Recurrence of severe symptomatic late-onset neutropenia on ocrelizumab.

BACKGROUND: Late-onset neutropenia (LON), defined as an absolute neutrophil count (ANC) < 1500/mm3 that develops between 4 weeks and 6 months after the last drug administration, is a rare side effect of anti-CD20 drugs including ocrelizumab. Although continuation of ocrelizumab after LON is not contraindicated, the risk of LON recurrence is not well known. CASES: We report three cases of recurrent symptomatic agranulocytosis (ANC < 500/mm3) occurring under ocrelizumab. CONCLUSION: Given the risk of recurrence of symptomatic agranulocytosis and the availability of other treatments, a therapeutic switch may be discussed after the first episode of LON.

Cancer and multiple sclerosis: 2023 recommendations from the French Multiple Sclerosis Society.

BACKGROUND: Epidemiological data reveal that 45% of persons with multiple sclerosis (PwMS) in France are more than 50 years. This population more than 50 is more susceptible to cancer, and this risk may be increased by frequent use of immunosuppressive drugs. Consequently, concerns have arisen about the potential increased risk of cancer in PwMS and how patients should be screened and managed in terms of cancer risk. OBJECTIVE: To develop evidence-based recommendations to manage the coexistence of cancer and multiple sclerosis (MS). METHODS: The French Group for Recommendations in MS collected articles from PubMed and university databases covering the period January 1975 through June 2022. The RAND/UCLA method was employed to achieve formal consensus. MS experts comprehensively reviewed the full-text articles and developed the initial recommendations. A group of multidisciplinary health care specialists then validated the final proposal. RESULTS: Five key questions were addressed, encompassing various topics such as cancer screening before or after initiating a disease-modifying therapy (DMT), appropriate management of MS in the context of cancer, recommended follow-up for cancer in patients receiving a DMT, and the potential reintroduction of a DMT after initial cancer treatment. A strong consensus was reached for all 31 recommendations. CONCLUSION: These recommendations propose a strategic approach to managing cancer risk in PwMS.

Pregnancy and multiple sclerosis: 2022 recommendations from the French multiple sclerosis society.

OBJECTIVE: The objective of this study was to develop evidence-based recommendations on pregnancy management for persons with multiple sclerosis (MS). BACKGROUND: MS typically affects young women in their childbearing years. Increasing evidence is available to inform questions raised by MS patients and health professionals about pregnancy issues. METHODS: The French Group for Recommendations in Multiple Sclerosis (France4MS) reviewed PubMed and university databases (January 1975 through June 2021). The RAND/UCLA appropriateness method was developed to synthesise the scientific literature and expert opinions on healthcare topics; it was used to reach a formal agreement. Fifty-six MS experts worked on the full-text review and initial wording of recommendations. A group of 62 multidisciplinary healthcare specialists validated the final proposal of summarised evidence. RESULTS: A strong agreement was reached for all 104 proposed recommendations. They cover diverse topics, such as pregnancy planning, follow-up during pregnancy and postpartum, delivery routes, locoregional analgesia or anaesthesia, prevention of postpartum relapses, breastfeeding, vaccinations, reproductive assistance, management of relapses and disease-modifying treatments. CONCLUSION: The 2022 recommendations of the French MS society should be helpful to harmonise counselling and treatment practice for pregnancy in persons with MS, allowing for better and individualised choices.

Pregnancy and neuromyelitis optica spectrum disorders: 2022 recommendations from the French Multiple Sclerosis Society.

