Immunology & Infectious Diseases HONOURS 2009

Welcome to Immunology & Infectious Diseases Honours Program 2010

The following is a guide to the structure of the honours year.

Immunology and Infectious Diseases Honours are research-based courses, commencing in early February and culminating in presentation of a thesis in early November. Candidates are assessed on all aspects of their research performance, by their supervisor and by two ‘internal’ examiners who will be scientists familiar with the general field of research, but not directly associated with the student or their research lab. Assessed components are: (i) a review of the literature pertaining to the particular research project undertaken by the candidate (formative), (ii) oral presentation of research in the form of a short seminar (twenty minutes talk and ten minutes for questions)(summative), (iii) the thesis, (summative), (iv) research performance (summative).

Honours Coordinator: Dr Allison Abendroth

Contact details: a.abendroth@usyd.edu.au

The Research Program

Candidates will work almost exclusively on their chosen research topic, independently, but under the guidance of their supervisor. At the end of the year, the research is written up in the form of an Honours Thesis and is examined by the supervisor and two other scientists appointed by the Honours committee. Each thesis is assessed on its overall quality and not simply on the quantity of results. ‘Getting results’ is not the sole aim of the research program.

In mid-October, 2-3 weeks before the thesis is submitted, candidates are required to present a seminar on their project. This will be a 20-minute presentation, followed by constructive feedback in the form of questions from the audience of peers, supervisors, examiners and interested colleagues.

The research program is further structured to help develop scientific writing skills in the following way:

1. Preparation of an approximately 2 page summary outlining the aims and experimental approach which the candidate understands to be their project and why the project has scientific merit in their opinion. (in other words, what they are going to do and why they are doing it). This is not formally assessed but will be discussed at interview with the supervisor.

2. Preparation of a ‘literature review’ essay that is the basis of the Introduction to the final thesis, and prior preparation of the results and methods chapters. These sections are read by the supervisor, and some corrections, suggestions

for improvements and other comments are made before submission of the thesis.

The Supplementary Program

The supplementary program has 2 main aims-

· to keep the candidate abreast of wider issues in Immunology and Infectious Diseases, while fostering their particular research interest, and

· to enable the candidate to develop strong skills in communication and critical appraisal of the literature.

To this end, in addition to research at the bench, students are required to attend

1. A Research Seminar Series at Centenary Institute, Westmead Millennium Institute or their equivalent in other locations.

2. Honours class meeting, where we create a ‘self-help’ program reviewing each other’s aims, results and relevant literature, solving technical problems and providing workshops in statistics and data analysis.

3. Meetings of their own research group which will focus on the particular interests of the group, and where the student is expected to participate both in presenting results and reviewing literature.

4. Journal Club in the Centenary Institute (or its equivalent), where each week a member of staff reviews one or two recent papers of more general interest.

Introductory Course on Animal Experimentation

An introductory course on Animal Experimentation will be presented by the Animal Care & Ethics Committee of the University and attendance and successful completion by all Honours students is compulsory.

Final Assessment

Assessment marks are considered at a Departmental Honours Committee meeting, which then forwards their recommendation to the Faculty of Science. The Faculty makes the final decision as to the grade awarded. The Faculty recently revised its Honours’ policy. The requirement for a minimum SCIWAM of 68 to be eligible for the award of First Class Honours was eliminated. Instead all Honours students are eligible for First Class Honours if their Honours performance justifies a mark of 80 or greater. However Departments are constrained in the average mark that can be awarded by the following: The rolling five year average mark difference (student Honours mark minus SCIWAM) should fall within the range 10 plus or minus 2. We do not anticipate any problem adhering to these guidelines. However if your WAM score was possibly affected by sickness, or other reasons, you should make sure that this is known to the Discipline Honours Committee, Allison Abendroth, in the first instance.

How to apply

1. Read and review the projects outlined in this document. Pay attention to the general thrust of the research and the techniques used. These should help you determine your level of interest in a particular proposal.

2. Attend the Information evening on Tuesday 15 September 2008, 4.30 – 6:00pm, in the ‘Dining Room’, level 6 of Centenary Institute. Here you may obtain more general information and meet supervisors and current students in an informal setting.

3. Choose 3-4 projects of special interest to yourself.

4. Arrange an interview with the supervisor of each of your selected projects. This will allow you to discuss these projects in detail on a one-to-one basis. (The supervisor needs to assess you, and you need to assess both the project and the supervisor!). You must take a copy of your academic transcript and a brief curriculum vitae to each interview.

5. Return the completed application form to Allison Abendroth by Friday 30 October 2009.

Qualifications for Admission

To qualify for admission to Honours the Faculty of Science requires that a student is qualified for the award of a pass degree, has a SCIWAM score of at least 60 and a credit in their chosen Honours topic. Further a student must be considered by the Faculty and the Head of Department concerned to have the requisite knowledge for an Honours course. To qualify for Honours in Immunology or in Infectious Diseases a student must satisfy the minimal conditions. The student should have performed well in all aspects of the senior Immunology units IMMU 3102 and 3202 or Infectious Diseases course INFD 3012 or Virology course VIRO3002 and achieved at least a credit. A SCIWAM score of 65 or above is also desirable. In special circumstances applicants who have studied subjects other than Immunology or Infectious Diseases may be considered.

Admission to Honours will be based upon these criteria and recommendations from the interview with the supervisor.

Please direct questions and submit your application together with your academic transcript and CV to Dr Allison Abendroth.

Application for Admission to Immunology or Infectious Diseases Honours 2010

Name: ………………………… Student ID ……………………….

Degree program (eg. BSc, BMEDSc) …………………………….

Contact Information:

Email address:………………………………………………………

Mailing Address & telephone number:

Term time………………………… Telephone… ……………….

Mobile……………………….

Vacation…………………………… Telephone…………

Mobile……………………..

Projects in which you have a special interest

(please list in order of preference)

1.-------------------------------------------------------------------------------------------------------

[Supervisor contacted Yes / No]

2.-------------------------------------------------------------------------------------------------------

[Supervisor contacted Yes / No]

3.-------------------------------------------------------------------------------------------------------

[Supervisor contacted Yes / No]

4.-------------------------------------------------------------------------------------------------------

[Supervisor contacted Yes / No]

Are you applying for Honours in Infectious Diseases?

Yes / No

Are you applying for Honours in Immunology?

Yes / No

IS INFECTIOUS DISEASES HONOURS YOUR FIRST PREFERENCE?

Yes / No

IS IMMUNOLOGY HONOURS YOUR FIRST PREFERENCE?

Yes / No

Are you applying for Honours courses in other subjects?

Yes / No

If yes, which subject(s) ……………………………………………………………….

Which is your FIRST preference Honours Program?

………………………………………………………………

ARE YOU APPLYING FOR OTHER COURSE EG. GRADUATE MEDICAL PROGRAM, MASTERS / GRAD DIPLOMA?

Yes / No

If yes, which course(s)

………………………………………………………………..

Please indicate when you will be notified if your application in other programs is successful.

……………………………………………….

SUPPORTING DOCUMENTATION

Please attach a copy of your academic transcript and a brief CV.

SUBMISSION

Please submit your application together with your academic transcript and CV to Dr Allison Abendroth, Infectious Diseases & Immunology, level 6 Blackburn Building DO6 on or before Friday 30 October 2009. Successful applicants will be notified in late December.

Infectious Diseases and Immunology Honours Projects

2010

Honours research projects are offered in different Sydney University locations viz. Central Campus, Westmead Campus and Nepean Campus and in various institutions within those areas. The research projects being offered for 2009 are listed here under each location and the laboratories within each location.

Should you require further information please contact Dr Allison Abendroth (a.abendroth@usyd.edu.au) in the first instance.

Central Campus………………………………………………… 7

Western Campus………………………………………………..44

Nepean Campus………………………………………………..64

VZV RESEARCH LAB

Title: Varicella zoster virus immunobiology and pathogenesis

Supervisor: Dr Allison Abendroth

Contact: email: a.abendroth@usyd.edu.au Phone: 93516867

BACKGROUND

Varicella zoster virus (VZV) is a medically important human herpesvirus which causes chickenpox (varicella) predominantly in childhood and shingles (herpes zoster) in middle to old age. VZV can establish a latent infection which is localized to sensory ganglia and may reactivate from this site and cause herpes zoster. The severity and morbidity associated with zoster is a major factor in its clinical impact for otherwise healthy people and among immunocompromised patients. The impact of both varicella and of zoster remain a major health issue and a means of prevention would offer important medical and economic benefits. To date, a much better understanding of VZV pathogenesis is needed for the development of better therapies to lessen the impact of VZV disease on the community.

The experimental techniques and intellectual stimulation generated from undertaking an Honours project in the lab will provide a tremendous base for enthusiastic and talented students keen to pursue a career in biomedical research in immunology/virology. The VZV lab is well equipped to undertake these studies and the projects outlined also have the potential to be continued and expanded into a PhD project.

Interaction of VZV with human dendritic cells (DCs)

There has been a tremendous new interest in DC as virus targets and in their role in antigen presentation and induction of both innate (NK cells via IFN) and adaptive cellular immunity. The skin is a major site of VZV replication yet it is also the site of immature DC (eg Langerhans cells) which sample the micro-environment for pathogens, and which are a pivotal cell type in the induction of anti-viral immune responses. The VZV lab has a major research focus on virus encoded immunomodulation and the infection and impact of VZV on human DCs. We were the first to show that VZV can infect immature and mature dendritic cells (DCs) and infection disrupts the ability of these cells to function properly in initiating an immune response. Thus, we have discovered that VZV has the ability to delay and/or evade the host immune system by infecting and altering the immune function of DCs.

Given the importance of the skin as a site of VZV infection and the role skin DC play in the induction of anti-viral immunity, there is good reason to study infection and modulation of DC in human skin during VZV infection. To date, we have determined the DC subsets that may participate in VZV pathogenesis by immunostaining sections of chickenpox and shingles skin lesions for immune cell markers. In VZV infected skin, Langerhans cells (LC), were decreased and plasmacytoid DC (PDC), DC that produces high levels of IFN-alpha, were increased in frequency compared to uninfected skin. We investigated whether these DC subsets support VZV infection in vivo by dual immunofluorescently staining sections of VZV infected skin lesions for LC/PDC markers and VZV proteins. Notably, a proportion of LC and PDC were positive for VZV proteins, suggesting these cells may be infected . Further assessment of skin DC infection, immune function and viability will define the mechanisms underlying cutaneous infection.

HONOURS PROJECT:VZV interactions with human Langerhan cells (LCs).

It is not known whether LC are permissive to VZV infection in vitro and what is the fate of virus infection on LC maturation, migration and function. This project will therefore seek to determine whether LC in vitro and in ex vivo human skin explants are infected and how the virus impacts LC functions and interferes with the expression of functionally important immune molecules and cell migration. The results obtained will provide valuable new information to our understanding of how VZV has evolved strategies to evade immune detection and become such a successful and ubiquitous human pathogen.

The project will involve a wide variety of molecular and cell biology techniques including specialized primary human dendritic cell culture, intact human skin explant culture, mammalian cell culture, virus culture, cell infection, multi-colour flow cytometry, immunofluorescent staining and confocal microscopy.

An investigation into how mast cell-activated regulatory B cells suppress immune responses

Supervisor: Dr. Scott Byrne

Location: Department of Infectious Diseases & Immunology,

Blackburn Building, University of Sydney, Camperdown – Main Campus

Contact details: email: scottb@med.usyd.edu.au ; phone: 9351 7308

Byrne lab website: http://web.me.com/scottbyrne/Byrne_Lab

Background and Significance:

Skin cancer incidence continues to rise despite awareness of the need to protect ourselves from the prime cause of skin cancer; sunlight. Clearly, preventing skin cancer by limiting the amount & type of sun exposure has been insufficient to reduce this incidence. In order to understand carcinogenesis we must study the mechanisms that allow tumours to establish: i.e. DNA damage and suppression of anti-tumour immunity. This project will investigate the mechanisms by which the ultraviolet (UV) wavelengths in sunlight suppress immune responses.

We recently discovered that UV suppresses immunity in two ways: (1) by activating regulatory B cells in lymph nodes (Byrne & Halliday 2005 J Invest Dermatol 124(3) 570-8), and (2) by altering mast cell migration patterns into and away from the skin as well as the draining lymph nodes (Byrne et al. 2008 J Immunol 180(7) 4648-55. These two events are linked because upon arrival in nodes, mast cells preferentially home towards and intimately associate with B cells. Both mechanisms depended on the release of UV-inflammatory mediators in the skin as pharmacologically interfering with the action of these mediators blocked UV mast cell migration and prevented immune suppression.

In an effort to identify novel UV-induced mediators involved in this process we discovered that UV upregulates both the mast cell chemoattractant CCL5 (RANTES) and the newly described cytokine, IL-33. This is likely to be important because we have found that UV attracts mast cells into the skin and IL-33 stimulates mast cells to produce the B cell activating cytokines IL-6 and IL-13. Furthermore, these IL-33-stimulated mast cells activate B cells with suppressor activity when transferred in vivo.

Project Summary:

This project will focus on two aspects: (1) The importance of UV-induced CCL5 in mediating mast cell migration and immune suppression as well as (2) the mechanism by which mast cell activated suppressor B cells regulate the immune response.

1. CCL5 works by binding to the chemokine receptor CCR5 on the surface of mast cells. We will use novel CCR5 antagonists to block the effect of UV-induced CCL5. Some of these drugs are currently in Phase III clinical trials for the treatment of HIV. If, as we suspect, UV-induced CCL5 is involved in the immune suppressive pathway, then blocking CCL5 binding to its receptor CCR5 will prevent both UV-mast cell accumulation in skin as well as immunosuppression.

2. We already know that UV-induced lymph node suppressor B cells secrete IL-10 (Matsumura & Byrne et al. 2006 J Immunol 177(7) 4810-17) and we have preliminary data that they also produce IL-13. However, we still do not know the exact mechanism by which mast cell-activated B cells suppress the induction of immunity. We also do not know whether this suppression is via the inhibition of effector T cell activation or via the induction of regulatory T cells. This part of the project will investigate these unknowns.

List of Important Immunological Techniques Employed in this Study:

1. Mouse handling techniques and their UV-radiation with a state of the art solar simulator

2. Isolation of primary and secondary lymphoid tissues from mice (in addition to skin)

3. Preparation of tissues for histology (including paraffin and frozen sections)

4. Immunohistochemistry staining and analysis (including fluorescence microscopy)

5. Tissue culture and aseptic techniques (incl. generation of mast cells from bone marrow stem cells)

6. ELISA, Flow cytometry acquisition and analysis, cell purification (magnetic beads) and sorting (FACSAria)

7. Important in vivo injection routes of cells and drugs – sub cutaneous, intravenous, & intraperitoneal

8. Molecular Biology techniques including qRT-PCR and possibly cDNA microarray

Modulators of Ross River Virus replication in vertebrate and invertebrate cells.

Dr Belinda L. Herring

Dept. of Infectious Diseases and Immunology

University of Sydney

Ph: 9036-6582, b.herring@usyd.edu.au

Location: Molecular Virology Laboratory, Blackburn Bld, Camperdown Campus

The specific aims of this project are:

1. To demonstrate the presence of miRNAs in RRV genomes and to define their role in mammalian cell infection.

2. To identify viRNAs in common vectors of RRV Aedes vigilax and Culex annulirostris infected with different strains of RRV, delineate the role of viRNAs in virus replication in these vectors and determine if these effects are strain dependent.

Arthritogenic disease caused by the arboviruses Ross River virus (RRV) and Barmah Forest virus (BFV) affect thousands of individuals in Australia. Annually, over 4000 cases of RRV disease and > 1000 cases of BFV disease are reported. Presentation of disease is variable, with some individuals experiencing no clinical signs of infection while others experience a mild arthralgia with or without rash and a small percentage suffer from a prolonged incapacitating polyarthritis predominantly affecting the peripheral joints. Arthritic pain can persist for weeks to months.. Disease significantly impacts quality of life with individuals experiencing difficulties with daily activities and possible inability to work.

