- PROFESSOR EMERITUS Microbiology
D.C. Sci., University of Paris
M.D., University of Bucharest
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virology, immunologyFor the future years, my laboratory has two major research goals. The first is related to the study of the enhancement of anti-viral immunity by using genetically or enzymatically engineered Igs and the second is related to study the effect of mature lymphocytes and skin immunity defect on TSK syndrome and the complementation of TSK mutation by collagen I and V defective genes. Advances in biotechnology have ushered in a new era of vaccine development. These developments are based on the utilization of peptides corresponding to protective epitopes, recombinant proteins, viral or bacteria vectors, naked DNA etc. Two promising avenues of research are maternal immunization which may confer protection to infants and neonatal immunization which may confer protection to children. During the past year, we have used engineered immunoglobulins as a delivery system for viral B and T cell epitopes. The overall goal is to use the influenza virus system to evaluate the effects of maternal and neonatal immunization of two types of new vaccines: antigenized Ig and naked DNA. Our aims are to understand the basic immune mechanisms of maternal and neonatal immunization with antigenized Ig and naked DNA which may led to protective response against influenza virus. In theses studies, we will use: a) genetically engineered Ig in which a B cell epitope was grafted in CDR2 and a T cell epitope in CDR3. b) Enzymatically engineered Ig in which the viral epitopes were specifically conjugated to sugar moiety of Ig molecule and c) Plasmids expressing influenza virus NP or HA genes. Transgenic mice expressing a TCR specific for influenza virus hemagglutinin dominant peptide will be used to study the molecular mechanisms of tolerance.
The second goal is to examine the development of tight skin syndrome in congenitally defective mice in lymphocyte receptor, skin immunity and in fibrillinogenesis. This study will be carried out on backcross progeny obtained by the crossing of TSK mice with: a) RAG2-/- and Ig-/- mice obtained by targeted deletion. The RAG2-/- mice lack both T and B mature lymphocytes, whereas, Ig-/- mice only B lymphocytes. b) vit/vit mice bearing a mutation responsible for the genetic defect in skin immunity. c) MOV13 mice which have complete transcriptional block of a1 collagen gene and pNA25 mice which have a defect of collagen type V gene. This study will provide definitive proof for the role of the immune system in TSK scleroderma-like syndrome and will provide information on the complementation of TSK gene defect by collagen defective genes. The ultimate goal of this project is to clone the TSK gene and use it to fish out a human gene which may be involved in scleroderma.
Saito S, Nishimura H, Brumeanu T, Casares S, Stan AC, Honjo T, Bona C. Characterization of mutated protein encoded by partially duplicated fibrillin-1 gene in tight skin (TSK). Mol Immunol 1999 Feb; 36(3): 169-176.Fibrillin-1 (Fbn-1) is a ubiquitous protein present in the extracellular matrix of various organs and it is a major component of microfibrils embedded in the core of elastic fibers. In humans, mutations or deletions of the Fbn-1 gene are associated with several genetic diseases. In addition, several microsatellite alleles near Fbn-1 gene were found associated with diffuse scleroderma. In TSK/+ mice, which develop a scleroderma-like syndrome, the Fbn-1 gene exhibits an inframe duplication of exons 17-40. In this study, we report that the synthesis and secretion of wild-type Fbn-1 in TSK/+ is higher than that of the mutated Fbn-1 protein excluding the possibility that TSK genetic defect is due to a loss of the wild allele. We also demonstrate for the first time that TGF-beta, which plays a crucial role in skin fibrosis, binds to both wild-type and mutated Fbn-1. The amount of bound TGF-beta was higher in mutated than wild-type Fbn-1 and appears related to the number of TGF-beta binding motifs.
Tan FK, Arnett FC, Antohi S, Saito S, Mirarchi A, Spiera H, Sasaki T, Shoichi O, Takeuchi K, Pandy JP, Silver RM, LeRoy C, Postlethwaite AE, Bona C. Autoantibodies to the extracellular matrix microfibrillar protein, fibrillin-1, in patients with scleroderma. J Immunol 1999 Jul; 163(2): 1066-1072.A duplication in the fibrillin-1 gene has been implicated as the cause of the tight skin 1 (tsk1) phenotype, an animal model of scleroderma or systemic sclerosis (SSc). In addition to the production of abnormal fibrillin-1 protein, the tsk1 mouse also produces autoantibodies to fibrillin-1. Among a population of Choctaw Native Americans with the highest prevalence of SSc yet described, a chromosome 15q haplotype containing the fibrillin-1 gene has been strongly associated with SSc. With a recombinant human fibrillin-1 protein, autoantibodies to fibrillin-1 were detected in the sera of Native American SSc patients that correlated significantly with disease. Abs to fibrillin-1 also were detected in sera from Japanese, Caucasian, and African-American SSc patients. Compared with other ethnic groups, Japanese and Native American SSc patients had significantly higher frequencies of anti-fibrillin-1 Abs. Sera from patients with diffuse SSc, calcinosis, Raynaud's, esophageal dysmotility, sclerodactyly, and telangiectasias syndrome and mixed connective tissue disease also had significantly higher frequencies of anti-fibrillin-1 Abs than sera from controls or patients with other non-SSc connective tissue diseases (lupus, rheumatoid arthritis, and Sjogren's syndrome). Ab specificity for fibrillin-1 was demonstrated by the lack of binding to a panel of other purified autoantigens. The results presented demonstrate for the first time the presence of high levels of anti-fibrillin-1 Abs in a significant portion of patients with SSc.
