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UNIVERSITY OF DUBLIN TRINITY COLLEGE DUBLIN SCHOOL OF BIOCHEMISTRY & IMMUNOLOGY SENIOR SOPHISTER IMMUNOLOGY COURSE DETAILS 2015-2016 1 This course is partially funded by the Irish government under the Human Capital Investment Operational Programme 2007-2013 and aided by the European Social Fund (ESF) under the 2007-2013 Community Support Framework (CSF) 2 IMMUNOLOGY SENIOR SOPHISTER MODULES (60 ECTS) BI4275 CELLS OF THE INNATE AND ADAPTIVE IMMUNE SYSTEM (5ECTS) This module outlines the differentiation and roles of specific leukocyte populations in mediating innate and adaptive immune responses BI4055 MICROBIAL DISEASES’ (5 ECTS) Protozoan, bacterial and viral diseases will be covered in detail in this module. BI4215 ORGAN-SPECIFIC IMMUNITY (5 ECTS) This module describes characteristic and distinctive features of the innate and adaptive immune responses at mucosal sites, the liver and the central nervous system. BI4045 AUTOIMMUNE AND INFLAMMATORY CONDITIONS (5 ECTS) This module covers basic and clinical aspects of autoinflammatory and autoimmune conditions, including rheumatoid arthritis and multiple sclerosis and immunodeficiency syndromes. BI4235 IMMUNE SIGNALLING (5 ECTS). This module covers immune-related signalling events including cell signalling in apoptotic cell death, cytokine signalling, the molecular basis for immune signalling, PI3 kinase and MTOR and T cell receptor signalling BI4245 IMMUNITY TO PATHOGENS AND VACCINATION (5 ECTS) This module covers immune responses to viruses and bacteria including tuberculosis and Bordetella pertussis, vaccines and the danger theory BI4255 IMMUNOTHERAPY, IMMUNOGENETICS AND IMMUNOMODULATION (5 ECTS) This module covers key concepts and specific examples in immunogenetics, immunotherapy and immunomodulation. It also covers transplantation, graft rejection and immunosuppressive therapies. BI4265 TUMOUR IMMUNOLOGY (5 ECTS) This module addresses cancer, invasion and tumour immunity including stem cell therapy, immunotherapy, vaccines and the immune response to tumours. BI4295 RESEARCH PROJECT IN IMMUNOLOGY (15 ECTS) The module comprises of an original research project in biochemistry and a research thesis. BI4015 DATA HANDLING (5 ECTS) This module covers quantitative biochemical problems, bioinformatics and molecular modelling. NOTE: Learning outcomes for each of the modules can be found on the School homepage: http://www.tcd.ie/Biochemistry/courses/senior_soph.php 3 Explanation of ECTS The European Credit Transfer and Accumulation System (ECTS) is an academic credit system based on the estimated student workload required to achieve the objectives of a module or programme of study. It is designed to enable academic recognition for periods of study, to facilitate student mobility and credit accumulation and transfer. The ECTS is the recommended credit system for higher education in Ireland and across the European Higher Education Area. The ECTS weighting for a module is a measure of the student input or workload required for that module, based on factors such as the number of contact hours, the number and length of written or verbally presented assessment exercises, class preparation and private study time, laboratory classes, examinations, clinical attendance, professional training placements, and so on as appropriate. There is no intrinsic relationship between the credit volume of a module and its level of difficulty. The European norm for full-time study over one academic year is 60 credits. The Trinity academic year is 40 weeks from the start of Michaelmas Term to the end of the annual examination period. 1 ECTS credit represents 20-25 hours estimated student input, so a 10-credit module will be designed to require 200-250 hours of student input including class contact time and assessments. ECTS credits are awarded to a student only upon successful completion of the course year. Progression from one year to the next is determined by the course regulations. Students who fail a year of their course will not obtain credit for that year even if they have passed certain component courses. Exceptions to this rule are one-year and part-year visiting students, who are awarded credit for individual modules successfully completed. For additional details see: http://www.tcd.ie/vp-cao/bd/vpbd3college_ects.php Examinations and Breakdown of Marks: Senior Sophister Module Name ECTS Weighting 1) Cells of the innate and adaptive immune system 2) Microbial diseases 3) Organ-specific immunity 4) Autoimmune and inflammatory conditions 5) Immune Signalling 6) Immunity to pathogens and vaccination 7) Immunotherapy, immunogenetics, immunomodulation 8) Tumour immunology 9) Research Project in Immunology 10) Data handling BI4275 BI4055 BI4215 BI4045 BI4235 BI4245 BI4255 BI4265 BI4295 BI4015 5 ECTS 5 ECTS 5 ECTS 5 ECTS 5 ECTS 5 ECTS 5 ECTS 5 ECTS 15 ECTS 5 ECTS 4 The Senior Sophister year is broken down into a total of 60 marks. Annual Examination Papers. Value: 40 marks or 66.66% of SS year There are four exam papers at the end of the SS year, each with equal weighting (total 40 marks or 66.66% of SS year) as follows: Paper 1 Value: 10 marks or 16.66% of SS year Specific questions related to modules 1-4 above. Exam paper divided up into 4 sections (one module per section). Answer 4 questions, one from each section. Paper 2 Value: 10 marks or 16.66% of SS year Specific questions related to modules 5-8 above. Exam paper divided up into 4 sections (one module per section). Answer 4 questions, one from each section. Papers 3 & 4 are indirectly linked to modules listed above, being general in nature. Paper 3 Value: 10 marks or 16.66% of SS year Exam paper divided up into two sections. Answer 4 questions at least one from each section. The quantitative problems given in Section A will be related to those practiced in the Junior Sophister AND Senior Sophister year. Paper 4 Value: 10 marks or 16.66% of SS year Exam paper divided up into two sections. Section A- question 1: short questions, answer 10 out of 18 (compulsory question) -worth 50% of paper. Section B-answer 2 questions -worth 50% of paper All answers from the above exam papers are double-marked. Research Project in Immunology 11-week research project and thesis. Value: 15 marks or 25% of SS year. Data Handling Value: 5 marks or 8.34% of SS year. This module covers quantitative biochemical problems, bioinformatics and molecular modelling, animal handling and a presentation by the student on a biochemical technique. Marks are awarded through continual assessment and in-course exams as follows: Quantitative biochemical problem topics: 3 sequence analysis exercises: 1 animal handling in-course exam: 1 biochemical techniques presentation: 2.5 marks 1.25 marks 0.625 mark 0.625 mark __________ 5 marks The overall degree mark is comprised of 80% of SS year and 20% of JS year. 5 On completion of their annual examinations, selected students sit a viva voce examination with the External Examiner. Students are considered ‘borderline’ if they are 2.5% or less off a grade and following the viva voce examination, the External Examiner may recommend at the Examiners’ meeting that the students’ degree mark be brought up to the next grade. Tutorials: Tutors have been chosen randomly (see page 16). Please contact your tutor during the first week of the first semester. You are expected to attend a tutorial every fortnight. Times and dates of tutorials given on timetable are a rough guide only. Your tutor will set various exercises and these should help you in your final examinations. Practise Vivas: Vivas (oral exams) are held approximately two weeks following the completion of your four exam papers. The maximum percentage marks that you can be brought up by is 2.5%. You cannot be marked down by a viva. You will not know your mark before sitting the viva. How can you prepare for the viva? Practise vivas will be held once in the first semester and once in the second semester. You will be assigned to a pair of academic staff members in the first semester and a different pair of staff in the second semester. The vivas take approximately 20 minutes and you will be asked a variety of questions. There are no marks going for this. Please regard these vivas not as a test of your knowledge but as useful practise. They are also good interview experience! When you are called for a viva in the summer, you should read over your project thesis as the Extern often starts off by asking you about your project. They will want to relax you and will generally start you off on a topic you know a lot about. The Extern will probably cover about 4-6 topics during the viva and it is impossible to second guess what they will ask. However, if you feel you did badly in one particular exam question, it is a good idea to revise this topic. The Extern has access to all your marks and if he/she sees a blip in an otherwise very consistent set of marks they may wish to follow this up. The Extern may also ask you if there is a topic in Biochemistry that you find particularly interesting and that you wish to talk about. It is therefore a good idea to have something prepared but ensure that it is a specific topic. Do not be too general and say that you’re interested in protein structure! The Extern may also ask you on your views of the course; was there a part of the course you really enjoyed or not as the case may be. The role of the Extern is not only to assess your performance but also to assess our teaching capabilities and to identify strengths/weaknesses and even omissions in the course so that they can make recommendations for the following year. Quantitative Problem Sessions: All Quantitative Problems will be given out at introductory sessions by different staff members (e.g. Prb 1 Intro on the timetable), at the times indicated. You will attempt the problem in your own time and must bring the problem with you to a timetabled 6 tutorial session (e.g. Prb 1 Tutorial). You must be able to demonstrate that you have attempted the problem to the staff member and failure to do so will result in being returned as non-satisfactory. In the tutorial session, the staff member will go through the solution with you. Later on that same week you will sit an exam on this particular problem topic (e.g. Prb 1 Exam). The mark for each problem topic will comprise of the in-course exam mark. Sequence Analysis Sessions: There will be three three-hour Sequence Analysis Sessions (Dr Jerrard Hayes). Each session will begin with a brief introduction in FRED (2pm). You will then move to the East End Public Access Mac Room for the practical session. Submission dates for the Sequence Analysis Exercises are indicated on the timetable. Dr Hayes will advise you as to how and where you submit the exercises. Course Feedback: A Feedback Form for each course will be given out at the beginning of the term. These (anonymous) forms are a mechanism whereby students can make comments and suggestions that will help us to maintain and indeed improve the quality of the teaching offered by the School of Biochemistry & Immunology. Please fill out the form upon completing each course, do not wait until the end of term (you will forget!). Put the forms into the box provided in the secretary’s office. Addresses and Phone No's: Please enter your College based address, e-mail address and telephone number (if any) on the sheet provided at the Introductory Lecture. Please also include a home (or other contact) address and telephone number. This will enable us to contact you in an emergency or with important changes in such details as timetables, exam venues, etc. If you do not enter these details you may not be informed of any changes. Careers Talk: Sean Gannon will give a Careers Talk tailored for Immunology students, on Tuesday 6th October at 11 am in FRED. Research Projects: At the start of first semester you will be given Project Summaries of all available projects. You then have two weeks to select your first choice for a project. Feel free to discuss these projects with Staff. You may not select a project offered by a Staff Member with whom you have done a Summer Project. Projects will then be allocated by the following procedure: any project with only one taker will be allocated to that student; any projects with more than one taker will be allocated by drawing one name out of a hat; losers and unallocated projects will then be placed into a second round; the allocation procedure will again take place, and will continue until all students and projects are exhausted. In this manner it is hoped than an element of 7 choice may operate in selection of projects. You may then, if you wish, change projects by mutual exchange between students. However, in the event you do not gain your project of choice no additional projects will be set. Once projects are selected you should then contact your project supervisor for discussions. Most importantly, you should check that essential chemicals and equipment are, or will be, available when required. Before projects begin, you will be expected to give a 15-20 min talk that explains your project, its intended aims and your experimental approach (week beginning 2th November in FRED). It is advisable to arrange at least one practise talk with your project supervisor. Project laboratory work will start on November 16th and terminate on the 26th of February. After the end of laboratory work, you will be given approximately 4 weeks to present a thesis on your project. In this period you will also present a Project Poster to the School at a poster session. There will be prizes for the best posters. A deadline for handing in projects will operate. It is 5.00 pm on Thursday the 24th of March. For every working day that your thesis is late, 2% of the mark that you would have obtained will be subtracted from your mark. Ms Roisin Cleere and Dr Audrey Carroll (Preparation Room) will advise you about the presentation of your poster and print it for you. Further details on Project write-ups and poster presentations will be given in the second semester. The Margaret Ciotti Prize Merit Award is awarded to a Senior Sophister student for excellence in undergraduate research. This award was initiated by Bruno Orsi to honour his wife's achievements in biochemistry and will now be a memorial to her. It is traditionally presented by Bruno on a date between the end of the exams and the vivas. You will be advised of the date closer to the time. This award is independent of the poster prizes. Biochemistry Personnel and Contact Details: The Senior Sophister Course Co-ordinator is Ed Lavelle (phone extension 2488, email ([email protected]). The names of tutors are provided on page 17. A complete list of staff in the school can be found at: http://www.tcd.ie/Biochemistry/people/index.php Health and Safety Management: 1) Registration with Safety Officer Preliminary safety registration takes place during two mandatory health and safety briefing sessions timetabled in Week 1 (see timetable). Later on you must register, in person, with the Safety Officer during the week after you have been assigned your project. This is necessary in order to record your next-of-kin details in the unlikely 8 event of an accident, to record where you will be working, to ascertain whether or not you have to work with major hazards during your project work (carcinogens, mutagens, cyto-toxics, biological agents, GMOs, radioactivity, etc), to ensure that you and your supervisor understand that you have to conduct a HIRAC review (hazard identification, risk assessment and risk control) of the proposed work and to discuss your response to the Science Faculty’s Health Questionnaire. 2) Formal Health and Safety Briefings Mr Liam McCarthy (Chief Technician) will describe the general management and security features of the building on the first day of term. Dr Nóirín Nic a’ Bháird, the School Safety Officer will give you a formal general Health and Safety briefing at 11 a.m. on Tuesday and Wednesday 29th & 30th of September. Dr Nic a’ Bháird will give two more detailed Health and Safety workshops just before you start your project work in the research laboratories. ATTENDANCE AT THESE BRIEFINGS AND ANY ADDITIONAL TRAINING SESSIONS (e.g. Radiological Protection Workshop, viewing safety videos, etc.) IS MANDATORY. Some of these actions are legal, license or College's insurer's requirements that have to be complied with. 3) Safety Lab Coat & Spectacles You must have at least one Howie-style laboratory safety coat, conforming to the NISO 1993, or better, standard, along with a pair of safety spectacles with you at all stages during active laboratory work. 4) Specific Aspects of Health and Safety Associated with Project Work You are required to attend two scheduled and compulsory short briefings on aspects of Health and Safety in laboratories given by the School Safety Officer, Dr. Audrey Carroll, a few days before project work starts. Any hazardous materials, steps or procedures (including off-site work connected with your research such as collecting samples from other laboratories, etc.) involved in your project will have been identified by, and discussed with you by your project supervisor. He/she is required, by law, to perform this hazard identification, risk assessment and risk control (HIRAC) on every experiment undertaken by you, but you have a role to play as well in making sure that you record the conclusions of this procedure in you notebook. The control measures necessary to reduce or eliminate risk must be written in your notebook for each hazardous step or procedure. The law requires this to be done. You are still in training so you cannot be classed as a competent biochemist and thus able to do this yourself to ensure your safety. If in doubt about the proper procedures for any experiment, do not perform that experiment. Senior Sophisters must make themselves aware of the College's and School’s Safety Statement which is displayed prominently in every laboratory in the School. [It can be downloaded from the School’s Local Home-Page at this URL: www.tcd.ie/biochemistry/]. You are still bound by the 'Science Faculty's Health and Safety Guidance Manual' and the associated Health Questionnaire which you completed at the start of JF year. In case your health status has changed since then in terms of the categories listed (including pregnancy or lactation) you have to complete a new Health Questionnaire. If your health status again changes during the 9 year you must consult, in confidence, with the Safety Officer. [This particularly applies in the case of pregnancy.] You are not permitted to work with unsealed radionuclide sources unless you have attended and satisfactorily completed a Radiological Protection Workshop to be held on Thursday 7th (9:30am-4:00pm) and Fri 8th (9:30am-1:00pm) of January 2014. Any student working with human materials (blood, buffy coats, semen, CSF, dialysis fluid, primary explants, etc.) must be vaccinated against Hepatitis B prior to commencing your project. You are not permitted to work with any Category 3 biological agents such as HIV, Hepatitis B and C, etc. or to culture Category 3 (or higher) pathogens. You must request or otherwise obtain Material Safety Data Sheets (MSDS) for any toxic or dangerous chemicals or preparations that you are using in your project. These MSDS's have to be requested at the point of ordering any material. The MSDS must be stuck into your laboratory notebook. The guidance must be followed. After 6:00 pm on working days, and at all times on weekends and public holidays, no Senior Sophister may work in any laboratory without the close presence of a member of the academic staff. It is the Senior Sophister's responsibility to ask that staff member if he/she will consent to act in a supervisory capacity for the time the student is working. During normal working hours no student may work alone in any laboratory. Failure to observe these rules/procedures will cause the offenders to be officially warned, and be reported to the Head of School, school safety officer and project supervisor. Normal College disciplinary procedures can be invoked (including fines being levied as well as withdrawal of student i.d. card, etc.) Persistent failure to observe these rules may result in that student being banned from laboratory work with loss of those marks available for project work. Students with Disabilities: The University Policy Relating to students with disabilities is available at www.tcd.ie/disability. The Student Disability Service is located in Room 2054 Arts Building, phone = 8963111, email = [email protected]. The Student Disability Services Committee provides the formal channel for raising issues affecting students with disabilities. Plagiarism: The full statement of College policy on plagiarism can be found at: http://tcd- ie.libguides.com/plagiarism We recommend that you familiarise yourself with these new regulations. The College Calendar defines plagiarism, gives examples of the kinds of actions that are deemed to constitute plagiarism, and elaborates on the procedures for dealing with plagiarism cases. It is essential that you read the Calendar entry that is relevant to you as an undergraduate or postgraduate student. You should also look at the matrix that explains the different levels of plagiarism and how they are dealt with. The calendar entry is presented below. 