Unit Contact
Professor
Nicholas Hunt
Discipline of Pathology
Medical Foundation Building (K25)
92-94 Parramatta Road
Camperdown, NSW, 2042
Phone: +61 2 9036 3242(Office)
Fax: +61 2 9036 3286
Email: nhunt@med.usyd.edu.au
Unit Personnel
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Research Overview
The
chief areas of research currently in the Unit are the immunopathology of infectious
disease (especially malaria), and the regulation of inflammatory responses.
We are interested in expression of host genes, in particular cytokines, chemokines
and haem enzymes. Some of our work is done with experimental models in
mice, some with cultured cell systems and some with material obtained post-mortem
from victims of malaria.
Overview
Malaria is one of the 'big three' diseases, along with HIV and tuberculosis.
More than half of the world's population lives in areas where malaria is endemic.
There are several hundred million cases each year, resulting in about 2 million
deaths. Most of the deaths occur in children aged 2 to 6 years living
in sub-Saharan Africa. Other groups at risk include pregnant women, adults
in South-East Asia and visitors to malaria-endemic areas.
Life-threatening complications of malaria infection include cerebral malaria, anaemia and lung complications. The mechanisms through which the malaria parasite causes illness in the host is a focus of research in this laboratory.
The malaria parasite spends part of its life cycle in a mosquito and part in another host. Different species of malaria parasite can infect humans, rodents or birds. The stage of infection during which malaria causes illness in the host is the one where it is living inside red blood cells circulating in the body. Many malaria researchers, including us, believe that it is the response of the immune system to the presence of the parasite that leads to many of the disease complications associated with infection. This is called "immunopathology."
Immunity against malaria parasites in the erythrocytic stage is complex. It certainly involves the white blood cells, or leukocytes, namely neutrophils, lymphocytes and monocytes/macrophages. These cells attempt to destroy malaria parasite through effector molecules such as perforin/granzyme, antibody and, possibly, nitric oxide. The immune and inflammatory responses are controlled and coordinated by families of molecules called cytokines and chemokines. Cytokines are proteins produced by cells involved in immune or inflammatory responses that act on other such cells to tell them to start carrying out a particular function, or to cease doing so. Chemokines are cytokines that are involved in attracting leukocytes to sites of infection and inactivating them.
Although the laboratory has some interest in the role of these factors in host immunity against malaria, its chief focus is in the role that they may play in the disease complications.
Specific Projects
Host
Response to Malaria
For many years the destruction of malaria
parasites during the host immune response was considered to be chiefly dependent
on the production of antibody and the phagocytosis of parasitised erythrocytes,
principally by macrophages in the spleen. However, more recently it
has become apparent that this scheme cannot explain all clinical observations
about malaria, particularly the presence within intact erythrocytes of degenerating
parasites and the extensive tissue damage seen in some host species even
when the parasite burden is very low.
Does cellular immunity contribute to the
host response against malaria parasites? It is believed that the cytokine
interferon-gamma plays a key role. This cytokine can be produced by
Thelper (CD4+) lymphocytes, CD8+ lymphocytes, natural
killer (NK) cells or natural killer T (NKT) cells. Interferon-gamma
may activate macrophages to produce a range of soluble mediators, some of
which may be deleterious to the parasite either directly or indirectly.
Two such effector molecules that have been suggested to be directly destructive
of malaria parasites are reactive oxygen species (ROS) and reactive nitrogen
intermediates (RNI).
Using a unique type of gene knockout mouse,
the gp91phox-/- mouse obtained from Dr Mary Dinauer (Indianapolis),
we have evaluated the role of phagocyte-derived ROS in malaria immunity
in mouse models. These gene knockout mice have been genetically engineered
to inactivate the key enzyme, NADPH oxidase, that generates the first in
a chain of ROS in phagocytes. Thus these cells cannot generate anti-microbial
ROS. We have found, in work submitted for publication, that the rate
of muliplication of 5 different strains of malaria parasites in these mice
is unchanged from normal ("wild type") mice that possess normal
phagocytes. Thus ROS are not critical for the host immune response
against primary or secondary malaria infections in mice.
Other researchers have questioned whether
there is a major role for nitric oxide in host anti-malarial responses.
