Journal: Journal of Neuroinflammation

Article Title: CNS-targeted NLRP3 Inhibition by NT-0527 confers therapeutic advantage in a CAPS mouse model

doi: 10.1186/s12974-026-03731-4

Figure Lengend Snippet: Pharmacological impact assessment in an acute LPS challenge model. A Schematic representation of the study protocol. B Core body temperature was assessed at T = 0 and T = 2 h post-LPS administration. Body temperature is shown as a function of treatment and time; bars represent SD. To evaluate whether each treatment caused a significant temperature drop within the groups, paired samples t-tests were performed on the before and after measurements on animals within each group (* p < 0.05, *** p < 0.001). C Peritoneal lavage fluid levels of IL-1β detected by ELISA are indicated as a function of treatment. For animals receiving only vehicle (no LPS), IL-1β levels were below the lower limit of quantitation of the ELISA and are plotted at 0 pg/ml; as such, this group was excluded from the statistical analysis. Differences were evaluated by one-way ANOVA and Tukey’s multiple comparisons test (*** p < 0.001). D Western blot analysis of autopod homogenates. The blots were probed with a polyclonal goat-anti-mouse IL-1β antibody which recognizes both pro- and mature IL-1β polypeptides. Lanes of the gel used to generate the lefthand “total” blot were loaded with 50 µg of homogenate protein. Lanes of the gel used to generate the righthand “mIL-1R-Fc pulldown” blot were loaded with mIL-1R1-Fc-captured isolates recovered from 1 mg of the corresponding autopod homogenate. Total and mIL-1R1-Fc pulldown homogenates were derived from the same homogenates with two mice from each indicated cohort profiled. Arrows point to the expected migration positions of pro- (35 kDa) and mature (17 kDa) IL-1β. Size of molecular weight markers (left lane on each blot) are indicated in kDa. Lanes 1 contained 10 pg of recombinant mature IL-1β (std). Additionally, one hNLRP3 extract was spiked with 10 pg of recombinant mature IL-1β (lanes 2, hNLRP3 + std). Levels of mature IL-1β were assessed by densitometry and are indicated in the righthand plot

Article Snippet: For soluble, mouse IL-1 receptor-Fc chimera protein (mIL-1R1-Fc) capture of mature IL-1β, 1 to 7 mg of each homogenate was transferred to a new microcentrifuge tube, the total volume was adjusted to 0.5 mL with homogenization buffer, and 2 μg of mIL-1R1-Fc (R&D Systems, #771-MR-100; 20 μL of a 100 μg/mL stock prepared in sterile PBS) were added.

Techniques: Enzyme-linked Immunosorbent Assay, Quantitation Assay, Western Blot, Derivative Assay, Migration, Molecular Weight, Recombinant

Journal: Journal of Neuroinflammation

Article Title: CNS-targeted NLRP3 Inhibition by NT-0527 confers therapeutic advantage in a CAPS mouse model

doi: 10.1186/s12974-026-03731-4

Figure Lengend Snippet: Pharmacological impact of NLRP3 inhibition on peripheral inflammatory biomarkers. A Schematic representation of the study design employed to compare therapeutic impact of CP-456,773 and NT-0527 in the CAPS disease model. hNLRP3 and hD305N mice were allowed ad libitum access to control chow or chow formulated with 600 mg/kg of CP-456,773 or NT-0527 for 7 weeks. At termination, tail homogenates were analyzed for MPO activity ( B ) and autopod homogenates were analyzed for IL-1β content by ELISA C. Individual plots comparing levels of the measured analyte between the two genotypes maintained on control chow as well as plots comparing pharmacological impact resulting from CP-456,773 or NT-0527 treatment within each genotype are shown. Horizontal lines indicate the mean value for each condition. In the case of MPO, significance was assessed using Welch’s one-way ANOVA and Dunnett’s T3 multiple comparisons test. For the IL-1β plot, all values for hNLRP3 autopods were below the lower limit of detection (LLQ = 15.6 pg/ml) and were thus plotted as 0. Significance was assessed using Welch’s t-test (strain comparison) or Welch’s one-way ANOVA and Dunnett’s T3 multiple comparisons test (compound effects). Denoted p values correspond to: * p < 0.05, ** p < 0.01; *** p < 0.001; **** p < 0.0001. D Western blot detection of IL-1β within autopod homogenates. Lefthand blot represents total homogenate protein (50 µg) while righthand blot represents mIL-1R1-Fc-captured isolates (derived from 1 mg of autopod homogenate); total and mIL-1R1-Fc pulldown were derived from the same autopod homogenates with two mice from each indicated cohort. Arrows point to the expected migration positions of pro- (35 kDa) and mature (17 kDa) IL-1β. Size of molecular weight markers (left lane on each blot) are indicated in kDa. Lanes 1 contained 10 pg of recombinant mature IL-1β (mIL-1β std). Additionally, one hNLRP3 extract was spiked with 10 pg of recombinant mature IL-1β (+ std)

