Expansion of memory Vδ2 T cells following SARS-CoV-2 vaccination revealed by temporal single-cell transcriptomics.

γδ T cells provide rapid cellular immunity against pathogens. Here, we conducted matched single-cell RNA-sequencing and γδ-TCR-sequencing to delineate the molecular changes in γδ T cells during a longitudinal study following mRNA SARS-CoV-2 vaccination. While the first dose of vaccine primes Vδ2 T cells, it is the second administration that significantly boosts their immune response. Specifically, the second vaccination uncovers memory features of Vδ2 T cells, shaped by the induction of AP-1 family transcription factors and characterized by a convergent central memory signature, clonal expansion, and an enhanced effector potential. This temporally distinct effector response of Vδ2 T cells was also confirmed in vitro upon stimulation with SARS-CoV-2 spike-peptides. Indeed, the second challenge triggers a significantly higher production of IFNγ by Vδ2 T cells. Collectively, our findings suggest that mRNA SARS-CoV-2 vaccination might benefit from the establishment of long-lasting central memory Vδ2 T cells to confer protection against SARS-CoV-2 infection.

Transcriptomic profile of TNFhigh MAIT cells is linked to B cell response following SARS-CoV-2 vaccination.

Introduction: Higher frequencies of mucosal-associated invariant T (MAIT) cells were associated with an increased adaptive response to mRNA BNT162b2 SARS-CoV-2 vaccine, however, the mechanistic insights into this relationship are unknown. In the present study, we hypothesized that the TNF response of MAIT cells supports B cell activation following SARS-CoV-2 immunization. Methods: To investigate the effects of repeated SARS-CoV-2 vaccinations on the peripheral blood mononuclear cells (PBMCs), we performed a longitudinal single cell (sc)RNA-seq and scTCR-seq analysis of SARS-CoV-2 vaccinated healthy adults with two doses of the Pfizer-BioNTech BNT162b2 mRNA vaccine. Collection of PBMCs was performed 1 day before, 3 and 17 days after prime vaccination, and 3 days and 3 months following vaccine boost. Based on scRNA/TCR-seq data related to regulatory signals induced by the vaccine, we used computational approaches for the functional pathway enrichment analysis (Reactome), dynamics of the effector cell-polarization (RNA Velocity and CellRank), and cell-cell communication (NicheNet). Results: We identified MAIT cells as an important source of TNF across circulating lymphocytes in response to repeated SARS-CoV-2 BNT162b2 vaccination. The TNFhigh signature of MAIT cells was induced by the second administration of the vaccine. Notably, the increased TNF expression was associated with MAIT cell proliferation and efficient anti-SARS-CoV-2 antibody production. Finally, by decoding the ligand-receptor interactions and incorporating intracellular signaling, we predicted TNFhigh MAIT cell interplay with different B cell subsets. In specific, predicted TNF-mediated activation was selectively directed to conventional switched memory B cells, which are deputed to high-affinity long-term memory. Discussion: Overall, our results indicate that SARS-CoV-2 BNT162b2 vaccination influences MAIT cell frequencies and their transcriptional effector profile with the potential to promote B cell activation. This research also provides a blueprint for the promising use of MAIT cells as cellular adjuvants in mRNA-based vaccines.

Persistence of KIRneg NK cells after haploidentical hematopoietic stem cell transplantation protects from human cytomegalovirus infection/reactivation.

