Combined lie's type D and type A agenesis of the left internal carotid artery diagnosed by magnetic resonance angiography.

PURPOSE: To report an unusual case of combined Lie's types A and D of internal carotid artery (ICA) agenesis, diagnosed by magnetic resonance angiography (MRA). METHODS: A 60-year-old woman with dizziness underwent cranial magnetic resonance imaging (MRI) and MRA of the intracranial region for the evaluation of brain and vascular lesions. The magnetic resonance machine was a 3.0-T scanner. RESULTS: MRI showed no abnormalities, except for multiple small white matter lesions. MRA showed that the left ICA was absent, except for the supraclinoid segment, and an anastomotic vessel was present between the paraclinoid segments of the bilateral ICAs, indicating Lie's type D ICA agenesis. The left posterior communicating artery (PCoA) was also present. Thus, there were also features of type A ICA agenesis. The anastomotic vessels between the bilateral ICAs and ipsilateral PCoA were relatively small in caliber. CONCLUSION: Lie's type D ICA agenesis usually does not communicate with the anterior and posterior circulations. We encountered a case of combined type D and type A ICA agenesis. To our knowledge, no similar case has been reported in the English literature. This is the second case of type D ICA agenesis with patent ipsilateral PCoA. We speculate that in case of type A ICA agenesis, when the development of the PCoA is insufficient to support collateral blood flow, an anastomotic vessel between bilateral ICAs may develop.

Visualization of the Internal Thoracic Arteries in Giant Cell Arteritis on 18 F-FDG Semiconductor PET/CT.

ABSTRACT: A 72-year-old woman presented with the fever and the pain of skull and face for 2 weeks. 18 F-FDG PET/CT equipped with semiconductor detectors revealed strong uptake not only in the temporal, cervical, subclavian arteries, and aorta, but also in the bilateral internal thoracic arteries. The diagnosis of giant cell arteritis was made. Semiconductor PET can visualize small arteries such as the internal thoracic artery. The patients with giant cell arteritis are at a high risk of ischemic heart disease, and inflammatory involvement of the internal thoracic arteries may affect the outcome of coronary artery bypass grafting.

State of the science in reconciling top-down and bottom-up approaches for terrestrial CO2 budget.

Robust estimates of CO2 budget, CO2 exchanged between the atmosphere and terrestrial biosphere, are necessary to better understand the role of the terrestrial biosphere in mitigating anthropogenic CO2 emissions. Over the past decade, this field of research has advanced through understanding of the differences and similarities of two fundamentally different approaches: "top-down" atmospheric inversions and "bottom-up" biosphere models. Since the first studies were undertaken, these approaches have shown an increasing level of agreement, but disagreements in some regions still persist, in part because they do not estimate the same quantity of atmosphere-biosphere CO2 exchange. Here, we conducted a thorough comparison of CO2 budgets at multiple scales and from multiple methods to assess the current state of the science in estimating CO2 budgets. Our set of atmospheric inversions and biosphere models, which were adjusted for a consistent flux definition, showed a high level of agreement for global and hemispheric CO2 budgets in the 2000s. Regionally, improved agreement in CO2 budgets was notable for North America and Southeast Asia. However, large gaps between the two methods remained in East Asia and South America. In other regions, Europe, boreal Asia, Africa, South Asia, and Oceania, it was difficult to determine whether those regions act as a net sink or source because of the large spread in estimates from atmospheric inversions. These results highlight two research directions to improve the robustness of CO2 budgets: (a) to increase representation of processes in biosphere models that could contribute to fill the budget gaps, such as forest regrowth and forest degradation; and (b) to reduce sink-source compensation between regions (dipoles) in atmospheric inversion so that their estimates become more comparable. Advancements on both research areas will increase the level of agreement between the top-down and bottom-up approaches and yield more robust knowledge of regional CO2 budgets.

Erythema Nodosum Masking Kawasaki Disease with an Initial Manifestation of Skin Lesions.

We report the first case demonstrating an association between Kawasaki disease (KD) and erythema nodosum (EN). A 3-year-old girl presented with EN as an initial manifestation of KD. At the initial visit, she showed high fever of 40°C, injection of the oropharynx, cervical lymphadenopathy, and red-purple cutaneous nodules, particularly on the lower limbs. She complained of severe pain in the neck and cutaneous lesions. Initially, the development of EN was attributed to Salmonella spp infection, which was detected in stool culture. However, the patient did not respond to high-dose ampicillin/sulbactam to which the Salmonella spp is sensitive. Echocardiography performed as screening for fever of unknown origin revealed medium-sized aneurysms of the left anterior descending artery. EN masked the diagnosis of KD, and the patient developed a coronary artery lesion. KD should be considered in the differential diagnosis of refractory EN in pediatric patients.

