Search

NOD.Cg- Prkdc scid Il2rg tm1Wjl /SzJ (NSG) mice, lacking many components of a mature immune system, are at increased risk of disease. General understanding of potential pathogens of these mice is limited. We describe a high mortality disease outbreak caused by an opportunistic bacterial infection in NSG mice. Affected animals exhibited perianal fecal staining, dehydration, and wasting. Histopathologic lesions included a primary necrotizing enterocolitis, with inflammatory and necrotizing lesions also occurring in the liver, kidneys, heart, and brain of some mice. All affected individuals tested negative for known opportunistic pathogens of immunodeficient mice. We initially identified a member of Enterobacter cloacae complex (ECC) in association with the outbreak by traditional diagnostics. ECC was cultured from extraintestinal organs, both with and without histopathologic lesions, suggesting bacteremia. Infrared spectroscopy and MALDI-TOF mass spectrometry demonstrated that isolates from the outbreak shared molecular features and likely a common origin. We subsequently hypothesized that advanced sequencing methods would identify a single species of ECC associated with clinical disease. Using a novel targeted amplicon-based next-generation sequencing assay, we identified Enterobacter hormaechei in association with this outbreak. Knowledge of this organism as a potential opportunistic pathogen in NSG mice is critical for preclinical studies to prevent loss of animals and confounding of research., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: KN, CW, and KSH are employees of Charles River Laboratories.

Accumulation of amyloid-β peptide (Aβ) aggregates in synapses may contribute to the profound synaptic loss characteristic of Alzheimer's disease (AD). The origin of synaptic Aβ aggregates remains elusive, but loss of endosomal proteostasis may trigger their formation. In this study, we identified the synaptic compartments where Aβ accumulates, and performed a longitudinal analysis of synaptosomes isolated from brains of TgCRND8 APP transgenic mice of either sex. To evaluate the specific contribution of Aβ-degrading protease endothelin-converting enzyme (ECE-1) to synaptic/endosomal Aβ homeostasis, we analyzed the effect of partial Ece1 KO in brain and complete ECE1 KO in SH-SY5Y cells. Global inhibition of ECE family members was used to further assess their role in preventing synaptic Aβ accumulation. Results showed that, before extracellular amyloid deposition, synapses were burdened with detergent-soluble Aβ monomers, oligomers, and fibrils. Levels of all soluble Aβ species declined thereafter, as Aβ42 turned progressively insoluble and accumulated in Aβ-producing synaptic endosomal vesicles with characteristics of multivesicular bodies. Accordingly, fibrillar Aβ was detected in brain exosomes. ECE-1-deficient mice had significantly increased endogenous synaptosomal Aβ42 levels, and protease inhibitor experiments showed that, in TgCRND8 mice, synaptic Aβ42 became nearly resistant to degradation by ECE-related proteases. Our study supports that Aβ accumulating in synapses is produced locally, within endosomes, and does not require the presence of amyloid plaques. ECE-1 is a determinant factor controlling the accumulation and fibrillization of nascent Aβ in endosomes and, in TgCRND8 mice, Aβ overproduction causes rapid loss of Aβ42 solubility that curtails ECE-mediated degradation. SIGNIFICANCE STATEMENT Deposition of aggregated Aβ in extracellular plaques is a defining feature of AD. Aβ aggregates also accumulate in synapses and may contribute to the profound synaptic loss and cognitive dysfunction typical of the disease. However, it is not clear whether synaptotoxic Aβ is mainly derived from plaques or if it is produced and aggregated locally, within affected synaptic compartments. Filling this knowledge gap is important for the development of an effective treatment for AD, as extracellular and intrasynaptic pools of Aβ may not be equally modulated by immunotherapies or other therapeutic approaches. In this manuscript, we provide evidence that Aβ aggregates building up in synapses are formed locally, within synaptic endosomes, because of disruptions in nascent Aβ proteostasis., (Copyright © 2023 the authors.)

Aggregation and accumulation of amyloid-β peptide (Aβ) are a critical trigger for the onset of Alzheimer's disease (AD). While the plaques are the most outstanding Aβ pathological feature, much of the recent research emphasis has been on soluble Aβ species because of their diffusible, proinflammatory, and toxic properties. The focus on soluble aggregated Aβ species has also increased the interest in antibodies that are selective for different Aβ conformations. In the current study, we developed and characterized a new class of monoclonal antibodies (referred to as mAbSL) that are selective for Aβ protofibrils. Cloning and sequencing of the heavy and light chain variable regions for multiple antibodies identified sequence characteristics that may impart the conformational selectivity by the antibodies. Transfection of FreeStyle 293F cells with the plasmids permitted in-house expression and purification of mAbSL antibodies along with non-conformation-selective Aβ monoclonal antibodies (Aβ mAbs). Several of the purified mAbSL antibodies demonstrated significant affinity and selectivity for Aβ42 protofibrils compared with Aβ42 monomers and Aβ42 fibrils. Competition ELISA assays assessing the best overall antibody, mAbSL 113, yielded affinity constants of 7 nM for the antibody-Aβ42 protofibril interaction, while the affinity for either Aβ42 monomers or Aβ42 fibrils was roughly 80 times higher. mAbSL 113 significantly inhibited Aβ42 monomer aggregation by a unique mechanism compared with the inhibition displayed by Aβ mAb 513. Aβ42 protofibril dynamics were also markedly altered in the presence of mAbSL 113, whereby insoluble complex formation and protofibril deposition were stimulated by the antibody at low substoichiometric molar ratios. As the field contemplates the therapeutic effectiveness of Aβ conformation-selective antibodies, the findings presented here demonstrate new information on a monoclonal antibody that selectively targets Aβ protofibrils and impacts Aβ dynamics., (© 2023 International Society for Neurochemistry.)

