Acoustic Radiation Forced Impulse of the Liver and the Spleen, Combined with Spleen Dimension and Platelet Count in New Ratio Scores, Identifies High-Risk Esophageal Varices in Well-Compensated Cirrhotic Patients.

Acoustic radiation forced impulse (ARFI) is an integrated ultrasound method, measuring stiffness by point shear wave elastography. To evaluate the diagnostic performance of the ARFI of the liver and the spleen, combined with spleen dimension and platelet count, in predicting high-risk esophageal varices (HRVs) in cirrhotic patients, a prospective and cross-sectional study was conducted between February 2017 and February 2021. The following ratio scores were calculated based on ARFI measurements: ALSDP (ARFI Liver-Spleen Diameter-to-Platelet Ratio Score), ASSDP (ARFI Spleen-Spleen Diameter-to-Platelet Ratio Score), ASSAP (ARFI Spleen-Spleen Area-to-Platelet Ratio Score), and ALSAP (ARFI Liver-Spleen Area-to-Platelet Ratio Score). In 100 enrolled subjects, spleen ARFI, ASSDP, and ASSAP were significantly associated with HRVs in the prospective short- and long-term follow-ups and in the cross-sectional study (p < 0.05), while ALSDP and ALSAP were associated with HRVs only in the prospective long-term follow-up and cross-sectional study (p< 0.05). ASSAP was the best ARFI ratio score for HRVs at the long-term follow-up [value of area under curve (AUC) = 0.88], although all the ARFI ratio scores performed better than individual liver and spleen ARFI (AUC > 0.7). In our study, ARFI ratio scores can predict, in well-compensated cirrhotic patients, the risk of developing HVRs in short- and long-term periods.

An Automated Visual Psychophysics Method to Measure Visual Function in Swine Preclinical Animal Model.

Purpose: The aim of this study was to develop and validate a test to assess visual function in pigs using the visual psychophysics contrast sensitivity function. Methods: We utilized a touchscreen along with a pellet reward dispenser to train three Göttingen pigs on a visual psychophysics test and determined their contrast sensitivity function. Images with different contrast resolutions were used as visual stimuli and presented against a control image in a two-choice test. Following animals' acclimatization and the first phase of training, the system was arranged such that animals could self-run multiple consecutive trials without human intervention. Results: All animals were trained within a week and remembered the task with 1 day of reinforcement when tested 1 month after the last visual assessment. All trained animals performed well during the trial with minimal screen side bias, especially at contrast threshold above 40%. Conclusions: Göttingen pigs are trainable for a visual psychophysics test and able to self-run the trial without human intervention. Translational Relevance: Contrast sensitivity is one of the key parameters to assess visual function in humans. The possibility of measuring the same parameters in a large animal model allows for a better translation and understanding of drug safety and efficacy in preclinical ophthalmology.

OCTN1 (SLC22A4) displays two different transport pathways for organic cations or zwitterions.

BACKGROUND: OCTN1 belongs to the SLC22 family, which includes transporters for cationic, zwitterionic, and anionic substrates. OCTN1 function and role in cells are still poorly understood. Not only cations, such as TEA, but also zwitterions, such as carnitine and ergothioneine, figure among transported molecules. METHODS: In this work, we carried out transport assays measuring [14C]-TEA and [3H]-Carnitine in proteoliposomes reconstituted with the recombinant human OCTN1 in the presence of Na+ or other cations. The homology model of OCTN1 was built using the structure of OCT3 as a template for docking analysis. RESULTS: TEA and carnitine did not inhibit each other. Moreover, carnitine uptake was not affected by the presence of Na+ and TEBA, whereas TEA was strongly inhibited by both compounds. Computational data revealed that TEA, Na+, and carnitine can interact with E381 in the OCTN1 substrate site. Differently from TEA, in the presence of Na+, carnitine is still able to interact with the binding site via R469. CONCLUSIONS: The lack of mutual inhibition of the two prototype substrates, the different effect of Na+ and TEBA on their transport reaction, together with the computational analysis supports the existence of two transport pathways for cations and zwitterions. GENERAL SIGNIFICANCE: The results shed new light on the transport mechanisms of OCTN1, helping to get further insights into the structure/function relationships. The described results correlate well with previous and very recent findings on the polyspecificity of the OCT group of transporters belonging to the same family.

