Detection of High-Risk Paraneoplastic Antibodies against TRIM9 and TRIM67 Proteins.

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.

Autoimmune Cerebellar Ataxia Associated with Anti-Glutamate Receptor δ2 Antibodies: a Rare but Treatable Entity.

We report two novel cases of autoimmune cerebellar ataxia (ACA) associated with anti-glutamate receptor δ2 antibodies (Gluδ2-Abs). The first case was confirmed by indirect immunofluorescence and cell-based assays: a 29-year-old woman presented after 5 days of headache and vomiting, a pancerebellar syndrome, downbeat nystagmus, decreased visual acuity linked to bilateral retrobulbar optic neuritis (RON), and lymphocytic pleocytosis in the cerebrospinal fluid (CSF) without any abnormality detected using cerebral magnetic resonance imaging (MRI). Second-line immunotherapy allowed progressive clinical improvement, with full recovery achieved after a 4-year follow-up. Thereafter, we retrospectively tested Gluδ2-Abs in 350 patients with a suspicion of autoimmune encephalitis without characterized autoantibody. We identified a second case, a 12-year-old boy who developed 10 days after a respiratory infection, a static cerebellar syndrome with lymphocytosis in the CSF, and right cerebellum hyperintensity in MRI. Five days of corticosteroid treatment allowed a quick clinical improvement. No tumor was identified in both cases, whereas laboratory analyses revealed autoimmune stigma. The present cases suggested that ACA associated with Gluδ2-Abs is an extremely rare but treatable disease. Therefore, testing for Gluδ2-Abs might be considered in the setting of suspected ACA and no initial antibody identification. The visual deficits and ocular motility abnormalities observed in the first reported case might be part of the clinical spectrum of Gluδ2-Abs ACA. Young age, infectious prodromes, lymphocytic pleocytosis, and autoimmune background usually appear together with this syndrome and should lead to discuss the initiation of immunotherapy (after ruling out differential diagnosis, especially infectious causes).

Microglia modulate gliotransmission through the regulation of VAMP2 proteins in astrocytes.

Vesicular release is one of the release mechanisms of various signaling molecules. In neurons, the molecular machinery involved in vesicular release has been designed through evolution to trigger fast and synchronous release of neurotransmitters. Similar machinery with a slower kinetic and a slightly different molecular assembly allows astrocytes to release various transmitters such as adenosine triphosphate (ATP), glutamate, and D-serine. Astrocytes are important modulators of neurotransmission through gliotransmitter release. We recently demonstrated that microglia, another type of glia, release ATP to modulate synaptic transmission using astrocytes as intermediate. We now report that microglia regulate astrocytic gliotransmission through the regulation of SNARE proteins in astrocytes. Indeed, we found that gliotransmission triggered by P2Y1 agonist is impaired in slices from transgenic mice devoid of microglia. Using total internal reflection fluorescence imaging, we found that the vesicular release of gliotransmitter by astrocytes was different in cultures lacking microglia compared to vesicular release in astrocytes cocultured with microglia. Quantification of the kinetic of vesicular release indicates that the overall release appears to be faster in pure astrocyte cultures with more vesicles close to the membrane when compared to astrocytes cocultured with microglia. Finally, biochemical investigation of SNARE protein expression indicates an upregulation of VAMP2 in absence of microglia. Altogether, these results indicate that microglia seems to be involved in the regulation of an astrocytic phenotype compatible with proper gliotransmission. The mechanisms described in this study could be of importance for central nervous system diseases where microglia are activated.

TRIM9 and TRIM67 Are New Targets in Paraneoplastic Cerebellar Degeneration.