Three decades of research, both in vitro and in vivo, have demonstrated the conformational heterogeneity that is displayed by the amyloid β peptide (Aβ) in Alzheimer's disease (AD). Understanding the distinct properties between Aβ conformations and how conformation may impact cellular activity remain open questions, yet still continue to provide new insights into protein misfolding and aggregation. In particular, there is interest in the group of soluble oligomeric prefibrillar Aβ species comprising lower molecular weight oligomers up to larger protofibrils. In the current study, a number of strategies were utilized to separate Aβ protofibrils and oligomers and show that the smaller Aβ oligomers have a much different conformation than Aβ protofibrils. The differences were consistent for both Aβ40 and Aβ42. Protofibrils bound thioflavin T to a greater extent than oligomers, and were highly enriched in β-sheet secondary structure. Aβ oligomers possessed a more open structure with significant solvent exposure of hydrophobic domains as determined by tryptophan fluorescence and bis-ANS binding, respectively. The protofibril-selective antibody AbSL readily discerned conformational differences between protofibrils and oligomers. The more developed structure for Aβ protofibrils ultimately proved critical for provoking the release of tumor necrosis factor α from microglial cells. The findings demonstrated a dependency on β-sheet structure for soluble Aβ aggregates to cause a microglial inflammatory response. The Aβ aggregation process yields many conformationally-varied species with different levels of β-structure and exposed hydrophobicity. The conformation elements likely determine biological activity and pathogenicity., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Buprenorphine is commonly used to control postoperative pain in rodents. Short-acting formulations of buprenorphine (bup-HCl) require frequent handling and restraint of animals for appropriate dosing, which can be stressful and confound research outcomes. Ethiqa XR (bup-ER) is an FDA-indexed extended-release buprenorphine formulation that is an alternative to bup-HCl in mice and rats. In the current study, we first evaluated the pharmacokinetics of bup-ER in male C57BL/6J mice by sampling blood at 10 time points, ranging from 30 min to 72 h after administration ( n = 3 mice per time point). Average plasma concentrations fell below therapeutic levels at 48 h after administration. We also evaluated the safety of bup-ER when administered prior to surgery in combination with common anesthetics and the efficacy of bup-ER in mouse laparotomy. Anesthetic safety was studied by measuring respiratory rate, rectal temperature, and recovery time in groups of mice ( n = 8) given bup-HCl, bup-ER, or saline in combination with isoflurane or ketamine-xylazine anesthesia. No differences were seen between analgesic treatment groups with either of the general anesthetics. To evaluate efficacy, mice ( n = 10) were randomly allocated to receive either bup-ER (3.25 mg/kg) once presurgically, bup-HCl (0.1 mg/kg) presurgically and then every 8 h, or saline once before surgery. Mice underwent a sham laparotomy and were assessed for pain based on changes in weight, cageside ethogram, nesting consolidation test, rearing frequency, and nociception to von Frey testing at 6, 12, 24, 48, and 72 h after surgery. Cageside ethogram, rearing frequency, and von Frey testing showed significant differences between bup-ER-treated mice and saline controls in the early postoperative period. No significant effects between treatment groups were seen in daily weights or nesting consolidation scores. This study demonstrates that bup-ER can be safely administered before surgery and provides analgesia for up to 48 h after administration based on pharmacokinetic and behavioral data.

Sepsis is a serious medical condition characterized by bacterial infection and a subsequent massive systemic inflammatory response. In an effort to identify compounds that block lipopolysaccharide (LPS)-induced inflammation reported herein is the development of simple Lipid-A analogues that lack a disaccharide core yet still possess potent antagonistic activity against LPS. The structure of the new lead compound was developed based on predictive computational experiments. LPS antagonism by the lead compound was not straightforward, and a biphasic effect was observed suggesting a possibility of more than one binding site. An IC 50 value of 13 nM for the new compound was determined for the possible high affinity site. The combination of computational, synthetic, and biological studies revealed new structural determinants of these simplified analogues. It is expected that the acquired information will aid future design of LPS targeting glycopharmaceuticals.