Transoral glossoepiglottopexy in the treatment of adult obstructive sleep apnoea: A surgical approach [Glossoepiglottoplastica con approccio trans-orale nel trattamento delle apnee ostruttive notturne nell\u2019adulto]

The treatment of obstructive sleep apnoea syndrome (OSAS) is still a matter of debate; among the different therapeutic alternatives, both surgical and conservative, treatment with continuous positive airway pressure (CPAP) is considered the \u201cgold standard\u201d. The recent scientific literature reports that even if CPAP represents an effective solution for sleep apnoeas, 12% of patients do not benefit from its use. In most cases, primary collapse of the epiglottis is responsible for failure. We developed a surgical technique that provides a stable support to the epiglottis without influencing its function during swallowing while preserving laryngeal anatomy and physiology. The procedure we propose is based on that conceived by Monnier for children affected by laryngomalacia. We analysed a group of 20 patients who underwent glossoepiglottopexy between January 2015 and September 2016 and compared data (AHI, ODI, t90, ESS, EAT10, etc.) collected before and 6 months after surgery to demonstrate the safety and effectiveness of our glossoepiglottopexy (GEP). The results allow us to consider GEP as a valid choice to treat adults who suffer from sleep apnoeas

A simplified sars-cov-2 pseudovirus neutralization assay

COVID-19 is an ongoing pandemic caused by the highly infectious coronavirus SARS-CoV-2 that is engaging worldwide scientific research to find a timely and effective eradication strategy. Great efforts have been put into anti-COVID-19 vaccine generation in an effort to protect the world population and block SARS-CoV-2 spread. To validate the protective efficacy of the vaccination campaign and effectively control the pandemic, it is necessary to quantify the induction of neutralizing antibodies by vaccination, as they have been established to be a correlate of protection. In this work, a SARS-CoV-2 pseudovirus neutralization assay, based on a replication-incompetent lentivirus expressing an adapted form of CoV-2 S protein and an ACE2/TMPRSS2 stably expressing cell line, has been minimized in terms of protocol steps without loss of accuracy. The goal of the present simplified neutralization system is to improve SARS-CoV-2 vaccination campaign by means of an easy and accessible approach to be performed in any medical laboratory, maintaining the sensitivity and quantitative reliability of classical serum neutralization assays. Further, this assay can be easily adapted to different coronavirus variants by simply modifying the pseudotyping vector

Redistribution of DAT/α-synuclein complexes visualized by “in situ” proximity ligation assay in transgenic mice modelling early Parkinson’s disease

Alpha-synuclein, the major component of Lewy bodies, is thought to play a central role in the onset of synaptic dysfunctions in Parkinson's disease (PD). In particular, α-synuclein may affect dopaminergic neuron function as it interacts with a key protein modulating dopamine (DA) content at the synapse: the DA transporter (DAT). Indeed, recent evidence from our "in vitro" studies showed that α-synuclein aggregation decreases the expression and membrane trafficking of the DAT as the DAT is retained into α-synuclein-immunopositive inclusions. This notwithstanding, "in vivo" studies on PD animal models investigating whether DAT distribution is altered by the pathological overexpression and aggregation of α-synuclein are missing. By using the proximity ligation assay, a technique which allows the "in situ" visualization of protein-protein interactions, we studied the occurrence of alterations in the distribution of DAT/α-synuclein complexes in the SYN120 transgenic mouse model, showing insoluble α-synuclein aggregates into dopaminergic neurons of the nigrostriatal system, reduced striatal DA levels and an altered distribution of synaptic proteins in the striatum. We found that DAT/α-synuclein complexes were markedly redistributed in the striatum and substantia nigra of SYN120 mice. These alterations were accompanied by a significant increase of DAT striatal levels in transgenic animals when compared to wild type littermates. Our data indicate that, in the early pathogenesis of PD, α-synuclein acts as a fine modulator of the dopaminergic synapse by regulating the subcellular distribution of key proteins such as the DAT

Simultaneous Combination of the CDK4/6 Inhibitor Palbociclib With Regorafenib Induces Enhanced Anti-tumor Effects in Hepatocarcinoma Cell Lines

