Defining the CSF cytokine/chemokine and injury biomarker signature of glial fibrillary acidic protein (GFAP) autoimmunity can inform immunopathogenesis. CSF GFAP-IgG-positive samples (N = 98) were tested for 17 cytokines/chemokines, neurofilament light chain (NfL), and GFAP (ELLA, Bio-Techne). Controls included non-inflammatory (N = 42), AQP4-IgG-positive (N = 83), CNS infections (N = 13), and neurosarcoidosis (N = 32). IL5, IL6, IL10, IL8/CXCL8, CXCL9, CXCL10, CXCL13, BAFF, GM-CSF, IFN-gamma, and TNF-alpha concentrations were higher compared to non-inflammatory controls (P < 0.01). GFAP concentrations were similar to those of AQP4-IgG-positive patients; NfL was higher (P < 0.001) and correlated with MRI changes and outcomes. CSF cytokine/chemokine findings in GFAP autoimmunity correlate with histopathology; GFAP and NfL hold promise as disease biomarkers.

OBJECTIVES:To investigate the value of cytokine, chemokine, and neurofilament light chain (NfL) concentrations in predicting relapse risk, chronic epilepsy, and functional impairment in LGI1 autoimmune encephalitis (AE). METHODS:Cytokines/chemokines (IL-1-beta, IL-2, IL-4, IL-5, IL-6, IL-8/CXCL8, IL-10, IL-12p70, IL-13, IL-17A, GM-CSF, TNF-alpha, IFN-gamma, CXCL9, CXCL10, CXCL13, BAFF) and NfL concentrations were measured in CSF and paired serum from LGI1-AE patients evaluated at Mayo Clinic (01/2015-02/2024), using a multiplex immunoassay system (ELLA, Bio-Techne) and correlated with clinical outcomes. A laboratory-based cohort of LGI1-IgG-positive patients and control cohorts, including patients with mixed non-inflammatory disorders (MNID), Alzheimer's disease (AD), and temporal lobe epilepsy (TLE) were analyzed. RESULTS:Forty-four patients with LGI1-AE were included; 29 (66%) were male, with a median age of 68.5 years (range, 8-85). Median time from symptom onset to CSF sampling was eight months (IQR, 3-17); 19/42 (45%) experienced a clinical relapse and 27% developed chronic epilepsy. Serum IL-6, serum and CSF IL-8/CXCL8, and IL-17A were higher in LGI1-IgG positive patients than MNID (p < 0.05). TLE cytokine/chemokine profiles were similar to LGI1 AE; AD patients had lower serum IL-6 and CSF IL-8/CXCL8 (p = 0.04; p = 0.01), and higher serum IL-17A and GM-CSF (p = 0.004; p = 0.01) than LGI1-AE. Higher CSF IL-6 and IL-8/CXCL8 in LGI1-AE associated with clinical relapse (p < 0.05) and higher CSF NfL associated with chronic epilepsy (p = 0.01). CONCLUSION:Elevations in IL-6, IL-8/CXCL8, and IL-17A were identified in this LGI1-AE cohort. CSF IL-6, IL-8/CXCL8, and NfL levels are potential prognostic biomarkers for risk of relapse and chronic epilepsy in LGI1-AE.

To describe the cytokine/chemokine profile in cerebrospinal fluid (CSF) of patients with neurosarcoidosis.

