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BioRxiv : the Preprint Server For... Jun 2024Identifying the origins and contributions of different immune cell populations following brain injury is crucial for understanding their roles in inflammation and tissue...
Identifying the origins and contributions of different immune cell populations following brain injury is crucial for understanding their roles in inflammation and tissue repair. This study investigated the infiltration and phenotypic characteristics of skull bone marrow-derived immune cells in the murine brain after TBI. We performed calvarium transplantation from GFP donor mice and subjected the recipients to controlled cortical impact (CCI) injury 14 days post-transplant. Confocal imaging at 3 days post-CCI revealed GFP+ calvarium-derived cells infiltrating the ipsilateral core lesional area, expressing CD45 and CD11b immune markers. These cells included neutrophil (Ly6G+) and monocyte (Ccr2+) identities. Calvarium-derived GFP+/Iba1+ monocyte/macrophages expressed the efferocytosis receptor MerTK and displayed engulfment of NeuN+ and caspase 3+ apoptotic cells. Phenotypic analysis showed that greater calvarium-derived monocyte/macrophages disproportionately express the anti-inflammatory arginase-1 marker than pro-inflammatory CD86. To differentiate the responses of blood- and calvarium-derived macrophages, we transplanted GFP calvarium skull bone into tdTomato bone marrow chimeric mice, then performed CCI injury 14 days post-transplant. Calvarium-derived GFP+ cells predominantly infiltrated the lesion boundary, while blood-derived TdTomato+ cells dispersed throughout the lesion and peri-lesion. Compared to calvarium-derived cells, more blood-derived cells expressed pro-inflammatory CD86 and displayed altered 3D morphologic traits. These findings uniquely demonstrate that skull bone-derived immune cells infiltrate the brain after injury and contribute to the neuroinflammatory milieu, representing a novel immune cell source that may be further investigated for their causal role in functional outcomes.
PubMed: 38948756
DOI: 10.1101/2024.06.21.597827 -
BioRxiv : the Preprint Server For... Jun 2024SARS-CoV-2 virus has continued to evolve over time necessitating the adaptation of vaccines to maintain efficacy. Monoclonal antibodies (mAbs) against SARS-CoV-2 were a...
UNLABELLED
SARS-CoV-2 virus has continued to evolve over time necessitating the adaptation of vaccines to maintain efficacy. Monoclonal antibodies (mAbs) against SARS-CoV-2 were a key line of defense for unvaccinated or immunocompromised individuals. However, these mAbs are now ineffective against current SARS-CoV-2 variants. Here, we tested three aspects of αSARS-CoV-2 therapeutics. First, we tested whether Fc engagement is necessary for clearance of SARS-CoV-2. Secondly, we tested bi-specific killer engagers (BiKEs) that simultaneously engage SARS-CoV-2 and a specific Fc receptor. Benefits of these engagers include the ease of manufacturing, stability, more cell-specific targeting, and high affinity binding to Fc receptors. Using both mAbs and BiKEs, we found that both neutralization and Fc receptor engagement were necessary for effective SARS-CoV-2 clearance. Thirdly, due to ACE2 being necessary for viral entry, ACE2 will maintain binding to SARS-CoV-2 despite viral evolution. Therefore, we used an ACE2 decoy Fc-fusion or BiKE, instead of an anti-SARS-CoV-2 antibody sequence, as a potential therapeutic that would withstand viral evolution. We found that the ACE2 decoy approach also required Fc receptor engagement and, unlike traditional neutralizing antibodies against specific variants, enabled the clearance of two distinct SARS-CoV-2 variants. These data show the importance of Fc engagement for mAbs, the utility of BiKEs as therapies for infectious disease, and the effectiveness of the ACE2 decoy approach. With further studies, we predict combining neutralization, the cellular response, and this ACE2 decoy approach will benefit individuals with ineffective antibody levels.
ABBREVIATIONS
ACE2, scFv, mAb, BiKE, COVID-19, Fc, CD16, CD32b, CD64, d.p.i.
KEY POINTS
With equal dosing, both neutralization and Fc engagement are necessary for the optimal efficacy of antibodies and bi-specific killer engagers (BiKEs) against SARS-CoV-2. BiKEs can clear SARS-CoV-2 virus and protect against severe infection in the hACE2-K18 mouse model. ACE2 decoys as part of Fc-fusions or BiKEs provide clearance of two disparate SARS-CoV-2 variants.
