Traumatic acute subdural haematoma is a debilitating condition. Laterality intuitively influences management and outcome. However, in contrast to stroke, this research area is rarely studied. The aim is to investigate whether the hemisphere location of the ASDH influences patient outcome.For this multicentre observational retrospective cohort study, patients were considered eligible when they were treated by a neurosurgeon for traumatic brain injury between 2008 and 2012, were > 16 years of age, had sustained brain injury with direct presentation to the emergency room and showed a hyperdense, crescent shaped lesion on the computed tomography scan. Patients were followed for a duration of 3-9 months post-trauma for functional outcome and 2-6 years for health-related quality of life. Main outcomes and measures included mortality, Glasgow Outcome Scale and the Quality of Life after Brain Injury score. The hypothesis was formulated after data collection.P = 0.07).Of the 187 patients included, 90 had a left-sided ASDH and 97 had a right-sided haematoma. Both groups were comparable at baseline and with respect to the executed treatment. Furthermore, both groups showed no significant difference in mortality and Glasgow Outcome Scale score. Health-related quality of life, assessed 59 months (IQR 43-66) post-injury, was higher for patients with a right-sided haematoma (Quality of Life after Brain Injury score: 80 vs 61, This study suggests patients with a right-sided acute subdural haematoma have a better long-term health-related quality of life compared to patients with a left-sided acute subdural haematoma.The online version contains supplementary material available at 10.1186/s12883-022-02790-3. Traumatic brain injury (TBI) is rising in incidence and is a major cause of morbidity and mortality. Those who survive often suffer from lifelong disability , 2. It iLaterality of the haematoma does not play a major role in the decision for surgery, as is reflected in the absence of consideration in TBI guidelines . FurtherThis is a retrospective observational cohort study of all ASDH patients treated consecutively between 2008 and 2012 by three Dutch neurosurgical departments in level 1 trauma centres . The twoPatients were included if they were older than 16 years, had a clinical diagnosis of blunt traumatic brain injury, presented directly to the emergency room and showed a hyperdense, crescent shaped lesion – indicative of an ASDH – on the computed tomography (CT) scan. Exclusion criteria were non-traumatic ASDH, ASDH secondary to an earlier procedure, penetrating injury and concomitant focal intracranial lesions  that required emergency surgery. Patients who were withheld from treatment because they were deemed unsalvageable or had severe comorbidity, were excluded as well.The study was approved by the medical ethics committee of the Leiden University Medical Centre. All patients and caregivers who participated gave written informed consent.Data on demographics, medical history, use of anticoagulants or antiplatelet agents, injury-related variables, radiological variables, treatment variables, complications, and outcome variables were collected from electronic patient files.Injury-related variables included trauma mechanism, first emergency room Glasgow Coma Scale (GCS) score, the severity of the patients’ initial GCS score , focal neurological symptoms , pupillary light reflex, clinical deterioration .The first CT scan was used to assess the radiological variables that included the thickness of the haematoma, the amount of midline shift (MLS), the presence of concomitant contusion and the patency of the basal cisterns. Furthermore, if a second preoperative CT scan was made, it was noted whether it showed radiological deterioration .Therapy-related variables consisted of the maximum ICP and the type of management . The treatment was considered conservative when the neurosurgeon on call decided not to operate acutely. It was considered surgically when a report was made on the indication and the surgical procedure was started after the last CT ordered or performed in the emergency room. Decompressive craniectomy was defined as such when the neurosurgeon did not reconstruct the bony defect after the craniotomy.Furthermore, all complications during admission that required medical attention (e.g. antibiotic treatment for infection) were noted. Complications were defined as intracranial , cardiovascular , respiratory , metabolic , infections , or other.Outcome measures were mortality at discharge, functional outcome and the Hr-QoL. Hr-QoL was the primary outcome measurement for this study. Functional outcome was judged according to the 5-point Glasgow Outcome Score (GOS)  derived from outpatient follow-up letters at three to 9 months post-trauma which were dichotomized into GOS favourable (GOS 4–5) and unfavourable (GOS 1–3). The Hr-QoL was assessed using the TBI-specific Qolibri questionnaire , 20–23. The statistical analysis was done using IBM SPSS Statistics 23. To assess differences between groups, appropriate tests were used according to distribution and scale of measurement.p-values and effect size r. A difference was considered significant with a p < 0.05. In order to provide a summary baseline prognostic score for both groups, a multivariable logistic regression model, based on all available variables featured by Corticosteroid Randomization After Significant Head Injury (CRASH) CT head injury prognosis model , was used to estimate a calculated predicted probabilities for mortality and unfavourable outcome [ and reported in  outcome . In sensOut of 612 screened consecutively admitted patients, 190 were included. For this study three patients were excluded due to unknown location of the ASDH. Of the remaining 187 patients, 90 had a left-sided ASDH and 97 had a right-sided ASDH  with a median age of 68 years (Interquartile range (IQR): 53-77). The right-sided ASDH cohort consisted of 46 patients (47.4%) were female with a median age of 68 years (IQR: 55-76). Furthermore, both groups showed no significant difference in TBI severity. Finally, the equal CRASH scores confirmed the short term  and long term (predicted unfavourable outcome) baseline comparability of both groups. There were no differences in proportions undergoing surgical treatment and DC or the proportion complications Table . Furtherp = 0.04, r = 0.32). The subgroup of patients without concomitant contusion consisted of 33 patients . In sensitivity analysis excluding these patients the Qolibri scores did not differ between left and right-sided ASDH .Seventy-seven patients (45%) died during hospital admission and 103 patients had a GOS < 4. The mortality and GOS did not differ between left- and right-sided haematoma patients. Subsequently, 24 patients (24%) died during the follow-up period , 13 (13%)) and 8 (4%) were still severely disabled , 4 (4%)), leaving 78 patients for the Qolibri assessment. Thirty-three did not respond, amounting to a loss to follow-up of 42%. The median duration of follow-up was 59 months (IQR: 43-66). Left-sided ASDH patients had a median Qolibri score of 61 (IQR: 52-74) whereas right-sided ASDH patients had a median Qolibri score of 80 (IQR: 56-94) (Table In this retrospective study patients with a right-sided ASDH compared to left-sided ASDH tended to have a better Hr-QoL while having similar mortality and functional outcome. Right-sided haematoma is associated with a Qolibri score comparable with healthy individuals whereas the Qolibri score of patients with a left-sided haematoma indicate some form of persistent impairment . Both grTo our knowledge, this is the first study to investigate the influence of laterality on functional outcome and Hr-QoL in patients with ASDH. The only previous study on hemispheric outcome difference in ASDH found a higher frequency of intractable brain swelling and mortality rate in patients with a left-sided ASDH. This difference was only significant if patients presented with a concomitant contusion , similarIn stroke, the impact of laterality on outcome and treatment effect has been investigated elaborately. Left-sided strokes seem to fare worse considering overall outcome and seem to have a two-fold increased chance of a good outcome after thrombolysis compared to patients with right-sided hemispheric stroke . PatientThe lower Hr-QoL in left-sided ASDH may be explained by the dominance of the left hemisphere and the linguistic centres. A TBI or stroke causing damage in these regions would cause dysphasia or aphasia and therefore have a more pronounced impact on Hr-QoL , 32. AnoResearch supports the notion that the perception and expression of emotion, as well as the autonomic arousal processes and awareness, are primarily represented in the right cerebral hemisphere. Patients who suffer damage of the right hemisphere often display aprosodia, asomatognosia, anosognosia, neglect, anosodiaphoria and have an impaired ability to recognise and express emotions , 42. PatThe results of this study can be applied to the majority ASDH patients presenting to a neurologist or neurosurgeon. However, some important study characteristics influence generalizability. The large proportion Dutch elderly reflects the rising age in TBI in developed countries. Furthermore, the applicability can differ between countries, since the Qolibri scores in healthy individuals differs.This study has numerous limitations of which some are mentioned here. The small sample and retrospective design hampers definite conclusions. Furthermore, laterality is used as a surrogate for the dominant hemisphere. Another limitation is the higher loss to follow-up for left-sided ASDH which may cause selection or attrition bias. However, the proportions of patients not able to respond to the questionnaire due to death or disability was comparable in both groups. Strengths are the baseline comparability of both groups and the long-term determination of Hr-QoL at 5 years.Future studies should re-estimate the association between ASDH laterality and outcome prospectively, while at the same time focus on the potential clinical relevance in terms of the changes over time. Long-term follow up of stroke patients has identified a possible mismatch between patients’ needs and delivered health care. Prolonged attention to healthcare needs may improve experienced Hr-QoL . In thisTraumatic ASDH is a debilitating condition with, in contrast to stroke, no known distinction in outcome between left-sided and right-sided ASDH. This multicentre observational study showed that patients with a right-sided ASDH have a higher Qolibri score compared to patients with a left-sided ASDH. This article is the first report on the existence of a difference in Hr-QoL based on location of the ASDH. Understanding the origins of this difference could provide additional insight on the laterality of brain functions, the extent to which subdural haemorrhage induces focal brain damage and ultimately prevention of the reduced Hr-QoL in patients with a left-sided ASDH.Additional file 1.