Stan AC, Radu D, Casares S, Bona C, Brumeanu T. Antineoplastic efficacy of doxorubicin enzymatically assembled on galactose residues of a monoclonal. Cancer Res 1999 Jan; 59(1): 115-121.We have developed a novel procedure to couple enzymatically the antineoplastic agent doxorubicin (Dox) on the galactose residues of a monoclonal antibody specific for the tumor-associated carcinoembryonic antigen. The synthesis of the immunoconjugate consists of covalent attachment of the NH2 terminus of Dox to oxidized galactose residues of desialylated monoclonal antibody, followed by concurrent stabilization of Schiff bases by mild reduction with pyridine borane. The immunoconjugate preserved both antibody specificity and drug cytotoxicity. At equimolar concentrations, the immunoconjugate was 8 times more cytotoxic against two carcinoembryonic antigen-expressing carcinoma cell lines, LoVo and SW-480, than Dox alone. The intracellular drug accumulation was 8-8.5 times higher than that obtained with free Dox, and >50% of the drug delivered by the conjugate was retained for 24 h in the tumor cells. Only 4 days after treatment with a single dose of immunoconjugate carrying 2.5 ng of Dox, LoVo and SW-480 tumor transplants on the chorioallantoic membrane of embryonated hen eggs showed reduced tumor-induced angiogenesis and tumor progression by half, with no detectable damage to surrounding tissues. In contrast, the same amount of free drug induced insignificant changes in tumor progression and tumor-induced angiogenesis. Enzymatically mediated, glycosidic coupling of antineoplastic agents to antibodies specific for tumor-associated antigens may represent a novel platform for the development of more efficient anticancer agents with reduced side effects.
Casares S, Zong C, Radu D, Miller A, Bona C, Brumeanu T. Antigen-specific signaling by a soluble, dimeric peptide/major histocompatibility complex class II/Fc. J Exp Med 1999 Aug; 190(4): 543-553.Interaction between a T cell receptor (TCR) and various ligands, i.e. , anti-TCR antibodies, superantigens, peptides, or altered peptide ligands in the context of major histocompatibility complex (MHC) molecules can trigger different T helper cell (Th) effector functions. Herein, we studied the T cell response induced by a soluble, dimeric peptide/MHC class II chimera, namely hemagglutinin (HA)110-120/I-E(d)alphabeta/Fcgamma2a (DEF). We have previously demonstrated that the soluble DEF molecule binds stably and specifically to HA110-120-specific TCRs expressed by a T cell hybridoma. Administration of DEF in vivo induced differentiation of resting and activated peptide-specific T cells toward a Th2 response, as indicated by the increase of interleukin (IL)-4, IL-10, and specific immunoglobulin (Ig)G1 antibodies and decrease of IL-2, specific IgG2a antibodies, and cytotoxic T lymphocyte activity. In contrast to HA110-120 peptide presented by the DEF molecule to T cells, the nominal synthetic peptide induced a predominant Th1 response, and the PR8 virus-derived HA110-120 peptides induced a mixed Th1/Th2 response. Independent of antigen processing, soluble DEF was almost 2 logs more potent in stimulating cognate T cells than the nominal peptide. Polarization of cognate T cells toward the Th2 response occurred upon interaction of soluble DEF with TCR and CD4 molecules followed by early activation of p56(lck) and ZAP-70 tyrosine kinases, and negative signaling of the signal transducer and activator of transcription (STAT)4 pathway of Th1 differentiation. DEF-like molecules may provide a new tool to study the mechanisms of signaling toward Th2 differentiation and may also provide a potential immunotherapeutic approach to modulate autoreactive T cells toward protective Th2 immune responses.
Casares S, Bona C, Brumeanu T. Enzymatically mediated engineering of multivalent MHC class II-peptide chimeras. Protein Eng 2001; 14: 195-200.
Stan AC, Casares S, Brumeanu T, Klinman DM, Bona C. CpG motifs of DNA vaccines induce the expression of chemokines and MHC class II molecules on myocytes. Eur J Immunol 2001 Jan; 31: 301-310.
Casares S, Stan AC, Bona C, Brumeanu T. Antigen-specific downregulation of T cells by doxorubicin delivered through a recombinant MHC II--peptide chimera. Nat Biotechnol 2001 Feb; 19: 142-147.
Brumeanu T, Bona C, Casares S. T-cell tolerance and autoimmune diabetes. (review) Int Rev Immunol 2001; 20(2): 301-31.
Casares S, Bona C, Brumeanu T. Modulation of CD4 T cell function by soluble MHC II-peptide chimeras. Int Rev Immunol 2001 Oct; 20(5): 547-73.
Casares S, Bona CA, Brumeanu TD. Immunoregulation of antigen-specific T cell by MHC-peptide chimeras. Int. Rev. Immunol 2001; 20: 547-574.
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