10 In addition members of staff of the School of Biochemistry & Immunology may scan your written assignments using plagiarism-detecting software such as Turnitin (additional information for which can be found at: http://turnitin.com/static/index.html). Calendar Statement on Plagiarism for Undergraduates - Part II, 82-91 Plagiarism 82 General It is clearly understood that all members of the academic community use and build on the work and ideas of others. It is commonly accepted also, however, that we build on the work and ideas of others in an open and explicit manner, and with due acknowledgement. Plagiarism is the act of presenting the work or ideas of others as one’s own, without due acknowledgement. Plagiarism can arise from deliberate actions and also through careless thinking and/or methodology. The offence lies not in the attitude or intention of the perpetrator, but in the action and in its consequences. It is the responsibility of the author of any work to ensure that he/she does not commit plagiarism. Plagiarism is considered to be academically fraudulent, and an offence against academic integrity that is subject to the disciplinary procedures of the University. 83 Examples of Plagiarism Plagiarism can arise from actions such as: (a) copying another student’s work; (b) enlisting another person or persons to complete an assignment on the student’s behalf; (c) procuring, whether with payment or otherwise, the work or ideas of another; (d) quoting directly, without acknowledgement, from books, articles or other sources, either in printed, recorded or electronic format, including websites and social media; (e) paraphrasing, without acknowledgement, the writings of other authors. Examples (d) and (e) in particular can arise through careless thinking and/or methodology where students: 11 (i) fail to distinguish between their own ideas and those of others; (ii) fail to take proper notes during preliminary research and therefore lose track of the sources from which the notes were drawn; (iii) fail to distinguish between information which needs no acknowledgement because it is firmly in the public domain, and information which might be widely known, but which nevertheless requires some sort of acknowledgement; (iv) come across a distinctive methodology or idea and fail to record its source. All the above serve only as examples and are not exhaustive. 84 Plagiarism in the context of group work Students should normally submit work done in co-operation with other students only when it is done with the full knowledge and permission of the lecturer concerned. Without this, submitting work which is the product of collusion with other students may be considered to be plagiarism. When work is submitted as the result of a group project, it is the responsibility of all students in the group to ensure, so far as is possible, that no work submitted by the group is plagiarised. 85 Self plagiarism No work can normally be submitted for more than one assessment for credit. Resubmitting the same work for more than one assessment for credit is normally considered self-plagiarism. 86 Avoiding plagiarism Students should ensure the integrity of their work by seeking advice from their lecturers, tutor or supervisor on avoiding plagiarism. All schools and departments must include, in their handbooks or other literature given to students, guidelines on the appropriate methodology for the kind of work that students will be expected to undertake. In addition, a general set of guidelines for students on avoiding plagiarism is available on http://tcdie.libguides.com/plagiarism. 87 If plagiarism as referred to in §82 above is suspected, in the first instance, the Director of Teaching and Learning (Undergraduate), or their designate, will write to the student, and the student’s tutor advising them of the concerns raised. The student and tutor (as an alternative to the tutor, students may nominate a representative from the Students’ Union) will be invited to attend an informal meeting with the Director of Teaching and Learning (Undergraduate), or their designate, and the lecturer concerned, in order to put their suspicions to the student and give the student the opportunity to respond. The student will be requested to respond in writing stating his/her agreement to attend such a meeting and confirming on which of the 12 suggested dates and times it will be possible for them to attend. If the student does not in this manner agree to attend such a meeting, the Director of Teaching and Learning (Undergraduate), or designate, may refer the case directly to the Junior Dean, who will interview the student and may implement the procedures as referred to under conduct and college regulations §2. 88 If the Director of Teaching and Learning (Undergraduate), or designate, forms the view that plagiarism has taken place, he/she must decide if the offence can be dealt with under the summary procedure set out below. In order for this summary procedure to be followed, all parties attending the informal meeting as noted in §87 above must state their agreement in writing to the Director of Teaching and Learning (Undergraduate), or designate. If the facts of the case are in dispute, or if the Director of Teaching and Learning (Undergraduate), or designate, feels that the penalties provided for under the summary procedure below are inappropriate given the circumstances of the case, he/she will refer the case directly to the Junior Dean, who will interview the student and may implement the procedures as referred to under conduct and college regulations §2. 89 If the offence can be dealt with under the summary procedure, the Director of Teaching and Learning (Undergraduate), or designate, will recommend one of the following penalties: (a) Level 1: Student receives an informal verbal warning. The piece of work in question is inadmissible. The student is required to rephrase and correctly reference all plagiarised elements. Other content should not be altered. The resubmitted work will be assessed and marked without penalty; (b) Level 2: Student receives a formal written warning. The piece of work in question is inadmissable. The student is required to rephrase and correctly reference all plagiarised elements. Other content should not be altered. The resubmitted work will receive a reduced or capped mark depending on the seriousness/extent of plagiarism; (c) Level 3: Student receives a formal written warning. The piece of work in question is inadmissible. There is no opportunity for resubmission. 90 Provided that the appropriate procedure has been followed and all parties in §87 above are in agreement with the proposed penalty, the Director of Teaching and Learning (Undergraduate) should in the case of a Level 1 offence, inform the course director and where appropriate the course office. In the case of a Level 2 or Level 3 offence, the Senior Lecturer must be notified and requested to approve the recommended penalty. The Senior Lecturer will inform the Junior Dean accordingly. The Junior Dean may nevertheless implement the procedures as referred to under conduct and college regulations §2. 91 If the case cannot normally be dealt with under the summary procedures, it is deemed to be a Level 4 offence and will be referred directly to the Junior Dean. Nothing provided for under the summary procedure diminishes or prejudices the disciplinary powers of the Junior Dean under the 2010 Consolidated Statutes. 13 School of Biochemistry & Immunology Guidelines on Marking: Scheme for marking of examination answers: I Excellent; full understanding of concepts with excellent knowledge of subject; evidence of outside reading and thought beyond the content of specific courses. II-I Very good answer demonstrating good understanding of concepts and broad knowledge of the subject. Lapse of content tolerated at the lower end of the scale. II-II Good answer that is generally sound but with limited scope. Lapses in detail. III Adequate but with significant shortcomings in content; containing errors in detail and with poor structure. F1 Weak answer containing some relevant information but lacking substance and understanding. F2 Poor answer; serious and absurd errors; contains few or no items relevant to the question. Scheme for marking of projects: The project mark is comprised of the Supervisor’s mark and one other Examiner’s marks for the project thesis. The Supervisor’s mark will be based on the student’s performance within the laboratory (technical ability, understanding of the project and literature pertaining to it, critical evaluation of results, demonstration of initiative and independent thought) and on the content and presentation of the first draft of the project thesis. The supervisor will also make the other Examiner of the project thesis aware of any unforeseen difficulties that arose during the course of the project. Class Mark Range 85100 I 70-84 Criteria Exceptional project report showing broad understanding of the project area and excellent knowledge of the relevant literature. Exemplary presentation and analysis of results, logical organisation and ability to critically evaluate and discuss results coupled with insight and originality. A very good project report showing evidence of wide reading, with clear presentation and thorough analysis of results and an ability to critically evaluate and discuss research findings. Clear indication of some insight and originality. A very competent and well presented report overall but falling short of excellence in each and every aspect. 14 II-1 60-69 II-2 50-59 III 40-49 20-39 Fail 0-19 A good project report, which shows a reasonably good understanding of the problem and some knowledge of the relevant literature. Mostly sound presentation and analysis of results but with occasional lapses. Some relevant interpretation and critical evaluation of results, though somewhat limited in scope. General standard of presentation and organisation adequate to good. A moderately good project report which shows some understanding of the problem but limited knowledge and appreciation of the relevant literature. Presentation, analysis and interpretation of the results at a basic level and showing little or no originality or critical evaluation. Insufficient attention to organization and presentation of the report. A weak project report showing only limited understanding of the problem and superficial knowledge of the relevant literature. Results presented in a confused or inappropriate manner and incomplete or erroneous analysis. Discussion and interpretation of result severely limited, including some basic misapprehensions, and lacking any originality or critical evaluation. General standard of presentation poor. An unsatisfactory project containing substantial errors and omissions. Very limited understanding, or in some cases misunderstanding of the problem and very restricted and superficial appreciation of the relevant literature. Very poor, confused and, in some cases, incomplete presentation of the results and limited analysis of the results including some serious errors. Severely limited discussion and interpretation of the results revealing little or no ability to relate experimental results to the existing literature. Very poor overall standard of presentation. A very poor project report containing every conceivable error and fault. Showing virtually no real understanding or appreciation of the problem and of the literature pertaining to it. Chaotic presentation of results, and in some cases incompletely presented and virtually non-existent or inappropriate or plainly wrong analysis. Discussion and interpretation seriously confused or wholly erroneous revealing basic misapprehensions. _________________ Dr Ed Lavelle September 2015 15 SS IMMUNOLOGY TUTORIAL GROUPS 2015-2016 STUDENT NUMBER NAME 12302743 Amet, Rebecca 12304512 Coleman, Amy 12303530 Diskin, Ciana 12302499 Delaney, Grainne Mary 12302659 Gorby, Claire 11500287 12305741 Case, Sarah Mahon, Fearghus McAlpine David 12300689 Kelly, Alanna 12300915 12306480 White, Graeme O Leary, Fionn Timothy Noel 12304832 Grennan, Olivia 12302931 12308123 Feehan, Karen MacDonald, James Fraser 12303030 O Hare, John 12307742 Barber, Gillian 12310699 Reid, Caoimhe 12308668 Shanahan, Katharine Ann 12327336 Fleming, Aaron 12310109 Hennessy, Naoise 12311515 Doran, Ciara EMAIL TUTOR LUKE O’NEILL CLAIR GARDINER KINGSTON MILLLS ANDREW BOWIE RACHEL MCLOUGHLIN ED LAVELLE CLIONA O’FARRELLY 16 SS IMMUNOLOGY PRACTICE VIVA VOCE GROUPS 2014-2015 First Semester Second Semester A. Bowie & C. Gardiner R McLoughlin & E. Lavelle Amet, Rebecca Coleman, Amy Diskin, Ciana Grainne Delaney Fionn O’Leary Sarah Case Fearghus Mahon Alanna Kelly Ciara Doran Graeme White Claire Gorby Karen Feehan James MacDonald R McLoughlin & E. Lavelle C. O’Farrelly & K Mills Fearghus Mahon Alanna Kelly Ciara Doran Graeme White Claire Gorby Karen Feehan James MacDonald John O Hare Gillian Barber Naoise Hennessy Katharine Shanahan Aaron Fleming Caoimhe Reid Olivia Grennan C. O’Farrelly & K Mills A. Bowie & C.Gardiner John O Hare Gillian Barber Naoise Hennessy Katharine Shanahan Aaron Fleming Caoimhe Reid Olivia Grennan Amet, Rebecca Coleman, Amy Diskin, Ciana Grainne Delaney Fionn O’Leary Sarah Case 17 Immunology: Breakdown of SS Papers I and II 2015-2016 Paper I Section 1: BI4275 ‘Cells of the innate and adaptive immune system’ Myeloid cells (EC) Natural killer cells (CG) T cells (KM) B cells and humoral immunity (MC) 2 questions Section 2: BI4055 ‘Microbial Diseases’ Trypanosomiases (DN) Prokaryotic pathogens (HW) Helminths (PF) 2 questions Section 3: BI4215 ‘Organ specific immunity’ Reproductive and liver immunology (COF) Gastrointestinal tract (EL) Respiratory tract (RMcL) Neuroimmunology (CC, AD) 2 questions Section 4: BI4225 ‘Autoimmune and inflammatory conditions’ Biochemistry of the inflammatory process (JB) Rheumatoid arthritis (LON) Autoinflammatory diseases (EC) Immunodeficiency/clinical immunology (DD) 2 questions Answer one question from each section (4 OUT OF 8) 18 Paper II Section 1: BI4235 ‘Immune signalling’ Apoptosis (DZ) Cytokine Signalling (LON) Molecular basis of immune signalling (AK) T cell receptor signalling (AB) PI3K and mTOR signalling (DF) 2 questions Section 2: BI4245 ‘Immunity to pathogens and vaccination’ Immunity to viruses (NS) Viral evasion of innate and adaptive immunity (AB) Antimicrobial resistance and host response to infection (RMcL) Immune response to tuberculosis (JK) Vaccines, adjuvants and danger hypothesis (EL) 2 questions Section 3: BI4225 ‘Immunogenetics, immunomodulation and immunotherapy Transgenics and animal models of disease (VK) Immunogenetics (RMcM) Immunotherapy and transplantation (COF) 2 questions Section 4: BI4265 ‘Tumour Immunology’ Initiation & Progression (VK) Metastasis & Treatment (VK/KM) Cellular imaging (DN) 2 questions Answer one question from each section (4 OUT OF 8) 19 SS Course Summaries: 2015-2016 BI4275 CELLS OF THE INNATE AND ADAPTIVE IMMUNE SYSTEM (5ECTS) Myeloid cells (3 lectures) Emma Creagh) Lecture 1: Summary of haematopoiesis from Myeloid progenitor cell; overview of Granulocyte, Macrophage and DC cell types, maturation & cell specific functions in innate immunity. Myeloid derived suppressor cells (MDSCs). Lecture 2: Pattern recognition receptors (PRRs), Complement Rs - recognition of infection & injury. Phagocytosis by cellular subtypes, vesicular trafficking, lysosome formation & degradation mechanisms. Lecture 3: Granulocyte migration, activation, degranulation. Exocytosis & Secretory pathways - release of preformed molecules from granules (eg. Histamine) and newly synthesised molecules (eg. TNFalpha). Natural Killer Cells (5 lectures) Clair Gardiner Lecture 1: General features of NK cells including cell surface phenotypes, cytokine production and main functions. Lecture 2: Cytotoxicity mechanisms used by NK cells: necrosis and apoptosis (fas-L, granzymes, TNF-α and LT). Benefits of these; NK cells as effectors of graft-rejection, GVHD and GVL. Recognition of target cells; missing self hypothesis Lecture 3: Receptors for MHC class I; human vs. mouse; lectin like receptors and Ig superfamily receptors. Inhibitory vs. stimulatory receptors. Diversity inherent within these receptors. Lecture 4: Ligand specificity of human NK cell receptors – KIR and CD94/NKG2. Role of peptide in ligand recognition; general mechanisms of NK cell recognition of virally inected cells; viral escape strategies. 20 Lecture 5: Role of NK cell and receptors in particular diseases - a research perspective. T cell differentiation and regulation (2 lectures) Kingston Mills Lecture 1. T cell subtypes, antigen presentation and T cell differentiation’. Lecture 2. Natural and induced regulatory T cells. Regulatory T cells in infectious diseases. Role of anti-inflammatory cytokines produced by innate cells and T cells in subversion of immunity to infection. T cell immunity to bacterial and viral infection (2 lectures) Kingston Mills Lecture 1: The bridge between innate and adaptive immunity. Pathogen activation of macrophages and dendritic cells through pattern recognition receptors. Role of dendritic cells in directing T cell subtypes. Lecture 2: Role of Th1/Th2 cells in immunity to infection, including HIV, hepatitis C virus and Bordetella pertussis. B cells and humoral immunity (4 lectures) Michael Carty Lecture 1: Discovery and history of B cells will be given as will a detailed description on the activation of B cells. B cell subtypes and regulation will also be described in detail. Lecture 2: A detailed description of antibody production will be given. Dysregulation of this system will be described in disease processes. Therapeutic manipulation of B cells and humoral immunity will also be provided in inflammatory diseases and other conditions. Lecture 3 Discovery of complement and the proteolytic cleavage pathways leading to the activation of the terminal effector proteins of the complement system. Activation of this system results in pathogen opsonisation, phagocytosis, recruitment of inflammatory cells 21 and pathogen killing. In addition mechanisms by which these pathways are regulated will also be described. Lecture 4: Diseases in which complement components are thought to be involved will be described. These diseases include those in which components of the complement system are altered by mutation such as Systemic lupus erythematosus and conditions where there is inappropriate and excessive activation of complement such as glomerulonephritis. Finally therapeutic opportunities which target proteins of the complement system will also be explored. 