Temporal Lobe Spikes Affect Distant Intrinsic Connectivity Networks

Objective: To evaluate local and distant blood oxygen level dependent (BOLD) signal changes related to interictal epileptiform discharges (IED) in drug-resistant temporal lobe epilepsy (TLE). Methods: Thirty-three TLE patients undergoing EEG–functional Magnetic Resonance Imaging (fMRI) as part of the presurgical workup were consecutively enrolled. First, a single-subject spike-related analysis was performed: (a) to verify the BOLD concordance with the presumed Epileptogenic Zone (EZ); and (b) to investigate the Intrinsic Connectivity Networks (ICN) involvement. Then, a group analysis was performed to search for common BOLD changes in TLE. Results: Interictal epileptiform discharges were recorded in 25 patients and in 19 (58%), a BOLD response was obtained at the single-subject level. In 42% of the cases, BOLD changes were observed in the temporal lobe, although only one patient had a pure concordant finding, with a single fMRI cluster overlapping (and limited to) the EZ identified by anatomo-electro-clinical correlations. In the remaining 58% of the cases, BOLD responses were localized outside the temporal lobe and the presumed EZ. In every patient, with a spike-related fMRI map, at least one ICN appeared to be involved. Four main ICNs were preferentially involved, namely, motor, visual, auditory/motor speech, and the default mode network. At the single-subject level, EEG–fMRI proved to have high specificity (above 65%) in detecting engagement of an ICN and the corresponding ictal/postictal symptom, and good positive predictive value (above 67%) in all networks except the visual one. Finally, in the group analysis of BOLD changes related to IED revealed common activations at the right precentral gyrus, supplementary motor area, and middle cingulate gyrus. Significance: Interictal temporal spikes affect several distant extra-temporal areas, and specifically the motor/premotor cortex. EEG–fMRI in patients with TLE eligible for surgery is recommended not for strictly localizing purposes rather it might be useful to investigate ICNs alterations at the single-subject level

A 3D Platform to Investigate Dynamic Cell-to-Cell Interactions Between Tumor Cells and Mesenchymal Progenitors

We here investigated the dynamic cell-to-cell interactions between tumor and mesenchymal stromal/stem cells (MSCs) by the novel VITVOⓇ 3D bioreactor that was customized to develop in vivo-like metastatic nodules of Ewing’s sarcoma (ES). MSCs are known to contribute to tumor microenvironment as cancer associated fibroblast (CAF) precursors and, for this reason, they have also been used as anti-cancer tools. Using dynamic conditions, the process of tissue colonization and formation of metastatic niches was recreated through tumor cell migration aiming to mimic ES development in patients. ES is an aggressive tumor representing the second most common malignant bone cancer in children and young adults. An urgent and unmet need exists for the development of novel treatment strategies to improve the outcomes of metastatic ES. The tumor-tropic ability of MSCs offers an alternative approach, in which these cells can be used as vehicles for the delivery of antitumor molecules, such as the proapoptotic TNF-related apoptosis inducing ligand (TRAIL). However, the therapeutic targeting of metastases remains challenging and the interaction occurring between tumor cells and MSCs has not yet been deeply investigated. Setting up in vitro and in vivo models to study this interaction is a prerequisite for novel approaches where MSCs affinity for tumor is optimized to ultimately increase their therapeutic efficacy. Here, VITVOⓇ integrating a customized scaffold with an increased inter-fiber distance (VITVO50) was used to develop a dynamic model where MSCs and tumor nodules were evaluated under flow conditions. Colonization and interaction between cell populations were explored by droplet digital PCR (ddPCR). VITVO50 findings were then applied in vivo. An ES metastatic model was established in NSG mice and biodistribution of TRAIL-expressing MSCs in mice organs affected by metastases was investigated using a 4-plex ddPCR assay. VITVOⓇ proved to be an easy handling and versatile bioreactor to develop in vivo-like tumor nodules and investigate dynamic cell-to-cell interactions with MSCs. The proposed fluidic system promises to facilitate the understanding of tumor-stroma interaction for the development of novel tumor targeting strategies, simplifying the analysis of in vivo data, and ultimately accelerating the progress towards the early clinical phase