Prediction of health effects of cross-border atmospheric pollutants using an aerosol forecast model.

Health effects of cross-border air pollutants and Asian dust are of significant concern in Japan. Currently, models predicting the arrival of aerosols have not investigated the association between arrival predictions and health effects. We investigated the association between subjective health symptoms and unreleased aerosol data from the Model of Aerosol Species in the Global Atmosphere (MASINGAR) acquired from the Japan Meteorological Agency, with the objective of ascertaining if these data could be applied to predicting health effects. Subjective symptom scores were collected via self-administered questionnaires and, along with modeled surface aerosol concentration data, were used to conduct a risk evaluation using generalized estimating equations between October and November 2011. Altogether, 29 individuals provided 1670 responses. Spearman's correlation coefficients were determined for the relationship between the proportion of the participants reporting the maximum score of two or more for each symptom and the surface concentrations for each considered aerosol species calculated using MASINGAR; the coefficients showed significant intermediate correlations between surface sulfate aerosol concentration and respiratory, throat, and fever symptoms (R = 0.557, 0.454, and 0.470, respectively; p < 0.01). In the general estimation equation (logit link) analyses, a significant linear association of surface sulfate aerosol concentration, with an endpoint determined by reported respiratory symptom scores of two or more, was observed (P trend = 0.001, odds ratio [OR] of the highest quartile [Q4] vs. the lowest [Q1] = 5.31, 95% CI = 2.18 to 12.96), with adjustment for potential confounding. The surface sulfate aerosol concentration was also associated with throat and fever symptoms. In conclusion, our findings suggest that modeled data are potentially useful for predicting health risks of cross-border aerosol arrivals.

Land use change and El Niño-Southern Oscillation drive decadal carbon balance shifts in Southeast Asia.

An integrated understanding of the biogeochemical consequences of climate extremes and land use changes is needed to constrain land-surface feedbacks to atmospheric CO2 from associated climate change. Past assessments of the global carbon balance have shown particularly high uncertainty in Southeast Asia. Here, we use a combination of model ensembles to show that intensified land use change made Southeast Asia a strong source of CO2 from the 1980s to 1990s, whereas the region was close to carbon neutral in the 2000s due to an enhanced CO2 fertilization effect and absence of moderate-to-strong El Niño events. Our findings suggest that despite ongoing deforestation, CO2 emissions were substantially decreased during the 2000s, largely owing to milder climate that restores photosynthetic capacity and suppresses peat and deforestation fire emissions. The occurrence of strong El Niño events after 2009 suggests that the region has returned to conditions of increased vulnerability of carbon stocks.

SV40 infection associated with rituximab treatment after kidney transplantation in nonhuman primates.

BACKGROUND: A regimen consisting of polyclonal anti-T-cell antibody, sirolimus (SRL), and donor bone marrow (DBM) infusion induces robust transplantation tolerance to skin allografts in mice. We investigated the effect of a similar regimen in a nonhuman primate (NHP) model. METHODS: Cynomolgus macaques (Macaca fascicularis) were transplanted with mismatched kidney allografts. Recipients were treated with 7 doses of antithymocyte globulin (Thymoglobulin, day 1 to 9), sirolimus, and DBM infusion (day 14). Anti-CD20 antibody, rituximab, was given on days 0 and 5. RESULTS: A regimen of Thymoglobulin, 30 days of SRL, and DBM infusion induced significantly greater prolongation of graft survival with a mean survival time of 88 days compared with the control regimen (no DBM) with an mean survival time of 53 days (P=0.022). Unlike the murine skin allograft model, all grafts were rejected within 111 days. A combination of Thymoglobulin, continuous SRL, and rituximab caused graft and systemic SV40 infection and failed to achieve further extension of graft survival. C4d deposition was observed in 50% of recipients as early as 18 days, suggesting antidonor antibody production. A transient, low-to-moderate degrees of multilineage chimerism was observed after DBM infusion. Treatment with Thymoglobulin resulted in profound depletion of CD4+ and CD8+ T cells, whereas addition of rituximab achieved prolonged (up to 3 months) depletion of CD20+ B cells. CONCLUSION: The Thymoglobulin, SRL, and DBM protocol is simple and produces long-term kidney allograft survival in NHP although additional treatment modalities may be necessary for induction of long-term tolerance.