22 BI4055 MICROBIAL DISEASES (5ECTS) African trypansomes (8 lectures) Derek Nolan The aim of these lectures is to provide an introduction to African trypanosomes, parasitic protozoans that cause sleeping sickness in humans and a related disease, Nagana, in cattle. These parasites are a major problem for human and veterinary health throughout sub Saharan Africa and serious barrier to economic development of the region. Perhaps the most striking feature of these parasites is that that they are exclusively extracellular. They grow and divide in the mammalian vasculature and consequently exposed the adaptive and innate defence responses of their mammalian hosts. In addition, for a variety of reasons, African trypanosomes have been come a favourite model organism for molecular and cell biologists and many discoveries of broad significance have emerged from studies on these model unicellular eukaryotes. Areas where such discoveries have been reported will be illustrated in the lectures where appropriate. The course is organized into two parts. Trypanosomes Part 1: Stealth strategies of an elusive parasite 1. How are trypanosomes, such as Trypanosoma brucei, able to evade the host humoral immune response given that they are constantly exposed to this arm of the immune response? 2. What other strategies do trypanosomes employ to circumvent the innate immune responses? 3. How are these parasites able to acquire essential macromolecular growth factors from their hosts without attracting a response? Trypanosomes Part 2: What is the molecular basis of human sleeping sickness? The focus in part II is on the innate immunity that humans and other primates have to infection by all but a few trypanosomes. In effect in this part we will consider the molecular basis of African human sleeping sickness. We will consider the nature of the trypanolytic toxin present in human serum and how this toxin kills these parasites. We will see an amazing link between the toxin and an unsuspected programmed cell death pathway. Finally, we will see how two strains of trypanosomes have responded by developing independent mechanisms to resist this toxin and how in turn certain human populations are able to overcome this resistance and the price they pay for this capacity. Reading List: Additional specific references for key experiments will be provided within the lectures which are available on the school website. Trypanosomes Part I (1) Cross, G.A.M. (2001) African trypanosomes in the 21st century: what is their future in science and health? Int. J. Parasitol. 31: 427-433 (2) Borst, P. (2002) Antigenic variation and alleleic exclusion Cell 109: -8. (3) Pays, E. (2005) Regulation of antigen gene expression in Trypanosoma brucei Trends Parasitol. 21: 517-520. 23 (4) Pays E. (2006) The variant surface glycoprotein as a tool for adaptation in African trypanosomes. Microbes and infection 8: 30-937. (5) Field, MC & Carrington, M. (2004) Intracellular membrane transport systems in Trypanosoma brucei. Traffic 5:1-9 (6) Nolan, DP, Garcia-Salcedo, J.A., Geuskens, M., Salmon, D., Paturiaux-Hanocq, F., Pays,A., Tebabi, P. and Pays, E. (2001) Endocytosis of macromolecules by African trypanosomes. pp127-141 In “World Class Parasites Volume 1: The African Trypanosomes” Eds. Seed, R. & Black, S.J. (Kluwer Academic Publishers) (7) Stockdale C. et al (2008) PLoS biology Vol6 issue 7 e185 “Antigenic Variation in Trypanosoma brucei: Joining the DOTs” (8) Navarro M. et al (2007)TRENDS in Microbiology Vol.15 No.6 doi:10.1016/j.tim.2007.04.004 Nuclear architecture underlying gene expression in Trypanosoma brucei Trypanosomes Part II (1) Pays, E. Vanhamme, L., Vanhollebeke, B., Nolan, D. P.. and Perez-Morga, D. (2006) The trypanolytic factor of human serum. Nat Rev Microbiol. 6: 477-86. (2) Vanhollebeke B & Pays E (2010) Mol. Microbiol. 76: 806-814 The trypanolytic factor of human serum, many ways to enter the parasites, a single way to kill it. (3) Pays E & Vanhollebeke B (2008) Microbes Infect 10: 985-989 Mutual self-defence: the trypanolytic story (4) Genovese et al. (2010) Science 329: 841-845. Association of trypanolytic ApoL1 variants with kidney disease in African Americans (5) Pays E. et al. (2014) The molecular arms race between African trypanosomes and humans Nature Reviews Microbiology VOLUME 12 575-584. (6) Vanwalleghem G. et al. (2015) NATURE COMMUNICATIONS | 6:8078 | DOI: 10.1038/ncomms9078 Coupling of lysosomal and mitochondrial membrane permeabilization in trypanolysis by APOL1 Helminths of Human Importance (4 lectures) Padraic Fallon A third of the world’s population is infected with parasitic worms. These lectures will address the major parasitic worms that are of medical importance. Lecture 1-2: Introduction to the major helminth parasites that infect man. Medical and economic impact of helminth parasites on society. Lecture 3-5: Wormy people: genetic predisposition to helminth infection. Co-evolution of man and parasitic worms: molecular and biochemical adaptation. The schistosome tegument or nematode 24 cuticle as model membranes. Helminth proteome and genome projects. Gastro-intestinal versus systemic (tissue or blood dwelling) worm infections. Endosymbiotic infections. Modulation of immunity by helminth parasites: implications for designing vaccines. Molecular and biochemical targets for current and future drugs to treat helminth infections. A reading list will be given out during the course Prokaryotic pathogens (4 lectures) Henry Windle Lecture 1:. Introduction to prokaryotic pathogens of medical importance. Emerging and reemerging diseases. Lecture 2: Overview of molecular mechanisms of bacterial induced disease - modulation of host cell signalling responses and pathogenesis. Strategies to identify vaccine candidates/therapeutic targets. Lecture 3: Bacterial pathogens as a paradigm for chronic infection I. Infection and cancer – the Helicobacter pylori connection: molecular basis of pathogenesis. Lecture 4: Bacterial pathogens as a paradigm for chronic infection II. Animal models of disease. Microbiomes, metagenomics and engineering microbes for our benefit. Mixed microbial populations and disease. General Reading: Insights into host responses against pathogens from transcriptional profiling. Jenner et al. Nat Rev Microbiol (2005)3 (4), 281-94 The impact of the microbiota on the pathogenesis of IBD: lessons from mouse infection models S Nell, S Suerbaum & C Josenhans NAT REV MICROBIOL (2010) 8 (8) 564-77. What are the consequences of the disappearing human microbiota? MJ. Blaser & S Falkow NAT REV MICROBIOL (2009) 7 (12) 887-94. Helicobacter pylori: gastric cancer and beyond. Polk DB, Peek RM (2010)10(6):403-14. Nat Rev Cancer. 25 BI4245 IMMUNITY TO PATHOGENS AND VACCINATION (5 ECTS) Immunity to viruses (3 lectures) Nigel Stephenson Lecture 1: Viruses and intracellular immunity This lecture will provide an introduction to viruses and the key intracellular innate immune events involved in viral detection and subsequent anti-viral Interferon induction. Lecture 2: Anti-viral Interferons This lecture will describe Interferons, their effects and signalling pathways required for anti-viral activity. The function of key Interferon Stimulated Genes will be outlined. The role of Interferons as therapeutics will be discussed with specific focus on Hepatitis C. Viral Evasion of innate and adaptive immunity (6 lectures) Dr Andrew Bowie) Lecture 1: Introduction to viruses and immune evasion. Virus classification and life cycle. Six key concepts in viral evasion: (1) Molecular recognition, (2) Nature of the viral-host interaction, (3) Viral evasion and pathogenicity – what makes a good virus? (4) Learning from viruses about the immune response, (5) Different types of viral proteins involved, and apparent contradictions, (6) Targetting innate vs adaptive immunity. Lecture 2: Theanti-viral immune response I: Viral PAMPs and Pattern Recognition Receptors(PRRs) – PKR, TLRs, RLRs, ALRs such as IFI16 and other DNA sensors (cGAS). How PRRs signal. Lecture 3: Theanti-viral immune response II: Anti-viral PRRs trigger type I IFN induction. The type I IFN system. IFN-stimulated genes (ISGs). Natural Killer Cells, Cytokines, Chemokines & Inflammation, Compliment, Antibody, T Cells. Lecture 4: Overview of evasion of innate and adaptive defense mechanisms. Avoiding and blocking recognition by PRRs. NFkB interactions – subversion or detection? Lecture 5: Interfering with Interferon. Inhibition of antigen presentation. Disrupting NK cell function 26 Lecture 6: Modulating chemokines and cytokines. Viral proteins and peptides as biotherapeutics. Example of vaccinia virus protein A46 and VIPER, a peptide derived from A46. Antimicrobial resistance and the host response to bacterial infection (2 lectures) Rachel McLoughlin Lecture 1: Mechanisms of antimicrobial resistance in bacteria. Epidemology of antimicrobial resistant bacterial infections. The limitations of antibiotics as therapeutic strategies. Lecture 2: Targeting the host immune response for the development of vaccines and immunomodulatory therapies to treat bacterial infection. Case study Staphylococcus aureus (MRSA) infections. The immune response to tuberculosis (3 lectures) Joe Keane) Lecture 1: The immune response to tuberculosis; a model for pathogen evasion of the human host response. The innate immune response to tuberculosis; macrophages and dendritic cells a. The first contact; Inhaled M. tuberculosis bacillus is phagocytosed by a macrophage; receptors for macrophage uptake; the signals of invasions; the phagolysosomal maturation arrest; TACO b. The host fights back; The ROI and RNI reactions to invasion; Cytokine and chemokine release; macrophage apoptosis and bug killing c. Time to go clonal; Antigen presentation; proliferation and recruitment of T and B cells; CMI; Delayed type hypersensitivity Lecture 2: The granuloma; prison for the live bug a. The granulomatous response; TB , sarcoid, leprosy, Wegeners and Schistosomiasis b. What holds it together? TNF, IFN gamma, somatostatin c. What breaks it apart?; Immunosupression, HIV, steroids, TNF blockers, antiinterferon Ab or interferon receptor defects Lecture 3: TNF blockers and reactivation of tuberculosis; more questions and clinical proof of scientific principle 27 The role of TNF in tuberculosis disease. The role of TNF in preventing reactivation of latent infection. Mouse evidence translated into human tuberculosis observations. The consequence of TNF blocking for the disease and the host. The differences in TNF blockers; sTNFR v. monoclonals. Other effects of monoclonals. Preventing reactivation with TNF blockade. Vaccines, adjuvants and the danger hypothesis (5 lectures) Dr Ed Lavelle) Lecture 1: Basic concepts in vaccine development. Traditional approaches to vaccination. Nature and mode of action of vaccines in current use. Lecture 2: Vaccine adjuvants. Inert particulate and live bacterial and viral delivery systems. Toxin/lectin and toll like receptor-based adjuvants. Lecture 3: Mucosal vaccines. Distinctive features of the mucosal immune system and implications for vaccination. Mucosal vaccine adjuvants and delivery systems. Lecture 4: Vaccines for neonatal immunisation. Therapeutic vaccines. Lecture 5: Danger theory. Endogenous danger signals in innate immune activation, role of danger signals in efficacy of vaccine adjuvants. BI4045 AUTOIMMUNE AND INFLAMMATORY CONDITIONS (5 ECTS) Biochemistry of the Inflammatory Process (4 lectures) Dr Jack Bloomfield) Lecture 1: Definition of inflammation and inflammatory diseases. Difference between inflammation in infection and inflammatory diseases.Mechanism of the inflammatory process: respiratory burst, prostaglandin and leukotriene production. Degranulation 28 causes release of inflammatory mediators. Treatment of inflammatory diseases. Anti-inflammatory drugs. Lecture 2: Psoriasis as a model of inflammatory skin disease. Role of neutrophil in the perpetuation of chronic inflammation. Role of leukotrienes in pathogenesis of psoriasis. Neutral proteinases in psoriasis. Lecture 3: Helicobacter pylori in peptic ulcer disease. Effect of H2-receptor antagonists on peptic ulcers. Study of prostaglandin and leukotriene production in peptic ulcer disease. Antiinflammatory effects of H2-receptor antagonists. Lecture 4: Role of prostaglandins and leukotrienes in inflammation. Effect of non-steroidal antiinflammatory drugs on prostaglandin and leukotriene production. Pathways of prostaglandin and leukotriene production. Rheumatoid Arthritis (2 lectures) Luke O’Neill Lecture 1: What is rheumatoid arthritis? Clinical, molecular and cellular definitions. Early concepts: connective tissue structure and degradation. Rheumatoid Factor. And B cells. HLA associations and the genetic component. Autoantigens. Role of inflammation – prostaglandins and tissue degrading enzymes. Lecture 2: Key role of cytokines – IL-1, TNF, IL6. Current therapies – NSAIDs, steroids, biologic therapies (anti-TNF, anti-IL-1, anti-IL-6, anti-CD20 and CTLA-4 Ig). Prospect for future therapies. Multiple Sclerosis and EAE (3 lectures) Jean Fletcher, Kingston Mills Lecture 1: Breakdown of tolerance in autoimmunity. Risk factors, pathogenesis, diagnosis and monitoring of MS Lecture 2. MS therapies: Mechanisms of action, efficacy, side effects. Lecture 3: EAE. Role of innate and adaptive immunity in pathogenesis of autoimmune diseases. Role of regulatory T cells in preventing autoimmune diseases. Autoinflammmatory diseases (2 lectures) Emma Creagh 29 Lecture 1: Key features of systemic autoinflammatory disorders. Classic hereditary 'Periodic Fever Syndromes' - FMF (Familial Mediterranean Fever), TRAPS (TNF Receptor Associated Periodic Syndrome) and HIDS (Hyperimmunoglobulinemia-D with periodic fever syndrome). Lecture 2: NLRP3/Cryopyrin-associated periodic syndromes (CAPS): Familial Cold Inflammatory Syndrome (FCAS); Muckle-Wells Syndrome (MWS) and Neonatal onset multisystem inflammatory disease (NOMID). Autoinflammatory disorders associated with skin pustules, such as DIRA (deficiency of IL-1R antagonist), CARD14 mediated psoriasis (CAMPS) and early onset inflammatory bowel diseases (EO-IBD). Immunodeficiency/ Clinical immunology (4 lectures) Derek Doherty Lecture 1: Primary immunodeficiencies This lecture will cover the genetic bases, clinical presentations, diagnoses and treatments of primary immunodeficiencies, including antibody, complement, MHC and lymphocyte deficiencies. Lecture 2: Acquired immunodeficiencies This lecture will cover the different causes of acquired immunodeficiencies but will focus mainly on HIV-associated disease, including the virology, immunology, clinical features and recent progress in vaccine development. The significance of HIV in the developing world, where many other infectious disease are also endemic, will be emphasized. Lecture 3: Autoimmune disease The lecture will start with an overview of autoimmune diseases, emphasizing the heterogeneity of such diseases and the roles of T cells, B cells and other cells of the immune system in the pathogenesis. We will then focus on the example of coeliac disease as an illustration of how multiple pathogenic hits can result in disease and how the disease can be diagnosed and treated. Lecture 4: Antibody-mediated autoimmune diseases This lecture will focus on autoimmune diseases whose pathologies are mainly mediated by antibodies, such as systemic lupus erythematosus, myasthenia gravis and autoimmune vasculitis. 30 BI4235 IMMUNE SIGNALLING (5 ECTS) Apoptosis (5 lectures) Daniela Zisterer Lecture 1: Introduction to apoptosis. Role in development, maturation of the immune system and in cell turnover. Morphological features of apoptosis. Comparison with necrosis. Biochemical methods used for examination of apoptosis e.g. Annexin V staining. Aberrations in apoptosis: implicated in cancer and neurodegenerative disesases e.g. Alzheimer's. Genetic studies into nematode C. elegans provides key insights into molecular mechanisms regulating apoptosis. Lecture 2: Caspases: family of cysteine proteases: 'death executioners' in apoptosis. 14 caspases identified to date. Caspases subdivided into 3 categories: substrate specificity, prodomain length and prodomain sequence. How are caspases activated? By autoactivation, transactivation or proteolysis by other proteases. Experimental evidence that caspases are important in apoptosis. Biochemical measurement of caspases: fluorigenic assays and Western Blotting assays. Lecture 3: Apoptotic signal linked to caspases through 'sensor' and 'adapter'. Model for regulation of apoptosis by APAF1: cytochrome c released from mitochondria as 'sensor' and APAF1 (apoptosis activating factor 1) as 'adapter'. Formation of apoptotsome. IAPs (inhibitor of apoptosis proteins) which bind to and inhibit caspase activity. Smac/DIABLO which binds to and neutralises IAPs inhibitory activity. Subcellular localisation of caspases: cytosol, nuclei, mitochondria and ER. Caspase substrates e.g. PARP (poly ADP ribose polymerase), Lamins and CAD (caspase activated DNAase). Caspase 12: link with Alzheimer's disease? Caspase-independent cell death. AIF, Endo G, Omi/HtrA2. Lecture 4: The Bcl-2 protein family. Primary structure. Subdivided into 'pro-survival' and 'pro-apoptotic' proteins. 3-D structure. Channel forming activity. Subcellular localisation. Model for regulation of apoptosis by Bcl-2. How is cytochrome c released from mitochondria during apoptosis? Two competing models: PTP (permeability transition pore) opening and movement through VDAC (voltage-dependent anion channel). Posttranslational modification of Bcl-2 family: 2 types- proteolysis and phophorylation. Example of each type. Regulation of cell survival by Akt pathway. Lecture 5: Death Receptors: signalling and modulation. Examples of death receptors and signalling mechanisms involved: Fas, TNFR1 (tumour necrosis factor receptor1), DR3 (death receptor3), DR4 and DR5. Death domains (DDs), Death effector domains (DEDs), caspase-recruitment domains (CARDs); DISC (death inducing signalling complex). Experimental evidence for TNFR1 signalling pathway. Modulation of apoptosis by decoy receptors e.g. DcR1 and DcR2 in TRAIL signalling. Apoptosis induction by Granzyme B. Mitogen-activated protein kinases and apoptosis. Induction of apoptosis by cancer chemotherapy. Mechanisms of evasion of apoptosis by tumour cells. 