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Objective: Volume status assessment of a patient by ultrasound (US) imaging of the inferior vena cava (IVC) is important for the diagnosis and prognosis of various clinical conditions. In order to improve the clinical investigation of IVC, which is mainly based on unidirectional US (in M-mode), automated processing of 2-D US scans (in B-mode) has enabled tissue movement tracking on the visualized plane and can average this in various directions. However, IVC geometry outside of the visualized plane is not under control and could result in errors that have not yet been evaluated., Methods: We used a method that integrates information from long- and short-axis IVC views (simultaneously acquired in the X-plane) to assess challenges in IVC diameter estimations using 2-D US scans in eight healthy subjects., Results: Relative movements between the US probe and IVC induced the following problems when assessing IVC diameter via 2-D view: a median error (i.e., absolute difference with respect to diameter measured in the X-plane) of 17% using 2-D US scans in the long-axis view of the IVC affected by medio-lateral displacements (median: 4 mm); and a median error of 7% and 9% when measuring the IVC diameter from a short-axis view in the presence of pitch angle (median: 0.12 radians) and cranio-caudal movement (median: 15 mm), respectively., Conclusion: Relative movements in the IVC that are out of view of B-mode scans cannot be detected, which results in challenges in IVC diameter estimation., Competing Interests: Conflict of interest An instrument implementing the 2-D algorithms used in this paper was patented by Politecnico di Torino and Università di Torino (patent number WO 2018/134726) and was taken in license by VIPER s.r.l., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Dendritic cells (DCs) are uniquely capable of transporting tumor antigens to tumor-draining lymph nodes (tdLNs) and interact with effector T cells in the tumor microenvironment (TME) itself, mediating both natural antitumor immunity and the response to checkpoint blockade immunotherapy. Using LIPSTIC (Labeling Immune Partnerships by SorTagging Intercellular Contacts)-based single-cell transcriptomics, we identified individual DCs capable of presenting antigen to CD4 + T cells in both the tdLN and TME. Our findings revealed that DCs with similar hyperactivated transcriptional phenotypes interact with helper T cells both in tumors and in the tdLN and that checkpoint blockade drugs enhance these interactions. These findings show that a relatively small fraction of DCs is responsible for most of the antigen presentation in the tdLN and TME to both CD4 + and CD8 + tumor-specific T cells and that classical checkpoint blockade enhances CD40-driven DC activation at both sites.

Re-exposure to an antigen generates abundant antibody responses and drives the formation of secondary germinal centers (GCs). Recall GCs in mice consist almost entirely of naïve B cells, whereas recall antibodies derive overwhelmingly from memory B cells. Here, we examine this division between cellular and serum compartments. After repeated immunization with the same antigen, tetramer analyses of recall GCs revealed a marked decrease in the ability of B cells in these structures to bind the antigen. Boosting with viral variant proteins restored antigen binding in recall GCs, as did genetic ablation of primary-derived antibody-secreting cells through conditional deletion of Prdm1, demonstrating suppression of GC recall responses by pre-existing antibodies. In hapten-carrier experiments in which B and T cell specificities were uncoupled, memory T cell help allowed B cells with undetectable antigen binding to access GCs. Thus, antibody-mediated feedback steers recall GC B cells away from previously targeted epitopes and enables specific targeting of variant epitopes, with implications for vaccination protocols., Competing Interests: Declaration of interests G.D.V. is a scientific advisor for Vaccine Company, Inc., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Sustained attention is pivotal for tasks like studying and working for which focus and low distractions are necessary for peak productivity. This study explores the effectiveness of adaptive transcranial direct current stimulation (tDCS) in either the frontal or parietal region to enhance sustained attention. The research involved ten healthy university students performing the Continuous Performance Task-AX (AX-CPT) while receiving either frontal or parietal tDCS. The study comprised three phases. First, we acquired the electroencephalography (EEG) signal to identify the most suitable metrics related to attention states. Among different spectral and complexity metrics computed on 3 s epochs of EEG, the Fuzzy Entropy and Multiscale Sample Entropy Index of frontal channels were selected. Secondly, we assessed how tDCS at a fixed 1.0 mA current affects attentional performance. Finally, a real-time experiment involving continuous metric monitoring allowed personalized dynamic optimization of the current amplitude and stimulation site (frontal or parietal). The findings reveal statistically significant improvements in mean accuracy (94.04 vs. 90.82%) and reaction times (262.93 vs. 302.03 ms) with the adaptive tDCS compared to a non-stimulation condition. Average reaction times were statistically shorter during adaptive stimulation compared to a fixed current amplitude condition (262.93 vs. 283.56 ms), while mean accuracy stayed similar (94.04 vs. 93.36%, improvement not statistically significant). Despite the limited number of subjects, this work points out the promising potential of adaptive tDCS as a tailored treatment for enhancing sustained attention.

