Download Immunology Course Booket 2016/17

UNIVERSITY OF DUBLIN
TRINITY COLLEGE DUBLIN
SCHOOL OF BIOCHEMISTRY &
IMMUNOLOGY
SENIOR SOPHISTER
IMMUNOLOGY
COURSE DETAILS
2016-2017
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.
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NOTE: Learning outcomes for each of the modules can be found in
blackboard (http://mymodule.tcd.ie) and also accessed through the school
homepage:
http://www.tcd.ie/Biochemistry/undergraduate/teach_immunol.php
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/vpcao/academic-development/ects.php
Examinations and Breakdown of Marks:
Senior Sophister Module Name
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
ECTS Weighting
BI4275
5 ECTS
BI4055
5 ECTS
BI4215
5 ECTS
BI4045
5 ECTS
BI4235
5 ECTS
BI4245
5 ECTS
BI4255
5 ECTS
BI4265
5 ECTS
BI4295
15 ECTS
BI4015
5 ECTS
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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.
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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. 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 during the year. You will be assigned to a pair of academic
staff members. 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. He/she 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
tutorial session (e.g. Prb 1 Tutorial). You must be able to demonstrate that you have
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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 Biochemistry students, on Tuesday
4th 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
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.
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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 31st
October in FRED). It is advisable to arrange at least one practise talk with your
project supervisor.
Project laboratory work will start on November 14th and terminate on the 24th
of February. After the end of laboratory work, you will be given approximately 3
weeks to present a thesis on your project. 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 4.00 pm on Monday 20th 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 represents 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 informed 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 Prof Ed Lavelle (4th floor TBSI, phone
extension 2488, email [email protected]). The names of tutors are provided on a
separate sheet. A complete list of the Staff in the school and their research interests
can be found at http://www.tcd.ie/Biochemistry/staff
Health and Safety Management:
1) Registration with Safety Officer
Preliminary safety registration takes place during one mandatory health and safety
briefing session timetabled in Week 1 (see timetable). Later on you must register, in
person, with the Safety Officer after you have been assigned your project. This is
necessary in order to record your next-of-kin details in the unlikely 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, cytotoxics, 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.
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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 10
a.m. on Friday 30th September. Dr Nic a’ Bháird will give another more detailed
Health and Safety workshop 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 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
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 Friday 8th (9:30am-1:00pm) January 2014.
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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’s policy on plagiarism (see Calendar, General
Regulations and Information, §82-§91 at http://tcd-ie.libguides.com/plagiarism are
reproduced below. 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). During your final year you will be expected to
prepare material for the Biochemical Techniques course and to write a report on the
research findings of your fourth year project. You will be provided with guidance
notes for the completion of these exercises. In the first and second semester, Prof.
Kingston Mills will give a tutorial class on how to prepare and write a report for your
research project.
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It is a college requirement that all students must complete an online tutorial on
avoiding
plagiarism
‘Ready,
Steady,
Write’,
located
at
http://tcdie.libguides.com/plagiarism/ready-steady-write.
In addition, students must complete cover sheets or include text containing
the following declaration when submitting assessed work in hard or soft copy
or via Blackboard:
I have read and I understand the plagiarism provisions in the General
Regulations of the University Calendar for the current year, found
at: http://www.tcd.ie/calendar
I have also completed the Online Tutorial on avoiding plagiarism ‘Ready,
Steady, Write’, located at http://tcd-ie.libguides.com/plagiarism/readysteady-write
§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.
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Examples (d) and (e) in particular can arise through careless thinking and/or
methodology where students:
(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://tcd-ie.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 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.
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§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.
§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.
§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.
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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.
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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
Criteria
Range
Exceptional project report showing broad understanding of the
project area and excellent knowledge of the relevant literature.
85Exemplary presentation and analysis of results, logical
100
organisation and ability to critically evaluate and discuss results
coupled with insight and originality.
I
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
70-84
indication of some insight and originality. A very competent and
well presented report overall but falling short of excellence in
each and every aspect.
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
II-1
60-69
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
II-2
50-59
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
III
40-49
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.
15
20-39
Fail
0-19
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 2016
16
Immunology: Breakdown of SS Papers I and II 2016-2017
Paper I
Section 1: BI4275 ‘Cells of the innate and adaptive immune system’
Myeloid cells (EC)
Natural killer cells (CG)
T cells (CF- 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 (NMW - 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)
17
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)
2 questions
Viral evasion of innate and adaptive immunity (AB)
Antimicrobial resistance and host response to infection (SL- RMcL)
Immune response to tuberculosis (JK)
Vaccines, adjuvants and danger hypothesis (EL)
Section 3: BI4225 ‘Immunogenetics, immunomodulation and
immunotherapy
Transgenics and animal models of disease (VK)
2 questions
Immunogenetics (RMcM)
Immunotherapy and transplantation (COF)
Section 4: BI4265 ‘Tumour Immunology’
Initiation & Progression (VK)
Metastasis & Treatment (VK/CF-KM)
Cellular imaging (DN)
2 questions
Answer one question from each section (4 OUT OF 8)
18
SS Course Summaries: 2016-2017
BI4275
(5ECTS)
CELLS OF THE INNATE AND ADAPTIVE IMMUNE SYSTEM
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- and LT). Benefits of these; NK cells as effectors of graftrejection, 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.
19
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 and pathogen killing. In addition mechanisms by which these
pathways are regulated will also be described.
20
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.
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
21
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.
(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
22
Helminths of Human Importance (4 lectures) Padraic Fallon
A third of the world’s population is infected with parasitic worms.
address the major parasitic worms that are of medical importance.
Lecture 1-2:
Introduction to the major helminth parasites that infect man.
impact of helminth parasites on society.
These lectures will
Medical and economic
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 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
re-emerging 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.
23
Helicobacter pylori: gastric cancer and beyond. Polk DB, Peek RM
(2010)10(6):403-14.
BI4245
Nat Rev Cancer.
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 viralhost 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.
24
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
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
phago-lysosomal 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,
Schistosomiasis
b. What holds it together? TNF, IFN gamma, somatostatin
Wegeners
and
25
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
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.
26
BI4045
AUTOIMMUNE AND INFLAMMATORY CONDITIONS (5 ECTS)
Biochemistry
Bloomfield)
of
the
Inflammatory
Process
(4
lectures)
Dr
Jack
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 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
anti-inflammatory drugs on prostaglandin and leukotriene production. Pathways
of prostaglandin and leukotriene production.
Rheumatoid Arthritis (2 lectures) Luke O’Neill
Lecture 1: What
definitions.
Early
Rheumatoid Factor.
Autoantigens. Role
enzymes.
is rheumatoid arthritis? Clinical, molecular and cellular
concepts: connective tissue structure and degradation.
And B cells. HLA associations and the genetic component.
of inflammation – prostaglandins and tissue degrading
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
27
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
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
28
mediated by antibodies, such as systemic lupus erythematosus, myasthenia
gravis and autoimmune vasculitis.
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). Post-translational modification of Bcl-2 family: 2 types- proteolysis and
phophorylation. Example of each type. Regulation of cell survival by Akt pathway.
29
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.
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.
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
30
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
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
(DKF)
(5 lectures)
Dr David Finlay
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.
31
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)
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.
32
BI4255
IMMUNOGENETICS,
IMMUNOTHERAPY (5 ECTS)
IMMUNOMODULATION
AND
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-ofthe-art 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
33
Inherited diseases cover a spectrum of disorders and can result from mutations in
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,
therapies) immunosuppressive agents (FK506, cyclosporine A).
personalised
34
Allo recognition; the major histocompatibility complex; NK cells; T cells; bone
marrow, liver, kidney transplantation
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
35
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.
36
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
37
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 two-hit 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
38
given along with 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 - anti-inflammatory 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
Lecture 1: Introduction to imaging and the concept of resolution. Application of
electron microscopy in cell imaging.
39
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.
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
40
*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.
#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.
41
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
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.
42
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
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
Wolefensohn,
M. Lloyd
Introduction to laboratory animal science and technology
Humane experimental technique
Burch
Experimental and surgical technique in the rat
Wayneforth,
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
J. Wagner
A. A. Tuffery
S.
J. Inglis
W. Russell, R.
H.
M. Balls, J. Bridges,
J. Southee
S. Cox Gad
T. Poole
P. Flecknell
K Laber-Laird,
J. Harkness
43
The laboratory animals, principles and practice
Petter,
A. Pearson
Man and mouse, animals in medical research
Lives in the balance;
Boyd
the ethics of using animals in biomedical research
Vivisection in historical prospective
W. LaneW. Paton
J. Smith,K.
R. Rupke
44
SENIOR SOPHISTER PROJECTS
2016-2017
45
SS PROJECT NO.1
TITLE: INVESTIGATING THE ROLE OF SOCS4 IN PROINFLAMMATORY
CYTOKINE SIGNALLING.
SUPERVISOR: DR. NIGEL STEVENSON
Summary: Cytokine receptor binding triggers intracellular signal transduction. Many
cytokines signal via the Janus Kinase/Signal Transducer and Activator of Transcription
(JAK/STAT) pathway, which induces a wide range of infammatory genes. To ensure an
appropriate response to infection the JAK/STAT pathway is regulated via the induction of
regulatory proteins called Suppressors Of Cytokine Signaling (SOCS) [1]. The SOCS family
is composed of eight members, SOCS1 to 7 and cytokine-inducible Src-homology 2 protein
(CIS) [2]. The role of CIS and SOCS1-3 in immune signalling has been well characterised,
however there is much less data regarding the targets and pathways regulated by SOCS4-7.
