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Gary A Levy, BSc, FRCPC, MD
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Senior Scientist
Division of Cellular & Molecular Biology
Toronto General Research Institute (TGRI) |
Keywords: hepatitis, immunology, procoagulant activity, fibroleukin, cellular, fgl2
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The focus of work in our laboratory has been to characterize unique molecules that participate in immune coagulation and examine their role in human disease.
We have identified three (3) molecules, tissue factor and fgl2/fibroleukin which are membrane associated serine proteases. These molecules play pivotal roles in the pathogenesis of viral hepatitis (fgl2/fibroleukin), allo and xenograft rejection (tissue factor and fgl2). The importance of these molecules can be best shown by the fact that treatment of mice with high titered neutralizing antibodies to fgl2 prevents the lethality of fulminant viral hepatitis caused by murine hepatitis virus strain 3 (MHV-3).
Our research will focus over the next five years on:- The role of FGL2/fibroleukin in human and experimental viral hepatitis
- The role of tissue factor and FGL2 in xenograft rejection (funded by the Heart and Stroke Foundation of Canada, co-investigator Dr. D. Grant)
- CIHR Group Grant 'Cellular and Molecular Basis of Organ Injury.' Dir: Dr. G. Levy and 5 principal investigators Drs. G. Downey, O. Rotstein, P. Marsden, L. Zhang, and M. Cybulsky. The group was first funded by CIHR 10 years ago and the focus of the work is to promote scientific excellence and advance the state of knowledge in organ injury; 2) to promote research to allow advances to move from the bench to the bedside and 3) to foster an environment for the training of future scientific leaders.
Molecular Characterization of the Proximal Human and Murine fgl2/Fibroleukin Promoter
The overall objective of this set of studies is to examine the basic regulatory features involved in viral induced fgl2 gene transcription. We have initiated studies to map the upstream promoter region of the murine and human fgl2 genes and defined a region from -372 to -306 upstream from the ATG translation initiation site to be responsive to the nucleocapsid protein of strains of MHV that induce fulminant hepatitis. Functional promoter activity will be assessed by transient transfection studies in which cells will be cotransfected with a construct containing a relevant viral protein and a set of fgl2 promoter/luciferase deletion constructs. Binding sites for putative transcriptional regulatory molecules will be assessed by electromobility shift assays and super shift assays (EMSA).
Structure/Function of fgl2/Fibroleukin
A recombinant murine fgl2 protein has now been generated using a baculovirus expression system. Fgl2 expressed in vesicles containing phospholipids has prothrombinase activity, whereas soluble fgl2 protein has potent immunosuppressive activity and can inhibit leukocyte-endothelial cell interactions. The experiments proposed will determine if these two functions are linked or can be assigned to different regions of the molecule.
To accomplish this, a set of truncated constructs and constructs in which candidate serine residues have been mutated have been generated. The active catalytic site of the serine protease (prothrombinase) will be determined by an assessing prothrombinase activity in:
a) fgl2 proteins generated by transient transfection studies using the fgl2 constructs in which candidate regions containing serine residues are deleted or b) by site directed mutagenesis of candidate serine residues. The importance of the transmembrane domain will similarly be assessed by expressing protein in which this region has been truncated. Similarly, the region of the fgl2 protein required for the immunosuppressive activity will be assessed using native and truncated proteins to study their ability to inhibit mixed lymphocyte reactions (MLR) cytotoxic T cell responses (CTL) and endothelial-leukocyte adherence.
Pathobiology of fgl2/Fibroleukin
The physiological role of fgl2 will be assessed in mice in which the fgl2 gene has been disrupted using targeted homologous recombination. The mice will be studied primarily, but not exclusively, for the effect of deletion of fgl2 on: a) viral replication (MHV-3); b) susceptibility/resistance to MHV-3 infection and c) ability of susceptible mice to mount a Th1/Th2 anti-viral response. Fgl2 deficient mice will also be used to assess the role of fgl2 on cytokine-induced fetal loss syndrome.
Significance
Collectively , these studies will not only characterize the role of fgl2/fibroleukin in fulminant viral hepatitis but will also provide insights into the regulation of this gene which will have important therapeutic implications for patients with a broad spectrum of diseases in which thrombosis plays an integral component.
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Pubmed Publications | | | |
Selected Publications | - Shalev I, Wong K, Foerester K, Zhu Y, Chan C, Maknojia A, He W, Liu MF, Clark DA, Phillips MJ, Zhang L, Cybulsky MI, Levy, GA. FGL2 the novel Treg effector molecule plays a critical role in the outcome of murine fulminant viral hepatitis. Hepatology 2009 in press.
- Han M, Yan W, Guo W, Xi D, Zhou Y, Li W, Gao S, Liu M, Levy G, Luo X, Ning Q. Hepatitis B virus induced HFGl2 transcription is dependent on c-Ets-2 and MAPK signal pathway. Journal of Biological Chemistry, 2008 Nov 21;283(47):32715-29.
