Molecular Mechanisms Laboratory
Leader: Dr Mark Hulett
The Molecular Mechanisms Laboratory of the Cancer and Vascular
Biology Group focuses on understanding the molecular basis of cell
invasion, with particular interest in inflammation, tumour metastasis
and new blood vessel growth (angiogenesis). The major barrier for
invading tumour cells, migrating leukocytes, and growing blood vessels
(endothelial cells) is the basement membrane (BM) that surrounds
the vessels, and the extracellular matrix (ECM), which forms a scaffold
in tissues to hold cells together. The BM and ECM are composed of
an interlocking network of proteins and complex carbohydrates, and
for cells to breach this barrier they deploy a battery of enzymes
that break down these proteins and carbohydrate components. The
major carbohydrate is heparan sulphate (HS), which acts as the glue
to maintain the integrity of the BM and ECM. The enzyme responsible
for cleaving HS, heparanase, has been shown to play a key role in
the degradation of the BM and ECM, and its activity strongly correlates
with the metastatic capacity of tumour cells and the migratory capacity
of leukocytes and endothelial cells. HS in the ECM also binds a
number of angiogenic growth factors, and the release of these by
heparanase, promotes angiogenesis and tumour growth. Following our
recent cloning of mammalian heparanase, we have been able to develop
the tools to investigate how heparanase functions at the molecular
level and to directly determine the role of heparanase in cell invasion,
angiogenesis and inflammation.
Over the last year our major research achievements include: (i)
demonstrating that mouse, rat and human heparanase are all processed
in a similar manner, from a ~65kDa inactive pro-form, to a 50kDa
active form; (ii) producing direct evidence that heparanse does
indeed play a critical role in tumour metastasis using a heparanase
mRNA antisense approach; (iii) the generation of a mouse heparanase
gene targeting construct for the inactivation of the heparanase
gene in mice; (iv) the identification of novel cell surface receptors
for human heparanase; and (v) making significant progress towards
understanding the molecular basis of heparanase gene regulation
by identifying key transcription factors and their DNA binding sites
in mammalian heparanase gene promoters. We are currently working
towards (i) further understanding the molecular basis of heparanase
function at the structural level, (ii) identifying the protease(s)
responsible for processing the enzyme to its active form, and (iii)
generating gene targeted mice that lack heparanase in specific cells
and tissues to further define its role in cell invasion and angiogenesis.
Matrix Biology Laboratory
Leader: Dr Craig Freeman
Research within the Matrix Biology Laboratory complements that
of the Molecular Mechanisms Laboratory by focusing on the biochemical
basis of cell invasion during cancer spread (tumor metastasis),
inflammation and during new blood vessel growth (angiogenesis).
We are also investigating the roles that the sulfated carbohydrate
heparan sulfate (HS) and its degradative enzymes play in health
and disease.
HS plays a vital role in many biological processes, including cell
growth and development, cell attachment to the surrounding matrix,
the breakdown of triglycerides and the entry of viruses and other
pathogens into cells. Many biologically important proteins, enzymes
and growth factors bind to cell surface HS which can regulate their
physiological actions. To ensure the specificity of their actions,
many of these proteins recognise unique sugar sequences within the
HS molecule, which is quite variable in its structure. We are therefore
developing procedures to determine these particular sugar sequences
which will allow us to design novel drugs (HS-mimetics) that mimic
that specific HS sugar sequence. These drugs may then be used to
specifically inhibit various physiological and pathological processes
involving HS-protein interaction. For example, since tumor growth
is critically dependent upon the development of a new blood supply,
the selective blocking of angiogenic growth factor binding to cell
surface HS has become a novel way to interfere with tumor development.
HS is also a key component of the extracellular matrix (ECM) and
the vascular basement membrane that surrounds the blood vessels
and acts as a barrier to cell invasion during tumor metastasis.
Malignant tumor cells have elevated levels of the enzyme heparanase,
which degrades HS, causing breakdown of the ECM structure and allowing
tumor cell invasion. Heparanase is normally involved in embryonic
development, angiogenesis, wound repair and inflammation, permitting
cell migration through the ECM and the release of growth factors
stored within the ECM that stimulate cell growth. However, heparanase
activity secreted by a growing tumor may also release these growth
factors, stimulating further tumor growth and new blood vessel growth
that can allow subsequent tumor cell escape. Similarly, the uncontrolled
invasion of leukocytes into the ECM can lead to inflammatory diseases
such as inflammatory bowel disease and the progression of autoimmune
diseases, such as multiple sclerosis and rheumatoid arthritis.
Previously, our group identified the HS-mimetic PI-88 which is
both an effective inhibitor of heparanase activity and angiogenenic
growth factor binding to HS. In animal models, PI-88 prevented the
growth and spread of cancer and it is currently undergoing clinical
trials in cancer patients. In conjunction with Dr Martin Banwell
at the Research School of Chemistry, ANU, we have developed a new
series of HS-mimetics. Preliminary studies have shown that some
of these compounds exhibit potent and selective inhibitory activity
against heparanase activity and the binding of various growth factors,
chemokines and proteins to HS. Such compounds may lead to the development
of a new series of drugs to prevent cancer, inflammation, viral
infection and to lower blood triglyceride levels.
Within the Cancer and Vascular Biology Group we are collaborating
with Professor Chris Parish and Dr Mark Hulett to investigate the
roles of heparanase in both health and disease, including the factors
which regulate its normal activity. Using a novel enzyme assay,
we were one of the first laboratories to purify and clone human
heparanase, demonstrating that only one heparanase activity is expressed.
Therefore heparanase represents an excellent target for the development
of anti-cancer and anti-inflammatory drugs. We are currently characterising
proteases that activate the enzyme as well searching for the presence
of endogenous inhibitors to determine if control of these factors
can also be used to inhibit heparanase activity. We are also studying
the interaction between tumor cells and blood platelets which occurs
during tumor metastasis as well as identifying important ligands
involved in this interaction. Our overall goal is to better understand
both the biology and structure of heparanase to enable the development
of inhibitors of the enzyme, which will hopefully lead to new drugs
to prevent cancer spread, angiogenesis, and inflammation.
Medical Genome
Centre
Leader: Professor Chris Goodnow
Many of the health problems we face today - cancer, autoimmune diseases,
allergy, cardiovascular disease, osteoporosis, - stem from a discordance
between genetics and environment. Our genetic code was selected
to survive for a shorter period of time in a very different world,
and it is inevitably out of step with the lifestyles we lead today.
In some cases we know what to do to fix this imbalance, like putting
on a hat and sunscreen to offset genetic susceptibility to the sun.
For many cancers and numerous other diseases such as diabetes, rheumatoid
arthritis, osteoporosis or obesity, the solutions are not so straightforward.
Rapid advances in gene technology are bringing us to the point
of visualizing the imbalance between our genetic code and lifestyle/environment.
