Antigen-specific regulatory T cells develop via the ICOS-ICOS-ligand pathway and inhibit allergen-induced airway hyperreactivity

Asthma is caused by T-helper cell 2 (Th2)-driven immune responses, but the immunological mechanisms that protect against asthma development are poorly understood. T-cell tolerance, induced by respiratory exposure to allergen, can inhibit the development of airway hyperreactivity (AHR), a cardinal feature of asthma, and we show here that regulatory T (T(R)) cells can mediate this protective effect. Mature pulmonary dendritic cells in the bronchial lymph nodes of mice exposed to respiratory allergen induced the development of T(R) cells, in a process that required T-cell costimulation via the inducible costimulator (ICOS-ICOS-ligand pathway. The T(R) cells produced IL-10, and had potent inhibitory activity; when adoptively transferred into sensitized mice, T(R) cells blocked the development of AHR. Both the development and the inhibitory function of regulatory cells were dependent on the presence of IL-10 and on ICOS-ICOS-ligand interactions. These studies demonstrate that T(R) cells and the ICOS-ICOS-ligand signaling pathway are critically involved in respiratory tolerance and in downregulating pulmonary inflammation in asthma.     

The Arabidopsis TONNEAU2 gene encodes a putative novel protein phosphatase 2A regulatory subunit essential for the control of the cortical cytoskeleton

In Arabidopsis ton2 mutants, abnormalities of the cortical microtubular cytoskeleton, such as disorganization of the interphase microtubule array and lack of the preprophase band before mitosis, markedly affect cell shape and arrangement as well as overall plant morphology. We present the molecular isolation of the TON2 gene, which is highly conserved in higher plants and has a vertebrate homolog of unknown function. It encodes a protein similar in its C-terminal part to B" regulatory subunits of type 2A protein phosphatases (PP2As). We show that the TON2 protein interacts with an Arabidopsis type A subunit of PP2A in the yeast two-hybrid system and thus likely defines a novel subclass of PP2A subunits that are possibly involved in the control of cytoskeletal structures in plants.     

Four-Dimensional Characterization of Thrombosis in a Live-Cell,Shear-Flow Assay: Development and Application to Xenotransplantation

Background

Porcine xenografts are a promising source of scarce transplantable organs, but stimulate intense thrombosis of human blood despite targeted genetic and pharmacologic interventions. Current experimental models do not enable study of the blood/endothelial interface to investigate adhesive interactions and thrombosis at the cellular level under physiologic conditions. The purpose of this study was to develop and validate a live-cell, shear-flow based thrombosis assay relevant to general thrombosis research, and demonstrate its potential in xenotransplantation applications.

Methodology/Principal Findings

Confluent wild-type (WT, n = 48) and Gal transferase knock-out (GalTKO, which resist hyperacute rejection; n = 11) porcine endothelia were cultured in microfluidic channels. To mimic microcirculatory flow, channels were perfused at 5 dynes/cm² and 37°C with human blood stained to fluorescently label platelets. Serial fluorescent imaging visualized percent surface area coverage (SA, for adhesion of labeled cells) and total fluorescence (a metric of clot volume). Aggregation was calculated by the fluorescence/SA ratio (FR). WT endothelia stimulated diffuse platelet adhesion (SA 65 ± 2%) and aggregation (FR 120 ± 1 a.u.), indicating high-grade thrombosis consistent with the rapid platelet activation and consumption seen in whole-organ lung xenotransplantation models. Experiments with antibody blockade of platelet aggregation, and perfusion of syngeneic and allo-incompatible endothelium was used to verify the biologic specificity and validity of the assay. Finally, with GalTKO endothelia thrombus volume decreased by 60%, due primarily to a 58% reduction in adhesion (P < 0.0001 each); importantly, aggregation was only marginally affected (11% reduction, P < 0.0001).

Conclusions/Significance

This novel, high-throughput assay enabled dynamic modeling of whole-blood thrombosis on intact endothelium under physiologic conditions, and allowed mechanistic characterization of endothelial and platelet interactions. Applied to xenogeneic thrombosis, it enables future studies regarding the effect of modifying the porcine genotype on sheer-stress-dependent events that characterize xenograft injury. This in-vitro platform is likely to prove broadly useful to study thrombosis and endothelial interactions under dynamic physiologic conditions.     

Functional Annotation,Genome Organization and Phylogeny of the Grapevine (<Emphasis Type="Italic">Vitis vinifera</Emphasis>) Terpene Synthase Gene Family Based on Genome Assembly,FLcDNA Cloning,and Enzyme Assays

Background  

Terpenoids are among the most important constituents of grape flavour and wine bouquet, and serve as useful metabolite markers in viticulture and enology. Based on the initial 8-fold sequencing of a nearly homozygous Pinot noir inbred line, 89 putative terpenoid synthase genes (VvTPS) were predicted by in silico analysis of the grapevine (Vitis vinifera) genome assembly [1]. The finding of this very large VvTPS family, combined with the importance of terpenoid metabolism for the organoleptic properties of grapevine berries and finished wines, prompted a detailed examination of this gene family at the genomic level as well as an investigation into VvTPS biochemical functions.     

