Nanoparticle delivery of mycophenolic acid upregulates PD-L1 on dendritic cells to prolong murine allograft survival.

Conventional immunosuppressive drug delivery requires high systemic drug levels to provide therapeutic benefit, but frequently results in toxic side effects. Novel drug delivery methods, such as FDA-approved poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs), are promising drug delivery platforms to reduce drug doses and minimize toxicity. Using murine models of skin transplantation, we investigated whether PLGA NPs would effectively deliver mycophenolic acid (MPA), a common clinical immunosuppressant, and avoid the toxicity of conventional drug delivery. We found that intermittent treatment with NPs encapsulated with MPA (NP-MPA) resulted in a significant extension of allograft survival than intermittent conventional MPA treatment even though the concentration of MPA within NP-MPA was a 1000-fold lower than conventional drug. Importantly, recipients who were administered NP-MPA intermittently avoided drug toxicity, whereas those treated with daily conventional drug manifested cytopenias. Dendritic cells (DCs) endocytosed NP-MPA to upregulate programmed death ligand-1 (PD-L1) and displayed a decreased ability to prime alloreactive T cells. Importantly, the ability of NP-MPA to promote allograft survival was partly PD-L1 dependent. Collectively, this study indicates that NPs are potent drug delivery tools that extend allograft survival without drug toxicity.

Aging augments IL-17 T-cell alloimmune responses.

As increasing numbers of elderly patients require solid organ transplantation, the need to better understand how aging modifies alloimmune responses increases. Here, we examined whether aged mice exhibit augmented, donor-specific memory responses prior to transplantation. We found that elevated donor-specific IL-17, but not IFN-gamma, responses were observed in aged mice compared to young mice prior to transplantation. Further characterization of the heightened IL-17 alloimmune response with aging demonstrated that memory CD4(+) T cells were required. Reduced IL-2 alloimmune responses with age contributed to the elevated IL-17 phenotype in vitro, and treatment with an anti-IL-17 antibody delayed the onset of acute allograft rejection. In conclusion, aging leads to augmented, donor-specific IL-17 immune responses that are important for the timing of acute allograft rejection in aged recipients. IL-17 targeting therapies may be useful for averting transplant rejection responses in older transplant recipients.

The role of hyaluronan degradation products as innate alloimmune agonists.

Dendritic cells (DCs) play a key role in initiating alloimmunity yet the substances that activate them during the host response to transplantation remain elusive. In this study we examined the potential roles of endogenous innate immune agonists in activating dendritic cell-dependent alloimmunity. Using a murine in vitro culture system, we show that 135 KDa fragments of the extracellular matrix glycosaminoglycan hyaluronan induce dendritic cell maturation and initiate alloimmunity. Priming of alloimmunity by hyaluronan-activated DCs was dependent on signaling via TIR-associated protein, a Toll-like receptor (TLR) adaptor downstream of TLRs 2 and 4. However, this effect was independent of alternate TLR adaptors, MyD88 or Trif. Using an in vivo murine transplant model, we show that hyaluronan accumulated during skin transplant rejection. Examination of human lung transplant recipients demonstrated that increased levels of intragraft hyaluronan were associated with bronchiolitis obliterans syndrome. In conclusion, our study suggests that fragments of hyaluronan can act as innate immune agonists that activate alloimmunity.

Peripheral vascular disease intervention in patients with end-stage renal disease: few complications in those treated with peritoneal dialysis.

BACKGROUND: We assessed the results of peripheral vascular surgery in patients with end-stage renal disease (ESRD) who were being treated with peritoneal dialysis. METHODS: Sixty-seven ESRD patients on peritoneal dialysis who had peripheral vascular surgery were assessed retrospectively for preoperative risk factors, primary and secondary patency rates, and mortalitv. The study group had 48 proximal femoral-popliteal bypasses, 12 distal femoral-popliteal bypasses, and 7 distal femoral-tibial and/or peroneal revascularizations. RESULTS: Among 67 peritoneal dialysis patients, 15 deaths (22%) occurred over 68 months (mean, 14 months). CONCLUSION: Patients on peritoneal dialysis had adequate patency rates and length of survival after peripheral vascular surgery when maintained on peritoneal dialysis.

