We previously demonstrated that intra-peritoneal delivery of adeno-associated virus serotype 8 (AAV8) stably transduces the pancreas, including the β-cells in the endogenous islets. We also demonstrated the ability to deliver and express genes specifically in β-cells for at least 6 months using a murine insulin promoter (mIP) in a double-stranded, self-complementary AAV vector (dsAAV8-mIP). Here we evaluated the effects of dsAAV8-mIP mediated delivery of interleukin 4 (mIL-4) to endogenous β-cells in NOD mice. In 4 week old NOD mice, the extent of gene transfer and expression in endogenous β-cells following i.p. delivery of dsAAV8-mIP-eGFP was comparable to normal BALB/c mice. Furthermore, following i.p. delivery of dsAAV8-mIP-IL4, expression of mIL-4 was detected in islets isolated and cultured from the treated mice. AAV8-mIP mediated gene expression of mIL-4 to endogenous β-cells of 4 and 8 week old NOD mice prevented the onset of hyperglycemia in NOD mice and reduced the severity of insulitis. Moreover, expression of mIL-4 also maintained the level of CD4+CD25+FoxP3+ cells and adoptive co-transfer of splenocytes from diabetes-free IL-4 vector recipients and splenocytes from wild type diabetic NOD mice prevented the onset diabetes. Taken together, these results demonstrate that local expression of mIL-4 in islets prevents islet destruction and blocks autoimmunity, in part, through regulation of T cell function. These results also demonstrate the utility of using dsAAV8-mIP gene transfer to endogenous NOD β-cells to examine the role of specific gene products in preventing or exacerbating the onset of type 1 diabetes.

Isolation and transplantation of rodent islets are frequently used as a tool for predicting the behavior of new protocols for islet allotransplants in type 1 diabetes patients. Bovine serum albumin (BSA) is recognized as a protease inhibitor possibly protecting function and viability in islets. For this study, the addition of 0.2% BSA to the isolation protocol resulted in a 30% increase in islet yields while other parameters, such as viability and function, retained high islet quality. In vivo, a minimal mass of 70 BSA treated islets showed their ability to control glycemia levels in diabetic mice by bringing the average blood glucose to 153 ± 13.2 mg/dL compared to 288 ± 22.6 mg/dL without BSA. Our results show that the simple addition of BSA to the isolation protocol constitutes a reliable and reproducible method for increasing islet yield. Also adding BSA to the transplantation medium improves islet function in vivo. The method outlined here can reduce the overall number of animals needed per experiment and also reduce the time and resources needed for islet preparation.

The major histocompatibility complex (MHC) on chromosome 6p is an established risk locus for ulcerative colitis (UC) and Crohn’s disease (CD). We aimed to better define MHC association signals in UC and CD by combining data from dense single nucleotide polymorphism (SNP) genotyping and from imputation of classical HLA types, their constituent SNPs and corresponding amino acids in 562 UC, 611 CD, and 1,428 control subjects. Univariate and multivariate association analyses were performed, controlling for ancestry. In univariate analyses, absence of the rs9269955 C allele was strongly associated with risk for UC (P = 2.67×10−13). rs9269955 is a SNP in the codon for amino acid position 11 of HLA-DRβ1, located in the P6 pocket of the HLA-DR antigen binding cleft. This amino acid position was also the most significantly UC-associated amino acid in omnibus tests (P = 2.68×10−13). Multivariate modeling identified rs9269955-C and 13 other variants in best predicting UC versus control status. In contrast, there was only suggestive association evidence between the MHC and CD. Taken together, these data demonstrate that variation at HLA-DRβ1, amino acid 11 in the P6 pocket of the HLA-DR complex antigen binding cleft is a major determinant of chromosome 6p association with ulcerative colitis.

