Longitudinal study on the influence of Nd:YAG laser irradiation on microleakage associated of two filling techniques.

Objective: This study investigates the effects of Nd:YAG laser irradiation on apical and coronal seals, when used prior to two root canal filling techniques. Background Data: Limited information exists regarding the effects of morphologic changes to dentin walls following Nd: YAG laser irradiation on the sealing ability of root fillings. Methods: Two hundred forty teeth were analyzed by observing coronal and apical leakage of Indian ink (DL), and 60 were analyzed for through-and-through leakage using the fluid transport model (FTM). The Nd: YAG laser parameters were 1.5W, 100mJ, and 15Hz (four times for 5s at 20s intervals). Each group consisted of a lased and a nonlased subgroup: each subgroup had root fills done by either cold lateral condensation (CLC) or hybrid condensation (HC). Leakage was assessed after 48 h, and then at 1, 6, and 12 months. The DL group was divided into four groups of 15 teeth for each evaluation point. Through-and-through leakage (L in microliters/day) was measured for 48h under a pressure of 1.2 atm using FTM, and recorded as L = 0 (L1), 0 10 (L3). Results: Apical and coronal dye leakage was observed in all groups. Significant differences (p < 0.05) in apical leakage were found between HC and HC + Nd after 1, 6, and 12 months, and between CLC and CLC + Nd at 6 and 12 months. No significant differences were found between laser-irradiated and non-laser-irradiated groups with FTM. Conclusion: Pulsed Nd: YAG laser irradiation following root canal preparation may reduce apical leakage in association with hybrid gutta-percha condensation

VHHs as tools for therapeutic protein delivery to the central nervous system

Background: The blood brain barrier (BBB) limits the therapeutic perspective for central nervous system (CNS) disorders. Previously we found an anti-mouse transferrin receptor (TfR) VHH (Nb62) that was able to deliver a biologically active neuropeptide into the CNS in mice. Here, we aimed to test its potential to shuttle a therapeutic relevant cargo. Since this VHH could not recognize the human TfR and hence its translational potential is limited, we also aimed to find and validate an anti-human transferrin VHH to deliver a therapeutic cargo into the CNS. / Methods: Alpaca immunizations with human TfR, and subsequent phage selection and screening for human TfR binding VHHs was performed to find a human TfR specific VHH (Nb188). Its ability to cross the BBB was determined by fusing it to neurotensin, a neuropeptide that reduces body temperature when present in the CNS but is not able to cross the BBB on its own. Next, the anti–β-secretase 1 (BACE1) 1A11 Fab and Nb62 or Nb188 were fused to an Fc domain to generate heterodimeric antibodies (1A11AM-Nb62 and 1A11AM-Nb188). These were then administered intravenously in wild-type mice and in mice in which the murine apical domain of the TfR was replaced by the human apical domain (hAPI KI). Pharmacokinetic and pharmacodynamic (PK/PD) studies were performed to assess the concentration of the heterodimeric antibodies in the brain over time and the ability to inhibit brain-specific BACE1 by analysing the brain levels of Aβ1–40. / Results: Selections and screening of a phage library resulted in the discovery of an anti-human TfR VHH (Nb188). Fusion of Nb188 to neurotensin induced hypothermia after intravenous injections in hAPI KI mice. In addition, systemic administration 1A11AM-Nb62 and 1A11AM-Nb188 fusions were able to reduce Aβ1-40 levels in the brain whereas 1A11AM fused to an irrelevant VHH did not. A PK/PD experiment showed that this effect could last for 3 days. / Conclusion: We have discovered an anti-human TfR specific VHH that is able to reach the CNS when administered systemically. In addition, both the currently discovered anti-human TfR VHH and the previously identified mouse-specific anti-TfR VHH, are both able to shuttle a therapeutically relevant cargo into the CNS. We suggest the mouse-specific VHH as a valuable research tool in mice and the human-specific VHH as a moiety to enhance the delivery efficiency of therapeutics into the CNS in human patients

