Real-world evidence of ocrelizumab-treated relapsing multiple sclerosis cohort shows changes in progression independent of relapse activity mirroring phase 3 trials.

Ocrelizumab is a B cell-depleting drug widely used in relapsing-remitting multiple sclerosis (RRMS) and primary-progressive MS. In RRMS, it is becoming increasingly apparent that accumulation of disability not only manifests as relapse-associated worsening (RAW) but also as progression independent of relapse activity (PIRA) throughout the disease course. This study's objective was to investigate the role of PIRA in RRMS patients treated with ocrelizumab. We performed a single-center, retrospective, cross-sectional study of clinical data acquired at a German tertiary multiple sclerosis referral center from 2018 to 2022. All patients with RRMS treated with ocrelizumab for at least six months and complete datasets were analyzed. Confirmed disability accumulation (CDA) was defined as a ≥ 12-week confirmed increase from the previous expanded disability status scale (EDSS) score of ≥ 1.0 if the previous EDSS was ≤ 5.5 or a ≥ 0.5-point increase if the previous EDSS was > 5.5. PIRA was defined as CDA without relapse since the last EDSS measurement and at least for the preceding 12 weeks. RAW was defined as CDA in an interval of EDSS measurements with ≥ 1 relapses. Cox proportional hazard models were used to analyze the probability of developing PIRA depending on various factors, including disease duration, previous disease-modifying treatments (DMTs), and optical coherence tomography-assessed retinal degeneration parameters. 97 patients were included in the analysis. Mean follow-up time was 29 months (range 6 to 51 months). 23.5% developed CDA under ocrelizumab therapy (n = 23). Of those, the majority developed PIRA (87.0% of CDA, n = 20) rather than RAW (13.0% of CDA, n = 3). An exploratory investigation using Cox proportional hazards ratios revealed two possible factors associated with an increased probability of developing PIRA: a shorter disease duration prior to ocrelizumab (p = 0.02) and a lower number of previous DMTs prior to ocrelizumab (p = 0.04). Our data show that in ocrelizumab-treated RRMS patients, the main driver of disability accumulation is PIRA rather than RAW. Furthermore, these real-world data show remarkable consistency with data from phase 3 randomized controlled trials of ocrelizumab in RRMS, which may increase confidence in translating results from tightly controlled RCTs into the real-world clinical setting.

The intertwining between lead and ethanol in the model organism Caenorhabditis elegans.

Caenorhabditis elegans (C. elegans) is a model organism widely used to evaluate the mechanistic aspects of toxicants with the potential to predict responses comparable to those of mammals. We report here the consequences of developmental lead (Pb) exposure on behavioral responses to ethanol (EtOH) in C. elegans. In addition, we present data on morphological alterations in the dopamine (DA) synapse and DA-dependent behaviors aimed to dissect the neurobiological mechanisms that underlie the relationship between these neurotoxicants. Finally, the escalation to superior animals that parallels the observed effects in both experimental models with references to EtOH metabolism and oxidative stress is also discussed. Overall, the literature revised here underpins the usefulness of C. elegans to evidence behavioral responses to a combination of neurotoxicants in mechanistic-orientated studies.

Sleep and conditioning of the siphon withdrawal reflex in Aplysia.

Sleep is essential for memory consolidation after learning as shown in mammals and invertebrates such as bees and flies. Aplysia californica displays sleep, and sleep in this mollusk was also found to support memory for an operant conditioning task. Here, we investigated whether sleep in Aplysia is also required for memory consolidation in a simpler type of learning, i.e. the conditioning of the siphon withdrawal reflex. Two groups of animals (Wake, Sleep, each n=11) were conditioned on the siphon withdrawal reflex, with the training following a classical conditioning procedure where an electrical tail shock served as the unconditioned stimulus (US) and a tactile stimulus to the siphon as the conditioned stimulus (CS). Responses to the CS were tested before (pre-test), and 24 and 48 h after training. While Wake animals remained awake for 6 h after training, Sleep animals had undisturbed sleep. The 24 h test in both groups was combined with extinction training, i.e. the extended presentation of the CS alone over two blocks. At the 24 h test, siphon withdrawal duration in response to the CS was distinctly enhanced in both Sleep and Wake groups with no significant difference between groups, consistent with the view that consolidation of a simple conditioned reflex response does not require post-training sleep. Surprisingly, extinction training did not reverse the enhancement of responses to the CS. On the contrary, at the 48 h test, withdrawal duration in response to the CS was even further enhanced across both groups. This suggests that processes of sensitization, an even simpler non-associative type of learning, contributed to the withdrawal responses. Our study provides evidence for the hypothesis that sleep preferentially benefits consolidation of more complex learning paradigms than conditioning of simple reflexes.

