Protein kinase C-inhibition reduces critical weight loss and improves functional outcome after experimental subarachnoid haemorrhage.

OBJECTIVES: Subarachnoid haemorrhage (SAH) carries a high burden of morbidity and mortality. One in three patients develop vasospasm, which is associated with Delayed Cerebral Ischemia. The pathophysiology includes vasoconstrictor receptor upregulation in cerebral arteries. The protein kinase C - inhibitor RO-31-7549 reduces the expression of several vasoconstrictor receptors and normalizes cerebral blood flow in experimental SAH but functional and behavioural effects are unknown. This study was undertaken to analyse functional outcomes up to 14 days after experimental SAH. MATERIALS AND METHODS: 54 male rats were randomised to experimental SAH or sham, using the pre-chiasmatic, single injection model, and subsequent treatment or vehicle. 42 remained for final analysis. The animals were euthanized on day 14 or when reaching a humane endpoint. The primary endpoint was overall survival, defined as either spontaneous mortality or when reaching a predefined humane endpoint. The secondary outcomes were differences in the rotating pole test, weight, open field test, novel object recognition and qPCR of selected inflammatory markers. RESULTS: In the vehicle group 6/15 rats reached the humane endpoint of >20 % weight loss compared to 1/14 in the treatment group. This resulted in a significant reduced risk of early euthanasia due to >20 % weight loss of HR 0.15 (0.03-0.66, p = 0.04). Furthermore, the treatment group did significantly better on the rotating pole test, RR 0.64 (0.47-0.91, p = 0.02). CONCLUSION: RO-31-7549 improved outcomes in terms >20 % weight loss and rotating pole performance after experimental SAH and could be investigated.

Progesterone distribution in the trigeminal system and its role to modulate sensory neurotransmission: influence of sex.

BACKGROUND: Women are disproportionately affected by migraine, representing up to 75% of all migraine cases. This discrepancy has been proposed to be influenced by differences in hormone levels between the sexes. One such hormone is progesterone. Calcitonin gene-related peptide (CGRP) system is an important factor in migraine pathophysiology and could be influenced by circulating hormones. The purpose of this study was to investigate the distribution of progesterone and its receptor (PR) in the trigeminovascular system, and to examine the role of progesterone to modulate sensory neurotransmission. METHODS: Trigeminal ganglion (TG), hypothalamus, dura mater, and the basilar artery from male and female rats were carefully dissected. Expression of progesterone and PR proteins, and mRNA levels from TG and hypothalamus were analyzed by immunohistochemistry and real-time quantitative PCR. CGRP release from TG and dura mater were measured using an enzyme-linked immunosorbent assay. In addition, the vasomotor effect of progesterone on male and female basilar artery segments was investigated with myography. RESULTS: Progesterone and progesterone receptor -A (PR-A) immunoreactivity were found in TG. Progesterone was located predominantly in cell membranes and in Aδ-fibers, and PR-A was found in neuronal cytoplasm and nucleus, and in satellite glial cells. The number of positive progesterone immunoreactive cells in the TG was higher in female compared to male rats. The PR mRNA was expressed in both hypothalamus and TG; however, the PR expression level was significantly higher in the hypothalamus. Progesterone did not induce a significant change neither in basal level nor upon stimulated release of CGRP from dura mater or TG in male or female rats when compared to the vehicle control. However, pre-treated with 10 µM progesterone weakly enhanced capsaicin induced CGRP release observed in the dura mater of male rats. Similarly, in male basilar arteries, progesterone significantly amplified the dilation in response to capsaicin. CONCLUSIONS: In conclusion, these results highlight the potential for progesterone to modulate sensory neurotransmission and vascular responses in a complex manner, with effects varying by sex, tissue type, and the nature of the stimulus. Further investigations are needed to elucidate the underlying mechanisms and physiological implications of these findings.

RNA therapeutics for epilepsy: An emerging modality for drug discovery.