The mitochondrial electron transport chain transforms energy satisfying cellular demand and generates reactive oxygen species (ROS) that act as metabolic signals or destructive factors. Therefore, knowledge of the possible modes and bifurcations of electron transport that affect ROS signaling provides insight into the interrelationship of mitochondrial respiration with cellular metabolism. Here, a bifurcation analysis of a sequence of the electron transport chain models of increasing complexity was used to analyze the contribution of individual components to the modes of respiratory chain behavior. Our algorithm constructed models as large systems of ordinary differential equations describing the time evolution of the distribution of redox states of the respiratory complexes. The most complete model of the respiratory chain and linked metabolic reactions predicted that condensed mitochondria produce more ROS at low succinate concentration and less ROS at high succinate levels than swelled mitochondria. This prediction was validated by measuring ROS production under various swelling conditions. A numerical bifurcation analysis revealed qualitatively different types of multistationary behavior and sustained oscillations in the parameter space near a region that was previously found to describe the behavior of isolated mitochondria. The oscillations in transmembrane potential and ROS generation, observed in living cells were reproduced in the model that includes interaction of respiratory complexes with the reactions of TCA cycle. Whereas multistationarity is an internal characteristic of the respiratory chain, the functional link of respiration with central metabolism creates oscillations, which can be understood as a means of auto-regulation of cell metabolism.

Author Summary

The mitochondrial respiratory chain shows a variety of modes of behavior. In living cells, flashes of ROS production and oscillations accompanied by a decrease of transmembrane potential can be registered. The mechanisms of such complex behavior are difficult to rationalize without a mathematical formalization of mitochondrial respiration. Our most complete model of mitochondrial respiration accounts for the details of electron transport, reproducing the observed types of behavior, which includes the existence of multiple steady states and periodic oscillations. This most detailed model contains hundreds of differential equations, and such complexity makes it difficult to grasp the main determinants of its behavior. Therefore the full model was reduced to a simplified description of complex III only, and numerical bifurcation analysis was used to study its behavior. Then the evolution of its behavior was traced in a sequence of models with increasing complexity leading back to the full model. This analysis revealed the mechanism of switching between the modes of behavior and the conditions for persistence in a given state, which defines ATP production, ROS signaling and destructive effects. This is important for understanding the biochemical basics of many systemic diseases.

OBJECTIVE

The safety of dendritic cells to selectively suppress autoimmunity, especially in type 1 diabetes, has never been ascertained. We investigated the safety of autologous dendritic cells, stabilized into an immunosuppressive state, in established adult type 1 diabetic patients.

RESEARCH DESIGN AND METHODS

A randomized, double-blind, phase I study was conducted. A total of 10, otherwise generally healthy, insulin-requiring type 1 diabetic patients between 18 and 60 years of age, without any other known or suspected health conditions, received autologous dendritic cells, unmanipulated or engineered ex vivo toward an immunosuppressive state. Ten million cells were administered intradermally in the abdomen once every 2 weeks for a total of four administrations. The primary end point determined the proportion of patients with adverse events on the basis of the physician’s global assessment, hematology, biochemistry, and immune monitoring for a period of 12 months.

RESULTS

The dendritic cells were safely tolerated. There were no discernible adverse events in any patient throughout the study. Other than a significant increase in the frequency of peripheral B220+ CD11c− B cells, mainly seen in the recipients of engineered dendritic cells during the dendritic cell administration period, there were no statistically relevant differences in other immune populations or biochemical, hematological, and immune biomarkers compared with baseline.

CONCLUSIONS

Treatment with autologous dendritic cells, in a native state or directed ex vivo toward a tolerogenic immunosuppressive state, is safe and well tolerated. Dendritic cells upregulated the frequency of a potentially beneficial B220+ CD11c− B-cell population, at least in type 1 diabetes autoimmunity.

Chronic pancreatitis is an inflammatory disease of the pancreas that causes permanent changes in the function and structure of the pancreas. It is most commonly a complication of cystic fibrosis or due to a genetic predisposition. Chronic pancreatitis generally presents symptomatically as recurrent abdominal pain, which becomes persistent over time. The pain eventually becomes disabling. Once specific medical treatments and endoscopic interventions are no longer efficacious, total pancreatectomy is the alternative of choice for helping the patient achieve pain control. While daily administrations of digestive enzymes cannot be avoided, insulin-dependent diabetes can be prevented by transplanting the isolated pancreatic islets back to the patient. The greater the number of islets infused, the greater the chance to prevent or at least control the effects of surgical diabetes. We present here a technical approach for the isolation and preservation of the islets proven to be efficient to obtain high numbers of islets, favoring the successful treatment of young patients.

