Biology subjects

Rossi, A. G.

Publications and source records attributed to Rossi, A. G..

6 recordsLinked to original sources

bioRxiv2025

Fluorescence Characterization of Extracellular Vesicles using Single-Molecule Confocal Microscopy

Extracellular vesicles (EVs) are small, membrane-bound particles released by cells into the extracellular environment. They play a pivotal role in cell communication and have recently gained prominence as biomarkers. However, their low abundance and high heterogeneity challenges their accurate characterization using conventional approaches. To enable the specific detection of individual EVs, we coupled EV-specific antibodies labeled with two different fluorophores with fast-flow microfluidics and single-molecule confocal microscopy. This allowed us to determine the concentration of EVs down to femtomolar levels ([~]107 EVs/mL), and we demonstrated the approachs capacity to detect EVs even in the presence of other lipid vesicles. We highlighted the ability to quantify EVs in serum and plasma samples, without the need for purification. Furthermore, we compared the yield of EVs extracted from both serum and plasma using ultracentrifugation and various size exclusion chromatography approaches. Overall, our method offers a highly specific, sensitive and easy-to-use solution for characterizing EVs from different sources.

biophysics↗

bioRxiv2024

Development of a pipeline to evaluate YAP-TEAD inhibitor potential - direct TEAD inhibition represses cancerous growth in a NF2-mutant model of mesothelioma

As the core, tumorigenic downstream effectors of the Hippo signalling pathway, YAP/TAZ and the TEAD family of transcription factors represent attractive targets for drug discovery efforts within cancer research. This is particularly true within the context of pleural mesothelioma, in which there are many recent preclinical developments and clinical trials evaluating the efficacy of TEAD inhibitors. The range of inhibitors have shown great promise, but comparisons of their performances are so far limited. Here we develop a high content pipeline that enables a comparative analysis of currently developed YAP/TAZ-TEAD inhibitors. We take advantage of isogenic cellular models that enable us to examine inhibitor specificity. We identify genetic compensation of the Hippo pathway transcriptional module, with implications for therapeutic targeting, and implement Cell Painting to develop a detailed morphological profiling pipeline that enables further characterisation, quantification, and analysis of off-target effects. Our pipeline is scalable and allows us to establish specificity and comparative potency within cancer relevant assays in a clinically relevant cellular model.

cancer biology↗

bioRxiv2021

Nucleated Fish Erythrocyte Extracellular Traps (FEETs) release is an evolutionary conserved immune defence process

Fish erythrocytes remain nucleated for their life-span, unlike mammalian erythrocytes which undergo enucleation. Asides transportation of oxygen, fish erythrocytes are capable of several immune defence processes. Nucleated fish erythrocytes represent prime candidates for carrying out ETotic responses. ETosis is an evolutionary conserved innate immune defence process found in both vertebrates and invertebrates, which involves the extrusion of DNA studded with antimicrobial proteins into the extracellular space serving to trap and kill microorganisms. In this report, we demonstrate that fish erythrocytes isolated from Danio rerio (zebrafish) produce ETotic-like responses when exposed to chemical and physiological stimuli. Furthermore, we found Salmo salar (Atlantic salmon) erythrocytes produce similar ETotic responses. We have termed these ET-like formations Fish Erythrocyte Extracellular Traps (FEETs). Interestingly, we discovered that mammalian inducers of NETosis, such as the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate and the calcium ionophore ionomycin, induced FEETs. Moreover, we found that FEETs are dependent upon activation of PKC and generation of mitochondrial reactive oxygen species. Thus, this brief report represents the first demonstration that fish erythrocytes can exhibit ETotic-like responses, unveiling a previously unknown function of nucleated erythrocytes, which sheds new light on the innate immune arsenal of erythrocytes.

evolutionary biology↗

bioRxiv2021

Macrophages stimulate epicardial VEGFaa to trigger cardiomyocyte proliferation in larval zebrafish heart regeneration

Cardiac injury induces a sustained macrophage response in both zebrafish and mammals. Macrophages perform a range of both beneficial and detrimental functions during mammalian cardiac repair, yet their precise roles in zebrafish cardiac regeneration are not fully understood. Here we characterise cardiac regeneration in the rapidly regenerating larval zebrafish laser injury model and use macrophage ablation and macrophage-null irf8 mutants to define the role of macrophages in key stages of regeneration. Live heartbeat-synchronised imaging and RNA sequencing revealed an early proinflammatory phase, marked by tnfa+ macrophages, which then resolved to an anti-inflammatory, profibrotic phase. Macrophages were required for cardiomyocyte proliferation but not for functional or structural recovery following injury. Importantly, we found that macrophages are specifically recruited to the epicardial-myocardial niche, triggering the expansion of the epicardium which upregulates VEGFaa expression to induce cardiomyocyte proliferation. Hence, revealing a novel mechanism by which macrophages facilitate cardiac regeneration.

immunology↗

bioRxiv2021

Prostaglandin E2 directly inhibits the conversion of inducible regulatory T cells through EP2 and EP4 receptors via antagonizing TGF-β signalling