KDM6B drives epigenetic reprogramming associated with lymphoid stromal cell early commitment and immune properties.

Mature lymphoid stromal cells (LSCs) are key organizers of immune responses within secondary lymphoid organs. Similarly, inflammation-driven tertiary lymphoid structures depend on immunofibroblasts producing lymphoid cytokines and chemokines. Recent studies have explored the origin and heterogeneity of LSC/immunofibroblasts, yet the molecular and epigenetic mechanisms involved in their commitment are still unknown. This study explored the transcriptomic and epigenetic reprogramming underlying LSC/immunofibroblast commitment. We identified the induction of lysine demethylase 6B (KDM6B) as the primary epigenetic driver of early immunofibroblast differentiation. In addition, we observed an enrichment for KDM6B gene signature in murine inflammatory fibroblasts and pathogenic stroma of patients with autoimmune diseases. Last, KDM6B was required for the acquisition of LSC/immunofibroblast functional properties, including the up-regulation of CCL2 and the resulting recruitment of monocytes. Overall, our results reveal epigenetic mechanisms that participate in the early commitment and immune properties of immunofibroblasts and support the use of epigenetic modifiers as fibroblast-targeting strategies in chronic inflammation.

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.

Retina and RPE lipid profile changes linked with ABCA4 associated Stargardt's maculopathy.

Stargardt maculopathy, caused predominantly by mutations in the ABCA4 gene, is characterized by an accumulation of non-degradable visual pigment derivative, lipofuscin, in the retinal pigment epithelium (RPE) - resulting in RPE atrophy. RPE is a monolayer tissue located adjacent to retinal photoreceptors and regulates their health and functioning; RPE atrophy triggers photoreceptor cell death and vision loss in Stargardt patients. Previously, ABCA4 mutations in photoreceptors were thought to be the major contributor to lipid homeostasis defects in the eye. Recently, we demonstrated that ABCA4 loss of function in the RPE leads to cell-autonomous lipid homeostasis defects. Our work underscores that an incomplete understanding of lipid metabolism and lipid-mediated signaling in the retina and RPE are potential causes for lacking treatments for this disease. Here we report altered lipidomic in mouse and human Stargardt models. This work provides the basis for therapeutics that aim to restore lipid homeostasis in the retina and the RPE.

A versatile laser-induced porcine model of outer retinal and choroidal degeneration for preclinical testing.

Over 30 million people worldwide suffer from untreatable vision loss and blindness associated with childhood-onset and age-related eye diseases caused by photoreceptor (PR), retinal pigment epithelium (RPE), and choriocapillaris (CC) degeneration. Recent work suggests that RPE-based cell therapy may slow down vision loss in late stages of age-related macular degeneration (AMD), a polygenic disease induced by RPE atrophy. However, accelerated development of effective cell therapies is hampered by the lack of large-animal models that allow testing safety and efficacy of clinical doses covering the human macula (20 mm2). We developed a versatile pig model to mimic different types and stages of retinal degeneration. Using an adjustable power micropulse laser, we generated varying degrees of RPE, PR, and CC damage and confirmed the damage by longitudinal analysis of clinically relevant outcomes, including analyses by adaptive optics and optical coherence tomography/angiography, along with automated image analysis. By imparting a tunable yet targeted damage to the porcine CC and visual streak - with a structure similar to the human macula - this model is optimal for testing cell and gene therapies for outer retinal diseases including AMD, retinitis pigmentosa, Stargardt, and choroideremia. The amenability of this model to clinically relevant imaging outcomes will facilitate faster translation to patients.

Uncovering transcriptomic landscape alterations of CAN-2409 in in vitro and in vivo glioma models.