We have previously reported data from clinical and laboratory animal observations which suggest that organ tolerance after transplantation depends on a state of balanced lymphodendritic cell chimerism between the host and donor graft. We have sought further evidence to support this hypothesis by investigating HLA-mismatched liver allograft recipients.

9 of 9 female recipients of livers from male donors had chimerism in their allografts and extrahepatic tissues, according to in-situ hybridisation and molecular techniques 10 to 19 years post-transplantation. In 8 women with good graft function, evidence of the Y chromosome was found in the blood (6/8), skin (8/8), and lymph nodes (7/8). A ninth patient whose transplant failed after 12 years from recurrent chronic viral hepatitis had chimerism in her lymph nodes, skin, jejunum, and aorta at the time of retransplantation.

Although cell migration is thought to take place after all types of transplantation, the large population of migratory cells in, and the extent of their seeding from, hepatic grafts may explain the privileged tolerogenicity of the liver compared with other organs.

Currently, for the patient with type 1 diabetes, a definitive treatment without resorting to the use of exogenous insulin can be achieved only with pancreas or islet cell transplantation. These means of restoring β-cell mass can completely maintain essentially normal long-term glucose homeostasis, although the need for powerful immunosuppressive regimens limits their application to only a subgroup of adult patients. Apart from the shortage of donors that has limited all kinds of transplantation, however, the widespread use of β-cell replacement has been precluded until recently by the drawbacks associated with both organ and islet cell transplantation. Although the study of recurrence of diabetes has generated attention, the fundamental obstacle to pancreas and islet transplantation has been, and remains, the alloimmune response. With a better elucidation of the mechanisms of alloengraftment achieved during the last 3 years, the stage has been set for further advances.

The enzyme α1,3-galactosyltransferase (α1,3GT or GCTA1) synthesizes α1,3-galactose (α1,3Gal) epitopes (Galα1,3Galβ1,4GlcNAc-R), which are the major xenoantigens causing hyperacute rejection in pig-to-human xenotransplantation. Complete removal of α1,3Gal from pig organs is the critical step toward the success of xenotransplantation. We reported earlier the targeted disruption of one allele of the α1,3GT gene in cloned pigs. A selection procedure based on a bacterial toxin was used to select for cells in which the second allele of the gene was knocked out. Sequencing analysis demonstrated that knockout of the second allele of the α1,3GT gene was caused by a T-to-G single point mutation at the second base of exon 9, which resulted in inactivation of the α1,3GT protein. Four healthy α1,3GT double-knockout female piglets were produced by three consecutive rounds of cloning. The piglets carrying a point mutation in the α1,3GT gene hold significant value, as they would allow production of α1,3Gal-deficient pigs free of antibiotic-resistance genes and thus have the potential to make a safer product for human use.

The therapy of type 1 diabetes is an open challenging problem. The restoration of normoglycemia and insulin independence in immunosuppressed type 1 diabetic recipients of islet allotransplantation has shown the potential of a cell-based diabetes therapy. Even if successful, this approach poses a problem of scarce tissue supply. Xenotransplantation can be the answer to this limited donor availability and, among possible candidate tissues for xenotransplantation, porcine islets are the closest to a future clinical application. Xenotransplantation, with pigs as donors, offers the possibility of using healthy, living, and genetically modified islets from pathogen-free animals available in unlimited number of islets. Several studies in the pig-to-nonhuman primate model demonstrated the feasibility of successful preclinical islet xenotransplantation and have provided insights into the critical events and possible mechanisms of immune recognition and rejection of xenogeneic islet grafts. Particularly promising results in the achievement of prolonged insulin independence were obtained with newly developed, genetically modified pigs islets able to produce immunoregulatory products, using different implantation sites, and new immunotherapeutic strategies. Nonetheless, further efforts are needed to generate additional safety and efficacy data in nonhuman primate models to safely translate these findings into the clinic.

