Stromal cells in chronic inflammation and tertiary lymphoid organ formation.

Inflammation is an unstable state. It either resolves or persists. Why inflammation persists and the factors that define tissue tropism remain obscure. Increasing evidence suggests that tissue-resident stromal cells not only provide positional memory but also actively regulate the differential accumulation of inflammatory cells within inflamed tissues. Furthermore, at many sites of chronic inflammation, structures that mimic secondary lymphoid tissues are observed, suggesting that chronic inflammation and lymphoid tissue formation share common activation programs. Similarly, blood and lymphatic endothelial cells contribute to tissue homeostasis and disease persistence in chronic inflammation. This review highlights our increasing understanding of the role of stromal cells in inflammation and summarizes the novel immunological role that stromal cells exert in the persistence of inflammatory diseases.

Inflammation-induced formation of fat-associated lymphoid clusters.

Fat-associated lymphoid clusters (FALCs) are a type of lymphoid tissue associated with visceral fat. Here we found that the distribution of FALCs was heterogeneous, with the pericardium containing large numbers of these clusters. FALCs contributed to the retention of B-1 cells in the peritoneal cavity through high expression of the chemokine CXCL13, and they supported B cell proliferation and germinal center differentiation during peritoneal immunological challenges. FALC formation was induced by inflammation, which triggered the recruitment of myeloid cells that expressed tumor-necrosis factor (TNF) necessary for signaling via the TNF receptors in stromal cells. Natural killer T cells (NKT cells) restricted by the antigen-presenting molecule CD1d were likewise required for the inducible formation of FALCs. Thus, FALCs supported and coordinated the activation of innate B cells and T cells during serosal immune responses.

Bioprinted 3D outer retina barrier uncovers RPE-dependent choroidal phenotype in advanced macular degeneration.

Age-related macular degeneration (AMD), a leading cause of blindness, initiates in the outer-blood-retina-barrier (oBRB) formed by the retinal pigment epithelium (RPE), Bruch's membrane, and choriocapillaris. The mechanisms of AMD initiation and progression remain poorly understood owing to the lack of physiologically relevant human oBRB models. To this end, we engineered a native-like three-dimensional (3D) oBRB tissue (3D-oBRB) by bioprinting endothelial cells, pericytes, and fibroblasts on the basal side of a biodegradable scaffold and establishing an RPE monolayer on top. In this 3D-oBRB model, a fully-polarized RPE monolayer provides barrier resistance, induces choriocapillaris fenestration, and supports the formation of Bruch's-membrane-like structure by inducing changes in gene expression in cells of the choroid. Complement activation in the 3D-oBRB triggers dry AMD phenotypes (including subRPE lipid-rich deposits called drusen and choriocapillaris degeneration), and HIF-α stabilization or STAT3 overactivation induce choriocapillaris neovascularization and type-I wet AMD phenotype. The 3D-oBRB provides a physiologically relevant model to studying RPE-choriocapillaris interactions under healthy and diseased conditions.

Distinct fibroblast subsets drive inflammation and damage in arthritis.

The identification of lymphocyte subsets with non-overlapping effector functions has been pivotal to the development of targeted therapies in immune-mediated inflammatory diseases (IMIDs)1,2. However, it remains unclear whether fibroblast subclasses with non-overlapping functions also exist and are responsible for the wide variety of tissue-driven processes observed in IMIDs, such as inflammation and damage3-5. Here we identify and describe the biology of distinct subsets of fibroblasts responsible for mediating either inflammation or tissue damage in arthritis. We show that deletion of fibroblast activation protein-α (FAPα)+ fibroblasts suppressed both inflammation and bone erosions in mouse models of resolving and persistent arthritis. Single-cell transcriptional analysis identified two distinct fibroblast subsets within the FAPα+ population: FAPα+THY1+ immune effector fibroblasts located in the synovial sub-lining, and FAPα+THY1- destructive fibroblasts restricted to the synovial lining layer. When adoptively transferred into the joint, FAPα+THY1- fibroblasts selectively mediate bone and cartilage damage with little effect on inflammation, whereas transfer of FAPα+ THY1+ fibroblasts resulted in a more severe and persistent inflammatory arthritis, with minimal effect on bone and cartilage. Our findings describing anatomically discrete, functionally distinct fibroblast subsets with non-overlapping functions have important implications for cell-based therapies aimed at modulating inflammation and tissue damage.

