Improving engraftment of hepatocyte transplantation using alpha-1 antitrypsin as an immune modulator.

For patients with non-cirrhotic liver-based metabolic disorders, hepatocyte transplantation can be an effective treatment. However, long-term function of transplanted hepatocytes following infusion has not been achieved due to insufficient numbers of hepatocytes reaching the liver cell plates caused by activation of the instant blood-mediated inflammatory reaction (IBMIR). Our aim was to determine if the natural immune modulator, alpha-1 antitrypsin (AAT), could improve engraftment of transplanted hepatocytes and investigate its mechanism of action. A tubing loop model was used to analyse activation of the IBMIR when human hepatocytes were in contact with ABO-matched blood and 4 mg/ml AAT. Platelet and white cell counts, complement and cytokine expression were analysed. To determine if AAT could improve short-term engraftment, female rats underwent tail vein injection of AAT (120 mg/kg) or water (control) prior to the intrasplenic transplantation of 2 × 107 male hepatocytes. At 48 h and 1 week, livers were collected for analysis. In our loop model, human hepatocytes elicited a significant drop in platelet count with thrombus formation compared to controls. Loops containing AAT and hepatocytes showed no platelet consumption and no thrombus formation. Further, AAT treatment resulted in reduced IL-1ß, IL-6 and IFN-γ and increased IL-1RA compared to untreated loops. In vivo, AAT significantly improved engraftment of rat hepatocytes compared to untreated at 48 h. AAT infusion may inhibit the IBMIR, thus improving short-term engraftment of donor hepatocytes and potentially improve the outcomes for patients with liver-based metabolic disease. KEY MESSAGES: • Alpha-1 antitrypsin (AAT) acts as an immune modulator to improve the efficacy of hepatocyte transplantation. • Treatment with AAT decreased thrombus formation and pro-inflammatory cytokine expression in a tubing loop model. • AAT significantly improved engraftment of donor hepatocytes within the first 48 h post transplantation.

Hematopoietic Stem/Progenitor Cell Dependent Participation of Innate Lymphoid Cells in Low-Intensity Sterile Inflammation.

Hematopoietic stem/progenitor cells (HSPC) are characterized by their unique capacities of self-renewal and multi-differentiation potential. This second property makes them able to adapt their differentiation profile depending on the local environment they reach. Taking advantage of an animal model of peritonitis, induced by injection of the TLR-2 ligand, zymosan, we sought to study the relationship between bone marrow-derived hematopoietic stem/progenitor cells (BM-HSPCs) and innate lymphoid cells (ILCs) regarding their emergence and differentiation at the site of inflammation. Our results demonstrate that the strength of the inflammatory signals affects the capacity of BM-derived HSPCs to migrate and give rise in situ to ILCs. Both low- and high-dose of zymosan injections trigger the appearance of mature ILCs in the peritoneal cavity where the inflammation occurs. Herein, we show that only in low-dose injected mice, the recovered ILCs are dependent on an in situ differentiation of BM-derived HSPCs and/or ILC2 precursors (ILC2P) wherein high-dose, the stronger inflammatory environment seems to be able to induce the emergence of ILCs independently of BM-derived HSPCs. We suggest that a relationship between HSPCs and ILCs seems to be affected by the strength of the inflammatory stimuli opening new perspectives in the manipulation of these early hematopoietic cells.

Alpha-1-antitrypsin in cell and organ transplantation.

Limited availability of donor organs and risk of ischemia-reperfusion injury (IRI) seriously restrict organ transplantation. Therapeutics that can prevent or reduce IRI could potentially increase the number of transplants by increasing use of borderline organs and decreasing discards. Alpha-1 antitrypsin (AAT) is an acute phase reactant and serine protease inhibitor that limits inflammatory tissue damage. Purified plasma-derived AAT has been well tolerated in more than 30 years of use to prevent emphysema in AAT-deficient individuals. Accumulating evidence suggests that AAT has additional anti-inflammatory and tissue-protective effects including improving mitochondrial membrane stability, inhibiting apoptosis, inhibiting nuclear factor kappa B activation, modulating pro- vs anti-inflammatory cytokine balance, and promoting immunologic tolerance. Cell culture and animal studies have shown that AAT limits tissue injury and promotes cell and tissue survival. AAT can promote tolerance in animal models by downregulating early inflammation and favoring induction and stabilization of regulatory T cells. The diverse intracellular and immune-modulatory effects of AAT and its well-established tolerability in patients suggest that it might be useful in transplantation. Clinical trials, planned and/or in progress, should help determine whether the promise of the animal and cellular studies will be fulfilled by improving outcomes in human organ transplantation.