31 Cytokine Signalling (5 lectures) Luke O'Neill Lecture 1: Cytokine families: interleukins, interferons, tumour necrosis factors, chemokines, colony stimulating factors. Properties and functions: inflammation, hemopoeisis, immune cell activation, anti-inflammatory cytokines. Class I cytokine receptors: JAKs and STATs. Specificity in signalling. WSWS motif. gp130 as second chain. Common and unique receptor chains. Complexity of IL2 signalling: PI3 kinase, IRS-1. Lecture 2: Type II cytokine receptors: Interferon receptor signalling: discovery of ISGFs and Tyk. Use of JAK and STAT nomenclature. JAK and STAT knock-out mice: key features. Interferon responsive genes and anti-viral effects. IL10 signalling. Suppresors of Cytokine signalling. Lecture 3: Type III cytokine receptor family: TNF receptors. Homology between TNFR, NGFR, Fas and CD40. TNF signalling: TRADD, RIP, FADD and caspases. TRAFs. Pathways to NFkB and apoptosis. Mechanism of activation of NFkB. IKK complex. CARD-containing proteins. Lecture 4: Type IV cytokine receptors: IL1 family. IL1 receptor signalling: IL1 pathway as prototypical 'stress' response in plants and animals. The TIR domain: structure and function. Toll-like receptors in mammals and innate immunity. LPS and IL18 receptors/ MyD88 as key adaptor. Roles of TLR-1 to TLR-10: recognition of PAMPs by PRRs. Primacy of TLRs in innate immunity. Lecture 5: Signal transduction pathways activated by the TIR domain. MyD88, IRAK1 – IRAK-4. TAB1/TAK-1. Traf-6 and ubiquitination. Regulation Stress activated protein kinases: p38 MAP kinase and JNK. Comparison to classical MAP kinases. IKK activation by TAK-1. Lessons from knock-out mice: Specific adapters for different TLRs? The role of Mal in LPS signalling. NALPs and NODs. Regulation of caspase-1 Molecular basis for immune signalling (2 lectures) Amir Khan Lecture 1: Structural properties of TCR/MHC complexes and the immunological synapse Lecture 2: Thermodynamics and kinetics of TcR/MHC complexes: models for immune signaling 32 T cell receptor signalling (2 lectures) Andrew Bowie Lecture 1 TCR signalling I: Signal transduction in T cells, overview of signalling molecules involved, T cell activation via the T cellreceptor: Src family kinases, adaptor proteins, PLCgamma, G proteins and GEFs, MAP kinases, transcription factors, feedback signals. Lecture 2. TCR signalling II: The contribution of signal 2 via co-stimulatory molecules to T cell activation, roles of CD28 co-stimulation, motifs on CD28 and key signallingmolecules involved in CD28 signalling, role of CTLA4 and ICOS, signalling basis of Th1 vs Th2 polarisation, role of lipid rafts in TCR signalling, NFkappaBactivation in T cells. PI3K, mTOR and immune cell metabolism (5 lectures) Dr David Finlay (DKF) Lecture 1: Phosphoinositide 3-kinase (PI3K) signalling (DKF) This lecture will introduce PI3K as a key signalling molecule in the immune system, describe PI3K’s enzymatic activity and discuss the various PI3K isoforms and their relative expression patterns. How the different PI3K isoforms are activated will also be discussed. Lecture 2: mammalian Target of Rapamycin (mTOR) signalling (DKF) The composition of the two distinct mTOR complexes, mTORC1 and mTORC2 will be described. Mechanisms that regulate mTORC1 activity will be explained. The relationship between PI3K and mTOR will be addressed using some experimental data. Lecture 3: PI3K and mTOR: immune cell function and metabolism (DKF) This lecture will discuss the role for PI3K and mTOR signalling in controlling the differentiation and function of immune cells. PI3K and mTORC1 control of immune cell metabolism will be introduced. Lecture 4: Cellular metabolism in immune cells (DKF) Recap on cellular metabolism, in particular glycolysis and oxidative phosphorylation. How cellular metabolism can be configured to match the demands of immune cells for energy, biosynthesis and longevity will be discussed. Lecture 5: Metabolic regulation of immune cell function (DKF) 33 This lecture will address the ways that cellular metabolism can directly impact upon immune cell function. Metabolic regulators and enzymes with important immunoregulatory functions will be described. Cellular metabolites that impact upon cellular signalling and epigenetic programming will be considered. 34 BI4255 IMMUNOGENETICS, IMMUNOMODULATION AND IMMUNOTHERAPY (5 ECTS) Transgenics and animal models of immune mediated disease (5 lectures) Vincent Kelly and Derek Nolan Lecture 1. Mutagenic, transgenic & cloning technology (VK): The concept of forward and reverse genetics in understanding gene function will be considered and how these mutations are physically introduced into the genome through random mutagenesis, viral mutagenesis, gene replacement and gene-targeting strategies. The process of microinjection to create transgenic animals, gene knockouts and cloned animal will be covered and the generation and use of induced pluripotant stem cells (iPS) in biomedical research applications. Lecture 2. Design and development of transgenic constructs (VK): The design of targeting vectors relies on a detailed structural/functional understanding of the gene under study. Various strategies for controlling the activity of the gene are available including the creation of knock-outs, knock-ins, conditional knockout and reporter systems. Gene-trap technology has, in recent times, gained significantly in popularity and the methodology will be examined in some detail. Lecture 3. Zinc Finger Nucleases and Talen Nucleases(VK): These state-of-theart technologies have the potential to revolutionise the manipulation of the eukaryotic genome, from cells in culture to mice, rats, rabbits, pigs etc. This lecture will cover the principles of this technology and how it is being currently exploited in research. Lectures 4 & 5. RNA interference (DN): The discovery of the classical RNA interference pathway involving siRNA will be described. The lectures will consider the concept of regulation of expression through siRNA and microRNAs along with the use and design of RNAi based approaches in functional genomics. The advantages and limitation of such approaches will investigated through the use of specific examples. The potential use of RNAi in therapeutic approaches will be outlined. Immunogenetics (5 lectures) Ross McManus Lecture 1. Identifying Disease Susceptibility Genes Lecture 2) Introduction to Genetic Diseases Inherited diseases cover a spectrum of disorders and can result from mutations in 35 single genes (Mendelian inheritance) or from the additive effects of common polymorphisms in complex networks of genes giving rise to complex genetic disorders. These lectures discuss the modes of inheritance of genetically determined diseases and the mechanisms used to identify chromosomal regions associated with disease susceptibility. Lecture 3. Genetics of Inflammatory Bowel Disease Inflammatory bowel disease afflicts almost 0.5% of western populations and is characterised by a chronic, relapsing, intestinal inflammation. It is subdivided primarily into two broad phenotypes, ulcerative colitis and Crohn’s disease. There is a clear tendency for these conditions to be inherited, although the pattern of inheritance is complex, indicating that both are multifactorial diseases resulting from the interplay of genetic and environmental factors. This lecture will review recent finding regarding the genetics of IBD. Lecture 4. Genetics of Coeliac Disease Coeliac Disease is a prevalent disease in Caucasian populations with a prevalence of susceptible individuals of approximately 1%, although the actual frequency of individuals displaying clinical symptoms is lower at about 1:300. Coeliac disease affects the small intestine where dietary proteins derived from cereals provoke an immunological reaction which leads to extensive damage to the gut wall and atrophy of the small intestinal villi. This lecture willcover what has been learned in the recent past regarding the genetics of this disease and what this tells us about the cell biological processes at play. Lecture 5. MHC & Inherited Diseases The Major Histocompatibility Complex is situated on the short arm of chromosome 6 and is one of the most gene- dense regions of the human genome. Many of these genes code for proteins which play a role in the function of the immune system with genes in both the HLA Class I and II regions involved in antigen presentation and genes in the Class III/IV encoding many other proteins with diverse functions in immunity and inflammation. Furthermore, many autoimmune or inflammatory diseases have been genetically linked to the MHC and it is clear the inherited variation in the MHC is a major determinant of susceptibility to these diseases. This lecture will describe the structure and content of the MHC and evidence of a causal association with inherited disease. Immunotherapy and transplantation (4 lectures) Cliona O’Farrelly Growth factors, interferons, monoclonal antibodies, steroids, personalised therapies) immunosuppressive agents (FK506, cyclosporine A). Allo recognition; the major histocompatibility complex; NK cells; T cells; bone marrow, liver, kidney transplantation 36 Immunotherapy and transplantation (4 lectures) Cliona O’Farrelly Growth factors, interferons, monoclonal antibodies, steroids, personalised therapies) immunosuppressive agents (FK506, cyclosporine A). Allo recognition; the major histocompatibility complex; NK cells; T cells; bone marrow, liver, kidney transplantation 37 BI4215 ORGAN-SPECIFIC IMMUNITY (5 ECTS) Introduction to regional immunity, reproductive and liver immunology (6 lectures) Cliona O’Farrelly) Reproductive Immunology This will introduce students to the basics of reproductive immunology. Against a background of some basic anatomy, physiology and endocrinology of the human male and female reproductive tracts, current understanding of local immune mechanisms and their regulation will be presented. The effects of immunoregulatory abnormalities on related pathologies will be introduced, in particular endometriosis, infertility, sexually transmitted infection and cervical cancer. In this context, the potential for immunotherapeutic interventions will be explored. Students will have the opportunity to visit the National Maternity Hospital at Holles St where the Director of the Merrion Fertility Clinic will give some insight into current major clinical challenges. Liver Immunology Cliona O’Farrelly This will introduce students to the fundamentals of liver immunology. Against a background of some basic anatomy and physiology of human liver, current understanding of local immune mechanisms and their regulation will be presented. The effects of immunoregulatory abnormalities on related pathologies will be introduced, in particular liver metastasis, transplant rejection and HCV infection. In this context, current immunotherapeutic interventions and the potential for new developments will be explored. Students will have the opportunity to visit the National Liver Transplant Centre at St.Vincent's University Hospital, where one of the hepatobiliary surgeons or pathologists will give some insight into current major clinical challenges facing hepatology. Gastrointestinal tract (3 lectures) Ed Lavelle Lecture 1: Overview of gut associated lymphoid tissue, Peyer’s patches, inductive and effector sites. Uptake of antigens across epithelial surfaces. Lecture 2: Dendritic cells and T cells in the gastrointestinal tract. Homing of gut T cells Lecture 3: Mucosal humoral immunity. IgA responses and their regulation. 38 Respiratory tract (3 lectures) Rachel McLoughlin Lecture 1: Introduction to the basic biology of the respiratory tract: conducting airways, mucosal surface, lung parenchyma and organization of the lung immune system. Understanding the concept that the lung is continually exposed to foreign antigens and must discriminate between recognition of innocuous environmental antigens and pathogenic antigens. Lecture 2: Roles played by individual cells in regulating immune response in the lung: airway epithelial cells, alveolar macrophages, regulatory T-cells, T-cell homing to lung, innate lymphoid cells Lecture 3: Immunological challenges faced by the lungs: Infection (???), Allergic disease (Asthma), inflammatory disease (COPD), toxin exposure (Cigarette smoke) Neuroimmunology (8 lectures) Aisling Dunne, Colm Cunningham I Colm Cunningham (4 lectures) Brain as an immune privileged organ, multiple sclerosis Multiple sclerosis and treatment Acute neuroinflammation in infection/stroke/TBI. CNS inflammation induced by systemic inflammation (Sickness Behaviour) II. Aisling Dunne (3 lectures) Aspects of microglial activation: downregulators microglial phenotypes, PAMPs, Microglial Sterile inflammation, DAMPs in the context of neurodegeneration (including ischaemic preconditioning). Discussion session 39 BI4265 TUMOUR IMMUNOLOGY (5 ECTS) Initiation & Progression (4 Lectures) Vincent Kelly Lecture 1. Underlying causes of cancer (VK): The characteristics that are used to classify cancers and their stage of development will be described. A number of examples will be given of how environmental factors, i.e. xenobiotics, radiation and oxidative damage contribute to multistep carcinogenesis. The means by which cancer is limited by DNA damage sensing, DNA repair and cellular adaptation to oxygen/radical damage will be covered. Lecture 2. Oncogenes and tumour suppressor genes (VK): Many of the original discoveries on oncogenes were derived from work on viruses. The concepts of onocgenes and proto-ocogenes will discussed such as src and the Rous sarcoma virus and there will be an in dept examination of the ras oncoprotein pathway an the function of other oncogenes including abl, sis, c-myc and how they influence cellular proliferation. Suppressor genes play an important role in limiting cancer formation and a number of models were put forward from original studies including Knodson’s twohit model and haploinsufficiency. The mode of action of tumour suppressors such as APC, MSH2, MLH1, BRCA1, p53 will be examined with particular focus on p53, Rb and APC. Lecture 3. Cancer epigenetics (VK): Changes in the genetic code is but one means to arrive at a pre-malignant crossroads. Epigenetics changes in gene expression have been found to alter tumor suppessor gene activity through. These epigenetic changes may occur as a consequence of altered DNA methylation status at CpG promoter regions of aberrant histone modification. In fact, cooperative suppression by both mechanisms has recently become the focus of new anti-cancer therapies through the development of DNMT and histone deacetylase inhibitors. Lecture 4. Cancer metabolism & the tumor microenvironment (VK): Many of the control points of cancer, oncogenes, tumor suppressor genes (including mTOR, PI3K, Akt, p53, AMPK) are intimately linked to metabolism, especially glycolysis, which provides the cancer with the building blocks for growth. The tumor cell microenvironment is invariably acidic and hypoxic causing the transcription factor HIF1a to set in place protective responses including unregulating the production of monocarboxylate transporters, VEGF, matrix metalloproteinases and angiogenic factors. Metastasis and Cancer Treatments (6 Lectures) Vincent Kelly & Kingston Mills Lecture 1. Angiogenesis and metastasis (VK): The process by which cancer cells develop new blood supplies (angiogensis) is reliant on being able to remodel the tumor environment and the extracellular matrix. A discussion of how this remodelling occurs through matrix metalloproteinases and plasminogen will be given along with 40 the cause and consequences of breaking cell-cell interactions. The means used by cancer cells to physically move from the primary tumor (e.g. epithelial-mesenchymal transition) and how the immune system promotes this process will be described. Breast cancer will be used as a model of how cancer cells choose secondary sites for proliferation, especially the bone marrow; ‘the vicious cycle’. Lecture 2. Colon cancer, genetics and epigenetics (VK): Arguably, colon cancer is one of the best studied cancers in terms of its formation and progression. This lecture will discuss the contribution of chromosomal instability in terms of changes to APC, COX2 and Smad4 and microsatellite instability caused by epigenetic suppression of mis-match repair enzymes including MSH2 & MLH1. The contribution of inflammation to colon caner will be considered and how NSAIDS and IL-10 mediate polyp formation. Lecture 3. Stem cell theory of cancer, focusing on colon cancer (VK): The intestinal crypt stem cells are maintained in a specialized compartment of the intestinal crypt through the Ephrin receptors. The maintenance and proliferation of these stems cells will be covered including the various signals used to control their proliferation, such as hedgehog, WNT, PDGF, Eph, NOTCH and BMP. The importance of the intestinal stems cells to cancer development and treatment will be considered. Lecture 4. Cancer treatment (VK): Classical anti-cancer drugs such as antimetabolites, alkylating agents and antimytotic agents are still widely used in therapy today despite severe side-effects. Newer ‘magic bullets, hold promise of more specific cancer treatment strategies such as Imatinab in the treatment of CML. However, drug resistance is a problem and has revealed the phenomenon of oncogene addition. Recent drug strategies have begun to focus on targeting tumor cell metabolism, its environment and the cancer initiating cells (cancer stem cells) that perpetuate proliferation even after treatment. Lecture 5. Cellular and humoral Immune responses to tumors (KM): These lectures include the role of antibody, cytotoxic T lymphocytes, macrophages, NK cells and Th1 cells; Evasion and subversion of immune responses by tumors - antiinflammatory cytokine production and regulatory T cell induction; Tumor-specific antigens and breaking tolerance to self antigens Lecture 6. Tumor immunotherapy (KM): Antibodies, Toll-like receptor agonists and cell-based therapies; Tumor vaccines - killed tumor cells, tumor specific peptides and antigens, heat shock proteins and dendritic cell vaccines Cellular Imaging (4 lectures) Derek Nolan 41 Lecture 1: Introduction to imaging and the concept of resolution. Application of electron microscopy in cell imaging. Lecture 2: EM tomography and specialized techniques. Introduction to light microscopy. Lecture 3: Advanced light microscopy: wide field and confocal microscopy. Lecture 4: Application of fluorescent proteins and probes in multidimensional imaging in fixed and live cells. 42 BI4015 DATA HANDLING (5 ECTS) SEQUENCE ANALYSIS & MOLECULAR MODELLING Sequence Analysis Jerrard Hayes The course will provide an introduction into Bioinformatics. Part I of the course consists of three lectures and three exercise sessions. Topics covered include: - DNA (including genomic) and protein databases Accessing sequence information from databases using the Internet Sequence similarity searches (i.e. BLAST, FASTA) Identification of homologous proteins Multiple sequence alignments (i.e. Clustal W) Searches for protein motifs, domain, patterns Students will carry out three exercises (marked as problems): Exercise 1: Accessing databases from the Internet, retrieval of sequences (DNA and protein), extracting relevant sequence information, presentation and annotation of a chosen sequence Exercise 2: Sequence similarity search (BLAST), identification of homologous proteins, multiple sequence alignment (Clustal W) Exercise 3: Sequence analysis of membrane proteins, identification of transmembrane helices and signal peptides hydrophobicity plots, Reading list: *essential reading # recommended *Bioinformatics: Sequence, structure, and databanks. A practical approach. D. Higgins and W. Taylor (eds.) Oxford University Press, 2000. *Trends guide to bioinformatics. Elsevier Science, 1998 #Benson, D. A. et al. 1999. GenBank. Nucleic Acid Research, 27: 12-17 #Bairoch, A. and R. Apweiler. 2000. The SWISS-PROT protein sequence database and its supplement TrEMBL in 2000. Nucleic Acid Research, 28: 45-48. 43 #Altschul et al. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403-410. #Needleman, S. B. and Wunsch, C. D. 1970. A general method applicable to the search for similarities in the amino acid sequence of two proteins. J. Mol. Biol., 48: 443-453. #Smith, T. F. and Waterman, M. S. 1981. Identification of common molecular subsequences. J. Mol. Biol., 147: 195-197. #Kyte, J. and Doolttle, R. F. 1982. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol., 157: 105-132. #Persson, B. and Argos, P. 1994. Prediction of transmembrane segments in proteins utilising multiple sequence alignments. J. Mol. Biol., 237: 182-192. #Rost, B. et al. 1995. Transmembrane helices predicted at 95% accuracy. Protein Science, 4: 521-533. #Von Heijne, G. 1992. Membrane protein structure prediction. Hydrophobicity analysis and the positive-inside rule. J. Mol. Biol., 225:487-494. #Sonnhammer, E. L. L. et al. 1998. A hidden Markov model for predicting transmembrane helices in protein sequences. In J. Glasgow et al. (eds.) Proc. Sixth Int. Conf. On Intelligent Systems for Molecular Biology, 175-182. AAAI Press. #Von Heijne, G. 1986. A new method for predicting signal sequence cleavage sites. Nucleic Acid Research, 14: 4683-90. #Nielsen, H. et al. 1997. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Engineering, 10:1-6. Molecular Modelling Dr Darren Fayne The course consists of one main exercise and multiple mini-problems during the workshop (Marked as one problem). Topics covered include: 1 - Introduction to the PDB database / information contained in crystal structure files. 2 - Use of the MOE software package for macromolecular visualisation of a protein/ inhibitor complex - here students will familiarise themselves with molecular displays, and will get used to 'thinking in 3D' there will be an associated (in session) task to do with displaying protein-ligand distances on screen. 3 - Use of the MOE software package – binding site analysis. - Students will use MOE to display the tertiary structure of the chosen protein complex in the popular `cartoon' rendering style. Here the students will see MOE in operation and will be walked through protein binding site analysis tools. 4 – Introduction to homology modelling in MOE 44 Exercise: a) Become familiarised with usage of the MOE software package. b) Use of MOE to create small drug-like molecules. c) Examination of protein-molecule binding complex d) Identifying potential drug binding sites on a novel protein e) Visualisation of the electrostatics of the protein binding site f) Compare the complementarity between the protein binding site and a potential new drug g) Homology modelling – generate the 3D structure of a new protein based on the known X-ray structure of a related protein h) A 30 minute exam based on the techniques covered during the workshop All the tasks are electronic in nature and will not require paper submissions. Background reading: http://www.chemcomp.com/software.htm BioResources Unit Introductory Animal Course Peter Nowlan The Purpose of this lecture course is to introduce Students to the basic requirements for working with animals. This is necessary if a full appreciation of animal related work is to be got from the projects. It is also a legal requirement that anybody involved in the use of animals for scientific purposes has appropriate training (EC directive 86/609) This module is not intended to be a comprehensive training course. To do this would require a much more detailed and extensive series of talks. Most of the training which will be required by students will be obtained by working in close contact with a technician and with experienced supervisors. The golden rule should be always 'if you don't know ask somebody'. The welfare of the animal and often the success of your Project will depend on using a correct approach to animals involved in your project. Even if you do not intend choosing a project, which involves live animals, you may do so in your future career. It will not be possible for anybody who does not pass the assessment to choose a project involving animals. Introduction to Laboratory Animal Science The Law and Application for a licence Animal House Design; Its effect on Research Characteristics of Individual species Experimental design Choice of species 45 Injections and tissue sampling Health Considerations Alternatives to live animal experimentation Handling Video, Safety, Local arrangements Video and discussion 'Ethics of Animal research' The Scientists Viewpoint Assessment Reading List: Laboratory animals an introduction for new experimenters Handbook of laboratory animal management and care M. Lloyd Introduction to laboratory animal science and technology Humane experimental technique Experimental and surgical technique in the rat P. Flecknell Animals and alternatives in toxicology ; present and future prospects In vitro toxicology UFAW handbook on the care & management of laboratory animals Laboratory animals anaesthesia Handbook of rodent and rabbit medicine M. Swindle, P. Flecknell The biology and medicine of rabbits and rodents Wagner The laboratory animals, principles and practice A. Pearson Man and mouse, animals in medical research Lives in the balance; the ethics of using animals in biomedical research Vivisection in historical prospective A. A. Tuffery S. Wolefensohn, J. Inglis W. Russell, R. Burch H. Wayneforth, M. Balls, J. Bridges, J. Southee S. Cox Gad T. Poole P. Flecknell K Laber-Laird, J. Harkness J. W. Lane-Petter, W. Paton J. Smith,K. Boyd R. Rupke 46 SENIOR SOPHISTER PROJECTS 2015-2016 47 SS PROJECT NO.1 Supervisor: Mark Hughes and Prof Luke O’Neill Title: EXPLORING THE ROLE OF GSTO1 AS A CRITICAL MEDIATOR OF TLR4 SIGNAL TRANSDUCTION Background: Antioxidants protect the cell from oxidative stress thereby maintaining the redox balance, utilizing glutathione as the main antioxidant in the cell. Glutathione maintains cytoplasmic redox potential and aids protein integrity via the glutathionylation of redox-sensitive amino acids1. Toll-like receptors (TLR) are critical pattern recognition receptors in innate immunity, playing pivotal roles in immune and infectious disease2. We have found evidence of glutathionylation as a regulator of TLR signaling via an enzyme termed glutathione transferase omega 1 (GSTO1). GSTO1 is able to both glutathionylate and deglutathionylate key substrates. We have found that GSTO1 is critical for signaling by the TLR4 agonist LPS, which is a critical mediator of septic shock3. Preliminary experiments on GSTO1 knockout bone marrow derived macrophages (BMDMs) have identified a strong phenotype in TLR4 signaling. Our evidence suggests GSTO1 may act upstream of NF-kB signaling, and we are currently assessing potential targets of GSTO1. Mal is the main adaptor protein utilized by TLR1/2, TLR2/6 and TLR4 along with MyD88 to elicit TLR signal transduction and induction of pro-inflammatory cytokines, including IL6, IL12p40 and TNFα4. Mal contains unusual free cysteines that are not in disulfide bonds and have the potential to be redox regulated. It is possible that GSTO1 is deglutathionylating these cysteines in order for Mal to signal. This project will examine this possibility, in order to determine the role of GSTO1 in signaling by TLR4. Project Aims: The aim of this project is to characterize the targeting of Mal by GSTO1 in order to further implicate GSTO1 as a critical regulator of TLR4 signaling. There will be 3 main aims: 1. Mutant forms of Mal in which critical amino acids that flank the key glutathionylated cysteines will be tested in various functional assays, notably the ability to bind another key adapter in TLR4 signaling MyD88, and also for NF-kB activation. In other proteins, flanking amino acids have been shown to be required for cysteine glutathionylation. 2. Whether the mutants are unable to undergo glutathionylation will be tested. 3. A GSTO1 inhibitor (ML175) will be tested in TLR4 signaling, particularly on the induction of cytokines by LPS. Methods: 48 Several techniques will be used, including cell culture with immortalized and primary cell lines, co-immunoprecipitations, NF-kB luciferase assays, bacterial cell culture, SDS-PAGE, western blotting, plasmid generation, cytotoxicity assays and qPCR. Predicted Outcome: This project could strongly support the critical role of GSTO1 in signaling by TLR4. Since TLR4 is a key therapeutic target in diseases such as sepsis and also inflammatory diseases such as rheumatoid arthritis, this project might therefore provide new possibilities for therapeutic intervention in these diseases where unmet medical need is great. References: 1. 2. 3. 4. Menon, D. et al. (2014). Free Radicals Biology and Medicine. 73, 318-327. O’Neill LA. et al. (2013). Nature Reviews Immunology. 13, 453-460. Menon, D. et al. (2015). Journal of Cell Science. 128, 1982-1990. Bernard, J and O’Neill LA. (2013). IUBMB Life. 65, 777-786 49 SS PROJECT NO.2 Supervisor: Dr. Zbigniew Zaslona and Prof. Luke O’Neill Title: REGULATION OF M2 MACROPHAGES BY BMAL1 – RELEVANCE TO ASTHMA/ALLERGY THE CIRCADIAN PROTEIN Background Our body clock drives rhythms in the expression of genes that have a 24 hour periodicity. A number of physiological processes including metabolism, cardiovascular and most importantly for this project immune function [1] have a time dependancy that is modulated by the circadian clock. The in-vivo response to an immune challenge, such as LPS (the gram negative bacterial product that acts via TLR4), varies greatly with time [2] and we recently published a paper demonstrating a reduction in macrophages in clock gene expression, specifically in the gene encoding Bmal1, with LPS challenge [2]. LPS stimulated macrophages are also called M1 macrophages, while those stimulated with the cytokine IL-4 are termed M2 macrophages. The molecular pathway responsible for M2 polarization is not fully understood, and the role of the body clock in the process of M2 regulation has not been explored. M2 macrophages are known to be critical for anti-parasitic responses and play roles in allergy and asthma [3]. Asthma is of particular relevance to the area of circadian control of immunity, since it is a disease with very strong clinical evidence suggesting regulation by ciracadian variation [4]. This project will explore the role of the clock protein Bmal1 in the regulation of M2 macrophages in order to provide added molecular insights into the control of macrophage function by the clock. We have preliminary evidence indicating that a model of allergic asthma in mice is more severe in Bmal1-deficient mice. This suggests that Bmal1 might limit M2 macrophage polarisation, since M2 macrophages are thought to play a role in the disease process in this model. We will therefore explore the role of Bmal1 in M2 macrophage polarisation to provide further information on the role of the circadian clock in asthma. Project Aims 1. Initially, we will use bone marrow derived macrophages from wildtype and Bmal1-deficient mice (BMAL1) and stimulate them with IL-4 for 24 h. Using qPCR we will estimate the expression of M2 markers such as ym-1 or arginase to determine the role of Bmal1 in the M2 phenotype. 2. We will also test for IL-4 signalling in the macrophages, measuring STAT6 phosphorylation as a key process regulated by IL-4. 3. M2 macrophages are also capable of phagocytosis and this will be also be measured in wild type and Bmal1-deficient cells. 4. If the availability of Bmal1-deficient mice is limiting, pharmacological approaches can be taken, in particular the use of a Rev-Erb-alpha agonist [5], since Rev-Erb-alpha is a possible downstream target for Bmal1 that could be responsible for the effects of Bmal1 here. 50 Methods: Key techniques that will be deployed will include cell culture, SDS-PAGE and Western blotting, RNA extraction and gene expression analysis by SybrGreen. Also flow cytometry analysis will be used to detect phagocytosis. Possible outcome: This project will aim to reveal an importance of Bmal1 in the activation of M2 macropages and their phagocytic capacity. This will offer new insights into the cross talk between inflammatory and circadian systems in diseases such as asthma/allergy. References 1. Curtis AM, Bellet MM, Sassone-Corsi P, O'Neill LA, Circadian clock proteins and immunity. Immunity. 2014 Feb 20;40(2):178-86 2. Curtis, A.M., et al., Circadian control of innate immunity in macrophages by miR-155 targeting Bmal1. Proc Natl Acad Sci U S A, 2015. 112(23): p. 7231-6. 3.Byers DE, Holtzman MJ. Alternatively activated macrophages and airway disease. Chest. 2011 Sep;140(3):768-74 4. Durrington, H.J., et al., The circadian clock and asthma. Thorax, 2014. 69(1): p. 90-2. 5. Gibbs JE, Blaikley J, Beesley S, Matthews L, Simpson KD, Boyce SH, Farrow SN, Else KJ, Singh D, Ray DW, Loudon AS. The nuclear receptor REV-ERBα mediates circadian regulation of innate immunity through selective regulation of inflammatory cytokines. Proc Natl Acad Sci U S A. 2012 Jan 10;109(2):582-7 51 SS PROJECT NO.3 Supervisor: Dr Marcin Baran and Prof. Andrew Bowie. Title: DETERMINING THE ROLE OF THE ELONGATOR COMPLEX IN CYTOKINE INDUCTION BY PATTERN RECOGNITION RECEPTORS. Background The Pyrin and HIN200 domain-containing (PYHIN) protein family consists of five proteins in human and 13 proteins in mouse. Mouse AIM2 and IFI204 have been previously described to be intracellular pattern recognition receptors (PRRs) for dsDNA, leading to inflammasome activation and IFNβ induction respectively1, 2. However the function of most mouse PYHIN proteins remains unknown. We have shown that the mouse PYHIN protein IFI207 is highly expressed in myeloid cells, and in contrast to IFI204, has an intrinsic role in regulation of gene induction of specific cytokines, independent of DNA sensing. Interestingly, IFI207 interactome analysis by mass spectrometry revealed that it interacted with the Elongator complex, a protein complex conserved from yeast to human which has been implicated in transcriptional regulation, but has not previously been shown to regualate inducible cytokines3. Using RNA interference to knock down the expression of Elongator subunits such as Elp2 in mouse cells we showed for the first time a role for Elongator in TNF induction. Preliminary results also showed a role for Elongator in LPS-stimulated TNF induction in the human monocytic cell line THP-1, even though there is no human ortholog of mouse IFI207. Thus the Elongator complex may have a general, but previously unappreciated, role in cytokine gene induction. Therefore the aim of this project is to charaterise the role of the Elongator complex in TNF gene induction in human monocytic cells. Specific Aims: 1. Use RNA interference (shRNA) to determine the role of Elongator subunits in PRRstimulated TNF induction in THP-1 cells. 2. Examine whether human PYHIN proteins cooperate with Elongator to induce the TNF gene. 3. Determine which human PYHIN proteins, if any, interact with the Elongator complex. Methods: THP-1 cells stably expressing control shRNA, or shRNA targeting Elp2 and Elp3 will be stimulated with TLR ligands, or transfected with RNA or DNA, and TNF mRNA induction and protein production measured by qRT-PCR and ELISA respectively. Cells will also be immunoblotted for Elp expression in order to demonstrate knockdown of shRNA targets. The effect of Elp knock-down on induction of other PRR-stimulated genes will also be determined. In a reporter gene assay which directly measures the induction of the TNF promoter, the ability of human PYHIN proteins, when overexpressed, to cooperate with Elps in inducing TNF will be determined. RNA interference can also be used to determine whether any of the four known human PYHIN proteins (AIM2, IFI16, MNDA and PYHIN1) are involved in PRR-stimulated TNF induction. Finally, co-immunoprecipitation assays will be used in order to determine whether, as in the case of mouse IFI207, human PYHIN proteins can interact with the Elongator complex. 52 Overall this project will reveal new mechanistic insights into how the pro-inflammatory cytokine TNF is regulated by PRR pathways in human cells. References: 1. Unterholzner, L., Keating, S.E., Baran, M., Horan, K.A., Jensen, S.B., Sharma, S., Sirois, C.M., Jin, T., Latz, E., Xiao, T.S., Fitzgerald, K.A., Paludan, S.R. & Bowie, A.G. 2010. Nat. Immunol. 11, 997-1005. 2. Connolly, D & Bowie A.G. 2014. The emerging role of human PYHIN proteins in innate immunity: implications for health and disease. Biochem Pharmacol. 92, 405-414. 3. Creppe, C & Buschbeck, M. 2011. Elongator: an ancestral complex driving transcription and migration through protein acetylation. J Biomed Biotechnol. doi: 10.1155/2011/924898. 53 SS PROJECT NO.4 Supervisor: Dr Dympna Connolly and Prof. Andrew Bowie Title: EXPLORING THE ROLE OF PYHIN-INTERACTING PROTEINS IN PYHIN FUNCTION AND INNATE IMMUNITY Background The innate immune response depends on the ability of immune cells to detect pathogens through germline-encoded pattern recognition receptors (PRRs). Recently discovered PRRs include some members of the Pyrin and HIN domain (PYHIN) family, which are encoded on an interferon-inducible gene cluster located on chromosome 1q231, 2. There are five human PYHIN proteins; Absent in melanoma 2 (AIM2), IFN-g inducible protein 16 (IFI16), Myeloid cell nuclear differentiation antigen (MNDA), Pyrin and HIN domain family member 1 (PYHIN1) and the recently identified Pyrin domain only protein 3 (POP3). Early studies reported roles for these proteins in cell cycle control, tumour suppression and transcriptional regulation. AIM2 and IFI16 have now been shown to be immune sensors of non-self DNA, such as that produced by viruses in infected cells2, 3. AIM2 binds DNA to activate the inflammasome, while IFI16 detection of DNA can lead to the up-regulation of type I interferons (IFNs). Furthermore, we have recently shown that MNDA is required for the type I IFN response to both pathogens, RNA and DNA in monocytes, since it transcriptionally regulates IRF7 expression. These diverse functions of PYHIN proteins are likely mediated by specific PYHIN interactions with distinct proteins, to regulate protein signalling and effector networks within cells. In order to begin to elucidate mechanisms whereby PYHIN proteins signal to altered gene expression, we performed unbiased affinity purification and mass spectroscopy to identify PYHIN-interacting proteins. Many of the proteins discovered in these PYHIN interactomes are predicted to have roles in gene regulation, and some such interactors have also been discovered in a recent paper using a similar approach4. Thus the aim of the project is to test the potential role of specific PYHIN interacting proteins in mediating PYHIN function. Specific Aims: 1. Select shortlist of PYHIN interactors to examine. 2. Determine which interactors are expressed in THP-1 and HT1080 cells (cells where PYHIN function can be examined). 3. Use loss-of-function approaches (siRNA) to examine the role of selected interactors in AIM2, IFI16 and MNDA functions. Methods: Up to five PYHIN interactors which have been found to interact with specific subsets of PYHIN proteins will be selected. For example SKIV2L2 interacts with MNDA and IFI16 but not AIM2, while LAMTOR1 interacts with MNDA and AIM2 but not IFI16, and VPRBP interacts with MNDA but not IFI16 nor AIM2. The expression of selected interactors in THP1 and HT1080 cells will then be examined, with and without cell stimulation, by qRT-PCR. In cells where these interactors are shown to be expressed, siRNA oligonucleotides targeting them will be designed and tested for efficacy. These siRNAs will then be used in loss-of- 54 function experiments to knock down specific interactors in order to assay their potential role in PYHIN functions. To assay AIM2 function, DNA-stimulated IL-1b release will be measured in THP-1 cells. To assay IFI16 function, DNA-stimulated type I IFN production will be measured in THP-1 and/or HT1080 cells by bioassay. To assay MNDA function, IRF7 protein expression and IFNa induction after cell stimulation of THP-1 cells will be measured by immunoblot and qRT-PCR respectively. Overall this project will provide novel mechanistic insights into how PYHIN proteins function in innate immunity. References: 1. Dempsey, A. and Bowie, A.G. (2015) Virology 479-480: 146-152 2. Connolly, D & Bowie A.G. (2014) Biochem Pharmacol. 92, 405-414. 3. Unterholzner, L. et al (2010) Nat. Immunol. 11, 997-1005. 4. Diner et al (2015) Molecular Systems Biology 11, 787. 