OX40 controls functionally different T cell subsets and their resistance to depletion therapy.

T cell depletion is a widely used approach in clinical transplantation. However, not all T cells are equally sensitive to depletion therapies and a significant fraction of T cells persists even after aggressive treatment. The functional attributes of such T cells and the mechanisms responsible for their resistance to depletion are poorly studied. In the present study, we showed that CD4(+) T cells that are resistant to polyclonal anti-lymphocyte serum (ALS) mediated depletion exhibit phenotypic features of memory cells and uniformly express OX40 on the cell surface. Studies using the foxp3gfp knockin mice revealed that the remaining CD4(+)OX40(+) cells consist of Foxp3(+) Tregs and Foxp3(-) T effector/memory cells. The ALS-resistant CD4(+)OX40(+) cells failed to mediate skin allograft rejection upon adoptive transferring into congenic Rag(-/-) mice, but removal of Foxp3(+) Tregs from the OX40(+) cells resulted in prompt skin allograft rejection. Importantly, OX40 is critical to survival of both Foxp3(+) Tregs and T effector/memory cells. However, OX40 exhibits opposing effects on the functional status of Foxp3(+) Tregs and T effector/memory cells, as stimulation of OX40 on T effector cells induced amplified cell proliferation but stimulation of OX40 on the Foxp3(+) Tregs impaired their suppressor functions. Our study demonstrates that OX40 is a critical molecule in regulating survival and functions of depletion-resistant T cells; and these findings may have important clinical implications.

Role of stromal-derived factor-1alpha in the induction of circulating CD34+CXCR4+ progenitor cells after cardiac surgery.

BACKGROUND: Cardioplegia and cardiopulmonary bypass (CP/CPB) leads to an increase in circulating progenitor cells. The role of stromal-derived factor-1alpha (SDF-1alpha), a key regulator of progenitor cell mobilization, and other cytokines in this process is not clear. METHODS AND RESULTS: Peripheral blood (n=24), atrial and skeletal tissue (n=6) samples were taken from patients undergoing CP/CPB before (pre-CP/CPB), 4 hours (post-CP/CPB), and 4 days (POD4) after CP/CPB. The number of circulating CD34+CXCR4+ cells increased post-CP/CPB (442+/-53 versus 286+/-27; P=0.04 versus pre-CP/CPB), but not at POD4 (382+/-50; P=0.28 versus pre-CP/CPB). Plasma levels of SDF-1alpha increased post-CP/CPB as compared with pre-CP/CPB (3325+/-325 versus 2911+/-165 pg/mL; P=0.046) but returned to baseline at POD4 (2838+/-224 pg/mL; P=0.90). Plasma levels of vascular endothelial growth factor were similar post-CP/CPB (P=0.90 versus pre-CP/CPB) but increased at POD4 (220+/-40 pg/mL versus 134+/-26 pg/mL; P=0.04 versus pre-CP/CPB). Serum levels of granulocyte-colony stimulating factor (G-CSF) increased early after CP/CPB as compared with pre-CP/CPB (265.0+/-41.7 versus 11.1+/-1.1 pg/mL; P<0.001) and returned to baseline at POD4 (P=0.84 versus pre-CP/CPB). The circulating CD34+CXCR4+ cells were positively correlated with plasma levels of SDF-1alpha early after CP/CPB (r=0.56, P<0.01), but not at other times. Protein expression of SDF-1alpha was elevated in the atrial myocardium after CP/CPB (9.4-fold; P=0.03). CONCLUSIONS: Exposure to CP/CPB leads to an increase in circulating CD34+CXCR4+ progenitor cells, which is associated with increased myocardial SDF-1alpha expression. The numbers of CD34+CXCR4+ progenitor cells positively correlate with the plasma levels of SDF-1alpha post-CP/CPB, suggesting an important role of SDF-1alpha in progenitor cell mobilization.

Short administration of polyclonal anti-T cell antibody (ALS) in NOD mice with extensive insulitis prevents subsequent development of autoimmune diabetes.

