Single cell sequencing in CNS autoimmune disease

Genomics data will be shared with other groups who have appropriate local ethical approval in place to use such data.

PATIENT CONSENT FORM: Immune Factors in Neurological Diseases Please Initial I confirm that I have read the information sheet dated (22-06-2018) version (4) for the above study. I have had the opportunity to consider the information, ask questions and have had these answered satisfactorily. I understand that my participation is voluntary and that I am free to withdraw at any time without giving any reason, without my medical care or legal rights being affected. I understand that the relevant sections of my medical notes and data collected during the study may be looked at by individuals from the University of Oxford, from regulatory authorities or from the relevant NHS trust, where it is relevant to my taking part in this research. I give permission for these individuals to have access to my records. I agree to give samples for research and/or allow samples already collected as part of my medical care to be used for this research. I consider these samples a gift to the University and I understand that I will not gain any direct personal benefit from them. I understand genetic information will not be fed back to me as the conditions are not known to be inherited. I give permission for study researchers to access my health care records in order to collect and store information necessary for this research. I give permission for results of any research tests that are relevant to my care to be shared with my clinical team. I agree to take part in this study. Turn over Optional: I agree to Fine Needle Aspiration (FNA) of cells in cervical lymph nodes being performed for research purposes I agree to bone marrow aspiration for research purposes I agree to stool being obtained for research purposes I agree to cerebrospinal fluid (CSF) being obtained for research purposes I agree to be videotaped and understand that I may be identifiable in videos documenting my illness. I agree to my videos being used in scientific publications or presentations. I agree for my anonymised samples to be used in future academic or commercial research, here or abroad, which has ethics approval. I agree to be contacted about related ethically approved research studies for which I may be suitable. I understand that agreeing to be contacted does not oblige me to participate in any further studies. I agree for researchers to use my blood or other samples available to study their effects on living cells or animals. Study Title: Immune Factors in Neurological Diseases IRAS Project ID: 195152 Ethics Ref: 16/YH/0013 Date and Version No: 22/06/2018 v4 Chief Investigator: Associate Professor Sarosh R Irani, Senior Clinical Fellow and Honorary Consultant Neurologist, Nuffield Department of Clinical Neurosciences, University of Oxford Investigators: Dr Patrick Waters, Senior Scientist, Nuffield Department of Clinical Neurosciences, University of Oxford Associate Professor Arjune Sen, Consultant Neurologist, Nuffield Department of Clinical Neurosciences, University of Oxford Dr Maria Isabel Leite, Senior Clinical Research Fellow and Honorary Consultant Neurologist, Nuffield Department of Clinical Neurosciences, University of Oxford Dr James Varley, Clinical Research Fellow, Nuffield Department of Clinical Neurosciences, University of Oxford Dr Adam Al-Diwani, Clinical Research Fellow, Department of Psychiatry, University of Oxford Dr Sophie Binks, Academic Clinical Fellow, Nuffield Department of Clinical Neurosciences, University of Oxford Dr Ronan McGinty, Clinical Research Fellow, Nuffield Department of Clinical Neurosciences, University of Oxford Dr Bettina Balint, Clinical Research Fellow, Nuffield Department of Clinical Neurosciences, University of Oxford Dr Sophia Michael, Clinical Research Fellow, Nuffield Department of Clinical Neurosciences, University of Oxford Dr Valentina Damato, Clinical Research Fellow, Nuffield Department of Clinical Neurosciences, University of Oxford Dr Adam Handel. Clinical Research Fellow, Nuffield Department of Clinical Neurosciences, University of Oxford Miss Archana Ramesh, Clinical Care Coordinator, Nuffield Department of Clinical Neurosciences, University of Oxford Ms Rhea Zambellas, Project Manager, Nuffield Department of Clinical Neurosciences, University of Oxford Sponsor: University of Oxford Funders: Wellcome Trust; British Medical Association (Vera Down Grant); UCB-Oxford Alliance; Association of British Neurologists; Epilepsy Research UK; UCB-Oxford Alliance funding to Dr Irani and Dr Sen, EuroImmun funding to Dr Waters; R&D funds to Dr Irani and Dr Waters Chief Investigator Signature: Potential conflicts of interest The Department of Clinical Neurology in Oxford receive royalties and payments for antibody assays: SRI and, BL and PW are coapplicants and receive royalties from patent application WO/2010/046716 entitled 'Neurological Autoimmune Disorders'. The patent has been licensed to Euroimmun AG for the development of assays for LGI1 and other VGKC-complex antibodies. Confidentiality Statement This document contains confidential information that must not be disclosed to anyone other than the Sponsor, the Investigator Team, host organisation, and members of the Research Ethics Committee, unless authorised to do so.   TABLE OF CONTENTS 1. SYNOPSIS 5 2. ABBREVIATIONS 6 3. BACKGROUND AND RATIONALE 7 4. OBJECTIVES AND OUTCOME MEASURES 8 5. STUDY DESIGN 10 6. PARTICIPANT IDENTIFICATION 10 6.1. Study Participants 10 6.2. Inclusion Criteria 10 6.3. Exclusion Criteria 10 7. STUDY PROCEDURES 11 7.1. Recruitment 11 7.2. Screening and Eligibility 12 7.3. Study Assessment 12 7.4. Subsequent Visits 14 7.5. Sample Handling 14 7.6. Discontinuation/Withdrawal of Participants from Study 16 7.7. Definition of End of Study 16 8. INTERVENTIONS 16 9. SAFETY REPORTING 16 9.1. Definition of Serious Adverse Events 16 9.2 Reporting Procedures for Serious Adverse Events 17 10. STATISTICS AND ANALYSIS 17 10.1. Description of Statistical Methods 17 10.2. The Number of Participants 17 10.3. Analysis of Outcome Measures 17 11. DATA MANAGEMENT 18 11.1. Access to Data 18 11.2. Data Recording and Record Keeping 18 12. QUALITY ASSURANCE PROCEDURES 18 13. ETHICAL AND REGULATORY CONSIDERATIONS 18 13.1. Declaration of Helsinki 18 13.2. Guidelines for Good Clinical Practice 18 13.3. Approvals 19 13.4. Reporting 19 13.5. Participant Confidentiality 19 13.6. Expenses and Benefits 19 13.7. Other Ethical Considerations 19 14. FINANCE AND INSURANCE 20 14.1. Funding 20 14.2. Insurance 20 15. PUBLICATION POLICY 20 16. REFERENCES 21 17. APPENDIX A: STUDY FLOW CHART 24 18. APPENDIX B: BNSU letter 26 APPENDIX C: AMENDMENT HISTORY 27   1. SYNOPSIS Study Title Immune Factors in Neurological Diseases Internal ref. no. / short title Immune Factors in Neurological Diseases Study Design Longitudinal observation and laboratory studies Study Participants Patients with neurological disease where autoimmunity is likely or being investigated as a component, and healthy and disease controls. Planned Sample Size 5000: 1500 participants with suspected or confirmed autoimmune component to their neurological disease. 500 anonymised samples from healthy volunteers, including environmental controls. 