Thus far, none of the preclinically successful and promising immunomodulatory agents for type 1 diabetes mellitus (T1DM) has conferred stable, long-term insulin independence to diabetic patients. The majority of these immunomodulators are humanised antibodies that target immune cells or cytokines. These as well as fusion proteins and inhibitor proteins all share varying adverse event occurrence and severity. Other approaches have included intact putative autoantigens or autoantigen peptides. Considerable logistical outlays have been deployed to develop and to translate humanised antibodies targeting immune cells, cytokines, and cytokine receptors to the clinic. Very recent phase III trials with the leading agent, a humanised anti-CD3 antibody, call into question whether further development of these biologics represents a step forward or more of the same. Combination therapies of one or more of these humanised antibodies are also being considered, and they face identical, if not more serious, impediments and safety issues. This paper will highlight the preclinical successes and the excitement generated by phase II trials while offering alternative possibilities and new translational avenues that can be explored given the very recent disappointment in leading agents in more advanced clinical trials.

OBJECTIVE

Because of reduced antioxidant defenses, β-cells are especially vulnerable to free radical and inflammatory damage. Commonly used antirejection drugs are excellent at inhibiting the adaptive immune response; however, most are harmful to islets and do not protect well from reactive oxygen species and inflammation resulting from islet isolation and ischemia-reperfusion injury. The aim of this study was to determine whether redox modulation, using the catalytic antioxidant (CA), FBC-007, can improve in vivo islet function post-transplant.

RESEARCH DESIGN AND METHODS

The abilities of redox modulation to preserve islet function were analyzed using three models of ischemia-reperfusion injury: 1) streptozotocin (STZ) treatment of human islets, 2) STZ-induced murine model of diabetes, and 3) models of syngeneic, allogeneic, and xenogeneic transplantation.

RESULTS

Incubating human islets with catalytic antioxidant during STZ treatment protects from STZ-induced islet damage, and systemic delivery of catalytic antioxidant ablates STZ-induced diabetes in mice. Islets treated with catalytic antioxidant before syngeneic, suboptimal syngeneic, or xenogeneic transplant exhibited superior function compared with untreated controls. Diabetic murine recipients of catalytic antioxidant–treated allogeneic islets exhibited improved glycemic control post-transplant and demonstrated a delay in allograft rejection. Treating recipients systemically with catalytic antioxidant further extended the delay in allograft rejection.

CONCLUSIONS

Pretreating donor islets with catalytic antioxidant protects from antigen-independent ischemia-reperfusion injury in multiple transplant settings. Treating systemically with catalytic antioxidant protects islets from antigen-independent ischemia-reperfusion injury and hinders the antigen-dependent alloimmune response. These results suggest that the addition of a redox modulation strategy would be a beneficial clinical approach for islet preservation in syngeneic, allogeneic, and xenogeneic transplantation.

OBJECTIVE

Type 1 diabetes is a chronic endocrine disorder in which enteroviruses, such as coxsackie B viruses and echoviruses, are possible environmental factors that can trigger or accelerate disease. The development or acceleration of type 1 diabetes depends on the balance between autoreactive effector T-cells and regulatory T-cells. This balance is particularly influenced by dendritic cells (DCs). The goal of this study was to investigate the interaction between enterovirus-infected human pancreatic islets and human DCs.

RESEARCH DESIGN AND METHODS

In vitro phagocytosis of human or porcine primary islets or Min6 mouse insuloma cells by DCs was investigated by flow cytometry and confocal analysis. Subsequent innate DC responses were monitored by quantitative PCR and Western blotting of interferon-stimulated genes (ISGs).