AIM 1

Recent discoveries indicating that viruses can encode micro-RNAs, which may modulate host gene expression to provide the virus with a replicative advantage may also impact the susceptibility of an individual to disease. Within a virus infected vertebrate cell, host encoded miRNA can play a role in viral replication as demonstrated with HCV infection, or virally encoded miRNAs can play a role in controlling viral replication by targeting either viral mRNA or host cell mRNA, as demonstrated with human cytomegalovirus infection. Scanning of the RRV genome (NC_100544) has identified 10 predicted miRNAs (http://alk.ibms.sinica.edu.tw/cgi-bin/miRNA/miRNA.cgi); however, the presence of these miRNA’s in the RRV genome and their function in vertebrate cell infection has not been established. The presence of these miRNAs in the RRV genome and their function in viral infection of vertebrate cells will be investigated in this project.

AIM 2

Central to the transmission of arboviruses is the mosquito vector; understanding the dynamics of viral replication in vectors may provide insights into disease transmission patterns. Recent data demonstrating that the RNA interference (RNAi) pathway, an intracellular anti-viral defence mechanism (Fig1), and virally encoded viRNAs play a role in the replication of a number of viruses (dengue and Sindbis) in mosquitoes suggest that this may dictate the ability of a mosquito species to be a viral vector. The following experiments will be conducted to achieve this aim.

i) identification of viRNAs in RRV infected mosquito cells (C6/36 cells) and common vectors of RRV.

ii) delineation of the role of viRNAs in virus replication in RRV vectors Aedes vigilax and Culex annulirostris.

determination of the effects of different virus strains and hence viRNAs on RNAi activity in Aedes vigilax and Culex annulirostris, common vectors of RRV.

Bio-Discovery Identification of novel viral agents

Dr Belinda L. Herring

Dept. of Infectious Diseases and Immunology

University of Sydney

Ph: 9036-6582, b.herring@usyd.edu.au

Prof. Richard Russell

Department of Medical Entomology

CIDM, Westmead Hospital

Ph: 9845-7279, rrussell@mail.usyd.edu.au

Location: Molecular Virology Laboratory, Blackburn Bld, Camperdown Campus

In the last decade the world has witnessed the emergence of novel biological agents such as SARS, Nipah virus, Highly pathogenic avian influenza (H5N1), and swine flu (H1N1). Discovery of new disease causing agents may pre-empt unpredicted disease outbreaks, significantly enhancing community preparedness for such events. The central aim of this project to is to analyse biological material from a variety of sources to identify new infectious agents that may be pathogenic to human, animal and wildlife health.

This project will address the following aims:

AIM 1 – Discovery of novel viral agents in mosquito isolates causing CPE in mammalian cells. To utilise molecular methods to identify and genetically characterise agents from mosquitos causing cytopathic effects in mammalian cell cultures as these agents may represent a pool of unrecognised viruses that may be associated with human, animal or wildlife disease. Once these techniques have been established this study will be expanded to examine ticks, urine and faecal matter from bats, tissue from native rodents.

Viruses that utilise arthropods as vectors, arboviruses, cause a large number of human and animal diseases. Arboviruses are transmitted by mosquitos, ticks and biting midges through biting of a susceptible host. Arboviruses replicate in both insect and animal with humans and animals being dead end hosts. Examples of pathogenic arboviruses include; yellow fever virus, Japanese encephalitis virus (JE), Chikungunya virus (CHIKV) and West Nile virus (WNV). The NSW Arbovirus Surveillance and Mosquito Monitoring Program traps mosquitos from over 16 sites in NSW to monitor mosquito vector populations and determine arbovirus activity. Virus isolation is attempted on mosquitos to provide information on the presence of arboviruses causing disease in Australia. Mosquito lysates are inoculated onto mammalian cell cultures and inspected for cell damage or death, a cytopathic effect (CPE). CPE positive cell cultures are further tested using an enzyme immunoassay to determine the arbovirus present. Cultures which exhibit CPE, but a virus cannot be identified are designated “unknown”, suggesting the presence of a novel viral agent. “Unknown agents” account for approximately 30% of isolations from trapped mosquitos (i.e. negative on the above EIA screening). These unknown agents could be possible pathogens of clinical importance, especially as a proportion of individuals (estimated to be approximately 15%) presenting with fever, rash and arthralgia in arbovirus endemic areas are seronegative for acute infections with a known aetiology. Twenty-two “unknown isolates” have been selected from the NSW Arbovirus Surveillance and Mosquito Monitoring Program repository. To identify 'unknown' agents, a sequence-independent single primer amplification method will be used in conjunction with cloning and sequencing of clones. Sequences generated will be subjected to computational search algorithms (BLAST) to identify viral sequence homologies. Once homology to a virus family has been identified directed PCR and sequencing approaches will be used to amplify and sequence complete viral genomes.

AIM 2 – Identification of the etiological agents causing disease in native fauna.

To examine tissue from diseased/deceased animals to identify the etiological cause of disease.

Two diseases of native bird species have been identified by the Wildlife Health and Conversation Centre, for which the aetiology is unknown. The first, clench-claw syndrome in lorikeets is a condition that primarily affects their ability to fly and perch. The second, “black and white bird disease” affects magpies and currawongs. This disease occurs in the summer months, coincident with mosquito activity, in coastal regions. Similar techniques to those used for Aim 1 will be utilised to determine the aetiologic origin of these conditions in native fauna.

Pathogenic potential of Ross River Virus variants in vitro.

Dr Belinda L. Herring

Dept. of Infectious Diseases and Immunology

University of Sydney

Ph: 9036-6582, b.herring@usyd.edu.au

Location: Molecular Virology Laboratory, Blackburn Bld, Camperdown Campus

The specific aims of this project are:

To genetically characterise isolates from RRV genogroups II and III.
To delineate if host cell factors or viral determinants such as genetic strain influence the development of Ross River Virus Disease (RRVD).

AIM 1

RRV is member of Togaviridae Family, genus Alphavirus. Both RRV and BFV have a positive sense, single stranded RNA genome (11.8kb), which encodes both non-structural and structural genes, including 2 envelope glycoproteins, E1 and E2. Relatively few studies have addressed genetic variation, geographic distribution and evolution of RRV in Australia. There is a paucity of genetic data for RRV, with only a handful of complete genome sequences in Genbank. RRV isolates (2-5) representative of each of the sub-clusters within genogroup II will be expanded by a single passage in C6/36 cells, viral RNA extracted, amplified and sequenced using established methods. Nucleotide and amino acid sequences will be aligned to each other and database sequences using ClustalX to identify nucleotide polymorphisms and synonymous and nonsynonymous substitutions. Phylogenetic analysis will be performed on genome sequences to identify unique clusters.

AIM 2

RRV disease occurs in individuals irrespective of age or sex. Infection can result in a spectrum of symptoms, which include a maculopapular rash, muscle and joint pain (>50% of patients) with peripheral joints predominantly affected, and development of debilitating polyarthritis (>50% of patients), limiting or precluding normal activities for months or years. Different RRV isolates display different levels of virulence in humans, mice and mosquitoes and variations in human antigenic response have been demonstrated. The observation that some individuals develop disease and others do not poses the question: is development of clinical disease due to viral or host cell genetic factors?

The following experiments will be preformed to address this question

i) Infection of monocytic cells, rhabdosarcoma cells (RD cells) and HeLa cells with diverse RRV isolates.

ii) Determination of viral replication and cell viability in permissive cells (HeLa, RD and MDM) one-step growth curves, indirect immunofluorescent staining, real time quantitative PCR).

iii) Measurement of cytokines released in infected cultures.

CYSTIC FIBROSIS MICROBIOLOGY RESEARCH GROUP

INFECTIOUS DISEASES AND IMMUNOLOGY

CENTRAL CLINICAL SCHOOL

LEVEL 6 BLACKBURN BUILDING

Supervisors: Associate Professor Colin Harbour, Dr Jim Manos and Dr Helen Hu

charbour@infdis.usyd.edu.au; jmanos@infdis.usyd.edu.au; honghuah@mail.usyd.edu.au

Background for Project 1:

Pseudomonas aeruginosa (P. aeruginosa) lung infection is a major cause of morbidity and mortality in cystic fibrosis (CF) patients. P. aeruginosa usually spreads to patients from the environment, and there are also epidemic clones, including two Australian epidemic strains (AES-1 and AES-2), spreading patient-to-patient which are an emerging threat to CF patients internationally. Our group has been working towards understanding the mechanisms of infectivity virulence and persistence in P. aeruginosa from CF patients in the following ways:

We have used microarrays designed for strain PAO1 to study gene expression in the clonal isolates (publications: Manos et al. J. Med Micro 2008 57: 1454-65. Manos et al. FEMS 2009 292; 107-114).
We have sequenced the genome of AES-1 (a virulent epidemic clone) and designed and manufactured a 70-mer oligonucleotide microarray (PANarray) in collaboration with Monash University. This array contains the genes of AES-1, PAO1 and six other sequenced genomes. We are now using this array to investigate expression and presence novel genes (i.e. not found in PAO1 but present in AES-1).
Developed a virulence assay in the C. elegans worm model to determine the LD₅₀ of clinical P. aeruginosa isolates.
Developed a medium that mimics lung sputum called Artificial Sputum Medium (ASMDM) and demonstrated that during growth in this medium AES-1 expresses specific genes that may play a role in its infectivity and persistence in CF sputum. Sharna Naughton (honours student in 2009) is using ASMDM as a model system to investigate P. aeruginosa virulence gene expression in CF lung by transcriptome profiling.

Project 1: Characterising the genetic determinants of chronic CF lung infection by P. aeruginosa

P. aeruginosa isolates that were present in CF patients several years apart (sequential isolates) have been transcriptionally profiled using the PANarray. This Honours project will investigate the importance of particular genes that were shown to be highly differentially expressed in the sequential isolates when grown in ASMDM. Genes of interest will be mutated (knocked out) and the effect of the mutant strain will be compared to the wildtype in their ability to infect and colonise mammalian cell lines and a mouse model of chronic lung infection, in order to identify of genes that might be important in infectivity and persistence.

Background for project 2:

Recently, a long-term nebulised hypertonic saline (HS) trial (7% NaCl inhaled twice daily for 48 weeks for CF patients over six years of age) coordinated by our group (Elkins et al, 2006 N. Engl. J. Med. 354, 229-240) showed that HS increased mucus clearance and improved lung function compared with normal saline used as control. The trial established the safety of HS as a novel therapy to treat CF lung infection with regard to bronchospasm and tolerability. Our overall findings have led to the incorporation of HS into mainstream treatment for adults with CF. However there have been no studies of the effects of HS on properties of CF P. aeruginosa isolates. Our preliminary work carried out on phenotypic characteristics of CF P. aeruginosa isolates from the long term HS trial showed that HS reduces virulence production and biofilm forming capacity and disrupts existing biofilms of P. aeruginosa isolates.

Project 2: The effect of Hypertonic Saline on the gene expression profiles of P. aeruginosa

This project will investigate the underlying mechanisms of HS on P. aeruginosa at the molecular level. Changes in P aeruginosa gene expression associated with HS usage will be assessed using saline-naïve CF P. aeruginosa isolates. Isolates will be grown for 96hr in our newly developed artificial sputum medium (ASMDM), with or without HS. RNA will be extracted and global expression of genes assessed using the PANarray (see description above). The differential expression of genes of interest will be confirmed by real-time PCR.

The molecular techniques used in these projects will include:

PCR and qualitative real-time PCR

Microarray

Genetic Mutation and complementation

Tissue culture infection studies

Other advanced techniques

Fluorescent and Confocal microscopy

Project Title: Generation of temperature-sensitive strains of Human Enterovirus 71 by clustered charge-to alanine mutagenesis of the viral RNA-dependent RNA polymerase gene

Supervision: Professor Peter McMinn and Dr Patchara Phuektes

Infectious Diseases and Immunology, Central Clinical School, Blackburn Building D06, Camperdown Campus

Contact: Peter McMinn, email p.mcminn@usyd.edu.au or phone 9354-2900

Project overview:

Human enterovirus 71 (HEV71) is a member of the Human Enterovirus A species within the Genus Enterovirus, Family Picornaviridae. Picornaviruses are small, non-enveloped viruses with a single stranded positive sense RNA genome (~7.5 kb). Since its discovery in 1969, HEV71 infection has been identified as an emerging cause of severe encephalitis affecting young children, particularly in the Asia-Pacific region. Since 1997, HEV71 has become the largest single cause of encephalitis in several countries, including Malaysia, Vietnam, Singapore, China and Taiwan. With the high level of endemic circulation of HEV71 in the Asia-Pacific region and lack of antiviral agents against HEV71, vaccination seems be the best approach for prevention and control of HEV71 infection. A major research goal of our laboratory is to generate potential live attenuated HEV71 vaccines.

This project will involve constructing a panel of temperature sensitive (ts) and virulence attenuated HEV71 viruses from the HEV71 infectious cDNA clone by using charge-to-alanine mutagenesis on the 3D^pol (RNA-dependent RNA polymerase) gene. 3D^pol plays a primary role in viral RNA synthesis during infection and mutations in this gene are associated with virulence attenuation in animal models of HEV71 infection. Charge-to alanine mutagenesis can be used to create ts mutant viruses by destabilizing hydrogen-bonding and/or electrostatic interactions at the surface of the targeted protein, rendering the function of such proteins more thermosensitive. This approach has been used successfully in the generation of temperature sensitive, attenuated mutants of other enteroviruses, including poliovirus. Mutant viruses generated during this study will be characterised in cell culture, including cells of neuronal and non-neuronal origin, in order to screen for the temperature sensitivity of viral growth and replication. Viral RNA synthesis will also be quantified in mutant and wild-type virus-infected cells using real time PCR.

Methods and techniques

1. Cell culture

2. Virus culture and titration

3. Molecular techniques, including RNA/DNA extraction and purification, Reverse transcription (RT)-PCR, real time PCR, cloning, nucleotide sequencing, site-directed mutagenesis

4. Transfection of DNA into cells

Project Title: Selection and genetic mapping of Guanidine hydrochloride-resistant mutants of Human enterovirus 71 (HEV71)

Supervision: Professor Peter McMinn and Dr Patchara Phuektes

Molecular Virology Laboratory, Infectious Diseases and Immunology, Central Clinical School, Blackburn Building D06, Camperdown Campus

Contact: Peter McMinn, email p.mcminn@usyd.edu.au or phone 9354-2900

Project overview:

Guanidine hydrochloride (GuHCl) is known to inhibit the growth of human polioviruses, the prototype members of the Genus Enterovirus. Research has shown that GuHCl specifically inhibits the initiation of viral RNA synthesis and that mutations in the 2C protein can confer resistance to GuHCl. In our laboratory, we have recently shown that GuHCl inhibits the growth of HEV71. In this project, the effect of GuHCl on the RNA replication of HEV71 will be further characterised in cell culture. In order to identify gene regions linked to guanidine resistance, mutant HEV71 strains will be selected in cell culture in the presence of GuHCl, and mutation/s responsible for altered guanidine sensitivity will be identified by nucleotide sequencing. The guanidine resistance phenotype associated with the observed mutations will be confirmed by mutagenesis of an infectious cDNA clone of wild-type HEV71 and testing of clone-derived virus populations in cell culture.

This project will enable us to better understand the mechanism of viral RNA replication by HEV71. Furthermore, we will be able to use the guanidine resistance mutations as a genetic marker to investigate the mutation frequency and the fidelity of replication of HEV71 in a GuHCl resistance assay. Altered replication fidelity of enteroviruses has been linked to virulence attenuation and may thus be of importance for the development of a live attenuated vaccine.