Treatment of overtly diabetic NOD mice with antilymphocyte serum (ALS), a polyclonal anti-T cell antibody, leads to cure of diabetes. Here, we investigated whether ALS-treatment of NOD mice after development of extensive insulitis prevents onset of diabetes. Female NOD mice were treated with two doses of ALS at 14, 19 or 23 weeks of age. No further treatment was given. In untreated female NOD mice, diabetes developed starting at 13 weeks and reached 68% by 37 weeks. ALS-treatment at 14, 19 or 23 weeks when histology showed progressive insulitis completely prevented onset of overt diabetes in 9/12, 11/12 or 12/12 mice, respectively. Intraperitoneal glucose tolerance tests in 43 week-old ALS-treated, diabetes-free mice showed a normal pattern. Co-adoptive transfer of lymphoid cells prepared from ALS-treated diabetes-free mice together with splenocytes from overtly diabetic NOD mice resulted in marked delay in diabetes onset in NOD.SCID mice, suggesting the presence of autoimmune regulatory cells in ALS-treated mice. Autoimmune regulatory cells were CD4(+)CD25(+), but not CD4(+)CD25(-), T cells. Thus, treatment of euglycemic individuals who already show signs of autoimmune diabetes with a short course of polyclonal anti-T cell antibody may effectively prevent onset of type 1 diabetes mellitus.

CD4+ regulatory T cells are spared from deletion by antilymphocyte serum, a polyclonal anti-T cell antibody.

Broad T cell depletion has been used as an integral part of treatment in transplantation and autoimmune diseases. Following depletion, residual T cells undergo homeostatic proliferation and convert to memory-like T cells. In this study, we investigated the effect of T cell depletion by antilymphocyte serum (ALS), a polyclonal anti-T cell Ab, on CD4(+) regulatory T cells. After ALS treatment, CD4(+)CD25(+) T cells underwent proliferation and expressed a memory T cell marker, CD44. One week after ALS treatment, both CD25(+) and CD25(-) T cells exhibited increased suppression of alloresponses in vitro, which waned thereafter to the levels mediated by naive CD25(+) and CD25(-) T cells. By real-time PCR analyses, ALS treatment of CD4-deficient mice adoptively transferred with Thy1.2(+)CD4(+)CD25(+)Foxp3(+) and Thy1.1(+)CD4(+)CD25(-)Foxp3(-) T cells resulted in the appearance of Thy1.2(+)CD4(+)CD25(-)Foxp3(+) and Thy1.1(+)CD4(+)CD25(+)Foxp3(+) T cells, suggesting the conversion between CD25(+) and CD25(-) T cells. Naive CD25(+) T cells expressed a higher level of intracellular Bcl-x(L) than CD25(-) T cells. Up-regulation of the Bcl-x(L) molecule during ALS-induced homeostatic expansion further promoted survival of CD25(+) and, to a lessor degree, CD25(-) cells. These results indicate that CD25(+) T cells are spared from ALS-mediated deletion, with some CD25(+) T cells converting to CD25(-) T cells, and continue to exhibit regulatory activity. The concomitant presence of T cell deletion and continuous regulatory T cell activity may underlie the therapeutic effect of ALS, particularly in treatment of autoimmune diseases.

Prevention and cure of autoimmune diabetes in nonobese diabetic mice by continuous administration of FTY720.

BACKGROUND: Treatment of nonobese diabetic (NOD) mice with FTY720 before the development of insulitis prevents the onset of diabetes. In this study, the authors investigated whether FTY720 treatment of NOD mice with established insulitis prevents the development of diabetes. METHODS: FTY720 (1 mg/kg) was administered continuously to euglycemic NOD mice starting at 14 or 23 weeks of age. A group of untreated, age-matched NOD mice served as controls. Mice with more than 300 mg/dL blood glucose on three consecutive measurements were considered diabetic. RESULTS: Diabetes developed in control mice starting at 13 weeks of age and reached 78% by 33 weeks of age. Mice at 14 and 23 weeks of age exhibited extensive insulitis that progressed with age. Continuous oral administration of FTY720 starting at either age completely prevented the development of diabetes. However, its withdrawal at 37 weeks of age led to abrupt diabetes onset. Pancreases of FTY720-treated diabetes-free mice showed peripheral insulitis, with strong insulin staining. The protection from diabetes was also achieved by intraperitoneal injection of FTY720 or sirolimus (1.5 mg/kg). Unlike FTY720, withdrawal of sirolimus did not induce diabetes. Continuous oral FTY720 (3 mg/kg) treatment in overtly diabetic NOD mice led to complete reversal of diabetes in 6 of 11 mice. The standard adoptive transfer study in NOD-severe combined immunodeficient mice showed that peripheral lymphoid organs of FTY720-treated mice contained diabetogenic cells but not dominant immunoregulatory cells. CONCLUSIONS: FTY720, which does not cause generalized immunosuppression, may be a safe and benign therapeutic agent for chronic use to prevent or cure type 1 diabetes.

