Matrix Metalloproteinase-9 (MMP-9) as a Potential Regulator of Blood–Brain Barrier Dysfunction in Pediatric Acute Neuropsychiatric Syndrome (PANS)

Matrix Metalloproteinase-9 (MMP-9) as a Potential Regulator of Blood–Brain Barrier Dysfunction in Pediatric Acute Neuropsychiatric Syndrome (PANS) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Matrix Metalloproteinase-9 (MMP-9) as a Potential Regulator of Blood–Brain Barrier Dysfunction in Pediatric Acute Neuropsychiatric Syndrome (PANS) Ayan Mondal, Tristan Chou, Agnieszka Kalinowski, Meiqian Ma, Bahare Farhadian, and 15 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9442808/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Background Pediatric Acute-onset Neuropsychiatric Syndrome (PANS) is characterized by the abrupt onset of obsessive–compulsive symptoms and/or restrictive eating accompanied by disturbances in sleep, affect regulation, behavior, motor function, and sensory processing. Increasing evidence implicates systemic immune activation and circulating autoantibodies in basal ganglia dysfunction. Blood–brain barrier (BBB) impairment has been proposed as a mechanism by which peripheral inflammatory mediators access the central nervous system; however, the molecular pathways linking systemic inflammation to BBB disruption in PANS remain incompletely defined. Methods To determine whether circulating factors contribute to BBB dysfunction, human brain endothelial cell (BEC) monolayers were exposed to plasma from PANS patients during symptomatic flare and recovery, as well as from matched healthy controls. Barrier integrity was assessed by paracellular permeability assays; transcriptomic changes were analyzed using bulk RNA sequencing. Junctional organization and cytoskeletal architecture were examined by immunofluorescence microscopy. Circulating and endothelial-derived mediators associated with barrier disruption were quantified using multiplex bead–based immunoassays and enzyme-linked immunosorbent assays. Results Plasma from PANS flare (n = 15 samples) significantly increased BEC monolayer permeability compared to plasma from matched controls (~ 50% change in permeability; p < 0.001), corresponding with increased concentration of S100B, an in vivo biomarker of BBB permeability (~ 2 fold increase vs controls; p < 0.01). Transcriptomic profiling of flare samples demonstrated downregulation of genes essential for endothelial stability, including those encoding tight junction, adherens junction, and extracellular matrix components. Immunofluorescence of flare samples confirmed disruption of zonula occludens-1 (ZO-1) and vascular endothelial cadherin (VE-Cadherin), accompanied by increased actin stress fiber formation, consistent with enhanced cytoskeletal tension and junctional disassembly. Matrix metalloproteinase-9 (MMP-9) concentration was elevated in flare plasma vs controls (> 2.5 fold increase; p < 0.001) and was highly correlated with BEC monolayer permeability (r = 0.84; p < 0.001). Inhibition of MMP-9 in flare samples (n = 6) resulted in significant decrease in BEC monolayer permeability ( p < 0.01), approaching that of matched controls. Conclusions Circulating factors present during PANS flares induce brain endothelial dysfunction in vitro , correlating with in vivo biomarker findings. Elevated MMP-9 functions as a key downstream effector linking systemic inflammation to endothelial barrier disruption, providing mechanistic insight into how peripheral immune activation may facilitate neuroinflammation in PANS. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Pediatric Acute-onset Neuropsychiatric Syndrome (PANS) and Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal infections (PANDAS) are characterized by the sudden onset of severe neuropsychiatric symptoms (obsessive-compulsive behaviors, eating restrictions, motor abnormalities, emotion and behavior dysregulation, sleep disturbances, cognitive dysfunction, and urinary symptoms) [ 1 – 8 ]. Emerging research suggests that the basal ganglia is the site of pathology, and the evidence includes: neuroimaging studies [ 9 – 12 ], neurological soft signs pertaining to the basal ganglia [ 13 ]; and REM sleep without atonia—seen in Parkinson’s and other basal ganglia disorders [ 14 – 17 ]. Additionally, autoantibodies have been shown to bind to cholinergic interneurons (CINs) within the basal ganglia and alter their electrical activity [ 18 – 20 ]. Autoantibodies to other neuronal antigens/targets (tubulin, lysoganglioside, dopamine receptors, etc) have also been reported [ 21 – 31 ]. Lastly, the hallmark symptoms of PANS align with basal ganglia dysfunction. Beyond central nervous system (CNS) inflammation, PANS is often associated with subtle signs of systemic inflammation including enthesitis, joint effusions, synovitis or capsulitis (demonstrated by joint ultrasound), vasculopathy signs, and a higher prevalence of comorbid autoimmune disorders [ 32 , 33 ]. Further evidence for systemic inflammation includes elevated proinflammatory cytokines and proinflammatory monocytes [ 34 – 38 ]. These observations support the hypothesis that both systemic and central immune processes contribute to the pathophysiology of PANS. For circulating immune factors, including autoantibodies, to access the CNS, they must first cross the blood-brain barrier (BBB). The BBB is a highly specialized and tightly regulated interface which is primarily composed of brain microvascular endothelial cells (BECs), which regulate the entry of solutes, immune cells, and macromolecules to the CNS. Under physiological conditions, the BBB maintains restrictive properties through complex intercellular junctions, including the BECs’ tight junctions (e.g., claudins, occludin, ZO-1) and adherens junctions (e.g., VE-cadherin), which are anchored to the actin cytoskeleton and are responsible for high trans-endothelial resistance [ 39 , 40 ]. However, systemic inflammation-driven disruption of this barrier may allow immune effectors to access and alter the CNS environment. Among the systemic inflammatory mediators, matrix metalloproteinases (MMPs) are important for their ability to degrade the junctions of endothelial cells and increase BBB permeability [ 41 , 42 ]. MMP-9 is recognized as the most effective among all the MMPs, due to its proteolytic specificity towards extracellular matrix and cell junctional proteins. MMP-9-associated BBB dysfunction has been described in other neuroinflammatory disorders [ 43 ], [ 29 ]. BBB disruption enables serum proteins, such as albumin and plasminogen, to enter the brain, activating neurotoxic pathways and facilitating the infiltration of peripheral immune cells, including monocytes and macrophages. These cells can exacerbate neuroinflammation and even acquire microglia-like phenotypes once within the CNS [ 44 ]. Notably, Rahman et al. recently demonstrated an increase in circulating proinflammatory monocytes in PANS flare (i.e., periods of abrupt symptom escalation) along with CNS-homing monocytes [ 37 , 38 ]. Another clinical observation that supports the hypothesis of BBB dysfunction in PANS is that patients exhibit sensitivity to psychotropic medications (during early flare state), a feature which is also described in autoimmune encephalitis [ 45 ]. Based on this evidence, we hypothesized that systemic inflammation during PANS flare contributes to BBB dysfunction. We tested the physiological effect of plasma from patients with PANS in their flare state (compared with healthy controls and patients with PANS in their recovered state) on a BEC monolayer. We observed that PANS flare plasma induced endothelial hyper-permeability, disrupted junctional integrity, and promoted actin stress fiber formation. Mechanistic studies implicated MMP-9 as a key effector molecule driving this dysfunction, and its inhibition successfully blocked this barrier breakdown. To our knowledge, this is the first mechanistic study using a BBB model and PANS patient plasma to demonstrate a direct link between plasma factors (likely to reflect systemic inflammation) and brain endothelial barrier disruption. These findings provide critical insight into how peripheral inflammation may facilitate neuroinflammation in PANS and support the role of the BBB as a therapeutic target. Materials and Methods The study protocol was approved by the Stanford Human Participants Institutional Review Board and covers all patients and procedures in this study. Immune Behavioral Health (IBH) Clinic clinicians cared for all patients. Written parental consent and child assent were obtained per study protocol (protocol number 26922). Diagnosis and disease state classification Psychiatric symptoms at clinic presentation and other demographics for samples used in each experiment are listed in Table 1 . Paired PANS flare and recovery samples were used whenever possible. A total of 15 PANS patients were enrolled in the study, of whom 10 provided paired flare–recovery samples for longitudinal analyses. For each experiment, PANS samples were matched to healthy control samples based on age (± 1 year) and sex assigned at birth to minimize demographic confounding. Blood samples were processed using Ficoll gradient to acquire peripheral blood mononuclear cells (PBMC) and plasma; these were stored in liquid nitrogen and − 80°C, respectively, and maintained by Stanford Biobank. Different sample sizes were used across assays depending on sample availability and specific experimental objectives. 15 plasma samples per group (PANS flare and healthy controls) were used for permeability and MMP-9 quantification; 10 per group were used for RNA-seq (selected for clinical uniformity and absence of recent medication to minimize heterogeneity); and 10 paired flare–recovery samples were included for the desired assays based on the availability. 6 heparinized plasmas with confirmed PANS flare were selected from the cohort to conduct exogenous MMP-9 inhibitor-based experiments. All study subjects were classified as PANS by the Stanford IBH psychiatry team (MS, YX, PT, MT) using PANS diagnosis and evaluation criteria [ 46 ]. Definitions of disease state and clinical course were based on previously published criteria and are summarized in Appendix Table 2 [ 47 ]. Table 1 Demographic and clinical presentation data of patients with PANS in flare state, recovered state, and healthy controls in this study. Note that patients in recovered state and heathy controls did not report neuropsychiatric symptoms at clinic presentation. Where data were not measured for a given subject group, the corresponding cell is empty. Age at the first clinic visit, mean (SD) Flare state (n = 15) Recovered state (n = 15) Healthy controls (n = 15) 9.7 (2.6) . 11.6 (2.9) Age at blood draw, mean (SD) 12.0 (3.2) . 11.7 (3.0) Male, n (%) 10 (67%) . 8 (53%) Race/ethnicity, n (%) White/European 13 (87%) . 6 (40%) Asian/Asian American (including biracial/mixed) 0 (0%) . 8 (53%) Hispanic/Latino (including biracial/mixed) 2 (13%) . 1 (7%) Clinical severity scores, mean (SD) Columbia Impairment Score 16.5 (11.5) 5.0 (4.1) 0.3 (0.5) Global Impairment 43.4 (19.6) 17.5 (11.7) 1.9 (5.4) Modified Overt Aggression Scale 6.2 (8.3) 2.0 (4.7) 0.0 (0.0) Children’s Global Assessment Scale 59.8 (9.1) 79.3 (11.5) . Children’s Yale-Brown Obsessive Compulsive Scale 21.1 (8.2) 13.0 (5.8) . Percent Baseline 55.8 (19.7) 83.0 (8.0) . Age at first neuropsychiatric decline, mean (SD) 8.1 (2.3) . . Age at deterioration leading to clinic visit, mean (SD) 9.4 (2.6) . . Neuropsychiatric symptoms at clinic presentation, n (%) Obsessions 13 (87%) . . Compulsions 12 (80%) . . Food refusal/avoidance 10 (67%) . . Fluid refusal/avoidance 3 (20%) . . Separation anxiety 11 (73%) . . Other anxiety 13 (87%) . . Mood swings/moodiness 9 (60%) . . Emotional lability 3 (20%) . . Suicidal ideation/behavior 4 (27%) . . Depression 10 (67%) . . Irritability 10 (67%) . . Aggression 8 (53%) . . Oppositional behavior 9 (60%) . . Hyperactivity 6 (40%) . . Trouble paying attention 9 (60%) . . Behavioral/developmental regression 12 (80%) . . Dysgraphia 6 (40%) . . Cognitive symptoms 8 (53%) . . Pain 10 (67%) . . Sleep problems 10 (67%) . . Enuresis 3 (20%) . . Urinary frequency 6 (40%) . . Sensory amplification 9 (60%) . . Hallucinations 1 (7%) . . Delusions or paranoid thoughts 3 (20%) . . Motor tics 5 (33%) . . Phonic tics 2 (13%) . . Endothelial cell monolayer permeability : BECs were grown to confluent monolayers on 24-well plate Transwell® inserts (Corning Costar, 0.4 µm) coated with fibronectin as described previously by Badawi et al. [ 48 ]. The cells were incubated with heat-inactivated plasma from patients with PANS flare patients (n = 15) and healthy controls (n = 15). The cell culture media was replaced for 60 min of each experiment with fresh dye-free culture media (Opti MEM, Life Technologies, Grand Island, NY) to avoid interfering agents during fluorescence measurement. The wells of the Transwell® plates were divided into 3 different groups: untreated control, healthy control and PANS flares. Endothelial cell permeability was measured following the protocol described by Robinson et al. [ 49 ]. Each sample was treated in duplicate, including the untreated control. Each experimental group was included in a 12-well/plate monolayer plate. FITC-dextran 10 kDa was applied to the luminal (upper) side of the Transwell® at a final concentration of approximately 500 µg/mL and allowed to equilibrate through the monolayer between the luminal and abluminal (lower) chambers for 30 mins. Cell culture media samples were obtained from the abluminal chambers of the Transwell® system and measured with a fluorometric plate reader (excitation, 485 nm; emission, 535 nm) to quantify the fluorescence intensity, which represents the permeability of FITC-dextran across the monolayer barrier. The fluorescence intensity values obtained with the PANS flares and healthy controls were normalized to the values obtained with the untreated samples performed on the same day and time, and expressed as percent permeability relative to untreated control (set to 100%). Bulk RNA sequencing of BECs BECs were treated with plasma from PANS flares (n = 10) and healthy controls (n = 10) for 12 hrs, after which the cells were washed with PBS and collected in TRIzol reagent (Invitrogen). Total RNA was isolated via a Direct-zol kit (Zymogen, Irvine, CA) according to the manufacturer’s instructions and sent to Novogene (Sacramento, CA) for bulk RNA sequencing on the Illumina Nova-seq X platform. The mRNA library was prepared using poly (A) mRNA enrichment. Paired-end RNA-seq reads were indexed and quantified via the pseudoalignment algorithm Kallisto, with an average read alignment of 88.8% [ 50 ]. The sequences were then annotated and mapped to the reference genome EnsDb.Hsapiens.v86 at the gene level [ 51 ]. The readings were normalized to transcripts per million (TPM), and principal component analysis was performed in R via built-in libraries. The R package edgeR was used to compile differential genes used to generate volcano plot and hierarchically clustered heatmaps. Immunofluorescence and F-actin Labeling : BECs were used for immunofluorescence localization of the tight junction protein ZO-1, adherens junction protein VE-cadherin, and actin stress fibers. The cells were grown as monolayers on chamber slides coated with fibronectin. The cells were incubated with heat-inactivated plasma from either PANS flares or healthy controls for 12 hrs as previously described to measure permeability. For stress fiber staining, the cells were fixed with 3.7% paraformaldehyde followed by subsequent washing with phosphate-buffered saline; ActinRed 555 solution was then added for 30 min. ActinRed 555 is a stable solution of rhodamine phalloidin at room temperature and used to stain actin stress fibers. To stain for ZO-1 and VE-cadherin, BECs were fixed in paraformaldehyde (3.7%) for 10 min following plasma treatment, followed by permeabilization with 0.5% Triton X-100 (Sigma‒Aldrich, Carlsbad, CA) for a maximum of 10 min. This was followed by blocking with bovine serum albumin (2%) (Sigma‒Aldrich) for 45–60 min and 60 min incubation with the primary antibodies anti-rabbit ZO-1 (ab221547; 1:200) and mouse monoclonal VE-cadherin (MAB9381; 1:150; R&D Systems, Minneapolis, MN). After primary antibody incubation, the cells were incubated with fluorescent-tagged secondary antibodies for a maximum of 60 min, followed by 5 washes with phosphate-buffered saline. The secondary antibodies used were Alexa Fluor 647-conjugated goat anti-mouse IgG (A21235; 1:1000) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (A11008; 1:500) (Invitrogen, Eugene, OR). All the slides were subsequently washed with PBS following fluorescence antibody incubation and mounted with ProLong™ Gold Antifade Mountant with DNA stain DAPI (Invitrogen, Eugene, OR). ZO-1, VE-cadherin and phalloidin fluorescence was visualized via a Zeiss LSM 980 with a super resolution Airyscan 2 confocal microscope at the Stanford Core facility. Images were taken and quantified via ImageJ, and the mean intensity value was calculated. MMP-9 