55 SS PROJECT NO.5 Supervisor: Dr Michael Carty & Prof Andrew Bowie Title: DOES SARM OLIGOMERISATION HAVE A ROLE IN INHIBITION OF HIV-1 TRANSCRIPTION? Background Toll-like receptors (TLRs) are pattern recognition receptors that recognise the presence of pathogen associated molecular patterns such as viral RNA and DNA within host cells1. TLRs signal to altered gene expression via Toll-IL-1R (TIR) domain containing adaptor proteins such as MyD88 and TRIF. Like MyD88 and TRIF, SARM is a cytosolic protein with a TIR domain2. SARM is the most evolutionarily conserved TIR domain protein, and has been shown to have roles both in TLR signaling but also more broadly in TLR-indpendent functions such as gene regulation and cell death2. For example, we showed a role for human SARM in the regulation of TRIF-dependent signalling3, while other roles for mammalian SARM include promotion of neuronal and axonal death, restriction of West Nile virus pathogenesis in the brain4, and in the induction of the chemokine CCL55. Other anti-viral roles for SARM are likely to emerge and very recently we showed that SARM also inhibits transcription of the HIV long terminal repeat (LTR). One mechanism whereby the HIV protein TAT fulfils an essential role in the HIV life cycle, is by transcriptionally regulating the HIV LTR, and we showed that overexpression of SARM inhibited TATdependent HIV LTR transcription, while knockdown of endogenous SARM by siRNA enhanced TAT-mediated LTR activity in HEK293 cells. Thus SARM may be a novel inhibitor of HIV infection, which may have relevance to virus-host interactions in HIV-1-infected cells. Work from other groups has suggested that SARM may oligomerise after cell activation in order to cause cell death2, however the mechanism whereby SARM inhibits TAT is unclear. Therefore the aim of this project is to explore the mechanism whereby SARM inhibits HIV-1 transcription and to test whether SARM oligomerisation is involved. Specific Aims: 1. Confirm the role of SARM in TAT inhibition using gain-of-function and loss-offunction approaches. 2. Determine the protein domains in SARM required to inhibit TAT. 3. Test whether the SARM domains required to inhibit TAT are required for SARM oligomerisation. Methods: The effect of SARM overexpression (gain of function) and RNA interference targeting endogenous SARM (loss of function) on the ability of TAT to enhance HIV transcription will be assessed in a reporter gene assay which examines TAT-stimulated activation of a HIV-1LTR-Luc reporter. This will be confirmed in HEK293 cells and tested in an independent cell type, namely HeLa. SARM has three types of defined protein motifs, namely two SAM motifs, ARM repeats and a TIR domain2. In order to map the domains of SARM required to inhibit TAT, different truncated forms of SARM will be tested for expression, and assessed 56 for the ability to inhibit TAT in the HIV LTR reporter gene assay. Thereafter, both full length and truncated forms of SARM will be tested for oligomerisation using a cross-linking assay, whereby protein crosslinking captures oligomeric protein complexes which can then be visualised on SDS-PAGE by immunoblotting. Overall this project will determine for the first time how SARM inhibits TAT-dependent HIV LTR transcription. References: 1. Gürtler, C. & Bowie, A.G. (2013) Trends Microbiol. 21: 413-20. 2. Panneerselvam and Ding (2015) International Reviews of Immunology, Early Online 113. 3. Carty, M. et al (2006) Nat Immunol 7: 1074-81. 4. Carty, M. et al (2014) Trends Immunol. 35: 79-87. 5. Gürtler, C. et al (2014) J Immunol. 192: 4821-32. 57 SS PROJECT NO.6 Supervisor: Mag Needham, Rachel McLoughlin and Cliona O’ Farrelly Title: IL8 AND HCV INFECTION Project Outline Background Irish women who have been exposed to hepatitis C virus through contaminated anti-D immunoglobulin products and who resisted infection provide a unique opportunity to study possible innate immune pathways involved in resistance to HCV infection. Previous work within the lab has discovered elevated levels of the IL8 cytokine in plasma samples from a small cohort of these uninfected anti-D women. We believe these women to be innately resistant to HCV infection and hypothesise that the increased expression of IL8 (a proinflammatory cytokine involved in neutrophil recruitment and activation) plays an integral role in this innate immune resistance. The human IL8 gene is well characterised and SNPs within its promoter region have previously been shown to directly influence gene expression and have been linked with the progression and outcome in different diseases. An IL8 gene promoter polymorphism (-251A/T ) has been associated with an increased risk of developing liver cirrhosis in people with hepatitis B (Qin et al, 2012) and with susceptibility to bacterial infection (Garey et al. 2010). Previously we have reported that genetic variation within the bovine IL8 promotor differentially regulates IL8 expression and may have an impact on bovine susceptibility to infectious disease and inflammation (Meade et al. 2012). Hypothesis We propose that IL8 has a role in HCV infection and that SNPs within the human IL8 promoter region are associated with HCV resistance possibly by influencing transcription factor binding sites Technologies We will use sequence analysis to identify any IL8 promoter SNPs in our sample cohort and to characterise the influence of any promoter haplotypes on IL8 expression levels using TLR agonist and interferon stimulation of PBMCs. This project will also use bioinformatics based studies to predict the possible impact of promoter region SNPs on transcriptional factor binding in the human IL8 gene. Finally, to explore the functional implications of elevated IL8 expression this project will investigate the impact of IL8 on neutrophil function, specifically the role IL8 plays in inducing neutrophil extracellular traps (NETs), which have recently been shown to have important antiviral roles (Jenne et al. 2015). Implications The findings from this project will help identify novel innate immune mechanisms responsible for resistance to HCV infection which could influence development of new vaccination and therapeutic strategies. 58 References: Qin X. et al. The IL-8 gene polymorphisms and the risk of the hepatitis B virus/infected patients. (2012) DNA Cell Biology 31, 1125-1130. Garey K. W. et al. A common polymorphism in the interleukin-8 gene promoter is associated with an increased risk for recurrent Clostridium difficile infection. (2010) Clinical Infectious Diseases (51)1406-1410. Meade K. G. et al. Functional characterisation of bovine interleukin 8 promoter haplotypes in vitro (2012) Molecular Immunology 50, 108-116. Jenne C. N. et al. Virus-Induced NETs – Critical Component of Host Defense or Pathogenic Mediator? (2015) PLoS Pathogens 11, e1004546. 59 SS PROJECT NO.7 Supervisor: Prof Cliona O’ Farrelly Title: THE EFFECT OF THE IMMUNOSUPPRESSIVE DRUG TACROLIMUS ON HEPATIC NATURAL KILLER CELL PHENOTYPE AND FUNCTION: RELEVANCE TO GRAFT TOLERANCE. Introduction: Liver transplantation is a life saving operation for patients with end stage liver disease. Recipients must take powerful immunosuppressive medication in order to prevent graft rejection, side effects of which include malignancy, cardiovascular disease and renal failure. Tacrolimus is the most commonly used of these drugs. Belonging to the calcineurin inhibitor family, it prevents T cell activation by blocking the phosphorylation of NFAT. However it is unclear how this drug affects innate lymphoid cells which play important roles in transplant rejection and tolerance. The adult human liver contains significant populations of innate immune cells, especially NK cells. The liver is a naturally tolerogenic organ and can be transplanted without HLA matching. It is hypothesised that immune cells in the donor organ mediate this tolerance. We believe hepatic NK cells from the donor organ are key. However, it is unclear how immunosuppression might interfere. Objectives: To identify the effects of tacrolimus on hepatic NK cell phenotype and function. Methodology: Molecular and cellular techniques will be used in the project, including cell culture, in vitro cytotoxicity assays and flow cytometry. These techniques will be optimised using NKL cells, a cell line derived from leukemic NK cells and then used to test stored hepatic NK cell populations from liver transplants. The effect of tacrolimus on hepatic NK cell cytotoxicity and IFN-g production will be assessed by a CD107a assay. The expression of natural cytotoxicity receptors (NCRs) will also be assessed to determine if immunosuppression has an effect on hepatic NK cell phenotype. Likely outcome: This is the first time that the effect of immunosuppression on liver resident NK cells has been assessed. The data will be an important component of an ongoing research project into the role of hepatic NK cells in graft tolerance post transplantation. References: 1. Harmon C et al. Am J Transplantation 2015 in press 2. Lysakova-Devine T, O’Farrelly C. J Leukoc Biol 2014 Dec 96(6):981–90. 3. Nemeth, E., Baird, A. W., & O’Farrelly, C. Seminars in Immunopathology, 2009 31(3), 333–43. 4. Moroso V et al. Liver Transplant 2010;16:895–908. 5. Benitez C et al. Hepatology. 2013;58(5):1824–35. 60 6. 7. Meehan AC et al.. PLoS One. 2013;8(3):e60144. Mukherjee & Mukherjee. Journal of Transplantation 2009 1-20 61 SS PROJECT NO.8 Supervisor: Ms. Anne Barry-Reidy and Prof. Cliona O’Farrelly Title: HOST DEFENCE PROTEINS IN THE FEMALE REPRODUCTIVE TRACT Introduction: Viral, bacterial, yeast and parasitic infection of the female reproductive tract is the most common cause of pathology in young females. The reproductive tract is equipped with a complex and diverse immune repertoire to provide protection against these challenges. Our group is particularly interested in the role of host defence peptides, important innate immune molecules, and have focused in the past on defensins The multifaceted nature of these peptides means their activity in the female reproductive tract (FRT) has received attention in recent years (Narciandi, Lloyd, Meade, & O’Farrelly, 2013; Tribe, 2015). The female tract is immunologically complex- it must tolerate the influx of foreign sperm and the implantation of an embryo, yet respond to pathogens which enter as a result of intercourse and labour. In addition, labour represents a significant level of tissue injury which must be resolved efficiently. Elafin, secretory leukocyte protease inhibitor (SLPI) and human epididymal protein 4 (HE4) members of the WFDC family are expressed throughout the human female reproductive tract (FRT) (Frew & Stock, 2011; Yarbrough, Winkle, & Herbst-Kralovetz, 2015). Elafin mRNA is increased in human endometrial epithelial cells treated with IL-1β and TNFα (King, Critchley, Sallenave, & Kelly, 2003). We aim to examine expression on these genes in endometrium from women with endometriosis. In the first instance, technologies will be optimised using bovine tissues. Our group has demonstrated that elafin and WFDC18 are upregulated in uterine biopsies taken from cows 7 days post-partum (dpp) relative to 21 dpp. Elafin is also upregulated in animals with a uterine infection relative to healthy animals 21 dpp. This suggests these proteins may be involved in pathogen clearance, wound repair and the re-establishment of homeostasis after labour. As prolonged post-partum inflammation is particularly common in the bovine and delays the return to a fertile state, we are interested in the role of WFDC proteins in the bovine FRT. Quantitative PCR will be used to detect expression of selected WFDC proteins in a biobank of bovine FRT tissues including vagina, cervix, uterus, fallopian tube and ovary. In addition, material will be made available from primary bovine endometrial stromal and epithelial culture experiments and from women with endometriosis. We also propose to use publiclyavailable genome databases to characterise the WFDC locus in the human and bovine genomes. Objectives: To determine the presence of WFDC proteins within a range of female reproductive tract tissues and within endometrial stromal and epithelial cells. Methodology: RNA extraction and cDNA synthesis from bovine reproductive tract tissues. 62 Primer design and qPCR for whey acidic protein genes. Likely outcome: Whey acid proteins are expressed in several tissues from the bovine female reproductive tract References: Frew, L., & Stock, S. J. (2011). Antimicrobial peptides and pregnancy. Reproduction, 141(6), 725–735. http://doi.org/10.1530/REP-10-0537 King, A. E., Critchley, H. O. D., Sallenave, J.-M., & Kelly, R. W. (2003). Elafin in human endometrium: an antiprotease and antimicrobial molecule expressed during menstruation. The Journal of Clinical Endocrinology and Metabolism, 88(9), 4426–4431. http://doi.org/10.1210/jc.2003-030239 Narciandi, F., Lloyd, A., Meade, K. G., & O’Farrelly, C. (2013). A novel subclass of bovine β-defensins links reproduction and immunology. Reproduction, Fertility and Development. Retrieved from http://dx.doi.org/10.1071/RD13153 Tribe, R. M. (2015). Small Peptides with a Big Role: Antimicrobial Peptides in the Pregnant Female Reproductive Tract. American Journal of Reproductive Immunology, 74(2), 123–125. http://doi.org/10.1111/aji.12379 Yarbrough, V. L., Winkle, S., & Herbst-Kralovetz, M. M. (2015). Antimicrobial peptides in the female reproductive tract: a critical component of the mucosal immune barrier with physiological and clinical implications. Human Reproduction Update, 21(3), 353–377. http://doi.org/10.1093/humupd/dmu065 63 SS PROJECT NO.9 Supervisor: Dr. Stephanie Longet and Dr. Ed Lavelle Title: ANALYSIS OF INTESTINAL IMMUNE RESPONSES FOLLOWING ORAL ADMINISTRATION OF THE INTK CELL ACTIVATOR Α-GALACTOSYLCERAMIDE Introduction: Enteric infections are a major worldwide health problem causing high levels of mortality and morbidity especially in developing countries (1). Oral vaccination is regarded as the optimal means to fight intestinal infections as it elicits local immune responses including the secretion of IgA, shown to be particularly important in protection from enteric infections. One of the most important challenges in oral vaccination is the generally poor immunogenicity of orally delivered antigens (2, 3). In order to increase immunogenicity, the presence of an adjuvant is required. Even though many oral adjuvants have been evaluated, as yet none has been licenced for human use (4). Consequently, it remains crucial to find suitable oral adjuvants capable of enhancing intestinal antigen-specific IgA responses that could be potentially used in humans. The glycolipid α-GalactosylCeramide (α-GalCer), an invariant natural killer cell activator, was shown to be a powerful mucosal adjuvant in animal models (5). In the lab, we have demonstrated the ability of α-GalCer to increase intestinal antigen-specific IgA responses in mice in the context of an oral vaccine against enterotoxigenic Escherichia Coli (ETEC) that we are developing (6-9). However, we lack a comprehensive understanding of how orally administered α-GalCer increases antibody responses and this project aims at addressing this deficiency. The effects of α-GalCer on B cells in the principal intestinal lymphoid tissues, Peyer’s patches and mesenteric lymph nodes will be determined in vivo and in vitro. In addition, the release of key cytokines involved in IgA class switching and secretion including IL-17 and TGF-β will be measured post-administration of oral α-GalCer in intestinal tissues and washes. Finally, the impact of oral administered α-GalCer on the expression and localisation of polymeric Ig receptor (pIgR) on intestinal epithelial cells will be assessed. This receptor is directly involved in IgA transcytosis into the intestinal lumen. Objectives: • To assess modifications in B cell activation in gut associated lymphoid tissues following oral administration of α-GalCer or in the presence of α-GalCer in vitro. • To examine changes in secretion of specific IgA promoting cytokines in intestinal tissues and washes post-administration of oral α-GalCer. • To evaluate the expression and localisation of pIgR on intestinal epithelial cells following oral administration of α-GalCer. Methodology: A range of state-of-the art immunological techniques will be used in the project. At several time-points after oral administration of α-GalCer in mice, cells will be isolated from Peyer’s patches and mesenteric lymph nodes. For comparison and to address systemic immune responses, the B cell analysis will also be performed with splenocytes. Modifications in 64 expression of B cell activation markers will be evaluated by FACS analysis. The same type of experiments will be performed with cells isolated from Peyer’s patches, mesenteric lymph nodes and spleen of naïve mice that will be stimulated with α-GalCer in vitro. IL-17 and TGF-β released in intestinal tissues and washes will be measured after oral administration of α-GalCer by ELISA. For comparison, the release of these cytokines will also be measured in serum. The expression of pIgR will be evaluated on intestinal tissues collected after oral administration of α-GalCer by immunohistochemistry. Likely outcome: The project will give us a better understanding of the effects of α-GalCer on intestinal responses related to IgA production/secretion after oral administration. It will be the first study towards a detailed understanding of how orally administered α-GalCer increases intestinal IgA responses. References: 1) Cheng, A.C. et al.Infectious diarrhea in developed and developing countries. J ClinGastroenterol, 2005. 39(9): p. 757-73. 2) Czerkinsky, C. and Holmgren, J. Enteric vaccines for the developing world: a challenge for mucosal immunology. Mucosal Immunol, 2009.2(4): p. 284-7. 3) Pasetti, M.F. et al. Immunology of gut mucosal vaccines. Immunol Rev, 2011.239(1): p. 125-48. 4) Rhee, J.H. et al.Mucosalvaccineadjuvantsupdate. ClinExpVaccine Res, 2012 Jul;1(1):5063. 5) Courtney, A.N. Characterization of alpha-galactosylceramide as a mucosal adjuvant. UT GSBS Dissertations and Theses (Open Access), Texas Medical Center Library, Digital Commons@The Texas Medical Center, 2010. Available online at:http://digitalcommons.library.tmc.edu/cgi/viewcontent.cgi?article=1085&context=utgsbs_di ssertations 6) Quadri, F. et al. EnterotoxigenicEscherichia coli in Developing Countries: Epidemiology, Microbiology, Clinical Features, Treatment, and Prevention, ClinMicrobiol Rev, 2005 Jul;18(3):465-83. 7) Croxen, M.A. andFinlay B.B.Molecularmechanisms of Escherichiacolipathogenicity.Nat Rev Microbiol, 2010 Jan;8(1):26-38. 8) Tobias, J., et al., Construction of non-toxic Escherichia coli and Vibrio cholerae strains expressing high and immunogenic levels of enterotoxigenic E. coli colonization factor I fimbriae. Vaccine, 2008.26(6): p. 743-52. 9) Lundgren, A., et al., Clinical trial to evaluate safety and immunogenicity of an oral inactivated enterotoxigenic Escherichia coli prototype vaccine containing CFA/I overexpressing bacteria and recombinantly produced LTB/CTB hybrid protein. Vaccine, 2013.31(8): p. 1163-70. 