SOCS4 is involved in regulating epidermal growth factor (EGF) signalling [3] and mice
lacking SOCS4 have increased susceptibility to Influenza virus infection. These mice also
have elevated proinflammatory cytokine expression, strongly indicating a role for SOCS4 in
more that just EGF signalling responses [4].
Project aim: This projects aims to determine if SOCS4 protein regulates responses to the key
inflammatory cytokine, IL-6.
1) SOCS4, SOCS3 and empty vector control plasmids will be transformed into bacteria,
selected for amplification and purified. 2) Basal SOCS4 protein levels will be determined in
Huh7 cells. 3) Cells will be transfected with SOCS4, SOCS3 or empty vector control plasmids
and overexpression will be confirmed by Immunoblotting. 4) Cells (-/+ SOCS4) will be
treated with IL-6 for 20min, before pSTAT3, STAT3 and B-actin protein levels will be
measured by Immunoblotting and densitometry. 5) Cells (-/+ SOCS4) will be treated with IL6 for 0, 4, 6h, before SOCS3 protein levels will be measured by Immunoblotting. (SOCS3
overexpression lysates will be used as positive controls) 6) Cells (-/+ SOCS4) will be treated
with IL-6 for 18h before supernatants are measured for CXCL10 by ELISA.
Techniques: 1) Bacterial transformation; 2) DNA construct purification; 3) Tissue Culture; 4)
Transfection; 5) Cytokine Stimulation; 6) Protein Analysis by Immunoblotting and
Densitometry and 7) ELISA.
References:
[1] Alexander WS (2002) Suppressors of cytokine signalling (SOCS) in the immune
system.Nat Rev Immunol 2: 410–416
[2] Hilton DJ, Richardson RT, Alexander WS, Viney EM, Willson TA, et al. (1998) Twenty
proteins containing a C-terminal SOCS box form five structural classes. Proc Natl Acad Sci U
S A 95: 114–119
[3] Kario E, Marmor MD, Adamsky K, Citri A, Amit I, et al. (2005) Suppressors of cytokine
signaling 4 and 5 regulate epidermal growth factor receptor signaling. J Biol Chem 280: 7038–
7048
46
[4] Kedzierski L, Clemens EB, Bird NL, Kile BT, Belz GT, et al. SOCS4 is dispensable for an
efficient recall response to influenza despite being required for primary immunity. Immunol
Cell Biol. 2015 Nov;93(10):909-13
47
SS PROJECT NO.2
TITLE: IS SOCS4 AN INHIBITOR OF ANTI-VIRAL TYPE 1 INTERFERON
SIGNALLING?
SUPERVISOR: DR. NIGEL STEVENSON
Summary: Cytokine receptor binding triggers intracellular signal transduction. Many
cytokines signal via the Janus Kinase/Signal Transducer and Activator of Transcription
(JAK/STAT) pathway, which induces a wide range of infammatory genes. To ensure an
appropriate response to infection the JAK/STAT pathway is regulated via the induction of
regulatory proteins called Suppressors Of Cytokine Signaling (SOCS) [1]. The SOCS family
is composed of eight members, SOCS1 to 7 and cytokine-inducible Src-homology 2 protein
(CIS) [2]. The role of CIS and SOCS1-3 in immune signalling has been well characterised,
however there is much less data regarding the targets and pathways regulated by SOCS4-7.
Mice lacking SOCS4 become rapidly infected with H1N1 influenza virus and are
hypersusceptible to infection with less virulent H3N2 influenza virus, indicating that SOCS4
is a critical regulator of anti-viral immunity [3].
Project aim: This projects aims to determine if SOCS4 protein regulates responses to the key
anti-viral cytokines, IFN-.
1) SOCS4, APOBEC3G and MxB and empty vector control plasmids will be transformed into
bacteria, selected for amplification and purified. 2) Basal SOCS4 protein levels will be
determined in Huh7 cells. 3) Cells will be transfected with SOCS4 or empty vector control
plasmids and overexpression will be confirmed by Immunoblotting. 4) Cells (-/+ SOCS4) will
be treated with IFN- for 20min, before pSTAT1, STAT1 and B-actin protein levels will be
measured by Immunoblotting and densitometry. 5) Cells (-/+ SOCS4) will be treated with
IFN- for 0, 4 and 6h, before APOBEC3G and MxB protein levels will be measured by
Immunoblotting (APOBEC3G and MxB overexpression lysates will be used as positive
controls). 6) Cells (-/+ SOCS4) will be treated with IFN- for 18h before supernatants are
measured for CXCL10 by ELISA.
Techniques: 1) Bacterial transformation; 2) DNA construct purification; 3) Tissue Culture; 4)
Transfection; 5) Cytokine Stimulation; 6) Protein Analysis by Immunoblotting and
Densitometry and 7) ELISA.
References:
[1] Alexander WS (2002) Suppressors of cytokine signalling (SOCS) in the immune
system.Nat Rev Immunol 2: 410–416
[2] Hilton DJ, Richardson RT, Alexander WS, Viney EM, Willson TA, et al. (1998) Twenty
proteins containing a C-terminal SOCS box form five structural classes. Proc Natl Acad Sci U
S A 95: 114–119
[3] Kedzierski L, Clemens EB, Bird NL, Kile BT, Belz GT, et al. SOCS4 is dispensable for an
efficient recall response to influenza despite being required for primary immunity. Immunol
Cell Biol. 2015 Nov;93(10):909-13
48
SS PROJECT NO.3
TITLE: NK CELL IMMUNOMETABOLISM: IMPACT FOR CANCER THERAPY
SUPERVISORS: CLAIR M. GARDINER AND ELENA WOODS
Natural Killer (NK) cells are lymphocytes that kill cancer cells and virally infected
cells. In terms of cancer treatments, NK cells can directly kill tumour cells and can be
activated ex vivo before infusion into patients. However, there is also a form of
immunotherapy based on identification of tumour cells using a specific antibody which NK
cells will then kill by a process of antibody dependent cellular cytotoxicity (ADCC). These
include anti-CD20 for lymphoma and anti-GD2 for neuroblastoma treatment (1).
We have recently demonstrated that human NK cells change their metabolism in
response to activating signals (2, 3). In particular, they upregulate both glycolysis and
oxidative phosphorylation (oxphos). We demonstrated that metabolism can impact on NK cell
function as interfering with e.g. glycolysis, impaired cytokine production by NK cells. We
have also shown defects in NK cell degranulation in response to inhibitors of oxphos.
However, degranulation is a surrogate for cytotoxicity and we wish to directly test if
interfering with glucose metabolism impacts on NK cell killing of tumour cells.
In this study, we will set up an assay using tumour target cells that allows
measurement of both direct cytotoxicity and ADCC by NK cells. These NK cells will be
activated by cytokines in the presence of absence of pharmacological modulators of
metabolism. We will measure both degranulation (CD107a) and cytotoxicity of these cells
and identify the contribution of metabolism to NK cell kiling of tumour cells.
Objectives:
o Using PBMC (from buffy coats and/or healthy donors), we will stimulate NK cells
with cytokines (IL2 or IL12/15) for various times (between 1-5 days) and optimise and
validate the direct cytotoxicity assay.
o We will use pharmacological agents e.g. oligomycin (to inhibit oxphos and drive
glycolysis), or 2-DG, limiting doses of glucose or culture in galactose (instead of
glucose) to investigate a potential role for these metabolic pathways in NK cell killing.
o Using PBMC (from buffy coats and/or healthy donors), we will stimulate NK cells
with cytokines (IL2 or IL12/15) and optimise and validate the ADCC cytotoxicity
assay using Daudi cells and anti-CD20 antibody.
o As above, we will use pharmacological agents to investigate a potential role for
glucose metabolism pathways in NK cell killing by ADCC.
Likely Outcome:
These data will likely demonstrate that NK cell cytotoxicity is impacted by NK cell
glucose metabolism. This may important implications for NK cells that are using in
immunotherapy regimens for the treatment of cancer.
References
49
1.
2.
3.
Wang, W., A. K. Erbe, J. A. Hank, Z. S. Morris, and P. M. Sondel. 2015. NK CellMediated Antibody-Dependent Cellular Cytotoxicity in Cancer Immunotherapy. Front
Immunol 6: 368.
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.
Keating, S. E., V. Zaiatz-Bittencourt, R. M. Loftus, C. Keane, K. Brennan, D. K.
Finlay, and C. M. Gardiner. 2016. Metabolic Reprogramming Supports IFN-gamma
Production by CD56bright NK Cells. Journal of immunology 196: 2552-2560.
50
SS PROJECT NO.4
TITLE: METABOLIC REGULATION OF NK CELL KILLING: SREBP TAKES
CENTRE STAGE
SUPERVISORS: CLAIR M. GARDINER, DAVID K. FINLAY, KATIE O’BRIEN AND
VANESSA ZAIATZ-BITTENCOURT
Natural Killer (NK) cells are lymphocytes that have important immune functions
including killing of cancer cells and virally infected cells. They use a number of effector
mechanisms for this including cytotoxicity and cytokine production (IFN).
We have recently demonstrated that NK cells change their metabolism in response to
activating signals (1, 2). In particular, they upregulate both glycolysis and oxidative
phosphorylation (oxphos) in response to cytokine. We have additional data (unpublished)
from murine NK cells that shows that SREBP, a transcription factor generally associated with
lipid metabolism, is important for NK cell glucose metabolism and effector function.
In this study, we will investigate if SREBP is important for human NK cell functions.
We will treat cytokine activated NK cells with inhibitors of SREBP (including 25hydroxycholesterol, cholesterol and PF429242) and investigate the effects on NK cell killing
and cytokine production.
