Neutrophils insert elastase into hepatocytes to regulate calcium signaling in alcohol-associated hepatitis.

Neutrophil infiltration occurs in a variety of liver diseases, but it is unclear how neutrophils and hepatocytes interact. Neutrophils generally use granule proteases to digest phagocytosed bacteria and foreign substances or neutralize them in neutrophil extracellular traps. In certain pathological states, granule proteases play a destructive role against the host as well. More recently, non-destructive actions of neutrophil granule proteins have been reported, such as modulation of tissue remodeling and metabolism. Here we report a completely different mechanism by which neutrophils act non-destructively, by inserting granules directly into hepatocytes. Specifically, elastase-containing granules were transferred to hepatocytes where elastase selectively degraded intracellular calcium channels to reduce cell proliferation without cytotoxicity. In response, hepatocytes increased expression of serpin E2 and A3, which inhibited elastase activity. Elastase insertion was seen in patient specimens of alcohol-associated hepatitis, and the relationship between elastase-mediated ITPR2 degradation and reduced cell proliferation was confirmed in mouse models. Moreover, neutrophils from patients with alcohol-associated hepatitis were more prone to degranulation and more potent in reducing calcium channel expression than neutrophils from healthy subjects. This non-destructive and reversible action on hepatocytes defines a previously unrecognized role for neutrophils in the transient regulation of epithelial calcium signaling mechanisms.

Burden of liver disease in Brazil, 1996-2022: a retrospective descriptive study of the epidemiology and impact on public healthcare.

Background: Liver disease is a major cause of mortality and morbidity worldwide and its epidemiology depends on the genetic background, exposure to risk factors, access to healthcare and other sociodemographic characteristics. Brazil is a large country with diverse multicultural and ethnic heritages and important socioeconomic inequalities. The burden of liver disease in Brazil, its regions and population is unknown. Methods: We retrieved data from the Unified Health System regarding liver diseases and analyzed the mortality and morbidity from 1996 to 2022 by gender, race/ethnicity, age, region and overall. We calculated the age-specific risk of deaths by liver disease, age-standardization of the data, mean hospitalization and liver transplant-associated costs. Findings: Malignant neoplasm of the liver and intrahepatic bile ducts, alcohol-associated liver disease, fibrosis, and cirrhosis of the liver, other diseases of the liver, hepatic failure, chronic viral hepatitis were identified as the major causes of death and morbidity in Brazil in the period analyzed. The epidemiology of these diseases was diverse, with variations according to geographic regions, gender and race/ethnicity. The major economic burden of liver disease is related to liver transplants, a common outcome of the progression of these diseases. Interpretation: Liver disease in Brazil is a serious issue for the public health system due to the high number of deaths and increasing mortality rate. Our study contributes as a necessary prerequisite for the development of tailored public health policies aimed at mitigating the increasing burden of liver diseases in specific populations and regions. Funding: CNPq, INCT, CAPES, FAPEMIG.

Long-term safety and efficacy of zilucoplan in patients with generalized myasthenia gravis: interim analysis of the RAISE-XT open-label extension study.

Background: Generalized myasthenia gravis (gMG) is a chronic, unpredictable disease associated with high treatment and disease burdens, with a need for more effective and well-tolerated treatments. Objectives: To evaluate the long-term safety, tolerability, and efficacy of zilucoplan in a mild-to-severe, acetylcholine receptor autoantibody-positive (AChR+) gMG population. Design: Ongoing, multicenter, phase III open-label extension (OLE) study. Methods: Eligible patients had completed a qualifying randomized, placebo-controlled phase II or phase III zilucoplan study and received daily, self-administered subcutaneous 0.3 mg/kg zilucoplan. The primary endpoint was incidence of treatment-emergent adverse events (TEAEs). Secondary efficacy endpoints included change from baseline in Myasthenia Gravis Activities of Daily Living (MG-ADL) score. Results: In total, 200 patients enrolled. At the cut-off date (8 September 2022), median (range) exposure to zilucoplan in RAISE-XT was 1.2 (0.11-4.45) years. Mean age at OLE baseline was 53.3 years. A total of 188 (94%) patients experienced a TEAE, with the most common being MG worsening (n = 52, 26%) and COVID-19 (n = 49, 25%). In patients who received zilucoplan 0.3 mg/kg in the parent study, further improvements in MG-ADL score continued through to Week 24 (least squares mean change [95% confidence interval] from double-blind baseline -6.06 [-7.09, -5.03]) and were sustained through to Week 60 (-6.04 [-7.21, -4.87]). In patients who switched from placebo in the parent study, rapid improvements in MG-ADL score were observed at the first week after switching to zilucoplan; further improvements were observed at Week 24, 12 weeks after switching (-6.46 [-8.19, -4.72]), and were sustained through to Week 60 (-6.51 [-8.37, -4.65]). Consistent results were observed in other efficacy endpoints. Conclusion: Zilucoplan demonstrated a favorable long-term safety profile, good tolerability, and sustained efficacy through to Week 60 with consistent benefits in a broad AChR+ gMG population. Additional long-term data will be available in future analyses. Trial registration: ClinicalTrials.gov identifier: NCT04225871 (https://clinicaltrials.gov/ct2/show/NCT04225871).