Methods and techniques

1. Cell culture

2. Virus culture and titration

3. Molecular techniques, including RNA/DNA extraction and purification, reverse transcription (RT)-PCR, real time PCR, cloning, nucleotide sequencing, site-directed mutagenesis

4. Transfection of DNA into cells

Investigating the immuno-suppressive effects of the Mycobacterium ulcerans toxin mycolactone

Supervisor: Dr Jamie Triccas Co-supervisor: Prof Wolfgang Weninger

Microbial Pathogenesis and Immunity Group Immune Imaging Group

Infectious Diseases & Immunology Centenary Institute

Blackburn Building, University of Sydney E-mail: w.weninger@centenary.org.au

Phone: 9351 6812

E-mail: jamiet@infdis.usyd.edu.au

Mycobacterium ulcerans is a slow-growing mycobacterium that causes skin infections in humans, typically ulcereated lesions (disease known as Buruli ulcer). Buruli ulcer is the third most common mycobacterial infection of humans, after tuberculosis and leprosy, and treatment of infection is extremely difficult. Much of the effects of Buruil ulcer is due to secretion by M. ulcerans of an immunosuppressive toxin called mycolactone. Evidence suggests that mycolacotne functions by suppressing the immune system, however the specific effect of the toxin on immune cells is not well characterised.

This project will investigate in detail the action of mycolatone on the immune system, making use of purified mycolactone or M. ulcerans that is unable to produce the toxin. Using a mouse model of infection, we will examine the effect of mycolactone on the priming, proliferation and cytokine secretion of T cells, and also determine how mycolactone influences the trafficking of T cells within the host. We will also determine if mycolactone exerts its immunosuppressive effects via its action on dendritic cells, as these cells are critical for the activation of T cells required to control mycobacterial infections. These experiments will make use of transgenic T cells that specifically recognise M. ulcerans, which will allow the detailed analysis of mycolactone on T cell function. In addition, in vivo models of live cell imaging have been established in the laboratory of Professor Wenigner, which will allow visualisation of the effects of mycolatone on immune cells.

This project will give the Honours student experience in culture of mycobacteria, animal handling, vaccination strategies, use of in vivo imaging systems (2-photon and confocal microscopy) and a number of immunological techniques (cell isolation, ELISPOT, multi-parameter flow cytometry).

Developing improved BCG-based vaccines for vaccination against tuberculosis

Supervisor: Dr Jamie Triccas

Microbial Pathogenesis and Immunity Group

Infectious Diseases and Immunology

Blackburn Building, University of Sydney

Phone: 9351 6812,

E-mail: jamiet@infdis.usyd.edu.au

Tuberculosis remains a major human infection and more effective immunisation strategies are urgently required. The existing vaccine, Mycobacterium bovis BCG, confers variable protective efficacy against tuberculosis. Despite this, BCG remains one of the safest and most widely used of all vaccines. BCG is a powerful adjuvant and elicits long lasting immune responses, making it an attractive vaccine vehicle for the delivery of heterologous antigens. Therefore much focus is now being directed at producing new recombinant strains of BCG with enhanced protective efficacy.

Our lab is currently developing strains of BCG that overexpress protective Mycobacterium tuberculosis antigens or immunomodulatory molecules. For example, we have previously developed recombinant (r) BCG strains that secrete the cytokine GM-CSF and shown this vaccine can protect against M. tuberculosis in mouse models. Protection afforded by this vaccine correlated with the effect of GM-CSF on the expansion and activation of dendritic cells (DCs), which are the major antigen presenting cells required for the development of anti-mycobacterial immunity. However, protection afforded by BCG secreting GM-CSF was short lived, and we hypothesise that improving the survival of memory T cell would be critical for improving the protective effect of this vaccine. In this project rBCG strains will be constructed that secrete GM-CSF together with the cytokines interleukin 7 and interleukin 15, which are important for the survival of CD4 and CD8 memory T cells. Experiments will investigate the influence on DC numbers and T cell responses by the recombinant vaccines, with a particular emphasis on the generation of memory T cells produced in response to BCG. These experiments will make use of transgenic T cells that specifically recognise CD4 and CD8 T cell epitopes expressed by the rBCG vaccines. The project will also investigate if the rBCG strains can protect mice against challenge with M. tuberculosis, using models of aerosol infection established in the laboratory.

This project will give the student experience in molecular biology techniques, animal handling, vaccination strategies and various immunological techniques (tissue culture, assays of T cell activation including ELISPOT, multi-parameter flow cytometry).

Mycobacterial Research Group

Centenary Institute of Cancer Medicine & Cell Biology

Dr Manuela Florido and Professor Warwick Britton

The Mycobacterial Research Group studies the interaction between the immune system and Mycobacterium tuberculosis and Mycobacterium leprae, the causes of tuberculosis and leprosy, at a number of levels. These include the cellular and cytokine responses to infection, the genetic control of host response and the development of new vaccines against TB. The group includes senior research scientists, research officers, research assistants, PhD and honours students. It is housed in modern laboratories in the Centenary Institute for Cancer Medicine and Cell Biology with a dedicated PC3 facility for studies with M. tuberculosis.

Project 1: Interaction of viral and mycobacterial infections in the lung

Tuberculosis remains a major cause of death and morbidity throughout the world. Protection against Mycobacterium tuberculosis infection in the lung is dependent on the activation of CD4 and CD8 T cells in mediastinal lymph nodes and their recruitment back to the lung. Many other viral respiratory pathogens stimulate T cell responses in the lung and the interaction between different types of infection in the lung is poorly understood. This interaction may influence the effectiveness of vaccines against either mycobacterial or viral pathogens during infection with different types of pathogen. We plan to address these questions by using recombinant viruses, which express dominant CD4 and CD8 T cell epitopes from mycobacterial secreted proteins. Recombinant Influenza virus which stimulates a limited lung infection, has been developed. TcR transgenic mice whose lymphocytes recognize the dominant CD4 T cell epitope in mycobacteria, and tetramers to recignise specific CD8 T cells are also available. These will allow us to determine how mycobacterium-specific CD4 and CD8 memory T cell responses, which are stimulated by short-lived or persistent viral infections, influence T cell responses to primary infection with M. tuberculosis or the vaccine strain M. bovis (BCG). The nature of the memory responses to the different pathogens will be examined and the effect of these responses on protection against subsequent mycobacterial challenge. These studies are relevant to the design and implementation of more effective vaccines against tuberculosis using recombinant viral and mycobacterial vectors.

Research Techniques: Cellular immunology, experimental influenza and BCG infection cytokine analysis, RT-PCR and flow cytometry with tetramer analysis

Contacts:

Dr Manuela Florido

Tel: 9565 6163

Email: m.florido@centenary.org.au

Level 3, Centenary Institute, Bld 93, RPAH

Prof Warwick Britton

Tel: 9515 5210

Email: wbritton@med.usyd.edu.au

Molecular regulation of morphology and dendrite extension in dendritic cells.

Supervisor: Dr Lois Cavanagh

Immune Imaging Program (PI: Prof Wolfgang Weninger)

Centenary Institute

9565 6245

l.cavanagh@centenary.org.au

Dendritic cells: shape and function.

Dendritic cells (DC) are a rare cell population that resides in virtually all tissues of the body, where they function as a link between the innate and adaptive immune systems. DC are notably present in those organs that form the interface of the body with the external environment, including the skin, gut and mucosal surfaces. In these locations DC are ideally situated to encounter threats to the body from the outside world such as microbes. In epithelial surfaces, such as the epidermis of the skin, DC are visualized as large cells, with prominent dendritic processes that form interconnecting networks that are thought to be involved in antigen sensing. Indeed, recent studies in our laboratory reveal that de novo dendrite formation is involved in uptake of the protozoan parasite Leishmania major during infection of the skin. Although the dendricity of DC is well appreciated at the anatomical level, little is known about the molecular regulation of this distinctive morphology. The actin filament system performs a plethora of functions in eukaryotic cells, including cell motility, cytokinesis, intracellular transport and determination of cell morphology and cell size. Tropomyosins (Tm) are a family of actin-binding proteins whose role in the regulation of actin-myosin interactions in skeletal muscle is well understood. Recent studies have suggested that these proteins may also play an important role in the organization of the cytoskeleton, such as dendrite formation in neurons. Thus, the hypothesis of this project is that Tm regulate DC shape, and, consequently, their ability to sense and capture invading microorganisms. Identification of molecular cues involved in dendrite formation and DC migration, may provide means to manipulate immune responses against pathogens, or to improve vaccination strategies.

Project outline:

Actin filaments are not a homogeneous system, but rather are comprised of heterogeneous filaments composed of various isoforms of both actin and Tm. Since actin filaments are involved in cell morphology it is likely that Tm may determine the unique DC morphology, as well as contributing to the rapid response of DC to invading pathogens. The aims here are: i. to identify DC-specific Tm using available antibodies (in collaboration with Prof Peter Gunning, UNSW); and ii. to determine whether Tm are involved in the detection or capture of invading L. major parasites by dermal DC in the skin. Firstly, to examine the role of Tm in determination of DC morphology, epidermal and dermal sheet preparations of mouse skin will be prepared and stained with a panel of antibodies against different Tm isoforms and DC markers. This will ascertain the distribution of Tm in DC in situ. Next, DC in Tm-deficient mice will be examined. Skin will be prepared from Tm5NM1-knockout mice, and the distribution and morphology of DC examined. To determine whether Tms are involved in the sensing and uptake of L. major parasites, CD11cYFP x Tm5NM1KO mice will be infected intradermally with L. major, then 2-photon intravital microscopy will be performed. Results from these studies may provide an important molecular mechanism by which the dendritic morphology of DC is maintained in situ, as well as remodeled during pathogen encounter.

Techniques used: mouse handling; flow cytometry; immunocytochemistry; confocal microscopy; Leishmania infection; confocal microscopy; 2-photon time-lapse video microscopy.

Title: DIPEPTIDYL PEPTIDASE PROTEOMICS

SUPERVISORS

· Assoc Prof Mark D. Gorrell. Molecular Hepatology, level one, Centenary Institute.

· Dr Fiona Keane. Molecular Hepatology, level one, Centenary Institute.

m.gorrell@centenary.usyd.edu.au, ph 95656152

f.keane@centenary.usyd.edu.au, ph 95656115

PROJECT DESCRIPTION

Chronic liver diseases lead to accumulation of liver scarring and often progress to liver cancer. Such diseases include hepatitis viruses, biliary injury and fatty liver. Enzymes, because of their essential roles in specialised functions, are biologically interesting and useful targets of pharmaceautical research. The dipeptidyl peptidase IV (DPIV) gene family of multifunctional enzymes includes DPIV, which has a variety of roles in metalbolism, immunology, liver and cancer biology; and fibroblast activation protein (FAP), which is important in liver disease and cancer biology. The novel enzymes DP8 and DP9, cloned in our lab, are potentially involved in functions overlapping with DPIV and FAP and have attracted great interest worldwide. This 2010 project seeks to better understand the DP IV gene family. Our research group is expert in liver disease pathogenesis and the DPIV gene family.

We have gene knockout (gko) mouse strains including the DPIV gko mouse. These mice resist liver damage. We would like to understand the mechanisms of this improved health outcome. The approach in this project is to identify proteins that alter with the absence of DPs in vitro. We will prepare cells from WT and DP GKO mice and use them to identify potential substrates of DPs. We will verify substrates in vivo and in vitro.

SIGNIFICANCE

These experiments will provide novel insights into the functioning of the DPIV family in chronic liver injury.

METHODS

§ Culture of non-hazardous cell lines and protein extraction from these cell lines for 2-D gels and mass spectroscopy.

§ in silico data mining.

§ Enzyme assays. Western blot. Recombinant protein production.

References

1. Gorrell 2005 DPIV and related peptidases in cell biology and liver disease. Clin Science. 108:277-292.

2. Levy M, McCaughan G, etal, Gorrell M 1999 Fibroblast activation protein: a cell surface dipeptidyl peptidase and gelatinase expressed by stellate cells at the tissue remodelling interface in human cirrhosis. Hepatology 29:1768-1778.

3. Wang, XM, DMT Yu, GW McCaughan, MD Gorrell 2005 Fibroblast activation protein increases apoptosis, cell adhesion, and migration by the LX-2 human stellate cell line. Hepatology, 42: 935-45.
Title: DIPEPTIDYL PEPTIDASE GENETICS

SUPERVISOR

· Assoc Prof Mark D. Gorrell. Molecular Hepatology, level one, Centenary Institute.

m.gorrell@centenary.usyd.edu.au, ph 95656152

PROJECT DESCRIPTION

Chronic liver diseases lead to accumulation of liver scarring and often progress to liver cancer. Such diseases include hepatitis viruses, autoimmunity, biliary injury and fatty liver. Our research group is expert in liver disease pathogenesis and the DPIV gene family. Fibroblast activation protein (FAP) and Dipeptidyl Peptidase (DP) IV are important for energy metabolism, tumour growth and cirrhosis pathogenesis. DP8 and DP9 were discovered in our lab so we are comparing them with the related enzymes DPIV and FAP. This 2010 project primarily investigates the therapeutic potential of the DP IV gene family members as targets for therapeutic reversal of liver scarring. Our hypothesis is such that such SNP bearing humans suffer less severe chronic liver disease.

Genomic Single Nucleotide Polymorphisms (SNPs) sometimes encode an amino acid change that results in an altered protein that has altered function. There are known to be three SNPs of human FAP that we predict produce no active FAP in humans. The project will develop methods to detect these SNPs in humans and will begin to uncover the SNP frequencies. The project will begin with FAP then mine the internet for more SNPs of this gene family. The most interesting new SNPs identified will be further studied by making a corresponding mutant protein and testing its enzyme activity.

METHODS WILL INCLUDE

§ DNA extraction from cell lines following culture.

§ DNA extraction from human white blood cells

§ PCR and DNA sequencing

§ Internet [in silico] data mining.

§ Cell transfection, enzyme assays, flow cytometry, immunostains.

References

1. Gorrell 2005 DPIV and related peptidases in cell biology and liver disease. Clin Science. 108:277-292.

3. Wang, XM, DMT Yu, GW McCaughan, MD Gorrell 2005 Fibroblast activation protein increases apoptosis, cell adhesion, and migration by the LX-2 human stellate cell line. Hepatology, 42: 935-45

Honours Projects in the T cell Biology Lab, Centenary Institute

Prof Barbara Fazekas de St. Groth

T Cell Biology Group

Centenary Institute of Cancer Medicine and Cell Biology

Email: b.fazekas@centenary.usyd.edu.au

Phone: 9565 6137

Aims of the laboratory research program

The long term aim of our group is to understand how the immune system chooses the appropriate response to mount under each different circumstance. This choice involves interactions between dendritic cells and T cells, which are the focus of our lab. A multitude of diseases, generally grouped under the heading autoimmune diseases, result from inappropriate immune responses. These include juvenile diabetes, rheumatoid arthritis, thyroid disease, systemic lupus erythematosis, multiple sclerosis, vitiligo, psoriasis, Crohn’s disease and ulcerative colitis. Current treatments are only partially effective and have very significant side effects. Our aim is to prevent such diseases and/or to cure them, rather than providing long term treatments that do not effectively treat the underlying condition.

We work in a number of related areas and tailor our honours projects to the interests of the student as well as the most exciting areas of current research. Please come and talk to us about possible projects within the areas listed below.

Areas of research interest

Human regulatory T cells in autoimmunity, allergy and cancer

Based on studies in mice, it has been proposed that abnormalities in the regulatory T cell network underlie human autoimmune disease. Our recent data has shown for the first time that patients with inflammatory bowel disease have deficiencies in regulatory T cell numbers. We plan to extend these studies to a number of other autoimmune diseases, using peripheral blood samples provided by our collaborators at Royal Prince Alfred hospital. We currently have access to peripheral blood samples from patients with vitiligo, psoriasis, inflammatory bowel disease, systemic lupus erythematosis, multiple sclerosis and many different types of cancer. Leukocytes will be purified and frozen. The number and phenotype of Treg cells will be measured on batches of thawed cells using 8-colour flow cytometry. Results will be correlated with clinical parameters.