Objectives:
o Using PBMC (from buffy coats and/or healthy donors), we will stimulate NK cells
with cytokines (IL2 or IL12/15) for various times (between 1-5 days) and optimise and
validate the cytotoxicity assay (fluorescent based), CD107a degranulation assay (by
flow cytometry) and IFN production assays (by flow cytometry).
o We will culture NK cells (in PBMC) in the presence of SREBP inhibitors and
investigate their impact on NK cell killing. As part of this, we will perform dose
response curves for the various inhibitors.
o We will also measure the impact of these inhibitors on IFN production by NK cells.
Likely Outcome:
These data will likely demonstrate that NK cell cytotoxicity and IFN production are
impacted by SREBP controlled processes. This will provide important information regarding
metabolic regulation of NK cell functions against tumour cells.
References
1.
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.
Keating, S. E., V. Zaiatz-Bittencourt, R. M. Loftus, C. Keane, K. Brennan, D. K.
Finlay, and C. M. Gardiner. 2016. Metabolic Reprogramming Supports IFN-gamma
Production by CD56bright NK Cells. Journal of immunology 196: 2552-2560.
51
SS PROJECT NO.5
TITLE: THE ROLE OF IL-17 IN INFLAMMASOME ACTIVATION AND IL-1Β
PRODUCTION
SUPERVISORS: DR CAROLINE SUTTON, AOIFE MCGINLEY AND PROF KINGSTON
MILLS
Introduction:
IL-17 production by Th17 and γδ T cells has been shown to be important for the control of
infections but can also be pathogenic in autoimmune diseases. We have reported that IL-1 and
IL-23 synergise to promote IL-17 production from Th17 and γδ T cells (1). However, we also
found that IL-17 can enhance TLR-induced IL-1 from bone marrow derived dendritic cells
(1), indicating a “feedback loop” for enhancement of IL-17 production. Mature IL-1β is
produced by innate immune cells following cleavage of pro-IL-1β by caspase-1 and the
NLRP3 inflammasome in response to TLR agonists (signal 1) and danger signals from ATP or
bacterial toxins (signal 2) [1]. It has recently been reported that IL-17 can induce
inflammasome activation in retinal epithelia cells [2] and it has also been suggested that CD3
T cells can produce IL-1β.
Multiple sclerosis (MS) is a debilitating autoimmune disease of the central nervous system.
Various T cell subsets have been implicated in pathogenesis of MS, including Th1 and Th17
cells, γδ T cells, as well as the cytokines IFN-γ and IL-17 [3] [4] [5]. However in the mouse
model, experimental autoimmune encephalomyelitis (EAE), we have shown that there is a
temporal role for IL-17 in the development of disease. Using the adoptive transfer model of
EAE we have shown that transfer of T cells from IL-17A-defetive mice can induce disease in
recipient wild-type mice following in vitro activation with autoantigen in the presence of IL1β and IL-23 (A McGinley, unpublished observation), suggesting that induction of IL-1β by
IL-17 may be crucial for the development of EAE. This project will examine the hypothesis
that IL-17 can induce IL-1 production, possibly by T cells, through inflammasome activation,
thus potentially enable better targeting of IL-17 or IL-1-based therapeutics for autoimmune
diseases.
Objectives:
1. To identify which cells (innate cells, T cells and endothelial cells) express the
receptor for IL-17 (IL-17R).
2. To isolate IL-17R-expressing cells (T cells, innate immune cells or epithelial cells)
and to examine IL-1 production following stimulate with IL-17 with or without
TNF-α and/or TLR stimulation.
3. To examine the hypothesis that IL-17 induces IL-1 production by caspase-1 and
inflammasome activation in T cells or other immune cell types, and if so by
providing which signal (signal 1 or signal 2).
Methodology:
52
FACS analysis and real time PCR will be used to examine IL-17R expression on various cell
subsets. IL-17R expressing populations will be isolated by FACS sorting and stimulated ex
vivo with IL-17. ELISA, Western blot and RT-PCR will be used to quantify IL-1 production
from each population. Caspase-1 activation will be assessed by FACS (FLICA assay) and
Western blotting and inflammasome activation will be determine using specific inhibitors of
NLRP3 and using cells from NLRP3 knockout mice.
Likely Outcome:
This project will help to elucidate the possible role of IL-17 in driving IL-1 production from
both innate and adaptive sources. It could provide crucial data to help understand the
pathogenic role of IL-17 at the early stage autoimmune disease and to identify new cellular
targets for IL-17-based drugs.
References:
1.
2.
3.
4.
5.
Strowig, T., et al., Inflammasomes in health and disease. Nature, 2012. 481(7381): p.
278-286.
Zhang, S., et al., Interleukin-17A Induces IL-1beta Secretion From RPE Cells Via the
NLRP3 Inflammasome. Invest Ophthalmol Vis Sci, 2016. 57(2): p. 312-9.
Sutton, C.E., et al., Interleukin-1 and IL-23 induce innate IL-17 production from
gammadelta T cells, amplifying Th17 responses and autoimmunity. Immunity, 2009.
31(2): p. 331-41.
Sutton, C., et al., A crucial role for interleukin (IL)-1 in the induction of IL-17producing T cells that mediate autoimmune encephalomyelitis. J Exp Med, 2006.
203(7): p. 1685-91.
Kroenke, M.A., et al., IL-12- and IL-23-modulated T cells induce distinct types of EAE
based on histology, CNS chemokine profile, and response to cytokine inhibition. J Exp
Med, 2008. 205(7): p. 1535-41.
53
SS PROJECT NO.6
TITLE: CHARACTERIZATION OF T CELL DIFFERENTIATION THROUGH
MODULATION OF INNATE CELLS ACTIVATION
SUPERVISORS: DR MATHILDE RAVERDEAU AND PROF KINGSTON MILLS
Introduction:
Cells of the innate immune system, especially dendritic cells and macrophages play a major role in
directing the adaptive immune response, largely through the secretion of T cells polarizing cytokines
in response to pathogen-associated molecular patterns (PAMPs) and other environmental stimuli (13). For example, M1 or classically activated macrophages are activated by TLR agonists, such as
LPS, in association with IFN-γ, and express highly levels of pro-inflammatory cytokines, reactive
nitrogen and oxygen species, which induce the differentiation of Th1 cells (4). Th1 cells are important
mediators of inflammation and host immunity to infection with bacteria and viruses. On the other
hand M2 or alternatively activated macrophage are activated by IL-4, IL-10 and IL-13 and express
low levels of pro-inflammatory cytokines but high levels of IL-10 (4). M2 macrophages are involved
in type 2 immune responses to parasitic infection and also play a role in maintaining immune
homeostasis, controlling inflammation and preventing pathogen-induced immunopathology (5).
While combinations of PAMPs and cytokines required for the activation of innate cells to induce Th1
and Th2 cells are well defined, the stimuli necessary for the differentiation of Th17 cells and
regulatory T cells (Treg) is less clear. We have shown that retinoic acid (RA) selectively activates
Treg, while inhibiting IL-17 production from Th17 and γδ T cells (6 and unpublished data) The goal
of this project is to examine the pathways involved in activating innate cells that promote or modulate
differentiation of naive T cells into Th1, Th2, Th17 and Treg cells. We will study the effect of TLR
agonists, cytokines on polarization of macrophages and dendritic cells (DCs) to type1 or type 2
phenotypes and how these cells, together with RA, can direct the differentiation of T cell subtypes.
Objectives:
 To examine the if M1 and M2 macrophage polarizing signals can promote activation of
equivalent phenotype in dendritic cells (DC1 and DC2)
 To examine the effect (activating or inhibitory) of M1/M2 macrophages and DC1/DC2 on
induction of Th1, Th2, Th17 or Treg cells
 To examine the direct versus indirect (via innate immune cells) of environmental factors (e.g.
RA) on T cell polarization
Methodology:
A range of state-of-the art molecular and cellular techniques will be used in the project, including cell
culture of primary macrophages and DCs cells and co-culture with T cells. The effect of different
TLR agonists and/or polarizing cytokines on the phenotype of innate cells and the subsequent
polarization of T cells will be examined by flow cytometry and RT-PCR. The cytokine production by
T cells and innate cells following stimulation or co-culture will be measured by ELISA. The pathways
54
and molecular intermediates identified in cells from the innate immune system and involved in T cells
differentiation will be tested using agonists, antagonists and/or blocking antibodies.
Likely outcome:
The production of the inflammatory cytokines IL-17 by Th17 cells and IFN-γ by Th1 cells are known
to play a major role in the pathology of many autoimmune and chronic inflammatory diseases,
including multiple sclerosis, psoriasis and Crohn’s disease (7-8). In contrast, the production of antiinflammatory cytokines such as IL-10 by Treg cells can dampen inflammation and is thus considered
a valuable immunotherapeutic approach for the treatment of autoimmune and chronic inflammatory
diseases. Therefore identifying the pathways in innate cells as well as environmental influences on the
differentiation of T cells towards pro-inflammatory Th1 and Th17 or anti-inflammatory Th2 and Treg
cells is an important step in a better understanding of the mechanisms involved in the development
and control of inflammatory diseases. Research from this project will give us a better understanding
of the roles innate immune cells in the differentiation of T cells and their potential use in the treatment
of autoimmune and chronic inflammatory diseases.
References:
1.
2.
3.
4.
5.
6.
7.
8.
Walsh KP and Mills KH, (2013). Trends Immunol. 34(11):521-530.
Pennock, N.D. et al., (2013). Adv Physiol Educ. 37(4):273-283.
Zhu J et al., (2010). Annu Rev Immunol. 28:445-489.
