CD4+ T lymphocytes are not necessary for the acute rejection of vascularized mouse lung transplants

Acute rejection continues to present a major obstacle to successful lung transplantation. Although CD4(+) T lymphocytes are critical for the rejection of some solid organ grafts, the role of CD4(+) T cells in the rejection of lung allografts is largely unknown. In this study, we demonstrate in a novel model of orthotopic vascularized mouse lung transplantation that acute rejection of lung allografts is independent of CD4(+) T cell-mediated allorecognition pathways. CD4(+) T cell-independent rejection occurs in the absence of donor-derived graft-resident hematopoietic APCs. Furthermore, blockade of the CD28/B7 costimulatory pathways attenuates acute lung allograft rejection in the absence of CD4(+) T cells, but does not delay acute rejection when CD4(+) T cells are present. Our results provide new mechanistic insight into the acute rejection of lung allografts and highlight the importance of identifying differences in pathways that regulate the rejection of various organs.     

U50,488H-induced internalization of the human kappa opioid receptor involves a beta-arrestin- and dynamin-dependent mechanism. Kappa receptor internalization is not required for mitogen-activated protein kinase activation

Agonist-promoted internalization of some G protein-coupled receptors has been shown to mediate receptor desensitization, resensitization, and down-regulation. In this study, we investigated whether opioids induced internalization of the human and rat kappa opioid receptors stably expressed in Chinese hamster ovary cells, the potential mechanisms involved in this process and its possible role in activation of mitogen-activated protein (MAP) kinase. Exposure of the human kappa receptor to the agonists U50,488H, U69,593, ethylketocyclazocine, or tifluadom, but not etorphine, promoted receptor internalization. However, none of these agonists induced significant internalization of the rat kappa opioid receptor. U50, 488H-induced human kappa receptor internalization was time- and concentration-dependent, with 30-40% of the receptors internalized following a 30-min exposure to 1 microM U50,488H. Agonist removal resulted in the receptors gradually returning to the cell surface over a 60-min period. The antagonist naloxone blocked U50, 488H-induced internalization without affecting internalization itself, while pretreatment with pertussis toxin had no effect on U50, 488H-induced internalization. In contrast, incubation with sucrose (0.4-0.8 M) significantly reduced U50,488H-induced internalization of the kappa receptor. While co-expression of the wild type GRK2, beta-arrestin, or dynamin I had no effect on kappa receptor internalization, co-expression of the dominant negative mutants GRK2-K220R, beta-arrestin (319-418), or dynamin I-K44A significantly inhibited receptor internalization. Whether receptor internalization is critical for MAP kinase activation was next investigated. Co-expression of dominant negative mutants of beta-arrestin or dynamin I, which greatly reduced U50,488H-induced internalization, did not affect MAP kinase activation by the agonist. In addition, etorphine, which did not promote human kappa receptor internalization, was able to fully activate MAP kinase. Moreover, U50,488H or etorphine stimulation of the rat kappa receptor, which did not undergo internalization, also effectively activated MAP kinase. Thus, U50,488H-induced internalization of the human kappa opioid receptor in Chinese hamster ovary cells occurs via a GRK-, beta-arrestin-, and dynamin I-dependent process that likely involves clathrin-coated pits. In addition, internalization of the kappa receptor is not required for activation of MAP kinase.     

Mouse vascular endothelium activates CD8+ T lymphocytes in a B7-dependent fashion

Despite several studies examining the contribution of allorecognition pathways to acute and chronic rejection of vascularized murine allografts, little data describing activation of alloreactive T cells by mouse vascular endothelium exist. We have used primary cultures of resting or IFN-gamma-activated C57BL/6 (H-2(b)) vascular endothelial cells as stimulators and CD8(+) T lymphocytes isolated from CBA/J (H-2(k)) mice as responders. Resting endothelium expressed low levels of MHC class I, which was markedly up-regulated after activation with IFN-gamma. It also expressed moderate levels of CD80 at a resting state and after activation. Both resting and activated endothelium were able to induce proliferation of unprimed CD8(+) T lymphocytes, with proliferation noted at earlier time points after coculture with activated endothelium. Activated endothelium was also able to induce proliferation of CD44(low) naive CD8(+) T lymphocytes. Activated CD8(+) T lymphocytes had the ability to produce IFN-gamma and IL-2, acquired an effector phenotype, and showed up-regulation of the antiapoptotic protein Bcl-x(L). Treatment with CTLA4-Ig led to marked reduction of T cell proliferation and a decrease in expression of Bcl-x(L). Moreover, we demonstrate that nonhemopoietic cells such as vascular endothelium induce proliferation of CD8(+) T lymphocytes in a B7-dependent fashion in vivo. These results suggest that vascular endothelium can act as an APC for CD8(+) direct allorecognition and may, therefore, play an important role in regulating immune processes of allograft rejection.     

Achieving Target 2 of the Global Strategy for Plant Conservation: building a preliminary assessment of vascular plant species using data from herbarium specimens

The Global Strategy for Plant Conservation calls for a preliminary assessment of the conservation status of all known plant species by the year 2010. To date insufficient progress has been made on meeting this target. New efforts to develop a preliminary list beyond using the full IUCN criteria in plant assessments are needed. Here we present an algorithm that provides a preliminary assessment of the conservation status of plant species using data from herbarium specimens. We use Hawaiian specimen data from the United States National Herbarium to calibrate the parameters of the algorithm and then use specimen data from the Arecaceae, Commelinaceae, Gesneriaceae and Heliconiaceae as examples of the application of the algorithm. The algorithm was calibrated to insure 95% accuracy in placing the Hawaiian plant species into previously and independently determined threatened categories. Our results indicate that 28% of the Hawaiian taxa, 27% of the species of Arecaceae, 45% of the species of Commelinaceae, 32% of the species of Gesneriaceae, and 35% of the species of Heliconiaceae are Not Threatened and will not need any further evaluation for the preliminary assessment. Species identified here as Potentially Extinct and Potentially Threatened can be further assessed by additional herbarium material and/or conservation specialists for final evaluation using other assessment strategies (e.g., regional and national lists, taxonomic expert assessment, etc.).     

