Vascular Immunology Laboratory - Honours projects available in 2009

An Honours project undertaken in this lab would be administered by the Discipline of Pathology.

General context: Fatal malaria is one of the most destructive and potentially correctable disease burdens in the world, affecting mainly children. A major complication is cerebral malaria (CM). Current therapies are limited by our lack of knowledge of the pathophysiological events. Our studies aim at increasing the understanding of pathogenetic mechanisms of CM, and may therefore lead to new approaches to the prevention or prompt treatment of potentially fatal malaria.

General background: We recently demonstrated that microparticles (MP), which we found to be present in greatly increased concentrations in the peripheral blood of Malawian children with CM (JAMA 2004), are crucial elements of pathogenesis in experimental murine CM (Am J Pathol 2005). Based on these findings, we now aim, in vitro and in vivo, to demonstrate that by modulating the process of vesiculation (i.e. by reducing the production or release of MPs, or by blocking their toxic effects) we can reduce the pathological processes characteristic of CM. The ultimate aim is to reach a novel intervention for preventing the progression of severe malaria towards a fatal outcome and for hastening recovery from severe malarial disease.

In our current projects, we therefore intend to unravel the mechanisms of MP production and action, to delineate pharmacological ways to interfere with these mechanisms, and to define the pathophysiological consequences of excessive MP production.

1. Assessing microparticles as effectors in the microvascular lesion of cerebral malaria

Supervisor + contact details:

• Professor Georges Grau
• Dr Valery Combes

Co-cultures of human brain microvascular endothelial cells with parasites and blood cells, in which purified microparticles will be added, to assess the functional consequences. Analysis of immunological and physiological parameters, such as monolayer permeability and trans-endothelial electrical resistance.

2. Investigating mechanisms of microparticle production by microvascular endothelial cells and defining clinically useful inhibitors.

Supervisor + contact details:

• Professor Georges Grau
• Dr Valery Combes

Co-cultures of human brain microvascular endothelial cells with parasites and blood cells, in which various inhibitors of intracellular pathways will be added, to assess their ability to reduce the number of MP released upon stimulation by parasites, or to alter their phenotype. Apart from classical inhibitors, we already have new candidate molecules with obvious effects on MP. Analysis of immunological and haemostatic parameters of the MP produced.

3. Defining the implication of hypoxia in the pathology of cerebral malaria

Supervisor + contact details:

• Professor Georges Grau
• Professor Nicholas Hunt

Using our well characterised model of cerebral malaria (Trends Immunol 2003), several immunological and pathological pathways have been unravelled but numerous questions remain unsolved. In particular, the extent to which the obstruction of brain microvessels by parasites and host cells lead to hypoxia in the surrounding parenchyma. Drs. V. Combes and S. Parekh in our lab have shown in preliminary experiments that tissue hallmarks of hypoxia can be detected in mice that are genetically susceptible to CM, but not in strains known to be resistant. Injection of probes in mice and follow up of tissue changes, notably in the brain, by immunohistochemistry. Comparison, using quantitative image analysis, of mice infected with the CM-inducing parasite, PbA or with the non-encephalitogenic parasite, K173. Also, comparison of brains from mouse strains exhibiting various genetic susceptibility towards CM.

4. Determining the interactions of MP with their target cells in the host

Supervisor + contact details:

• Dr Valery Combes
• Professor Georges Grau

Microparticles (MP) are known to not only bind to cells but also interact with them and are responsible for various downstream effects such as activation (as evidenced by up-regulation of surface molecules, or triggering of signalling pathways), induction of apoptosis, increase binding to other cells, release of soluble molecules. We will address the following questions:

• What is the outcome of a pathogen-induced MP once it reaches its target cell and is there a difference according to the vesiculation agonist?
  
• What is the consequence of the interaction between MP and target cells at the level of the latter?

We have preliminary data, generated by confocal microscopy and flow cytometry, which show that platelet-derived MP are able to bind to endothelial cells and activate them.

For this project we will use two cell types (endothelial cells and monocytes) and study the interactions of their derived-MP with either endothelial cells or monocytes.

The techniques will include cell culture, flow cytometry, fluorescence microscopy and electron microscopy. The ImageStream® will also be used to visualise the interactions MP-target cell. The ImageStream® is a benchtop imaging flow cytometer designed for quantitative image-based analyses. It allows the collection of large numbers of digital images and combines cell information provided by fluorescence microscopy with the statistical significance of large sample size common to flow cytometry.

5. Deciphering the involvement of TCTP proteins in malarial pathogenesis

Supervisor + contact details:

• Professor Georges Grau

During cerebral malaria, the alteration of endothelium in cerebral vessels is a major element of pathogenesis. Several mechanisms have been described, leading to apoptosis of endothelial cells and/or to the production of pro-inflammatory microparticles. In culture, these alterations can be induced by P. falciparum-parasited red blood cells, but the proteins and the mechanisms involved in this process are not identified. Two approaches are proposed within this framework:

1. Study of the role of histamine and TCTP proteins in endothelial alterations.
   The role of parasitic proteins and of mediators of allergy in the formation of the lesions (especially in the opening of the tight junctions) will be studied in cultures of the human microvascular endothelial cell line, HBEC-5i, using electrophysiology, microscopy and molecular approaches.
   
2. Study of the production of microparticles by cells, upon stimulation by TCTP proteins.
   The in vitro production of microparticles by endothelial cells, monocytes and platelets will be analysed by flow cytometry after stimulation of the cells by recombinant parasitic proteins. In vivo studies also will be undertaken in mice infected by P. berghei ANKA associated or not with the injection of recombinant proteins.

6. Defining intracellular signalling pathways of microvascular endothelium stimulation by parasite and host cells

Supervisor + contact details:

• Dr Angeles Sanchez-Perez
• Professor Georges Grau

Adherence of Plasmodium falciparum-infected erythrocytes (iRBC) to brain microvessels is believed to be a critical step in the development of cerebral malaria (CM), an illness that kills around 2 million people, mostly children, every year. The malarial parasite modifies the infected erythrocyte by transporting proteins into its membrane. PfEMP1, a parasite protein expressed on the erythrocyte surface, binds to receptors in endothelial cells and promotes sequestration of the iRBCs.

Co-cultures of human brain microvascular endothelial cells with parasites and blood cells will be used to mimic the conditions found in CM and to investigate the intracellular pathways triggered by the parasite in the endothelial cells.

7. Dissecting the molecular mechanisms of platelet-endothelial interactions

Supervisor + contact details:

• Dr Angeles Sanchez-Perez
• Professor Georges Grau

In cerebral malaria PBRCs and platelets accumulate and induce deleterious functional alterations in the endothelium of brain microvessels. S-1-P is a sphingolipid that is released in high amounts by activated platelets and is involved in the regulation of the endothelial barrier function. The role of this molecule in the induction of microvessel lesions in the context of cerebral malaria will be studied using an in vitro platelet-endothelial cell co-culture system already established in this lab. We will analyze the effect of the modulation of the S-1-P signalling pathway by selective chemical agonists/inhibitors or by RNA interference in the co-cultured cells.