inhibition We used MMP-9 inhibitor I (Abcam, Waltham, MA) to block MMP-9 activity. The inhibitor binds Zn ions at the active site of the MMP-9 pro enzyme and blocks its activation. To measure permeability, the inhibitor was pretreated for an hour at a minimum concentration of 5 µM in an immunofluorescence assay, and subsequently plasma was added. ELISAs : MMP-9, TIMP-1, IL-6, IL-8, S100B, and CCL11 ELISAs were performed according to the manufacturer’s instructions. The plasma was diluted according to the following: 1:50 for MMP-9; and 1:100 for TIMP-1, IL-6, IL-8, and S100B. The human MMP-9, TIMP-1, IL-6, and IL-8 ELISA kits were purchased from Proteintech (Rosemont, IL), CCL11 from R and D Systems (Minneapolis, MN), and S100B from Millipore (Hayward, CA). Cytokine multiplex assay : This assay was performed with Luminex-EMD Millipore Human 48 Plex kits (Millipore, Burlington, MA) by the Human Immune Monitoring Center at Stanford University. Kits were run according to the manufacturer’s recommendations with modifications by the team. The H48 kits include one panel: Milliplex HCYTA-60K-PX48. The assay setup adhered to the recommended protocol. The culture supernatants were undiluted in a 96-well plate. The plasma samples were diluted 3-fold, and the samples were incubated overnight at 4°C with shaking on an orbital shaker at 500–600 rpm. Following incubation, the plates were washed twice with wash buffer using a BioTek ELx405 washer (BioTek Instruments, Winooski, VT) and incubated with detection antibody for 1 hr at room temperature, followed by the addition of Streptavidin-PE for 30 min with shaking. Finally, the plates were washed as described above, and wash buffer was added to the wells for reading in the Luminex FLEXMAP 3D instrument, ensuring a lower bound of 50 beads per sample per cytokine. Each sample was measured in duplicate. Custom AssayChex control beads (Radix BioSolutions, Georgetown, TX) were added to all the wells. Wells with a bead count < 50 were flagged, and data with a bead count < 20 were excluded. Statistical analysis Data are presented as median values with individual data points. Statistical comparisons between two groups were performed using the Mann–Whitney U test for unpaired data (e.g., healthy controls vs. PANS flare) and the Wilcoxon signed-rank test for paired data (e.g., PANS flare vs. recovery). Correlation analyses were performed using Spearman’s rank correlation coefficient. A p-value < 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism (version 11). Results PANS flare plasma induces BEC hyperpermeability and this is strongly correlated with clinical severity To evaluate the impact of plasma from PANS patients on BBB integrity, we cultured primary BECs as confluent monolayers on Transwell® inserts. Heat-inactivated plasma from PANS flare patients, those same patients during recovery (partial and full), and healthy controls (age- and sex-matched) was applied to the apical compartment, and endothelial permeability was assessed using a FITC–dextran flux assay (Fig. 1A). Plasma was incubated for approximately 12 h, a duration previously determined to yield maximal biological activity following neutralization of cytotoxic effects on cells (permeability kinetics and cytotoxicity measurement displayed in Appendix Fig. 1). Exposure to PANS flare plasma resulted in a significant increase in BEC monolayer permeability compared with application of healthy control plasma (p < 0.001; n = 15) (Fig. 1B), indicating that circulating factors in PANS plasma compromise the BEC monolayer integrity. We also assessed Spearman’s rank correlation between BEC monolayer permeability and clinical severity scores: PANS 31-Item Symptom Rating Scale, Columbia Impairment Scale, PANS Global Impairment Scale, and the Modified Overt Agression Scale [ 52 – 56 ]. Figure 1C depicts the correlation between permeability percentage and clinical severity scores for all subjects with recorded scores. There were modest-to-strong correlations between the BEC permeability and severity measures. PANS flare plasma alters BEC transcriptional programs related to cell junctions, immune signaling and cytoskeletal organizations To explore the molecular basis of this hyperpermeability, we performed bulk RNA sequencing on BECs exposed to PANS flare plasma (n = 10) and age- and sex-matched healthy control plasma (n = 10) (equal numbers of males and females). Principal component analysis (PCA) revealed clear separation between PANS flare state and controls (Fig. 2A). The first two principal components, PC1 (37.3%) and PC2 (17.2%), accounted for the majority of the total variance. Samples clustered distinctly along PC1, with healthy controls (green) on the left and PANS flare samples (red) on the right, suggesting that PANS plasma induces a unique and consistent endothelial transcriptomic signature. Differential expression analysis (PANS flare versus matched healthy control, Fig. 2B) further identified a broad transcriptional shift in genes known to regulate endothelial barrier structures and inflammation. The result showed significantly increased expression of several genes in the BEC known to be involved in extracellular matrix remodeling, immune-endothelial signaling, and cellular adhesion that include APOE, FN1, and NECTIN2 (gene abbreviations provided in Appendix Table 3). Conversely, ANGPT1, CLDN12, and GJA1 were markedly downregulated, indicating loss of barrier integrity due to destabilization of tight and adherens junctions. A focused hierarchical clustering heatmap of endothelial junctional and adhesion-related genes (Fig. 2C) demonstrated robust group-specific expression patterns, where PANS flares are marked as “P” and healthy controls as “H”. PANS flare plasma caused reduced expression of canonical tight junction markers (CLDN3, TJP1, TJP2, GJA1) and adherens junction components (CDH5, ANGPT1), as well as genes maintaining extracellular matrix integrity and cytoskeletal regulation (CDC42, HIF1A, HEG1, NECTIN1, ITGB1). In contrast, leukocyte adhesion and inflammatory genes (ICAM1, ICAM2, CCL2, ANGPT2) were consistently upregulated in the BEC with the plasma from PANS flare, indicating an activated endothelial phenotype during active disease state. Altogether, the bulk RNA-seq results (from BECs exposed to PANS flare plasma) indicate strong transcriptomic evidence that circulating inflammatory mediators in PANS flare plasma compromise BBB integrity by remodeling endothelial junctional architecture and inducing inflammatory signaling pathways, thereby promoting a hyperpermeable and pro-inflammatory vascular phenotype consistent with neuroimmune dysregulation in PANS. Immunofluorescence shows plasma from PANS flare disrupts endothelial junctional integrity and BEC cytoskeleton To validate the above findings obtained in the bulk RNA sequencing, particularly the changes in cell junction and extracellular matrix remodeling genes (induced by PANS flare plasma), we performed immunofluorescence labeling of ZO-1 (tight junction) and VE-cadherin (adherens junction), along with rhodamine–phalloidin labeling for actin stress fibers. Consistent with the RNA-seq results, treatment with PANS flare plasma led to fragmented, discontinuous staining of ZO-1 and VE-cadherin, and the appearance of stress fiber-like actin rearrangements, indicating substantial cytoskeletal remodeling and loss of cell–cell junction integrity in the BEC (Fig. 3A). Healthy control-treated BECs exhibited continuous and well-defined staining of these junctional markers and a lack of stress fiber formation, which is consistent with cellular and barrier stability. Specifically, mean fluorescence intensity analysis showed significantly reduced expression of VE-cadherin (3-fold) and ZO-1 (nearly 3-fold; Fig. 3B). Rhodamine–phalloidin labeling showed 4-fold increase in actin stress fibers in BECs exposed to PANS flare plasma compared with healthy controls (p < 0.01; n = 10). These findings indicate that distinct plasma protein factors during the PANS flare induce endothelial junctional disruption and contribute to BBB permeability. PANS recovery plasma preserves BEC barrier integrity and junctional organization To assess whether recovery plasma induced barrier abnormalities, BECs were treated with plasma from the same patients after recovery and pairwise comparison with flare state was performed. We compared the permeability assay (Fig. 4A), and immunofluorescence for ZO-1, VE-cadherin, and phalloidin for actin-stress fibers (Fig. 4B), as previously done. FITC-dextran permeability assay showed a significant difference between flare and recovery (p < 0.01; n = 10) and was consistent across subjects. Moreover, recovery plasma did not induce discontinuous VE-cadherin and ZO-1 labeling along the cell borders, nor did it induce actin-stress fiber formation. Quantitative image analysis confirmed junctional protein intensity and lack of actin-stress fiber formation with consistent results across subjects. (p < 0.05–0.001, n = 10; Fig. 4C). Collectively, these findings support the presence of cell-disrupting factors in the plasma of PANS flare, causing endothelial cell disassembly and disruption of the actin cytoskeleton. This leads to BBB hyperpermeability in PANS flare samples, which is not induced by plasma from PANS recovery samples. Elevated plasma matrix metalloprotease-9 (MMP-9) is associated with the BEC hyperpermeability in PANS flare We measured MMP-9 concentration in the plasma of PANS flare and in the matched healthy controls using ELISA. MMP-9 was selected due to its established function as a pro-inflammatory matrix metalloprotease secreted by activated leukocytes, and its known capacity to degrade extracellular matrix components and disrupt endothelial junctions, including VE-cadherin and tight junction proteins [ 57 , 58 ]. A comparative analysis revealed that MMP-9 concentrations were more than 2.5-fold higher in the plasma of PANS flare patients compared with healthy controls (p < 0.001; n = 15; Fig. 5). These elevated plasma MMP-9 levels strongly correlated with the degree of BEC permeability measured by FITC–dextran (r = 0.84; p < 0.001). PANS flare samples (red, n = 15) exhibited higher MMP-9 concentrations (up to ~ 1000 ng/mL) and increased permeability (~ 180%), whereas healthy controls (green, n = 14) clustered around lower MMP-9 levels (< 200 ng/mL) and baseline permeability (~ 100%). These results indicate a strong association between systemic MMP-9 and BBB leakiness (Fig. 5A). To evaluate whether MMP-9 levels reflect disease state, we analyzed paired plasma samples collected from the same patients during flare and recovery phases. MMP-9 concentrations were approximately 2-fold lower during recovery compared to the flare state. This reduction was statistically significant (p < 0.01; n = 10) and consistent across patients (Fig. 5B). Overall, these findings suggest that plasma MMP-9 levels are dynamic, reflect disease state, and may serve as a key mediator of BBB disruption during PANS flare, highlighting its potential as a therapeutic target in PANS. Increased plasma MMP-9 in PANS flare may induce an inflammatory feedback loop in BECs To identify immune mediators released by BECs in response to PANS plasma, we performed a multiplex assay of cell culture supernatants. We used heparinized plasma for this assay as it preserves proteolytic activity (e.g., MMP-9 activity) more effectively than EDTA plasma. Several analytes were below the detection limit; however, PDGF-AA, IL-6, and IL-8 (CXCL-8) were consistently elevated in PANS flare-treated BEC culture supernatant in comparison to the healthy controls (Appendix Fig. 2). Based on these findings, IL-6 and IL-8 (CXCL-8) were selected for targeted ELISA validation. We measured IL-6 and IL-8 (CXCL-8) levels in BEC supernatants by ELISA using heat-inactivated heparinized plasma from PANS flare and matched controls. Both cytokines are known for their established role in compromising endothelial junctional integrity and increasing barrier permeability [ 59 , 60 ]. ELISA analysis revealed a significant increase in IL-6 levels in BEC supernatants exposed to PANS flare plasma, approximately 2.5-fold higher than those in the healthy control group (Fig. 6A). Although IL-8 (CXCL-8) levels also showed a trend toward elevation, the difference did not reach statistical significance. These findings suggest that plasma factors (potentially MMP-9) may stimulate IL-6 release from BECs. Notably, previous studies have shown that elevated IL-6 and IL-8 (CXCL-8) can further induce MMP-9 expression and activation, amplifying endothelial disruption [ 61 , 62 ]. In addition to inducing BEC production of cytokines, PANS-flare-treated BECs produced MMP-9 at a 6-fold increase over healthy controls. The active form of MMP-9 was assessed by determining the MMP-9/TIMP-1 ratio, where TIMP-1 serves as an endogenous inhibitor of MMP-9 [ 63 ]. An elevated MMP-9/TIMP-1 ratio previously associated with BBB disruption and neurovascular injury in conditions such as stroke, cerebral ischemia, and systemic sclerosis [ 64 – 66 ] was also observed in PANS flare-treated BEC supernatant (Fig. 6B). Collectively, these results indicate that elevated MMP-9 in the PANS flare state induces a self-reinforcing inflammatory loop that exacerbates BEC dysfunction. High MMP-9 levels stimulate IL-6 and IL-8 (CXCL-8) secretion, which, in turn, promote further MMP-9 activation, driving endothelial barrier degradation. The elevated MMP-9/TIMP-1 ratio further supports the presence of a proteolytic inflammatory cascade contributing to BBB dysfunction in PANS. An exogenous MMP-9 inhibitor prevents BEC barrier disruptions induced by PANS flare plasma To further validate the contribution of MMP-9 to BEC disruptions, we tested the impact of direct application of MMP-9 and co-treatment with a pharmacological inhibitor of MMP-9. We used a specific MMP-9 inhibitor, MMP-9 inhibitor-I, 2-[benzyl-(4-methoxyphenyl)sulfonylamino]-5-(diethylaminomethyl)-N-hydroxy-3-methylbenzamide. To confirm the functional role of MMP-9, we first added recombinant human MMP-9 (500 ng/mL) to normal human serum, which increased BEC permeability compared to the serum alone, mimicking the effects observed with PANS flare plasma (Fig. 7A). Co-treatment with the MMP-9 inhibitor attenuated this effect in a dose-dependent manner. A similar protective effect was observed when PANS flare plasma was co-treated with the MMP-9 inhibitor. Specifically, inhibition of MMP-9 significantly reduced PANS flare plasma-induced hyperpermeability, with 5 µM partially reversing barrier disruption and 50 µM restoring barrier integrity to near-baseline levels (p < 0.01; n = 6; Fig. 7B). These results demonstrate a strong and dose-dependent protective effect of MMP-9 inhibition on endothelial barrier function. Next, we assessed the influence of co-treatment with the MMP-9 inhibitor on IL-6 and IL-8 (CXCL-8). ELISA analysis of BEC supernatants revealed MMP-9 inhibition markedly reduced IL-6 and IL-8 (CXCL-8) concentration in the cell culture supernatant (Fig. 7C). Immunofluorescence further confirmed that MMP-9 inhibition preserved endothelial junctional architecture (Fig. 7D). BECs treated with PANS flare plasma alone showed disrupted localization of VE-cadherin and ZO-1 (loss of junctional continuity) and enhanced phalloidin staining (actin stress-fiber formation). In contrast, co-treatment with the MMP-9 inhibitor restored continuous VE-cadherin and ZO-1 staining along cell borders and markedly reduced stress fibers. Quantitative analysis verified these findings (p < 0.05 for all markers, n = 5; Fig. 7E), thus validating the protective effect of MMP-9 inhibition at the cellular level. This finding suggests a feedback loop in which MMP-9 not only disrupts endothelial integrity but also sustains cytokine production, possibly through downstream inflammatory signaling pathways or extracellular matrix remodeling. Pharmacological inhibition of MMP-9 effectively reverses these pathological effects, supporting its potential as a therapeutic target for restoring BBB integrity in PANS. Elevated plasma S100B provides indirect in vivo evidence of BBB disruption during PANS flare Indirect evidence of BBB disruption during PANS flare was assessed by measuring plasma levels of S100B, which is a well-recognized peripheral biomarker of BBB permeability. Plasma S100B concentrations were significantly elevated in patients during PANS flare compared with age- and sex-matched healthy controls and were reduced upon clinical recovery in paired samples (Appendix 1). These findings support the presence of BBB vulnerability in vivo during active PANS disease states. Impact of PANS flare on endothelial monolayer permeability is specific to BECs To determine whether the effects of PANS flare plasma on endothelial permeability were specific to the brain vasculature, primary human endothelial cells derived from lung, umbilical vein, and brain were cultured as confluent monolayers on Transwell® inserts. Exposure to PANS flare plasma did not significantly alter permeability in lung or umbilical vein endothelial monolayers