Stromal cells in tertiary lymphoid structures: Architects of autoimmunity.

The molecular mediators present within the inflammatory microenvironment are able, in certain conditions, to favor the initiation of tertiary lymphoid structure (TLS) development. TLS is organized lymphocyte clusters able to support antigen-specific immune response in non-immune organs. Importantly, chronic inflammation does not always result in TLS formation; instead, TLS has been observed to develop specifically in permissive organs, suggesting the presence of tissue-specific cues that are able to imprint the immune responses and form TLS hubs. Fibroblasts are tissue-resident cells that define the anatomy and function of a specific tissue. Fibroblast plasticity and specialization in inflammatory conditions have recently been unraveled in both immune and non-immune organs revealing a critical role for these structural cells in human physiology. Here, we describe the role of fibroblasts in the context of TLS formation and its functional maintenance in the tissue, highlighting their potential role as therapeutic disease targets in TLS-associated diseases.

A randomized, double-blind, placebo-controlled, parallel group study on the effects of a cathepsin S inhibitor in primary Sjögren's syndrome.

OBJECTIVES: Primary SS (pSS) is a chronic autoimmune disorder characterized by mucosal dryness and systemic symptoms. We tested the effects of inhibition of cathepsin S using the potent and selective inhibitor RO5459072 on disease activity and symptoms of pSS. METHODS: This was a randomized, double-blind, placebo-controlled, parallel-group, Phase IIA study to investigate the effects of RO5459072 (100 mg twice daily; 200 mg per day). Seventy-five patients with pSS were randomized 1:1 to receive either RO5459072 or placebo for 12 weeks. The primary outcome was the proportion of patients with a ≥3 point reduction from baseline in EULAR SS Disease Activity Index (ESSDAI) score. We also investigated the effects of RO5459072 on quality of life, exocrine gland function, biomarkers related to SS, and safety and tolerability. RESULTS: The proportion of patients showing an improvement in ESSDAI score was not significantly different between the RO5459072 and placebo arms. No clinically meaningful treatment effects were observed in favour of RO5459072 for all secondary outcomes. Analysis of soluble biomarkers indicated target engagement between RO5459072 and cathepsin S. There were modest decreases in the number of circulating B cells and T cells in the RO5459072 group, although these did not reach significance. RO5459072 was safe and well-tolerated. CONCLUSIONS: There was no clinically relevant improvement in ESSDAI score (primary endpoint), and no apparent benefit in favour of RO5459072 in any of the secondary clinical endpoints. Further work is needed in order to understand the mechanisms of MHC-II-mediated immune stimulation in pSS. TRIAL REGISTRATION: ClinicalTrials.gov; NCT02701985.

Multiscale Entropy Algorithms to Analyze Complexity and Variability of Trunk Accelerations Time Series in Subjects with Parkinson's Disease.

The aim of this study was to assess the ability of multiscale sample entropy (MSE), refined composite multiscale entropy (RCMSE), and complexity index (CI) to characterize gait complexity through trunk acceleration patterns in subjects with Parkinson's disease (swPD) and healthy subjects, regardless of age or gait speed. The trunk acceleration patterns of 51 swPD and 50 healthy subjects (HS) were acquired using a lumbar-mounted magneto-inertial measurement unit during their walking. MSE, RCMSE, and CI were calculated on 2000 data points, using scale factors (τ) 1-6. Differences between swPD and HS were calculated at each τ, and the area under the receiver operating characteristics, optimal cutoff points, post-test probabilities, and diagnostic odds ratios were calculated. MSE, RCMSE, and CIs showed to differentiate swPD from HS. MSE in the anteroposterior direction at τ4 and τ5, and MSE in the ML direction at τ4 showed to characterize the gait disorders of swPD with the best trade-off between positive and negative posttest probabilities and correlated with the motor disability, pelvic kinematics, and stance phase. Using a time series of 2000 data points, a scale factor of 4 or 5 in the MSE procedure can yield the best trade-off in terms of post-test probabilities when compared to other scale factors for detecting gait variability and complexity in swPD.