3000 anonymised samples and characterising data from research participants (who consented to use of their samples in further research). Planned Study Period 15/12/2015-15/12/2025 Objectives Outcome Measures Primary Clinical and immunological characterisation of antibody positive patients with neurological conditions that may have an autoimmune component. Clinical data from medical notes, including measures of neurological function, cognition and mood. Clinical data may be supplemented with standard questionnaires where this is necessary to achieve a standard minimal dataset. More detailed patient interviews in selected patients. Immunological findings (through routine diagnostics and research assays) from blood, CSF, lymph node aspirate, bone marrow aspirate, stool, urine and saliva. Correlations between clinical and immunological findings to determine whether there are clinical or immunological features which predict a good response to treatments [1-3]. Comparisons with healthy and disease control samples will determine cut-offs and syndrome specificity of immunological findings. Secondary Novel antibody discovery in patients with similar phenotypes. Correlations between clinical and immunological findings, as performed in previous studies [1, 4], to describe phenotypes associated with novel antibodies, and their response to treatments. Comparisons with disease and healthy control samples will determine antibody specificity. Identify other immunological characteristics and markers in patients with neurological conditions. Immunological findings, including lymphocyte types, immune-system related genes, proteins, microbes and cytokine levels, in bodily fluids, including bone marrow aspirate, and stool. The presence of serum antibodies in urine and saliva. Comparisons with disease and healthy control samples will determine assay specificity and cut-offs to allow understanding of whether these markers segregate with autoimmune neurological diseases. Characterise the antibodies and the cells that produce antibodies in order to understand the biology of neurological conditions and identify possible drug targets. Cell phenotypes (protein and transcripts), responses of cells to factors in culture. In vitro and in vivo models to study the functional effects of identified patient immune factors. Comparisons with disease and healthy control samples will determine assay cut-offs to allow understanding of whether these markers segregate with autoimmune neurological diseases. Non-heritable genetic factors which predispose to the condition. HLA-associations with clinical phenotype – especially in patients with LGI1-antibodies. 2. ABBREVIATIONS ABN Association of British Neurologists BNSU British Neurological Surveillance Unit CI Chief Investigator CRF Case Report Form CSF Cerebrospinal Fluid CTRG Clinical Trials & Research Governance, University of Oxford ERUK Epilepsy Research UK FNA Fine Needle Aspiration GCP Good Clinical Practice GP General Practitioner ICF Informed Consent Form NHS National Health Service NIHR National Institute for Health Research NRES National Research Ethics Service OUH Oxford University Hospitals NHS Trust PI Principal Investigator PIL Participant/ Patient Information Leaflet PLEX Plasma Exchange R&D NHS Trust R&D Department REC Research Ethics Committee SOP Standard Operating Procedure 3. BACKGROUND AND RATIONALE Neurological disorders are often difficult to treat. We, and other clinical researchers, have shown an autoimmune, usually antibody mediated, cause for some such conditions in several studies.[5-10] Affected patients are of both sexes, all ages and all races. Examples include, myasthenia gravis and, most recently, novel forms of encephalitis and epilepsy. The diagnosis is usually confirmed with (serological) blood and cerebrospinal fluid (CSF) tests. Until recently CSF was not required for diagnosis, but recent publications suggest that it should be done in parallel with serum since both can be informative.[11] After the diagnosis is confirmed, these conditions are often effectively treated with immunomodulatory drugs.[2, 12, 13] There is an appreciated need to determine more fully the number of conditions that respond to immunomodulatory therapies. One way to achieve this aim is to detect more patients with autoantibodies and related abnormal immunological parameters, which will help define conditions with an immunological component, and that are often immunotherapy-responsive. Such conditions now include a variety of degenerative and other neurological conditions, in which it is beginning to be appreciated that the immune system may play a role in determining patient progression or response to treatments[14-17]. In summary, immunological parameters determined within this study can be useful in defining an immune component to these diseases, and may also suggest a role for immunotherapies in these patients. This research builds on a previous project, Immune and genetic factors in neurological disorders (REC Ref: 07/Q1604/28) where consent was obtained to use clinical details and research blood and cerebrospinal fluid (CSF), sent for routine clinical purposes to the University of Oxford Department of Clinical Neurosciences Neuroimmunology Laboratory (often through the Oxford University Hospitals NHS Trust Clinical Neuroimmunology Service), for novel and established immunological markers. Also, additional blood, CSF, urine and saliva were obtained in order to enable us to study immunological markers in more detail. In addition, patients were able to consent to having these bodily fluids collected directly from clinical encounters (typically outpatient appointments). The previous project has been successful in defining new antibody targets, generating associated patents, translating rapidly to clinical practice and offering patients worldwide appropriate treatments with good outcomes: many of these outputs are described in the reference list. [2, 6, 8, 18-20] This current study aims to examine clinical and immunological laboratory features in patients for whom autoimmunity is a part of their neurological disease. In particular, we are interested in symptoms, signs, investigation results, autoantibodies, cytokine and lymphocyte profiles of these patients. The presence of an autoantibody often means that the patient receives a diagnosis of an autoimmune neurological condition and that the patient may respond to immunologically-directed therapies (‘immunotherapies’). We intend to continue to characterise the detailed clinical profile of patients with these disorders in order to allow identification of immunotherapy-responsive diseases. This approach has been successful in us identifying novel clinical phenotypes which respond to immunotherapies. Identification is markedly enhanced with videos of patients and accounts of disease from their spouses, relatives, or friends. [1, 18, 19, 21] Since some patients will not have the defined autoantibodies, systematic clinical characterisation may help define a syndrome that may respond to known therapies. These clinical-laboratory correlations are a major focus of our research and we, and others within the field, have already helped define new phenotypes and offered many patients new, immunotherapy-responsive diagnoses using these approaches, as reviewed in our published work. [8, 9, 20] Our research aims to discover immune markers, including immune-cell and genetic signatures, which define immunotherapy-responsive diseases, predict responses to treatment, inform causation, and help characterise the clinical features of the affected patients. Our laboratory investigations of patient samples – with and without antibodies – will help us understand the biology of these neurological conditions with an autoimmune component by identifying the mechanisms which operate in vitro and in vivo [22]and the cells which produce the antibodies,[23] and which may prove to be viable targets for future drug development. Indeed, our recent published studies have improved our understanding of cells in circulation which make the autoantibodies. Therefore as a natural extension of this work, we aim to: 1. Characterize if these cells are present in the bone marrow, a potential niche for cells which make antibodies, [24] which will have implications on identifying the cell types and their naïve precursors which produce antibodies, and future targeted therapies. 