Studies in mouse models

We use TCR transgenic mouse models to study how CD4 T cell responses are controlled.

Current projects include:

· Collaboration of T and B cells in the generation of IgE and allergic asthma

· A new paradigm of organ graft rejection and its suppression by regulatory T cells

· Control of DC activation by regulatory T cells

Techniques

These projects will give the student experience in research on patient samples, lymphocyte purification and labelling, tissue culture, 8-colour flow cytometry, use of mouse models in research.

Preventing breast tumour growth using oncogene-induced senescence of angiogenic blood vessels.

Contact details of supervisor:

Dr Matthew Grimshaw,

Vascular Biology, Centenary Institute, University of Sydney.

Tel: 02 9565 6226. Email: M.Grimshaw@centenary.org.au

Outline of project:

To grow and spread, tumours need to develop blood vessels by a process known as angiogenesis; drugs that stop angiogenesis can prevent or delay tumour growth.

We have identified a novel way in which the body attempts to prevent angiogenesis and tumour growth - the tumour environment stimulates ‘oncogene-induced senescence’ of the blood vessels, thus preventing their growth, which in turn stops tumour growth. We have recently identified a novel gene - SENEX - that may control this response in breast cancer. The aim of this project, therefore, is to test whether SENEX controls breast tumour angiogenesis via oncogene-induced senescence of endothelial (blood vessel) cells.

To do this, we shall over-express or deplete SENEX in human endothelial cells and then test how these cells with altered SENEX levels produce blood vessels in vitro. As well as modifying SENEX expression, the cells will be labelled with a fluorescent protein so that we can use confocal microscopy to ‘watch’ the cells migrate, line-up and form vessels in real-time.

Methods:

Cell culture of human umbilical vein endothelial cells, viral transfection of SENEX, RNAi knockdown of SENEX, in vitro angiogenesis assays, and confocal microscopy with real-time imaging. Quantative real-time PCR and Western blotting will be used to test the levels of SENEX in the endothelial cells.

Role of micro-RNAs in B cell differentiation and cancer

Dr Chris Jolly, DNA Repair Lab, Centenary Institute

c.jolly@centenary.org.au

ph 9565 6188

Project Overview

Micro-RNAs are a newly-discovered class of gene regulators, and play critical roles in tissue differentiation and cancer. miRNAs regulate gene expression by binding to the 3’-untranslated regions of mRNAs; thereby suppressing translation and/or inducing mRNA degradation. Abnormal expression of particular miRNAs is now known to play a key role in the development of cancers derived from B cells. One micro-RNA can regulate expression of many hundreds of genes and there are now known to be many hundreds of micro-RNAs. At present, the standard way to identify roles for miRNAs in development and cancer is to use gene-targeting to knock out each miRNA one-by-one. It will take a very long time indeed to study all miRNAs using this approach!

An alternative, higher-throughput way to determine the roles of miRNAs in cell differentiation and cancer is to use retroviral-mediated over-expression of miRNAs in vivo to identify phenotypic changes and to identify target genes. My lab routinely uses recombinant retroviruses to express a gene of interest (plus green fluorescent protein - GFP) in B cells in vivo. We use GFP expression to track transduced cells and see how expression of the gene of interest alters cell differentiation in vivo.

The honours student will set out to achieve 4 goals:

(1) use micro-array analysis to identify miRNAs that are differentially-expressed in B cells at various stages of B cell development in vivo.

(2) clone a number of miRNAs selected from the experiment in (1) into retroviral vectors and transduce B cells in vivo.

(3) use flow cytometry and histochemistry to identify changes in B cell development induced by specific miRNA over-expression

(4) use micro-array analysis to identify target genes whose expression is altered in the B cells by miRNA over-expression

Methods

· micro-array analysis

· gene cloning and production of recombinant protein

· transduction of primary mouse B cells with recombinant retrovirus

· adoptive transfer of transduced cells into host mice

· analysis of host mice using histochemistry and flow cytometry

Mutation of Antibody and Cancer Genes and the Cell Cycle

Dr Chris Jolly

02 9565 6188

c.jolly@centenary.org.au

Somatic hypermutation of antibody (Ig) V regions, and recombination (class switching) between Ig S regions, occur in B cells activated in response to infection. This physiological process diversifies the infection-specific antibody produced, which ultimately leads to the production of more effective antibodies, but it is also linked to cancer. Hypermutation and class switching are both dependent on the activation-induced cytidine deaminase (AID) protein. AID directly deaminates deoxycytidine (dC) bases in genes, converting the targeted dCs to deoxyuracil (dU). dU bases are not usually tolerated in DNA because they mimic dT bases. Mutation of dC to dU is a common event in all cells, regardless of AID activity and is normally repaired with high fidelity. However, processing of the dU bases produced by AID either converts the “temporary” dC to dU mutation into a permanent mutation, or leads to mutation of nearby dA•dT base pairs. Both outcomes involve DNA breakage and recombination. Although the antibody gene mutation induced by AID is vital for health, AID contributes to cancer (1) by occasionally initiating translocation of proto-oncogenes to the Ig loci, and (2) by directly mutating oncogenes.

My lab has just produced exciting data showing that AID-induced mutations become permanent in activated B cells because DNA repair pathways are recruited in abnormal phases of the cell cycle. This discovery provides a key to understanding why AID activity causes mutation and cancer. Activated B cells are suspected to cycle extremely quickly in vivo, although there is scarce data to prove this. New technologies now allow us to watch cells move through the cell cycle in real time. This project will track B cell division and cell cycle progression in vivo using unique GFP-expressing retroviruses and state-of-the-art dual photon microscopy.

Techniques that will be used in conducting the project:

· construction of new GFP-expressing retroviruses and transduction of primary mouse B cells

· adoptive transfer of GFP-tagged mouse B cells to immunised hosts

· analysis of cell proliferation and cell cycle progression using flow cytometry, confocal microscopy and dual-photon microscopy

Visualisation of extracellular matrix remodeling during inflammatory responses in the skin.

Supervisors: Dr Paul Mrass

Immune Imaging Program (PI: Prof Wolfgang Weninger)

Centenary Institute

9565 6247

p.mrass@centenary.org.au

2-photon microscopy – a novel view of tissues and the immune system.

Innovative 2-photon (2P) imaging technology has recently expanded the horizons of immunology, by facilitating the understanding of how, when and where cells interact within living tissues. Using 2P microscopy, the organization of immune cell interactions in space and time can be examined in a variety of tissues. Data from previous studies have elucidated how immune cells interact during the initiation of immune responses, and during delivery of effector function at peripheral sites, for example tumours. A unique feature of 2P microscopy is the direct visualization of highly ordered extracellular matrix (ECM) fibres, such as collagen, by virtue of second harmonic generation signals. This process requires no labeling of the tissue, yet enables very clear images of the organization of the ECM at the structural level. The Immune Imaging lab at Centenary Institute is currently setting up a state-of-the-art 2P microscope, the first of its kind in Australia. Senior lab personnel have substantial experience in imaging of various organs in a number of disease and inflammatory models. Our setup includes a new laser with emission in the infrared wavelength range (up to 1200 nm). This enables the highlighting of ECM fibres at a detail that has not been reached before – thus, we are capable of generating a novel insight into the three-dimensional organisation of macromolecules that will provide a new understanding of the ECM both in steady state and disease.

Project outline:

The ECM serves several purposes: 1. It provides the structural backbone that holds tissues together; 2. It acts as storage for proinflammatory mediators, such as cytokines and chemokines; and 3. It serves as a guiding scaffold for migrating leukocytes. Despite these important functions, we have little understanding as to the three-dimensional organization of ECM fibres. Moreover, inflammation or tissue injury is known to alter the structure of the ECM. For example, chronic UV-irradiation of the skin leads to disorganization or loss of ECM fibres, and chronic exposure to corticosteroids leads to thinning of the skin as well as collagen reorganization. This knowledge is based mainly on histologic sections of the skin in various conditions, which does not provide any information on the 3D organization of the ECM. In this project, the ECM within the skin of mice will be examined by 2P microscopy. The structure of the skin will be studied in normal mice or mice that were exposed to UV irradiation, corticosteroids or during delayed type hypersensitivity responses. The ECM will be imaged, then reconstructed on the computer. In addition, interactions of immune cells with ECM under these conditions can be observed by the use of various mice that express fluorescent proteins in different immune cell subsets, such as T cells or dendritic cells. This will allow us to determine whether the nature of immune cell-ECM interactions change in the different experimental conditions. Results from these studies may not only provide important insights into inflammatory diseases that affect the skin, but may serve as a paradigm of tissue architecture remodeling during inflammation.

Techniques used: mouse handling; induction of skin inflammation; DTH responses; flow cytometry; 2-photon time-lapse video microscopy; off-line video analysis.

Mycobacterial Research Programme

Tuberculosis (TB) is an enormous global health problem, with 9 million new cases of TB a year, and almost 2 million deaths. Our group is focused on investigating the Host response to TB infection, in particular inflammatory responses and how macrophages kill TB.

Project: Characterization of human-specific anti-TB pathways

Macrophages are key cellular components of the innate immune system that activate anti-microbial pathways upon recognizing pathogens through Toll-like Receptors (TLRs), as well as other recognition systems. We have identified genes that are regulated by TLR ligands in macrophages from humans, but not mice (and vice versa).

AIM: This project will assess the expression of “human-specific” genes in human macrophages in response to M. tuberculosis and study the function of selected genes in these macrophages during infection.

Human macrophages (HMDM) will be infected with M. tuberculosis or M. bovis BCG, or treated with heat-killed bacteria. Expression of “human-specific” TLR-regulated genes will be assessed over a time course by microarray, qPCR (mRNA) and western blotting (protein). We have some preliminary data showing that a number of novel genes in the autophagy and ion transport pathways are upregulated in TB infected macrophages. These two pathways are known to be important in the anti-microbial response of macrophages to TB infection.

From these initial studies we will investigate 1 or 2 highly upregulated “human-specific genes induced by TB infection. We will characterize the function of these genes using siRNA or antibodies to inhibit expression in HMDM. Intracellular bacterial loads, macrophage survival and production of cytokines and chemokines will be assessed.

OUTCOMES The characterization of novel anti-microbial pathways in human macrophages may lead to the identification of mechanisms for enhancing host anti-microbial responses against this important human pathogen.

This project will give an honours student experience in both cellular and molecular techniques. Including; isolation of human and murine macrophages, purification of single cell suspensions, analysis of cell activation through; Flow cytometry, ELISA, biological assays measuring macrophage activation and apoptosis and changes in gene expression using real-time quantitative PCR and microarrays, and with siRNA technology.

Contact:

Dr Bernadette Saunders Centenary Institute

Ph 9565-6114

Email: B.Saunders@centenary.org.au

Liver Immunobiology Laboratory, Centenary Institute

Project Title: Genetic factors that predict liver disease amongst excessive alcohol drinkers: a pilot genome wide analysis using Illumina single nucleotide polymorphism (SNP) chips

Name and contact details of Supervisor (location, telephone and email address)
Dr Devanshi Seth

Drug Health Services & Centenary Institute, Missenden Rd., Camperdown 2050.

Ph: 9515 7201; Fax: 95158970; Email: Devanshi.seth@email.cs.nsw.gov.au

Progression of multi-stage liver disease in heavy drinkers.

A BRIEF overview of the project: In Australia, 61% of all alcohol consumed is at risky/high risk levels. Alcoholic cirrhosis (ALC) is the most prominent endpoint and contributes to 5% of the total disease burden, 50% of the total liver disease burden and 15% of liver transplants with an estimated total cost of AUD3.8 billion per year. Despite the recognition of alcohol as an important cause of liver cirrhosis, treatment remains unsatisfactory with abstinence being the only effective treatment. It is unknown why a small proportion (8-15%) of heavy and prolonged alcohol users develop ALC (Fig). Environmental co-factors fail to explain the ‘between individual’ variability in the development of ALC. Twin studies reveal that underlying genetic factors are associated with risk for ALC, but search for single gene changes has been inconclusive. We hypothesize that susceptibility to ALC, like other multi-factorial complex diseases, is controlled by a number of genes each of which makes a small overall contribution and therefore, a genome-wide association (GWA) approach is more likely than the single gene approach to identify small to moderate risk genetic variants associated with ALC. It is important to determine (i) whether there is a genetic component to susceptibility between individuals (ii) which genes are responsible for the susceptibility and (iii) whether identification of such genes will lead to discovery of novel diagnostic and/or therapeutic targets. This project will use HumanOmni1-Quad bead chip from Illumina in a high-throughput genome-wide search for genetic changes called single nucleotide polymorphisms (SNPs) to identify multiple genetic risk factors associated with ALC. Blood DNA from clinically characterised Controls (heavy drinkers without ALC) matched for age, gender, alcohol consumption to Cases (heavy drinkers with ALC) will be subjected to SNP-GWA analysis. Statistical analysis will be performed to define p values, false positive rates and identify novel and confirm known gene associations. SNP data will be analysed using multivariate logistic regression to test for association between genotype and phenotype using age and gender as covariates. This is the first study of its kind in the world. It will provide a basis for the discovery of novel genetic risk factors associated with progression of ALC.

Techniques to be used in this project

· DNA: isolation from blood, quality check

· GWA: Illumina SNP chip hybridization

· Statistical analysis

Liver Immunobiology Laboratory, Centenary Institute

Project Title: To investigate Osteopontin mediated MAP Kinase and Akt signaling in liver cells in response to alcohol

Name and contact details of Supervisor (location, telephone and email address)
Dr Devanshi Seth

Drug Health Services & Centenary Institute, Missenden Rd., Camperdown 2050.

Ph: 9515 7201; Fax: 95158970; Email: Devanshi.seth@email.cs.nsw.gov.au

Opn-Opn receptor mediated signaling and cellular functions. Opn can initiate Arg-Gly-Asp (RGD)-dependent and RGD-independent interactions with integrin and CD44, respectively. This mediates anti-apoptotic signals on CD44 binding, through Akt phosphorylation, leading to cell survival. Binding of Opn to integrins (avb3) , enhances Erk phosphorylation and activates AP-1 dependent expression of uPA, plasmin and MMPs leading to increased motility. ∆ Thrombin (IIa) cleavage exposes and increases Opn binding to integrins (a9b, a4b). Blocking antibodies/inhibitors () will be used to investigate pathways in this project.

A BRIEF overview of the project

We have shown for the first time that Osteopontin (Opn) is over-expressed in response to alcohol in human, in vitro and in vivo models. Our research findings strongly suggest that Opn is an important and essential part of alcoholic liver injury. Studies in breast cancer cells reveal that Opn-Opn receptor interactions invoke several signaling pathways leading to increased cellular and biological functions (see Fig). The mechanism of Opn signaling has not been defined in liver disease and is an important precursor to developing therapeutic strategies. The interactions of Opn with its receptors (CD44, several integrins) are complex. This project will utilize alcohol as a model of liver injury (in vitro) to investigate and delineate signaling pathways responsible for specific biological and cellular functions (described in the figure) important in the development of disease. Identification of these specific mechanisms/pathways will guide the development of a therapeutic strategy.

Techniques to be used in this project

· Liver cell culture: Alcohol treatment, Blocking antibody/Inhibitor treatment

· RNA: siRNA, Q-PCR

· Protein: Western blot, Signaling, Immunohistochemistry, Kinase assay, ELISA

· Functional assays: Cell migration, Plasmin activity, Fibrinolysis

Role of Bone-Marrow Derived Cells in Progressive Liver Injury and Carcinogenesis

Supervisors: Dr N Shackel and Dr F Warner

Liver Cell Biology http://www.centenary.org.au/p/whatwedo/liver/liverimmunobiology/LiverCellBiology/

Contact:

Dr. Nick Shackel n.shackel@centenary.usyd.edu.au 95656286

Dr. Fiona Warner f.warner@centenary.usyd.edu.au 95656268

Background: The promise of hepatic stem cells is impressive given their potential as therapy for a variety of liver diseases(1). There has been a recent explosion of research in the area of both general and liver stem cell biology and this has led to considerable debate about the origin and function of hepatic stem cells. Further, basic concepts surrounding stem cell biology including - “plasticity” and “transdifferentiation” has been introduced in an attempt to explain the role of resident and bone marrow derived stem cells in hepatocyte repopulation of the liver. These concepts have an underlying assumption that is not proven, that there are distinct liver specific stem cell precursors that are important in liver regeneration following injury(1).

