ABSTRACT
Non-opioid single-chain variable fragment (scFv) small antibodies were generated as pain-reducing block of P2X4R receptor (P2X4R). A panel of scFvs targeting an extracellular peptide sequence of P2X4R was generated followed by cell-free ribosome display for recombinant antibody selection. After three rounds of bio-panning, a panel of recombinant antibodies was isolated and characterized by ELISA, cross-reactivity analysis, and immunoblotting/immunostaining. Generated scFv antibodies feature binding activity similar to monoclonal antibodies but with stronger affinity and increased tissue penetrability due to their ~30% smaller size. Two anti-P2X4R scFv clones (95, 12) with high specificity and affinity binding were selected for in vivo testing in male and female mice with trigeminal nerve chronic neuropathic pain (FRICT-ION model) persisting for several months in untreated BALBc mice. A single dose of P2X4R scFv (4 mg/kg, i.p.) successfully, completely, and permanently reversed chronic neuropathic pain-like measures in male mice only, providing retention of baseline behaviors indefinitely. Untreated mice retained hypersensitivity, and developed anxiety- and depression-like behaviors within 5 weeks. In vitro P2X4R scFv 95 treatment significantly increased the rheobase of larger-diameter (>25 µm) trigeminal ganglia (TG) neurons from FRICT-ION mice compared to controls. The data support use of engineered scFv antibodies as non-opioid biotherapeutic interventions for chronic pain.
Subject(s)
Chronic Pain/drug therapy , Purinergic P2X Receptor Antagonists/therapeutic use , Single-Chain Antibodies/therapeutic use , Animals , Antibody Affinity , Cells, Cultured , Chronic Pain/immunology , Female , Male , Mice , Peptide Library , Purinergic P2X Receptor Antagonists/chemistry , Purinergic P2X Receptor Antagonists/immunology , Receptors, Purinergic P2X4/chemistry , Receptors, Purinergic P2X4/immunology , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , Recombinant Proteins/therapeutic use , Single-Chain Antibodies/chemistry , Single-Chain Antibodies/immunologyABSTRACT
Studies conducted during the COVID-19 Pandemic have reported increased rates of mental illnesses including depression, anxiety, and post-traumatic stress disorder (PTSD) [1]. A common symptom of mental illness is change in Rapid Eye Movement (REM) sleep, the phase of sleep associated with dreaming and nightmares. The COVID-19 pandemic offers a unique opportunity to evaluate the effects of systemic stress on nightmares. In this study, we investigate whether the COVID-19 pandemic affects nightmare frequency and content using a web-based survey within the state of New Mexico. The survey returned 197 responses showing an increase in the quantity of both bad dreams and nightmares. Furthermore, significant changes in nightmare themes were reported compared to relative rates prior to the pandemic (RR 1,42, p < 0.01; RR 5, p < 0.001). This novel data supports that increased stress from the COVID-19 pandemic has altered dream and nightmare content and frequency.