Mycophenolate and methotrexate are better tolerated than azathioprine in myasthenia gravis.

Azathioprine is recommended as the first-line steroid-sparing immunosuppressive agent for myasthenia gravis. Mycophenolate and methotrexate are often considered as second-line choices despite widespread consensus on their efficacy. We aimed to gather real-world data comparing the tolerability and reasons for discontinuation for these agents, by performing a national United Kingdom survey of side effects and reasons for discontinuation of immunosuppressants in myasthenia gravis. Of 235 patients, 166 had taken azathioprine, 102 mycophenolate, and 40 methotrexate. The most common side effects for each agent were liver dysfunction for azathioprine (23 %), diarrhoea for mycophenolate (14 %), and fatigue for methotrexate (18 %). Women were generally more likely to experience side effects of immunosuppressants. Azathioprine was significantly more likely to be discontinued than mycophenolate and methotrexate due to side effects. There was no significant difference in treatment cessation due to lack of efficacy. This study highlights the significant side-effect burden of treatment for myasthenia gravis. Mechanisms to reduce azathioprine toxicity should be utilised, however mycophenolate and methotrexate appear to be good treatment choices if teratogenicity is not a concern. Women are disadvantaged due to higher frequency of side effects and considerations around pregnancy and breastfeeding. Treatments with improved tolerability are needed.

Improvement of fatigue in generalised myasthenia gravis with zilucoplan.

BACKGROUND: Fatigue is a debilitating symptom of myasthenia gravis (MG). The impact of fatigue on MG can be assessed by Quality of Life in Neurological Disorders (Neuro-QoL) Short Form Fatigue scale. Transformation of raw Neuro-QoL fatigue scores to T-scores is a known approach for facilitating clinical interpretation of clinically meaningful and fatigue severity thresholds. METHODS: In the Phase 3, double-blind, placebo-controlled RAISE study (NCT04115293), adults with acetylcholine receptor autoantibody-positive generalised MG (MG Foundation of America Disease Class II-IV) were randomised 1:1 to daily subcutaneous zilucoplan 0.3 mg/kg or placebo for 12 weeks. Patients completing RAISE could opt to receive zilucoplan 0.3 mg/kg in an ongoing, open-label extension study, RAISE-XT (NCT04225871). In this post-hoc analysis, we evaluated the long-term effect of zilucoplan on fatigue in RAISE patients who entered RAISE-XT. We report change in Neuro-QoL Short Form Fatigue T-scores and fatigue severity levels from RAISE baseline to Week 60. RESULTS: Mean Neuro-QoL Short Form Fatigue T-scores improved from baseline to Week 12 in the zilucoplan group (n = 86) with a clinically meaningful difference versus placebo (n = 88; least squares mean difference: - 3.61 (nominal p-value = 0.0060]), and these improvements continued further to Week 60. At Week 12, more patients on zilucoplan (n = 34, 47.2%) experienced improvements in ≥ 1 fatigue severity level from baseline versus placebo (n = 23, 28.4%; p = 0.017). At Week 60, most (n = 55, 65.5%) patients had mild fatigue or none. CONCLUSION: Treatment with zilucoplan demonstrated statistical and clinically meaningful improvements in fatigue scores and severity versus placebo during RAISE, which were sustained to Week 60 in RAISE-XT.

Pre-germination treatments of melon seeds for the production of seedlings irrigated with biosaline water / Tratamentos pré-germinativos de sementes de melão para a produção de mudas irrigadas com água biossalina

Abstract Melon production in the Brazilian semi-arid region is subject to the use of marginal waters with high salinity. However, the use of regulators and bioactivators in seed treatment can mitigate the harmful effects of salts in irrigation water. In this context, the objective was to evaluate the effect of pre-germination treatments with plant regulators and bioactivator in melon seeds for the production of seedlings irrigated with biosaline water from fish farming effluent. For this, two trials with the Goldex and Grand Prix hybrids were carried out separately. A completely randomized design was used in a 4 × 3 factorial scheme (pre-germination treatments × water dilutions). In addition to the control, the seeds were treated with salicylic and gibberellic acids and thiamethoxam. The waters used for irrigation were local-supply water, fish farming effluent (biosaline water) and these diluted to 50%. Physiological and biochemical analyses were performed for fourteen days. Biosaline water (5.0 dS m-1) did not affect the emergence of Goldex melon seedlings, but compromised the establishment of the Grand Prix cultivar. Seed pre-treatments with salicylic and gibberellic acids attenuate the effects of water salinity and promote growth modulations, resulting in more vigorous melon seedlings.