Indefinite allograft survival mediated by donor bone marrow is dependent on the presence of a functional CD95 (Fas) gene in recipients.

It is well documented that donor bone marrow in combination with peri-transplant anti-thymocyte globulin (ATG) administration induces transplantation tolerance in a variety of animal models. Our previous work showed that the ability of donor marrow to induce tolerance was dependent on the presence of CD95 ligand (Fas-ligand) on the donor cells. In this study we investigate whether CD95 (Fas) on the recipient cells is required. By comparing skin allograft survival times between wild-type C57BL/6 ATG-treated recipients and C57BL/6(lpr/lpr) ATG-treated recipients (which do not have a functional CD95 gene), we show that donor bone marrow could induce indefinite transplant survival (median survival time >200 days) only in recipients with a functional CD95 gene. Thus, we conclude that the CD95 ligand-CD95 apoptotic pathway plays a major role in donor bone marrow-induced transplantation tolerance.

An essential role for natural killer cells in augmentation of allograft survival mediated by donor spleen cells.

BACKGROUND: Previous studies have shown that skin allograft survival can be augmented by the administration of donor spleen or donor bone marrow in antithymocyte serum (ATS) treated recipients. Because natural killer cells (NK) have been reported to possess immunoregulatory properties, we investigated whether the ability of donor spleen or bone marrow cells to enhance allograft survival was dependent on the presence of donor NK cells. METHODS: Recipient (C57BL/6 x A/J)F1 strain mice (H2 haplotypes Kb/k, Ab/k, E-/k, Db/d) were treated with ATS on days -1 and +2 relative transplantation of a C3H (H-2k) skin allograft. On day +7, each recipient was randomly assigned to one of the following groups that received i.v. donor C3H cell infusions via the tail vein: 1) 5.0x10(7) wild-type donor spleen cells (SPC); 2) 5.0x10(7) spleen cells from C3H/HeJ-Lystbg-2J/+ mice (commonly called beige mice and have selectively impaired NK cell function); 3) 2.5x10(7) wild-type donor bone marrow cells (BMC); 4) 2.5x10(7)beige C3H bone marrow cells; and 5) no donor cell infusion (ATS controls). In another experiment, each recipient was randomly assigned to one of the following groups that received injections of: 1) 4.75x10(7) spleen cells depleted of NK cells; 2) 2.5x10(6) purified splenic NK cells; 3) a coinfusion of 5.0x10(7) beige spleen cells and 2.5x10(6) purified wild-type splenic NK cells. RESULTS: Recipients infused with wild-type SPC exhibited significant augmentation of allograft survival compared with ATS controls. However, graft survival was reduced in recipients that were infused with spleen cells from beige mice compared with recipients infused with wild-type SPC (median survival time (MST): 38 vs. 92 days, P=0.02). In contrast, infusions of beige BMC augmented allograft survival as well as wild-type BMC (MST: 47 vs. 49 days, P=0.76). Furthermore, the ability of wild-type SPC to augment allograft survival was abrogated by the depletion of NK cells (MST=92 vs. 34 days, respectively, P=0.005). The co-infusion of beige SPC and purified splenic NK cells enhanced allograft survival as well as wild-type SPC (MST=56 days, P=0.65). Finally, recipients infused with purified NK cells did not experience increased graft survival compared to recipients that received no infusion (MST=29 vs. 33 days, respectively, P=0.6). CONCLUSIONS: Donor splenic NK cells are necessary, but not sufficient, for the extension of graft survival by infusion of donor splenocytes, suggesting that they may work in concert with another cell-type. In contrast, the extension of graft survival by donor bone marrow does not depend on the presence of donor NK cells.