OBJECTIVE:To evaluate the cerebrospinal fluid (CSF) cytokine/chemokine profile of central nervous system (CNS) neurosarcoidosis (NS), and its utility in differential diagnosis, treatment, and prognostication. METHODS:In this case-control study, we validated 17 cytokines/chemokines (interleukin [IL]-1-beta, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, IL-17A, BAFF, IL-8/CXCL8, CXCL9, CXCL10, CXCL13, GM-CSF, interferon-gamma, and tumor necrosis factor [TNF]-alpha) in a multiplexed automated immunoassay system (ELLA; Bio-Techne, Minneapolis, MN, USA), and assessed them in CSF and serum of symptomatic patients with probable or definite CNS NS (01/2011-02/2023) with gadolinium enhancement and/or CSF pleocytosis. Patients with multiple sclerosis, primary CNS lymphoma, aquaporin-4 immunoglobulin G positivity, non-inflammatory disorders, and healthy individuals were used as controls. RESULTS:A total of 32 NS patients (59% women; median age, 59 years [19-81]) were included; concurrent sera were available in 12. CSF controls consisted of 26 multiple sclerosis, 8 primary CNS lymphoma, 84 aquaporin-4 immunoglobulin G positive, and 34 patients with non-inflammatory disorders. Gadolinium enhancement was present in 31 of 32 NS patients, and CSF pleocytosis in 27 of 32 (84%). CSF IL-2, IL-6, IL-10, IL-13, BAFF, IL-8/CXCL8, CXCL9, CXCL10, CXCL13, GM-CSF, interferon-gamma, and TNF-alpha levels were significantly higher in NS patients compared with non-inflammatory controls (p ≤ 0.02); elevations were more common in CSF than serum. Concurrent elevation of IL-6, CXCL9, CXCL10, GM-CSF, interferon-gamma, and TNF-alpha was present in 18 of 32 NS patients, but only in 1 control. Elevated IL-6, IL-10, IL-13, CXCL9, CXL10, GM-CSF, and TNF-alpha associated with measures of disease activity. INTERPRETATION:NS CSF cytokine/chemokine profiles suggest T cell (mainly T helper cell type 1), macrophage, and B-cell involvement. These signatures aid in NS diagnosis, indicate disease activity, and suggest therapeutic avenues. ANN NEUROL 2024;96:704-714.

ObjectiveThe aim of this study was to assess the diagnostic utility of cerebrospinal fluid (CSF) myelin oligodendrocyte glycoprotein antibodies (MOG‐IgG) testing.MethodsWe retrospectively identified patients for CSF MOG‐IgG testing from January 1, 1996, to May 1, 2023, at Mayo Clinic and other medical centers that sent CSF MOG‐IgG for testing including: controls, 282; serum MOG‐IgG positive MOG antibody‐associated disease (MOGAD), 74; serum MOG‐IgG negative high‐risk phenotypes, 73; serum false positive MOG‐IgG with alternative diagnoses, 18. A live cell‐based assay assessed CSF MOG‐IgG positivity (IgG‐binding‐index [IBI], ≥2.5) using multiple anti‐human secondary antibodies and end‐titers were calculated if sufficient sample volume. Correlation of CSF MOG‐IgG IBI and titer was assessed.ResultsThe pan‐IgG Fc‐specific secondary was optimal, yielding CSF MOG‐IgG sensitivity of 90% and specificity of 98% (Youden's index 0.88). CSF MOG‐IgG was positive in: 4/282 (1.4%) controls; 66/74 (89%) serum MOG‐IgG positive MOGAD patients; and 9/73 (12%) serum MOG‐IgG negative patients with high‐risk phenotypes. Serum negative but CSF positive MOG‐IgG accounted for 9/83 (11%) MOGAD patients, and all fulfilled 2023 MOGAD diagnostic criteria. Subgroup analysis of serum MOG‐IgG low‐positives revealed CSF MOG‐IgG positivity more in MOGAD (13/16[81%]) than other diseases with false positive serum MOG‐IgG (3/15[20%]) (p = 0.01). CSF MOG‐IgG IBI and CSF MOG‐IgG titer (both available in 29 samples) were correlated (Spearman's r = 0.64, p < 0.001).InterpretationCSF MOG‐IgG testing has diagnostic utility in patients with a suspicious phenotype but negative serum MOG‐IgG, and those with low positive serum MOG‐IgG results and diagnostic uncertainty. These findings support a role for CSF MOG‐IgG testing in the appropriate clinical setting. ANN NEUROL 2024;96:34–45