Collaborators:
Dr Mary Dinaeur, Indianapolis, USA
Funding: NHMRC
Pertinent Publications:
Potter, S.M., Sanni, L., Simasathiansophon, S., Dinauer, M.C. &
Hunt, N.H. (2001). Phagocyte-derived reactive oxygen species and the immunology
and pathology of murine malaria. Redox Report 6, 200-203.
Potter, S.M., Mitchell, A.J., Cowden, W., Sanni, L.A., Dinauer, M., de Haan,
J. & Hunt, N.H. Do phagocyte-derived reactive oxygen species influence
the progress of blood-stage malaria infections in mice? Under revision.
Roles
of Cytokines and ROS in Cerebral Malaria
Several aspects of the pathology of malaria
infection seem inexplicable in terms of immunopathological reactions.
In other words, an over-vigorous, excessively prolonged or not specifically
targeted host immune response may cause "bystander" damage to
host cells and tissues. The endothelium (the cells lining the blood
vessels) may be particularly susceptible in this regard. Immune system
regulatory and effector molecules such as ROS, nitric oxide and cytokines
have been implicated in the mechanisms leading to the pathological complications
of malaria infection.
For some years we have been studying the
pathogenesis of cerebral malaria using a mouse model. Previous indirect
evidence had suggested that ROS might be important in the pathogenesis of
murine cerebral malaria. However, experiments with the gp91phox-/-
mice show that phagocyte-derived ROS are not important in this process.
Like other researchers, we have shown that the cytokines interferon-gamma
and lymphotoxin-alpha are important in the pathogenesis of cerebral malaria
in the mouse, but another cytokine, tumour necrosis factor is not.
Collaborators: Dr Christian
Engwerda, Brisbane; Dr Jonathon Sedgwick, DNAX, USA; Professor Georges Grau,
Marseille, France
Funding: NHMRC
Pertinent Publications:
Hansen, A.M., Chaudhri, G. & Hunt, N.H. (1999). Role of immune mediators
in the pathology of experimental murine cerebral malaria. Redox Report 4, 321-322.
Sanni, L.A., Fu, S., Dean, R.T., Bloomfield, G., Stocker, R., Chaudhri,
G., Dinauer, M.C. & Hunt, N.H. (1999). Are reactive oxygen species involved
in the pathogenesis of murine cerebral malaria? J. Infect. Dis. 179, 217-222.
Biochemistry
of Cerebral Malaria
We have found that the kynurenine pathway
of tryptophan metabolism is activated in the brain during cerebral and
non-cerebral malaria in mice. In this cerebral malaria model, an
imbalance occurs in the production of the neuroexcitotoxin quinolinic
acid relative to that of its antagonist kynurenic acid. This may
underlie the hyperexcitable phase of cerebral malaria in mice. Following
on from this, we found that there was a disturbance in the profile of
the biologically active metabolites of this pathway in the cerebrospinal
fluid of cerebral malaria patients from Vietnam and Malawi.
These studies are being carried further
using a unique gene knockout mouse, the indoleamine 2,3-dioxygenase gene
knockout mouse from Drs Mellor and Munn in the USA.
Our studies using Nuclear Magnetic Resonance
have shown that there is a change in the metabolic profiles of brain in
the later stages of murine cerebral malaria, but not in the brains of
mice with severe malaria that does not involve the brain complications.
There is a build up of lactate and alanine, which classically are considered
to be markers of a shift to a hypoxic pattern of metabolism. These
changes are similar to previous observations in human cerebral malaria.
However, the mechanisms involved are not clear in either the mouse model
or in the human disease and we are continuing our investigations of these.
The mouse model allows us to test hypotheses about the mechanisms underlying
these metabolic changes.
Collaborators: Dr
Caroline Rae, Department of Biochemistry, University of Sydney; Dr Isabelle
Medana, Oxford, UK; Dr Terrie Taylor, Blantyre, Malawi; Dr Roland Stocker,
University of New South Wales, Sydney
Funding: NHMRC, ARC
Pertinent Publications:
Sanni, L.A., Tattam, B., Thomas, S.R., Moore, D., Chaudhri, G., Stocker,
R. & Hunt, N.H. (1998). Dramatic changes in oxidative tryptophan metabolism
along the kynurenine pathway in cerebral and non-cerebral malaria. Am.