Identification and characterization of nanobodies targeting the EphA4 receptor

The ephrin receptor A4 (EphA4) is one of the receptors in the ephrin system that plays a pivotal role in a variety of cell-cell interactions, mostly studied during development. In addition, EphA4 has been found to play a role in cancer biology as well as in the pathogenesis of several neurological disorders such as stroke, spinal cord injury, multiple sclerosis, amyotrophic lateral sclerosis (ALS), and Alzheimer's disease. Pharmacological blocking of EphA4 has been suggested to be a therapeutic strategy for these disorders. Therefore, the aim of our study was to generate potent and selective Nanobodies against the ligand-binding domain of the human EphA4 receptor. Weidentified two Nanobodies, Nb 39 and Nb 53, that bind EphA4 with affinities in the nanomolar range. These Nanobodies were most selective for EphA4, with residual binding to EphA7 only. Using Alphascreen technology, we found that both Nanobodies displaced all known EphA4-binding ephrins from the receptor. Furthermore, Nb39 andNb53 inhibited ephrin-induced phosphorylationoftheEphA4proteininacell-basedassay. Finally, in a cortical neuron primary culture, both Nanobodies were able to inhibit endogenous EphA4-mediated growth-cone collapse induced by ephrin-B3. Our results demonstrate the potential of Nanobodies to target the ligand-binding domain of EphA4. These Nanobodiesmaydeservefurtherevaluationaspotentialtherapeutics in disorders in which EphA4-mediated signaling plays a role

Selective inhibitors of the PSEN1–gamma-secretase complex

Clinical development of γ-secretases, a family of intramembrane cleaving proteases, as therapeutic targets for a variety of disorders including cancer and Alzheimer’s disease was aborted because of serious mechanism-based side effects in the phase III trials of unselective inhibitors. Selective inhibition of specific γ-secretase complexes, containing either PSEN1 or PSEN2 as the catalytic subunit and APH1A or APH1B as supporting subunits, does provide a feasible therapeutic window in preclinical models of these disorders. We explore here the pharmacophoric features required for PSEN1 versus PSEN2 selective inhibition. We synthesized a series of brain penetrant 2-azabicyclo[2,2,2]octane sulfonamides and identified a compound with low nanomolar potency and high selectivity (>250-fold) toward the PSEN1–APH1B subcomplex versus PSEN2 subcomplexes. We used modeling and site-directed mutagenesis to identify critical amino acids along the entry part of this inhibitor into the catalytic site of PSEN1. Specific targeting one of the different γ-secretase complexes might provide safer drugs in the future

Selective inhibitors of the PSEN1-gamma-secretase complex

Clinical development of Y-secretases, a family of intramembrane cleaving proteases, as therapeutic targets for a variety of disorders including cancer and Alzheimer’s disease was aborted because of serious mechanism-based side effects in the phase III trials of unselective inhibitors. Selective inhibition of specific Y-secretase complexes, containing either PSEN1 or PSEN2 as the catalytic subunit and APH1A or APH1B as supporting subunits, does provide a feasible therapeutic window in preclinical models of these disorders. We explore here the pharmacophoric features required for PSEN1 versus PSEN2 selective inhibition. We synthesized a series of brain penetrant 2-azabicyclo[2,2,2]octane sulfonamides and identified a compound with low nanomolar potency and high selectivity (>250-fold) toward the PSEN1–APH1B subcomplex versus PSEN2 subcomplexes. We used modeling and site-directed mutagenesis to identify critical amino acids along the entry part of this inhibitor into the catalytic site of PSEN1. Specific targeting one of the different Y-secretase complexes might provide safer drugs in the future.The work was supported by an AIO-project (no. HBC.2016.0884). This project received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 Research and Innovation Programme (grant agreement no. ERC-834682 CELLPHASE_AD). This work was supported by the Flanders Institute for Biotechnology (VIB vzw), a Methusalem grant from KU Leuven and the Flemish Government, the Fonds voor Wetenschappelijk Onderzoek, KU Leuven, The Queen Elisabeth Medical Foundation for Neurosciences, the Opening the Future campaign of the Leuven Universitair Fonds, the Belgian Alzheimer Research Foundation (SAO-FRA), and the Alzheimer’s Association USA.Peer ReviewedPostprint (published version

AAV-mediated delivery of an anti-BACE1 VHH alleviates pathology in an Alzheimer's disease model

