Endosomal MR1 Trafficking Plays a Key Role in Presentation of Mycobacterium tuberculosis Ligands to MAIT Cells

Mucosal-Associated Invariant T (MAIT) cells, present in high frequency in airway and other mucosal tissues, have Th1 effector capacity positioning them to play a critical role in the early immune response to intracellular pathogens, including Mycobacterium tuberculosis (Mtb). MR1 is a highly conserved Class I-like molecule that presents vitamin B metabolites to MAIT cells. The mechanisms for loading these ubiquitous small molecules are likely to be tightly regulated to prevent inappropriate MAIT cell activation. To define the intracellular localization of MR1, we analyzed the distribution of an MR1-GFP fusion protein in antigen presenting cells. We found that MR1 localized to endosomes and was translocated to the cell surface upon addition of 6-formyl pterin (6-FP). To understand the mechanisms by which MR1 antigens are presented, we used a lentiviral shRNA screen to identify trafficking molecules that are required for the presentation of Mtb antigen to HLA-diverse T cells. We identified Stx18, VAMP4, and Rab6 as trafficking molecules regulating MR1-dependent MAIT cell recognition of Mtb-infected cells. Stx18 but not VAMP4 or Rab6 knockdown also resulted in decreased 6-FP-dependent surface translocation of MR1 suggesting distinct pathways for loading of exogenous ligands and intracellular mycobacterially-derived ligands. We postulate that endosome-mediated trafficking of MR1 allows for selective sampling of the intracellular environment.     

Dendritic Cells Loaded with Tumor Antigens and a Dual Immunostimulatory and Anti-Interleukin 10-Specific Small Interference RNA Prime T Lymphocytes against Leukemic Cells

Vaccines using dendritic cells (DCs) harboring leukemic antigens to stimulate T cells is a possible treatment of acute myeloid leukemia (AML). Limitations of breaking tolerance to leukemic cells and lack of specific activation of T cell-mediated cytotoxicity may explain the discouraging clinical results with this approach. To break self-tolerance against AML cells, we loaded DCs with AML antigens and a bifunctional small interference (si) RNA targeting interleukin (IL) 10 and simultaneously activating toll-like receptors (TLRs). In vitro, this active siRNA inhibited (P < .05) IL-10 production by silencing the IL-10 gene in DCs. The active siRNA stimulated production of tumor necrosis factor α, implying activation of TLRs. Vaccination in a nonimmunogenic rat model mimicking human AML with the loaded DCs induced a substantial and specific T-cell cytotoxicity. Leukemic rats treated with the active siRNA lived longer and had markedly less leukemic cell mass infiltrating their bone marrow compared with rats given inactive siRNA (P < .05). Furthermore, compared with inactive siRNA treatment, the active siRNA led to significantly less extramedullar leukemic dissemination, evidenced by reduced matrix metalloproteinase activity and smaller spleens. Our data demonstrate that this bifunctional siRNA may work as an immunomodulatory drug with antileukemic properties.     

Chemical strategies for the global analysis of protein function

As the human genome project nears completion, biological research is entering a new era in which experimental focus will shift from identifying novel genes to determining the function of gene products. Rising to this challenge, several technologies have emerged that aim to characterise genes and/or proteins collectively rather than individually. Of particular interest is a new breed of strategies that employs synthetic chemistry to enrich our understanding of protein function on a global scale.     

Differences in natural infections of two mortality-related trematodes in lesser scaup and American coot

Populations of North American waterbirds, particularly lesser scaup, have been declining due to habitat disturbance, changing food resources, contaminants, bad water quality, and competition. However, epizootic diseases, including parasitism, may also play an important role in further decline. Trematode-associated mortality of migrating waterbirds, mainly American coot and lesser scaup, has been occurring in the Upper Mississippi River National Wildlife and Fish Refuge since 2002. We examined the levels of infective stages of Cyathocotyle bushiensis and Sphaeridiotrema globulus in the invasive, intermediate host snail, Bithynia tentaculata, during the fall of 2005 and compared these to infection levels in moribund or dead bird hosts. Our results show different infection levels of these 2 parasites in the 2 bird species; C. bushiensis is found more frequently in coot, and S. globulus is more common in scaup. This result is interesting because both bird species are presumed to forage on the same snail population and thus should be experiencing the same extent of exposure. These differences in infections could be attributed to differences in resources of gastrointestinal tracts of coot and scaup, or host resistance. Alternatively, differences in feeding behaviors of coot and scaup may also contribute to differential infections of the 2 trematodes.     

The dilemma of foraging herbivores: dealing with food and fear

For foraging herbivores, both food quality and predation risk vary across the landscape. Animals should avoid low-quality food patches in favour of high-quality ones, and seek safe patches while avoiding risky ones. Herbivores often face the foraging dilemma, however, of choosing between high-quality food in risky places or low-quality food in safe places. Here, we explore how and why the interaction between food quality and predation risk affects foraging decisions of mammalian herbivores, focusing on browsers confronting plant toxins in a landscape of fear. We draw together themes of plant–herbivore and predator–prey interactions, and the roles of animal ecophysiology, behaviour and personality. The response of herbivores to the dual costs of food and fear depends on the interplay of physiology and behaviour. We discuss detoxification physiology in dealing with plant toxins, and stress physiology associated with perceived predation risk. We argue that behaviour is the interface enabling herbivores to stay or quit food patches in response to their physiological tolerance to these risks. We hypothesise that generalist and specialist herbivores perceive the relative costs of plant defence and predation risk differently and intra-specifically, individuals with different personalities and physiologies should do so too, creating individualised landscapes of food and fear. We explore the ecological significance and emergent impacts of these individual-based foraging outcomes on populations and communities, and offer predictions that can be clearly tested. In doing so, we provide an integrated platform advancing herbivore foraging theory with food quality and predation risk at its core.     