Power and robustness of a score test for linkage analysis of quantitative traits using identity by descent data on sib pairs.

Identification of genes involved in complex traits by traditional (lod score) linkage analysis is difficult due to many complicating factors. An unfortunate drawback of non-parametric procedures in general, though, is their low power to detect genetic effects. Recently, Dudoit and Speed [2000] proposed using a (likelihood-based) score test for detecting linkage with IBD data on sib pairs. This method uses the likelihood for theta, the recombination fraction between a trait locus and a marker locus, conditional on the phenotypes of the two sibs to test the null hypothesis of no linkage (theta = (1/2)). Although a genetic model must be specified, the approach offers several advantages. This paper presents results of simulation studies characterizing the power and robustness properties of this score test for linkage, and compares the power of the test to the Haseman-Elston and modified Haseman-Elston tests. The score test is seen to have impressively high power across a broad range of true and assumed models, particularly under multiple ascertainment. Assuming an additive model with a moderate allele frequency, in the range of p = 0.2 to 0.5, along with heritability H = 0.3 and a moderate residual correlation rho = 0.2 resulted in a very good overall performance across a wide range of trait-generating models. Generally, our results indicate that this score test for linkage offers a high degree of protection against wrong assumptions due to its strong robustness when used with the recommended additive model.

Pedigree selection and tests of linkage in a Hutterite asthma pedigree.

We explored methods for kinship and linkage analysis in a Hutterite pedigree comprising 1,544 individuals, 72 of whom were diagnosed with asthma. Subpedigrees were selected by (a) identifying nuclear families containing asthmatics, (b) identifying couples with many asthmatic descendants in an ad hoc manner, and (c) finding the most recent common ancestors of all asthmatics. Markov chain Monte Carlo (MCMC) methods were used to estimate allele sharing in the larger subpedigrees and transmission/disequilibrium tests were performed on nuclear families. On chromosome 5q near the cytokine cluster, modest evidence for linkage to asthma was obtained. Using MCMC, we were able to evaluate the evidence for linkage in complex subpedigrees of several hundred individuals, and hence, incorporate some of the co-ancestry of this founder population.

A differential requirement for CD8+ donor cells in the augmentation of allograft survival by posttransplantation administration of donor spleen cells and donor bone marrow cells.

BACKGROUND: Peritransplant treatment with antithymocyte serum (ATS) and posttransplantation administration of donor bone marrow or donor splenocytes results in extended skin allograft survival. In this study, we examined the molecular basis of the tolerance promoting effect of donor bone marrow (BMC) cells and splenocytes with emphasis on the role of CD8 expression on the donor cells. METHODS: (C57BL/6J x A/J)F1 mice were treated on days -1 and +2 with ATS relative to transplantation with C3H/HeJ skin. On day +7, they were infused with CD8+ BMC, CD8- BMC, CD8+ splenocytes, or CD8- splenocyte donor subpopulations isolated by magnetic or fluorescence-based sorting. In additional experiments, B10.D2(R107) mice were treated in the same manner with C57BL/6 skin and BMC or splenocytes from C57BL/6 mice in which the CD8alpha gene had been inactivated. RESULTS: CD8+ donor bone marrow cells induced operational tolerance (defined as graft acceptance in the absence of chronic immunosuppression) in skin graft recipients at a dose that was reduced by 250-fold relative to unfractionated bone marrow cells (1.0x10(5) cells per recipient, median survival time (MST)=41 days vs. 2.5x10(7) cells per recipient, MST=49 days, P=0.40). Similarly, donor bone marrow cells from CD8 knockout mice did not promote graft acceptance (MST=98 days vs. animals not treated with bone marrow cells, MST=70 days, P=0.16). In contrast, the extension of graft survival by donor splenocytes did not require the presence of CD8+ donor cells because splenocytes depleted of CD8+ cells extended graft survival (MST=55 days) as well as unsorted splenocytes (44 days, P=0.2), and splenocytes from CD8 knockout animals (MST=145 days) extended graft survival at least as well as unsorted splenocytes (MST=74 days, P=0.4) CONCLUSIONS: These results suggest that the prolongation of graft survival by donor bone marrow is dependent on the presence of the CD8 molecule, whereas prolongation by donor splenocytes is not. Therefore, we suggest that the prolongation of graft survival by these cell types occurs via distinct molecular mechanisms probably mediated by different cell types.

