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Pramod K Srivastava - One of the best experts on this subject based on the ideXlab platform.
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cloning and expression of the escherichia coli k1 outer membrane protein a receptor a gp96 homologue
Infection and Immunity, 2003Co-Authors: Nemani V Prasadarao, Pramod K Srivastava, Rajyalakshmi S Rudrabhatla, Kwang Sik Kim, Shenghe Huang, Sunil K SukumaranAbstract:Escherichia coli is one of the most common gram-negative bacteria that cause meningitis in neonates. Our previous studies have shown that outer membrane protein A (OmpA) of E. coli interacts with a 95-kDa human brain microvascular endothelial cell (HBMEC) glycoprotein, Ecgp, for invasion. Here, we report the identification of a gene that encodes Ecgp by screening of an HBMEC cDNA expression library as well as by 5′ rapid amplification of cDNA ends. The sequence of the Ecgp gene shows that it is highly similar to gp96, a Tumor Rejection Antigen-1, and contains an endoplasmic reticulum retention signal, KDEL. Overexpression of either Ecgp or gp96 in both HBMECs and CHO cells increases E. coli binding and invasion. We further show that Ecgp gene-transfected HBMECs express Ecgp on the cell surface despite the presence of the KDEL motif. Northern blot analysis of total RNA from various eukaryotic cells indicates that Ecgp is significantly expressed in HBMECs. Recombinant His-tagged Ecgp blocked E. coli invasion efficiently by binding directly to the bacteria. These results suggest that OmpA of E. coli K1 interacts with a gp96-like molecule on HBMECs for invasion.
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heat shock proteins come of age primitive functions acquire new roles in an adaptive world
Immunity, 1998Co-Authors: Pramod K Srivastava, Antoine Menoret, Sreyashi Basu, Robert J Binder, Kristi L McquadeAbstract:During the analysis of representation of HSP-peptide complexes by APCs, another facet of HSP-APC interaction came to light. Exposure of APCs to gp96 preparations was observed to lead to stimulation of APCs to secrete low levels of cytokines regardless of the peptides associated with gp96 (Suto and Srivastava 1995xA mechanism for the specific immunogenicity of heat shock protein-chaperoned peptides. Suto, R and Srivastava, P.K. Science. 1995; 269: 1585–1588Crossref | PubMedSee all ReferencesSuto and Srivastava 1995; Figure 3Figure 3). This was surprising but consistent with the observation that immunization with cancer-derived HSP-peptide complexes elicits, in addition to the CD8+ and CD4+ T cell response, an NK response that is crucial for eliciting protective immunity (Tamura et al. 1997xImmunotherapy of Tumors with autologous Tumor-derived heat shock protein preparations. Tamura, Y, Peng, P, Kang, L, Daou, M, and Srivastava, P.K. Science. 1997; 278: 117–120Crossref | PubMed | Scopus (572)See all ReferencesTamura et al. 1997). In light of the fact that HSPs are abundant components of pathogens as well as hosts, this observation may indicate a basic role for HSP-APC interaction in innate immunity in addition to the crucial role of such interactions in adaptive, peptide-dependent immunity discussed thus far (73xHeat shock proteins transfer peptides during Antigen processing and CTL priming. Srivastava, P.K, Udono, H, Blachere, N.E, and Li, Z. Immunogenetics. 1994; 39: 93–98Crossref | PubMed | Scopus (456)See all References, 81xCellular requirements for Tumor-specific immunity elicited by heat shock proteins (Tumor Rejection Antigen gp96 primes CD8+ T cells in vivo) . Udono, H, Levey, D.L, and Srivastava, P.K. Proc. Natl. Acad. Sci. USA. 1994; 91: 3077–3081Crossref | PubMedSee all References, 75xA mechanism for the specific immunogenicity of heat shock protein-chaperoned peptides. Suto, R and Srivastava, P.K. Science. 1995; 269: 1585–1588Crossref | PubMedSee all References). Contemplation of these ideas leads to some interesting insights. HSPs are among the most primitive proteins in living systems, and macrophage-like cells of one kind or another are present in the earliest multicellular living systems (Beck and Habicht 1996xImmunity and invertebrates. Beck, G and Habicht, G.S. Sci. Am. 1996; 275: 60–63Crossref | PubMedSee all ReferencesBeck and Habicht 1996). It is conceivable that in a less polymorphic era, when adaptive immune response was but a distant gleam in the evolutionary eye, the interaction of HSPs with macrophage-like cells, leading to stimulation of the macrophage-like cells to secrete IL-1 and other messengers, was the primary “innate” defense mechanism. As HSPs are the most abundant soluble molecules in cells, they can be reasonably expected to be the most reliable messengers of cell death or, at any rate, cell lysis, which would result if the organism were in stress or danger, e.g., being consumed by a predator. This chain of events would also explain the otherwise strange presence of a surface receptor for HSPs, which are normally intracellular. In this view, the HSPs would have been transformed from being protectors of cells from cellular stress in the single-celled organisms to being messengers of stress and protectors against such stress in the first multicellular organisms or, at any rate, among those in which a differentiated macrophage-like cell had evolved. By all accounts, this happened quite early, as phagocytes of echinoderms, mollusks, annelids, and tunicates had already acquired the ability to secrete IL-1 like molecules (seeOttaviani and Franceschi 1997xThe invertebrate phagocytic immunocyte (clues to a common evolution of immune and neuroendocrine systems) . Ottaviani, E and Franceschi, C. Immunol. Today. 