Biology subjects

Da Silva, J.

Publications and source records attributed to Da Silva, J..

6 recordsLinked to original sources

bioRxiv2025

Blockade of mTOR ameliorates IgA Nephropathy by correcting CD89 and CD71 dysfunctions in humanized mice

Background and hypothesisIgA nephropathy (IgAN) is the most common glomerulonephritis and major cause of renal failure worldwide. Pathogenesis involves galactose deficient IgA1-immune complexes containing soluble IgA Fc receptor (sCD89). Both IgA1 and sCD89 bind independently to transferrin receptor (TfR1, CD71), a mesangial IgA1 receptor. Here, we hypothesize that sCD89 plays a pathogenic role in IgAN by driving a tri-partite IgA1-sCD89-CD71 complex inducing activation in situ of the mTOR (mammalian target of rapamycin) signaling pathway in mesangial cells and contributing to disease progression. mTOR inhibition may disrupt this pathogenic axis, reducing IgA1 and sCD89 deposits, modulating CD71 expression, and alleviating disease manifestations. Here, we investigated the role of sCD89 and a mTOR inhibitor using humanized mouse models of IgAN expressing CD89 and/or IgA1. MethodsSingle cell and RNAseq were obtained from IgAN public dataset and immunostaining from childhood IgAN (cIgAN) biopsies. Human mesangial cell (HMC) stimulation by recombinant sCD89 (rsCD89) was followed by western blot analysis. Pre-clinical assays with mTOR inhibitor (Everolimus) by oral gavage were performed using young 1KI mice injected with rsCD89 for 25 days (preventive protocol) and adult 1KICD89Tg mice (treated protocol) for 75 days. Proteinuria, renal function, and circulating immune-complexes (CIC) were analysed and kidneys harvested for histology. Results: RNAseq revealed increased TfR1 and mTOR mesangial cell expression in IgAN patients. TfR1 upregulation was confirmed in cIgAN biopsies. sCD89 stimulation induced HMC TfR1 expression and phosphorylation of mTOR, Akt and p70S6K1. Everolimus treatment prevented or reverted mesangial IgA1 and C3 deposits but also decreased mesangial TfR1 and cell proliferation. Everolimus impaired levels of sCD89- and IgA-CIC, proteinuria, as well as renal function. ConclusionThese findings highlight the critical role of the sCD89-TfR1-mTOR axis in IgAN pathogenesis and support the use of mTOR inhibitors as a novel therapeutic approach. This approach could significantly improve outcomes by slowing disease progression and minimizing the systemic toxic effects of current immunosuppressive therapies. This is particularly crucial for pediatric patients, where the only approved treatment - steroids - has severe side effects, including detrimental impacts on bone health and growth.

immunology↗

bioRxiv2024

Temporal and anteriorly positioned mitotic zones drive asymmetric microtubule patterns needed for Left-Right Organizer development.

Cellular proliferation is vital for tissue development, including the Left-Right Organizer (LRO), a transient organ critical for establishing the vertebrate LR body plan. This study investigates cell redistribution and the role of specific progenitor cells in LRO formation, focusing on cell lineage and behavior. Using zebrafish as a model, we mapped all mitotic events in Kupffers Vesicle (KV), revealing an FGF-dependent, anteriorly enriched mitotic pattern. With a KV-specific fluorescent microtubule (MT) line, we observed that mitotic spindles align along the KVs longest axis until the rosette stage, spindles that form after spin, and are excluded from KV. Early aligned spindles assemble cytokinetic bridges that point MT bundles toward a tight junction where a rosette will initially form. Post-abscission, repurposed MT bundles remain targeted at the rosette center, facilitating actin recruitment. Additional cells, both cytokinetic and non-cytokinetic, are incorporated into the rosette, repurposing or assembling MT bundles before actin recruitment. These findings show that initial divisions are crucial for rosette assembly, MT patterning, and actin remodeling during KV development.

developmental biology↗

bioRxiv2022

Rab8-, Rab11-, and Rab35-dependent mechanisms coordinating lumen and cilia formation during Left-Right Organizer development