Objective. The wide detection volume of surface electromyogram (EMG) makes it prone to crosstalk, i.e. the signal from other muscles than the target one. Removing this perturbation from bipolar recordings is an important open problem for many applications. Approach. An innovative nonlinear spatio-temporal filter is developed to estimate the EMG generated by the target muscle by processing noisy signals from two bipolar channels, placed over the target and the crosstalk muscle, respectively. The filter is trained on some calibration data and then can be applied on new signals. Tests are provided in simulations (considering different thicknesses of the subcutaneous tissue, inter-electrode distances, locations of the EMG channels, force levels) and experiments (from pronator teres and flexor carpi radialis of 8 healthy subjects). Main results. The proposed filter allows to reduce the effect of crosstalk in all investigated conditions, with a statistically significant reduction of its root mean squared of about 20%, both in simulated and experimental data. Its performances are also superior to those of a blind source separation method applied to the same data. Significance. The proposed filter is simple to be applied and feasible in applications in which single bipolar channels are placed over the muscles of interest. It can be useful in many fields, such as in gait analysis, tests of myoelectric fatigue, rehabilitation with EMG biofeedback, clinical studies, prosthesis control., (Creative Commons Attribution license.)

The inferior vena cava (IVC) is the largest vein in the body. It returns deoxygenated blood to the heart from the tissues placed under the diaphragm. The size and dynamics of the IVC depend on the blood volume and right atrial pressure, which are important indicators of a patient's hydration and reflect possible pathological conditions. Ultrasound (US) assessment of the IVC is a promising technique for evaluating these conditions, because it is fast, non-invasive, inexpensive, and without side effects. However, the standard M-mode approach for measuring IVC diameter is prone to errors due to the vein movements during respiration. B-mode US produces two-dimensional images that better capture the IVC shape and size. In this review, we discuss the pros and cons of current IVC segmentation techniques for B-mode longitudinal and transverse views. We also explored several scenarios where automated IVC segmentation could improve medical diagnosis and prognosis.

The aging process is a multifaceted phenomenon that affects cognitive-affective and physical functioning as well as interactions with the environment. Although subjective cognitive decline may be part of normal aging, negative changes objectified as cognitive impairment are present in neurocognitive disorders and functional abilities are most impaired in patients with dementia. Electroencephalography-based brain-machine interfaces (BMI) are being used to assist older people in their daily activities and to improve their quality of life with neuro-rehabilitative applications. This paper provides an overview of BMI used to assist older adults. Both technical issues (detection of signals, extraction of features, classification) and application-related aspects with respect to the users' needs are considered.

Understanding how different areas of the human brain communicate with each other is a crucial issue in neuroscience. The concepts of structural, functional and effective connectivity have been widely exploited to describe the human connectome, consisting of brain networks, their structural connections and functional interactions. Despite high-spatial-resolution imaging techniques such as functional magnetic resonance imaging (fMRI) being widely used to map this complex network of multiple interactions, electroencephalographic (EEG) recordings claim high temporal resolution and are thus perfectly suitable to describe either spatially distributed and temporally dynamic patterns of neural activation and connectivity. In this work, we provide a technical account and a categorization of the most-used data-driven approaches to assess brain-functional connectivity, intended as the study of the statistical dependencies between the recorded EEG signals. Different pairwise and multivariate, as well as directed and non-directed connectivity metrics are discussed with a pros-cons approach, in the time, frequency, and information-theoretic domains. The establishment of conceptual and mathematical relationships between metrics from these three frameworks, and the discussion of novel methodological approaches, will allow the reader to go deep into the problem of inferring functional connectivity in complex networks. Furthermore, emerging trends for the description of extended forms of connectivity (e.g., high-order interactions) are also discussed, along with graph-theory tools exploring the topological properties of the network of connections provided by the proposed metrics. Applications to EEG data are reviewed. In addition, the importance of source localization, and the impacts of signal acquisition and pre-processing techniques (e.g., filtering, source localization, and artifact rejection) on the connectivity estimates are recognized and discussed. By going through this review, the reader could delve deeply into the entire process of EEG pre-processing and analysis for the study of brain functional connectivity and learning, thereby exploiting novel methodologies and approaches to the problem of inferring connectivity within complex networks.

The protective efficacy of serum antibodies results from the interplay of antigen-specific B cell clones of different affinities and specificities. These cellular dynamics underlie serum-level phenomena such as original antigenic sin (OAS)-a proposed propensity of the immune system to rely repeatedly on the first cohort of B cells engaged by an antigenic stimulus when encountering related antigens, in detriment to the induction of de novo responses 1-5 . OAS-type suppression of new, variant-specific antibodies may pose a barrier to vaccination against rapidly evolving viruses such as influenza and SARS-CoV-2 6,7 . Precise measurement of OAS-type suppression is challenging because cellular and temporal origins cannot readily be ascribed to antibodies in circulation; its effect on subsequent antibody responses therefore remains unclear 5,8 . Here we introduce a molecular fate-mapping approach with which serum antibodies derived from specific cohorts of B cells can be differentially detected. We show that serum responses to sequential homologous boosting derive overwhelmingly from primary cohort B cells, while later induction of new antibody responses from naive B cells is strongly suppressed. Such 'primary addiction' decreases sharply as a function of antigenic distance, allowing reimmunization with divergent viral glycoproteins to produce de novo antibody responses targeting epitopes that are absent from the priming variant. Our findings have implications for the understanding of OAS and for the design and testing of vaccines against evolving pathogens., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