RESULTS

In this study, we show that both mock- and coxsackievirus B3 (CVB3)-infected human and porcine pancreatic islets were efficiently phagocytosed by human monocyte–derived DCs. Phagocytosis of CVB3-infected, but not mock-infected, human and porcine islets resulted in induction of ISGs in DCs, including the retinoic acid–inducible gene (RIG)-I–like helicases (RLHs), RIG-I, and melanoma differentiation–associated gene 5 (Mda5). Studies with murine Min6 insuloma cells, which were also efficiently phagocytosed, revealed that increased ISG expression in DCs upon encountering CVB-infected cells resulted in an antiviral state that protected DCs from subsequent enterovirus infection. The observed innate antiviral responses depended on RNA within the phagocytosed cells, required endosomal acidification, and were type I interferon dependent.

CONCLUSIONS

Human DCs can phagocytose enterovirus-infected pancreatic cells and subsequently induce innate antiviral responses, such as induction of RLHs. These responses may have important consequences for immune homeostasis in vivo and may play a role in the etiology of type 1 diabetes.

Non-human primates (NHPs) are a very valuable experimental model for diabetes research studies including experimental pancreatic islet transplantation. In particular NHPs are the recipients of choice to validate pigs as possible source of pancreatic islets. The aim of this study was to quantify glycated hemoglobin percentage in NHPs and to assess whether changes in values reflect the metabolic trends after diabetes induction and islet transplantation. Sera from 15 NHPs were analyzed. 9 NHPs were rendered diabetic with streptozotocin (STZ), and 3 of them received porcine islet transplants. Hemoglobin A1c (HbA1c) percentage was measured with an assay based on a latex immunoagglutination inhibition methodology. Whereas diabetes and its duration were associated with increasing HbA1c levels, postislet transplantation blood glucose normalization was paralleled by a decrease in the HbA1c percentage. Our data provide evidence that HbA1c is a useful tool to monitor glucose metabolism in NHPs.

Epistasis could be an important source of risk for disease. How interacting loci might be discovered is an open question for genome-wide association studies (GWAS). Most researchers limit their statistical analyses to testing individual pairwise interactions (i.e., marginal tests for association). A more effective means of identifying important predictors is to fit models that include many predictors simultaneously (i.e., higher dimensional models).

We explore a procedure called screen and clean (SC) for identifying liability loci, including interactions, by using the lasso procedure, which is a model selection tool for high dimensional regression. We approach the problem by using a varying dictionary consisting of terms to include in the model. In the first step the lasso dictionary includes only main effects. The most promising SNPs are identified using a screening procedure. Next the lasso dictionary is adjusted to include these main effects and the corresponding interaction terms. Again, promising terms are identified using lasso screening. Then significant terms are identified through the cleaning process. Implementation of SC for GWAS requires algorithms to explore the complex model space induced by the many SNPs genotyped and their interactions. We propose and explore a set of algorithms and find that SC successfully controls Type I error while yielding good power to identify risk loci and their interactions. When the method is applied to data obtained from the Wellcome Trust Case Control Consortium study of Type 1 Diabetes it uncovers evidence supporting interaction within the HLA class II region as well as within Chromosome 12q24.

Reactive oxygen species (ROS) produced in the mitochondrial respiratory chain (RC) are primary signals that modulate cellular adaptation to environment, and are also destructive factors that damage cells under the conditions of hypoxia/reoxygenation relevant for various systemic diseases or transplantation. The important role of ROS in cell survival requires detailed investigation of mechanism and determinants of ROS production. To perform such an investigation we extended our rule-based model of complex III in order to account for electron transport in the whole RC coupled to proton translocation, transmembrane electrochemical potential generation, TCA cycle reactions, and substrate transport to mitochondria. It fits respiratory electron fluxes measured in rat brain mitochondria fueled by succinate or pyruvate and malate, and the dynamics of NAD+ reduction by reverse electron transport from succinate through complex I. The fitting of measured characteristics gave an insight into the mechanism of underlying processes governing the formation of free radicals that can transfer an unpaired electron to oxygen-producing superoxide and thus can initiate the generation of ROS. Our analysis revealed an association of ROS production with levels of specific radicals of individual electron transporters and their combinations in species of complexes I and III. It was found that the phenomenon of bistability, revealed previously as a property of complex III, remains valid for the whole RC. The conditions for switching to a state with a high content of free radicals in complex III were predicted based on theoretical analysis and were confirmed experimentally. These findings provide a new insight into the mechanisms of ROS production in RC.

