Transient spurious intrathecal immunoglobulin synthesis in neurological patients after therapeutic apheresis

Background The analysis of cerebrospinal fluid (CSF) is usually done under steady-state conditions, when proteins (e.g., immunoglobulins) reach diffusion equilibrium between blood and CSF. However, little data has been published on CSF analysis under non-steady-state conditions after therapeutic apheresis. By reducing serum proteins (e.g., immunoglobulins), while leaving CSF unchanged, therapeutic apheresis might cause spuriously altered intrathecal immunoglobulin fractions. Methods Based on the incidental finding of plasma exchange-induced increased intrathecal immunoglobulin fractions in a cohort of 12 unsystematically selected patients with various neurological disorders, we retrospectively investigated CSF results that had been raised during routine diagnostic work-up from 41 consecutive neurological patients (predominantly Guillain-Barré syndrome and autoimmune encephalitis) treated with plasmapheresis or immunoadsorption in a tertiary care university hospital in whom lumbar puncture (LP) was performed after a varying number of treatments of therapeutic apheresis. Results Only when LP was performed 1 day after therapeutic apheresis, spurious quantitative intrathecal immunoglobulin (Ig) synthesis of at least one subclass (IgG, IgA and/or IgM) was found in 68.4 % of the patients, irrespective of the number of treatments, in all age groups and independent of other previous immunotherapies (e.g., steroids). This phenomenon occurred only transiently and was almost always accompanied by an elevation of the IgG index. In one patient, an elevated IgG index was noticed even 2 days after plasmapheresis. Neither quantitative Ig synthesis, nor elevated IgG index was observed when the LP was performed three or more days after therapeutic apheresis. Conclusions Spurious quantitative intrathecal Ig synthesis and increased IgG index are common findings shortly after plasmapheresis or immunoadsorption due to altered serum immunoglobulin levels. Knowledge of this phenomenon is needed for clinicians to prevent false interpretations leading to unnecessary diagnostic and therapeutic procedures. Misdiagnoses can be avoided by considering the characteristic CSF constellation including absence of oligoclonal bands and the close temporal relation to therapeutic apheresi

Human gestational N‐methyl‐d‐aspartate receptor autoantibodies impair neonatal murine brain function

Objective: Maternal autoantibodies are a risk factor for impaired brain development in offspring. Antibodies (ABs) against the NR1 (GluN1) subunit of the N-methyl-d-aspartate receptor (NMDAR) are among the most frequently diagnosed anti-neuronal surface ABs, yet little is known about effects on fetal development during pregnancy. Methods: We established a murine model of in utero exposure to human recombinant NR1 and isotype-matched nonreactive control ABs. Pregnant C57BL/6J mice were intraperitoneally injected on embryonic days 13 and 17 each with 240μg of human monoclonal ABs. Offspring were investigated for acute and chronic effects on NMDAR function, brain development, and behavior. Results: Transferred NR1 ABs enriched in the fetus and bound to synaptic structures in the fetal brain. Density of NMDAR was considerably reduced (up to -49.2%) and electrophysiological properties were altered, reflected by decreased amplitudes of spontaneous excitatory postsynaptic currents in young neonates (-34.4%). NR1 AB-treated animals displayed increased early postnatal mortality (+27.2%), impaired neurodevelopmental reflexes, altered blood pH, and reduced bodyweight. During adolescence and adulthood, animals showed hyperactivity (+27.8% median activity over 14 days), lower anxiety, and impaired sensorimotor gating. NR1 ABs caused long-lasting neuropathological effects also in aged mice (10 months), such as reduced volumes of cerebellum, midbrain, and brainstem. Interpretation: The data collectively support a model in which asymptomatic mothers can harbor low-level pathogenic human NR1 ABs that are diaplacentally transferred, causing neurotoxic effects on neonatal development. Thus, AB-mediated network changes may represent a potentially treatable neurodevelopmental congenital brain disorder contributing to lifelong neuropsychiatric morbidity in affected children

Platform trial design for neurofibromatosis type 1, NF2-related schwannomatosis and non-NF2-related schwannomatosis:A potential model for rare diseases