μ-H-Bridged Bicyclo[3.3.3]undecyl Cations. Theoretical Calculations of Physical and Chemical Properties

7-H-Bridged carbocations 1 and 2, which are still unknown experimentally, are structures with fascinating possibilities as intermediates for the synthesis of very strained in-bicyclic and tricyclic alkanes and alkenes. They are also expected to possess record high pK_a values. In conjunction with our experimental program to try to prepare 1 and 2, we have carried out ab initio calculations on these structures and various reference compounds, with the aim of assessing just how stable these cations might be, and what physical and chemical properties they might possess. The results of these studies confirm that 1 and 2 should be viable species; they have energies similar to those of the conventional out-cations, and the 7-H bond distances are not very different from those calculated for known 7-H cations. The calculated ¹H NMR chemical shift for the mu-H of 1 is -12 - 0.5 and -9.5 - 0.5 for 2, both values considerably more negative than in known 7-H cations. The possible effect of large amplitude motions of the 7-H on the ¹H NMR chemical shift was investigated and not found to be significant. The pK_as for 1 and 2 are estimated to be 17-18 with respect to an alkene conjugate base, a virtually unimaginable size for a formally alkyl cation. The paper also discusses the possibility of the 7-H being involved in the acid-conjugate base chemistry, since this is shown to be a hugely exothermic reaction. Finally, the alkenes and alkanes associated with cations 1 and 2 have been calculated, the in-alkene 7 from cation 1 is shown, for example, to possess a large steric strain, yet this structure should be accessible via deprotonation of 1 with a strong base.     

Surfactant protein D is proatherogenic in mice

Surfactant protein D (SP-D) is an important innate immune defense molecule that mediates clearance of pathogens and modulates the inflammatory response. Moreover, SP-D is involved in lipid homeostasis, and pulmonary accumulation of phospholipids has previously been observed in SP-D-deficient (Spd-/-) mice. Atherogenesis involves both inflammation and lipid deposition, and we investigated the role of SP-D in the development of atherosclerosis. SP-D synthesis was localized to vascular endothelial cells. Atherosclerotic lesion areas were 5.6-fold smaller in the aortic roots in Spd-/- mice compared with wild-type C57BL/6N mice on an atherogenic diet. HDL cholesterol (HDL-C) was significantly elevated in Spd-/- mice. Treatment of Spd-/- mice with a recombinant fragment of human SP-D resulted in decreases of HDL-C (21%) as well as total cholesterol (26%), and LDL cholesterol (28%). Plasma TNF-alpha was reduced in Spd-/- mice (45% difference). SP-D was proatherogenic in the mouse model used. The effect is likely to be due to the observed disturbances of plasma lipid metabolism and alteration of the inflammatory process, which underlie the reduced susceptibility to atherosclerosis in Spd-/- mice.     

Inhibition of hydroxyproline synthesis by palladium ions.

Palladium ions, administered as PdSO4, markedly affect the incorporation of L-[3,4-3H2] proline into non-dialyzable fractions in 10-day chick embryo cartilage explants with a 55-65% reduction in the concentration range 0.06-0.6 mM. Under these conditions the synthesis of [3H]hydroxyproline was nearly completely inhibited. Experiments with prolyl hydroxylase (EC 1.14.11.2) indicated a strong irreversible inhibition of the enzyme with a competition between Fe2+ and Pd2+. The Ki for the inhibition was 0.02 mM. Pd2+-treated enzyme remained inactive after extensive dialysis. These studies suggest that Pd2+ may inhibit collagen synthesis by replacing Fe2+ in the active site of prolyl hydroxylase and forming strong complexes with the enzyme. These studies also point to a potential mechanism of Pd2+ toxicity.     

Identification and characterization of a suppressor T cell hybridoma specifically inducible by L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT)

In vitro activation of naive spleen cells from C57BL/10 mice with GAT and the monoclonal GAT-TsF1, 372B3.5, followed by fusion with BW5147 resulted in generation of a hybridoma that fails to produce GAT-TsF constitutively, but upon reexposure to GAT and 372B3.5 is induced to secrete GAT-TsF2. The induction is GAT specific and requires de novo RNA, protein synthesis, and DNA synthesis. Although both GAT and 372B3.5 are required for induction, they may be added sequentially, provided the GAT is added first. The GAT-TsF produced by the induced cell is antigen specific and composed of two polypeptide chains: one capable of binding antigen, the other bearing determinants encoded by the I-J region of the MHC. The utility of this inducible GAT-TsF2 cell line for molecular biology and other studies is discussed.