Hyperlipidemia Promotes Anti-Donor Th17 Responses That Accelerate Allograft Rejection.

Hyperlipidemia occurs in 95% of organ transplant recipients, however its effect on organ allograft rejection has not been investigated. We found that induction of hyperlipidemia in mice caused a significant acceleration of rejection of cardiac allografts. Accelerated rejection was associated with an aggressive T cell infiltrate that mediated significant tissue damage as well as increased serum levels of the proinflammatory cytokines IL-2, IL-6, and IL-17. Hyperlipidemic mice had an increased number of Th17 cells in their periphery and rejecting allografts from hyperlipidemic mice contained significant numbers of IL-17 producing T cells that were not detectable in transplants harvested from controls. Neutralization or genetic ablation of IL-17 prolonged survival of cardiac allografts transplanted into hyperlipidemic recipients, suggesting that IL-17 production promotes accelerated rejection. Analysis of alloreactive T cell frequencies directly ex vivo in naïve mice revealed that the frequency of donor reactive IL-17 producing cells in hyperlipidemic was increased prior to antigen exposure, suggesting that hyperlipidemia was sufficient to alter T cell alloreactivity and promote anti-donor Th17 responses on first exposure to antigen. Together, our data suggest that hyperlipidemia alters rejection by altering the types of T cell subsets that respond to donor antigen by promoting Th17 biased anti-donor reactivity.

Hyperlipidemia Alters Regulatory T Cell Function and Promotes Resistance to Tolerance Induction Through Costimulatory Molecule Blockade.

Recent work from our laboratory has shown that hyperlipidemia promotes accelerated rejection of vascularized cardiac allografts in mice by inducing anti-donor Th17 reactivity and production of IL-17. Here, we show that hyperlipidemia also affects FoxP3(+) regulatory T cells (Tregs). Hyperlipidemia promotes the development of Tregs that express low levels of CD25. Hyperlipidemia also promotes a decrease in central Tregs and an increase in effector Tregs that appears to account for the increase in the frequency of CD25(low) Tregs. Alterations in Treg subsets also appear to lead to alterations in Treg function. The ability of FoxP3(+) , CD25(high) , CD4(+) Tregs from hyperlipidemic mice to inhibit proliferation of effector T cells stimulated with anti-CD3 and CD28 was reduced when compared with Tregs from control mice. Regulatory T cells isolated from hyperlipidemic recipients exhibit increased activation of Akt, and a reduction in Bim levels that permits the expansion of FoxP3(+) CD25(low) CD4(+) T cells. Hyperlipidemic mice were also resistant to tolerance induction using costimulatory molecule blockade consisting of anti-CD154 and CTLA4Ig, a strategy that requires Tregs. Together, our data suggest that hyperlipidemia profoundly affects Treg subsets and function as well as the ability to induce tolerance.

MicroRNA-494 promotes cyclosporine-induced nephrotoxicity and epithelial to mesenchymal transition by inhibiting PTEN.

A major complication associated with cyclosporine (CsA) treatment is nephrotoxicity. In this study, we examined whether microRNAs play a role in cyclosporine-induced nephrotoxicity. Treatment of mice with CsA resulted in nephrotoxicity that was associated with an early increase in expression of microRNA mmu-miR-494 (miR-494). Similarly, tubular epithelial cell epithelial-mesenchymal transition (EMT) induced by CsA toxicity resulted in the upregulation of microRNA-494 and a decrease in PTEN levels in vitro. miR-494 directly targeted Pten and negatively regulated its expression. Preventing Pten targeting by miR-494 was sufficient to prevent CsA induced EMT. Knockdown of miR-494 prevented the downregulation of PTEN in tubular epithelial cells following CsA treatment and also prevented CsA induced EMT. Thus, miR-494 plays a major role in promoting CsA induced nephrotoxicity through its ability to target Pten thereby contributing to EMT. We suggest that manipulating miR-494 expression may represent a novel approach to preventing EMT associated with CsA induced nephrotoxicity.