Drug discovery in epilepsy began with the finding of potassium bromide by Sir Charles Locock in 1857. The following century witnessed the introduction of phenotypic screening tests for discovering antiseizure medications (ASMs). Despite the high success rate of developing ASMs, they have so far failed in eliminating drug resistance and in delivering disease-modifying treatments. This emphasizes the need for new drug discovery strategies in epilepsy. RNA-based drugs have recently shown promise as a new modality with the potential of providing disease modification and counteracting drug resistance in epilepsy. RNA therapeutics can be directed either toward noncoding RNAs, such as microRNAs, long noncoding RNAs (ncRNAs), and circular RNAs, or toward messenger RNAs. The former show promise in sporadic, nongenetic epilepsies, as interference with ncRNAs allows for modulation of entire disease pathways, whereas the latter seem more promising in monogenic childhood epilepsies. Here, we describe therapeutic strategies for modulating disease-associated RNA molecules and highlight the potential of RNA therapeutics for the treatment of different patient populations such as sporadic, drug-resistant epilepsy, and childhood monogenic epilepsies.

Heterogeneous vasomotor responses in segments from Göttingen Minipigs coronary, cerebral, and mesenteric artery: A comparative study.

Göttingen Minipigs (GM) are used as an important preclinical model for cardiovascular safety pharmacology and for evaluation of cardiovascular drug targets. To improve the translational value of the GM model, the current study represents a basic characterization of vascular responses to endothelial regulators and sympathetic, parasympathetic, and sensory neurotransmitters in different anatomical origins. The aim of the current comparative and descriptive study is to use myography to characterize the vasomotor responses of coronary artery isolated from GM and compare the responses to those obtained from parallel studies using cerebral and mesenteric arteries. The selected agonists for sympathetic (norepinephrine), parasympathetic (carbachol), sensory (calcitonin gene-related peptide, CGRP), and endothelial pathways (endothelin-1, ET-1, and bradykinin) were used for comparison. Further, the robust nature of the vasomotor responses was evaluated after 24 h of cold storage of vascular tissue mimicking the situation under which human biopsies are often kept before experiments or grafting is feasible. Results show that bradykinin and CGRP consistently dilated, and endothelin consistently contracted artery segments from coronary, cerebral, and mesenteric origin. By comparison, norepinephrine and carbachol, had responses that varied with the anatomical source of the tissues. To support the basic characterization of GM vasomotor responses, we demonstrated the presence of mRNA encoding selected vascular receptors (CGRP- and ETA-receptors) in fresh artery segments. In conclusion, the vasomotor responses of isolated coronary, cerebral, and mesenteric arteries to selected agonists of endothelial, sympathetic, parasympathetic, and sensory pathways are different and the phenotypes are similar to sporadic human findings.

Intraocular Adeno-Associated Virus-Mediated Transgene Endothelin-1 Delivery to the Rat Eye Induces Functional Changes Indicative of Retinal Ischemia-A Potential Chronic Glaucoma Model.

Endothelin-1 (ET-1) overactivity has been implicated as a factor contributing to glaucomatous neuropathy, and it has been utilized in animal models of retinal ischemia. The functional effects of long-term ET-1 exposure and possible compensatory mechanisms have, however, not been investigated. This was therefore the purpose of our study. ET-1 was delivered into rat eyes via a single intravitreal injection of 500 µM or via transgene delivery using an adeno-associated viral (AAV) vector. Retinal function was assessed using electroretinography (ERG) and the retinal expression of potentially compensatory genes was evaluated by means of qRT-PCR. Acute ET-1 delivery led to vasoconstriction and a significant reduction in the ERG response. AAV-ET-1 resulted in substantial transgene expression and ERG results similar to the acute ET-1 injections and comparable to other models of retinal ischemia. Compensatory changes were observed, including an increase in calcitonin gene-related peptide (CGRP) gene expression, which may both counterbalance the vasoconstrictive effects of ET-1 and provide neuroprotection. This chronic ET-1 ischemia model might be especially relevant to glaucoma research, mimicking the mild and repeated ischemic events in patients with long-term vascular dysfunction. The compensatory mechanisms, and particularly the role of vasodilatory CGRP in mitigating the retinal damage, warrant further investigation with the aim of evaluating new therapeutic strategies.

