Lymphotoxin β Receptor (LtβR): Dual Roles in Demyelination and Remyelination and Successful Therapeutic Intervention Using LtβR-Ig Protein

Inflammation mediated by macrophages is increasingly found to play a central role in diseases and disorders that affect a myriad of organs, prominent among these are diseases of the CNS. The neurotoxicant-induced, cuprizone model of demyelination is ideally suited for the analysis of inflammatory events. Demyelination on exposure to cuprizone is accompanied by predictable microglial activation and astrogliosis, and, after cuprizone withdrawal, this activation reproducibly diminishes during remyelination. This study demonstrates enhanced expression of lymphotoxin beta receptor (Lt betaR) during the demyelination phase of this model, and Lt betaR is found in areas enriched with microglial and astroglial cells. Deletion of the Lt betaR gene (Lt betaR-/-) resulted in a significant delay in demyelination but also a slight delay in remyelination. Inhibition of Lt betaR signaling by an Lt betaR-Ig fusion decoy protein successfully delayed demyelination in wild-type mice. Unexpectedly, this Lt betaR-Ig decoy protein dramatically accelerated the rate of remyelination, even after the maximal pathological disease state had been reached. This strongly indicates the beneficial role of Lt betaR-Ig in the delay of demyelination and the acceleration of remyelination. The discrepancy between remyelination rates in these systems could be attributed to developmental abnormalities in the immune systems of Lt betaR-/- mice. These findings bode well for the use of an inhibitory Lt betaR-Ig as a candidate biological therapy in demyelinating disorders, because it is beneficial during both demyelination and remyelination

NLRP3 Plays a Critical Role in the Development of Experimental Autoimmune Encephalomyelitis by Mediating Th1 and Th17 Responses

The interplay between innate and adaptive immunity is important in multiple sclerosis (MS). The inflammasome complex, which activates caspase-1 to process pro–IL-1β and pro–IL-18, is rapidly emerging as a pivotal regulator of innate immunity, with nucleotide-binding domain, leucine-rich repeat containing protein family, pyrin domain containing 3 (NLRP3) (cryopyrin or NALP3) as a prominent player. Although the role of NLRP3 in host response to pathogen associated molecular patterns and danger associated molecular patterns is well documented, its role in autoimmune diseases is less well studied. To investigate the role of NLRP3 protein in MS, we used a mouse model of MS, experimental autoimmune encephalomyelitis (EAE). Nlrp3 expression was elevated in the spinal cords during EAE, and Nlrp3−/− mice had a dramatically delayed course and reduced severity of disease. This was accompanied by a significant reduction of the inflammatory infiltrate including macrophages, dendritic cells, CD4, and CD8+ T cells in the spinal cords of the Nlrp3−/− mice, whereas microglial accumulation remained the same. Nlrp3−/− mice also displayed improved histology in the spinal cords with reduced destruction of myelin and astrogliosis. Nlrp3−/− mice with EAE produced less IL-18, and the disease course was similar to Il18−/− mice. Furthermore, Nlrp3−/− and Il18−/− mice had similarly reduced IFN-γ and IL-17 production. Thus, NLRP3 plays a critical role in the induction of the EAE, likely through effects on capase-1–dependent cytokines which then influence Th1 and Th17

Criteria for effective design, construction, and gene knockdown by shRNA vectors-0

<p><b>Copyright information:</b></p><p>Taken from "Criteria for effective design, construction, and gene knockdown by shRNA vectors"</p><p>BMC Biotechnology 2006;6():7-7.</p><p>Published online 24 Jan 2006</p><p>PMCID:PMC1409772.</p><p>Copyright © 2006 Taxman et al; licensee BioMed Central Ltd.</p>everse strand containing a one bp mutation within both the sense and antisense copy of the target sequence (shown in red). The double stranded hybrid is ligated into the retroviral vector 5' of an H1 promoter and transformed into competent bacteria. Since replication is semi-conservative, the daughter bacteria will be of two different populations that carry either a double-stranded wild-type or a double-stranded mutant vector and can be isolated by preparing and sequencing individual colonies

Criteria for effective design, construction, and gene knockdown by shRNA vectors-4

<p><b>Copyright information:</b></p><p>Taken from "Criteria for effective design, construction, and gene knockdown by shRNA vectors"</p><p>BMC Biotechnology 2006;6():7-7.</p><p>Published online 24 Jan 2006</p><p>PMCID:PMC1409772.</p><p>Copyright © 2006 Taxman et al; licensee BioMed Central Ltd.</p> decision threshold varied from minimum to maximum scores (see Materials and Methods for details) for the Hsieh et al. (A), 5' ΔΔG (free energy differential) (B), Reynolds et al. (C), Amarzguioui et al. (D), Ui-Tei et al. (E) and Takasaki et al. (F) algorithms using an efficacy threshold of 50% knockdown. ROC curves for modified Amarzguioui et al. (G), Ui-Tei et al. (H) and Takasaki et al. (I) algorithms are also shown. A set of 38 published shRNAs (Table 5) was analyzed using the modified Amarzguioui et al. (J), Ui-Tei et al. (K) and Takasaki et al. (L) algorithms to confirm the utility of the modified algorithms. The area under the curve (AUC) and the probability (p) that the AUC is significantly different from 0.5, the area under diagonal, is indicated for each ROC curve