A worldwide effort has already produced a map and determined the
spelling of the forty-thousand genes in our genetic 'dictionary'-
collectively referred to as our genome. The epic challenge for the
next decade is to decipher what these gene words mean. How are they
combined into the molecular language that guides the cells in our
body and determines how well we cope with different environmental
stresses? Which gene products are good targets for new drugs to
prevent or cure common diseases? Differences in the spelling of
those gene words underlie differences in susceptibility to modern
diseases and divergent responses to particular therapies, and gene-fingerprinting
technology now allows a list of spelling differences to be compiled
from each person's unique genetic dictionary. This great opportunity
to advance health care hinge on establishing the function of genes
and how they interact.
To decipher the functional meaning of genes and their contextual
interactions, laboratory mice represent a crucial Rosetta stone.
The mouse genome has also now been sequenced, and almost all of
the genes in mice match a human counterpart with only small changes
in their spelling. While surprising given the large differences
in external appearance, the mouse and human genetic languages are
in fact no more different than Chaucer's English and modern English.
This underlying similarity makes it possible to define the function
of human health genes in the mouse in a way that is impossible in
humans.
The strategy being followed is genome-wide mutagenesis in the mouse
at an unparalleled scale. In the last year, the Medical Genome Centre
has successfully analysed a large panel of new mutant strains to
reveal functions of many key genes. These strains were derived from
libraries of laboratory mice in which single letters in the spelling
of many of the genes in the mouse genetic dictionary have been changed
by random chemical mutagenesis. The ENU1 library comprised 185 pedigrees
of ENU mutagenised C57BL/6 mice, bred over three generations to
yield at least 25 potentially recessive homozyogous offspring in
each pedigree. Collectively, we estimate that this library scanned
15,000 loss of function mutations. By visual inspection and high-throughput
screening of blood, this library yielded many new mutant mouse models
to discover and understand genes controlling obesity and diabetes,
cardiovascular disease, kidney disease, limb and spine abnormalities,
eye function, melanocyte function, coordination, seizures, dermatitis,
colitis, and disorders of lymphocyte development such as autoimmunity,
immunodeficiency, and leukemia/lymphoma.
State-of-the-art facilities for transgenic mouse production, sperm
freezing, and database tracking of genetic and phenotypic data have
been developed to support this resource and provide a service to
researchers. The chromosomal location of the defective gene in each
of the mutant strains has been determined, employing an efficient
strategy for gene mapping many mutant strains in parallel. In most
of the strains, the defective gene has been identified through resequencing
of mRNA, revealing critical functional domains in the proteins these
genes encode. In parallel, we have defined the precise cellular
abnormalities brought about by each mutation by cell and biochemical
analysis. From the immunological screen on blood cells, we have
shown that the genome-wide mutagenesis approach yields clusters
of mutant strains from a library of 185 families, to reveal key
functional elements at many successive steps in the molecular pathway
of T and B cell activation. To use the dictionary analogy, this
approach is ideally suited to the problem of how individual genes
are linked together to form a molecular language, because it identifies
many of the key genes in the molecular sentences that cells use
to communicate and respond to stress, rather than individual gene
words in isolation.
To build on our genome-wide mutagenesis technology, more sophisticated
screens are underway. With Professor John Bell and Dr Richard Cornall
at Oxford University, a collaborative Programme Grant from The Wellcome
Trust employs sophisticated immune cell challenges and sensitization
with a T cell receptor transgene to identify a broad range of genes
controlling the immune response to antigen. A Special Programme
funded by the Juvenile Diabetes Research Foundation and the NH&MRC
is using a pair of transgenes in the mutagenized mouse stock to
sensitize the discovery of genes that control diabetes. A consortium
of the ANU, Monash University, the Garvan Institute, and the University
of Queensland was awarded funds from the Commonwealth Government
Major National Research Facilities program to establish a much larger
National Facility, the Australian Phenomics Facility. Design and
construction of the facility on the ANU campus is underway with
expected completion in February 2004.
Mining the genome for disease genes
During 2001 and 2002 the sequence of our DNA, the human genome,
was completed, along with the genome sequence for the laboratory
mouse. These accomplishments are unquestionably major milestones
in the history of biomedical research. Nevertheless, completion
of the human genome sequence represents only the first step in unlocking
the information encoded in our DNA. The challenge we now face is
to identify all genes, the unique pieces of DNA in the genome that
encode proteins, within the vast genome sequence and to decipher
how these genes enable our bodies to grow, develop and fight disease.
This is a tremendous challenge. It is estimated that the genomes
of humans and mice contain at least 40,000 genes and currently we
only understand the importance of a fraction of these genes. At
the Medical Genome Centre of the John Curtin School of Medical Research
we have developed a process that represents a powerful, new approach
for discovering genes important in disease. This process in effect
pinpoints genes in the genome based on their importance in specific
physiological, developmental and disease processes. It is difficult
to accurately predict the importance of genes to specific physiological
and disease processes from computer analyses or in vitro methods
such as tissue culture. Because of these limitations, our process
utilises the gold-standard laboratory model for human physiology
and disease - the mouse.
The process we have developed, called genome-wide chemical mutagenesis,
is initiated by subtly altering the DNA code of hundreds of genes
simultaneously in a laboratory mouse by treating it with the chemical
ethyl-nitroso-urea (ENU). The ENU has its effect on genes by inducing
random point changes or mutations in the chemical sequence of DNA
across the genome. These point mutations have the potential to affect
the function of proteins encoded by the chemically altered genes
and ultimately cause significant changes in physiology or disease
susceptibility. These gene altering point mutations are not apparent
in the initial ENU-treated mouse, but they can be detected in his
offspring by screening them for specific characteristics or 'phenotypes'
that indicate that a gene critical to a physiological or disease
process has been mutated. Many of the screens we use are very similar
to screens done in a hospital on human patients. Once a mouse is
identified with a disease phenotype or resistance to diseases, we
are then able to identify the gene that was mutated in that mouse
by using a combination of genetic mapping and DNA sequencing techniques.
Most importantly, by starting with a mouse that exhibits a disease
phenotype we know that the gene mutated in that mouse is critical
to the development of that disease. Thus, this process allows us
to unequivocally identify genes involved in different disease processes.
Using this approach, we have identified over 50 new strains of
mice that exhibit phenotypes directly related to human disease.
These phenotypes include immunodeficiency, late and early onset
obesity, diabetes, neurological disorders and cancer. We have now
mapped and characterised many of the mutated genes that result in
the observed disease phenotypes with collaboration in San Diego
and Houston, we have also formed a biotechnology start-up company,
Phenomix to accelerate the process of developing better treatments
from this technological approach. From these studies it is clear
that many of the genes identified using these mice have never been
characterised. In addition, this approach has led to the discovery
of important new functions of previously identified genes in critical
processes such as red blood cell development. Genome-wide chemical
mutagenesis is allowing us to mine deeply into the genome for genes
that are critical to many different disease processes. The information
derived from this genetic excavation will be critical in the development
of new therapeutic strategies for the treatment of a number of human
diseases.