The bone marrow response to liver injury is not well understood. While it has been shown that there is a bone marrow stem/progenitor cell contribution to liver injury, the responding cells are poorly characterized and their physiological relevance remains unclear.

Hypothesis and aims: We hypothesize that there is a specific bone marrow stem cell mobilization with liver injury. Furthermore we hypothesize that bone marrow derived stem cells that mobilize with liver injury may contribute to the development of liver cancer.

Methods: The initial phase of this project will compare the expression of stem cells markers on a rodent stem cell line using immunohistochemistry/immunofluoresence and flow cytometry. Further we plan to use conditioned media to study the events involved in the differentiation of the stem cell line into hepatocytes, endothelial and cholangiocyte cell lineages. The final part of this project will study the effects of the stem cells administered to rodents in models of liver injury.

Skills: This project will utilise real-time RT-PCR, cell isolation, magnetic bead separation and flow cytometry techniques. It is envisaged that the student who undertakes this project will become proficient in all of these methods whilst being exposed to a number of other general laboratory techniques.

Conclusion and Significance: Our results to-date support the hypothesis that a population of liver-specific stem cells reside within the bone marrow and are recruited to the liver with injury. It now needs to be determined what role these recruited cells have in liver pathobiology.

This project has great significance in determining the nature of the molecular events responsible for the differentiation of stem cells into liver cell lineages. The results will have widespread implications for the majority of human disease, as this is likely to be applicable to general stem cell responses in liver injury. Importantly, the project as outlined will be suitable for publication. This project embodies a number of techniques and builds on established knowledge and expertise with our laboratory. We look forward to attracting a student to what we believe is exciting and significant work!

1. N. A. Shackel and D. C. Rockey (2005) In pursuit of the "Holy Grail"- Stem cells, hepatic injury, fibrogenesis and repair. Hepatology 41(1) p16-8.

The role of Cyclophilin and Extracellular Matrix Metalloproteinase (EMMPRIN) interactions in liver injury.

Supervisors: Dr N Shackel and Dr F Warner

Liver Cell Biology http://www.centenary.org.au/p/whatwedo/liver/liverimmunobiology/LiverCellBiology/

Dr. Nick Shackel n.shackel@centenary.usyd.edu.au 95656286

Dr. Fiona Warner f.warner@centenary.usyd.edu.au 95656268

Background: Human liver disease is a common cause of morbidity and an increasing cause of mortality in our community. Liver disease, from various causes, is characterised by a remarkably consistent sequence of events in which inflammation drives extracellular matrix (ECM) remodelling of the liver, to give the scar formation that is the hallmark of cirrhosis. The pivotal cell in this process is the hepatic stellate cell (HSC). However, the dominant cell type in the human liver is the hepatocyte. Further, liver injury is always driven by an inflammatory response. The role of the inflammatory infiltrate in driving hepatocyte injury and the effect of this process on fibrosis development is largely unknown.

EMMRPIN is an abundant glycoprotein and a potent inducer of matrix metalloproteinases (MMPs) in malignant cells and fibroblasts. Cyclophilins are the ligands for EMMPRIN and powerful chemoattracants for inflammatory cells. Following gene array analysis of human liver we identified increased expression of EMMPRIN in cirrhosis(1, 2). Further, we have shown inflammatory cells up-regulate EMMPRIN expression and cyclophilin production. The proposed project will address the inter-relationship between EMMPRIN and cyclophilins in liver inflammation and fibrogenesis.

Hypothesis and aims: We hypothesize that Cyclophilin and EMMPRIN interactions are important determinant of liver fibrosis and inflammation. Our specific aims with this project are; (1) characterise EMMPRIN expression on the surface of inflammatory cells and its functional interaction with cyclophilins and (2) characterise inflammatory cell EMMPRIN and cyclophilin interactions with hepatocytes.

Methods: EMMRPIN and cyclophilin expression and function will be studied in both in-vivo in animal models of liver injury and in-vitro in cell culture systems.

Skills: We plan to utilize a number of cell isolation and culture techniques including study of primary cells, cell lines and knockout animals This project will utilise real-time RT-PCR, cell culture, transfection, flow cytometry, immunohistochemistry and western blot techniques. It is envisaged that the student who undertakes this project will become proficient in all of these methods whilst being exposed to a number of other general laboratory techniques.

Conclusion and significance: This project will answer important questions about liver injury development whilst exposing the student to a broad range of laboratory skills. Importantly, the project as outlined will be suitable for publication. This project embodies a number of techniques and builds on established knowledge and expertise within our laboratory. We look forward to attracting a student to this exciting and significant work!

1. Shackel NA, McGuinness PH, Abbott CA, Gorrell MD, McCaughan GW. Identification of novel molecules and pathogenic pathways in primary biliary cirrhosis: cDNA array analysis of intrahepatic differential gene expression. Gut 2001;49:565-576.

2. Shackel NA, McGuinness PH, Abbott CA, Gorrell MD, McCaughan GW. Insights into the pathobiology of hepatitis C virus-associated cirrhosis: analysis of intrahepatic differential gene expression. Am J Pathol 2002;160:641-654.

The role of Extracellular Matrix Metalloproteinase (EMMPRIN) binding partners in the development of human liver injury.

Supervisors: Dr N Shackel and Dr F Warner

Liver Cell Biology http://www.centenary.org.au/p/whatwedo/liver/liverimmunobiology/LiverCellBiology/

Dr. Nick Shackel n.shackel@centenary.usyd.edu.au 9565 6286

Dr. Fiona Warner f.warner@centenary.usyd.edu.au 9565 6268

EMMPRIN is an abundant glycoprotein and a potent inducer of MMPs in malignant cells and fibroblasts. Following gene array analysis of human liver we identified increased expression of EMMPRIN in primary biliary cirrhosis (PBC) and hepatitis C virus (HCV) associated cirrhosis (1, 2). In addition to its role in induction of MMPs EMMPRIN binds proteins both in the circulation and on the cell surface to increase their bioavailability. In other organ systems EMMRPIN binding partners determine many of the proteins function. Therefore, in the liver these binding partners are likely to affect hepatic EMMPRIN function and ECM remodelling.

Hypothesis and aims: We hypothesize that EMMPRIN is an important intrahepatic mediator of ECM remodelling and that its interaction with other proteins are pivotal in the development of cirrhosis. Our aim with this project is to identify proteins, which bind to EMMPRIN and investigate the cell biology of interactions between EMMPRIN and its binding proteins.

Methods: EMMPRIN binding partners will be identified using proteomic techniques, immunoprecipitation, SDS PAGE, Western blotting and Mass Spectrometry. EMMPRIN and its protein-binding partners will be studied in hepatocytes using Western blotting, immunofluorescent labelling and confocal microscopy techniques.

Skills: This project will utilise real-time RT-PCR, cell culture, transfection, primary cell isolation, immunohistochemistry, microscopy, Western blotting techniques and Mass Spectometry. It is envisaged that the student who undertakes this project will become proficient in all of these methods whilst being exposed to a number of other general laboratory techniques.

Conclusion and significance: This project will answer important questions about EMMPRIN and its role in cirrhosis development whilst exposing the student to a broad range of laboratory skills. Importantly, the project as outlined will be suitable for publication. This project embodies a number of techniques and builds on established knowledge and expertise with our laboratory. We look forward to attracting a student to, what we believe, is exciting and significant work!

THE IMMUNE RESPONSE AGAINST TUMOURS:

Role of antigen-presenting dendritic cells and antigen-specific CD4 T cells in controlling tumour growth in vivo

Supervisor: Dr. Elena Shklovskaya

Senior Research Officer

T Cell Biology Group (group head, Prof B Fazekas de St Groth)

Centenary Institute of Cancer Medicine and Cell Biology

Email: e.shklovskaya@centenary.org.au

Phone 02 9565 6198

You are interested in basic immunology and want to do some state-of-the-art research? OR you really want to focus on cancer? With this project, you can do both. You will gain a good understanding of how the immune system works, how to manipulate immune responses in vivo and why tumours don’t get rejected, by performing original research in a mouse model of cancer. Many aspects of anti-cancer immunity are still poorly understood.

The AIM of this project is to understand the three-way relationship between antigen-presenting dendritic cells (DCs) that acquire and present a tumour-derived antigen (Tag), CD4 T cells specific for this Tag and the tumour that makes Tag. The GOAL is to manipulate DCs such that better immune responses against the tumour are achieved.

This project is built on our expertise in the areas of dendritic cell biology and CD4 T cell immune responses. This year, we have made several tumour cell lines producing different versions of model Tag, and confirmed that Tag-specific T cells recognise Tag-expressing tumours in vitro and in vivo. Next year, this system will be applied to study the role of DCs in activating Tag-specific T cells in tumour-bearing mice. There are 9 different subsets of DCs in mice. We believe that many of the 9 DC subsets induce immunity, however some DCs can induce tolerance (antigen-specific unresponsiveness). Part of your project will be to find out which subset(s) of DCs are good at presenting Tag and turning on the immune response against the tumour, and which DCs are suppressing the immune response. DC subsets will be then specifically targeted for activation or elimination, as appropriate.

The project will involve:

- subcutaneous injection of tumour cells into mice and monitoring tumour growth

- injection of T cells specific for the model antigen into mice and monitoring T cell responses

- assessment of DC phenotype and activation

- in vivo and in vitro experiments analysing T cell activation and proliferation

List of techniques that will be used in conducting the research

In vitro culture of tumour cell lines

Mouse in vivo techniques (vaccinations, intravenous injections, isolation of mouse lymph nodes and spleens, labelling of cell suspensions with CFSE)

Flow cytometry 7-10 colour – remember we have the best Flow facility in Australia!

FACS sorting

Monoclonal antibody purification and conjugation (optional)

Confocal microscopy (possibly)

Vascular Biology Research Program.

One of the most exciting scientific findings in recent years has been the discovery of small regulatory molecules known as microRNAs. These molecules control various biological pathways such as cell death growth and differentiation.

The Vascular Biology Research Program is interested in how these microRNAs are involved in the management of blood vessels. The cells lining blood vessels are known as endothelial cells and the creation of new blood vessels from pre-existing vasculature is defined as angiogenesis.  We have identified a subset of microRNAs which are rapidly expressed in proliferating endothelial cells. We have shown that these microRNAs are able to regulate angiogenesis particularly in the setting of tumour angiogenesis. Understanding how and what controls the expression of these miRNAs will aid in their development as drugs for the inhibition of tumour growth.

MicroRNA production occurs in three distinct stages, nuclear processing, export and cytoplasmic cleavage. There is now evidence that various miRNAs are processed differently in the nucleus. Moreover, many miRNAs are retained in the nucleus and are not exported to the cytoplasm for final cleavage. However the mechanisms for this selective processing and block in export remain unknown.

 We will investigate the role of the processing of these specific miRNAs in endothelial cells. We will elucidate their post-transcriptional regulation and the protein factors controlling the expression of the miRNAs.

The project methods include cell culture of endothelial cells, Real-Time PCR, nuclear run on assays, primer extension, Northern blots, siRNA knock down and cell biology methods.

Contact details of Supervisors:

The Centenary Institute, Sydney University.

Phone: 9565 6226

§         Dr. Nham Tran                        n.tran@centenary.org.au

§         Prof. Jenny Gamble               j.gamble@centenary.org.au

The Role of Biliary Epithelial to Mesenchymal Transition (EMT) in Liver Fibrosis

Liver Cell Biology http://www.centenary.org.au/p/whatwedo/liver/liverimmunobiology/LiverCellBiology/

Supervisors: Dr F Warner & Dr. N Shackel

Contact: f.warner@centenary.usyd.edu.au
Ph 9565 6268

Liver fibrosis leads to considerable morbidity and mortality in our community. Most liver diseases result from injury to hepatocytes (the main liver epithelial cell) and cholangiocytes (bile duct epithelial cells). The tissue repair response that ensues following liver injury to epithelia involves inflammatory cells, and mesenchymal cells, phenotypically transformed interstitial fibroblasts (hepatic stellate cells (HSC) and myofibroblasts), that produce transforming growth factor-b1 and are responsible for collagen deposition and fibrous tissue formation. Traditional studies of fibrosis have focused on fibroblasts as the critical cell in the production of extracellular matrix, but recently studies suggest epithelia contribute to the process by creating new fibroblasts.

Of interest to our group is the cholangiocyte, which is important in monitoring bile composition. Newly formed bile ducts resulting from proliferation of pre-existing bile duct epithelial cells are surrounded by smooth muscle a-actin (a-SMA) positive myofibroblasts. Controversy exists as to the origin of these cells and whether these cells differ from myofibroblastic HSC. Recently, a sub-population of HSC isolated from fibrotic liver, were shown to display cell markers of both fibroblast and cholangiocytes. These observations pose the question of whether fibroblast populations of cells can originate from cholangiocytes.

Emerging evidence suggest that fibroblasts can derive from epithelial cells in adult tissues by a process known as Epithelial-Mesenychmal Transition (EMT). Studies of renal fibrosis suggest that more than 30% of all disease-related fibroblasts originate from tubular epithelia at the site of injury. Cholangiocytes show many similarities to renal tubular cells in that they display a simple columnar epithelial phenotype and have a basement membrane. Whether cholangiocytes in adult liver are capable of undergoing EMT remains unknown.

Central hypothesis: Cholangiocytes have the potential to undergo EMT to become fibroblasts in response to injury and therefore contribute to liver fibrosis. The aim of this proposal is: To determine if primary cultures of normal rat cholangiocytes in the presence of TGF-b1 undergo key EMT events to attain a functional fibroblast phenotype. (Fig 1)

Skills: This project will utilise primary cell isolation, real-time RT-PCR, flow cytometry, enzyme zymography and tissue immunohistochemistry/histology. It is envisaged that the student who undertakes this project will also become proficient in general laboratory techniques.

Conclusion and Significance: This project has great significance in identifying if cholangiocytes can undergo EMT to an fibroblast phenotype through a series of defining events; de novo expression of a-SMA, loss of E-cadherin, transformation of a fibroblastic morphology and production of extracellular matrix components. This proposal will provide information towards ultimately understanding the role of cholangiocytes in fibrogenesis. This project embodies a number of techniques and builds on established knowledge and expertise with our laboratory.

The Role of Inflammatory Response in Regulating the Hepatic Renin-Angiotensin System

Liver Cell Biology http://www.centenary.org.au/p/whatwedo/liver/liverimmunobiology/LiverCellBiology/

Supervisors: Dr F Warner and Dr. N Shackel

Contacts: f.warner@centenary.usyd.edu.au 9565 6268

n.shackel@centenary.usyd.edu.au 9565 6286

The renin angiotensin system (RAS) is a fundamental hormonal system that has been shown to contribute to the pathogenesis of liver fibrosis^1,2.

The response following liver injury involves the infiltration of inflammatory cells and matrix remodelling. Each of these cellular events in the microenvironment of repair is associated with molecular events that lead to the de-novo generation of Angiotensin II. Following liver injury, both classical and counter-regulatory arms of the RAS are up-regulated and characterised by distinct temporal patterns of expression. The classical pathway is activated within 24 hrs of injury whilst the counter-regulatory pathway lags by 3 wks^A7, suggesting differential regulation of these pathways that parallels the progression of inflammation.

Therefore, the Hypothesis of this Project is that following liver injury the classical and counter-regulatory pathways of the RAS are differentially regulated by the inflammatory response.