OBJECTIVES:To report an autoimmune paraneoplastic encephalitis characterized by immunoglobulin G (IgG) antibody targeting synaptic protein calmodulin kinase-like vesicle-associated (CAMKV). METHODS:Serum and cerebrospinal fluid (CSF) samples harboring unclassified antibodies on murine brain-based indirect immunofluorescence assay (IFA) were screened by human protein microarray. In 5 patients with identical cerebral IFA staining, CAMKV was identified as top-ranking candidate antigen. Western blots, confocal microscopy, immune-absorption, and mass spectrometry were performed to substantiate CAMKV specificity. Recombinant CAMKV-specific assays (cell-based [fixed and live] and Western blot) provided additional confirmation. RESULTS:Of 5 CAMKV-IgG positive patients, 3 were women (median symptom-onset age was 59 years; range, 53-74). Encephalitis-onset was subacute (4) or acute (1) and manifested with: altered mental status (all), seizures (4), hyperkinetic movements (4), psychiatric features (3), memory loss (2), and insomnia (2). Paraclinical testing revealed CSF lymphocytic pleocytosis (all 4 tested), electrographic seizures (3 of 4 tested), and striking MRI abnormalities in all (mesial temporal lobe T2 hyperintensities [all patients], caudate head T2 hyperintensities [3], and cortical diffusion weighted hyperintensities [2]). None had post-gadolinium enhancement. Cancers were uterine adenocarcinoma (3 patients: poorly differentiated or neuroendocrine-differentiated in 2, both demonstrated CAMKV immunoreactivity), bladder urothelial carcinoma (1), and non-Hodgkin lymphoma (1). Two patients developed encephalitis following immune checkpoint inhibitor cancer therapy (atezolizumab [1], pembrolizumab [1]). All treated patients (4) demonstrated an initial response to immunotherapy (corticosteroids [4], IVIG [2]), though 3 died from cancer. INTERPRETATION:CAMKV-IgG is a biomarker of immunotherapy-responsive paraneoplastic encephalitis with temporal and extratemporal features and uterine cancer as a prominent oncologic association. ANN NEUROL 2024;96:21-33.

Objective Co‐occurring anti‐tripartite motif‐containing protein 9 and 67 autoantibodies (TRIM9/67‐IgG) have been reported in only a very few cases of paraneoplastic cerebellar syndrome. The value of these biomarkers and the most sensitive methods of TRIM9/67‐IgG detection are not known. Methods We performed a retrospective, multicenter study to evaluate the cerebrospinal fluid and serum of candidate TRIM9/67‐IgG cases by tissue‐based immunofluorescence, peptide phage display immunoprecipitation sequencing, overexpression cell‐based assay (CBA), and immunoblot. Cases in which TRIM9/67‐IgG was detected by at least 2 assays were considered TRIM9/67‐IgG positive. Results Among these cases (n = 13), CBA was the most sensitive (100%) and revealed that all cases had TRIM9 and TRIM67 autoantibodies. Of TRIM9/67‐IgG cases with available clinical history, a subacute cerebellar syndrome was the most common presentation (n = 7/10), followed by encephalitis (n = 3/10). Of these 10 patients, 70% had comorbid cancer (7/10), 85% of whom (n = 6/7) had confirmed metastatic disease. All evaluable cancer biopsies expressed TRIM9 protein (n = 5/5), whose expression was elevated in the cancerous regions of the tissue in 4 of 5 cases. Interpretation TRIM9/67‐IgG is a rare but likely high‐risk paraneoplastic biomarker for which CBA appears to be the most sensitive diagnostic assay. ANN NEUROL 2023;94:1086–1101