Resumo A produção de meloeiro no semiárido brasileiro está sujeita a utilização de águas marginais com salinidade elevada. Entretanto, a utilização de reguladores e bioativadores no tratamento de sementes podem mitigar os efeitos nocivos dos sais na água de irrigação. Nesse sentido, objetivou-se avaliar o efeito de tratamentos pré-germinativos com fitorreguladores e bioativador em sementes de melão para a produção de mudas irrigadas com água biossalina de efluente de piscicultura. Para isso, dois ensaios com os híbridos Goldex e Grand Prix foram realizados separadamente. Utilizou-se delineamento inteiramente casualizado em esquema fatorial 4 × 3 (tratamentos pré-germinativos × diluições de água). Além do controle, as sementes foram tratadas com os ácidos salicílico e giberélico, e tiametoxam. As águas utilizadas para irrigação foram a de abastecimento local, efluente de piscicultura (água biossalina) e estas diluídas a 50%. Durante quatorze dias foram realizadas as análises fisiológicas e bioquímicas. A água biossalina (5,0 dS m-1) não afetou a emergência de plântulas de meloeiro Goldex, mas prejudicou o estabelecimento da cultivar Grand Prix. Os pré-tratamentos de sementes com os ácidos salicílico e giberélico atenuam os efeitos da salinidade da água e promovem modulações no crescimento, proporcionando mudas de meloeiro mais vigorosas.

Pre-germination treatments of melon seeds for the production of seedlings irrigated with biosaline water

Abstract Melon production in the Brazilian semi-arid region is subject to the use of marginal waters with high salinity. However, the use of regulators and bioactivators in seed treatment can mitigate the harmful effects of salts in irrigation water. In this context, the objective was to evaluate the effect of pre-germination treatments with plant regulators and bioactivator in melon seeds for the production of seedlings irrigated with biosaline water from fish farming effluent. For this, two trials with the Goldex and Grand Prix hybrids were carried out separately. A completely randomized design was used in a 4 × 3 factorial scheme (pre-germination treatments × water dilutions). In addition to the control, the seeds were treated with salicylic and gibberellic acids and thiamethoxam. The waters used for irrigation were local-supply water, fish farming effluent (biosaline water) and these diluted to 50%. Physiological and biochemical analyses were performed for fourteen days. Biosaline water (5.0 dS m-1) did not affect the emergence of Goldex melon seedlings, but compromised the establishment of the Grand Prix cultivar. Seed pre-treatments with salicylic and gibberellic acids attenuate the effects of water salinity and promote growth modulations, resulting in more vigorous melon seedlings.

Resumo A produção de meloeiro no semiárido brasileiro está sujeita a utilização de águas marginais com salinidade elevada. Entretanto, a utilização de reguladores e bioativadores no tratamento de sementes podem mitigar os efeitos nocivos dos sais na água de irrigação. Nesse sentido, objetivou-se avaliar o efeito de tratamentos pré-germinativos com fitorreguladores e bioativador em sementes de melão para a produção de mudas irrigadas com água biossalina de efluente de piscicultura. Para isso, dois ensaios com os híbridos Goldex e Grand Prix foram realizados separadamente. Utilizou-se delineamento inteiramente casualizado em esquema fatorial 4 × 3 (tratamentos pré-germinativos × diluições de água). Além do controle, as sementes foram tratadas com os ácidos salicílico e giberélico, e tiametoxam. As águas utilizadas para irrigação foram a de abastecimento local, efluente de piscicultura (água biossalina) e estas diluídas a 50%. Durante quatorze dias foram realizadas as análises fisiológicas e bioquímicas. A água biossalina (5,0 dS m-1) não afetou a emergência de plântulas de meloeiro Goldex, mas prejudicou o estabelecimento da cultivar Grand Prix. Os pré-tratamentos de sementes com os ácidos salicílico e giberélico atenuam os efeitos da salinidade da água e promovem modulações no crescimento, proporcionando mudas de meloeiro mais vigorosas.

Adaptation to volumetric compression drives hepatoblastoma cells to an apoptosis-resistant and invasive phenotype.

