the rate of acute rejection episodes or increase graft survival compared to a concentration-controlled CsA-Pred regimen.

Regulation of T- and B-lymphocyte survival and death is crucial for maintaining immune homeostasis. Unresponsiveness to death signals can result in lymphoproliferative disorders including cancer and autoimmunity, whereas lymphocytes hypersensitive to such signals can be manifested as immunodeficiencies. Often within these cells, cytokines and their receptors regulate the critical balance between life and death. It is becoming ever more apparent that within these effector cascades, Janus tyrosine kinases (Jak) and signal transducers and activators of transcription (Stat) act as key regulatory components. Invaluable knowledge about Jaks and Stats has arisen from mice made genetically deficient in these mqlecules, tumor models, and proteomics/genomics, which has begun to define their role in survival versus apoptosis. These findings have also suggested how Jaks and Stats might be manipulated for therapeutic intervention in lymphoid-derived diseases. This review seeks to focus on the role of Jak tyrosine kinases and Stat transcription factors in mediating the lymphocyte life cycle.

Background— Mechanical unloading of the heart results in atrophic remodeling. In skeletal muscle, atrophy is associated with inactivation of the mammalian target of rapamycin (mTOR) pathway and upregulation of critical components of the ubiquitin proteosome proteolytic (UPP) pathway. The hypothesis is that mechanical unloading of the mammalian heart has differential effects on pathways of protein synthesis and degradation. Methods and Results— In a model of atrophic remodeling induced by heterotopic transplantation of the rat heart, we measured gene transcription, protein expression, polyubiquitin content, and regulators of the mTOR pathway at 2, 4, 7, and 28 days. In atrophic hearts, there was an increase in polyubiquitin content that peaked at 7 days and decreased by 28 days. Furthermore, gene and protein expression of UbcH2, a ubiquitin conjugating enzyme, was also increased early in the course of unloading. Transcript levels of TNF-α, a known regulator of UbcH2-dependent ubiquitin conjugating activity, were upregulated early and transiently in the atrophying rat heart. Unexpectedly, p70S6K and 4EBP1, downstream components of mTOR, were activated in atrophic rat heart. This activation was independent of Akt, a known upstream regulator of mTOR. Rapamycin treatment of the unloaded rat hearts inhibited the activation of p70S6K and 4EBP1 and subsequently augmented atrophy in these hearts compared with vehicle-treated, unloaded hearts. Conclusions— Atrophy of the heart, secondary to mechanical unloading, is associated with early activation of the UPP. The simultaneous activation of the mTOR pathway suggests active remodeling, involving both protein synthesis and degradation.

Background, Ten different highly polymorphic amino acids (AAs) are located in the al (α 1h ) and a2 (α 2h ) helical regions of the class I major histocompatibility complex RT1.A n rat alloantigen. We examined the potential of α 1h -RT1.A n versus α 2h -RT1.A n polymorphic AAs to induce accelerated rejection or tolerance of heart allografts. Methods. The allochimeric α 1h 52-90 n -RT1.A c and α 2h 148-179 n -RT1.A c cDNAs were produced by the substitution of nucleotides encoding recipient RT1.A c AAs for donor RT1.A n AAs. Allochimeric and wild-type (WT)-RT1.A n proteins were generated in an Escherichia coli expression system. Results. A single portal vein administration of 100 μg α 1h 52-90 n -RT1.A c protein in combination with a 7-day course of oral cyclosporine A (4 mg/kg) induced tolerance to Brown Norway (BN) (RT1 n ) heart allografts in PVG (RT1 c ) recipients more effectively than did WT-RT1.A n protein; α 2h 148-179 n -RT1.A c protein was ineffective. However, subcutaneous injection of 100 μg WT-RT1.A n (but neither α 1h 52-90 n -RT1.A c nor α 2h 148-179 n -RT1.A c ) protein induced accelerated rejection of BN heart allografts. Untreated PVG recipients of BN heart allografts displayed activation of both interleukin (IL)-2- and interferon-γ-producing T helper (Th) 1 cells and IL-4- and IL-10-producing Th2 cells on days 5, 7, and 14 postgrafting, as measured by an enzyme-linked immunospot assay. In contrast, in comparison with rejectors, tolerant recipients showed down-regulation of Th1 cells and up-regulation of Th2 cells on days 5, 7, 14, and 200 postgrafting. Histology of heart allografts showed that tolerant BN heart allografts had no evidence of acute or chronic rejection when examined on day 100 after transplantation. Conclusions. The poorly immunogenic α 1h 52-90 n -RT1.A c allochimeric protein induces tolerance by selective activation of regulatory Th2 cells.