The aim of this work was to monitor the effects of extracellular α-synuclein on the firing activity of midbrain neurons dissociated from substantia nigra TH-GFP mice embryos and cultured on microelectrode arrays (MEA). We monitored the spontaneous firing discharge of the network for 21 days after plating and the role of glutamatergic and GABAergic inputs in regulating burst generation and network synchronism. Addition of GABA A , AMPA and NMDA antagonists did not suppress the spontaneous activity but allowed to identify three types of neurons that exhibited different modalities of firing and response to applied L-DOPA: high-rate (HR) neurons, low-rate pacemaking (LR-p), and low-rate non-pacemaking (LR-np) neurons. Most HR neurons were insensitive to L-DOPA, while the majority of LR-p neurons responded with a decrease of the firing discharge; less defined was the response of LR-np neurons. The effect of exogenous α-synuclein (α-syn) on the firing discharge of midbrain neurons was then studied by varying the exposure time (0-48 h) and the α-syn concentration (0.3-70 μM), while the formation of α-syn oligomers was monitored by means of AFM. Independently of the applied concentration, acute exposure to α-syn monomers did not exert any effect on the spontaneous firing rate of HR, LR-p, and LR-np neurons. On the contrary, after 48 h exposure, the firing activity was drastically altered at late developmental stages (14 days in vitro , DIV, neurons): α-syn oligomers progressively reduced the spontaneous firing discharge (IC 50 = 1.03 μM), impaired burst generation and network synchronism, proportionally to the increased oligomer/monomer ratio. Different effects were found on early-stage developed neurons (9 DIV), whose firing discharge remained unaltered, regardless of the applied α-syn concentration and the exposure time. Our findings unravel, for the first time, the variable effects of exogenous α-syn at different stages of midbrain network development and provide new evidence for the early detection of neuronal function impairment associated to aggregated forms of α-syn., 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 © 2023 Tomagra, Franchino, Cesano, Chiarion, de lure, Carbone, Calabresi, Mesin, Picconi, Marcantoni and Carabelli.)

Germinal centers (GCs) form in secondary lymphoid organs in response to infection and immunization and are the source of affinity-matured B cells. The duration of GC reactions spans a wide range, and long-lasting GCs (LLGCs) are potentially a source of highly mutated B cells. We show that rather than consisting of continuously evolving B cell clones, LLGCs elicited by influenza virus or SARS-CoV-2 infection in mice are sustained by progressive replacement of founder clones by naive-derived invader B cells that do not detectably bind viral antigens. Rare founder clones that resist replacement for long periods are enriched in clones with heavily mutated immunoglobulins, including some with very high affinity for antigen, that can be recalled by boosting. Our findings reveal underappreciated aspects of the biology of LLGCs generated by respiratory virus infection and identify clonal replacement as a potential constraint on the development of highly mutated antibodies within these structures., Competing Interests: Declaration of interests G.D.V. is an advisor for Vaccine Company, Inc., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Antibody responses are characterized by increasing affinity and diversity over time. Affinity maturation occurs in germinal centers by a mechanism that involves repeated cycles of somatic mutation and selection. How antibody responses diversify while also undergoing affinity maturation is not as well understood. Here, we examined germinal center (GC) dynamics by tracking B cell entry, division, somatic mutation, and specificity. Our experiments show that naive B cells continuously enter GCs where they compete for T cell help and undergo clonal expansion. Consistent with late entry, invaders carry fewer mutations but can contribute up to 30% or more of the cells in late-stage germinal centers. Notably, cells entering the germinal center at later stages of the reaction diversify the immune response by expressing receptors that show low affinity to the immunogen. Paradoxically, the affinity threshold for late GC entry is lowered in the presence of high-affinity antibodies., Competing Interests: Declaration of interests The Rockefeller University has filed a provisional patent application in connection with C135 and C144 antibodies used in this work, on which M.C.N. is an inventor (US patent 63/021,387)., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Efficient mouse models to study SARS-CoV-2 infection are critical for the development and assessment of vaccines and therapeutic approaches to mitigate the current pandemic and prevent reemergence of COVID-19. While the first generation of mouse models allowed SARS-CoV-2 infection and pathogenesis, they relied on ectopic expression and non-physiological levels of human angiotensin-converting enzyme 2 (hACE2). Here we generated a mouse model carrying the minimal set of modifications necessary for productive infection with multiple strains of SARS-CoV-2. Substitution of only three amino acids in the otherwise native mouse Ace2 locus ( Ace2 TripleMutant or Ace2 ™), was sufficient to render mice susceptible to both SARS-CoV-2 strains USA-WA1/2020 and B.1.1.529 (Omicron). Infected Ace2 ™ mice exhibited weight loss and lung damage and inflammation, similar to COVID-19 patients. Previous exposure to USA-WA1/2020 or mRNA vaccination generated memory B cells that participated in plasmablast responses during breakthrough B.1.1.529 infection. Thus, the Ace2 ™ mouse replicates human disease after SARS-CoV-2 infection and provides a tool to study immune responses to sequential infections in mice., Competing Interests: NP is named on a patent describing the use of nucleoside-modified mRNA in lipid nanoparticles as a vaccine platform. He has disclosed those interests fully to the University of Pennsylvania and has an approved plan in place for managing any potential conflicts arising from the licensing of that patent. PJCL is an employee of Acuitas Therapeutics, a company involved in the development of mRNA-LNP therapeutics. GDV is a scientific advisor for Vaccine Company, Inc. 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 © 2022 Nakandakari-Higa, Parsa, Reis, de Carvalho, Mesin, Hoffmann, Bortolatto, Muramatsu, Lin, Bilate, Rice, Pardi, Mucida, Victora and Canesso.)

