Lack of functional selectin ligand interactions compromises long term tumor protection by CD8+ T cells

Central memory CD8⁺ T cells expressing the adhesion molecule CD62L (L-selectin) are potent mediators of anti-cancer immunity due to their ability to proliferate extensively upon antigen re-stimulation. The interaction of selectin with its ligands mediates leukocyte rolling along high endothelial venules. Mice deficient in α(1,3) Fucosyltransferase IV and VII (FtDKO) lack functional L, P and E selectin ligands. Thus, we addressed whether the lack of selectin ligand interactions alters tumor protection by CD8⁺ T cells in FtDKO mice. Listeria monocytogenes-OVA (LM-OVA) infection evoked potent OVA-specific CD8⁺ T cells that proliferated and contracted at similar kinetics and phenotype in FtDKO and wild-type mice. Additionally, OVA-specific CD8⁺ T cells in both mouse strains exhibited similar phenotypic differentiation, in vivo cytolytic activity and IFN-γ expression. However, FtDKO mice succumbed to B16-OVA tumors significantly earlier than wild-type mice. In contrast, FtDKO mice evoked strong recall memory CD8⁺ T cell responses and protection to systemic LM-OVA re-challenge. The diminished tumor protection in FtDKO mice was not related to defective antigen presentation by dendritic cells or reduced proliferation of antigen-specific CD8⁺ T cells. However, WT or FtDKO OVA-specific CD8⁺ T cells showed significantly reduced ability to traffic to lymph nodes upon adoptive transfer into naïve FtDKO recipients. Furthermore, FtDKO OVA-specific CD8⁺ T cells displayed poor ability to infiltrate tumors growing in WT mice. These results reveal that selectin ligand expression on host endothelium as well CD8⁺ T cells may be important for their efficient and continued extravasation into peripheral tumors.     

Evaluation of Anti-A/Udorn/307/1972 Antibody Specificity to Influenza A/H3N2 Viruses Using an Evanescent-Field Coupled Waveguide-Mode Sensor

Discrimination of closely related strains is a key issue, particularly for infectious diseases whose incidence fluctuates according to variations in the season and evolutionary changes. Among infectious diseases, influenza viral infections are a worldwide cause of pandemic disease and mortality. With the emergence of different influenza strains, it is vital to develop a method using antibodies that can differentiate between viral types and subtypes. Ideally, such a system would also be user friendly. In this study, a polyclonal antibody generated against A/Udorn/307/1972 (H3N2) was used as a probe to distinguish between influenza H3N2 viruses based on the interaction between the antibody and hemagglutinin, demonstrating its applicability for viral discrimination. Clear discrimination was demonstrated using an evanescent-field-coupled waveguide-mode sensor, which has appealing characteristics over other methods in the viewpoint of improving the sensitivity, measurement time, portability and usability. Further supporting evidence was obtained using enzyme-linked immunosorbent assays, hemagglutination-inhibition assays, and infectivity neutralization assays. The results obtained indicate that the polyclonal antibody used here is a potential probe for distinguishing influenza viruses and, with the aid of a handheld sensor it could be used for influenza surveillance.     

Pathogen proliferation governs the magnitude but compromises the function of CD8 T cells

CD8+ T cell memory is critical for protection against many intracellular pathogens. However, it is not clear how pathogen virulence influences the development and function of CD8+ T cells. Salmonella typhimurium (ST) is an intracellular bacterium that causes rapid fatality in susceptible mice and chronic infection in resistant strains. We have constructed recombinant mutants of ST, expressing the same immunodominant Ag OVA, but defective in various key virulence genes. We show that the magnitude of CD8+ T cell response correlates directly to the intracellular proliferation of ST. Wild-type ST displayed efficient intracellular proliferation and induced increased numbers of OVA-specific CD8+ T cells upon infection in mice. In contrast, mutants with defective Salmonella pathogenicity island II genes displayed poor intracellular proliferation and induced reduced numbers of OVA-specific CD8+ T cells. However, when functionality of the CD8+ T cell response was measured, mutants of ST induced a more functional response compared with the wild-type ST. Infection with wild-type ST, in contrast to mutants defective in pathogenicity island II genes, induced the generation of mainly effector-memory CD8+ T cells that expressed little IL-2, failed to mediate efficient cytotoxicity, and proliferated poorly in response to Ag challenge in vivo. Taken together, these results indicate that pathogens that proliferate rapidly and chronically in vivo may evoke functionally inferior memory CD8+ T cells which may promote the survival of the pathogen.     