BACKGROUND: In 2020, the French Multiple Sclerosis (MS) Society (SFSEP) decided to develop a national evidence-based consensus on pregnancy in MS. As neuromyelitis optica spectrum disorders (NMOSD) shares a series of commonalities with MS, but also some significant differences, specific recommendations had to be developed. OBJECTIVES: To establish recommendations on pregnancy in women with NMOSD. METHODS: The French Group for Recommendations in Multiple Sclerosis (France4MS) reviewed PubMed and universities databases (January 1975 through June 2021). The RAND/UCLA appropriateness method, which was developed to synthesise the scientific literature and expert opinions on health care topics, was used to reach a formal agreement. Fifty-six MS experts worked on the full-text review and initial wording of recommendations. A sub-group of nine NMOSD experts was dedicated to analysing available data on NMOSD. A group of 62 multidisciplinary healthcare specialists validated the final proposal of summarised evidence. RESULTS: A strong agreement was reached for all 66 proposed recommendations. They cover diverse topics, such as pregnancy planning, follow-up during pregnancy and postpartum, delivery routes, loco-regional analgesia or anaesthesia, prevention of postpartum relapses, breastfeeding, vaccinations, reproductive assistance, management of relapses, and disease-modifying treatments. CONCLUSION: Physicians and patients should be aware of the new and specific evidence-based recommendations of the French MS Society for pregnancy in women with NMOSD. They should help harmonise counselling and treatment practise, allowing for better individualised choices.

G protein-coupled receptors can control the Hippo/YAP pathway through Gq signaling.

The Hippo pathway is an evolutionarily conserved kinase cascade involved in the control of tissue homeostasis, cellular differentiation, proliferation, and organ size, and is regulated by cell-cell contact, apical cell polarity, and mechanical signals. Miss-regulation of this pathway can lead to cancer. The Hippo pathway acts through the inhibition of the transcriptional coactivators YAP and TAZ through phosphorylation. Among the various signaling mechanisms controlling the hippo pathway, activation of G12/13 by G protein-coupled receptors (GPCR) recently emerged. Here we show that a GPCR, the ghrelin receptor, that activates several types of G proteins, including G12/13, Gi/o, and Gq, can activate YAP through Gq/11 exclusively, independently of G12/13. We revealed that a strong basal YAP activation results from the high constitutive activity of this receptor, which can be further increased upon agonist activation. Thus, acting on ghrelin receptor allowed to modulate up-and-down YAP activity, as activating the receptor increased YAP activity and blocking constitutive activity reduced YAP activity. Our results demonstrate that GPCRs can be used as molecular switches to finely up- or down-regulate YAP activity through a pure Gq pathway.

Nanobody-Based Quantification of GTP-Bound RHO Conformation Reveals RHOA and RHOC Activation Independent from Their Total Expression in Breast Cancer.

As key regulators of the actin cytoskeleton, RHO GTPase expression and/or activity are deregulated in tumorigenesis and metastatic progression. Nevertheless, the vast majority of experiments supporting this conclusion was conducted on cell lines but not on human tumor samples that were mostly studied at the expression level only. Up to now, the activity of RHO proteins remains poorly investigated in human tumors. In this article, we present the development of a robust nanobody-based ELISA assay, with a high selectivity that allows an accurate quantification of RHO protein GTP-bound state in the nanomolar range (1 nM; 20 µg/L), not only in cell lines after treatment but also in tumor samples. Of note, we present here a fine analysis of RHOA-like and RAC1 active state in tumor samples with the most comprehensive study of RHOA-GTP and RHOC-GTP levels performed on human breast tumor samples. We revealed increased GTP-bound RHOA and RHOC protein activities in tumors compared to normal tissue counterparts, and demonstrated that the RHO active state and RHO expression are two independent parameters among different breast cancer subtypes. Our results further highlight the regulation of RHO protein activation in tumor samples and the relevance of directly studying RHO GTPase activities involvement in molecular pathways.

The Study to Investigate COVID-19 Infection in People Living in Ireland (SCOPI): A seroprevalence study, June to July 2020.

