Research Grants Progress Reports 2006

Establishment of an improved model of relapsing MS and the characterisation of the role of Peroxisome Proliferator – activated receptors alpha and beta.

Dr Peter J. Cabot, Dr Sarah J. Roberts-Thomson, Dr Gregory R. Monteith and Ms Madusha Peiris.

School of Pharmacy, The University of Queensland, St Lucia, QLD, 4072.

Lay Description:

This project involves the development and characterisation of a unique mouse model of relapsing-remitting MS. Multiple relapsing models provide a distinct advantage by the nature of the relapse phenomena. Its possible therefore with predictable and consistent relapses to introduce a drug once disease is established and observe drug action in a mouse model reflective of Relapsing Remitting MS. The second component of the study was to assess the role of a relatively newly discovered receptor class, the PPAR receptors. These receptors have been shown to have roles in inflammation, dietary fat, muscle development and immune system function. Preliminary studies have identified agents of this class as potentially useful in treating MS both in animal models and in some case studies in patients. Our studies have shown that intervention with PPAR receptor agonists can reduce the symptoms seen in the mouse model of MS. These correlated with both histological and inflammatory markers. Moreover in a genetically modified animal the influence of PPAR receptors was evident on differentiating between the mouse model having episodes of relapse and remittance. Future efforts are being focused at determining how this influence occurs as a possible target for MS treatment.

Progress Report:

Hypothesis:

Relapsing-Remitting Multiple Sclerosis can be mimicked more effectively using a modified EAE model and can be used to assess the effects of intervention on disease relapse.

Aims (overall study aims, knockout models are not asked to be funded in the seeding grant)

1) Investigate the effects of bolus MOG35-55/Quil A for the purpose of developing a multiple relapsing model of MS.

To this end we have found that the combination of MOG35-55/quilA produce a multiple relapsing remitting EAE model of MS, clinical symptoms were low in comparison to chronic models in the literature but clearly evident as all studies were blinded and histological samples verified both lymphocyte infiltration and IFNgamma increase. (paper 1)

2) Investigate the behavioural changes in the disease progression of multiple sclerosis in control, wildtype, (long term: PPARα and β knockout mice).

To this end PPARbeta mice were examined versus wildtype, which revealed an interesting difference in disease profile. PPAR beta receptor knockout mice have lost the ability also to undergo relapse and remitting disease profiles. This may provide insight into the PPARbeta receptors having an integral role in the prevention of disease progression, this is being drafted into paper 2. The PPAR colony could not be studied due to its unavailability from the specified source.

3) Investigate the effects of the pharmacological intervention with PPAR γ agonist on established EAE.

The PPARgamma agonist pioglitazone was able to block the symptoms in a blinded control intervention study, this has been incorporated into paper 1. Submitted to J immunology.

4) Investigate the consequences of multiple relapsing EAE in controls (long term: and PPARα and β knockouts) on the proliferation of isolated T-cells from mice with an experimental model of MS (EAE).

Full histological analysis has been performed on the spinal tissue from mice in the PPAR gamma treated group, controls as well as PPARbeta knockouts and wildtypes, different stages of the disease have been included so the analysis here is much more in depth than first proposed, it appears that the ppargamma ligands does indeed block or reduce infiltration of lymphocytes into spinal tissue and this has been incorporated into paper 1.

5) Investigate the consequences of multiple relapsing EAE in controls (long term: as well as PPARα and β knockouts) on the release of cytokines from splenocytes from mice with EAE.

Due to extension of this study additional kits had to be purchased, however the results were very valuable with IFNgamma levels being modulated by drug intervention and significant differences were evident between pparbeta knockout and wildtype disease animals. These results are incorporated into paper 1 and paper 2.

6) Investigate the changes in cell adhesion molecule expression in the spinal cord of multiple relapsing EAE in controls (long term: as well as PPARα and β knockout mice).

This study was altered slightly to look at a better downstream target indiciative of EAE, which is the chemokine MCP-1, this was not altered in the model and future efforts will look at changes in expression instead as it appears that protein modulation is very limited in this chemokine.

Publications:

Submitted:

Peroxisome Proliferator-Activated Receptor- agonist reduces relapse in a novel model of Relapsing-Remitting Experimental Autoimmune Encephalomyelitis (EAE). Madusha Peiris, Gregory R. Monteith, Sarah J. Roberts-Thomson and Peter J. Cabot. Submitted, J.Immunol, Jan, 2006.