Background: Post-COVID Clinics were recommended for patients with persistent symptoms following COVID-19, but no specific tests were suggested for evaluation. This study aimed to present a post-COVID clinic patient cohort and evaluate the use of a post-COVID symptom questionnaire (PCQ) score. Methods: Patients were referred from a population of approximately 1 million citizens. PCQ and standardized health scales were registered. Descriptive analyses were performed to assess the prevalence of symptoms, and correlation analyses was undertaken to asses convergent and discriminant trends between PCQ scores and health scales. Results: Of 547 patients, 447 accepted inclusion. The median age was 47 years and 12% of the patients were hospitalized. At a median of 6.3 (IQR 4.4–9.9) months after the onset of symptoms, 82% of the patients reported both physical exhaustion and concentration difficulties. Functional disability and extreme fatigue were reported as moderate to severe by 33% and 62% of the patients, respectively. The PCQ score correlated significantly with each of the standardized health scales. Conclusion: Patients referred to a Post-COVID Clinic were previously generally healthy. At the time of diagnosis, they reported multiple symptoms with severely affected health. The PCQ score could be used as valid measure of Post-COVID severity. Long-term symptoms following coronavirus disease COVID-19) (long COVID) were recognized early in the epidemic [9  and to include recovery. “Muscle and joint symptoms and conditions” did not meet an a priori consensus criteria for inclusion in the COS. However, these were rated of high importance among people with long COVID [The World Health Organization (WHO) published a clinical case definition of post-COVID condition , but theng COVID .In Denmark, concerns of how to care for patients with long COVID led to the establishment of national post-COVID-19 services in the Departments of Pulmonology and Infectious Diseases. During the summer of 2020, the Danish Health Authority gathered experts to draft recommendations. In October 2020, it was recommended to establish post-COVID clinics in all of the five Danish regions to care for patients with this poorly defined disease, and to gather data for quality assurance and research . MultiplThe objective of this study was to describe patients included during the first nine months in a post COVID clinic in a Danish region, Central Denmark Region (approx. 1.3 million inhabitants), using a post-COVID-19 symptom questionnaire (PCQ) and standardized health scale measures, and to compare PCQ scores with standardized health scales. We hypothesized that the severity of symptoms reported in the PCQ correlate to the severity measured by other standardized measures. This is the first description of a Danish post-COVID cohort including all patients referred for examination in a regional post-COVID-19 clinic due to persistent symptoms.In the beginning of 2021, a post-COVID clinic was established and a multidisciplinary research project on long-term health consequences of COVID-19 illness (MULTICOV), registering patient symptoms was initiated at Aarhus University Hospital in Central Denmark Region, covering an uptake area with 17% (1.0 of 5.8 million) of the Danish Population.As early reports described possible long-term cognitive disturbances, dyspnea and fatigue and a number of other less frequent symptoms ,3, we aiThe selection of symptoms  and valiOrgan-specific and general symptom assessment: Selection of acute and long-term symptoms was based on initial interviews of hospitalized patients ,11, clinCognitive assessment: During the start of the pandemic, we registered cognitive impairment by telephone interviews ,11. For Dyspnea assessment: A previously well-validated standardized scale was chosen for the registration of how dyspnea affected level of functioning. The Medical Research Council (MRC) Dyspnea Scale is commonly used for patients with chronic dyspnea . DyspneaFatigue assessment: As fatigue was a common manifestation in long COVID patients and the mental or physical component was unknown, we used the Fatigue Assessment Scale (FAS) , which iGeneral health assessment: Health-related quality of life and functional disability were recognized markers of morbidity and health outcomes ,24,29. TMental health assessment: To rule out psychiatric illness, we wished to register anxiety, as well as depression and general strain. For this purpose, we used the Symptom Checklist , in which all three conditions could be registered using 13 questions ,30. We fBackground information: Data on demographic characteristics and medical history in the post-COVID cohort were retrieved from the patient file. An interview guide was used by the physician including the variables shown in The post-COVID clinics in Central Denmark Region were established 1st of February 2021 and located at two hospitals. One in the Department of Infectious Diseases at Aarhus University Hospital (AUH), which covers an uptake area of 1 million people, equivalent to ¾ of the population in the region . In thisPatients aged 18 and over experiencing unexpected or complex and prolonged symptoms for more than 12 weeks were eligible for referral by their GP. Persistent symptoms were to have occurred in timely correlation after COVID-19 infection and we recommended a screening for other concurrent conditions before referral. Referral letters were declined in case of obvious referral for other reasons, such as referrals on suspicion of adverse effects of vaccination. Before March 2021, the Danish Health Authority recommended patients to be referred as early as six weeks following SARS-CoV-2 infection. However, during February and March 2021, patients were not assessed until three months after onset of symptoms due to a waiting list. Patients on the waiting list were phoned before they were scheduled for assessment. Patients who improved spontaneously during the 12 weeks after onset of symptoms were thus not assessed in the clinic. According to guidelines from the Danish Health Authority, patients with simple or spontaneously improving health issues following COVID-19 were treated by a GP . PatientPatients were included in the post-COVID cohort at their first consultation in the Post-COVID Clinic, where they underwent a thorough medical examination by a multidisciplinary team using PCQ/standardized health scales as a part of the examination and assessment. Objective examination, broad biochemical analyses and other paraclinical examination depending on indications, such as lung function test in patients with dyspnea, were performed. Data on demography, medical history and results from paraclinical investigations, along with patient reported data from the PCQ and standardized health scales , were reAcute symptoms: Symptoms during the acute phase of COVID-19 were registered as present or not present. In case of registration of “unchanged compared to before the acute phase of COVID-19” or “don’t know”, the response was categorized as “not present” in analyses. Long COVID symptoms: The severity of each symptom during a four-week time period before the evaluation in the clinic was graded between 0 and 4 . Symptoms “unchanged compared to before the acute phase of COVID-19” were counted as none. In binary analyses, symptoms were considered present in case the answer was some, a lot or very much. A PCQ score was calculated by adding all 31 scores of long COVID symptoms (0–124), as subjective fever was reported as yes or no , and thus, not included in the PCQ score. According to COS recommendations , which rStandardized scores: The MRC Dyspnea Scale is a 5-point scale 1–5, where 3–5 scores are considered severe . An OMC p-values using Bonferroni correction were presented.Data were analyzed using Stata Intercooled version 17. Median (interquartile range (IQR)), means (standard deviation (sd)) and frequencies were reported. Missing values in PCQ and organ-specific and FAS scores were counted as 0 unless all answers were missing. Symptom scores were compared in two-way graphs with linear prediction of correlation, and statistical significance of correlation was calculated. Spearman correlation coefficients and Of the 547 patients, 447 (82%) accepted participation in the post-COVID cohort study . ParticiPatients reported several new onset symptoms. During the acute phase of COVID-19, the most reported new onset symptoms were physical exhaustion (94%), headache (82%), myalgia (80%), cough (76%), fever (74%) and altered sense of smell (76%) and taste (73%) . Less thStandardized scales indicated impaired cognition in 26% (OMC ≤ 24), severe disability due to dyspnea in 28% (MRC 3–5), functional disability in 33% (PCFS 3–4), extreme fatigue in 62% (FAS ≥ 35) and indication of BDS was found in 11% (4–5 domains ≥ 4) of the patients. The patients rated their health on an EQ VAS scale (1–100) at a mean of 58 (sd 19) and had a mean quality of life according to the EQ-5D-5L index of 0.72 (sd 0.19). Fifteen percent had a depression score of SCL >8 and 28 % had an anxiety score SCL > 5 .Comparing patients hospitalized during the acute phase of COVID-19 with non-hospitalized patients, odds were higher of long COVID dyspnea  and odds ratio for MRC ≥ 3 was 3.19 (1.64–6.22)). Otherwise, long COVID symptoms did not differ significantly when comparing hospitalized with non-hospitalized patients.p-value was < 0.0001 for all correlations. The EQ-5D-5L had the highest correlation coefficient of 0.45 (The median PCQ score was 32 (IQR 23–44)  and the  of 0.45 .p < 0.000, p < 0.001 for all correlations except EQ-5D-5L anxiety item vs. PCFS).We exploratively examined the correlation between the organ-specific symptom scores of CNS, CP, GI and MS symptoms  and fouA total of 1.1% of SARS-CoV-2 positive individuals from the hospital catchment area were referred to the Post-COVID Clinic. The patients were previously healthy and had severely affected health at time of referral. Patients reported multiple symptoms; above 80% of them reported concentration difficulties and physical fatigue, two thirds reported extreme fatigue and one third reported reduced level of functioning 6.3 months after onset of symptoms. The PCQ score correlated with PCFS, FAS and quality of life scores , and we suggest the PCQ score to be used as a measure of the individual burden of symptoms.Despite few comorbidities and previous health risk factors, new onset symptoms were reported by more than 80% of patients with respect to disturbed sleep, concentration difficulties and physical exhaustion. Several other symptoms were common  . In systUsing the Montreal Cognitive Assessment, cognitive impairment was registered in 17% of hospitalized patients in a UK multicenter study (PHOS-COVID)  and cognSeverely affected functioning due to dyspnea (MRC dyspnea score 3–5) was found in 28% of our patients, even though asthma and COPD were only reported in 14% and 2% of the patients, respectively, and only 12% were hospitalized during the acute phase of COVID-19 and had more severe long COVID dyspnea. Huang et al. reported an mMRC ≥ 1 in 26% of patients six months after hospitalization . Thus, patients in our cohort reported dyspnea more frequently than hospitalized patients six months after onset of symptoms . COPD paFatigue is the most prevalently reported symptom in long COVID ,8,11,13.The PCFS was now validated against EQ-5D-5L and SF-36 ,50, and We found an EQ-5D-5L index of 0.72 among our patients, which is a better health-related quality of life score compared with most published data on chronic diseases (−0.20–1.00) , but a lIn conclusion, assessment of patients in a post-COVID clinic using graded symptom questions in the PCQ and standardized health scales showed that patients were affected in all areas suggested in COS recommendations  and stanWe found that the PCQ score correlated with all standardized health scales. Construct validity evaluation of PCFS was previously found to correlate with long COVID symptoms  , and wasExploratively, we constructed organ-specific scores, which were in agreement with NICE, UK guidelines and COS. Only in NICE guidelines, are musculoskeletal symptoms categorized separately, as in this study ,17,19. WPatients in this study were mainly female (72%). An equal number of females and males were infected with SARS-CoV-2 during the pandemic ; howeverReturning back to normal life after infection has previously been shown to exacerbate even subtle cognitive symptoms , howeverMultiple factors may affect the selection of patients referred to the Post-COVID Clinic, such as the GP or patients’ suspicion of long COVID, knowledge of the existence of a post-COVID clinic, level of health literacy or the presence of symptoms affecting daily functioning. Patients were primarily women, middle aged, often of higher education, which may affect patient-reported outcomes. Recall bias may also have affected the registration of symptoms during acute COVID-19. While interviewer bias may have caused the registration of more symptoms while filling in a questionnaire used in the clinical assessment, we have no reason to think this would have caused differences in registration in the PCQ vs. standardized health scores. Missing values considered as zero and “unchanged symptom” considered not present might have underestimated severity of disease, but our cohort, however, still appeared to have numerous symptoms. In this study, we use the unique opportunity to describe some of the more severely affected long COVID patients referred due to unexpected, complex and prolonged symptoms. The participation rate was high (82%) and non-participants’ age and gender did not differ from participants. We registered new onset symptoms and SARS-CoV-2 test results to ensure the condition described was, in fact, long COVID.Collectively, results indicate that our patient cohort included patients who were previously healthy and who were among some of the more severely affected long COVID patients. Post-COVID clinics in other regions of Denmark used the same referral criteria, and a number of post-COVID clinics worldwide report seeing patients with multiple symptoms and low functional level ,15,16,17No specific diagnostic or paraclinical tests are suggested in national or international guidelines, and the various assessment tools used in different settings underline the complexity of the numerous symptoms described in patients with long COVID. We did not aim to cover the complexity of long COVID-19 disease, and the PCQ was prepared as a supplement to the anamnesis. Other studies registered up to 115 different symptoms and the registration of more symptoms might have detected overlooked phenomena . Even soThe symptoms reported in the PCQ occur after other infectious diseases, but little is known of the prevalence and duration. Future studies might benefit from focusing on the systematic collection of data following epidemics.The post-COVID clinics were established to receive patients with persistent symptoms after COVID-19 infection with regard to diagnostics and treatment. This made it possible to create a cohort of patients with severe symptoms. Multiple symptoms and extreme fatigue, as well as reduced functional level, were reported. A PCQ questionnaire including a wide spectrum of symptoms significantly correlated with burden of disease in standardized health scales and may be used for evaluation of severity of symptoms in long COVID. Follow-up studies are needed to evaluate whether symptom scales can predict recovery. We hope this study will encourage the development of common validated screening and outcome measures for use in prospective cohort studies and treatment trials.