Criteria for effective design, construction, and gene knockdown by shRNA vectors-3

<p><b>Copyright information:</b></p><p>Taken from "Criteria for effective design, construction, and gene knockdown by shRNA vectors"</p><p>BMC Biotechnology 2006;6():7-7.</p><p>Published online 24 Jan 2006</p><p>PMCID:PMC1409772.</p><p>Copyright © 2006 Taxman et al; licensee BioMed Central Ltd.</p>rved knockdown efficiency for the Hsieh et al. (A), 5' ΔΔG (free energy differential) (B), Reynolds et al. (C), Amarzguioui et al. (D), Ui-Tei et al. (E) and Takasaki et al. (F) algorithms. The 5' ΔΔG score is plotted on a reverse horizontal axis since knockdown efficacy is predicted to correlate with negative 5' ΔΔG value. A trend line is shown along with the Rvalue for each plot. Knockdown of less than 10% is plotted as zero

Criteria for effective design, construction, and gene knockdown by shRNA vectors-1

<p><b>Copyright information:</b></p><p>Taken from "Criteria for effective design, construction, and gene knockdown by shRNA vectors"</p><p>BMC Biotechnology 2006;6():7-7.</p><p>Published online 24 Jan 2006</p><p>PMCID:PMC1409772.</p><p>Copyright © 2006 Taxman et al; licensee BioMed Central Ltd.</p>t were prepared simultaneously as detailed in Figure 1. (B) Realtime analysis of shRNA knockdown, and loss of knockdown by mutant shRNA vectors from (A). Values are standardized to 100% in non-transduced THP1 cells. The expression in THP1 cells transduced with an empty vector (EV) is shown as an additional control. Values represent average +SEM for at least three assays performed in duplicate

Criteria for effective design, construction, and gene knockdown by shRNA vectors-2

<p><b>Copyright information:</b></p><p>Taken from "Criteria for effective design, construction, and gene knockdown by shRNA vectors"</p><p>BMC Biotechnology 2006;6():7-7.</p><p>Published online 24 Jan 2006</p><p>PMCID:PMC1409772.</p><p>Copyright © 2006 Taxman et al; licensee BioMed Central Ltd.</p>cated by the row of numbers in each panel and the Y axis is the signal intensity. Sequencing reaction conditions shown are BigDye v1.1 (BD) chemistry (A), 0.83 M Betaine + 1 ×PCRx Enhancer in BD chemistry (B), 10:1 BD:dGTP chemistries (C), 0.83 M Betaine + 1 ×PCRx Enhancer in 10:1 BD:dGTP chemistries (D), and 1 × ThermoFidelase I in 10:1 BD:dGTP chemistries (E). The drop in signal (step in peak height) at the hairpin is highlighted by an arrow in the 10:1 BD:dGTP chemistries panel

Oral once-daily berotralstat for the prevention of hereditary angioedema attacks: A randomized, double-blind, placebo-controlled phase 3 trial

Background: Berotralstat (BCX7353) is an oral, once-daily inhibitor of plasma kallikrein in development for the prophylaxis of hereditary angioedema (HAE) attacks. Objective: Our aim was to determine the efficacy, safety, and tolerability of berotralstat in patients with HAE over a 24-week treatment period (the phase 3 APeX-2 trial). Methods: APeX-2 was a double-blind, parallel-group study that randomized patients at 40 sites in 11 countries 1:1:1 to receive once-daily berotralstat in a dose of 110 mg or 150 mg or placebo (Clinicaltrials.gov identifier NCT03485911). Patients aged 12 years or older with HAE due to C1 inhibitor deficiency and at least 2 investigator-confirmed HAE attacks in the first 56 days of a prospective run-in period were eligible. The primary efficacy end point was the rate of investigator-confirmed HAE attacks during the 24-week treatment period. Results: A total of 121 patients were randomized; 120 of them received at least 1 dose of the study drug (n = 41, 40, and 39 in the 110-mg dose of berotralstat, 150-mg of dose berotralstat, and placebo groups, respectively). Berotralstat demonstrated a significant reduction in attack rate at both 110 mg (1.65 attacks per month; P = .024) and 150 mg (1.31 attacks per month; P < .001) relative to placebo (2.35 attacks per month). The most frequent treatment-emergent adverse events that occurred more with berotralstat than with placebo were abdominal pain, vomiting, diarrhea, and back pain. No drug-related serious treatment-emergent adverse events occurred. Conclusion: Both the 110-mg and 150-mg doses of berotralstat reduced HAE attack rates compared with placebo and were safe and generally well tolerated. The most favorable benefit-to-risk profile was observed at a dose of 150 mg per day