Biology subjects

Ottaviani, A.

Publications and source records attributed to Ottaviani, A..

6 recordsLinked to original sources

bioRxiv2022

From genome wide SNPs to genomic islands of differentiation: the quest for species diagnostic markers in two scleractinian corals, Pocillopora and Porites

Coral reefs are of paramount importance in marine ecosystems, where they provide support for a large part of the biodiversity. Being quite sensitive to global changes, they are therefore the prime targets for biodiversity conservation policies. However, such conservation goals require accurate species identification, which are notoriously difficult to get in these highly morphologically variable organisms, rich in cryptic species. There is an acute need for easy-to-use and resolutive species diagnostic molecular markers. The present study builds on the huge sequencing effort developed during the TARA Pacific expedition to develop a genotyping strategy to assign coral samples to the correct species within two coral genera (Porites and Pocillopora). For this purpose, we developed a technique that we called "Divergent Fragment" based on the sequencing of a less than 2kb long diagnostic genomic fragment determined from the metagenomic data of a subset of the corals collected. This method has proven to be rapid, resolvable and cost-effective. Sequencing of PCR fragments nested along the species diagnostic fragment allowed us to assign 232 individuals of the genus Pocillopora and 247 individuals of the genus Porites to previously identified independent genetic lineages (i.e. species). This genotyping method will allow to fully analyze the coral samples collected across the Pacific during the Tara Pacific expedition and opens technological perspectives in the field of population genomics-guided conservation.

evolutionary biology↗

bioRxiv2022

Design, implementation, and functional validation of a new generation of microneedle 3D high-density CMOS multi-electrode array for brain tissue and spheroids

In the last decades, planar multi-electrode arrays (MEAs) have been widely used to record activity from in vitro neuronal cell cultures and tissue slices. Though successful, this technique bears some limitations, particularly relevant when applied to three-dimensional (3D) tissue, such as brain slices, spheroids or organoids. For example, planar MEAs signals are informative on just one side of a 3D-organized structure. This limits the interpretation of the results in terms of network functions in a complex structured and hyperconnected brain tissue. Moreover, the side in contact with the MEAs often shows lower oxygenation rates and related vitality issues. To overcome these problems, we empowered a CMOS high-density multi-electrode array (HD-MEA) with thousands of microneedles (needles) of 65-90 m height, able to penetrate and record in-tissue signals, providing for the first time a 3D HD-MEA chip. We propose a CMOS-compatible fabrication process to produce arrays of needles of different widths mounted on large pedestals to create microchannels underneath the tissue. By using cerebellar and cortico-hippocampal slices as a model, we show that the needles efficiently penetrate the 3D tissue while the microchannels allow the flowing of maintenance solutions to increase tissue vitality in the recording sites. These improvements are reflected by the increase in electrodes sensing capabilities, the number of sampled neuronal units (compared to matched planar technology), and the efficiency of compound effects. Importantly, each electrode can also be used to stimulate the tissue with optimal efficiency due to the 3D structure. Furthermore, we demonstrate how the 3D HD-MEA can efficiently penetrate and get outstanding signals from in vitro 3D cellular models as brain spheroids. In conclusion, we describe a new recording device characterized by the highest spatio-temporal resolution reported for a 3D MEA and significant improvements in the quality of recordings, with a high signal-to-noise ratio and improved tissue vitality. The applications of this game-changing technique are countless, opening unprecedented possibilities in the neuroscience field and beyond.

neuroscience↗

bioRxiv2021

ANTIBODY-INDEPENDENT ANTITUMOR EFFECTS OF CD32A-CHIMERIC RECEPTOR T CELLS: IMPLICATIONS FOR BREAST CANCER PROGNOSIS AND TREATMENT.

