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RATIONALE - Lung natural killer cells (NKs) kill a greater percentage of autologous lung parenchymal cells in chronic obstructive pulmonary disease (COPD) than in nonobstructed smokers. To become cytotoxic, NKs require priming, typically by dendritic cells (DCs), but whether priming occurs in the lungs in COPD is unknown.
METHODS - We used lung tissue and in some cases peripheral blood from patients undergoing clinically indicated resections to determine in vitro killing of CD326 lung epithelial cells by isolated lung CD56 NKs. We also measured the cytotoxicity of unprimed blood NKs after preincubation with lung DCs. To investigate mechanisms of DC-mediated priming, we used murine models of COPD induced by cigarette smoke (CS) exposure or by polymeric immunoglobulin receptor (pIgR) deficiency, and blocked IL-15Rα (IL-15 receptor α subunit) trans-presentation by genetic and antibody approaches.
RESULTS - Human lung NKs killed isolated autologous lung epithelial cells; cytotoxicity was increased (P = 0.0001) in COPD, relative to smokers without obstruction. Similarly, increased lung NK cytotoxicity compared with control subjects was observed in CS-exposed mice and pIgR mice. Blood NKs both from smokers without obstruction and subjects with COPD showed minimal epithelial cell killing, but in COPD, preincubation with lung DCs increased cytotoxicity. NKs were primed by CS-exposed murine DCs in vitro and in vivo. Inhibiting IL-15Rα trans-presentation eliminated NK priming both by murine CS-exposed DCs and by lung DCs from subjects with COPD.
CONCLUSIONS - Heightened NK cytotoxicity against lung epithelial cells in COPD results primarily from lung DC-mediated priming via IL-15 trans-presentation on IL-15Rα. Future studies are required to test whether increased NK cytotoxicity contributes to COPD pathogenesis.
Helicobacter pylori colonizes the human stomach and is a potential cause of peptic ulceration or gastric adenocarcinoma. H. pylori secretes a pore-forming toxin known as vacuolating cytotoxin A (VacA). The 88 kDa secreted VacA protein, composed of an N-terminal p33 domain and a C-terminal p55 domain, assembles into water-soluble oligomers. The structural organization of membrane-bound VacA has not been characterized in any detail and the role(s) of specific VacA domains in membrane binding and insertion are unclear. We show that membrane-bound VacA organizes into hexameric oligomers. Comparison of the two-dimensional averages of membrane-bound and soluble VacA hexamers generated using single particle electron microscopy reveals a structural difference in the central region of the oligomers (corresponding to the p33 domain), suggesting that membrane association triggers a structural change in the p33 domain. Analyses of the isolated p55 domain and VacA variants demonstrate that while the p55 domain can bind membranes, the p33 domain is required for membrane insertion. Surprisingly, neither VacA oligomerization nor the presence of putative transmembrane GXXXG repeats in the p33 domain is required for membrane insertion. These findings provide new insights into the process by which VacA binds and inserts into the lipid bilayer to form membrane channels.
© 2016 John Wiley & Sons Ltd.
Staphylococcus aureus (S. aureus) is a human pathogen that relies on the subversion of host phagocytes to support its pathogenic lifestyle. S. aureus strains can produce up to five beta-barrel, bi-component, pore-forming leukocidins that target and kill host phagocytes. Thus, preventing immune cell killing by these toxins is likely to boost host immunity. Here, we describe the identification of glycine-rich motifs within the membrane-penetrating stem domains of the leukocidin subunits that are critical for killing primary human neutrophils. Remarkably, leukocidins lacking these glycine-rich motifs exhibit dominant-negative inhibitory effects toward their wild-type toxin counterparts as well as other leukocidins. Biochemical and cellular assays revealed that these dominant-negative toxins work by forming mixed complexes that are impaired in pore formation. The dominant-negative leukocidins inhibited S. aureus cytotoxicity toward primary human neutrophils, protected mice from lethal challenge by wild-type leukocidin, and reduced bacterial burden in a murine model of bloodstream infection. Thus, we describe the first example of staphylococcal bi-component dominant-negative toxins and their potential as novel therapeutics to combat S. aureus infection.
© 2016 The Authors.