Rationale: CAN-2409 is a locally delivered oncolytic therapy, which results in vaccination against the injected tumor. CAN-2409 consists of a non-replicating adenovirus armed with the Herpes virus thymidine kinase, which metabolizes ganciclovir into a phosphorylated nucleotide that is incorporated into the tumor cell's genome, thereby inflicting immunogenic cancer cell death. While CAN-2409's immunological impact has been well characterized, its effects on the tumor cells transcriptome remains unknown. We compared the transcriptomic landscape after treatment of glioblastoma models with CAN-2409 in vitro and in vivo to assess how the interplay with the tumor microenvironment influences CAN-2409-mediated transcriptome alterations. Methods: We performed RNA-Seq with CAN-2409 treated patient-derived glioma stem-like cells and tumors of C57/BL6 mice and compared KEGG pathway usage and differential gene expression focusing on immune cell and cytokine profiles. T-cell -killing assays were performed to assess candidate effectors. Results: PCA analysis showed distinct clustering of control and CAN-2409 samples under both conditions. KEGG pathway analysis revealed significant enrichment for p53 signaling and cell cycle pathway, with similar dynamics for key regulators of both pathways in vitro and in vivo, including MYC, CCNB1, PLK1 and CDC20. Selected alterations (PLK1 and CCNB1) were validated at the protein level. Cytokine expression analysis revealed upregulation of pro-inflammatory IL12a under both conditions; immune cell gene profiling showed reduction of myeloid associated genes. T-cell-killing assays showed increased killing in the presence of IL-12. Conclusion: CAN-2409 significantly alters the transcriptome both in vitro and in vivo. Comparison of pathway enrichment revealed mutual and differential utilization of pathways under both conditions, suggesting a modulating influence on the cell cycle in tumor cells, and of the tumor microenvironment on the transcriptome in vivo. IL-12 synthesis likely depends on interactions with the tumor microenvironment, and it facilitates CAN-2409 cell killing. This dataset provides potential to understand resistance mechanisms and identify potential biomarkers for future studies.

Salmonella infection induces the reorganization of follicular dendritic cell networks concomitant with the failure to generate germinal centers.

Germinal centers (GCs) are sites where plasma and memory B cells form to generate high-affinity, Ig class-switched antibodies. Specialized stromal cells called follicular dendritic cells (FDCs) are essential for GC formation. During systemic Salmonella Typhimurium (STm) infection GCs are absent, whereas extensive extrafollicular and switched antibody responses are maintained. The mechanisms that underpin the absence of GC formation are incompletely understood. Here, we demonstrate that STm induces a reversible disruption of niches within the splenic microenvironment, including the T and B cell compartments and the marginal zone. Alongside these effects after infection, mature FDC networks are strikingly absent, whereas immature FDC precursors, including marginal sinus pre-FDCs (MadCAM-1+) and perivascular pre-FDCs (PDGFRß+) are enriched. As normal FDC networks re-establish, extensive GCs become detectable throughout the spleen. Therefore, the reorganization of FDC networks and the loss of GC responses are key, parallel features of systemic STm infections.

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.

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.

A randomized, phase II study of sequential belimumab and rituximab in primary Sjögren's syndrome.

BACKGROUNDPrimary Sjögren's syndrome (pSS) is characterized by B cell hyperactivity and elevated B-lymphocyte stimulator (BLyS). Anti-BLyS treatment (e.g., belimumab) increases peripheral memory B cells; decreases naive, activated, and plasma B cell subsets; and increases stringency on B cell selection during reconstitution. Anti-CD20 therapeutics (e.g., rituximab) bind and deplete CD20-expressing B cells in circulation but are less effective in depleting tissue-resident CD20+ B cells. Combined, these 2 mechanisms may achieve synergistic effects.METHODSThis 68-week, phase II, double-blind study (GSK study 201842) randomized 86 adult patients with active pSS to 1 of 4 arms: placebo, s.c. belimumab, i.v. rituximab, or sequential belimumab + rituximab.RESULTSOverall, 60 patients completed treatment and follow-up until week 68. The incidence of adverse events (AEs) and drug-related AEs was similar across groups. Infections/infestations were the most common AEs, and no serious infections of special interest occurred. Near-complete depletion of minor salivary gland CD20+ B cells and a greater and more sustained depletion of peripheral CD19+ B cells were observed with belimumab + rituximab versus monotherapies. With belimumab + rituximab, reconstitution of peripheral B cells occurred, but it was delayed compared with rituximab. At week 68, mean (± standard error) total EULAR Sjögren's syndrome disease activity index scores decreased from 11.0 (1.17) at baseline to 5.0 (1.27) for belimumab + rituximab and 10.4 (1.36) to 8.6 (1.57) for placebo.CONCLUSIONThe safety profile of belimumab + rituximab in pSS was consistent with the monotherapies. Belimumab + rituximab induced enhanced salivary gland B cell depletion relative to the monotherapies, potentially leading to improved clinical outcomes.TRIAL REGISTRATIONClinicalTrials.gov NCT02631538.FUNDINGFunding was provided by GSK.