Reproductive mode evolution in lizards revisited: updated analyses examining geographic, climatic and phylogenetic effects support the cold-climate hypothesis.

Viviparity, the bearing of live young, has evolved well over 100 times among squamate reptiles. This reproductive strategy is hypothesized to allow maternal control of the foetus' thermal environment and thereby to increase the fitness of the parents and offspring. Two hypotheses have been posited to explain this phenomenon: (i) the cold-climate hypothesis (CCH), which advocates low temperatures as the primary selective force; and (ii) the maternal manipulation hypothesis (MMH), which advocates temperature variability as the primary selective force. Here, we investigate whether climatic and geographic variables associated with the CCH vs. the MMH best explain the current geographical distributions of viviparity in lizards while incorporating recent advances in comparative methods, squamate phylogenetics and geospatial analysis. To do this, we compared nonphylogenetic and phylogenetic models predicting viviparity based on point-of-capture data from 20,994 museum specimens representing 215 lizard species in conjunction with spatially explicit bioclimatic and geographic (elevation and latitude) data layers. The database we analysed emphasized Nearctic lizards from three species-rich genera (Phrynosoma, Plestiodon and Sceloporus); however, we additionally analysed a less substantial, but worldwide sample of species to verify the universality of our Nearctic results. We found that maximum temperature of the warmest month (and, less commonly, elevation and maximum temperature of the driest quarter) was frequently the best predictor of viviparity and showed an association consistent with the CCH. Our results strongly favour the CCH over the MMH in explaining lizard reproductive mode evolution.

Tolerance to MHC class II disparate allografts through genetic modification of bone marrow.

Induction of molecular chimerism through genetic modification of bone marrow is a powerful tool for the induction of tolerance. Here, we demonstrate for the first time that expression of an allogeneic MHC class II gene in autologous bone marrow cells, resulting in a state of molecular chimerism, induces tolerance to MHC class II mismatched skin grafts, a stringent test of transplant tolerance. Reconstitution of recipients with syngeneic bone marrow transduced with retrovirus encoding H-2I-A(b) (I-A(b)) resulted the long-term expression of the retroviral gene product on the surface of MHC class II-expressing bone marrow-derived cell types. Mechanistically, tolerance was maintained by the presence of regulatory T cells, which prevented proliferation and cytokine production by alloreactive host T cells. Thus, the introduction of MHC class II genes into bone marrow-derived cells through genetic engineering results in tolerance. These results have the potential to extend the clinical applicability of molecular chimerism for tolerance induction.

Dynamic imaging reveals that brain-derived neurotrophic factor can independently regulate motility and direction of neuroblasts within the rostral migratory stream.

Neuronal precursors generated in the subventricular zone (SVZ) migrate through the rostral migratory stream (RMS) to the olfactory bulb (OB). Although, the mechanisms regulating this migration remain largely unknown. Studies have shown that molecular factors, such as brain-derived neurotrophic factor (BDNF) emanating from the OB, may function as chemoattractants drawing neuroblasts toward their target. To better understand the role of BDNF in RMS migration, we used an acute slice preparation from early postnatal mice to track the tangential migration of GAD65-GFP labeled RMS neuroblasts with confocal time-lapse imaging. By quantifying the cell dynamics using specific directional and motility criteria, our results showed that removal of the OB did not alter the overall directional trajectory of neuroblasts, but did reduce their motility. This suggested that additional guidance factors present locally within the RMS region also contribute to this migration. Here we report that BDNF and its high affinity receptor, tyrosine kinase receptor type 2 (TrkB), are indeed heterogeneously expressed within the RMS at postnatal day 7. By altering BDNF levels within the entire pathway, we showed that reduced BDNF signaling changes both neuroblast motility and direction, while increased BDNF levels changes only motility. Together these data reveal that during this early postnatal period BDNF plays a complex role in regulating both the motility and direction of RMS flow, and that BDNF comes from sources within the RMS itself, as well as from the olfactory bulb.