2. Further study causality, which was identified as the major unmet research need by our patients and relatives from our recent PPI day. We propose investigating environmental factors, including gut microbes (the microbiome), which is known to influence many major diseases [37]. ]We have also very recently ascertained HLA genetic factors which will have implications for disease causation, as originally proposed (Binks, Varley…and Irani, in press), and we would like to extend these studies to study further patients. 4. OBJECTIVES AND OUTCOME MEASURES Objectives Outcome Measures Primary Objective Clinical and immunological characterisation of patients with neurological conditions that may have an autoimmune component. Clinical data from medical notes, including measures of neurological function, functional capacity, quality of life, cognition, mood, and environmental exposures. Clinical data may be supplemented with standard questionnaires where this is necessary to achieve a standard minimal dataset. More detailed patient interviews may be performed in selected consenting patients. Immunological findings (through routine diagnostics and research assays, including in vitro and in vivo models) from blood, lymph node aspirate, bone marrow aspirate, stool, urine, saliva and CSF. Correlations between clinical and immunological findings in particular autoantibody detection to determine whether there are clinical or immunological features which predict a good response to treatments. Comparisons with healthy and disease control samples will determine cut-offs and syndrome specificity of immunological findings. Secondary Objectives Novel antibody discovery in patients with similar phenotypes. Correlations between clinical and immunological findings, to describe phenotypes associated with novel antibodies and their response to treatments. Comparisons with disease and healthy control samples will determine antibody specificity. Identify other immunological characteristics and markers in patients with neurological conditions. Immunological findings, including lymphocyte types, immune-related proteins, genes, microbes and cytokine levels, in bodily fluids, including bone marrow aspirate, and stool. The presence of serum antibodies in urine and saliva. Comparisons with disease and healthy control samples will determine assay specificity and cut-offs to allow understanding of whether these markers segregate with autoimmune neurological diseases. Characterise the antibodies and the cells that produce antibodies in order to understand the biology of neurological conditions and identify possible drug targets. Cell phenotypes (protein and transcripts), responses of cells to factors in culture. Examination of the bone marrow compartment may further inform on the cells which produce the antibodies, and further define pathogenic mechanisms. Use of in vitro and in vivo models to understand effects of immune factors, especially antibodies. Comparisons with disease and healthy control samples will determine assay cut-offs to allow understanding of whether these markers segregate with autoimmune neurological diseases. Genetic studies to understand non-heritable genes which may predispose to the neurological condition. Presence of genetic factors including polymorphisms or mutations which are found in patients more frequently than controls (data from publically available databases). Our recent finding of strong HLA associations in patients with LGI1/ CASPR2 autoantibodies demonstrates a need for this line of investigation (Binks, Varley….Irani, in press). 5. STUDY DESIGN This is an exploratory study correlating longitudinal observational clinical data from patients with a possible autoimmune component to their neurological condition, with sequential immunological findings from standard and novel assays. It is designed to minimise burden on participants by drawing primarily on information from medical notes and either diagnostic samples or samples taken at the same time as those obtained for clinical purposes. This clinical and immunological characterisation will allow description of associated phenotypes, biomarkers and responses to treatments. This will be true for both well-established and novel antibodies or immunological parameters. Further cellular and other immunogenetic laboratory work will seek to understand the biology of neurological conditions, predisposing factors and to identify possible targets for future drug development. 6. PARTICIPANT IDENTIFICATION 6.1. Study Participants 1. Patients in whom their clinician has established or suspects an autoimmune component to their neurological disease: this is anticipated to be 1500 participants over 10 years. Healthy controls: 500 samples from healthy volunteers will come from clinic, word-of-mouth, e-mail, and cohorts available from collaborators and biobanks (e.g. www.oxfordbiobank.org.uk) and be consented within our study. 2. 3000 anonymised samples and characterising data from research participants (who consented to use of their samples in further research). 6.2. Inclusion Criteria Patients: • There is or may be an autoimmune component to their neurological disease. • Individual and/or parent/guardian willing to give informed consent for participation in the study OR: • where an individual is deemed to lack capacity to consent, a declaration from a designated consultee. Controls (from biobanks and within this study): • Disease controls with a range of neurological conditions that share some overlapping clinical features to the patients with autoimmune neurological diseases. These will act as stringent controls. • Healthy volunteers - age-matched as closely as possible to adult patients who are recruited to studies and those who have lived in close contact with/ a similar environment to a patient. 6.3. Exclusion Criteria An individual may not enter the study if ANY of the following apply: • For competent adults: unwilling to provide informed consent • For children or those lacking capacity: expressing dissent • For designated consultees: unwilling to provide a declaration in support of participation. • For controls: consent not present for further research use of samples. 