Cell-autonomous lipid-handling defects in Stargardt iPSC-derived retinal pigment epithelium cells.

Stargardt retinopathy is an inherited form of macular degeneration caused by mutations in gene ABCA4 and characterized by the accumulation of lipid-rich deposits in the retinal pigment epithelium (RPE), RPE atrophy, and photoreceptor cell death. Inadequate mechanistic insights into pathophysiological changes occurring in Stargardt RPE have hindered disease treatments. Here, we show that ABCA4 knockout and induced pluripotent stem cell-derived RPE (STGD1-iRPE) from patients with Stargardt differentiate normally but display intracellular lipid and ceramide deposits reminiscent of the disease phenotype. STGD1-iRPE also shows defective photoreceptor outer segment (POS) processing and reduced cathepsin B activity-indicating higher lysosomal pH. Lipid deposits in STGD1-iRPE are lowered by increasing the activity of ABCA1, a lipid transporter, and ABCA4 ortholog. Our work suggests that ABCA4 is involved in POS and lipid handling in RPE cells and provides guidance for ongoing gene therapy approaches to target both RPE and photoreceptor cells for an effective treatment.

The p-ERG spatial acuity in the biomedical pig under physiological conditions.

Pigs are becoming an important pre-clinical animal species for translational ophthalmology, due to similarities with humans in anatomical and physiological patterns. Different models of eye disorders have been proposed, and they are good candidates to assess biocompatibility/functionality of retinal prostheses. Electroretinography is a common tool allowing to gain information on retinal function, with several types of electroretinogram (ERG) been implemented including full field (ff-ERG), multifocal (mf-ERG) and pattern (p-ERG). p-ERG represents a valuable tool to monitor Retinal Ganglion Cells (RGCs) activity and can be used to calculate p-ERG spatial acuity. Unfortunately, scarce methodological data are available regarding recording/interpretation of p-ERG and retinal acuity in biomedical pigs yet enhancing knowledge regarding pig vision physiology will allow for more refined and responsible use of such species. Aim of this study was to record p-ERG in juvenile pigs to functionally assess visual acuity. Six female hybrid pigs underwent two p-ERG recording sessions at 16 and 19 weeks of age. Photopic ff-ERG were also recorded; optical coherence tomography (OCT) and histology were used to confirm retinal integrity. ff-ERG signals were repeatable within/across sessions. All p-ERG traces consistently displayed characterizing peaks, and the progressive decrease of amplitude in response to the increment of spatial frequency revealed the reliability of the method. Mean p-ERG spatial acuities were 5.7 ± 0.14 (16 weeks) and 6.2 ± 0.15 cpd (19 weeks). Overall, the p-ERG recordings described in the present work seem reliable and repeatable, and may represent an important tool when it comes to vision assessment in pigs.

Characterization of CD41+ cells in the lymph node.

Lymph nodes (LNs) are the critical sites of immunity, and the stromal cells of LNs are crucial to their function. Our understanding of the stromal compartment of the LN has deepened recently with the characterization of nontraditional stromal cells. CD41 (integrin αIIb) is known to be expressed by platelets and hematolymphoid cells. We identified two distinct populations of CD41+Lyve1+ and CD41+Lyve1- cells in the LNs. CD41+Lyve1- cells appear in the LN mostly at the later stages of the lives of mice. We identified CD41+ cells in human LNs as well. We demonstrated that murine CD41+ cells express mesodermal markers, such as Sca-1, CD105 and CD29, but lack platelet markers. We did not observe the presence of platelets around the HEVs or within proximity to fibroblastic reticular cells of the LN. Examination of thoracic duct lymph fluid showed the presence of CD41+Lyve1- cells, suggesting that these cells recirculate throughout the body. FTY720 reduced their trafficking to lymph fluid, suggesting that their egress is controlled by the S1P1 pathway. CD41+Lyve1- cells of the LNs were sensitive to radiation, suggestive of their replicative nature. Single cell RNA sequencing data showed that the CD41+ cell population in naïve mouse LNs expressed largely stromal cell markers. Further studies are required to examine more deeply the role of CD41+ cells in the function of LNs.