To describe autoantibodies (Abs) against tripartite motif-containing (TRIM) protein 9 and 67 in two patients with paraneoplastic cerebellar degeneration (PCD) associated with lung adenocarcinoma. Abs were characterized using immunohistochemistry, Western blotting, cultures of murine cortical, and hippocampal neurons, immunoprecipitation, mass spectrometry, knockout mice for Trim9 and 67, and cell-based assay. Control samples included sera from 63 patients with small cell lung cancer without any paraneoplastic neurological syndrome, 36 patients with lung adenocarcinoma and PNS, CSF from 100 patients with autoimmune encephalitis, and CSF from 165 patients with neurodegenerative diseases. We found Abs targeting TRIM9 and TRIM67 at high concentration in the serum and the cerebrospinal fluid (CSF) of a 78-year-old woman and a 65-year-old man. Both developed subacute severe cerebellar ataxia. Brain magnetic resonance imaging found no abnormality and no cerebellar atrophy. Both had CSF inflammation with mild pleiocytosis and a few oligoclonal bands. We identified a pulmonary adenocarcinoma, confirming the paraneoplastic neurological syndrome in both patients. They received immunomodulatory and cancer treatments without improvement of cerebellar ataxia, even though both were in remission of their cancer (for more than 10 years in one patient). Anti-TRIM9 and anti-TRIM67 Abs were specific to these two patients. All control serum and CSF samples tested were negative for anti-TRIM9 and 67. Anti-TRIM9 and anti-TRIM67 Abs appeared to be specific biomarkers of PCD and should be added to the panel of antigens tested when this is suspected.

Neurological autoimmunity in melanoma patients: a comparison between those exposed and non-exposed to immune checkpoint inhibitors.

BACKGROUND: The clinical spectrum of melanoma-associated neurological autoimmunity, whether melanoma-associated paraneoplastic neurological syndromes (PNS) or induced by immune checkpoint inhibitors (ICI), is not well characterized. We aim to describe the clinical spectrum of melanoma-associated neurological autoimmunity. METHODS: A systematic review of the literature combined with patients from French databases of paraneoplastic neurological syndromes was conducted. All melanoma patients with a possible immune-mediated neurologic syndrome were included and classified according to whether they had previously been exposed to ICI (ICI-neurotoxicity) or not (ICI-naïve) at first neurological symptoms. RESULTS: Seventy ICI-naïve (literature: n = 61) and 241 ICI-neurotoxicity patients (literature: n = 180) were identified. Neuromuscular manifestations predominated in both groups, but peripheral neuropathies were more frequent in ICI-neurotoxicity patients (39.4% vs 21.4%, p = 0.005) whereas myositis was more frequent in ICI-naïve patients (42.9% vs 18.7%, p < 0.001). ICI-naïve patients had also more frequent central nervous system (CNS) involvement (35.7% vs 23.7%, p = 0.045), classical paraneoplastic syndrome (25.7% vs 5.8%, p < 0.001), and more frequently positive for anti-neuron antibodies (24/32, 75.0% vs 38/90, 42.2%, p = 0.001). Although more ICI-neurotoxicity patients died during the acute phase (22/202, 10.9% vs 1/51, 2.0%, p = 0.047), mostly myositis patients (14/22, 63.6%), mortality during follow-up was higher in ICI-naïve patients (58.5% vs 29.8%, p < 0.001). There was no significant difference in the frequency of life independence (mRS ≤ 2) in the surviving patients in both groups (95.5% vs 91.0%, p = 0.437). CONCLUSIONS: Melanoma-associated PNS appear remarkably rare. The clinical similarities observed in neurological autoimmunity between ICI-treated and ICI-naïve patients, characterized predominantly by demyelinating polyradiculoneuropathy and myositis, suggest a potential prior immunization against melanoma antigens contributing to ICI-related neurotoxicity.

Phospholipases of mineralization competent cells and matrix vesicles: roles in physiological and pathological mineralizations.

