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Renato Mantegazza, Chiara Cordiglieri, Alessandra Consonni, Fulvio Baggi Neurology IV Unit, Neuroimmunology and Neuromuscular Disorders, Foundation IRCCS Neurological Institute “Carlo Besta”, Milan, Italy Abstract: Myasthenia gravis (MG) is a chronic autoimmune disease caused by the immune attack of the neuromuscular junction. Antibodies directed against the acetylcholine receptor (AChR) induce receptor degradation, complement cascade activation, and postsynaptic membrane destruction, resulting in functional reduction in AChR availability. Besides anti-AChR antibodies, other autoantibodies are known to play pathogenic roles in MG. The experimental autoimmune MG (EAMG) models have been of great help over the years in understanding the pathophysiological role of specific autoantibodies and T helper lymphocytes and in suggesting new therapies for prevention and modulation of the ongoing disease. EAMG can be induced in mice and rats of susceptible strains that show clinical symptoms mimicking the human disease. EAMG models are helpful for studying both the muscle and the immune compartments to evaluate new treatment perspectives. In this review, we concentrate on recent findings on EAMG models, focusing on their utility and limitations. Keywords: myasthenia gravis, autoimmunity, neuroimmunology, AChR

Interleukin 10 (IL-10) is an antiinflammatory cytokine, but also promotes B cell responses and plays a pathogenic role in systemic lupus erythematosus (SLE). CD4+CCR6+IL-7R+T cells from human tonsils produced IL-10 following stimulation by naïve B cells, which promoted B cell immunoglobulin G (IgG) production. These tonsillar CCR6+B helper T cells were phenotypically distinct from follicular helper T (TFH) cells and lacked BCL6 expression. In peripheral blood, a CCR6+T cell population with similar characteristics was identified, which lacked Th17- and TFH-associated gene signatures and differentiation-associated surface markers. CD4+CCR6+T cells expressing IL-10, but not IL-17, were also detectable in the spleens of cytokine reporter mice. They provided help for IgG production in vivo, and expanded systemically in pristane-induced lupus-like disease. In SLE patients, CD4+CCR6+IL-7R+T cells were associated with the presence of pathogenic anti-dsDNA (doublestranded DNA) antibodies, and provided spontaneous help for autoantibody production ex vivo. Strikingly, IL-10-producing CCR6+T cells were highly abundant in lymph nodes of SLE patients, and colocalized with B cells at the margins of follicles. In conclusion, we identified a previously uncharacterized population of extrafollicular B helper T cells, which produced IL-10 and could play a prominent pathogenic role in SLE. [ABSTRACT FROM AUTHOR]

Long-term culture of primary lymphocytes and hematopoietic stem and progenitor cells (HSPCs) is pivotal to their expansion and study. Furthermore, genetic engineering of the above-mentioned primary human cells has several safety needs, including the requirement of efficient in vitro assays for unwanted tumorigenic events. In this work, we tested and optimized the Miniaturized Optically Accessible Bioreactor (MOAB) platform. The MOAB consists of a millifluidic cell culture device with three optically-accessible culture chambers. Inside the MOAB, we inserted a silk-based framework that resembles some properties of the bone marrow environment and cultivated in this device either CD4 + T lymphocytes isolated from healthy donor buffy coat or cord blood-derived hematopoietic CD34 + cells. A fraction of these cells is viable for up to 3 months. Next, we tested the capability of the MOAB to detect tumorigenic events. Serial dilutions of engineered fluorescent tumor cells were mixed with either CD4 + or CD34 + primary cells, and their growth was followed. By this approach, we successfully detected as little as 100 tumorigenic cells mixed with 100,000 primary cells. We found that non-tumorigenic primary cells colonized the silk environment, whereas tumor cells, after an adaptation phase, expanded and entered the circulation. We conclude that the millifluidic platform allows the detection of rare tumorigenic events in the long-term culture of human cells., Competing Interests: MR is a co-founder of the university spinoff company MOAB srl and holds shares. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Ritter, Oliveto, Cordiglieri, Fasciani, Di Buduo, della Volpe, Bocconi, Conci, Miguel, Di Micco, Balduini, Raimondi and Biffo.)

Synaptic dysfunction is an early pathogenic event leading to cognitive decline in Huntington's disease (HD). We previously reported that the active ADAM10 level is increased in the HD cortex and striatum, causing excessive proteolysis of the synaptic cell adhesion protein N-Cadherin. Conversely, ADAM10 inhibition is neuroprotective and prevents cognitive decline in HD mice. Although the breakdown of cortico-striatal connection has been historically linked to cognitive deterioration in HD, dendritic spine loss and long-term potentiation (LTP) defects identified in the HD hippocampus are also thought to contribute to the cognitive symptoms of the disease. The aim of this study is to investigate the contribution of ADAM10 to spine pathology and LTP defects of the HD hippocampus. We provide evidence that active ADAM10 is increased in the hippocampus of two mouse models of HD, leading to extensive proteolysis of N-Cadherin, which has a widely recognized role in spine morphology and synaptic plasticity. Importantly, the conditional heterozygous deletion of ADAM10 in the forebrain of HD mice resulted in the recovery of spine loss and ultrastructural synaptic defects in CA1 pyramidal neurons. Meanwhile, normalization of the active ADAM10 level increased the pool of synaptic BDNF protein and activated ERK neuroprotective signaling in the HD hippocampus. We also show that the ADAM10 inhibitor GI254023X restored LTP defects and increased the density of mushroom spines enriched with GluA1-AMPA receptors in HD hippocampal neurons. Notably, we report that administration of the TrkB antagonist ANA12 to HD hippocampal neurons reduced the beneficial effect of GI254023X, indicating that the BDNF receptor TrkB contributes to mediate the neuroprotective activity exerted by ADAM10 inhibition in HD. Collectively, these findings indicate that ADAM10 inhibition coupled with TrkB signaling represents an efficacious strategy to prevent hippocampal synaptic plasticity defects and cognitive dysfunction in HD., (© 2024. The Author(s).)

