Flow cytometry analysis of T cell subsets in cerebrospinal fluid and pheriperal blood of narcolepsy type 1 patients with long lasting disease

Background: Type 1 Narcolepsy (NT1) is a central hypersomnia linked to the destruction of hypocretin-producing neurons. A great body of genetic and epidemiological data points to a likely autoimmune disease aetiology. Recent reports have characterized peripheral blood T-cell subsets in NT1, whereas data regarding the cerebrospinal fluid (CSF) immune cell composition are lacking. In this study, we aim to identify the T- and NK- cell subsets in NT1 patients with long-term disease course. Methods: Immune cell subsets from CSF and peripheral blood mononuclear cell (PBMC) samples were analysed by flow cytometry in two age-, sex and BMI-balanced groups of 14 NT1 patients versus 14 healthy controls. The frequency of CSF cell groups were compared with PBMCs. Non-parametric tests were used for statistical analyses. Results: NT1 patients did not show significant differences of CSF immune cell subsets compared to controls, despite a trend towards higher CD4+ terminally differentiated effector memory T cells. T cells preferentially displayed a memory phenotype in the CSF compared to PBMCs. Furthermore, a reduced frequency of CD4+ terminally differentiated effector memory T cells and an increased frequency of Natural Killer CD56bright cells were observed in PBMCs from patients compared to controls. Finally, the ratio between CSF and peripheral CD4+ terminally differentiated effector memory T cells showed a two-fold increase in NT1 patients versus controls. Conclusions: Significant differences in PBMCs and CSF immune cell profile were found between NT1 patients versus controls. These differences may reflect differences in HLA status, or be primary or secondary to hypocretin deficiency in narcolepsy

Non-24-hour sleep-wake rhythm disorder and melatonin secretion impairment in a patient with pineal cyst

We report the case of a 14-year-old girl with a wide non-compressive pineal cyst, associated with the inability to control her sleep-wake schedule. Actigraphic monitoring showed a 24-hour free-running disorder (tau 26.96 hours). A 24-hour serum melatonin curve assay, with concomitant video-polysomnographic and body-core temperature monitoring, was performed. Melatonin curve showed a blunted nocturnal peak, lower total quantity of melatonin, and prolonged melatonin secretion in the morning, with normal temperature profile and sleep parameters. Treatment with melatonin up to 14 mg at bedtime was initiated with complete realignment of the sleep-wake rhythm (tau 23.93 hours). The role of the pineal cyst in the aforementioned alteration of melatonin secretion and free-running disorder remains controversial, but our case supports the utility of monitoring sleep/wake, temperature, and melatonin rhythms in the diagnostic work-up of pineal cysts associated with free-running disorder

Pre-treatment of blood samples reveal normal blood hypocretin/orexin signal in narcolepsy type 1

The hypocretin/orexin system regulates arousal through central nervous system mechanisms and plays an important role in sleep, wakefulness and energy homeostasis. It is unclear whether hypocretin peptides are also present in blood due to difficulties in measuring reliable and reproducible levels of the peptides in blood samples. Lack of hypocretin signalling causes the sleep disorder narcolepsy type 1, and low concentration of cerebrospinal fluid hypocretin-l/oreadn-A peptide is a hallmark of the disease. This measurement has high diagnostic value, but performing a lumbar puncture is not without discomfort and possible complications for the patient. A blood-based test to assess hypocretin-1 deficiency would therefore be of obvious benefit. We here demonstrate that heating plasma or scrum samples to 65 degrees C for 30 min at pH 8 significantly increases hypocretin-1 immunoreactivity enabling stable and reproducible measurement of hypocretin-1 in blood samples. Specificity of the signal was verified by high-performance liquid chromatography and by measuring blood samples from mice lacking hypocretin. Unspecific background signal in the assay was high. Using our method, we show that hypocretin-1 immunoreactivity in blood samples from narcolepsy type 1 patients does not differ from the levels detected in control samples. The data presented here suggest that hypocretin-1 is present in the blood stream in the low picograms per millilitres range and that peripheral hypocretin-1 concentrations are unchanged in narcolepsy type 1

Case report: Chorea and cognitive decline in a young woman: instrumental and genetic assessment of a case originally diagnosed as multiple sclerosis