The case-control study is a widely used method for investigating the genetic landscape of binary traits. However, the health-related outcome or disease status of participants in long-term, prospective cohort studies such as the UK Biobank are subject to change. Here, we develop an approach for the genetic association study leveraging disease liabilities computed from a deep patient phenotyping framework (AI-based liability). Analyzing 44 common traits in 261,807 participants from the UK Biobank, we identified novel loci compared to the conventional case-control (CC) association studies. Our results showed that combining liability scores with CC status was more powerful than the CC-GWAS in detecting independent genetic loci across different diseases. This boost in statistical power was further reflected in increased SNP-based heritability estimates. Moreover, polygenic risk scores calculated from AI-based liabilities better identified newly diagnosed cases in the 2022 release of the UK Biobank that served as controls in the 2019 version (6.2% percentile rank increase on average). These findings demonstrate the utility of deep neural networks that are able to model disease liabilities from high-dimensional phenotypic data in large-scale population cohorts. Our pipeline of genome-wide association studies with disease liabilities can be applied to other biobanks with rich phenotype and genotype data. The UK Biobank (UKBB) database has genotype data for about 500,000 participants and their socio-demographic, lifestyle and health-related phenotypes4. It provides a cohort of unparalleled size and scope, allowing the studies of human genetics research6. Over the past 15 years, genome-wide association studies (GWAS) of continuous and binary traits have provided new insights into the genetic architecture of a wide range of complex diseases5. However, while large-scale genotyping helps identify and localize common-to-rare variant signals, the proportion of phenotypic variance captured is limited7. In addition, recent studies have reported that the heritability estimates 9. This gap also labeled as “missing heritability” is substantial and can be due to multiple factors, such as sample size, omitting rare and structural variants, as well as gene-environment interactions10.The advent of biobanks has greatly reduced the cost and difficulty of collecting large genome-wide data13. Time-to-event analyses are computationally expensive and alternative approaches have focused on integrating family history to capture the underlying disease liability. Genome-wide association study by proxy (GWAX) where individuals who reported cases within family members are included as cases themselves can identify new risk loci14. GWAX has been particularly popular in the study of Alzheimer’s disease where 46,828 proxy-cases in addition to 2,447 diagnosed cases in the UKBB contributed to increased statistical power16. Going beyond a binary phenotype, more refined models have used family history to derive a continuous genetic liability phenotype where CC and family history configurations are differentiated more appropriately17.To unravel genetics’ role in disease studies, researchers typically compare patients with the disease (the cases) to individuals who are not (the controls). However, patient cohorts may progress beyond a dichotomous setting of “diseased”and “non-diseased”. Healthy patients can become diseased years down the line and GWAS studies explicitly modelling age-at-onset showed an increase in statistical power compared to case-control (CC) studies19. Our study seeks to improve genetic prediction with deep learning-based estimates of disease liability probabilities that leverage joint semantic and structure-based embeddings of phenotypes in the UKBB. To the best of our knowledge, this is the first demonstration of leveraging deep neural networks to present disease phenotypes quantitatively for genetic studies of complex traits.Although previous studies have identified thousands of disease-associated loci, researchers have yet to deeply explore the joint contribution of family history, clinical measures, and lifestyle factors to model disease liabilities. Machine learning techniques have advanced in recent years, allowing to reveal patterns in massive, high-dimensional datasets and capturing non-linear relationships20 to recognize a comprehensive set of phenotype labels in UK Biobank. Based on this framework, we present a methodology to improve the genetic prediction of complex traits using a continuous representation of the phenotypes based on individual-level data in the UKBB. Applied to 44 complex traits, our method improved statistical power in GWASs as demonstrated through higher heritability estimates, and higher polygenic risk score (PRS) for newly diagnosed cases. We also found many putative disease susceptibility variants for phenotypes such as essential hypertension, breast neoplasm, uterine leiomyoma, hypothyroidism, type 2 diabetes, alcoholism, myocardial infarction, and brain cancer compared to traditional CC studies.Recently, we developed a framework POPDx20 to extract multiple layers of information, including electronic medical records, laboratory tests, physical measurements, answered questionnaires and interviews about the socio-demographic status, lifestyle, and family history of 392,246 individuals in the UK Biobank. The overview of our artificial intelligence (AI)-based genetic analysis is illustrated in 20 for patient phenotyping in UK Biobank. The performance of the patient phenotyping is shown in We used a deep learning framework21. According to earlier research, CABLES2 has a crucial function in colorectal carcinogenesis22. 23. ZC3H4 (zinc finger CCCH domain-containing protein 4) is a candidate gene associated with susceptibility to type 2 diabetes (T2D) identified via the combined approach alone. zinc finger protein. Previous research indicates ZC3H4 having an essential role in mediating non-coding transcription in human cells24. It was also previously reported the association ZC3H4 and uncontrolled eating25. Additional results from the GWAS studies are provided in the We leveraged AI-based disease liabilities to conduct GWAS analyses and discovered independent risk loci for a wide range of complex and polygenic traits. Three methods of patient characterization were used to identify disease-associated risk loci: (1) cases and controls defined by the in-patient diagnosis record of the UK Biobank dataset (CC), 2) AI-based disease liabilities, (3) a combination of AI-based disease liabilities and case-control status (combined CC-liability).  AI-based28. For breast cancer , rs13281615 (PCAT1) is highly associated in both AI-based and combined approach. It was not identified in our CC analysis but was reported in other breast cancer studies30. Some SNPs such as rs10889338 (DOCK7), rs12916 (HMGCR), rs10455872 (LPA), rs15285 (LPL), rs507666 (ABO), and rs11057830 (SCARB1) were identified to be associated with hypercholesterolemia using the CC-liability method. These SNPs were found to be significant in the other GWAS studies of the UKBB31.We performed an in-depth investigation of our GWAS results (Supplementary Table GWAS Significant Loci). Most of the significant loci identified by the liability and CC-liability methods have been either reported in our CC analysis or other previous studies. Some SNPs are missed in the CC study but could be validated in the literature. For example, rs6544713 (ABCG8) and rs12190287 (TCF21) identified in our combined approach for coronary atherosclerosis were previously found to be associated with the risk for coronary artery disease32  to infer phecodes and calculate disease liability probabilities (section POPDx). Genetic data analyses were restricted to unrelated white British individuals that passed additional genetic quality control (QC) filters according to the UKBB . Furthermore, samples were excluded if the genetically inferred sex did not match the self-reported sex and if they had withdrawn their consent. Analyses were performed on autosomal genotype data provided by the UK Biobank with MAF > 0.01 . Further quality filters and overlapping with external datasets  could reduce this number to 577,395 markers. All reported genetic analyses are for 261,807 unrelated white British individuals on geq 577,395 genetic markers. Sex-specific traits were analysed on 142,844 females and 118,963 males, respectively.The UKBB is a cohort with rich phenotype data including International Classification of Diseases, Tenth Revision (ICD-10) codes from which we could derive phecodes38. In this study, we retrieved 44 specific phecodes that associate with more than 10,000 patients to represent 44 common diseases from the 2019 download of phenotype data. To obtain diagnostic labels for all the individuals in the UK Biobank, we mapped International Classification of Diseases Tenth Revision (ICD-10) codes to phecodes40. From the 2019 downloads, we received phenotype data from the UK Biobank. Again, we obtained an updated release of phenotype data in early 2022. On average, participants have more ICD-10 codes in their records. For each of the 44 complex traits, we have identified the individuals that are no longer part of the control group. For PRS validation, the new cases are compared to the controls that do not have the diagnostic code for both data lookups.ICD billing codes are commonly utilized to select individuals from large observational datasets such as the UK Biobank. Also, cases of multiple ICD codes are often put together as a group of patients to study a specific disease. Phecodes, combinations of relevant ICD codes used to describe phenotypes, have worked well in clinical research20. We trained the POPDx framework on the phenotypic and health-related patient data. The 1538 phecodes as gold standard labels correspond to 12,803 ICD-10 diagnosis codes from UKBB. We trained and tested POPDx on all the individuals based on the 2019 version of phenotype data.POPDx is an existing AI-based methodology that outputs the phenotype liability probabilities for each UK Biobank participantwww.ldak.org41). Prior regression, phenotypes were corrected for sex, age, age2, and PCs 1–40. The analysis was performed on 649,644 genetic markers with minor allele frequencies (MAF) ≥ 0.01.We performed GWAS analyses on all 44 complex traits for three phenotype definitions: i) original case-control status, ii) POPDx probability, iii) combined case-POPDx phenotype. GWAS were performed using linear regression as implemented in the LDAK software . Clumping was performed using LDAK and as reference panel we used the genotypes of 20,000 randomly selected individuals among the included 261,807 samples.We performed clumping on SNPs passing the genome-wide significance threshold of 5e-8 to obtain independent SNP associations  of all the protein-coding genes and non-coding RNA genes. The SNPs need to lie within the target genes to be annotated.We mapped the SNPs to genes under the guidance of a comprehensive reference annotation (GENCODE GRCh37 release 42)32. BLD-LDAK estimates We calculated SNP heritability ions see  using thFor each of the 261,807 individuals included in our analyses we calculated polygenic risk scores (PRS) for each trait. Per trait, two PRS were assigned to each person - the first based on original case-control status and the second based on POPDx probabilities. Since our aim was to compare which of the two phenotype definitions assigns a lower percentile rank  to emerging disease cases, estimation of per-predictor effect sizes and phenotype prediction were performed on the same individuals. Information as to whether an individual turns into a case is not available to either phenotype definition, and thus comparison at a later time point constitutes an independent validation analysis.33. First, we estimated per-predictor heritabilities which requires the summary statistic of the trait and phenotype definition of interest  following the LDAK-Bolt-Predict procedure as implemented in the LDAK software v5.2rest see , a SNP trest see  and the Per trait, we compared the two phenotype definitions by concentrating on the newly emerged cases (control in the earlier release and case in the new release). To this end, we first ranked the individuals relative to their PRS in the entire dataset. Each new case would thus have two ranks, once calculated from POPDx probabilities and once from the original case-control classification. For each trait, the difference in ranks of the new cases were then compared through a two-sided Wilcoxon signed-rank test.Supplement 1Supplement 2