Background and Objectives Neural antibodies are detected by tissue-based indirect immunofluorescence assay (IFA) in Mayo Clinic's Neuroimmunology Laboratory practice, but the process of characterizing and validating novel antibodies is lengthy. We report our assessment of human protein arrays. Methods Assessment of arrays (81% human proteome coverage) was undertaken using diverse known positive samples (17 serum and 14 CSF). Samples from patients with novel neural antibodies were reflexed from IFA to arrays. Confirmatory assays were cell-based (CBA) or line blot. Epitope mapping was undertaken using phage display immunoprecipitation sequencing (PhiPSeq). Results Control positive samples known to be reactive with linear epitopes of intracellular antigens (e.g., ANNA-1 [anti-Hu]) were readily identified by arrays in 20 of 21 samples. By contrast, 10 positive controls known to be enriched with antibodies against cell surface protein conformational epitopes (e.g., GluN1 subunit of NMDA-R) were indistinguishable from background signal. Three antibodies, previously characterized by other investigators (but unclassified in our laboratory), were unmasked in 4 patients using arrays (July-December 2022): Neurexin-3α, 1 patient; regulator of gene protein signaling (RGS)8, 1 patient; and seizure-related homolog like 2 (SEZ6L2), 2 patients. All were accompanied by previously reported phenotypes (encephalitis, 1; cerebellar ataxia, 3). Patient 1 had subacute onset of seizures and encephalopathy. Neurexin-3α ranked high in CSF (second ranked neural protein) but low in serum (660th overall). Neurexin-3α CBA was positive in both samples. Patient 2 presented with rapidly progressive cerebellar ataxia. RGS8 ranked the highest neural protein in available CSF sample by array (third overall). RGS8-specific line blot was positive. Patients 3 and 4 had rapidly progressive cerebellar ataxia. SEZ6L2 was the highest ranked neural antigen by arrays in all samples (CSF, 1, serum, 2; Patient 3, ranked 9th overall in CSF, 11th in serum; Patient 4, 6th overall in serum]). By PhIPSeq, diverse neurexin-3α epitopes (including cell surface) were detected in CSF from patient 1, but no SEZ6L2 peptides were detected for serum or CSF samples from Patient 3. Discussion Individualized autoimmune neurologic diagnoses may be accelerated using protein arrays. They are optimal for detection of intracellular antigen-reactive antibodies, though certain cell surface-directed antibodies (neurexin-3α and SEZ6L2) may also be detected.

Autoimmune movement disorders are increasingly recognized, but isolated tremor is extremely rare. We describe a 70-year-old male with rapidly progressive, severe postural and intention tremor and weight loss. His cerebrospinal fluid was inflammatory and harbored a neural tissue-restricted antibody. The autoantigen was identified by immunoprecipitation and mass spectrometry and confirmed by antigen-specific assays to be specific for tenascin-R. He was investigated for cancer and diagnosed with follicular lymphoma that expressed tenascin-R suggesting a paraneoplastic origin; cancer treatment and immunotherapy led to complete recovery. With this individualized patient approach and antibody discovery, we expand the spectrum of antibodies accompanying autoimmune hyperkinetic movement disorders. ANN NEUROL 2023;94:502-507.

Adenylate kinase 5 (AK5) antibodies are biomarkers of a poorly responsive to immunotherapy, non-paraneoplastic, autoimmune limbic encephalitis. We detected 6 patients (all female, median age: 72 years [49-80]) with identical CSF antibody staining by indirect immunofluorescence on mouse tissues. We identified AK5 as the antigen and confirmed with standardized assays. Three patients with clinical information had limbic encephalitis, inflammatory CSF and mesiotemporal lobe T2 hyperintensities that evolved to atrophy on brain MRI. One patient had burning smell sensation with no evidence of seizures. Despite immunotherapy, minimal improvement was noticed in one patient; all had severe memory deficits remaining.