Anti-GD2 CAR MSCs against metastatic Ewing's sarcoma

Background: Ewing's sarcoma (ES) is an aggressive cancer affecting children and young adults. We pre-clinically demonstrated that mesenchymal stromal/stem cells (MSCs) can deliver tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) against primary ES after local injection. However, ES is often metastatic calling for approaches able to support MSC targeting to the ES multiple remote sites. Considering that the disialoganglioside GD2 is expressed by ES and to optimise MSC tumour affinity, bi-functional (BF) MSCs expressing both TRAIL and a truncated anti-GD2 chimeric antigen receptor (GD2 tCAR) were generated and challenged against ES. Methods: The anti-GD2 BF MSCs delivering a soluble variant of TRAIL (sTRAIL) were tested in several in vitro ES models. Tumour targeting and killing by BF MSCs was further investigated by a novel immunodeficient ES metastatic model characterized by different metastatic sites, including lungs, liver and bone, mimicking the deadly clinical scenario. Findings: In vitro data revealed both tumour affinity and killing of BF MSCs. In vivo, GD2 tCAR molecule ameliorated the tumour targeting and persistence of BF MSCs counteracting ES in lungs but not in liver. Interpretation: We here generated data on the potential effects of BF MSCs within a complex ES metastatic in vivo model, exploring also the biodistribution of MSCs. Our BF MSC-based strategy promises to pave the way for potential improvements in the therapeutic delivery of TRAIL for the treatment of metastatic ES and other deadly GD2-positive malignancies

fMRI-Based Effective Connectivity in Surgical Remediable Epilepsies: A Pilot Study

Simultaneous EEG-fMRI can contribute to identify the epileptogenic zone (EZ) in focal epilepsies. However, fMRI maps related to Interictal Epileptiform Discharges (IED) commonly show multiple regions of signal change rather than focal ones. Dynamic causal modeling (DCM) can estimate effective connectivity, i.e. the causal effects exerted by one brain region over another, based on fMRI data. Here, we employed DCM on fMRI data in 10 focal epilepsy patients with multiple IED-related regions of BOLD signal change, to test whether this approach can help the localization process of EZ. For each subject, a family of competing deterministic, plausible DCM models were constructed using IED as autonomous input at each node, one at time. The DCM findings were compared to the presurgical evaluation results and classified as: "Concordant" if the node identified by DCM matches the presumed focus, "Discordant" if the node is distant from the presumed focus, or "Inconclusive" (no statistically significant result). Furthermore, patients who subsequently underwent intracranial EEG recordings or surgery were considered as having an independent validation of DCM results. The effective connectivity focus identified using DCM was Concordant in 7 patients, Discordant in two cases and Inconclusive in one. In four of the 6 patients operated, the DCM findings were validated. Notably, the two Discordant and Invalidated results were found in patients with poor surgical outcome. Our findings provide preliminary evidence to support the applicability of DCM on fMRI data to investigate the epileptic networks in focal epilepsy and, particularly, to identify the EZ in complex cases

Mapping the Effect of Interictal Epileptic Activity Density During Wakefulness on Brain Functioning in Focal Childhood Epilepsies With Centrotemporal Spikes

