Extracellular Vesicles from a Helminth Parasite Suppress Macrophage Activation and Constitute an Effective Vaccine for Protective Immunity

Recent studies have demonstrated that many parasites release extracellular vesicles (EVs), yet little is known about the specific interactions of EVs with immune cells or their functions during infection. We show that EVs secreted by the gastrointestinal nematode Heligmosomoides polygyrus are internalized by macrophages and modulate their activation. EV internalization causes downregulation of type 1 and type 2 immune-response-associated molecules (IL-6 and TNF, and Ym1 and RELMα) and inhibits expression of the IL-33 receptor subunit ST2. Co-incubation with EV antibodies abrogated suppression of alternative activation and was associated with increased co-localization of the EVs with lysosomes. Furthermore, mice vaccinated with EV-alum generated protective immunity against larval challenge, highlighting an important role in vivo. In contrast, ST2-deficient mice are highly susceptible to infection, and they are unable to clear parasites following EV vaccination. Hence, macrophage activation and the IL-33 pathway are targeted by H. polygyrus EVs, while neutralization of EV function facilitates parasite expulsion

Dynamic Analysis of Selected Firing Mechanisms of Firearms

This bachelor thesis strives to evaluate the dynamic analysis of firing mechanisms of different types of firearms. In the present research is analyzed the matter of firearms and ammunitions, followed by the description of distribution of firearm mechanisms, as well as the detailed scrutiny of the initiation mechanism. The thesis also marginally explores the testing of primers. The last part of the research documents the possible solutions for the analyzed issues. The practical part of the thesis is devoted to the geometry of firing mechanisms, followed by the description of the physical principles, which are relevant to this matter. Based on the analysis, a theoretical calculation of the initiation energies for all of the analyzed types of firearms is established. Lastly, the calculation of the initiation energy is calculated, and the security in secure firing of the bullet's primer is determined

Reductions in HeV titre via pre-treatment with combined RNAi.

<p>HeLa cells were transfected with 40 nM combined HeV targeting (white bars) or control (black bars) siRNAs. Transfection media was replaced after 4 hours and cells were infected with HeV clinical isolate at an MOI of 0.1. Infected cells were incubated for 24 hours before supernatant removed and processed for TCID₅₀ assay. Cells infected with supernatant were incubated for 3 days before TCID₅₀ calculation and are shown as the mean ± S.E.M. of six biological replicates from two independent experiments. Differences between the combination treatments and expected values based on the average of singular siRNA treatments (grey bars) are shown.</p

Reductions in HeV titre via treatment with lower RNAi concentrations.

<p>HeLa cells were transfected with 1 µg/ml Poly IC, 1 nM of control or HeV targeting siRNAs. Transfection media was replaced after 4 hours and cells were infected with HeV clinical isolate at an MOI of 0.1. Infected cells were incubated for 24 hours before supernatant removed and processed for TCID₅₀ assay. Cells infected with supernatant were incubated for 3 days before TCID₅₀ calculation and are shown as the mean ± S.E.M. of three biological replicates.</p

Ability of siRNAs to knockdown recombinant HeV-luciferase and cause immune stimulation.

<p>HeLa cells were transfected with 40 µg/ml Poly IC, 40 nM control or HeV targeting siRNAs. siLuciferase was a positive control (grey bar), while scrambled (ScrM7) siRNA and Oligofectamine were negative controls (black bars). <b>A)</b> Transfection media was replaced after 4 hours and cells were infected with recombinant HeV-luciferase at an MOI of 0.5. Infected cells were incubated for 24 hours before luciferase activity was measured. Levels of luciferase luminescence were normalized to Oligofectamine control levels and are the mean ± S.D. of six biological replicates from two independent experiments. <b>B)</b> Cells were incubated for 12 hours before RNA extraction. Gene expression levels of IL-6 were quantified by qRT-PCR as relative expression to B-actin housekeeping gene. Results are the mean ± S.D. of six biological replicates from two independent experiments. Significant differences between Oligofectamine control and siRNAs are indicated (**p = <0.01, ***p = <0.001, ****p = <0.0001; two-sided t-test).</p

Reductions in HeV via pre-treatment with RNAi.

<p>HeLa cells were transfected with 40 µg/ml Poly IC, 40 nM control or HeV targeting siRNAs. Transfection media was replaced after 4 hours and cells were infected with HeV clinical isolate at an MOI of 0.1. Infected cells were incubated for 24 hours before <b>A)</b> Infected cells were fixed and labelled for HeV N, P or M protein and DAPI stain. Cells were imaged with a Leica confocal microscope. Indicative images are shown. Scale bar = 50 µM. <b>B)</b> Supernatant was removed and titrated for TCID₅₀ assay. Cells infected with supernatant were incubated for 3 days before TCID₅₀ was calculated and shown as the mean ± S.E.M. of six biological replicates from two independent experiments. Significant differences between Oligofectamine control and siRNAs are indicated (***p = <0.001, two-sided t-test).</p

Reductions in HeV titre via pre-treatment with RNAi and Poly IC.

<p>HeLa cells were transfected with 40 µg/ml Poly IC, 40 nM control (black bars), HeV targeting siRNA, or combined Poly IC/siN15. Transfection media was replaced after 4 hours and cells were infected with HeV clinical isolate at an MOI of 0.1. <b>A)</b> Infected cells were incubated for 24 hours before RNA extraction. Gene expression levels of N were quantified by qRT-PCR as relative expression to B-actin housekeeping gene. Results are the mean ± SEM of six biological replicates from two independent experiments. Significant differences between Oligofectamine control and siRNAs are indicated (*p = <0.05; two-sided t-test). <b>B)</b> Infected cells were incubated for 24 hours before supernatant removed and processed for TCID₅₀ assay. Cells infected with supernatant were incubated for 3 days before TCID₅₀ calculation and are shown as the mean ± S.E.M. of six biological replicates from two independent experiments.</p

Sequence characterisation of HeV siRNAs and D-siRNAs targeting N, P and M genes.

<p>Nucleocapsid siRNAs are conventional 21 bp siRNAs. All M, P and ScrM7 are D-siRNAs that are 25+27 bp long, with a two RNA nucleotide overhang only on the antisense strand and two DNA nucleotides on the sense strand depicted in lowercase letters.</p

Reductions in HeV gene expression and protein caused by RNAi targeting HeV.

<p>HeLa cells were transfected with 40 µg/ml Poly IC, 40 nM control or HeV targeting siRNAs. Transfection media was replaced after 4 hours and cells were infected with HeV clinical isolate at an MOI of 0.1. <b>A)</b> Infected cells were incubated for 24 hours before RNA extraction. Gene expression levels of N were quantified by qRT-PCR as relative expression to B-actin housekeeping gene. Results are the mean ± SEM of six biological replicates from two independent experiments. Significant differences between Oligofectamine control and siRNAs are indicated (*p = <0.05; two-sided t-test). <b>B)</b> Infected cells were incubated for 24 hours and were subsequently fixed and labelled for HeV N protein and DAPI nuclear stain. Cells were imaged with an EVOS Microscope at 10x objective and analysed for the presence or absence of GFP indicating N protein. Indicative images are shown.</p