Wang et al., (2014). Front Immunol. 5: 614.
Biswas SK, Mantovani A (2010). Nat Immunol. 11(10):889-896.
Raverdeau M et al., (2016). Immunol Cell Biol. 2016 May 10. [Epub ahead of print]
Damsker, JM et al., (2010), Ann N Y Acad Sci. 1183: 211–221.
Mills KH, (2011). Nature Reviews Immunology. 11(12):807-22
55
SS PROJECT NO.7
TITLE: THE ROLE OF NOD1/2 AGONISTS IN INNATE IMMUNE TRAINING
SUPERVISORS: DR MARK LYNCH AND PROF KINGSTON MILLS
Introduction: NOD-like receptors (NLRs) are intracellular pattern recognition receptors that
recognise pattern- and danger-associated molecular patterns. NOD1 and NOD2 are a class of
NLR responsible for recognising bacterial cell wall components, especially derivatives of
peptidoglycans, such as muramyl dipeptide (MDP)1. Despite their role in inflammation,
NOD2 has been shown to play a protective role in Crohn’s Disease, with mutations in NOD2
associated with increased disease severity2. Furthermore, we have shown a protective role for
NOD1/2 agonists (NODa) in experimental autoimmune encephalomyelitis (EAE), a mouse
model for multiple sclerosis. This was associated with suppression of pathogenic Th17 cells.
Despite the observed protective effects of NODa in autoimmune diseases, little is known
about the functional mechanism that provides protection. We have shown that NOD1 agonists
can enhance anti-inflammatory IL-10 production by innate immune cells. Furthermore, we
have evidence that the anti-inflammatory effect of NODa may be mediated by innate immune
training. This concept describes a form of innate “memory”, whereby innate immune cells can
confer memory traits through epigenetic changes as a result of previous encountered
challenges3,4. This process is thought to be distinct from LPS-induced tolerance, where
multiple stimulations of LPS can downregulate pro-inflammatory gene expression5.
This project will utilise a panel of novel NOD1/2 agonists, examining their ability to alter
gene expression under different treatment regimes. The ability of NODa to induce discrete
tolerised and trained states will be examined in bone marrow-derived macrophages (BMDM),
with gene expression and cytokine secretion as the main readout. The function of trained
versus tolerised macrophages will be examined by assessing their ability to act as antigen
presenting cells (APCs) for, or modulators of, Th17 cell activation.
Objectives
 To determine kinetics of gene expression in activated, trained, and tolerised
macrophages.
 To discover unique gene expression profiles in trained versus tolerised states.
 To determine the influence of trained or tolerised macrophages on activation of Th17
cells as an explanation for the protective role for NODa in autoimmune diseases.
Methodology
A range of molecular and cellular techniques will be used in this project, such as cell culture,
RNA isolation, PCR and ELISA. Cells will be activated, trained, or tolerised via multiple
stimulations of NODa, with supernatants and cell lysates taken at different time points. CD4 T
cell that secrete IL-17 (Th17 cells) will be purified from spleen of mice with EAE and
cultured with NODa-stimulated macrophages (spleens will be prepared by Postdoc
supervisor). Gene expression will be analysed by PCR. Cytokine protein concentrations will
be analysed by ELISA and Western blot.
Likely Outcomes
This project will be invaluable in helping to determine the mechanism by which NODa are
protective against autoimmune disease in vivo by examining how these agonists can alter gene
expression and function of innate immune cells and consequently pathogenic Th17 cells.
56
References
1.
Caruso, R., Warner, N., Inohara, N. & Núñez, G. NOD1 and NOD2: signaling, host
defense, and inflammatory disease. Immunity 41, 898–908 (2014).
2.
Inohara, N. et al. Host recognition of bacterial muramyl dipeptide mediated through
NOD2. Implications for Crohn’s disease. J. Biol. Chem. 278, 5509–12 (2003).
3.
Saeed, S. et al. Epigenetic programming of monocyte-to-macrophage differentiation
and trained innate immunity. Science (80-. ). 345, 1251086–1251086 (2014).
4.
Gardiner, C. M. & Mills, K. H. G. The cells that mediate innate immune memory and
their functional significance in inflammatory and infectious diseases. Semin. Immunol.
(2016). ePub ahead of print.
5.
Fan, H. & Cook, J. A. Molecular mechanisms of endotoxin tolerance. J. Endotoxin Res.
10, 71–84 (2004).
57
SS PROJECT NO.8
TITLE: DETERMINING THE ROLE OF PYHIN PROTEINS AND ELONGATOR
COMPLEX IN REGULATION OF CYTOKINE INDUCTION BY TLR4
SUPERVISORS: DR MARCIN BARAN AND PROF. ANDREW BOWIE.
Background
Innate immunity is initiated by the recognition of Pathogen-associated Molecular Patterns
(PAMPS such as LPS) or Danger-associated Molecular Patterns (DAMPS such as self DNA)
by Pattern Recognition Receptors (PRRs). 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 IFN 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
such as TNF, independent of DNA sensing. Thus stimulation of cells with PAMPs such as
LPS requires IFI207 for maximal TNF production. 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 regulate inducible cytokines3. Using RNA
interference to knock down the expression of Elongator subunits (ELPs) such as ELP2 in
mouse cells we showed for the first time a role for Elongator in TNF and IL10 induction.
Further studies showed a role for ELPs in LPS-stimulated cytokine induction in the human
monocytic cell line THP-1. Thus the Elongator complex may have a general, but previously
unappreciated, role in cytokine gene induction. Even though there is no human ortholog of
mouse IFI207, preliminary studies showed that knock down of the expression of human
PYHIN protein PYHIN1 also affects induction of multiple cytokines in response to PRR
stimulation suggesting that it might act as a functional equivalent of mouse IFI207. The aim
of this project is to further examine the role of murine IFI207, human PYHIN1 and
Elongator complex in regulation of cytokine induction in response to LPS stimulation.
Specific Aims:
1. Examine the effects of transient overexpression of Ifi207, Pyhin1 and Elps on
regulation of cytokine induction by LPS stimulation
2. Test whether Ifi207 and Pyhin1 proteins can cooperate with Elps in mediating the
effects on cytokine induction
3. Determine if lentiviral expression of Ifi207 in murine macrophages and/or Pyhin1 in
human monocytes can affect the LPS induction of endogenous cytokines
4. Create lentiviral constructs for stable overexpression of Elps in cells in order to further
study their function in regulation of cytokine induction in murine and human
monocytic cells
Methods:
58
Reporter gene assays which directly measure the induction of chosen cytokine promoters
(TNF, IFN, CCL5, IL6 etc.) will be performed in HEK293 cells stably expressing TLR4
receptor to determine the ability of overexpressed IFI207, PYHIN1 and components of
Elongator complex (ELPs) to affect the LPS induced cytokine induction. Next, the ability of
IFI207 and Pyhin1 proteins to cooperate with Elps in mediating those effects will be
determined. Western blot will be used to inspect the obtained expression levels of PYHIN
proteins and Elps in performed experiments. J774 mouse macrophages stably expressing
IFI207 or THP1 human monocytes stably expressing Pyhin1 will be stimulated with LPS and
cytokine protein production will be measured by ELISA. In the meantime, PCR will be
performed to clone Elps into the retroviral expression vector for creation of murine and human
cell lines stably expressing Flag tagged versions of those proteins study their effects on
cytokine induction in monocytic cells.
Overall this project will reveal new mechanistic insights into how the expression of proinflammatory cytokines is regulated in response to TLR4 stimulation and might create
useful tools for the future studies of this topic.
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.
59
SS PROJECT NO.9
TITLE: INHIBITION OF HOST DNA SENSING BY MOLLUSCUM CONTAGIOSUM
VIRUS PROTEIN MC008
SUPERVISORS: DR GARETH BRADY AND PROFESSOR ANDREW BOWIE
Pattern recognition receptors (PRRs) of the innate immune system sense virus and activate the
host anti-viral response. Poxviruses have evolved a wide-spectrum of open-reading frames
(ORFs) encoding immunoregulatory proteins which bind host proteins and disrupt this antiviral response (1). These ORFs are acquired from the host and incorporated into the terminal
regions of the viral genome. The acquisition of this ‘toolbox’ of immunoevasive sequences
over long periods of host-virus co-evolution make them highly incisive tools for revealing
novel facets of a host immune system under the continual selective pressure of pathogens.
Studying how poxviral proteins antagonize innate immunity has revealed insights into how
PRR signaling functions. Molluscum Contagiosum virus (MCV) is the only known human–
adapted poxvirus and the only poxvirus to commonly cause human disease since the
eradication of smallpox yet very little is known about how it overcomes the human innate
immune response. MCV appears rigidly adapted to human infection and as such is likely to be
an effective suppressor of human anti-viral immunity. Furthermore, whilst many of the
centrally located housekeeping genes in the MCV genome are conserved with other vertebrate
poxviruses, the majority of terminally located ORFs, where poxviral immunoregulators
typically localise, are unique to MCV and currently have no known function. Elucidating how
such MCV proteins antagonise innate immune signaling in human cells will likely reveal new
information about the human innate immune response (2). We now know a key event in the
anti-viral response is the sensing of viral nucleic acids by PRRs. Viral DNA is sensed by
cGAS which generates the secondary messenger cGAMP. cGAMP is sensed by ER-resident
protein STING which then activates the transcription factors NFκB and IRF3. These
transcription factors collaborate to drive the expression of anti-viral genes like Interferon-β
thereby limiting virus spread and eventually leading to its clearance. Previous work identified
MC008 as a potent inhibitor of STING by targeting its regulator TRIM32. Here we will
investigate precisely how MC008 inhibits this vital virus-sensing machinery.