Dr Keats Nelms
Australian Cancer Research
Foundation Genetics Laboratory
A one million dollar grant from the Australian Cancer Research
Foundation in 1997 made possible the establishment of the ACRF Genetics
Laboratory in the Medical Genome Centre. The laboratory's focus
is on developing genome-wide mutagenesis resources for studying
candidate human cancer genes and novel laboratory mouse models to
accelerate cancer research at both the basic and clinical ends of
the spectrum. Four new mouse strains to illuminate cancer genes
have been obtained from the ENU1 forward genetics library. One of
these strains, Plastic, carries a single dominant gene defect that
results in acute T cell lymphoblastic leukemia, providing a valuable
model for understanding this important form of childhood leukemia.
Peter Papathanasiou has mapped and identified the mutation in this
strain, which creates a single change in a protein that appears
to be frequently defective in human T cell lymphoblastic leukemia.
The cancer protein normally regulates expression of many other genes
in blood cells. He has shown that the protein is critical for the
production of red blood cells, because it is necessary to maintain
expression of receptors for stem cell growth factors as the cells
differentiate.
Adele Loy has mapped the mutation in another cancer-prone strain,
Bblast, which carries a semi-dominant gene defect that results in
leukemia, lymphoma, osteosarcoma, chondrosarcoma and teratocarcinoma.
These cancers arise because the mice carry a single misspelling
of a protein called Tumor suppressor p53. Tsp53 is the most frequent
defect in a wide range of human cancers, and controls a range of
tumour and ageing processes. The Bblast strain therefore provides
a valuable model for investigating cancers of many types. A third
strain, carrying a purely recessive susceptibility to lymphoma and
primitive myeloid leukemias, is currently in the gene mapping and
identification phase.
Progress continued on a separate discovery project aimed at illuminating
genes regulating solid tumours of the skin and cervix. This project
involves a consortium with Dr Douglas Hanahan at the University
of California in San Francisco, who has developed a sensitized mouse
strain for skin and cervical cancer research, and Dr Simon Foote
at the Walter and Eliza Hall Institute. The project is mapping cancer
inhibitory genes between two inbred mouse strains, C57BL/6 and FVB,
and conducting genome-wide mutagenesis to illuminate skin cancer
inhibitory genes.
Genes and functions discovered by these efforts will be available
to the research community to help in three key areas: a) primary
prevention, where illuminating patterns of inherited susceptibility
will help to resolve environmental risk factors and target preventative
measures, b) early diagnosis, where alterations in gene spelling
and expression pattern will help to distinguish cancer cells early
and predict the best course for treatment-therapy and c) prevention
of secondary tumours, where the genes identified will provide new
targets for drugs and new avenues for chemotherapeutic or immunological
intervention.
Goodnow Laboratory
Leader: Professor Chris Goodnow
Our research aims to understand how immune cells make a fundamental
decision: either to fight or to disarm. The process of deciding
which immune cells should fight and which should disarm is key to
our ability to resist infection and parasitism. Mistakes in this
process result in autoimmune diseases, allergy, lymphoma, and leukemia.
Moreover, drugs and other ways to alter fight or disarm decisions
are sorely needed to improve the success of organ transplantation
and treatment of autoimmune diseases and metastatic cancer.
The immune system is made up of billions of immune cells called
lymphocytes. By a remarkable gene shuffling process akin to a poker
machine, each lymphocyte carries a unique receptor, enabling every
lymphocyte to detect a different set of molecules termed antigens.
Some lymphocytes have receptors for foreign antigens that are unique
parts of the molecular makeup of different infectious organisms.
When these rare lymphocytes bind a foreign antigen during an infection,
they receive a signal to fight. The lymphocyte first multiplies
to make many clones of itself, and then the cells elaborate destructive
compounds that neutralize the infectious antigen.
By chance, other lymphocytes carry receptors for self antigens,
ie. parts of our own normal tissues and body fluids. When a lymphocyte
binds a self antigen it normally receives a signal to disarm. Instead
of multiplying and producing destructive compounds, the lymphocyte
either commits cell suicide by apoptosis or the cell disarms itself
by becoming functionally tolerant, ie. less responsive to antigens
and less able to multiply or produce destructive compounds.
For a long time it was not possible to see how self-reactive lymphocytes
disarm themselves. Our laboratory has developed ways to visualize
this process in genetically modified laboratory mice called transgenic
mice. By studying cells in the transgenic mice, we have discovered
that each immune cell must run through a complex series of fight
or disarm checkpoints before it can be fully launched into an immune
response. In some ways, the process resembles the sequence of fight/disarm
decisions in a military missile launch, which serve a similar purpose
of preventing friendly fire.
Members of the laboratory are deciphering different fight/disarm
checkpoint processes, using a combination of biochemistry, cellular
immunology, genetic analysis, and transgenesis. At each of these
checkpoints, we are focusing much of our work on elucidating how
it is that antigen receptors on lymphocytes can trigger several
different cell fates ranging from cell proliferation to cell death.
Three examples of our work that have been published this year are
summarized below.
T cell tolerance and destruction of pancreatic islets
The cellular and molecular processes regulating CD4 T cell responses
to self antigens that are restricted to specific organs (organ-specific
tolerance and autoimmunity) are being investigated through an NIH
and JDRF-funded project involving a unique transgenic mouse model.
The core of this model is a "TCR-transgenic" mouse where
most CD4 T cells react with a model antigen, and where the model
antigen is made by pancreatic islet beta cells. The islet-reactive
T cells are normally regulated so that they can cause inflammation
of the pancreatic islets, but this inflammation does not progress
to diabetes or autoantibody production. This regulation is broken
by one or more genes from the NOD strain, an excellent animal model
for spontaneous Type 1 diabetes in humans, but it has been difficult
to identify what regulatory processes are primarily affected. Dr
Sylvie Lesage has discovered that one of the main actions of the
diabetes susceptibility genes is to dramatically reduce the efficiency
of islet-reactive T cell deletion in the thymus. Some of the diabetes
genes act within the islet reactive T cells, interfering with the
normal processes that censor these forbidden clones of cells. By
revealing the primary cell regulatory processes that are disturbed
by diabetes genes, this experimental model provides a unique opportunity
to define the molecular pathways regulating diabetes and other autoimmune
diseases.