Abstracts

M Peiris, S Roberts-Thomson, GR Monteith & PJ Cabot Pioglitazone treatment prevents relapse in a multiple relasping0remitting experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. Proceedings of the Australasian Pharmaceutical Scientists Association, Melbourne. Dec, (2005).

Pioglitazone Treatment Prevents Relapse In A Multiple Relapsing-Remitting Experimental Autoimmune Encephalomyelitis (Eae) In C57bl/6 Mice.

M Peiris, S Roberts-Thomson, GR Monteith & PJ Cabot. The School of Pharmacy, University of Queensland, QLD, 4072.

Multiple sclerosis (MS) is a chronic, neurodegenerative disease characterised by axonal demyelination caused by autoimmune mediated inflammation and myelin degradation (McDonald 1974). While MS can assume different forms, 85% of all patients suffer relapsing-remitting MS (RRMS) at some point (Revel, 2003). The aim of this study was to assess Pioglitazone as an affective intervention therapy in Relapsing-remitting Experimental Autoimmune Encephalomyelitis (RR-EAE), the animal model of MS. Mice were anesthetised with 3% isoflurane and injected with MOG protein and Saponin extract adjuvant to induce EAE. Mice treated with Pioglitazone at 50mg/kg/day over 10 days did not show any symptoms associated with a relapse during this period, while those receiving 10mg/kg/day of Pioglitazone had a modest decrease in symptom severity. Upon the completion of treatment, both groups of mice suffered a relapse that was more severe than those previously incurred, suggesting treatment withdrawal had a negative impact. Those animals treated with vehicle, however, proceeded to develop relapse symptoms and a RR-EAE disease pattern was observed throughout. Furthermore, neuronal inflammation as measured by monocyte infiltration was decreased in treated mice compared to controls. Therefore, Pioglitazone treatment may have decreased immune cell infiltration which result in myelin protein degradation. Albeit, demyelination was no altered, all groups had similar levels of myelin loss indicating that although a relapse event was prevented, previous and consequent episodes had damaged myelin integrity. Pioglitazone was able to reversibly block RR-EAE symptoms in a dose-dependent manner by inhibiting immune cell infiltration, but was not able to reverse the damage produced by earlier insult. In conclusion, this study highlights Pioglitazone as a putative therapeutic intervention capable of ameliorating relapse in MS.

References:

1. McDonald, WI, (1974), Brain, 97(1), 179-96.

2. Revel, M, (2003), Pharmacol Ther, 100(1),49-62

Project Title: Determination of Gene Expression Profiles in Multiple Sclerosis Affected Brain Tissue

Names of All Researchers

Mr Attila L Szvetko Jr., BHSc(Hons), PhD Candidate

Professor Lyn R Griffiths, Principal Supervisor, Director

Genomics Research Centre

School of Medical Science

Gold Coast campus, Griffith University

PMB 50 Gold Coast Mail Centre

Queensland 9726, Australia

Lay Summary

MS is the leading cause of acquired neurological disability excluding trauma arising in early to middle adulthood. It is a paradigm of complex disease demonstrating widely variable clinical expression with unpredictable course. Pathology is characterized by chronic inflammation and selective destruction of nerve insulation in the brain and spinal cord. The cause of MS is unknown; however circumstantial evidence suggests that an environmental agent (such as a virus) triggers the disease in genetically susceptible individuals. Immunomodulatory treatments have demonstrated some success, suggesting that MS is in part an autoimmune disorder. The ultimate goal of MS research is to determine the processes leading to pathology. Better understanding of the pathologic state should lead to improved diagnostic and treatment strategies. This research intends to contribute to our understanding of the genetics of MS by investigating gene expression, genetic variations, and the viral hypothesis. A brain-tissue repository and genetic material (DNA) from blood have been established and are being expanded. We have investigated 8 candidate genes yielding significant results for 6 of those genes. We are currently investigating another 2 genes bringing the total number of candidates to date to 10. These investigations have yielded 1 first-author publication as a direct result of these investigations and 1 co-authored article investigating another gene involving a hormone receptor, this year. A second first-author manuscript is now being prepared in light of significant findings within a group of 3 genes while follow-up investigations are being conducted with a view to manuscript preparation for a second set of 3 genes once validation studies are complete. The next phase of our research entails undertaking an array-based technology to investigate viral hypotheses.