Background Neurofibromatosis type 1, NF2-related schwannomatosis and non-NF2-related schwannomatosis (grouped under the abbreviation "NF") are rare hereditary tumor predisposition syndromes. Due to the low prevalence, variability in the range, and severity of manifestations, as well as limited treatment options, these conditions require innovative trial designs to accelerate the development of new treatments.Methods Within European Patient-Centric Clinical Trial Platforms (EU-PEARL), we designed 2 platform-basket trials in NF. The trials were designed by a team of multidisciplinary NF experts and trial methodology experts.Results The trial will consist of an observational and a treatment period. The observational period will serve as a longitudinal natural history study. The platform trial design and randomization to a sequence of available interventions allow for the addition of interventions during the trial. If a drug does not meet the predetermined efficacy endpoint or reveals unacceptable toxicities, participants may stop treatment on that arm and re-enter the observational period, where they can be re-randomized to a different treatment arm if eligible. Intervention-specific eligibility criteria and endpoints are listed in intervention-specific-appendices, allowing the flexibility and adaptability needed for highly variable and rare conditions like NF.Conclusions These innovative platform-basket trials for NF may serve as a model for other rare diseases, as they will enhance the chance of identifying beneficial treatments through optimal learning from a small number of patients. The goal of these trials is to identify beneficial treatments for NF more rapidly and at a lower cost than traditional, single-agent clinical trials

Optimizing expert and patient input in pediatric trial design : lessons learned and recommendations from a collaboration between conect4children and European Patient‐CEntric ClinicAl TRial PLatforms

Advice from multiple stakeholders is required to design the optimal pediatric clinical trial. We present recommendations for acquiring advice from trial experts and patients/caregivers, derived from advice meetings that were performed through a collaboration of the Collaborative Network for European Clinical Trials for Children (c4c) and the European Patient‐CEntric ClinicAl TRial PLatforms (EU‐PEARL). Three advice meetings were performed: (1) an advice meeting for clinical and methodology experts, (2) an advice meeting for patients/caregivers, and (3) a combined meeting with both experts and patients/caregivers. Trial experts were recruited from c4c database. Patients/caregivers were recruited through a patient organization. Participants were asked to provide input on a trial protocol, including endpoints, outcomes, and the assessment schedule. Ten experts, 10 patients, and 13 caregivers participated. The advice meetings resulted in modification of eligibility criteria and outcome measures. We have provided recommendations for the most effective meeting type per protocol topic. Topics with limited options for patient input were most efficiently discussed in expert advice meetings. Other topics benefit from patient/caregiver input, either through a combined meeting with experts or a patients/caregivers‐only advice meeting. Some topics, such as endpoints and outcome measures, are suitable for all meeting types. Combined sessions profit from synergy between experts and patients/caregivers, balancing input on protocol scientific feasibility and acceptability. Both experts and patients/caregivers provided critical input on the presented protocol. The combined meeting was the most effective methodology for most protocol topics. The presented methodology can be used effectively to acquire expert and patient feedback

Aberrant phase separation and nucleolar dysfunction in rare genetic diseases

Thousands of genetic variants in protein-coding genes have been linked to disease. However, the functional impact of most variants is unknown as they occur within intrinsically disordered protein regions that have poorly defined functions1-3. Intrinsically disordered regions can mediate phase separation and the formation of biomolecular condensates, such as the nucleolus4,5. This suggests that mutations in disordered proteins may alter condensate properties and function6-8. Here we show that a subset of disease-associated variants in disordered regions alter phase separation, cause mispartitioning into the nucleolus and disrupt nucleolar function. We discover de novo frameshift variants in HMGB1 that cause brachyphalangy, polydactyly and tibial aplasia syndrome, a rare complex malformation syndrome. The frameshifts replace the intrinsically disordered acidic tail of HMGB1 with an arginine-rich basic tail. The mutant tail alters HMGB1 phase separation, enhances its partitioning into the nucleolus and causes nucleolar dysfunction. We built a catalogue of more than 200,000 variants in disordered carboxy-terminal tails and identified more than 600 frameshifts that create arginine-rich basic tails in transcription factors and other proteins. For 12 out of the 13 disease-associated variants tested, the mutation enhanced partitioning into the nucleolus, and several variants altered rRNA biogenesis. These data identify the cause of a rare complex syndrome and suggest that a large number of genetic variants may dysregulate nucleoli and other biomolecular condensates in humans.© 2023. The Author(s)