Liver cancer involves tumor cells rapidly growing within a packed tissue environment. Patient tumor tissues reveal densely packed and deformed cells, especially at tumor boundaries, indicative of physical crowding and compression. It is not well understood how these physical signals modulate tumor evolution and therapeutic susceptibility. Here we investigate the impact of volumetric compression on liver cancer (HepG2) behavior. We find that conditioning cells under a highly compressed state leads to major transcriptional reprogramming, notably the loss of hepatic markers, the epithelial-to-mesenchymal transition (EMT)-like changes, and altered calcium signaling-related gene expression, over the course of several days. Biophysically, compressed cells exhibit increased Rac1-mediated cell spreading and cell-extracellular matrix interactions, cytoskeletal reorganization, increased YAP and ß-catenin nuclear translocation, and dysfunction in cytoplasmic and mitochondrial calcium signaling. Furthermore, compressed cells are resistant to chemotherapeutics and desensitized to apoptosis signaling. Apoptosis sensitivity can be rescued by stimulated calcium signaling. Our study demonstrates that volumetric compression is a key microenvironmental factor that drives tumor evolution in multiple pathological directions and highlights potential countermeasures to re-sensitize therapy-resistant cells. Significance statement: Compression can arise as cancer cells grow and navigate within the dense solid tumor microenvironment. It is unclear how compression mediates critical programs that drive tumor progression and therapeutic complications. Here, we take an integrative approach in investigating the impact of compression on liver cancer. We identify and characterize compressed subdomains within patient tumor tissues. Furthermore, using in vitro systems, we induce volumetric compression (primarily via osmotic pressure but also via mechanical force) on liver cancer cells and demonstrate significant molecular and biophysical changes in cell states, including in function, cytoskeletal signaling, proliferation, invasion, and chemoresistance. Importantly, our results show that compressed cells have impaired calcium signaling and acquire resistance to apoptosis, which can be countered via calcium mobilization.

Nanozymes with Peroxidase-like Activity for Ferroptosis-Driven Biocatalytic Nanotherapeutics of Glioblastoma Cancer: 2D and 3D Spheroids Models.

Glioblastoma (GBM) is the most common primary brain cancer in adults. Despite the remarkable advancements in recent years in the realm of cancer diagnosis and therapy, regrettably, GBM remains the most lethal form of brain cancer. In this view, the fascinating area of nanotechnology has emerged as an innovative strategy for developing novel nanomaterials for cancer nanomedicine, such as artificial enzymes, termed nanozymes, with intrinsic enzyme-like activities. Therefore, this study reports for the first time the design, synthesis, and extensive characterization of innovative colloidal nanostructures made of cobalt-doped iron oxide nanoparticles chemically stabilized by a carboxymethylcellulose capping ligand (i.e., Co-MION), creating a peroxidase-like (POD) nanozyme for biocatalytically killing GBM cancer cells. These nanoconjugates were produced using a strictly green aqueous process under mild conditions to create non-toxic bioengineered nanotherapeutics against GBM cells. The nanozyme (Co-MION) showed a magnetite inorganic crystalline core with a uniform spherical morphology (diameter, 2R = 6-7 nm) stabilized by the CMC biopolymer, producing a hydrodynamic diameter (HD) of 41-52 nm and a negatively charged surface (ZP~-50 mV). Thus, we created supramolecular water-dispersible colloidal nanostructures composed of an inorganic core (Cox-MION) and a surrounding biopolymer shell (CMC). The nanozymes confirmed the cytotoxicity evaluated by an MTT bioassay using a 2D culture in vitro of U87 brain cancer cells, which was concentration-dependent and boosted by increasing the cobalt-doping content in the nanosystems. Additionally, the results confirmed that the lethality of U87 brain cancer cells was predominantly caused by the production of toxic cell-damaging reactive oxygen species (ROS) through the in situ generation of hydroxyl radicals (·OH) by the peroxidase-like activity displayed by nanozymes. Thus, the nanozymes induced apoptosis (i.e., programmed cell death) and ferroptosis (i.e., lipid peroxidation) pathways by intracellular biocatalytic enzyme-like activity. More importantly, based on the 3D spheroids model, these nanozymes inhibited tumor growth and remarkably reduced the malignant tumor volume after the nanotherapeutic treatment (ΔV~40%). The kinetics of the anticancer activity of these novel nanotherapeutic agents decreased with the time of incubation of the GBM 3D models, indicating a similar trend commonly observed in tumor microenvironments (TMEs). Furthermore, the results demonstrated that the 2D in vitro model overestimated the relative efficiency of the anticancer agents (i.e., nanozymes and the DOX drug) compared to the 3D spheroid models. These findings are notable as they evidenced that the 3D spheroid model resembles more precisely the TME of "real" brain cancer tumors in patients than 2D cell cultures. Thus, based on our groundwork, 3D tumor spheroid models might be able to offer transitional systems between conventional 2D cell cultures and complex biological in vivo models for evaluating anticancer agents more precisely. These nanotherapeutics offer a wide avenue of opportunities to develop innovative nanomedicines for fighting against cancerous tumors and reducing the frequency of severe side effects in conventionally applied chemotherapy-based treatments.