Relatively slow hematopoietic recovery after isolated bone marrow (I-BM) engraftment is probably caused by a disrupted microenvironment of stromal and stem cells. Thus, we compared the kinetics of hematopoietic recovery of lethally irradiated rats that received I-BM versus vascularized BM (V-BM). Total body irradiated (TBI; 8 Gy) Lewis (LEW; RT11) rats were either injected IV with syngeneic sex-mismatched 80 × 106 I-BM or transplanted with 80 × 106 V-BM in orthotopic hind limb grafts. Ten days later, peripheral blood (PB) and mesenteric lymph nodes (MLN) of these recipients were examined for the presence of donor-derived hematopoietic cells with a panel of monoclonal antibodies by FACS. To detect male cells in sex-mismatched female recipients, PCR was performed using male Y chromosome primers. When examined in PB and MLN, recipients transplanted with V-BM displayed significantly faster recovery of leukocytes (CD43+), monocytes (CD14+), and T cells (CD5+) in comparison with I-BM recipients. In addition, only V-BM (but not I-BM) groups contained stroma-like male-positive cells in PB and MLN. Our results suggest that V-BM transplants provided superior hematopoietic recovery in comparison to I-BM transplants. We postulated that close proximity between stromal and stem cells in V-BM is essential for efficient repopulation with progenitors of different lines of leukocytes.

Signal transducer and activator of transcription (Stat)5a and Stat5b are critical for normal immune function. Progression of T cells through G1-S phase of cell cycle requires T cell receptor (TCR)- and/or cytokine-inducible tyrosine phosphorylation of Stat5a/b. Stat5a/b may also, in a cell-dependent manner, be constitutively or cytokine-inducibly phosphorylated on a Pro-Ser-Pro (PSP) motif located within the transcriptional activation domain. Phosphorylation of the PSP motif is needed for maximal transcriptional activation by Stat5, at least in certain promoter contexts. The basal and cytokine-inducible serine phosphorylation state of Stat5a/b has not been determined in T cells. Using primary human T cells and T lymphocytic cell lines coupled with novel phospho-specific antibodies to this conserved phosphoserine motif in Stat5a or Stat5b, we report that: Stat5a and Stat5b were unphosphorylated on the PSP motif under basal conditions and became markedly phosphorylated in response to several T cell growth factor stimuli, including interleukin (IL)-2, -7, -9, and -15 and phorbol ester 12-myristate 13-acetate but not TCR engagement; inducible Stat5a/b serine phosphorylation differed quantitatively and temporally; and Stat5a/b serine phosphorylation was, in contrast to inducible Stat3 serine phosphorylation, insensitive to inhibitors of mitogen-activated protein kinase, phosphatidylinositol-3 kinase, and mammalian target of rapamycin or deletion of Raf-A, -B, or -C by antisense oligonucleotides. We conclude that IL-2 family cytokines tightly control Stat5 serine phosphorylation through a kinase distinct from the Stat3 serine kinase.

Background. The various toxicities associated with the general immune suppression resulting from current clinical immunosuppressive therapies continue to plague transplant recipients as well as jeopardize allograft survival. Methods. The present study utilized allochimeric class I MHC antigens (α 1h u 70-77-RT1.A a ) bearing only four donor RT1.A u polymorphic amino acids (a.a.; His 70 , Val 73 , Asn 74 , and Asn 77 ) superimposed on the recipient RT1.A a background to induce transplantation tolerance in the rat cardiac transplant model. Results. Oral delivery of α 1h u 70-77-RT1.A a protein alone (days 0-6) induced tolerance, as evidenced by inhibition of both acute and chronic rejection processes. Delivery of α 1h u 70-77-RT1.A a with therapeutic doses of cyclosporine (CsA) also prevented chronic rejection, otherwise readily developed after treatment with CsA alone. A polymerase chain reaction (PCR)-based analysis showed that tolerant recipients had reduced numbers of interleukin (IL)-2/interferon (IFN)-γ-producing T helper (Th)1 cells and elevated numbers of IL-4/IL-10-producing Th2 cells. Adoptive transfer experiments revealed that potent regulatory T cells mediated tolerance. The same T cells displayed diminished T cell receptor (TCR)-driven signaling via extracellular regulated kinase, AP-1, and NF-κB. as well as the common γ-chain (γ c ) cytokine-receptor-induced signaling by Janus kinase 3 (Jak3)/stimulators and activators of transcription Stat/5 pathways. Tolerance induction was prevented in vivo by inhibition of signal 2 by CTL4Ig or of signal 3 by either rapamycin, which disrupts the mammalian target of rapamycin, or AG490, which inhibits Jak3. Finally, partial or complete tyrosine phosphorylation of Zap70 was observed in alloantigen-specific T cell clones in response to tolerogenic versus immunogenic peptides, respectively. Conclusions. Tolerance induction by allochimeric proteins is achieved by partial TCR activation in the presence of signals 2 and 3, resulting in a skewed Th2 phenotype.