Immunofibroblasts are pivotal drivers of tertiary lymphoid structure formation and local pathology.

Resident fibroblasts at sites of infection, chronic inflammation, or cancer undergo phenotypic and functional changes to support leukocyte migration and, in some cases, aggregation into tertiary lymphoid structures (TLS). The molecular programming that shapes these changes and the functional requirements of this population in TLS development are unclear. Here, we demonstrate that external triggers at mucosal sites are able to induce the progressive differentiation of a population of podoplanin (pdpn)-positive stromal cells into a network of immunofibroblasts that are able to support the earliest phases of TLS establishment. This program of events, that precedes lymphocyte infiltration in the tissue, is mediated by paracrine and autocrine signals mainly regulated by IL13. This initial fibroblast network is expanded and stabilized, once lymphocytes are recruited, by the local production of the cytokines IL22 and lymphotoxin. Interfering with this regulated program of events or depleting the immunofibroblasts in vivo results in abrogation of local pathology, demonstrating the functional role of immunofibroblasts in supporting TLS maintenance in the tissue and suggesting novel therapeutic targets in TLS-associated diseases.

The role of stroma and epithelial cells in primary Sjögren's syndrome.

Primary SS (pSS) is a chronic autoimmune condition characterized by infiltration of the exocrine glands and systemic B cell hyperactivation. This glandular infiltration is associated with loss of glandular function, with pSS patients primarily presenting with severe dryness of the eyes and mouth. Within the affected glands, the infiltrating lymphocytes are organized in tertiary lymphoid structures. Tertiary lymphoid structures subvert normal tissue architecture and impact on organ function, by promoting the activation and maintenance of autoreactive lymphocytes. This review summarizes the current knowledge about the role of stromal cells (including endothelium, epithelium, nerves and fibroblasts) in the pathogenesis of pSS, in particular the interactions taking place between stromal cells and infiltrating lymphocytes. We will provide evidences pointing towards the driving role of stromal cells in the orchestration of the local inflammatory milieu, thus highlighting the need for therapies aimed at targeting this compartment alongside classical immunosuppression in pSS.

A phase 2 randomized, double-blind, placebo-controlled, proof-of-concept study of oral seletalisib in primary Sjögren's syndrome.

OBJECTIVES: This phase 2 proof-of-concept study (NCT02610543) assessed efficacy, safety and effects on salivary gland inflammation of seletalisib, a potent and selective PI3Kδ inhibitor, in patients with moderate-to-severe primary Sjögren's syndrome (PSS). METHODS: Adults with PSS were randomized 1:1 to seletalisib 45 mg/day or placebo, in addition to current PSS therapy. Primary end points were safety and tolerability and change from baseline in EULAR Sjögren's Syndrome Disease Activity Index (ESSDAI) score at week 12. Secondary end points included change from baseline at week 12 in EULAR Sjögren's Syndrome Patient Reported Index (ESSPRI) score and histological features in salivary gland biopsies. RESULTS: Twenty-seven patients were randomized (seletalisib n = 13, placebo n = 14); 20 completed the study. Enrolment challenges led to early study termination with loss of statistical power (36% vs 80% planned). Nonetheless, a trend for improvement in ESSDAI and ESSPRI [difference vs placebo: -2.59 (95% CI: -7.30, 2.11; P=0.266) and -1.55 (95% CI: -3.39, 0.28), respectively] was observed at week 12. No significant changes were seen in saliva and tear flow. Serious adverse events (AEs) were reported in 3/13 of patients receiving seletalisib vs 1/14 for placebo and 5/13 vs 1/14 discontinued due to AEs, respectively. Serum IgM and IgG concentrations decreased in the seletalisib group vs placebo. Seletalisib demonstrated efficacy in reducing size and organisation of salivary gland inflammatory foci and in target engagement, thus reducing PI3K-mTOR signalling compared with placebo. CONCLUSION: Despite enrolment challenges, seletalisib demonstrated a trend towards clinical improvement in patients with PSS. Histological analyses demonstrated encouraging effects of seletalisib on salivary gland inflammation and organisation. TRIAL REGISTRATION: https://clinicaltrials.gov, NCT02610543.