- Selzner N, Girgrah N, Lilly L, Guindi M, Selzner S, Therapondos G, Adeyi O, McGilvray ID, Cattral M, Greig PD, Grant DR, Levy, GA, Renner EL. The difference in the fibrosis progression of recurrent hepatitis C after live donor liver transplantation versus deceased donor liver transplantation is attributable to the difference in donor age. Liver Transpl. 2008 Nov 24;14(12):1778-1786.
- Liu H, Shalev I, Manuel J, He W, Leung E, Crookshank J, Liu MF, Diao J, Cattral MS, Clark DA, Gorczynski RM, Zhang L, Grant DR, Phillips MJ, Isenman DE, Cybulsky MI, and Levy GA. The FGL2-FcgRIIB pathway: a potent immunosuppressive mechanism and its role in prevention of allograft rejection. European Journal of Immunology, 2008:38:3114-3126.
- Dubay D, Smith RJ, Qiu KG, Levy GA, Lilly L, Therapondos G. Sirolimus in liver transplant recipients with renal dysfunction offers no advantage over low dose calcineurin inhibitor regiments. Liver Transplantation 2008 May;14(5):651-9.
- Yu K, Yang S, Foerster K, Manuel J, Molina H, Levy GA, Gorczynski RM, Clark DA. LPS-induced murine abortions require C5 but not C3 and are prevented by upregulating expression of the CD200 tolerance signalling molecule. American Journal of Reproductive Immunology, 2008:60:135-140.
- Kovarik JM, Villamil F, Otero A, Levy GA, Lynch S, Cillo U, Fischer L, Nashan B, Pollard S, De Carlis LCyclosporine pharmacokinetics and blood pressure responses after conversion to once-daily dosing in maintenance liver transplant patients. Clin Transplant. 2008 Jan-Feb;22(1):68-75.
- Shalev I, Liu H, Koscik C, Bartczak A, Javadi M, Wong KM, Maknojia A, He W, Liu MF, Diao J, Winter E, Cattral M, Gommerman J, Clark DA, Phillips MJ, Gorczynski RM, Zhang L, Downey GP, Grant DR, Cybulsky MI, Levy, GA. Targeted Deletion of fgl2 Leads to Impaired Regulatory T Cell Activity and Development of Autoimmune Disease. Journal of Immunology 2008:1;180(1):249-60.
- Foerster K, He W, Manuel J, Bartczak A, Liu M, Markert UR, Levy GA, Clark DA LPS-induced occult loss in mice requires FGL2. American Journal of Reproductive Immunology, 58:524-529, 2007.
- Mu J, Qu D, Bartczak A, Phillips MJ, Manuel J, He W, Koscik C, Mendicino M, Zhang L, Clark DA, Grant DR, Backx PH, Levy GA, Adamson L. fgl2 deficiency causes neonatal death and cardiac dysfunction during embryonic and postnatal development in mice. Physiol Genomics. 2007 Sep 19;31(1):53-62.
- Shah SA, Tan JC, McGilvray ID, Cattral MS, Levy GA, Greig PD, Grant DR. Does Microvascular Invasion Affect Outcomes After Liver Transplantation for HCC? A Histopathological Analysis of 155 Consecutive Explants. J Gastrointest Surg. 2007 Apr;11(4):464-71.
- Ross K, Abbey S, Levy GA, Grant D. Living Anonymous Liver Donation: Case Report and Ethical Justification. American Journal of Transplantation 2007 April 7(4):1032-5.
- Shah SA, Levy GA, Greig, PD, Smith, R, McGilvray ID, Lilly LB, Girgrah NT, Cattral MS, Grant DR. Reduced Mortality with Right-Lobe Living Donor Compared to Deceased-Donor Liver Transplantation When Analyzed From the Time of Listing. American Journal of Transplantation, 2007 April 7(4):998-1002.
- Shah SA, Cattral MS, McGilvray ID, Adcock LD, Gallagher G, Smith R, et al. Selective use of older adults in right lobe living donor liver transplantation. American Journal of Transplantation, 2007 January 7(1):142-50.
- Shah SA et al. Biliary Strictures in 130 Consecutive Right Lobe Living Donor Liver Transplant Recipients: Results of a Western Center. American Journal of Transplantation, 2007 Jan;7(1):161-7.
- McCluskey SA, Karkouti K, Wijeysundera DN, Kakizawa K, Ghannam M, Hamdy A, Grant D, Levy GA. Derivation of a risk index for massive transfusion in liver transplantation. Liver Transpl. 2006 Nov;12(11):1584-93.
- De Albuquerque N, Baig E, Ma MX, Zhang J, He W et al. Murine hepatitis virus strain 1 (MHV-1) produces a clinically relevant model of SARS in A/J Mice. Journal of Virology, 2006:80(21):10382-10394.
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