Memory receptor tail
Our ability to resist infection stems from a cardinal property
of the immune system, namely an ability to mobilise a much higher
and faster antibody response when the antigens that make up a virus
or bacteria are seen a second time. This phenomenon of immunological
memory lasts for years after infection or immunisation, but its
cellular and molecular basis is still poorly understood. Mr Stephen
Martin has defined a key role for the 'BCR Tail sequence' in promoting
memory-level antibody responses. It has been known for many years
that B cells switch portions of the antibodies they produce from
IgM to IgG as part of the memory process. Among the many changes
that result from switching to IgG, one intriguing change is in the
sequence and length of the antibody segment that serves as a receptor
for antigen - the transmembrane and cytoplasmic tail. Through a
sophisticated combination of transgenic mice and in vivo
immunological analyses, Steve has shown that the memory-type IgG
tail dramatically increases the number of progeny cells that are
formed by proliferation of initially rare B cells that react to
antigen. This finding explains the memory phenomenon by localizing
it to a precise sequence of amino acids and a specific process -
augmenting the clonal burst. This finding has wide implications
for vaccination, allergy and autoimmunity, where B cells bearing
the memory tail are formed.
Maintaining an army reserve
Infectious disease agents are constantly changing, with new or
variant forms arising constantly, so that it is impossible to anticipate
the form or fingerprint of an infection in advance. To tackle this
problem, the immune system must keep on hand a reserve of billions
of B lymphocytes marching about the body. Each cell carries a different
antibody so that by chance at least one of these antibodies will
be able to recognize and destroy any possible infectious agent.
A central issue is how the size of this reserve population is regulated:
immunodeficiency diseases result from it becoming too small, while
common forms of lymphoma and leukemia result from it growing out
of control. Through analysis of an ENU-induced mouse mutation, Ms
Lisa Miosge has defined a critical regulator of this process, NfkappaB2
showing that this DNA-binding transcription factors acts within
each B cell to promote persistence in the reserve population of
recirculating mature B cells. This result helps explain how activating
mutations in NfkappaB2, which have previously been demonstrated
in B cell lymphoma, can contribute to the uncontrolled accumulation
of these cells. Together with other research published at the same
time, Lisa's work establishes that NfkappaB2 mediates the survival-promoting
effects of a newly discovered B cell growth factor, BAFF/Blyss.
Immunity
and Immunopathology in Infectious Disease
Dr Eva Lee, Dr Mario Lobigs and Dr Arno Müllbacher
 |
The general aims of the program are first, to
generate new knowledge relevant to our understanding of the
fundamental properties of immune responses at the molecular,
cellular and whole system level with particular emphasis on
immune responses against viruses and second, to study virus/host
interactions at the cellular and molecular level and through
this devise strategies for the prevention of viral disease.
Dr Mario Lobigs |
The members of the program have wide expertise in immunology, immunopathology
and molecular virology. Our current investigations focus on the
different functions of cytolytic effector molecules, MHC class I
antigen presentation, T lymphocyte responses against infection with
viruses, bacteria and fungi, the cytotoxic T memory response, virus/host
interactions in flavivirus assembly and replication, and viral immune
evasion strategies. A large number of virus models including flaviviruses,
poxviruses, influenza and parainfluenza viruses, alphaviruses, herpes
viruses and adenoviruses are employed in these studies. The availability
and establishment by our laboratories of gene targeted mice defective
in immune effector molecules including perforin, the granzymes,
and Fas receptor/ligand has allowed us to elucidate important host/parasite
relationships in the context of the host immune response. Another
important approach which is currently applied widely in the group
is that of reverse genetics using full-length cDNA copies of flavivirus
RNA genomes. These allow the in vitro synthesis of infectious
viral RNA and thus structure/function studies in flavivirus replication
and pathogenesis.
Progress in our research in the last year
Antigen dependent release of interferon-gamma by cytotoxic T
cells upregulates Fas on target cells and facilitates exocytosis
independent specific target cell lysis.
Effector cytolytic T (Tc) lymphocytes, deficient in the exocytosis-mediated
pathway of target cell lysis, induce Fas on target cells and, in
turn, delayed cell death and apoptosis via the FasL-Fas interaction.
The induction of Fas can be blocked by anti- IFN-g
antibodies. This Fas upregulation on initially Fas negative target
cells is not mediated by T cell receptor-MHC/peptide signalling
per se but by secreted interferon-gamma from Tc cells after
antigen engagement. The Fas upregulation by Tc cells can be mimicked
by treatment of target cells with rIFN-g.
Tc cells from IFN-g ko mice do not induce
Fas expression on target cells. Tc cell mediated Fas expression
on third party, bystander, target cells does not enhance their susceptibility
to lysis by these nominal effector cells. The results are discussed
as to the possible relevance of the phenomenon in efficiency and
regulation of the Tc cell response to infections by viruses.
Cell-mediated cytotoxicity in recovery from poxvirus infections
The availability of mutant and gene targeted knock out mice with
defects in components of cellular cytotoxicity mediated by either
the Fas or the exocytosis pathway permitted an analysis of their
role in recovery from poxvirus infections. Ectromelia (EV), a natural
mouse pathogen causing mouse pox, the closely related orthopoxviruses
cow pox (CPV) and vaccinia virus (VV), each encode serpins which
inhibit Fas mediated apoptosis and lysis of target cells. Nevertheless,
differences were seen when the three viruses were inoculated into
perforin-deficient mice: highly resistant C57Bl/6 mice became susceptible
to low doses of EV; resistance to CPV increased whereas there was
no effect on VV infections. Absence of the cytolytic granule associated
granzymes (gzm) A and B rendered C57Bl/6 mice increasingly more
susceptible to EV infections. Lack of both gzms rendered them as
susceptible as perforin deficient mice, despite the presence of
functionally active perforin. Elevated EV titres in liver and spleen
of gzmAxB deficient mice, early after infection and before cytotoxic
T cells were detectable, strongly suggests that these two gzms exert
an anti-viral effect by a mechanism distinct from effector molecules
of NK and cytotoxic T cells.
Lack of both Fas ligand and perforin protects from flavivirus-mediated
encephalitis in mice
The mechanism by which encephalitic flaviviruses enter the brain
to inflict a life-threatening encephalomyelitis in a small percentage
of infected individuals is obscure. We investigated this issue in
a mouse model for flavivirus encephalitis in which the virus was
administered to six-week-old animals by the intravenous route, analogous
to the portal of entry in natural infections, using a virus dose
in the range experienced following the bite of an infectious mosquito.
In this model infection with 0.1- 105 PFU of virus gave
mortality in ~50% of animals despite low or undetectable virus growth
in extraneural tissues. We have shown that the cytolytic effector
functions play a crucial role in invasion of the encephalitic flavivirus
into the brain. Mice deficient in either the granule exocytosis-
or Fas-mediated pathways of cytotoxicity showed delayed and reduced
mortality. Mice deficient in both cytotoxic effector functions were
resistant to a low dose peripheral infection with the neurotropic
virus.
|
Role of type I and type II interferon responses in the
recovery from infection with an encephalitic flavivirus
We have investigated the contribution of the interferon (IFN)-a/b
system, IFN-g, and nitric oxide
to recovery from infection with Murray Valley encephalitis
virus, using a mouse model for flaviviral encephalitis where
a small dose of virus was administered to 6-week-old wild-type
and gene knockout animals by the intravenous route. We show
that a defect in the IFN-a/b responses
results in uncontrolled extraneural virus growth, rapid virus
entry into the brain, and 100% mortality. In contrast, mice
deficient in IFN-g or nitric oxide
production display an only marginally increased susceptibility
to infection with the neurotropic virus.