The human lymph node microenvironment unilaterally regulates T-cell activation and differentiation.

The microenvironment of lymphoid organs can aid healthy immune function through provision of both structural and molecular support. In mice, fibroblastic reticular cells (FRCs) create an essential T-cell support structure within lymph nodes, while human FRCs are largely unstudied. Here, we show that FRCs create a regulatory checkpoint in human peripheral T-cell activation through 4 mechanisms simultaneously utilised. Human tonsil and lymph node-derived FRCs constrained the proliferation of both naïve and pre-activated T cells, skewing their differentiation away from a central memory T-cell phenotype. FRCs acted unilaterally without requiring T-cell feedback, imposing suppression via indoleamine-2,3-dioxygenase, adenosine 2A Receptor, prostaglandin E2, and transforming growth factor beta receptor (TGFßR). Each mechanistic pathway was druggable, and a cocktail of inhibitors, targeting all 4 mechanisms, entirely reversed the suppressive effect of FRCs. T cells were not permanently anergised by FRCs, and studies using chimeric antigen receptor (CAR) T cells showed that immunotherapeutic T cells retained effector functions in the presence of FRCs. Since mice were not suitable as a proof-of-concept model, we instead developed a novel human tissue-based in situ assay. Human T cells stimulated using standard methods within fresh tonsil slices did not proliferate except in the presence of inhibitors described above. Collectively, we define a 4-part molecular mechanism by which FRCs regulate the T-cell response to strongly activating events in secondary lymphoid organs while permitting activated and CAR T cells to utilise effector functions. Our results define 4 feasible strategies, used alone or in combinations, to boost primary T-cell responses to infection or cancer by pharmacologically targeting FRCs.

Unique expansion of IL-21+ Tfh and Tph cells under control of ICOS identifies Sjögren's syndrome with ectopic germinal centres and MALT lymphoma.

OBJECTIVES: To explore the relevance of T-follicular-helper (Tfh) and pathogenic peripheral-helper T-cells (Tph) in promoting ectopic lymphoid structures (ELS) and B-cell mucosa-associated lymphoid tissue (MALT) lymphomas (MALT-L) in Sjögren's syndrome (SS) patients. METHODS: Salivary gland (SG) biopsies with matched peripheral blood were collected from four centres across the European Union. Transcriptomic (microarray and quantitative PCR) analysis, FACS T-cell immunophenotyping with intracellular cytokine detection, multicolor immune-fluorescence microscopy and in situ hybridisation were performed to characterise lesional and circulating Tfh and Tph-cells. SG-organ cultures were used to investigate functionally the blockade of T-cell costimulatory pathways on key proinflammatory cytokine production. RESULTS: Transcriptomic analysis in SG identified Tfh-signature, interleukin-21 (IL-21) and the inducible T-cell co-stimulator (ICOS) costimulatory pathway as the most upregulated genes in ELS+SS patients, with parotid MALT-L displaying a 400-folds increase in IL-21 mRNA. Peripheral CD4+CXC-motif chemokine receptor 5 (CXCR5)+programmed cell death protein 1 (PD1)+ICOS+ Tfh-like cells were significantly expanded in ELS+SS patients, were the main producers of IL-21, and closely correlated with circulating IgG and reduced complement C4. In the SG, lesional CD4+CD45RO+ICOS+PD1+ cells selectively infiltrated ELS+ tissues and were aberrantly expanded in parotid MALT-L. In ELS+SG and MALT-L parotids, conventional CXCR5+CD4+PD1+ICOS+Foxp3- Tfh-cells and a uniquely expanded population of CXCR5-CD4+PD1hiICOS+Foxp3- Tph-cells displayed frequent IL-21/interferon-γ double-production but poor IL-17 expression. Finally, ICOS blockade in ex vivo SG-organ cultures significantly reduced the production of IL-21 and inflammatory cytokines IL-6, IL-8 and tumour necrosis factor-α (TNF-α). CONCLUSIONS: Overall, these findings highlight Tfh and Tph-cells, IL-21 and the ICOS costimulatory pathway as key pathogenic players in SS immunopathology and exploitable therapeutic targets in SS.

CXCL13 as biomarker for histological involvement in Sjögren's syndrome.

