Antiinflammatory Therapy with Canakinumab for Atherosclerotic Disease

Background: Experimental and clinical data suggest that reducing inflammation without affecting lipid levels may reduce the risk of cardiovascular disease. Yet, the inflammatory hypothesis of atherothrombosis has remained unproved. Methods: We conducted a randomized, double-blind trial of canakinumab, a therapeutic monoclonal antibody targeting interleukin-1β, involving 10,061 patients with previous myocardial infarction and a high-sensitivity C-reactive protein level of 2 mg or more per liter. The trial compared three doses of canakinumab (50 mg, 150 mg, and 300 mg, administered subcutaneously every 3 months) with placebo. The primary efficacy end point was nonfatal myocardial infarction, nonfatal stroke, or cardiovascular death. RESULTS: At 48 months, the median reduction from baseline in the high-sensitivity C-reactive protein level was 26 percentage points greater in the group that received the 50-mg dose of canakinumab, 37 percentage points greater in the 150-mg group, and 41 percentage points greater in the 300-mg group than in the placebo group. Canakinumab did not reduce lipid levels from baseline. At a median follow-up of 3.7 years, the incidence rate for the primary end point was 4.50 events per 100 person-years in the placebo group, 4.11 events per 100 person-years in the 50-mg group, 3.86 events per 100 person-years in the 150-mg group, and 3.90 events per 100 person-years in the 300-mg group. The hazard ratios as compared with placebo were as follows: in the 50-mg group, 0.93 (95% confidence interval [CI], 0.80 to 1.07; P = 0.30); in the 150-mg group, 0.85 (95% CI, 0.74 to 0.98; P = 0.021); and in the 300-mg group, 0.86 (95% CI, 0.75 to 0.99; P = 0.031). The 150-mg dose, but not the other doses, met the prespecified multiplicity-adjusted threshold for statistical significance for the primary end point and the secondary end point that additionally included hospitalization for unstable angina that led to urgent revascularization (hazard ratio vs. placebo, 0.83; 95% CI, 0.73 to 0.95; P = 0.005). Canakinumab was associated with a higher incidence of fatal infection than was placebo. There was no significant difference in all-cause mortality (hazard ratio for all canakinumab doses vs. placebo, 0.94; 95% CI, 0.83 to 1.06; P = 0.31). Conclusions: Antiinflammatory therapy targeting the interleukin-1β innate immunity pathway with canakinumab at a dose of 150 mg every 3 months led to a significantly lower rate of recurrent cardiovascular events than placebo, independent of lipid-level lowering. (Funded by Novartis; CANTOS ClinicalTrials.gov number, NCT01327846.

FIX-ΔLUNA virus does not increase viral genome copy number after IL6 induced differentiation of CD14⁺ cells.

<p>Viral DNA was quantified by qPCR. A) Total DNA was extracted from FIX-WT, FIX-ΔLUNA, and FIX-Rev infected HF cells over a 20 d time course. B) Total DNA was extracted from FIX-WT, FIX-ΔLUNA, and FIX-Rev infected CD14⁺ cells at 1 dpi, 3 dpi, 5 dpi, 10 dpi, 20 dpi. Differentiation was induced in CD14⁺ cells at 10 dpi by the addition of IL6 and at 5 days (sample termed as 15dpIL6) or 10 days (samples termed as 20dpIL6) total DNA was collected. All samples were run in triplicate. Statistical analysis was done using a Student <i>t</i> test comparing FIX-WT vs. FIXΔLUNA or FIX-Rev vs. FIX-ΔLUNA. The * indicates a <i>P</i>-value<0.01, and the ** indicates a <i>P</i>-value<0.001.</p

HCMV Protein LUNA Is Required for Viral Reactivation from Latently Infected Primary CD14⁺ Cells