J. Pathol. 152, 611-619.
Rae, C., Maitland, A., Bubb, W.A. & Hunt, N.H. (2000). Dichloroacetate
(DCA) reduces brain lactate but increases brain glutamate in experimental
cerebral malaria: a 13C NMR study. Redox Report 5, 141-143.
Hansen, A.M., Driussi, C., Turner, V., Takikawa, O. & Hunt, N.H. (2000).
Tissue distribution of indoleamine 2,3-dioxygenase in normal and malaria-infected
tissue. Redox Report 5, 112-115.
Medana, I.M., Hien, T.T., Day, N.P., Phu, N.H., Mai, N.T.H., Chu’ong,
L.V., Chau, T.T.H., Taylor, A., Salahifar, H., Stocker, R., Smythe, G.,
Turner, G.D.H., Farrar, J., White, N.J. & Hunt, N.H. (2002). The clinical
significance of cerebrospinal fluid levels of kynurenine pathway metabolites
and lactate in severe malaria. J. Infect. Dis. 185, 650-656
Medana, I.M., Day, N., Salahifar, H., Stocker, R., Smythe, G., Bwanaisa,
L., Njobvu, A., Kayira, K., Turner, G.D.H., Taylor, T.E. & Hunt, N.H.
(2003). Metabolites of the kynurenine pathway of tryptophan metabolism
in the cerebrospinal fluid of Malawian children with malaria. J. Infect.
Dis. 188, 844-849
Rae, C., McQuillan, J.A., Parekh, S.P., Bubb, W.A., Weiser, S., Balcar,
V.J., Hansen, A., Ball, H. & Hunt, N.H. (2004). Brain gene expression,
metabolism and bioenergetics: Interrelationships in murine models of cerebral
and non-cerebral malaria. FASEB J. 18, 499-510
Hansen, A.M., Ball, H.J., Mitchell, A., Miu, J.M., Takikawa, O. &
Hunt, N.H. (2004). Increased expression of indoleamine 2,3-dioxygenase
in murine malaria infection is predominantly localized to the vascular
endothelium. Int J Parasitol 34, 1309-1319
Other
Immunopathological Mechanisms in Cerebral Malaria
It has been shown by others that CD8+ T lymphocytes play an essential role in the pathogenesis of cerebral malaria.
We have been further investigating this, concentrating on the role of effector
molecules used by these cells to kill cellular targets.
The expression of chemokines and chemokine receptors
at the mRNA level in the brain has been thoroughly documented and intervention
studies using appropriate gene knockout mice are under way to evaluate whether
these changes are essential for the pathogenesis of cerebral malaria in
this mouse model.
Also of interest to the laboratory are the roles
of the haem enzymes haem oxygenase-1 and cyclooxgenase-2. We have
shown that a selective inhibitor of cyclooxgenase-2 exacerbates the onset
of cerebral malaria in the mouse model, which could be relevant to the known
deleterious effects of ingesting aspirin in humans infected with malaria.
We have started on a program of evaluating the
interaction of cytokines and malaria antigens with the individual cell components
that make up the brain: neurons, astrocytes, microglia and endothelial cells.
This work will largely be carried out in cell culture using mouse cells.
A unique approach to studying cell-cell interactions
and pathological mechanisms employed in our laboratory has been the use
of retinal wholemounts. Insights have been gained into the roles of
adhesion molecules, astrocytes and microglia in murine cerebral malaria
in collaboration with Dr Tailoi Chan-Ling, University of Sydney.
Collaborators: Dr Tailoi
Chan-Ling, Department of Anatomy and Histology, University of Sydney; Professor
Iain Campbell, Department of Biochemistry, University of Sydney
Funding: NHMRC, University of Sydney Sesqui Scheme
Pertinent Publications:
Chan-Ling, T., Neill, A.L. & Hunt, N.H. (1992). Early microvascular
changes in murine cerebral malaria detected using retinal wholemounts. Am.
J. Pathol. 140, 1121-1130
Neill, A.L., Chan-Ling, T. & Hunt, N.H. (1993). Comparisons between
microvascular changes in cerebral malaria in mice, using the retinal wholemount
technique. Parasitology 107, 477-487
Medana, I.M., Chan-Ling, T. & Hunt, N.H. (1996). Redistribution and
degeneration of retinal astrocytes in experimental murine cerebral malaria:
relationship to disruption of the blood-retinal barrier. Glia 16,
51-64. [Front cover photograph].