Power of a score test for quantitative trait linkage analysis of relative pairs.

The score test of Dudoit and Speed [(2000) Biostatistics 1:1-26] to detect linkage between a trait locus and a marker locus, using identity by descent data on sib pairs, is extended to other types of relative pairs (grandparent/grandchild, avuncular, and half-sib relationships). The test is based on the likelihood of the recombination fraction theta between trait and marker loci, conditional on phenotypes of the relatives. We present results of simulation studies characterizing power and robustness properties of this linkage score test, and compare the power of the score test to that of the classical and modified Haseman-Elston tests. The score test has considerable power, particularly under sampling schemes where selection is on double probands. Use of a generic additive model [Goldstein et al., submitted] with allele frequency p = 0.2, heritability H = 0.3, and a moderate residual correlation of rho = 0.2 resulted in a very good overall performance across a wide range of trait-generating models.

A familial risk profile for osteoporosis.

PURPOSE: To describe genetic epidemiologic aspects of osteoporosis. METHODS: 69 patients with osteoporosis were interviewed regarding personal and family histories of osteoporosis and related fractures. Family history information was obtained on 421 first degree and 748 second degree relatives. RESULTS: 45% of cases reported a family history of osteoporosis. Familial cases were characterized neither by an earlier age of diagnosis nor by a greater degree of phenotypic severity. Empiric risks for osteoporosis were highest for mothers, 33%, and were 19% for sisters. CONCLUSION: These results provide an initial genetic epidemiologic profile for osteoporosis and information useful for genetic counseling.

Enhanced allograft survival induced by posttransplant donor spleen cell infusion occurs via a mechanism that is distinct from the mechanism of enhancement by donor bone marrow.

BACKGROUND: Our previous studies have shown that the ability of donor bone marrow to augment skin graft survival in antithymocyte serum (ATS)-treated recipients is dependent on the presence of functional CD95-ligand (Fas-ligand) molecules on donor cells. Because donor spleen cells can augment graft survival to a similar degree in the same model, we investigated whether the donor spleen cell effect was also dependent on the presence of CD95-ligand on donor cells and CD95 on recipient cells. METHODS: Mutant mice bearing defects in the expression of CD95 (lpr mutation) and CD95-ligand (gld mutation) were used as recipients and cell donors, respectively. Recipients were injected with rabbit ATS on days -1 and +2, and then were injected with 5x10(7) spleen cells on day +7. Skin graft survival was compared and correlated with the use of mutant mice as recipients and cell donors. RESULTS: The combination of ATS and infusions of wild-type [median survival (MST)=44 days, P=0.0004] and gld (mutant CD95-ligand, MST=37 days, P=0.02) donor spleen cells enhanced C3H graft survival, compared with (C57BL/6 x A)F1 recipients treated with ATS alone (MST=27 days). Furthermore, C57BL/6 lpr (CD95-deficient) strain recipients treated with ATS and donor spleen cells demonstrated enhanced B10.D2(R107) strain skin graft survival (MST=44 days, P=0.003), compared with C57BL/6 lpr recipients treated with ATS alone (MST=31 days). Wild-type C57BL/6 recipients treated in the same manner also exhibited an extension of graft survival (MST=64 days) versus controls treated with ATS alone (MST=31 days). CONCLUSION: The data demonstrate that the ability of donor spleen cells to augment allograft survival is not dependent on the CD95/CD95-ligand pathway; therefore the deletion of allospecific cells by donor spleen cells may be induced via a pathway other than deletion by donor bone marrow cells.