Background and PurposeRegulatory T (Treg) cells are essential for control of inflammatory processes by suppressing Th1 and Th17 cells. The bioactive lipid mediator prostaglandin E2 (PGE2) promotes inflammatory Th1 and Th17 cells and exacerbates T cell-mediated autoimmune diseases. However, the actions of PGE2 on the development and function of Treg cells, particularly under inflammatory conditions, are debated. In this study, we examined whether PGE2 had a direct action on T cells to modulate de novo differentiation of Treg cells. Experimental ApproachWe employed an in vitro T cell culture system of TGF-{beta}-dependent Treg induction from naive T cells. PGE2 and selective agonists for its receptors, and other small molecular inhibitors were used. Mice with specific lack of EP4 receptors in T cells were used to assess Treg cell differentiation in vivo. Human peripheral blood T cells from healthy individuals were used to induce differentiation of inducible Treg cells. Key ResultsTGF-{beta}-induced Foxp3 expression and Treg cell differentiation in vitro was markedly inhibited by PGE2, which was due to interrupting TGF-{beta} signalling. EP2 or EP4 agonism mimicked suppression of Foxp3 expression in WT T cells, but not in T cells deficient in EP2 or EP4, respectively. Moreover, deficiency of EP4 in T cells impaired iTreg cell differentiation in vivo. PGE2 also appeared to inhibit the conversion of human iTreg cells. Conclusion and ImplicationsOur results show a direct, negative regulation of iTreg cell differentiation by PGE2, highlighting the potential for selectively targeting the PGE2-EP2/EP4 pathway to control T cell-mediated inflammation. What is already knownPGE2 promotes inflammatory Th1 and Th17 cells and facilitates T cell-mediated immune inflammation, but the action of PGE2 on Treg cells is debated. What does this study addPGE2 directly acts on T cells to inhibit inducible Treg cell differentiation in vitro and in vivo through its receptors EP2 and EP4 and by antagonising TGF-{beta} signalling. What is the clinical significanceTherapeutically blocking the EP4 receptor may be beneficial for management of T cell-mediated autoimmune inflammation.

immunology↗

bioRxiv2019

Eosinophil deficiency promotes aberrant repair and adverse remodelling following acute myocardial infarction

BackgroundEosinophil count predicts outcome following myocardial infarction (MI) and eosinophils regulate tissue repair and regeneration in extra-cardiac settings.\n\nObjectivesTo investigate the role of eosinophils in regulating inflammation, repair and remodelling following MI.\n\nMethodsBlood eosinophil count was assessed in 732 patients undergoing primary percutaneous coronary intervention for ST-segment elevation MI (STEMI). Experimental MI was induced in wild-type (WT) and eosinophil-depleted mice ({Delta}dblGATA or anti-Siglec F antibody treated). Cardiac function was characterised by high-resolution ultrasound and immune cell infiltration by flow cytometry of infarct digests.\n\nResultsBlood eosinophil count declined in the hours following STEMI in patients and following MI induction in mice. Eosinophils were subsequently identified in the myocardium of patients and mice. Genetic eosinophil depletion in mice increased LV dilatation (end-systolic area: 29.0{+/-}2.2cm2 v 21.6{+/-}1.6cm2; p=0.02) and reduced ejection fraction (22.0{+/-}3.6% v 34.3{+/-}4.0% in p=0.04,) in {Delta}dblGATA v WT following MI (n=8-9/group), an outcome reproduced by pharmacological depletion. {Delta}dblGATA mice had increased scar size with disrupted collagen deposition and altered expression of the collagen cross-linking genes plod2 and lox. CD206+pro-repair macrophages were less prevalent in the infarct zone of {Delta}dblGATA mice (27.0{+/-}2.3% v WT: 39.2.4{+/-}3.5%; p=0.01, n=9-11/group) but were restored by replenishment with bone marrow-derived eosinophils. Anti-inflammatory cytokine concentrations were reduced in {Delta}dblGATA mice and IL-4 complex administration 24h after MI rescued adverse remodelling.\n\nConclusionsEosinophils are recruited to the heart following MI and are required for effective repair and to prevent adverse remodelling. IL-4 therapy has potential to limit detrimental outcomes when eosinophil availability is low.\n\nHIGHLIGHTSA drop in eosinophil blood count is associated with recruitment of eosinophils to the heart during repair following clinical and experimental myocardial infarction.\n\nGenetic & pharmacological eosinophil depletion leads to increased adverse remodelling in experimental MI.\n\nEosinophils are required for aquisition of an anti-inflammatory macrophage phenotype and a shift to resolution of inflammation during infarct repair.\n\nInterleukin-4 therapy is able to rescue the adverse remodelling phenotype in conditions of eosinophil deficiency.\n\nCondensed abstractIn ST-segment elevation myocardial infarction (STEMI) of both patients and mice, there was a decline in blood eosinophil count, with activated eosinophils recruited to the infarct zone. Eosinophil deficiency resulted in attenuated anti-inflammatory pro-repair macrophage polarization, enhanced myocardial inflammation, increased scar size and deterioration of myocardial structure and function. Adverse cardiac remodelling in the setting of eosinophil deficiency was prevented by IL-4 therapy.

immunology↗