Calcium signaling in chemotherapy-induced neuropathy.

Alterations in calcium (Ca2+) signaling is a major mechanism in the development of chemotherapy-induced peripheral neuropathy (CIPN), a side effect caused by multiple chemotherapy regimens. CIPN is associated with numbness and incessant tingling in hands and feet which diminishes quality of life during treatment. In up to 50% of survivors, CIPN is essentially irreversible. There are no approved, disease-modifying treatments for CIPN. The only recourse for oncologists is to modify the chemotherapy dose, a situation that can compromise optimal chemotherapy and impact patient outcomes. Here we focus on taxanes and other chemotherapeutic agents that work by altering microtubule assemblies to kill cancer cells, but also have off-target toxicities. There have been many molecular mechanisms proposed to explain the effects of microtubule-disrupting drugs. In neurons, an initiating step in the off-target effects of treatment by taxane is binding to neuronal calcium sensor 1 (NCS1), a sensitive Ca2+ sensor protein that maintains the resting Ca2+ concentration and dynamically enhances responses to cellular stimuli. The taxane/NCS1 interaction causes a Ca2+ surge that starts a pathophysiological cascade of consequences. This same mechanism contributes to other conditions including chemotherapy-induced cognitive impairment. Strategies to prevent the Ca2+ surge are the foundation of current work.

A systematic literature review to examine the considerations around pregnancy in women of child-bearing age with myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD) or aquaporin 4 neuromyelitis optica spectrum disorder (AQP4+ NMOSD).

BACKGROUND: Aquaporin-4 antibody positive (AQP4+) neuromyelitis optica spectrum disorder (NMOSD) and myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD) are rare autoimmune diseases with overlapping phenotypes. Understanding their clinical manifestation prior to, during and after pregnancy may influence the management of women of child-bearing age (WOCBA) with these diseases. METHODS: This systematic review identified relevant MEDLINE-indexed publications dated between 01 January 2011 and 01 November 2021, and congress materials from key conferences between 01 January 2019 and 01 November 2021. These were manually assessed for relevance to AQP4+ NMOSD and/or MOGAD in WOCBA, with selected data extracted and considered. RESULTS: In total, 107 articles were retrieved and reviewed for relevancy, including 65 clinical studies. Limited evidence was found regarding a conclusive impact of either disease on female fertility, sexual function or menarche, and impact on maternal outcomes requires further investigation in both conditions to establish risk for pre-eclampsia, gestational diabetes and other complications relative to the general population. Collated data for pregnancy outcomes show clear risks in AQP4+ NMOSD to healthy delivery and a rise in annualised relapse rate postpartum that may require adaptation of treatment regimens. Disease activity appears to be attenuated during pregnancy in MOGAD patients with an increased risk of relapse during the postpartum months, but strong conclusions cannot be made due to a paucity of available data. CONCLUSIONS: This review brings together the literature on AQP4+ NMOSD and MOGAD in WOCBA. The potential impact of pregnancy and the postpartum period on disease activity suggest a proactive management strategy early on may improve maternal and infant outcomes, but more clinical data are needed, particularly for MOGAD.

Discussing the possibility of fertility treatment being unsuccessful as part of routine care offered at clinics: patients' experiences, willingness, and preferences.

