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Partial correction of immunodeficiency by lentiviral vector gene therapy in mouse models carrying Rag1 hypomorphic mutations
No full textRecombination activating genes (RAG) 1 and 2 defects are the most frequent form of severe combined immunodeficiency (SCID). Patients with residual RAG activity have a spectrum of clinical manifestations ranging from Omenn syndrome to delayed-onset combined immunodeficiency, often associated with granulomas and/or autoimmunity (CID-G/AI). Lentiviral vector (LV) gene therapy (GT) has been proposed as an alternative treatment to the standard hematopoietic stem cell transplant and a clinical trial for RAG1 SCID patients recently started. However, GT in patients with hypomorphic RAG mutations poses additional risks, because of the residual endogenous RAG1 expression and the general state of immune dysregulation and associated inflammation. In this study, we assessed the efficacy of GT in 2 hypomorphic Rag1 murine models (Rag1F971L/F971L and Rag1R972Q/R972Q), exploiting the same LV used in the clinical trial encoding RAG1 under control of the MND promoter.
Starting 6 weeks after transplant, GT-treated mice showed a decrease in proportion of myeloid cells and a concomitant increase of B, T and total white blood cells. However, counts remained lower than in mice transplanted with WT Lin- cells. At euthanasia, we observed a general redistribution of immune subsets in tissues, with the appearance of mature recirculating B cells in the bone marrow. In the thymus, we demonstrated correction of the block at double negative stage, with a modest improvement in the cortical/medullary ratio. Analysis of antigen-specific IgM and IgG serum levels after in vivo challenge showed an amelioration of antibody responses, suggesting that the partial immune correction could confer a clinical benefit. Notably, no overt signs of autoimmunity were detected, with B-cell activating factor decreasing to normal levels and autoantibodies remaining stable after GT. On the other hand, thymic enlargement was frequently observed, although not due to vector integration and insertional mutagenesis.
In conclusion, our work shows that GT could partially alleviate the combined immunodeficiency of hypomorphic RAG1 patients and that extensive efficacy and safety studies with alternative models are required before commencing RAG gene therapy in these highly complex patients
68Ga-PSMA and 68Ga-DOTA-RM2 PET/MRI in Recurrent Prostate Cancer: Diagnostic Performance and Association with Clinical and Histopathological Data
No full textRaw data to reprlicate the analyses conducted in "68Ga-PSMA and 68Ga-DOTA-RM2 PET/MRI in Recurrent Prostate Cancer: Diagnostic Performance and Association with Clinical and Histopathological Data
Tolerogenic IL-10-engineered dendritic cell-based therapy to restore antigen-specific tolerance in T cell mediated diseases
No full textIn the manuscript "Tolerogenic IL-10-engineered dendritic cell-based therapy to restore antigen-specific tolerance in T cell mediated diseases" published in Journal of Autoimmunity on May 22 2023 (doi: 10.1016/j.jaut.2023.103051). We describe the in vitro generation of dendritic cells genetically modified to render them pro-tolerogenic and able to modulate unwanted immune responses to auto-antigens both in vitro and in vivo. Raw data for each main figure published in the manuscript are available in the relative.csv file. Read the Read_me.rtf file for more details and for the accession numbers of NGS data deposited at GEO
Interfering with the ERC1–LL5beta interaction disrupts plasma membrane–associated platforms and affects tumor cell motility
No full textDOI: 10.1371/journal.pone.0287670
Cell migration requires a complex array of molecular events to promote protrusion at the front of motile cells. The scaffold protein LL5beta interacts with the scaffold ERC1, and recruits it at plasma membrane–associated platforms that form at the front of migrating tumor cells. LL5beta and ERC1 proteins support protrusion during migration as shown by the finding that depletion of either endogenous protein impairs tumor cell motility and invasion. In this study we have tested the hypothesis that interfering with the interaction between LL5beta and ERC1 may be used to interfere with the function of the endogenous proteins to inhibit tumor cell migration. For this, we identified ERC1(270-370) and LL5beta(381-510) as minimal fragments required for the direct interaction between the two proteins. The biochemical characterization demonstrated that the specific regions of the two proteins, including predicted intrinsically disordered regions, are implicated in a reversible, high affinity direct heterotypic interaction. NMR spectroscopy further confirmed the disordered nature of the two fragments and also support the occurrence of interaction between them. We tested if the LL5beta protein fragment interferes with the formation of the complex between the two full-length proteins. Coimmunoprecipitation experiments showed that LL5beta(381-510) hampers the formation of the complex in cells. Moreover, expression of either fragment is able to specifically delocalize endogenous ERC1 from the edge of migrating MDA-MB-231 tumor cells. Coimmunoprecipitation experiments show that the ERC1-binding fragment of LL5beta interacts with endogenous ERC1 and interferes with the binding of endogenous ERC1 to full length LL5beta. Expression of LL5beta(381-510) affects tumor cell motility with a reduction in the density of invadopodia and inhibits transwell invasion. These results provide a proof of principle that interfering with heterotypic intermolecular interactions between components of plasma membrane–associated platforms forming at the front of tumor cells may represent a new approach to inhibit cell invasion
Genotoxic effects of base and prime editing in human hematopoietic stem cells
No full textRaw data associated to the manuscript by Fiumara et al. Base and prime editors (BEs and PEs) may provide more precise genetic engineering than nuclease-based approaches because they bypass the dependence on DNA double-strand breaks. However, little is known about their cellular responses and genotoxicity. Here, we compared state-of-the-art BEs and PEs and Cas9 in human hematopoietic stem and progenitor cells with respect to editing efficiency, cytotoxicity, transcriptomic changes and on-target and genome-wide genotoxicity. BEs and PEs induced detrimental transcriptional responses that reduced editing efficiency and hematopoietic repopulation in xenotransplants and also generated DNA double-strand breaks and genotoxic byproducts, including deletions and translocations, at a lower frequency than Cas9. These effects were strongest for cytidine BEs due to suboptimal inhibition of base excision repair and were mitigated by tailoring delivery timing and editor expression through optimized mRNA design. However, BEs altered the mutational landscape of hematopoietic stem and progenitor cells across the genome by increasing the load and relative proportions of nucleotide variants. These findings raise concerns about the genotoxicity of BEs and PEs and warrant further investigation in view of their clinical application
Aryl hydrocarbon receptor activity downstream of IL-10 signaling is required to promote regulatory functions in human dendritic cells
No full textIn the manuscript "Aryl hydrocarbon receptor activity downstream of IL-10 signaling is required to promote regulatory functions in human dendritic cells" published in Cell Reports on March 03, 2023 https://doi.org/10.1016/j.celrep.2023.112193 we describe a novel role for AHR in controlling the establishment of tolerogenic gene expression patterns and functional features down-stream IL-10 signaling in human dendritic cells.