Introduction: Glioblastoma IDH -wildtype (GBM) is the most malignant brain tumor in adults, with a poor prognosis of approximately 15 months after diagnosis. Most patients suffer from a recurrence in <1 year, and this renders GBM a life-threatening challenge. Among molecular mechanisms driving GBM aggressiveness, angiogenesis mediated by GBM endothelial cells (GECs) deserves consideration as a therapeutic turning point. In this scenario, calpains, a family of ubiquitously expressed calcium-dependent cysteine proteases, emerged as promising targets to be investigated as a novel therapeutic strategy and prognostic tissue biomarkers., Methods: To explore this hypothesis, GECs were isolated from n=10 GBM biopsies and characterized phenotypically by immunofluorescence. The expression levels of calpains were evaluated by qRT-PCR and Western blot, and their association with patients' prognosis was estimated by Pearson correlation and Kaplan-Meier survival analysis. Calpain targeting efficacy was assessed by a time- and dose-dependent proliferation curve, MTT assay for viability, caspase-3/7 activity, migration and angiogenesis in vitro , and gene and protein expression level modification., Results: Immunofluorescence confirmed the endothelial phenotype of our primary GECs. A significant overexpression was observed for calpain-1/2/3 (CAPN) and calpain-small-subunits-1/2 (CAPNS1), whereas calpastatin gene, the calpain natural inhibitor, was reported to be downregulated. A significant negative correlation was observed between CAPN1/CAPNS1 and patient overall survival. GEC challenging revealed that the inhibition of calpain-1 exerts the strongest proapoptotic efficacy, so GEC mortality reached the 80%, confirmed by the increased activity of caspase-3/7. Functional assays revealed a strong affection of in vitro migration and angiogenesis. Gene and protein expression proved a downregulation of MAPK, VEGF/VEGFRs, and Bcl-2, and an upregulation of caspases and Bax-family mediators., Conclusion: Overall, the differential expression of calpains and their correlation with patient survival suggest a novel promising target pathway, whose blockade showed encouraging results toward precision medicine strategies., Competing Interests: Authors EB, GA, LR have been involved, as researchers, thanks to the participation of Andremacon Srl in the study. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Guarnaccia, Navone, Begani, Barilla, Garzia, Campanella, Miozzo, Fontana, Alotta, Cordiglieri, Gaudino, Schisano, Ampollini, Riboni, Locatelli and Marfia.)

Huntington's disease (HD) causes selective degeneration of striatal and cortical neurons, resulting in cell mosaicism of coexisting still functional and dysfunctional cells. The impact of non-cell autonomous mechanisms between these cellular states is poorly understood. Here we generated telencephalic organoids with healthy or HD cells, grown separately or as mosaics of the two genotypes. Single-cell RNA sequencing revealed neurodevelopmental abnormalities in the ventral fate acquisition of HD organoids, confirmed by cytoarchitectural and transcriptional defects leading to fewer GABAergic neurons, while dorsal populations showed milder phenotypes mainly in maturation trajectory. Healthy cells in mosaic organoids restored HD cell identity, trajectories, synaptic density, and communication pathways upon cell-cell contact, while showing no significant alterations when grown with HD cells. These findings highlight cell-type-specific alterations in HD and beneficial non-cell autonomous effects of healthy cells, emphasizing the therapeutic potential of modulating cell-cell communication in disease progression and treatment., (© 2024. The Author(s).)

Malignant mesothelioma is a type of cancer that affects the mesothelium. It is an aggressive and deadly form of cancer that is often caused by exposure to asbestos. At the molecular level, it is characterized by a low number of genetic mutations and high heterogeneity among patients. In this work, we analyzed the plasticity of gene expression of primary mesothelial cancer cells by comparing their properties on 2D versus 3D surfaces. First, we derived from primary human samples four independent primary cancer cells. Then, we used Nichoids, which are micro-engineered 3D substrates, as three-dimensional structures. Nichoids limit the dimension of adhering cells during expansion by counteracting cell migration between adjacent units of a substrate with their microarchitecture. Tumor cells grow effectively on Nichoids, where they show enhanced proliferation. We performed RNAseq analyses on all the samples and compared the gene expression pattern of Nichoid-grown tumor cells to that of cells grown in a 2D culture. The PCA analysis showed that 3D samples were more transcriptionally similar compared to the 2D ones. The 3D Nichoids induced a transcriptional remodeling that affected mainly genes involved in extracellular matrix assembly. Among these genes responsible for collagen formation, COL1A1 and COL5A1 exhibited elevated expression, suggesting changes in matrix stiffness. Overall, our data show that primary mesothelioma cells can be effectively expanded in Nichoids and that 3D growth affects the cells' tensegrity or the mechanical stability of their structure.