In-home consumer and shear force evaluation of steaks from the M. serratus ventralis thoracis.

The M. serratus ventralis thoracis was obtained from US Select arm chucks (n=87) to investigate if this underutilized muscle can be used as a steak alternative. Muscles were assigned randomly into three treatment groups: (1) control; (2) blade tenderization; and (3) injection, containing salt, phosphate, and papain. Steaks were cut from each muscle for in-home consumer evaluation (n=136) and Warner-Bratzler shear (WBS) force determination. The WBS values for injected steaks (13.1N) were lower (P<0.05) than for blade-tenderized (18.4N) and control (19.9N) steaks. Tenderness ratings for the injected steaks were higher (P<0.05) compared to the other treatments when steaks were grilled, oven prepared or were cooked in a skillet; however, this improvement did not significantly influence overall like scores for steaks that were oven prepared or cooked in a skillet. For the most part, degree of doneness did not significantly impact consumer evaluations of steaks prepared by the various cooking methods. However, there was a treatment x degree of doneness interaction for grilled-cooked steaks where increased doneness for blade-tenderized and injected steaks resulted in increased palatability ratings, whereas increased doneness for control steaks generally resulted in lowered palatability ratings. Consumer ratings and WBS values for the M. serratus ventralis thoracis indicate that merchandising steaks from this muscle may be a viable option in the marketplace, especially if blade tenderization or injection processes are used for further enhancement.

Electromagnetic fields and modal excitations on a thin silver film.

In this paper we extend the fast-all-modes method and the numerical modified steepest-descent-path method to the optical frequency range by finding all modes and solving the total electric field in three dimensions that is due to a point source above a lossy thin metal film with a negative permittivity situated between two dissimilar dielectric materials. We show that up to four proper surface wave modes may propagate on the film surface, including both backward and forward waves. We also solve for the electric field below the lossy thin metal film and verify the existence of superlensing of the electric field, comparing that case to the case of a dielectric film where no superlensing occurs. The CPU time using the fast-all-modes method and the numerical modified steepest-descent-path method is considerably less than that using the conventional method of integration along the Sommerfeld integration path.

Activated polymorphonuclear cells promote injury and excitability of dorsal root ganglia neurons.

Therapies aimed at depleting or blocking the migration of polymorphonuclear leukocytes (PMN or neutrophils) are partially successful in the treatment of neuroinflammatory conditions and in attenuating pain following peripheral nerve injury or subcutaneous inflammation. However, the functional effects of PMN on peripheral sensory neurons such as dorsal root ganglia (DRG) neurons are largely unknown. We hypothesized that PMN are detrimental to neuronal viability in culture and increase neuronal activity and excitability. We demonstrate that isolated peripheral PMN are initially in a relatively resting state but undergo internal oxidative burst and activation by an unknown mechanism within 10 min of co-culture with dissociated DRG cells. Co-culture for 24 h decreases neuronal count at a threshold<0.4:1 PMN:DRG cell ratio and increases the number of injured and apoptotic neurons. Within 3 min of PMN addition, fluorometric calcium imaging reveals intracellular calcium transients in small size (<25 microm diam) and large size (>25 microm diam) neurons, as well as in capsaicin-sensitive neurons. Furthermore, small size isolectin B4-labeled neurons undergo hyperexcitability manifested as decreased current threshold and increased firing frequency. Although co-culture of PMN and DRG cells does not perfectly model neuroinflammatory conditions in vivo, these findings suggest that activated PMN can potentially aggravate neuronal injury and cause functional changes to peripheral sensory neurons. Distinguishing the beneficial from the detrimental effects of PMN on neurons may aid in the development of more effective drug therapies for neurological disorders involving neuroinflammation, including painful neuropathies.

Homeostatic expansion permits T cells to re-enter the thymus and deliver antigen in a tolerogenic fashion.