The present review aims to systematically and critically analyze the current knowledge on phospholipases and their role in physiological and pathological mineralization undertaken by mineralization competent cells. Cellular lipid metabolism plays an important role in biological mineralization. The physiological mechanisms of mineralization are likely to take place in tissues other than in bones and teeth under specific pathological conditions. For instance, vascular calcification in arteries of patients with renal failure, diabetes mellitus or atherosclerosis recapitulates the mechanisms of bone formation. Osteoporosis-a bone resorbing disease-and rheumatoid arthritis originating from the inflammation in the synovium are also affected by cellular lipid metabolism. The focus is on the lipid metabolism due to the effects of dietary lipids on bone health. These and other phenomena indicate that phospholipases may participate in bone remodelling as evidenced by their expression in smooth muscle cells, in bone forming osteoblasts, chondrocytes and in bone resorbing osteoclasts. Among various enzymes involved, phospholipases A1 or A2, phospholipase C, phospholipase D, autotaxin and sphingomyelinase are engaged in membrane lipid remodelling during early stages of mineralization and cell maturation in mineralization-competent cells. Numerous experimental evidences suggested that phospholipases exert their action at various stages of mineralization by affecting intracellular signaling and cell differentiation. The lipid metabolites-such as arachidonic acid, lysophospholipids, and sphingosine-1-phosphate are involved in cell signaling and inflammation reactions. Phospholipases are also important members of the cellular machinery engaged in matrix vesicle (MV) biogenesis and exocytosis. They may favour mineral formation inside MVs, may catalyse MV membrane breakdown necessary for the release of mineral deposits into extracellular matrix (ECM), or participate in hydrolysis of ECM. The biological functions of phospholipases are discussed from the perspective of animal and cellular knockout models, as well as disease implications, development of potent inhibitors and therapeutic interventions.

Anti-LGI1 Encephalitis With Co-occurring IgLON5 Antibodies: Clinical Features and Human Leukocyte Antigen Haplotypes.

OBJECTIVES: Autoimmune encephalitis (AE) with antibodies against LGI1 and IgLON5 are clinically distinctive but share some particularities such as a strong association with specific human leukocyte antigen (HLA) class II alleles. METHODS: We clinically describe a patient with double positivity for LGI1 and IgLON5 antibodies. In addition, we conducted specific immunodepletion with the patient's serum and HLA typing and investigated the presence of serum IgLON5 antibodies in a cohort of 23 anti-LGI1 patients carrying the HLA predisposing for anti-IgLON5 encephalitis. RESULTS: A 70-year-old woman with a history of lymphoepithelial thymoma presented with subacute cognitive impairment and seizures. Investigations included MRI and EEG showing medial temporal involvement, increased CSF protein content, and polysomnography with REM and non-REM motor activity, along with obstructive apnea. Neural antibody testing revealed both LGI1 and IgLON5 antibodies in serum and CSF, and serum immunodepletion ruled out cross-reactivity. The patient carried DRB1*07:01 and DQA1*01:01∼DQB1*05:01, but no other IgLON5-positive case was identified in a cohort of anti-LGI1 patients carrying DQA1*01∼DQB1*05. Nearly full therapeutic response was obtained after intensified immunosuppressive treatment. DISCUSSION: We present a case of anti-LGI1 encephalitis with concomitant IgLON5 antibodies. Co-occurring IgLON5 antibodies in anti-LGI1 encephalitis are exceptional, but may appear in genetically predisposed individuals.

VEGF controls microglial phagocytic response to amyloid-ß.

Microglial cells are well known to be implicated in the pathogenesis of Alzheimer's disease (AD), due to the impaired clearance of amyloid-ß (Aß) protein. In AD, Aß accumulates in the brain parenchyma as soluble oligomers and protofibrils, and its aggregation process further give rise to amyloid plaques. Compelling evidence now indicate that Aß oligomers (Aßo) are the most toxic forms responsible for neuronal and synaptic alterations. Recently, we showed that the Vascular Endothelial Growth Factor (VEGF) counteracts Aßo-induced synaptic alterations and that a peptide derived from VEGF is able to inhibit Aß aggregation process. Moreover, VEGF has been reported to promote microglial chemotaxis to Aß brain deposits. We therefore investigated whether VEGF could influence microglial phagocytic response to Aß, using in vitro and ex vivo models of amyloid accumulation. We report here that VEGF increases Aßo phagocytosis by microglial cells and further characterized the molecular basis of the VEGF effect. VEGF is able to control α-secretase activity in microglial cells, resulting in the increased cleavage of the Triggering Receptor Expressed on Myeloid cells 2 (TREM2), a major microglial Aß receptor. Consistently, the soluble form sTREM2 also increases Aßo phagocytosis by microglial cells. Taken together, these findings propose VEGF as a new regulator of Aß clearance and suggest its potential role in rescuing compromised microglial function in AD.