OBJECTIVES: SS is an autoimmune condition characterized by systemic B-cell activation, autoantibody production and ectopic germinal centres' formation within the salivary gland (SG). The extent of SG infiltrate has been proposed as a biomarker of disease severity. Plasma levels of CXCL13 correlate with germinal centres' activity in animal models and disease severity in SS, suggesting its potential use as a surrogate serum marker to monitor local B-cell activation. The aim of this study was to evaluate the potential role of CXCL13 as a biomarker of SG pathology in two independent SS cohorts. METHODS: 109 patients with SS were recruited at Sapienza University of Rome (Italy) (n = 60), or at Queen Elizabeth Hospital in Birmingham and Barts Health NHS Trust in London (n = 49). Both sera and matched minor SG biopsy were available. Sicca (n = 57) and healthy subjects' (n = 19) sera were used as control. RESULTS: CXCL13 serum level was higher in SS patients compared with controls. Correlations between its serum levels and a series of histomorphological parameters, including size of the aggregates and the presence germinal centres', were observed. CONCLUSION: Our data foster the use of CXCL13 to monitor the extent of local pathology in SS and its validation in longitudinal clinical studies.

The porcine iodoacetic acid model of retinal degeneration: Morpho-functional characterization of the visual system.

Porcine models of ophthalmological diseases are often used in pre-clinical translational studies due to pigs' similarities to humans. In particular, the iodoacetic acid (IAA) model of photoreceptor degeneration seems to mimic well the endstage phenotype of human pathologies as retinitis pigmentosa and age-related macular degeneration, with high potential for prosthesis/retinal devices testing. IAA is capable of inducing photoreceptor death by blockage of glycolysis, and its effects on the retina have been described. Nonetheless, up to date, literature lacks of a comprehensive morpho-functional characterization of the entire visual system of this model. This gap is particularly critical for prosthesis testing as inner retinal structures and optic pathways must be preserved to elicit cortical responses and restore vision. In this study, we investigated the functional and anatomical features of the visual system of IAA-treated pigs and compared them to control animals. IAA was administered intravenously at 12 mg/kg; control animals received saline solution (NaCl 0.9% w/v). Electrophysiological analyses included full-field (ffERGs) and pattern (PERGs) electroretinograms and flash visually evoked potentials (fVEPs). Histological evaluations were performed on the retina and the optic pathways and included thickness of the different retinal layers, ganglion cells count, and immunohistochemistry for microglial cells, macroglial cells, and oligodendrocytes. The histological results indicate that IAA treatment does not affect the morphology of the inner retina and optic pathways. Electrophysiology confirms the selective rod and partial cone degeneration, but is ambiguous as to the functionality of the optic pathways, seemingly preserved as indicated by the still detectable fVEPs. Overall, the work ameliorates the characterization of such rapid and cost-effective model, providing more strength and reliability for future pre-clinical translational trials.

Persistent treatment resistance in genetic generalized epilepsy: A long-term outcome study in a tertiary epilepsy center.

OBJECTIVE: To assess prognostic patterns and investigate clinical and electroencephalography (EEG) variables associated with persistent treatment resistance in a population of genetic generalized epilepsy (GGE) patients with a long-term follow-up. METHODS: Data from GGE patients followed from 1975 to 2019 were reviewed retrospectively. Subjects with a follow-up >10 years, starting from epilepsy diagnosis, were included. Persistent treatment resistance was defined as the absence of any period of remission ≥1 year despite treatment with two appropriate and adequate antiepileptic drugs (AEDs). RESULTS: One hundred ninety-nine patients were included. The median age was 39.5 years (interquartile range [IQR] 30-49) and the median follow-up was 27 years (IQR 18-35). The most common syndrome was juvenile myoclonic epilepsy (JME), diagnosed in 44.2% of patients. During follow-up, 163 subjects (81.9%) experienced 3-year remission from any seizure type, whereas 5- and 10-year remission occurred in 141 (70.8%) and 92 (46.2%) cases, respectively. The most common prognostic pattern was a relapsing-remitting course, observed in 80 patients (40.2%), whereas 29 (14.6%) displayed persistent treatment resistance. According to multivariable logistic regression analysis, febrile seizures (FS), specific EEG patterns (namely generalized paroxysmal fast activity, GPFA) and valproate (VPA) resistance were the only variables significantly associated with persistent treatment resistance. JME was the only epilepsy syndrome statistically associated with persistent treatment resistance in univariable logistic regression analysis. SIGNIFICANCE: Persistent treatment resistance was observed in almost 15% of GGE patients followed in a tertiary epilepsy center. A worse outcome was associated with specific clinical variables (JME, FS) and EEG patterns (GPFA).