The Non-Coding RNA Journal Club: Highlights on Recent Papers-12.

We are delighted to share with you our twelfth Journal Club and highlight some of the most interesting papers published recently [...].

Knockdown of Circular RNAs Using LNA-Modified Antisense Oligonucleotides.

Circular RNAs (circRNAs) constitute an abundant class of covalently closed noncoding RNA molecules that are formed by backsplicing from eukaryotic protein-coding genes. Recent studies have shown that circRNAs can act as microRNA or protein decoys, as well as transcriptional regulators. However, the functions of most circRNAs are still poorly understood. Because circRNA sequences overlap with their linear parent transcripts, depleting specific circRNAs without affecting host gene expression remains a challenge. In this study, we assessed the utility of LNA-modified antisense oligonucleotides (ASOs) to knock down circRNAs for loss-of-function studies. We found that, while most RNase H-dependent gapmer ASOs mediate effective knockdown of their target circRNAs, some gapmers reduce the levels of the linear parent transcript. The circRNA targeting specificity can be enhanced using design-optimized gapmer ASOs, which display potent and specific circRNA knockdown with a minimal effect on the host genes. In summary, our results demonstrate that LNA-modified ASOs complementary to backsplice-junction sequences mediate robust knockdown of circRNAs in vitro and, thus, represent a useful tool to explore the biological roles of circRNAs in loss-of-function studies in cultured cells and animal models.

Knockdown of the long noncoding RNA PURPL induces apoptosis and sensitizes liver cancer cells to doxorubicin.

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer with increasing incidence in western countries. Most HCC patients have advanced cancer at the time of diagnosis due to the asymptomatic nature of early-stage HCC and do not qualify for potentially curative surgical treatment, thus, highlighting the need for new therapeutic strategies. Long noncoding RNAs (lncRNAs) comprise a large and heterogeneous group of non-protein coding transcripts that play important regulatory roles in numerous biological processes in cancer. In this study, we performed RNA sequencing of liver biopsies from ten HCC, ten hepatitis C virus-associated HCC, and four normal livers to identify dysregulated lncRNAs in HCC. We show that the lncRNA p53-upregulated-regulator-of-p53-levels (PURPL) is upregulated in HCC biopsies and that its expression is p53-dependent in liver cancer cell lines. In addition, antisense oligonucleotide-mediated knockdown of PURPL inhibited cell proliferation, induced apoptosis, and sensitized HepG2 human HCC cells to treatment with the chemotherapeutic agent doxorubicin. In summary, our findings suggest that PURPL could serve as a new therapeutic target for reversing doxorubicin resistance in HCC.

The evolutionarily conserved miRNA-137 targets the neuropeptide hypocretin/orexin and modulates the wake to sleep ratio.

Hypocretin (Hcrt), also known as orexin, neuropeptide signaling stabilizes sleep and wakefulness in all vertebrates. A lack of Hcrt causes the sleep disorder narcolepsy, and increased Hcrt signaling has been speculated to cause insomnia, but while the signaling pathways of Hcrt are relatively well-described, the intracellular mechanisms that regulate its expression remain unclear. Here, we tested the role of microRNAs (miRNAs) in regulating Hcrt expression. We found that miR-137, miR-637, and miR-654-5p target the human HCRT gene. miR-137 is evolutionarily conserved and also targets mouse Hcrt as does miR-665. Inhibition of miR-137 specifically in Hcrt neurons resulted in Hcrt upregulation, longer episodes of wakefulness, and significantly longer wake bouts in the first 4 h of the active phase. IL-13 stimulation upregulated endogenous miR-137, while Hcrt mRNA decreased both in vitro and in vivo. Furthermore, knockdown of miR-137 in zebrafish substantially increased wakefulness. Finally, we show that in humans, the MIR137 locus is genetically associated with sleep duration. In conclusion, these results show that an evolutionarily conserved miR-137:Hcrt interaction is involved in sleep­wake regulation.

