Natalizumab treatment shows low cumulative probabilities of confirmed disability worsening to EDSS milestones in the long-term setting.

Abstract Background Though the Expanded Disability Status Scale (EDSS) is commonly used to assess disability level in relapsing-remitting multiple sclerosis (RRMS), the criteria defining disability progression are used for patients with a wide range of baseline levels of disability in relatively short-term trials. As a result, not all EDSS changes carry the same weight in terms of future disability, and treatment benefits such as decreased risk of reaching particular disability milestones may not be reliably captured. The objectives of this analysis are to assess the probability of confirmed disability worsening to specific EDSS milestones (i.e., EDSS scores ≥3.0, ≥4.0, or ≥6.0) at 288 weeks in the Tysabri Observational Program (TOP) and to examine the impact of relapses occurring during natalizumab therapy in TOP patients who had received natalizumab for ≥24 months. Methods TOP is an ongoing, open-label, observational, prospective study of patients with RRMS in clinical practice. Enrolled patients were naive to natalizumab at treatment initiation or had received ≤3 doses at the time of enrollment. Intravenous natalizumab (300 mg) infusions were given every 4 weeks, and the EDSS was assessed at baseline and every 24 weeks during treatment. Results Of the 4161 patients enrolled in TOP with follow-up of at least 24 months, 3253 patients with available baseline EDSS scores had continued natalizumab treatment and 908 had discontinued (5.4% due to a reported lack of efficacy and 16.4% for other reasons) at the 24-month time point. Those who discontinued due to lack of efficacy had higher baseline EDSS scores (median 4.5 vs. 3.5), higher on-treatment relapse rates (0.82 vs. 0.23), and higher cumulative probabilities of EDSS worsening (16% vs. 9%) at 24 months than those completing therapy. Among 24-month completers, after approximately 5.5 years of natalizumab treatment, the cumulative probabilities of confirmed EDSS worsening by 1.0 and 2.0 points were 18.5% and 7.9%, respectively (24-week confirmation), and 13.5% and 5.3%, respectively (48-week confirmation). The risks of 24- and 48-week confirmed EDSS worsening were significantly higher in patients with on-treatment relapses than in those without relapses. An analysis of time to specific EDSS milestones showed that the probabilities of 48-week confirmed transition from EDSS scores of 0.0–2.0 to ≥3.0, 2.0–3.0 to ≥4.0, and 4.0–5.0 to ≥6.0 at week 288 in TOP were 11.1%, 11.8%, and 9.5%, respectively, with lower probabilities observed among patients without on-treatment relapses (8.1%, 8.4%, and 5.7%, respectively). Conclusions In TOP patients with a median (range) baseline EDSS score of 3.5 (0.0–9.5) who completed 24 months of natalizumab treatment, the rate of 48-week confirmed disability worsening events was below 15%; after approximately 5.5 years of natalizumab treatment, 86.5% and 94.7% of patients did not have EDSS score increases of ≥1.0 or ≥2.0 points, respectively. The presence of relapses was associated with higher rates of overall disability worsening. These results were confirmed by assessing transition to EDSS milestones. Lower rates of overall 48-week confirmed EDSS worsening and of transitioning from EDSS score 4.0–5.0 to ≥6.0 in the absence of relapses suggest that relapses remain a significant driver of disability worsening and that on-treatment relapses in natalizumab-treated patients are of prognostic importance

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Identification of interaction sites for dimerization and adapter recruitment in Toll/interleukin-1 receptor (TIR) domain of Toll-like receptor 4

Toll-like receptor signaling requires interactions of the Toll/IL-1 receptor (TIR) domains of the receptor and adapter proteins. Using the mammalian protein-protein interaction trap strategy, homology modeling, and site-directed mutagenesis, we identify the interaction surfaces in the TLR4 TIR domain for the TLR4-TLR4, TLR4-MyD88 adapter-like (MAL), and TLR4-TRIF-related adapter molecule (TRAM) interaction. Two binding sites are equally important for TLR4 dimerization and adapter recruitment. In a model based on the crystal structure of the dimeric TLR10 TIR domain, the first binding site mediates TLR4-TLR4 TIR-TIR interaction. Upon dimerization, two identical second binding sites of the TLR4 TIR domain are juxtaposed and form an extended binding platform for both MAL and TRAM. In our mammalian protein-protein interaction trap assay, MAL and TRAM compete for binding to this platform. Our data suggest that adapter binding can stabilize the TLR4 TIR dimerization

The Development of RNA-KISS, a Mammalian Three-Hybrid Method to Detect RNA-Protein Interactions in Living Mammalian Cells

RNA-protein interactions are essential for the regulation of mRNA and noncoding RNA functions and are implicated in many diseases, such as cancer and neurodegenerative disorders. A method that can detect RNA-protein interactions in living mammalian cells on a proteome-wide scale will be an important asset to identify and study these interactions. Here we show that a combination of the mammalian two-hybrid protein-protein detection method KISS (kinase substrate sensor) and the yeast RNA three-hybrid method, utilizing the specific interaction between the MS2 RNA and MS2 coat protein, is capable of detecting RNA-protein interactions in living mammalian cells. For conceptional proof we used the subgenomic flavivirus RNA (sfRNA) of the dengue virus (DENV), a highly structured noncoding RNA derived from the DENV genome known to target host cell proteins involved in innate immunity and antiviral defense, as bait. Using RNA-KISS, we could confirm the previously established interaction between the RNA-binding domain of DDX6 and the DENV sfRNA. Finally, we performed a human proteome-wide screen for DENV sfRNA-binding host factors, identifying several known flavivirus host factors such as DDX6 and PACT, further validating the RNA-KISS method as a robust and high-throughput cell-based RNA-protein interaction screening tool.status: publishe