Bilirubin is thought to exert anti-inflammatory effects by inhibiting vascular cell adhesion molecule-1 (VCAM-1)-dependent leukocyte migration and by suppressing the expression of inducible nitric oxide synthase (iNOS). As VCAM-1 and iNOS are important mediators of tissue injury in the dextran sodium sulfate (DSS) murine model of inflammatory colitis, we examined whether bilirubin prevents colonic injury in DSS-treated mice. Male C57BL/6 mice were administered 2.5% DSS in the drinking water for 7 days, while simultaneously receiving intraperitoneal injections of bilirubin (30 mg/kg) or potassium phosphate vehicle. Disease activity was monitored, peripheral blood counts and serum nitrate levels were determined, and intestinal specimens were analyzed for histological injury, leukocyte infiltration, and iNOS expression. The effect of bilirubin on IL-5 production by HSB-2 cells and on Jurkat cell transendothelial migration also was determined. DSS-treated mice that simultaneously received bilirubin lost less body weight, had lower serum nitrate levels, and exhibited reduced disease severity than vehicle-treated animals. Concordantly, histopathological analyses revealed that bilirubin-treated mice manifested significantly less colonic injury, including reduced infiltration of eosinophils, lymphocytes, and monocytes, and diminished iNOS expression. Bilirubin administration also was associated with decreased eosinophil and monocyte infiltration into the small intestine, with a corresponding increase in peripheral blood eosinophilia. Bilirubin prevented Jurkat migration but did not alter IL-5 production. In conclusion, bilirubin prevents DSS-induced colitis by inhibiting the migration of leukocytes across the vascular endothelium and by suppressing iNOS expression.
Copyright © 2015 the American Physiological Society.
Despite the importance of Staphylococcus aureus as a common invasive bacterial pathogen, the humoral response to infection remains inadequately defined, particularly in children. The purpose of this study was to assess the humoral response to extracellular staphylococcal virulence factors, including the bicomponent leukotoxins, which are critical for the cytotoxicity of S. aureus toward human neutrophils. Children with culture-proven S. aureus infection were prospectively enrolled and stratified by disease type. Fifty-three children were enrolled in the study, of which 90% had invasive disease. Serum samples were obtained during the acute (within 48 h) and convalescent (4 to 6 weeks postinfection) phases, at which point both IgG titers against S. aureus exotoxins were determined, and the functionality of the generated antibodies was evaluated. Molecular characterization of clinical isolates was also performed. We observed a marked rise in antibody titer from acute-phase to convalescent-phase sera for LukAB, the most recently described S. aureus bicomponent leukotoxin. LukAB production by the isolates was strongly correlated with cytotoxicity in vitro, and sera containing anti-LukAB antibodies potently neutralized cytotoxicity. Antibodies to S. aureus antigens were detectable in healthy pediatric controls but at much lower titers than in sera from infected subjects. The discovery of a high-titer, neutralizing antibody response to LukAB during invasive infections suggests that this toxin is produced in vivo and that it elicits a functional humoral response.
BACKGROUND & AIMS - Helicobacter pylori infection is a risk factor for gastric cancer. Ammonia/ammonium (A/A) is a cytotoxin generated by H pylori that kills gastric epithelial cells. We investigated whether A/A cytotoxicity occurs by activating N-methyl d-aspartate (NMDA) channels, which results in Ca(2+) permeation and epithelial cell death.
METHODS - Gastric epithelial cells were cultured to confluence and then incubated with A/A and NMDA channel or cell signaling antagonists. Cells were incubated with wild-type H pylori or mutant strains that do not produce A/A. Changes in intracellular Ca(2+) were examined in living cells by confocal microscopy. Biochemical and histochemical techniques were used to examine the relationship between A/A-induced cell death and intracellular levels of Ca(2+).
RESULTS - A/A increased Ca(2+) permeation in gastric epithelial cells; the increase was blocked by NMDA receptor and cell signaling antagonists. Wild-type, but not mutant H pylori, also caused extensive Ca(2+) permeation of gastric epithelial cells, which was blocked when NMDA-receptor expression was repressed. Ca(2+) that entered cells was initially cytoplasmic and activated proteases. Later, the Ca(2+) was sequestered to cytoplasmic vacuoles that are dilatations of the endoplasmic reticulum. Inositol-3-phosphate-dependent release of Ca(2+) from the endoplasmic reticulum and protease activity damaged mitochondria, reduced levels of adenosine triphosphate, and transcriptionally up-regulated cell death effectors. Expression of the NMDA receptor was altered in stomachs of mice infected with H pylori.
CONCLUSIONS - A/A affects gastric epithelial cell viability by allowing excessive Ca(2+) permeation through NMDA channels. NMDA channels might thereby regulate cell survival and death pathways during development of gastric cancers associated with H pylori infection.
Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.