Anti-AGO1 Antibodies Identify a Subset of Autoimmune Sensory Neuronopathy.

BACKGROUND AND OBJECTIVES: Autoantibodies (Abs) improve diagnosis and treatment decisions of idiopathic neurologic disorders. Recently, we identified Abs against Argonaute (AGO) proteins as potential autoimmunity biomarkers in neurologic disorders. In this study, we aim to reveal (1) the frequency of AGO1 Abs in sensory neuronopathy (SNN), (2) titers and IgG subclasses, and (3) their clinical pattern including response to treatment. METHODS: This retrospective multicentric case/control study screened 132 patients with SNN, 301 with non-SNN neuropathies, 274 with autoimmune diseases (AIDs), and 116 healthy controls (HCs) for AGO1 Abs through ELISA. Seropositive cases were also tested for IgG subclasses, titers, and conformation specificity. RESULTS: AGO1 Abs occurred in 44 patients, comprising significantly more of those with SNN (17/132 [12.9%]) than those with non-SNN neuropathies (11/301 [3.7%]; p = 0.001), those with AIDs (16/274 [5.8%]; p = 0.02), or HCs (0/116; p < 0.0001). Ab titers ranged from 1:100 to 1:100,000. IgG subclass was mainly IgG1, and 11/17 AGO1 Ab-positive SNN (65%) had a conformational epitope. AGO1 Ab-positive SNN was more severe than AGO1 Ab-negative SNN (e.g., SNN score: 12.2 vs 11.0, p = 0.004), and they more frequently and more efficiently responded to immunomodulatory treatments than AGO1 Ab-negative SNN (7/13 [54%] vs 6/37 [16%], p = 0.02). Regarding the type of treatments more precisely, this significant difference was confirmed for the use of IV immunoglobulins (IVIg) but not for steroids or second-line treatments. Multivariate logistic regression adjusted for potential confounders showed that AGO1 Ab positivity was the only predictor of response to treatment (OR 4.93, 1.10-22.24 95% CI, p = 0.03). DISCUSSION: Although AGO Abs are not specific for SNN, based on our retrospective data, they may identify a subset of cases with SNN with more severe features and a possibly better response to IVIg. The significance of AGO1 Abs in clinical practice needs to be explored on a larger series.

Direct determination of phospholipase D activity by infrared spectroscopy.

To determine phospholipase D (PLD) activity, an infrared spectroscopy assay was developed, based on the phosphate vibrational mode of phospholipids such as dimyristoylphophatidylcholine (DMPC), lysophosphatidylglycerol (lysoPG), dipalmitoylphosphatidylethanolamine (DPPE), and lysophosphatidylserine (lysoPS). The phosphate bands served to monitor the hydrolysis rates of phospholipids with PLD. The measurements could be performed within less than 20min with 10µl of buffer containing 2 to 40mM DMPC and 10 to 200ng of Streptomyces chromofuscus PLD (corresponding to 350-7000pmol of DMPC hydrolyzed per minute). The limit of sensitivity was approximately 10ng of PLD at 100mM Tris-HCl (pH 8.0) with 10mM Ca(2+) and 2.5mgml(-1) Triton X-100. Reproducible specific activity of PLD (35±5nmol of hydrolyzed DMPCmin(-1)µg(-1) PLD) measured by the infrared assay remained stable over 50 to 200ng of PLD and over 5 to 40mM DMPC. The feasibility of this assay to determine the hydrolysis rate of other phospholipids such as lysoPG, DPPE, and lysoPS was confirmed. The IC(50) of cobalt (800±200µM), a known S. chromofuscus PLD inhibitor, was measured by means of the infrared assay, demonstrating that this assay can be used to screen PLD activity and/or the specificity of its inhibitors.