Pro- and anti-inflammatory macrophages express a sub-type specific purinergic receptor profile.

Extracellular nucleotides act as danger signals that orchestrate inflammation by purinergic receptor activation. The expression pattern of different purinergic receptors may correlate with a pro- or anti-inflammatory phenotype. Macrophages function as pro-inflammatory M1 macrophages (M1) or anti-inflammatory M2 macrophages (M2). The present study found that murine bone marrow-derived macrophages express a unique purinergic receptor profile during in vitro polarization. As assessed by real-time polymerase chain reaction (PCR), Gαs-coupled P1 receptors A2A and A2B are upregulated in M1 and M2 compared to M0, but A2A 15 times higher in M1. The ionotropic P2 receptor P2X5 is selectively upregulated in M1- and M2-polarized macrophages. P2X7 is temporarily expressed in M1 macrophages. Metabotropic P2Y receptors showed a distinct expression profile in M1 and M2-polarized macrophages: Gαq coupled P2Y1 and P2Y6 are exclusively upregulated in M2, whereas Gαi P2Y13 and P2Y14 are overexpressed in M1. This consequently leads to functional differences between M1 and M2 in response to adenosine di-phosphate stimulation (ADP): In contrast to M1, M2 showed increased cytoplasmatic calcium after ADP stimulation. In the present study we show that bone marrow-derived macrophages express a unique repertoire of purinergic receptors. We show for the first time that the repertoire of purinergic receptors is highly flexible and quickly adapts upon pro- and anti-inflammatory macrophage differentiation with functional consequences to nucleotide stimulation.

[Muscle weakness and CK elevation: is it myositis?] / Muskelschwäche und CK­Erhöhung: Ist es immer eine Myositis?

Rheumatologists are often confronted by patients with muscle weakness and elevated creatine kinase (CK) levels. Myositis cannot always be determined to be the cause of the complaints. This article presents two cases from our hospital where the diagnosis could only be determined by muscle biopsy. In the first case the patient presented with muscle weakness, pathological weight loss and a significant increase in CK levels. A muscle biopsy revealed an immune-mediated necrotizing myopathy (IMNM) caused by anti-3-hydroxy-3-methyl-gulatryl-CoA reductase (HMG-CoA reductase) autoantibodies due to the intake of statins. The second patient presented with cramp-like and burning muscle pain and weakness of the extremities without a relevant increase in CK level. Myoadenylate deaminase deficiency was also detected by muscle biopsy, and further confirmed by genetic testing.

Wakefulness rather than sleep benefits extinction of an inhibitory operant conditioning memory in Aplysia.

Sleep enhances memory consolidation which has been shown in mammals as well as in invertebrates, like bees and Drosophila. The current study is part of a series of experiments examining whether this memory function of sleep is preserved in Aplysia with an even simpler nervous system. Previous work showed that Aplysia sleep and that sleep after training supports memory on an inhibitory conditioning task ('learning that food is inedible', LFI). Here, we tested whether sleep in Aplysia would also support memory for an extinction learning on the LFI task. Following Acquisition in which animals learned that netted food is inedible, two groups of animals, a Sleep group (n = 15) and a Wake group (n = 16) underwent extinction training. After a 17-hour Retention interval which contained either regular nocturnal sleep or daytime wakefulness (supported by sleep deprivation) animals were retested on the LFI task. Contrary to our hypothesis, the Wake animals showed significantly prolonged food intake behavior on the LFI, indicating that extinction memory in these animals was better than in the Sleep animals. Performance of a control group not subjected to extinction training, ruled out that the superior extinction performance of Wake animals merely reflected forgetting over time of the LFI memory, and also excluding a possible circadian confound. We speculate that wakefulness mainly acts by accelerating active forgetting of the LFI memory after it was labialized through extinction training, thereby facilitating the re-emergence of the original innate behavior of food intake.

Cerebrospinal fluid findings in reversible cerebral vasoconstriction syndrome: a way to differentiate from cerebral vasculitis?