7. STUDY PROCEDURES 7.1. Recruitment Patients: Recruitment of patients with established or suspected autoimmune component to their neurological disease is summarised in Appendix A Flowchart. As many of these diseases are rare (estimated to be around 100 patients per year in the UK), identification and recruitment will incorporate three methods: 1. Clinician Identification. Patients with autoimmune neurological conditions may be identified by their UK clinician. Where appropriate arrangements are in place (collaboration, delegation, Trust approval) clinicians may obtain consent from their patient for participation in the research. Alternatively, they can obtain verbal consent for their patient’s details to be passed on to Oxford researchers who will contact the patient to provide the PIS, discuss the study in greater detail, and obtain consent if the patient wishes to participate. 2. British Neurological Surveillance Unit (BNSU). British Neurological Surveillance Unit (BNSU). The BNSU (http://www.theabn.org/what-we-do/bnsu.html) currently provides a portal by which UK neurologists can declare having seen a patient with a specified condition. This is especially useful in rare diseases. Via the BNSU, we have permission to survey UK neurologists with patients that have NMDAR or VGKC-complex antibodies (Appendix B). Clinicians can provide the PIS and/or obtain permission to pass on patient details to Oxford researchers to discuss potential research involvement and consent the interested patients. Telephone numbers and patient details will be conveyed using a secure email address and patients will be coded as rapidly as possible to maximize confidentiality. 3. Diagnostic Referral. Patients with suspected autoimmune neurological conditions may be identified after their blood/CSF test positive after NHS-based testing for autoimmune neurological conditions, frequently performed in our Oxford NHS or University laboratories. We would contact the clinician referring the sample, as above. Disease/ Healthy Controls - Disease controls will be approached via Neurology clinics and ward inpatients within Oxford University Hospital. - Healthy controls may be approached though word-of-mouth, or inter-departmental advertising through, for instance, e-mail. We anticipate that a large number of healthy controls will be recruited alongside patients in clinic (eg. friends or relatives), and in the case of partners/ spouse/ relatives who have lived with the patient for a substantial period of time, recruitment of such individuals may serve to act as important environmental controls. In addition, we aim to study: 3000 anonymised samples and characterising data from research participants (who consented to use of their samples in further research). These patients will have a range of neurological disorders, with clinical or immunological features which overlap with the patients. Healthy control anonymised samples recruited from cohorts available from collaborators and biobanks (n=500). 7.2. Screening and Eligibility Patients are eligible if their clinician has established or suspects an autoimmune component to their neurological disease. Results will be compared to age and sex-matched adult healthy controls and disease controls throughout, to generate reference values. Disease controls will be those with a low likelihood of having an autoimmune component to their neurological disease but often have a few clinical features that are comparable to the patients. Control samples will be obtained from cohorts consented elsewhere, such as biobanks (e.g. http://www.oxfordbiobank.org.uk) or via collaborators who hold research samples for which there is consent for their further research use. Informed Consent The participant and individual obtaining consent will personally sign and date the latest approved version of the Informed Consent form before any study-specific procedures are performed. The participant will be allowed as much time as wished to consider the information, and the opportunity to question the investigator or other independent parties to decide whether they will participate in the study. This may occur in person or over the telephone. Subsequently, written and verbal versions of the Participant Information Sheet and Consent Form will be presented to the participants. These will detail the exact nature of the study; what it will involve for the participant; the implications and constraints of the protocol; any risks involved in taking part. Informed written consent to study participation will be obtained by a member of the participant’s clinical team who is suitably qualified and experienced, and has been authorised to do so by the Chief Investigator. Individuals obtaining consent will ensure that the study is explained in a manner and at a level appropriate for the potential participant. Where participants are minors, informed consent will be sought from the responsible parent or guardian. A separate information sheet will be provided for children aged 10 upwards. Children under age 16 will be asked for assent. For adults with cognitive impairments so severe as to render them incapable of consenting for themselves, advice will be sought from a designated consultee, as per the provisions of the Mental Capacity Act 2005. The consent form clearly states that the participant is free to withdraw from the study at any time for any reason without prejudice to current or future care, and with no obligation to give the reason for withdrawal. A copy of the signed Informed Consent will be given to the participant. The original signed form will be retained at the study site and a copy sent by secure email to AProf Irani to retain in Oxford. 7.3. Study Assessment The clinical details and immunological findings from blood, CSF, lymph node aspirate, bone marrow aspirate, urine, saliva and stool are the parameters to be measured. These will be compared to healthy and disease control samples, although CSF and bone marrow will not be requested from healthy and disease controls. Clinical / in vivo data Clinical data obtained as part of clinical care will be collected from the patient medical record. These will include the patient’s demographics, presenting symptoms and signs, subsequent clinical features, test results, treatment and responses to treatment. If clinical features are assessed using established tools and questionnaires (such as the modified Rankin Scale (mRS), Addenbrooke’s cognitive examination revised (ACER)[25, 26], Hospital anxiety and depression scale (HADS)[27], formal neuropsychology, and Quality of life scores (e.g. Quality of life in epilepsy 31 (QOLIE-31))[28], this will also be recorded. Where possible, in order to obtain this minimal dataset, consenting participants will be asked complete assessments which had not been completed as part of routine practice. In addition to routine practice, patients with unusual problems will be consented to longer interviews to understand certain complaints, environmental exposures or qualitative symptoms in greater depth. These are anticipated to last 30-90 minutes. Occasionally, if it is inconvenient for the patient to visit researchers, the researcher will ask to visit them with their permission. Video recordings for research purposes will be used to document the precise clinical phenotypes of selected patients and controls, as has been essential in previous studies.