compared with healthy control plasma. In contrast, a marked increase in permeability was observed exclusively in BEC monolayers (Appendix 2C), indicating tissue-specific susceptibility of the BBB to circulating factors present during PANS flare. Exploratory plasma multiplex profiling identifies CCL11 as a flare-associated chemokine in PANS To explore whether circulating inflammatory mediators beyond MMP-9 are altered during PANS disease activity, we performed an exploratory multiplex cytokine and chemokine analysis of PANS plasma (flare and recovery), along with matched healthy controls. Among the analytes assessed, CCL11 was selectively elevated in plasma during PANS flare compared with healthy controls and showed a consistent reduction upon clinical improvement in paired samples (Appendix 4 ). In contrast, other chemokines, cytokines, and growth factors showed variability within groups and failed to exhibit a consistent, flare-specific, state-dependent pattern. We followed up by measuring plasma CCL11 concentration using ELISA in an expanded sample set, which strongly supported the multiplex results (Appendix 5). Altogether, the exploration multiplex plasma profiling provides a useful direction for hypothesis generating and identifies CCL11 as a candidate flare-associated chemokine in PANS. Discussion PANS is increasingly recognized as an immune-mediated condition caused by infections and systemic inflammation. BBB dysfunction has been implicated based on preliminary studies which show elevated protein and/or albumin quotient in the cerebral spinal fluid of patients, hypersensitivity to psychotropics, and brain homing cells [ 38 , 45 , 67 ]. Our present findings provide direct mechanistic evidence supporting the role of systemic inflammatory factors in the possible BBB dysfunction that occurs during a PANS flare. Plasma from flare patients caused disruption of structure and function of BECs which form the BBB. Exposure to PANS flare plasma led to rapid and pronounced hyperpermeability over time, accompanied by loss of tight and adherens junctional proteins and enhanced actin stress fiber formation. These effects were not observed with BECs treated with plasma from healthy controls nor plasma from the same patients collected during the recovered state, thus indicating that the BBB disruption in PANS reflects a dynamic and disease state-dependent process. The potential for BBB disruption during PANS flare was also supported by one of our crucial preliminary findings: an increased circulatory S100B protein concentration during flare, measured in the plasma by ELISA (Appendix Fig. 4). S100B is considered a promising biomarker for BBB leakiness as it does not cross the BBB in a healthy physiological state [ 68 , 69 ]. These early findings provide a critical clue that BBB dysfunction may not just be a bystander, but an active contributor to neuropsychiatric dysfunction during PANS flares. Our study supports the need to investigate BBB breakdown as both a mechanistic link and a promising therapeutic target in the care of youth affected by this debilitating condition. We conducted bulk RNA-seq analysis of BECs treated with PANS flare plasma that revealed marked downregulation of genes encoding critical junctional proteins. These include genes that form and stabilize the tight junction structure, such as TJP1 (encoding ZO-1), claudins, and JAMs. Downregulation of these genes likely compromises BBB integrity by disrupting intercellular adhesion and cytoskeletal structure. The tight junction protein ZO-1, in particular, plays a central role in organizing the endothelial tight junctions and stabilizing adherens junctions [ 70 , 71 ]. ZO‑1 is a critical scaffolding protein that links TJ proteins, claudins and occludin to the actin cytoskeleton. Therefore, downregulation of TJP1 is presumed to have a significant impact on the stability of endothelial junctions, disrupting both tight junctions and adherens junctions. The immunofluorescence of ZO-1 with PANS flare plasma showed a decrease in the fluorescence intensity around the BEC junctions, indicating ZO-1 instability in the BECs. Beyond TJP1, we also identified reduced expression of vinculin (VCL) and ANGPT1, which are known to stabilize adherens junction VE-cadherin. VCL is known to stabilize cadherin–catenin complex formation, while ANGPT1 stabilizes VE-cadherin–actin interactions through its receptor, Tie2, on endothelial cells [ 72 – 75 ]. Decreased expression of both VCL and ANGPT1 weakens cytoskeletal support at the adherens junctions, destabilizing and further compromising endothelial barrier integrity. Although decreased CDH5 expression was not observed in the volcano plot (Fig. 2B), the heat map in Fig. 2C showed consistent reduction of CDH5 (encoding the VE-cadherin) in the BECs with PANS flare plasma. The decrease in expression of VE-cadherin in immunofluorescence staining with the PANS flare plasma also indicates the disruptions of adherens junctions and destabilization of barrier integrity. In addition to downregulation of canonical junctional genes such as TJP1 and CDH5, the present findings reveal suppression of ITGB1 and CDC42, both of which are essential for cytoskeletal anchoring of endothelial cells to the basement membrane and for maintaining barrier stability [ 76 , 77 ]. β1-integrin (ITGB1) is more consistently reported to promote actin stability and support endothelial structure and its downregulation destabilizes the endothelial junctions and increases the paracellular permeability by stress fiber actin formation [ 77 – 79 ]. The immunofluorescence data were also corroborated by RNA-seq data, which showed increased actin stress fibers with PANS flare plasma. Notably, our RNA-seq data for BEC strongly align with recent findings from a mouse model of repeated group A streptococcus (GAS) infection, in which the BBB-associated genes in the BECs were transcriptionally suppressed, including the genes stabilizing multiple junctional proteins. Further analysis revealed transcriptional elevation of inflammatory microglial phenotype in GAS-induced mouse models, strongly supporting BBB predominant BBB dysfunction in PANS. The study further identified robust inflammatory microglial activation following GAS infection, strongly supporting the contribution of BBB dysfunction in PANS/PANDAS-like conditions (Wayne et al, 2026 bioRxiv Preprint doi: 10.64898/2026.02.04.703836 ). Simultaneously, upregulation of ICAM1, ICAM2, and CCL2 indicates a shift toward a pro-adhesive, inflammatory endothelial state that may promote leukocyte trafficking across the BBB [ 80 – 83 ]. Elevated ANGPT2 alongside reduced ANGPT1 further supports vascular destabilization through imbalance of the Tie2 signaling axis, a well-established hallmark of endothelial dysfunction [ 84 , 85 ]. Together, these results strongly implicate that PANS flare plasma not only compromises the structural integrity of the brain endothelial barrier but also primes it for increased immune cell trafficking. This dual impact reflects the classic hallmarks of BBB dysfunction. The impact of PANS flare plasma on permeability appears to be limited to BECs, rather than primary lung or umbilical vein endothelial cells (Appendix Fig. 1). Interestingly, according to a recent publication by Ma et al. [ 33 ], 96% of patients with PANS have clinical evidence of low-grade vascular inflammation using a limited clinical evaluation panel (high D-dimer, high von Willebrand factor antigen, livedo reticularis, and periungual erythema). BECs are complex, with a highly restrictive tight junction architecture. Due to the unique structure, they are susceptible to inflammatory perturbations, commonly observed in neuroinflammatory disorders. A subtle disruption in junctional integrity proteins (occludin, claudins, and ZO-1) results in paracellular leakage and immune cell infiltration, leading to increased CNS inflammation. Given that PANS is a relapsing–remitting disorder as described by Masterson et al. [ 47 ], we also compared the impact of PANS flare plasma versus recovery plasma on ZO-1, VE-cadherin, and actin stress fiber formation. We observed that plasma from the recovered state did not disrupt the BEC barrier and cell structure (compared to flare) and was similar to the healthy control data, suggesting the role of circulatory inflammatory mediators that disrupt the BEC barrier is reduced or absent in the recovered state. We identified MMP-9 as a central effector in PANS-associated BBB dysfunction. MMP-9 is a proteolytic enzyme known to degrade tight junction proteins and extracellular matrix components, thereby compromising endothelial junctional integrity [ 43 ]. In our study, MMP-9 levels were significantly elevated in the PANS flare plasma and showed a strong positive correlation with increased endothelial permeability. Interestingly, MMP-9 levels in plasma collected from the recovered state are significantly lower than the flare state (and similar to healthy control levels). This observation aligns with previous research demonstrating that MMP-9 is quickly activated and released into the circulation during acute inflammation, primarily by inflammatory leukocytes (e.g., activated neutrophils) in response to proinflammatory cytokines and chemokines. Elevated circulating MMP-9 has been associated with disease severity, tissue remodeling, and neuronal injury across a range of neuroinflammatory-associated conditions such as multiple sclerosis, cerebral aneurysm, stroke, Alzheimer’s disease, Parkinson’s disease, epilepsy, schizophrenia, and neuro-invasive infections (tic borne encephalitis, West Nile virus, measles, human immunodeficiency virus and bacterial meningitis) [ 86 – 93 ]. Given the ability of MMP-9 to degrade both tight and adherens junction proteins, its activity likely contributes directly to the barrier dysfunction and hyperpermeability observed in our in vitro model, supporting a mechanistic link between systemic inflammation and BBB compromise in PANS. We also observed elevated levels of several proinflammatory mediators, including IL-6 and IL-8 (also known as CXCL8), in the PANS flare-treated BEC supernatant. These proinflammatory mediators are known to disrupt endothelial junctions and have been shown to amplify MMP-9 transcription [ 94 , 95 ]. Supporting these findings, we also observed a significant increase in active MMP-9 in the flare-treated BEC supernatant. Compared with healthy control-treated BEC supernatant, the MMP/TIMP-1 ratio in PANS flare-treated supernatant plasma was markedly greater, supporting the presence of active MMP-9. These findings suggest a self-perpetuating inflammatory loop in which MMP-9 triggers the release of cytokines from BECs that further enhance MMP-9 expression in BECs, thereby exacerbating barrier disruption. We employed a specific extracellular MMP-9 antagonist, MMP-9 inhibitor-I, with PANS flare plasma, which significantly rescued BEC permeability in a dose-dependent manner. The results showed that 50 µM of the inhibitor prevented loss of junctional proteins, reduced actin stress fibers and attenuated the secretion of IL-6 and IL-8 (CXCL-8). These findings confirm that MMP-9 is not only necessary for flare-induced endothelial cell disruption but also sufficient to exacerbate the inflammatory response. This finding reinforces the dual role of MMP-9 as a structural disruptor and immune mediator that promotes inflammation. In summary, the present study provides evidence for dysfunction of BECs, which are a key component forming the BBB. This study identifies elevated circulating MMP-9 as a central mediator for dysfunction, linking systemic inflammation to brain endothelial injury and neuroimmune activation. Notably, we demonstrate for the first time that plasma from active PANS flare patients induces acute, time-dependent hyperpermeability in BECs, mediated by MMP-9–driven structural degradation and cytokine amplification loops. These findings underscore MMP-9 as a potential therapeutic target to mitigate BBB breakdown and downstream neuroinflammatory cascades. In line with our findings, MMP-9 inhibitors (e.g., GM6001, MMP-9 inhibitor I) have been shown to have neuroprotective effects on various neuropsychiatric and neurodegenerative disorders, including amyotrophic lateral sclerosis and Parkinson's disease [ 96 , 97 ]. This study, therefore, provides additional rationale for targeting MMP-9 in the context of acute neuroinflammatory conditions such as PANS. Beyond the MMP-9 mediated effects, our data suggest a role for chemokine CCL11 modulating the BBB integrity during a PANS flare. A pilot multiplex array of plasma showed marked elevation of CCL11 during flare episodes compared to the healthy controls and confirmed similar observation with follow up ELISA studies. (Appendix 4 and 5). CCL11 is recognized as a mediator of neuroinflammation and BBB disruption, acting via CCR3 receptors to promote microglial activation and neuronal injury [ 98 – 100 ]. It can also be secreted by endothelial cells in response to Th2 cytokines and further amplifies immune cell infiltration and local inflammation [ 101 , 102 ]. Together, these findings suggest that CCL11, alongside MMP-9, functions as a co-effector driving BBB hyperpermeability and neuroimmune activation in PANS flares. Four medication classes have emerged as therapeutic options for patients with PANS and all appear to impact the aforementioned pathways. Non-steroidal anti-inflammatories, corticosteroids and azithromycin are known to reduce MMP-9, IL 6 and IL 8 in many clinical conditions [ 103 – 110 ]. Moreover, these drugs have also been shown to improve PANS flare symptoms and flare duration in observational studies [ 111 , 112 ]. Finally, serotonin reuptake inhibitors also lower MMP-9 and Il-6 and have shown beneficial impacts in PANS, though patients in early flare episodes may respond better to lower doses than typical due to increased risk of side-effects [ 45 ]. A summary of medications is shown in Appendix Table 4. This study provides a foundation for employing multicellular BBB models incorporating astrocytes, pericytes, and microglia to better capture neurovascular complexity and further validate the role of systemic inflammation in PANS. Such models will be essential to understand the full impact of MMP-9 antagonists currently being used and may help identify additional mediators driving BBB disruption and microglial activation in neuroinflammation. Limitations of the study: A primary limitation of this study is the use of BEC monolayers as an in vitro model of the BBB. While BECs represent a fundamental component, this monoculture system cannot fully recapitulate the complex microenvironment of the neurovascular unit, which includes dynamic interactions with pericytes, astrocytes, microglia, the basement membrane, and physiological hemodynamic shear stress. Consequently, future investigations utilizing more complex systems, such as multicellular co-cultures or microfluidic "BBB-on-a-chip" platforms, are warranted to enhance the physiological relevance of the findings. Secondly, while the data implicate MMP-9 as a key mediator of BEC hyperpermeability, it is important to acknowledge that plasma is a complex biological matrix. It is therefore plausible that additional circulating factors, such as cytokines, chemokines, and autoantibodies, act synergistically with or upstream of MMP-9 to promote BBB dysfunction. Although our exploratory analysis identified CCL11 as a potential co-effector, this preliminary observation requires validation in larger cohorts and through pathway-specific inhibition studies. Thirdly, the matching of patient and control cohorts was limited to age and sex, introducing the potential for confounding from unmatched demographic characteristics like race and ethnicity. To confirm the generalizability of these findings, a larger cohort with more comprehensive matching will be necessary. Finally, the study cohort was heterogeneous regarding disease course, including patients with both new-onset flares and persistent PANS presentations. As disease duration may influence the plasma inflammatory profile and its effect on BEC permeability, future studies with larger, well-stratified cohorts are required to determine whether distinct clinical trajectories are associated with differential impacts on BBB integrity. Declarations Funding The current project was supported by the Brain Foundation, the Stanford Institute for Immunity, Transplantation & Infection and the Stanford Autoimmune & Allergy Supergroup. Funding for the infrastructure of our research program, training, and overlapping projects came from: 1) Lucile Packard Foundation for Children's Health; 2) National Institute of Mental Health- Pediatrics and Developmental Neuroscience Branch which supported the initial creation of the Stanford IBH Program; 3) The Neuroimmune Foundation for education/training, microbial sequencing, and medication analyses; 4) The Brain Foundation and O’Sullivan Foundation for Autism research; 5) The Tara & Dave Dollinger PANS Biomarker Discovery Core for patient sample collection; 6) Stanford Maternal and Child Health Research Institute (MCHRI) for HLA research; 7) Stanford SPARK, SPARK Pisa, and International OCD Foundation for immunophenotyping research; 8) Oxnard Foundation for imaging research; 9) Caudwell Children’s Foundation for genetics and immunology research; 10) The Louisa Adelynn Johnson Fund for Complex Disease for expanding the Illuminate PANS project; 11) Stanford COMET, DRIVE, and HBREX programs for student research in our program. Authorship contribution statement Conceptualization: AM, JF, EDM Methodology: AM, JF, EDM, BT, AK, TC Patient classification based on review of records, multiple clinical interviews, and physical exam: MS, PT, MT, YX, BF, MM (supervised by MM and JF). Disease state classification: BF, MM, MS, JF (supervised by JF) Healthy control project supervision and matching: JH, LC, JF Sample acquisition: CM Investigation: AM Data analysis: AM, SHG Supervision of investigation and review of data throughout the project: JF, EDM. Senior oversight of project continuity, laboratory resources, and critical intellectual revision of the manuscript: MK Writing – original draft: AM, JF Writing – review & editing: All Funding acquisition: JF, EDM, AM Declaration of competing interests Authors declare that they have no competing interests. Acknowledgments We are grateful for our patients and families who understand treatment limitations and continue to lend their time and cooperation to research participation. We would also like to thank Claudia Macaubas (for her critical input), the Stanford IBH Clinic Team, Stanford core facilities (Cell Sciences Imaging Facility, Neuroscience Microscopy Service, Human Immune Monitoring Core). Data and materials availability All data are available in the main text or the appendices. The RNA sequencing datasets generated and analyzed in this study have been deposited in the CBI Gene Expression Omnibus under accession number GSE324385. All materials used in the analysis can be available upon request. References Swedo SE, Leckman JF, Rose NR. From research subgroup to clinical syndrome: modifying the PANDAS criteria to describe PANS (pediatric acute-onset neuropsychiatric syndrome). Pediatr Therapeut. 