Objectives: Inferior vena cava (IVC) pulsatility quantified by the Caval Index (CI) is characterized by poor reliability, also due to the irregular magnitude of spontaneous respiratory activity generating the major pulsatile component. The aim of this study was to test whether the IVC cardiac oscillatory component could provide a more stable index (Cardiac CI-CCI) compared to CI or respiratory CI (RCI)., Methods: Nine healthy volunteers underwent long-term monitoring in supine position of IVC, followed by 3 minutes passive leg raising (PLR). CI, RCI, and CCI were extracted from video recordings by automated edge-tracking and CCI was averaged over each respiratory cycle (aCCI). Cardiac output (CO), mean arterial pressure (MAP) and heart rate (HR) were also recorded during baseline (1 minutes prior to PLR) and PLR (first minute)., Results: In response to PLR, all IVC indices decreased (P < .01), CO increased by 4 ± 4% (P = .055) while HR and MAP did not vary. The Coefficient of Variation (CoV) of aCCI (13 ± 5%) was lower than that of CI (17 ± 5%, P < .01), RCI (26 ± 7%, P < .001) and CCI (25 ± 7%, P < .001). The mutual correlations in time of the indices were 0.81 (CI-RCI), 0.49 (CI-aCCI) and 0.2 (RCI-aCCI)., Conclusions: Long-term IVC monitoring by automated edge-tracking allowed us to evidence that 1) respiratory and averaged cardiac pulsatility components are uncorrelated and thus carry different information and 2) the new index aCCI, exhibiting the lowest CoV while maintaining good sensitivity to blood volume changes, may overcome the poor reliability of CI and RCI., (© 2021 American Institute of Ultrasound in Medicine.)

Aortic stiffness is an important clinical parameter for predicting cardiovascular events. Carotid-femoral pulse wave velocity (cf-PWV) has been proposed for performing this evaluation non-invasively; however, it requires dedicated equipment and experienced operators. We explored the possibility of measuring aortic stiffness using ultrasound scans of the abdominal aorta coupled with the Bramwell-Hill equation. Healthy subjects were investigated; measurements of cf-PWV were taken by arterial tonometry and aortic systo-diastolic pressure difference was estimated using a validated model. Pulsatility of an abdominal tract of aorta was assessed by automated processing of ultrasound scans. Through a Bland-Altmann analysis, we found large biases when estimating each parameter by applying the Bramwell-Hill equation to the measured values of the other two paramters (bias, ± 1.96 SD; PWV, about 2.1 ± 2.5 m/s; pulsatility, 12 ± 14%; pressure jump, 47 ± 55 mmHg). These results indicate that the two measures are not interchangeable, and that a large part of the bias is attributable to blood pressure estimation. Further studies are needed to identify the possible sources of bias between cf-PWV and aortic pulsatility.

The non-invasive estimation of right atrial pressure (RAP) would be a key advancement in several clinical scenarios, in which the knowledge of central venous filling pressure is vital for patients’ management. The echocardiographic estimation of RAP proposed by Guidelines, based on inferior vena cava (IVC) size and respirophasic collapsibility, is exposed to operator and patient dependent variability. We propose novel methods, based on semi-automated edge-tracking of IVC size and cardiac collapsibility (cardiac caval index—CCI), tested in a monocentric retrospective cohort of patients undergoing echocardiography and right heart catheterization (RHC) within 24 h in condition of clinical and therapeutic stability (170 patients, age 64 ± 14, male 45%, with pulmonary arterial hypertension, heart failure, valvular heart disease, dyspnea, or other pathologies). IVC size and CCI were integrated with other standard echocardiographic features, selected by backward feature selection and included in a linear model (LM) and a support vector machine (SVM), which were cross-validated. Three RAP classes (low < 5 mmHg, intermediate 5−10 mmHg and high > 10 mmHg) were generated and RHC values used as comparator. LM and SVM showed a higher accuracy than Guidelines (63%, 71%, and 61% for LM, SVM, and Guidelines, respectively), promoting the integration of IVC and echocardiographic features for an improved non-invasive estimation of RAP.