[21] Samples for Laboratory/experimental studies Blood is routinely obtained for clinical purposes. We aim to obtain additional volumes of blood at the time of clinical blood sampling during a single venepuncture. We anticipate blood (up to 80mls), will be sampled at all baseline visits. Baseline visits will also involve CSF for clinical purposes in around 20% of patients, and for all consenting patients additional CSF will be obtained during the same procedure (up to 30mls). In addition, CSF will be sampled in about 10% of adult patients with capacity to consent for themselves, for research purposes. For participants unable to come to Oxford or for convenience there is the option to have a blood sample taken at their GP practice. Consent would have been obtained prior to this. The study team will send the participant an EDTA tube labelled with their participant ID, a covering letter for their GP to explain the study, and a stamped-addressed envelope for its return to the research team. We will advise participants to show the phlebotomist their study consent form, and the GP practice may wish to keep a copy if that is their policy. Self-collection of faecal samples will follow IHMS standard procedures (SOP 05 V1), and online YouTube videos are available to assist participants (see: www.goo.gl/iq9Nm4). For convenience, participants will have the option of collecting their sample from home and posting their sample to us. Participants will be sent a stool collection pack via post in advance, including a stool container labelled with their study ID, and a stamped addressed envelope for return of their sample. Additionally, for research purposes in adult patients with capacity to consent, we will request a donation of cells from cervical lymph nodes using a technique called ultrasound-guided Fine Needle Aspiration (FNA). A radiologist experienced in head and neck ultrasound-guided FNA will obtain cells from cervical lymph nodes which can then be characterised by immunophenotyping, genetic sequencing and cell culture. FNA is used routinely in the assessment of diseases of the head and neck, and has a very low complication rate, equivalent to routine peripheral venous blood sampling and is lower than lumbar puncture. It has been used successfully to investigate the role of cervical lymph node cells in Multiple Sclerosis for example [36]. The main risks are local discomfort, infection, bleeding/bruising and damage to local structures. These are offset by aseptic technique, targeting nodes far removed from major blood vessels or nerves, and precise ultrasound-guidance. Similarly, for research purposes around 40 adult patients with capacity to consent, bone marrow aspiration will be possible. A trained Haematologist will obtain the bone marrow aspirate, which will be used to study the immune cells which are known to make antibodies. The procedure is used routinely in Haematology practice, and has few major risks. Urine (up to 50mls), saliva (up to 5mls), and stool are non-invasive collection procedures and may be requested from patients with 20 minutes extra to spare (anticipated 30%). We aim to understand whether 20 patients with high and low levels of serum antibodies have detectable antibodies in non-invasive bodily fluids (urine and saliva). If successful, we may extend this to other patients, especially children in whom routine invasive procedures can be difficult. Stool samples will be used to explore the gut microbiome. Samples from disease and healthy controls (prospectively recruited to our study and from biobanks or collaborators with permissions to share samples) will be used to determine cut-offs for the assays, typically at a stringent level of the mean plus three standard deviations. The samples requested will reflect the phenotypes of the participants found to have immune markers. For example, if a patient with a new antibody had an Alzheimer’s like presentation, we may ask to test cohorts with Alzheimer’s disease. Clinical-laboratory correlations and impact on stated objectives Correlations between clinical and laboratory observations are ongoing projects with constant evaluations which guide our subsequent thinking, and future directions. [8, 29, 30] Typically, after 10 patients with similar clinical or immunological findings, we will begin to analyse the data in batches. The recruitment for a particular analysis is estimated to take 1-2 years. We will use the clinical assessments to evaluate whether there has been a response to treatment over time and observe whether the presence of antibodies or other immune factors are enriched within the responders (primary objective). In patients with suspected autoimmune neurological diseases and novel antibodies or other immune factors including cytokines and immune cells, we will observe whether clinical assessments improve with immune-system directed therapies (second objective, first three points). This will help us understand whether the immune factors we detect segregate closely with clinical phenotypes. We will further explore HLA and other genetic associations in patients and by comparison to controls. 7.4. Subsequent Visits Patient participants will typically have clinical appointments at 3-monthly intervals with a major aim being to understand whether there has been a clear response to treatments. Further clinical details (including neurological follow-up, the tools and questionnaires mentioned above, and videos) would be obtained at these times to assess clinical progress and allow us to determine the effect of treatment in patients with autoimmune neurological diseases. At these follow-up appointments, we will also obtain repeat biological samples to assess the alterations in measured immunological parameters in parallel with the change in the clinical features. The aim is to longitudinally assess the correlation of immune factors and clinical features, especially in patients administered immune-system directed therapies as part of their clinical care (primary objective). We intend to follow them for the duration of their routine clinical follow-up, typically between 2 and 5 years. Occasionally, when inconvenient for the patient to visit Oxford, we will ask to visit the patient at home, or conduct follow-up via the telephone. 7.5. Sample Handling We anticipate samples will be analysed using a variety of immunological and cellular/molecular techniques including immunochemistry (including cell-based assays), western blotting, flow-cytometry, ELISAs, cell culture, sequencing and polymerase chain reaction. See table 1, below. The samples, which will be stored in the John Radcliffe Hospital, Nuffield Department of Clinical Neurosciences. Biological samples will be frozen at -20˚C, -80˚C or on liquid nitrogen for use in current and future studies. Disease control and healthy control samples will be stored under the same conditions. A selection of the samples will be sent to companies who can perform the above assays. A selection of samples including sera, plasma, CSF, urine and lymph node/bone marrow aspirates may be used to study their effects in in vivo and in vitro systems comprising cellular and live animal models. Table 1. Samples Sample Purpose Volume and potential burden Blood For serum, plasma, cytokines and immune-cell studies. A single sample may be taken for genetic studies. Up to 80mls. Obtaining this volume in addition to any clinical sampling causes no adverse effects and is usually possible from a single needle. We may require large volumes (e.g. 80mls) in order to study rare antibody-secreting cell populations in selected patients. Previous research practice has established that this amount can be obtained without additional adverse affect to the participant. CSF To examine antibodies, cytokines and cells found within the central nervous system, not just in the periphery (blood) Up to 30 mls. Obtaining this additional