Mitofusin-2 (MFN2) is an outer mitochondrial membrane protein essential for mitochondrial networking in most cells. Autosomal dominant mutations in the MFN2 gene cause Charcot-Marie-Tooth type 2A disease (CMT2A), a severe and disabling sensory-motor neuropathy that impacts the entire nervous system. Here, we propose a novel therapeutic strategy tailored to correcting the root genetic defect of CMT2A. Though mutant and wild-type MFN2 mRNA are inhibited by RNA interference (RNAi), the wild-type protein is restored by overexpressing cDNA encoding functional MFN2 modified to be resistant to RNAi. We tested this strategy in CMT2A patient-specific human induced pluripotent stem cell (iPSC)-differentiated motor neurons (MNs), demonstrating the correct silencing of endogenous MFN2 and replacement with an exogenous copy of the functional wild-type gene. This approach significantly rescues the CMT2A MN phenotype in vitro, stabilizing the altered axonal mitochondrial distribution and correcting abnormal mitophagic processes. The MFN2 molecular correction was also properly confirmed in vivo in the MitoCharc1 CMT2A transgenic mouse model after cerebrospinal fluid (CSF) delivery of the constructs into newborn mice using adeno-associated virus 9 (AAV9). Altogether, our data support the feasibility of a combined RNAi and gene therapy strategy for treating the broad spectrum of human diseases associated with MFN2 mutations., (© 2023. The Author(s).)

Colorectal cancer (CRC) is a leading cause of cancer-associated death. In the tumor site, the interplay between effector immune cells and cancer cells determines the balance between tumor elimination or outgrowth. We discovered that the protein TMEM123 is over-expressed in tumour-infiltrating CD4 and CD8 T lymphocytes and it contributes to their effector phenotype. The presence of infiltrating TMEM123+ CD8+ T cells is associated with better overall and metastasis-free survival. TMEM123 localizes in the protrusions of infiltrating T cells, it contributes to lymphocyte migration and cytoskeleton organization. TMEM123 silencing modulates the underlying signaling pathways dependent on the cytoskeletal regulator WASP and the Arp2/3 actin nucleation complex, which are required for synaptic force exertion. Using tumoroid-lymphocyte co-culture assays, we found that lymphocytes form clusters through TMEM123, anchoring to cancer cells and contributing to their killing. We propose an active role for TMEM123 in the anti-cancer activity of T cells within tumour microenvironment., Competing Interests: Author Renata Grifantini is currently employed by CheckmAb Srl. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Pesce, Cordiglieri, Bombaci, Eppenberger-Castori, Oliveto, Manara, Crosti, Ercan, Coto, Gobbini, Campagnoli, Donnarumma, Martinelli, Bevilacqua, De Camilli, Gruarin, Sarnicola, Cassinotti, Baldari, Viale, Biffo, Abrignani, Terracciano and Grifantini.)

Metastatic brain disease (MBD) has seen major advances in clinical management, focal radiation therapy approaches and knowledge of biological factors leading to improved prognosis. Extracellular vesicles (EVs) have been found to play a role in tumor cross-talk with the target organ, contributing to the formation of a premetastatic niche. Human lung and breast cancer cell lines were characterized for adhesion molecule expression and used to evaluate their migration ability in an in vitro model. Conditioned culture media and isolated EVs, characterized by super resolution and electron microscopy, were tested to evaluate their pro-apoptotic properties on human umbilical vein endothelial cells (HUVECs) and human cerebral microvascular endothelial cells (HCMEC/D3) by annexin V binding assay. Our data showed a direct correlation between expression of ICAM1, ICAM2, β3-integrin and α2-integrin and the ability to firmly adhere to the blood-brain barrier (BBB) model, whereas the same molecules were down-regulated at a later step. Extracellular vesicles released by tumor cell lines were shown to be able to induce apoptosis in HUVEC while brain endothelial cells showed to be more resistant.

Gold nanoparticles (GNPs) are considered promising candidates for healthcare applications, however, their toxicity after long-term exposure to the material remains uncertain. Since the liver is the main filter organ for nanomaterials, this work was aimed at evaluating hepatic accumulation, internalisation and overall safety of well-characterised and endotoxin-free GNPs in healthy mice from 15 minutes to 7 weeks after a single administration. Our data demonstrate that GNPs were rapidly segregated into lysosomes of endothelial cells (LSEC) or Kupffer cells regardless of coating or shape but with different kinetics. Despite the long-lasting accumulation in tissues, the safety of GNPs was confirmed by liver enzymatic levels, as they were rapidly eliminated from the blood circulation and accumulated in the liver without inducing hepatic toxicity. Our results demonstrate that GNPs have a safe and biocompatibile profile despite their long-term accumulation.