Perturbing DDR signaling enhances cytotoxic effects of local oncolytic virotherapy and modulates the immune environment in glioma.

CAN-2409 is a replication-deficient adenovirus encoding herpes simplex virus (HSV) thymidine kinase (tk) currently in clinical trials for treatment of glioblastoma. The expression of tk in transduced cancer cells results in conversion of the pro-drug ganciclovir into a toxic metabolite causing DNA damage, inducing immunogenic cell death and immune activation. We hypothesize that CAN-2409 combined with DNA-damage-response inhibitors could amplify tumor cell death, resulting in an improved response. We investigated the effects of ATR inhibitor AZD6738 in combination with CAN-2409 in vitro using cytotoxicity, cytokine, and fluorescence-activated cell sorting (FACS) assays in glioma cell lines and in vivo with an orthotopic syngeneic murine glioma model. Tumor immune infiltrates were analyzed by cytometry by time of flight (CyTOF). In vitro, we observed a significant increase in the DNA-damage marker γH2AX and decreased expression of PD-L1, pro-tumorigenic cytokines (interleukin-1ß [IL-1ß], IL-4), and ligand NKG2D after combination treatment compared with monotherapy or control. In vivo, long-term survival was increased after combination treatment (66.7%) compared with CAN-2409 (50%) and control. In a tumor re-challenge, long-term immunity after combination treatment was not improved. Our results suggest that ATR inhibition could amplify CAN-2409's efficacy in glioblastoma through increased DNA damage while having complex immunological ramifications, warranting further studies to determine the ideal conditions for maximized therapeutic benefit.

Cross-tissue, single-cell stromal atlas identifies shared pathological fibroblast phenotypes in four chronic inflammatory diseases.

BACKGROUND: Pro-inflammatory fibroblasts are critical for pathogenesis in rheumatoid arthritis, inflammatory bowel disease, interstitial lung disease, and Sjögren's syndrome and represent a novel therapeutic target for chronic inflammatory disease. However, the heterogeneity of fibroblast phenotypes, exacerbated by the lack of a common cross-tissue taxonomy, has limited our understanding of which pathways are shared by multiple diseases. METHODS: We profiled fibroblasts derived from inflamed and non-inflamed synovium, intestine, lungs, and salivary glands from affected individuals with single-cell RNA sequencing. We integrated all fibroblasts into a multi-tissue atlas to characterize shared and tissue-specific phenotypes. FINDINGS: Two shared clusters, CXCL10+CCL19+ immune-interacting and SPARC+COL3A1+ vascular-interacting fibroblasts, were expanded in all inflamed tissues and mapped to dermal analogs in a public atopic dermatitis atlas. We confirmed these human pro-inflammatory fibroblasts in animal models of lung, joint, and intestinal inflammation. CONCLUSIONS: This work represents a thorough investigation into fibroblasts across organ systems, individual donors, and disease states that reveals shared pathogenic activation states across four chronic inflammatory diseases. FUNDING: Grant from F. Hoffmann-La Roche (Roche) AG.

Immunofibroblasts regulate LTα3 expression in tertiary lymphoid structures in a pathway dependent on ICOS/ICOSL interaction.

Immunofibroblasts have been described within tertiary lymphoid structures (TLS) that regulate lymphocyte aggregation at sites of chronic inflammation. Here we report, for the first time, an immunoregulatory property of this population, dependent on inducible T-cell co-stimulator ligand and its ligand (ICOS/ICOS-L). During inflammation, immunofibroblasts, alongside other antigen presenting cells, like dendritic cells (DCs), upregulate ICOSL, binding incoming ICOS + T cells and inducing LTα3 production that, in turn, drives the chemokine production required for TLS assembly via TNFRI/II engagement. Pharmacological or genetic blocking of ICOS/ICOS-L interaction results in defective LTα expression, abrogating both lymphoid chemokine production and TLS formation. These data provide evidence of a previously unknown function for ICOSL-ICOS interaction, unveil a novel immunomodulatory function for immunofibroblasts, and reveal a key regulatory function of LTα3, both as biomarker of TLS establishment and as first driver of TLS formation and maintenance in mice and humans.