Alpha-1 antitrypsin treatment of new-onset type 1 diabetes: An open-label, phase I clinical trial (RETAIN) to assess safety and pharmacokinetics.

OBJECTIVE: To determine the safety and pharmacokinetics of alpha-1 antitrypsin (AAT) in adults and children. RESEARCH DESIGN AND METHODS: Short-term AAT treatment restores euglycemia in the non-obese mouse model of type 1 diabetes. A phase I multicenter study in 16 subjects with new-onset type 1 diabetes studied the safety and pharmacokinetics of Aralast NP (AAT). This open-label, dose-escalation study enrolled 8 adults aged 16 to 35 years and 8 children aged 8 to 15 years within 100 days of diagnosis, to receive 12 infusions of AAT: a low dose of 45 mg/kg weekly for 6 weeks, followed by a higher dose of 90 mg/kg for 6 weeks. RESULTS: C-peptide secretion during a mixed meal, hemoglobin A1c (HbA1c), and insulin usage remained relatively stable during the treatment period. At 72 hours after infusion of 90 mg/kg, mean levels of AAT fell below 2.0 g/L for 7 of 15 subjects. To identify a plasma level of AAT likely to be therapeutic, pharmacodynamic ex vivo assays were performed on fresh whole blood from adult subjects. Polymerase chain reaction (PCR) analyses were performed on inhibitor of IKBKE, NOD1, TLR1, and TRAD gene expression, which are important for activation of nuclear factor-κB (NF-κB) and apoptosis pathways. AAT suppressed expression dose-dependently; 50% inhibition was achieved in the 2.5 to 5.0 mg/mL range. CONCLUSIONS: AAT was well tolerated and safe in subjects with new-onset type 1 diabetes. Weekly doses of AAT greater than 90 mg/kg may be necessary for an optimal therapeutic effect.

Contrasting Roles of Islet Resident Immunoregulatory Macrophages and Dendritic Cells in Experimental Autoimmune Type 1 Diabetes.

The innate immune system critically shapes diabetogenic adaptive immunity during type 1 diabetes (T1D) pathogenesis. While the role of tissue-infiltrating monocyte-derived macrophages in T1D is well established, the role of their tissue-resident counterparts remains undefined. We now demonstrate that islet resident macrophages (IRMs) from non-autoimmune mice have an immunoregulatory phenotype and powerfully induce FoxP3+ Tregs in vitro. The immunoregulatory phenotype and function of IRMs is compromised by TLR4 activation in vitro. Moreover, as T1D approaches in NOD mice, the immunoregulatory phenotype of IRMs is diminished as is their relative abundance compared to immunostimulatory DCs. Our findings suggest that maintenance of IRM abundance and their immunoregulatory phenotype may constitute a novel therapeutic strategy to prevent and/or cure T1D.

Kidney Versus Islet Allograft Survival After Induction of Mixed Chimerism With Combined Donor Bone Marrow Transplantation.

We have previously reported successful induction of transient mixed chimerism and long-term acceptance of renal allografts in MHC mismatched nonhuman primates. In this study, we attempted to extend this tolerance induction approach to islet allografts. A total of eight recipients underwent MHC mismatched combined islet and bone marrow (BM) transplantation after induction of diabetes by streptozotocin. Three recipients were treated after a nonmyeloablative conditioning regimen that included low-dose total body and thymic irradiation, horse Atgam (ATG), six doses of anti-CD154 monoclonal antibody (mAb), and a 1-month course of cyclosporine (CyA) (Islet A). In Islet B, anti-CD8 mAb was administered in place of CyA. In Islet C, two recipients were treated with Islet B, but without ATG. The results were compared with previously reported results of eight cynomolgus monkeys that received combined kidney and BM transplantation (Kidney A) following the same conditioning regimen used in Islet A. The majority of kidney/BM recipients achieved long-term renal allograft survival after induction of transient chimerism. However, prolonged islet survival was not achieved in similarly conditioned islet/BM recipients (Islet A), despite induction of comparable levels of chimerism. In order to rule out islet allograft loss due to CyA toxicity, three recipients were treated with anti-CD8 mAb in place of CyA. Although these recipients developed significantly superior mixed chimerism and more prolonged islet allograft survival (61, 103, and 113 days), islet function was lost soon after the disappearance of chimerism. In Islet C recipients, neither prolonged chimerism nor islet survival was observed (30 and 40 days). Significant improvement of mixed chimerism induction and islet allograft survival were achieved with a CyA-free regimen that included anti-CD8 mAb. However, unlike the kidney allograft, islet allograft tolerance was not induced with transient chimerism. Induction of more durable mixed chimerism may be necessary for induction of islet allograft tolerance.