Clinical advances of RNA therapeutics for treatment of neurological and neuromuscular diseases.

RNA therapeutics comprise a diverse group of oligonucleotide-based drugs such as antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), and short hairpin RNAs (shRNAs) that can be designed to selectively interact with drug targets currently undruggable with small molecule-based drugs or monoclonal antibodies. Furthermore, RNA-based therapeutics have the potential to modulate entire disease pathways, and thereby represent a new modality with unprecedented potential for generating disease-modifying drugs for a wide variety of human diseases, including central nervous system (CNS) disorders. Here, we describe different strategies for delivering RNA drugs to the CNS and review recent advances in clinical development of ASO drugs and siRNA-based therapeutics for the treatment of neurological diseases and neuromuscular disorders.Abbreviations 2'-MOE: 2'-O-(2-methoxyethyl); 2'-O-Me: 2'-O-methyl; 2'-F: 2'-fluoro; AD: Alzheimer's disease; ALS: Amyotrophic lateral sclerosis; ALSFRS-R: Revised Amyotrophic Lateral Sclerosis Functional Rating Scale; ARC: Antibody siRNA Conjugate; AS: Angelman Syndrome; ASGRP: Asialoglycoprotein receptor; ASO: Antisense oligonucleotide; AxD: Alexander Disease; BBB: Blood brain barrier; Bp: Basepair; CNM: Centronuclear myopathies; CNS: Central Nervous System; CPP: Cell-penetrating Peptide; CSF: Cerebrospinal fluid; DMD: Duchenne muscular dystrophy; DNA: Deoxyribonucleic acid; FAP: Familial amyloid polyneuropathy; FALS: Familial amyotrophic lateral sclerosis; FDA: The United States Food and Drug Administration; GalNAc: N-acetylgalactosamine; GoF: Gain of function; hATTR: Hereditary transthyretin amyloidosis; HD: Huntington's disease; HRQOL: health-related quality of life; ICV: Intracerebroventricular; IT: Intrathecal; LNA: Locked nucleic acid; LoF: Loss of function; mRNA: Messenger RNA; MS: Multiple Sclerosis; MSA: Multiple System Atrophy; NBE: New Biological Entity; NCE: New Chemical Entity; NHP: Nonhuman primate; nt: Nucleotide; PD: Parkinson's disease; PNP: Polyneuropathy; PNS: Peripheral nervous system; PS: Phosphorothioate; RISC: RNA-Induced Silencing Complex; RNA: Ribonucleic acid; RNAi: RNA interference; s.c.: Subcutaneous; siRNA: Small interfering RNA; SMA: Spinal muscular atrophy; SMN: Survival motor neuron; TTR: Transthyretin.

Development of siRNA Therapeutics for the Treatment of Liver Diseases.

Small interfering RNA (siRNA)-based therapeutics holds the promise to treat a wide range of human diseases that are currently incurable using conventional therapies. Most siRNA therapeutic efforts to date have focused on the treatment of liver diseases due to major breakthroughs in the development of efficient strategies for delivering siRNA drugs to the liver. Indeed, the development of lipid nanoparticle-formulated and GalNAc-conjugated siRNA therapeutics has resulted in recent FDA approvals of the first siRNA-based drugs, patisiran for the treatment of hereditary transthyretin amyloidosis and givosiran for the treatment of acute hepatic porphyria, respectively. Here, we describe the current strategies for delivering siRNA drugs to the liver and summarize recent advances in clinical development of siRNA therapeutics for the treatment of liver diseases.

New paths for sustainable solutions to tackle global and emerging infectious threats.