Haploidentical hematopoietic stem cell transplantation (h-HSCT) is a therapeutic option to cure patients affected by hematologic malignancies. The kinetics and the quality of immune-reconstitution (IR) impact the clinical outcome of h-HSCT and limit the onset of life-threatening Human Cytomegalovirus (HCMV) infection/reactivation. Natural Killer (NK) cells are the first lymphocytes that recover after h-HSCT and they can provide rapid innate immune responses against opportunistic pathogens. By performing a longitudinal single-cell analysis of multiparametric flow-cytometry data, we show here that the persistence at high frequencies of CD158b1b2jneg/NKG2Apos/NKG2Cneg/NKp30pos/NKp46pos (KIRneg) NK cells is associated with HCMV infection/reactivation control. These KIRneg NK cells are "unlicensed", and are not terminal-differentiated lymphocytes appearing early during IR and mainly belonging to CD56bright/CD16neg and CD56bright/CD16pos subsets. KIRneg NK cells are enriched in oxidative and glucose metabolism pathways, produce interferon-γ, and are endowed with potent antiviral activity against HCMV ex vivo. Decreased frequencies of KIRneg NK cells early during IR are associated with clinically relevant HCMV replication. Taken together, our findings indicate that the prolonged persistence of KIRneg NK cells after h-HSCT could serve as a biomarker to better predict HCMV infection/reactivation. This phenomenon also paves the way to optimize anti-viral immune responses by enriching post-transplant donor lymphocyte infusions with KIRneg NK cells.

Development of Human ILCs and Impact of Unconventional Cytotoxic Subsets in the Pathophysiology of Inflammatory Diseases and Cancer.

Innate lymphoid cells (ILCs) were firstly described by different independent laboratories in 2008 as tissue-resident innate lymphocytes mirroring the phenotype and function of T helper cells. ILCs have been subdivided into three distinct subgroups, ILC1, ILC2 and ILC3, according to their cytokine and transcriptional profiles. Subsequently, also Natural Killer (NK) cells, that are considered the innate counterpart of cytotoxic CD8 T cells, were attributed to ILC1 subfamily, while lymphoid tissue inducer (LTi) cells were attributed to ILC3 subgroup. Starting from their discovery, significant advances have been made in our understanding of ILC impact in the maintenance of tissue homeostasis, in the protection against pathogens and in tumor immune-surveillance. However, there is still much to learn about ILC ontogenesis especially in humans. In this regard, NK cell developmental intermediates which have been well studied and characterized prior to the discovery of helper ILCs, have been used to shape a model of ILC ontogenesis. Herein, we will provide an overview of the current knowledge about NK cells and helper ILC ontogenesis in humans. We will also focus on the newly disclosed circulating ILC subsets with killing properties, namely unconventional CD56dim NK cells and cytotoxic helper ILCs, by discussing their possible role in ILC ontogenesis and their contribution in both physiological and pathological conditions.

Biomechanical behavior of Hoffa's fat pad in healthy and osteoarthritic conditions: histological and mechanical investigations.

The Infrapatellar Fat Pad (IFP) lies between patella, femur, meniscus and tibia and properly fills the space between these structures. This fatty structure facilitates distribution of synovial fluid and may act to absorb impulsive actions generated through the joint. In case of Osteoarthritis (OA), IFP is found to be affected by inflammation, hypertrophy and fibrosis. The aim of the present study is to analyze the correlation between microscopic characteristics and mechanical properties of the IFP in healthy and OA conditions. The microscopic anatomy of the IFP was analyzed through histological methods, whose results showed that the IFP affected by OA maintains similar lobules configuration but thicker interlobular septa. Geometrical data together with the morphological analysis of lobules and septa represented the basic data to provide numerical micro-models of the IFP. Numerical analyses were developed to evaluate the mechanical behavior considering the characteristic loading conditions as compressive, torsion and shear actions. The results were applied to identify the parameters of a homogenized hyperelastic constitutive formulation that interprets the IFP mechanics. The constitutive formulation was implemented within a finite element model of the knee, which was applied to evaluate the overall mechanical functionality of the knee structures. The results pointed out the actual mechanical relevance of IFP and the loss of proper stress-strain behavior of the OA IFP under mechanical loads.

A Procedure for the Automatic Analysis of High-Resolution Manometry Data to Support the Clinical Diagnosis of Esophageal Motility Disorders.