Specific aims:
1. Generate a monocyte cell line expressing MC008.
2. Determine if MC008 can also inhibit cGAS-driven DNA sensing in these cells.
3. Determine how MC008 and TRIM32 interact.
Methods
Retroviral expression is an important tool for studying the function of proteins in cells to
which is difficult to deliver genes by traditional means. MC008 will be sub-cloned into a
retroviral expression vector using molecular cloning techniques. Retrovirus will then be
produced in a HEK293T packaging line and used to transduce THP-1 cells to generate MC008
stable-expressing lines. Once the expression of MC008 has been demonstrated, THP-1 stable
lines will be stimulated with DNA and control ligands after which immunoblotting for
activated, phosphorylated NFkB and IRF3 will be performed. In addition, type I Interferon
60
bioassays and ELISA assays will determine if MC008 can inhibit STING-driven anti-viral
gene expression. Interaction of MC008 with endogenous TRIM32 and STING will then be
assessed in these cells and TRIM32 truncations will be used to map the region of this protein
through which MC008 exerts its inhibitory effect.
This project will enhance our knowledge of MC008 function and possibly answer a long
standing question about the nature of poxvirus-host adaptation.
References:
1. B. T. Seet et al., Poxviruses and immune evasion. Annual review of immunology 21,
377 (2003).
2. Brady G and Bowie AG Innate immune activation of NFκB and its antagonism by
poxviruses. Cytokine Growth Factor Rev. 2014 Oct;25 (5):611-20.
61
SS PROJECT NO.10
TITLE: THE IMPACT OF GUT MICROBE-DERIVED FACTORS ON DENDRITIC
CELL INDUCED ADAPTIVE HUMORAL IMMUNITY
SUPERVISORS: CRAIG MCENTEE, PROF. ED LAVELLE
Introduction: Due to our physiological need to respire and ingest food, the mucosal surfaces
of the respiratory and gastrointestinal (GI) tracts are constantly exposed to a plethora of
exogenous antigens. In addition, humans are colonised by an estimated 100 trillion
prokaryotic organisms, a process which begins in utero and is affected throughout life by
external factors including the environment in which one lives and the composition of one’s
diet. While the relationship which exists between man and microbe is typically symbiotic and
mutually beneficial, perturbations in the immune status of the host or a failure to induce oral
tolerance to dietary antigens can sometimes result in the generation of highly inflammatory
responses. Central to the maintenance of immune-quiescence within the gastrointestinal tract
(GIT) are regulatory T cells (TREGS) and secretory, immunoglobulin A (S-IgA) responses, both
of which are elicited only in the presence of gut microbial communities (Kawamoto et al.,
2014; Palm et al., 2014; D. Ruane et al., 2016). Both of these responses require antigen
uptake, processing and presentation by tolerogenic mucosal dendritic cells (DCs), specifically
CD103+ DCs, which are a highly specialised DC subset present at the mucosae and which are
capable of metabolising dietary Vitamin A into its metabolite retinoic acid (RA) (D. T. Ruane
& Lavelle, 2011). Recently, the role of microbial metabolites in the prevention of gut
inflammation has been the subject of intensive research (Singh et al., 2014). Moreover, several
murine studies have demonstrated that specific ablation of S-Ig responses alters the
composition of the host’s microbiota, which can have deleterious impacts on overall gut health
(Cao, Yao, Gong, Elson, & Cong, 2012). In addition, recent work by Maria Rescigno and
colleagues has demonstrated that natural IgA levels can be predictive of mucosal vaccine
efficacy, suggesting that the identification of factors capable of boosting such responses may
prove to be correlates of protective immune responses in the GI tract. This project will aim to
identify factors, both intrinsic to the host and of microbial origin, which impact on mucosal SIg responses and elucidate the mechanisms by which these effects are elicited.
Objectives- Elucidate the effects of microbial metabolites on BM-DCs in vitro. Isolate primary
mucosal DCs from various mouse strains to compare their relative abilities to induce naïve B
cells to produce IgA. Determine whether supplementation with such metabolites can boost
murine IgA responses in vivo.
Methodology: The student will gain first-hand experience in the generation and culture of
DCs from bone marrow (BM-DCs) and will stimulate these cells with various microbial
products including short and long chain fatty acids, RA and known toll-like receptor (TLR)
ligands such as lipopolysaccharide (LPS) and flagellin. Primary DCs as well as intestinal
epithelial cells will also be isolated and purified by flow cytometry for co-culture assays.
ELISA and real-time PCR will be used to quantify factors which are critically involved in IgA
class switch recombination.
Likely Outcome: The student will gain insight into the complex interactions between the host
and their resident gut microbes as well as developing an understanding of the importance of
62
microbial-products in the context of both innate and adaptive immune responses. Moreover,
this project will contribute to work being carried out in the Lavelle lab, which is being
prepared for publication, on the impact of the gut microbiota on mucosal IgA responses.
References:
Cao, A. T., Yao, S., Gong, B., Elson, C. O., & Cong, Y. (2012). Th17 cells upregulate
polymeric Ig receptor and intestinal IgA and contribute to intestinal homeostasis. Journal
of
Immunology
(Baltimore,
Md. :
1950),
189(9),
4666–73.
doi:10.4049/jimmunol.1200955
Kawamoto, S., Maruya, M., Kato, L., Suda, W., Atarashi, K., Doi, Y., … Fagarasan, S.
(2014). Foxp3+ T Cells Regulate Immunoglobulin A Selection and Facilitate
Diversification of Bacterial Species Responsible for Immune Homeostasis. Immunity,
41(1), 152–165. doi:10.1016/j.immuni.2014.05.016
Palm, N. W., Zoete, M. R. De, Cullen, T. W., Barry, N. a, Stefanowski, J., Hao, L., … Flavell,
R. a. (2014). Immunoglobulin A Coating Identifies Colitogenic Bacteria in Inflammatory
Bowel Disease. Cell, 158(5), 1000–1010. doi:10.1016/j.cell.2014.08.006
Ruane, D., Chorny, A., Lee, H., Faith, J., Pandey, G., Shan, M., … Mehandru, S. (2016).
Microbiota regulate the ability of lung dendritic cells to induce IgA class-switch
recombination and generate protective gastrointestinal immune responses. The Journal of
Experimental Medicine, 213(1), 53–73. doi:10.1084/jem.20150567
Ruane, D. T., & Lavelle, E. C. (2011). The role of CD103+ dendritic cells in the intestinal
mucosal
immune
system.
Frontiers
in
Immunology,
2(July),
1–6.
doi:10.3389/fimmu.2011.00025
Singh, N., Gurav, A., Sivaprakasam, S., Brady, E., Padia, R., Shi, H., … Ganapathy, V.
(2014). Activation of Gpr109a, receptor for niacin and the commensal metabolite
butyrate, suppresses colonic inflammation and carcinogenesis. Immunity, 40(1), 128–139.
doi:10.1016/j.immuni.2013.12.007
63
SS PROJECT NO.11
TITLE: ANALYSIS OF INTESTINAL IMMUNE RESPONSES FOLLOWING ORAL
ADMINISTRATION
OF
THE
INTK
CELL
ACTIVATOR
ΑGALACTOSYLCERAMIDE
SUPERVISORS: DR. STEPHANIE LONGET AND PROF. ED LAVELLE
Introduction:
Enteric infections are a major worldwide health problem causing high levels of mortality and
morbidity especially in developing countries. 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. In order to increase immunogenicity, the presence of an adjuvant is
required. Even though many oral adjuvants have been evaluated, as yet none have been
licenced for human use. Consequently, it remains crucial to find suitable oral adjuvants
capable of enhancing intestinal antigen-specific IgA responses that could be potentially used
in humans (1). The glycolipid α-GalactosylCeramide (α-GalCer), an invariant natural killer
cell activator, was shown to be a powerful mucosal adjuvant in animal models (2). In the lab,
we have demonstrated the ability of α-GalCer to increase intestinal antigen-specific IgA and
systemic antigen-specific IgG responses in mice in the context of an oral vaccine against
enterotoxigenic Escherichia Coli (ETEC) that we are developing (3-4). However, we lack a
comprehensive understanding of how orally administered α-GalCer increases antibody
responses and this project aims at addressing this deficiency.
We have recently shown that oral α-GalCer was able to increase the expression of some
activation markers on splenic, mesenteric lymph node (MLN) and Peyer’s patches (PP) B cells
24hr post-administration. The effects of α-GalCer on B cell activation in these tissues will be
compared in vivo at later time-points and we will determine which subsets of B cells are
activated in these tissues. We will also analyse the impacts of oral α-GalCer administration on
the induction of key B cell activating cytokines including IFN-γ and IL-4 in intestinal tissues,
draining lymph nodes and blood (5). In addition, we will examine the effects of oral α-GalCer
on the production of total IgA in intestinal tissues. Finally, as we know that dendritic cells
(DC) may play a direct or indirect role in B cell responses, we will determine the effects of
oral α-GalCer administration on splenic, MLN and PP DC activation.
Objectives:
 To assess B cell and DC activation in gut associated lymphoid tissues following oral
administration of α-GalCer.
 To evaluate the secretion of key cytokines following oral administration of α-GalCer.
 To examine changes in production of IgA in intestinal tissues.
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
64
patches and mesenteric lymph nodes. For comparison and to address systemic immune
responses, the B cell and DC analyses will also be performed with splenocytes. Modifications
in expression of B cell and DC activation markers will be evaluated by FACS analysis.
Cytokines and total IgA produced will be measured by ELISA.