Raw data for each main figure published in the manuscript are available in the relative folder containing a .csv file for each figure panel.
Read the Description.rtf file for more details and for the accession numbers of NGS data deposited at GEO
Role of Machine Learning (ML)-Based Classification Using Conventional 18F-FDG PET Parameters in Predicting Postsurgical Features of Endometrial Cancer Aggressiveness
No full textPurpose: to investigate the preoperative role of ML-based classification using conventional 18F-FDG PET parameters and clinical data in predicting features of EC aggressiveness.
Methods: retrospective study, including 123 EC patients who underwent 18F-FDG PET (2009-2021) for preoperative staging. Maximum standardized uptake value (SUVmax), SUVmean, metabolic tumour volume (MTV), and total lesion glycolysis (TLG) were computed on the primary tumour. Age and BMI were collected. Histotype, myometrial invasion (MI), risk group, lymph-nodal involvement (LN), and p53 expression were retrieved from histology. The population was split into a train and a validation set (80-20%). The train set was used to select relevant parameters (Mann-Whitney U test; ROC analysis) and implement ML models, while the validation set was used to test prediction abilities.
Results: on the validation set, the best accuracies obtained with individual parameters and ML were: 61% (TLG) and 87% (ML) for MI; 71% (SUVmax) and 79% (ML) for risk groups; 72% (TLG) and 83% (ML) for LN; 45% (SUVmax; SUVmean) and 73% (ML) for p53 expression.
Conclusions: ML-based classification using conventional 18F-FDG PET parameters and clinical data demonstrated ability to characterize the investigated features of EC aggressiveness, providing a non-invasive way to support preoperative stratification of EC patients
NGR-TNF Engineering with an N‑Terminal Serine Reduces Degradation and Post-Translational Modifications and Improves Its Tumor-Targeting Activity
No full textRaw data and original images for the article entitled "NGR-TNF Engineering with an N-Terminal Serine Reduces Degradation and Post-Translational Modifications and Improves Its Tumor-Targeting Activity" by Angelo Corti, Anna Maria Gasparri, Angelina Sacchi, Barbara Colombo, Matteo Monieri, Eltjona Rrapaj, Andrés J. M. Ferreri, and Flavio Curnis
Molecular Pharmaceutics 2020 17 (10), 3813-3824, DOI: 10.1021/acs.molpharmaceut.0c0057
A fluorescent reporter model for the visualization and characterization of TDC
No full textTDC are hematopoietic cells that combine dendritic cell (DC) and conventional T cell markers and functional properties. They were identified in secondary lymphoid organs (SLOs) of naïve mice as cells expressing CD11c, major histocompatibility molecule (MHC)-II, and the T cell receptor (TCR) chain. Despite thorough characterization as to their potential functional properties, a physiological role for TDC remains to be determined. Unfortunately, using CD11c as a marker for TDC has the caveat of its upregulation on different cells, including T cells, upon activation. Therefore, a more specific marker is needed to further investigate TDC functions in peripheral organs in different pathological settings. Here we took advantage of Zbtb46-GFP reporter mice to explore the frequency and localization of TDC in peripheral tissues at steady state and upon viral infection. RNA sequencing analysis confirmed that TDC identified with this reporter model have a gene signature that is distinct from conventional T cells and DC. In addition, frequency and total numbers of TDC in the SLOs recapitulated those found using CD11c as a marker. This reporter model allowed for identification of TDC in situ not only in SLOs but also in the liver and lung of naïve mice. Interestingly, we found that TDC numbers in the SLOs increased upon viral infection, suggesting that TDC might play a role during viral infections. In conclusion, we propose a visualization strategy that might shed light on the physiological role of TDC in several pathological contexts, including infection and cancer
Ceruloplasmin-deficient mice show dysregulation of lipid metabolism in liver and adipose tissue reduced by a protein replacement
No full textRaw data, not included in the pubblished supplemental materials, that generated the figures of the manuscript by Raia et al. "Ceruloplasmin-deficient mice show dysregulation of lipid metabolism in liver and adipose tissue reduced by a protein replacement" International Journal of Molecular Sciences, 2023, 24, 1150 (https://doi.org/10.3390/ijms24021150)