BackgroundRobust data on SARS-CoV-2 population seroprevalence supplement surveillance data in providing evidence for public health action.AimTo conduct a SARS-CoV-2 population-based seroprevalence survey in Ireland.MethodsUsing a cross-sectional study design, we selected population samples from individuals aged 12-69 years in counties Dublin and Sligo using the Health Service Executive Primary Care Reimbursement Service database as a sampling frame. Samples were selected with probability proportional to the general population age-sex distribution, and by simple random sampling within age-sex strata. Antibodies to SARS-CoV-2 were detected using the Abbott Architect SARS-CoV-2 IgG Assay and confirmed using the Wantai Assay. We estimated the population SARS-CoV-2 seroprevalence weighted for age, sex and geographic area.ResultsParticipation rates were 30% (913/3,043) and 44% (820/1,863) in Dublin and Sligo. Thirty-three specimens had detectable SARS-CoV-2 antibodies (1.9%). We estimated weighted seroprevalences of 3.12% (95% confidence interval (CI): 2.05-4.53) and 0.58% (95% CI: 0.18-1.38) for Dublin and Sligo, and 1.69% (95% CI: 1.13-2.41) nationally. This equates to an estimated 59,482 (95% CI: 39,772-85,176) people aged 12-69 years nationally having had infection with SARS-CoV-2, 3.0 (95% CI: 2.0-4.3) times higher than confirmed notifications. Ten participants reported a previous laboratory-confirmed SARS-CoV-2 -infection; eight of these were antibody-positive. Twenty-five antibody-positive participants had not reported previous laboratory-confirmed infection.ConclusionThe majority of people in Ireland are unlikely to have been infected with SARS-CoV-2 by June-July 2020. Non-pharmaceutical public health measures remained key pending widespread availability of vaccination, and effective treatments.

An alternative mechanism of early nodal clustering and myelination onset in GABAergic neurons of the central nervous system.

In vertebrates, fast saltatory conduction along myelinated axons relies on the node of Ranvier. How nodes assemble on CNS neurons is not yet fully understood. We previously described that node-like clusters can form prior to myelin deposition in hippocampal GABAergic neurons and are associated with increased conduction velocity. Here, we used a live imaging approach to characterize the intrinsic mechanisms underlying the assembly of these clusters prior to myelination. We first demonstrated that their components can partially preassemble prior to membrane targeting and determined the molecular motors involved in their trafficking. We then demonstrated the key role of the protein ß2Nav for node-like clustering initiation. We further assessed the fate of these clusters when myelination proceeds. Our results shed light on the intrinsic mechanisms involved in node-like clustering prior to myelination and unravel a potential role of these clusters in node of Ranvier formation and in guiding myelination onset.

Clinical, neuropathological, and genetic characterization of STUB1 variants in cerebellar ataxias: a frequent cause of predominant cognitive impairment.

PURPOSE: Pathogenic variants in STUB1 were initially described in autosomal recessive spinocerebellar ataxia type 16 and dominant cerebellar ataxia with cerebellar cognitive dysfunction (SCA48). METHODS: We analyzed a large series of 440 index cerebellar ataxia cases, mostly with dominant inheritance. RESULTS: STUB1 variants were detected in 50 patients. Age at onset and severity were remarkably variable. Cognitive impairment, predominantly frontal syndrome, was observed in 54% of STUB1 variant carriers, including five families with Huntington or frontotemporal dementia disease-like phenotypes associated with ataxia, while no STUB1 variant was found in 115 patients with frontotemporal dementia. We report neuropathological findings of a STUB1 heterozygous patient, showing massive loss of Purkinje cells in the vermis and major loss in the cerebellar hemispheres without atrophy of the pons, hippocampus, or cerebral cortex. This screening of STUB1 variants revealed new features: (1) the majority of patients were women (70%) and (2) "second hits" in AFG3L2, PRKCG, and TBP were detected in three families suggesting synergic effects. CONCLUSION: Our results reveal an unexpectedly frequent (7%) implication of STUB1 among dominantly inherited cerebellar ataxias, and suggest that the penetrance of STUB1 variants could be modulated by other factors, including sex and variants in other ataxia-related genes.

The pharmacologic characterization of allosteric molecules: Gq protein activation.

