User:Marios F. Sardis: Difference between revisions

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In 1967, a puzzling observation was made by Calne and his laboratory team, during liver transplantation experiments on pigs. One animal survived for months, after receiving an allograft, without any immunosupression. Later, it was found that the soluble factors, which were secreted by the liver allograft and were responsible for the development of tolerance to the recipient animal, were soluble MHC molecules(1,2). From this point, many laboratories around the world entered the quest, for the elucidation of the immunoregulatory effects of the various soluble forms of MHC molecules. Today, it is widely accepted that body fluids, isolated from healthy individuals contain various amounts of soluble MHC molecules(1,3,4,5,6). Deviations from the concentration of these molecules in the body fluids of healthy individuals, have been recorded in numerous pathological conditions, such as viral encephalitis5, rheumatoid arthritis(7,8,9), pathologic pregnancies(10), asthma(11) and AIDS(5). These results do not clarify whether soluble MHC molecules have a special role in the immune responses, during the course of these diseases. Additional research has confirmed that soluble forms of MHC molecules can have various immunomodulating effects, such as induction of apoptosis of CD4+ T cells(12); activation of CD8+ T cells(13) and down-regulation of NK activity(13). Soluble dimeric MHC class II molecules built on Fcg2a scaffold can cause activation of T cells and switching towards a TH2 response(14). According to another report, soluble dimeric IEk molecules can act as potent activators for T cells, especially in the presence of the CD4 coreceptor(15).
Soluble MHC molecules
The main focus of my undergraduate research project was the isolation of soluble MHC molecules and thecollection of data about their biological function. We have collected protein solutions that have been isolated from the serum of male and pregnant Balb/c mice, which show anti-I-Ad activity in ELISA, antibody binding inhibition and Western blot assays. After the isolation process was complete, I proceeded with functional assays in order to gather information about the biological role of these molecules. When spleen cells from syngeneic mice were treated with the protein solutions, we had isolated, a significant increase in the incorporation of radioactivity was recorded. However, in some assays, allogeneic spleen cells (C3H/HeN) responded also positively to the soluble molecules we had isolated. Spleen cells that were treated with Concanavalin A and soluble MHC molecules increased the incorporation of radioactivity in an additive manner. Furthermore, during one way, Mixed Lymphocyte Reactions when Balb/c spleen cells that responded to inactivated with mitomycin C3H/HeN spleen cells, were treated with solution of soluble MHC II molecules, their responses were significantly boosted. These results led me to investigate whether there is a specific cell population that responds maximally when these soluble molecules are added to the medium. After separating various cell populations from spleens, according to their surface markers I found that cells bearing the CD4 and CD25 markers and adherent cells tend to respond with maximum efficiency to soluble MHC molecules. I decided to focus more to the CD4 receptor. In order to test whether this molecular structure is important for the effects, I tried to interfere it by adding anti-L3T4 monoclonal antibody to the medium. When CD4 was blocked, the spleen cells did not respond to the soluble molecules.
Furthermore, I decided to test whether the soluble molecules I had isolated, could cause any deviations in the production of immunoglobulins from B-cells. I started by causing polyclonal proliferation of B-cells. This was achieved by adding LPS from E.coli (Type-1 thymus independent antigen) to cultures of freshly isolated spleen cells, from Balb/c mice. To continue, I immunized in vitro the spleen cells with DNP-HSA and in some samples i added soluble MHC molecules. The supernatants were screened for IgM level, with ELISAs. Soluble MHC molecules caused a significant decrease in the level of IgM in the supernatants. This effect could be the result of interference of cell-cell interactions or of the profile of cytokines produced by the spleen cells.   
 