Surface electromyography (sEMG) has been the subject of thousands of scientific articles, but many barriers limit its clinical applications. Previous work has indicated that the lack of time, competence, training, and teaching is the main barrier to the clinical application of sEMG. This work follows up and presents a number of analogies, metaphors, and simulations using physical and mathematical models that provide tools for teaching sEMG detection by means of electrode pairs (1D signals) and electrode grids (2D and 3D signals). The basic mechanisms of sEMG generation are summarized and the features of the sensing system (electrode location, size, interelectrode distance, crosstalk, etc.) are illustrated (mostly by animations) with examples that teachers can use. The most common, as well as some potential, applications are illustrated in the areas of signal presentation, gait analysis, the optimal injection of botulinum toxin, neurorehabilitation, ergonomics, obstetrics, occupational medicine, and sport sciences. The work is primarily focused on correct sEMG detection and on crosstalk. Issues related to the clinical transfer of innovations are also discussed, as well as the need for training new clinical and/or technical operators in the field of sEMG.

Objective: Myoelectric control requires fast and stable identification of a movement from data recorded from a comfortable and straightforward system., Methods: We consider a new real-time pre-processing method applied to a single differential surface electromyogram (EMG): deconvolution, providing an estimation of the cumulative firings of motor units. A 2 channel-10 class finger movement problem has been investigated on 10 healthy subjects. We have compared raw EMG and deconvolution signals, as sources of information for two specific classifiers (based on either Support Vector Machines or k-Nearest Neighbours), with classical time-domain input features selected using Mutual Component Analysis., Results: Using the proposed pre-processing technique, classification performances statistically improve. For example, the true positive rates of the best-tested configurations were 80.9% and 86.3% when using the EMG and its deconvoluted signal, respectively., Conclusion: Even considering the limited dataset and range of classification approaches investigated, our preliminary results indicate the potential usefulness of the deconvolution pre-processing., Significance: Deconvolution of EMG is a fast pre-processing that could be easily embedded in different myoelectric control applications.

B cells, which are critical for intestinal homeostasis, remain understudied in ulcerative colitis (UC). In this study, we recruited three cohorts of patients with UC (primary cohort, n = 145; validation cohort 1, n = 664; and validation cohort 2, n = 143) to comprehensively define the landscape of B cells during UC-associated intestinal inflammation. Using single-cell RNA sequencing, single-cell IgH gene sequencing and protein-level validation, we mapped the compositional, transcriptional and clonotypic landscape of mucosal and circulating B cells. We found major perturbations within the mucosal B cell compartment, including an expansion of naive B cells and IgG + plasma cells with curtailed diversity and maturation. Furthermore, we isolated an auto-reactive plasma cell clone targeting integrin αvβ6 from inflamed UC intestines. We also identified a subset of intestinal CXCL13-expressing TFH-like T peripheral helper cells that were associated with the pathogenic B cell response. Finally, across all three cohorts, we confirmed that changes in intestinal humoral immunity are reflected in circulation by the expansion of gut-homing plasmablasts that correlates with disease activity and predicts disease complications. Our data demonstrate a highly dysregulated B cell response in UC and highlight a potential role of B cells in disease pathogenesis., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Background: Freezing of Gait (FOG) is one of the most disabling motor complications of Parkinson's disease, and consists of an episodic inability to move forward, despite the intention to walk. FOG increases the risk of falls and reduces the quality of life of patients and their caregivers. The phenomenon is difficult to appreciate during outpatients visits; hence, its automatic recognition is of great clinical importance. Many types of sensors and different locations on the body have been proposed. However, the advantages of a multi-sensor configuration with respect to a single-sensor one are not clear, whereas this latter would be advisable for use in a non-supervised environment., Methods: In this study, we used a multi-modal dataset and machine learning algorithms to perform different classifications between FOG and non-FOG periods. Moreover, we explored the relevance of features in the time and frequency domains extracted from inertial sensors, electroencephalogram and skin conductance. We developed both a subject-independent and a subject-dependent algorithm, considering different sensor subsets., Results: The subject-independent and subject-dependent algorithms yielded accuracies of 85% and 88% in the leave-one-subject-out and leave-one-task-out test, respectively. Results suggest that the inertial sensors positioned on the lower limb are generally the most significant in recognizing FOG. Moreover, the performance impairment experienced when using a single tibial accelerometer instead of the optimal multi-modal configuration is limited to 2-3%., Conclusions: The achieved results disclose the possibility of getting a good FOG recognition using a minimally invasive set-up made of a single inertial sensor. This is very significant in the perspective of implementing a long-term monitoring of patients in their homes, during activities of daily living.

The influence of large vessels on near infrared spectroscopy (NIRS) measurement is generally considered negligible. Aim of this study is to test the hypothesis that changes in the vessel size, by varying the amount of absorbed NIR light, could profoundly affect NIRS blood volume indexes. Changes in haemoglobin concentration (tHb) and in tissue haemoglobin index (THI) were monitored over the basilic vein (BV) and over the biceps muscle belly, in 11 subjects (7 M - 4 F; age 31 ± 8 year) with simultaneous ultrasound monitoring of BV size. The arm was subjected to venous occlusion, according to two pressure profiles: slow (from 0 to 60 mmHg in 135 s) and rapid (0 to 40 mmHg maintained for 30 s). Both tHb and THI detected a larger blood volume increase (1.7 to 4 fold; p < 0.01) and exhibited a faster increase and a greater convexity on the BV than on the muscle. In addition, NIRS signals from BV exhibited higher correlation with changes in BV size than from muscle (r = 0.91 vs 0.55, p < 0.001 for THI). A collection of individual relevant recordings is also included. These results challenge the long-standing belief that the NIRS measurement is unaffected by large vessels and support the concept that large veins may be a major determinant of blood volume changes in multiple experimental conditions., (© 2022. The Author(s).)