Context: Drugs such as positive allosteric modulators (PAMs) produce complex behaviors when acting on tissues in different physiological contexts in vivo. Objective: This study describes the use of functional assays of varying receptor sensitivity to unveil the various behaviors of PAMs and thus quantify allosteric effect through system independent scales. Materials and methods: Muscarinic receptor activation with acetylcholine (ACh) was used to the demonstrate activity of the PAM agonist 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, Benzyl quinolone carboxylic acid (BQCA) in terms of direct agonism, potentiation of ACh affinity, and ACh efficacy. Concentration-response curves were fit to the functional allosteric model to yield indices of agonism (τB), effects on affinity (α cooperativity), and efficacy (ß cooperativity). Results: It is shown that a highly sensitive functional assay revealed the direct efficacy of BQCA as an agonist and relatively insensitive cells (produced by chemical alkylation of muscarinic receptor with phenoxybenzamine) revealed a positive allosteric effect of BQCA on ACh efficacy. A wide range of functional assay sensitivities produced a complex pattern of behavior for BQCA all of which was accurately quantified through the system-independent parameters of the functional allosteric model. Conclusions: The study of complex allosteric molecules in a range of functional assays of varying sensitivity allows the measurement of the complete array of activities of these molecules on receptors and also better predicts which will be seen with these in vivo where a range of tissue sensitivities is encountered.

HTS-compatible FRET-based conformational sensors clarify membrane receptor activation.

Cell surface receptors represent a vast majority of drug targets. Efforts have been conducted to develop biosensors reporting their conformational changes in live cells for pharmacological and functional studies. Although Förster resonance energy transfer (FRET) appears to be an ideal approach, its use is limited by the low signal-to-noise ratio. Here we report a toolbox composed of a combination of labeling technologies, specific fluorophores compatible with time-resolved FRET and a novel method to quantify signals. This approach enables the development of receptor biosensors with a large signal-to-noise ratio. We illustrate the usefulness of this toolbox through the development of biosensors for various G-protein-coupled receptors and receptor tyrosine kinases. These receptors include mGlu, GABAB, LH, PTH, EGF and insulin receptors among others. These biosensors can be used for high-throughput studies and also revealed new information on the activation process of these receptors in their cellular environment.

Equilibrium Assays Are Required to Accurately Characterize the Activity Profiles of Drugs Modulating Gq-Protein-Coupled Receptors.

This paper discusses the process of determining the activity of candidate molecules targeting Gq-protein activation through G-protein-coupled receptors for possible therapeutic application with two functional assays; calcium release and inositol phosphate metabolism [inositol monophosphate (IP1)]. While both are suitable for detecting ligand activity (screening), differences are seen when these assays are used to quantitatively measure ligand parameters for therapeutic activity. Specifically, responses for Gq-related pathways present different and dissimulating patterns depending on the functional assay used to assess them. To investigate the impact of functional assays on the accuracy of compound pharmacological profiles, five exemplar molecules [partial agonist, antagonist, inverse agonist, positive allosteric modulator (PAM) agonist, and positive ß-PAM] targeting either muscarinic M1 or ghrelin receptors were tested using two functional assays (calcium release and IP1) and the results were compared with theoretical pharmacological models. The IP1 assay is an equilibrium assay that is able to determine the correct (i.e., internally consistent) pharmacological profiles of all tested compounds. In contrast, the nonequilibrium nature of calcium assays yields misleading classification of most of the tested compounds. Our study suggests that the use of an equilibrium assay, such as IP1, is mandatory for the optimal use of pharmacological models that can both identify mechanisms of action and also convert descriptive-to-predictive data for therapeutic systems. Such assays allow the identification of consistent and simple scales of activity that can guide medicinal chemistry in lead optimization of candidate molecules for therapeutic use.

Rituximab in chronic inflammatory demyelinating polyradiculoneuropathy with associated diseases.

We aimed to analyse the response to rituximab in a cohort of chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) patients with associated disorders. We conducted a clinical and electrophysiological retrospective monocentric study in 28 CIDP patients. Response to rituximab was defined as (a) a five-point increase in the Medical Research Council sum score or a one-point decrease in the Overall Neuropathy Limitations Scale score, compared to the score at the first rituximab infusion, or (b) the discontinuation of, or reduced need for, the last treatments before rituximab initiation. Twenty-one patients (75%) were responders to rituximab. The median time before response was 6 months (1-10 months). Only two patients needed to be treated again during a median follow-up of 2.0 years (0.75-9 years). Interestingly, the response rate was good in patients with associated autoimmune disease (5/8) and similar to the response rate observed in patients with a haematological disease (16/20) (P = 0.63). A shorter disease duration was associated with a better clinical response to rituximab (odds ratio 0.81, P = 0.025) and the response rate was better (P = 0.05) in common forms (83.3%) than in sensory forms (42.9%). No major adverse events were recorded. Rituximab is efficacious in CIDP patients with haematological or autoimmune disease. It improves clinical response and decreases dependence on first-line treatments.