Advanced hepatocarcinoma (HCC) is an aggressive malignancy with poor prognosis and limited treatment options. Alterations of the cyclin D-CDK4/6-Rb pathway occur frequently in HCC, providing the rationale for its targeting at least in a molecular subset of HCC. In a panel of HCC cell lines, we investigated whether the CDK4/6 inhibitor palbociclib might improve the efficacy of regorafenib, a powerful multi-kinase inhibitor approved as second-line treatment for advanced HCC after sorafenib failure and currently under clinical investigation as first-line therapy in combination with immunotherapy. In Rb-proficient cells, the simultaneous drug combination, but not the sequential schedules, inhibited cell proliferation, either in short or in long-term experiments, and induced cell death more strongly than individual treatments. Moreover, the combination significantly reduced spheroid cell growth and inhibited cell migration/invasion. The superior efficacy of palbociclib plus regorafenib emerged also under hypoxia and was associated with a significant down-regulation of CDK4/6-Rb-myc and mTORC1/p70S6K signaling. Moreover, regorafenib suppressed palbociclib-induced expression of cyclin D1 contributing to the cytotoxic effects of the combination. Besides these inhibitory effects on cell viability/proliferation, palbociclib and regorafenib reduced glucose uptake, although this effect was dependent on the cell model and on the oxygen availability (normoxia or hypoxia). Palbociclib and regorafenib combination impaired glucose uptake and utilization, down-regulating basal and hypoxia-induced expression of HIF-1α, HIF-2α, GLUT-1, and MCT4 proteins as well as the activity/expression of glycolytic enzymes (HK2, PFKP, aldolase A, PKM2). In addition, regorafenib alone reduced mitochondrial respiration. The combined treatment impaired glucose metabolism and respiration without enhancing the effects of the single agents. Our findings provide pre-clinical evidence for the effectiveness of palbociclib and regorafenib combination in HCC cell models

Retinoic acid stimulates meningioma cell adhesion to the extracellular matrix and inhibits invasion

Meningiomas are tumours derived from the arachnoid and pia mater. During embryogenesis, these membranes develop from the migrating craniofacial neural crest. We have previously demonstrated that meningiomas have characteristic features of embryonic meninges. Craniofacial neural crest derivatives are affected during normal development and migration by retinoic acid. We speculated, therefore, that meningioma cell migration and invasion would be affected in a similar way. In this study we investigated the mechanisms of invasion and migration in meningiomas and the effects of retinoic acid (RA). We found that low doses of RA inhibit in vitro invasion in meningioma cells, without affecting cell proliferation or viability. The matrix metalloproteinases MMP-2 (72 kDa gelatinase) and MMP-9 (92 kDa gelatinase), which play a key role in invasion in other tumours, are not affected by RA. RA inhibits cell migration on collagen I and fibronectin. A possible mechanism for these effects is provided by the fact that RA strongly stimulates adhesion of meningioma cells to extracellular matrix substrates. As in vitro invasion, migration and decreased adhesion to the extracellular matrix correlate with the clinical manifestation of tumour invasion, we conclude that RA induces a non-invasive phenotype in meningioma cells. © 1999 Cancer Research Campaig

Intratumor Regulatory Noncytotoxic NK Cells in Patients with Hepatocellular Carcinoma

Previous studies support the role of natural killer (NK) cells in controlling hepatocellular carcinoma (HCC) progression. However, ambiguity remains about the multiplicity and the role of different NK cell subsets, as a pro-oncogenic function has been suggested. We performed phenotypic and functional characterization of NK cells infiltrating HCC, with the corresponding nontumorous tissue and liver from patients undergoing liver resection for colorectal liver metastasis used as controls. We identified a reduced number of NK cells in tumors with higher frequency of CD56(BRIGHT)CD16(-) NK cells associated with higher expression of NKG2A, NKp44, and NKp30 and downregulation of NKG2D. Liver-resident (CXCR6(+)) NK cells were reduced in the tumors where T-bet(hi)Eomes(lo) expression was predominant. HCCs showed higher expression of CD49a with particular enrichment in CD49a(+)Eomes(+) NK cells, a subset typically represented in the decidua and playing a proangiogenic function. Functional analysis showed reduced TNF-alpha production along with impaired cytotoxic capacity that was inversely related to CXCR6(-), T-bet(hi)Eomes(lo), and CD49a(+)Eomes(+) NK cells. In conclusion, we identified a subset of NK cells infiltrating HCC, including non-liver-resident cells that coexpressed CD49a and Eomes and showed reduced cytotoxic potential. This NK cell subset likely plays a regulatory role in proangiogenic function

FluoroSpot assay to analyze SARS-CoV-2-specific T cell responses

Monitoring antigen-specific T cell frequency and function is essential to assess the host immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Here, we present a FluoroSpot assay for concurrently detecting ex vivo antiviral cytokine production by SARS-CoV-2-specific T cells following peptide stimulation. We then detail intracellular cytokine staining by flow cytometry to further validate the FluoroSpot assay results and define the specific T cell subpopulations. For complete details on the use and execution of this protocol, please refer to Tiezzi et al. (2023).