Cellbased assay for detection of NMDA-receptor-antibodies

Die Anti-N-Methyl-D-Aspartat-Rezeptor (NMDAR)-Encephalitis wurde 2007 erstmals beschrieben und ist eine der häufigsten Ursachen für eine Encephalitis. Im Unterschied zu anderen Formen autoimmuner Encephalitiden folgt die Anti-NMDAR- Encephalitis häufig einem rasanten und schweren Verlauf einhergehend mit Bewusstseinseintrübung, epileptischen Anfällen, autonomer Dysregulation und Hypopnoe bis hin zu Intensivpflichtigkeit mit teils tödlichem Ausgang. Die Diagnostik erfolgt bei typischer Klinik durch den spezifischen Nachweis der pathogenen Antikörper aus Liquor oder Serum gegen den NMDA-Rezeptor. Eine Vorhersage der Krankheitsschwere ist derzeit nicht möglich, wäre aber für die frühzeitige und angemessene Behandlung sehr hilfreich. Eine denkbare Rolle für die Prognose könnte die Affinität der NMDAR-Antikörper spielen. Daher entwickelten wir zellbasierte Testverfahren, mit denen sich geringe Antikörpertiter mit hoher Sensitivität nachweisen lassen und ein Vergleich der Bindungsaffinitäten von NMDAR-Antikörpern unterschiedlicher Patienten in Folgeuntersuchungen möglich ist. Zu diesem Zweck wurde die cDNA der NR1-Untereinheit des humanen NMDA-Rezeptors mittels Polymerase-Kettenreaktion amplifiziert, in verschiedene Vektoren kloniert und aus Escheria coli aufgereinigt. Im Folgenden wurden transient transfizierte, und stabile NMDAR- exprimierende HEK-Zelllinien entwickelt. Hiermit konnten in der indirekten Immunfluoreszenzfärbung humane NMDAR-Antikörper aus Liquor und Serum von Patienten in deutlich niedrigerer Konzentration als mit kommerziell verfügbaren Assays nachgewiesen werden. Eine Verwendung der transfizierten HEK-Zellen in der Durchflusszytometrie ermöglichte eine exakte Quantifizierung der Antikörpertiter, welches zur Prüfung des Behandlungserfolges genutzt werden könnte und somit zur Verlaufskontrolle geeignet wäre. Weiterhin koppelten wir Liquorantikörper von Patienten mit dem Fluorochrom Alexa 594. Dies ermöglichte humane NMDAR-Antikörper auch ohne Zweitantikörper nachzuweisen. Weiterhin konnten mit diesem Verfahren erstmals nicht-humane NMDAR-Liquorantikörper detektiert werden. Die post mortem Liquoranalyse eines jungen Eisbären (Ursus maritimus) erbrachte den Nachweis von hochtitrigen Antikörpern gegen die NR1-Untereinheit des NMDA-Rezeptors. Diese Antikörper zeigten in der Immunhistochemie ein typisches neuropiles Verteilungsmuster in Hippocampus und Kleinhirn, wie es bei menschlichen Patienten mit Anti-NMDAR- Encephalitis typisch ist. In Übereinstimmung mit der Klinik, insbesondere epileptischen Anfällen, und dem histopathologischen Nachweis einer floriden Encephalitis mit Plasmazellinfiltration lässt dies darauf schließen, dass dieser Eisbär an einer Anti-NMDAR-Encephalitis gestorben ist. Dies ist weltweit der erste dokumentierte nicht-menschliche Fall und legt nahe, dass auch andere Säugetiere von dieser behandelbaren Erkrankung betroffen sein könnten. Autoimmunität gegen neuropile Strukturen könnte somit ein neuer spezies-übergreifender und damit grundlegender Pathomechanismus bei Säugetieren sein.Discovered in 2007, anti-N-Methyl-D-Aspartat receptor (NMDAR) encephalitis is one of the most commonly identified causes for encephalitis. The disease often runs a severe and rapid course showing reduced state of consciousness, epileptic seizures, autonomic dysregulation and hypopnea leading to intensive care unit treatment and even death. Clinical diagnosis is made by detection of pathogenic NMDAR antibodies from a patient’s cerebrospinal fluid (CSF) or serum. Little is known about prognosis factors for the disease severity, but would be helpful for providing early and appropriate treatment. We suppose the affinity of NMDAR antibodies might be an important predicting factor. We developed highly sensitive methods to detect low concentrations of NMDAR antibodies, which can be used to analyze antibody affinity of different patients in future. Therefore, cDNA of the NR1 subunit of the NMDA receptors was amplified by polymerase chain reaction, cloned into different vectors and purified from Escheria coli for transient and stable transfection. Immunofluorescence staining on transfected HEK-cells detected lower concentrations of human NMDAR antibodies than commercial assays and flow cytometry showed exact quantification of the antibody titer, which can be used to monitor clinical progress and evaluate therapeutic success in follow-up examinations. Further CSF was conjugated with Alexa Fluor 594 to detect human and non-human NMDA receptor antibodies without secondary antibodies. The post mortem CSF analysis of a young polar bear (Ursus maritimus) suffering epileptic seizures showed strong binding to HEK cells expressing NMDA receptors. Tissue immunohistochemistry exploration demonstrated a typical neuropil signal in hippocampus and cerebellum as in human patients with NMDAR encephalitis and histopathological examination showed an encephalitis with infiltration of plasma cells. We conclude, death was caused by NMDAR encephalitis. This is the first reported non-human case and suggests, that other mammals might suffer from this treatable disease. Autoimmune response against neuropil structures might be a basic pathomechanism across mammal species