Ultrasound (US)-based measurements of the inferior vena cava (IVC) diameter are widely used to estimate right atrial pressure (RAP) in a variety of clinical settings. However, the correlation with invasively measured RAP along with the reproducibility of US-based IVC measurements is modest at best. In the present manuscript, we discuss the limitations of the current technique to estimate RAP through IVC US assessment and present a new promising tool developed by our research group, the automated IVC edge-to-edge tracking system, which has the potential to improve RAP assessment by transforming the current categorical classification (low, normal, high RAP) in a continuous and precise RAP estimation technique. Finally, we critically evaluate all the clinical settings in which this new tool could improve current practice.

Assessment of vascular size and of its phasic changes by ultrasound is important for the management of many clinical conditions. For example, a dilated and stiff inferior vena cava reflects increased intravascular volume and identifies patients with heart failure at greater risk of an early death. However, lack of standardization and sub-optimal intra- and inter- operator reproducibility limit the use of these techniques. To overcome these limitations, we developed two image-processing algorithms that quantify phasic vascular deformation by tracking wall movements, either in long or in short axis. Prospective studies will verify the clinical applicability and utility of these methods in different settings, vessels and medical conditions., Competing Interests: RB was employed by company Chirurgia Generale e Trauma Team GOM Niguarda. 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 © 2022 Mesin, Albani, Policastro, Pasquero, Porta, Melchiorri, Leonardi, Albera, Scacciatella, Pellicori, Stolfo, Grillo, Fabris, Bini, Giannoni, Pepe, Ermini, Seddone, Sinagra, Antonini-Canterin and Roatta.)

A deconvolution method is proposed for conduction block (CB) estimation based on two compound muscle action potentials (CMAPs) elicited by stimulating a nerve proximal and distal to the region in which the block is suspected. It estimates the time delay distributions by CMAPs deconvolution, from which CB is computed. The slow afterwave (SAW) is included to describe the motor unit potential, as it gives an important contribution in case of the large temporal dispersion (TD) often found in patients. The method is tested on experimental signals obtained from both healthy subjects and pathological patients, with either Chronic Inflammatory Demyelinating Polyneuropathy (CIDP) or Multifocal Motor Neuropathy (MMN). The new technique outperforms the clinical methods (based on amplitude and area of CMAPs) and a previous state-of-the-art deconvolution approach. It compensates phase cancellations, allowing to discriminate among CB and TD: estimated by the methods of amplitude, area and deconvolution, CB showed a correlation with TD equal to 39.3%, 29.5% and 8.2%, respectively. Moreover, a significant decrease of percentage reconstruction errors of the CMAPs with respect to the previous deconvolution approach is obtained (from a mean/median of 19.1%/16.7% to 11.7%/11.2%). Therefore, the new method is able to discriminate between CB and TD (overcoming the important limitation of clinical approaches) and can approximate patients' CMAPs better than the previous deconvolution algorithm. Then, it appears to be promising for the diagnosis of demyelinating polyneuropathies, to be further tested in the future in a prospective clinical trial.

Besides antigen-specific responses to viral antigens, humoral immune response in virus infection can generate polyreactive and autoreactive antibodies. Dengue and Zika virus infections have been linked to antibody-mediated autoimmune disorders, including Guillain-Barré syndrome. A unique feature of flaviviruses is the secretion of nonstructural protein 1 (NS1) by infected cells. NS1 is highly immunogenic, and antibodies targeting NS1 can have both protective and pathogenic roles. In the present study, we investigated the humoral immune response to Zika virus NS1 and found NS1 to be an immunodominant viral antigen associated with the presence of autoreactive antibodies. Through single B cell cultures, we coupled binding assays and BCR sequencing, confirming the immunodominance of NS1. We demonstrate the presence of self-reactive clones in germinal centers after both infection and immunization, some of which present cross-reactivity with NS1. Sequence analysis of anti-NS1 B cell clones showed sequence features associated with pathogenic autoreactive antibodies. Our findings demonstrate NS1 immunodominance at the cellular level as well as a potential role for NS1 in ZIKV-associated autoimmune manifestations., Competing Interests: Disclosures: The authors declare no competing financial interests., (© 2021 Cavazzoni et al.)