Islet Oxygen Consumption Rate (OCR) Dose Predicts Insulin Independence in Clinical Islet Autotransplantation.

BACKGROUND: Reliable in vitro islet quality assessment assays that can be performed routinely, prospectively, and are able to predict clinical transplant outcomes are needed. In this paper we present data on the utility of an assay based on cellular oxygen consumption rate (OCR) in predicting clinical islet autotransplant (IAT) insulin independence (II). IAT is an attractive model for evaluating characterization assays regarding their utility in predicting II due to an absence of confounding factors such as immune rejection and immunosuppressant toxicity. METHODS: Membrane integrity staining (FDA/PI), OCR normalized to DNA (OCR/DNA), islet equivalent (IE) and OCR (viable IE) normalized to recipient body weight (IE dose and OCR dose), and OCR/DNA normalized to islet size index (ISI) were used to characterize autoislet preparations (n = 35). Correlation between pre-IAT islet product characteristics and II was determined using receiver operating characteristic analysis. RESULTS: Preparations that resulted in II had significantly higher OCR dose and IE dose (p<0.001). These islet characterization methods were highly correlated with II at 6-12 months post-IAT (area-under-the-curve (AUC) = 0.94 for IE dose and 0.96 for OCR dose). FDA/PI (AUC = 0.49) and OCR/DNA (AUC = 0.58) did not correlate with II. OCR/DNA/ISI may have some utility in predicting outcome (AUC = 0.72). CONCLUSIONS: Commonly used assays to determine whether a clinical islet preparation is of high quality prior to transplantation are greatly lacking in sensitivity and specificity. While IE dose is highly predictive, it does not take into account islet cell quality. OCR dose, which takes into consideration both islet cell quality and quantity, may enable a more accurate and prospective evaluation of clinical islet preparations.

Innate immunity for better or worse govern the allograft response.

PURPOSE OF REVIEW: To update knowledge concerning the cause and consequences of the detrimental forms of innate immunity that inevitably occurs in peritransplant period tissue and cellular transplants. In addition, we review the information that a newly discovered, engraftment-promoting, and tolerance-inducing macrophage population is identified and characterized. RECENT FINDINGS: The allograft response mounted by adaptive immune cells is shaped by innate immunity. The early allograft response is uniquely intense as a result of activation of the innate immune response created by ischemia reperfusion injury in organ transplants, delayed revascularization of cell transplants, and hypoxia. Inflammation is created by both cellular 'debris' and cytokines. However, a newly discovered prominent, albeit fragile, tissue-resident, noninvasive, and immunoregulatory macrophage promotes engraftment and tolerance. The role of intracellular 'debris' as well as inflammation in evoking detrimental rejection-provoking peritransplant inflammation is emphasized as well as characterization of a prominent and highly immunoregulatory albeit fragile macrophage population that is tissue-resident and does not circulate is characterized. SUMMARY: Opportunity lies in the ability to rein in detrimental peri-transplant inflammation and in the ability to promote the longevity of a subpopulation of highly potent tissue-resident immunoregulatory macrophages.

Hand transplants and the mandate for tolerance.

