Interactions between CD8+ T cells and endothelial cells are important in both protective and pathologic immune responses. Endothelial cells regulate the recruitment of CD8 + T cells into tissues, and the activation of CD8 + T cells by antigen presentation and costimulatory signals. PD-L1 and PD-L2 are recently described B7-family molecules which bind to PD-1 on activated lymphocytes and down-regulate T cell activation. We found that PD-L1 is expressed on interferon-+ stimulated cultured human and mouse endothelial cells, while PD-L2 was found on stimulated human but not mouse endothelial cells. Expression was further upregulated by TNF- § . Antibody blockade of endothelial cell PD-L1 and PD-L2 enhanced endothelial cell costimulation of PHA-activated human CD8 + T cells. Antibody blockade of mouse endothelial cell PD-L1 enhanced both IFN-+ secretion and cytolytic activity of CD8 + T cells in response to endothelial cell antigen presentation. These results show that IFN-+ activated endothelial cells can inhibit T cell activation via expression of the immunoinhibitory PD-L1 and PD-L2 molecules. Endothelial expression of PD-ligands would allow activation and extravasation of T cells without excessive vessel damage. Our findings highlight a potentially important pathway by which endothelial cells down-regulate CD8 + T cellmediated immune responses.
Neuropilins (NRPs) are trans-membrane receptors involved in axon guidance and vascular development. Many growth factors and other signaling molecules bind to NRPs through a C-terminal, basic sequence motif (C-end Rule or CendR motif). Peptides with this motif (CendR peptides) are taken up into cells by endocytosis. Tumor-homing CendR peptides penetrate through tumor tissue and have shown utility in enhancing drug delivery into tumors. Here we show, using RNAi screening and subsequent validation studies, that NRP1-mediated endocytosis of CendR peptides is distinct from known endocytic pathways. Ultrastructurally, CendR endocytosis resembles macropinocytosis, but is mechanistically different. We also show that nutrient-sensing networks such as mTOR signaling regulate CendR endocytosis and subsequent intercellular transport of CendR cargo, both of which are stimulated by nutrient depletion. As CendR is a bulk transport pathway, our results suggest a role for it in nutrient transport; CendR-enhanced drug delivery then makes use of this natural pathway.
Background— PD-L1 and PD-L2 are ligands for the inhibitory receptor programmed death-1 (PD-1), which is an important regulator of immune responses. PD-L1 is induced on cardiac endothelial cells under inflammatory conditions, but little is known about its role in regulating immune injury in the heart. Methods and Results— Cytotoxic T-lymphocyte–mediated myocarditis was induced in mice, and the influence of PD-L1 signaling was studied with PD-L1/L2–deficient mice and blocking antibodies. During cytotoxic T-lymphocyte–induced myocarditis, the upregulation of PD-L1 on cardiac endothelia was dependent on T-cell–derived interferon-γ, and blocking of interferon-γ signaling worsened disease. Genetic deletion of both PD-1 ligands [ PD-L1/2 (−/−) ], as well as treatment with PD-L1 blocking antibody, transformed transient myocarditis to lethal disease, in association with widespread polymorphonuclear leukocyte–rich microabscesses but without change in cytotoxic T-lymphocyte recruitment. PD-L1/2 (−/−) mice reconstituted with bone marrow from wild-type mice remained susceptible to severe disease, which demonstrates that PD-L1 on non–bone marrow–derived cells confers the protective effect. Finally, depletion of polymorphonuclear leukocytes reversed the enhanced susceptibility to lethal myocarditis attributable to PD-L1 deficiency. Conclusions— Myocardial PD-L1, mainly localized on endothelium, is critical for control of immune-mediated cardiac injury and polymorphonuclear leukocyte inflammation.
There is considerable interest in using nanoparticles as labels or to deliver drugs and other bioactive compounds to cells in vitro and in vivo. Fluorescent imaging, commonly used to study internalization and subcellular localization of nanoparticles, does not allow unequivocal distinction between cell surface-bound and internalized particles, since there is no methodology to turn particles ‘off.’ We have developed a simple technique to rapidly remove silver nanoparticles outside living cells leaving only the internalized pool for imaging or quantification. The silver nanoparticle (AgNP) etching is based on the sensitivity of Ag to a hexacyanoferrate/thiosulfate redox-based destain solution. In demonstration of the technique we present a new class of multicolored plasmonic nanoprobes comprising dye-labeled AgNPs that are exceptionally bright and photostable, carry peptides as model targeting ligands, can be etched rapidly and with minimal toxicity in mice and that show tumour uptake in vivo.