Germinal centers (GCs) are the site of immunoglobulin somatic hypermutation and affinity maturation, processes essential to an effective antibody response. The formation of GCs has been studied in detail, but less is known about what leads to their regression and eventual termination, factors that ultimately limit the extent to which antibodies mature within a single reaction. We show that contraction of immunization-induced GCs is immediately preceded by an acute surge in GC-resident Foxp3 + T cells, attributed at least partly to up-regulation of the transcription factor Foxp3 by T follicular helper (T FH ) cells. Ectopic expression of Foxp3 in T FH cells is sufficient to decrease GC size, implicating the natural up-regulation of Foxp3 by T FH cells as a potential regulator of GC lifetimes., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

During affinity maturation, germinal center (GC) B cells alternate between proliferation and somatic hypermutation in the dark zone (DZ) and affinity-dependent selection in the light zone (LZ). This anatomical segregation imposes that the vigorous proliferation that allows clonal expansion of positively selected GC B cells takes place ostensibly in the absence of the signals that triggered selection in the LZ, as if by "inertia." We find that such inertial cycles specifically require the cell cycle regulator cyclin D3. Cyclin D3 dose-dependently controls the extent to which B cells proliferate in the DZ and is essential for effective clonal expansion of GC B cells in response to strong T follicular helper (Tfh) cell help. Introduction into the Ccnd3 gene of a Burkitt lymphoma-associated gain-of-function mutation (T283A) leads to larger GCs with increased DZ proliferation and, in older mice, clonal B cell lymphoproliferation, suggesting that the DZ inertial cell cycle program can be coopted by B cells undergoing malignant transformation., Competing Interests: Disclosures: A. Melnick reports grants from Janssen, Sanofi, and Daiichi Sankyo, and personal fees from KDAC, Epizyme, Constellation, Jubilant, and ExoTherapeutics outside the submitted work. J. Ordovas-Montanes reports personal fees from Cellarity outside the submitted work. No other disclosures were reported., (© 2020 Pae et al.)

A new approach is introduced for non-rigid registration of a pair of magnetic resonance images (MRI). It is a generalization of the demons algorithm with low computational cost, based on local information augmentation (by integrating multiple images) and balanced implementation. Specifically, a single deformation that best registers more pairs of images is estimated. All these images are extracted by applying different operators to the two original ones, processing local neighbors of each pixel. The following five images were found to be appropriate for MRI registration: the raw image and those obtained by contrast-limited adaptive histogram equalization, local median, local entropy and phase symmetry. Thus, each local point in the images is supplemented by augmented information coming by processing its neighbor. Moreover, image pairs are processed in alternation for each iteration of the algorithm (in a balanced way), computing both a forward and a backward registration. The new method (called balanced multi-image demons) is tested on sagittal MRIs from 10 patients, both in simulated and experimental conditions, improving the performances over the classical demons approach with minimal increase of the computational cost (processing time around twice that of standard demons). Specifically, a simulated deformation was applied to the MRIs (either original or corrupted by additive Gaussian or speckle noises). In all tested cases, the new algorithm improved the estimation of the simulated deformation (squared estimation error decreased by about 65% in the average). Moreover, statistically significant improvements were obtained in experimental tests, in which different brain regions (i.e., brain, posterior fossa and cerebellum) were identified by the atlas approach and compared to those manually delineated (in the average, Dice coefficient increased of about 6%). The conclusion is that a balanced method applied to multiple information extracted from neighboring pixels is a low cost approach to improve registration of MRIs., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Germinal centres, the structures in which B cells evolve to produce antibodies with high affinity for various antigens, usually form transiently in lymphoid organs in response to infection or immunization. In lymphoid organs associated with the gut, however, germinal centres are chronically present. These gut-associated germinal centres can support targeted antibody responses to gut infections and immunization 1 . But whether B cell selection and antibody affinity maturation take place in the face of the chronic and diverse antigenic stimulation characteristic of these structures under steady state is less clear 2-8 . Here, by combining multicolour 'Brainbow' cell-fate mapping and sequencing of immunoglobulin genes from single cells, we find that 5-10% of gut-associated germinal centres from specific-pathogen-free (SPF) mice contain highly dominant 'winner' B cell clones at steady state, despite rapid turnover of germinal-centre B cells. Monoclonal antibodies derived from these clones show increased binding, compared with their unmutated precursors, to commensal bacteria, consistent with antigen-driven selection. The frequency of highly selected gut-associated germinal centres is markedly higher in germ-free than in SPF mice, and winner B cells in germ-free germinal centres are enriched in 'public' clonotypes found in multiple individuals, indicating strong selection of B cell antigen receptors even in the absence of microbiota. Colonization of germ-free mice with a defined microbial consortium (Oligo-MM 12 ) does not eliminate germ-free-associated clonotypes, yet does induce a concomitant commensal-specific B cell response with the hallmarks of antigen-driven selection. Thus, positive selection of B cells can take place in steady-state gut-associated germinal centres, at a rate that is tunable over a wide range by the presence and composition of the microbiota.

The pulsatility of the inferior vena cava (IVC) reflects the volume status of patients. It can be investigated by ultrasounds (US), offering an important non-invasive tool supporting fluid management. However, the method has limitations attributable to many confounding factors, e.g., related to IVC movements and non-regular shapes. Short- or long-axis views have been used, both having advantages and limitations in counteracting such confounding factors, depending on the specific condition. The aim of this study is to investigate IVC pulsatility in the different directions on the transverse plane and to assess its variability. Moreover, different components of this pulsatility (induced by either respiratory or cardiac activity) are investigated. The method is tested on 10 healthy patients, with large variations across them of IVC section (mean diameters in the range 1 cm to 3 cm), shape and pulsatility (average caval index [CI] ranging from approximately 20% to 70%). The average coefficient of variation of the CI estimated on 10 different directions was 13% (21% and 20% for the respiratory and cardiac components, respectively), with a range that was approximately 50% of the mean CI across different directions (approximately the same for the 2 different components). The minimum and maximum CI were found close to the directions of maximum and minimum IVC diameter, respectively. The investigation of IVC dynamics in the entire cross-section is crucial to obtain a more repeatable and reliable characterization of IVC pulsatility. The calculation of a CI based on the "equivalent" diameter (proportional to the square root of the IVC cross-sectional area) is encouraged., (Copyright © 2020 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