Despite the progress made in the diagnoses and therapy of gastrointestinal cancers, these diseases are still plagued by a high mortality. Thus, novel therapeutic approaches are urgently required. In this regard, small interfering RNA (siRNA), double-stranded RNA molecules able to specifically target the mRNA of pathological genes, have the potential to be of therapeutic value. To be effective in the human body, siRNAs need to be protected against degradation. Additionally, they need to target the tumor, leaving the normal tissue untouched in an effort to preserve organ function. To accomplish these tasks, siRNAs have been formulated with smart delivery systems such has polymers and lipids. While siRNA protection is not particularly difficult to achieve, their targeting of tumor cells remains problematic. Here, after introducing the general features of gastrointestinal cancers, we describe siRNA characteristics together with representative delivery systems developed for gastrointestinal cancers. Afterward, we present a selection of research papers employing siRNAs against upper- and lower- gastrointestinal cancers. For the liver, we also consider papers using siRNAs to combat liver cirrhosis, a relevant risk factor for liver cancer development. Finally, we present a brief description of clinical trials employing siRNAs for gastrointestinal cancers.  In this review, we describe the potential use of small interfering RNAs (siRNAs) for the treatment of gastrointestinal (GI) tumors focusing on gastric cancer, hepatocellular carcinoma, pancreatic cancer and colorectal cancer. Additionally, as liver fibrosis/cirrhosis represents a major risk for the development of hepatocellular carcinoma, we also describe works employing siRNAs against liver fibrosis/cirrhosis.The adult human gastrointestinal tract has an average length of seven meters. Furthermore, it comprises additional organs including the pancreas, liver, gallbladder and biliary ducts. As a result of substantial cellular mass and the rapid turnover, gastrointestinal cancers are among the most frequent malignancies, responsible for roughly half of all cancer-related deaths.The distinct tissues of origin give rise to a diverse set of diseases, such as colorectal cancer, pancreatic carcinoma and gastric cancers, each with specific clinical features. In all cases, an altered activation or deactivation of tumor suppressor or proto-oncogenes genes occurs. These common themes can enhance our collective understanding of these malignancies and could perhaps be exploited therapeutically.Gastric cancer is the fourth most common cancer and the second leading cause of cancer-related mortality in the world. Late diagnosis and classical therapeutic approaches such as surgery, chemotherapy and radiotherapy make this disease a constantly threatening tumor. In Europe, the five-year survival rate is 25% ,2.Hepatocellular carcinoma (HCC) is the most frequent primary liver malignancy and one of the most common malignancies worldwide. According to the National Cancer Institute’s SEER database, the average five-year survival rate of HCC patients in the US is 19.6% but can be as low as 2.5% for advanced, metastatic disease ,4. When Pancreatic cancer is uniformly fatal unless it can be surgically resected. Pancreatic cancer remains one with the poorest prognosis among all cancers with a five-year survival of 8.1%. The ten-year survival is 3% . PancreaTo conclude, colorectal cancer (CRC) is the third most common type of non-skin cancer in both men and women. It is the second leading cause of cancer death in the United States after lung cancer. In 2016, an estimated 134,490 people in the United States were diagnosed with colorectal cancer and 49,190 people died from it . IncidenThe common characteristic among the above pathologies is that they have a poor prognosis, and the majority of therapies could often be useless. Thus, novel therapeutic approaches are urgently needed. In this regard, siRNAs hold great promise, mainly due to the ability to specifically target disease genes and due to the flexibility in modulating a large range of targets. However, because of challenges in stability and delivery, it may take time for the clinical practice to become reality. https://www.ncbi.nlm.nih.gov/myncbi/ (accessed on 3 July 2022).Optimal drugs for the treatment of human cancers, including those of the GI tract, still need to be developed. Among molecules of potential interest, there are nucleic acid-based drugs” (NABDs). These potential “drugs” that include ribozymes, DNAzymes, antisense oligonucleotides (ASOs), aptamers, and small interfering RNAs (siRNAs) , base thsiRNA consists of two RNA molecules containing 21- and 22-nucleotides; these two RNA strands, sense and anti-sense, are bound together according to Watson and Crick base pairing. In mammalian cells, siRNA is generated by ribonuclease III cleavage from longer double-stranded RNA (dsRNAs) . This clThe therapeutic potential of double stranded siRNA stems from the fact that they can be chemically synthesized and subsequently introduced into the target cells to hit virtually any deleterious RNA. Additionally, siRNAs have a highly sequence specific mode of action: indeed, being partially complementary of the antisense strand to the target RNA prevents RISC action and thus target RNA degradation. This implies that siRNA have an extraordinary discriminatory power with regard to the target RNA. Thus, compared to conventional chemotherapy drugs, they are far more specific and potentially have negligible toxicity, a problem that plagues most chemotherapy drugs. Finally, the generation of synthetic siRNAs is relatively easy, being based on a mathematical algorithm and/or experimental approaches which consider different parameters, including the energy profiles , the theAs all NABDs ,23,24,25Appropriate delivery vectors can defend siRNA from body elimination and can promote a certain degree of cell targeting. Among the most employed delivery materials, there are lipids and synthetic/natural polymers ,21,28. TThe most well-known type of lipid-based delivery material is the liposome. Liposomes are spherical vesicles constituted by an inner aqueous space surrounded by a bilayer membrane composed of amphiphilic lipids. The aqueous hydrophilic phase can host siRNA, conferring them protection from degradation. Because the outer side of lipids have commonly positive charged moieties, the negatively charged siRNAs can electrostatically bind the outer part of liposomes. Other helpful aspects of liposomes include the fact that they are relatively cheap to produce, can be conjugated with cell targeting moieties and are biodegradable, so they can be easily metabolized and eliminated by the human body. Among the limitations, there is the tendency to accumulate in the liver and lung when administered systemically. However, this feature can be positive in the case of liver/lung targeting. Finally, depending on the specific type of lipids that compose liposomes, a certain attitude to promote inflammation may be present.A novel class of natural lipid particles termed exosomes became of interest for siRNA delivery. Exosomes, constituted by lipidic nanovesicles 40–100 nm in size, are synthesized by different cells and subsequently released into blood circulation . As theyNowadays, many different types of polymers both of natural and synthetic origin have been tested as siRNA delivery materials ,31,32. TThe most evident advantage in polymer use depends on the possibility to finely tuning their function(s) via the addition of specific chemical moieties . In thisIn addition to the above delivery systems, others can be used, such as dendrimers and inorganic nanoparticles. Dendrimers  are formed by a central core which acts as the root from which a number of highly branched, tree-like arms originate in an ordered and symmetrical fashion . For siRDifferent inorganic materials have been tested for siRNA delivery. Among these, mesoporous silica are solid materials composed by a honeycomb-like porous structure able to absorb/encapsulate relatively large amounts of siRNA. The high surface area, the large pore volume, the tunable pore size and the good chemical and thermal stability make mesoporous silica very interesting for controlled drug release . Mesoporhttps://www.fda.gov/drugs/news-events-human-drugs/fda-approves-add-therapy-lower-cholesterol-among-certain-high-risk-adults (accessed on 14 October 2022). Finally, the most recent addition to the array of GalNac conjugates in the market is vutrisiran  used to treat polyneuropathy caused by hereditary amyloidosis in adults.In addition to the above mentioned delivery systems, another strategy adopted to promote siRNA delivery and targeting is based on the use of bioconjugation. Bioconjugation consists of the addition to siRNA of biological molecules able to promote cellular uptake, siRNA cell targeting and/or reduce clearance from the circulation. A number of different molecules have ben used in this regard: lipids, peptides, aptamers and sugar moieties . Among lTargeting drugs to the GI tract can be accomplished via oral, rectal, or endoscopic methods, which can decrease the unnecessary systemic exposure and associated adverse effects of parenteral medications. Despite this, siRNAs delivery to the GI tract encounters the barriers above reported. For this reason, different carriers have been investigated, including biodegradable polymers and lipid-based carriers as below reported.Here, we focus on the potential use of siRNAs in the tumor diseases of upper- and lower-GI and of liver fibrosis/cirrhosis due to its role in HCC development. Given the huge number of papers published in the field of GI/siRNAs, here we have selected some of them to give a general overview knowing, however, that other works could also have been mentioned and described. The mRNAs chosen as siRNA targets and described in this review have been summarized in The research papers reported in this section have been summarized in It has been demonstrated that the proto-oncogene astrocyte elevated gene 1 (AEG-1) is involved in many biological processes, including cell proliferation, survival, apoptosis, invasion and metastasis . AEG-1 rThe B lymphocyte/leukemia-2 (Bcl-2) gene is an oncogene that effectively inhibits cell apoptosis and prolongs cell vitality. Liu et al. delivered the commercial lipid Lipofectamine 2000 an anti BCL-2 siRNA to cultured BGC-823 gastric cancer cells . The autCao et al. recently also demonstrated that siRNA-mediated knockdown of the phosphatase of regenerating liver-3 (PRL-3) gene effectively inhibited gastric carcinoma invasion and metastasis . PRL-3 iThe work reported in this section has been summarized in G12D. Kamerkar et al. [G12D (Exo-si KRASG12D). In vitro, in Panc-1 cells bearing the KRASG12D mutation, Exo-si KRASG12D efficiently reduced KRASG12D mRNA; notably, no effect was observed in Panc-1 cells with-wild type KRAS (KRASwt), indicating the specificity of the system. In an orthotopic mouse model of pancreatic cancer, Exo-si KRASG12D reduced tumor growth compared to controls following i.v. administration. Importantly, animal survival increase was also observed. In line with the in vitro data, Exo-si KRASG12D had no effects in an orthotopic model of pancreatic cancer with KRASwt. Worth mentioning is the fact that Exo-si KRASG12D could down-regulate metastasis and prolong animal survival when administered in the advanced state of a pancreatic tumor. This is particularly relevant for potential future application in humans, which often present with advanced forms of the disease.The K-Ras protein, encoded by the Kirsten rat sarcoma virus (KRAS) gene, transmits the signal from outside the cells to the cytoplasm triggering cell proliferation or differentiation, depending on the specific biological condition. Mutation(s) in KRAS are commonly observed in pancreatic cancer