Conformation-stabilizing ELISA and cell-based assays reveal patient subgroups targeting three different epitopes of AGO1 antibodies.

Autoantibodies (Abs) are biomarkers for many disease conditions and are increasingly used to facilitate diagnosis and treatment decisions. To guarantee high sensitivity and specificity, the choice of their detection method is crucial. Via cell-based assays, we recently found 21 patients with neurological diseases positive for antibodies against argonaute (AGO), 10 of which having a neuropathy (NP). Here, we established a simple and conformation-sensitive ELISA with the aim to distinguish between AGO1 Abs against conformational epitopes and non-conformational epitopes and to reveal further characteristics of AGO1 antibodies in NP and autoimmune disease (AID). In a retrospective multicenter case/control and observational study, we tested 434 patients with NP, 274 disease controls with AID, and 116 healthy controls (HC) for AGO1 Abs via conformation-stabilizing ELISA. Seropositive patients were also tested for conformation-specificity via comparative denaturing/stabilizing ELISA (CODES-ELISA), CBA positivity, AGO1 titers and IgG subclasses, and AGO2 reactivity. These parameters were statistically compared among different epitope-specific patient groups. We found Abs in 44 patients, including 28/434 (6.5%) NP, 16/274 (5.8%) AID, and 0/116 (0%) HC. Serum reactivity was consistently higher for AGO1 than AGO2. Globally among the 44 AGO1 Abs-positive patients, 42 were also tested in CBA for AGO1 Abs positivity and 15 (35.7%) were positive. Furthermore, 43 were tested for conformation-specificity and 32 (74.4%) bound a conformational epitope. Among the subgroups of highly positive patients (ELISA z-score >14) with sera binding conformational epitopes (n=23), 14 patient sera were also CBA positive and 9 bound a second conformational but CBA-inaccessible epitope. A third, non-conformational epitope was bound by 11/43 (15.6%). Among the epitope-specific patient subgroups, we found significant differences regarding the Abs titers, IgG subclass, and AGO2 reactivity. When comparing AGO1 Abs-positive NP versus AID patients, we found the conformation-specific and CBA inaccessible epitope significantly more frequently in AID patients. We conclude that 1) conformational ELISA was more sensitive than CBA in detecting AGO1 Abs, 2) serum reactivity is higher for AGO1 than for AGO2 at least for NP patients, 3) AGO1 Abs might be a marker-of-interest in 6.5% of NP patients, 4) distinguishing epitopes might help finding different patient subgroups.

Cerebellar Ataxia With Anti-DNER Antibodies: Outcomes and Immunologic Features.

BACKGROUND AND OBJECTIVES: There is no report on the long-term outcomes of ataxia with antibodies against Delta and Notch-like epidermal growth factor-related (DNER). We aimed to describe the clinical-immunologic features and long-term outcomes of patients with anti-DNER antibodies. METHODS: Patients tested positive for anti-DNER antibodies between 2000 and 2020 were identified retrospectively. In those with available samples, immunoglobulin G (IgG) subclass analysis, longitudinal cerebellum volumetry, human leukocyte antigen isotyping, and CSF proteomic analysis were performed. Rodent brain membrane fractionation and organotypic cerebellar slices were used to study DNER cell-surface expression and human IgG binding to the Purkinje cell surface. RESULTS: Twenty-eight patients were included (median age, 52 years, range 19-81): 23 of 28 (82.1%) were male and 23 of 28 (82.1%) had a hematologic malignancy. Most patients (27/28, 96.4%) had cerebellar ataxia; 16 of 28 (57.1%) had noncerebellar symptoms (cognitive impairment, neuropathy, and/or seizures), and 27 of 28 (96.4%) became moderately to severely disabled. Half of the patients (50%) improved, and 32.1% (9/28) had no or slight disability at the last visit (median, 26 months; range, 3-238). Good outcome significantly associated with younger age, milder clinical presentations, and less decrease of cerebellar gray matter volumes at follow-up. No human leukocyte antigen association was identified. Inflammation-related proteins were overexpressed in the patients' CSF. In the rodent brain, DNER was enriched in plasma membrane fractions. Patients' anti-DNER antibodies were predominantly IgG1/3 and bound live Purkinje cells in vitro. DISCUSSION: DNER ataxia is a treatable condition in which nearly a third of patients have a favorable outcome. DNER antibodies bind to the surface of Purkinje cells and are therefore potentially pathogenic, supporting the use of B-cell-targeting treatments.