PubMed: 38948747
DOI: 10.1101/2024.06.20.599956 -
BioRxiv : the Preprint Server For... Jun 2024Beckwith-Wiedemann Syndrome (BWS) is an epigenetic overgrowth syndrome caused by methylation changes in the human 11p15 chromosomal locus. Patients with BWS exhibit...
Beckwith-Wiedemann Syndrome (BWS) is an epigenetic overgrowth syndrome caused by methylation changes in the human 11p15 chromosomal locus. Patients with BWS exhibit tissue overgrowth, as well as an increased risk of childhood neoplasms in the liver and kidney. To understand the impact of these 11p15 changes, specifically in the liver, we performed single-nucleus RNA sequencing (snRNA-seq) and single-nucleus assay for transposase-accessible chromatin with sequencing (snATAC-seq) to generate paired, cell-type-specific transcriptional and chromatin accessibility profiles of both BWS-liver and nonBWS-liver nontumorous tissue. Our integrated RNA+ATACseq multiomic approach uncovered hepatocyte-specific enrichment and activation of the peroxisome proliferator-activated receptor α (PPARA) - a liver metabolic regulator. To confirm our findings, we utilized a BWS-induced pluripotent stem cell (iPSC) model, where cells were differentiated into hepatocytes. Our data demonstrates the dysregulation of lipid metabolism in BWS-liver, which coincided with observed upregulation of PPARA during hepatocyte differentiation. BWS liver cells exhibited decreased neutral lipids and increased fatty acid β-oxidation, relative to controls. We also observed increased reactive oxygen species (ROS) byproducts in the form of peroxidated lipids in BWS hepatocytes, which coincided with increased oxidative DNA damage. This study proposes a putative mechanism for overgrowth and cancer predisposition in BWS liver due to perturbed metabolism.
PubMed: 38948745
DOI: 10.1101/2024.06.14.599077 -
BioRxiv : the Preprint Server For... Jun 2024Poor intervertebral disc (IVD) healing causes IVD degeneration (IVDD) and progression to herniation and back pain. This study identified distinct roles of...
UNLABELLED
Poor intervertebral disc (IVD) healing causes IVD degeneration (IVDD) and progression to herniation and back pain. This study identified distinct roles of TNFα-receptors (TNFRs) in contributing to poor healing in painful IVDD. We first isolated IVDD tissue of back pain subjects and determined the complex pro-inflammatory mixture contained many chemokines for recruiting inflammatory cells. Single-cell RNA-sequencing of human IVDD tissues revealed these pro- inflammatory cytokines were dominantly expressed by a small macrophage-population. Human annulus fibrosus (hAF) cells treated with IVDD-conditioned media (CM) underwent senescence with greatly reduced metabolic rates and limited inflammatory responses. TNFR1 inhibition partially restored hAF cell metabolism sufficiently to enable a robust chemokine and cytokine response to CM. We showed that the pro-reparative TNFR2 was very limited on hIVD cell membranes so that TNFR2 inhibition with blocking antibodies or activation using Atsttrin had no effect on hAF cells with CM challenge. However, TNFR2 was expressed in high levels on macrophages identified in scRNA-seq analyses, suggesting their role in repair responses. Results therefore point to therapeutic strategies for painful IVDD involving immunomodulation of TNFR1 signaling in IVD cells to enhance metabolism and enable a more robust inflammatory response including recruitment or delivery of TNFR2 expressing immune cells to enhance IVD repair.
SUMMARY STATEMENT
TNFR1 signaling drives cells towards senesce and muted inflammatory response in painful intervertebral disc degeneration, while limited TNFR2 signaling may limit disc cell repair responses.
PubMed: 38948728
DOI: 10.1101/2024.02.22.581620 -
BioRxiv : the Preprint Server For... Jun 2024Chronic obstructive pulmonary disease (COPD) is the most prevalent lung disease, and macrophages play a central role in the inflammatory response in COPD. We here report...