Aim Our Kidney Paired Donation (KPD) program (called the Alliance for Paired Donation; APD) arranges exchanges among patients with incompatible live donors. We evaluated the sensitization of APD patients. Methods Since 2010, the APD registered 1122 patients and arranged 418 transplants (Tx). APD patients had 40% highly sensitized (HS; cPRA of 80–100%) and 30% very highly sensitized (VHS; cPRA of 95–100%) patients. Since Tx patients had only 22% HS with half of them VHS, there was an accumulation of HS/VHS patients. Results We examined the HLA sensitization pattern: >85% HS had IgG to HLA-A; >90% to -B, 60% to -C; 82% to -DR; 85% to -DQB, 49% to -DPB, and 30% to -DQA. Sensitization was lower in HS Tx patients (Fig. 1A): the median cPRA was reduced for each HLA locus in HS Tx patients compared to the waiting HS patients (all p Conclusions (1) APD accumulates HS/VHS patients; (2) cross-reactivities within CREG groups for class I and cross-reactive HLAs for class II contribute to sensitization; and (3) shared epitopes among HLAs are involved in the sensitization. Download : Download high-res image (172KB) Download : Download full-size image

BACKGROUND Clinical application of islet transplantation to treat type 1 diabetes has been limited by islet allograft destruction by both allogeneic and autoimmune diabetogenic T-cell responses. The current study aims at determining whether an anti-T-cell receptor (TCR) monoclonal antibody (mAb) has potential as a novel and potent induction immunotherapy for islet transplantation. METHODS We have investigated the therapeutic efficacy and mechanisms of action of anti-TCR therapy in four different murine models, which comprise either allo- or autoimmune responses alone or both together. RESULTS T-cell response to islet allografts was potently abrogated by a brief treatment with an anti-TCRβ mAb (clone H57-597), resulting in long-term survival of BALB/c islet allografts in streptozotocin-induced diabetic B6 mice. Moreover, transient anti-TCR treatment permanently prevented BALB/c skin allograft rejection on Rag1 B6 recipients that were reconstituted with Foxp3 cell-depleted B6 splenocytes, but did not impair the reconstituted cells' ability to reject the later transplanted C3H skin allografts (transplanted at 120 days after BALB/c skin grafting). Transient anti-TCR treatment was also able to completely prevent diabetes onset in NOD.SCID.γc mice that were transferred with lymphocytes from diabetic NOD mice. Next, transient anti-TCR treatment significantly prolonged the survival of transplanted BALB/c islets in overtly diabetic NOD mice, which comprise both allogeneic and autoimmune diabetogenic T-cell responses to the transplanted islets. CONCLUSIONS Overall, anti-TCR mAb induced peripheral tolerance to specific alloantigens even in the absence of Foxp3-expressing natural regulatory T cells. These findings reveal the potential for using TCR-targeting mAbs as induction immunotherapy for islet transplantation.

Purpose. We tested the effects of selective inhibition of interleukin (IL)-2 gene expression by IL-2 antisense oligonucleotide (oligo) with phosphorothioate (PS)/ phosphodiester (PD)/2'-methoxyethyl (ME) modifications (17359) on T-cell function and the survival of heart allografts in mice. Methods. The PS- (17328) or PS/PD/ME- (17359) IL-2 oligo was electroporated to mouse T cell lymphoma cells (TIB 155) stimulated with concanavalin A (Con A). Expression of IL-2 was analyzed by an ELISA spot assay and a reverse transcript polymerase chain reaction method. C3H (H-2 k ) mice transplanted with BALB/c (H-2 d ) heart grafts were treated i.v. with a 7-day osmotic pump with 20 mg/kg 17359 alone or in combination with sirolimus (SRL). Results. In comparison with untreated controls, 500 to 2000 nM 17328 inhibited IL-2 protein production by 21.8% to 47.2%, whereas 500 to 2000 nM 17359 did so by 35.5% to 83.5% (both P