We previously have shown that delivery of alloantigen on T cells can be used to induce tolerance through central deletion. Here, we analyzed the requirements for tolerance induced by T cells. Adoptively transferred allogeneic T cells undergo extensive homeostatic proliferation in the periphery of lethally irradiated hosts receiving a syngeneic bone marrow transplant, and acquire a memory-like cell surface phenotype. Analysis of the kinetics of thymic re-entry of transferred T cells revealed that T cells undergo homeostatic proliferation in the periphery prior to re-entry into the thymus. Prevention of homeostatic proliferation results in a failure of transferred T cells to re-enter the thymus. In the absence of homeostatic proliferation, adoptively transferred T cells were unable to induce tolerance. These date suggest that homeostatic proliferation of T cells resulting in an activated cell surface phenotype is required for thymic re-entry and is mechanistically linked to the ability of T cells to induce tolerance.

Persistence of antigen is required to maintain transplantation tolerance induced by genetic modification of bone marrow stem cells.

Genetic modification of hematopoietic stem cells (HSCs) resulting in a state of molecular chimerism can be used to induce donor-specific tolerance to allografts. However, the requirements for maintaining tolerance in molecular chimeras remain unknown. Here, we examined whether long-term expression of a retrovirally encoded alloantigen in hematopoietic cells is required to maintain donor-specific tolerance in molecular chimeras. To this end, mice were reconstituted with syngeneic bone marrow transduced with retroviruses carrying the gene encoding the allogeneic MHC class I molecule Kb. Following induction of molecular chimerism, mice were depleted of cells expressing Kb by administration of the anti-Kb monoclonal antibody Y-3. Mice that were effectively depleted of cells expressing the retrovirally encoded MHC class I antigen rejected Kb disparate skin allografts. In contrast, control molecular chimeras accepted Kb disparate skin allografts indefinitely. These data suggest maintenance of tolerance in molecular chimeras requires long-term expression of retrovirally transduced alloantigen on the progeny of retrovirally transduced HSCs.

Inhibition of CD26 peptidase activity significantly improves engraftment of retrovirally transduced hematopoietic progenitors.

It has previously been shown that inhibition of CD26 (DPPIV/dipeptidylpeptidase IV) peptidase activity improves homing of hematopoietic stem cells (HSCs) to the bone marrow and increases engraftment efficiency. Here, we demonstrate that treatment of retrovirally transduced mouse bone marrow cells with the tri-peptide Diprotin A (Ile-Pro-Ile), a specific inhibitor of CD26, significantly enhances engraftment of retrovirally transduced HSCs. Treatment of transduced bone marrow cells with Diprotin A permitted long-term expression of a retrovirally encoded MHC class I gene on multiple hematopoietic cell lineages after transplantation of a suboptimal number of transduced cells. Secondary transfer experiments revealed that expression of the transduced MHC class I gene resulted from engraftment of transduced HSCs. Expression of the allogeneic MHC class I antigen on bone marrow-derived cells following transplantation of Diprotin A-treated cells was sufficient to induce transplantation tolerance. Therefore, inhibition of CD26 activity significantly enhances engraftment of limited numbers of genetically modified HSCs, resulting in physiologically relevant levels of gene transfer.

Gene therapy progress and prospects: gene therapy in organ transplantation.

One major complication facing organ transplant recipients is the requirement for life-long systemic immunosuppression to prevent rejection, which is associated with an increased incidence of malignancy and susceptibility to opportunistic infections. Gene therapy has the potential to eliminate problems associated with immunosuppression by allowing the production of immunomodulatory proteins in the donor grafts resulting in local rather than systemic immunosuppression. Alternatively, gene therapy approaches could eliminate the requirement for general immunosuppression by allowing the induction of donor-specific tolerance. Gene therapy interventions may also be able to prevent graft damage owing to nonimmune-mediated graft loss or injury and prevent chronic rejection. This review will focus on recent progress in preventing transplant rejection by gene therapy.

Cytokine-augmented culture of haematopoietic progenitor cells in a novel three-dimensional cell growth matrix.