Likely outcome:
The project will give us a better understanding of the effects of α-GalCer on intestinal immune
responses after oral administration. It will be the first study towards a detailed understanding
of how orally administered α-GalCer may increase intestinal immune responses.
References:
1) Rhee, J.H. et al.Mucosal vaccine adjuvants update. Clin Exp Vaccine Res, 2012
Jul;1(1):50-63.
2) 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_diss
ertations
3) 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.
4) Davitt CJ, McNeela EA, Longet S, Tobias J, Aversa V, McEntee CP, Rosa M, Coulter IS,
Holmgren J, Lavelle EC A novel adjuvanted capsule based strategy for oral vaccination
against infectious diarrhoeal pathogens. J Control Release. 2016 Jul 10;233:162-73. doi:
10.1016/j.jconrel.2016.05.001.
5) Kitamura H, Ohta A, Sekimoto M, Sato M, Iwakabe K, Nakui M, Yahata T, Meng H, Koda
T, Nishimura S, Kawano T, Taniguchi M, Nishimura T. Alpha-galactosylceramide induces
early B-cell activation through IL-4 production by NKT cells. Cell Immunol. 2000 Jan
10;199(1):37-42.
65
SS PROJECT NO.12
TITLE: DEXAMETHASONE LOADED MAGNETIC NANOPARTICLES AS A
NOVEL TOOL TO SUPPRESS INFLAMMATORY RESPONSES
SUPERVISORS: DR DULCE BENTO, DR FILIPA LEBRE AND PROF. ED LAVELLE
Introduction:
The use of nanoparticles to modulate the immune system is a recent but rapidly growing field.
Nanoengineered systems have been extensively used as immunostimulatory agents, namely as
vaccine adjuvants. However less attention has been given to its use for therapeutic antiinflammatory strategies [1]. Reformulation of immunosuppressive agents using
nanotechnology platforms helps to reduce undesirable side effects. This makes the design of
smart multifunctional drug delivery platforms an interesting approach to control inflammation.
Recently, mesoporous silica nanoparticles have received significant attention for drug and
vaccine delivery. 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 antigen molecules and increased stability compared with other
polymer-based carriers [2].
In this project we aim to study the application of magnetic nanoparticles carrying the
immunosuppressive drug dexamethasone, to suppress inflammatory responses following
foreign material implantation. For that, we are going to use mesoporous magnetic silica
nanoparticles loaded with dexamethasone. These particles can be activated to release the drug
after reaching the target by an external stimulus.
Objectives:
 To assess whether the dexamethasone loaded magnetic mesoporous nanoparticles can
decrease the inflammatory response on LPS-stimulated murine dendritic cells and
macrophages
 To test how different dexamethasone release profiles can affect the anti-inflammatory
effects
 To assess if the dexamethasone loaded nanoparticles can efficiently reduce the
inflammatory response to endotoxin in mice
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 death will be analysed
by flow cytometry (FACs). The ability of the nanoparticle system to inhibit LPS-induced
secretion of TNF-α and IL-6 and upregulation of activation markers in BMDCs will be
assessed by ELISA and FACS, respectively.
The immunosuppressive effect will be evaluated in vivo after intraperitoneal immunization in
a LPS-induced acute inflammation mouse model. Serum levels of TNF-α, IL-6, and IL-1β will
be determined by ELISA. Peritoneal exudate cells and spleens will be collected and innate
66
immune response will be assessed by flow cytometry analysis and cytokine production after
restimulation with various stimuli (e.g. TLR agonists) by ELISA.
Likely outcome:
This study will provide valuable insights about how a controlled release of
immunosuppressive drugs can modulate the inflammatory response. The fact that these
nanoparticles can be activated to release the drug after reaching their target enhances the cargo
performance, allows a reduction of the inflammatory response but decreasing potential side
effects. This platform could be applied for many therapies in different fields such as
pharmacology, oncology, immunology, tissue engineering an regenerative medicine.
References
[1] Lebre F, Hearnden CH, Lavelle EC. Modulation of Immune Responses by Particulate
Materials. Adv Mater. 2016;28:5525-41.
[2] Vallhov H, Kupferschmidt N, Gabrielsson S, Paulie S, Stromme M, Garcia-Bennett AE, et
al. Adjuvant properties of mesoporous silica particles tune the development of effector T cells.
Small. 2012;8:2116-24.
67
SS PROJECT NO.13
TITLE: IL-22 INDUCTION OF HOST-DEFENCE PEPTIDE EXPRESSION IN BOVINE
ENDOMETRIAL EPITHELIAL AND STROMAL CELLS
SUPERVISORS: PAUL KELLY AND PROF. CLIONA O’FARRELLY
Introduction:
Health defence peptides (HDPs) are critical molecules of the innate immune response (Mansour, Pena
et al. 2014) that include broad families of effector molecules, including defensins, cathelicidins, whey
acid proteins and S100 proteins. They have been shown to be involved in tissue repair, bacterial
opsonisation and chemotaxis along with displaying immunomodulatory properties (Yarbrough,
Winkle et al. 2015).
Our group has been investigating beta-defensins, particularly in the bovine at genomic, transcriptomic
and protein levels for almost two decades (Cormican, Meade et al. 2008, Davies, Meade et al. 2008).
Our recent discovery of a whole cluster of these genes expressed only in the bovine reproductive tract
along with the discovery that these genes are upregulated in postpartum cows experiencing a
pathological inflammatory immune response (termed endometritis) has led us to hypothesise that they
have a critical role in this complex inflammatory environment (Narciandi, Lloyd et al. 2011, Foley,
Chapwanya et al. 2015). We propose that their regulation/dysregulation will determine whether cows
recover normally post-partum or develop endometritis which compromises their productivity and
fertility.
We have developed a primary cell culture model of endometrial epithelial and stromal cells allowing
us to investigate the expression and regulation of these beta defensins. Recent studies have
demonstrated a role for IL-22 in inducing human beta defensin expression at mucosal surfaces (Li,
Gan et al. 2015, Mulcahy, Leech et al. 2016) and in promoting tissue repair, making its study in the
endometrium extremely relevant.
Objectives:
a. To investigate the expression of IL-22 and its receptor within a range of bovine female
reproductive tract tissues
b. To determine if it can drive HDP expression within endometrial stromal and epithelial cells.
68
Methodology:
Quantitative PCR will be used to detect expression of IL-22 and its receptor in a biobank of
bovine FRT tissues including vagina, cervix, uterus, fallopian tube and ovary. In addition,
primary bovine endometrial stromal and epithelial cells will be stimulated with IL-22 to
investigate HDP expression.
Likely outcomes:
a. Demonstration of IL-22 in bovine reproductive tract; co-localisation with HDP expression
b. Effect of IL22 on HDP expression in the bovine female reproductive tract
References:
Cormican, P., K. G. Meade, S. Cahalane, F. Narciandi, A. Chapwanya, A. T. Lloyd and C.
O'Farrelly (2008). "Evolution, expression and effectiveness in a cluster of novel bovine betadefensins." Immunogenetics 60(3-4): 147-156.
Davies, D., K. G. Meade, S. Herath, P. D. Eckersall, D. Gonzalez, J. O. White, R. S. Conlan,
C. O'Farrelly and I. M. Sheldon (2008). "Toll-like receptor and antimicrobial peptide
expression in the bovine endometrium." Reprod Biol Endocrinol 6: 53.
Foley, C., A. Chapwanya, J. J. Callanan, R. Whiston, R. Miranda-CasoLuengo, J. Lu, W. G.
Meijer, D. J. Lynn, O. F. C and K. G. Meade (2015). "Integrated analysis of the local and
systemic changes preceding the development of post-partum cytological endometritis." BMC
Genomics 16(1): 811.
Li, A., Y. Gan, R. Wang, Y. Liu, T. Ma, M. Huang and X. Cui (2015). "IL-22 Up-Regulates
beta-Defensin-2 Expression in Human Alveolar Epithelium via STAT3 but Not NF-kappaB
Signaling Pathway." Inflammation 38(3): 1191-1200.
Mansour, S. C., O. M. Pena and R. E. Hancock (2014). "Host defense peptides: front-line
immunomodulators." Trends Immunol 35(9): 443-450.
Mulcahy, M. E., J. M. Leech, J. C. Renauld, K. H. Mills and R. M. McLoughlin (2016).
"Interleukin-22 regulates antimicrobial peptide expression and keratinocyte differentiation to
control Staphylococcus aureus colonization of the nasal mucosa." Mucosal Immunol.
Narciandi, F., A. T. Lloyd, A. Chapwanya, O. F. C and K. G. Meade (2011). "Reproductive
tissue-specific expression profiling and genetic variation across a 19 gene bovine betadefensin cluster." Immunogenetics 63(10): 641-651.
Yarbrough, V. L., S. Winkle and M. M. Herbst-Kralovetz (2015). "Antimicrobial peptides in
the female reproductive tract: a critical component of the mucosal immune barrier with
physiological and clinical implications." Hum Reprod Update 21(3): 353-377.
69
SS PROJECT NO.14
TITLE: IMMUNOSUPPRESSIVE GRANULOCYTIC MYELOID CELLS IN HUMAN
LIVER METASTASES
SUPERVISORS: DR DALAL ALMUAILI, PROF CLIONA O’FARRELLY
The liver is at constant risk of malignancy because of its blood supply and exposure to toxins,
pathogens and carcinogenic dietary components. A diverse repertoire of immune cells protects
the healthy liver from metastases and tumorigenesis. Hepatic resident anti-tumour immune
Kupffer cells(KC), dendritic cells (DC), neutrophils (1, 2). In patients with primary liver
cancer or liver metastases, the phenotype and activity of these cells is compromised thus
facilitating tumour growth (3). Our group was the first to observe an expansion of granulocytic
cells in metastatic human liver (4). Here we hypothesise that these myeloid cells have
immunosuppressive activity, perhaps similar to myeloid derived suppressor cells (5); we think
they compromise local anti-tumour activity by secreting factors that suppress NK and CD8 T
cell activity. We propose that quantification of these cells and their products may provide new
prognostic biomarkers for liver metastases progression.