Background: The COVID-19 pandemic is an infectious disease caused by SARS-CoV-2. The first step of SARS-CoV-2 infection is the recognition of angiotensin-converting enzyme 2 (ACE2) receptors by the receptor-binding domain (RBD) of the viral Spike (S) glycoprotein. Although the molecular and structural bases of the SARS-CoV-2-RBD/hACE2 interaction have been thoroughly investigated in vitro, the relationship between hACE2 expression and in vivo infection is less understood., Methods: Here, we developed an efficient SARS-CoV-2-RBD binding assay suitable for super resolution microscopy and simultaneous hACE2 immunodetection and mapped the correlation between hACE2 receptor abundance and SARS-CoV-2-RBD binding, both in vitro and in human lung biopsies. Next, we explored the specific proteome of SARS-CoV-2-RBD/hACE2 through a comparative mass spectrometry approach., Findings: We found that only a minority of hACE2 positive spots are actually SARS-CoV-2-RBD binding sites, and that the relationship between SARS-CoV-2-RBD binding and hACE2 presence is variable, suggesting the existence of additional factors. Indeed, we found several interactors that are involved in receptor localization and viral entry and characterized one of them: SLC1A5, an amino acid transporter. High-resolution receptor-binding studies showed that co-expression of membrane-bound SLC1A5 with hACE2 predicted SARS-CoV-2 binding and entry better than hACE2 expression alone. SLC1A5 depletion reduces SARS-CoV-2 binding and entry. Notably, the Omicron variant is more efficient in binding hACE2 sites, but equally sensitive to SLC1A5 downregulation., Interpretation: We propose a method for mapping functional SARS-CoV-2 receptors in vivo. We confirm the existence of hACE2 co-factors that may contribute to differential sensitivity of cells to infection., Funding: This work was supported by an unrestricted grant from "Fondazione Romeo ed Enrica Invernizzi" to Stefano Biffo and by AIRC under MFAG 2021 - ID. 26178 project - P.I. Manfrini Nicola., Competing Interests: Declaration of interests The authors report no conflict of interest., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Stem cell engineering of striatal medium spiny neurons (MSNs) is a promising strategy to understand diseases affecting the striatum and for cell-replacement therapies in different neurological diseases. Protocols to generate cells from human pluripotent stem cells (PSCs) are scarce and how well they recapitulate the endogenous fetal cells remains poorly understood. We have developed a protocol that modulates cell seeding density and exposure to specific morphogens that generates authentic and functional D1- and D2-MSNs with a high degree of reproducibility in 25 days of differentiation. Single-cell RNA sequencing (scRNA-seq) shows that our cells can mimic the cell-fate acquisition steps observed in vivo in terms of cell type composition, gene expression, and signaling pathways. Finally, by modulating the midkine pathway we show that we can increase the yield of MSNs. We expect that this protocol will help decode pathogenesis factors in striatal diseases and eventually facilitate cell-replacement therapies for Huntington's disease (HD)., Competing Interests: All authors declare no competing interests., (© 2022 The Authors.)

Tuberculosis (TB) causes millions of deaths every year, ranking as one of the most dangerous infectious diseases worldwide. Because several pathogenic strains of M. tuberculosis ( Mtb ) have developed resistance against most of the established anti-TB drugs, new therapeutic options are urgently needed. An attractive target for the development of new anti-TB agents is the salicylate synthase MbtI, the first enzyme of the mycobacterial siderophore biochemical machinery, absent in human cells. In this work, a set of analogues of 5-(3-cyanophenyl)furan-2-carboxylic acid ( I ), the most potent MbtI inhibitor identified to date, was synthesized, characterized, and tested to further elucidate the structural requirements for achieving an efficient MbtI inhibition and potent antitubercular activity. The structure-activity relationships (SAR) discussed herein evidenced the importance of the side chain linked to the phenyl moiety to improve the in vitro antimycobacterial activity. In detail, 1f emerged as the most effective analogue against the pathogen, acting without cytotoxicity issues. To deepen the understanding of its mechanism of action, we established a fluorescence-based screening test to quantify the pathogen infectivity within host cells, using MPI-2 murine cells, a robust surrogate for alveolar macrophages. The set-up of the new assay demonstrates significant potential to accelerate the discovery of new anti-TB drugs.

The cellular heterogeneity of the tumor environment of breast cancer (BC) is extremely complex and includes different actors such as neoplastic, stromal, and immunosuppressive cells, which contribute to the chemical and mechanical modification of the environment surrounding the tumor-exasperating immune-escaping mechanisms. In addition to molecular signals that make the tumor microenvironment (TME) unacceptable for the penetrance of the immune system, the physical properties of tumoral extracellular matrix (tECM) also have carved out a fundamental role in the processes of the protection of the tumor niche. Tumor-associated macrophages (TAMs), with an M2 immunosuppressive phenotype, are important determinants for the establishment of a tumor phenotype excluded from T cells. NF-κB transcription factors orchestrate innate immunity and represent the common thread between inflammation and cancer. Many studies have focused on canonical activation of NF-κB; however, activation of non-canonical signaling predicts poor survival and resistance to therapy. In this scenario, we demonstrated the existence of an unusual association of NF-κB components in TAMs that determines the deposition of HSPG2 that affects the stiffness of tECM. These results highlight a new mechanism counterbalanced between physical factors and a new perspective of mechano-pathology to be targeted to counteract immune evasion in BC.