The antigen specificity of Anti‐Neuronal Nuclear Antibody‐type 3 (ANNA3)‐IgG is unknown. We identified Dachshund‐homolog 1 (DACH1) as the ANNA3 autoantigen and confirmed it by antigen‐specific assays, immunohistochemical colocalization and immune absorption experiments. Patients' median age was 63.5 years (range, 49–88); 67% were female. Neurological manifestations (information available for 30 patients) included one or more of neuropathy, 12; cognitive difficulties, 11; ataxia, 8; dysautonomia, 7. Evidence of a neoplasm was present in 27 of 30 (90%), most of neuroendocrine lineage. DACH1‐IgG is rare and represents a novel proposed biomarker of neurological autoimmunity and cancer. ANN NEUROL 2022;91:670–675

Significance Statement Fractalkine receptor 1 (CX3CR1) mediates macrophage infiltration into the vasculature. In this study, we used humanized mice knocked in with the human CX3CR1 gene and inhibited CX3CR1 signaling using a variable domains of camelid heavy-chain-only molecule (BI 655088) to test the hypothesis that blockade of CX3CR1 results in less of the venous neointimal hyperplasia formation that is associated with arteriovenous fistula (AVF) failure. We also used human samples removed from failed AVFs combined with cell culture experiments. Our results demonstrate a novel role for CX3CR1 in reducing venous stenosis formation in AVFs. Background Fractalkine receptor 1 (CX3CR1) mediates macrophage infiltration and accumulation, causing venous neointimal hyperplasia (VNH)/venous stenosis (VS) in arteriovenous fistula (AVF). The effect of blocking CX3CR1 using an anti–human variable VHH molecule (hCX3CR1 VHH, BI 655088) on VNH/VS was determined using a humanized mouse in which the human CX3CR1 (hCX3CR1) gene was knocked in (KI). Methods Whole-transcriptomic RNA sequencing with bioinformatics analysis was used on human stenotic AVF samples, C57BL/6J, hCX3CR1 KI mice with AVF and CKD, and in in vitro experiments to identify the pathways involved in preventing VNH/VS formation after hCX3CR1 VHH administration. Results Accumulation of CX3CR1 and CD68 was significantly increased in stenotic human AVFs. In C57BL/6J mice with AVF, there was increased Cx3cr1, Cx3cl1, Cd68, and Tnf-α gene expression, and increased immunostaining of CX3CR1 and CD68. In hCX3CR1-KI mice treated with hCX3CR1 VHH molecule (KI-A), compared with vehicle controls (KI-V), there was increased lumen vessel area and patency, and decreased neointima in the AVF outflow veins. RNA-seq analysis identified TNF-α and NF-κB as potential targets of CX3CR1 inhibition. In KI-A–treated vessels compared with KI-V, there was decreased gene expression of Tnf- α, Mcp-1, and Il-1 β; with reduction of Cx3cl1, NF-κB, and Cd68; decreased M1, Ly6C, smooth muscle cells, fibroblast-activated protein, fibronectin, and proliferation; and increased TUNEL and M2 staining. In cell culture, monocytes stimulated with PMA and treated with hCX3CR1 VHH had decreased TNF- α, CD68, proliferation, and migration. Conclusions CX3CR1 blockade reduces VNH/VS formation by decreasing proinflammatory cues.

Hyperoxia exposure in premature infants increases the risk of subsequent lung diseases, such as asthma and bronchopulmonary dysplasia. Fibroblasts help maintain bronchial and alveolar integrity. Thus, understanding mechanisms by which hyperoxia influences fibroblasts is critical. Cellular senescence is increasingly recognized as important to the pathophysiology of multiple diseases. We hypothesized that clinically relevant moderate hyperoxia (<50% O2) induces senescence in developing fibroblasts. Using primary human fetal lung fibroblasts, we investigated effects of 40% O2 on senescence, endoplasmic reticulum (ER) stress, and autophagy pathways. Fibroblasts were exposed to 21% or 40% O2 for 7 days with etoposide as a positive control to induce senescence, evaluated by morphological changes, β-galactosidase activity, and DNA damage markers. Senescence-associated secretory phenotype (SASP) profile of inflammatory and profibrotic markers was further assessed. Hyperoxia decreased proliferation but increased cell size. SA-β-gal activity and DNA damage response, cell cycle arrest in G2/M phase, and marked upregulation of phosphorylated p53 and p21 were noted. Reduced autophagy was noted with hyperoxia. mRNA expression of proinflammatory and profibrotic factors (TNF-α, IL-1, IL-8, MMP3) was elevated by hyperoxia or etoposide. Hyperoxia increased several SASP factors (PAI-1, IL1-α, IL1-β, IL-6, LAP, TNF-α). The secretome of senescent fibroblasts promoted extracellular matrix formation by naïve fibroblasts. Overall, we demonstrate that moderate hyperoxia enhances senescence in primary human fetal lung fibroblasts with reduced autophagy but not enhanced ER stress. The resulting SASP is profibrotic and may contribute to abnormal repair in the lung following hyperoxia.