cells, a fact that promotes uncontrolled cell proliferation and metastasis. A common mutation is KRASr et al.  developeProgrammed death-ligand 1 (PD-L1) is a 40kDa type 1 transmembrane protein that plays a relevant role in down-regulating the adaptive arm of immune systems. Thus, its targeting may be considered a potential strategy to promote antitumor immunity. Based on this concept, Jung et al.  targetedGemcitabine is used to treat pancreatic cancer. However, often patients develop resistance to Gemcitabine (GEM). Thus, it is relevant to find strategies to reverse the GEM resistance. CXCR4 is a chemokine receptor overexpressed in pancreatic cancer that can promote tumor cell migration. Polo-like kinase 1 (PLK1) represents a family member of serine and threonine kinases highly expressed in pancreatic cancer; moreover, increased PLK1 expression correlates with GEM resistance. Thus, CXCR4 and PLK1 targeting may represent an interesting approach to overcome GEM resistance. Tang et al.  developeA particular type of non-coding RNA is represented by CircRNAs, characterized by a covalently closed circular loop structure . It has Chronic infection of hepatitis B/C virus, alcohol abuse and non-alcoholic fatty liver disease are the major causes of liver fibrosis (LF) that invariably lead to the progressive impairment of liver function (cirrhosis) . LF reprBecause of the above reported considerations, LF and HCC represent relevant challenges in the field of gastroenterology for which novel therapeutic approaches are urgently needed. Here we describe some siRNA-based approaches that, in our opinion, are promising for the development of future novel therapeutic approaches.The work reported in this section has been summarized in Most of the genes targeted with the purpose to down-regulate LF are implicated in HSC activation and/or are involved in ECM synthesis/degradation. In this regard, transforming growth factor (TGF)-β1 has been considered an interesting target to down-regulate LF. TGF-β1, considered among the most powerful pro-fibrogenic molecules, is heavily implicated in cirrhosis development, being able to regulate ECM gene expression and matrix degradation. Kim et al. studied the effects of TGF-β1 targeting by siRNA in a CCl4-induced murine model of liver fibrosis . The antThe effectiveness of targeting TGF-β1 by siRNA was confirmed in another study by Cheng et al. . In thisAnother potential target for LF down-regulation is represented by platelet-derived growth factor (PDGF). The PDGF family is composed of four different components named PDGF-A, PDGF-B, PDGF-C and PDGF-D. A component can aggregate with another one, giving rise to five homo-/heterogeneous dimers . Notably, PDGF-B is considered among the most potent mitogen for HSCs, acting via the interaction with PDGF receptor-β subunit (PDGFR-β). Chen et al.  developeLim et al.  studied Following activation, HSCs increase the synthesis of type I collagen, which is produced from the COL1A1 and COL1A2 genes. Calvente et al.  generateIn another work , COL1A1 The work reported in this section has been summarized in Fragile X mental retardation protein (FMRP) is an RNA-binding protein relevant for the biology of postsynaptic neurons. Despite its clear involvement in the nervous system, evidence is emerging about an involvement in HCC metastasis as well. Therefore, FMRP has been considered an interesting target to combat HCC. Zhao et al.  developeVEGF promotes Survivin expression and promotes HCC neovascularization. Following oral administration in a xenograft mouse model of HCC, GTC-Surv-VEGF-siRNA increased tumor apoptosis with no significant un-specific toxicity.Asialoglycoprotein receptor (ASGP-R) recognizes and binds galactose and galactosamines residues. While normal hepatocytes express it at low levels, in HCC cells it is over-expressed . Thus, iRecently, we  developeIn a recent work  GalNac, Epidermal growth factor receptor (EGFR) is a transmembrane receptor tyrosine kinase that, when activated by specific ligands, promotes cell proliferation and survival. Notably, it has a relevant role in HCC . Gao et TERT) and EGFR, both involved in tumor growth [EGFR and TERT expression paralleled by a reduction of tumor growth in a xenograft mouse model of HCC.The transferrin receptor (TfR) is a transmembrane glycoprotein necessary for the uptake of transferrin, the iron-carrying protein circulating in the blood. The expression of TfR is augmented in many cancer cells such as HCC cells . The humr growth ,91. siRNAn articulated siRNA delivery system for HCC was developed by Han et al. . The autg-PEI) and loaded with an anti-Survivin siRNA (RGD-PEG-g-PEI-siSurv). Following tail vein injection of RGD-PEG-g-PEI-siSurv in a subcutaneous mouse model of HCC, the authors observed a clear inhibition of tumor growth with increased tumor HCC cell apoptosis/necrosis. The fact that no significant down-regulation of tumor angiogenesis was observed promotes the speculation that mechanisms different from those predicted could have contributed to the effects of RGD-PEG-g-PEI-siSurv.Integrins constitute a group of transmembrane receptors regulating the adhesion of cells to ECM. Following binding to the ligand, they trigger different signal transduction pathways that control several cellular processes, including cell proliferation. The expression of the integrin subtype αvβ3/αvβ5 is increased in the angiogenic endothelium in many malignant tumors , includiThe scientific works reported in this section have been summarized in Fatty acid oxidation (FAO) plays a relevant role in cancer, as it can efficiently produce ATP employed in many cellular processes, including cell growth and survival . CarnitiPD-1 is an immunosuppressive receptor able to down-regulate T cell activation via the binding to its ligand Programmed death receptor ligand (PD-L1). While PD-1 is mostly expressed on the surface of immune cells, PD-L1 is predominately expressed by tumor cells . Thus, tThe upregulation of CD47 by tumor cells represents a defense mechanism against clearance by the immune system as it provides a “self” signal which allows the escape from immune cells elimination . The optCancer stem cells (CSCs) represent the faction of cells thought to confer the ability to initiate/maintain tumors and to be responsible for tumor recurrence and drug resistance. CD44 is considered to be one of the most common CSC surface markers, responsible for the regulation of cancer cell stemness . MoreoveThe efflux of therapeutic drugs from the tumor cells is one of the most generally acknowledged mechanisms of drug resistance. Among other enzymes, ATPase copper efflux transporter A (ATP7A) mediates drug efflux from the cancer cell. ATP7A is one of the key regulators of the intracellular level of platinum-based drug such as Oxaliplatin (OXA) , commonlSo far, the number of clinical trials involving siRNA for GI tumors are very limited. Additionally, often the patients included are affected by different GI tumors. Thus, we thought it was more logical to group them in a separate paragraph at the end of the present review. The work reported in this section has been summarized in Atu027 is a chemically stabilized siRNA delivered via liposomal particles that targets the expression of protein kinase N3 (PKN3) in the vascular endothelium. PKN3 is a protein kinase C-related molecule thought to be an effector mediating malignant cell growth downstream of activated phosphoinositide 3-kinase (PI3K). The chronic activation of PI3K signal transduction pathway contributes to metastatic cell growth. A phase 1 study of Atu027 was tested in patients with advanced solid tumors, including colon cancer ,107. TheCALAA-01 is a cyclodextrin-based polymeric nanoparticle loaded with a siRNA that targets the mRNA of the M2 subunit of ribonucleotide reductase (R2). CALAA-01 is the first targeted, polymer-based nanoparticle carrying siRNA to be systemically administered to humans. A phase Ia ,109 studIn the phase 1 clinical study identified with number NCT02227459 , the purTKM-080301 is a lipid nanoparticle (LNP) formulation containing siRNA against the PLK1 (polo-like kinase-1) gene product. More specifically, TKM-080301 is a type of LNP formulation, referred to as SNALP (Stable Nucleic Acid Lipid Particles). PLK1 has been validated as a molecular target and a prognostic factor in a variety of cancers. Inhibition of PLK1 activity in proliferating cancer cells rapidly induces mitotic arrest and apoptosis. TKM-080301 was administered to patients with unresectable colorectal, pancreatic, gastric, breast, ovarian and esophageal cancers with hepatic metastases; additionally, patients with primary liver cancer which relapsed after first line treatment were also included. This study (NCT01437007) is upcoming, and no results have been published.G12D siRNA (siG12D) drug, designed to release the drug regionally within a pancreatic tumor, at a prolonged rate of 12-16 weeks. As most pancreatic ductal adenocarcinomas involve mutations in the KRAS oncogene (the most common is G12D), the administration of KRASG12D siRNA has the potential to be of therapeutic value. In phase I/IIa clinical trials (NCT01188785) [TM and Gemcitabine was well tolerated, safe and demonstrated a potential efficacy. A Phase 2 Study of KRAS-LODER in combination with chemotherapy in patients with locally advanced pancreatic cancer (NCT01676259), is ongoing.The investigational agent siG12D LODER  is a miniature biodegradable polymeric matrix that encompasses the anti KRAS1188785) , patientThe purpose of the Atu027 Plus Gemcitabine trials is to evaluate a novel treatment strategy for advanced pancreatic cancer disease by combining Atu027 with the standard chemotherapeutic gemcitabine. The objectives of this clinical trial (NCT018086389) were to evaluate safety and activity of two Atu027 schedules in combination with standard gemcitabine treatment in patients with advanced or metastatic pancreatic adenocarcinoma. The results indicate that Atu027 plus gemcitabine is safe and well tolerated . MoreoveIn this review, we summarized some scientific research describing the potential of siRNA-based drugs for GI tumors. So far, the major challenge for siRNA use concerns appropriate delivery to the target cells/tissues. In this regard, some aspects have to be considered. The first deals with the ability of the delivery system to protect siRNA and to deliver it with optimal timing. This task does not seem to be dramatically difficult, judging from the works so far published in oncology regarding GI tract tumors. The second, and more complex task, deals with the ability to reach a specific targeting of tumor cells, leaving the normal cells untouched as much as possible. This aspect is particularly relevant when the systemic delivery of siRNA is necessary, such as in many cases of GI tumors. Only for localized tumors may it be possible to think of an in-situ administration. Obviously, the possibility to detect tumors in the initial phase depends on an effective screening campaign which, for GI tumors such as those of colorectal cancer, is nowadays a reality. The specificity of targeting could be achieved via two approaches: one based on the targeting of antigens exclusively/predominantly expressed on GI tumor cells; the other using siRNAs directed against GI cancer related oncogenes. The identification of optimal molecules to be targeted is, however, not easy since tumor cells may change the surface antigens over time. Additionally, cancer cells frequently share with the normal counterpart similar antigens. Also, the strategy to direct siRNA against cancer related oncogenes may be difficult, as often they are also expressed in normal cells, although to a lesser extent. Regardless of the above difficulties, we think that an ideal level of specificity may be achieved by combining the targeting of specific surface cancer antigens with the use of siRNAs directed against genes predominantly overexpressed in cancer cells, but not in the normal counterpart. In conclusion, despite the as yet unresolved issues in siRNA delivery, we are confident that by considering the above aspects, together with the encouraging works described here, siRNAs have the potential to become novel molecules for GI cancer treatment in the future.