HIV integrates into the host genome, creating a viral reservoir of latently infected cells that persists despite effective antiretroviral treatment. CD4-positive (CD4 + ) T cells are the main contributors to the HIV reservoir. CD4 + T cells are a heterogeneous population, and the mechanisms of latency establishment in the different subsets, as well as their contribution to the reservoir, are still unclear. In this study, we analyzed HIV latency establishment in different CD4 + T cell subsets stimulated with interleukin 15 (IL-15), a cytokine that increases both susceptibility to infection and reactivation from latency. Using a dual-reporter virus that allows discrimination between latent and productive infection at the single-cell level, we found that IL-15-treated primary human CD4 + T naive and CD4 + T stem cell memory (T SCM ) cells are less susceptible to HIV infection than CD4 + central memory (T CM ), effector memory (T EM ), and transitional memory (T TM ) cells but are also more likely to harbor transcriptionally silent provirus. The propensity of these subsets to harbor latent provirus compared to the more differentiated memory subsets was independent of differential expression of pTEFb components. Microscopy analysis of NF-κB suggested that CD4 + T naive cells express smaller amounts of nuclear NF-κB than the other subsets, partially explaining the inefficient long terminal repeat (LTR)-driven transcription. On the other hand, CD4 + T SCM cells display similar levels of nuclear NF-κB to CD4 + T CM , CD4 + T EM , and CD4 + T TM cells, indicating the availability of transcription initiation and elongation factors is not solely responsible for the inefficient HIV gene expression in the CD4 + T SCM subset. IMPORTANCE The formation of a latent reservoir is the main barrier to HIV cure. Here, we investigated how HIV latency is established in different CD4 + T cell subsets in the presence of IL-15, a cytokine that has been shown to efficiently induce latency reversal. We observed that, even in the presence of IL-15, the less differentiated subsets display lower levels of productive HIV infection than the more differentiated subsets. These differences were not related to different expression of pTEFb, and modest differences in NF-κB were observed for CD4 + T naive cells only, implying the involvement of other mechanisms. Understanding the molecular basis of latency establishment in different CD4 + T cell subsets might be important for tailoring specific strategies to reactivate HIV transcription in all the CD4 + T subsets that compose the latent reservoir.

Background: von Willebrand factor propeptide (VWFpp) plays an important role in VWF multimerization and storage. VWFpp mutations have been previously associated with types 1, 3 and 2A/IIC von Willebrand disease (VWD)., Aims: To characterize the novel p.Thr274Pro variant identified in two unrelated type 1 VWD patients., Methods: Phenotype tests were performed to evaluate patients' plasma and platelets following the current ISTH-SSC guidelines. Molecular analysis was performed using next-generation sequencing. The pcDNA3.1-VWF-WT and mutant pcDNA3.1-VWF-Thr274Pro expression vectors were transiently transfected into HEK293 cells to evaluate recombinant (r)VWF constitutive and regulated secretion. For the latter, the transfected cells were stimulated with phorbol-12-myristate-13-acetate. Immunofluorescence staining was performed to assess the localization of WT-rVWF and Thr274Pro-rVWF in endoplasmic reticulum, lysosomes, cis-/trans-Golgi and pseudo-Weibel Palade bodies., Results: Biochemical characterization of patients' plasma samples indicated a type 1 VWD diagnosis. Both patients were heterozygous for the p.Thr274Pro variant. Hybrid Thr274Pro/WT-rVWF showed a secretion reduction of 36±4% according to patients' plasma VWF:Ag levels, whereas Thr274Pro-rVWF secretion was strongly impaired (21±2%). The amount of rVWF in cell lysates was nearly normal for both Thr274P (62±17%) and Thr274Pro/WT-rVWF (72±23%). The regulated secretion was impaired for Thr274Pro/WT-rVWF, whereas Thr274Pro-rVWF was not released at all. Immunofluorescence staining revealed no particular differences between WT and Thr274Pro-rVWF, although Thr274Pro-rVWF showed less pseudo-Weibel Palade bodies with a rounder shape than WT-rVWF., Conclusions: The novel p.Thr274Pro mutation has a dominant effect and it is responsible of patients' type 1 VWD phenotype through a combined mechanism of reduced synthesis, impaired secretion and multimerization., (© 2022 The Authors. Haemophilia published by John Wiley & Sons Ltd.)

Coronavirus disease 2019 (COVID-19) is an infectious disease caused by beta-coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that has rapidly spread across the globe starting from February 2020. It is well established that during viral infection, extracellular vesicles become delivery/presenting vectors of viral material. However, studies regarding extracellular vesicle function in COVID-19 pathology are still scanty. Here, we performed a comparative study on exosomes recovered from the plasma of either MILD or SEVERE COVID-19 patients. We show that although both types of vesicles efficiently display SARS-CoV-2 spike-derived peptides and carry immunomodulatory molecules, only those of MILD patients are capable of efficiently regulating antigen-specific CD4 + T-cell responses. Accordingly, by mass spectrometry, we show that the proteome of exosomes of MILD patients correlates with a proper functioning of the immune system, while that of SEVERE patients is associated with increased and chronic inflammation. Overall, we show that exosomes recovered from the plasma of COVID-19 patients possess SARS-CoV-2-derived protein material, have an active role in enhancing the immune response, and possess a cargo that reflects the pathological state of patients in the acute phase of the disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Pesce, Manfrini, Cordiglieri, Santi, Bandera, Gobbini, Gruarin, Favalli, Bombaci, Cuomo, Collino, Cricrì, Ungaro, Lombardi, Mangioni, Muscatello, Aliberti, Blasi, Gori, Abrignani, De Francesco, Biffo and Grifantini.)