Reversible cerebral vasoconstriction syndrome (RCVS) is characterized by an acute onset of severe headache and multi-focal segmental vasoconstriction of cerebral arteries resolving within 12 weeks. Diagnostic criteria include normal or near-normal findings in cerebrospinal fluid (CSF) analysis, especially leucocyte levels < 10/mm³. Distinguishing RCVS from primary angiitis of the central nervous system (PACNS) is essential to avoid unnecessary and sometimes unfavourable immunosuppressive treatment. We reviewed retrospectively the clinical and diagnostic data of 10 RCVS patients who presented in our neurological department from 1 January 2013 to February 2017. The main purpose was to verify whether CSF leucocyte counts < 10/mm³ serve to discriminate RCVS from PACNS. Five of six patients who underwent lumbar puncture presented with CSF leucocyte levels ≥ 10/mm³. Two patients had a history of misinterpretation of CSF pleocytosis as cerebral vasculitis and of immunosuppressive treatment. A complete restitution of cerebral vasoconstriction was evident in all. No patient had further cerebral strokes or bleedings without immunosuppressive treatment over more than 12 weeks. Despite the established diagnostic criteria, RCVS can manifest with CSF leucocyte levels > 10/mm³. Careful anamnesis and the response of 'vasculitis-like angiography' to nimodipine given as a test during angiography and as oral medication are key to differentiate RCVS from cerebral vasculitis.

Sleep supports inhibitory operant conditioning memory in Aplysia.

Sleep supports memory consolidation as shown in mammals and invertebrates such as bees and Drosophila. Here, we show that sleep's memory function is preserved in Aplysia californica with an even simpler nervous system. Animals performed on an inhibitory conditioning task ("learning that a food is inedible") three times, at Training, Retrieval 1, and Retrieval 2, with 17-h intervals between tests. Compared with Wake animals, remaining awake between Training and Retrieval 1, Sleep animals with undisturbed post-training sleep, performed significantly better at Retrieval 1 and 2. Control experiments testing retrieval only after ∼34 h, confirmed the consolidating effect of sleep occurring within 17 h after training.

Gastroesophageal Reflux Disease in Children with Interstitial Lung Disease.

Gastroesophageal reflux disease is common in adult patients with interstitial lung disease. However, no data currently exist regarding the prevalence and characteristics of the disease in pediatric patients with interstitial lung disease. The aim of the present study was to prospectively assess the incidence of gastroesophageal reflux disease and characterize its features in children with interstitial lung disease. Gastroesophageal reflux disease was established based on 24 h pH-impedance monitoring (MII-pH). Gastroesophageal reflux episodes (GERs) were classified according to widely recognized criteria as acid, weakly acid, weakly alkaline, or proximal. Eighteen consecutive patients (15 boys, aged 0.2-11.6 years) were enrolled in the study. Gastroesophageal reflux disease was diagnosed in a half (9/18) of children. A thousand GERs were detected by MII-pH (median 53.5; IQR 39.0-75.5). Of these, 585 (58.5 %) episodes were acidic, 407 (40.7 %) were weakly acidic, and eight (0.8 %) were weakly alkaline. There were 637 (63.7 %) proximal GERs. The patients in whom gastroesophageal reflux disease was diagnosed had a significantly higher number of proximal and total GERs. We conclude that the prevalence of gastroesophageal reflux disease in children with interstitial lung disease is high; thus, the disease should be considered regardless of presenting clinical symptoms. A high frequency of non-acid and proximal GERs makes the MII-pH method a preferable choice for the detection of reflux episodes in this patient population.

Indirect effects of a 7 year PCV7/PCV13 mass vaccination program in children on the incidence of pneumonia among adults: a comparative study based on two Polish cities.

BACKGROUND AND OBJECTIVES: In 2006 the city of Kielce, Poland, introduced a mandatory PCV7 (replaced by PCV13 in 2011) vaccination program against S. pneumoanie for all children under 2 years old. At that time, the neighboring city of Ostrowiec Swietokrzyski had no such large-scale vaccination program in place. This created an opportunity to observe the results of the vaccination by comparing the incidence of pneumonia in these two cities. The aim of this study was to analyze how the incidence of pneumonia among adults was indirectly affected by the PCV7/PCV13 vaccination program in children during the 7 year follow-up period. METHODS: We performed a retrospective study. PCV7/PCV13 vaccinations were delivered according to a 2 + 1 schedule. The vaccination rate in the analyzed period amounted to almost 99%. The following age groups were analyzed: 30-49, 50-64 and 65+. The Cochran-Armitage test was used to investigate the significance of the observed trend in pneumonia morbidity. The significance of deviations from a linear trend was also tested. In addition, the importance of the trend (in the case of deviations from linearity) was confirmed with the use of the Mantel test. RESULTS: In the 65+ age group there was a decrease of 66.5% in the incidence of diagnosed pneumonia (p < 0.0001). This was followed by smaller, but statistically significant, declines in the other age groups: 30.75% in the 30-49 age group (p = 0.001) and 56.8% in the 50-64 age group (p < 0.0001). This decreasing trend continued for seven consecutive years of observation. In addition, we demonstrated a statistically significant higher rate of pneumonia in all age groups in the City of Ostrowiec Swietokrzyski. CONCLUSIONS: The results clearly indicate that the indirect effectiveness of the PCV7/PCV13 vaccine program, performed according to the 2 + 1 schedule and applied in Kielce, Poland, is statistically significant.