65 SS PROJECT NO. 10 Supervisor: Dulce Bento, Filipa Lebre and Dr. Ed Lavelle Title: ACTIVATION OF DENDRITIC CELLS USING POROUS NANOPARTICLES TO TARGET IMMUNOMODULATORS Introduction The use of particles as vaccine adjuvants offers several advantages including enhanced uptake by antigen presenting cells, protection of antigen from degradation, promotion of a depot effect with gradual release of the antigen and the ability to facilitate antigen cross-presentation and co-deliver of antigens and immunomodulators to the same cell population [1]. Properties including particle size, surface chemistry, charge and shape are important for the immunomodulatory effects of particulates [2]. Recently, mesoporous silica nanoparticles (MSNs) have received significant attention for drug and vaccine delivery [3]. Their advantages include biocompatibility, surface functionalization, the ability to fine-tune particle and pore size and geometry, a high surface area that allows the efficient loading of drugs and antigens molecules and increased stability when compared with other polymer-based carriers. However, the effect of particle pore size in the induction of innate and adaptive immune responses has not been addressed. In this project, the influence of pore size of silica nanoparticles on the innate immune response will be assessed using 200 nm porous and non-porous particles. Additionally, the ability of these nanoparticles to improve the immunomodulatory properties of TLR agonists will be tested. Nanoparticle size was selected based on data from our lab showing that showed that of a range of particle sizes (50 nm to 100 µm), the smaller particles were optimal for induction of both CD4+ T and CD8+ T cell response. Objectives: • To assess whether MSN can activate dendritic cells and macrophages and determine the impact of pore size of MSN on cytokine production and maturation • To evaluate the capacity of MSN to efficiently delivery TLR ligands to dendritic cells and to compare ligands for plasma membrane and endosomal TLRs • To assess the innate immune response induced by the injection of unmodified or TLR ligand-loaded MSN Methodology: Cell culture techniques will be employed to stimulate murine bone-marrow derived dendritic cells (BMDCs) and bone-marrow derived macrophages (BMDMs) with particles. The cell supernatants will be collected and cytokines measured by ELISA. Cell maturation and cell death will be analysed by flow cytometry. The innate immunomodulatory effects of the most promising strategies will be evaluated 24 h after intraperitoneal injection in vivo. Peritoneal exudate and cells, draining lymph nodes and spleens will be collected and innate immune response will be assessed by flow cytometry analysis assessed by FACs, cytokine production after restimulation with various stimuli (e.g. TLR agonists) by ELISA. 66 Likely Outcome: This study will provide valuable new information on how the pore size of porous particulate adjuvants impacts on the induction of innate immune responses and of how mesoporous particles synergize with immunomodulators. This has relevance for the rational design of novel particle based adjuvants. References [1] V.E. Schijns, E.C. Lavelle, Trends in vaccine adjuvants, Expert review of vaccines, 10 (2011) 539-550. [2] D.M. Smith, J.K. Simon, J.R. Baker, Jr., Applications of nanotechnology for immunology, Nature reviews. Immunology, 13 (2013) 592-605. [3] H. Vallhov, N. Kupferschmidt, S. Gabrielsson, S. Paulie, M. Stromme, A.E. GarciaBennett, A. Scheynius, Adjuvant properties of mesoporous silica particles tune the development of effector T cells, Small, 8 (2012) 2116-2124. 67 SS PROJECT NO. 11 Supervisor: Dr Natalia Muñoz-Wolf and Dr Ed Lavelle Title: INFLUENCE OF CHARGE IN THE ADJUVANTICITY OF NANOCONJUGATES BASED ON TLR LIGANDS. Introduction: Vaccine industry has shifted from attenuated or inactivated vaccines towards safer subunit formulations based on purified antigens. Although safer, subunit vaccines are less immunogenic and require adjuvants to boost its efficacy. Particularly, there is a pressing need for generation of adjuvants able to trigger robust Th1 and Th17 cell-mediated immunity necessary to protect against pathogens like TB, HIV or pneumococcus [1]. Nanotechnology is a valuable tool for adjuvant development since nanoparticles (NPs) can display intrinsic adjuvanticity, act as antigen delivery systems and be tailored for specific cell-targeting. These systems are attractive because they allow the delivery of antigens to specific immune cells enhancing cellular responses [2]. Particulates can activate the NLRP3 inflammasome inducing secretion of the Th17-polarising cytokine IL-1 [3]. Pathogen-associated molecular patterns (PAMPS) such as nucleic acid sensor, Toll-like or NOD-like receptor agonists can also act as immunopotentiators [4]. Therefore, generation of tailored NPs which can simultaneously target and activate immune cells is of great interest for adjuvant development. We have generated a new candidate adjuvant based on conjugation of the TLR5/NLRC4 agonist Flagellin (FliC) to negatively charged nanoparticles. The nanoconjugate NP-FliC showed enhanced ability to activate antigen-presenting cells by enhancing secretion of proinflammatory cytokines, expression of costimulatory molecules and inflammasome activation. NP-FliC also activated human mononuclear cells, pointing to the potential of this novel nanoparticulate adjuvant for injectable and mucosal vaccines. Physicochemical characteristics of particles such as size, charge, shape, polymer composition, hydrophobicity, define hoe the particles interact with the immune system [5] and understanding this is crucial for rational design of adjuvants and vaccines. Our current understanding is that cationic NPs are more immunogenic than negative particles. On the other hand, positively charged NPs are generally more toxic. Hence we aim to explore how NP charge can affect adjuvanticity and immunostimulatory properties of our novel adjuvant NPFliC. Objectives: 1. To study the effect of surface charge on adjuvant potential of flagellin-coated nanoparticles by assessing their capacity to induce maturation and activation of antigen presenting cells (APCs). 2. To study the importance of conjugation vs adsorption of flagellin in the immunostimulatory effect of the candidate adjuvant. 3. To evaluate functional capacity of APCs stimulated with particles of different charges to drive differentiation of Th1 and Th17 cells in vitro. 68 Methodology: This project will involve a number of state-of-the-art techniques including cell culture of primary cells, evaluation of cytokine gene expression by retrotranscription/real-time PCR and flow cytometry for evaluation of surface marker expression and cell death. Secretion of cytokines will be also measured by ELISA in culture supernatants. Likely outcome: The results obtained from this project will contribute to our understanding in the molecular mechanisms involved in adjuvanticity of particulate adjuvants. Also they will complement the in vivo studies that will be carried out in parallel to address the adjuvanticity of FliC-NPs when administered in combination with relevant antigens. References: 1. Rappuoli, R. and A. Aderem, A 2020 vision for vaccines against HIV, tuberculosis and malaria. Nature, 2011. 473(7348): p. 463-9. 2. Zhao, L., et al., Nanoparticle vaccines. Vaccine, 2014. 32(3): p. 327-37. 3. McNeela, E.A. and E.C. Lavelle, Recent advances in microparticle and nanoparticle delivery vehicles for mucosal vaccination. Curr Top Microbiol Immunol, 2012. 354: p. 75-99. 4. Fujita, Y. and H. Taguchi, Overview and outlook of Toll-like receptor ligand-antigen conjugate vaccines. Ther Deliv, 2012. 3(6): p. 749-60. 5. Yue, H. and G. Ma, Polymeric micro/nanoparticles: Particle design and potential vaccine delivery applications. Vaccine, 2015. 69 SS PROJECT NO. 12 Supervisor: Vanessa Ziaitz Bittencourt, Dr David Finlay and Dr Clair Gardiner Title: TO DEFINE THE IMPACT OF INHIBITOR RECEPTOR SIGNALLING ON CYTOKINE INDUCED GLYCOLYSIS IN NK CELLS Background: It is becoming clear that immune cells undergo dynamic and dramatic metabolic changes in response to stimulation. Furthermore, metabolism can impact on the immune response of cells and regulate their effector functions(1). Natural Killer (NK) cells are lymphocytes that kill virally infected and cancer cells. Their functions are regulated by cytokines, and by a range of cell surface receptors that serve to either activate or inhibit NK cells. Our recent work (and that of others) on NK cells has shown that they can upregulate both glycolysis and oxidative phosphorylation (oxphos) in response to cytokine stimulation and that mTORC1 is important in the glycolytic response (2, 3). We have also recently found that in the mouse, co-stimulation of activating receptors alongside cytokine primes NK cells for IL2 activation of metabolism (unpublished). In this project, we now to wish to investigate the impact of engaging activating receptors on cytokine induced metabolic changes in human NK cells. Objectives: To investigate the hypothesis that engagement of activating receptors on NK cells attenuates cytokine induced changes in glycolysis. Methods: Peripheral blood mononuclear cells (PBMC) containing NK cells will be isolated from buffy coat preparation. Cells will be stimulated with different cytokine combinations in the presence or absence of antibody to engage activating receptors. The effect on phenotype and metabolism will be investigated. Outputs measured will include CD71 and CD98 expression, glucose uptake, glucose receptor expression, IL2R expression and potentially some detailed metabolic analysis using the Seahorse analyser. Outputs: These experiments will define if specific activating signals to NK cells modulate metabolic changes induced in response to cytokine. References: 1. Pearce, E. L., and E. J. Pearce. 2013. Metabolic pathways in immune cell activation and quiescence. Immunity 38: 633-643. 2. Donnelly, R. P., R. M. Loftus, S. E. Keating, K. T. Liou, C. A. Biron, C. M. Gardiner, and D. K. Finlay. 2014. mTORC1-dependent metabolic reprogramming is a prerequisite for NK cell effector function. Journal of immunology 193: 4477-4484. 3. Marcais, A., J. Cherfils-Vicini, C. Viant, S. Degouve, S. Viel, A. Fenis, J. Rabilloud, K. Mayol, A. Tavares, J. Bienvenu, Y. G. Gangloff, E. Gilson, E. Vivier, and T. Walzer. 2014. 70 The metabolic checkpoint kinase mTOR is essential for IL-15 signaling during the development and activation of NK cells. Nature immunology 15: 749-757. 71 SS PROJECT NO. 13 Supervisor: Clair Gardiner, Orla Sheils and John O’Leary Title: ELUCIDATING THE ROLE OF CD96 IN HUMAN NK CELLS Natural Killer (NK) cells are lymphocytes that kill cancer cells and virally infected cells. Their functions are regulated by activating and inhibiting receptors that they express. Among the recently described signalling pathways described is the CD155/CD112CD226/CD96/TIGIT axis. CD155 and CD112 are expressed on target cells while CD226, CD96 and TIGIT are expressed on NK cells (1). These molecules provide ligands for CD226, CD96 and TIGIT. CD226 is known to function as an activating receptor after ligation with CD155 while evidence supports that TIGIT inhibits NK cell functions. However, it is not yet clear the role that CD96 plays in human NK cells. It has been described as a co-stimulatory receptor in human NK cells and as an inhibitory receptor in murine NK cells (1). In this series of experiments, we aim to define the function of CD96 in human NK cells and investigate if it can be regulated by the pleiotropic cytokine, TGFβ. Objectives: o Using PBMC from buffy coats, we will cross-link the CD96 receptor, CD226 or TIGIT on human NK cells and measure the impact on direct activation using CD69 and functional read outs. o We will titre cytokine to determine suboptimal IL2 or IL12 cytokine stimulation of IFNγ production by NK cells and then look for the ability of CD96 to co-activate or indeed, inhibit NK cells. CD226 and TIGIT will function as control molecules. o Use of rCD155 will be used to confirm results obtained with cross-linking antibodies. o A role of TGFβ in the system will be investigated by testing if rTGFβ modulates CD96 (or other important receptors) expression on human NK cells. Likely Outcome: These data will identify a function for CD96 as either activating or inhibitory in human NK cells. References 1. Chan, C. J., L. Martinet, S. Gilfillan, F. Souza-Fonseca-Guimaraes, M. T. Chow, L. Town, D. S. Ritchie, M. Colonna, D. M. Andrews, and M. J. Smyth. 2014. The receptors CD96 and CD226 oppose each other in the regulation of natural killer cell functions. Nat Immunol 15: 431-438. 72 SS PROJECT NO. 14 Supervisor: Dr Caroline Sutton, Dr Annie Curtis and Prof Kingston Mills Title: THE ROLE OF THE CLOCK GENE BMAL1 IN DENDRITIC CELL ACTIVATION Introduction: Our internal body clock drives expression of cassettes of mRNA and protein that have a 24 h periodicity, and this is termed circadian expression. The central clock resides in the suprachiasmatic nucleus but functioning clocks have been identified in the heart, liver, vasculature and more recently in immune compartments such as the spleen. It has recently been demonstrated that macrophages (Mθ) have circadian gene expression controlled by clock genes and that these 24 h cycles within the Mθ regulate the inflammatory nature of these cells (1), inflammatory cytokines and the pro-inflammatory microRNA MiR-155 (2). These oscillations in innate immune cell activation coincide with fluctuations in pro-inflammatory markers such as IL-1 and IL-6. One such clock gene, Bmal1 appears to limit pro-inflammatory signalling in Mθ. Bmal1Lox/Lox Lyz2Cre mice (Bmal1-/-), in which the clock gene Bmal1 has been knocked out of innate immune cells, show a hyper inflammatory environment associated with accumulation of inflammatory monocytes (1). Furthermore, another clock gene has been shown to be important for the development of inflammatory adaptive Th17 responses (3). We have shown that under inflammatory conditions, such as stimulation with Mycobacterium tuberculosis (Mtb), that the absence of Bmal1 in innate cells leads to hyper activation of both Mθ and dendritic cells (DC) and in the case of DC that this is associated with a significant increase in the Th1 and Th17 polarising cytokines IL-12p70 and IL-23p19 (Sutton, Mills and Curtis unpublished). Furthermore, this increase promotes IL-17 and IFN-γ production from CD4 T cells. We have also found that the loss of Bmal1 from innate immune cells can lead to the exacerbation of experimental autoimmune encephalomyelitis (EAE), a mouse model for multiple sclerosis. Objectives: • To examine whether bone marrow derived dendritic cells (BMDC) from Bmal1-/- have an enhanced pro-inflammatory phenotype, such as upregulated co-stimulatory molecule expression and chemokine production, compared with WT BMDC at both a basal level and under inflammatory conditions. • To evaluate whether anti-inflammatory pathways, such as PD-L1 and IL-10 expression, is down regulated in Bmal1-/- BMDC. Methodology: FACS analysis, ELISA and real time PCR will be used to examine CD11c+ BMDC for their expression of both pro and anti-inflammatory associated markers under inflammatory and non-inflammatory conditions. The project with require cell culture, RNA isolation, FACS staining as well as FACS sorting of CD11c+ cells. Likely outcome; The results of this study should provide new information on how clock genes may influence the immune response to infection and will have implications on best timing of treatment with anti-inflammatory therapeutics. 73 References: 1) Nguyen, K. D. et al, Circadian gene Bmal1 regulates diurnal oscillations of Ly6Chi inflammatory monocytes. Science, Vol 341; 1483-1488. 2013. 2) Curtis, A.M et al, Circadian control of innate immunity in macrophages by miR-155 targeting Bmal1. PNAS, Vol 112; 7231-7236. 2015 3) Yu, X et al, TH17 cell differentiation is regulated by the circadian clock. Science. Vol 342; 727-730. 2013. 74 SS PROJECT NO. 15 Supervisor: Dr Alicja Misiak, Ms Aideen Allen and Professor Kingston Mills Title: SYNERGY BETWEEN TLR AND STING AGONISTS IN PROMOTING INNATE RESPONSES, WHICH DRIVE TH1 AND TH17 CELLS THAT PROTECT AGAINST BACTERIAL INFECTION Introduction: Innate immune responses that help to control infection are activated through pattern recognition receptors (PRR) that recognize a range of pathogen-associated molecular patterns (PAMPs) expressed by microbial pathogens (1). These PPRs include Toll-like receptors (TLR), such as TLR4 that recognizes lipopolysaccharides (LPS) from Gram-negative bacteria, TLR9 that binds bacterial CpG, TLR2 that bind bacterial lipoproteins, and intracellular nucleic acid receptors, including stimulator of IFN genes protein (STING), that bind nucleic acids and their derivatives. STING plays a critical role in response to cytosolic DNA and unique bacterial nucleic acids called cyclic dinucleotides and is involved in promoting type I IFN production during infection (2, 3). The bacterial pathogen Bordetella pertussis causes whooping cough (pertussis), a severe respiratory infection in infants and young children. The incidence of infection has been increasing in recent years with epidemics in many countries including Ireland (4). This is thought to reflect a failure of the current acellular pertussis (aP) vaccine to induce optimum immunity, especially Th1 and Th17 cells that are crucial for clearing bacteria from the respiratory tract (5). The current aP vaccine is composed of 3 pertussis antigens formulated with alum as the adjuvant. Studies in the Mills lab have shown that formulating the antigens with TLR9 or TLR2 agonists enhances efficacy of the vaccine by promoting Th1 responses (5, 6). Preliminary data has suggested that it may be possible to enhance this further by combining TLR and STING agonists as adjuvants (Allen et al unpublished). Although activation of nucleic acid sensing pathway has traditionally been associated with type I IFN production, we have evidence that STING, in combination with TLR agonists, can induce potent IL-1, IL-23 and IL-12 production and co-stimulatory molecule expression on dendritic cells (DCs) and thereby promote Th1 and Th17 responses. Objectives: • To examine the synergy between STING and TLR agonists in activating DCs and macrophages to produce IL-1, IL-12 and IL-23 production and CD80 and MHC class II expression. • To examine the signalling pathways in DCs and macrophages activated by TLR and STING agonists. • To examine the capacity of TLR and STING co-activated DCs to promote Th1 and Th17 responses in vitro Methodology: A range of immunology techniques will be used in the project, including culture of dendritic cells and macrophages, ELISA, real time PCR, Western blotting and intracellular staining and 75 FACS analysis. Small molecule inhibitors or siRNA specific for signalling molecules/pathways (NFκB, ERK, MAP kinase, IRF1, IRF8, NLRP3) will be utilized to assess the possible pathway involved in the synergy between TLR and STING agonism. T cells from T cell receptor transgenic mice (cells prepared by licensed Postdoc in the lab) will be used to assess the capacity of TLR and STING activated DCs to promote antigen-specific Th1 and Th17 responses. Likely outcome: The long term objective is to design a more effective vaccine against whopping cough. This project will assist in this goal by providing important information on the mechanism of activity of two PAMPs that work in synergy as adjuvants to promote protective Th1 and Th17 responses against B. pertussiss. References 1. Kawai, T., and S. Akira. 2009. The roles of TLRs, RLRs and NLRs in pathogen recognition. Int Immunol. 2. Burdette, D. L., K. M. Monroe, K. Sotelo-Troha, J. S. Iwig, B. Eckert, M. Hyodo, Y. Hayakawa, and R. E. Vance. 2011. STING is a direct innate immune sensor of cyclic di-GMP. Nature 478: 515-518. 3. Barker, J. R., B. J. Koestler, V. K. Carpenter, D. L. Burdette, C. M. Waters, R. E. Vance, and R. H. Valdivia. 2013. STING-dependent recognition of cyclic di-AMP mediates type I interferon responses during Chlamydia trachomatis infection. MBio 4: e00018-00013. 4. Mills, K. H., P. J. Ross, A. C. Allen, and M. M. Wilk. 2014. Do we need a new vaccine to control the re-emergence of pertussis? Trends Microbiol 22: 49-52. 