Fc{gamma} RIIA (CD32A) and their ligands, including the immunoglobulin Fc fragment and pentraxins, are key players in a variety of innate immune responses. Still unclear is whether additional ligands of CD32A do exist. The objective of this study is to demonstrate that CD32A-chimeric receptor (CR) can be utilized for the identification of CD32A cell surface ligand(s). Among fifteen cancer cell lines tested, CD32A-CR T cells recognized three of breast cancer (BC) including the MDA-MB-468 and one colorectal carcinoma (HT29) in the absence of targeting antibodies. Conjugation of sensitive BC cells with CD32A-CR T cells induced CD32A polarization and down-regulation, CD107 release, and mutual cell elimination in vitro. Conversely, normal fibroblasts and myoblasts were not affected while normal HUVEC cells promoted CD32A down-regulation. CD32A-CR T cell activity was not inhibited by human IgGs or human serum, but; it was rather enhanced by cetuximab antibody. RNAseq analysis of sensitive vs resistant BC cells identified a fingerprint of 42 genes predicting the sensitivity of BC cells to CD32A-CR T cells and their association with favorable prognostic significance in advanced BC patients. Our data also identify ICAM 1 as a major regulator of CD32A-CR T cell-mediated cytotoxicity. Finally, CD32A-CR T cell administration protected immunodeficient mice from subcutaneous growth of MDA-MB-468 cells in the absence of tumor-specific antibodies. These data indicate that CD32A-CR can be utilized for the identification of (1) cell surface CD32A ligand(s); (2) rational therapeutic strategies to target BC; and (3) novel transcriptomic signatures prognostically relevant for advanced BC patients.

immunology↗

bioRxiv2020

Cerebellar Golgi cell models predict dendritic processing and mechanisms of synaptic plasticity

The Golgi cells are the main inhibitory interneurons of the cerebellar granular layer. Although recent works have highlighted the complexity of their dendritic organization and synaptic inputs, the mechanisms through which these neurons integrate complex input patterns remained unknown. Here we have used 8 detailed morphological reconstructions to develop multicompartmental models of Golgi cells, in which Na, Ca, and K channels were distributed along dendrites, soma, axonal initial segment and axon. The models faithfully reproduced a rich pattern of electrophysiological and pharmacological properties and predicted the operating mechanisms of these neurons. Basal dendrites turned out to be more tightly electrically coupled to the axon initial segment than apical dendrites. During synaptic transmission, parallel fibers caused slow Ca-dependent depolarizations in apical dendrites that boosted the axon initial segment encoder and Na-spike backpropagation into basal dendrites, while inhibitory synapses effectively shunted backpropagating currents. This oriented dendritic processing set up a coincidence detector controlling voltage-dependent NMDA receptor unblock in basal dendrites, which, by regulating local calcium influx, may provide the basis for spike-timing dependent plasticity anticipated by theory. Author SummaryThe Golgi cells are the main inhibitory interneurons of the cerebellum granular layer and play a fundamental role in controlling cerebellar processing. However, it was unclear how spikes are processed in the dendrites by specific sets of ionic channels and how they might contribute to integrate synaptic inputs and plasticity. Here we have developed detailed multicompartmental models of Golgi cells that faithfully reproduced a large set of experimental findings and revealed the nature of signal interchange between dendrites and axo-somatic compartments. A main prediction of the models is that synaptic activation of apical dendrites can effectively trigger spike generation in the axonal initial segment followed by rapid spike backpropagation into basal dendrites. Here, incoming mossy fiber inputs and backpropagating spikes regulate the voltage-dependent unblock of NMDA channels and the induction of spike timing-dependent plasticity (STDP). STDP, which was predicted by theory, may therefore be controlled by contextual information provided by parallel fibers and integrated in apical dendrites, supporting the view that spike timing is fundamental to control synaptic plasticity at the cerebellar input stage.

neuroscience↗

bioRxiv2019

CD16-158-VALINE CHIMERIC RECEPTOR T CELLS OVERCOME THE RESISTANCE OF KRAS-MUTATED COLORECTAL CARCINOMA CELLS TO CETUXIMAB