Active role of chimerism in transplantation tolerance induced by antilymphocyte serum, sirolimus, and bone-marrow-cell infusion.

BACKGROUND: A regimen consisting of antilymphocyte serum (ALS), sirolimus, and donor bone-marrow-cell (BMC) infusion induces indefinite skin allograft survival across fully mismatched mouse strain combinations. We investigated the role of chimerism in this transplantation tolerance model. MATERIALS: B10.A (H-2a) mice were treated with ALS on day -1 and 2, sirolimus, and infusion of (C57BL/6xDBA/2)F1 (B6D2F1, H-2(b/d)) BMCs on day 7 relative to DBA/2 (D2) skin grafting on day 0. At postgraft days 30, 50 and 120, the recipient mice were injected intravenously with splenocytes prepared from either naive or D2 mixed chimeric B10.A mice that had been sensitized in vivo to B6. Changes in chimerism and graft survival were monitored. RESULTS: Although D2 skin grafts were rejected with a median survival time of 63.8 days in B10.A mice given ALS and sirolimus alone, they survived more than 200 days in all B10.A mice given ALS, sirolimus, and B6D2F1 BMCs. Chimerism became evident 21 days postgrafting and progressively increased thereafter to 20% at postgraft day 200. Infusion of anti-B6 presensitized cells resulted in depletion of chimeric donor cells and subsequent graft rejection regardless of the timing of injection. Injection of presensitized cells in mice given ALS and sirolimus alone had no effect on graft survival. Injection of presensitized cells that were cytotoxic to alloantigen expressed by BMCs but tolerant to skin reduced, but did not deplete, established chimerism and allowed continued allograft survival. CONCLUSIONS: Chimeric donor cells play a major role in both the early and late phases of transplantation tolerance induced by the ALS, sirolimus, and BMC regimen.

Penasulfate A, a new alpha-glucosidase inhibitor from a marine sponge Penares sp.

A new alpha-glucosidase inhibitor, penasulfate A, has been isolated from a marine sponge Penares sp.(1) Its structure was elucidated by spectral and chemical methods to be a scalemic mixture of methyl pipecolates acylated with a novel sulfated fatty acid.

Cure of overt diabetes in NOD mice by transient treatment with anti-lymphocyte serum and exendin-4.

Treatment of overtly diabetic NOD mice with anti-lymphocyte serum (ALS), a polyclonal anti-T-cell antibody, abrogates autoimmunity and achieves partial clinical remission. Here we investigated whether the addition of exendin-4, a hormone that stimulates insulin secretion and beta-cell replication and differentiation, improves induction of remission by ALS. Transient treatment of overtly diabetic NOD mice with ALS and exendin-4 achieved complete remission in 23 of 26 mice (88%) within 75 days, accompanied by progressive normalization of glucose tolerance, improved islet histology, increased insulin content in the pancreas, and insulin release in response to a glucose challenge. Syngeneic islets transplanted into mice cured by treatment with ALS plus exendin-4 remained intact, and cotransfer of lymphocytes from cured mice delayed diabetes induction by adoptive transfer, suggesting the long-lasting presence of autoimmune regulatory cells. Although ALS alone also achieved reversal of diabetes, the frequency of remission was low (40%). No treatment or exendin-4 alone failed to produce remission. These results show that exendin-4 synergistically augments the remission-inducing effect of ALS. The addition of beta-cell growth factors, such as exendin-4, to immunotherapy protocols with anti-T-cell antibodies presents a potential novel approach to the cure of patients with new-onset type 1 diabetes.

Chimeric donor cells play an active role in both induction and maintenance phases of transplantation tolerance induced by mixed chimerism.