Inspiratory loading does not accelerate dystrophy in mdx mouse diaphragm: implications for regenerative therapy.

Since the finding that the mdx mouse diaphragm, in contrast to limb muscles, undergoes progressive degeneration analogous to that seen in Duchenne muscular dystrophy, the relationship between the workload on a muscle and the pathogenesis of dystrophy has remained controversial. We increased the work performed by the mdx mouse diaphragm in vivo by tracheal banding and evaluated the progression of dystrophic changes in that muscle. Despite the establishment of dramatically increased respiratory workload and accelerated myofiber damage documented by Evans blue dye, no change in the pace of progression of dystrophy was seen in banded animals vs. unbanded, sham-operated controls. At the completion of the study, more centrally nucleated fibers were evident in the diaphragms of banded mdx mice than in sham-operated mdx controls, indicating that myofiber regeneration increases to meet the demands of the work-induced damage. These data suggest that there is untapped regenerative capacity in dystrophin-deficient muscle and validates experimental efforts aimed at augmenting regeneration within skeletal muscle as a therapeutic strategy in the treatment of dystrophinopathies.     

Herbarium-Based Preliminary Conservation Assessments of Lesser Antillean Endemic Seed Plants Reveal a Flora at Risk

Conservation assessments present a formidable challenge for regions of the developing world such as the Lesser Antilles. Of the 2652 seed plant taxa recorded from this archipelago (Acevedo-Rodríguez & Strong, 2012, 2016), only 150 taxa (including 14 endemics) are listed in the IUCN Red List. Using only herbarium data, we carried out a preliminary conservation assessment of the 263 seed plant taxa unique to these islands by two methods. Using the algorithm originally developed to assess the Hawaiian flora (Krupnick et al. in Biodiversity and Conservation 18: 1459–1474, 2009), 8% of the taxa were deemed to be Potentially Extinct and 62% Potentially Threatened while 30% were assessed as Not Threatened. Using a Red List criteria approach, 70% were deemed Threatened (Critically Endangered, Endangered and Vulnerable), while 30% were assessed as Near Threatened, Least Concern or Data Deficient. The congruence between the assessments by these distinct methods and for the 14 Red Listed species validate this approach as a first step in prioritising plant conservation efforts.     

Quantitative monitoring of mouse lung tumors by magnetic resonance imaging

Primary lung cancer remains the leading cause of cancer-related death in the Western world, and the lung is a common site for recurrence of extrathoracic malignancies. Small-animal (rodent) models of cancer can have a very valuable role in the development of improved therapeutic strategies. However, detection of mouse pulmonary tumors and their subsequent response to therapy in situ is challenging. We have recently described MRI as a reliable, reproducible and nondestructive modality for the detection and serial monitoring of pulmonary tumors. By combining respiratory-gated data acquisition methods with manual and automated segmentation algorithms described by our laboratory, pulmonary tumor burden can be quantitatively measured in approximately 1 h (data acquisition plus analysis) per mouse. Quantitative, analytical methods are described for measuring tumor burden in both primary (discrete tumors) and metastatic (diffuse tumors) disease. Thus, small-animal MRI represents a novel and unique research tool for preclinical investigation of therapeutic strategies for treatment of pulmonary malignancies, and it may be valuable in evaluating new compounds targeting lung cancer in vivo.     

Murine vascular endothelium activates and induces the generation of allogeneic CD4+25+Foxp3+ regulatory T cells

Unlike graft-resident donor-derived hemopoietic APCs, which decrease in number over time after transplantation, vascular endothelial cells are lifelong residents of a vascularized allograft. Endothelial cells are potent APCs for allogeneic CD8+ T lymphocytes but are unable to induce proliferation of allogeneic CD4+ T lymphocytes. Although the reason for this differential response has been poorly understood, here we report that alloantigen presentation by vascular endothelium to CD4+ T lymphocytes activates and induces CD4+25+Foxp3+ regulatory T cells, which can inhibit proliferation of alloreactive T cells both in vitro and in vivo. This process occurs independently of B7.1 costimulation but is dependent on programmed death ligand 1 (B7-H1). This finding may have important implications for tolerance induction in transplantation.     

Non-hematopoietic allograft cells directly activate CD8+ T cells and trigger acute rejection: an alternative mechanism of allorecognition

Despite evidence that human non-hematopoietic cells, such as vascular endothelium, can activate allogeneic T lymphocytes in vitro, the prevailing view has been that hematopoietic antigen-presenting cells are required to trigger alloimmune responses in vivo. Here we report that mouse non-hematopoietic cells activate alloreactive CD8+ T lymphocytes in vitro and in vivo. We also show that vascularized cardiac allografts are acutely rejected via CD8+ direct allorecognition even if the alloantigen is not presented by hematopoietic professional antigen-presenting cells. Because activation of alloreactive CD8+ T cells by donor-type non-hematopoietic cells can continue for the life of the allograft, these findings present a new clinically relevant mechanism of allorecognition and should be taken into consideration when developing strategies to prevent allograft vasculopathy or to induce tolerance.