Background. We aimed to identify the polymorphic epitopes that mitigate graft-versus-host disease (GvHD) and host-versus-graft response (HvGR) toward rat small bowel allografts in rats. Methods. We tailored class I major histocompatibility complex (MHC) allochimeric antigens encoding 10 α1-helical (α 1h 158-80-RT1.A a ) or 4 (α 1h 1/u62-69-RT1.A a ) polymorphic amino acids. In the GvHD model, ACI (RT1 a ) donors were pretreated (day -14) with an intrathymic injection of α 1h 158-80-RT1.A a , α 1h 1/u62-69-RT1.A a , or RT1.A 1 protein, with or without simultaneous intravenous injection of anti-T-cell receptor R73 monoclonal antibodies. Wistar-Furth (WF; RT1 u ) donors were tested with a similar protocol. In the HvGR model, ACI recipients were treated with a protocol designed to induce transplantation tolerance toward WF heart allografts: a portal vein injection of α 1h 1/u62-69-RT1.A a protein and cyclosporine (4 mg/kg, intramuscular; days 0-6). Results. GvHD was prevented in all (ACI x LEW) F 1 recipients (RT1 a/l ) by pretreating ACI donors with R73 monoclonal antibody and recipient RT1.A 1 or α 1h 158-80-RT1.A a protein. Similarly, pretreatment of WF donors with RT1.A a protein also prevented GvHD in (ACI x WF) F 1 recipients. However, in a combined GvHD/HvGR model, ACI recipient perioperative treatment designed to prevent HvGR only modestly prolonged WF small bowel allograft survival (27.7±5.3 days compared to 17.4±4.6 days in the cyclosporine-alone group). In contrast, application of the two protocols significantly prolonged WF allograft survival (55.6±34.6 days), with two of seven recipients surviving more than 100 days. Conclusion. Simultaneous inhibition of GvHD and HvGR significantly prolongs small bowel allograft survival.

Background. C-raf is a well-characterized serine/ threonine (Ser/Thr) protein kinase that is involved in the transduction of multiple signals of T cells. We demonstrate that the inhibition of C-raf mRNA expression prolongs heart allograft survival. Methods. Three 20-mer C-raf antisense oligonucleotides, each with identical sequences, were synthesized with different chemical modifications: one as a uniform phosphorothioate oligodeoxynucleotide (PS oligo), a second with a PS backbone and 2*-methoxyethyl (ME) substitutions at the 2*-sugar positions in the first and last five nucleotides, and a third with a mixed PS and phosphodiester (PD) backbone and ME modifications on the first and last five nucleotides. Results. Both ME-modified C-raf antisense oligos were at least 5-fold more effective than the PS C-raf antisense oligo in blocking C-raf mRNA expression in two cell lines. Similarly, each of the ME C-raf antisense oligos produced better heart allograft survival rates than did PS C-raf oligo. Furthermore, although the combination of PS C-raf antisense oligo with sirolimus (SRL) acted synergistically to extend heart allograft survival, the effect was potentiated by either of the ME-modified oligos. Conclusions. C-raf inhibition extends heart allograft survival, and ME-modification potentiates antisense activity. The T cell receptor (TCR) complex is involved in the initiation of signal transduction from the T cell membrane to the nucleus (1). After the engagement of TCR ab chains by alloantigen, the TCR signal is transduced by the CD3 gdeezz chains in association with the Fyn, Lck, and ZAP70 kinase network, leading to the initiation of the Ras/Raf/Mek mitogen-activated protein (MAP) kinase cascade (2‐ 4). In addition to TCR signaling, full activation of T cells requires a second membrane signal delivered through the interaction of CD28 molecules (expressed on T cells) with B7‐1/B7‐2 ligands that are present on antigen presenting cells (APC) (5). The CD28/B7 signal transduction pathway utilizes the MAP kinase pathway to stabilize and increase the production of cytokines via the CD28 response element present in the