Main Report

This research is based on the premise that particular genetic variations and gene expression patterns underlie the molecular pathology of MS. In addition, it is possible that a viral pathogen may contribute to the natural history of the disease.

Quantitation of Novel Candidate Genes in MS:

Investigation of post-mortem brain tissue ribonucleic acid (RNA) from MS sufferers versus health controls by relative quantitative real-time polymerase chain reaction (rt-PCR) methods.

We have conducted sensitive case-control gene expression studies of the genes glial cell derived neurotrophic factor (GDNF), neuregulin 1 (NRG1), insulin receptor (INSR), protein tyrosine phosphatase receptor type C (PTPRC), toll-like receptor 3 (TLR3), and claudin 11 oligodendrocyte transmembrane protein (CLDN11) in secondary-progressive multiple sclerosis affected brains investigating expression differences between frontally and temporally obtained gray-matter and white-matter by relative qualitative realtime polymerase chain reaction (qRT-PCR). Genomic techniques have been used to validate particular aspects of the gene expression studies. The investigations have yielded significant results for 4 of the 6 investigated genes (p=<0.05). A manuscript is currently being prepared for 3 of the 6 significant genes while follow-up validation studies investigating protein from MS brains is conducted for the second set of 3 genes. It is envisaged that a second manuscript will be prepared for the second set of 3 genes once the validation studies are complete.

Investigation of Genomic Variations Associated with MS:

Investigation of blood-derived lymphocytic deoxyribonucleic acid (DNA) from MS sufferers versus age, gender, and ethnicity matched healthy controls. Association between particular genotypes and MS will be investigated.

This past year, two polymorphisms: MTRR A66G and MTHFR A1298C were investigated in an Australian age and gender matched case-control study. Significant allele frequency differences were not observed between cases and controls at the α=0.05 level (MTRR χ2=0.005, P=0.95, MTHFR χ2=1.15, P=0.28). Our preliminary findings suggest that methionine synthase reductase (MTRR A66G), and methylenetetrahydrofolate reductase (MTHFR A1298C), genes that are known to regulate enzymes controlling plasma homocysteine levels, which have been suggested to play a role in CNS dysfunction, neurodegenerative, and cerebrovascular diseases via vasotoxic and neurotoxic mechanisms, are not necessarily involved with MS pathology. These findings were presented at the 11th International Congress of Human Genetics (Brisbane, Qld, August 2006) and then published in the Journal of the Neurological Sciences 252 (2007) 49-52. Another investigation examining allelic variation within 2 estrogen receptor (ESR) markers (G594A and T938C) in relapsing-remitting, secondary-progressive, and primary-progressive MS patients yielded no evidence of association with MS (p=0.50 and p=0.45, respectively). A co-authored article was published in the Journal of the Neurological Sciences 252 (2007) 9-12.

Genomic investigation of another 2 markers by a novel technique (highresolution melt analysis; HRM) is currently being conducted.

Investigation of the Viral Hypothesis Associated with MS:

A viral microarray will be undertaken to determine and characterize the possible involvement of numerous MS viral candidates.

We are pursing enquiries as to the feasibility of designing custom arrays. We have identified commercial entities that provide array-scanning services should they be required. In the meantime, we have obtained several primer sequences spanning the open reading frames (ORFs) of several candidate viruses; human T-cell lymphocytotrophic virus I & II (HTLVI, HTLVII), hepatitis C virus (HCV), Epstein-barr virus (EBV), human herpesvirus 4 (HHV-4), human herpesviruses 6A & 6B (HHV-6A/HHV6B), Kaposi’s sarcoma associated herpesvirus (KSHV), and human herpesvirus 8 (HHV-8). The microarray experiments will complement the real-time PCR expression studies, and we are conserving brain tissues and cDNA samples for future use for those arrays.

Publications

Szvetko AL., Fowdar J., Nelson J., Colson N., Tajouri L., Csurhes PA., Pender MP., Griffiths LR. J Neurol Sci 252 (2007) 49-52. No Association Between MTHFRA1298C and MTRR A66G polymorphisms, and MS in an Australian Cohort.