Astrocytic tumor with large cells and worrisome features in two patients with tuberous sclerosis: drastically different diagnoses and prognoses.

INTRODUCTION: Tuberous sclerosis complex (TSC) is a rare autosomal dominant genetic disease, characterized by the development of benign tumors in several organs. During infancy, 6 - 20% of patients develop brain tumors called subependymal giant cell astrocytoma (SEGA). CASE REPORTS: Here we present two patients with TSC, who displayed acute intracranial tumors requiring surgery. Although both tumors shared similar histological aspects with large astrocytic cells and worrisome features, immunohistochemical and genetic analysis successfully distinguished an opposite diagnosis for the two patients.
.

Rapid sensing of circulating ghrelin by hypothalamic appetite-modifying neurons.

To maintain homeostasis, hypothalamic neurons in the arcuate nucleus must dynamically sense and integrate a multitude of peripheral signals. Blood-borne molecules must therefore be able to circumvent the tightly sealed vasculature of the blood-brain barrier to rapidly access their target neurons. However, how information encoded by circulating appetite-modifying hormones is conveyed to central hypothalamic neurons remains largely unexplored. Using in vivo multiphoton microscopy together with fluorescently labeled ligands, we demonstrate that circulating ghrelin, a versatile regulator of energy expenditure and feeding behavior, rapidly binds neurons in the vicinity of fenestrated capillaries, and that the number of labeled cell bodies varies with feeding status. Thus, by virtue of its vascular connections, the hypothalamus is able to directly sense peripheral signals, modifying energy status accordingly.

Evidence of disease activity during pregnancy and post-partum in MS patients treated with high-efficacy therapies.

BACKGROUND: Multiple sclerosis (MS) predominantly affects women of childbearing age. Due to the risk of teratogenicity, women with active multiple sclerosis (MS) who require high-efficacy therapies (HET) may need to discontinue treatment during pregnancy. Fingolimod and Natalizumab withdrawal increases the risk of disease reactivation, a risk not commonly associated with anti-CD20 therapies. However, comparative data are limited during pregnancy and post-partum. Our aim was to compare evidence of disease activity during pregnancy and post-partum in women treated with HET (anti-CD20 therapies, Natalizumab or Fingolimod) before conception, whether or not exposed during pregnancy. METHODS: In this single-center retrospective study, we included consecutive pregnancies of relapsing-remitting MS patients and classified them in three groups according to the last HET used before conception: « anti-CD20 ¼ « Natalizumab (NTZ) ¼ and « Fingolimod (FGD) ¼. The main outcome was annualized relapse rate (ARR) during pregnancy and post-partum. RESULTS: We included 66 pregnancies: 21, 24 and 21 in anti-CD20, NTZ and FGD groups respectively. Overall, mean ARR (SD) increased from 0.36 (0.6) during the preconception year to 0.60 (1.3) during pregnancy and to 1.03 (2.0) in the first 3 months post-partum. Mean ARR in anti-CD20 group (0.09 (0.3)) during pregnancy and the first 3 months post-partum was lower compared with NTZ (0.48 (0.6); p = 0,09) and FGD (1.50 (1.8); p = 0.001) groups. Proportion of pregnancies with radiological activity during pregnancy and post-partum in anti-CD20 group (5.2 %) was lower compared with NTZ (63.1 %; p < 0.001) and FGD (72.2 %; p < 0.001) groups. There was no significant difference in the evolution of EDSS score from conception to post-partum between each group (p = 0.75). CONCLUSION: Evidence of disease activity was significantly lower in patients exposed to anti-CD20 therapies before conception. This study suggests that use of anti-CD20 therapies is an efficient option to prevent disease reactivation during pregnancy and post-partum.