2012;2(2):113. 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Verleden SE, Vandooren J, Vos R, Willems S, Dupont LJ, Verleden GM, Van Raemdonck DE, Opdenakker G, Vanaudenaerde BM. Azithromycin decreases MMP-9 expression in the airways of lung transplant recipients. Transpl Immunol. 2011;25(2–3):159–62. Park A, Anderson D, Battaglino RA, Nguyen N, Morse LR. Ibuprofen use is associated with reduced C-reactive protein and interleukin-6 levels in chronic spinal cord injury. J Spinal Cord Med. 2022;45(1):117–25. Quante T, Ng YC, Ramsay EE, Henness S, Allen JC, Parmentier J, Ge Q, Ammit AJ. Corticosteroids reduce IL-6 in ASM cells via up-regulation of MKP-1. Am J Respir Cell Mol Biol. 2008;39(2):208–17. Haydar D, Cory TJ, Birket SE, Murphy BS, Pennypacker KR, Sinai AP, Feola DJ. Azithromycin Polarizes Macrophages to an M2 Phenotype via Inhibition of the STAT1 and NF-kappaB Signaling Pathways. J Immunol. 2019;203(4):1021–30. Evrard A, Cuq P, Ciccolini J, Vian L, Cano JP. Increased cytotoxicity and bystander effect of 5-fluorouracil and 5-deoxy-5-fluorouridine in human colorectal cancer cells transfected with thymidine phosphorylase. Br J Cancer. 1999;80(11):1726–33. Sticherling M, Baisch C, Bornscheuer E, Schroder JM, Christophers E. The role of the Duffy antigen-related chemokine receptor in psoriasis vulgaris. Cytokine. 2002;18(6):329–33. Spartz EJ, Freeman GM Jr., Brown K, Farhadian B, Thienemann M, Frankovich J. Course of Neuropsychiatric Symptoms After Introduction and Removal of Nonsteroidal Anti-Inflammatory Drugs: A Pediatric Observational Study. J Child Adolesc Psychopharmacol. 2017;27(7):652–9. Brown K, Farmer C, Farhadian B, Hernandez J, Thienemann M, Frankovich J. Pediatric Acute-Onset Neuropsychiatric Syndrome Response to Oral Corticosteroid Bursts: An Observational Study of Patients in an Academic Community-Based PANS Clinic. J Child Adolesc Psychopharmacol. 2017;27(7):629–39. Additional Declarations No competing interests reported. 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01:38:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9442808/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9442808/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108725498,"identity":"c22526f8-2e5d-4d6c-a173-2eece1ac76b4","added_by":"auto","created_at":"2026-05-07 16:55:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":151034,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePANS flare plasma induces BECs hyperpermeability and correlates with clinical severity: \u003c/strong\u003e(A) Schematic of the \u003cem\u003ein vitro\u003c/em\u003e BEC permeability assay. BEC monolayers were cultured on Transwell® inserts and exposed to 1% heat-inactivated plasma from healthy controls or PANS flare patients. FITC–dextran (10 kDa) was added to the apical chamber, and fluorescence intensity in the basolateral chamber (Ex 485 nm/Em 535 nm) was measured as an indicator of monolayer permeability. (B) Quantification of BECs permeability 12 h post-treatment, data expressed as percentage of fold change compared to untreated. Each dot represents an individual sample (healthy controls, green; PANS flare, red; n=15), and the data are represented as median values ***p\u0026lt;0.001 using Mann–Whitney U test. (C) Spearman’s rank correlation analysis between BEC permeability and clinical severity scores (healthy controls, green; PANS flare, red).\u003c/p\u003e","description":"","filename":"Slide1.png","url":"https://assets-eu.researchsquare.com/files/rs-9442808/v1/35087993eb474591751ad061.png"},{"id":108806103,"identity":"98ac9e95-a86c-4968-bfc7-2d0ad59a1ebe","added_by":"auto","created_at":"2026-05-08 15:27:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":214480,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePANS flare plasma alters BECs transcriptional programs related to cell junctions, immune signaling and cytoskeletal organizations:\u003c/strong\u003e (A) Principal component analysis of RNA-seq data from BECs treated with plasma (HC, green; PANS flare, red). Symbols represent biological sex of donors (female or male). (B) Volcano plot illustrating differential gene expression in PANS flare compared to healthy controls. Significantly upregulated (blue) and downregulated (red) genes are defined by Log2 fold change≥1.5 and adjusted p\u0026lt;0.05 (n=10 per group). Genes implicated in the permeability have been marked. (C) Hierarchical clustering heat map of genes interfering in the permeability functions (cell junctions, morphology and inflammatory mediators), depicting z-score normalized expression values.\u003c/p\u003e","description":"","filename":"Slide2.png","url":"https://assets-eu.researchsquare.com/files/rs-9442808/v1/56c7574f16ef6759fb61d6ed.png"},{"id":108725499,"identity":"290baa5d-13fe-4fab-9ee0-7ba8dcbe5961","added_by":"auto","created_at":"2026-05-07 16:55:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":508416,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePANS flare plasma disrupts endothelial junctional integrity and cytoskeletal organization: \u003c/strong\u003e(A) Representative immunofluorescence images showing adherens junction VE-cadherin (green), tight junction ZO-1 (red), and labeling of actin stress fiber phalloidin (yellow). Nuclei were counterstained with DAPI (blue). Scale bars correspond to 20 µm. (B) Quantification of VE-cadherin, ZO-1 and phalloidin mean fluorescence intensities expressed as median (n=10 per group). *p\u0026lt;0.05 using Mann–Whitney U test.\u003c/p\u003e","description":"","filename":"Slide3.png","url":"https://assets-eu.researchsquare.com/files/rs-9442808/v1/b17d66414fef256e3a0fc850.png"},{"id":108805994,"identity":"65c96c1a-b670-428c-a95e-6953f9a2bac2","added_by":"auto","created_at":"2026-05-08 15:27:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":461686,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePANS recovery plasma preserves BEC barrier integrity and junctional organization: \u003c/strong\u003e(A) FITC–dextran permeability assay of BEC monolayers treated with paired plasma samples from PANS flare (red) and PANS recovered (blue) phases. Permeability is expressed as percentage relative to untreated control as baseline. Lines connect paired samples (n=10). *p\u0026lt;0.05, **p\u0026lt;0.01 using Wilcoxon signed-rank test. (B) Representative confocal images showing partial improvement of VE-cadherin and ZO-1 junctional continuity and reduced actin stress fiber formation in BECs treated with plasma from PANS recovered patients. Scale bars correspond to 20 µm. (C) Quantitative analysis of junctional and cytoskeletal markers in BECs treated with PANS flare (red) versus PANS recovered (blue) plasma (n=10 paired). Data are presented as median values. *p\u0026lt;0.05 using Wilcoxon signed-rank test. Lines connecting the paired samples are displayed in the panel below.\u003c/p\u003e","description":"","filename":"Slide4.png","url":"https://assets-eu.researchsquare.com/files/rs-9442808/v1/36ba7fa461064b3de7c1a1e7.png"},{"id":108725503,"identity":"da590f42-b179-4b31-8ed2-69a1b5bb13ae","added_by":"auto","created_at":"2026-05-07 16:55:54","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":98672,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eElevated plasma matrix metalloprotease-9 (MMP-9) is associated with BECs hyperpermeability in PANS flare:\u003c/strong\u003e(A)Left panel: Dot plot showing plasma MMP-9 concentrations (ng/mL), measured by quantitative sandwich ELISA, in healthy controls (green) and PANS flare patients (red). Each dot represents an individual sample. Data are presented as median values. Right panel: Spearman’s rank correlation between plasma MMP-9 levels and BEC permeability (% of control). (B) Dot plot comparison of plasma MMP-9 levels in PANS subjects during flare (red) and recovery (blue). Lines connect paired samples. Data are presented as median values. *p\u0026lt;0.05 using Wilcoxon signed-rank test.\u003c/p\u003e","description":"","filename":"Slide5.png","url":"https://assets-eu.researchsquare.com/files/rs-9442808/v1/84a2052040cf1052a5e2b3a6.png"},{"id":108807129,"identity":"58e5a431-f91c-460f-8574-d943ce2c00ae","added_by":"auto","created_at":"2026-05-08 15:30:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":91592,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePANS flare plasma induces pro-inflammatory cytokine release and increased MMP-9 activity in BECs: \u003c/strong\u003eDot plots showing ELISA measurements of BEC culture supernatants following exposure to plasma from healthy controls (green) or PANS flare patients (red). (A) Interleukin-6 (IL-6) and CXCL-8 (IL-8) concentrations. (B) MMP-9 concentrations. (C) Active MMP-9 levels, expressed as the MMP-9/TIMP-1 ratio. Each dot represents an individual plasma-treated sample. Data are presented as median values. ***p\u0026lt;0.001; **p\u0026lt;0.01; *p\u0026lt;0.05 using the Mann-Whitney U test.\u003c/p\u003e","description":"","filename":"Slide6.png","url":"https://assets-eu.researchsquare.com/files/rs-9442808/v1/1618b4e1510fe5ad3d80c8dc.png"},{"id":108725505,"identity":"62a1bc69-a16a-4622-ba95-3d4bda8ae1ae","added_by":"auto","created_at":"2026-05-07 16:55:54","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":330202,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePharmacological inhibition of MMP-9 attenuates PANS flare–induced BECs dysfunction and cytoskeletal remodeling:\u003c/strong\u003e (A) BEC permeability relative to untreated controls following exposure to human serum supplemented with recombinant MMP-9, in the absence or presence of MMP-9 inhibitor I. Serum was used in this control experiment to assess the direct effect of MMP-9 on endothelial permeability independent of patient-derived plasma factors. (B) Paired dot plot showing BECs permeability compared to untreated control following exposure to PANS flare plasma alone or PANS flare plasma supplemented with MMP-9 inhibitor I in two different concentrations (5 µM or 50 µM). Lines connect matched samples. (C) Paired dot plot showing the levels of interleukin-6 (IL-6) and CXCL-8 (IL-8) measured by ELISA in BEC culture supernatants following treatment with PANS flare plasma with or without MMP-9 inhibitor I. (D) Representative immunofluorescence images of BECs treated with PANS flare plasma alone or in the presence of MMP-9 inhibitor I (50 µM), stained for VE-cadherin (green), ZO-1 (red), and F-actin (phalloidin, orange), with nuclei counterstained with DAPI (blue). Scale bar corresponds to 20 µm. (E) Paired dot plot showing quantification of mean fluorescence intensity corresponding to panel D. Each dot represents an individual sample. *p\u0026lt;0.05, **p\u0026lt;0.01 using Wilcoxon signed-rank test.\u003c/p\u003e","description":"","filename":"Slide7.png","url":"https://assets-eu.researchsquare.com/files/rs-9442808/v1/7ef1a1c73da3f44d691c227f.png"},{"id":108809996,"identity":"3a45eac5-91b6-4449-811f-8b524868577f","added_by":"auto","created_at":"2026-05-08 15:56:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2302466,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9442808/v1/2ec98560-646a-4c17-b706-a169e985d07f.pdf"},{"id":108806146,"identity":"5f3d3971-007e-4b7e-ae54-8102047b3cf3","added_by":"auto","created_at":"2026-05-08 15:27:48","extension":"pptx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3264410,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFiguresJNIAMJF2026.pptx","url":"https://assets-eu.researchsquare.com/files/rs-9442808/v1/97fd7de87f2d980873c81119.pptx"},{"id":108805977,"identity":"4162a08e-12b3-4e51-b8ab-2e24353f3bb6","added_by":"auto","created_at":"2026-05-08 15:27:21","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":21799,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryJNIAMJF2026.docx","url":"https://assets-eu.researchsquare.com/files/rs-9442808/v1/c230d53dcb9b8b8b5acc8cf8.docx"},{"id":108725508,"identity":"55cf8976-e53e-4bb7-842c-b475d0ec8b2d","added_by":"auto","created_at":"2026-05-07 16:55:55","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":31728,"visible":true,"origin":"","legend":"","description":"","filename":"AppendixTablesJNIAMJF.docx","url":"https://assets-eu.researchsquare.com/files/rs-9442808/v1/1858674b103a549d2dc97624.docx"},{"id":108806598,"identity":"51125136-a582-40e0-8ca5-9abb234b1d67","added_by":"auto","created_at":"2026-05-08 15:29:01","extension":"pptx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1250665,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalabstractJNIAMJF2026.pptx","url":"https://assets-eu.researchsquare.com/files/rs-9442808/v1/8ec5b6ee76ffd08894649d3a.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Matrix Metalloproteinase-9 (MMP-9) as a Potential Regulator of Blood–Brain Barrier Dysfunction in Pediatric Acute Neuropsychiatric Syndrome (PANS)","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePediatric Acute-onset Neuropsychiatric Syndrome (PANS) and Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal infections (PANDAS) are characterized by the sudden onset of severe neuropsychiatric symptoms (obsessive-compulsive behaviors, eating restrictions, motor abnormalities, emotion and behavior dysregulation, sleep disturbances, cognitive dysfunction, and urinary symptoms) [\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6 CR7\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Emerging research suggests that the basal ganglia is the site of pathology, and the evidence includes: neuroimaging studies [\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], neurological soft signs pertaining to the basal ganglia [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]; and REM sleep without atonia\u0026mdash;seen in Parkinson\u0026rsquo;s and other basal ganglia disorders [\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Additionally, autoantibodies have been shown to bind to cholinergic interneurons (CINs) within the basal ganglia and alter their electrical activity [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Autoantibodies to other neuronal antigens/targets (tubulin, lysoganglioside, dopamine receptors, etc) have also been reported [\u003cspan additionalcitationids=\"CR22 CR23 CR24 CR25 CR26 CR27 CR28 CR29 CR30\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Lastly, the hallmark symptoms of PANS align with basal ganglia dysfunction.\u003c/p\u003e \u003cp\u003eBeyond central nervous system (CNS) inflammation, PANS is often associated with subtle signs of systemic inflammation including enthesitis, joint effusions, synovitis or capsulitis (demonstrated by joint ultrasound), vasculopathy signs, and a higher prevalence of comorbid autoimmune disorders [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Further evidence for systemic inflammation includes elevated proinflammatory cytokines and proinflammatory monocytes [\u003cspan additionalcitationids=\"CR35 CR36 CR37\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. These observations support the hypothesis that both systemic and central immune processes contribute to the pathophysiology of PANS.