Doing without valproate in women of childbearing potential with idiopathic generalized epilepsy: Implications on seizure outcome.

OBJECTIVE: Valproate (VPA) use in women with idiopathic generalized epilepsy (IGE) who are of reproductive age has been a matter of concern and debate, which eventually led to the recent restrictions by regulatory agencies. The aim of our study was to investigate the relationship between VPA avoidance/switch and seizure outcome in women of childbearing potential. METHODS: We retrospectively reviewed data from female patients with IGE, 13-50 years of age, followed since 1980. We evaluated the prescription habits, and the rate of VPA switch for other antiepileptic drugs (AEDs) and its prognostic implications. Seizure remission (SR) was defined as the absence of any seizure type more than 18 months before the last medical observation. The main aim of the study was to assess (a) possible changes in seizure outcome related to VPA switch for other AEDs, especially in patients planning a pregnancy; and (b) possible differences in SR based on the presence/absence of VPA at last observation. RESULTS: One hundred ninety-eight patients were included in the study. Overall SR at last medical observation was 62.7%. SR significantly differed between subjects taking and those not taking VPA (P < .001) at last visit. Multiple regression models showed that taking VPA at last medical observation was strongly associated with SR in both the general population (P < .001) and the juvenile myoclonic epilepsy (JME) group (P < .001). Thirty-six (70.6%) of 51 patients who switched from VPA during follow-up experienced a clinical worsening. Switching back to VPA was more frequently associated with SR at last observation (P < .001). In those patients who substituted VPA in view of a pregnancy, SR and drug burden (monotherapy vs polytherapy) differed significantly before and after the switch. SIGNIFICANCE: Our study suggests that VPA avoidance/switch might be associated with unsatisfactory seizure control in women with IGE who are of childbearing potential. Our findings further highlight the complexity of the therapeutic management of female patients of reproductive age.

Mediterranean diet and risk of Sjögren's syndrome.

OBJECTIVES: Non-genetic risk factors for Sjögren's syndrome (SS) are poorly understood. Adherence to a Mediterranean diet has been associated with reduction in other autoimmune diseases. We examined the association of Mediterranean diet with SS. METHODS: New patients attending a single centre warranting investigation for primary SS (pSS) were recruited into the Optimising Assessment in Sjögren's Syndrome cohort established in Birmingham, UK (2014-2018). Participants were classified into pSS and non-SS sicca, considered as cases and non-cases, respectively, and asked to complete an optional food frequency questionnaire on their diet before onset of symptoms. A semi-quantitative Mediterranean diet score (MDS) was calculated (possible range=0 to 18). Using multivariate logistic regression, corrected for energy intake, body-mass index, sex, age, symptom duration, and smoking status, we examined the association of MDS with SS. RESULTS: Dietary data were available for 133/243 (55%) eligible patients (n=82 pSS and n=51 sicca). In the adjusted model, a higher total MDS (mean ± SD, 9.41±2.31 points) was associated with lower odds of pSS (OR 0.81, 95% CI 0.66-0.99; p=0.038) per one unit of MDS. Among MDS components, the strongest association was seen with fish with OR 0.44 (95% CI 0.24-0.83; p=0.01) in the comparison between <1 portion/week and 1 to 2.5 portions/week. Higher galactose, vitamin A-retinol-equivalents and vitamin C showed associations with lower odds of pSS in multivariate analysis, where the association of vitamin C was attenuated when adjusted for MDS. CONCLUSIONS: When adjusted for potential confounders, adherence to the Mediterranean diet was associated with lower likelihood of having pSS.

A Training Tool to support the management and diagnosis of Sjögren's syndrome.