Clinical and Prognostic Value of Immunogenetic Characteristics in Anti-LGI1 Encephalitis.

OBJECTIVE: Antibodies against leucine-rich glioma-inactivated 1 (LGI1-Abs) characterize a limbic encephalitis (LE) strongly associated with HLA-DRB1*07:01, although some patients lack LGI1-Abs in CSF or do not carry this allele. Whether they represent a different subtype of disease or have different prognoses is unclear. METHODS: Retrospective analysis of clinical features, IgG isotypes, and outcome according to LGI1-Ab CSF positivity and DRB1*07:01 in a cohort of anti-LGI1 LE patients. RESULTS: Patients with LGI1-Abs detected in both CSF and serum (105/134, 78%) were compared with those who were CSF negative (29/134, 22%). Both groups had similar clinical features and serum levels, but CSF-positive patients had shorter diagnostic delay, more frequently hyponatremia, inflammatory CSF, and abnormal MRI (p < 0.05). Human leukocyte antigen (HLA) genotyping was performed in 72/134 (54%) patients and 63/72 (88%) carried DRB1*07:01. Noncarriers (9/72, 12%) were younger, more commonly women, and had less frequently psychiatric and frontal symptoms (p < 0.05). No difference in IgG isotypes according to CSF positivity or HLA was found (p > 0.05). HLA and IgG isotypes were not associated with poor outcome (mRS >2 at last follow-up) in univariate analyses; CSF positivity was only identified as a poor outcome predictor in the multivariate analysis including the complete follow-up, whereas age and female sex also remained when just the first year was considered. CONCLUSIONS: LE without CSF LGI1-Abs is clinically indistinguishable and likely reflects just a lesser LGI1-Ab production. HLA association is sex and age biased and presents clinical particularities, suggesting subtle differences in the immune response. Long-term outcome depends mostly on demographic characteristics and the intensity of the intrathecal synthesis.

Argonaute Autoantibodies as Biomarkers in Autoimmune Neurologic Diseases.

OBJECTIVE: To identify and characterize autoantibodies (Abs) as novel biomarkers for an autoimmune context in patients with central and peripheral neurologic diseases. METHODS: Two distinct approaches (immunoprecipitation/mass spectrometry-based proteomics and protein microarrays) and patients' sera and CSF were used. The specificity of the identified target was confirmed by cell-based assay (CBA) in 856 control samples. RESULTS: Using the 2 methods as well as sera and CSF of patients with central and peripheral neurologic involvement, we identified Abs against the family of Argonaute proteins (mainly AGO1 and AGO2), which were already reported in systemic autoimmunity. AGO-Abs were mostly of immunoglobulin G 1 subclass and conformation dependent. Using CBA, AGO-Abs were detected in 21 patients with a high suspicion of autoimmune neurologic diseases (71.4% were women; median age 57 years) and only in 4/856 (0.5%) controls analyzed by CBA (1 diagnosed with small-cell lung cancer and the other 3 with Sjögren syndrome). Among the 21 neurologic patients identified, the main clinical presentations were sensory neuronopathy (8/21, 38.1%) and limbic encephalitis (6/21, 28.6%). Fourteen patients (66.7%) had autoimmune comorbidities and/or co-occurring Abs, whereas AGO-Abs were the only autoimmune biomarker for the remaining 7/21 (33.3%). Thirteen (61.9%) patients were treated with immunotherapy; 8/13 (61.5%) improved, and 3/13 (23.1%) remained stable, suggesting an efficacy of these treatments. CONCLUSIONS: AGO-Abs might be potential biomarkers of autoimmunity in patients with central and peripheral nonparaneoplastic neurologic diseases. In 7 patients, AGO-Abs were the only biomarkers; thus, their identification may be useful to suspect the autoimmune character of the neurologic disorder. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that AGO-Abs are more frequent in patients with autoimmune neurologic diseases than controls.