Filarial parasites induce NK cell activation, type 1 and type 2 cytokine secretion, and subsequent apoptotic cell death

NK cells are an important source of early cytokine production in a variety of intracellular viral, bacterial, and protozoan infections; however, the role of NK cells in extracellular parasitic infections such as filarial infections is not well-defined. To investigate the role of NK cells in filarial infections, we have used an in vitro model system of culturing live infective-stage larvae (L3) or live microfilariae (Mf) of Brugia malayi, a causative agent of human lymphatic filariasis, with PBMC of normal individuals. We found that NK cells undergo early cell activation and produce IFN-gamma and TNF-alpha within 24 h after stimulation with both live L3 and Mf. Interestingly, NK cells also express IL-4 and IL-5 at this time point in response to live Mf but not L3. This is accompanied by significant alterations in NK cell expression of costimulatory molecules and natural cytotoxicity receptors. This activation is dependent on the presence of monocytes in the culture, IL-12, and direct contact with live parasites. The early activation event is subsequently followed by apoptosis of NK cells involving a caspase-dependent mechanism in response to live L3 but not live Mf. Thus, the NK cell-parasite interaction is complex, with filarial parasites inducing NK cell activation and cytokine secretion and finally NK cell apoptosis, which may provide an additional mechanism of down-regulating the host immune response.     

Rapid clonal expansion and prolonged maintenance of memory CD8+ T cells of the effector (CD44highCD62Llow) and central (CD44highCD62Lhigh) phenotype by an archaeosome adjuvant independent of TLR2

Vaccines capable of eliciting long-term T cell immunity are required for combating many diseases. Live vectors can be unsafe whereas subunit vaccines often lack potency. We previously reported induction of CD8(+) T cells to Ag entrapped in archaeal glycerolipid vesicles (archaeosomes). In this study, we evaluated the priming, phenotype, and functionality of the CD8(+) T cells induced after immunization of mice with OVA-Methanobrevibacter smithii archaeosomes (MS-OVA). A single injection of MS-OVA evoked a profound primary response but the numbers of H-2K(b)OVA(257-264)-specific CD8(+) T cells declined by 14-21 days, and <1% of primarily central phenotype (CD44(high)CD62L(high)) cells persisted. A booster injection of MS-OVA at 3-11 wk promoted massive clonal expansion and a peak effector response of approximately 20% splenic/blood OVA(257-264)-specific CD8(+) T cells. Furthermore, contraction was protracted and the memory pool (IL-7Ralpha(high)) of approximately 5% included effector (CD44(high)CD62L(low)) and central (CD44(high)CD62L(high)) phenotype cells. Recall response was observed even at >300 days. CFSE-labeled naive OT-1 (OVA(257-264) TCR transgenic) cells transferred into MS-OVA-immunized recipients cycled profoundly (>90%) within the first week of immunization indicating potent Ag presentation. Moreover, approximately 25% cycling of Ag-specific cells was seen for >50 days, suggesting an Ag depot. In vivo, CD8(+) T cells evoked by MS-OVA killed >80% of specific targets, even at day 180. MS-OVA induced responses similar in magnitude to Listeria monocytogenes-OVA, a potent live vector. Furthermore, protective CD8(+) T cells were induced in TLR2-deficient mice, suggesting nonengagement of TLR2 by archaeal lipids. Thus, an archaeosome adjuvant vaccine represents an alternative to live vectors for inducing CD8(+) T cell memory.     

A reduced antigen load in vivo, rather than weak inflammation, causes a substantial delay in CD8+ T cell priming against Mycobacterium bovis (bacillus Calmette-Guérin)

Regardless of the dose of Ag, Ag presentation occurs rapidly within the first few days which results in rapid expansion of the CD8+ T cell response that peaks at day 7. However, we have previously shown that this rapid priming of CD8+ T cells is absent during infection of mice with Mycobacterium bovis (bacillus Calmette-Guérin (BCG)). In this study, we have evaluated the mechanisms responsible for the delayed CD8+ T cell priming. Because BCG replicates poorly and survives within phagosomes we considered whether 1) generation of reduced amounts of Ag or 2) weaker activation by pathogen-associated molecular patterns (PAMPs) during BCG infection is responsible for the delay in CD8+ T cell priming. Using rOVA-expressing bacteria, our results indicate that infection of mice with BCG-OVA generates greatly reduced levels of OVA, which are 70-fold lower in comparison to the levels generated during infection of mice with Listeria monocytogenes-expressing OVA. Furthermore, increasing the dose of OVA, but not PAMP signaling during BCG-OVA infection resulted in rapid Ag presentation and consequent expansion of the CD8+ T cell response, indicating that the generation of reduced Ag levels, not lack of PAMP-associated inflammation, was responsible for delayed priming of CD8+ T cells. There was a strong correlation between the relative timing of Ag presentation and the increase in the level of OVA in vivo. Taken together, these results reveal that some slowly replicating pathogens, such as mycobacteria, may facilitate their chronicity by generating reduced Ag levels which causes a substantial delay in the development of acquired immune responses.     