We describe the case of a young woman affected by debilitating chorea and rapidly progressive cognitive decline. While her original diagnosis was multiple sclerosis, we performed a full instrumental and genetic assessement, though which we identified multiple genetic variants, including a novel variant of the APP gene. We propose some possible mechanisms by which such variants may contribute to neuroinflammation and ultimately lead to this devastating clinical course

Sodium oxybate treatment in pediatric type 1 narcolepsy

Background: Narcolepsy type 1 (NT1) is a chronic neurologic disorder defined by excessive daytime sleepiness, cataplexy, sleep paralysis, hallucinations and disrupted nocturnal sleep, typically with onset during child-hood/adolescence. Pediatric NT1 is associated with limitations on children\u2019s activities and achievements, especially poor performance at school, difficulty with peers due to disease symptoms and comorbidities including depression, obesity, and precocious puberty. Sodium oxybate (SO) is a sodium salt of \u3b3-hydroxybutyric (GHB) acid and is greatly effective in treating cataplexy and excessive daytime sleepiness in NT1 and it can be helpful also for sleep disruption, hypnagogic hallucination and sleep paralysis in these patients. Method: We conducted a research of literature into bibliographic databases regarding NT1 features in childhood and the possible option treatment with SO in this kind of patient population. Results: We reported sixteen papers focusing on symptom presentation and on clinical and metabolic features of children affected with NT1. Furthermore, we reported 24 manuscripts focusing on SO biological actions and pharmacological properties and on the few but important available studies (8) conducted in NT1 children under SO therapy. Conclusion: Although in the majority of patients develop NT1 during childhood, there are no approved treatments for pediatric NT1. However, SO has been widely used off-label to treat narcolepsy symptoms in children and adolescents with NT1 in non-controlled studies, showing a similar safety profile and therapeutic response to adult patients. Ongoing pediatric therapy is based only on observational data shared among sleep disorders clinicians

Development and validation of volumetric absorptive microsampling coupled with UHPLC-MS/MS for the analysis of gamma-hydroxybutyric acid in human blood

A volumetric microsampling (VAMS) device (20 \u3bcl) was evaluated and validated for the analysis of \u3b3-hydroxybutyric acid (GHB) in venous blood using a simple ultra-high-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method. GHB was extracted from VAMS device by acetonitrile, after a re-hydration step in a temperature-controlled ultrasonic bath at 60\ub0C for 10\u2009min. Chromatographic analysis was carried out on a Kinetex C18 column using 0.1% formic acid in water and acetonitrile as binary gradient mobile phase (from 5 to 95% of acetonitrile from 1 to 2.5\u2009min) at a flow rate of 0.3\u2009ml/min. The VAMS method was fully validated according to current guidelines with satisfactory results in terms of linearity, selectivity, precision, absolute recovery, matrix effect and stability. The linearity was determined from 0.5 to 200\u2009\u3bcg/ml and the lower limit of quantitation was 0.5 \u3bcg/ml. The novel VAMS-UHPLC-MS/MS method was successfully compared with plasma-based method in a GHB-treated patient as a proof of concept

The importance of social zeitgeber in pediatric type 1 narcolepsy: What we can learn from the COVID‐19 restrictions adopted in Italy?

The lockdown due to the new coronavirus pandemic (COVID-19) has led to unparalleled changes in several aspects of human behaviour. During the lockdown, the general population delayed sleep timing and spent more time in bed. Very little is known on the effects of COVID-19 restriction on children and adolescents suffering type 1 narcolepsy. At the end of 2019, we performed follow-up actigraphy in 18 type 1 narcolepsy children and adolescents under stable pharmacological treatment with sodium oxybate. Patients were contacted for a follow-up actigraphy during the first Italian lockdown. Actigraphs and the Epworth Sleepiness Scale for children and adolescents (ESS-CHAD) have been sent to participants’ homes. Differences in motor activity were analyzed through functional linear modelling. During lockdown, type 1 narcolepsy children and adolescents went to bed and woke up later, slept more during the daytime and napped more frequently. No difference emerged in time in bed, estimated total sleep time and nocturnal sleep quality. Similarly, no difference emerged in ESS-CHAD and body mass index. The time-series analysis of motor activity documented reduced activity during the early morning and in the evening during the lockdown period compared with pre-lockdown. Our study objectively showed that type 1 narcolepsy children and adolescents delayed the sleep phase and slept more during the daytime during the lockdown. The analysis of type 1 narcolepsy children and adolescents’ behaviour during the lockdown has provided new information that could pave the way to a personalized school schedule