Ma, N., Hunt, N.H., Madigan, M.C. & Chan-Ling, T. (1996). Correlation
between enhanced vascular permeability, upregulation of cell adhesion molecules
and monocyte adhesion in retina during fatal murine cerebral malaria. Am.
J. Pathol. 149, 1745-1762.
Medana, I.M., Hunt, N.H. & Chan-Ling, T. (1997). Early activation of
microglia in the pathogenesis of fatal murine cerebral malaria. Glia 19, 91-103. [Front cover photograph].
Ma, N., Madigan, M.C., Chan-Ling, T. & Hunt, N.H. (1997). Compromised
blood-nerve barrier, astrogliosis, and myelin disruption in optic nerves
during fatal murine cerebral malaria. Glia 19, 135-151.
Medana, I.M., Chan-Ling, T. & Hunt, N.H. (2000). Reactive changes of
retinal microglia during fatal murine cerebral malaria: effects of dexamethasone
and experimental permeabilisation of the blood-brain barrier. Am. J.
Pathol. 156, 1055-1065.
Medana, I.M., Chaudhri, G., Chan-Ling, T. & Hunt, N.H. (2001). Central
nervous system in cerebral malaria: "innocent bystander" or active
participant in the induction of pathology? (Review). Immunol. Cell Biol. 79, 101- 120.
Ball, H.J., MacDougall, H.G., McGregor, I.S. & Hunt, N.H. (2004). Cyclooxygenase
2 in the pathogenesis of cerebral malaria. J. Infect. Dis. 189,
751-758.
Gene
Expression in Cerebral Malaria
As noted earlier, the host immune response
has been found to contribute to the pathogenesis of cerebral malaria.
Determining which components of the immune response are induced might lead
to additional therapies. We are investigating changes in gene expression
in specific cell types in murine malaria infection, and carrying out correlative
studies where possible on human brain tissue obtained post-mortem from malaria
victims. A feature of cerebral malaria is damage to the endothelial
cells leading to the breakdown of the blood-brain barrier. We have
isolated endothelial cells from mouse brain using an advanced technique
called laser capture microdissection. This allows individual cell
types to be captured from frozen sections of tissue then extracted for analysis
of mRNA and, thereby, gene expression. We hope to extend this technique
to human post-mortem tissue in collaboration with Dr Terrie Taylor, Malawi.
Collaborators: Dr Terrie
Taylor, Blantyre, Malawi
Funding: NHMRC, University of Sydney Sesqui Scheme
Pertinent Publications:
Ball, H.J., McParland, B., Driussi, C. & Hunt, N.H. (2002). Isolating
vessels from the mouse brain for gene expression analysis using laser capture
microdissection. Brain Research Protocols 9, 206-213.
Rae, C., McQuillan, J.A., Parekh, S.P., Bubb, W.A., Weiser, S., Balcar,
V.J., Hansen, A., Ball, H. & Hunt, N.H. (2004). Brain gene expression,
metabolism and bioenergetics: Interrelationships in murine models of cerebral
and non-cerebral malaria. FASEB J. 18, 499-510.
Gene
Expression in Asthma
In collaboration with Professor Judy Black,
Drs Jeanette Burgess and Peter Johnson from Pharmacology, University of
Sydney and Dr Margaret Hughes, Pharmacy, University of Sydney, we have examined
the expression of genes in airways smooth muscle in patients with asthma.
This led to the identification of a role for the cell surface molecule OX40L.
The therapeutic possibilities of this have been explored in collaboration
with a major pharmaceutical company.
Collaborators: Professor
Judy Black, Dr Janette Burgess, Dr Peter Johnson, Department of Pharmacology,
University of Sydney; Dr Margaret Hughes, Pharmacy, University of Sydney
Funding: Australian Research Council SPIRT, NHMRC (Prof J Black)
Pertinent Publications:
Burgess, J.K., Carlin, S., Pack, R.A., Arndt, G.M., Au, W. W., Johnson,
P.R.A., Black, J.L. & Hunt., N.H. (2004). Detection and characterisation
of OX40 ligand expression in human airway smooth muscle cells: A possible
role in asthma? J. Allergy Clin. Immunol. 113, 683-689.