Childhood epilepsy with centrotemporal spikes (CECTS) is the most common type of \u201cself-limited focal epilepsies.\u201d In its typical presentation, CECTS is a condition reflecting non-lesional cortical hyperexcitability of rolandic regions. The benign evolution of this disorder is challenged by the frequent observation of associated neuropsychological deficits and behavioral impairment. The abundance (or frequency) of interictal centrotemporal spikes (CTS) in CECTS is considered a risk factor for deficits in cognition. Herein, we captured the hemodynamic changes triggered by the CTS density measure (i.e., the number of CTS for time bin) obtained in a cohort of CECTS, studied by means of video electroencephalophy/functional MRI during quite wakefulness. We aim to demonstrate a direct influence of the diurnal CTS frequency on epileptogenic and cognitive networks of children with CECTS. A total number of 8,950 CTS (range between 27 and 801) were recorded in 23 CECTS (21 male), with a mean number of 255 CTS/patient and a mean density of CTS/30 s equal to 10,866 \ub1 11.46. Two independent general linear model models were created for each patient based on the effect of interest: \u201cindividual CTS\u201d in model 1 and \u201cCTS density\u201d in model 2. Hemodynamic correlates of CTS density revealed the involvement of a widespread cortical\u2013subcortical network encompassing the sensory-motor cortex, the Broca's area, the premotor cortex, the thalamus, the putamen, and red nucleus, while in the CTS event-related model, changes were limited to blood\u2013oxygen-level-dependent (BOLD) signal increases in the sensory-motor cortices. A linear relationship was observed between the CTS density hemodynamic changes and both disease duration (positive correlation) and age (negative correlation) within the language network and the bilateral insular cortices. Our results strongly support the critical role of the CTS frequency, even during wakefulness, to interfere with the normal functioning of language brain networks

Mapping the Effect of Interictal Epileptic Activity Density During Wakefulness on Brain Functioning in Focal Childhood Epilepsies With Centrotemporal Spikes

Childhood epilepsy with centrotemporal spikes (CECTS) is the most common type of \u201cself-limited focal epilepsies.\u201d In its typical presentation, CECTS is a condition reflecting non-lesional cortical hyperexcitability of rolandic regions. The benign evolution of this disorder is challenged by the frequent observation of associated neuropsychological deficits and behavioral impairment. The abundance (or frequency) of interictal centrotemporal spikes (CTS) in CECTS is considered a risk factor for deficits in cognition. Herein, we captured the hemodynamic changes triggered by the CTS density measure (i.e., the number of CTS for time bin) obtained in a cohort of CECTS, studied by means of video electroencephalophy/functional MRI during quite wakefulness. We aim to demonstrate a direct influence of the diurnal CTS frequency on epileptogenic and cognitive networks of children with CECTS. A total number of 8,950 CTS (range between 27 and 801) were recorded in 23 CECTS (21 male), with a mean number of 255 CTS/patient and a mean density of CTS/30 s equal to 10,866 \ub1 11.46. Two independent general linear model models were created for each patient based on the effect of interest: \u201cindividual CTS\u201d in model 1 and \u201cCTS density\u201d in model 2. Hemodynamic correlates of CTS density revealed the involvement of a widespread cortical\u2013subcortical network encompassing the sensory-motor cortex, the Broca's area, the premotor cortex, the thalamus, the putamen, and red nucleus, while in the CTS event-related model, changes were limited to blood\u2013oxygen-level-dependent (BOLD) signal increases in the sensory-motor cortices. A linear relationship was observed between the CTS density hemodynamic changes and both disease duration (positive correlation) and age (negative correlation) within the language network and the bilateral insular cortices. Our results strongly support the critical role of the CTS frequency, even during wakefulness, to interfere with the normal functioning of language brain networks

Neoantigen-specific T-cell immune responses: The paradigm of NPM1-mutated acute myeloid leukemia