 
References
 
[1]van Rood JJ, van Leeuwen A, van Santen MCT: Anti-HL-A2 inhibitor in normal human serum. Nature (1970) Vol. 226(5243):366<br/>
[2] Jon J. van Rood and Aad van Leeuwen, Soluble HLA Antigens: How It All Started, Human Immunology (1999) Vol. 60:412–413<br/>
[3]Francesco Puppo Francesco Indiveri, Marco Scudeletti and Soldano Femme, ., Soluble HLA
antigens: new roles and uses, Trends in Immunology (1997), April (unspecified pages)<br/>
[4] Masao Hagihara, Tatsuo Shimura, Kozue Yamamoto, Kentaro Takebe, Batmunkh Munkhbat, and Kimiyoshi Tsuji, Soluble HLA Class I and Class II in Japanese, Human Immunology(1994) Vol. 40:
171-173<br/>
[5] Donnie Aultman, Irena Adamashvili, Kamalakar Yaturu, Marlyn Langford, Frank Gelder, Michael Gautreaux, G. E. Ghali, and John McDonald , Soluble HLA in Human Body Fluids, Human Immunology(1999) Vol. 60: 239–244<br/>
[6] Kerstin A. Pfeiffer, Vera Rebmann, Monika Passler, Kathrin van der Ven, Hans van der Ven,
Dieter Krebs, and Hans Grosse-Wilde , Soluble HLA Levels in Early Pregnancy After In Vitro
Fertilization, Human Immunology (2000) Vol. 61: 559–564<br/>
[7] Leon A. Verbruggen, Hendrik Versaen, Vera Rebmann, William Duquet, Seija De Cock, Hans
Grosse-Wilde, and Christian Demanet, Soluble HLA-DR Levels in Serum are Associated With Therapy and Genetic Factors in Rheumatoid Arthritis, Human Immunology (2002) Vol. 63: 758–764<br/>
[8] Wolf RE, Adamashvili IM, Gelder FB, Hall VC, Fraser PA, McDonald JC., Soluble HLA-I in Rheumatic Diseases Human Immunology(1998) Vol. 59: 644–649<br/>
[9] L.A. Verbruggen, N. Dumarey, H. Van de Velde,V. Rebmann, J. Flament, C. Van Wayenberge, H. Grosse-Wilde, C. Demanet, Soluble HLA-DR antigen levels in serum correlate with rheumatoid arthritis disease activity and the presence of disease-associated epitopes, Tissue Antigens (2000) Vol. 56: 436–440<br/>
[10] Andrea Steinborn,  Vera Rebmann, Alexander Scharf, Christof Sohn, Hans Grosse-Wilde,, Soluble HLA-DR levels in the maternal circulation of normal and pathologic pregnancy, Am J Obstet Gynecol (2003) February:  473-479<br/>
[11] Roberta Rizzo, Cristina E. Mapp, Loredana Melchiorri, Piero Maestrelli,  Annalisa Visentin,  Stefano Ferretti, Ilaria Bononi, Deborah Miotto,  Olavio R. Baricordi, Defective production of soluble HLA-G molecules by peripheral blood monocytes in patients with asthma, J Allergy Clin Immunol (2004) Vol. 115: 508-513<br/>
[12] Bishwajit Nag, Teresa Kendrick, Subhashini Arimilli, Sheue Ching T. Yu, Subramaniam Sriram, Soluble MHC II–Peptide Complexes Induce Antigen-Specific Apoptosis in T Cells, Cellular Immunology (1996) Vol. 170: 25 –33<br/>
[13] Webb BJ, Bochan MR, Montel A, Padilla LM, Brahmi Z., The lack of NK cytotoxicity associated with fresh HUCB may be due to the presence of soluble HLA in the serum, Cellular Immunology (1994) Vol. 159: 246-261<br/>
[14]Sofia Casares, Cong S. Zong, Dorel L. Radu, Alexander Miller, Constantin A. Bona, and Teodor-Doru Brumeanu, Antigen-specific Signaling by a Soluble, Dimeric Peptide/Major Histocompatibility Complex Class II/Fc Chimera Leading to T Helper Cell Type 2 Differentiation J. Exp. Med. (1999) Vol. 190: 543–553<br/>
[15]Abdel Rahim A. Hamad, Sean M. O’Herrin, Michael S. Lebowitz, Ananth Srikrishnan, Joan Bieler, Jonathan Schneck, Drew Pardoll Potent T Cell Activation with Dimeric Peptide–Major Histocompatibility Complex Class II Ligand: The Role of CD4 Coreceptor, J. Exp. Med. (1998) Vol. 188: 1633–1640<br/>


In 1967, a puzzling observation was made by Calne and his laboratory team, during liver transplantation experiments on pigs. One animal survived for months, after receiving an allograft, without any immunosupression. Later, it was found that the soluble factors, which were secreted by the liver allograft and were responsible for the development of tolerance to the recipient animal, were soluble MHC (sMHC) molecules.From this point, many laboratories around the world entered the quest, for the elucidation of the immunoregulatory effects of the various soluble forms of MHC molecules. Today, it is widely accepted that body fluids, isolated from healthy individuals contain various amounts of soluble MHC molecules. The origin of these molecules has not been clearly determined and it could be the result of shedding, alternative splicing or active secretion, whereas it has been demonstrated that trophoblast cells secrete to the medium soluble HLA-DR molecules when stimulated by IFNγ. In the 70s and 80s during the study of T suppressors (some of which might correspond to the modern T regulatory cells), it was suggested that they affected nearby cells secreting various factors with MHC determinants. Deviations from the concentration of these molecules in the body fluids of healthy individuals, have been recorder in numerous pathological conditions, such as viral encephalitis, rheumatoid arthritis, pathologic pregnancies, asthma and AIDS. These results imply that soluble MHC molecules have a special role, in the pathology of these diseases but it is not clarified whether they contribute to the pathology or if they are by-products of the pathological condition (they could have a potential role in the diagnosis of the disease stage). Additional research has confirmed that soluble forms of MHC molecules can have various immunomodulating effects, such as induction of apoptosis of CD4+ activation of CD8+ T cells and down-regulation of NK activity.
The aim of this study was to perform a downstream investigation of the possible immunomodulating action of soluble MHC molecules isolated from the serum of male and pregnant Balb/c mice. We selected the 12th day of gestation for the isolation of the serum, according to Ranella A. (The role and the regulatory mechanisms of growth factors, membrane and secreted MHC class II molecules in the survival of the hemiallogeneic embryo during gestation, Ph.D. thesis, University of Crete, unpublished data), because the titer of the serum reaches a maximum. During the study we used affinity chromatography and immunoprecipitation for the isolation of sMHC II from the serum and cell culture techniques, in order to reveal any effect on freshly isolated spleen cells. Pregnancy is often called immune paradox, because the immune system of the mother allows to the fetus to survive although it is an allotransplant. Generally, pregnancy is largely considered to be a TH2 phenomenon that is characterized by downregulation of the immune system in the point where the mothers body meets the fetus. Soluble MHC class II molecules secreted by trophoblast cells could contribute to the development of tolerance, because the ability of antigen presentation from the trophoblast is lost along with the secreted MHC II. However, it has not been determined whether the molecules we isolate from the serum originate from the trophoblast. The presence of the same molecules in the serum of male mice suggests an alternative source.
   