Absence of p53-dependent apoptosis leads to UV radiation hypersensitivity,enhanced immunosuppression and cellular senescence

Genotoxic stress triggers the p53 tumor suppressor network to activate cellular responses that lead to cell cycle arrest, DNA repair, apoptosis or senescence. This network functions mainly through transactivation of different downstream targets, including cell cycle inhibitor p21, which is required for short-term cell cycle arrest or long-term cellular senescence, or proapoptotic genes such as p53 upregulated modulator of apoptosis (PUMA) and Noxa. However, the mechanism that switches from cell cycle arrest to apoptosis is still unknown. In this study, we found that mice harboring a hypomorphic mutant p53, R172P, a mutation that abrogates p53-mediated apoptosis while keeping cell cycle control mostly intact, are more susceptible to ultraviolet-B (UVB)-induced skin damage, inflammation and immunosuppression than wild-type mice. p53^R172P embryonic fibroblasts (MEFs) are hypersensitive to UVB and prematurely senesce after UVB exposure, in stark contrast to wild-type MEFs, which undergo apoptosis. However, these mutant cells are able to repair UV-induced DNA lesions, indicating that the UV-hypersensitive phenotype results from the subsequent damage response. Mutant MEFs show an induction of p53 and p21 after UVR, while wild-type MEFs additionally induce PUMA and Noxa. Importantly, p53^R172P MEFs failed to downregulate anti-apoptotic protein Bcl-2, which has been shown to play an important role in p53-dependent apoptosis. Taken together, these data demonstrate that in the absence of p53-mediated apoptosis, cells undergo cellular senescence to prevent genomic instability. Our results also indicate that p53-dependent apoptosis may play an active role in balancing cellular growth.Key words: UVB irradiation, p53, DNA damage, DNA damage responses, apoptosis, senescence     

Vitamin D receptor expression by the lung micro-environment is required for maximal induction of lung inflammation

Mice lacking the vitamin D receptor (VDR) are resistant to airway inflammation. Pathogenic immune cells capable of transferring experimental airway inflammation to wildtype (WT) mice are present and primed in the VDR KO mice. Furthermore, the VDR KO immune cells homed to the WT lung in sufficient numbers to induce symptoms of asthma. Conversely, WT splenocytes, Th2 cells and hematopoetic cells induced some symptoms of experimental asthma when transferred to VDR KO mice, but the severity was less than that seen in the WT controls. Interestingly, experimentally induced vitamin D deficiency failed to mirror the VDR KO phenotype suggesting there might be a difference between absence of the ligand and VDR deficiency. Lipopolysaccharide (LPS) induced inflammation in the lungs of VDR KO mice was also less than in WT mice. Together the data suggest that vitamin D and the VDR are important regulators of inflammation in the lung and that in the absence of the VDR the lung environment, independent of immune cells, is less responsive to environmental challenges.     

Structural Basis for Antibody Recognition of Lipid A: INSIGHTS TO POLYSPECIFICITY TOWARD SINGLE-STRANDED DNA*

Septic shock is a leading cause of death, and it results from an inflammatory cascade triggered by the presence of microbial products in the blood. Certain LPS from Gram-negative bacteria are very potent inducers and are responsible for a high percentage of septic shock cases. Despite decades of research, mAbs specific for lipid A (the endotoxic principle of LPS) have not been successfully developed into a clinical treatment for sepsis. To understand the molecular basis for the observed inability to translate in vitro specificity for lipid A into clinical potential, the structures of antigen-binding fragments of mAbs S1–15 and A6 have been determined both in complex with lipid A carbohydrate backbone and in the unliganded form. The two antibodies have separate germ line origins that generate two markedly different combining-site pockets that are complementary both in shape and charge to the antigen. mAb A6 binds lipid A through both variable light and heavy chain residues, whereas S1–15 utilizes exclusively the variable heavy chain. Both antibodies bind lipid A such that the GlcN-O6 attachment point for the core oligosaccharide is buried in the combining site, which explains the lack of LPS recognition. Longstanding reports of polyspecificity of anti-lipid A antibodies toward single-stranded DNA combined with observed homology of S1–15 and A6 and the reports of several single-stranded DNA-specific mAbs prompted the determination of the structure of S1–15 in complex with single-stranded DNA fragments, which may provide clues about the genesis of autoimmune diseases such as systemic lupus erythematosus, thyroiditis, and rheumatic autoimmune diseases.