Donor hemopoietic cell engraftment is considered to be an indicator of allograft tolerance. We depleted chimerism with cells specifically presensitized to the bone marrow donor to investigate its role in mixed chimera-induced tolerance. Three experimental models were used: model A, B10.A cells presensitized to B6 (a anti-b cells) were injected into (B6 x D2)F(1) --> B10.A mixed chimeras grafted with DBA/2 skin; model B, anti-B6 presensitized cells prepared in DBA/2 --> B10.A mixed chimeras, thus unresponsive to DBA/2 (a anti-b/tol-d cells), were injected into (B6 x D2)F(1) --> B10.A mixed chimeras grafted with DBA/2 skin; and model C, (BALB/c x B6)F(1) cells presensitized to CBA (d/b anti-k cells) were injected into (B6 x CBA)F(1) --> BALB/c mixed chimeras grafted with B6 skin. Skin was grafted on day 30. Injection of each cell type before skin grafting abolished hemopoietic cell engraftment and prevented allograft acceptance. Injection of presensitized cells after skin grafting resulted in different outcomes depending on the models. In model A, injection of a anti-b cells completely depleted chimerism and caused allograft rejection. In model B, injection of a anti-b/tol-d cells markedly reduced, but did not deplete, peripheral chimerism and maintained skin allograft survival. In model C, d/b anti-k cells reduced chimerism to the background levels but failed to cause graft rejection, probably due to persistence of injected cells which share MHC with skin grafts. Together, the results show that presence of chimeric donor cells is essential in both the induction and maintenance phases of tolerance induced by mixed chimerism.

Prevention of autoimmune diabetes by FTY720 in nonobese diabetic mice.

BACKGROUND: FTY720 prevents allograft rejection with remarkable potency without inducing generalized immunosuppression. We determined the effect of FTY720 on development of autoimmune diabetes in nonobese diabetic (NOD) mice. METHODS: NOD mice were given FTY720 (0.5 mg/kg, orally) five times per week starting from 4 weeks of age. RESULTS: Daily FTY720 prevented development of diabetes in 15 of 16 treated mice, whereas 70% of untreated NOD mice became diabetic by 35 weeks of age. Withdrawal of FTY720 at 35 weeks of age led to development of diabetes within 2 weeks in five mice, whereas the remaining mice maintained diabetes-free conditions for up to 44 weeks of age. No side effect of the drug was seen throughout the treatment period. FTY720 also prevented cyclophosphamide-induced diabetes in NOD mice. CONCLUSIONS: FTY720 is a safe and benign therapeutic agent that may be used chronically in prediabetic individuals.

Immunologic mechanisms in tolerance produced in mice with nonradiation-based lymphoablation and donor-specific bone marrow.

BACKGROUND: These experiments evaluate the mechanisms associated with tolerance in mice treated with sirolimus, antilymphocyte serum (ALS), and donor-specific bone marrow (BM). METHODS: Tolerance to fully MHC-incompatible skin allografts was induced as follows: C57Bl/10 (H2b) recipients received 0.5 mL of rabbit anti-mouse polyclonal ALS on days -1, +2, and +5 relative to B10.A (H2k) donor skin grafting on day 0. Sirolimus was given in a single dose (24 mg/kg intraperitoneally) on day 6. Freshly harvested B10.A (H2k) donor-specific BM was administered at a dose of 25 x 10(6) (ALS/BM25/sirolimus) or 150 x 10(6) (ALS/BM150/sirolimus) cells intravenously on day 7. Skin allograft survival was correlated with the recipient's immunologic status. Recipients were assayed for suppressor cell activity (mixed lymphocyte coculture assays), clonal deletion (T-cell receptor Vbeta11 assay), peripheral and thymic chimerism (flow cytometry and reverse transcriptase polymerase chain reaction), and anergy (response to exogenous interleukin 2). RESULTS: Mice treated with ALS/BM25/sirolimus showed specifically prolonged but not indefinite allograft survival (median survival time 116 days). Allograft survival correlated with donor-specific clonal deletion and the presence of donor class II mRNA in the recipient's thymus. Mice given the ALS/BM150/sirolimus protocol showed indefinite donor-specific tolerance. Tolerance could not be broken with the administration of high doses of interleukin 2. Splenocytes taken from mice 14 days after tolerance induction inhibited donor-specific and third-party mixed lymphocyte culture proliferation in a dose-dependent fashion. This suppression could be ablated by depleting splenocytes of cells of donor origin before use in coculture. Clonal deletion was detectable 30 days after tolerance induction in mice treated with ALS/BM150/sirolimus and was maintained indefinitely. CONCLUSION: Induction of tolerance by ALS, BM, and sirolimus results in a state of donor-specific tolerance, and multilineage chimerism evolves that is permanent and associated with clonal deletion of alloreactive T cells.