Dr Eva Lee
|
 |
Cross-protective and infection-enhancing immunity in mice vaccinated
against flaviviruses belonging to the Japanese encephalitis virus
serocomplex
The Japanese encephalitis virus serocomplex is a group of mosquito-borne
flaviviruses that cause severe encephalitic disease in humans. The
recent emergence of several members of this serocomplex in geographic
regions where other closely related flaviviruses are endemic has
raised urgent human health issues. Thus, the impact of vaccination
against one neurotropic virus on the outcome of infection with a
second, serologically related virus is unknown. We show here that
immunity against Murray Valley encephalitis virus in vaccinated
mice can cross-protect but also augment disease severity following
challenge with Japanese encephalitis virus. Immunepotentiation of
heterologous flavivirus disease was apparent in animals immunized
with a ‘killed’ virus preparation when humoral antiviral
immunty of low magnitude was elicited.
Viral Immunology Group
Leader: Professor RV Blanden
In adult humans, many of the B lymphocytes responsible for memory
and secondary antibody responses display mutations in the genes
encoding the protein chains of the antibody molecule. These mutations
contribute to the phenomenon of affinity maturation of antibody
responses in which secondary and later responses show improvement
in affinity over early responses, thus improving the protective
potential of the antibodies.
Conditions under which hypermutation of antibody genes is induced
in B lymphocytes and the mechanisms of selection of B cells with
mutated antibodies of high affinity against the immunizing antigen
have been well defined. However the molecular mechanism of somatic
hypermutation remains one of the major unsolved mysteries in immunology.
We are testing an hypothesis involving pre-mRNA from rearranged
variable regions of antibody genes as the primary target of sequence
variation. Reverse transcription then produces mutated cDNA which
replaces the original gene by homologous recombination. Predictions
of this molecular model are being tested using transgenic mice.
Immunopathology Research Group
Dr Bill Cowden and Dr Brett Charlton
 |
Work in the Immunopathology Research Group is
focused on developing novel treatments for and understanding
key biochemical processes underlying debilitating cell-mediated
disease processes. As the term immunopathology suggests, the
Group is interested in the pathological (tissue damaging) changes
that occur as a direct result of either normal or abnormal immunological
processes.
Dr Bill Cowden |
Autoimmune diseases such as multiple sclerosis (MS) and rheumatoid
arthritis are examples of pathologies that occur as the result of
an abnormal immunological process because in these cases cells of
the immune system appear to 'attack' what seems to be healthy tissue
rather than performing their intended role of dealing with invading
pathogens. Such cell-mediated autoimmune diseases are characterised
by an accumulation of leucocytes, white blood cells that are the
mainstays of the immune system, in the affected tissue. Thus, in
the case of MS an abnormal accumulation of leucocytes is found in
the brain and spinal cord while in arthritis this build-up occurs
in the diseased joint.
The usual role for these cells is in fighting infection but in
the case of autoimmunity the cells appear to 'recognise' normal
tissue as being 'foreign' and as a consequence of this 'misinterpretation'
these cells attack the affected tissue. This results in damage to
the tissue and this is reflected in clinical symptoms. In the case
of MS, symptoms range in type and severity depending upon the areas
of the brain affected. Thus, transient mild weakness or tingling
in the limbs may be the only symptom in some MS patients while others
may suffer paralysis or blindness. In the case of arthritis, symptoms
are invariably directly associated with the inflamed joint and once
again the clinical signs range in severity from mild discomfort
to crippling disfiguration.
Since an essential component of these diseases is the accumulation
of inflammatory cells, a potential approach to treating them is
to reduce or prevent the accumulation of leucocytes in the affected
tissue (inflammatory site). White blood cells normally circulate
through the body, along with erythrocytes (red blood cells), inside
the vascular system (blood vessels). The mechanism by which leucocytes
arrive at the inflammatory site is both complex and incompletely
understood. There are, however, many potential points at which this
process may be interfered with. Work carried out in collaboration
with others within the School and at the Neurosciences Research
Group at The Canberra Hospital has identified a promising biochemical
target for preventing cell migration. Thus, research in our Group
is currently targeting one of the final biochemical steps involved
in the migration of the leucocytes from blood vessels into the adjacent
tissue. The importance of translating basic findings into clinical
outcomes is central to our efforts and since experimental results
have been promising we hope to trial the potential treatment in
the clinic. Therefore, one of our goals over the next 12 months
is to initiate and facilitate a multi-centre clinical trial in the
treatment of relapsing-remitting MS.
Another major interest of the Group is the role of a molecule,
called nitric oxide, in autoimmune processes. This molecule is produced
in large amounts by certain leukocytes that have been stimulated
by invading pathogens, especially bacteria and protozoan parasites
(malaria for example) that live and replicate inside host cells.
Infection with some viruses also elicits the production of nitric
oxide. This molecule is chemically reactive and this attribute may
be responsible for its ability to inhibit the growth and spread
of certain infectious agents. This property may also contribute
to its widely reported immunopathological properties in both infectious
and in autoimmune diseases. Another way of looking at this is that
cell of the immune system produce nitric oxide in order to damage
invading micro-organisms but in the process, normal tissue in the
immediate vicinity can also be damaged by this chemically reactive
molecule. On the other hand, nitric oxide is also produced in small
quantities by cells other than those of the immune system. In this
situation, nitric oxide has some roles that are directly related
to normal, non-immunological processes such as the control of high
blood pressure and neurotransmission. Other groups within the School
study some aspects of these activities.
The Immunopathology Group has investigated the potential pathological
role of nitric oxide in some autoimmune diseases such as type-1
or juvenile-onset insulin-dependent diabetes and in a multiple sclerosis-like
disease. In contrast to many reports in the medical and scientific
literature, our group has discovered that in some autoimmune disease
settings, nitric oxide has a down-regulating effect on the disease.
In other words, in addition to possibly having a localised tissue
damaging role in these diseases, it may, in contrast, actually slow
down or reverse the disease process. In this regard our group has
found that in the absence of nitric oxide production by the immune
system, some autoimmune diseases are actually more severe and protracted
than would otherwise be the case. This finding implies that nitric
oxide may function in a "feed-back" manner to signal the
immune system to reduce or halt its attack. This was further supported
by our discovery that by increasing systemic levels of nitric oxide,
either through immunological manipulation or drug treatment, these
diseases were less severe. This finding is perhaps important from
a therapeutic perspective, and because of this the group is actively
studying potential means by which nitric oxide production can be
elevated in the hopes that such therapy may be of benefit in the
treatment of autoimmune diseases.