Studies aimed at the in vitro expansion of haematopoietic progenitor cells (HPCs) have suffered from the conflict of increasing cell numbers while maintaining long-term repopulating ability. We have developed a long-term bone marrow bioreactor culture system resembling the marrow-microenvironment that cultures HPCs in an inert, three-dimensional, porous biomatrix termed Cellfoam. Previous studies have shown that the short-term culture of CD34(+)cells in Cellfoam improved the maintenance and multipotency of haematopoietic stem cells compared to cells cultured on plastic dishes. In this study, we examined the effects of low concentrations of cytokines including stem cell factor (SCF), IL-3, and Flk-2/Flt-3 ligand, on the maintenance, preservation and multipotency of CD34(+) cells cultured for 3 or 6 weeks in Cellfoam. Analysis of cell yields using flow cytometry showed that in SCF and Flk-2/Flt-3 ligand-supplemented cultures as well as cytokine-free cultures, a higher number of CD45(+)34(+) and CD45(+)34(+)38(-) cells is observed in Cellfoam cultures as compared to plastic cultures. The function of cultured cells was evaluated in colony-forming assays. The data demonstrate that Cellfoam cultures supplemented with SCF and Flk-2/Flt-3 ligand resulted in a higher output of colony activity compared to plastic cultures. Analysis of CAFC (29 days) activity also demonstrated that primitive progenitors were maintained to a greater extent in Cellfoam cultures containing either no cytokines or low concentrations of early-acting cytokines. These data suggest that culture of HPCs in three-dimensional bioreactors such as Cellfoam for extended periods may benefit from the addition of low levels of early-acting cytokines, including SCF and Flk-2/Flt-3 ligand, resulting in high yields of cells that are enriched for multipotent haematopoietic progenitors. These findings demonstrate that a three-dimensional matrix promotes the survival of primitive HPCs in culture and may modulate the in vitro effects of cytokines.

Defining the requirements for peptide recognition in gene therapy-induced T cell tolerance.

Expression of a retrovirally transduced MHC class I Ag, H-2K(b) (K(b)), in bone marrow-derived cells leads to specific prolongation of K(b) disparate skin grafts. To examine the extent to which peptides derived from K(b) contribute to the induction of tolerance, retroviruses carrying mutant K(b) genes designed to enter separate pathways of Ag presentation were constructed. Thymectomized and CD8 T cell-depleted mice that had been irradiated and reconstituted with bone marrow cells expressing a secreted form of K(b) showed prolongation of K(b) disparate skin graft survival. Skin graft prolongation was not observed when similar experiments were performed using mice that were not CD8 T cell depleted. This suggests that hyporesponsiveness can be induced in CD4 T cells, but not CD8 T cells by Ags presented via the exogenous pathway of Ag processing. Modest prolongation of skin allografts was observed in mice reconstituted with bone marrow cells transduced with retroviruses carrying a gene encoding a mutant K(b) molecule expressed only in the cytoplasm. Prolongation was also observed in similar experiments in mice that were thymectomized and CD4 T cell depleted following complete reconstitution, but not in mice that were reconstituted and then thymectomized and CD8 T cell depleted. Thus, hyporesponsiveness can be induced in a subset of CD8 T cells by recognition of peptides derived from K(b) through both the direct and indirect pathways of Ag recognition, while CD4 T cell hyporesponsiveness to MHC class I disparate grafts occurs only through the indirect pathway of Ag recognition.

A critical role for interleukin 4 in activating alloreactive CD4 T cells.

To generate antigen-specific responses, T cells and antigen presenting cells (APCs) must physically associate with each other and elaborate soluble factors that drive the full differentiation of each cell type. Immediately after T cell activation, CD4 T cells can produce both interferon gamma (IFN-gamma) and interleukin 4 (IL-4) before polarization into distinct T helper subsets. Inhibition of IL-4 during mixed allogeneic lymphocyte culture resulted in a defect in the ability of APCs to generate sufficient costimulatory signals for activation of alloreactive T cells. In vivo, a deficiency in IL-4 production inhibited the activation of alloreactive IL-2-, IL-4- and IFN-gamma-producing CD4 T cells in mice challenged with allogeneic skin grafts, resulting in prolonged skin graft survival. Thus, production of IL-4 by CD4T cells helps activate alloreactive T cells by affecting APC function.