The gut epithelium is populated by intraepithelial lymphocytes (IELs), a heterogeneous T cell population with cytotoxic and regulatory properties, which can be acquired at the epithelial layer. However, the role of T cell receptor (TCR) in this process remains unclear. Single-cell transcriptomic analyses revealed distinct clonal expansions between cell states, with CD4 + CD8αα + IELs being one of the least diverse populations. Conditional deletion of TCR on differentiating CD4 + T cells or of major histocompatibility complex (MHC) class II on intestinal epithelial cells prevented CD4 + CD8αα + IEL differentiation. However, TCR ablation on differentiated CD4 + CD8αα + IELs or long-term cognate antigen withdraw did not affect their maintenance. TCR re-engagement of antigen-specific CD4 + CD8αα + IELs by Listeria monocytogenes did not alter their state but correlated with reduced bacterial invasion. Thus, local antigen recognition is an essential signal for differentiation of CD4 + T cells at the epithelium, yet differentiated IELs are able to preserve an effector program in the absence of TCR signaling., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The herniation of cerebellum through the foramen magnum may block the normal flow of cerebrospinal fluid determining a severe disorder called Chiari I Malformation (CM-I). Different surgical options are available to help patients, but there is no standard to select the optimal treatment. This paper proposes a fully automated method to select the optimal intervention. It is based on morphological parameters of the brain, posterior fossa and cerebellum, estimated by processing sagittal magnetic resonance images (MRI). The processing algorithm is based on a non-rigid registration by a balanced multi-image generalization of demons method. Moreover, a post-processing based on active contour was used to improve the estimation of cerebellar hernia. This method allowed to delineate the boundaries of the regions of interest with a percentage of agreement with the delineation of an expert of about 85%. Different features characterizing the estimated regions were then extracted and used to develop a classifier to identify the optimal surgical treatment. Classification accuracy on a database of 50 patients was about 92%, with a predictive value of 88% (tested with a leave-one-out approach).

Surface electromyogram (EMG) has a relatively large detection volume, so that it could include contributions both from the target muscle of interest and from nearby regions (i.e., crosstalk). This interference can prevent a correct interpretation of the activity of the target muscle, limiting the use of surface EMG in many fields. To counteract the problem, selective spatial filters have been proposed, but they reduce the representativeness of the data from the target muscle. A better solution would be to discard only crosstalk from the signal recorded in monopolar configuration (thus, keeping most information on the target muscle). An inverse modelling approach is here proposed to estimate the contributions of different muscles, in order to focus on the one of interest. The method is tested with simulated monopolar EMGs from superficial nearby muscles contracted at different force levels (either including or not model perturbations and noise), showing statistically significant improvements in information extraction from the data. The median over the entire dataset of the mean squared error in representing the EMG of the muscle under the detection electrode was reduced from 11.2% to 4.4% of the signal energy (5.3% if noisy data were processed); the median bias in conduction velocity estimation (from 3 monopolar channels aligned to the muscle fibres) was decreased from 2.12 to 0.72 m/s (1.1 m/s if noisy data were processed); the median absolute error in the estimation of median frequency was reduced from 1.02 to 0.67 Hz in noise free conditions and from 1.52 to 1.45 Hz considering noisy data., Competing Interests: Declaration of Competing Interest All authors must disclose any financial and personal relationships with other people or organisations that could inappropriately influence (bias) their work. Examples of potential conflicts of interest include employment, consultancies, stock ownership, honoraria, paid expert testimony, patent applications/registrations, and grants or other funding., (Copyright © 2020 IPEM. Published by Elsevier Ltd. All rights reserved.)

Slow biphasic complexes (SBC) have been identified in the EEG of patients suffering for inflammatory brain diseases. Their amplitude, location and frequency of appearance were found to correlate with the severity of encephalitis. Other characteristics of SBCs and of EEG traces of patients could reflect the grade of pathology. Here, EEG rhythms are investigated together with SBCs for a better characterization of encephalitis. EEGs have been acquired from pediatric patients: ten controls and ten encephalitic patients. They were split by neurologists into five classes of different severity of the pathology. The relative power of EEG rhythms was found to change significantly in EEGs labeled with different severity scores. Moreover, a significant variation was found in the last seconds before the appearance of an SBC. This information and quantitative indexes characterizing the SBCs were used to build a binary classification decision tree able to identify the classes of severity. True classification rate of the best model was 76.1% (73.5% with leave-one-out test). Moreover, the classification errors were among classes with similar severity scores (precision higher than 80% was achieved considering three instead of five classes). Our classification method may be a promising supporting tool for clinicians to diagnose, assess and make the follow-up of patients with encephalitis.