volume during procedure causes no adverse effects. Previous research practice has established that this amount can be obtained without additional adverse affect to the participant. Lymph node aspirate To examine extra-CNS development of CNS Antigen-specific lymphocytes. Ultrasound-guided Fine Needle Aspiration of cervical lymph nodes. Volume is negligible, cells fill tip of 23 gauge needle during aspiration. Procedure is routinely performed by radiology colleagues to assess head and neck disease. Local complication rate, largely localised bruising <5% is equivalent to phlebotomy. Bone Marrow Aspirate To study the bone marrow compartment to answer questions generated by our recent studies about whether the earliest and latest B-lineage cells (both found in bone marrow) have capacity to produce autoantibodies in patients. Up to 20mls Procedure performed as a routine Haematological investigation for assessment of many Haematological conditions. Complications include localised pain, and infrequently, infection, and major bleeding (1 in 1000). See ‘Appendix D’. Urine To study the presence of immunological markers, especially antibodies, in non-invasively obtained fluids Up to 50mls Saliva Up to 5mls Stool To explore the effect of the gut microbiome on antibody production, cytokines, and T cell interactions, and longitudinal alterations in individuals. One unit, requested on up to 3 occasions per consenting patients Additional residual samples Patients with immune related neurological disorders often have plasma exchange (PLEX) as part of their treatment. This involves removal of plasma, and this is a useful resource for large quantities of antibodies which could be used in laboratory studies. The plasma filtrate would otherwise be discarded. Similarly, consent will be obtained for any samples or tissue surplus to diagnostic need resulting from other clinical treatments or investigations. 7.6. Discontinuation/Withdrawal of Participants from Study Each participant has the right to withdraw from the study at any time. Their data and samples may be withdrawn from the study and destroyed at their request. In addition, the Investigator may discontinue a participant from the study at any time if the Investigator considers it necessary for any reason including: • Ineligibility (either arising during the study or retrospectively having been overlooked at screening) • Withdrawal of Consent • Loss to follow up The reason for withdrawal will be recorded in the CRF. 7.7. Definition of End of Study The end of study is the date of the last visit of the last participant. 8. INTERVENTIONS Lumbar puncture. This will be taken once or twice in most cases for clinical purposes and, in about 10% of consenting adult patients, specifically for research. 9. SAFETY REPORTING 9.1. Definition of Serious Adverse Events A lumbar puncture can result in the potentially life−threatening conditions of subdural haematoma (requiring craniotomy to relieve intracranial pressure), or meningitis (requiring intravenous antibiotics). Such severe adverse events are very rare (based on the practice from an experienced neurological ward where such procedures are carried out on a daily basis typically without any complications). A bone marrow aspiration may result in potentially life-threatening major haemorrhage, which may require resuscitation and blood transfusion. This is extremely rare, occurring at a rate of 1 in 1000 cases. Patients will be advised in advance to be familiar with their aftercare leaflet, avoid any strenuous activity post-procedure, and in the instance of any noted bleeding through their dressing site, to apply pressure and seek medical attention immediately. All necessary expertise is available on site to deal promptly with such outcomes, if they are required. Other ‘important medical events’ may also be considered serious if they jeopardise the participant or require an intervention to prevent one of the above consequences. 9.2 Reporting Procedures for Serious Adverse Events A serious adverse event (SAE) occurring to a participant should be reported to the REC that gave a favourable opinion of the study where in the opinion of the Chief Investigator the event was ‘related’ (resulted from administration of any of the research procedures) and ‘unexpected’ in relation to those procedures. Reports of related and unexpected SAEs should be submitted within 15 working days of the Chief Investigator becoming aware of the event, using the HRA report of serious adverse event form (see HRA website). 10. STATISTICS AND ANALYSIS 10.1. Description of Statistical Methods Databases will be maintained on Microsoft Excel and Access. T-tests, ANOVA, correlation coefficients and other statistical analyses, typically available using GraphPadPRISM software or SPSS will be used to compare patients with disease and healthy controls. Ongoing analyses are planned, for the observational data collected. 10.2. The Number of Participants We plan to study: 1. 1500 patients in whom their clinician has established or suspects an autoimmune component to their neurological disease. Healthy controls: 500 anonymised samples from healthy volunteers will come from clinic, word-of-mouth, e-mail, and cohorts available from collaborators and biobanks (e.g. www.oxfordbiobank.org.uk). 2. 3000 anonymised samples and characterising data from research participants (who consented to use of their samples in further research). Novel antibodies will be looked at on a case-by-case basis for around the first 10 cases, to establish clinical correlations. For questions examining established antibodies, other immunological markers, cells or genetic findings at least 10 patients are typically required to understand trends. [2, 31] An equal number of age- and sex-matched controls will be compared throughout. For DNA assessments, we anticipate 300 patients will be required and control data derived from available public databases. 10.3. Analysis of Outcome Measures The laboratory work will help determine the presence of an autoantibody (using brain tissue staining, cell based assays and other related techniques), the properties of the patient white blood cells from blood and lymph and bone marrow aspirates (especially using flow cytometry and cell culture techniques), the cytokines found in patients’ blood and CSF, and DNA signatures found in the patient’s blood. Throughout, healthy control and disease controls samples will be used to calibrate the baselines for assays, and determine rigorous cut-offs. We hypothesize that, in addition to stool samples, saliva and urine may be non-invasive fluids from which to determine antibody levels. We aim to trial this on 20 patients with high and low levels of serum antibodies, and 20 healthy controls to compare antibody levels, and proceed to further collections if successful. The clinical outcome measures will be analysed using the statistical methods described above, as already established in the literature4,9,11,17. These are often simply descriptive but often utilize statistical techniques such as Chi-Squared, correlation coefficients and t-tests available in GraphPad PRISM. Values may be compared to those available from disease and healthy controls to establish unique features of the participants. Data from participants who have withdrawn will be excluded from analyses. 