Effects of a novel fentanyl derivative on drug discrimination and learning in rhesus monkeys.

Three monkeys discriminated 1.78 mg/kg of mirfentanil while responding under a fixed-ratio 5 schedule of stimulus-shock termination. Two mirfentanil derivatives, OHM3295 and OHM10579, substituted for mirfentanil in all subjects. However, other drugs produced variable effects among monkeys; for example, mu and kappa opioid agonists and clonidine substituted for mirfentanil on some occasions in two monkeys. Cocaine, amphetamine, and ketamine did not substitute in any subject. Opioid antagonists did not attenuate the effects of mirfentanil. In monkeys responding under a repeated acquisition and performance procedure, errors increased only during the acquisition phase at doses of mirfentanil that decreased response rates. Thus, unlike fentanyl, the discriminative stimulus effects of mirfentanil do not appear to be mediated exclusively through opioid receptors. Finally, mirfentanil does not appear to disrupt complex behavioral processes.

Extended culture of multipotent hematopoietic progenitors without cytokine augmentation in a novel three-dimensional device.

The ability to culture multipotent hematopoietic progenitor cells for extended periods is of practical importance to both clinical and research efforts involving these cells. Conventional techniques for the extended culture of hematopoietic progenitor cells (HPCs) have proven largely ineffective in sustaining these cells and preserving their multipotency over protracted periods. To overcome barriers to extended HPC culture, numerous alternative approaches, including cytokine augmentation and co-culture with bone marrow stroma, have been explored to enhance HPC maintenance but have generally yielded mixed results. The present study examined the ability of a novel, three-dimensional, tantalum-coated porous biomaterial (TCPB) to support HPC maintenance and multipotency in long-term cultures to which no exogenous cytokines have been added. As a follow-up to previously published short-term HPC cultures in TCPB, we examined the maintenance, phenotype and multipotency of HPCs cultured for up to 6 weeks in the TCPB matrix compared to control systems, including fibronectin-coated plastic, bone marrow stroma cocultures and other three-dimensional materials. These studies indicated that TCPB supports the maintenance of immature progenitors for up to 6 weeks in the absence of supplemented cytokines. Further, the results demonstrate that the TCPB matrix facilitates and enhances HPC maintenance and leads to a 1.5-fold expansion of HPC numbers following 1 week in culture and a 6.7-fold increase in colony-forming ability following 6 weeks in culture in the absence of exogenous cytokines. Under the same conditions, control systems were less able to support progenitor viability and multipotency. These findings point to new approaches that may improve the in vitro preservation of progenitors and may have important implications in clinical areas such as progenitor expansion, bone marrow transplantation and gene therapy.

Daily mirfentanil induces (cross-) tolerance to the rate-decreasing effects of morphine and not mirfentanil.

The fentanyl derivative mirfentanil has a novel set of behavioral effects in non-humans including low-efficacy opioid actions and non-opioid antinociceptive actions. This study evaluated the rate-decreasing effects of mirfentanil, morphine, naltrexone and ketamine in pigeons both prior to and during a period of chronic treatment with mirfentanil (3.2-17.8 mg/kg/day). Daily treatment with mirfentanil did not modify the rate-decreasing effects of mirfentanil or ketamine; however, daily treatment decreased sensitivity to the rate-decreasing effects of morphine and increased sensitivity to naltrexone. These results demonstrate a lack of tolerance to an apparently non-opioid action (rate-decreasing effect) of mirfentanil, which might predict a lack of tolerance to the non-opioid antinociceptive actions of this compound. These results further indicate that cross-tolerance (to morphine) and dependence (increased sensitivity to naltrexone) can occur in the absence of tolerance (to mirfentanil).