\u003c/p\u003e \u003cp\u003eFor circulating immune factors, including autoantibodies, to access the CNS, they must first cross the blood-brain barrier (BBB). The BBB is a highly specialized and tightly regulated interface which is primarily composed of brain microvascular endothelial cells (BECs), which regulate the entry of solutes, immune cells, and macromolecules to the CNS. Under physiological conditions, the BBB maintains restrictive properties through complex intercellular junctions, including the BECs\u0026rsquo; tight junctions (e.g., claudins, occludin, ZO-1) and adherens junctions (e.g., VE-cadherin), which are anchored to the actin cytoskeleton and are responsible for high trans-endothelial resistance [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. However, systemic inflammation-driven disruption of this barrier may allow immune effectors to access and alter the CNS environment.\u003c/p\u003e \u003cp\u003eAmong the systemic inflammatory mediators, matrix metalloproteinases (MMPs) are important for their ability to degrade the junctions of endothelial cells and increase BBB permeability [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. MMP-9 is recognized as the most effective among all the MMPs, due to its proteolytic specificity towards extracellular matrix and cell junctional proteins. MMP-9-associated BBB dysfunction has been described in other neuroinflammatory disorders [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. BBB disruption enables serum proteins, such as albumin and plasminogen, to enter the brain, activating neurotoxic pathways and facilitating the infiltration of peripheral immune cells, including monocytes and macrophages. These cells can exacerbate neuroinflammation and even acquire microglia-like phenotypes once within the CNS [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Notably, Rahman et al. recently demonstrated an increase in circulating proinflammatory monocytes in PANS flare (i.e., periods of abrupt symptom escalation) along with CNS-homing monocytes [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAnother clinical observation that supports the hypothesis of BBB dysfunction in PANS is that patients exhibit sensitivity to psychotropic medications (during early flare state), a feature which is also described in autoimmune encephalitis [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBased on this evidence, we hypothesized that systemic inflammation during PANS flare contributes to BBB dysfunction. We tested the physiological effect of plasma from patients with PANS in their flare state (compared with healthy controls and patients with PANS in their recovered state) on a BEC monolayer. We observed that PANS flare plasma induced endothelial hyper-permeability, disrupted junctional integrity, and promoted actin stress fiber formation. Mechanistic studies implicated MMP-9 as a key effector molecule driving this dysfunction, and its inhibition successfully blocked this barrier breakdown.\u003c/p\u003e \u003cp\u003eTo our knowledge, this is the first mechanistic study using a BBB model and PANS patient plasma to demonstrate a direct link between plasma factors (likely to reflect systemic inflammation) and brain endothelial barrier disruption. These findings provide critical insight into how peripheral inflammation may facilitate neuroinflammation in PANS and support the role of the BBB as a therapeutic target.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eThe study protocol was approved by the Stanford Human Participants Institutional Review Board and covers all patients and procedures in this study. Immune Behavioral Health (IBH) Clinic clinicians cared for all patients. Written parental consent and child assent were obtained per study protocol (protocol number 26922).\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eDiagnosis and disease state classification\u003c/strong\u003e \u003cp\u003ePsychiatric symptoms at clinic presentation and other demographics for samples used in each experiment are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Paired PANS flare and recovery samples were used whenever possible. A total of 15 PANS patients were enrolled in the study, of whom 10 provided paired flare\u0026ndash;recovery samples for longitudinal analyses. For each experiment, PANS samples were matched to healthy control samples based on age (\u0026plusmn;\u0026thinsp;1 year) and sex assigned at birth to minimize demographic confounding. Blood samples were processed using Ficoll gradient to acquire peripheral blood mononuclear cells (PBMC) and plasma; these were stored in liquid nitrogen and \u0026minus;\u0026thinsp;80\u0026deg;C, respectively, and maintained by Stanford Biobank. Different sample sizes were used across assays depending on sample availability and specific experimental objectives. 15 plasma samples per group (PANS flare and healthy controls) were used for permeability and MMP-9 quantification; 10 per group were used for RNA-seq (selected for clinical uniformity and absence of recent medication to minimize heterogeneity); and 10 paired flare\u0026ndash;recovery samples were included for the desired assays based on the availability. 6 heparinized plasmas with confirmed PANS flare were selected from the cohort to conduct exogenous MMP-9 inhibitor-based experiments. All study subjects were classified as PANS by the Stanford IBH psychiatry team (MS, YX, PT, MT) using PANS diagnosis and evaluation criteria [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Definitions of disease state and clinical course were based on previously published criteria and are summarized in Appendix Table\u0026nbsp;2 [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDemographic and clinical presentation data of patients with PANS in flare state, recovered state, and healthy controls in this study. Note that patients in recovered state and heathy controls did not report neuropsychiatric symptoms at clinic presentation. Where data were not measured for a given subject group, the corresponding cell is empty.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAge at the first clinic visit, mean (SD)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFlare state\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;15)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRecovered state\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;15)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHealthy controls\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;15)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.7 (2.6)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.6 (2.9)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge at blood draw, mean (SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.0 (3.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.7 (3.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMale, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 (67%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8 (53%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRace/ethnicity, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWhite/European\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13 (87%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6 (40%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAsian/Asian American (including biracial/mixed)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8 (53%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHispanic/Latino (including biracial/mixed)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2 (13%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 (7%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClinical severity scores, mean (SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eColumbia Impairment Score\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16.5 (11.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.0 (4.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.3 (0.5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlobal Impairment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e43.4 (19.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17.5 (11.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.9 (5.4)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eModified Overt Aggression Scale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.2 (8.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.0 (4.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0 (0.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChildren\u0026rsquo;s Global Assessment Scale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e59.8 (9.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e79.3 (11.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChildren\u0026rsquo;s Yale-Brown Obsessive Compulsive Scale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21.1 (8.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13.0 (5.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePercent Baseline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e55.8 (19.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e83.0 (8.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge at first neuropsychiatric decline, mean (SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.1 (2.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge at deterioration leading to clinic visit, mean (SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.4 (2.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNeuropsychiatric symptoms at clinic presentation, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eObsessions\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13 (87%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompulsions\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12 (80%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFood refusal/avoidance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 (67%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFluid refusal/avoidance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3 (20%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSeparation anxiety\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11 (73%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOther anxiety\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13 (87%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMood swings/moodiness\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9 (60%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEmotional lability\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3 (20%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSuicidal ideation/behavior\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 (27%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDepression\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 (67%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIrritability\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 (67%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAggression\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8 (53%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOppositional behavior\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9 (60%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHyperactivity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6 (40%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTrouble paying attention\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9 (60%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBehavioral/developmental regression\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12 (80%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDysgraphia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6 (40%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCognitive symptoms\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8 (53%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 (67%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSleep problems\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 (67%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEnuresis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3 (20%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUrinary frequency\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6 (40%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSensory amplification\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9 (60%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHallucinations\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 (7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDelusions or paranoid thoughts\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3 (20%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMotor tics\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5 (33%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhonic tics\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2 (13%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/p\u003e \u003cp\u003e\u003cb\u003eEndothelial cell monolayer permeability\u003c/b\u003e: BECs were grown to confluent monolayers on 24-well plate Transwell\u0026reg; inserts (Corning Costar, 0.4 \u0026micro;m) coated with fibronectin as described previously by Badawi et al. [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The cells were incubated with heat-inactivated plasma from patients with PANS flare patients (n\u0026thinsp;=\u0026thinsp;15) and healthy controls (n\u0026thinsp;=\u0026thinsp;15). The cell culture media was replaced for 60 min of each experiment with fresh dye-free culture media (Opti MEM, Life Technologies, Grand Island, NY) to avoid interfering agents during fluorescence measurement. The wells of the Transwell\u0026reg; plates were divided into 3 different groups: untreated control, healthy control and PANS flares. Endothelial cell permeability was measured following the protocol described by Robinson et al. [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Each sample was treated in duplicate, including the untreated control. Each experimental group was included in a 12-well/plate monolayer plate. FITC-dextran 10 kDa was applied to the luminal (upper) side of the Transwell\u0026reg; at a final concentration of approximately 500 \u0026micro;g/mL and allowed to equilibrate through the monolayer between the luminal and abluminal (lower) chambers for 30 mins. Cell culture media samples were obtained from the abluminal chambers of the Transwell\u0026reg; system and measured with a fluorometric plate reader (excitation, 485 nm; emission, 535 nm) to quantify the fluorescence intensity, which represents the permeability of FITC-dextran across the monolayer barrier. The fluorescence intensity values obtained with the PANS flares and healthy controls were normalized to the values obtained with the untreated samples performed on the same day and time, and expressed as percent permeability relative to untreated control (set to 100%).\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eBulk RNA sequencing of BECs\u003c/strong\u003e \u003cp\u003eBECs were treated with plasma from PANS flares (n\u0026thinsp;=\u0026thinsp;10) and healthy controls (n\u0026thinsp;=\u0026thinsp;10) for 12 hrs, after which the cells were washed with PBS and collected in TRIzol reagent (Invitrogen). Total RNA was isolated via a Direct-zol kit (Zymogen, Irvine, CA) according to the manufacturer\u0026rsquo;s instructions and sent to Novogene (Sacramento, CA) for bulk RNA sequencing on the Illumina Nova-seq X platform. The mRNA library was prepared using poly (A) mRNA enrichment. Paired-end RNA-seq reads were indexed and quantified via the pseudoalignment algorithm Kallisto, with an average read alignment of 88.8% [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. The sequences were then annotated and mapped to the reference genome EnsDb.Hsapiens.v86 at the gene level [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The readings were normalized to transcripts per million (TPM), and principal component analysis was performed in R via built-in libraries. The R package edgeR was used to compile differential genes used to generate volcano plot and hierarchically clustered heatmaps.