Author Summary

Respiration at the level of mitochondria is considered as delivery of electrons and protons from NADH or succinate to oxygen through a set of transporters constituting the respiratory chain (RC). Mitochondrial respiration, dealing with transfer of unpaired electrons, may produce reactive oxygen species (ROS) such as O2− and subsequently H2O2 as side products. ROS are chemically very active and can cause oxidative damage to cellular components. The production of ROS, normally low, can increase under stress to the levels incompatible with cell survival; thus, understanding the ways of ROS production in the RC represents a vital task in research. We used mathematical modeling to analyze experiments with isolated brain mitochondria aimed to study relations between electron transport and ROS production. Elsewhere we reported that mitochondrial complex III can operate in two distinct steady states at the same microenvironmental conditions, producing either low or high levels of ROS. Here, this property of bistability was confirmed for the whole RC. The associations between measured ROS production and computed individual free radical levels in complexes I and III were established. The discovered phenomenon of bistability is important as a basis for new strategies in organ transplantation and therapy.

Background

Prior to organ harvesting, an attempt was made to modulate the donor’s immune responses against prospective xenogeneic recipients by infusion of “recipient-type” bone marrow.

Methods

For this purpose, baboons conditioned with total lymphoid irradiation were given 6 × 108 unmodified human bone marrow cells/kg body weight with no subsequent treatment.

Results

Animals survived until they were euthanized at 18 months. Using primers specific for human chorionic gonadotropin gene, the presence of human DNA was confirmed by polymerase chain reaction in the blood of one animal for up to 18 months after cell transplantation; in the other animal, xenogeneic chimerism became undetectable in the blood at 6 months after bone marrow infusion. However, tissue samples obtained from both animals at the time they were euthanized had evidence of donor (human) DNA. Additionally, the presence of donor DNA in individually harvested colonies of erythroid and myeloid lineages suggested that infused human bone marrow cells had engrafted across the xenogeneic barrier in both baboons.

Conclusions

Bone marrow transplantation from human to baboon leads to establishment of chimerism and modulation of donor-specific immune reactivity, which suggests that this strategy could be reproducibly employed to create “surrogate” tolerogenesis in prospective donors for subsequent organ transplantation across xenogeneic barriers.

Galactose-α1,3-galactose(αGal) epitopes, the synthesis of which requires the enzyme product of α1,3-galactosyltransferase (α1,3GT), are sugar chains on the cell surface of most mammalian species. Notable exceptions are higher primates including Old World monkeys, apes, and humans. The αGal-negative species as well as mice with deletion of the α1,3GT gene produce abundant anti-αGal antibodies. The evolutionary loss of αGal epitopes has been attributed to point mutations in the coding region of the gene. Because no transcripts could be found in the higher primate species with Northern blot analysis, a potential alternative explanation has been loss of upstream regulation of the gene. Here, we have demonstrated that the rhesus promoter is functional. More importantly, a variety of full-length transcripts were detected with sensitive PCR-based methods in the tissues of rhesus monkeys, orangutans, and humans. Five crucial mutations were delineated in the coding region of the human and rhesus and three in the orangutan, any one of which could be responsible for inactivation of the α1,3GT gene. Two of the mutations were shared by all three higher primates. These findings, which elucidate the molecular basis for the evolutionary loss of αGal expression, may have implications in medical research.