STUDY QUESTION: Are patients willing to discuss the possibility of treatment being unsuccessful as part of routine care offered at clinics, and what are the factors associated with this willingness? SUMMARY ANSWER: Nine in every 10 patients are willing to discuss this possibility as part of routine care, with willingness being associated with higher perceived benefits, lower barriers, and stronger positive attitudes towards it. WHAT IS KNOWN ALREADY: Fifty-eight percent of patients who complete up to three cycles of IVF/ICSI in the UK do not achieve a live birth. Offering psychosocial care for unsuccessful fertility treatment (PCUFT), defined as assistance and guidance on the implications of treatment being unsuccessful, could reduce the psychosocial distress patients experience when it happens, and promote positive adjustment to this loss. Research shows 56% of patients are willing to plan for an unsuccessful cycle, but little is known about their willingness and preferences towards discussing the possibility of definitive unsuccessful treatment. STUDY DESIGN, SIZE, DURATION: The study was of cross-sectional design, comprising a theoretically driven and patient-centred bilingual (English, Portuguese) mixed-methods online survey. The survey was disseminated via social media (April 2021-January 2022). Eligibility criteria included being aged 18 or older, waiting to or undergoing an IVF/ICSI cycle, or having completed a cycle within the previous 6 months without achieving a pregnancy. Out of 651 people accessing the survey, 451 (69.3%) consented to participate. From these, 100 did not complete 50% of the survey questions, nine did not report on the primary outcome variable (willingness), and 342 completed the survey (completion rate 75.8%, 338 women). PARTICIPANTS/MATERIALS, SETTING, METHODS: The survey was informed by the Health Belief Model (HBM) and Theory of Planned Behaviour (TPB). Quantitative questions covered sociodemographic characteristics and treatment history. Quantitative and qualitative questions gathered data on past experiences, willingness, and preferences (with whom, what, how and when) to receive PCUFT, as well as theory-informed factors hypothesized to be associated with patients' willingness to receive it. Descriptive and inferential statistics were used on quantitative data about PCUFT experiences, willingness, and preferences, and thematic analysis was applied to textual data. Two logistic regressions were used to investigate the factors associated with patients' willingness. MAIN RESULTS AND THE ROLE OF CHANCE: Participants were, on average, 36 years old and most resided in Portugal (59.9%) and the UK (38.0%). The majority (97.1%) were in a relationship for around 10 years, and 86.3% were childless. Participants were undergoing treatment for, on average, 2 years [SD = 2.11, range: 0-12 years], with most (71.8%) having completed at least one IVF/ICSI cycle in the past, almost all (93.5%) without success. Around one-third (34.9%) reported having received PCUFT. Thematic analysis showed participants received it mainly from their consultant. The main topic discussed was patients' low prognosis, with the emphasis being put on achieving a positive outcome. Almost all participants (93.3%) would like to receive PCUFT. Reported preferences indicated that 78.6% wanted to receive it from a psychologist/psychiatrist/counsellor, mostly in case of a bad prognosis (79.4%), emotional distress (73.5%), or difficulties in accepting the possibility of treatment being unsuccessful (71.2%). The preferred time to receive PCUFT was before initiating the first cycle (73.3%), while the preferred format was in an individual (mean = 6.37, SD = 1.17; in 1-7 scale) or couple (mean = 6.34, SD = 1.24; in 1-7 scale) session. Thematic analysis showed participants would like PCUFT to provide an overview of treatment and all possible outcomes tailored to each patient's circumstances and to encompass psychosocial support, mainly focused on coping strategies to process loss and sustain hope towards the future. Willingness to receive PCUFT was associated with higher perceived benefit of building psychosocial resources and coping strategies (odds ratios (ORs) 3.40, 95% CI 1.23-9.38), lower perceived barrier of triggering negative emotions (OR 0.49, 95% CI 0.24-0.98), and stronger positive attitudes about PCUFT being beneficial and useful (OR 3.32, 95% CI 2.12-5.20). LIMITATIONS, REASONS FOR CAUTION: Self-selected sample, mainly composed of female patients who had not yet achieved their parenthood goals. The small number of participants unwilling to receive PCUFT reduced statistical power. The primary outcome variable was intentions, and research shows a moderate association between intentions and actual behaviour. WIDER IMPLICATIONS OF THE FINDINGS: Fertility clinics should provide patients with early opportunities to discuss the possibility of their treatment being unsuccessful as part of routine care. PCUFT should focus on minimizing suffering associated with grief and loss by reassuring patients they can cope with any treatment outcome, promoting coping resources, and signposting to additional support. STUDY FUNDING/COMPETING INTEREST(S): M.S.-L. holds a doctoral fellowship from the Portuguese Foundation for Science and Technology, I.P. [Fundação para a Ciência e a Tecnologia] (FCT; SFRH/BD/144429/2019). R.C. holds a post-doctoral fellowship supported by the European Social Fund (ESF) and FCT (SFRH/BPD/117597/2016). The EPIUnit, ITR and CIPsi (PSI/01662) are also financed by FCT through the Portuguese State Budget, in the scope of the projects UIDB/04750/2020, LA/P/0064/2020 and UIDB/PSI/01662/2020, respectively. Dr Gameiro reports consultancy fees from TMRW Life Sciences and Ferring Pharmaceuticals A/S, speaker fees from Access Fertility, SONA-Pharm LLC, Meridiano Congress International and Gedeon Richter, grants from Merck Serono Ltd, an affiliate of Merck KgaA, Darmstadt, Germany. TRIAL REGISTRATION NUMBER: N/A.

International Delphi Consensus on the Management of AQP4-IgG+ NMOSD: Recommendations for Eculizumab, Inebilizumab, and Satralizumab.