Burgess, J.K., Blake, A.E., Boustany, S., Johnson, P.R.A., Armour, C.L.,
Black, J.L., Hunt, N.H. & Hughes, J.M. (2005). Increased expression
of CD40 and OX40L on asthmatic airway smooth muscle following stimulation
with TNFa: Modulation by IL1b. J. Allergy Clin. Immunol. 115, 302-308.
Inflammation
We are collaborating with Dr Bob Bao of
this Department in his work on the pathogenesis of inflammatory bowel disease
using mouse models.
Collaborators: A/Prof Bob Bao, Mucosal Immunology Unit,
Department of Pathology, University of Sydney
Honours Students Projects Available in 2009
Understanding a key new enzyme in the kynurenine pathway
Supervisors: Dr Helen Ball (Pathology), Professor
Nick Hunt (Pathology)
Contact Details: Level 1, Medical Foundation Building helenb@med.usyd.edu.au; phone: 9036 3238
Project
Description: Indoleamine 2,3-dioxygenase (IDO)
catalyses the first step in the kynurenine pathway of tryptophan metabolism.
This pathway is involved in numerous physiological and pathophysiological
processes including immunomodulation and central nervous system disorders.
We recently discovered a second enzyme (IDO2) that performs the same reaction
as IDO (now IDO1) but which is found in different anatomical locations. This project will further investigate the expression pattern of IDO2 by looking at subcellular localisation and expression during development and disease states. The biochemical properties of IDO2 will be examined by determining whether IDO2 enzymatic activity in intact cells is inhibited by nitric oxide, an important mechanism for regulation of IDO1 activity. This project will possibly also include investigating phenotypical characteristics of an IDO2 gene knockout mouse e.g. susceptibility to infectious disease (malaria and meningitis). Laboratory techniques involved in this project include immunohistochemistry, immunofluorescence, quantitative RT-PCR, cell culture, transfection, biochemical assays, mouse models of disease.
What causes death and disability in bacterial meningitis?
Supervisors: Professor Nick Hunt and Dr Helen Ball
(Pathology)
Contact Details: Level 1, Medical Foundation Building
(K25); email: nhunt@pathology.usyd.edu.au; helenb@med.usyd.edu.au
Project
Description: Bacterial Meningitis kills over 150,000 people every year and many more suffer neurological sequelae following infection. We have exetensive experience in uncovering the immunopathological mechanisms in cerebral malaria. We have recently established a mouse model of bacterial meningitis and our preliminary evidence suggests that the cytokine interferon gamma (IFNg) plays an important role in determining whether mice with bacterial meningitis succumb to the disease. IFNg is a key regulator of indoleamine 2,3-dioxygenase-1 (IDO1) expression and thus activity of the kynurenine pathway. Activation of this pathway is observed in a number of central nervous system disorders including cerebral malaria.
This project will further investigate the role of IFNg in bacterial meningitis. Gene expression during bacterial meningitis in IFNg gene knockout and wildtype mice will be compared using gene arrays to identify pathways regulated by IFNg in bacterial meningitis. IDO1 expression is upregulated in bacterial meningitis in wildtype mice and IDO1 expression will also be investigated in the IFNg gene knockout mice. The levels of kynurenine pathway metabolites during bacterial meningitis will be measured in both mouse strains. The neurological sequelae in IFNg gene knockout mice that recover from bacterial meningitis will be measured in behavioural studies that compare these mice with both uninfected and antiobiotic-treated mice. The progression and outcome of bacterial meningitis in IDO1 gene knockout mice will also be investigated. Laboratory techniques involved in the project include mouse models of disease, mouse behavioural studies, quantitative RT-PCR and gene arrays, histopathological analysis, biochemical assays.
Recent Publications
Click here for full list of publications
Current Student Research Projects
Mr
James McQuillan
Severe malaria is an infection tht poses a two pronged problem
for the patient. The parasite continues to replicate, invading host
red blood cells and destroying them. At the same time the immune
system, in an attempt to eradicate the parasite can often have deleterious
side-effects. We plan to investigate the outcome of combining immune
modulation strategies with anti-malarial drugs in an attempt to improve
treatment for malaria. We will also study the biological mechanisms
underlying these actions.
Unit Resources
Grants Awarded