5. Ross, P. J., C. E. Sutton, S. Higgins, A. C. Allen, K. Walsh, A. Misiak, E. C. Lavelle, R. M. McLoughlin, and K. H. Mills. 2013. Relative contribution of Th1 and Th17 cells in adaptive immunity to Bordetella pertussis: towards the rational design of an improved acellular pertussis vaccine. PLoS Pathog 9: e1003264. 6. Dunne, A., L. A. Mielke, A. C. Allen, C. E. Sutton, R. Higgs, C. C. Cunningham, S. C. Higgins, and K. H. Mills. 2015. A novel TLR2 agonist from Bordetella pertussis is a potent adjuvant that promotes protective immunity with an acellular pertussis vaccine. Mucosal Immunol 8: 607-617. 76 SS PROJECT NO. 16 Supervisor: Dr Joseph DeCourcey, Dr Mieszko Wilk and Prof Kingston Mills Title: THE ROLE OF EPITHELIAL CELLS IN THE IMMUNE RESPONSE TO BACTERIA AND THEIR PRODUCTS. Introduction: The mucosal surfaces in the body are lined with epithelial cells that are often the targets for binding of invading pathogens but also play a role in protective immunity to infection. They recognise bacteria or their product via pattern recognition receptors (PRRs) located on, or within the cell. PRRs recognise pathogen associated molecular patterns (PAMPs) which are highly conserved structures from the cell wall or nucleic acids of invading pathogens1, 2. Upon activation of PRRs, a number of signalling pathways are stimulated leading to activation of innate immune cells and secretion of cytokines and chemokines. These innate immune responses also direct the induction of the adaptive immune system which mediates clearance of the invading pathogen. During inflammation these pathways can become dysregulated leading to prolonged inflammation and inflammatory disease3. Bordetella pertussis is a Gram negative bacterium that infects the respiratory system. It is the cause of pertussis (whooping cough), which mainly affects young children. WHO estimated that in 2008, about 16 million cases of pertussis occurred worldwide, 95% of which were in developing countries, and that some 195 000 patients died from this disease4. B. pertussis binds to epithelial cells, infects the the cell and may also modulate the immune response in order to hinder its clearance. Our lab has shown that B. pertussis subvert immune responses by modulating activation of dendritic cells and macrophages and thereby subvert protective Th1 and Th17 responses that are required to eliminate the bacteria4. It has also been reported that tracheal cytotoxin (a product of bacterial peptidoglycan) is an agonist for the NOD-like receptor (NLR) NOD-1 and can bind to epithelial cells and enhance IL-1 and nitric oxide (NO) production, thereby causing damage to the epithelial lining of the respiratory tract5. We have also shown that synthetic NOD-1 agonists promote IL-10 by macrophages thereby exerting anti-inflammatory function (Unpublished). However, the effect of B. pertussis and the immunomodulatory effects of NOD-1 agonists on epithelial cells has received little attention. This project will investigate the effect of live versus heat killed B. pertussis on epithelial cells in order to investigate the possible role of these cells in the immune response and their modulation by B. pertussis. It will also assess the effects of synthetic PAMPs, such as the cell wall component lipopolysaccharide (LPS), CpG, lipoproteins or peptidoglycans and their derivatives, NOD-1 and NOD-2 agonists, on epithelial cell activation. Objectives: • Investigate the effect of live versus heat killed B. pertussis on cytokine and chemokine production by epithelial cells. • To examine the effect of TLR and NOD agonists on epithelial cell activation • To identify the PRR pathways in epithelial cells activated, or modulated by exposure to bacteria or their products 77 Methodology: The student will receive training in a range of immunology techniques, including culture of cell line and primary cells, ELISA, intracellular cytokine staining and flow cytometry, quantitative real time PCR and Western blotting. Likely outcome: This work will lead to a better understanding of the mechanisms by which B. pertussis is able to infect the host, modulate the immune response and persist in the respiratory tract. It could also generate vital new information on how NOD-like receptors may be involved in antinflammatory responses and can be exploited as therapeutics for inflammatory disorders. References: 1. De Nardo D. Toll-like receptors: Activation, signalling and transcriptional modulation. Cytokine. 2015 Aug;74(2):181-9. 2. Caruso R, Warner N, Inohara N, Núñez G. NOD1 and NOD2: signaling, host defense, and inflammatory disease. Immunity. 2014 Dec 18;41(6):898-908. 3. Mills KH. TLR-dependent T cell activation in autoimmunity. Nat Rev Immunol. 2011 Nov 18;11(12):807-22. 4. Higgs R, Higgins SC, Ross PJ, Mills KH. Immunity to the respiratory pathogen Bordetella pertussis. Mucosal Immunol. 2012 Sep;5(5):485-500. 5. Flak TA, Heiss LN, Engle JT, Goldman WE. Synergistic epithelial responses to endotoxin and a naturally occurring muramyl peptide. Infect Immun. 2000 Mar;68(3):123542. 78 SS PROJECT NO. 17 Supervisor: Dr Rachel McLoughlin Title: THE ROLE OF IL-22 IN REGULATING STAPHYLOCOCCUS AUREUS ADHERENCE TO HUMAN KERATINOCYTES. This project will investigate the regulatory role of the proinflammatory cytokine IL-22 during Staphylococcus aureus colonisation of the epithelium. S. aureus is an opportunistic pathogen that is responsible for a wide variety of diseases, ranging from superficial skin and soft tissue infections to invasive diseases such as endocarditis, osteomyelitis and sepsis. In addition to its invasive infective potential, S. aureus is commonly found as part of the normal human microflora and is carried persistently and asymptomatically by 20% of the population with the remainder colonised intermittently1. Colonisation is an important risk factor for subsequent infection particularly in a nosocomial setting and persistent carriers are most likely to be infected by their resident strain2. Due to the increasing prevalence of meticilin-resistant S. aureus (MRSA) carriage by at-risk individuals, novel therapies that activate or enhance the local host immune response to S. aureus in the nose are needed and may provide new strategies for patient decolonisation. S. aureus nasal colonisation is a multifactorial process with many host and bacterial factors implicated. Bacterial attachment to keratinocytes in the nasal epithelium is facilitated by a specific interaction between S. aureus and structural keratinocyte cell envelope proteins, most notably loricrin and cytokeratin 103, 4. As part of the host tissue healing response, the proinflammatory cytokine IL-22 can downregulate the expression of loricrin and cytokeratin 10 at the epithelium5, 6. Unpublished data from our lab indicates that IL-22 is expressed at the nasal epithelium during S. aureus nasal colonisation in mice and may mediate the downregulation of murine loricrin and cytokeratin 10 protein expression in vivo, facilitating a host defence mechanism whereby staphylococcal attachment to the nasal epithelium is limited. Based on these results, this project aims to investigate the direct ability of IL-22 to influence attachment of S. aureus to human keratinocytes in vitro. Specifically, this research project will: 1. Investigate the ability of S. aureus to adhere to human keratinocytes. In vitro cultures of the HaCaT human keratinocyte cell line will be established and co-cultured with fluorescently-labelled S. aureus. Bacterial adherence will be visualised using fluorescent microscopy and will quantified by enumerating colony forming units (CFU) per field of view and by measuring mean fluorescence. 2. Examine the effect of IL-22 on bacterial adherence. Bacterial adherence to HaCaT cells (as outlined in 1) will be assessed following incubation of HaCaT cells with increasing concentrations of recombinant IL-22. Changes in bacterial adherence will be determined visually using fluorescent microscopy and by measuring mean fluorescence. Cell culture supernatants will be collected and pro-inflammatory cytokine (IL6/IL-8) production quantified by ELISA. 79 3. Determine the availability of staphylococcal ligands in the presence of IL-22. HaCaT cells will be co-cultured with S. aureus with and without the presence of IL-22 (as outlined in 2). HaCaT cells will be collected and proteins extracted. The presence of loricrin and cytokeratin 10 proteins will be determined using Western immunoblotting and will be quantified using densitometry. References: 1. van Belkum A, Verkaik NJ, de Vogel CP, Boelens HA, Verveer J, Nouwen JL et al. Reclassification of Staphylococcus aureus nasal carriage types. The Journal of infectious diseases 2009; 199(12): 1820-1826. 80 SS PROJECT NO. 18 Supervisor: Dr Rachel McLoughlin Title: THE ROLE OF STAPHYLOCOCCUS AUREUS SURFACE PROTEINS IN ATTACHMENT AND IMMUNE ACTIVATION DURING SKIN INFECTION. This project will investigate the ability of Staphyloccocus aureus to activate the host innate immune system at the initial stages of skin infection. S. aureus is one of the leading causes of skin and soft tissue infections (SSTIs) globally. Meticilin-resistant S. aureus (MRSA) accounted for 59% of purulent SSTIs presented in emergency departments across North America in 20081. The host innate response is the first line of defence against staphylococcal infection in the skin. Antimicrobial peptides are expressed upon bacterial insult and promote bacterial elimination as well as amplifying the immune response by mediating proinflammatory cytokine production2. The incidence of skin infection relies on the ability of S. aureus to attach successfully to the epithelium and evade host innate immune defences. S. aureus expresses several multifunctional and functionally redundant surface proteins that act as virulence factors to mediate these processes at the onset of infection. These include clumping factor (Clf)A and ClfB which may facilitate adherence to matrix molecules in the epithelium such as fibrinogen, loricrin and cytokeratin3, 4, 5 and staphylococcal protein A (Spa) which plays an important role in staphylococcal immune evasion6. ClfA, ClfB and Spa are covalently linked to the bacterial cell wall in a process facilitated by the enzyme sortase A9. Recent evidence suggests that Spa can bind to tumor necrosis factor receptor-1 (TNFR1) on airway epithelial cells and inhibit the activity of TNF-α7. TNF-α has been shown to mediate the production of antimicrobial peptides and inflammatory factors in keratinocyte cultures in vitro8. This study aims to examine the role of staphylococcal surface proteins in attachment to the epithelial surface and activation of the innate immune response (specifically the production of antimicrobial peptides) at the onset of skin infection. Specifically, this project will: 1. Assess the adherence and virulence potential of mutant strains of MRSA to human keratinocytes. In vitro cultures of the HaCaT human keratinocyte cell line will be established and infected with bioluminescent MRSA USA300 strain LAC:lux and compared to mutants generated in this strain that are deficient in ClfA, ClfB, Spa and sortase A. Bacterial attachment will be compared and quantified visually using fluorescent microscopy and by measuring mean fluorescence. Keratinocyte cell survival will be determined by collecting cell culture supernatants and measuring LDH release. 2. Compare the ability of each strain to activate the kerainocyte innate immune response. Following infection with mutant MRSA strains (outlined in 1), infected HaCaT cells will be isolated and RNA extracted. RNA expression of antimicrobial peptides will be determined using quantitative real-time PCR. Cell culture supernatants will be collected and proinflammatory cytokine producion (IL-8, IL-6, TNFa) quantified by ELISA. 81 3. Investigate the virulence of mutant strains in an in vivo model of staphylococcal subcutaneous infection. Based on results obtained from 1 and 2, mutant MRSA strains that are defective in epithelial adherence and/or innate immune activation will be assessed in vivo in a murine model of subcutaneous skin infection and virulence will be compared to a WT strain. Bacterial burden will be measured by quantifying bacterial bioluminescence using a photon imager over the course of infection. References: 1. Edelsberg, J., C. Taneja, M. Zervos, N. Haque, C. Moore, K. Reyes, J. Spalding, J. Jiang, and G. Oster. 2009. Trends in US hospital admissions for skin and soft tissue infections. Emerging infectious diseases 15: 1516-1518. 2. Schauber J, Gallo RL. Antimicrobial peptides and the skin immune defense system. J Allergy Clin Immunol. 2008;122:261–266. 3. Mcdevitt, D., Nanavaty, T., House-Pompeo, K., Bell, E., Turner, N., Mcintire, L., Foster, T. and HööK, M. (1997), Characterization of the Interaction Between the Staphylococcus Aureus Clumping Factor (ClfA) and Fibrinogen. European Journal of Biochemistry, 247: 416–424. doi: 10.1111/j.1432-1033.1997.00416.x 4. Mulcahy ME, Geoghegan JA, Monk IR, O'Keeffe KM, Walsh EJ, Foster TJ et al. Nasal colonisation by Staphylococcus aureus depends upon clumping factor B binding to the squamous epithelial cell envelope protein loricrin. PLoS pathogens 2012; 8(12): e1003092. 5. O'Brien LM, Walsh EJ, Massey RC, Peacock SJ, Foster TJ. Staphylococcus aureus clumping factor B (ClfB) promotes adherence to human type I cytokeratin 10: implications for nasal colonization. Cellular microbiology 2002; 4(11): 759-770 6. Gemmell, C., Tree, R., Patel, A., O’Reilly, M., Foster, T. J. Susceptibility to opsonophagocytosis of protein A, α-haemolysin and β-toxin deficient mutants of Staphylococcus aureus isolated by allele-replacement. Zentralbl. Bakteriol. 21 (Suppl.), 273– 277 (1991). 7. Gomez, M. I., A. Lee, B. Reddy, A. Muir, G. Soong, A. Pitt, A. Cheung, and A. Prince. 2004. Staphylococcus aureus protein A induces airway epithelial inflammatory responses by activating TNFR1. Nat. Med. 10:842-848. 8. Guilloteau, K., Paris, I., Pedretti, N., Boniface, K., Juchaux, F., Huguier, V., Guillet, G., Bernard, F. X., Lecron, J. C., and Morel, F. (2010) Skin inflammation induced by the synergistic action of IL-17A, IL-22, oncostatin M, IL-1, and TNF-a recapitulates some features of psoriasis. J. Immunol. 184, 5263–5270 9. Mazmanian S. K., Liu G., Jensen E. R., Lenoy E. & Schneewind O. Staphylococcus aureus sortase mutants defective in the display of surface proteins and in the pathogenesis of animal infections. Proc. Natl. Acad. Sci. U. S. A. 97, 5510–5515 (2000). 82 SS PROJECT NO. 19 Supervisor: Dr. Nigel Stevenson Title: DOES HEPATITIS B VIRUS (HBV) ENHANCE ANTI-VIRAL RESPONSES VIA THE IFN-Α JAK/STAT PATHWAY? Summary: Hepatitis B Virus (HBV) infects the liver and is a major global health problem, with ~360 million people chronically infected worldwide and >1 million dying from associated liver disease per year [1]. The cytokine interferon-alpha (IFN-α) signals via the Janus kinase/Signal transducer and activator of transcription (JAK/STAT) pathway, leading to the upregulation of numerous anti-viral mediators [2]. Interestingly, it is estimated that up to 95% of patients clear HBV naturally, for reasons that remain undetermined. We have found that several viruses, including Respiratory syncytial virus (RSV) [3] and HCV [4] block the IFN pathway by targeting STAT proteins for degradation via the proteasome, but our preliminary discoveries indicate that HBV actually enhances STAT signalling, which may explain why so many patients spontaneously clear infection. Therefore, to further investigate the effect of HBV upon anti-viral responses this project will analyse the effect of specific HBV proteins upon IFN-α-mediated anti-viral gene induction and the activation of the IFN-stimulated response element (ISRE). Techniques: 1) Tissue culture, 2) Transfection 3) Real-Time-PCR, 4) Reporter gene assays. References: [1] Thimme R, Dandri M. Dissecting the divergent effects of interferon-alpha on immune cells: time to rethink combination therapy in chronic hepatitis B? J Hepatol. 2013 Feb;58(2):205-9. doi: 10.1016/j.jhep.2012.11.007. Epub 2012 Nov 14. [2] Horner S, Gale MJ (2009) Intracellular innate immune cascades and interferon defenses that control hepatitis C virus. J Interferon CytokineRes29:489-498. [3] Elliott J, Lynch OT, Suessmuth Y, Qian P, Boyd CR ,Burrows JF, Buick R, Stevenson NJ, Touzelet O, Gadina M, Power UF, and Johnston JA. Respiratory Syncytial Virus NS1 Protein Degrades STAT2 by Using the Elongin-Cullin E3 Ligase. J Virol, 2007; 81 (7): 3428–3436 [4] Stevenson NJ, Bourke NM, Ryan EJ, Binder M, Fanning L, Johnston JA, Hegarty JE, Long A, and O’Farrelly C. Hepatitis C Virus targets the Interferon-α JAK/STAT pathway by promoting proteosomal degradation in immune cells and hepatocytes. FEBS Lett. 2013; 587(10):1571-8. 83 SS PROJECT NO. 20 Supervisor: Dr. Nigel Stevenson Title: DOES TRIAD3A PLAY A ROLE IN HIV-VIF-MEDIATED STAT DEGRADATION? Summary: HIV is a major world health problem, affecting more than 40 million people worldwide. Although anti-retroviral drugs are used to control the spread of the virus there is currently no cure for the disease. The anti-viral cytokine, Interferon (IFN)-α signals via the Janus kinase/Signal transducer and activator of transcription (JAK/STAT) pathway, leading to the production of anti-viral mediators, such as protein kinase R (PKR), OAS, MxA and MxB, which disrupt viral replication [1]. Even though IFN-α is induced upon HIV infection it is not understood why this natural anti-viral drug fails to clear the virus. We have found that several viruses, including Respiratory syncytial virus (RSV) [2] and Hepatitis C Virus (HCV) [3] block the IFN pathway by targeting STAT proteins for ubiquitination and subsequent degradation by the proteasome. Our preliminary investigations indicate that HIV protein Vif is also targeting STAT proteins for degradation. TRIAD3A is a ubiquitin ligase which has a role in innate immune signaling pathways and has been shown to interact with HIV Vif [4]. TRIAD3A has been shown to ubiquitinate TRAF3, TLR4 and TLR9 and promote their proteasomal degradation [5]. To determine the mechanism by which Vif degrades STAT proteins we will investigate if TRIAD3A plays a role. This will be achieved by investigating if over-expression of TRIAD3A can affect Vif-mediated STAT degradation. In parallel to the over-expression studies, STAT degradation by Vif will be investigated in cells where endogenous TRIAD3A expression is suppressed by shRNA. Finally, the effect of Vif and TRIAD3A on downstream IFN stimulated genes (ISGs) will be assessed by quantitative real time PCR. Techniques: 1) Tissue culture, 2) Transfection, 3) Western blotting and 4) Real-Time-PCR. References: [1] Horner S, Gale MJ (2009) Intracellular innate immune cascades and interferon defenses that control hepatitis C virus. J Interferon CytokineRes29:489-498. [2] Elliott J, Lynch OT, Suessmuth Y, Qian P, Boyd CR ,Burrows JF, Buick R, Stevenson NJ, Touzelet O, Gadina M, Power UF, and Johnston JA. Respiratory Syncytial Virus NS1 Protein Degrades STAT2 by Using the Elongin-Cullin E3 Ligase. J Virol, 2007; 81 (7): 3428–3436 [3] Stevenson NJ, Bourke NM, Ryan EJ, Binder M, Fanning L, Johnston JA, Hegarty JE, Long A, and O’Farrelly C. Hepatitis C Virus targets the Interferon-α JAK/STAT pathway by promoting proteosomal degradation in immune cells and hepatocytes. FEBS Lett. 2013; 587(10):1571-8. [4] Feng F., Davis A., Lake JA., Carr J., Xia W., Burrell C., Li P. Ring finger protein ZIN interacts with human immunodeficiency virus type 1 Vif. J. Virol. 2004. 78(19):10574-81 84 [5] Nakhaei P1, Mesplede T, Solis M, Sun Q, Zhao T, Yang L, Chuang TH, Ware CF, Lin R, Hiscott J. The E3 ubiquitin ligase Triad3A negatively regulates the RIG-I/MAVS signaling pathway by targeting TRAF3 for degradation. 2009. PLoS. Pathog. 85