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e\u003cb\u003eImmunofluorescence and F-actin Labeling\u003c/b\u003e: BECs were used for immunofluorescence localization of the tight junction protein ZO-1, adherens junction protein VE-cadherin, and actin stress fibers. The cells were grown as monolayers on chamber slides coated with fibronectin. The cells were incubated with heat-inactivated plasma from either PANS flares or healthy controls for 12 hrs as previously described to measure permeability. For stress fiber staining, the cells were fixed with 3.7% paraformaldehyde followed by subsequent washing with phosphate-buffered saline; ActinRed 555 solution was then added for 30 min. ActinRed 555 is a stable solution of rhodamine phalloidin at room temperature and used to stain actin stress fibers. To stain for ZO-1 and VE-cadherin, BECs were fixed in paraformaldehyde (3.7%) for 10 min following plasma treatment, followed by permeabilization with 0.5% Triton X-100 (Sigma‒Aldrich, Carlsbad, CA) for a maximum of 10 min. This was followed by blocking with bovine serum albumin (2%) (Sigma‒Aldrich) for 45\u0026ndash;60 min and 60 min incubation with the primary antibodies anti-rabbit ZO-1 (ab221547; 1:200) and mouse monoclonal VE-cadherin (MAB9381; 1:150; R\u0026amp;D Systems, Minneapolis, MN). After primary antibody incubation, the cells were incubated with fluorescent-tagged secondary antibodies for a maximum of 60 min, followed by 5 washes with phosphate-buffered saline. The secondary antibodies used were Alexa Fluor 647-conjugated goat anti-mouse IgG (A21235; 1:1000) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (A11008; 1:500) (Invitrogen, Eugene, OR). All the slides were subsequently washed with PBS following fluorescence antibody incubation and mounted with ProLong\u0026trade; Gold Antifade Mountant with DNA stain DAPI (Invitrogen, Eugene, OR). ZO-1, VE-cadherin and phalloidin fluorescence was visualized via a Zeiss LSM 980 with a super resolution Airyscan 2 confocal microscope at the Stanford Core facility. Images were taken and quantified via ImageJ, and the mean intensity value was calculated.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eMMP-9 inhibition\u003c/strong\u003e \u003cp\u003eWe used MMP-9 inhibitor I (Abcam, Waltham, MA) to block MMP-9 activity. The inhibitor binds Zn ions at the active site of the MMP-9 pro enzyme and blocks its activation. To measure permeability, the inhibitor was pretreated for an hour at a minimum concentration of 5 \u0026micro;M in an immunofluorescence assay, and subsequently plasma was added.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e\u003cb\u003eELISAs\u003c/b\u003e: MMP-9, TIMP-1, IL-6, IL-8, S100B, and CCL11 ELISAs were performed according to the manufacturer\u0026rsquo;s instructions. The plasma was diluted according to the following: 1:50 for MMP-9; and 1:100 for TIMP-1, IL-6, IL-8, and S100B. The human MMP-9, TIMP-1, IL-6, and IL-8 ELISA kits were purchased from Proteintech (Rosemont, IL), CCL11 from R and D Systems (Minneapolis, MN), and S100B from Millipore (Hayward, CA).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCytokine multiplex assay\u003c/b\u003e: This assay was performed with Luminex-EMD Millipore Human 48 Plex kits (Millipore, Burlington, MA) by the Human Immune Monitoring Center at Stanford University. Kits were run according to the manufacturer\u0026rsquo;s recommendations with modifications by the team. The H48 kits include one panel: Milliplex HCYTA-60K-PX48. The assay setup adhered to the recommended protocol. The culture supernatants were undiluted in a 96-well plate. The plasma samples were diluted 3-fold, and the samples were incubated overnight at 4\u0026deg;C with shaking on an orbital shaker at 500\u0026ndash;600 rpm. Following incubation, the plates were washed twice with wash buffer using a BioTek ELx405 washer (BioTek Instruments, Winooski, VT) and incubated with detection antibody for 1 hr at room temperature, followed by the addition of Streptavidin-PE for 30 min with shaking. Finally, the plates were washed as described above, and wash buffer was added to the wells for reading in the Luminex FLEXMAP 3D instrument, ensuring a lower bound of 50 beads per sample per cytokine. Each sample was measured in duplicate. Custom AssayChex control beads (Radix BioSolutions, Georgetown, TX) were added to all the wells. Wells with a bead count\u0026thinsp;\u0026lt;\u0026thinsp;50 were flagged, and data with a bead count\u0026thinsp;\u0026lt;\u0026thinsp;20 were excluded.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eStatistical analysis\u003c/strong\u003e \u003cp\u003eData are presented as median values with individual data points. Statistical comparisons between two groups were performed using the Mann\u0026ndash;Whitney U test for unpaired data (e.g., healthy controls vs. PANS flare) and the Wilcoxon signed-rank test for paired data (e.g., PANS flare vs. recovery). Correlation analyses were performed using Spearman\u0026rsquo;s rank correlation coefficient. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism (version 11).\u003c/p\u003e \u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cstrong\u003ePANS flare plasma induces BEC hyperpermeability and this is strongly correlated with clinical severity\u003c/strong\u003e \u003cp\u003eTo evaluate the impact of plasma from PANS patients on BBB integrity, we cultured primary BECs as confluent monolayers on Transwell\u0026reg; inserts. Heat-inactivated plasma from PANS flare patients, those same patients during recovery (partial and full), and healthy controls (age- and sex-matched) was applied to the apical compartment, and endothelial permeability was assessed using a FITC\u0026ndash;dextran flux assay (Fig.\u0026nbsp;1A). Plasma was incubated for approximately 12 h, a duration previously determined to yield maximal biological activity following neutralization of cytotoxic effects on cells (permeability kinetics and cytotoxicity measurement displayed in Appendix Fig.\u0026nbsp;1). Exposure to PANS flare plasma resulted in a significant increase in BEC monolayer permeability compared with application of healthy control plasma (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; n\u0026thinsp;=\u0026thinsp;15) (Fig.\u0026nbsp;1B), indicating that circulating factors in PANS plasma compromise the BEC monolayer integrity.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eWe also assessed Spearman\u0026rsquo;s rank correlation between BEC monolayer permeability and clinical severity scores: PANS 31-Item Symptom Rating Scale, Columbia Impairment Scale, PANS Global Impairment Scale, and the Modified Overt Agression Scale [\u003cspan additionalcitationids=\"CR53 CR54 CR55\" citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Figure\u0026nbsp;1C depicts the correlation between permeability percentage and clinical severity scores for all subjects with recorded scores. There were modest-to-strong correlations between the BEC permeability and severity measures.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003ePANS flare plasma alters BEC transcriptional programs related to cell junctions, immune signaling and cytoskeletal organizations\u003c/strong\u003e \u003cp\u003eTo explore the molecular basis of this hyperpermeability, we performed bulk RNA sequencing on BECs exposed to PANS flare plasma (n\u0026thinsp;=\u0026thinsp;10) and age- and sex-matched healthy control plasma (n\u0026thinsp;=\u0026thinsp;10) (equal numbers of males and females). Principal component analysis (PCA) revealed clear separation between PANS flare state and controls (Fig.\u0026nbsp;2A). The first two principal components, PC1 (37.3%) and PC2 (17.2%), accounted for the majority of the total variance. Samples clustered distinctly along PC1, with healthy controls (green) on the left and PANS flare samples (red) on the right, suggesting that PANS plasma induces a unique and consistent endothelial transcriptomic signature.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eDifferential expression analysis (PANS flare versus matched healthy control, Fig.\u0026nbsp;2B) further identified a broad transcriptional shift in genes known to regulate endothelial barrier structures and inflammation. The result showed significantly increased expression of several genes in the BEC known to be involved in extracellular matrix remodeling, immune-endothelial signaling, and cellular adhesion that include APOE, FN1, and NECTIN2 (gene abbreviations provided in Appendix Table\u0026nbsp;3). Conversely, ANGPT1, CLDN12, and GJA1 were markedly downregulated, indicating loss of barrier integrity due to destabilization of tight and adherens junctions.\u003c/p\u003e \u003cp\u003eA focused hierarchical clustering heatmap of endothelial junctional and adhesion-related genes (Fig.\u0026nbsp;2C) demonstrated robust group-specific expression patterns, where PANS flares are marked as \u0026ldquo;P\u0026rdquo; and healthy controls as \u0026ldquo;H\u0026rdquo;. PANS flare plasma caused reduced expression of canonical tight junction markers (CLDN3, TJP1, TJP2, GJA1) and adherens junction components (CDH5, ANGPT1), as well as genes maintaining extracellular matrix integrity and cytoskeletal regulation (CDC42, HIF1A, HEG1, NECTIN1, ITGB1). In contrast, leukocyte adhesion and inflammatory genes (ICAM1, ICAM2, CCL2, ANGPT2) were consistently upregulated in the BEC with the plasma from PANS flare, indicating an activated endothelial phenotype during active disease state.\u003c/p\u003e \u003cp\u003eAltogether, the bulk RNA-seq results (from BECs exposed to PANS flare plasma) indicate strong transcriptomic evidence that circulating inflammatory mediators in PANS flare plasma compromise BBB integrity by remodeling endothelial junctional architecture and inducing inflammatory signaling pathways, thereby promoting a hyperpermeable and pro-inflammatory vascular phenotype consistent with neuroimmune dysregulation in PANS.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eImmunofluorescence shows plasma from PANS flare disrupts endothelial junctional integrity and BEC cytoskeleton\u003c/strong\u003e \u003cp\u003eTo validate the above findings obtained in the bulk RNA sequencing, particularly the changes in cell junction and extracellular matrix remodeling genes (induced by PANS flare plasma), we performed immunofluorescence labeling of ZO-1 (tight junction) and VE-cadherin (adherens junction), along with rhodamine\u0026ndash;phalloidin labeling for actin stress fibers.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eConsistent with the RNA-seq results, treatment with PANS flare plasma led to fragmented, discontinuous staining of ZO-1 and VE-cadherin, and the appearance of stress fiber-like actin rearrangements, indicating substantial cytoskeletal remodeling and loss of cell\u0026ndash;cell junction integrity in the BEC (Fig.\u0026nbsp;3A). Healthy control-treated BECs exhibited continuous and well-defined staining of these junctional markers and a lack of stress fiber formation, which is consistent with cellular and barrier stability. Specifically, mean fluorescence intensity analysis showed significantly reduced expression of VE-cadherin (3-fold) and ZO-1 (nearly 3-fold; Fig.\u0026nbsp;3B). Rhodamine\u0026ndash;phalloidin labeling showed 4-fold increase in actin stress fibers in BECs exposed to PANS flare plasma compared with healthy controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; n\u0026thinsp;=\u0026thinsp;10). These findings indicate that distinct plasma protein factors during the PANS flare induce endothelial junctional disruption and contribute to BBB permeability.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003ePANS recovery plasma preserves BEC barrier integrity and junctional organization\u003c/strong\u003e \u003cp\u003eTo assess whether recovery plasma induced barrier abnormalities, BECs were treated with plasma from the same patients after recovery and pairwise comparison with flare state was performed. We compared the permeability assay (Fig.\u0026nbsp;4A), and immunofluorescence for ZO-1, VE-cadherin, and phalloidin for actin-stress fibers (Fig.\u0026nbsp;4B), as previously done. FITC-dextran permeability assay showed a significant difference between flare and recovery (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; n\u0026thinsp;=\u0026thinsp;10) and was consistent across subjects. Moreover, recovery plasma did not induce discontinuous VE-cadherin and ZO-1 labeling along the cell borders, nor did it induce actin-stress fiber formation. Quantitative image analysis confirmed junctional protein intensity and lack of actin-stress fiber formation with consistent results across subjects. (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u0026ndash;0.001, n\u0026thinsp;=\u0026thinsp;10; Fig.\u0026nbsp;4C).\u003c/p\u003e \u003c/p\u003e \u003cp\u003eCollectively, these findings support the presence of cell-disrupting factors in the plasma of PANS flare, causing endothelial cell disassembly and disruption of the actin cytoskeleton. This leads to BBB hyperpermeability in PANS flare samples, which is not induced by plasma from PANS recovery samples.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eElevated plasma matrix metalloprotease-9 (MMP-9) is associated with the BEC hyperpermeability in PANS flare\u003c/strong\u003e \u003cp\u003eWe measured MMP-9 concentration in the plasma of PANS flare and in the matched healthy controls using ELISA. MMP-9 was selected due to its established function as a pro-inflammatory matrix metalloprotease secreted by activated leukocytes, and its known capacity to degrade extracellular matrix components and disrupt endothelial junctions, including VE-cadherin and tight junction proteins [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. A comparative analysis revealed that MMP-9 concentrations were more than 2.5-fold higher in the plasma of PANS flare patients compared with healthy controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; n\u0026thinsp;=\u0026thinsp;15; Fig.\u0026nbsp;5). These elevated plasma MMP-9 levels strongly correlated with the degree of BEC permeability measured by FITC\u0026ndash;dextran (r\u0026thinsp;=\u0026thinsp;0.84; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). PANS flare samples (red, n\u0026thinsp;=\u0026thinsp;15) exhibited higher MMP-9 concentrations (up to ~\u0026thinsp;1000 ng/mL) and increased permeability (~\u0026thinsp;180%), whereas healthy controls (green, n\u0026thinsp;=\u0026thinsp;14) clustered around lower MMP-9 levels (\u0026lt;\u0026thinsp;200 ng/mL) and baseline permeability (~\u0026thinsp;100%). These results indicate a strong association between systemic MMP-9 and BBB leakiness (Fig.\u0026nbsp;5A).\u003c/p\u003e \u003c/p\u003e \u003cp\u003eTo evaluate whether MMP-9 levels reflect disease state, we analyzed paired plasma samples collected from the same patients during flare and recovery phases. MMP-9 concentrations were approximately 2-fold lower during recovery compared to the flare state. This reduction was statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; n\u0026thinsp;=\u0026thinsp;10) and consistent across patients (Fig.\u0026nbsp;5B).\u003c/p\u003e \u003cp\u003eOverall, these findings suggest that plasma MMP-9 levels are dynamic, reflect disease state, and may serve as a key mediator of BBB disruption during PANS flare, highlighting its potential as a therapeutic target in PANS.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eIncreased plasma MMP-9 in PANS flare may induce an inflammatory feedback loop in BECs\u003c/strong\u003e \u003cp\u003eTo identify immune mediators released by BECs in response to PANS plasma, we performed a multiplex assay of cell culture supernatants. We used heparinized plasma for this assay as it preserves proteolytic activity (e.g., MMP-9 activity) more effectively than EDTA plasma. Several analytes were below the detection limit; however, PDGF-AA, IL-6, and IL-8 (CXCL-8) were consistently elevated in PANS flare-treated BEC culture supernatant in comparison to the healthy controls (Appendix Fig.\u0026nbsp;2). Based on these findings, IL-6 and IL-8 (CXCL-8) were selected for targeted ELISA validation.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eWe measured IL-6 and IL-8 (CXCL-8) levels in BEC supernatants by ELISA using heat-inactivated heparinized plasma from PANS flare and matched controls. Both cytokines are known for their established role in compromising endothelial junctional integrity and increasing barrier permeability [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. ELISA analysis revealed a significant increase in IL-6 levels in BEC supernatants exposed to PANS flare plasma, approximately 2.5-fold higher than those in the healthy control group (Fig.\u0026nbsp;6A). Although IL-8 (CXCL-8) levels also showed a trend toward elevation, the difference did not reach statistical significance. These findings suggest that plasma factors (potentially MMP-9) may stimulate IL-6 release from BECs. Notably, previous studies have shown that elevated IL-6 and IL-8 (CXCL-8) can further induce MMP-9 expression and activation, amplifying endothelial disruption [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition to inducing BEC production of cytokines, PANS-flare-treated BECs produced MMP-9 at a 6-fold increase over healthy controls. The active form of MMP-9 was assessed by determining the MMP-9/TIMP-1 ratio, where TIMP-1 serves as an endogenous inhibitor of MMP-9 [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. An elevated MMP-9/TIMP-1 ratio previously associated with BBB disruption and neurovascular injury in conditions such as stroke, cerebral ischemia, and systemic sclerosis [\u003cspan additionalcitationids=\"CR65\" citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e] was also observed in PANS flare-treated BEC supernatant (Fig.\u0026nbsp;6B).