Human Genetics Group
Leader: Professor Simon Easteal
My research is aimed at understanding human genetic variation as
it relates to disease. I take a broad view, recognising the importance
of the evolutionary forces that have shaped the structure of the
human genome and the nature of human genetic diversity. Our investigations
fall into two areas: Mechanisms of gene and genome evolution, and
the nature of human genetic and genomic variation.
Gene and genome evolution
In mammals genetic novelty arises through genetic and evolutionary
processes such as mutation, gene duplication, gene conversion, genetic
drift and natural selection, occurring in large and structurally
complex genomes. Information relating to genome and protein function,
the processes by which functional novelty has arisen and patterns
of species evolution are contained within DNA and protein sequences.
We use a comparative approach to the interpretation of this information,
investigating differences in genomes, genes and the proteins they
encode, among related species, particularly humans and other primates.
Human genetic and genomic variation
Medical genetics has largely been focused on understanding single
gene defects. Less attention has been given to genetic disorders
that have a polygenic basis, such as mental illness, cardiovascular
disease and diabetes. Although these are more important from a public
health point of view, they are technically more difficult to investigate.
This situation is changing largely through the technological advances
of the human genome project. Associated with this change is an increased
recognition of the importance of the normal range of human genetic
variation, and its importance in relation to disease prevention.
Understanding the relationship between molecular genotypes and disease
phenotypes is becoming more dependent on population-based rather
than family-based investigations. Our research is directed at understanding
the basis of a number of genetic disorders, and of the patterns
of normal genetic variation in human populations.One particular
focus is the genetic basis of personality variation, which is associated
with a predisposition to common forms of mental illness involving
depression and anxiety. This work is done in collaboration with
the NH&MRC Centre for Mental Health Research.
Vaccine
Immunology Group
Leader: Professor Ian Ramshaw
After two decades of intense research in HIV vaccine development,
recent successes with several commercially viable vaccine candidates
now offer hope. Studies in animal models, including our own, demonstrate
that DNA prime: poxvirus boost strategies can induce potent T cell
responses. Furthermore, these responses confer significant protection
against both HIV and SHIV challenge. With the support of a US National
Institutes of Health award for A$30 million to a consortium of Australian
researchers we will test this AIDS vaccine concept in clinical trial
in early 2003. Our Group has been responsible for construction and
testing of the DNA component of this vaccine. This is the largest
grant yet given to an Australian research team from an international
agency. Several significant obstacles remain, however, for eventual
wide scale application of these approaches in humans. Importantly,
since the HIV epidemic is global, and the different isolates of
HIV differ significantly in sequence homology, an effective vaccine
must be able to protect a genetically diverse human population against
a wide range of viral isolates. We are currently developing the
next generation of HIV vaccines to address this issue (see details
below).
Immune Regulation and Vaccine
Development Laboratory
Leader: Professor Ian Ramshaw
The focus of our research is to study the factors important in
generating high levels of protective immunity to vaccination. In
particular, we are concentrating on the use of prime boost immunisation
to stimulate high levels of cell-mediated immunity. We have been
studying why this vaccination strategy generates such a powerful
response. Using a tetramer staining technique we have been able
to show that DNA vaccines are able to induce very effective or high
avidity T cells which are then expanded by boosting with a recombinant
fowlpoxvirus vaccine encoding the same vaccine antigen. The quality
of an immune response generated by a particular vaccine strategy
may therefore be as important as the levels of immunity induced.
Vaccines that generate T cell populations of high avidity, optimising
the early detection of infected cells, offer new hope for development
of effective prophylaxis against pathogens such as HIV, which have
presented major problems for vaccine development.
Synthetic Vaccines Laboratory
Leader: Dr Scott Thomson
Research in the laboratory focuses on developing new vaccine and
therapeutic strategies for a broad range of human diseases including
viral infections eg HIV-1, Hepatitis C virus, and tuberculosis,
and cancer eg Cervical Cancer and Nasopharyngeal Carcinoma. The
laboratory is closely involved with the Australian HIV Vaccine Consortium,
which has a large NIH funded design and development contract to
carryout two HIV-1 clinical trials in Australia and SE Asia. The
HIV consortium work includes ongoing development work on the two
DNA vaccines we constructed which will be used as the first component
of the immunisations in the clinical trials for two major HIV-1
subtypes, A/E and B.
The laboratory is also continuing to develop the new scrambled
antigen vaccine (SAVINE) strategy. This strategy is particularly
suited for inducing killer T cells or cytotoxic T cells (CTL) which
patrol the body and kill other cells that have been infected or
have become cancerous. This technology which is a major breakthrough
in T cell vaccine design, is essentially a genetic-based delivery
strategy for overlapping peptide sets. Overlapping peptide sets
that span a virus or protein antigen are often used to identify
T cell epitopes but have far too many components to be used as a
vaccine. The SAVINE strategy means that the long unsolved problem
of population coverage can now be catered for while retaining the
safety characteristics of short peptides. The lab has already synthesised
and is testing an HIV-1 vaccine that incorporates sequence from
the entire virus. We are currently synthetising a hepatitis C virus
SAVINE (whole virus) and Tuberculosis SAVINE (13 key immunogenic
antigens). We are also considering constructing a SAVINE using cancer
antigens since such antigens cannot be used whole due to their onogenic
potential.
Older technology based on polyepitope vaccines, where multiple
contiguous T cell epitopes are used to design synthetic antigens,
is continuing to be developed through collaborations with the EBV
unit and CRC for vaccine technology (Queensland Institute of Medical
Research) and the Sir Albert Sakzewski Virus Research Center (University
of Queensland). These collaborations are developing vaccines against
Epstein-Barr virus which causes glandular fever and Human Papilloma
virus which is the leading cause of cervical cancer.
The laboratory also has a strong interest in various immunomodulatory
molecules and delivery vectors which when combined with our various
antigen technologies may enhance vaccines in the future and add
to our understanding on how vaccines operate.
Initiators and Regulators
of Immunity Laboratory
Leader: Dr Joanne Banyer
The nature of work in the Initiators and Regulators of Immunity Laboratory
aims to further our understanding of how immune responses are initiated
or avoided by cytokines, pathogens, and vaccine agents.
|
This knowledge may be used to design more effective vaccines
and to identify immunoregulatory molecules that may be used
as vaccine adjuvants, or immunotherapeutics that re-direct
inappropriate or enhance immune responses to pathogens.
Dendritic cells (DC) are the immuno-interpretors of the innate
immune cell system that identify, process, and present antigens
from micro-organisms to adaptive immune cells.
|
Dr Joanne Banyer
|
Whether the DC initiates an adaptive cell mediated immune (CMI)
or humoral immune (HI) response is dependent on the nature of the
pathogen and the types of cytokines that are released into the microenvironment
by innate immune cells at the site of infection. These combinations
of factors act on DC's to develop immunoregulatory properties which
communicate to adaptive immune cells the identity of the pathogen
and the type of immune response which should be generated against
the pathogen. How the DC communicates this information and regulates
adaptive immunity is not completely understood. To address this
issue our laboratory is investigating the effects of CMI and HI
cytokines on the immunoregulatory properties of DC's. Our approach
utilises a combination of biological and molecular analysis of ex
vivo DC's and DC cell lines to identify immunoregulatory molecules
that are differentially regulated by these cytokines.
