Biology subjects

Daily, J.

Publications and source records attributed to Daily, J..

3 recordsLinked to original sources

bioRxiv2025

Exploring GPCR-mediated optogenetic modulation of seizure network in a pig model of Temporal Lobe Epilepsy

RationaleOptogenetics offers unmatched cellular specificity and control over cellular activity. Various opsins have been tested in animal models of epilepsy, each contributing to our understanding of seizure circuit dynamics. However, inhibitory optogenetic tools based on microbial rhodopsins have low light sensitivity and, thus, are less suitable for applications involving larger brains. We evaluated eOPN3, a red-shifted, highly sensitive inhibitory G-protein coupled receptor opsin in a porcine seizure model using integrated electro-optical sensing and modulation. The results demonstrated the feasibility of eOPN3 circuit modulation in a large animal epilepsy model. MethodsMRI-guided stereotactic surgery was used to deliver 20-60 {micro}L of AAV-eOPN3 (AAV5-/AAV9-CaMKII-eOPN3-mScarlet) into the hippocampus (HPC) of three Gottingen minipigs. Each hemisphere received either an active or a control viral vector (AAV5/9-CaMKII-mScarlet) with gadolinium to visualize the injection sites and diffusion volume via post-operative MRI. Two to three months post-injection, bilateral deep brain stimulation electrodes integrated with optic fibers were stereotactically implanted into the anterior nucleus of the thalamus (ANT) and HPC to assess: 1) opsin expression using fiber photometry, 2) optogenetic modulation of stimulation evoked response potentials (SERPs), 3) induction and propagation of seizure-like activity via intrahippocampal kainic acid (KA) injection, and 4) optogenetic modulation of KA-induced seizure activity. After the electrophysiology recording, brains were harvested for histological analysis to evaluate injection target precision, eOPN3 expression, and estimate eOPN3-modulated volume. ResultseOPN3 expression was confirmed during surgery via fiber photometry. ANT electrical stimulation elicited robust SERPs in the HPCs, which were attenuated by HPC light illumination. HPC stimulation similarly induced SERPs in the ipsilateral ANT and the contralateral HPC. The HPC stimulation-induced SERPs were significantly reduced by illuminating the site of the recording areas, the ipsilateral ANT and the contralateral HPC, demonstrating the optogenetic inhibition of the synaptic release from the HPC. KA injection into the HPC induced 20-30 Hz seizure-like activity. The ANT and HPC light illumination suppressed the localized KA-induced seizure activity in the early stage. However, after the generalization of KA-induced seizures, the ANT-HPC illumination lost efficacy for the control of seizures. Histological analysis confirmed eOPN3 expression in the HPC, ANT and other Papez circuit nodes. ConclusionOur pilot study highlights that eOPN3-mediated inhibition alters SERP and the latency and spread of KA-induced seizure-like activity. We developed a platform incorporating pre- and postoperative MRI for precise viral vector delivery, real-time fiber photometry for quantifying opsin expression, and integrated electro-optical sensing and stimulation to assess optogenetic efficacy in a large animal model. The large animal model provides a solid foundation for future translational research to develop electro-optical devices and cellular therapies for human epilepsy.

neuroscience↗

bioRxiv2021

Cytolytic memory CD4+ T cell clonotypes are expanded during Plasmodium falciparum infection