11. DATA MANAGEMENT 11.1. Access to Data Direct access will be granted to authorised representatives from the Sponsor and host institution for monitoring and/or audit of the study to ensure compliance with regulations. 11.2. Data Recording and Record Keeping Participant details will be held on a password-protected Microsoft Excel file within the CI’s laboratory / offices on University of Oxford premises. Participants will be identified by a unique, study-specific code number. Paper records (including consent forms) will be held in locked cabinets in the CI’s laboratory/offices. Retrieval of data from medical records will generally be effected by means of NHS numbers and is subject to participant consent. Participant identifying data will be retained for the duration of the study’s ethical approval and then destroyed. 12. QUALITY ASSURANCE PROCEDURES The study may be monitored, or audited in accordance with the current approved protocol, GCP, relevant regulations and standard operating procedures. 13. ETHICAL AND REGULATORY CONSIDERATIONS 13.1. Declaration of Helsinki The Investigator will ensure that this study is conducted in accordance with the principles of the Declaration of Helsinki. 13.2. Guidelines for Good Clinical Practice The Investigator will ensure that this study is conducted in accordance with relevant regulations and with Good Clinical Practice. Dr Irani has completed his GCP certification. 13.3. Approvals The protocol, informed consent form, participant information sheet and any proposed advertising material will be submitted to an appropriate Research Ethics Committee (REC), and host institution(s) for written approval. The Investigator will submit and, where necessary, obtain approval from the above parties for all substantial amendments to the original approved documents. 13.4. Reporting Upon request, the CI shall submit a Progress report to the REC Committee, host organisation and Sponsor. In addition, an End of Study notification and final report will be submitted to the same parties. 13.5. Participant Confidentiality The study staff will ensure that the participants’ anonymity is maintained. The participants will be identified only by a participant ID number on all study documents and any electronic database, with the exception of the CRF, where participant initials may be added. The ID number will contain the initials of hospital of recruitment (e.g. JR for John Radcliffe) followed by a randomly generated six-digit number, starting from 000000. As few researchers as possible in Dr Irani's team will have access to the name and identification numbers of the patients for initial data inputting and code generation. All documents will be stored securely and only be accessible by study staff and authorised personnel. The study will comply with the Data Protection Act, which requires data to be anonymised as soon as it is practical to do so. Identifiable videos will be kept on laboratory computers used only by the research team. Consent forms will be emailed using Dr Irani’s secure nhs.net email address. 13.6. Expenses and Benefits Reasonable travel expenses for any visits additional to normal care will be reimbursed on production of receipts, or a mileage allowance provided as appropriate. 13.7. Other Ethical Considerations Vulnerable Participants: As many patients with autoimmune neurological diseases have cognitive impairment, their participation will be supported, where appropriate, through involvement of a designated consultee, in keeping with the provision of the Mental Capacity Act, 2005. Children will be included in this study as patients where there is suspicion of an autoimmune component to their neurological disease. We have developed information aimed at children aged 10 and above only. This was decided on the basis of the study's theoretical complexity balanced against the minimal anticipated intervention (at most, a blood, urine or saliva sample, if these have not already been collected in clinical care). Age 10 will be used as a guide; attempts will be made to engage all children in decisions about participation as appropriate to their understanding. Incidental Findings: Findings considered to be of clinical significance for patients will be discussed with their referring clinician / GP and fed back to collaborators. For control samples, feedback pathways will comply with biobank and source study protocols. Genetic findings will not be fed back to participants because the conditions under investigation are not known to be heritable. 14. FINANCE AND INSURANCE 14.1. Funding Dr Irani has a Wellcome-Trust funded intermediate fellowship, BMA Vera Down and Margaret Temple, ONO pharmaceuticals, and UCB-Oxford University Alliance grants to study neurological diseases with an autoimmune component. Dr Irani and Dr Waters are supervising two DPhil students with ABN fellowships to study autoimmunity in neurology and have R&D funds. Dr Waters is supported by EuroImmun AG, and by the NIHR Oxford BRC. Dr Sen and Dr Irani are co-PIs on a grant from the UCB-Oxford Alliance. Dr Varley is supported by an ABN Clinical Research Training Fellowship. Dr Al-Diwani has a Wellcome Trust DPhil Training Fellowship supported by the NIHR Oxford BRC and Oxford University Medical Sciences Doctoral Training Centre. Dr Binks is supported by the Oxford University Clinical Academic Graduate School and a Wellcome / BRC DPhil. Dr McGinty and Dr Michael are supported by the UCB-Oxford University Alliance. The lab is supported by BRC Oxford. 14.2. Insurance The University has a specialist insurance policy in place, which would operate in the event of any participant suffering harm as a result of their involvement in the research (Newline Underwriting Management Ltd, at Lloyd’s of London). NHS indemnity operates in respect of the clinical treatment that is provided. 15. PUBLICATION POLICY The Investigators will be involved in reviewing drafts of the manuscripts, abstracts, press releases and any other publications arising from the study. Authors will acknowledge that the study was funded by the Wellcome Trust. Authorship will be determined in accordance with the ICMJE guidelines and other contributors will be acknowledged. To ensure results are available to study participants, they are often presented to patient charities/groups (e.g. myaware and encephalitis society) and published as open access journals. Links to our scientific publications can be find via our group’s online website: https://www.ndcn.ox.ac.uk/research/autoimmune-neurology-group  16. REFERENCES 1. Irani SR, Stagg CJ, Schott JM, et al (2013) Faciobrachial dystonic seizures: the influence of immunotherapy on seizure control and prevention of cognitive impairment in a broadening phenotype. Brain 136:3151–3162. doi: 10.1093/brain/awt212 2. Irani SR, Alexander S, Waters P, et al (2010) Antibodies to Kv1 potassium channel-complex proteins leucine-rich, glioma inactivated 1 protein and contactin-associated protein-2 in limbic encephalitis, Morvan's syndrome and acquired neuromyotonia. Brain 133:2734–2748. doi: 10.1093/brain/awq213 3. Dalmau J, Gleichman AJ, Hughes EG, et al (2008) Anti-NMDA-receptor encephalitis: case series and analysis of the effects of antibodies. Lancet Neurol 7:1091–1098. doi: 10.1016/S1474-4422(08)70224-2 4. Hacohen Y, Dlamini N, Hedderly T, et al (2013) N-methyl-D-aspartate receptor antibody-associated movement disorder without encephalopathy. Developmental Medicine & Child Neurology 56:190–193. doi: 10.1111/dmcn.12321 5. Irani SR, Vincent A, Schott JM (2011) Autoimmune