Interactions between the pathogen and the DC provide the perfect
opportunity for the pathogen to modify the type of immune response
that develops. Pathogens such as HIV and Hepatitis C utilize these
interactions to reduce the immunostimulatory properties of DC's
thereby disrupting clearance of the virus and supporting viral persistence.
The immune avoidance mechanisms elicited by these viruses are also
not completely understood. Our laboratory is investigating these
interactions using Hepatitis C infection of human monocyte derived
DC's as a model system. Previous studies of chronically infected
patients have indicated Hepatitis C persistently infects and disrupts
the immunoregulatory function of these cells. To establish how the
virus achieves this we are targeting the initial interaction between
the virus and non-infected DC's utilising both biological and molecular
analysis.
Many vaccines are based on the use of pathogen-derived vectors
and some pathogen molecules, such as CpG sequences, are used as
vaccine adjuvants to enhance immunoresponsiveness to vaccines. The
nature of these vaccine agents, however also influences the type
of immune response that a DC directs. In addition, some of these
vaccine agents have been shown to elicit immune avoidance mechanisms
that target the immunoregulatory properties of DC's. To further
understand the effects of vaccine agents on the immunoregulatory
properties of DC's our laboratory has examined the effects of DNA
and viral vaccine vectors in combination with CMI and HI associated
cytokine stimulus of DC's. This work established that in the presence
of CMI-associated cytokines DC's are more efficient at taking up
and processing these vaccine agents. Also, in combination with these
cytokines the vaccine agents can enhance the immunoregulatory properties
displayed by the DC's and therefore are likely to trigger stronger
immune responses. Similar to other recent studies, we found that
particular viral vaccine agents, vaccinia and fowl-pox, affect the
viability of the DC, however we established that particular CMI
associated cytokines enable the DC to process and present the viral
antigen prior to cell death. In addition, preliminary studies in
our laboratory suggest that DC's utilise other mechanisms, such
as cross-presentation, to present virally encoded antigen. These
studies have highlighted the important role of cytokines that enable
DC's to handle pathogen-derived antigen appropriately and thereby
support their potential use in effective immunization strategies.
Design and engineering of highly effective vaccines in the future
will benefit from a more thorough understanding of the immune stimulatory
and inhibitory properties of vaccine agents so these properties
may be altered to better support generation of strong and appropriate
immune responses.
Infection and Immunity Group
Leader: Dr Gunasegaran Karupiah
 |
Viral diseases are a major health, social and economic problem
worldwide. Effective vaccines have been successfully developed
for only a small minority of disease-causing viruses and available
antiviral agents are of limited efficacy and high cost. Furthermore,
little effective intervention is currently available to minimize
the often, serious immunopathology in established infections.
Dr Guna Karupiah
|
The immune system uses several strategies to fight virus infections.
These include the activation of a complex network in which numerous
cell types and soluble factors of the immune system participate.
Our hypothesis is that the host's own immune system is inadvertently
responsible for the cause of pathological states through its over-vigorous
attempts to destroy the virus, resulting in damage to the host itself.
The research interests of the Infection and Immunity Group are
in the broad area of virus-host interactions and we are pursuing
this goal using a range of viral and animal models. An integral
component of our research program involves basic studies that attempt
to dissect the roles of leukocyte subsets, cytokines, chemokines,
antibody and a number of signaling molecules in viral infection
and disease. The effector mechanisms that are generated to control
and clear virus instead often cause immunopathology that has serious,
sometimes lethal, consequences for the host. As a consequence we
have directed our research effort toward dissecting out the immunological
parameters that allow the rapid resolution of virus infection with
minimum pathology. These studies are being carried out in parallel
with others that attempt to reveal the many strategies that viruses
have evolved to subvert the host immune response. We believe that
understanding virus-host interactions is the most promising route
to the development of effective vaccines and of selective treatments
that would minimize the damaging effects of an established infection.
The Group moved from the University of Sydney to the JCSMR at the
end of 2001. There was an anticipated and expected transition phase
through which the laboratories were set up and personnel recruited.
In the last year, we have made substantial progress in 5 key areas
of our research. First, we now have a better understanding of whether
the cytolytic potential or the ability to produce cytokines by CD8+
lymphocytes (a key population of white blood cells necessary for
virus control) is important for virus clearance. Second, we have
successfully established an animal model for studying the consequences
of 'reverse signaling' via membrane bound tumor necrosis factor,
a key proinflammatory cytokine and one that is involved in leukocyte
recruitment. We have found that this is a critical cytokine for
recovery from some viral infections. Third, we have some useful
insights into how antibody that is produced during a primary viral
infection controls virus proliferation and spread. Fourth, we have
evidence that granulocytes are critical for helping to shape the
protective immune response to viral infection. Finally, we believe
we have some pertinent clues as to why the gas, nitric oxide, an
important molecule involved in normal physiology, can also be a
critical mediator of influenza pneumonia.
Diabetes/Transplantation
Immunobiology Laboratory
Leader: Dr Charmaine Simeonovic
Studies in the laboratory have focused on the immunobiology of
pancreatic islet xenotransplantation and allotransplantation in
mice.
Immunomodulatory effects of QFA (Coxiella burnetii, Phase 1)
treatment of mice
We have been examining the capacity for non-specific antigen stimulation
of the host immune system with QFA (formalin-killed, complement
fixing antigen of Coxiella burnetii) to induce a regulatory
immune response which protects against islet allograft rejection.
Previous studies by others have shown that QFA treatment of pre-diabetic,
adult NOD mice prevents the induction of clinical diabetes i.e.
QFA therapy appears to regulate the destructive autoimmune response.
In an islet allograft model, we have shown that QFA treatment modulates
the cellular immune response to allografts of adult BALB/c islets
in C57BL/6J (B6) mice; recipient animals pretreated with QFA (150ml)
i.p. at 7 days prior to transplant showed prolonged survival of
islet tissue, often in the presence of a mild cellular infiltrate.