Article Snippet: For soluble, mouse IL-1 receptor-Fc chimera protein (mIL-1R1-Fc) capture of mature IL-1β, 1 to 7 mg of each homogenate was transferred to a new microcentrifuge tube, the total volume was adjusted to 0.5 mL with homogenization buffer, and 2 μg of mIL-1R1-Fc (R&D Systems, #771-MR-100; 20 μL of a 100 μg/mL stock prepared in sterile PBS) were added.

Techniques: Inhibition, Control, Activity Assay, Enzyme-linked Immunosorbent Assay, Comparison, Western Blot, Derivative Assay, Migration, Molecular Weight, Recombinant

Journal: Journal of Neuroinflammation

Article Title: CNS-targeted NLRP3 Inhibition by NT-0527 confers therapeutic advantage in a CAPS mouse model

doi: 10.1186/s12974-026-03731-4

Figure Lengend Snippet: Pharmacological impact of NLRP3 inhibitors on brain inflammatory biomarkers. A Total RNA recovered from brains harvested at termination (study depicted in Fig. A) was analyzed by RT-qPCR for mRNA transcripts encoding ChiL3 , H2-Aa , Nlrp12 , Il1b , Gfap , and NLRP3 . Transcript levels are indicated as a function of genotype and treatment. Horizontal lines indicate the geometric mean of measured fold change values. Significance testing was performed using ordinary one-way ANOVA assuming log-normal distribution of fold-change values and Tukey’s multiple comparisons test in GraphPad Prism. Denoted p values correspond to: * p < 0.05, ** p < 0.01; *** p < 0.001; **** p < 0.0001. B Western blot detection of IL-1β within brain homogenates. Lanes of the gel used in generating the lefthand blot were loaded with 50 µg of total homogenate protein. Lanes of the gel used to generate to the righthand blot were loaded with mIL-1R1-Fc-captured isolates recovered from 7 mg of the corresponding brain homogenates. The total and mIL-1R1-Fc pulldown homogenates were derived from the same brain homogenates with two mice from each indicated cohort profiled. Both blots were probed with a goat anti-mouse IL-1β antiserum. The arrows point to the expected migration positions of pro- (35 kDa) and mature (17 kDa) IL-1β. Size of molecular weight markers (left lane on each blot) are indicated in kDa. Lanes denoted “S” contained 10 pg of recombinant mature IL-1β. Levels of mature IL-1β recovered by IL-1R-Fc capture from hD305N cohorts were assessed by densitometry and are indicated in the adjacent plot

Article Snippet: For soluble, mouse IL-1 receptor-Fc chimera protein (mIL-1R1-Fc) capture of mature IL-1β, 1 to 7 mg of each homogenate was transferred to a new microcentrifuge tube, the total volume was adjusted to 0.5 mL with homogenization buffer, and 2 μg of mIL-1R1-Fc (R&D Systems, #771-MR-100; 20 μL of a 100 μg/mL stock prepared in sterile PBS) were added.

Techniques: Quantitative RT-PCR, Western Blot, Derivative Assay, Migration, Molecular Weight, Recombinant