Purpose: To develop a clinically relevant model of percutaneous transluminal angioplasty (PTA) of venous stenosis in mice with arteriovenous fistula (AVF) and test the hypothesis, that there is increased wall shear stress (WSS) after PTA and histologically characterize the vessels. Methods: Twenty C57BL/6J male mice, 6–8 weeks old underwent partial nephrectomy to create CKD. Twenty-eight days later, an AVF was created from the right external jugular vein to the left carotid artery. Fourteen days later, an angioplasty or sham procedure was performed and the mice were sacrificed 14 days later for histologic evaluation to identify the cells contributing to the vascular remodeling ( α -SMA, FSP-1, CD31, CD68), proliferation (Ki-67), cell death (TUNEL), and hypoxia staining (HIF-1 α ). Histomorphometric analysis was performed to assess lumen area, neointima area and cellular density. Ultrasound was performed weekly after creation of the AVF. Results: Venous stenosis occurs 14 days after creation of AVF. PTA treated vessels have significantly higher WSS, average peak systolic velocity, with increased lumen Conclusion: A clinically relevant model of PTA of venous stenosis in mice was created. PTA treated vessels have increased lumen vessel area and wall shear stress. The alterations in tissue markers of vascular remodeling, tissue hypoxia, proliferation, and cell death may be implications for future design of drug and device development.

Reactive airway diseases are significant sources of pulmonary morbidity in neonatal and pediatric patients. Supplemental oxygen exposure in premature infants contributes to airway diseases such as asthma and promotes development of airway remodeling, characterized by increased airway smooth muscle (ASM) mass and extracellular matrix (ECM) deposition. Decreased plasma membrane caveolin-1 (CAV1) expression has been implicated in airway disease and may contribute to airway remodeling and hyperreactivity. Here, we investigated the impact of clinically relevant moderate hyperoxia (50% O 2 ) on airway remodeling and caveolar protein expression in a neonatal mouse model. Within 12 h of birth, litters of B6129SF2J mice were randomized to room air (RA) or 50% hyperoxia exposure for 7 days with or without caveolin-1 scaffolding domain peptide (CSD; caveolin-1 mimic; 10 µl, 0.25 mM daily via intraperitoneal injection) followed by 14 days of recovery in normoxia. Moderate hyperoxia significantly increased airway reactivity and decreased pulmonary compliance at 3 wk. Histologic assessment demonstrated airway wall thickening and increased ASM mass following hyperoxia. RNA from isolated ASM demonstrated significant decreases in CAV1 and cavin-1 in hyperoxia-exposed animals while cavin-3 was increased. Supplementation with intraperitoneal CSD mitigated both the physiologic and histologic changes observed with hyperoxia. Overall, these data show that moderate hyperoxia is detrimental to developing airway and may predispose to airway reactivity and remodeling. Loss of CAV1 is one mechanism through which hyperoxia produces these deleterious effects. Supplementation of CAV1 using CSD or similar analogs may represent a new therapeutic avenue for blunting hyperoxia-induced pulmonary damage in neonates.