In this aim we plan to dissect the inflammatory response from fibrogenesis and determine its role in regulating the classical (angiotensinogen, ACE, Ang II, AT1R) and counter-regulatory (ACE2, Ang(1-7), Mas) pathways using two mouse models that develop acute hepatic inflammation in the absence of fibrosis. Two models will be used. Concanavalin A, a model of acute liver inflammation which is predominately CD4 T-cell mediated. The other model, a transgenic mouse model of CD8 cytotoxic lymphocyte-initiated hepatitis. Comparison of these models will determine whether the counter-regulatory arm of the RAS is modulated by acute hepatic inflammation and the role of CD4 versus CD8 T-lymphocytes in this process.

Skills: This project will involve using two mouse models of liver injury. Real-time PCR and in-situ hybridization will be used to detect and localise enzyme and receptor expression. Cytokines (interferon ã, TNFá, IL-4, IL-5, IL-17, IL-22) & chemokines (RANTES, MCP-1, IP-10) will be measured by ELIZA and flow cytometry. It is envisaged that the student who undertakes this project will also become proficient in general laboratory techniques.

Overall Outcome The basic mechanisms and cell biology factors regulating the expression of hepatic ACE2 and the counter-regulatory pathway remains poorly characterised. Ultimately, understanding the mechanisms regulating ACE2 and its role in the counter regulation of the classical RAS pathway will allow us to dissect the roles of these pathways in normal and injured liver and to develop novel strategies to minimise or resolve inflammation and fibrosis.

1. Herath CB., Warner F.J., (2007) J Hepatology 47:387

2. Warner F.J, et al. (2007) Clin Science 113: 109

Regulation of the Bone Marrow Microenvironment by G-CSF: Effects on Normal and Malignant Lymphopoiesis

Dr Linda Bendall, Westmead Millennium Institute, Ph-9845 9069.

Email-linda_bendall@wmi.usyd.edu.au

The Leukemia Cell Biology Group is offering an honours project examining the potential risks and benefits of modulating the bone marrow microenvironment by the administration of the cytokine, granulocyte colony-stimulating factor (G-CSF). G-CSF is used to support patients suffering immuno-suppression as a result of therapy they are receiving for treatment for malignancies including acute lymphoblastic leukemia (ALL). G-CSF supports the innate immune system, reducing the length and severity of neutropenia. However, G-CSF suppresses bone marrow lymphopoiesis. Immature lymphoid cells die as a result of indirect effects of G-CSF on the bone marrow. The mechanism responsible for the death of these cells is currently unknown but is thought to relate to changes in the stem and progenitor cell microenvironmental niches in the bone marrow. In contrast, using a NOD/SCID mouse model of human ALL, we found that G-CSF treatment resulted in an increase in the extent of the disease. G-CSF cannot be acting directly on the ALL cells because they do not express the G-CSF receptor. This raises two major questions. 1. Why do ALL cells increase in number in a G-CSF treated microenvironment, while normal lymphoid progenitors die? Does the administration of G-CSF to ALL patients increase their risk of disease relapse? This project will determine the prevalence of this response of ALL to G-CSF, and investigate why G-CSF suppresses normal but enhances malignant lymphopoiesis.

The NOD/SCID mouse model of ALL, which is established in our laboratory, will be used to determine the proportion of ALL samples respond to G-CSF with increased progression. In vitro studies will be used to investigate the mechanisms responsible for the altered ALL cell growth. Since the effects of G-CSF are clearly indirect this will involve co-culture studies of normal bone marrow with ALL cells. In addition, the bone marrow of G-CSF and control treated mice will be compared to determine what factors are changed in response to G-CSF.

This project will provide important information regarding the safety of G-CSF administration to patients with ALL. It will also provide training in a number of techniques listed below.

Techniques

Animal model of human ALL including injections and blood collection.

Flow cytometry – up to 7 colour.

Tissue Culture

Quantitative RT-PCR.

The Role of Sphingosine-1-Phosphate (S1P) in Acute Lymphoblastic Leukaemia

Dr Linda Bendall and Dr Nadia Harun, Westmead Millennium Institute, Ph-9845 9069.

Email-linda_bendall@wmi.usyd.edu.au

The Leukaemia Cell Biology group is offering the opportunity to participate in a clinically-relevant project investigating the role of sphingosine-1-phosphate (S1P) in the progression of acute lymphoblastic leukaemia. This project would suit an enthusiastic, diligent student with an interest in immunology.

S1P is a key chemoattractant for haematopoietic cells that has recently generated much interest in the research world for its role in several diseases. S1P has a demonstrated role in lymphocyte trafficking. The blockage of S1P activity is reportedly beneficial in the suppression of autoimmune diseases such as colitis. The overexpression of S1P is thought to aid cancer progression by increasing tumour cell migration and metastasis. Furthermore, S1P can influence cell survival and proliferation. The role of S1P in leukaemia is currently unknown, but may be similar to that seen in solid malignancies. Our laboratory has previously demonstrated that leukaemic cells respond to S1P. The impact on the progression of leukaemia, however, remains to be elucidated. This project aims to determine the role of S1P in a mouse model of acute lymphoblastic leukaemia and will test the hypothesis that S1P will exert a migratory effect on leukaemic cells, thus promoting the progression of the disease.

Our laboratory has established a mouse model in which the conditional knock-out of the S1P receptor can be achieved. Acute lymphoblastic leukaemia will be specifically induced by the forced expression of the Bcr-Abl gene in both S1P receptor wild-type and conditional knock-out mice. The resultant effect on leukaemia progression will be analysed via molecular biology techniques such as flow cytometry. This project will also involve in vitro work to elucidate the mechanisms via which S1P mediates it effects.

This project is designed to equip students with several skills important for a career in research. Honours students will perform both in vivo and in vitro experiments and learn essential tissue culture and molecular biology techniques. A willingness to work with animals is essential.

Techniques involved: Induction of leukaemia in a mouse model; Retroviral transduction of haematopoietic stem cells; Animal tissue harvesting and blood collection; Flow cytometry; Leukaemic cell line maintenance; Chemotaxis assays.

Analysis of the cytotoxic activity of γδ T-cells against myeloid and lymphoid leukemia cells: a potential for use in leukemia therapy

Supervisors: Dr. Leighton Clancy, Ms. Shivashni Gaundar and Prof. David Gottlieb

Sydney Cellular Therapies Group, Westmead Institute for Cancer Research, Westmead Millennium Institute, Westmead 2145, NSW.

Contacts:

Dr Leighton Clancy leighton_clancy@wmi.usyd.edu.au 9845 6212

Prof David Gottlieb david_gottlieb@wmi.usyd.edu.au 9845 6033

Overview and Significance

γδ T-cells represent a small population of T-cells, which together with NK and NK-T cells, have a critical role in immune surveillance and innate response to cancer processes. The majority of γδ T-cells express a T-cell receptor comprising the variable segments Vγ9 and Vδ2 (Vγ9Vδ2 T-cells) and recognize both peptide and non-peptide ligands in an MHC unrestricted fashion. Vδ2 negative γδ T-cells (including those that are CMV specific) have been associated with a reduction in cancer risk. Vγ9Vδ2 T-cells exert potent cytotoxicity against a range of solid tumor cell-lines and adoptive transfer of these cells is an attractive proposition for cell based immunotherapy for cancer patients. The mechanisms that may be relevant for the activity of γδ T-cells include utilization of NKG2D, CD16 (FcγRIII), and possibly other yet unidentified receptors.

Aims of project

Generate γδ T-cells in culture from peripheral blood mononuclear cells and assess their role combined with dendritic cells in generating antigen specific immune cells.
Test the in vitro activity of γδ T-cells on cells derived from myeloid and lymphoid malignancies.

3. Assess the effect of combining γδ T-cells with clinically used chemotherapy agents.

This project will provide training in the following techniques

Cell culture using sterile techniques- handling and processing small volumes of blood, primary T-cell culture and maintenance of cell-cultures
Flow cytometry
Cytotoxicity assays

4. Apoptosis assays

Additional procedures may include

ELISA

2. Western blotting

This project will be conducted at the Westmead Millennium Institute. The student will have the opportunity to attend fortnightly Institute-wide meetings to keep abreast of research being conducted at Westmead. They will also have the opportunity to attend and participate in fortnightly lab and journal club meetings conducted in a friendly setting with other members of the Leukemia Research and Immunotherapy team.

Development and validation of an ELISPOT immunoassay for monitoring of immunity to BK virus

Supervisor
Professor David Gottlieb
Department of Haematology
Wesmtead Clinical School
Westmead NSW
Ph: +61 2 9845 6033
david_gottlieb@wmi.usyd.edu..au

Co-supervisor
Dr Leighton Clancy
Westmead Institute of Cancer Research

Westmead Millennium Institute
Darcey Rd

Westmead

Ph: +61 2 9845 6212

leighton_clancy@wmi.usyd.edu.au

Project
To develop and validate a method for measuring the BKV specific immunity in normal donors and patients with immune deficiency.

Background

BK virus is a ubiquitous polyomavirus that asymptomatically infects >90% of individuals. It remains in a latent state and can reactivate causing disease in the patients with dysfunctional immune systems. In blood stem cell transplant recipients BKV can cause haemorrhagic cystitis and in renal transplant BKV associated disease is one of the leading causes of renal transplant failure. Identifying and monitoring cellular immunity in patients at risk of disease is an important research tool in understanding disease pathogenesis, and will also be required for post-T cell infusion monitoring when clinical trials commence. This project will involve using blood samples from normal donors and transplant patients to develop the ELISPOT immunoassay to monitor the immune response to BKV.

Our group has a successful clinical program of adoptive immunotherapy where cellular immune deficiency is treated with infusion of antigen specific T cells. We work closely with the bone marrow and renal transplant units at Westmead Hospital. This project offers the opportunity for a student to gain exposure to the exciting field of clinical cellular therapy.

Techniques

Sterile tissue culture techniques

Ficoll-paque gradient centrifugation of peripheral blood Cell count using manual haemocytometer Basic flow cytometry methods for cell phenotype Manual cell separation techniques using magnetic nanoparticles ELISPOT immunoassay

Analysis of The Role of Rab Proteins in HIV Trafficking in Dendritic Cells

Supervisor: Anthony L. Cunningham (02 9845 9005, tony_cunningham@wmi.usyd.edu.au)

Co-supervisor: Andrew Harman (02 9845 9110, andrew_harman@wmi.usyd.edu.au)

Centre for Virus Research, Westmead Millennium Institute

Research Background

Dendritic cells (DC) are potent antigen presenting cells that form a link between the innate and adaptive immune systems. HIV uses DCs to get from the site of infection to the draining lymph nodes where it establishes chronic infection in CD4 T-lymphocytes. DCs therefore play a vital role in the establishment of HIV infection. After initial exposure, HIV enters DCs and manipulates them to avoid its own destruction and to transfer itself to T-lymphocytes upon arrival to the lymph node. We have performed microarray experiments to gain a global view of the effects of HIV on DC gene expression during various phases of the virus life cycle. During the early phase of the HIV replication cycle a group of genes encoding Rab proteins were shown to be changed in their expression. These proteins are involved in regulating the formation and function of endocytic pathways and in vesicle trafficking. We believe that HIV may manipulate these genes in order to aid its transport through the DC and its subsequent transfer to T-lymphocytes in the lymph nodes.

Research Aim

This project will involve following up microarray studies of HIV treated DCs to confirm the differential expression of Rab genes and investigating associated protein expression and function. Of particular interest is the effect of Rab proteins of HIV trafficking in DCs.

Research Plan

Initially quantitative PCR (QPCR) will be used to confirm and extend the observation by microarrays that genes encoding Rab proteins are differentially expressed in DCs is response to treatment with HIV. Next changes in the expression of the Rab proteins themselves will be determined by western blot and flow cytometry. In addition their subcellular location and possible co-localisation with HIV virus particles will be investigated by confocal microscopy. Peripheral blood mononuclear cells (PBMC) will be isolated from whole blood using density gradient separation. CD14+ monocytes will be selected from the PBMCs using magnetic bead separation and differentiated to DCs using cytokines ready for HIV-1 infection. This project will provide a comprehensive education in the use of QPCR in determining changes in gene expression levels and also the use of cutting edge technologies in immunology such as multicolour confocal microscopy.

Title: Role of secreted proteases in cell wall integrity and secretion of virulence determinants in the pathogenic yeast, Cryptococcus neoformans

Location: Centre for Infectious Diseases & Microbiology, Westmead Millennium Institute & Sydney Medical School-Western, Westmead Hospital Westmead 2145 NSW

Supervisor: Dr Julie Djordjevic 02 9845 7367, email: julie_djordjevic@wmi.usyd.edu.au

Assoc. Supervisors: Dr Xiaoming Zuo, Prof Tania Sorrell

Background

The model yeast pathogen, Cryptococcus neoformans, infects individuals with AIDS causing life-threatening illness. Multiple key virulence phenotypes, including the ability to replicate at 37^oC, produce a protective melanized cell wall and a capsule, and secrete phospholipase B (Plb1), all facilitate the infection process. Although a family of secreted aspartyl proteases (SAPs) and metaloproteases are candidate antifungal drug targets due to their ability to influence all of these virulence determinants, the mechanism by which they achieve this has never been investigated in C. neoformans. A subset of glycosylphosphatidylinositol (GPI) anchored mannoproteins that attach to the outer layer of the cell wall, are essential for cell wall integrity during fungal growth and infection. For these proteins to translocate to the cell wall, they must be cleaved from their membrane GPI anchor and/or be activated by proteases. We demonstrated that the secreted virulence determinant, Plb1, is also a GPI anchored mannoprotein localizing in the membrane and cell wall and is therefore an excellent marker protein for assessing the role of secreted proteases in fungal cell wall integrity. We found that one of the five cryptococcal SAPs colocalizes with Plb1 in membrane rafts suggesting that SAPs are involved in the transfer of Plb1 and other GPI anchored proteins to the cell wall.

Hypothesis: Secreted proteases contribute to cell wall integrity, high temperature growth and virulence of C. neoformans, by regulating the release of GPI anchored mannoproteins from yeast cell membrane and therefore the retention of melanin and capsule in the cell wall and the secretion of proteins involved in host invasion.

Aim The aim of this project is to use targeted gene disruption to elucidate the role of SAPs and metaloproteases in (A) cell wall integrity and host temperature growth (B) melanin and capsule retention in the cell wall, (C) secretion of host-invading enzymes (Plb1) and (D) virulence in animal models.

Methods Genes encoding SAPs and metaloproteases will be inactivated singly or in combination, in C. neoformans, using overlap PCR and targeted biolistic gene disruption. The gene disruptions will be verified by PCR and the knockout strains will be assessed for protease secretion, cell wall integrity by comparing growth in the presence of cell wall disrupting agents, growth at physiological temperature (relative to WT) and the ability to localize melanin in the cell wall and attach a polysaccharide capsule. Plb1 distribution/activity in membranes, cell walls and culture supernatants will be assessed using Western blotting/radiometric enzyme assays. Virulence will be tested in mouse models.

Techniques: Yeast cell culture and testing for capsule and melanin production and cell wall integrity, overlapPCR/PCR, DNA biolistic transformation, western blotting, radiometric enzyme assays, virulence testing

Project Outcomes

The expected outcomes of this work will be:

(A) The identification of mechanisms used by pathogenic yeast to establish cell wall integrity (allowing host colonization) and allow secretion of enzymes involved in host invasion

(B) The identification of a novel antifungal drug target and/or target of the aspartyl protease component of HIV HAART medication which has antifungal properties

Cytomegalovirus Research Laboratory at the Westmead Millennium Institute

The Cytomegalovirus Research Laboratory seeks enthusiastic students with a strong interest in determining how cytomegalovirus causes life-threatening disease in transplant recipients

What is human cytomegalovirus?