Sexual Well-Being in Adult Male Patients with Congenital Adrenal Hyperplasia

Introduction. Men with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency show impaired fecundity due to testicular adrenal rest tumors and/or suppression of the gonadal axis. Sexual well-being might be an additional factor; however, no data exists. Patients and Methods. Prospective longitudinal monocentric study included 20 male CAH patients (14 salt wasting, 6 simple virilizing; age 18–49 yr). Clinical assessment, testicular ultrasound, biochemical and hormonal parameters, three validated self-assessment questionnaires (SF-36, GBB-24, and HADS), and male Brief Sexual Function Inventory (BSFI) were analyzed at baseline and after two years. Results. Basal LH and testosterone levels suggested normal testicular function. LH and FSH responses to GnRH were more pronounced in patients with a good therapy control according to androstenedione/testosterone ratio < 0.2. This group had significant higher percentage of patients on dexamethasone medication. GBB-24, HADS, and SF-36 showed impaired z-scores and no changes at follow-up. BSFI revealed impairments in dimensions “sexual drive,” “erections,” and “ejaculations,” whereas “problem assessment” and “overall satisfaction” revealed normal z-scores. Androstenedione levels correlated (P=0.036) inversely with z-scores for “sexual drive” with higher levels associated with impaired “sexual drive.” Conclusion. Male CAH patients showed a partly impaired sexual well-being which might be an additional factor for reduced fecundity

Real-time Probing the Formation of [M(2,2−bipyridine)2(NO3)3][M(2,2-bipyridine)_{2}(NO_{3})_{3}] (M=Ce, La, Tb) Complexes and Influence of Synthesis Parameters

Despite the strong technological importance of lanthanide complexes, their formation processes are rarely investigated. This work is dedicated to determining the influence of synthesis parameters on the formation of [Ce(bipy)2(NO3)3] as well as Ce3+‐ and Tb3+‐substituted [La(bipy)2(NO3)3] (bipy = 2,2′‐bipyridine) complexes. To this end, we performed in situ luminescence measurements, synchrotron‐based X‐ray diffraction (XRD) analysis, infrared spectroscopy (IR), and measured pH value and/or ion conductivity during their synthesis process under real reaction conditions. For the [Ce(bipy)2(NO3)3] complex, the in situ luminescence measurements initially presented a broad emission band at 490 nm, assigned to the 5d→4f Ce3+ ions within the ethanolic solvation shell. Upon the addition of bipy, a red shift to 700 nm was observed. This shift was attributed to the changes in the environment of the Ce3+ ions, indicating their desolvation and incorporation into the [Ce(bipy)2(NO3)3] complex. The induction time was reduced from 8 to 3.5 min, by increasing the reactant concentration by threefold. In contrast, [La(bipy)2(NO3)3] crystallized within days instead of minutes, unless influenced by high Ce3+ and Tb3+ concentrations. Monitoring and controlling the influence of the reaction parameters on the structure of emissive complexes is important for the development of rational synthesis approaches and optimization of their structure‐related properties like luminescence

Solvent Impact on the Properties of Benchmark Metal–Organic Frameworks: Acetonitrile‐Based Synthesis of CAU‐10, Ce‐UiO‐66, and Al‐MIL‐53

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