OBJECTIVE: Degenerative phenomena may affect esophageal motility as a relevant social-health problem. The diagnosis of such disorders is usually performed by the analysis of data from high-resolution manometry (HRM). Inter- and intraobserver variability frequently affects the diagnosis, with potential interpretative and thus therapeutic errors, with unnecessary or worse treatments. This may be avoided with automatic procedures that minimize human intervention in data processing. METHODS: In order to support the traditional diagnostic process, an automatic procedure was defined considering a specific physiomechanical model that is able to objectively interpret data from HRM. A training set (N = 226) of healthy volunteers and pathological subjects was collected in order to define the model parameters distributions of the different groups of subjects, providing a preliminary database. A statistical algorithm was defined for an objective identification of the patient's healthy or pathological condition by comparing patient parameters with the database. RESULTS: A collection of HRMs including subjects of the training set has been built. Statistical relationships between parameters and pathologies have been established leading to a preliminary database. An automatic diagnosis procedure has been developed to compare model parameters of a specific patient with the database. The procedure was able to match the correct diagnosis up to 86% of the analyzed subjects. CONCLUSION: The success rate of the automatic procedure addresses the suitability of the developed algorithms to provide a valid support to the clinicians for the diagnostic activity. SIGNIFICANCE: The objectivity of developed tools increases the reliability of data interpretation and, consequently, patient acceptance.

A biomechanical approach to the analysis of methods and procedures of bariatric surgery.

Bariatric surgery includes a variety of procedures that are performed on obese people and aim at decreasing the intake of food and calories. This goal is usually pursued by reducing stomach capacity and/or absorbing capability. Adjustable gastric banding is the most common and successful operation. In general, bariatric surgical procedures are effective, but are often associated with major complications. Surgical procedure and post-surgical conformation of the stomach are usually defined on clinical and surgical basis only. Instead, the optimal configuration should be identified by analyzing the mechanical functionality of the stomach and the surrounding structures, and the relationship between food intake, nutrient adsorption, mechanical stimulation of stomach wall and feeling of satiety. A novel approach to bariatric surgery is required, integrating competences in the areas of biomechanics, physiology and surgery, based on a strong interaction between engineers and clinicians. Preliminary results from coupled experimental and computational investigations are here reported. The analyses aim to develop computational tools for the investigation of stomach mechanical functionality in pre- and post-surgical conformations.

Experimental investigation of the structural behavior of equine urethra.

BACKGROUND AND OBJECTIVE: An integrated experimental and computational investigation was developed aiming to provide a methodology for characterizing the structural response of the urethral duct. The investigation provides information that are suitable for the actual comprehension of lower urinary tract mechanical functionality and the optimal design of prosthetic devices. METHODS: Experimental activity entailed the execution of inflation tests performed on segments of horse penile urethras from both proximal and distal regions. Inflation tests were developed imposing different volumes. Each test was performed according to a two-step procedure. The tubular segment was inflated almost instantaneously during the first step, while volume was held constant for about 300s to allow the development of relaxation processes during the second step. Tests performed on the same specimen were interspersed by 600s of rest to allow the recovery of the specimen mechanical condition. Results from experimental activities were statistically analyzed and processed by means of a specific mechanical model. Such computational model was developed with the purpose of interpreting the general pressure-volume-time response of biologic tubular structures. The model includes parameters that interpret the elastic and viscous behavior of hollow structures, directly correlated with the results from the experimental activities. RESULTS: Post-processing of experimental data provided information about the non-linear elastic and time-dependent behavior of the urethral duct. In detail, statistically representative pressure-volume and pressure relaxation curves were identified, and summarized by structural parameters. Considering elastic properties, initial stiffness ranged between 0.677 ± 0.026kPa and 0.262 ± 0.006kPa moving from proximal to distal region of penile urethra. Viscous parameters showed typical values of soft biological tissues, as τ1=0.153±0.018s, τ2=17.458 ± 1.644s and τ1=0.201 ± 0.085, τ2= 8.514 ± 1.379s for proximal and distal regions respectively. DISCUSSION: A general procedure for the mechanical characterization of the urethral duct has been provided. The proposed methodology allows identifying mechanical parameters that properly express the mechanical behavior of the biological tube. The approach is especially suitable for evaluating the influence of degenerative phenomena on the lower urinary tract mechanical functionality. The information are mandatory for the optimal design of potential surgical procedures and devices.

Investigation of biomechanical response of Hoffa's fat pad and comparative characterization.