AMPK Promotes Xenophagy through Priming of Autophagic Kinases upon Detection of Bacterial Outer Membrane Vesicles

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A Point-of-Care Immunosensor for Human Chorionic Gonadotropin in Clinical Urine Samples Using a Cuneated Polysilicon Nanogap Lab-on-Chip

Human chorionic gonadotropin (hCG), a glycoprotein hormone secreted from the placenta, is a key molecule that indicates pregnancy. Here, we have designed a cost-effective, label-free, in situ point-of-care (POC) immunosensor to estimate hCG using a cuneated 25 nm polysilicon nanogap electrode. A tiny chip with the dimensions of 20.5 × 12.5 mm was fabricated using conventional lithography and size expansion techniques. Furthermore, the sensing surface was functionalized by (3-aminopropyl)triethoxysilane and quantitatively measured the variations in hCG levels from clinically obtained human urine samples. The dielectric properties of the present sensor are shown with a capacitance above 40 nF for samples from pregnant women; it was lower with samples from non-pregnant women. Furthermore, it has been proven that our sensor has a wide linear range of detection, as a sensitivity of 835.88 μA mIU^-1 ml^-2 cm^-2 was attained, and the detection limit was 0.28 mIU/ml (27.78 pg/ml). The dissociation constant K_d of the specific antigen binding to the anti-hCG was calculated as 2.23 ± 0.66 mIU, and the maximum number of binding sites per antigen was B_max = 22.54 ± 1.46 mIU. The sensing system shown here, with a narrow nanogap, is suitable for high-throughput POC diagnosis, and a single injection can obtain triplicate data or parallel analyses of different targets.     

Titanium Dioxide Nanoparticle-Based Interdigitated Electrodes: A Novel Current to Voltage DNA Biosensor Recognizes E. coli O157:H7

Nanoparticle-mediated bio-sensing promoted the development of novel sensors in the front of medical diagnosis. In the present study, we have generated and examined the potential of titanium dioxide (TiO₂) crystalline nanoparticles with aluminium interdigitated electrode biosensor to specifically detect single-stranded E.coli O157:H7 DNA. The performance of this novel DNA biosensor was measured the electrical current response using a picoammeter. The sensor surface was chemically functionalized with (3-aminopropyl) triethoxysilane (APTES) to provide contact between the organic and inorganic surfaces of a single-stranded DNA probe and TiO₂ nanoparticles while maintaining the sensing system’s physical characteristics. The complement of the target DNA of E. coli O157:H7 to the carboxylate-probe DNA could be translated into electrical signals and confirmed by the increased conductivity in the current-to-voltage curves. The specificity experiments indicate that the biosensor can discriminate between the complementary sequences from the base-mismatched and the non-complementary sequences. After duplex formation, the complementary target sequence can be quantified over a wide range with a detection limit of 1.0 x 10^-13M. With target DNA from the lysed E. coli O157:H7, we could attain similar sensitivity. Stability of DNA immobilized surface was calculated with the relative standard deviation (4.6%), displayed the retaining with 99% of its original response current until 6 months. This high-performance interdigitated DNA biosensor with high sensitivity, stability and non-fouling on a novel sensing platform is suitable for a wide range of biomolecular interactive analyses.     

Induction of hsp70, alterations in oxidative stress markers and apoptosis against dichlorvos exposure in transgenic Drosophila melanogaster: modulation by reactive oxygen species

We examined a hypothesis that reactive oxygen species (ROS) generated by organophosphate compound dichlorvos modulates Hsp70 expression and anti-oxidant defense enzymes and acts as a signaling molecule for apoptosis in the exposed organism. Dichlorvos (0.015-15.0 ppb) without or with inhibitors of Hsp70, superoxide dismutase (SOD) and catalase (CAT) were fed to the third instar larvae of Drosophila melanogaster transgenic for hsp70 (hsp70-lacZ) Bg(9) to examine Hsp70 expression, oxidative stress and apoptotic markers. A concentration- and time-dependent significant increase in ROS generation accompanied by a significant upregulation of Hsp70 preceded changes in antioxidant defense enzyme activities and contents of glutathione, malondialdehyde and protein carbonyl in the treated organisms. An inhibitory effect on SOD and CAT activities significantly upregulated ROS generation and Hsp70 expression in the exposed organism while inhibition of Hsp70 significantly affected oxidative stress markers induced by the test chemical. A comparison made among ROS generation, Hsp70 expression and apoptotic markers showed that ROS generation is positively correlated with Hsp70 expression and apoptotic cell death end points indicating involvement of ROS in the overall adversity caused by the test chemical to the organism. The study suggests that (a) Hsp70 and anti-oxidant enzymes work together for cellular defense against xenobiotic hazard in D. melanogaster and (b) free radicals may modulate Hsp70 expression and apoptosis in the exposed organism.