OBJECTIVES: The objective of this work is to present a Training Tool designed to support healthcare professionals involved in the diagnosis and management of Sjögren's syndrome. METHODS: The Training Tool aims to fulfil the gap of targeted education by providing a structured protocol of training including state of the art guidelines and practices. For the development of the Training Tool, latest relevant technologies have been used to assure efficiency and usability. Core functionalities include training by a series of multimedia courses, testing during the learning process, and profiling for monitoring the progress. An iterative requirement analysis process was established involving a large number of clinical experts, with the objective to identify user's training needs. RESULTS: Comprehensive usability evaluation was performed by applying, an Unmoderated Remote Usability Test resulting to 97.2% Success Rate; and the well-established System Usability Scale, reaching a score of 90.4 which classifies the Training Tool as "A" graded-excellent. CONCLUSIONS: The Training Tool offers open-online training of healthcare professionals involved in the diagnosis and management of Sjögren's syndrome, using a well-designed training protocol in highly usable manner. To our knowledge, this is the first such tool for Sjögren's syndrome.

Autonomic effects induced by pharmacological activation and inhibition of Raphe Pallidus neurons in anaesthetized adult pigs.

The Raphe Pallidus (RPa) is a region of the brainstem that was shown to modulate the sympathetic outflow to many tissues and organs involved in thermoregulation and energy expenditure. In rodents, the pharmacological activation of RPa neurons was shown to increase the activity of the brown adipose tissue, heart rate, and expired CO2 , whereas their inhibition was shown to induce cutaneous vasodilation and a state of hypothermia that, when prolonged, leads to a state resembling torpor referred to as synthetic torpor. If translatable to humans, this synthetic torpor-inducing procedure would be advantageous in many clinical settings. A first step to explore such translatability, has been to verify whether the neurons within the RPa play the same role described for rodents in a larger mammal such as the pig. In the present study, we show that the physiological responses inducible by the pharmacological stimulation of RPa neurons are very similar to those observed in rodents. Injection of the GABAA agonist GABAzine in the RPa induced an increase in heart rate (from 99 to 174 bpm), systolic (from 87 to 170 mm Hg) and diastolic (from 51 to 98 mm Hg) arterial pressure, and end-tidal CO2 (from 49 to 62 mm Hg). All these changes were reversed by the injection in the same area of the GABAA agonist muscimol. These results support the possibility for RPa neurons to be a key target in the research for a safe and effective procedure for the induction of synthetic torpor in humans.

Phosphatidylinositol 3-kinase delta pathway: a novel therapeutic target for Sjögren's syndrome.

BACKGROUND: The phosphatidylinositol 3-kinase delta isoform (PI3Kδ) belongs to an intracellular lipid kinase family that regulate lymphocyte metabolism, survival, proliferation, apoptosis and migration and has been successfully targeted in B-cell malignancies. Primary Sjögren's syndrome (pSS) is a chronic immune-mediated inflammatory disease characterised by exocrine gland lymphocytic infiltration and B-cell hyperactivation which results in systemic manifestations, autoantibody production and loss of glandular function. Given the central role of B cells in pSS pathogenesis, we investigated PI3Kδ pathway activation in pSS and the functional consequences of blocking PI3Kδ in a murine model of focal sialoadenitis that mimics some features of pSS. METHODS AND RESULTS: Target validation assays showed significant expression of phosphorylated ribosomal protein S6 (pS6), a downstream mediator of the phosphatidylinositol 3-kinase delta (PI3Kδ) pathway, within pSS salivary glands. pS6 distribution was found to co-localise with T/B cell markers within pSS aggregates and the CD138+ plasma cells infiltrating the glands. In vivo blockade of PI3Kδ activity with seletalisib, a PI3Kδ-selective inhibitor, in a murine model of focal sialoadenitis decreased accumulation of lymphocytes and plasma cells within the glands of treated mice in the prophylactic and therapeutic regimes. Additionally, production of lymphoid chemokines and cytokines associated with ectopic lymphoneogenesis and, remarkably, saliva flow and autoantibody production, were significantly affected by treatment with seletalisib. CONCLUSION: These data demonstrate activation of PI3Kδ pathway within the glands of patients with pSS and its contribution to disease pathogenesis in a model of disease, supporting the exploration of the therapeutic potential of PI3Kδ pathway inhibition in this condition.