The infrapatellar adipose body (Hoffa's fat pad, IFP) is situated between the patellar tendon, the femoral condyle and the tibial plateau. The IFP consists of lobules of white adipose tissue delimited by thin connective septa. The actual structural functionality of the IFP is debated and should pertain to a cushioning role in the knee joint, providing to distribute and to damp mechanical stresses during articular activity. The present study is aimed to analyze the correlation between histological configuration and mechanical properties of the IFP, compared to other adipose tissues, partially differentiated by composition and conformation. Histological and ultrastructural methods were exploited to analyze the microscopic anatomies of IFP, knee (KSF) and abdominal (ASF) subcutaneous fat tissues. Numerical micro-models of the different tissues were developed by using histo-morphometric data, as the size of adipose lobules, the thickness of the septa and their composition. Numerical analyses made it possible to evaluate the mechanical functionality of the different fat tissues considering the characteristic loading conditions, as compressive and shear actions. The results pointed out the actual mechanical relevance of IFP and KSF, while ASF exhibited different mechanical properties. Furthermore, the contribution of connective septa and adipose lobules to compressive and shear mechanical behavior was elucidated. This preliminary investigation represents the basis for biomechanical interpretation and the definition of more refined model to be developed on the acquisition of additional histological and morphometric data.

Mechanics of the urethral duct: tissue constitutive formulation and structural modeling for the investigation of lumen occlusion.

Urinary incontinence, often related to sphincter damage, is found in male patients, leading to a miserable quality of life and to huge costs for the healthcare system. The most effective surgical solution currently considered for men is the artificial urinary sphincter that exerts a pressure field on the urethra, occluding the duct. The evaluation of this device is currently based on clinical and surgical competences. The artificial sphincter design and mechanical action can be investigated by a biomechanical model of the urethra under occlusion, evaluating the interaction between tissues and prosthesis. A specific computational approach to urethral mechanics is here proposed, recalling the results of previous biomechanical experimental investigation. In this preliminary analysis, the horse urethra is considered, in the light of a significant correlation with human and in consideration of the relevant difficulty to get to human samples, which anyway represents the future advance. Histological data processing allow for the definition of a virtual and a subsequent finite element model of a urethral section. A specific hyperelastic formulation is developed to characterize the nonlinear mechanical behavior. The inverse analysis of tensile tests on urethra samples leads to the definition of preliminary constitutive parameters. The parameters are further refined by the computational analysis of inflation tests carried out on the entire urethral structure. The results obtained represent, in the light of the correlation reported, a valid preliminary support for the information to be assumed for prosthesis design, integrating surgical and biomechanical competences.

A physiological model for the investigation of esophageal motility in healthy and pathologic conditions.

Recent technological advances in esophageal manometry allowed the definition of new classification methods for the diagnosis of disorders of esophageal motility and the implementation of innovative computational tools for the autonomic, reliable and unbiased detection of different disorders. Computational models can be developed aiming to interpret the mechanical behavior and functionality of the gastrointestinal tract and to summarize the results from clinical measurements, as high-resolution manometry pressure plots, into model parameters. A physiological model was here developed to interpret data from esophageal high-resolution manometry. Such model accounts for parameters related to specific physiological properties of the biological structures involved in the peristaltic mechanism. The identification of model parameters was performed by minimizing the discrepancy between clinical data from high-resolution manometry and model results. Clinical data were collected from both healthy volunteers ( n = 35) and patients with different motor disorders, such as achalasia patterns 1 ( n = 13), 2 ( n = 20) and 3 ( n = 5), distal esophageal spasm ( n = 69), esophago-gastric junction outflow obstruction ( n = 25), nutcracker esophagus ( n = 11) and normal motility ( n = 42). The physiological model that was formulated in this work can properly explain high-resolution manometry data, as confirmed by the evaluation of the coefficient of determination R2 = 0.83 - 0.96. The study finally led to identify the statistical distributions of model parameters for each healthy or pathologic conditions considered, addressing the applicability of the achieved results for the implementation of autonomic diagnosis procedures to support the medical staff during the traditional diagnostic process.