Smooth- and rough-form lipopolysaccharides from phenol-water extraction of cells from Helicobacter pylori type strain NCTC 11637 were isolated as the water-soluble component of high-M(r) and water-insoluble low-M(r) gel. Structural investigations were performed on the intact water-soluble smooth-form lipopolysaccharide, various oligosaccharides formed as chemical and enzymic degradation products, and three oligosaccharide fractions liberated by acetic acid hydrolysis from the water-insoluble rough-form lipopolysaccharide. A structure is proposed for the complete polysaccharide component of the smooth-form lipopolysaccharide comprising the O antigen chain, an intervening region, and the inner core oligosaccharide on the basis of 1H and 13C NMR experiments, fast atom bombardment/mass spectrometry, and methylation linkage analysis of permethylated oligo- and polysaccharide derivatives. The most striking feature of the O antigen region in the lipopolysaccharide is the presence of extended chains with fucosylated and nonfucosylated N-acetyllactosamine (LacNAc) units that mimic human cell surface glycoconjugates in normal human granulocytes. The chains are terminated by di- or trimeric Lewis(x) (Le(x)) determinants, which are also found in tumor-associated carbohydrate antigens in many adenocarcinomas.
Lipopolysaccharides from phenol-water extraction of cells of Campylobacter jejuni serotype O:19 were separated into a water-soluble gel of low M(r) and a water-soluble component of high M(r). Acetic acid hydrolysis of the ketosidic linkages to lipid A furnished respectively a core oligosaccharide, the structure of which is reported herein, and an O antigenic polysaccharide. Structural investigations were performed on the O-deacetylated lipopolysaccharide of low M(r), the liberated core oligosaccharide and the various products from removal of neuraminic acid and phosphate residues, and from the Smith degradation. It is concluded that the lipopolysaccharide from the serostrain has a core region with two types of closely related oligosaccharide chains showing striking homologies with gangliosides, the first with a single N-acetylneuraminic acid residue in an outer chain resembling GM1 and the second with two N-acetyl-neuraminic acid residues with a terminal region resembling GD1a. Similar experiments were carried out on lipopolysaccharides of low M(r) from bacterial isolates OH 4384 and OH 4382 serotyped as O:19 that had been obtained from two patients who subsequently developed the Guillain-Barré syndrome. The core oligosaccharide region of lipopolysaccharide from the former isolate differed only slightly from that of the serostrain, whereas that from the latter isolate was distinctly shorter.
The incidence of adverse effects and pathogen resistance encountered with small molecule antibiotics is increasing. As such, there is mounting focus on immunogene therapy to augment the immune system’s response to infection and accelerate healing. A major obstacle to in vivo gene delivery is that the primary uptake pathway, cellular endocytosis, results in extracellular excretion and lysosomal degradation of genetic material. Here we show a nanosystem that bypasses endocytosis and achieves potent gene knockdown efficacy. Porous silicon nanoparticles containing an outer sheath of homing peptides and fusogenic liposome selectively target macrophages and directly introduce an oligonucleotide payload into the cytosol. Highly effective knockdown of the proinflammatory macrophage marker IRF5 enhances the clearance capability of macrophages and improves survival in a mouse model of Staphyloccocus aureus pneumonia.
Complete structures, including the location of N-acetylneuraminic acid (NeuSAc) residues, were assigned for the core regions of Campylobacter jejuni serotypes 0:1, 0:4, and 0:23 and 0 : 3 6 lipopolysaccharides (LPS). In continuation of earlier studies, structure determinations of liberated oligosaccharides and, where necessary, of intact LPS, were by 'H-NMR spectroscopy, Smith degradation, chromium trioxide and enzymic degradations, in conjunction with methylation studies supported by fast-atom-bombardment mass spectrometry and linkage analyses by gas chromatography/ mass spectrometry. It was concluded on the basis of the following structures, in which each was linked 1-5 to a terminal 3-deoxy-~-manno-octu~osonic acid residue, that the core regions with qualititatively similar sugar compositions showed serotypic differences in one or more of their sequences, linkage types, and anomeric configurations : PEtn
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