Single domain antibodies (VHHs) are potentially disruptive therapeutics, with important biological value for treatment of several diseases, including neurological disorders. However, VHHs have not been widely used in the central nervous system (CNS), largely because of their restricted blood–brain barrier (BBB) penetration. Here, we propose a gene transfer strategy based on BBB-crossing adeno-associated virus (AAV)-based vectors to deliver VHH directly into the CNS. As a proof-of-concept, we explored the potential of AAV-delivered VHH to inhibit BACE1, a well-characterized target in Alzheimer’s disease. First, we generated a panel of VHHs targeting BACE1, one of which, VHH-B9, shows high selectivity for BACE1 and efficacy in lowering BACE1 activity in vitro. We further demonstrate that a single systemic dose of AAV-VHH-B9 produces positive long-term (12 months plus) effects on amyloid load, neuroinflammation, synaptic function, and cognitive performance, in the AppNL-G-F Alzheimer’s mouse model. These results constitute a novel therapeutic approach for neurodegenerative diseases, which is applicable to a range of CNS disease targets

Cost Effectiveness of a CYP2C19 Genotype-Guided Strategy in Patients with Acute Myocardial Infarction:Results from the POPular Genetics Trial

INTRODUCTION: The POPular Genetics trial demonstrated that a CYP2C19 genotype-guided P2Y12 inhibitor strategy reduced bleeding rates compared with standard treatment with ticagrelor or prasugrel without increasing thrombotic event rates after primary percutaneous coronary intervention (PCI). OBJECTIVE: In this analysis, we aimed to evaluate the cost effectiveness of a genotype-guided strategy compared with standard treatment with ticagrelor or prasugrel. METHODS: A 1-year decision tree based on the POPular Genetics trial in combination with a lifelong Markov model was developed to compare costs and quality-adjusted life-years (QALYs) between a genotype-guided and a standard P2Y12 inhibitor strategy in patients with myocardial infarction undergoing primary PCI. The cost-effectiveness analysis was conducted from a Dutch healthcare system perspective. Within-trial survival and utility data were combined with lifetime projections to evaluate lifetime cost effectiveness for a cohort of 1000 patients. Costs and utilities were discounted at 4 and 1.5%, respectively, according to Dutch guidelines for health economic studies. Besides deterministic and probabilistic sensitivity analyses, several scenario analyses were also conducted (different time horizons, different discount rates, equal prices for P2Y12 inhibitors, and equal distribution of thrombotic events between the two strategies). RESULTS: Base-case analysis with a hypothetical cohort of 1000 subjects demonstrated 8.98 QALYs gained and €725,550.69 in cost savings for the genotype-guided strategy (dominant). The deterministic and probabilistic sensitivity analysis confirmed the robustness of the model and the cost-effectiveness results. In scenario analyses, the genotype-guided strategy remained dominant. CONCLUSION: In patients undergoing primary PCI, a CYP2C19 genotype-guided strategy compared with standard treatment with ticagrelor or prasugrel resulted in QALYs gained and cost savings. TRIAL REGISTRATION: Clinicaltrials.gov number: NCT01761786, Netherlands trial register number: NL2872

Exploring the Fate of Antibody-Encoding pDNA after Intramuscular Electroporation in Mice

DNA-based antibody therapy seeks to administer the encoding nucleotide sequence rather than the antibody protein. To further improve the in vivo monoclonal antibody (mAb) expression, a better understanding of what happens after the administration of the encoding plasmid DNA (pDNA) is required. This study reports the quantitative evaluation and localization of the administered pDNA over time and its association with corresponding mRNA levels and systemic protein concentrations. pDNA encoding the murine anti-HER2 4D5 mAb was administered to BALB/c mice via intramuscular injection followed by electroporation. Muscle biopsies and blood samples were taken at different time points (up to 3 months). In muscle, pDNA levels decreased 90% between 24 h and one week post treatment (p p < 0.0001). Evaluation of pDNA localization revealed that extranuclear pDNA was cleared fast, whereas the nuclear fraction remained relatively stable. This is in line with the observed mRNA and protein levels over time and indicates that only a minor fraction of the administered pDNA is ultimately responsible for the observed systemic mAb levels. In conclusion, this study demonstrates that durable expression is dependent on the nuclear uptake of the pDNA. Therefore, efforts to increase the protein levels upon pDNA-based gene therapy should focus on strategies to increase both cellular entry and migration of the pDNA into the nucleus. The currently applied methodology can be used to guide the design and evaluation of novel plasmid-based vectors or alternative delivery methods in order to achieve a robust and prolonged protein expression