Impact of Immune Checkpoint Inhibitors on the Course of Multiple Sclerosis.

OBJECTIVES: Immune checkpoint inhibitors (ICIs) are increasingly used in cancer treatment. Their mechanism of action raises the question of possible exacerbation of preexisting multiple sclerosis (MS). The aim of our study was to assess the risk of increased MS activity, defined by the occurrence of a relapse and/or a new MRI lesion, after ICI initiation. METHODS: This French multicentric study collected retrospective and prospective data on patients with MS treated with ICIs after a cancer diagnosis. RESULTS: We identified 18 patients with a median age of 48 years. Three of them (17%), all aged 50 years or younger, with a relapsing-remitting course, showed clinical and/or radiologic signs of MS activity 3 to 6 months after ICI initiation. They had stopped disease-modifying treatment (DMT) several months earlier, at the time of cancer diagnosis. Only one had both clinical and MRI activity, with mild severity and complete recovery. DISCUSSION: Our study suggests that the overall risk of MS activity under ICI is low and could be mainly driven by DMT discontinuation, as in MS in general. Although larger studies are needed for better risk assessment in younger patients with more active disease, ICI should be considered when needed in patients with MS.

A single glycine in extracellular loop 1 is the critical determinant for pharmacological specificity of dopamine D2 and D3 receptors.

Subtype-selective agents for the dopamine D3 receptor (D3R) have been considered as potential medications for drug addiction and other neuropsychiatric disorders. Medicinal chemistry efforts have led to the discovery of 4-phenylpiperazine derivatives that are >100-fold selective for the dopamine D3 receptor over dopamine D2 receptor (D2R), despite high sequence identity (78% in the transmembrane domain). Based on the recent crystal structure of D3R, we demonstrated that the 4-phenylpiperazine moiety in this class of D3R-selective compounds binds to the conserved orthosteric binding site, whereas the extended aryl amide moiety is oriented toward a divergent secondary binding pocket (SBP). In an effort to further characterize molecular determinants of the selectivity of these compounds, we modeled their binding modes in D3R and D2R by comparative ligand docking and molecular dynamics simulations. We found that the aryl amide moiety in the SBP differentially induces conformational changes in transmembrane segment 2 and extracellular loop 1 (EL1), which amplify the divergence of the SBP in D3R and D2R. Receptor chimera and site-directed mutagenesis studies were used to validate these binding modes and to identify a divergent glycine in EL1 as critical to D3R over D2R subtype selectivity. A better understanding of drug-dependent receptor conformations such as these is key to the rational design of compounds targeting a specific receptor among closely related homologs, and may also lead to discovery of novel chemotypes that exploit subtle differences in protein conformations.

Design and validation of a homogeneous time-resolved fluorescence-based leptin receptor binding assay.

The pleiotropic cytokine hormone leptin, by activating its receptor OB-R, plays a major role in many biological processes, including energy homeostasis, immune function, and cell survival and proliferation. Abnormal leptin action is associated with obesity, autoimmune diseases, and cancer. The pharmacological characterization of OB-R and the development of synthetic OB-R ligands are still in their infancy because currently available binding assays are not compatible with ligand saturation binding experiments and high-throughput screening (HTS) approaches. We have developed here a novel homogeneous time-resolved fluorescence-based binding assay that overcomes these limitations. In this assay, fluorescently labeled leptin or leptin antagonist binds to the SNAP-tagged OB-R covalently labeled with terbium cryptate (Tb). Successful binding is monitored by measuring the energy transfer between the Tb energy donor and the fluorescently labeled leptin energy acceptor. Ligand binding saturation experiments revealed high-affinity dissociation constants in the subnanomolar range with an excellent signal-to-noise ratio. The assay performed in a 384-well format shows high specificity and reproducibility, making it perfectly compatible with HTS applications to identify new OB-R agonists or antagonists. In addition, fluorescently labeled leptin and SNAP-tagged OB-R will be valuable tools for monitoring leptin and OB-R trafficking in cells and tissues.