PURPOSE OF REVIEW: The field of vascularized composite allograft (VCA) to achieve its full potential will require induction of tolerance. This review will introduce a new method of potential inducing tolerance in hand transplantation. RECENT FINDINGS: Hand transplantation is never a life-extending transplant. This fact resulted in considerable debate both for and against the use of immunosuppression for nonlife-extending transplants. There is considerable debate about the ethics of hand transplantation. There is now consensus that nonlife-extending transplants are acceptable in properly selected patients. However, ideally, hand transplants should not receive life-long immunosuppression. Therefore, attempts to achieve drug-free tolerance through nonlife-endangering therapies are warranted. To this end, we propose implementation of tolerizing therapy long after periinflammation has subsided and drug minimization has proven successful. Evidence that short-term treatment with low doses of IL-2 or a long-lived IL-2 immunoglobulin (Ig) can tilt the balance of immunity from tissue destructive to tolerance come from preclinical demonstrations in mouse and nonhuman primate models of autoimmunity and/or transplantation and even more recent clinical trials. SUMMARY: We believe that with the proper use of low-dose IL-2 given at an opportune time in the inflammatory process of transplant that reduce immunosuppression and even tolerance can be induced in hand transplantation. We propose that tolerance can be inducted after a long period of conventional treatment to avoid 'tolerance-hindering' adverse inflammation that occurs in the posttransplant period. With abatement of posttransplant inflammation and with time, we will institute low-dose IL-2-based therapy to support the proliferation, viability and functional phenotype of regulatory T cells.

Fragile TIM-4-expressing tissue resident macrophages are migratory and immunoregulatory.

Macrophages characterized as M2 and M2-like regulate immune responses associated with immune suppression and healing; however, the relationship of this macrophage subset to CD169+ tissue-resident macrophages and their contribution to shaping alloimmune responses is unknown. Here we identified a population of M2-like tissue-resident macrophages that express high levels of the phosphatidylserine receptor TIM-4 and CD169 (TIM-4hiCD169+). Labeling and tracking of TIM-4hiCD169+ macrophages in mice revealed that this population is a major subset of tissue-resident macrophages, homes to draining LNs following oxidative stress, exhibits an immunoregulatory and hypostimulatory phenotype that is maintained after migration to secondary lymphoid organs, favors preferential induction of antigen-stimulated Tregs, and is highly susceptible to apoptosis. Moreover, CD169+ tissue-resident macrophages were resistant to oxidative stress-induced apoptosis in mice lacking TIM-4. Compared with heart allografts from WT mice, Tim4-/- heart allografts survived much longer and were more easily tolerized by non-immunosuppressed recipients. Furthermore, Tim4-/- allograft survival was associated with the infiltration of Tregs into the graft. Together, our data provide evidence that M2-like TIM-4hiCD169+ tissue-resident macrophages are immunoregulatory and promote engraftment of cardiac allografts, but their influence is diminished by TIM-4-dependent programmed cell death.

Role of myeloid-derived suppressor cells in mouse pre-sensitized cardiac transplant model.

Harness of sensitized transplantation remains a clinical challenge particularly in parallel with prolonged cold ischemia time (PCI)-mediated injury. Our present study was to test the role of myeloid-derived suppressor cells (MDSCs) in mouse pre-sensitized transplantation. Our findings revealed that CD11b+Gr1(low) MDSC was shown to have strong suppressive activity. MDSCs subsets from the tolerated mice exhibited higher suppressive capacities compared with counterparts from naive (untreated) mice. Depletion of Tregs could not affect splenic CD11b+Gr1(-low) MDSC frequency, but increase peripheral and intragraft CD11b+Gr1(-low) frequency. Intriguingly, boost of Tregs remarkably caused an increase of CD11b+Gr1(-low) frequency in the graft, peripheral blood, and spleen. Furthermore, peripheral CD11b+Gr1(-low) cells were massively accumulated at the early stage when allogeneic immune response was enhanced. Taken together, MDSCs could prevent grafts from PCI-mediated injury independent on Tregs in the pre-sensitized transplant recipients. Utilization of MDSC subset particularly CD11b+Gr1(-low) might provide a novel insight into improving graft outcome under such clinical scenarios.

Alpha 1-antitrypsin reduces inflammation and enhances mouse pancreatic islet transplant survival.

The promise of islet cell transplantation cannot be fully realized in the absence of improvements in engraftment of resilient islets. The marginal mass of islets surviving the serial peritransplant insults may lead to exhaustion and thereby contribute to an unacceptably high rate of intermediate and long-term graft loss. Hence, we have studied the effects of treatment with alpha 1-antitrypsin (AAT) in a syngeneic nonautoimmune islet graft model. A marginal number of syngeneic mouse islets were transplanted into nonautoimmune diabetic hosts and islet function was analyzed in control and AAT treated hosts. In untreated controls, marginal mass islet transplants did not restore euglycemia. Outcomes were dramatically improved by short-term AAT treatment. Transcriptional profiling identified 1,184 differentially expressed transcripts in AAT-treated hosts at 3 d posttransplantation. Systems-biology-based analysis revealed AAT down-regulated regulatory hubs formed by inflammation-related molecules (e.g., TNF-α, NF-κB). The conclusions yielded by the systems-biology analysis were rigorously confirmed by QRT-PCR and immunohistology. These data suggest that short-term AAT treatment of human islet transplant recipients may be worthy of a clinical trial.