<div><p>Human cytomegalovirus (HCMV) is a member of the <em>Herpesviridae</em> family that infects individuals throughout the world. Following an initial lytic stage, HCMV can persist in the individual for life in a non-active (or latent) form. During latency, the virus resides within cells of the myeloid lineage. The mechanisms controlling HCMV latency are not completely understood. A latency associated transcript, UL81-82ast, encoding the protein LUNA (Latency Unique Natural Antigen) was identified from latently infected donors <em>in vivo</em>. To address the role of the UL81-82ast protein product LUNA, in the context of the viral genome, we developed a recombinant HCMV bacterial artificial chromosome (BAC) that does not express LUNA. This construct, LUNA knockout FIX virus (FIX-ΔLUNA), was used to evaluate LUNA's role in HCMV latency. The FIX-ΔLUNA virus was able to lytically infect Human Fibroblast (HF) cells, showing that LUNA is not required to establish a lytic infection. Interestingly, we observed significantly higher viral copy numbers in HF cells infected with FIX-ΔLUNA when compared to FIX-WT virus. Furthermore, FIX-WT and FIX-ΔLUNA genomic DNA and transcription of UL81-82ast persisted over time in primary monocytes. In contrast, the levels of UL138 transcript expression in FIX-ΔLUNA infected HF and CD14⁺ cells was 100 and 1000 fold lower (respectively) when compared to the levels observed for FIX-WT infection. Moreover, FIX-ΔLUNA virus failed to reactivate from infected CD14⁺ cells following differentiation. This lack of viral reactivation was accompanied by a lack of lytic gene expression, increase in viral copy numbers, and lack of the production of infectious units following differentiation of the cells. Our study suggests that the LUNA protein is involved in regulating HCMV reactivation, and that in the absence of LUNA, HCMV may not be able to enter a proper latent state and therefore cannot be rescued from the established persistent infection in CD14⁺ cells.</p> </div

Primers qPCR, and qRT-PCR.

*<p>primers also used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052827#pone.0052827.s002" target="_blank">Figure S2</a>.</p

FIX-ΔLUNA viral infection of CD14⁺ cells fail to produce infectious virus after IL6 differentiation.

<p>A) Cell lysates plus supernatant from FIX-WT, FIX-ΔLUNA, or FIX-Rev infected cells were collected at 10 dpi and 20 dpi. IL6 was added at 10 dpi; at 5 (15dpIL6) and 10 (20dpIL6) days after differentiation, cell lysates and sups were also collected. The collected lysate/supernatant mix was serially diluted and used to infect HF cells. Two days post infection, cells were fixed with 4× paraformaldehyde, then stained with mouse anti-IE1/2 (1∶250) overnight. Cells were then incubated with Alexa Fluor 488 anti-mouse IgG for 1 hr, and incubated with DAPI for 30 min. IE positive cells were counted to determine the number of infectious units (IU). Only the time points post reactivation showed any significance when comparing FIX-WT or FIX-Rev to FIX-ΔLUNA (10dpIL6: p<3×10⁻⁵; 20dpIL6: p<0.0006). The ** indicates a <i>P</i>-value<0.001.</p

LUNA deletion mutants do not disrupt pp71.

<p>A) Sequence of the start codon region for UL8-82ast in FIX-WT and FIX-ΔLUNA viruses. The upper strand represents the coding frame for UL82 and its translation, the bottom strand represents the coding frame for UL81-82ast and its translation. Red-labeled nucleotides and amino acids indicate those that changed after mutagenesis. B) Hind III digest of FIX-BAC-WT (lane 1), FIX-BAC-(GalK-KAN)-A (lane 2), FIX-BAC-ΔLUNA (lane 3), FIX-BAC-(GalK-KAN)-B (lane 4) and FIX-BAC-Rev (lane 5). FIX-BAC-(GalK-KAN)-A contains the Galk-Kan insert before the mutation; FIX-BAC-(GalK-KAN)-B is the re-introduction of the insert in the process of creating the revertant. C) Southern blot of the digest in (B) with a GalK specific probe. The Galk-Kan insert is indicated by the arrow. D) HF cells were infected with FIX-WT, FIX-ΔLUNA or FIX-Rev respectively over a 20day time course. The presence of the pp71 protein was detected via western blotting. 20 µg of protein was added per well, samples were loaded in the following order for each time point: FIX-WT, FIX-ΔLUNA and FIX-Rev. Lane 1: Mock HF, lanes 2–4: HF 1 dpi, lanes 5–7: HF 3 dpi, lanes 8–10: HF 5 dpi, lanes 11–13: HF 10 dpi, lanes 14–16: HF 20 dpi. Blots were incubated with primary antibodies goat anti-pp71 (1∶500) and mouse anti-actin (1∶10,000) and secondary antibodies anti-goat or anti-mouse IgG HRP (1∶1000).</p

Primers for BAC mutagenesis and RT-PCR.

a<p>F: Forward Primer; R: Reverse Primer.</p