KRAS mutation hinders the therapeutic efficacy of epidermal-growth-factor-receptor (EGFR) mAb (cetuximab and panitumumab)-based immunotherapy of EGFR+ cancers. Although, cetuximab controls KRAS-mutated cancer cell growth in vitro utilizing a NK cell-mediated antibody-dependent-cellular-cytotoxicity-(ADCC) mechanism, KRAS-mutated colorectal carcinoma (CRC) cells can still escape NK cell immunosurveillance. To overcome this limitation, we used cetuximab and panitumumab to redirect Fc{gamma} chimeric receptor (CR) T cells against KRAS-mutated HCT116 CRC cells. We compared 4 polymorphic Fc{gamma}-CR constructs including CD16158F-CR, CD16158V-CR, CD32131H-CR, and CD32131R-CR which were transduced into T cells utilizing retroviral transduction. Percentages of transduced T cells expressing CD32131H-CR (83.5{+/-}9.5) and CD32131R-CR (77.7.{+/-}13.2) were significantly higher than those expressing with CD16158F-CR (30.3{+/-}10.2) and CD16158V-CR (51.7{+/-}13.7) (p<0.003). CD32131R-CR T cells specifically bound soluble cetuximab and panitumumab. However, only CD16158V-CR T cells released significantly higher levels of interferon gamma (IFN{gamma}=1145.5 pg/ml {+/-}16.5 pg/ml, p<0.001) and tumor necrosis factor alpha (TNF=614 pg/ml {+/-} 21 pg/ml, p<0.001) than non-transduced T cells when incubated with KRAS-mutated HCT116 cells opsonized with cetuximab. Only CD16158V-CR T cells combined with cetuximab controlled the growth of HCT116 cells subcutaneously engrafted in CB17-SCID mice. These results suggest that CD16158V-CR T cells combined with cetuximab represent useful reagents to develop an effective immunotherapy of EGFR+KRAS-mutated cancer.

immunology↗

bioRxiv2019

ELIMINATION OF EGFR-OVEREXPRESSING CANCER CELLS BY CD32 CHIMERIC RECEPTOR T CELLS IN COMBINATION WITH CETUXIMAB OR PANITUMUMAB

Cetuximab and panitumumab bind the human epidermal growth factor receptor (EGFR). While the chimeric cetuximab (IgG1) triggers antibody-dependent-cellular-cytotoxicity (ADCC) of EGFR positive target cells, panitumumab (a human IgG2) does not. The inability of panitumumab to trigger ADCC reflects a poor binding affinity of human IgG2 Fc for the Fc{gamma}RIII (CD16) on NK cells. However, both human IgG1 and IgG2 bind the Fc{gamma}RII (CD32) to a similar extent. Here, we have compared the ability of T cells, engineered with a novel low-affinity CD32131R -chimeric receptor (CR), and those engineered with the low-affinity CD16158F-CR T cells in eliminating EGFR positive epithelial cancer cells (ECCs) in combination with cetuximab or panitumumab. Following T cell transduction, the percentage of CD32131R-CR T cells was (74{+/-}10) significantly higher than that of CD16158F-CR T cells (46{+/-}15). Only CD32131R-CR T cells bound panitumumab. CD32131R-CR T cells combined with the mAb 8.26 (anti-CD32) and CD16158F-CR T cells combined with the mAb 3g8 (anti-CD16) eliminated colorectal carcinoma (CRC), HCT116Fc{gamma}R+ cells, in a reverse ADCC assay in vitro. Cross-linking of CD32131R-CR on T cells by cetuximab or panitumumab and CD16158F-CR T cells by cetuximab induced elimination of triple negative breast cancer (TNBC) MDA-MB-468 cells, and secretion of IFN gamma (IFN{gamma}) and tumor necrosis factor alpha (TNF). Neither cetuximab nor panitumumab induced Fc{gamma}-CR T anti-tumor activity against KRAS-mutated HCT116, non-small-cell-lung-cancer, A549 and TNBC, MDA-MB-231 cells. ADCC of Fc{gamma}-CR T cells was significantly associated with the over-expression of EGFR on ECCs. In conclusion, CD32131R-CR T cells are efficiently redirected by cetuximab or panitumumab against BC cells overexpressing EGFR.\n\nArticle categoryTumor Immunology and Microenvironment\n\nNovelty and ImpactMonoclonal antibody-redirected Fc{gamma}-CR T cell immunotherapy represents a promising approach in the fight against cancer. Here, we expand the application of this methodology to TNBC overexpressing the EGFR utilizing a novel CD32A131R-CR in combination with anti-EGFR mAbs. Our study supports the use of CD32A131R-CR T cells combined with panitumumab or cetuximab for targeting TNBC cells overexpressing the EGFR. Our results may be utilized as a platform for the rational design of therapies targeting TNBC overexpressing EGFR.

immunology↗