Meta-analysis by combining parameter estimates: simulated linkage studies.

Several meta-analytic techniques have been developed for combining information from multiple studies in contexts other than linkage detection. We apply the technique of combining parameter estimates to the problem of finding disease loci in the simulated data and compare results with those obtained by reanalyzing pooled raw data. To facilitate the combination of study results, we highly recommend that parameter estimates and their standard errors be reported in published studies. If different research groups were to make original data available, progress toward disease gene location and characterization may be more quickly made.

Meta-analysis by combining p-values: simulated linkage studies.

Meta-analysis has been little explored to make an overall assessment of linkage from different studies. In practice, it is likely that published linkage studies will only report p-values. We compared the performance of the widely used Fisher method for combining p-values with that of pooling raw data. More loci were consistently found by pooling raw data. In the absence of further information, combining p-values can provide an overall, but limited, assessment of different linkage studies. However, meta-analysis would be better viewed as a preliminary step toward the goal of analyzing the pooled raw data.

Donor bone marrow and transplantation tolerance: historical perspectives, molecular mechanisms and future directions (review).

The induction of transplantation tolerance, defined as the indefinite existence of an intact, functioning allograft, has been a goal of transplantation immunologists for decades because of the morbid effects of immunosuppressive drugs required to maintain graft function. One promising method of tolerance induction involves the peritransplant administration of cytoablative drugs followed by administration of a low dose of donor bone marrow cells. A subpopulation of donor bone marrow cells bearing the CD8 accessory molecule cause the functional deletion of graft reactive T cells via a CD95 dependent mechanism that also involves the cytokine TGFbeta. This interaction is bi-directional in which the bone marrow cell deletes the graft reactive T cell that recognizes it. Therefore, only T cells that recognize the donor antigens are deleted, leaving the immune response to other antigens intact. This protocol has been successfully used in rodents and an outbred large animal model. Clinical trials of donor bone marrow administration are now underway and initially indicate that administration of donor bone marrow is clinically safe.

Clenbuterol and anabolic steroids: a previously unreported cause of myocardial infarction with normal coronary arteriograms.

During the last 10 years, several cases of myocardial infarction associated with anabolic steroid use have been reported. Postulated mechanisms to explain this association have included changes in lipid levels, the fibrinolytic system, and platelet aggregation. Clenbuterol is a beta 2-agonist with anabolic properties that has not been seen previously with myocardial infarction. We report a case of myocardial infarction in an otherwise healthy 26-year-old body-builder who recently used clenbuterol and anabolic steroids. In this case, synergistic effects of the two agents seem likely to have played a role in the infarct. The normal coronary arteriograms before any anticoagulant or thrombolytic therapy strongly suggest coronary spasm as the mechanism of the infarct.

An essential role for Fas ligand in transplantation tolerance induced by donor bone marrow.

Medawar and co-workers originally demonstrated that injection of donor bone marrow (DBM) into immuno-incompetent neonatal rodents could induce tolerance to grafts from animals of the same strain as the bone marrow donor. Induction of tolerance in this manner can also be accomplished in mature mice, dogs and monkeys if the resident T-cell populations in the recipient are depleted by a polyclonal antithymocyte globulin or an anti-T cell immunotoxin. The molecular mechanisms by which bone marrow cells mediate the induction of tolerance remain uncertain. Here we examined a well-established adult mouse model of antithymocyte globulin and DBM treatment and show that expression of functional Fas ligand (FasL, also CD95L) on the injected bone marrow cells is required for tolerance induction. The results indicate that a state of microchimerism per se is insufficient for the induction of tolerance in T cell-depleted transplant recipients. Moreover, the results are consistent with the hypothesis that tolerance induced by DBM involves an apoptotic process leading to deletion of graft-reactive cells.