Background: The emergence of acute-onset functional neurological symptoms, the focus of this study, is one of three stress responses related to immunisation. This case–control study documents the experience of 61 young people with past or current functional neurological disorder (FND) in relation to the COVID-19 vaccination program in Australia. Methods: Information about the young person’s/parent’s choice and response pertaining to COVID-19 vaccination was collected as part of routine clinical care or FND research program follow-up. Results: 61 young people treated for FND  and 46 healthy controls  were included in the study. Vaccination rates were high: 58/61 (95.1%) in the FND group and 45/46 (97.8%) in the control group. In the FND group, 2 young people  presented with new-onset FND following COVID-19 vaccination; two young people with resolved FND reported an FND relapse ; and two young people with unresolved FND  reported an FND exacerbation. In the control group no FND symptoms were reported. Conclusions: Acute-onset FND symptoms following COVID-19 vaccination are uncommon in the general population. In young people prone to FND, COVID-19 vaccination can sometimes trigger new-onset FND, FND relapse, or FND exacerbation. Diagnostic and Statistical Manual of Mental Disorders  no longer requires identification of a precipitating stressor, young people with FND—and their families—commonly report a triggering stressor or event against the background of cumulative stress or adverse childhood experiences (ACEs) .,35.2 areBy contrast, the mechanisms underpinning the second and third clusters—functional neurological symptoms and post-immunisation illness—are less well understood, poten-tially much more complex, and the focus of current research efforts .In this article we document the experience of 61 young people with past or current treatment for FND in the Mind–Body Program (2018–2022) in relation to the New South Wales COVID-19 vaccination program and the occurrence—new onset, exacerbation, or recurrence—of acute-onset functional neurological symptoms (cluster 2 above). We also report the experience of healthy controls who volunteered to take part in the FND research program.Participants were 61 young people who had been referred for treatment for FND in the Mind–Body Program at The Children’s Hospital at Westmead (October 2018 to September 2022) and who had agreed to participate in the FND research program. All young people with FND had undergone a comprehensive neurology assessment and had been given a positive diagnosis of FND (DSM-5 criteria) by a paediatric neurologist  see  and the The Sydney Children’s Hospital Network Ethics Committee approved the study (HREC/18/SCHN/232). Participants and their legal guardians provided written informed consent.In August/September 2022, information about the young person’s/parent choice per-taining to COVID-19 vaccination was collected as part of routine clinical care or as part of follow-up—looking at outcomes over time—for the FND research program. For young people treated in the past, questions were asked over the phone  and age (t(84.40) = −0.303; p = 0.763).The final study group interviewed about their vaccination experience (August/September 2022) comprised 61 young people (47 girls and 14 boys) aged 12.08 to 21.83 years  who had been or were being treated for FND and 46 healthy controls (34 girls and 12 boys) aged 10.83 to 21.5 years . The groups were matched for sex  and depression  were the most common). In addition, 21 (34.4%) had reported suicidal ideation, and 16 (26.2%) had suffered from one or more comorbid functional syndromes: functional gut disorder , postural orthostatic tachycardia syndrome (POTS) , and frequent vasovagal events .In the FND group, the clinical presentations of the 61 young people—at the time of their admission for treatment into the Mind–Body Program—had been diverse see . They prp < 0.001)) (see p < 0.001)).On admission to the research program, young people in the FND group reported a greater number of ACEs  compared to healthy controls  (t(70.57) = 8.22; 01)) see . On admin = 59), their medical status at the time of vaccination—or when pre-sented with the option of vaccination—was as follows: fully resolved FND ; resolved FND with a pattern of short-lived relapses in the face of stress ; and unresolved FND   were referred for treatment to the Mind–Body Program after their FND illness was triggered by COVID-19 vaccination. For the rest of the cohort (.8%) see .n = 34, 55.7%), depression , and PTSD  being the most common (see Forty-seven (77.0%) young people in the FND group were experiencing mental health symptoms at the time of vaccination (or option of vaccination), with anxiety .The vaccination rate in the FND group was 58/61 (95.1%). The vaccination rate in healthy control group was 45/46 (97.8%). There was no statistical difference between groups on vaccination rate —and her family—reported that she had not had the vaccine because her mother had experienced neurological symptoms  and cardiac symptoms  fol-lowing COVID-19 vaccination. In this context the adolescent (and her parents) were con-cerned that the vaccine could potentially trigger cardiac and neurological symptoms in the adolescent.The rates of FND relapse, FND exacerbation, and first-presentation FND following COVID-19 vaccination in our FND cohort are reported in Case 1. Relapse of FND symptoms post COVID-19 vaccination in a child whose FND had resolvedAn 18-year-old female with a past history of FND , coupled with comor-bid back pain, nausea, fatigue, dizziness, and functional gut symptoms, presented with FND relapse. Three years earlier, the initial episode of FND was triggered by a sporting injury . Other stressors at the time had included bullying and the death of her grandfather from a long illness. The young woman had recovered from the FND illness more than two years earlier. At the time of vaccination, she had current diag-noses of POTS and anxiety. Two weeks after her first COVID-19 Pfizer vaccination, she developed recurrence of functional seizures, which continued every 2–3 days for six months before resolving with further treatment. She subsequently had two further vac-cinations (Pfizer and Moderna) with no issues see .Case 2. Relapse of FND symptoms post COVID-19 vaccination in a young woman whose FND had resolved but who had experienced a number of short relapses with stressA 17-year-old female with developmental disability and severe anxiety developed her first episode of FND at age 15. That episode had emerged in the following context: influenza vaccination; chest infection (that same week); allergic reaction (after eating an egg sandwich) with urticarial rash and difficulty breathing (week 3); extreme fatigue and a collapse event at school, with hypokalaemia on blood screen treated with potassium (week 4); and emergence of FND in the form of jerky movements (week 5). Over time, the patient’s FND symptoms came to include back arching (dystonic movements in her back), functional seizures, loss of coordination in the legs (astasia abasia), and shaking and tremoring, coupled with episodes of sudden stabbing pain and panic attacks (with long periods of hyperventilation). The death of a grandfather was an additional, concurrent stressor. Following full recovery, the patient had had short-lived FND relapses in the con-text of stress at school . She presented two days after her first COVID-19 Pfizer vaccination with dysphagia, sialorrhea, leg weakness, and exacerbation of functional seizures. She was admitted for nine days of rehabilitation as her dysphagia resulted in restriction of oral intake to the degree of becoming medically unstable. Her FND symptoms resolved with treatment. She subsequently had two further COVID-19 Pfizer vaccinations with no issues see .Case 3. Worsening of FND symptoms in unresolved FNDA 19-year-old birth-assigned female who identifies as male suffered with chronic FND from age 15: functional seizures, leg weakness , and time-limited episodes of whole-body stiffening and functional blindness that occurred on a weekly basis. The patient’s FND had occurred in the setting of cumulative stressors over many years. These included: bullying, exposure to violence, and exposure to a severe mental illness in an older sibling. Comorbid diagnoses at the time of vaccination included dissociative identity disorder, depression, and gender dysphoria. Three days after the second COVID-19 Pfizer vaccination, the patient experienced a recurrence of bilateral leg weakness and required a wheelchair to mobilise. The leg weakness continued for six months while the patient underwent outpatient rehabilitation. The time-limited FND episodes of whole-body stiffening and blindness continued to occur. She subsequently had a further COVID-19 Moderna vaccination with no issues see .Case 4. Worsening of FND symptoms in unresolved FNDA 15-year-old adolescent female aspiring to be a doctor first experienced FND symp-toms at age 13 while under academic stress. Her FND included weakness and dragging of the left leg, loss of coordination in the legs, tremor, loss of hearing and tinnitus, blotchy vision, light-headedness, nausea, and cognitive symptoms of being unable to think clearly. In the patient’s own words, “The root cause of my symptoms was my tendency to put pressure on myself. I just challenged myself so much that I eventually burnt out.” At the time of her first COVID-19 Pfizer vaccination, the patient was making excellent progress toward a full recovery. Her remaining symptoms included functional seizures (of decreasing frequency), tinnitus, and visual loss, as well as comorbid POTS and functional gut symptoms. Immediately following the first vaccination, the patient experienced a functional seizure. Following the functional seizure, her legs were weak and unsteady, she experienced ticcing a new symptom , and she needed to use a wheelchair. The leg weakness recurred following the second vaccination. On both occasions the symptoms resolved in a few days.Case 5. New-onset FND following COVID-19 vaccinationA 17-year-old female experienced new-onset FND following COVID-19 vaccination that led to admission into the Mind–Body Program for treatment. Immediately after her first Pfizer vaccination, she experienced pain and itchiness at the injection site. She woke the next day with a circular, raised, erythematous rash with central blistering that ex-tended along the entire right arm. The rash remained for a week. The day following the vaccination she also experienced nausea and vomiting as well as an unresponsive epi-sode that lasted for approximately 40 min.Three weeks following the vaccination, she had increased frequent collapses. She was admitted for further investigation: an electroencephalogram did not show any evidence of epileptiform activity. Her collapses continued for six months before the diagnosis of FND was made by a neurologist after further investigations (lumber puncture and magnetic resonance imaging). By this time the patient was experiencing new symptoms of limb weakness, muscle spasms (causing a shoulder subluxation), and functional tics, which were both verbal and motor. She was also experiencing irritable bladder, functional gut symptoms, and dizziness on standing secondary to POTS.Predisposing risk factors included the following: the death of four grandparents, death of an aunt, death of a close friend, death of