The role of TNF-α in mice with type 1- and 2- diabetes.

BACKGROUND: Previously, we have demonstrated that short-term treatment of new onset diabetic Non-obese diabetic (NOD) mice, mice that are afflicted with both type 1 (T1D) and type 2 (T2D) diabetes with either Power Mix (PM) regimen or alpha1 antitrypsin (AAT) permanently restores euglycemia, immune tolerance to self-islets and normal insulin signaling. METHODOLOGY AND PRINCIPAL FINDINGS: To search for relevant therapeutic targets, we have applied genome wide transcriptional profiling and systems biology oriented bioinformatics analysis to examine the impact of the PM and AAT regimens upon pancreatic lymph node (PLN) and fat, a crucial tissue for insulin dependent glucose disposal, in new onset diabetic non-obese diabetic (NOD) mice. Systems biology analysis identified tumor necrosis factor alpha (TNF-α) as the top focus gene hub, as determined by the highest degree of connectivity, in both tissues. In PLNs and fat, TNF-α interacted with 53% and 32% of genes, respectively, associated with reversal of diabetes by previous treatments and was thereby selected as a therapeutic target. Short-term anti-TNF-α treatment ablated a T cell-rich islet-invasive and beta cell-destructive process, thereby enhancing beta cell viability. Indeed anti-TNF-α treatment induces immune tolerance selective to syngeneic beta cells. In addition to these curative effects on T1D anti-TNF-α treatment restored in vivo insulin signaling resulting in restoration of insulin sensitivity. CONCLUSIONS: In short, our molecular analysis suggested that PM and AAT both may act in part by quenching a detrimental TNF-α dependent effect in both fat and PLNs. Indeed, short-term anti-TNF-α mAb treatment restored enduring euglycemia, self-tolerance, and normal insulin signaling.

Effects of an agonist interleukin-2/Fc fusion protein, a mutant antagonist interleukin-15/Fc fusion protein, and sirolimus on cardiac allograft survival in non-human primates.

BACKGROUND: To tilt the immunologic balance toward tolerance and away from rejection, non-human primate recipients of cardiac allografts were treated with interleukin (IL)-2/Fc, mutant (m) antagonist type mIL-15/Fc, and sirolimus. METHODS: Heterotopic heart transplants were performed on 8 fully mismatched cynomolgus macaques. An untreated control recipient rejected its graft by post-operative Day 6. The remaining 7 animals received oral or intramuscular immunosuppression with sirolimus. A recipient treated with sirolimus alone rejected at the end of 28 days of immunosuppression. The remaining 6 monkeys also received IL-2/Fc and mIL-15/Fc intramuscularly until 28 days after transplant. One animal received a second 28-day course of fusion protein starting at Day 50. In these 6 animals, sirolimus was continued for 28 days (n = 4) or until protein levels were low (n = 2). RESULTS: In the 4 monkeys treated with a 28-day course of sirolimus and fusion proteins, mean graft survival was 51.5 days (range, 28-76 days). The animal receiving a second course of fusion protein rejected its graft on Day 177, despite detectable levels of the fusion proteins and sirolimus. The central memory, effector memory, and naïve CD4(+) and CD8(+) T-cell populations in the peripheral blood did not change significantly during fusion protein administration. A 2.5-fold expansion in CD4(+)CD25(+) lymphocytes occurred in recipients treated with fusion proteins and sirolimus that was not observed in the recipient treated with sirolimus alone. CONCLUSIONS: Although IL-2/Fc, mIL-15/Fc, and sirolimus administered in this manner permitted modest prolongation of graft survival and expansion of CD4(+)CD25(+) T cells, tolerance was not achieved.

Towards cytoprotection in the peritransplant period.