BACKGROUND: Ingested interferon (IFN)-alpha is a biological response modifier in experimental autoimmune encephalomyelitis and multiple sclerosis, and prevents type 1 diabetes in nonobese diabetic mice. Islet transplantation possesses significant potential advantages over whole-gland transplantation because it is simple, may achieve insulin independence, and has clear advantages over exogenous insulin therapy. Therefore, we examined whether ingested IFN-alpha, administered to islet allograft recipients, could prevent islet allograft rejection. METHODS: Recipient C3H mice (H2k) were made diabetic and either untreated or treated with 10-1000 international units (IU) of ingested murine IFN-alpha daily from day -7 through day +14 after transplantation for a total of 21 days. Seven days after diabetes induction, recipients received allograft islets isolated from C57BL.10 donors (H2b) under the kidney capsule and were followed for overt diabetes via elevated blood glucose. RESULTS: Control recipients and recipients fed 1000 IU all became diabetic by day 13, whereas mice ingesting IFN-alpha had delayed rejection for up to 27 (10 IU) to 29 days (100 IU) after islet transplantation. Treatment of recipients of islet allografts with ingested IFN-alpha doubles the time period before rejection compared with control mice. The feeding period with daily IFN-alpha was doubled from 21 days to 42 days in total, 7 days before transplantation and 35 days after transplantation. CONCLUSION: Treatment of recipients of islet allografts with prolonged ingested IFN-alpha prevents rejection in a subset of recipients. Ingested IFN-alpha may prevent rejection if given continuously after transplantation.

Background. In rodent transplant models, FTY720 exerts a synergistic affect with cyclosporine (CsA) to prolong allograft survival. The present experiments sought to test this combination in subhuman primates. Methods. Cynomolgus monkeys were transplanted with kidney allografts that were incompatible in mixed lymphocyte culture reactions. The animals were treated with daily intramuscular injections of CsA using doses selected to maintain whole blood trough concentrations at therapeutic values between 40 and 200 ng/ml. The 4 experimental groups included CsA without or with 0.1, 0.3, or 1 mg/kg/day FTY720 delivered daily by intravenous bolus injection. Therapeutic effects were suggested both by the graft histology of biopsy within the first 10 posttransplant days and by the length of host survival. Results. Whereas recipients treated with CsA alone rejected kidney allografts at a median survival time of 8.5 days (n=4), those treated with either 0.1 or 0.3 mg/kg/day FTY720 in addition to CsA showed significant prolongation of kidney allograft survival to 71 days (n=3; P

BACKGROUND: We designed an antisense phosphorothioate oligodeoxynucleotide (oligo) to specifically inhibit the expression of rat intercellular adhesion molecule-1 (ICAM-1) mRNA (IP-9125). METHODS: IP-9125 oligo was delivered intravenously by osmotic pump alone or in combination with cyclosporine (CsA) to recipients in order to prevent the rejection of kidney or heart allografts. In additional experiments, kidney allografts were perfused with IP-9125 before grafting. RESULTS: IP-9125 inhibited ICAM-1 mRNA and ICAM-1 protein expression in rat aortic endothelial cells; scrambled controls IP-12140 and IP-13944 were ineffective. Untreated ACI (RT1a) recipients rejected Lewis (RT1l) kidney allografts at a mean survival time of 8.5+/-1.1 days. A 14-day intravenous administration of 2.5 mg/kg/day IP-9125 prolonged the survival of kidney allografts to 39.2+/-16.4 days; 5.0 mg/kg/day, to 43.0+/-17.5 days; and 10.0 mg/kg/day, to 50.4+/-21.6 days. In contrast, a scrambled control IP-12140 was not effective. A combination of 10 mg/kg/day IP-9125 and 1.0 mg/kg/day CsA delivered for 14 days synergistically extended kidney allograft survival times 88.5+/-7.5 days. In contrast, the combination of 10.0 mg/kg/day control IP-12140 with CsA was ineffective (20.7+/-3.2 days) when compared with CsA alone (20.2+/-4.0 days). Similar results were obtained for heart transplants in recipients treated with IP-9125 alone or in combination with CsA. Furthermore, in situ immunostaining showed that IP-9125 significantly reduced the expression of ICAM-1 protein in kidney allografts. Finally, perfusion of kidney grafts alone with 20.0 mg per 2 ml of IP-9125 protected kidney allografts from rejection (37.5+/-7.5 days; P < 0.001), whereas perfusion with 20 mg per 2 ml of control IP-12140 was ineffective (12.6+/-5.0 days). CONCLUSIONS: Rat ICAM-1 IP-9125 oligo inhibits ICAM-1 protein expression in vitro and in vivo as well as blocks allograft rejection when used for pretreatment of donors, graft perfusion, or postoperative treatment of recipients.