Brain cholesterol is produced mainly by astrocytes and is important for neuronal function. Its biosynthesis is severely reduced in mouse models of Huntington's disease. One possible mechanism is a diminished nuclear translocation of the transcription factor sterol regulatory element-binding protein 2 (SREBP2) and, consequently, reduced activation of SREBP2-controlled genes in the cholesterol biosynthesis pathway. Here we evaluated the efficacy of a gene therapy based on the unilateral intra-striatal injection of a recombinant adeno-associated virus 2/5 (AAV2/5) targeting astrocytes specifically and carrying the transcriptionally active N-terminal fragment of human SREBP2 (hSREBP2). Robust hSREBP2 expression in striatal glial cells in R6/2 Huntington's disease mice activated the transcription of cholesterol biosynthesis pathway genes, restored synaptic transmission, reversed dopamine receptor D2 (Drd2) transcript levels decline, cleared mutant huntingtin aggregates and attenuated behavioural deficits. We conclude that glial SREBP2 participates in Huntington's disease brain pathogenesis in vivo and that AAV-based delivery of SREBP2 to astrocytes counteracts key features of the disease., (© The Author(s) (2021). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

A postprandial increase of translation mediated by eukaryotic Initiation Factor 6 (eIF6) occurs in the liver. Its contribution to steatosis and disease is unknown. In this study we address whether eIF6-driven translation contributes to disease progression. eIF6 levels increase throughout the progression from Non-Alcoholic Fatty Liver Disease (NAFLD) to hepatocellular carcinoma. Reduction of eIF6 levels protects the liver from disease progression. eIF6 depletion blunts lipid accumulation, increases fatty acid oxidation (FAO) and reduces oncogenic transformation in vitro. In addition, eIF6 depletion delays the progression from NAFLD to hepatocellular carcinoma, in vivo. Mechanistically, eIF6 depletion reduces the translation of transcription factor C/EBPβ, leading to a drop in biomarkers associated with NAFLD progression to hepatocellular carcinoma and preserves mitochondrial respiration due to the maintenance of an alternative mTORC1-eIF4F translational branch that increases the expression of transcription factor YY1. We provide proof-of-concept that in vitro pharmacological inhibition of eIF6 activity recapitulates the protective effects of eIF6 depletion. We hypothesize the existence of a targetable, evolutionarily conserved translation circuit optimized for lipid accumulation and tumor progression., (© 2021. The Author(s).)

The etiology of intervertebral disc degeneration is largely unknown, but local neuroinflammation may exert a crucial role through activation of cells as microglia and pro-inflammatory cytokines production. We aimed to compare the effect of degenerated and normal intervertebral disc microenvironment on microglial cells and the potential role of sphingosine-1-phosphate, a pro-inflammatory sphingolipid, in their crosstalk. Human degenerated intervertebral discs (Pfirrmann grade IV) were obtained at surgery for spondylolisthesis. Normal intervertebral discs were collected from cadaveric normal lumbar spines. Normal and degenerated-intervertebral discs were kept in culture to obtain media conditioning. Then, microglial cells were cocultured with conditioned media and viability, proliferation, migration, chemotaxis, and inflammatory gene expression were evaluated. The results demonstrate that conditioned media from degenerated intervertebral discs activate microglial cells, increasing chemotaxis, migration, and pro-inflammatory mediators release to a great extent than normal discs. In addition, we show that the administration of sphingosine-1-phosphate to normal intervertebral disc/microglia coculture mimicked degenerative effects. Interestingly, sphingosine-1-phosphate content in conditioned media from degenerated discs was significantly higher than that from normal ones. In addition, FTY720, a functional antagonist of sphingosine-1-phosphate, potently inhibited the effect of degenerated intervertebral discs on microglial inflammatory factor transcription and migration. Our data report, for the first time, that sphingosine-1-phosphate is involved as signal in the microenvironment of human degenerated intervertebral discs. Sphingosine-1-phosphate signaling modulation by FTY720 may induce beneficial effects in counteracting microglial activation during intervertebral disc degeneration., (© 2020 Orthopaedic Research Society. Published by Wiley Periodicals LLC.)

Regulatory T (T reg ) cells are a barrier for tumor immunity and a target for immunotherapy. Using single-cell transcriptomics, we found that CD4 + T cells infiltrating primary and metastatic colorectal cancer and non-small-cell lung cancer are highly enriched for two subsets of comparable size and suppressor function comprising forkhead box protein P3 + T reg and eomesodermin homolog (EOMES) + type 1 regulatory T (Tr1)-like cells also expressing granzyme K and chitinase-3-like protein 2. EOMES + Tr1-like cells, but not T reg cells, were clonally related to effector T cells and were clonally expanded in primary and metastatic tumors, which is consistent with their proliferation and differentiation in situ. Using chitinase-3-like protein 2 as a subset signature, we found that the EOMES + Tr1-like subset correlates with disease progression but is also associated with response to programmed cell death protein 1-targeted immunotherapy. Collectively, these findings highlight the heterogeneity of T reg cells that accumulate in primary tumors and metastases and identify a new prospective target for cancer immunotherapy.

The immune system is a fine modulator of the tumor biology supporting or inhibiting its progression, growth, invasion and conveys the pharmacological treatment effect. Tumors, on their side, have developed escaping mechanisms from the immune system action ranging from the direct secretion of biochemical signals to an indirect reaction, in which the cellular actors of the tumor microenvironment (TME) collaborate to mechanically condition the extracellular matrix (ECM) making it inhospitable to immune cells. TME is composed of several cell lines besides cancer cells, including tumor-associated macrophages, cancer-associated fibroblasts, CD4 + and CD8 + lymphocytes, and innate immunity cells. These populations interface with each other to prepare a conservative response, capable of evading the defense mechanisms implemented by the host's immune system. The presence or absence, in particular, of cytotoxic CD8 + cells in the vicinity of the main tumor mass, is able to predict, respectively, the success or failure of drug therapy. Among various mechanisms of immunescaping, in this study, we characterized the modulation of the phenotypic profile of CD4 + and CD8 + cells in resting and activated states, in response to the mechanical pressure exerted by a three-dimensional in vitro system, able to recapitulate the rheological and stiffness properties of the tumor ECM.