· Human cytomegalovirus (CMV) is a medically important virus that affects millions of people worldwide

· CMV is a member of the Herpesvirus family of viruses

· CMV is carried by the vast majority of the human population (up to 90%)

· After initial (primary) productive infection, the virus remains in your body for the rest of your life in a dormant (latent) state, but can reawaken (reactivate) years later to cause devastating disease if you become immunosuppressed

· The host immune response clears the initial (primary) productive infection, but cannot eliminate the latent virus from the body.

The good news...

· Infection usually causes mild or asymptomatic disease in most healthy adults

The bad news...

· CMV infection is the most common congenitally acquired infection in infants where it is the leading viral cause of neurological defects eg mental retardation, deafness.

· CMV is a major cause of life-threatening disease in immunocompromised individuals including AIDS patients and allogeneic transplant recipients. Infection during immunosuppression results in disseminated CMV which can lead to severe infections of the GI tract, hepatitis, pneumonia, accelerated atherosclerosis, rejection in solid organ transplant recipients and graft-versus-host disease in bone marrow recipients.

· CMV disease in transplant recipients is associated with high mortality and increased health care costs.

Reactivation from latent infection causes most of the serious CMV disease in immunosuppressed individuals such as transplant recipients. There is no vaccine or drug that prevents viral latency, nor is it known how the virus is able to establish latency to successfully persist within the human host

Projects:

· We have also discovered that CMV expresses a number of viral genes during the latent phase of infection, including those that encode immunosuppressive properties.

· We hypothesise that viral genes expressed during the latent phase of infection are likely to encode functions which enable the virus to successfully persist within the human host. Therefore, viral genes identified as playing an important role in latency would serve as ideal targets for the development of therapies aimed at reducing the devastating disease resulting from reactivation of latent virus in immunosuppressed individuals.

· This project will examine the functions of viral genes expressed by CMV during viral latency using a number of molecular and cellular biology approaches.

· There is also scope to undertake additional projects examining control of immune function by CMV

· All Honours projects in the CMV lab are designed so as to provide a solid basis for extension into a project suitable for those seeking to undertake a PhD

For more information contact:

Dr Barry Slobedman, Deputy Director, Centre for Virus Research, Westmead Millennium Institute, Head, CMV Research Group and Senior Research Fellow, University of Sydney

Phone: 9845 9122, Email: barry_slobedman@wmi.usyd.edu.au

Dr Allison Abendroth, Senior Lecturer in Immunology and Head, VZV Research Group

Phone: 93516867, Email: a.abendroth@usyd.edu.au
Project Title: Characterisation of the virological synapse between HIV exposed Dendritic cells and CD4-T cells

Name and contact details of Supervisor (location, telephone and email address):

Stuart Turville

Centre For Virus Research

HIV Biology Lab

Westmead Millennium Institute

Bld C24, Darcy Rd Westmead

9845 9115

s.turville@usyd.edu.au

A BRIEF overview of the project Dendritic cells represent an important link between the innate and acquired immune systems. Observation of early events in HIV transmission, support the current hypothesis that a large population of memory CD4 T cells is infected and provides the means by which viral dissemination proceeds throughout the body (ie. Threshold theory). The natural interaction and potential synergy between other cells of the immune system has not been readily considered in this scenario. Several studies have observed that interactions between CD4 T cells and HIV exposed dendritic cells results in explosive levels of viral replication. Therefore several have hypothesised that this interaction is also crucial for establishing the viral threshold needed for transmission. Using a collection of HIV clones that have been genetically tagged, we are now in a position to characterise the important interaction between HIV exposed dendritic cells and CD4 T cells.

The above subject is currently funded by a NHMRC grant. Other projects maybe considered and viewed at http://www.usyd.edu.au/research/opportunities/opportunities/1015

A list of techniques that will be used in conducting the project

Techniques used in this project are complementary and involve several assays that are routinely used in cell biology and virology.

Cell sorting (magnetic and/or elutriation)
Primary cell culture and passaging of various cell lines
Mutagenesis of HIV clones for tracking
Passaging and titering HIV
Transfection and generation of stocks of genetically manipulated HIV
Fluorescence microscopy (both confocal and wide field systems are available).
Flow cytometry

Project Name: Construction and Screening of a Genomic Library from a Miltefosine Resistant Yeast Strain

Host School/Institute: Centre for Infectious Diseases & Microbiology, Westmead Millennium Institute & Sydney Medical School-Western, Westmead Hospital Westmead 2145 NSW

URL: http://www.wmi.usyd.edu.au/research/infectiousdisease.htm

Supervisors: Dr. Xiaoming Zuo & Dr. Julianne Djordjevic

Contact phone: (02) 9845 5819/(02)9845 7367

Contact email: xiaoming_zuo@wmi.usyd.edu.au

Description of Project:

Our laboratory has reported that the anti-cancer drug miltefosine (MI) has broad range of antifungal activity both in vitro and in vivo. Based on the chemical structure of MI, development of a new class of antifungal drug is in progress. However, the fungicidal mechanism of the drug still remains unknown. By means of chemical mutagenesis, we have obtained a stable dominant MI resistant strain of Saccharomyces cerevisiae. To gain insight into the modes of action of the drug at the molecular level, a single copy genomic expression library will be constructed from the resistant strain and screened for gene(s) involved in the drug response.

We plan to extract genomic DNA from the resistant yeast strain. The DNA will be fragmented by restriction enzymes and fragments cloned into a bacterial-yeast shuttle vector. The resulting plasmids will then be propagated in bacteria, forming a genomic library. Yeast cells that are sensitive to miltefosine will be used as recipients of the library. Miltefosine-resistant transformants will be isolated and screened for single plasmids carrying gene(s) responsible for the resistance. DNA fragments will be retrieved from the plasmids and their identity and biological function determined by blast searching the genomic database. Since the library is constructed from a dominant drug resistant strain, we expect to isolate and identify gene(s) with gain-of-function in critical metabolism pathways.

Techniques used in the project are listed below:

· Yeast genetic analysis

· Drug susceptibility test

· DNA extractions/purifications and electrophoresis

· Genomic library construction (massive cloning)

· Bacterial and yeast transformations

· Subcloning and plasmid construction

· DNA sequencing

· Bioinformatic analyses
AXONAL TRANSPORT, ASSEMBLY AND EXIT OF HERPES SIMPLEX VIRUS FROM NERVE CELLS

Supervisor: Dr. Monica Miranda Saksena

Centre For Virus Research, Westmead Millennium Institute

Email: monica_miranda@wmi.usyd.edu.au

Phone: 9845 9114

BACKGROUND: HERPES SIMPLEX INFECTION OF HUMANS

Herpes simplex viruses (HSV) types 1 and 2 are important human pathogens, which commonly cause recurrent infection affecting the skin, mouth, lips, eyes, and genitals. Most often, HSV-1 causes herpes labialis, and herpes keratitis while HSV-2 usually causes genital lesions. HSV can also cause serious life-threatening infections including neonatal herpes, encephalitis, and meningitis. Transmission of HSV occurs from close contact with an individual who is actively shedding virus. Viral shedding generally occurs from lesions but can occur even when lesions are not apparent. . Herpes infection can be treated but not cured, as the virus lies dormant in nerve cells. Virus reactivation occurs frequently during the lifetime of the human host and can be triggered by a number of factors including stress or illness.

PROJECT OVERVIEW

Understanding the mechanisms used by HSV for entry, transport along nerves, and assembly in nerve cells will assist in the development of new strategies for antivirals for control of recurrent herpes and also in the use of HSV as gene therapy vector to deliver drugs to the nervous system. Our lab has established several models for culture and HSV-1 infection of primary human fetal and rat neurons in vitro that has resulted in publications scientific journals of high impact in the field. We have an Honours project available that has the potential to be expanded into a PhD project for a student interested in continuing a career in biomedical research.

PROJECT

The aim of the project is to explore the mechanism(s) used by HSV to exit nerve cells. This project will establish whether the exit of the virus from nerve cells is dependent on calcium influx by in vitro modulation of cellular calcium concentrations. In addition, this project will examine the effects that HSV infection has on the expression of key proteins called SNARE proteins involved in the release of neurotransmitters and hormones from nerve cells. This project will utilize primary cultures of nerve cells and fluorescently tagged viruses in order to visualize the process of viral exocytosis in vitro using real time imaging and electron microscopy.

LABORATORY TECHNIQUES

These projects utilize a variety of molecular and cell biology techniques including specialized neuronal culture models, real-time fluorescent imaging of viral particles in nerve cells, FRAP (fluorescence recovery after photobleaching), real time PCR, Western blot, confocal microscopy, electron microscopy and mammalian cell and virus culture techniques.

Project choices at the Retroviral Genetics laboratory, Westmead Millennium Institute, University of Sydney, Westmead

Dr Nitin Saksena

Project 1: Studying primary CD14 monocyte proteome during different stages of plasma viremia in HIV patients

It is now widely accepted that the key to resistance to HIV infection and its progression lies within the host immune system that consists of innate and adaptive immune components (Reviewed in Arriaga et al., 2006; Saksena 2004, and 2007). Innate immunity controls HIV and other infections through a rapid host response (Levy et al., 2003), which involves intracellular signaling pathway activated by pattern recognition receptors on the surface of variety of cells, most of them antigen-presenting cells (dendritic cells, monocytes and macrophages). Innate immune system comprises of wide variety of cells, each playing a unique role in infectious immunity. In contrast, the adaptive immune system comprises mainly of T-lymphocytes, B-cells and T cell lineage. It is important to emphasize that DCs, monocytes and macrophages can play a prominent role in both innate and adaptive immunity as antigen presenting cells. HIV tampers with antigen presentation and the antigen presentation gets severely impaired during HIV infection at different stages of plasma viremia. Therefore, the aims of the project will be to characterize CD14+ monocytes directly from HIV+ patients. To address this, we will adopt an approach to stratify HIV patients into 5 different groups. 1. HIV+ patients with below detection plasma viremia, patients with low viremia (<5000 copies/ml plasma), patients with intermediate viremia (<50,000 copies/ml plasma), patients with high plasma viremia (>100,000 copies/ml) and therapy naïve non-progressing patients. The goal will be to assess how the proteome of CD14 monocytes undergoes changes in response to viremic levels in plasma. This will lead to the detection of crucial monocyte proteins that may be necessary in various virus-specific activities and will define impairment at different levels of viremia, as opposed to patients who maintain below detectable levels of viremia with or without antiretroviral drugs. This will be compared against the HIV negative controls. Since it involves handling of HIV-infected cells, the processing of blood samples and cell separation will be carried out in the PC3 containment facility. The CD14 monocytes will be separated from whole blood using CD14 magnetic beads. These CD14+ monocytes will be processed for whole protein extraction. The protein extracts collected from CD14+ monocytes from patients at different stages of viremia will be analyzed by 2-D gel electrophoresis, followed by differential protein gel elctrophoresis to identify proteins differentially expressed between diseased and non-diseased CD14+ monocytes. The relevant protein bands will be eluted from the gels following accurate identification of differentially expressed proteins. Once the proteins have been identified, they will be chemically identified using mass spectrometry, along with a clear functional annotation of each protein through existing protein databases. Further biological, virological or biochemical assays will be designed to define the functional and biological relevance of these proteins. Overall, these analyses will define the proteome of primary CD14+ monocytes at different viremic stages of HIV disease. In addition, it will provide insights into differentially expressed proteins at different viremic stages of HIV disease, clues about impairment in the activity of these potent cells during viremic stages and will also provide insights into possible new biomarkers, which may be highly relevant in HIV pathogenesis.

Project 2: NK cells proteomics, genomics Natural killer (NK) cells are a subset of lymphoid cells that function as important mediators of the innate immune defence mechanisms against viruses and tumour cells. As a crucial component of the innate immune system, NK cells might have an important role in host defence against HIV infection, as well as in the control of HIV replication in vivo, along with various other pathogens. NK cells mediate suppression of viral replication in both a cytolytic manner and a non-cytolytic manner. The balance between the activation of inhibitory NK-cell receptors (iNKRs) and activating NK-cell receptors is fundamental to the regulation of NK-cell cytotoxic activity. HIV is known to selectively downregulate the expression of MHC class I molecules at the surface of infected cells in vitro, thereby escaping recognition and lysis by CD8⁺T cells and lysis by NK cells. NK cells produce abundant amounts of CC-chemokines and have been shown to suppress HIV replication in vitro by inhibiting HIV entry to target cells. However, NK cells from individuals with HIV viraemia secrete reduced amounts of CC-chemokines and cannot adequately suppress HIV replicationFunctional defects in NK cells during HIV-1 infection have been described and such defects could be a consequence of decreasing numbers (which impinges on cytotoxic activity of these cells). In addition, functionally defective CD56^-/CD16⁺population of NK cells in viremic versus aviremic patients has also been shown. In HIV-infected viremic patients, expression of iNKRs is well conserved and that in most cases, there is a trend toward increased expression on NK cells as compared with healthy donors. Functional tests confirmed that the abnormal expression of the activating receptors and of iNKRs was associated with a markedly impaired NK cytolytic function. This phenomenon is not attributed to a direct HIV-1 infection of NK cells; thus, this study may provide insight into the mechanisms of impaired host defenses in HIV-1 viremic patients. Given these roles of NK cells, our main objective is to tease out the transcriptome and proteome of the NK cells at various stages of viremia in HIV patients, to clearly define impairment in the phylsiology of this cell type at the level of genome and proteome. A clear definition of these deficits will lead to a comprehensive understanding of these crucial cell type, which so effectively controls HIV and other viral infection. These studies will also clearly establish the impairment in their activity by HIV directly or indirectly. We will use a variety of proteomic and gene microarray techniques to define the regulation of NK cell proteome and genome during HIV infection. Overall, these analyses will provide insights into antiviral arsenal of NK cells and how it is impaired at different viremic stages, which can provide clues to viral load threshold at which time the impairment begins. If such an event is identified, therapeutic interventions can be designed to halt this event.

Project 3: Antigen sensing, proteomics and genomics Response of a given cell type to HIV antigen can reveal how immune cells sense the antigen in order to defend themselves from a possible attack. A snapshot of these innate and adaptive signals is necessary for a clear understanding of antiviral protection the immune generates at a given time. A wholesome understanding of such responses may also reveal how virus subverts human immune system and how host and virus fight battle against each other during infectious process. In general such responses are weakened in patients experiencing viremia and to some extent they are restored in patients who receive timely therapy, i.e., before the onset of high plasma viremia. Interestingly, the responses to gag peptides are extremely strong in patients who survive therapy and virus-free for >20 years. Our studies show that responses to gag p24 are invariably strong in such HIV patients (Wang et al., 2002, Zaunders et al., 2004). Due to the strength of these responses, they can maintain a non-progressive HIV disease. To date, what these immune responses comprise of is not known. This project proposes to characterize such responses primary in CD4+T cells from HIV patients upon exposure to HIV p24 antigen and compared against non-stimulated cells and cells from HIV-negative control patients. Primary CD4+ T cells will be exposed to p24 antigen and whole RNA will be extracted. This RNA will be used in gene expression studies. We plan to carry out gene expression using RT2 profiler assay, which quantifies gene expression levels in real time. We will characterize genes, which are involved in innate and adaptive immune function, apoptosis and host genes modulated during HIV infection. Followed by that, we also wish to study proteomic differences in stimulated and unstimulated CD4+ T cells from patients. A clear determination of such responses in patients who survive for longer periods of time, such as HIV+ non-progressors, will lead to a identification of antiviral arsenal used in defending against HIV. Quantitative gene expression can accurately determine the fold changes in innate, adaptive and antiviral genes, which may further assist in proteomic studies, which are planned in a similar fashion as described in project 1 above. Overall, these studies will determine the biochemical components involved in antiviral activity during HIV infection. Some of these can be useful in natural therapeutics and some will lead to novel biomarker discovery.

