Dose reconstruction including dynamic six-degree of freedom motion during prostate radiotherapy

© Published under licence by IOP Publishing Ltd. An in-house developed program for real-time reconstruction of motion-induced dose errors, DoseTracker, was extended to handle rotational target motion in addition to the previously implemented translational motion, and applied offline for prostate VMAT treatments. For translational motion, the motion-induced errors of DoseTracker were in good agreement with ground truth dose reconstructions performed in a commercial treatment planning system. For rotational motion, no ground truth was available, but DoseTracker showed that the VMAT dose is highly robust against static interfractional rotations but quite sensitive to dynamic intrafraction rotations due to interplay effects between target motion and machine motion

Roskilde Minder 1863-67

Roskilde Minder 1863-6

STING agonists enable antiviral cross-talk between human cells and confer protection against genital herpes in mice

<div><p>In recent years, there has been an increasing interest in immunomodulatory therapy as a means to treat various conditions, including infectious diseases. For instance, Toll-like receptor (TLR) agonists have been evaluated for treatment of genital herpes. However, although the TLR7 agonist imiquimod was shown to have antiviral activity in individual patients, no significant effects were observed in clinical trials, and the compound also exhibited significant side effects, including local inflammation. Cytosolic DNA is detected by the enzyme cyclic GMP-AMP (2’3’-cGAMP) synthase (cGAS) to stimulate antiviral pathways, mainly through induction of type I interferon (IFN)s. cGAS is activated upon DNA binding to produce the cyclic dinucleotide (CDN) 2’3’-cGAMP, which in turn binds and activates the adaptor protein Stimulator of interferon genes (STING), thus triggering type I IFN expression. In contrast to TLRs, STING is expressed broadly, including in epithelial cells. Here we report that natural and non-natural STING agonists strongly induce type I IFNs in human cells and in mice <i>in vivo</i>, without stimulating significant inflammatory gene expression. Systemic treatment with 2’3’-cGAMP reduced genital herpes simplex virus (HSV) 2 replication and improved the clinical outcome of infection. More importantly, local application of CDNs at the genital epithelial surface gave rise to local IFN activity, but only limited systemic responses, and this treatment conferred total protection against disease in both immunocompetent and immunocompromised mice. In direct comparison between CDNs and TLR agonists, only CDNs acted directly on epithelial cells, hence allowing a more rapid and IFN-focused immune response in the vaginal epithelium. Thus, specific activation of the STING pathway in the vagina evokes induction of the IFN system but limited inflammatory responses to allow control of HSV2 infections <i>in vivo</i>.</p></div

Systemic treatment with STING agonists confers protection against genital HSV2 infection.

<p>Wildtype and <i>cGas</i>^-/- mice were treated with 2’3’-cGAM(PS)₂ (Rp/Sp) (125 μg/mouse) and infected intravaginally with HSV2 (6.7×10⁴ p.f.u.). (<b>A</b>) Illustration of the timeline for the treatment regimens. (<b>B, C</b>) Overall survival for HSV2-infected and treated wildtype (<b>B</b>) or <i>cGas</i>^<i>-</i>/- (<b>C</b>) mice. n = 6–10. * = p<0,05 compared to mock. <b>(D</b>) HSV2 titer (TCID50) in vaginal washes collected 48 hours post infection. n = 6–10. * = p<0,05 compared to mock in the same genotype. Statistics, (<b>B</b>, <b>C</b>) Log-rank test with Holm-Bonferroni correction. (<b>D</b>) One-way ANOVA of log₁₀-transformed data with Dunnett’s multiple comparisons test.</p

HSV2 and STING agonists induce antiviral genes in vaginal epithelium.

<p>Mice were injected i.p. with 2’3’-cGAM(PS)₂ (125 μg/mouse) or infected with HSV2 (6.7×10⁴ p.f.u.). Tissues were isolated from mice 6 h and 24 h after CDN stimulation and HSV2 infection, respectively. Paraffin sections of the vaginal tissues were stained for viperin (red) and HSV2 (green). DAPI (blue) marks the nuclei and the dotted white lines mark basal membrane between the epithelium and stroma. White arrows highlight examples of viperin positive cells, and arrowheads mark examples of HSV2-infected cells. L = lumen, E = epithelium, S = stroma. n = 3. One representative picture is shown for each staining and treatment group.</p

STING agonists induce IFN responses in the vaginal epithelium faster and more efficiently than TLR agonists.

<p>(<b>A</b>) HaCaT cells were treated with imiquimod (1μg/ ml), ODN2216 (1μg/ ml), and 3’3’-cAIMP (100μg/ ml) for 24 h. Levels of ISG15 and viperin were determined in the cell lysate by Western blotting. (<b>B</b>) Mice were treated intravaginally with imiquimod or ODN1826 (25 μg per mouse) 12 h prior to infection with HSV2. Vaginal washes were collected 48 h p.i. and viral load was determined. n = 5 per group. (<b>C-D</b>) Mice were anesthetized for 30 min and imiquimod, ODN1826, or 3’3’-cAIMP was applied to the vagina. Tissues were isolated (<b>C</b>) 6 h or (<b>D</b>) 36 h after treatment. Paraffin sections of the vaginal tissues were stained for viperin (red). DAPI (blue) marks the nuclei and the dotted white lines mark basal membrane between the epithelium and stroma. White arrows highlight examples of viperin positive cells. L = lumen, E = epithelium, S = stroma. n = 4. One representative picture is shown for each staining. (<b>E-G</b>) RNA was isolated from vaginal tissue treated as indicated for 6 h, and levels of <i>Ifnb</i>, <i>Mx1</i>, and <i>Tnfa</i> mRNA were determined by RT-qPCR. n = 4–5. mRNA levels were normalized to <i>Gapdh</i> and shown as relative levels of expression compared to mock-treated mice. (<b>B, E-G</b>) Statistics, Kruskal-Wallis test with Dunn’s multiple comparisons test. * = p<0,05 compared to mock treated.</p