encephalitis. BMJ 342:d1918–d1918. doi: 10.1136/bmj.d1918 6. Irani SR, Bera K, Waters P, et al (2010) N-methyl-d-aspartate antibody encephalitis: temporal progression of clinical and paraclinical observations in a predominantly non-paraneoplastic disorder of both sexes. Brain 133:1655–1667. doi: 10.1093/brain/awq113 7. Rosenfeld MR, Dalmau J (2011) Anti-NMDA-Receptor Encephalitis and Other Synaptic Autoimmune Disorders. Curr Treat Options Neurol 13:324–332. doi: 10.1007/s11940-011-0116-y 8. Irani SR, Gelfand JM, Al-Diwani A, Vincent A (2014) Cell-surface central nervous system autoantibodies: clinical relevance and emerging paradigms. Ann Neurol 76:168–184. doi: 10.1002/ana.24200 9. Vincent A, Bien CG, Irani SR, Waters P (2011) Autoantibodies associated with diseases of the CNS: new developments and future challenges. Lancet Neurol 10:759–772. doi: 10.1016/S1474-4422(11)70096-5 10. Dalmau J, Lancaster E, Martinez-Hernandez E, et al (2011) Clinical experience and laboratory investigations in patients with anti-NMDAR encephalitis. Lancet Neurol 10:63–74. doi: 10.1016/S1474-4422(10)70253-2 11. Gresa-Arribas N, Titulaer MJ, Torrents A, et al (2014) Antibody titres at diagnosis and during follow-up of anti-NMDA receptor encephalitis: a retrospective study. Lancet Neurol 13:167–177. doi: 10.1016/S1474-4422(13)70282-5 12. Byrne S, Lim M (2014) N-methyl- d-aspartate receptor antibody encephalitis: how much treatment is enough? Developmental Medicine & Child Neurology 57:14–15. doi: 10.1111/dmcn.12559 13. Carvajal-González A, Leite MI, Waters P, et al (2014) Glycine receptor antibodies in PERM and related syndromes: characteristics, clinical features and outcomes. Brain 137:2178–2192. doi: 10.1093/brain/awu142 14. Angus-Leppan H, Rudge P, Mead S, et al (2013) Autoantibodies in Sporadic Creutzfeldt-Jakob Disease. JAMA Neurol 70:919–4. doi: 10.1001/jamaneurol.2013.2077 15. Dahm L, Ott C, Steiner J, et al (2014) Seroprevalence of autoantibodies against brain antigens in health and disease. Ann Neurol 76:82–94. doi: 10.1002/ana.24189 16. Zandi MS, Irani SR, Lang B, et al (2011) Disease-relevant autoantibodies in first episode schizophrenia. J Neurol 258:686–688. doi: 10.1007/s00415-010-5788-9 17. Doss S, Wandinger K-P, Hyman BT, et al (2014) High prevalence of NMDA receptor IgA/IgM antibodies in different dementia types. Ann Clin Transl Neurol 1:822–832. doi: 10.1002/acn3.120 18. Kitley J, Leite MI, Nakashima I, et al (2012) Prognostic factors and disease course in aquaporin-4 antibody-positive patients with neuromyelitis optica spectrum disorder from the United Kingdom and Japan. Brain 135:1834–1849. doi: 10.1093/brain/aws109 19. Leite MI, Jacob S, Viegas S, et al (2008) IgG1 antibodies to acetylcholine receptors in “seronegative” myasthenia gravis. Brain 131:1940–1952. doi: 10.1093/brain/awn092 20. Varley J, Vincent A, Irani SR (2015) Clinical and experimental studies of potentially pathogenic brain-directed autoantibodies: current knowledge and future directions. J Neurol 262:1081–1095. doi: 10.1007/s00415-014-7600-8 21. Irani SR, Michell AW, Lang B, et al (2011) Faciobrachial dystonic seizures precede Lgi1 antibody limbic encephalitis. Ann Neurol 69:892–900. doi: 10.1002/ana.22307 22. Wright S, Hashemi K, Stasiak L, et al (2015) Epileptogenic effects of NMDAR antibodies in a passive transfer mouse model. Brain 138:3159–3167. doi: 10.1093/brain/awv257 23. Chihara N, Aranami T, Oki S, et al (2013) Plasmablasts as migratory IgG-producing cells in the pathogenesis of neuromyelitis optica. PLoS ONE 8:e83036. doi: 10.1371/journal.pone.0083036 24. Halliley JL, Tipton CM, Liesveld J, et al (2015) Long-Lived Plasma Cells Are Contained within the CD19(-)CD38(hi)CD138(+) Subset in Human Bone Marrow. Immunity 43:132–145. doi: 10.1016/j.immuni.2015.06.016 25. Elamin M, Holloway G, Bak TH, Pal S (2015) The Utility of the Addenbrooke's Cognitive Examination Version Three in Early-Onset Dementia. Dement Geriatr Cogn Disord 41:9–15. doi: 10.1159/000439248 26. Tsoi KKF, Chan JYC, Hirai HW, et al (2015) Cognitive Tests to Detect Dementia: A Systematic Review and Meta-analysis. JAMA Intern Med 175:1450–1458. doi: 10.1001/jamainternmed.2015.2152 27. Spurgeon L, James G, Sackley C (2015) The Hospital Anxiety and Depression Scale: a pilot study to examine its latent structure and the link between psychological state and symptom severity in transient ischaemic attack patients. Psychol Health Med 1–7. doi: 10.1080/13548506.2015.1074711 28. Fiest KM, Sajobi TT, Wiebe S (2014) Epilepsy surgery and meaningful improvements in quality of life: results from a randomized controlled trial. Epilepsia 55:886–892. doi: 10.1111/epi.12625 29. Waters PJ, McKeon A, Leite MI, et al (2012) Serologic diagnosis of NMO: a multicenter comparison of aquaporin-4-IgG assays. Neurology 78:665–671. doi: 10.1212/WNL.0b013e318248dec1 30. Brenner T, Sills GJ, Hart Y, et al (2013) Prevalence of neurologic autoantibodies in cohorts of patients with new and established epilepsy. Epilepsia 54:1028–1035. doi: 10.1111/epi.12127 31. Irani SR, Buckley C, Vincent A, et al (2008) Immunotherapy-responsive seizure-like episodes with potassium channel antibodies. Neurology 71:1647–1648. doi: 10.1212/01.wnl.0000326572.93762.51 36. Fabriek BO, Zwemmer JN, Teunissen CE, et al. (2005) In vivo detection of myelin proteins in cervical lymph nodes of MS patients using ultrasound-guided fine-needle aspiration cytology. J Neuroimmunol. 2005 Apr;161(1-2):190-4. DOI: 10.1016/j.jneuroim.2004.12.018 37. Qin J, Li Y, Cai Z, et al (2012) A metagenome-wide association study of gut microbiota in type 2 diabetes. Nature 490(7418):55-60. doi: 10.1038/nature11450.   17. APPENDIX A: STUDY FLOW CHART *the grey text indicates the routine NHS laboratory work which will be a source of patient recruitment for NDCN researchers. It will be a route by which researchers will retrospectively and prospectively identify clinicians looking after patients who may be eligible research participants. In addition, the data generated by the NHS service provided by NDCN researchers will be regularly audited to understand service provision, outside of the auspices of this research project. OUH = Oxford University Hospitals; OUHCI = Oxford University Hospitals Clinical Immunology laboratory; NDCN = Nuffield department of Clinical Neurosciences; BNSU = British Neurological Surveillance Unit. * the text in red indicates the direct research 18. APPENDIX B: BNSU letter APPENDIX C: AMENDMENT HISTORY Amendment No. Protocol Version No. Date issued Author(s) of changes Details of Changes made 1 3 19/01/2017 Dr Adam Al-Diwani Addition of Lymph node aspiration Addition of option of participant blood sampling at GP practices Addition of investigators 2 4 22/06/2018 Dr Sophia Michael AProf Sarosh Irani Removal and addition of investigators, and amendment of current investigators’ titles Updated CTRG telephone number – Participant and Parental Information Sheets Inclusion of use of in vitro and in vivo models to study the effects of patient immune factors. Adjustment of planned sample size to account for anticipated increased number of samples Amended specificity of lymph node sample site from ‘submental’ to ‘cervical’ Addition of bone marrow aspiration and stool collection and creation of ‘Appendix D’. Expansion of control groups, with new information and consent sheets for ‘Non-autoimmune participants’ Study funders updated to include ONO Pharmaceuticals