Characteristics in limbic encephalitis with anti-adenylate kinase 5 autoantibodies.

OBJECTIVE: To report 10 patients with limbic encephalitis (LE) and adenylate kinase 5 autoantibodies (AK5-Abs). METHODS: We conducted a retrospective study in a cohort of 50 patients with LE with uncharacterized autoantibodies and identified a specific target using immunohistochemistry, Western blotting, immunoprecipitation, mass spectrometry, and cell-based assay. RESULTS: AK5 (a known autoantigen of LE) was identified as the target of antibodies in the CSFs and sera of 10 patients with LE (median age 64 years; range 57-80), which was characterized by subacute anterograde amnesia without seizure and sometimes preceded by a prodromal phase of asthenia or mood disturbances. Anterograde amnesia can be isolated, but some patients also complained of prosopagnosia, paroxysmal anxiety, or abnormal behavior. No associated cancer was observed. All 10 patients had bilateral hippocampal hypersignal on a brain MRI. CSF analysis generally showed a mild pleiocytosis with elevated immunoglobulin G index and oligoclonal bands, as well as high levels of tau protein with normal concentration of Aß42 and phospho-tau, suggesting a process of neuronal death. Except for one patient, clinical response to immunotherapy was unfavorable, with persistence of severe anterograde amnesia. Two patients evolved to severe cognitive decline. Hippocampal atrophy was observed on control brain MRI. Using in vitro tests on hippocampal neurons, we did not identify clues suggesting a direct pathogenic role of AK5-Abs. CONCLUSIONS: AK5-Abs should be systematically considered in aged patients with subacute anterograde amnesia. Recognition of this disorder is important to develop new treatment strategies to prevent irreversible limbic damage.

Direct determination of phosphatase activity from physiological substrates in cells.

A direct and continuous approach to determine simultaneously protein and phosphate concentrations in cells and kinetics of phosphate release from physiological substrates by cells without any labeling has been developed. Among the enzymes having a phosphatase activity, tissue non-specific alkaline phosphatase (TNAP) performs indispensable, multiple functions in humans. It is expressed in numerous tissues with high levels detected in bones, liver and neurons. It is absolutely required for bone mineralization and also necessary for neurotransmitter synthesis. We provided the proof of concept that infrared spectroscopy is a reliable assay to determine a phosphatase activity in the osteoblasts. For the first time, an overall specific phosphatase activity in cells was determined in a single step by measuring simultaneously protein and substrate concentrations. We found specific activities in osteoblast like cells amounting to 116 ± 13 nmol min(-1) mg(-1) for PPi, to 56 ± 11 nmol min(-1) mg(-1) for AMP, to 79 ± 23 nmol min(-1) mg(-1) for beta-glycerophosphate and to 73 ± 15 nmol min(-1) mg(-1) for 1-alpha-D glucose phosphate. The assay was also effective to monitor phosphatase activity in primary osteoblasts and in matrix vesicles. The use of levamisole--a TNAP inhibitor--served to demonstrate that a part of the phosphatase activity originated from this enzyme. An IC50 value of 1.16 ± 0.03 mM was obtained for the inhibition of phosphatase activity of levamisole in osteoblast like cells. The infrared assay could be extended to determine any type of phosphatase activity in other cells. It may serve as a metabolomic tool to monitor an overall phosphatase activity including acid phosphatases or other related enzymes.