BAX inhibitor-1 is a Ca(2+) channel critically important for immune cell function and survival.

The endoplasmic reticulum (ER) serves as the major intracellular Ca(2+) store and has a role in the synthesis and folding of proteins. BAX (BCL2-associated X protein) inhibitor-1 (BI-1) is a Ca(2+) leak channel also implicated in the response against protein misfolding, thereby connecting the Ca(2+) store and protein-folding functions of the ER. We found that BI-1-deficient mice suffer from leukopenia and erythrocytosis, have an increased number of splenic marginal zone B cells and higher abundance and nuclear translocation of NF-κB (nuclear factor-κ light-chain enhancer of activated B cells) proteins, correlating with increased cytosolic and ER Ca(2+) levels. When put into culture, purified knockout T cells and even more so B cells die spontaneously. This is preceded by increased activity of the mitochondrial initiator caspase-9 and correlated with a significant surge in mitochondrial Ca(2+) levels, suggesting an exhausted mitochondrial Ca(2+) buffer capacity as the underlying cause for cell death in vitro. In vivo, T-cell-dependent experimental autoimmune encephalomyelitis and B-cell-dependent antibody production are attenuated, corroborating the ex vivo results. These results suggest that BI-1 has a major role in the functioning of the adaptive immune system by regulating intracellular Ca(2+) homeostasis in lymphocytes.

Sleep and memory in mammals, birds and invertebrates.

Sleep supports memory consolidation. Based on studies in mammals, sleep-dependent consolidation has been conceptualized as 'active system consolidation'. During waking, information is encoded into an initial store (hippocampus). During subsequent sleep, some of the newly encoded memories are selected to be reactivated and redistributed toward networks serving as long-term store (e.g., neocortex), whereby memories become transformed into more general, schema-like representations. Here we asked whether sleep in non-mammalian species might play a comparable role for memory. The literature review revealed that sleep produces enhancing effects on memory in all non-mammalian species studied. Furthermore, across species some of the hallmarking features of active system consolidation were identified: Studies of filial imprinting in chicks suggest that a redistribution of imprinting memory toward long-term storage sites occurs during sleep; song learning in birds appears to be driven by reactivations of song representations during sleep; studies of bees demonstrated the selectivity of sleep-dependent consolidation, benefiting extinction but not original classical conditioning. Although overall fragmentary, first evidence in non-mammalian species suggests active system consolidation might be an evolutionary conserved function of sleep.

Quality control for retinal OCT in multiple sclerosis: validation of the OSCAR-IB criteria.

BACKGROUND: Retinal optical coherence tomography (OCT) permits quantification of retinal layer atrophy relevant to assessment of neurodegeneration in multiple sclerosis (MS). Measurement artefacts may limit the use of OCT to MS research. OBJECTIVE: An expert task force convened with the aim to provide guidance on the use of validated quality control (QC) criteria for the use of OCT in MS research and clinical trials. METHODS: A prospective multi-centre (n = 13) study. Peripapillary ring scan QC rating of an OCT training set (n = 50) was followed by a test set (n = 50). Inter-rater agreement was calculated using kappa statistics. Results were discussed at a round table after the assessment had taken place. RESULTS: The inter-rater QC agreement was substantial (kappa = 0.7). Disagreement was found highest for judging signal strength (kappa = 0.40). Future steps to resolve these issues were discussed. CONCLUSION: Substantial agreement for QC assessment was achieved with aid of the OSCAR-IB criteria. The task force has developed a website for free online training and QC certification. The criteria may prove useful for future research and trials in MS using OCT as a secondary outcome measure in a multi-centre setting.

Characterization of sleep in Aplysia californica.

STUDY OBJECTIVE: To characterize sleep in the marine mollusk, Aplysia californica. DESIGN: Animal behavior and activity were assessed using video recordings to measure activity, resting posture, resting place preference, and behavior after rest deprivation. Latencies for behavioral responses were measured for appetitive and aversive stimuli for animals in the wake and rest states. SETTING: Circadian research laboratory for Aplysia. PATIENTS OR PARTICIPANTS: A. californica from the Pacific Ocean. INTERVENTIONS: N/A. MEASUREMENTS AND RESULTS: Aplysia rest almost exclusively during the night in a semi-contracted body position with preferential resting locations in the upper corners of their tank. Resting animals demonstrate longer latencies in head orientation and biting in response to a seaweed stimulus and less frequent escape response steps following an aversive salt stimulus applied to the tail compared to awake animals at the same time point. Aplysia exhibit rebound rest the day following rest deprivation during the night, but not after similar handling stimulation during the day. CONCLUSIONS: Resting behavior in Aplysia fulfills all invertebrate characteristics of sleep including: (1) a specific sleep body posture, (2) preferred resting location, (3) reversible behavioral quiescence, (4) elevated arousal thresholds for sensory stimuli during sleep, and (5) compensatory sleep rebound after sleep deprivation.