With the dramatic background of a newly emerged virus (SARS-CoV-2) spreading around the world, Coronavirus and other infectious health threats for the human and animal populations were illustrated and debated in excellent presentations at the IABS meeting 26-28 of February 2020. Historical evidence of pandemics and lessons learned from recent epidemics or epizootics caused by many pathogens (e.g., Ebola, Zika, and African Swine Fever viruses) illustrated the overarching need for close international cooperation. New and old technologies in vaccine development and their use were presented, resulting in a call for greater interaction between the human and the veterinary fields in order to leverage the expertise and knowledge in both human and animal medicine. The One Health concept was also emphasized for eliminating the 59,000 fatal human rabies cases annually attributed to unvaccinated dogs. For preventable, infectious diseases commonly spreading in the poorer regions of the world, a new regulatory approach and governance structure was called for to give access to affordable vaccines. Vaccines were touted as one of the most successful health invention ever introduced; on a similar level to health improvements due to clean water.

Obstacles to vaccination of animals and prospective solutions.

On the 17th of October 2019, a workshop was held at Wageningen Bioveterinary Research in Lelystad, the Netherlands, to discuss the obstacles to vaccination in the veterinary field. Participants from academia, OIE, FAO, EC, EMA, USDA, national regulatory and veterinary health authorities, and the animal health industry discussed how availability and access to animal vaccines can be improved not just in the EU and US but also in Low to Middle Income Countries (LMIC) across the world and agreed that this requires innovations in both the scientific and the regulatory field. The workshop called for engaging all stakeholders to improve regulatory acceptance of novel vaccine technologies and encourage their registration. There is a need for better mutual understanding between academia, industry and regulators, and more openness to discuss framework, requirements, and product authorisations, and to converge the regulatory rules between regions. The next leap forward could be a broader application of novel technologies using RNA- or DNA-based vaccine platforms, where the "backbone" is maintained, while the gene of interest coding for an immunogenic protein can be exchanged in a standardised manner. This approach enables rapid response in outbreak situations and should lower the risk and cost of vaccine development.

Diagnostics in the veterinary field: The role in health surveillance and disease identification.

An international workshop, held in Wiesbaden, Germany on 15-17 May 2019 provided an overview of existing and new methods and approaches to diagnostics in animal health and their benefits and challenges. The variability in quality and authority review of test kits across the world is a concern for the reliability of test results and the decisions that are based on the diagnostic data. In countries or regions without regulatory oversight, there is an urgent need for international harmonisation of quality requirements and licensing procedures. This would increase the validity of the diagnostic methods and allow mutual recognition of test results within the network of official control laboratories and amongst animal health officials. Regional cooperation, as well as the OIE Laboratory Network, should be used to support licensing procedures, pool resources for serum and sample banks, survey outbreak responses, and coordinate research and development of new veterinary diagnostics. The end-users must have clear information on a test's performance, limitations, and interpretation of results.

CD8+ T cells from patients with narcolepsy and healthy controls recognize hypocretin neuron-specific antigens.

Narcolepsy Type 1 (NT1) is a neurological sleep disorder, characterized by the loss of hypocretin/orexin signaling in the brain. Genetic, epidemiological and experimental data support the hypothesis that NT1 is a T-cell-mediated autoimmune disease targeting the hypocretin producing neurons. While autoreactive CD4+ T cells have been detected in patients, CD8+ T cells have only been examined to a minor extent. Here we detect CD8+ T cells specific toward narcolepsy-relevant peptides presented primarily by NT1-associated HLA types in the blood of 20 patients with NT1 as well as in 52 healthy controls, using peptide-MHC-I multimers labeled with DNA barcodes. In healthy controls carrying the disease-predisposing HLA-DQB1*06:02 allele, the frequency of autoreactive CD8+ T cells was lower as compared with both NT1 patients and HLA-DQB1*06:02-negative healthy individuals. These findings suggest that a certain level of CD8+ T-cell reactivity combined with HLA-DQB1*06:02 expression is important for NT1 development.

Absence of autoreactive CD4+ T-cells targeting HLA-DQA1*01:02/DQB1*06:02 restricted hypocretin/orexin epitopes in narcolepsy type 1 when detected by EliSpot.