Objectives:
 To quantify and localise granulocytic myeloid cells in human liver tissue samples from
patients undergoing resection for liver metastases;
 To measure levels of suppressive factors (eg. IL10 & TGF beta) in metastatic liver
tissue; compare with levels in healthy human liver tissue obtained from donor organs
 To determine whether granulocytic cells in metastatic liver tissue are the primary
sources of these suppressive cytokines
 To determine whether liver granulocytic cell counts are predictive of patient outcome
Methodologies:
Liver tissue samples from healthy donors and patients undergoing liver resection have been
collected through collaboration with the National Liver Transplant Centre and the Metastatic
Liver Group at St.Vincent’s University Hospital. These samples will be sectioned and stained
with eosin and haematoxylin. Granulocytic cells will be identified and quantified. Protein will
be prepared from human liver tissue samples and quantified using BCA assays; enzyme-linked
immunosorbent assays (ELISAs) will be used to quantify levels of IL10 and TGFbeta in these
protein preparations. Immunohistochemistry will be performed on the previously collected and
fixed paraffin embedded tissues (6) to determine whether suppressive cytokine production can
be localised to granulocytic cells.
Likely outcomes:
Granulocytic cells will be identified and quantified in metastatic liver tissue. The
immunosuppressive potential of hepatic granulocytes will be defined. The possibility that
granulocytic cell counts might provide a new prognostic indicator for liver metastases will be
explored.
References:
1. Nemeth, E. et al (2009) Microanatomy of the liver immune system. Seminars in
immunopathology, 31, 333-343.
70
2. Crispe, I. (2009) The liver as a lymphoid organ. Annual Review of Immunology, 27,
147-163.
3. Robinson, M.W et al (2016) Liver immunology and its role in inflammation and
homeostasis. Cellular and Molecular Immunology, 13, 267-676.
4. Golden Mason, L. et al (2000) Differential expression of lymphoid and myeloid
markers on differentiating hematopoietic stem cells in normal and tumor-bearing adult
human liver. Hepatology, 31(6), 1251-1256.
5. Kumar, V. et al (2016) The nature of myeloid-derived suppressor cells in the tumor
microenvironment. Trends in Immunology, 37(3), 208-220.
6. Smith, F. et al (2003) Hepato-Gastroenterology, 50, 1311-1315.
71
SS PROJECT NO.15
TITLE: THE IMMUNOREGULATORY POTENTIAL OF HUMAN HEPATIC
MESENCHYMAL STEM CELLS (MSCS)
SUPERVISORS: DR LENA FISCHER & PROF CLIONA O’FARRELLY
Background



Current treatment for end-stage liver disease is liver transplantation; however there is
limited availability of donor livers and risk of organ rejection.
Need for new therapies particularly deriving from mesenchymal stem cells. However,
autologous transplantation of MSCs has been shown to suppress the pro-inflammatory
environment in the liver leading to the hypothesis that hepatic MSCs have potent
immunomodulatory activity.
MSCs are multipotent stem cells which can differentiate into cells of the mesenchymal
lineage (adipocytes, osteocytes and chondrocytes) found in tissues that are capable of
self-regeneration (1) Preliminary work of the group has detected MSCs in human liver
perfusates and expanded them in vitro. MSCs have previously been shown to be
immunoregulatory in the liver where they suppress local lymphoid activity (2,3). Pilot
studies from our group showed that MSCs from human liver perfusate suppress the
proliferation of activated T-cells in the presence of IFN-γ. We propose that factors
such as GM-CSF may be responsible for the suppressive effect of MSCs which may
signficantly impact on the function of NK cells the predominant lymphoid cells in the
liver.
Aim
The overall aims of this project are to
a. determine expression of GM-CSF (and other immunoregulatory factors) by MSCs
b. Determine whether expression of GM-CSF affects NK-cell function?
Methodologies





Isolation of MSCs from human liver perfusate/frozen liver perfusates
In vitro expansion and characterization by plastic adherence, morphology and qPCR
for stem-cell markers (Sox2, Klf4, Nanog) as well as absence of hepatocyte-markers
such as albumin.
Measure of GM-CSF in supernatants of in vitro cultured MSCs by ELISA
Co-culture of MSCs and NKs
Functional studies to test effect of MSC-secreted GM-CSF on NKs using flow
cytometry and by qPCR for NK-receptors
72
Outcomes: The immunosuppressive potential of human liver MSCs and their effect NK
function will be defined
References
(1) Pittenger M.F., Mackay A.M., Beck S.C., Jaiswal R.K., Douglas R., Mosca J.D.,
Moorman M.A., Simonetti D.W., Craig S., Marshak D.R., (1999). Multilineage
potential of adult human mesenchymal stem cells. Science 284:143-7.
(2) Pan Q., Fouraschen S.M.G., Kaya F.S.F.A., Verstegen M.M., Pescatori M.,
Stubbs A.P., van IJcken W., van der Sloot A., Smits R., Kwekkeboom J.,
Metselaar H.J., Kazemier G., de Jonge J., Tilanus H.W., Wagemaker G., Janssen
H.L.A., van der Laan L.J.W. (2001). Mobilization of Hepatic Mesenchymal Stem
Cells From Human Liver Grafts. Liver Transpl. 17(5):596-609
(3) de Mare-Bredemeijer E.L., Mancham S., Verstegen M.M., de Ruiter P.E., van
Gent R., O'Neill D., Tilanus H.W., Metselaar H.J., de Jonge J., Kwekkeboom J.,
Hall S.R.4, van der Laan L.J. (2015). Human graft-derived mesenchymal stromal
cells potently suppress alloreactive T-cell responses. Stem Cells Dev. 24(12):1436-47
73
SS PROJECT NO.16
TITLE: INVESTIGATING THE ROLE OF ITACONATE ON THE MODULATION OF
INFLAMMATORY GENES
SUPERVISORS: DR. DEEPTHI MENON AND PROF. LUKE O’NEILL
Innate immunity is considered as the first line of defense against invading pathogens in
mammalian cells. In macrophages, activation of TLR4 by LPS has been shown to recruit
downstream signaling proteins and induce the generation of reactive oxygen species (ROS)
and pro-inflammatory cytokines such as IL-1β [1-5]. Metabolic reprogramming is emerging as
a key hallmark ofinnate immunity. Previous studies have established that macrophages
activated via the LPS/TLR4 pathway acquire a glycolytic phenotype characterized by the
increased accumulation of succinate resulting in the stabilization of HIF1α and
induction/maintenance of in IL-1β [6].Recent studies have elucidated the role of other
metabolites such as itaconate in the accumulation of succinate in response to LPS [7-9]. The
regulatory role of metabolites such as succinate and itaconate in regulating inflammatory
genes is not yet fully understood.
On-going
work
in
our
lab
(unpublished) has identified a long
non coding RNA, lncRNA p21 as a
novel regulator of IL-1β. Our data
show that LPS induces the expression
of lncRNA p21 in macrophages and
this induction is further heightened in
the presence of a succinate analogue,
diethyl succinate. We are currently
working towards characterizing the
modulation of lncRNA p21 by other
TCA metabolites including itaconate.
This study would help establish a previously unexplored link between lncRNA p21 and
metabolism in macrophages and lay the foundation for in vivo studies elaborating the role of
non coding RNAs in infection models.
Project aims
1. To characterize the mechanism by which lncRNA p21 regulates pro-inflammatory gene
expression in macrophages. This
aim will employ techniques such as cell culture, gene knockdown and DNA plasmid
transfections.
2. To determine the effect of metabolites such as itaconate and glutamine on lncRNA p21
expression in macrophages.
3. To determine the effect of metabolite itaconate on potential targets of lncRNA p21
including HIF1α and histone demethylase JMJD3 in primary BMDMs. This aim will be
addressed by measuring mRNA and protein expression (qPCRS, western blotting).
74
Methods: A wide spectrum of molecular and cellular techniques will be employed in this
project including cell culture of human, murine and genetically modified (knockout) cells,
transfection of DNA plasmids, siRNA knockdown, SDS-PAGE, western blotting, EnzymeLinked Immuno Sorbent Assays (ELISAs), RNA isolation, cDNA synthesis and real time
Polymerase Chain
Reactions (qPCR).
Expected outcomes: This project aims to characterize the metabolic modulation of lncRNA
p21 in macrophages. We expect itaconate to attenuate lncRNA p21 mediated proinflammatory responses in macrophages. The experimental design will also help elucidate the
mechanism by which lncRNA p21 targets gene transcription in response to TCA metabolites
and identify
Significance: LncRNAs are fast gaining recognition as key regulators of inflammatory
responses. The identification of lncRNA p21 and its modulation by TCA metabolites has the
potential to reveal novel insights into the metabolic re-programming of proinflammatory
macrophages.
References
1. Stoll, L.L., G.M. Denning, and N.L. Weintraub, Endotoxin, TLR4 signaling and vascular
inflammation: potential
therapeutic targets in cardiovascular disease. Current pharmaceutical design, 2006. 12(32): p. 42294245.