It has been postulated that the resident “passenger” leukocytes of hematolymphoid origin that migrate from whole organ grafts and subsequently establish systemic chimerism are essential for graft acceptance and the induction of donor-specific nonreactivity. This phenomenon was augmented by infusing 3×108 unmodified donor bone-marrow cells into 40 patients at the time of organ transplantation. Fifteen of the first 18 analyzable patients had sequential immunological evaluation over postoperative intervals of 5 to 17 months, (which included 7 kidney (two with islets), 7 liver (one with islets), and one heart recipient). The evolution of changes was compared with that in 16 kidney and liver nonmarrow controls followed for 4 to 5 months. The generic immune reactivity of peripheral blood mononuclear cells (PBMC) was determined by their proliferative responses to mitogens (PHA, ConA). Alloreactivity was measured by the recipient mixed lymphocyte reaction (MLR) to donor and HLA-mismatched third-party panel cells. Based on all 3 tests, the recipients were classified as donor-specific hyporeactive, intermediate, and responsive; patients who were globally suppressed made up a fourth category. Eight (53%) of the 15 marrow-treated recipients exhibited progressive modulation of donor-specific reactivity (3 hyporeactive and 5 intermediate) while 7 remained antidonor-responsive. In the nonmarrow controls, 2 (12.5%) of the 16 patients showed donor-specific hyporeactivity, 10 (62.5%) were reactive, and 4 (25%) studied during a CMV infection had global suppression of responsiveness to all stimuli.

Summary

Background

Insight into the mechanisms of organ engraftment and acquired tolerance has made it possible to facilitate these mechanisms, by tailoring the timing and dosage of immunosuppression in accordance with two therapeutic principles: recipient pretreatment, and minimum use of post-transplant immunosuppression. We aimed to apply these principles in recipients of renal and extrarenal organ transplants.

Methods

82 patients awaiting kidney, liver, pancreas, or intestinal transplantation were pretreated with about 5 mg/kg of a broadly reacting rabbit antithymocyte globulin during several hours. Post-transplant immunosuppression was restricted to tacrolimus unless additional drugs were needed to treat breakthrough rejection. After 4 months, patients on tacrolimus monotherapy were considered for dose-spacing to every other day or longer intervals.

Findings

We frequently saw evidence of immune activation in graft biopsy samples, but unless this was associated with graft dysfunction or serious immune destruction, treatment usually was not intensified. Immunosuppression-related morbidity was virtually eliminated. 78 (95%) of 82 patients survived at 1 year and at 13–18 months. Graft survival was 73 (89%) of 82 at 1 year and 72 (88%) of 82 at 13–18 months. Of the 72 recipients with surviving grafts, 43 are on spaced doses of tacrolimus monotherapy: every other day (n=6), three times per week (11), twice per week (15), or once per week (11).

Interpretation

The striking ability to wean immunosuppression in these recipients indicates variable induction of tolerance. The simple therapeutic principles are neither drug-specific nor organ-specific. Systematic application of these principles should allow improvements in quality of life and long-term survival after organ transplantation.

A sample of 492 full heritage, unrelated residents of the Gila River Indian Community (GRIC) of Arizona were characterized for their high resolution DNA alleles at the HLA-A, B, C, DRB1, DQA1, and DQB1 loci. Only 5 allelic categories are found at HLA-A, 10 at HLA-B, 8 at HLA-C and HLA-DR, and 4 at DQA1 and DQB1. There is little evidence for population structure at the 6 loci. Two “private” alleles, B*5102 and B*4005, that are found nearly exclusively in American Indian populations in the desert southwest and northern Mexico, are likely new mutations after the first inhabitation of the area, the evolution of which are reflected in the contemporary distribution of their respective haplotypes. DRB1*1402 has the highest reported frequency of any specificity at the DRB1 locus, 0.7461, and serves as a sensitive probe for locating related east Asian populations. The haplotypes in this population also exhibit a highly restricted distribution and strong genetic disequilibria, which has important implications for matching solid organ and bone marrow allografts. It is shown that, when one considers HLA-A-B-DRB1 homozygotes as allograft donors for all full heritage members of the GRIC, 50% of the community would find a non-mismatched organ within the homozygotes for the 6 most common haplotypes. This raises questions about transplantation policy and whether, in the presence of high frequency private alleles and a restricted number of haplotypes, the full heritage American Indian community of the desert southwest should act as its own pool of donors for its affected members.