The C-terminal aminoacidic sequence from NPM1-mutated protein, absent in normal human tissues, may serve as a leukemia-specific antigen and can be considered an ideal target for NPM1-mutated acute myeloid leukemia (AML) immunotherapy. Different in silico instruments and in vitro/ex vivo immunological platforms have identified the most immunogenic epitopes from NPM1-mutated protein. Spontaneous development of endogenous NPM1-mutated-specific cytotoxic T cells has been observed in patients, potentially contributing to remission maintenance and prolonged survival. Genetically engineered T cells, namely CAR-T or TCR-transduced T cells, directed against NPM1-mutated peptides bound to HLA could prospectively represent a promising therapeutic approach. Although either adoptive or vaccine-based immunotherapies are unlikely to be highly effective in patients with full-blown leukemia, these strategies, potentially in combination with immune-checkpoint inhibitors, could be promising in maintaining remission or preemptively eradicat-ing persistent measurable residual disease, mainly in patients ineligible for allogeneic hematopoietic stem cell transplant (HSCT). Alternatively, neoantigen-specific donor lymphocyte infusion derived from healthy donors and targeting NPM1-mutated protein to selectively elicit graft-versus-leukemia effect may represent an attractive option in subjects experiencing post-HSCT relapse. Future studies are warranted to further investigate dynamics of NPM1-mutated-specific immunity and explore whether novel individualized immunotherapies may have potential clinical utility in NPM1-mutated AML patients

Motor and Limbic System Contribution to Emotional Laughter across the Lifespan

Laughter is a universal human behavior generated by the cooperation of different systems toward the construction of an expressive vocal pattern. Given the sensitivity of neuroimaging techniques to movements, the neural mechanisms underlying laughter expression remain unclear. Herein, we characterized the neural correlates of emotional laughter using the onsets and the duration of laughter bursts to inform functional magnetic resonance imaging. Laughter-related blood oxygen level-dependent (BOLD) increases involved both the motor (motor cortex, supplementary motor area, frontal operculum) and the emotional/limbic (anterior cingulate cortex, amygdala, n. accumbens, hippocampus) systems, as well as modulatory circuitries encompassing the basal ganglia, thalamus, and cerebellum. BOLD changes related to the 2 s preceding the laughter outbreak were selectively observed at the temporo-occipital junction and the periaqueductal gray matter, supporting the role of the former in the detection of incongruity and the gating role of the latter in the initiation of spontaneous laughter. Moreover, developmental changes were identified in laughter processing, consisting in a greater engagement of the reward circuitry in younger subjects; conversely, the default mode network appears more activated in older participants. Our findings contribute valuable information about the processing of real-life humorous materials and suggest a close link between laughter-related motor, affective, and cognitive elements, confirming its complex and multi-faceted nature

The neuronal network of laughing in young patients with untreated narcolepsy

Objective To investigate the neuronal correlates of spontaneous laughter in drug-naive pediatric patients with narcolepsy type I (NT1) compared to healthy controls by means of blood oxygen leveldependent (BOLD) MRI. Methods Twenty-one children/adolescents with recent onset of NT1 and 21 age- and sex-matched healthy controls were studied with fMRI while viewing funny videos using a naturalistic paradigm. Whole-brain hemodynamic correlates of spontaneous laughter were investigated in each group and compared by use of appropriate second-level general linear model analyses. If recorded, cataplexy events were treated as the effect of no interest at the single-participant level. Correlations analyses between these contrasts and behavioral findings were performed. Results Emotion-induced laughter occurred in 16 patients (294 events) and 21 controls (357 events). In controls, laughter-related BOLD increases involved a widespread cortical and subcortical network including the bilateral motor and premotor areas, cingulated cortex, insula, and amygdala. In NT1, laughter induced BOLD signal increments in the motor cortex, right thalamus, and left subthalamic nucleus/zona incerta (STN/ZI). STN/ZI and thalamic changes were significantly higher during fMRI sessions with laughter without cataplexy compared to sessions in which laughter was associated with cataplexy. Conclusion Laughter expression in individuals with NT1 involves different brain circuits compared to controls by means of overactivation of cortical and subcortical regions belonging to the volitional control of laughter. The activation of the STN/ZI region observed predominantly in patients with NT1 during laugh episodes without cataplexy suggests that the ZI could act to prevent cataplexy