Chronic obstructive pulmonary disease (COPD) is the most prevalent lung disease, and macrophages play a central role in the inflammatory response in COPD. We here report a comprehensive characterization of circulating short non-coding RNAs (sncRNAs) in plasma from patients with COPD. While circulating sncRNAs are increasingly recognized for their regulatory roles and biomarker potential in various diseases, the conventional RNA-seq method cannot fully capture these circulating sncRNAs due to their heterogeneous terminal structures. By pre-treating the plasma RNAs with T4 polynucleotide kinase, which converts all RNAs to those with RNA-seq susceptible ends (5'-phosphate and 3'-hydroxyl), we comprehensively sequenced a wide variety of non-microRNA sncRNAs, such as 5'-tRNA halves containing a 2',3'-cyclic phosphate. We discovered a remarkable accumulation of the 5'-half derived from tRNA in plasma from COPD patients, whereas the 5'-tRNA half is predominant in healthy donors. Further, the 5'-tRNA half activates human macrophages via Toll-like receptor 7 and induces cytokine production. Additionally, we identified circulating rRNA-derived fragments that were upregulated in COPD patients and demonstrated their ability to induce cytokine production in macrophages. Our findings provide evidence of circulating, immune-active sncRNAs in patients with COPD, suggesting that they serve as inflammatory mediators in the pathogenesis of COPD.
PubMed: 38948719
DOI: 10.1101/2024.06.19.599707 -
BioRxiv : the Preprint Server For... Jun 2024Distinct basolateral amygdala (BLA) cell populations influence emotional responses in manners thought important for anxiety and anxiety disorders. The BLA contains...
Distinct basolateral amygdala (BLA) cell populations influence emotional responses in manners thought important for anxiety and anxiety disorders. The BLA contains numerous cell types which can broadcast information into structures that may elicit changes in emotional states and behaviors. BLA excitatory neurons can be divided into two main classes, one of which expresses (encoding protein phosphatase 1 regulatory inhibitor subunit 1B) which is downstream of the genes encoding the D1 and D2 dopamine receptors ( and respectively). The role of or BLA neurons in learned and unlearned emotional responses is unknown. Here, we identified that the + and + BLA neuron populations form two parallel pathways for communication with the ventral striatum. These neurons arise from the basal nucleus of the BLA, innervate the entire space of the ventral striatum, and are capable of exciting ventral striatum neurons. Further, through three separate behavioral assays, we found that the + and + parallel pathways bidirectionally influence both learned and unlearned emotional states when they are activated or suppressed, and do so depending upon where they synapse in the ventral striatum - with unique contributions of + and + circuitry on negative emotional states. Overall, these results contribute to a model whereby parallel, genetically-distinct BLA to ventral striatum circuits inform emotional states in a projection-specific manner.
PubMed: 38948716
DOI: 10.1101/2024.06.19.599749 -
BioRxiv : the Preprint Server For... Jun 2024Uveal coloboma, a developmental eye defect, is caused by failed development of the optic fissure, a ventral structure in the optic stalk and cup where axons exit the eye...
BACKGROUND
Uveal coloboma, a developmental eye defect, is caused by failed development of the optic fissure, a ventral structure in the optic stalk and cup where axons exit the eye and vasculature enters. The Hedgehog (Hh) signaling pathway regulates optic fissure development: loss-of-function mutations in the Hh receptor produce overactive Hh signaling and can result in coloboma. We previously proposed a model where overactive Hh signaling disrupts optic fissure formation by upregulating transcriptional targets acting both cell- and non-cell-autonomously. Here, we examine the Netrin family of secreted ligands as candidate Hh target genes.
RESULTS
We find multiple Netrin ligands upregulated in the zebrafish mutant during optic fissure development. Using a gain-of-function approach to overexpress Netrin in a spatiotemporally specific manner, we find that or overexpression is sufficient to cause coloboma and disrupt wild-type optic fissure formation. We used loss-of-function alleles, CRISPR/Cas9 mutagenesis, and morpholino knockdown to test if loss of Netrin can rescue coloboma in the mutant: loss of genes does not rescue the mutant phenotype.
CONCLUSION
These results suggest that Netrin is sufficient but not required to disrupt optic fissure formation downstream of overactive Hh signaling in the mutant.
KEY FINDINGS
Overactive Hedgehog signaling in the mutant causes increased netrin expression Spatiotemporally specific overexpression of netrin1a and netrin1b can cause coloboma Spatiotemporally specific overexpression of netrin1a can disrupt optic fissure and stalk formation as well as optic stalk cell morphology, similar to the mutant Loss of netrin ligands in the mutant does not rescue the phenotype.
PubMed: 38948711
DOI: 10.1101/2024.06.18.599642 -
Journal of Family Medicine and Primary... May 2024The Coronavirus disease 2019 (COVID-19) pandemic has been the biggest threat to humankind during the last 3 years. It has caused the loss of more than 6.9 million...