   


Revision as of 06:46, 6 October 2006

Sardis Marios Frantzeskos
Department of Biology
University of Crete
Herakleion, Crete
Greece

hartigan.sc@gmail.com

Education


2006-........M.Sc. in Protein Biotechnology, Department of Biology,University of Crete
2002-2006 B.Sc. in Biology (Biomolecular science and Biotechnology), University of Crete.
2001-2002 Certificate of Higher Education, University of Cardiff, Wales, U.K.

Awards

2003-2004 Award granted from the National Institute of Studentships of Greece (IKY) for excellent academic performance.
2003-2004 Studentship granted from the National Institute of Studentships of Greece (IKY) for top academic performance of the year.

Research experience

Soluble MHC molecules

In 1967, a puzzling observation was made by Calne and his laboratory team, during liver transplantation experiments on pigs. One animal survived for months, after receiving an allograft, without any immunosupression. Later, it was found that the soluble factors, which were secreted by the liver allograft and were responsible for the development of tolerance to the recipient animal, were soluble MHC (sMHC) molecules.From this point, many laboratories around the world entered the quest, for the elucidation of the immunoregulatory effects of the various soluble forms of MHC molecules. Today, it is widely accepted that body fluids, isolated from healthy individuals contain various amounts of soluble MHC molecules. The origin of these molecules has not been clearly determined and it could be the result of shedding, alternative splicing or active secretion, whereas it has been demonstrated that trophoblast cells secrete to the medium soluble HLA-DR molecules when stimulated by IFNγ. In the 70s and 80s during the study of T suppressors (some of which might correspond to the modern T regulatory cells), it was suggested that they affected nearby cells secreting various factors with MHC determinants. Deviations from the concentration of these molecules in the body fluids of healthy individuals, have been recorder in numerous pathological conditions, such as viral encephalitis, rheumatoid arthritis, pathologic pregnancies, asthma and AIDS. These results imply that soluble MHC molecules have a special role, in the pathology of these diseases but it is not clarified whether they contribute to the pathology or if they are by-products of the pathological condition (they could have a potential role in the diagnosis of the disease stage). Additional research has confirmed that soluble forms of MHC molecules can have various immunomodulating effects, such as induction of apoptosis of CD4+ activation of CD8+ T cells and down-regulation of NK activity. The aim of this study was to perform a downstream investigation of the possible immunomodulating action of soluble MHC molecules isolated from the serum of male and pregnant Balb/c mice. We selected the 12th day of gestation for the isolation of the serum, according to Ranella A. (The role and the regulatory mechanisms of growth factors, membrane and secreted MHC class II molecules in the survival of the hemiallogeneic embryo during gestation, Ph.D. thesis, University of Crete, unpublished data), because the titer of the serum reaches a maximum. During the study we used affinity chromatography and immunoprecipitation for the isolation of sMHC II from the serum and cell culture techniques, in order to reveal any effect on freshly isolated spleen cells. Pregnancy is often called immune paradox, because the immune system of the mother allows to the fetus to survive although it is an allotransplant. Generally, pregnancy is largely considered to be a TH2 phenomenon that is characterized by downregulation of the immune system in the point where the mothers body meets the fetus. Soluble MHC class II molecules secreted by trophoblast cells could contribute to the development of tolerance, because the ability of antigen presentation from the trophoblast is lost along with the secreted MHC II. However, it has not been determined whether the molecules we isolate from the serum originate from the trophoblast. The presence of the same molecules in the serum of male mice suggests an alternative source.


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1st Joint Meeting of European National Societies of Immunology 6-9 September, 2006, Paris

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