2. Zhang, G. and S. Ghosh, Toll-like receptor–mediated NF-κB activation: a phylogenetically
conserved paradigm in
innate immunity. Journal of Clinical Investigation, 2001. 107(1): p. 13-19.
3. Doyle, S.L. and L.A. O’Neill, Toll-like receptors: from the discovery of NFκB to new insights into
transcriptional
regulations in innate immunity. Biochemical pharmacology, 2006. 72(9): p. 1102-1113.
4. Gill, R., A. Tsung, and T. Billiar, Linking oxidative stress to inflammation: Toll-like receptors. Free
Radical Biology
and Medicine, 2010. 48(9): p. 1121-1132.
5. Zhou, R., et al., Thioredoxin-interacting protein links oxidative stress to inflammasome activation.
Nature
immunology, 2009. 11(2): p. 136-140.
6. Tannahill, G.M., et al., Succinate is an inflammatory signal that induces IL-1beta through HIF1alpha. Nature, 2013.
496(7444): p. 238-42.
7. Cordes, T., et al., Immunoresponsive Gene 1 and Itaconate Inhibit Succinate Dehydrogenase to
Modulate Intracellular
Succinate Levels. J Biol Chem, 2016. 291(27): p. 14274-84.
8. Lampropoulou, V., et al., Itaconate Links Inhibition of Succinate Dehydrogenase with Macrophage
Metabolic
Remodeling and Regulation of Inflammation. Cell Metab, 2016. 24(1): p. 158-66.
9. Meiser, J., et al., Pro-inflammatory Macrophages Sustain Pyruvate Oxidation through Pyruvate
Dehydrogenase for
the Synthesis of Itaconate and to Enable Cytokine Expression. J Biol Chem, 2016. 291(8): p. 3932-46.
75
SS PROJECT NO.17
TITLE: ANALYSIS OF CLIC1 AND CLIC4 AS A REGULATOR OF THE NLRP3
INFLAMMASOME
SUPERVISORS: DR RAQUEL DOMINGO-FERNANDEZ AND PROF LUKE O’NEILL
The NLRP3 inflammasome is a critical regulator of inflammation, driving production of the
pro-inflammatory cytokine IL-1beta (1). It is of great interest to immunologists investigating
the basis for inflammatory diseases as it has been implicated in multiple inflammatory
diseases, including osteoarthritis, MS and neuroinflammatory conditions such as Alzheimer’s
disease and Parkinson’s disease. How NLRP3 is activated is still not fully understood. Agents
such as ATP or beta-amyloid activate it and there is evidence for potassium efflux being
required (2). We have recently found a role for chloride efflux in NLRP3 activation. We
therefore examined the role of of chloride channels and we have evidence that the channels
CLIC1 and CLIC4 might critically involved.
Our evidence is partly based on an inhibitor of NLRP3 termed MCC950 (3), which has been
shown to interact with CLIC1 and possibly prevent chloride efflux. We have also found
evidence for CLIC1 and CLIC4 translocation to the plasma membrane in response to NLRP3
activators, and have evidence that knocking down CLIC1 and especially CLIC4 prevents
NLRP3 activation. In this project we will further examine the role of CLIC1 and CLIC4 in
the regulation of NLRP3.
Project aims
1. The first aim of the project is to examine the effect of NLRP3 activators on CLIC1 and
CLIC4 localisation in the cell.
2. The second aim is to determine the effect of knocking down CLIC1 and CLIC4 on
NLRP3 activation, and to examine inhibitors of chloride channels.
3. The third aim is to examine the effect of MCC950 on NLRP3 activation in cells where
CLIC1 and CLIC4 have been knocked down. This is to further explore whether
MCC950 is targetting NLRP3.
Methods : A wide spectrum of molecular and cellular techniques will be employed in this
project including cell culture of human and murine cells, cell transfection with siRNA, SDSPAGE, Western blotting, Enzyme-Linked Immuno Sorbent Assays (ELISAs) and confocal
microscopy.
Likely outcomes: This project has the potential to reveal a role for CLICs in the regulation of
NLRP3. Given the importance of NLRP3 for inflammatory diseases we may therefore
uncover a key component that could be critical for inflammatory disease pathogenesis.
Furthermore, it may point towards novel therapeutic approaches to treat inflmamatory
diseases.
References
76
1. Próchnicki T, Mangan MS, Latz E. Recent insights into the molecular mechanisms of
the NLRP3 inflammasome activation. F1000Res. 2016 Jun 22;5. pii: F1000 Faculty
Rev-1469. doi: 10.12688/f1000research.8614.1. eCollection 2016
2. Muñoz-Planillo R, Kuffa P, Martínez-Colón G, Smith BL, Rajendiran TM, Núñez G.
K⁺ efflux is the common trigger of NLRP3 inflammasome activation by bacterial
toxins and particulate matter. Immunity. 2013 Jun 27;38(6):1142-53
3. Coll RC, Robertson AA, Chae JJ, Higgins SC, Muñoz-Planillo R, Inserra MC, Vetter I,
Dungan LS, Monks BG, Stutz A, Croker DE, Butler MS, Haneklaus M, Sutton CE,
Núñez G, Latz E, Kastner DL, Mills KH, Masters SL, Schroder K, Cooper
MA, O'Neill LA. A small-molecule inhibitor of the NLRP3 inflammasome for the
treatment of inflammatory diseases. Nat Med. 2015 Mar;21(3):248-55
77
SS PROJECT NO. 18
TITLE: REGULATION OF GLYCOLYTIC INFLAMMATION BY MIR-21 DURING
MACROPHAGE MYCOBACTERIAL RESPONSES.
SUPERVISOR: DR FRED SHEEDY
Introduction:
Macrophages are a phagocytic innate immune cell which play a key role in the recognition and
containment of the bacillus Mycobacterium tuberculosis (Mtb) and organising the subsequent
inflammatory response1. Recently, we demonstrated that infection with Mtb alters macrophage
glucose metabolism, with an increase in aerobic glycolysis linked to containment of the bacillus2.
This is achieved through promoting production of the pro-inflammatory mediator pro-IL-1β3 which
promotes macrophage containment through regulation of prostaglandin synthesis. In more recent
unpublished studies, we observed that infection of Mtb also leads to an up-regulation of the small
molecule non-coding negative regulator of inflammation, microRNA-21 (miR-21)4 in macrophages.
This negatively regulates host responses against Mtb and loss of miR-21 leads to increased bacterial
containment and pro-inflammatory cytokine production, including an increase in pro-IL-1β levels. We
hypothesise that this increase in pro-IL-1β corresponds to increased aerobic glycolysis in cells lacking
miR-21 and that miR-21 controls the induction of aerobic glycolysis to negatively regulate host
responses following Mtb infection.
Objectives:
This project will test whether miR-21 regulates the switch to aerobic glycolysis in Mtb
infected macrophages and subsequent macrophages responses by:
 Analysis of glucose metabolic pathways in Mtb-infected macrophages from wild-type (WT)
and miR-21-deficient (miR-21-/-) animals.
 Targeting glycolysis using competitive inhibitors to determine whether miR-21 functional
effects proceed through negative regulation of metabolism
 Identification of potential miR-21 target genes and processes which regulate glycolysis in
activated macrophages
Methodology:
Bone-marrow derived macrophages (BMDM) will be isolated and generated from WT and miR-21-/mice by standard cell culture techniques. Infection of macrophages with Mtb will be carried out using
an inactivated non-viable form of Mtb (iMtb) which triggers aerobic glycolysis to a similar extent as
live Mtb, through activation of TLR signaling. This will be used to interrogate glucose metabolism
pathways in WT & miR-21-/- BMDM using Seahorse technology and through biochemical
measurement of lactate production. Subsequent effects of pro-inflammatory cytokine induction and
prostaglandin synthesis will be assessed using qPCR technology, Western blotting and ELISA. A
competitive inhibitors of glucose, 2-deoxglucose (2-DG) will be used to block glycolysis, alongside
more specific inhibitors like TEPP-46 (which blocks the enzyme PKM2) which will be used to
78
examine the functional outcome of miR-21-mediated aerobic glyolsysis using the assays outlined
above. In addition, the effect on production of nitric oxide species and reactive oxygen species will be
assessed using biochemical techniques and flow cytometry. These studies will identify potential
pathways and processes regulated by miR-21 through aerobic glycolysis. To identify more specific
direct miR-21 target genes involved in this process, in silico analysis will be carried out alongside invitro 3’UTR luciferase and RNA-immunoprecipitation assays.
Likely outcome:
This project will identify novel processes regulated by miR-21 relevant to mycobacterial infection. It
will extend our understanding of the intersection of inflammatory responses with macrophage
metabolism and highlight how this novel phenomenon can be regulated epigenetically using
endogenous small RNA species, which can be targeted by Mtb to subvert host immune responses.
References:
9. O'Garra, A. et al. The immune response in tuberculosis. Annual review of immunology 31,
475-527, doi:10.1146/annurev-immunol-032712-095939 (2013).
10. Gleeson, L. E. et al. Cutting Edge: Mycobacterium tuberculosis Induces Aerobic Glycolysis in
Human Alveolar Macrophages That Is Required for Control of Intracellular Bacillary
Replication. J Immunol 196, 2444-2449, doi:10.4049/jimmunol.1501612 (2016).
11. Tannahill, G. M. et al. Succinate is an inflammatory signal that induces IL-1beta through HIF1alpha. Nature 496, 238-242, doi:10.1038/nature11986 (2013).
12. Sheedy, F.J. et al. Negative regulation of TLR4 via targeting of the proinflammatory tumor
suppressor PDCD4 by the microRNA miR-21. Nat Immunol 11, 141-147 (2010).
79