INTRODUCTION
The Coronavirus disease 2019 (COVID-19) pandemic has been the biggest threat to humankind during the last 3 years. It has caused the loss of more than 6.9 million precious lives across the world. The only method by which the massacre could be stopped was by mass vaccination or mass immunization. The patients suffering from autoimmune rheumatic disorders (AIRDs) and treated with immunosuppressants were the high-priority candidates for vaccination. However, the data regarding the efficacy of COVID-19 vaccines in this group of patients are very less. Hence, this study was planned to study the immunogenicity of Covishield in patients with AIRDs attending the rheumatology OPD at DMCH, Ludhiana.
MATERIALS AND METHODS
It was a prospective cohort study and was planned by the Department of Biochemistry and Department of Clinical Immunology and Rheumatology at Dayanand Medical College and Hospital, Ludhiana. Fifty patients with AIRDs attending the DMCH rheumatology OPD and 52 age and sex-matched healthy controls who had received two doses of Covishield vaccine were included in this study. Patients having any other immunosuppressive conditions like uncontrolled diabetes, hepatitis, malignancy or HIV were excluded. Patients who had suffered from previous laboratory-confirmed COVID-19 infection (by RT-PCR) were also excluded. Blood samples were collected following all aseptic precautions from patients and controls on the 28 day after administration of a second dose of Covishield vaccine and total antibodies to the severe acute respiratory syndrome coronavirus 2 spike (S) protein receptor binding domain was measured using Elecsys Anti-SARS-CoV-2 S kit from Roche.
RESULTS
It was observed that no significant difference was there in antibody titre between cases and controls (6213 ± 4418 vs. 8331 ± 7979, = 0.1022). It was also observed that no statistically significant difference in antibody titre in cases without prednisolone and those taking treatment with prednisolone was found ( = 0.7058). A similar observation was found in terms of methotrexate also ( = 0.457). No significant difference in antibody titres was there when compared with controls (for prednisolone, = 0.169, for methotrexate, = 0.078). We found that only the patients receiving mycophenolate mofetil showed a statistically significant decrease in antibody titre in comparison to healthy controls ( = 0.03). Our study showed no statistically significant difference in antibody titres between patients suffering from different AIRDs.
CONCLUSION
Our study supplements the fact that patients with AIRDs in India can receive Covishield as the primary vaccine against COVID-19 without concerns regarding decreased immunogenicity or increased adverse effects.
PubMed: 38948615
DOI: 10.4103/jfmpc.jfmpc_1021_23 -
Frontiers in Endocrinology 2024
Editorial: Underlying molecular interconnections of the estrogen receptor alpha and associated factors involved in breast cancer development: the way to new therapeutic approaches, volume II.
PubMed: 38948523
DOI: 10.3389/fendo.2024.1427468 -
Frontiers in Endocrinology 2024Lu-oxodotreotide peptide receptor therapy (LuPRRT) is an efficient treatment for midgut neuroendocrine tumors (NETs) of variable radiological response. Several clinical,...
BACKGROUND
Lu-oxodotreotide peptide receptor therapy (LuPRRT) is an efficient treatment for midgut neuroendocrine tumors (NETs) of variable radiological response. Several clinical, biological, and imaging parameters may be used to establish a relative disease prognosis but none is able to predict early efficacy or toxicities. We investigated expression levels for mRNA and miRNA involved in radiosensitivity and tumor progression searching for correlations related to patient outcome during LuPRRT therapy.
METHODS
Thirty-five patients received LuPRRT for G1/G2 midgut NETs between May 2019 and September 2021. Peripheral blood samples were collected prior to irradiation, before and 48 h after the second and the fourth LuPRRT, and at 6-month follow-up. Multiple regression analyses and Pearson correlations were performed to identify the miRNA/mRNA signature that will best predict response to LuPRRT.
RESULTS
Focusing on four mRNAs and three miRNAs, we identified a miRNA/mRNA signature enabling the early identification of responders to LuPRRT with significant reduced miRNA/mRNA expression after the first LuPRRT administration for patients with progressive disease at 1 year ( < 0.001). The relevance of this signature was reinforced by studying its evolution up to 6 months post-LuPRRT. Moreover, nadir absolute lymphocyte count within the first 2 months after the first LuPRRT administration was significantly related to low miRNA/mRNA expression level ( < 0.05) for patients with progressive disease.
CONCLUSION
We present a pilot study exploring a miRNA/mRNA signature that correlates with early hematologic toxicity and therapeutic response 12 months following LuPRRT. This signature will be tested prospectively in a larger series of patients.
PubMed: 38948517
DOI: 10.3389/fendo.2024.1385079