CTF3 Design Report: Preliminary Phase

The design of CLIC is based on a two-beam scheme, where the short pulses of high power 30 GHz RF are extracted from a drive beam running parallel to the main beam. The 3rd generation CLIC Test Facility (CTF3) will demonstrate the generation of the drive beam with the appropriate time structure, the extraction of 30 GHz RF power from this beam, as well as acceleration of a probe beam with 30 GHz RF cavities. The project makes maximum use of existing equipment and infrastructure of the LPI complex, which became available after the closure of LEP. In the first stage of the project, the "Preliminary Phase", the existing LIL linac and the EPA ring, both modified to suit the new requirements, are used to investigate the technique of frequency multiplication by means of interleaving bunches from subsequent trains. This report describes the design of this phase

Genomic HIV RNA Induces Innate Immune Responses through RIG-I-Dependent Sensing of Secondary-Structured RNA

Contains fulltext : 108031.pdf (publisher's version ) (Open Access)BACKGROUND: Innate immune responses have recently been appreciated to play an important role in the pathogenesis of HIV infection. Whereas inadequate innate immune sensing of HIV during acute infection may contribute to failure to control and eradicate infection, persistent inflammatory responses later during infection contribute in driving chronic immune activation and development of immunodeficiency. However, knowledge on specific HIV PAMPs and cellular PRRs responsible for inducing innate immune responses remains sparse. METHODS/PRINCIPAL FINDINGS: Here we demonstrate a major role for RIG-I and the adaptor protein MAVS in induction of innate immune responses to HIV genomic RNA. We found that secondary structured HIV-derived RNAs induced a response similar to genomic RNA. In primary human peripheral blood mononuclear cells and primary human macrophages, HIV RNA induced expression of IFN-stimulated genes, whereas only low levels of type I IFN and tumor necrosis factor alpha were produced. Furthermore, secondary structured HIV-derived RNA activated pathways to NF-kappaB, MAP kinases, and IRF3 and co-localized with peroxisomes, suggesting a role for this organelle in RIG-I-mediated innate immune sensing of HIV RNA. CONCLUSIONS/SIGNIFICANCE: These results establish RIG-I as an innate immune sensor of cytosolic HIV genomic RNA with secondary structure, thereby expanding current knowledge on HIV molecules capable of stimulating the innate immune system

The innate immune response induced by HIV genomic RNA or RNA oligos is dependent on RIG-I and MAVS.

<p>(A) PBMCs were treated with bafilomycin A1 (0.5 µM) as indicated 15 min prior to stimulation with genomic HIV RNA, RNA oligos, or ssRNA40 (all 2 µg/ml). IFN-α was included as a positive control (10 ng/ml). Supernatants were harvested 18 h post stimulation for measurement of CXCL10. (B) BMMs from C57BL/6 wildtype and MAVS−/− mice were stimulated with genomic HIV RNA (2 µg/ml), Tar (2 µg/ml), Sendai virus (MOI 1), IFN-α (10 ng/ml), ssRNA40 (2 µg/ml), or R848 (2 µg/ml). Supernatants were harvested after 16 h and CXCL10 was measured by ELISA. UT, untreated cells. Data are shown as means of triplicates +/− st.dev. (C) Huh7, Huh7.5 (RIG-I mutant), Huh7.5 EV (empty vector), and Huh7.5 RIG-I cells were transfected with Tar RNA (2 µg/ml), or subjected to mock transfection with Lipofectamine 2000. Total RNA was harvested 6 h later and CXCL10 mRNA levels were analysed by qPCR. Data are shown as means of triplicates +/− st.dev. Similar results were obtained in two or three independent experiments. Mock, Lipofectamine 2000 alone. RU, relative units *, p<0.05.</p

Genomic HIV RNA induces innate immune responses dominated by ISGs.

<p>PBMCs were stimulated for 16 h with HIV genomic RNA in increasing doses (from 0.3 to 3.0 µg/ml). Positive and negative controls included ssRNA40 (2 µg/ml) and ssRNA41 (2 µg/ml), respectively. Supernatants were harvested for measurement of (A) CXCL10, (B) IFN-β, (C) IFN-α, (D) TNF-α, and (E) IL-6. Data are shown as means of triplicates +/− st.dev. Similar results were obtained in two or three independent experiments. Mock, Lipofectamine 2000 alone.</p

HIV RNA activates the NF-κB, p38, and IRF signaling pathways.

<p>PBMCs were stimulated with HIV Tar (oligo 2, 2 µg/ml) and in panel A also with oligo 4 (2 µg/ml). (A–B) Whole-cell lysates were isolated 2 h post treatment, and (C–E) nuclear extracts were isolated at the indicated time points (IFN-γ, 10 ng/ml, 6 h; SeV, MOI 1, 6 h; R848, 500 ng/ml, 6 h). (A–B) P-IκBα and P-p38 were measured by Luminex technology and (C–E) DNA binding of IRF-1, 3, and 7 to an ISRE consensus sequence was measured by TransAM. Data are shown as means of duplicates or triplicates +/− st.dev. Similar results were obtained in two or three independent experiments. RU, relative units. Mock, Lipofectamine 2000 alone. *, p<0.05.</p