\u003c/p\u003e \u003cp\u003eCollectively, these results indicate that elevated MMP-9 in the PANS flare state induces a self-reinforcing inflammatory loop that exacerbates BEC dysfunction. High MMP-9 levels stimulate IL-6 and IL-8 (CXCL-8) secretion, which, in turn, promote further MMP-9 activation, driving endothelial barrier degradation. The elevated MMP-9/TIMP-1 ratio further supports the presence of a proteolytic inflammatory cascade contributing to BBB dysfunction in PANS.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eAn exogenous MMP-9 inhibitor prevents BEC barrier disruptions induced by PANS flare plasma\u003c/strong\u003e \u003cp\u003eTo further validate the contribution of MMP-9 to BEC disruptions, we tested the impact of direct application of MMP-9 and co-treatment with a pharmacological inhibitor of MMP-9. We used a specific MMP-9 inhibitor, MMP-9 inhibitor-I, 2-[benzyl-(4-methoxyphenyl)sulfonylamino]-5-(diethylaminomethyl)-N-hydroxy-3-methylbenzamide.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eTo confirm the functional role of MMP-9, we first added recombinant human MMP-9 (500 ng/mL) to normal human serum, which increased BEC permeability compared to the serum alone, mimicking the effects observed with PANS flare plasma (Fig.\u0026nbsp;7A). Co-treatment with the MMP-9 inhibitor attenuated this effect in a dose-dependent manner. A similar protective effect was observed when PANS flare plasma was co-treated with the MMP-9 inhibitor. Specifically, inhibition of MMP-9 significantly reduced PANS flare plasma-induced hyperpermeability, with 5 \u0026micro;M partially reversing barrier disruption and 50 \u0026micro;M restoring barrier integrity to near-baseline levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; n\u0026thinsp;=\u0026thinsp;6; Fig.\u0026nbsp;7B). These results demonstrate a strong and dose-dependent protective effect of MMP-9 inhibition on endothelial barrier function.\u003c/p\u003e \u003cp\u003eNext, we assessed the influence of co-treatment with the MMP-9 inhibitor on IL-6 and IL-8 (CXCL-8). ELISA analysis of BEC supernatants revealed MMP-9 inhibition markedly reduced IL-6 and IL-8 (CXCL-8) concentration in the cell culture supernatant (Fig.\u0026nbsp;7C).\u003c/p\u003e \u003cp\u003eImmunofluorescence further confirmed that MMP-9 inhibition preserved endothelial junctional architecture (Fig.\u0026nbsp;7D). BECs treated with PANS flare plasma alone showed disrupted localization of VE-cadherin and ZO-1 (loss of junctional continuity) and enhanced phalloidin staining (actin stress-fiber formation). In contrast, co-treatment with the MMP-9 inhibitor restored continuous VE-cadherin and ZO-1 staining along cell borders and markedly reduced stress fibers. Quantitative analysis verified these findings (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for all markers, n\u0026thinsp;=\u0026thinsp;5; Fig.\u0026nbsp;7E), thus validating the protective effect of MMP-9 inhibition at the cellular level.\u003c/p\u003e \u003cp\u003eThis finding suggests a feedback loop in which MMP-9 not only disrupts endothelial integrity but also sustains cytokine production, possibly through downstream inflammatory signaling pathways or extracellular matrix remodeling. Pharmacological inhibition of MMP-9 effectively reverses these pathological effects, supporting its potential as a therapeutic target for restoring BBB integrity in PANS.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eElevated plasma S100B provides indirect \u003cem\u003ein vivo\u003c/em\u003e evidence of BBB disruption during PANS flare\u003c/strong\u003e \u003cp\u003eIndirect evidence of BBB disruption during PANS flare was assessed by measuring plasma levels of S100B, which is a well-recognized peripheral biomarker of BBB permeability. Plasma S100B concentrations were significantly elevated in patients during PANS flare compared with age- and sex-matched healthy controls and were reduced upon clinical recovery in paired samples (Appendix 1). These findings support the presence of BBB vulnerability \u003cem\u003ein vivo\u003c/em\u003e during active PANS disease states.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eImpact of PANS flare on endothelial monolayer permeability is specific to BECs\u003c/strong\u003e \u003cp\u003eTo determine whether the effects of PANS flare plasma on endothelial permeability were specific to the brain vasculature, primary human endothelial cells derived from lung, umbilical vein, and brain were cultured as confluent monolayers on Transwell\u0026reg; inserts. Exposure to PANS flare plasma did not significantly alter permeability in lung or umbilical vein endothelial monolayers compared with healthy control plasma. In contrast, a marked increase in permeability was observed exclusively in BEC monolayers (Appendix 2C), indicating tissue-specific susceptibility of the BBB to circulating factors present during PANS flare.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eExploratory plasma multiplex profiling identifies CCL11 as a flare-associated chemokine in PANS\u003c/strong\u003e \u003cp\u003eTo explore whether circulating inflammatory mediators beyond MMP-9 are altered during PANS disease activity, we performed an exploratory multiplex cytokine and chemokine analysis of PANS plasma (flare and recovery), along with matched healthy controls. Among the analytes assessed, CCL11 was selectively elevated in plasma during PANS flare compared with healthy controls and showed a consistent reduction upon clinical improvement in paired samples (Appendix 4 ). In contrast, other chemokines, cytokines, and growth factors showed variability within groups and failed to exhibit a consistent, flare-specific, state-dependent pattern. We followed up by measuring plasma CCL11 concentration using ELISA in an expanded sample set, which strongly supported the multiplex results (Appendix 5). Altogether, the exploration multiplex plasma profiling provides a useful direction for hypothesis generating and identifies CCL11 as a candidate flare-associated chemokine in PANS.\u003c/p\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePANS is increasingly recognized as an immune-mediated condition caused by infections and systemic inflammation. BBB dysfunction has been implicated based on preliminary studies which show elevated protein and/or albumin quotient in the cerebral spinal fluid of patients, hypersensitivity to psychotropics, and brain homing cells [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. Our present findings provide direct mechanistic evidence supporting the role of systemic inflammatory factors in the possible BBB dysfunction that occurs during a PANS flare. Plasma from flare patients caused disruption of structure and function of BECs which form the BBB. Exposure to PANS flare plasma led to rapid and pronounced hyperpermeability over time, accompanied by loss of tight and adherens junctional proteins and enhanced actin stress fiber formation. These effects were not observed with BECs treated with plasma from healthy controls nor plasma from the same patients collected during the recovered state, thus indicating that the BBB disruption in PANS reflects a dynamic and disease state-dependent process. The potential for BBB disruption during PANS flare was also supported by one of our crucial preliminary findings: an increased circulatory S100B protein concentration during flare, measured in the plasma by ELISA (Appendix Fig.\u0026nbsp;4). S100B is considered a promising biomarker for BBB leakiness as it does not cross the BBB in a healthy physiological state [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. These early findings provide a critical clue that BBB dysfunction may not just be a bystander, but an active contributor to neuropsychiatric dysfunction during PANS flares. Our study supports the need to investigate BBB breakdown as both a mechanistic link and a promising therapeutic target in the care of youth affected by this debilitating condition.\u003c/p\u003e \u003cp\u003eWe conducted bulk RNA-seq analysis of BECs treated with PANS flare plasma that revealed marked downregulation of genes encoding critical junctional proteins. These include genes that form and stabilize the tight junction structure, such as TJP1 (encoding ZO-1), claudins, and JAMs. Downregulation of these genes likely compromises BBB integrity by disrupting intercellular adhesion and cytoskeletal structure. The tight junction protein ZO-1, in particular, plays a central role in organizing the endothelial tight junctions and stabilizing adherens junctions [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. ZO‑1 is a critical scaffolding protein that links TJ proteins, claudins and occludin to the actin cytoskeleton. Therefore, downregulation of TJP1 is presumed to have a significant impact on the stability of endothelial junctions, disrupting both tight junctions and adherens junctions. The immunofluorescence of ZO-1 with PANS flare plasma showed a decrease in the fluorescence intensity around the BEC junctions, indicating ZO-1 instability in the BECs.\u003c/p\u003e \u003cp\u003eBeyond TJP1, we also identified reduced expression of vinculin (VCL) and ANGPT1, which are known to stabilize adherens junction VE-cadherin. VCL is known to stabilize cadherin\u0026ndash;catenin complex formation, while ANGPT1 stabilizes VE-cadherin\u0026ndash;actin interactions through its receptor, Tie2, on endothelial cells [\u003cspan additionalcitationids=\"CR73 CR74\" citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. Decreased expression of both VCL and ANGPT1 weakens cytoskeletal support at the adherens junctions, destabilizing and further compromising endothelial barrier integrity. Although decreased CDH5 expression was not observed in the volcano plot (Fig.\u0026nbsp;2B), the heat map in Fig.\u0026nbsp;2C showed consistent reduction of CDH5 (encoding the VE-cadherin) in the BECs with PANS flare plasma. The decrease in expression of VE-cadherin in immunofluorescence staining with the PANS flare plasma also indicates the disruptions of adherens junctions and destabilization of barrier integrity.\u003c/p\u003e \u003cp\u003eIn addition to downregulation of canonical junctional genes such as TJP1 and CDH5, the present findings reveal suppression of ITGB1 and CDC42, both of which are essential for cytoskeletal anchoring of endothelial cells to the basement membrane and for maintaining barrier stability [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. β1-integrin (ITGB1) is more consistently reported to promote actin stability and support endothelial structure and its downregulation destabilizes the endothelial junctions and increases the paracellular permeability by stress fiber actin formation [\u003cspan additionalcitationids=\"CR78\" citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. The immunofluorescence data were also corroborated by RNA-seq data, which showed increased actin stress fibers with PANS flare plasma. Notably, our RNA-seq data for BEC strongly align with recent findings from a mouse model of repeated group A streptococcus (GAS) infection, in which the BBB-associated genes in the BECs were transcriptionally suppressed, including the genes stabilizing multiple junctional proteins. Further analysis revealed transcriptional elevation of inflammatory microglial phenotype in GAS-induced mouse models, strongly supporting BBB predominant BBB dysfunction in PANS. The study further identified robust inflammatory microglial activation following GAS infection, strongly supporting the contribution of BBB dysfunction in PANS/PANDAS-like conditions (Wayne et al, 2026 bioRxiv Preprint doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.64898/2026.02.04.703836\u003c/span\u003e\u003cspan address=\"10.64898/2026.02.04.703836\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSimultaneously, upregulation of ICAM1, ICAM2, and CCL2 indicates a shift toward a pro-adhesive, inflammatory endothelial state that may promote leukocyte trafficking across the BBB [\u003cspan additionalcitationids=\"CR81 CR82\" citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e]. Elevated ANGPT2 alongside reduced ANGPT1 further supports vascular destabilization through imbalance of the Tie2 signaling axis, a well-established hallmark of endothelial dysfunction [\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e, \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e]. Together, these results strongly implicate that PANS flare plasma not only compromises the structural integrity of the brain endothelial barrier but also primes it for increased immune cell trafficking. This dual impact reflects the classic hallmarks of BBB dysfunction.\u003c/p\u003e \u003cp\u003eThe impact of PANS flare plasma on permeability appears to be limited to BECs, rather than primary lung or umbilical vein endothelial cells (Appendix Fig.\u0026nbsp;1). Interestingly, according to a recent publication by Ma et al. [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], 96% of patients with PANS have clinical evidence of low-grade vascular inflammation using a limited clinical evaluation panel (high D-dimer, high von Willebrand factor antigen, livedo reticularis, and periungual erythema).\u003c/p\u003e \u003cp\u003eBECs are complex, with a highly restrictive tight junction architecture. Due to the unique structure, they are susceptible to inflammatory perturbations, commonly observed in neuroinflammatory disorders. A subtle disruption in junctional integrity proteins (occludin, claudins, and ZO-1) results in paracellular leakage and immune cell infiltration, leading to increased CNS inflammation.\u003c/p\u003e \u003cp\u003eGiven that PANS is a relapsing\u0026ndash;remitting disorder as described by Masterson et al. [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], we also compared the impact of PANS flare plasma versus recovery plasma on ZO-1, VE-cadherin, and actin stress fiber formation. We observed that plasma from the recovered state did not disrupt the BEC barrier and cell structure (compared to flare) and was similar to the healthy control data, suggesting the role of circulatory inflammatory mediators that disrupt the BEC barrier is reduced or absent in the recovered state.\u003c/p\u003e \u003cp\u003eWe identified MMP-9 as a central effector in PANS-associated BBB dysfunction. MMP-9 is a proteolytic enzyme known to degrade tight junction proteins and extracellular matrix components, thereby compromising endothelial junctional integrity [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. In our study, MMP-9 levels were significantly elevated in the PANS flare plasma and showed a strong positive correlation with increased endothelial permeability. Interestingly, MMP-9 levels in plasma collected from the recovered state are significantly lower than the flare state (and similar to healthy control levels). This observation aligns with previous research demonstrating that MMP-9 is quickly activated and released into the circulation during acute inflammation, primarily by inflammatory leukocytes (e.g., activated neutrophils) in response to proinflammatory cytokines and chemokines. Elevated circulating MMP-9 has been associated with disease severity, tissue remodeling, and neuronal injury across a range of neuroinflammatory-associated conditions such as multiple sclerosis, cerebral aneurysm, stroke, Alzheimer\u0026rsquo;s disease, Parkinson\u0026rsquo;s disease, epilepsy, schizophrenia, and neuro-invasive infections (tic borne encephalitis, West Nile virus, measles, human immunodeficiency virus and bacterial meningitis) [\u003cspan additionalcitationids=\"CR87 CR88 CR89 CR90 CR91 CR92\" citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGiven the ability of MMP-9 to degrade both tight and adherens junction proteins, its activity likely contributes directly to the barrier dysfunction and hyperpermeability observed in our \u003cem\u003ein vitro\u003c/em\u003e model, supporting a mechanistic link between systemic inflammation and BBB compromise in PANS. We also observed elevated levels of several proinflammatory mediators, including IL-6 and IL-8 (also known as CXCL8), in the PANS flare-treated BEC supernatant. These proinflammatory mediators are known to disrupt endothelial junctions and have been shown to amplify MMP-9 transcription [\u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e, \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e]. Supporting these findings, we also observed a significant increase in active MMP-9 in the flare-treated BEC supernatant. Compared with healthy control-treated BEC supernatant, the MMP/TIMP-1 ratio in PANS flare-treated supernatant plasma was markedly greater, supporting the presence of active MMP-9. These findings suggest a self-perpetuating inflammatory loop in which MMP-9 triggers the release of cytokines from BECs that further enhance MMP-9 expression in BECs, thereby exacerbating barrier disruption.