Project 4: Plasma proteome at different stages of HIV disease

BACKGROUND: Plasma is a biomarker discovery opportunity because it is easily available and because it comprehensively and regularly samples the human condition and in all states of health and/or disease. This blood component is integral to both prognosis and diagnosis of HIV and in defining therapy regimen for HIV patients. Drilling deeply into the human plasma proteome with state-of-the-art technologies holds enormous promise in developing new markers for disease prognosis, diagnosis, disease progression/nonprogression, response

to therapy monitoring and stratification of patients. Unfortunately, like many protein-rich bio-fluids (saliva, tears, and urine) plasma has an extraordinary protein concentration range. The top 20 proteins occupy the vast major of discovery space. Great discovery opportunities exist if we can "drill far more deeply into the human plasma proteome". A lot is known about the major proteins in plasma in the context of HIV disease, yet the underlying reasons for the progression and non-progression of HIV disease remain poorly understood, to date. Therefore, we believe that without the removal of these abundant proteins, visualization of the low abundance (i.e., rare) proteins is just not feasible. Therefore, the minor protein species may be more valuable in the discovery new biomarkers for efficient HIV prognosis and diagnosis, which can also be used in predicting the disease outcome. There are 2 specific aims of the project over 3 years duration.

To deplete human plasma proteome to unveil low-abundance proteins for the discovery of new generation of biomarkers in HIV disease. To validate low-abundance plasma proteins through the development of sensitive molecular assays and develop new generation of cost-effective diagnostics for HIV.

This project will investigate the utility of a novel cyclic immuno-depletion technique for the removal of the high abundant human plasma proteins using IgY antibodies and assess other minor proteins using various proteomic techniques including DIGE (Differential Gel Electrophoresis), mass spectrometry, which are revealed following depletion of abundant plasma proteins. These proteins will comprise host and viral proteins in plasma, which are difficult to visualize in the presence of background of major plasma proteins. The identified proteins will be validated as described below and sensitive molecular technologies will be developed for the detection of these biomarker proteins by using Rolling Circle Amplification.

Validation of low-abundance plasma proteins through the development of novel technologies, which can directly detect low-abundant proteins in plasma: At present, plasma is being routinely used in HIV testing because it is a dynamic compartment and a variety of biomarkers are available. There are no effective molecular

tools, which can provide rapid, sensitive and cost-effective estimation and detection of low abundance plasma proteins. In the proposed study we will apply our newly developed technologies, real-time PCR, ligase chain reaction (LCR) and the Rolling Circle Amplification (RCA) (Wang et al., 2005) to address the aforementioned issues. By using these technologies and upon unambiguous identification of low abundant proteins in plasma, relevant to HIV disease, we will investigate the effectiveness of our newly developed molecular technologies to identify various relevant low abundance plasma proteins through the use of molecular technologies we have developed. This will be mainly to develop cost effective tests, which will be extremely useful in predicting HIV disease stages and the success or failure of therapy. Further, this will be complemented with multiplexed tandem PCR (MT-PCR), a novel high-throughput method, which provides sensitive and quantitative analysis of populations of infectious agents and their copy numbers. This will give a precise estimate of the resistance prevalence in quick time, viral copy estimation and detection of recombinant HIV strains. Together, it may not only serve as a diagnostic tool but will also form a solid platform for the analysis of multiple proteins. The primary objective of this proposed work involves depletion of the plasma proteome with an overall view of the discovery of biomarkers of healthy HIV negative individuals and HIV+ progressors and non-progressors, along with HIV patients who are therapy naive and therapy experienced at different stages of viremia. Secondly, the discovery of these biomarkers would explain the underlying reasons for HIV disease progression and non-progression and the possible reasons for success and failure of therapy. Thirdly, this may lead to the development of better tools for predicting HIV disease and success and failure of therapy, HIV diagnosis, clinical management of patients and defining therapeutic strategies. In addition, the outcome could lead to a renaissance in the discovery, evaluation and deployment of novel clinical diagnostics in the HIV therapeutic realm.

Expression and attenuation of resistance genes in common mobile genetic elements

A number of gene capture systems are important in the transmission of mobile antibiotic resistance genes in the Enterobacteriaceae, and the associations between them are relatively consistent once developed. An important aspect of this is the expression of the genes so captured, and the responsible promotor may be present in a variety of strengths which have potentially important influences on phenotype. A number of promotors of differing strengths are to be found in the 5'CS of class 1 integrons, for example, and in various insertion sequences (IS) commonly associated with mobile genes. The student will use standard cloning methods to construct a number of expression/ promotor detection vectors and use these to study the phenotypes of important but not well-characterised antibiotic resistance genes in isogenic strain sets.

In this project, high and low-copy vectors will be constructed with a number of common promotors (eg IS26, ISEcp1, ISCR1, ISAba1, int Pc promotors of various strength) upstream of a promotor detection system (eg GFP) and several of the most troublesome antibiotic resistance genes (eg blaIMP, blaKPC, blaVIM, rmtC, aacA4) with and without transcriptional attenuators cloned in from common gene cassettes.

This will allow us to directly compare the power of these promotors and their impact in a number of bacterial host contexts, including E. coli, K pneumoniae with and without discrete porin deletions (OmpK36 and OmpK37) and P. aeruginosa with and without oprD- and/or mex-mediated resistance.

The project will give the student experience in practical genetic engineering in bacteria, in promotor detection studies and in the use of shuttle vectors in different systems, in the laboratory determination of antibiotic resistance and in the roles of these important mobile resistance elements in the bacterial gene pool.

supervisors J Iredell*/ A Ginn/ S Partridge

*02 9845 6255

Centre for Infectious Diseases and Microbiology

Westmead Hospital

jon.iredell@swahs.health.nsw.gov.au

jiredell@usyd.edu.au
Project Title: CORRELATION BETWEEN GENOTYPE AND VIRULENCE OF A NEW SUPER KILLER – CRYPTOCOCCUS NEOFORMANS

Research Group Leader: A/Prof. Wieland Meyer

Co-supervisor: Dr Fabian Carriconde

Research Group Address: Molecular Mycology Research Laboratory

Westmead Hospital, Westmead, NSW 2145

Research Interests/Focus: The Molecular Mycology Laboratory at Westmead Hospital is home to a group of enthusiastic young researchers who are interested in a better understanding of pathogenesis and phylogeny of yeast species within the two imperfect fungal genera Candida and Cryptococcus and in applying the findings of these studies to molecular fungal identification in the clinical laboratory.

Research Centre/Institute:

Centre for Infectious Diseases and Microbiology, Westmead Millennium Institute, Department of Medicine, University of Sydney, Western Clinical School, Westmead Hospital

E-mail: w.meyer@usyd.edu.au / fcarriconde@gmail.com

Phone: 02-98456895/02-98456332

Webpage: www.mmrl.med.usyd.edu.au

PROJECT SYNOPSIS: Members of the Cryptococcus species complex (C. neoformans and C. gattii) are recognized as important pathogenic fungi of humans and other mammals throughout the world. Isolates of the Cryptococcus species complex have been grouped into 8 major molecular types based on the PCR-fingerprinting, AFLP analysis, and URA5- and PLB1-RFLP analysis. Highly virulent strains have recently emerged in several parts of the world, leading to a number of outbreaks such as the recent ongoing one on Vancouver Island, BC, Canada, resulting in an increased number of deaths. This makes it important to establish the global molecular epidemiology of the Cryptococcus species complex in order to investigate the emergence and the origin of highly virulent strains, to enable better predictions and public health responses for possible outbreaks. With this aim we have established in a Multilocus Sequence Typing (MLST) scheme and started a population genetic analysis to investigate the global molecular epidemiology of 220 selected isolates representing the 8 major molecular types of the Cryptococcus species complex. Future studies need to increase the number of isolates as well as to investigated if there is a link between genotype and virulence using animal models.

Aims: (1) To conduct a global analysis of the population genetic structure using MLST to identify specific subgenotypes. (2) To correlate the different genotypes obtained by MLST with virulence in a mice model.

Techniques: DNA extraction, PCR, sequencing, bioinformatics analysis, phylogenetic analysis, animal models.

Project title: FINDING THE LINK BETWEEN THE ENVIRONMENT AND THE PATIENT – ENVIRONMENTAL SAMPLING AND PHYLOGENETIC STUDIES WITH THE ARTIFICIAL GENUS CANDIDA

Supervisor/Research Group Leader: A/Prof. Wieland Meyer

Co-supervisor: Dr. Fabian Carriconde

Research Group Address: Molecular Mycology Research Laboratory

Westmead Hospital, Westmead, NSW 2145

Research Centre/Institute:

Centre for Infectious Diseases and Microbiology, Westmead Millennium Institute, Department of Medicine, University of Sydney, Western Clinical School, Westmead Hospital

E-mail: w.meyer@usyd.edu.au / fcarriconde@gmail.com

Phone: 02-98456895/02-98456332

Webpage: www.mmrl.med.usyd.edu.au

PROJECT SYNOPSIS:

Yeasts of the artiﬁcial genus Candida include plant endophytes, insect symbionts, and opportunistic human pathogens. A better knowledge on the evolutionary relationships of Candida species is vital to understand the ecology, clinical relevance, and diagnosis of these yeasts. Recently, we studied the phylogeny of selected Candida species, with special emphasis on clinical isolates, using the sequence of five genes. We showed six major clades resolving part of the Candida phylogeny. However, the genus Candida is complex and need more study. Now our next objective is to increase the number of strains obtained from the environment to gain a better robustness of the phylogeny of this genus and also to understand the relationship between environment and clinical infections.

For this reason, the first part of this project will be to collect as many samples as possible from a number of environmental sources: soil, flowers, plants, hospital environment and isolate the cultures in specific media.

The second part of this project will be to identify these strains to the species level, using molecular techniques, find new species and to study their phylogeny relationships within the artificial genus Candida, to improve the phylogenetic overall resolution.

Aims: (1) To obtain samples, containing yeasts from a number of environmental sources: woods, trees, flowers, soil; (2) Grow these samples on different culture media and isolate pure Candida cultures (3) Identify those cultures to the species level using molecular techniques (4) and to perform phylogenetic analysis.

Techniques: Environmental sampling, fungal culturing, microscopy, DNA estarction, PCR, sequencing, use of phylogenetic programs as CLUSTALW, MEGA, BIOEDIT and PAUP.

Project Title: Autoantibodies against the glutamate receptor NMDA-R in paediatric encephalitis

Supervisors: Dr. Fabienne Brilot-Turville, Dr Russell Dale

Contact details: Neuroinflammation group, Institute for Neuroscience and Muscle Research, The Kids Research Institute at the Children’s Hospital at Westmead

Email: fabiennb@chw.edu.au, russelld@chw.edu.au

Phone: 9845 0133 (Fabienne Brilot-Turville) or 9845 3404 (Russell Dale)

Project description:

Encephalitis is defined as “inflammation of the brain”. Encephalitis results in acute neurological dysfunction including drowsiness, confusion, seizures, movement disorders, and behavioural alteration. Encephalitis is potentially devastating and life threatening, and remains a leading cause of acquired neurological disability with an annual incidence of 10 per 100,000 people, highest in young children. Encephalitis has many causes or precipitants. Viruses are common causes accounting for 5-10% of encephalitis. The other common group is autoimmune encephalitis, which is important because it is treatable with immune suppressant therapies.

Recently, we and others have described a subgroup of autoimmune encephalitis with autoantibodies that bind to N-methyl-D-aspartate receptor (NMDA-R). NMDA-R is a glutamate receptor that plays an essential role in the regulation of neuronal communication and function in the brain. NMDA-R is constituted of four sub-units; two NR1 and two NR2, either A, B, C, D. The different subunits follow a developmental pattern of regional expression, and are highly expressed in the young brain.

This project aims at understanding whether how frequent is encephalitis with autoantibodies targeting NMDA-R, and whether these autoantibodies impair neuronal function.

A cellular model will be engineered to express subunits of NMDA-R. The expression of these proteins will be determined using western blotting and immunocytochemistry. The binding of autoantibodies from patient and control sera will be determined using flow cytometry and confocal fluorescence microscopy. Their potential action on neuronal physiology will be examined using primary cultures of neurons and live cell imaging.

Interested students are strongly advised to contact the project supervisor to discuss potential honours and Ph.D. projects (fabiennb@chw.edu.au).

Information:

Laboratory Heads: Dr. Fabienne Brilot-Turville and Dr. Russell C. Dale

Website: http://www.inmr.com.au/ourteam_section.asp?id=9

Recent publications: Dale RC, Irani, SR, Brilot F, Pillai S, Webster R, Gill D, Lang B & Vincent A. Pediatric dyskinetic encephalitis lethargica is an NMDA receptor encephalitis. Accepted for publication in Annals of Neurology on July 2009.

F. Brilot, T. Strowig, F. Array, S. M. Roberts & C. Münz. NK cell survival mediated through the regulatory synapse with dendritic cells requires IL-15Ra. The Journal of Clinical Investigation. 2007; 117 (11): 3316-3329

Assoc. Prof. Cheryl Jones MBBS(Hons) PhD FRACP

DIsc. Paediatrics & Child Health, Uni. of Sydney

Text Box: CENTRE FOR PERINATAL INFECTION RESEARCH
Honours Projects available 2010 Paediatric Infectious Diseases Consultant

Head, Centre for Perinatal Unit

The Children's Hospital at Westmead

Locked Bag 4001

WESTMEAD NSW 2145

Contact details:

Ph: 61-2-9845-3382 (sec)

Fax: 61-2-9845 3389

E-mail: cherylj@chw.edu.au

Dr. Marian Fernandez, PhD

Senior Research Officer,

Centre for Perinatal Infection Research Unit

Ph: 61-2-9845 3113

Email: MarianF@chw.edu.au

We will offer one Honours Project in 2010

Defining the role of resident skin gdT cells (DETCs) in the immune response to cutaneous HSV infection

The skin is the largest organ of the body and provides the first line of defence against many infections including HSV, a pathogen of global importance. HSV enters the body through breaks in the skin or mucosa where it first encounters immune effectors within the skin. Understanding the immunological mechanisms that govern protective cutaneous immunity e is critical to the development of vaccines, antivirals and topical microbicides. Our laboratory uses a murine model of herpes simplex virus (HSV), to understand the parameters for immune protection against viruses in the skin and to define age-dependant differences in neonatal innate and adaptive antiviral immunity.

T cell receptor gd cells (gd T cells) are a minor population of circulating T cells. Although they have been found in increased numbers in tissues during the course of several viral infections including HSV, their precise role remains unknown. A subset of gd T cells, called dendritic epidermal T cells (DETC) express a unique Vg5/Vd1 TCR, and are largely resident in the skin. They are in intimate contact with neighbouring epidermal cells such as keratinocytes and Langerhans cells. They recognise antigen expressed by damaged keratinocytes and produce cytokines (TNF-a, TGF-b, IL-1, Il-3 and GMCSF), keratinocyte growth factors, (IGF-1, KGF-1), and chemokines. They have been therefore shown to play a role in wound repair. Soluble factors, IL-7 and IL-15 are important for long term survival of DETC in the epidermis. DETC produce chemokines such as lymphotactin, MIP 1-a, MIP 1-b and RANTES

Recently, we have made the key observation using fluorescent HSV strains that γδ T cells are infected HSV shortly after skin infection. In this project, we will extend these studies to define the morphology, density, kinetics of HSV Ag uptake, migration and viability of HSV infected and bystander DETC in the ear skin after cutaneous HSV infection in mice. We will examine the effect of cutaneous HSV infection on expression of IGF-1, KGF-1, IL-7 and IL-15 by RT-PCR to understand the effect of HSV on normal DETC regulated skin homeostatic mechanisms. This information may allow development of novel therapies that harness the ability of DETC to promote resolution of cutaneous HSV lesions.

Techniques to be used: Virus preparation, cell culture, mouse inoculation, preparation of single cell suspensions and epidermal sheets from mouse skin, multi-colour flow cytometry, immunofluorescence and confocal microscopy of murine skin, RT-PCR, in vivo adoptive cell transfer, intracellular cytokine staining.