three dogs, and sexual assault, all within the period of two years. Prior to the development of FND, this patient had been given a diagnosis of depression with posttraumatic stress disorder—related her traumatic expe-riences—and she was being managed by a community mental health team.The patient was admitted for a three-week rehabilitation admission. On discharge she was mobilising on a walking frame, and her tics had largely subsided. She had en-gaged in the tasks of learning physiological regulation strategies  and learCase 6. New-onset FND following COVID-19 vaccinationA 12-year-old girl experienced new onset of three different medical conditions—atypical narcolepsy  ,51,52,53The child presented to the emergency department with an 11-day history of hyper-somnolence that started two hours after her second vaccination. She was treated with dexamphetamine. Four weeks following the vaccination, she developed acute episodes of nausea, vomiting, and abdominal pain, coupled with an inability to tolerate oral intake. The patient’s pain was managed with simple analgesics; her distress, with olanzapine (as needed); and her nutritional needs, with feeds via a nasogastric tube. Two weeks follow-ing the vaccination, she developed bilateral lower limb weakness (necessitating a wheel-chair to mobilise). She also developed functional seizures that showed semiology changes across time: zoning out; episodes of collapse; generalised shaking; generalised shaking coupled with the patient hitting herself; episodes of aggression while the patient was in an altered state; and episodes where the patient ran around and around her hospital room while in an altered state . The child’s FND symptoms made the clinical picture very complex and excluded the possibility of clearly diagnosing cataplexy.The patient was discharged from hospital on day 97 with ongoing outpatient man-agement of narcolepsy (by the sleep team), functional gut disorder (by the family doctor), and FND, depression, and anxiety .Predisposing risk factors for FND and a functional gut disorder included the follow-ing: the stress of home schooling during COVID-19 lockdowns; increased stress (conflict) in the parental relationship, which had previously been amicable; conflict with peers in Year 6; loss of the opportunity to go to a performing arts high school; stress around transi-tion to high school; and a medical history of functional cough, functional abdominal pain, and untreated depression.In the FND group, 24 (39.3%%) reported that they had contracted COVID-19. Of these, 23 (37.7%) reported that they had contracted COVID following one or more vaccinations. No exacerbations of FND were reported in association with COVID-19 infections, and no prolonged illness (long COVID) was reported.In the control group, 31 (67.4%) reported that they had contracted COVID-19. Of these, 30 (96.8%) reported that they had been vaccinated, and they had contracted COVID-19 after one or more vaccinations. In this vaccinated subgroup, no neurological symptoms and no prolonged illness (long COVID) were reported.Additionally, in the control group, the adolescent girl who had not been vaccinated—because her mother had experienced neurological symptoms after being vaccinated—reported a prolonged illness (long COVID). After falling ill with COVID-19, she developed POTS—reflecting activation and dysregulation of the autonomic nervous system—alongside high temperatures and cold-like symptoms. The POTS symptoms slowly resolved after more than two months and recurred three times with subsequent viral infections (not COVID-19). In each case the autonomic symptoms preceded the adolescent’s cold-like symptoms. The symptoms were severe enough to require management by a cardiologist.The current study documents the experience of a cohort of 61 young people admitted to the Mind–Body Program for the treatment of FND (October 2018 to September 2022)—and 46 healthy controls—with respect to the COVID-19 vaccination program in New South Wales, Australia. Vaccination rates across the two groups were high. In the FND group see , two youThree previous follow-up studies of young people with FND at 4 years, 12 months, and 18 months have shown that 17.5%, 11.5%, and 4%, respectively, experienced relapse in response to subsequent stress. In the current study, of the 51 young people with past or current FND who were vaccinated for COVID-19, 4/51 (7.84%) experienced a relapse or a worsening of their symptoms with vaccination see . As abovStress-related responses to vaccination are well documented in the literature (see the three clusters described in the introduction) ,31,32,33hypothesis based on emerging research.In the clinical setting, the understanding of underlying biological mechanisms that underpin stress-related responses to vaccination is limited. For example, the 2019 updated World Health Organization user manual—Causality Assessment of an Adverse Event Following Immunization —FND presA large body of research highlights that stressors—whether they be physical or psy-chological—activate the stress response in a coordinated manner see  59]. By. By59]. Vaccination as a physical (bottom-up) stressor involves activation of the immune-inflammatory system via vaccine constituents and the mounting of an antibody response. The antibody response also involves activation of the sympathetic nervous system (a component of the autonomic nervous system) and the HPA axis . BecauseVaccination as a psychological (top-down) stressor involves activation of the stress system via mental processes (thoughts and feelings) ,62,63,64Previous literature has noted that young people are susceptible to communications of threat  from family members, peers, and social media. A group-contagion effect pertaining to mass outbreaks of FND and other functional somatic symptoms (see clusters 2 and 3 in the Introduction) is well documented. Such outbreaks appear to occur during times of threat or stress, including war and pandemics ,66. The The hypothesised mechanisms—working bottom-up and working top down—are not mutually exclusive. The mechanisms can occur in tandem and may reflect a set of neuroimmune–endocrine–psychological cascade of threat-related responses—the body–brain–mind response to signals of danger —that interact with each other in a mutually reinforcing way.Importantly, in most healthy young people, stress-system activation is short-lived. The stressor is dealt with, and body and brain systems return to normal function . In young people who de-velop FND, however, various components of the stress system—the HPA axis , the au-In this final section of the discussion we consider three potential biological mecha-nisms via which vaccination may trigger a cascade of stress-related responses and con-tribute to the process by which FND symptoms are triggered or exacerbated.First, the nervous system and the immune system are interdependent systems that communicate in a bidirectional manner—from the brain to the immune system and from the immune system to the brain . Any altSecond, neural networks are now conceptualised as neuron–glia networks—where microglia engage in reciprocal signalling with neurons to underpin brain function in health and disease ,83,84,85Third, young people with FND ,89 reporOnce vaccination or another stressor  has triggered the FND symptoms, a number of factors may contribute to symptom maintenance see  3,6,7,8,94,95,96A prospective design using a whole-population approach is needed to provide inci-dence statistics of acute-onset FND following COVID-19 vaccination in the general popu-lation. Such data will also tell us what FND symptoms are most common post COVID-19 vaccination. Such a design would also provide incidence data about the two other stress responses related to immunisation: acute anxiety-related responses (cluster 1) and a post-immunisation illness characterised by nonspecific functional somatic symptoms (cluster 3) (see introduction for the description of the three clusters). The lack of longitudinal outcome data for case 5 and case 6—who were still experiencing FND symptoms at the time this paper was written—is another limitation.Acute-onset FND symptoms following COVID-19 vaccination are uncommon in the general population. Notwithstanding, in young people prone to FND, COVID-19 vaccina-tion can trigger new-onset FND, FND relapse, or FND exacerbation. The biological mech-anisms involved are thought to be complex: a cascade of stress-related responses (includ-ing epigenetic reprograming) that lead to a sustained activation of neural  networks ,74,75 co


Cerebrospinal fluid (CSF) and interstitial fluid exchange along a brain-wide network of perivascular spaces (PVS) termed the ‘glymphatic system’. The aquaporin-4 (AQP4) water channels abundantly expressed on astrocytic endfeet play a key role in the CSF circulation in the glymphatic system. Neuromyelitis optica spectrum disorder (NMOSD) is an inflammatory demyelinating autoimmune disease of the central nervous system (CNS) featured with a specific autoantibody directed against AQP4 in most of patients. Anti-AQP4 antibodies are likely resulting in the impairment of the brain glymphatic system and the enlargement of PVS in NMOSD patients. In the current study, we aimed to demonstrate the features of EPVS detected by MRI and its association with the CSF anti-AQP4 antibody titer, CNS inflammatory markers, and disease severity in NMOSD patients.We conducted a retrospective review of a consecutive cohort of 110 patients with NMOSD who had brain MRI. We assessed the correlation of EPVS with markers of neuroinflammation, blood-brain barrier (BBB) function and severity of neurological dysfunction in patients. We used multivariate logistic regression analysis to determine the independent variables associated with disease severity.The median number of total-EPVS was 15.5  in NMOSD patients. The number of total-EPVS was significantly related to EDSS score after correcting for the effects of age and hypertension . The number of total-EPVS was also significantly associated with the titer of CSF anti-AQP4 antibody, the albumin rate , the CSF albumin, IgG and IgA levels. Logistic regression analysis showed that total-EPVS and serum albumin level were two independent factors to predict disease severity in NMOSD patients . Furthermore, ROC analysis achieved AUC of 0.736  for total-EPVS to determine severe NMOSD (EDSS 4.5-9.5).In our cohort, we found a relationship between EPVS and neuroinflammation and BBB function in NMOSD. Moreover, EPVS might independently predict neurological dysfunction in patients with NMOSD. The brain was long believed to be devoid of a lymphatic vascular system. In 1970s, Cserr and colleagues has suggested a fluid-transport system in the brain; however, only late in 2012, this lymphatic transport system was designated the glial-associated lymphatic system, or the ‘glymphatic system . AstrocyIt has been shown that the glymphatic system plays a role in neuroinflammation and immune responses within the brain . And theStudies demonstrated that the number and size of PVS may change in the course of inflammatory diseases such as multiple sclerosis (MS) –13. HistNeuromyelitis optica spectrum disorder (NMOSD) is an inflammatory demyelinating autoimmune disease of the central nervous system (CNS) mainly affected optic nerve, spinal cord and brain . The disThe autoantibodies that attack the glial AQP4 water channels are likely resulting in the impairment of the brain glymphatic system in NMOSD patients . We hypoin situ while EPVS can be easily visualized on magnetic resonance imaging (MRI). And EPVS could partially reflect the function of glymphatic system. Therefore, in the current study, we aimed to demonstrate the features of EPVS detected by MRI and its association with the CSF anti-AQP4 antibody, CNS inflammatory markers, blood-brain barrier (BBB) function, and disease severity in NMOSD patients.So far, the clinical significance of the glymphatic system and EPVS in NMOSD is unknown. We suggested they might play key roles in the immune process of NMOSD. However, glymphatic system is difficult to learn The medical records of consecutive patients admitted to our center from April 2013 to January 2022 with the diagnosis of NMOSD were reviewed. Patients who fulfilled the diagnostic criteria established by Wingerchuk et al. in 2015  were incThe study was approved by the Ethics Committee of RenJi Hospital. Informed consent was obtained from all the included patients.