\u003c/p\u003e \u003cp\u003eWe employed a specific extracellular MMP-9 antagonist, MMP-9 inhibitor-I, with PANS flare plasma, which significantly rescued BEC permeability in a dose-dependent manner. The results showed that 50 \u0026micro;M of the inhibitor prevented loss of junctional proteins, reduced actin stress fibers and attenuated the secretion of IL-6 and IL-8 (CXCL-8). These findings confirm that MMP-9 is not only necessary for flare-induced endothelial cell disruption but also sufficient to exacerbate the inflammatory response. This finding reinforces the dual role of MMP-9 as a structural disruptor and immune mediator that promotes inflammation.\u003c/p\u003e \u003cp\u003eIn summary, the present study provides evidence for dysfunction of BECs, which are a key component forming the BBB. This study identifies elevated circulating MMP-9 as a central mediator for dysfunction, linking systemic inflammation to brain endothelial injury and neuroimmune activation. Notably, we demonstrate for the first time that plasma from active PANS flare patients induces acute, time-dependent hyperpermeability in BECs, mediated by MMP-9\u0026ndash;driven structural degradation and cytokine amplification loops. These findings underscore MMP-9 as a potential therapeutic target to mitigate BBB breakdown and downstream neuroinflammatory cascades.\u003c/p\u003e \u003cp\u003eIn line with our findings, MMP-9 inhibitors (e.g., GM6001, MMP-9 inhibitor I) have been shown to have neuroprotective effects on various neuropsychiatric and neurodegenerative disorders, including amyotrophic lateral sclerosis and Parkinson's disease [\u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e, \u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e]. This study, therefore, provides additional rationale for targeting MMP-9 in the context of acute neuroinflammatory conditions such as PANS.\u003c/p\u003e \u003cp\u003eBeyond the MMP-9 mediated effects, our data suggest a role for chemokine CCL11 modulating the BBB integrity during a PANS flare. A pilot multiplex array of plasma showed marked elevation of CCL11 during flare episodes compared to the healthy controls and confirmed similar observation with follow up ELISA studies. (Appendix 4 and 5). CCL11 is recognized as a mediator of neuroinflammation and BBB disruption, acting via CCR3 receptors to promote microglial activation and neuronal injury [\u003cspan additionalcitationids=\"CR99\" citationid=\"CR98\" class=\"CitationRef\"\u003e98\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e100\u003c/span\u003e]. It can also be secreted by endothelial cells in response to Th2 cytokines and further amplifies immune cell infiltration and local inflammation [\u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e101\u003c/span\u003e, \u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e102\u003c/span\u003e]. Together, these findings suggest that CCL11, alongside MMP-9, functions as a co-effector driving BBB hyperpermeability and neuroimmune activation in PANS flares.\u003c/p\u003e \u003cp\u003eFour medication classes have emerged as therapeutic options for patients with PANS and all appear to impact the aforementioned pathways. Non-steroidal anti-inflammatories, corticosteroids and azithromycin are known to reduce MMP-9, IL 6 and IL 8 in many clinical conditions [\u003cspan additionalcitationids=\"CR104 CR105 CR106 CR107 CR108 CR109\" citationid=\"CR103\" class=\"CitationRef\"\u003e103\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR110\" class=\"CitationRef\"\u003e110\u003c/span\u003e]. Moreover, these drugs have also been shown to improve PANS flare symptoms and flare duration in observational studies [\u003cspan citationid=\"CR111\" class=\"CitationRef\"\u003e111\u003c/span\u003e, \u003cspan citationid=\"CR112\" class=\"CitationRef\"\u003e112\u003c/span\u003e]. Finally, serotonin reuptake inhibitors also lower MMP-9 and Il-6 and have shown beneficial impacts in PANS, though patients in early flare episodes may respond better to lower doses than typical due to increased risk of side-effects [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. A summary of medications is shown in Appendix Table\u0026nbsp;4.\u003c/p\u003e \u003cp\u003eThis study provides a foundation for employing multicellular BBB models incorporating astrocytes, pericytes, and microglia to better capture neurovascular complexity and further validate the role of systemic inflammation in PANS. Such models will be essential to understand the full impact of MMP-9 antagonists currently being used and may help identify additional mediators driving BBB disruption and microglial activation in neuroinflammation.\u003c/p\u003e\n\u003ch3\u003eLimitations of the study:\u003c/h3\u003e\n\u003cp\u003eA primary limitation of this study is the use of BEC monolayers as an \u003cem\u003ein vitro\u003c/em\u003e model of the BBB. While BECs represent a fundamental component, this monoculture system cannot fully recapitulate the complex microenvironment of the neurovascular unit, which includes dynamic interactions with pericytes, astrocytes, microglia, the basement membrane, and physiological hemodynamic shear stress. Consequently, future investigations utilizing more complex systems, such as multicellular co-cultures or microfluidic \"BBB-on-a-chip\" platforms, are warranted to enhance the physiological relevance of the findings.\u003c/p\u003e \u003cp\u003eSecondly, while the data implicate MMP-9 as a key mediator of BEC hyperpermeability, it is important to acknowledge that plasma is a complex biological matrix. It is therefore plausible that additional circulating factors, such as cytokines, chemokines, and autoantibodies, act synergistically with or upstream of MMP-9 to promote BBB dysfunction. Although our exploratory analysis identified CCL11 as a potential co-effector, this preliminary observation requires validation in larger cohorts and through pathway-specific inhibition studies.\u003c/p\u003e \u003cp\u003eThirdly, the matching of patient and control cohorts was limited to age and sex, introducing the potential for confounding from unmatched demographic characteristics like race and ethnicity. To confirm the generalizability of these findings, a larger cohort with more comprehensive matching will be necessary.\u003c/p\u003e \u003cp\u003eFinally, the study cohort was heterogeneous regarding disease course, including patients with both new-onset flares and persistent PANS presentations. As disease duration may influence the plasma inflammatory profile and its effect on BEC permeability, future studies with larger, well-stratified cohorts are required to determine whether distinct clinical trajectories are associated with differential impacts on BBB integrity.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe current project was supported by the Brain Foundation, the Stanford Institute for Immunity, Transplantation \u0026amp; Infection and the Stanford Autoimmune \u0026amp; Allergy Supergroup. \u0026nbsp;Funding for the infrastructure of our research program, training, and overlapping projects came from: 1) Lucile Packard Foundation for Children\u0026apos;s Health; 2) National Institute of Mental Health- Pediatrics and Developmental Neuroscience Branch which supported the initial creation of the Stanford IBH Program; 3) The Neuroimmune Foundation for education/training, microbial sequencing, and medication analyses; \u0026nbsp;4) The Brain Foundation and O\u0026rsquo;Sullivan Foundation for Autism research; 5) The Tara \u0026amp; Dave Dollinger PANS Biomarker Discovery Core for patient sample collection; 6) Stanford Maternal and Child Health Research Institute (MCHRI) for HLA research; 7) Stanford SPARK, SPARK Pisa, and International OCD Foundation for immunophenotyping research; 8) Oxnard Foundation for imaging research; 9) Caudwell Children\u0026rsquo;s Foundation for genetics and immunology research; 10) The Louisa Adelynn Johnson Fund for Complex Disease \u0026nbsp;for expanding the Illuminate PANS project; 11) Stanford COMET, DRIVE, and HBREX programs for student research in our program.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: AM, JF, EDM\u003c/p\u003e\n\u003cp\u003eMethodology: AM, JF, EDM, BT, AK, TC\u003c/p\u003e\n\u003cp\u003ePatient classification based on review of records, multiple clinical interviews, and physical exam: MS, PT, MT, YX, BF, MM (supervised by MM and JF).\u003c/p\u003e\n\u003cp\u003eDisease state classification: BF, MM, MS, JF (supervised by JF)\u003c/p\u003e\n\u003cp\u003eHealthy control project supervision and matching: JH, LC, JF\u003c/p\u003e\n\u003cp\u003eSample acquisition: CM\u003c/p\u003e\n\u003cp\u003eInvestigation: AM\u003c/p\u003e\n\u003cp\u003eData analysis: AM, SHG\u003c/p\u003e\n\u003cp\u003eSupervision of investigation and review of data throughout the project: JF, EDM.\u003c/p\u003e\n\u003cp\u003eSenior oversight of project continuity, laboratory resources, and critical intellectual revision of the manuscript: MK\u003c/p\u003e\n\u003cp\u003eWriting \u0026ndash; original draft: AM, JF\u003c/p\u003e\n\u003cp\u003eWriting \u0026ndash; review \u0026amp; editing: All\u003c/p\u003e\n\u003cp\u003eFunding acquisition: JF, EDM, AM\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful for our patients and families who understand treatment limitations and continue to lend their time and cooperation to research participation. We would also like to thank Claudia Macaubas (for her critical input), the Stanford IBH Clinic Team, Stanford core facilities (Cell Sciences Imaging Facility, Neuroscience Microscopy Service, Human Immune Monitoring Core).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and materials availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are available in the main text or the appendices. The RNA sequencing datasets generated and analyzed in this study have been deposited in the CBI Gene Expression Omnibus under accession number GSE324385. All materials used in the analysis can be available upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSwedo SE, Leckman JF, Rose NR. From research subgroup to clinical syndrome: modifying the PANDAS criteria to describe PANS (pediatric acute-onset neuropsychiatric syndrome). 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Cytokine. 2002;18(6):329\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSpartz EJ, Freeman GM Jr., Brown K, Farhadian B, Thienemann M, Frankovich J. Course of Neuropsychiatric Symptoms After Introduction and Removal of Nonsteroidal Anti-Inflammatory Drugs: A Pediatric Observational Study. J Child Adolesc Psychopharmacol. 2017;27(7):652\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrown K, Farmer C, Farhadian B, Hernandez J, Thienemann M, Frankovich J. Pediatric Acute-Onset Neuropsychiatric Syndrome Response to Oral Corticosteroid Bursts: An Observational Study of Patients in an Academic Community-Based PANS Clinic. J Child Adolesc Psychopharmacol. 2017;27(7):629\u0026ndash;39.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-neuroinflammation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jneu","sideBox":"Learn more about [Journal of Neuroinflammation](http://jneuroinflammation.biomedcentral.com)","snPcode":"12974","submissionUrl":"https://submission.nature.com/new-submission/12974/3","title":"Journal of Neuroinflammation","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-9442808/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9442808/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003ePediatric Acute-onset Neuropsychiatric Syndrome (PANS) is characterized by the abrupt onset of obsessive\u0026ndash;compulsive symptoms and/or restrictive eating accompanied by disturbances in sleep, affect regulation, behavior, motor function, and sensory processing. Increasing evidence implicates systemic immune activation and circulating autoantibodies in basal ganglia dysfunction. Blood\u0026ndash;brain barrier (BBB) impairment has been proposed as a mechanism by which peripheral inflammatory mediators access the central nervous system; however, the molecular pathways linking systemic inflammation to BBB disruption in PANS remain incompletely defined.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eTo determine whether circulating factors contribute to BBB dysfunction, human brain endothelial cell (BEC) monolayers were exposed to plasma from PANS patients during symptomatic flare and recovery, as well as from matched healthy controls. Barrier integrity was assessed by paracellular permeability assays; transcriptomic changes were analyzed using bulk RNA sequencing. Junctional organization and cytoskeletal architecture were examined by immunofluorescence microscopy. Circulating and endothelial-derived mediators associated with barrier disruption were quantified using multiplex bead\u0026ndash;based immunoassays and enzyme-linked immunosorbent assays.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003ePlasma from PANS flare (n\u0026thinsp;=\u0026thinsp;15 samples) significantly increased BEC monolayer permeability compared to plasma from matched controls (~\u0026thinsp;50% change in permeability; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), corresponding with increased concentration of S100B, an \u003cem\u003ein vivo\u003c/em\u003e biomarker of BBB permeability (~\u0026thinsp;2 fold increase vs controls; \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.01). Transcriptomic profiling of flare samples demonstrated downregulation of genes essential for endothelial stability, including those encoding tight junction, adherens junction, and extracellular matrix components. Immunofluorescence of flare samples confirmed disruption of zonula occludens-1 (ZO-1) and vascular endothelial cadherin (VE-Cadherin), accompanied by increased actin stress fiber formation, consistent with enhanced cytoskeletal tension and junctional disassembly. Matrix metalloproteinase-9 (MMP-9) concentration was elevated in flare plasma vs controls (\u0026gt;\u0026thinsp;2.5 fold increase; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and was highly correlated with BEC monolayer permeability (r\u0026thinsp;=\u0026thinsp;0.84; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Inhibition of MMP-9 in flare samples (n\u0026thinsp;=\u0026thinsp;6) resulted in significant decrease in BEC monolayer permeability (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), approaching that of matched controls.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eCirculating factors present during PANS flares induce brain endothelial dysfunction \u003cem\u003ein vitro\u003c/em\u003e, correlating with \u003cem\u003ein vivo\u003c/em\u003e biomarker findings. Elevated MMP-9 functions as a key downstream effector linking systemic inflammation to endothelial barrier disruption, providing mechanistic insight into how peripheral immune activation may facilitate neuroinflammation in PANS.\u003c/p\u003e","manuscriptTitle":"Matrix Metalloproteinase-9 (MMP-9) as a Potential Regulator of Blood–Brain Barrier Dysfunction in Pediatric Acute Neuropsychiatric Syndrome (PANS)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-07 16:55:19","doi":"10.21203/rs.3.rs-9442808/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"155560135898954319550102010426578409322","date":"2026-05-09T02:09:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"292987352890712073954664866539857819134","date":"2026-05-08T00:56:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"270814455609161598742195739492777403888","date":"2026-05-06T16:32:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"34175805184327744863489544185465588232","date":"2026-05-04T14:03:17+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-21T22:05:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-18T18:30:44+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-18T07:45:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Neuroinflammation","date":"2026-04-17T01:26:11+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-neuroinflammation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jneu","sideBox":"Learn more about [Journal of Neuroinflammation](http://jneuroinflammation.biomedcentral.com)","snPcode":"12974","submissionUrl":"https://submission.nature.com/new-submission/12974/3","title":"Journal of Neuroinflammation","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"27af8f66-6a6a-45b2-870e-f5a5554b3841","owner":[],"postedDate":"May 7th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"155560135898954319550102010426578409322","date":"2026-05-09T02:09:06+00:00","index":53,"fulltext":""},{"type":"reviewerAgreed","content":"292987352890712073954664866539857819134","date":"2026-05-08T00:56:09+00:00","index":52,"fulltext":""},{"type":"reviewerAgreed","content":"270814455609161598742195739492777403888","date":"2026-05-06T16:32:30+00:00","index":50,"fulltext":""},{"type":"reviewerAgreed","content":"34175805184327744863489544185465588232","date":"2026-05-04T14:03:17+00:00","index":46,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-07T16:55:22+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-07 16:55:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9442808","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9442808","identity":"rs-9442808","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}