In contrast, control grafts from PBS-treated mice showed an intense
inflammatory response and only damaged islet tissue present at the
graft site. Similar prolongation in allograft survival was achieved
in iNOS-/- mice but not in IFN-g-/- mice
or TNF-/- mice, indicating that the protective mechanism is IFN-g-dependent,
TNF-dependent but not iNOS-dependent nor NO-dependent. To further
examine the mechanism of IFN-g-dependent
protection, quantitative RT-PCR analysis of cytokine and iNOS gene
expression in allografts from QFA-treated and PBS-treated iNOS-/-,
iNOS+/+, IFN-g-/- and B6 mice showed
that islet allograft protection in QFA-treated mice correlated with
enhanced intragraft expression of TNF and strong expression of IFNg_transcripts;
protection also correlated with lack of IL-4 gene expression. Prolonged
islet allograft survival following QFA treatment therefore correlates
with IFNg gene expression, enhanced TNF
mRNA levels and inhibition of IL-4 gene transcription. RT-PCR analyses
of cytokine transcripts in liver mononuclear cells (MNCs) from QFA-treated
and control B6 mice suggested that there was enhanced expression
of IFN-g and TNF transcripts in QFA-treated
liver MNCs. Together these studies suggested that allograft protection
may be mediated by one or more subpopulations of liver MNCs. Four-colour
flow cytometry staining (NK1.1, abTCR,
CD4/CD8 and to _- galactosyl- ceramide-loaded CD1 (a-
GalCer- CD1) tetramer) indicated that QFA treatment did not lead
to amplification of NKT cells in liver or spleen. In addition, B6.CD1-/-
mice (which lack functional NKT cells) pre-treated with QFA (150ml,
i.p.) but not PBS (controls) remained intact at 1 week post-transplant,
confirming that NKT cells do not contribute to QFA-mediated islet
allograft protection. Flow cytometry studies of liver MNCs from
B6 mice at 2 weeks post-QFA treatment showed that CD11b+F4/80+
macrophages increased 241-fold and TCRab+
NK1.1- T cells increased 69-fold compared to liver MNCs
from control PBS-treated B6 mice. It is therefore possible that
liver CD11b+F4/80+ macrophages and/or liver
TCR + NK1.1- T cells are responsible for QFA-mediated
allograft protection; these sub-populations may also play a role
in QFA-induced protection from clinical diabetes in NOD mice. Adoptive
transfer studies in the islet allograft and autoimmune diabetes
models are now warranted.
Charmaine J. Simeonovic, Sarah K. Popp, Jodie Zarb and Alan
Baxter
Evaluation of Fowlpox viral vector for gene delivery to pancreatic
islet
transplants
The feasibility of using avipox virus as a vector for gene
delivery into fetal pig proislets, fetal mouse proislets and adult
mouse islets was examined using a recombinant fowlpox virus engineered
to express the lacZ gene (FPV-LacZ). The efficiency of in vitro
transduction was tissue-specific and species-specific: the optimal
infective dose of FPV-LacZ for pig proislets, mouse proislets, adult
mouse islets and NIT-1 cells (pancreatic beta cell line) was 1 x
105pfu / 100ml (mouse proislets
only) -1 x 108pfu/ 100ml.
Following transplantation to immuno-incompetent nude mice, reporter
gene expression in the FPV-treated islet tissue was transient (3-7
days). In contrast, FPV-LacZ- infected NIT-1 cells maintained lacZ
gene expression beyond 18 days. Silencing of transgene expression
which occurred in vivo but not in vitro, therefore
did not require a T cell immune response. Treatment of transplanted
nude mice with anti-IFN-g mAb failed
to prolong transgene expression. Following transplantation to immunocompetent
mouse strains, immunological destruction of isografts of FPV-LacZ-infected
adult mouse islets suggested that either an immune response to FPV
proteins or to the reporter gene product, b-galactosidase
had been generated. Co-delivery of the immunoregulatory cytokine
gene bioactive rat TGFb to adult mouse
islets by in vitro infection with FPV-TGFb
failed to prevent immunological injury, despite intragraft expression
of TGFb mRNA for up to 3 days post-transplant.
The Fowlpox viral vector is therefore a suitable mechanism for gene
delivery to pancreatic isle tissue in vitro but not in
vivo, due to an unidentified pathway of transgene silencing.
Michelle Solomon, Ian Ramshaw, Jodie Zarb, Celina-Anne Lynch,
Karla Harris and Charmaine Simeonovic
Cancer and Human Immunology
Group
Leader: Dr Hilary Warren
Human Natural Killer Cells
Natural Killer (NK) cells are cells of the innate immune response
that attack virally infected cells or cells that have undergone
malignant transformation. To regulate these activities, NK cells
express both activating and inhibitory receptors. The inhibitory
receptors bind MHC class I molecules, such that alterations in cells
that lower the level of MHC class I at the cell surface result in
permissive NK cell activity. The identity of various activating
receptors is known, but the ligands they interact with remain largely
unknown.
During 2002, studies have been completed on the expression and
function of MHC class I receptors in patients with NK lymphocytosis,
and on the expression and function of the KIR2DL4 receptor in donors
expressing different alleles of this receptor. Both these projects
are a collaborative study with immunogeneticists at Royal Perth
Hospital.
NK lymphocytosis patients have on average 17-fold higher numbers
of NK cells in their peripheral blood compared to normal donors.
We showed that the CD94/NKG2A inhibitory receptor is expressed by
all NK cells in these patients, compared to 50% in normal donors,
and that patients' NK cells have a higher level of expression of
this receptor. Other inhibitory receptors such as the Killer Ig-like
Receptors are rarely expressed on patients' NK cells. CD94/NKG2A
inhibits NK cell activity by binding to the non classical class
I molecule HLA-E. Interestingly, HLA-E is only cell surface expressed
when it binds in its peptide groove the signal sequence peptide
from a range of classical class I molecules. Thus CD94/NKG2A can
monitor the level of expression of many class I molecules to assess
whether a cell is normal or abnormal. We showed that in NK lymphocytosis
patients CD94/NKG2A is functional. Normal functioning of this receptor
may account for the fact that many patients are asymptomatic, such
that the enlarged NK cell pool remains functionally silent.
The KIR2DL4 receptor is expressed (at the mRNA level) by all NK
cells, yet its cell surface expression and function remain controversial.
KIR2DL4 probably recognises HLA-G, a non classical class I molecule
expressed on placental trophoblasts, implicating a role for this
receptor in pregnancy. Interestingly, there are several alleles
of the KIR2DL4 gene. Of the two most common alleles, one results
in a truncated form of the receptor lacking a cytoplasmic domain.
Twenty five percent of the population are homozygous for this defective
KIR2DL4 allele. We have established that the presence of at least
one allele encoding the full length receptor is required for expression
and function of KIR2DL4, and that there is no expression and function
of KIR2DL4 in individuals homozygous for the defective allele. Interestingly,
even for individuals with the full length allele, KIR2DL4 is expressed
only on the minor NK cell subset characterised by high expression
of CD56 and which is a potent producer of IFN . The major NK cell
subset, characterised by low expression of CD56, does not express
KIR2DL4, but expression is induced following culture. KIR2DL4 functions
as an activating receptor in experimental conditions, yet has the
capacity to function also as an inhibitory receptor. Our studies
have clarified conflicting results in the literature on the expression
and function of KIR2DL4.