-3, which indicates the disruption of blood-brain barrier.The clinical, laboratory, and radiology records of all the included patients were retrospectively reviewed. The clinical information collected including age, sex, age at onset, past medical history, total disease duration, time from last relapse, annualized relapse rate (ARR), Expanded Disability Status Scale (EDSS) score, serum status and titers of anti-AQP4 antibody, cerebrospinal fluid results , presentation of optic neuritis (ON), myelitis, and longitudinally extensive transverse myelitis (LETM). The patient was regarded as being in the acute phase of the attack if the time from last relapse was less than 30 days . EDSS scPatients underwent MRI according to a standardized protocol as part of routine clinical assessments. The protocol included T1- and T2-weighted, fluid-attenuated inversion recovery (FLAIR), axial trace diffusion-weighted imaging (DWI) with 2 b-values (0 and 1000), and apparent diffusion coefficient (ADC) sequences. All studies were performed on 3.0 T scanners. Images were 2D sequences. Sequences typically included 20–30 slices of 5-mm thickness. The imaging parameters were as follows: T1 ; T2 ; FLAIR ; diffusion tensor imaging .Enlarged PVS (EPVS) are commonly seen in the centrum semiovale (CSO-EPVS), basal ganglia (BG-EPVS) and midbrain (MB-EPVS) .EPVS were rated on axial T2-weighted MRI using a validated visual rating scale –24. EPVSEPVS were separated and rated in the BG, CSO and MB regions. They were counted in both sides on the brain slice showing the greatest extent of EPVS. A total number of EPVS  was calculated as the sum of EPVS in the three regions.The following rating categories were used: 0 = no EPVS, 1 = 1 to 10 EPVS, 2 = 11 to 20 EPVS, 3 = 21 to 40 EPVS, and 4 = more than 40 EPVS. For the purpose of this analysis, EPVS were categorized into 0–2 vs 3–4 grades (mild vs severe EVPS).Two trained neurologists evaluated MRI scans and when inconsistency existed between the reported results both neurologists discussed the case and reached an accord.2(X+1) was used. The Kolmogrov-Smirnov Z test was used to verify the normal distribution of the data. Categorical variables were summarized as counts (percentage) and continuous variables as the means  or medians , if not distributed normally. Statistical comparisons between two groups were performed using Student’s t-test or the Mann–Whitney U test for continuous variables, as well as the χ2 and Fisher’s exact tests for categorical variables, as deemed appropriate. Correlations between continuous variables were assessed by Spearman’s correlation coefficient. Logistic regression analysis was used to detect the independent factors associated with severe EPVS (EPVS grade 3-4) or severe NMOSD (EDSS 4.5-9.5). Receiver operating characteristic (ROC) curve and area under curve (AUC) were used to test the accuracy for EPVS to determine disease severity. A two-tailed probability value < 0.05 was considered significant. We did not correct for multiple comparisons.Data analysis was performed using SPSS version 21.0 . For the statistical analysis on the anti-AQP4 antibody titer X, a logarithmic value of logAs The median numbers of BG-EPVS, CSO-EPVS and MB-EPVS were 7.0 , 8.0  and 0  respectively. The median number of total-EPVS was 15.5  in NMOSD patients.We define EPVS grade 0-2 as mild category of EPVS and grade 3-4 as severe category of EPVS. There were 39 (35.5%) patients had severe EPVS. NMOSD patients with severe EPVS as compared to those with mild EVPS were older  and had a higher percentage of hypertension . Severe EPVS patients tended to have higher levels of CSF albumin and CSF IgG, higher titers of CSF anti-AQP4 antibody, and a lower level of serum albumin. The median albumin rate was higher in patients with severe EPVS, which indicates a more severe blood-brain barrier disruption. Moreover, EDSS score was significantly higher in severe EPVS group as compared to mild EPVS group . Table 1 Sensitivity analyses were conducted by comparing lowest EPVS group (EVPS grade 0-1) with highest EPVS group (EPVS grade 4) and similar results were shown. , which means NMOSD patients with more numbers of EPVS had more severe neurological dysfunction. . However, after correcting for the effects of age and hypertension, only the positive relationship between the numbers of CSO-, BG- EPVS and EDSS score remained significant .The number of total-EPVS was significantly associated with the titer of CSF, but not serum, anti-AQP4 antibody . , which indicates NMOSD patients with more numbers of EPVS were accompanied with more serious blood-brain barrier disruption. , which implied that more numbers of total-EPVS were related to more severe central nervous inflammation. (Two logistic regression models were conducted to find out the independent factors associated with severe EPVS (EPVS grade 3-4). As In order to clarify the independent predictive effect of total-EPVS with disease severity in NMOSD, patients were divided into two groups: mild NMOSD group (EDSS 0-4.0) (N=72) and severe NMOSD group (EDSS 4.5-9.5) (N=38). The variates of total-EPVS, as well as age, age of onset, numbers of all attacks, and serum albumin were entered into the logistic model. The result showed that total-EPVS and serum albumin level were two independent factors in the model to predict disease severity  Table 3.Our study demonstrated that more total-EPVS was significantly correlated with higher CSF anti-AQP4 antibody titer, more severe blood-brain barrier disruption and intenser neuroinflammation. Moreover, total-EPVS was an independent predictor of severe neurological dysfunction in NMOSD.PVS is part of the structure of glymphatic system. Studies have already proved that PVS are important in immune responses and inflammatory processes within the brain , 27. EPVPathological examination showed abundant antibody-secreting cells were noted in perivascular spaces in autoimmune glial fibrillary acidic protein (GFAP) astrocytopathy, a CNS inflammatory disease targeting astrocytes . In a caAstrocytic endfeet and their dense expression of the aquaporin-4 water channels promote fluid exchange between the perivascular spaces and the neuropil. Thus, anti-AQP4 antibody targeting the main water channels on asctrocytes will impair CSF influx from PVS to neuropil in NMOSD . TherefoOur results showed that patients with higher CSF titer of anti-AQP4 antibody had more EPVS on MRI. Higher level of CSF antibody will impair more water channels on astrocyte endfeet of PVS, which in turn block the influx of CSF and lead to the enlargement of PVS.We also found that EPVS are associated with neuroinflammation and BBB disruption in NMOSD. As our data showed, severe EPVS are related to higher CSF albumin, IgG and IgA levels, which implied an intense inflammatory reaction in CNS; severe EPVS are also correlated with a greater impairment of BBB indicated by higher CSF/serum ratios for albumin. It has already been proved that glymphatic impairment aggravates CNS inflammation by suppressing cytokine clearance from the brain . The BBBOur study also demonstrated that EPVS was independently associated with neurological dysfunction in NMOSD. Since EPVS can indicate inflammation in CNS, thereby more EPVS are related to more severe clinical presentation. Previous studies in MS have already demonstrated that increased numbers of EPVS are associated with clinical disability , 35.The results of the current study prompt us to raise the following hypothesis. Anti-AQP4 antibodies originate in peripheral and enter the PVS through endothelial transcytosis or at areas of increased BBB permeability. Then anti-AQP4 antibodies in PVS bind selectively to AQP4 on astrocyte endfeet. This interaction results in down-regulation of surface AQP4 and less clearance of waste and pro-inflammatory cytokines in the CNS. Moreover, it activates complement produced locally by astrocytes, which in turn leads to increased BBB permeability and massive infiltration of leukocytes and cytokines. And inflammation in PVS will increase the numbers and volumes of EPVS. The neurological dysfunction will also aggravate in the condition of more severe neuroinflammation.There are several limitations in the current study. One limitation is the relatively small number of patients included and the retrospective design of the study; although the statistical analyses were significant, the correlations were relatively weak. Therefore, large-scale prospective studies should be conducted with longer periods of follow-up to confirm the results drawn from the current study. Secondly, more biomarkers of neuroinflammation and BBB function such as CSF pro-inflammatory cytokines and radiological studies should be used to further prove the current results. Thirdly, the glymphatic system has been proved to be existed in brain and optic nerves; however, whether it exists in spinal cord need further studies. It is an important question to be explored because spinal cord is an end organ frequently attacked in NMOSD. Finally, we also found a relationship between EVPS and disease severity in anti-AQP4 antibody negative NMOSD patients and it could not be explained by the above-mentioned hypothesis. There might be other mechanisms involved.In conclusion, we found a relationship between EPVS and neuroinflammation and BBB function in NMOSD. Moreover, EPVS might independently predict neurological dysfunction in patients with NMOSD.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by ethics committee of Ren Ji Hospital. The patients/participants provided their written informed consent to participate in this study.X-YY: conception, methodology, data analysis, and writing original draft. M-CG: resources, data collection. S-WB: resources, data collection. LX: data analysis. Y-YS: resources, data collection. C-RX: resources, data collection. Y-FW: resources, data collection. YH: data curation. YZ: validation, methodology. Y-TG: conception. supervision, writing – review and editing. All authors contributed to the article and approved the submitted version.This study was supported by the National Natural Science Foundation of China (No. 81801211) and the Outstanding Youth Training Funds of Shanghai Ren Ji Hospital (No. PYII-17-001). Innovative research team of high-level local universities in Shanghai (SHSMU-ZDCX20211901).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.