A review of the effects of some endocrinological factors on respiratory mechanics.

CONTEXT: Endocrinological factors have been recently described to affect respiratory mechanics. OBJECTIVE: To review recent literature data, most of all obtained by the end-inflation occlusion method, describing the effects of molecules of endocrinological interest such as endothelin, erythropoietin and renin-angiotensin, on respiratory mechanics parameters. METHODS: The papers considered in this review were found by inserting in Pubmed/Medline the following indexing terms: hormones, endothelin, erythropoietin, angiotensin and respiratory mechanics. RESULTS: It was found that the above cited molecules, beside their well known physiological main effects, exhibit influences on respiratory mechanics, most of all on the airflow resistance, which was described to be increased by endothelin and angiotensin, and decreased by erythropoietin. CONCLUSIONS: A number of molecules of biological interest exhibit unexpected influences on respiratory mechanics. The clinical effects depend on the consequences of modified inspiratory pressure values the respiratory muscles have to perform for a given breathing pattern.

Experimental investigation of the biomechanics of urethral tissues and structures.

NEW FINDINGS: What is the central question of this study? Prostheses for treatment of urinary incontinence elicit complications associated with an inadequate mechanical action. This investigation aimed to define a procedure addressed to urethral mechanical characterization. Experimental tests are the basis for constitutive formulation, with a view to numerical modelling for investigation of the interaction between the tissues and a prosthesis. What is the main finding and its importance? Horse urethra, selected for its histomorphometric similarity to human urethra, was characterized by integrated histological analysis and mechanical tests on the biological tissue and structure, leading to constitutive formulation. A non-linear, anisotropic and time-dependent response was found, representing a valid basis for development of a numerical model to interpret the functional behaviour of the urethra. Urinary dysfunction can lead to incontinence, with an impact on the quality of life. Severe dysfunction can be overcome surgically by the use of an artificial urinary sphincter. Nonetheless, several complications may result from inappropriate functioning of the prosthesis, in many instances resulting from an unsuitable mechanical action of the device on the urethral tissues. Computational models allow investigation of the mechanical interaction between biological tissues and biomedical devices, representing a potential support for surgical practice and prosthesis design. The development of such computational tools requires experimental data on the mechanics of biological tissues and structures, which are rarely reported in the literature. The aim of this study was to provide a procedure for the mechanical characterization of urethral tissues and structures. The experimental protocol included the morphometric and histological analysis of urethral tissues, the mechanical characterization of the response of tissues to tensile and stress-relaxation tests and evaluation of the behaviour of urethral structures by inflation tests. Results from the preliminary experiments were processed, adopting specific model formulations, and also providing the definition of parameters that characterize the elastic and viscous behaviour of the tissues. Different experimental protocols, leading to a comprehensive set of experimental data, allow for a reciprocal assessment of reliability of the investigation approach.

Analysis of the structural behaviour of colonic segments by inflation tests: Experimental activity and physio-mechanical model.

A coupled experimental and computational approach is provided for the identification of the structural behaviour of gastrointestinal regions, accounting for both elastic and visco-elastic properties. The developed procedure is applied to characterize the mechanics of gastrointestinal samples from pig colons. Experimental data about the structural behaviour of colonic segments are provided by inflation tests. Different inflation processes are performed according to progressively increasing top pressure conditions. Each inflation test consists of an air in-flow, according to an almost constant increasing pressure rate, such as 3.5 mmHg/s, up to a prescribed top pressure, which is held constant for about 300 s to allow the development of creep phenomena. Different tests are interspersed by 600 s of rest to allow the recovery of the tissues' mechanical condition. Data from structural tests are post-processed by a physio-mechanical model in order to identify the mechanical parameters that interpret both the non-linear elastic behaviour of the sample, as the instantaneous pressure-stretch trend, and the time-dependent response, as the stretch increase during the creep processes. The parameters are identified by minimizing the discrepancy between experimental and model results. Different sets of parameters are evaluated for different specimens from different pigs. A statistical analysis is performed to evaluate the distribution of the parameters and to assess the reliability of the experimental and computational activities.