Narcolepsy type 1, a neurological sleep disorder strongly associated with Human Leukocyte Antigen (HLA-)DQB1*06:02, is caused by the loss of hypothalamic neurons producing the wake-promoting neuropeptide hypocretin (hcrt, also known as orexin). This loss is believed to be caused by an autoimmune reaction. To test whether hcrt itself could be a possible target in the autoimmune attack, CD4+ T-cell reactivity towards six different 15-mer peptides from prepro-hypocretin with high predicted affinity to the DQA1*01:02/DQB1*06:02 MHC class II dimer was tested using EliSpot in a cohort of 22 narcolepsy patients with low CSF hcrt levels, and 23 DQB1*06:02 positive healthy controls. Our ELISpot assay had a detection limit of 1:10,000 cells. We present data showing that autoreactive CD4+ T-cells targeting epitopes from the hcrt precursor in the context of MHC-DQA1*01:02/DQB1*06:02 are either not present or present in a frequency is <1:10,000 among peripheral CD4+ T-cells from narcolepsy type 1 patients.

Cerebrospinal Fluid Biomarkers of Neurodegeneration Are Decreased or Normal in Narcolepsy.

Objectives: To investigate whether cerebrospinal fluid (CSF) biomarkers of neurodegeneration are altered in narcolepsy in order to evaluate whether the hypocretin deficiency and abnormal sleep-wake pattern in narcolepsy leads to neurodegeneration. Methods: Twenty-one patients with central hypersomnia (10 type 1 narcolepsy, 5 type 2 narcolepsy, and 6 idiopathic hypersomnia cases), aged 33 years on average and with a disease duration of 2-29 years, and 12 healthy controls underwent CSF analyses of the levels of ß-amyloid, total tau protein (T-tau), phosphorylated tau protein (P-tau181), α-synuclein, neurofilament light chain (NF-L), and chitinase 3-like protein-1 (CHI3L1). Results: Levels of ß-amyloid were lower in patients with type 1 narcolepsy (375.4 ± 143.5 pg/mL) and type 2 narcolepsy (455.9 ± 65.0 pg/mL) compared to controls (697.9 ± 167.3 pg/mL, p < .05). Furthermore, in patients with type 1 narcolepsy, levels of T-tau (79.0 ± 27.5 pg/mL) and P-tau181 (19.1 ± 4.3 pg/mL) were lower than in controls (162.2 ± 49.9 pg/mL and 33.8 ± 9.2 pg/mL, p < .05). Levels of α-synuclein, NF-L, and CHI3L1 in CSF from narcolepsy patients were similar to those of healthy individuals. Conclusion: Six CSF biomarkers of neurodegeneration were decreased or normal in narcolepsy indicating that taupathy, synucleinopathy, and immunopathy are not prevalent in narcolepsy patients with a disease duration of 2-29 years. Lower CSF levels of ß-amyloid, T-tau protein, and P-tau181 in narcolepsy may indicate that hypocretin deficiency and an abnormal sleep-wake pattern alter the turnover of these proteins in the central nervous system.

Survey of 800+ data sets from human tissue and body fluid reveals xenomiRs are likely artifacts.

miRNAs are small 22-nucleotide RNAs that can post-transcriptionally regulate gene expression. It has been proposed that dietary plant miRNAs can enter the human bloodstream and regulate host transcripts; however, these findings have been widely disputed. We here conduct the first comprehensive meta-study in the field, surveying the presence and abundances of cross-species miRNAs (xenomiRs) in 824 sequencing data sets from various human tissues and body fluids. We find that xenomiRs are commonly present in tissues (17%) and body fluids (69%); however, the abundances are low, comprising 0.001% of host human miRNA counts. Further, we do not detect a significant enrichment of xenomiRs in sequencing data originating from tissues and body fluids that are exposed to dietary intake (such as liver). Likewise, there is no significant depletion of xenomiRs in tissues and body fluids that are relatively separated from the main bloodstream (such as brain and cerebro-spinal fluids). Interestingly, the majority (81%) of body fluid xenomiRs stem from rodents, which are a rare human dietary contribution but common laboratory animals. Body fluid samples from the same studies tend to group together when clustered by xenomiR compositions, suggesting technical batch effects. Last, we performed carefully designed and controlled animal feeding studies, in which we detected no transfer of plant miRNAs into rat blood, or bovine milk sequences into piglet blood. In summary, our comprehensive computational and experimental results indicate that xenomiRs originate from technical artifacts rather than dietary intake.