Soluble PD-L1 and Circulating CD8+PD-1+ and NK Cells Enclose a Prognostic and Predictive Immune Effector Score in Immunotherapy Treated NSCLC patients

Introduction: Upfront criteria to foresee immune checkpoint inhibitors (ICIs) efficacy are far from being identified. Thus, we integrated blood descriptors of pro-inflammatory/immunosuppressive or effective anti-tumor response to non-invasively define predictive immune profiles in ICI-treated advanced non-small cell lung cancer (NSCLC). Methods: Peripheral blood (PB) was prospectively collected at baseline from 109 consecutive NSCLC patients undergoing ICIs as first or more line treatment. Soluble PD-L1 (sPD-L1) (immunoassay), CD8+PD-1+ and NK (FACS) cells were assessed and interlaced to generate an Immune effector Score (IeffS). Lung Immune Prognostic Index (LIPI) was computed by LDH levels and derived Neutrophil-to-Lymphocyte Ratio (dNLR). All these parameters were correlated with survival outcome and treatment response. Results: High sPD-L1 and low CD8+PD-1+ and NK number had negative impact on PFS (P < 0.001), OS (P < 0.01) and ICI-response (P < 0.05). Thus, sPD-L1high, CD8+PD-1+low and NKlow were considered as risk factors encompassing IeffS, whose prognostic power outperformed that of individual features and slightly exceeded that of LIPI. Accordingly, the absence of these risk factors portrayed a favorable IeffS characterizing patients with significantly (P < 0.001) prolonged PFS (median NR vs 2.3 months) and OS (median NR vs 4.1) and greater benefit from ICIs (P < 0.01). We then combined each risk parameter composing IeffS and LIPI (LDHhigh, dNLRhigh), thus defining three distinct prognostic classes. A remarkable impact of IeffS-LIPI integration was documented on survival outcome (PFS, HR = 4.61; 95%CI = 2.32-9.18; P < 0.001; OS, HR=4.03; 95%CI=1.91-8.67; P < 0.001) and ICI-response (AUC=0.90, 95%CI=0.81-0.97, P < 0.001). Conclusion: Composite risk models based on blood parameters featuring the tumor-host interaction might provide accurate prognostic scores able to predict ICI benefit in NSCLC patients

Alpha-synuclein/synapsin III pathological interplay boosts the motor response to methylphenidate

16siLoss of dopaminergic nigrostriatal neurons and fibrillary α-synuclein (α-syn) aggregation in Lewy bodies (LB) characterize Parkinson's disease (PD). We recently found that Synapsin III (Syn III), a phosphoprotein regulating dopamine (DA) release with α-syn, is another key component of LB fibrils in the brain of PD patients and acts as a crucial mediator of α-syn aggregation and toxicity. Methylphenidate (MPH), a monoamine reuptake inhibitor (MRI) efficiently counteracting freezing of gait in advanced PD patients, can bind α-syn and controls α-syn-mediated DA overflow and presynaptic compartmentalization. Interestingly, MPH results also efficient for the treatment of attention deficits and hyperactivity disorder (ADHD), a neurodevelopmental psychiatric syndrome associated with Syn III and α-syn polymorphisms and constituting a risk factor for the development of LB disorders. Here, we studied α-syn/Syn III co-deposition and longitudinal changes of α-syn, Syn III and DA transporter (DAT) striatal levels in nigrostriatal neurons of a PD model, the human C-terminally truncated (1−120) α-syn transgenic (SYN120 tg) mouse, in comparison with C57BL/6J wild type (wt) and C57BL/6JOlaHsd α-syn null littermates. Then, we analyzed the locomotor response of these animals to an acute administration of MPH (d-threo) and other MRIs: cocaine, that we previously found to stimulate Syn III-reliant DA release in the absence of α-syn, or the selective DAT blocker GBR-12935, along aging. Finally, we assessed whether these drugs modulate α-syn/Syn III interaction by fluorescence resonance energy transfer (FRET) and performed in silico studies engendering a heuristic model of the α-syn conformations stabilized upon MPH binding. We found that only MPH was able to over-stimulate a Syn III-dependent/DAT-independent locomotor activity in the aged SYN120 tg mice showing α-syn/Syn III co-aggregates. MPH enhanced full length (fl) α-syn/Syn III and even more (1–120) α-syn/Syn III interaction in cells exhibiting α-syn/Syn III inclusions. Moreover, in silico studies confirmed that MPH may reduce α-syn fibrillation by stabilizing a protein conformation with increased lipid binding predisposition. Our observations indicate that the motor-stimulating effect of MPH can be positively fostered in the presence of α-syn/Syn III co-aggregation. This evidence holds significant implications for PD and ADHD therapeutic management.openopenFaustini G.; Longhena F.; Bruno A.; Bono F.; Grigoletto J.; La Via L.; Barbon A.; Casiraghi A.; Straniero V.; Valoti E.; Costantino G.; Benfenati F.; Missale C.; Pizzi M.; Spillantini M.G.; Bellucci A.Faustini, G.; Longhena, F.; Bruno, A.; Bono, F.; Grigoletto, J.; La Via, L.; Barbon, A.; Casiraghi, A.; Straniero, V.; Valoti, E.; Costantino, G.; Benfenati, F.; Missale, C.; Pizzi, M.; Spillantini, M. G.; Bellucci, A