Role of CC-chemokine receptor 6 in the targeting of autoantigen-specific T cells to the CNS

Names of all researchers involved in the project: Julie Webster, Bsc Hon, Assoc/Prof Heinrich Koerner PhD

Lay summary: The migration of autoimmune cells to the central nervous system is a fundamental event in the establishment of the autoimmune disease Multiple Sclerosis (MS). The mechanisms governing the navigation of these cells have not yet been resolved. We proposed to investigate the role of one receptor molecule that helps autoimmune cells to navigate to the brain in an experimental disease, experimental autoimmune encephalomyelitis (EAE), which resembles in some aspects the human disease MS. The experiments were performed at the Comparative Genomics Centre, James Cook University in Townsville by Julie Webster and Heinrich Koerner.   Currently, further work was carried out by Johannes Ploetzl, a German PhD student.

The first projects were to analyze the role of this molecule in the start of a T cell response and to induce the experimental model disease EAE in mice that miss this receptor molecule. We found that T cells had no defect that prevented them from functioning. This indicates that the problems we see were due to downstream defects. For the second approach, we used a brain autoantigen to immunize the mice and to start the autoimmune process, which normally leads to a transient paralysis. In these mice the paralysis was slightly less pronounced than in wildtype mice. Furthermore, we analysed differences in the cells, which enter the brain and cause disease. We found that the organization of these cells is different in the receptor-negative mice. We are currently investigating the role this group of cells has in normal EAE.

Main report:

Aim 1: To dissect the role of CCR6 in the differentiation of T cells from naïve to effector T cells.

CCR6 is expressed on T cells after activation and its ligand CCL20 can be secreted by dendritic cells. The presence of CCR6 could therefore influence the efficiency of autoantigen presentation. In 2005 the antigen-specific proliferation of T cells from CCR6-/- mice has been compared to WT mice in two out of three suggested ways.

1) WT and CCR6-negative CD4-positive T cells were purified by flow cytometry and proliferation of 2 x 105 T cells was induced using anti-CD3 and -CD28 mAbs coated to the plastic surface of the tissue culture dish. The cells behaved normally and divided at a comparable rate. With this approach we excluded any influence of antigen or any deficiency of an antigen presenting cell and focused at the T cell. We were able to rule out any intrinsic defect of CCR6-negative T cells.

2) WT and CCR6-negative CD4-positive T cells were purified by flow cytometry and proliferation of 2 x 105 T cells was induced by the addition of 1 x 106 irradiated spleen cells plus ConA. This approach was also antigen-independent and was supposed to detect deficiencies on the cellular side of the antigen presentation process eg. costimulatory problems of the dendritic cells. Also in this experimental approach the CCR6-/- T cells proliferated normally and comparable to the WT cells and we could exclude intrinsic deficiencies of the antigen presenting cells.

After these negative results the third approach, the immunization with antigen of CCR6-/- mice and the subsequent isolation and analysis of T cells will be most interesting. In attempts to isolate MOG-specific T cells (see Aim 2) we were so far not able to derive antigen-specific T cells from CCR6-/- mice which points to a substantial contribution of this receptor to antigen presentation.

Aim 2: To analyse the function of CCR6 in the migration of autoantigen-specific T cells from the site of their priming to the CNS in MOG-induced active and passive EAE in CCR6-deficient mice.

The induction of active EAE was successful and resulted in a slightly but significantly attenuated form of disease (Figure 1). Currently experiments are underway to determine the composition of the cellular infiltrate in the CNS of WT compared to CCR6-/- mice. First results have shown that the absence of CCR6 influences the ability of different cell populations to enter the brain.

The initiation of passive EAE has not been successful. Pre-requisite is an encephalitogenic T cell that is usually established by immunizing mice, isolating lymph node cells and restimulation the T cell component of these cells in vitro. In our hands this restimulation has not been successful and we were so far unable to establish any T cell line regardless of the specificity. It will be interesting to determine the underlying cause (see Aim 1) of this unexpected problem of antigen presentation.

Figure 1: Course of disease in WT and CCR6-/- mice. Mice were immunized in the tail base with 50microgram of MOG peptide 35-55. The intensity of disease (y-axis) versus the time after immunization are depicted. Two independent experiments were combined for this figure. For every point in time the standard error of the mean is shown.