Variazioni del profilo motorio circadiano in funzione di dati clinici, neurofisiologici e biochimici nella Narcolessia di tipo 1 pediatrica

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Quantifying the impact of age, sleepiness and hypocretin on activity profile in pediatric NT1

Study Objectives: Pediatric type 1 Narcolepsy (NT1) is a chronic neurological disorder characterized by hypersomnolence, cataplexy, and dissociated rapid eye movement sleep manifestations. Recently several studies highlighted a peculiar nycthemeral profile, characterized by enhanced motor activity throughout the nocturnal period and blunted motor activity in the first afternoon, which is already detectable in children close to disease onset [1]. In the present study we analyzed the features of motor activity profile of pediatric NT1 patients applying of a novel statistical framework to stratify the motor profile with respect to demographical, clinical and biomedical data. Methods: Fifty drug-naïve NT1 children and adolescents (mean age = 12.31 ± 3.41) underwent seven days of actigraphy prior to the hospitalization finalized to diagnosis. Activity profiles were processed by mean of functional linear modeling (FLM) to analyze raw activity values with respect to age, BMI, ESS-CHAD scores and Hcrt-1 levels [2]. Results: FLM showed that age display a significant impact on NT1 children activity profile with younger children presenting with higher nocturnal motor activity from 2:00 to 6:00 (global F values on permutations <0.05) coupled with higher diurnal motor activity from 11:00 to 22:00 (point-wise F values on permutations <0.05). Higher ESS-CHAD scores are associated with lower motor activity from 10:00 to 12:00 (critical F) and from 16:00 to 18:00 (point-wise F). Conversely, higher sleep latency at MSLT are associated with higher motor activity during morning (7:30-10:00) and early afternoon (14:00-16:00). ESS-CHAD scores also significantly impact children activity profile, with higher scores associated with higher motor activity during the first part of nocturnal period (24:00-3:00, global F) and lower motor activity from 9:00-12:00 and from 16:00-18:00 (global F). Finally higher Hctr-1 values were associated with higher motor activity from 14:00-18:00 (global F) and lower motor activity levels throughout the nocturnal period (point-wise F). BMI does not significantly impact on children’s nycthemeral profile. Conclusion: Results highlight that subjective and objective sleepiness severity influence NT1 children’s activity profile in specific time windows corresponding to the morning and the first afternoon. The influence of age on circadian activity profile appears to be especially evident during the nocturnal period, which suggests that in younger NT1 children the nocturnal sleep disruption is more severe

Variation of the motor activity profile according to sleep macro and microstructure in pediatric type 1 narcolepsy

Objectives/Introduction: Disrupted nighttime sleep (DNS) is a core symptom of type 1 narcolepsy (NT1), resulting from hypocretin neu- ronal loss. In this study we quantified the influence of DNS on NT1 children motor activity profile. Methods: Fifty-nine drug-naïve NT1 children and adolescents (age = 12.47 ± 3.56) underwent two weeks of actigraphy immedi- ately prior to the diagnostic hospitalization. The second PSG night was analyzed and the following variables extracted: percentage of N1, N2, SWS and REM sleep, SE and PLMi. For a subsamples of pa- tients (n = 47) we also computed sleep transitions index including: a) Wake index, b) NREM/2-3 to Wake/N1, c) REM/N2/N3 to Wake/ N1 and d) Wake/N1. Activity profiles were processed by mean of functional linear modeling (FLM) to convert and analyze raw activity in functional form. Results: The analysis show that the N1 sleep percentage has a sig- nificant influence on NT1 children's motor profile with higher per- centages of N1 sleep being associated with lower motor intensity between 12:00-14:00 (critical F < 0.05) and 18:00–20:00 (point- wise F < 0.05). Similarly, lower REM sleep percentage is associated with lower motor activity intensity between 11:00-13:00 (critical F < 0.05). On the contrary N2, SWS percentage and sleep transition index does not significantly influence the motor activity profile. Conclusions: These findings show that the N1 sleep percentage is linked to lower daytime activity levels. Combining information deriv- ing from actigraphy and PSG in a single analytical framework allows to characterize the effects of DNS on NT1 children's everyday life. Disclosure: Nothing to disclose