Hemophilia is an X-linked recessive bleeding disorder. In pregnant women carrier of hemophilia, the fetal sex can be determined by non-invasive analysis of fetal DNA circulating in the maternal blood. However, in case of a male fetus, conventional invasive procedures are required for the diagnosis of hemophilia. Fetal cells, circulating in the maternal bloodstream, are an ideal target for a safe non-invasive prenatal diagnosis. Nevertheless, the small number of cells and the lack of specific fetal markers have been the most limiting factors for their isolation. We aimed to develop monoclonal antibodies (mAbs) against the ribosomal protein RPS4Y1 expressed in male cells. By Western blotting, immunoprecipitation and immunofluorescence analyses performed on cell lysates from male human hepatoma (HepG2) and female human embryonic kidney (HEK293) we developed and characterized a specific monoclonal antibody against the native form of the male RPS4Y1 protein that can distinguish male from female cells. The availability of the RPS4Y1-targeting monoclonal antibody should facilitate the development of novel methods for the reliable isolation of male fetal cells from the maternal blood and their future use for non-invasive prenatal diagnosis of X-linked inherited disease such as hemophilia.

Friedreich's ataxia (FRDA) is an autosomal-recessive neurodegenerative and cardiac disorder which occurs when transcription of the FXN gene is silenced due to an excessive expansion of GAA repeats into its first intron. Herein, we generate dorsal root ganglia organoids (DRG organoids) by in vitro differentiation of human iPSCs. Bulk and single-cell RNA sequencing show that DRG organoids present a transcriptional signature similar to native DRGs and display the main peripheral sensory neuronal and glial cell subtypes. Furthermore, when co-cultured with human intrafusal muscle fibers, DRG organoid sensory neurons contact their peripheral targets and reconstitute the muscle spindle proprioceptive receptors. FRDA DRG organoids model some molecular and cellular deficits of the disease that are rescued when the entire FXN intron 1 is removed, and not with the excision of the expanded GAA tract. These results strongly suggest that removal of the repressed chromatin flanking the GAA tract might contribute to rescue FXN total expression and fully revert the pathological hallmarks of FRDA DRG neurons.

: Circulating platelets (PLTs) are able to affect glioblastoma (GBM) microenvironment by supplying oncopromoter and pro-angiogenic factors. Among these mediators, sphingosine-1-phophate (S1P) has emerged as a potent bioactive lipid enhancing cell proliferation and survival. Here, we investigated the effect of "tumor education", characterizing PLTs from GBM patients in terms of activation state, protein content, and pro-angiogenic potential. PLTs from healthy donors (HD-PLTs) and GBM patients (GBM-PLTs) were collected, activated, and analyzed by flow cytometry, immunofluorescence, and Western blotting. To assess the pro-angiogenic contribution of GBM-PLTs, a functional cord formation assay was performed on GBM endothelial cells (GECs) with PLT-releasate. GBM-PLTs expressed higher positivity for P-selectin compared to HD-PLTs, both in basal conditions and after stimulation with adenosine triphosphate (ADP) and thrombin receptor activating peptide (TRAP). PLTs showed higher expression of VEGFR-1, VEGFR-2, VWF, S1P, S1PR1, SphK1, and SPNS. Interestingly, increased concentrations of VEGF and its receptors VEGFR1 and VEGFR2, VWF, and S1P were found in GBM-PLT-releasate with respect to HD-PLTs. Finally, GBM-PLT-releasate showed a pro-angiogenic effect on GECs, increasing the vascular network's complexity. Overall, our results demonstrated the contribution of PLTs to GBM angiogenesis and aggressiveness, advancing the potential of an anti-PLT therapy and the usefulness of PLT cargo as predictive and monitoring biomarkers., Competing Interests: The authors declare the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Stem cell-derived neurons are generally obtained in mass cultures that lack both spatial organization and any meaningful connectivity. We implement a microfluidic system for long-term culture of human neurons with patterned projections and synaptic terminals. Co-culture of human midbrain dopaminergic and striatal medium spiny neurons on the microchip establishes an orchestrated nigro-striatal circuitry with functional dopaminergic synapses. We use this platform to dissect the mitochondrial dysfunctions associated with a genetic form of Parkinson's disease (PD) with OPA1 mutations. Remarkably, we find that axons of OPA1 mutant dopaminergic neurons exhibit a significant reduction of mitochondrial mass. This defect causes a significant loss of dopaminergic synapses, which worsens in long-term cultures. Therefore, PD-associated depletion of mitochondria at synapses might precede loss of neuronal connectivity and neurodegeneration. In vitro reconstitution of human circuitries by microfluidic technology offers a powerful system to study brain networks by establishing ordered neuronal compartments and correct synapse identity., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Beneficial effects of probiotics on gut microbiota homeostasis and inflammatory immune responses suggested the investigation of their potential clinical efficacy in experimental models of autoimmune diseases. Indeed, administration of two bifidobacteria and lactobacilli probiotic strains prevented disease manifestations in the Lewis rat model of Myasthenia Gravis (EAMG). Here, we demonstrate the clinical efficacy of therapeutic administration of vital bifidobacteria (i.e., from EAMG onset). The mechanisms involved in immunomodulation were investigated with ex vivo and in vitro experiments. Improvement of EAMG symptoms was associated to decreased anti-rat AChR antibody levels, and differential expression of TGFβ and FoxP3 immunoregulatory transcripts in draining lymph nodes and spleen of treated-EAMG rats. Exposure of rat bone marrow-derived dendritic cells to bifidobacteria or lactobacilli strains upregulated toll-like receptor 2 mRNA expression, a key molecule involved in bacterium recognition via lipotheicoic acid. Live imaging experiments of AChR-specific effector T cells, co-cultured with BMDCs pre-exposed to bifidobacteria, demonstrated increased percentages of motile effector T cells, suggesting a hindered formation of TCR-peptide-MHC complex. Composition of gut microbiota was studied by 16S rRNA gene sequencing, and α and β diversity were determined in probiotic treated EAMG rats, with altered ratios between Tenericutes and Verrucomicrobia (phylum level), and Ruminococcaceae and Lachnospiraceae (family level). Moreover, the relative abundance of Akkermansia genus was found increased compared to healthy and probiotic treated EAMG rats. In conclusion, our findings confirms that the administration of vital bifidobacteria at EAMG onset has beneficial effects on disease progression; this study further supports preclinical research in human MG to evaluate probiotic efficacy as supplementary therapy in MG., (Copyright © 2019 Rinaldi, Consonni, Cordiglieri, Sacco, Crasà, Fontana, Morelli, Elli, Mantegazza and Baggi.)