Plasmodium falciparum (Pf) malaria causes high rates of morbidity and mortality and lacks a sufficiently effective vaccine. Clinical immunity develops in residents of malaria endemic regions which confers reduced clinical symptoms during infection and protection against severe disease. We hypothesized that understanding the immune mechanisms of clinical immunity could inform vaccine design to improve efficacy. We compared the peripheral blood cellular and humoral immune responses during a mild episode of Pf malaria infection. Participants were classified as either clinically susceptible or clinically protected, based on the number of recurrent clinical infections over an 18-month longitudinal study in a malaria endemic region in Malawi. Susceptible participants had three or more recurrent clinical episodes while clinically immune individuals had one or none. Protected participants exhibited higher plasma immunoglobulin G (IgG) breadth and titers against Pf antigens, and greater antibody (Ab)-dependent Pf opsonization compared to susceptible participants. Using high dimensional mass cytometry (CyTOF), spectral flow cytometry and single-cell transcriptomic analyses, we identified expanded memory CD4+ T cell clones sharing identical T cell receptor clonotypes in the blood of protected participants during malaria infection. These cells express a strong cytolytic T helper 1 effector program with transcripts encoding granzymes (A, B, H, M), granulysin, NKG7 and the Zeb2 master transcriptional regulator of terminally differentiated effector T cells. Memory CD4+ T cells expressing Zeb2+ were CD39hiTIGIThi and expressed multiple chemotactic and checkpoint inhibitory receptors, although the cellular levels of several of these receptors were reduced in protected compared to susceptible individuals. We propose that clonally expanded Zeb2+ cytolytic memory CD4+ Th1 cells could represent essential contributors to clinical immunity against Pf malaria. One Sentence SummaryA population of cytolytic memory CD4+ T cells is clonally expanded in patients with Plasmodium falciparum malaria and has reduced chemotactic and inhibitory receptor expression in patients with naturally acquired clinical malaria immunity.

immunology↗

bioRxiv2020

Blockade of LAG-3 in PD-L1-deficient mice enhances clearance of blood stage malaria independent of humoral responses

T cells expressing high levels of inhibitory receptors such as PD-1 and LAG-3 are a hallmark of chronic infections and cancer. Checkpoint blockade therapies targeting these receptors have been largely validated as promising strategies to restore exhausted T cell functions and clearance of chronic infections and tumors. The inability to develop long-term natural immunity in malaria-infected patients has been proposed to be at least partially accounted for by sustained expression of high levels of inhibitory receptors on T and B lymphocytes. While blockade or lack of PD-1/PD-L1 and/or LAG-3 was reported to promote better clearance of Plasmodium parasites in mice, how exactly these pathways contributes to protection is not known. Herein, using a mouse model of non-lethal P. yoelii (Py) infection, we reveal that the kinetics of blood parasitemia is indistinguishable between PD-1-/-, PD-L1-/- and WT mice. Yet, monoclonal antibody (mAb) blockade of LAG-3 in PD-L1-/- mice promoted accelerated control of blood parasite growth and clearance. We also report that i) the majority of LAG-3+ cells are T cells, ii) selective depletion of CD8+ T cells did not prevent anti-LAG-3-mediated protection, and iii) production of effector cytokines by CD4+ T cells is increased in anti-LAG-3-treated versus control mice. In addition, parasite-specific Ab serum titers and their ability to transfer protection from both groups of mice was comparable and depletion of CD4+ T cells prevented protection. Thus, taken together, these results are consistent with a model in which disruption of PD-L1 and LAG-3 on parasite-specific CD4+ T cells unleashes their ability to effectively clear blood parasites, independently from humoral responses. Author SummaryMalaria, caused by Plasmodium parasites, is a global burden for which an efficacious vaccine is urgently needed. The development of long-term immunity against malaria is unclear, but we know that both T and B (that produce antibodies, Ab) lymphocytes, that are subsets of white blood cells, are required. Studies in mouse models of malaria have suggested that sets of inhibitory receptors, namely LAG-3 and PD-1, expressed on cytotoxic and helper T lymphocytes hamper the development of effective immunity against malaria. Therapeutic blockade of these receptors was reported to enhance blood parasite clearance through the development of more protective parasite-specific helper T lymphocytes and Abs. Herein, we reveal that, while mice genetically deficient for the PD-1 pathway fail to clear blood parasites better than WT counterparts, anti-LAG-3 treatment does. Importantly, we found comparable parasite-specific Ab responses between all mouse groups, and Ab transfers conferred similar protection to newly infected mice. We also show that LAG-3 is mostly expressed on T lymphocytes, and that cytotoxic T lymphocytes are not involved in anti-LAG-3 accelerated clearance of parasites. Our results suggest that LAG-3 blockade acts on helper T lymphocytes to unleash their effector responses and enhance the control of blood-stage malaria, independently from parasite-specific Abs.

immunology↗