Publications:

1) Lechner A., Ritter U., Varona R., Marquez G., Bogdan C. and H. Körner (2007) Protective Immunity and Delayed Type Hypersensitivity Reaction are Uncoupled in Experimental Leishmania major Infection of CCR6-negative Mice. Microbes Infect. 9: 291-299.

Progress report on Trish Foundation funded research carried out in 2006

Project title: Studies on the functional role of antibodies in multiple sclerosis

Names of researchers: Dr Judith Greer, Prof Michael Pender, Ms Shannon Beasley (PhD student)

Lay summary:

Almost the only common feature shared by people with multiple sclerosis (MS) is that they usually have immune proteins known as antibodies in the CSF, the fluid that surrounds the brain and spinal cord. This suggests that these antibodies might play a role in the development of MS. Many people with MS also have similar antibodies in their blood. The hypothesis that we set out to test in this project was that people with MS who have increased levels of these antibodies in their blood and CSF might develop more severe disease than people with MS who do not have such high levels of antibodies. Also, having high levels of antibody in the blood or CSF might influence which parts of the brain and spinal cord are attacked in MS, which in turn will affect the types of symptoms that people with MS develop.

We tested blood from 100 people with MS, 30 people with other neurological disorders, and 40 healthy controls and found that many people with MS have increased levels of antibodies specific for the most abundant myelin protein, proteolipid protein (PLP), compared to healthy controls and people with other neurological diseases. When these antibodies (directed against PLP) from people with MS were added to human myelin in a test tube, they allowed the myelin to be more readily ingested by macrophages. Macrophages are the cells that eat up the myelin and cause a lot of the damage in MS. This result therefore suggests that the presence of the antibodies against PLP might lead to the macrophages attacking myelin more than they would normally. Antibodies against other components of myelin could also potentially have similar effects, and we aim to measure the effects of these in the future.

One problem with testing myelin proteins in the test tube is that when you extract the proteins, their shape and conformation can change. Antibodies often recognize a very specific conformation of a protein, and so, in order to test if the antibodies can bind to the PLP, as it would be found in the myelin membrane, we need to see if the antibodies from people with MS bind directly to human brain tissue. It has taken longer than we anticipated to obtain some well-preserved human brain tissue, but we have now found a source of this, and these experiments will shortly take place. We will also use cells that have been genetically engineered to make PLP and express it in the correct conformation in these experiments. If the genetically engineered cells show the same reactivity as the human brain tissue, then we will be able to use them in future experiments, without the need to obtain human tissues.

We were also interested in the question of whether people who had more rapidly progressive MS had increased levels of antibodies. We found that people who had an average rate of progression in their MS (approximately 0.25 EDSS increase per year) generally had less of the PLP antibodies in their blood than those people who had more rapidly progressive MS (increase of greater than 0.5 EDSS per year). We need to test a larger number of people with rapidly progressive MS to verify this result, but it appears that increased levels of antibodies against myelin correlate with and may actually be the cause of a more rapidly progressive form of MS. If so, antibodies against PLP may be a good therapeutic target in rapidly progressive MS.

The last question that we have been addressing is whether antibodies play a role in determining where lesions occur. Using a mouse model of MS, we can show that the antibodies can have an effect on where in the nervous system the demyelinating lesions occur. It is not so clear-cut in MS. At the moment, it appears that antibodies may be necessary, but not sufficient, to determine where lesions occur. This will be followed up over the next 3 years.

Thanks to the assistance of the grant from the Trish MS Research Foundation for this project, we were able to generate sufficient preliminary data to successfully apply for further funds from the NHMRC to continue this project looking at the role of antibodies in MS.

Publications:

Greer JM, Pender MP. 2005. Antibodies specific for myelin proteolipid protein are of potential pathogenic relevance in myelin opsonization in multiple sclerosis. Clinical Immunology 115 (Suppl. 1): S91 (Meeting abstract)

Pender MP, Greer JM. (in press) Immunology of multiple sclerosis. Current Allergy & Asthma Reports

Greer JM, Pender MP. (in preparation) Potential pathogenicity of autoantibodies against myelin proteolipid protein in multiple sclerosis.