Sjögren's and non-Sjögren's sicca share a similar symptom burden but with a distinct symptom-associated proteomic signature.

OBJECTIVES: Given the similarity in symptoms between primary Sjogren's syndrome (SjS) and non-SjS sicca syndrome (sicca), we sought to characterise clinical and proteomic predictors of symptoms in both groups in order to better understand disease mechanisms and help guide development of immunomodulatory treatments. These have not, to date, unequivocally improved symptoms in SjS clinical trials. METHODS: Serum proteomics was performed using O-link inflammation and cardiovascular II panels. SjS (n=53) fulfilled 2016 ACR/European Alliance of Associations for Rheumatology (EULAR) criteria whereas sicca (n=60) were anti-Ro negative, displayed objective or subjective dryness, and either had a negative salivary gland biopsy or, in the absence of a biopsy, it was considered that a biopsy result would not change classification status. Linear regression analysis was performed to identify the key predictors of symptoms. Cluster analysis was completed using protein expression values. RESULTS: EULAR-Sjögren's-Syndrome-Patient-Reported-Index (ESSPRI), EuroQoL-5 Dimension utility values, and anxiety and depression did not differ between SjS and sicca. Correlations between body mass index (BMI) and ESSPRI were found in sicca and to a lesser extent in SjS. Twenty proteins positively associated with symptoms in sicca but none in SjS. We identified two proteomically defined subgroups in sicca and two in SjS that differed in symptom burden. Within hierarchical clustering of the SjS and sicca pool, the highest symptom burden groups were the least distinct. Levels of adrenomedullin (ADM), soluble CD40 (CD40) and spondin 2 (SPON2) together explained 51% of symptom variability in sicca. ADM was strongly correlated with ESSPRI (spearman's r=0.62; p<0.0001), even in a multivariate model corrected for BMI, age, objective dryness, depression and anxiety scores. CONCLUSIONS: Obesity-related metabolic factors may regulate symptoms in sicca. Further work should explore non-inflammatory drivers of high symptom burden in SjS to improve clinical trial outcomes.

Generalized Fast Discharges Along the Genetic Generalized Epilepsy Spectrum: Clinical and Prognostic Significance.

Objective: To investigate the electroclinical characteristics and the prognostic impact of generalized fast discharges in a large cohort of genetic generalized epilepsy (GGE) patients studied with 24-h prolonged ambulatory electroencephalography (paEEG). Methods: This retrospective multicenter cohort study included 202 GGE patients. The occurrence of generalized paroxysmal fast activity (GPFA) and generalized polyspike train (GPT) was reviewed. GGE patients were classified as having idiopathic generalized epilepsy (IGE) or another GGE syndrome (namely perioral myoclonia with absences, eyelid myoclonia with absences, epilepsy with myoclonic absences, generalized epilepsy with febrile seizures plus, or GGE without a specific epilepsy syndrome) according to recent classification proposals. Results: GPFA/GPT was found in overall 25 (12.4%) patients, though it was significantly less frequent in IGE compared with other GGE syndromes (9.3 vs. 25%, p = 0.007). GPFA/GPT was found independently of seizure type experienced during history, the presence of mild intellectual disability/borderline intellectual functioning, or EEG features. At multivariable analysis, GPFA/GPT was significantly associated with drug resistance (p = 0.04) and with a higher number of antiseizure medications (ASMs) at the time of paEEG (p < 0.001) and at the last medical observation (p < 0.001). Similarly, GPFA/GPT, frequent/abundant generalized spike-wave discharges during sleep, and a higher number of seizure types during history were the only factors independently associated with a lower chance of achieving 2-year seizure remission at the last medical observation. Additionally, a greater number of GPFA/GPT discharges significantly discriminated between patients who achieved 2-year seizure remission at the last medical observation and those who did not (area under the curve = 0.77, 95% confidence interval 0.57-0.97, p = 0.02). Conclusion: We found that generalized fast discharges were more common than expected in GGE patients when considering the entire GGE spectrum. In addition, our study highlighted that GPFA/GPT could be found along the entire GGE continuum, though their occurrence was more common in less benign GGE syndromes. Finally, we confirmed that GPFA/GPT was associated with difficult-to-treat GGE, as evidenced by the multivariable analysis and the higher ASM load during history.