Background and Aims: Primary biliary cholangitis is an autoimmune biliary disease characterized by injury of bile ducts, eventually leading to cirrhosis and death. In most cases, anti-mitochondrial antibodies and persistently elevated serum alkaline phosphatase are the basis for the serological diagnosis. Anti-nuclear antibodies are also useful and may indicate a more aggressive diseases course. In patients in which anti-mitochondrial antibodies are not detected, an accurate diagnosis requires liver histology. This study aims at identifying specific biomarkers for the serological diagnosis of primary biliary cholangitis., Methods: Sera from patients affected by primary biliary cholangitis, primary sclerosing cholangitis, hepatitis C virus (with and without cryoglobulinemia), hepatocarcinoma and healthy donors were tested on a protein array representing 1658 human proteins. The most reactive autoantigens were confirmed by DELFIA analysis on expanded cohorts of the same mentioned serum classes, and on autoimmune hepatitis sera, using anti-PDC-E2 as reference biomarker., Results: Two autoantigens, SPATA31A3 and GARP, showed high reactivity with primary biliary cholangitis sera, containing or not anti-mitochondrial antibodies. Their combination with PDC-E2 allowed to discriminate primary biliary cholangitis from all tested control classes with high sensitivity and specificity. We found that GARP expression is upregulated upon exposure to biliary salts in human cholangiocytes, an event involving EGFR and insulin pathways. GARP expression was also detected in biliary duct cells of PBC patients., Conclusions: This study highlighted SPATA31A3 and GARP as new biomarkers for primary biliary cholangitis and unravelled molecular stimuli underlying GARP expression in human cholangiocytes., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Glioblastoma (GBM) is the most common and fatal human brain tumor, with the worst prognosis. The aberrant microenvironment, enhanced by the activation of proangiogenic mediators such as hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), and their downstream effectors, sustain GBM malignancy. Proangiogenic signaling represents an attractive chemotherapeutic target. Recent evidence suggests a therapeutic benefit from aspirin (acetylsalicylic acid, or ASA) intake in reducing risk and cancer progression., Methods: In the present study, human primary GBM-endothelial cells (ECs) were used to ascertain whether ASA could inhibit angiogenesis and improve cell sensitivity to drugs. The impact of ASA was observed by measuring cell viability, tube-like structure formation, migration, VEGF production, and proliferative, proangiogenic, and apoptotic modulators expression, such as HIF-1α/VEGF/vascular endothelial growth factor receptor/(VEGFR)-1/VEGFR-2, Ras/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase, phosphoinositide 3-kinase/AKT signaling axis, and Bcl-2-associated X protein/B-cell lymphoma 2 (BCL-2) ratio. Furthermore, we evaluated the effect of ASA alone or in combination with temozolomide (TMZ), bevacizumab (BEV), and sunitinib (SUN)., Results: Our data reported that ASA affected GBM-EC viability, tube-like structure formation, cell migration, and VEGF releasing in a dose-dependent manner and that combined treatments with TMZ, BEV, and SUN synergized to counteract proangiogenic cell ability. mRNA expression analysis displayed a marked effect of ASA in reducing VEGF, VEGFR-1, HIF-1α, RAS, mitogen-activated protein kinase kinase, AKT, and BCL-2, as well a combined anticancer effect of ASA together with TMZ, BEV, and SUN. Levels of HIF-1α, VEGFR-2, Bcl-2-associated X protein, and BCL-2 protein expression confirmed a positive trend., Conclusions: ASA and antiangiogenic therapies showed synergetic anticancer efficacy in human primary GBM-ECs. Thus, the combination of conventional chemotherapy with ASA may offer a new strategy to counteract tumor malignancy., (Copyright © 2018 Elsevier Inc. All rights reserved.)