Normal Morning Melanin-Concentrating Hormone Levels and No Association with Rapid Eye Movement or Non-Rapid Eye Movement Sleep Parameters in Narcolepsy Type 1 and Type 2.

STUDY OBJECTIVES: Other than hypocretin-1 (HCRT-1) deficiency in narcolepsy type 1 (NT1), the neurochemical imbalance of NT1 and narcolepsy type 2 (NT2) with normal HCRT-1 levels is largely unknown. The neuropeptide melanin-concentrating hormone (MCH) is mainly secreted during sleep and is involved in rapid eye movement (REM) and non-rapid eye movement (NREM) sleep regulation. Hypocretin neurons reciprocally interact with MCH neurons. We hypothesized that altered MCH secretion contributes to the symptoms and sleep abnormalities of narcolepsy and that this is reflected in morning cerebrospinal fluid (CSF) MCH levels, in contrast to previously reported normal evening/afternoon levels. METHODS: Lumbar CSF and plasma were collected from 07:00 to 10:00 from 57 patients with narcolepsy (subtypes: 47 NT1; 10 NT2) diagnosed according to International Classification of Sleep Disorders, Third Edition (ICSD-3) and 20 healthy controls. HCRT-1 and MCH levels were quantified by radioimmunoassay and correlated with clinical symptoms, polysomnography (PSG), and Multiple Sleep Latency Test (MSLT) parameters. RESULTS: CSF and plasma MCH levels were not significantly different between narcolepsy patients regardless of ICSD-3 subtype, HCRT-1 levels, or compared to controls. CSF MCH and HCRT-1 levels were not significantly correlated. Multivariate regression models of CSF MCH levels, age, sex, and body mass index predicting clinical, PSG, and MSLT parameters did not reveal any significant associations to CSF MCH levels. CONCLUSIONS: Our study shows that MCH levels in CSF collected in the morning are normal in narcolepsy and not associated with the clinical symptoms, REM sleep abnormalities, nor number of muscle movements during REM or NREM sleep of the patients. We conclude that morning lumbar CSF MCH measurement is not an informative diagnostic marker for narcolepsy.

Use of colistin-containing products within the European Union and European Economic Area (EU/EEA): development of resistance in animals and possible impact on human and animal health.

Since its introduction in the 1950s, colistin has been used mainly as a topical treatment in human medicine owing to its toxicity when given systemically. Sixty years later, colistin is being used as a last-resort drug to treat infections caused by multidrug-resistant (MDR) Pseudomonas aeruginosa, Acinetobacter baumannii and Enterobacteriaceae (e.g., Escherichia coli, Klebsiella pneumoniae), for which mortality can be high. In veterinary medicine, colistin has been used for decades for the treatment and prevention of infectious diseases. Colistin has been administered frequently as a group treatment for animal gastrointestinal infections caused by Gram-negative bacteria within intensive husbandry systems. Given the ever-growing need to retain the efficacy of antimicrobials used to treat MDR infections in humans, the use of colistin in veterinary medicine is being re-evaluated. Despite extensive use in veterinary medicine, there is limited evidence for the development of resistance to colistin and no evidence has been found for the transmission of resistance in bacteria that have been spread from animals to humans. Since surveillance for colistin resistance in animals is limited and the potential for such transmission exists, there is a clear need to reinforce systematic monitoring of bacteria from food-producing animals for resistance to colistin (polymyxins). Furthermore, colistin should only be used for treatment of clinically affected animals and no longer for prophylaxis of diseases, in line with current principles of responsible use of antibiotics.