BACKGROUND AND OBJECTIVES: Neuromyelitis optica spectrum disorder (NMOSD) is a rare debilitating autoimmune disease of the CNS. Three monoclonal antibodies were recently approved as maintenance therapies for aquaporin-4 immunoglobulin G (AQP4-IgG)-seropositive NMOSD (eculizumab, inebilizumab, and satralizumab), prompting the need to consider best practice therapeutic decision-making for this indication. Our objective was to develop validated statements for the management of AQP4-IgG-seropositive NMOSD, through an evidence-based Delphi consensus process, with a focus on recommendations for eculizumab, inebilizumab, and satralizumab. METHODS: We recruited an international panel of clinical experts in NMOSD and asked them to complete a questionnaire on NMOSD management. Panel members received a summary of evidence identified through a targeted literature review and provided free-text responses to the questionnaire based on both the data provided and their clinical experience. Responses were used to generate draft statements on NMOSD-related themes. Statements were voted on over a maximum of 3 rounds; participation in at least 1 of the first 2 rounds was mandatory. Panel members anonymously provided their level of agreement (6-point Likert scale) on each statement. Statements that failed to reach a predefined consensus threshold (≥67%) were revised based on feedback and then voted on in the next round. Final statements were those that met the consensus threshold (≥67%). RESULTS: The Delphi panel comprised 24 experts, who completed the Delphi process in November 2021 after 2 voting rounds. In round 1, 23/25 statements reached consensus and were accepted as final. The 2 statements that failed to reach consensus were revised. In round 2, both revised statements reached consensus. Twenty-five statements were agreed in total: 11 on initiation of or switching between eculizumab, inebilizumab, and satralizumab; 3 on monotherapy/combination therapy; 7 on safety and patient population considerations; 3 on biomarkers/patient-reported outcomes; and 1 on research gaps. DISCUSSION: An established consensus method was used to develop statements relevant to the management of AQP4-IgG-seropositive NMOSD. These international statements will be valuable for informing individualized therapeutic decision-making and could form the basis for standardized practice guidelines.

The emerging spectrum of fetal acetylcholine receptor antibody-related disorders (FARAD).

In utero exposure to maternal antibodies targeting the fetal acetylcholine receptor isoform (fAChR) can impair fetal movement, leading to arthrogryposis multiplex congenita (AMC). Fetal AChR antibodies have also been implicated in apparently rare, milder myopathic presentations termed fetal acetylcholine receptor inactivation syndrome (FARIS). The full spectrum associated with fAChR antibodies is still poorly understood. Moreover, since some mothers have no myasthenic symptoms, the condition is likely underreported, resulting in failure to implement effective preventive strategies. Here we report clinical and immunological data from a multicentre cohort (n = 46 cases) associated with maternal fAChR antibodies, including 29 novel and 17 previously reported with novel follow-up data. Remarkably, in 50% of mothers there was no previously established myasthenia gravis (MG) diagnosis. All mothers (n = 30) had AChR antibodies and, when tested, binding to fAChR was often much greater than that to the adult AChR isoform. Offspring death occurred in 11/46 (23.9%) cases, mainly antenatally due to termination of pregnancy prompted by severe AMC (7/46, 15.2%), or during early infancy, mainly from respiratory failure (4/46, 8.7%). Weakness, contractures, bulbar and respiratory involvement were prominent early in life, but improved gradually over time. Facial (25/34; 73.5%) and variable peripheral weakness (14/32; 43.8%), velopharyngeal insufficiency (18/24; 75%) and feeding difficulties (16/36; 44.4%) were the most common sequelae in long-term survivors. Other unexpected features included hearing loss (12/32; 37.5%), diaphragmatic paresis (5/35; 14.3%), CNS involvement (7/40; 17.5%) and pyloric stenosis (3/37; 8.1%). Oral salbutamol used empirically in 16/37 (43.2%) offspring resulted in symptom improvement in 13/16 (81.3%). Combining our series with all previously published cases, we identified 21/85 mothers treated with variable combinations of immunotherapies (corticosteroids/intravenous immunoglobulin/plasmapheresis) during pregnancy either for maternal MG symptom control (12/21 cases) or for fetal protection (9/21 cases). Compared to untreated pregnancies (64/85), maternal treatment resulted in a significant reduction in offspring deaths (P < 0.05) and other complications, with treatment approaches involving intravenous immunoglobulin/ plasmapheresis administered early in pregnancy most effective. We conclude that presentations due to in utero exposure to maternal (fetal) AChR antibodies are more common than currently recognized and may mimic a wide range of neuromuscular disorders. Considering the wide clinical spectrum and likely diversity of underlying mechanisms, we propose 'fetal acetylcholine receptor antibody-related disorders' (FARAD) as the most accurate term for these presentations. FARAD is vitally important to recognize, to institute appropriate management strategies for affected offspring and to improve outcomes in future pregnancies. Oral salbutamol is a symptomatic treatment option in survivors.