As a consequence of ischemia-reperfusion injury of whole organ transplants and hypoxia-anoxia of cell transplants, transplantation unavoidably triggers adverse, cytodestructive inflammation within the allograft. Interventions that dampen adverse inflammation may limit the extent and duration of this injury, and preserve tissue function. Moreover, these interventions should create a milieu that guides many donor-activated T cells into a tissue-protective phenotype, thus promoting graft acceptance or even tolerance. Hence, it is useful, maybe crucial, to identify the measures that minimize deleterious consequences of acute and chronic inflammation upon allograft. Several therapies that inhibit activity of certain proinflammatory cytokines or expression of tissue "danger signals", while sustaining or potentially enhancing the expression of tissue-intrinsic anti-inflammatory and cytoprotective genes, are awaiting clinical trials or are already approved for the treatment of immuno-inflammatory disorders. If applied in the peritransplant period, such cytoprotective regimens may increase the pool of donor organs suitable for transplantation, reduce the overall requirements for maintenance immunosuppression and perhaps foster transplant tolerance.

Inflammation and the balance of Treg and Th17 cells in transplant rejection and tolerance.

PURPOSE OF REVIEW: Inflammation of the allograft, occurring as a consequence of hypoxia and ischemia/reperfusion injury, adversely influences short-term and long-term transplant outcomes. Thus far, imbalance of tissue-protective Treg and tissue-destructive Th17 cells has been confirmed in a number of tissue-inflammatory states, including autoimmune disease. Hence, benefits of tilting Treg-Th17 equilibrium toward dominance of Tregs may promote transplant tolerance. RECENT FINDINGS: Adverse graft inflammation creates extreme resistance to the induction of donor-specific tolerance. Proinflammatory cytokines, when abundantly expressed within the graft and draining lymph nodes, prevent commitment of donor-activated T cells into graft-protective, T-regulatory phenotype, while fostering generation of donor-reactive Th1, Th2 or Th17 effector subsets. In addition, the inflammatory milieu may destabilize the program of both natural and induced Tregs, converting them into inflammatory, effector-like phenotypes. Therefore permanent, Treg-dependent acceptance of an allograft may not be achieved without limiting adverse tissue inflammation. SUMMARY: Balance of graft-protective regulatory and graft-destructive effector T cells largely depends on the balance of proinflammatory and anti-inflammatory cytokines in the milieu, in which donor-directed T-cell response occurs. In the absence of proinflammatory cytokines, the constitutive expression of TGF-beta may guide recipient T cells into a tissue-protective, pro-tolerant mode. Therefore, targeting adverse tissue inflammation may represent a powerful means to tilt antidonor immunity towards tolerance.

In vivo tracking of 'color-coded' effector, natural and induced regulatory T cells in the allograft response.

Here we present methods to longitudinally track islet allograft-infiltrating T cells in live mice by endoscopic confocal microscopy and to analyze circulating T cells by in vivo flow cytometry. We developed a new reporter mouse whose T cell subsets express distinct, 'color-coded' proteins enabling in vivo detection and identification of effector T cells (T(eff) cells) and discrimination between natural and induced regulatory T cells (nT(reg) and iT(reg) cells). Using these tools, we observed marked differences in the T cell response in recipients receiving tolerance-inducing therapy (CD154-specific monoclonal antibody plus rapamycin) compared to untreated controls. These results establish real-time cell tracking as a powerful means to probe the dynamic cellular interplay mediating immunologic rejection or transplant tolerance.

Prediction of marginal mass required for successful islet transplantation.

Islet quality assessment methods for predicting diabetes reversal (DR) following transplantation are needed. We investigated two islet parameters, oxygen consumption rate (OCR) and OCR per DNA content, to predict transplantation outcome and explored the impact of islet quality on marginal islet mass for DR. Outcomes in immunosuppressed diabetic mice were evaluated by transplanting mixtures of healthy and purposely damaged rat islets for systematic variation of OCR/DNA over a wide range. The probability of DR increased with increasing transplanted OCR and OCR/DNA. On coordinates of OCR versus OCR/DNA, data fell into regions in which DR occurred in all, some, or none of the animals with a sharp threshold of around 150-nmol/min mg DNA. A model incorporating both parameters predicted transplantation outcome with sensitivity and specificity of 93% and 94%, respectively. Marginal mass was not constant, depended on OCR/DNA, and increased from 2,800 to over 100,000 islet equivalents/kg body weight as OCR/DNA decreased. We conclude that measurements of OCR and OCR/DNA are useful for predicting transplantation outcome in this model system, and OCR/DNA can be used to estimate the marginal mass required for reversing diabetes. Because human clinical islet preparations in a previous study had OCR/DNA.