The plasma membrane channel ORAI1 mediates detrimental calcium influx caused by endogenous oxidative stress.

The mouse hippocampal cell line HT22 is an excellent model for studying the consequences of endogenous oxidative stress. Addition of extracellular glutamate depletes the cells of glutathione (GSH) by blocking the glutamate-cystine antiporter system x(c)(-). GSH is the main antioxidant in neurons and its depletion induces a well-defined program of cell death called oxytosis, which is probably synonymous with the iron-dependent form of non-apoptotic cell death termed ferroptosis. Oxytosis is characterized by an increase of reactive oxygen species and a strong calcium influx preceding cell death. We found a significant reduction in store-operated calcium entry (SOCE) in glutamate-resistant HT22 cells caused by downregulation of the Ca(2+) channel ORAI1, but not the Ca(2+) sensors STIM1 or STIM2. Pharmacological inhibition of SOCE mimicked this protection similarly to knockdown of ORAI1 by small interfering RNAs. Long-term calcium live-cell imaging after induction of the cell death program showed a specific reduction in Ca(2+)-positive cells by ORAI1 knockdown. These results suggest that dysregulated Ca(2+) entry through ORAI1 mediates the detrimental Ca(2+) entry in programmed cell death induced by GSH depletion. As this detrimental Ca(2+) influx occurs late in the course of the cell death program, it might be amenable to therapeutic intervention in diseases caused by oxidative stress.

Evaluation of laryngopharyngeal reflux in pediatric patients with asthma using a new technique of pharyngeal pH-monitoring.

There is a debate about the association between asthma and gastroesophageal and/or laryngopharyngeal reflux (LPR). Pharyngeal pH-monitoring is a new technique that allows a physician to assess whether reflux passes the upper esophageal sphincter barrier. The aim of the study was to assess the prevalence of LPR in children with difficult-to-treat asthma. The present study was an open, prospective one. A total of 21 subjects of the mean age 12.7 years were enrolled in the study. All children were asked to fill out a Reflux Symptoms Index questionnaire and a 24-h pharyngeal pH monitoring was performed, using the Dx-pH Measurement System. The LPR was diagnosed in 13 (61.9%) children. There was a positive correlation between LPR diagnosis and the degree of asthma control. The LPR was more frequent in children treated with a higher than lower doses of fluticasone (p = 0.019, OR = 17.3) and in those using montelukast compared with non-users (p = 0.008, OR = 19.0). The mean Reflux Symptoms Index score was almost twice greater in children with LPR than in those without it (13.2 vs. 6.8, respectively, p = 0.003). We conclude that the prevalence of laryngopharyngeal reflux in children with difficult-to-treat asthma is substantial.

Indirect population impact of universal PCV7 vaccination of children in a 2 + 1 schedule on the incidence of pneumonia morbidity in Kielce, Poland.

The aim of this study was an analysis of the population effects of a seven-valent pneumococcal conjugate vaccine (PCV7) on pneumonia incidence rates in the 5-year follow-up period after the introduction in 2006 of a universal PCV7 vaccination programme in the city of Kielce, Poland. Vaccinations were carried out according to a 2 + 1 schedule. The vaccination compliance rate amounted to approximately 99 %. The age groups 0-2, 30-49, 50-65 and 65+ years were analysed. The Cochran-Armitage test was used to investigate the significance of observed trends in pneumonia morbidity. The significance of deviations from a linear trend was also tested. The importance of the trend was confirmed by the Mantel test. Between 2005 and 2010, the greatest decline, 82.9 % (2005, 25.31/1,000; 2010, 4.34/1,000), in pneumonia morbidity was observed for children <2 years of age. In the 65+ years age group, this amounted to 43.5 %. Lesser declines, but still of statistical significance, were observed for the other age groups: 16.5 % in the 30-49 years group and 40.4 % in the 50-64 years group. All reductions are statistically significant and confirmed by the Mantel test. Five years after the introduction of a universal PCV7 vaccination programme in Kielce, Poland, its effectiveness in pneumonia prevention has been demonstrated in both the <2 years of age group and indirectly for other groups.