Safety and efficacy of zilucoplan in patients with generalised myasthenia gravis (RAISE): a randomised, double-blind, placebo-controlled, phase 3 study.

BACKGROUND: Generalised myasthenia gravis is a chronic, unpredictable, and debilitating rare disease, often accompanied by high treatment burden and with an unmet need for more efficacious and well tolerated treatments. Zilucoplan is a subcutaneous, self-administered macrocyclic peptide complement C5 inhibitor. We aimed to assess safety, efficacy, and tolerability of zilucoplan in patients with acetylcholine receptor autoantibody (AChR)-positive generalised myasthenia gravis. METHODS: RAISE was a randomised, double-blind, placebo-controlled, phase 3 trial that was done at 75 sites in Europe, Japan, and North America. We enrolled patients (aged 18-74 years) with AChR-positive generalised myasthenia gravis (Myasthenia Gravis Foundation of America disease class II-IV), a myasthenia gravis activities of daily living (MG-ADL) score of least 6, and a quantitative myasthenia gravis score of at least 12. Participants were randomly assigned (1:1) to receive subcutaneous zilucoplan 0·3 mg/kg once daily by self-injection, or matched placebo, for 12 weeks. The primary efficacy endpoint was change from baseline to week 12 in MG-ADL score in the modified intention-to-treat population (all randomly assigned patients who received at least one dose of study drug and had at least one post-dosing MG-ADL score). Safety was mainly assessed by the incidence of treatment-emergent adverse events (TEAEs) in all patients who had received at least one dose of zilucoplan or placebo. This trial is registered at ClinicalTrials.gov, NCT04115293. An open-label extension study is ongoing (NCT04225871). FINDINGS: Between Sept 17, 2019, and Sept 10, 2021, 239 patients were screened for the study, of whom 174 (73%) were eligible. 86 (49%) patients were randomly assigned to zilucoplan 0·3 mg/kg and 88 (51%) were assigned to placebo. Patients assigned to zilucoplan showed a greater reduction in MG-ADL score from baseline to week 12, compared with those assigned to placebo (least squares mean change -4·39 [95% CI -5·28 to -3·50] vs -2·30 [-3·17 to -1·43]; least squares mean difference -2·09 [-3·24 to -0·95]; p=0·0004). TEAEs occurred in 66 (77%) patients in the zilucoplan group and in 62 (70%) patients in the placebo group. The most common TEAE was injection-site bruising (n=14 [16%] in the zilucoplan group and n=8 [9%] in the placebo group). Incidences of serious TEAEs and serious infections were similar in both groups. One patient died in each group; neither death (COVID-19 [zilucoplan] and cerebral haemorrhage [placebo]) was considered related to the study drug. INTERPRETATION: Zilucoplan treatment showed rapid and clinically meaningful improvements in myasthenia gravis-specific efficacy outcomes, had a favourable safety profile, and was well tolerated, with no major safety findings. Zilucoplan is a new potential treatment option for a broad population of patients with AChR-positive generalised myasthenia gravis. The long-term safety and efficacy of zilucoplan is being assessed in an ongoing open-label extension study. FUNDING: UCB Pharma.

Anesthesia or seizure-like behavior? The effects of two Amazonian plants, Acmella oleracea and Piper alatabaccum in zebrafish (Danio rerio).

Currently, available fish anesthetics can produce important side effects, including respiratory arrest and distress. Easy-to-implement alternatives with low toxicity are needed to ensure fish health as well as to help artisanal fisheries and fish sellers in handling and transporting fishes, and native plants seems to be the best alternative. We aimed to implement an anesthetic protocol using crude ethanolic extracts from flowers and leaves of two Amazonian plants, the Acmella oleracea and Piper alatabaccum. We first tested the extracts for anesthesia, using the zebrafish as model. Even though in some treatments the animals apparently entered deep anesthesia, many of them presented aberrant behaviors and even died. Thus, we performed new experiments testing the extracts effects on seizure-like behaviors of the fish. Only the leaf extract of A. oleracea has potential effects for fish anesthesia. Both the flower extract from this plant and the leaf extract from P. alatabaccum induced seizure-like behavior in the animals. In conclusion, besides bringing a possible new anesthetic protocol for fish, our work draws attention for the neurotoxic effects the anesthetic solutions may cause, since several studies defend other Piper species as anesthetic for fish and A. oleracea flowers' extract was already pointed as fish anesthetic.