An essential process during Danio rerios left-right organizer (Kupffers Vesicle, KV) development is for the majority of developing KV cells to form a motile cilium that extend into the KV lumen. Left-right beating of motile cilia within the KV lumen directs fluid flow to establishment the embryos left-right axis. However, when KV cells start to form cilia and how cilia formation is coordinated with KV lumen formation has not been examined. We identified that nascent KV cells form cilia at their centrosomes at random intracellular positions that then move towards a forming apical membrane containing cystic fibrosis transmembrane conductance regulator (CFTR). Using optogenetic clustering approaches, we found that Rab35 positive membranes recruit Rab11 to modulate CFTR delivery to the apical membrane, which is required for lumen opening, and subsequent cilia extension into the lumenal cavity. Once the intracellular cilia reach the CFTR positive apical membrane, Arl13b-positive cilia extend and elongate in a Rab8 dependent manner into the forming lumen once the lumen reaches an area of 300 m2. These studies demonstrate the need to acutely coordinate Rab8, Rab11, and Rab35-mediated membrane trafficking events to ensure appropriate timing in lumen and cilia formation during KV development.

cell biology↗

bioRxiv2022

Lipid Droplets Fuel Small Extracellular Vesicle Biogenesis

Despite an increasing gain of knowledge regarding small extracellular vesicle (sEV) composition and functions in cell-cell communication, the mechanism behind their biogenesis remains unclear. Here, we revealed for the first time that the sEV biogenesis and release into the microenvironment are tightly connected with another important organelle: Lipid Droplets (LD). We have observed this correlation using different human cancer cell lines as well as patient-derived colorectal cancer stem cells (CR-CSCs). Our results showed that the use of external stimuli such as radiation, pH, hypoxia, or lipid interfering drugs, known to affect the LD content, had a similar effect in terms of sEV secretion. Additional validations were brought using multiple omics data, at the mRNA and protein levels. Altogether, the possibility to fine-tune sEV biogenesis by targeting LDs, could have a massive impact on the amount, the cargos and the properties of those sEVs, paving the way for new clinical perspectives. Significance Statement

cancer biology↗

bioRxiv2021

High genetic barrier to escape from human polyclonal SARS-CoV-2 neutralizing antibodies

The number and variability of the neutralizing epitopes targeted by polyclonal antibodies in SARS-CoV-2 convalescent and vaccinated individuals are key determinants of neutralization breadth and, consequently, the genetic barrier to viral escape. Using chimeric viruses and antibody-selected viral mutants, we show that multiple neutralizing epitopes, within and outside the viral receptor binding domain (RBD), are variably targeted by polyclonal plasma antibodies and coincide with sequences that are enriched for diversity in natural SARS-CoV-2 populations. By combining plasma-selected spike substitutions, we generated synthetic polymutant spike proteins that resisted polyclonal antibody neutralization to a similar degree as currently circulating variants of concern (VOC). Importantly, by aggregating VOC-associated and plasma-selected spike substitutions into a single polymutant spike protein, we show that 20 naturally occurring mutations in SARS-CoV-2 spike are sufficient to confer near-complete resistance to the polyclonal neutralizing antibodies generated by convalescents and mRNA vaccine recipients. Strikingly however, plasma from individuals who had been infected and subsequently received mRNA vaccination, neutralized this highly resistant SARS-CoV-2 polymutant, and also neutralized diverse sarbecoviruses. Thus, optimally elicited human polyclonal antibodies against SARS-CoV-2 should be resilient to substantial future SARS-CoV-2 variation and may confer protection against future sarbecovirus pandemics.

microbiology↗

bioRxiv2021

Development of potency, breadth and resilience to viral escape mutations in SARS-CoV-2 neutralizing antibodies

Antibodies elicited in response to infection undergo somatic mutation in germinal centers that can result in higher affinity for the cognate antigen. To determine the effects of somatic mutation on the properties of SARS-CoV-2 spike receptor-binding domain (RBD)-specific antibodies, we analyzed six independent antibody lineages. As well as increased neutralization potency, antibody evolution changed pathways for acquisition of resistance and, in some cases, restricted the range of neutralization escape options. For some antibodies, maturation apparently imposed a requirement for multiple spike mutations to enable escape. For certain antibody lineages, maturation enabled neutralization of circulating SARS-CoV-2 variants of concern and heterologous sarbecoviruses. Antibody-antigen structures revealed that these properties resulted from substitutions that allowed additional variability at the interface with the RBD. These findings suggest that increasing antibody diversity through prolonged or repeated antigen exposure may improve protection against diversifying SARS-CoV-2 populations, and perhaps against other pandemic threat coronaviruses.

microbiology↗