Superinfections from Staphylococcus aureus following influenza are an increasing concern. We assessed several laboratory and clinical strains in a mouse coinfection model with influenza virus. A methicillin-resistant USA300 clone and several recent clinical strains from patients with necrotizing pneumonia caused high mortality following influenza virus infection in mice. Both viral and bacterial lung titers were enhanced during coinfections compared with single infections. However, differences in titers did not correspond with differences in disease outcomes in a comparison of superinfections from a highly pathogenic strain with those from a poorly pathogenic strain. These strains did differ, however, in expression of Panton-Valentine leukocidin and in the degree of inflammatory lung damage each engendered. The viral cytotoxin PB1-F2 contributed to the negative outcomes. These data suggest that additional study of specific bacterial virulence factors involved in the pathogenesis of inflammation and lung damage during coinfections is needed.
Levuglandins and their stereo- and regio-isomers (termed isolevuglandins or isoketals) are gamma-ketoaldehydes (IsoK) that rapidly react with lysines to form stable protein adducts. IsoK protein adduct levels increase in several pathological conditions including cardiovascular disease. IsoKs can induce ion channel dysfunction and cell death, potentially by adducting to cellular proteins. However, IsoKs also adduct to phosphatidylethanolamine (PE) in vitro, and whether PE adducts form in cells or contribute to the effects of IsoKs is unknown. When radiolabeled IsoK was added to HEK293 cells, 40% of the radiolabel extracted into the chloroform lower phase suggesting the possible formation of PE adducts. We therefore developed methods to measure IsoK-PE adducts in cells. IsoK-PE was quantified by LC/MS/MS after hydrolysis to IsoK-ethanolamine by Streptomyces chromofuscus phospholipase D. In HEK293 and human umbilical vein endothelial cells (HUVEC), IsoK dose-dependently increased PE adduct concentrations to a greater extent than protein adduct. To test the biological significance of IsoK-PE formation, we treated HUVEC with IsoK-PE. IsoK-PE dose dependently induced cytotoxicity (LC(50) 2.2 muM). These results indicate that cellular PE is a significant target of IsoKs, and that formation of PE adducts may mediate some of the biological effects of IsoKs relevant to disease.
Oxidative stress, defined as an increase in reactive oxygen species, leads to peroxidation of polyunsaturated fatty acids and generates a vast number of biologically active molecules, many of which might contribute in some way to health and disease. This chapter will focus on one specific class of peroxidation products, the levuglandins and isoketals (also called isolevuglandins). These gamma-ketoaldehydes are some of the most reactive products derived from the peroxidation of lipids and exert their biological effects by rapidly adducting to primary amines such as the lysyl residues of proteins. The mechanism of their formation and remarkable reactivity will be described, along with evidence for their increased formation in disease conditions linked with oxidative stress and inflammation. Finally, the currently known effects of these gamma-ketoaldehydes on cellular function will then be discussed and when appropriate compared to the effects of alpha,beta-unsaturated fatty aldehydes, in order to illustrate the significant differences between these two classes of peroxidation products that modify proteins.
Activation of the extracellular signal-regulated kinase1/2 (ERK1/2) signaling cascade mediates human multiple myeloma (MM) growth and survival triggered by cytokines and adhesion to bone marrow stromal cells (BMSCs). Here, we examined the effect of AZD6244 (ARRY-142886), a novel and specific MEK1/2 inhibitor, on human MM cell growth in the bone marrow (BM) milieu. AZD6244 blocks constitutive and cytokine-stimulated ERK1/2 phosphorylation and inhibits proliferation and survival of human MM cell lines and patient MM cells, regardless of sensitivity to conventional chemotherapy. Importantly, AZD6244 (200 nM) induces apoptosis in patient MM cells, even in the presence of exogenous interleukin-6 or BMSCs associated with triggering of caspase 3 activity. AZD6244 sensitizes MM cells to both conventional (dexamethasone) and novel (perifosine, lenalidomide, and bortezomib) therapies. AZD6244 down-regulates the expression/secretion of osteoclast (OC)-activating factors from MM cells and inhibits in vitro differentiation of MM patient PBMCs to OCs induced by ligand for receptor activator of NF-kappaB (RANKL) and macrophage-colony stimulating factor (M-CSF). Finally, AZD6244 inhibits tumor growth and prolongs survival in vivo in a human plasmacytoma xenograft model. Taken together, these results show that AZD6244 targets both MM cells and OCs in the BM microenvironment, providing the preclinical framework for clinical trials to improve patient outcome in MM.