1997; 18: 169–174Abstract | Full Text PDF | PubMed | Scopus (130)See all ReferencesOttaviani and Franceschi 1997). From this initial point, it is relatively straightforward to see how the HSP-APC interaction could have been incorporated into future versions of the developing defense mechanisms until, with the arrival of specific immunity and its paraphernalia of specificity of peptide-binding and T cell receptors, the primal peptide-binding proteins, i.e., the HSPs, would become accessories in Antigen presentation by MHC molecules. The evolutionary implications of a convergence of the peptide-binding functions of the HSPs and the MHC molecules has been discussed in detail elsewhere (Srivastava and Heike 1991xTumor-specific immunogenicity of stress-induced proteins (convergence of two evolutionary pathways of Antigen presentation?) . Srivastava, P.K and Heike, M. Semin. Immunol. 1991; 3: 57–64PubMedSee all ReferencesSrivastava and Heike 1991).In the proposed role of HSPs as harbingers of cell death and, therefore, of danger to an organism (seeMatzinger 1994xTolerance, danger, and the extended family. Matzinger, P. Annu. Rev. Immunol. 1994; 12: 991–1045Crossref | PubMedSee all ReferencesMatzinger 1994), a kind of death that results in an encounter of HSPs with the immune system will be more productive, i.e., communicative of danger, and protective than a “silent death” that does not lead to HSP-APC interaction. It is therefore our belief that immunogenicity of cells, infectious agents, and cancers is defined to a significant degree upon the extent to which cell death leads to induction of HSPs and consequent HSP-APC interaction. Studies that show a cosegregation of immunogenicity of cancers with the expression of molecules of the hsp60 and hsp70 family (39xCo-segregation of Tumor immunogenicity with expression of inducible but not constitutive hsp70 in rat colon carcinomas. Menoret, A, Patry, Y, Burg, C, and Le Pendu, J. J. Immunol. 1995; 155: 740–747PubMedSee all References, 85xRestoration of MHC class I surface expression and endogenous Antigen presentation by a molecular chaperone. Wells, A.D, Rai, S.K, Salvato, M.S, Band, H, and Malkovsky, M. Scand. J. Immunol. 1997; 45: 605–612Crossref | PubMedSee all References, 38xTumor immunogenicity is determined by the mechanism of cell death via induction of heat shock protein expression. Melcher, A, Todryk, A, Hardwick, N, Ford, M, Jacobson, M, and Vile, R.G. Nat. Med. 1998; 4: 581–587Crossref | PubMed | Scopus (368)See all References) are consistent with this belief. It may be tempting in this context to imagine that immunologically unproductive and productive modes of death will correlate neatly with apoptotic and necrotic types of death; however, recent results indicate that this is not necessarily so (7xProcessing of engulfed apoptotic bodies yields T cell epitopes. Bellone, M, Iezzi, G, Rovere, P, Galati, G, Ronchetti, A, Protti, M.P, Davoust, J, Rugarli, C, and Manfredi, A.A. J. Immunol. 1997; 159: 5391–5399PubMedSee all References, 1xDendritic cells acquire Antigen from apoptotic cells and induce class I-restricted CTLs. Albert, M.L, Sauter, B, and Bhardwaj, N. Nature. 1998; 392: 86–89Crossref | PubMed | Scopus (1839)See all References, 38xTumor immunogenicity is determined by the mechanism of cell death via induction of heat shock protein expression. Melcher, A, Todryk, A, Hardwick, N, Ford, M, Jacobson, M, and Vile, R.G. Nat. Med. 1998; 4: 581–587Crossref | PubMed | Scopus (368)See all References). Other factors yet to be discovered must influence the immunological productivity of dying cells. In any case, it is safe to suggest that the recent fascination of immunologists with death has atavistic origins in the evolutionary past, when the presence of extracellular HSPs signaled physical disintegration.*To whom correspondence should be addressed (e-mail: srivastava@nso2.uchc.edu).
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molecular heterogeneity of Tumor Rejection Antigen heat shock protein gp96
International Journal of Cancer, 1995Co-Authors: Anna M Feldweg, Pramod K SrivastavaAbstract:: Glycoproteins of 96 kDa (gp96) have been shown to mediate Tumor-specific immunogenicity of a number of murine sarcomas. The purity of gp96 preparations used in these studies was originally demonstrated by their homogeneity on silver-stained gels and the observation that a single amino acid terminus was detected by micro-sequencing. Results reported here show that gp96 preparations consist of 3-4 closely spaced bands. However, each of the bands is recognized by well-characterized monoclonal or monospecific antibodies to gp96. We have obtained purified preparations of a slowly migrating 110-kDa and a faster migrating 96-kDa band of the gp96 cluster from the Meth A sarcoma and have observed both species to be immunogenic in a dose-restricted manner. In addition to the size-based heterogeneity, purified 96-kDa molecules are found to contain 2 major populations, which share the same amino and carboxy termini but differ in the region recognized by an anti-grp94 monoclonal antibody (MAb). The heterogeneity in gp96 preparations does not result from differences in glycosylation but may result from differential phosphorylation, conformation or the Antigenic peptides associated with gp96. It has been suggested that the Tumor-specific immunogenicity of gp96 preparations does not derive from gp96 but from a 110-kDa protein which co-purifies with gp96 and is distinct from it. Our observations show instead that the presence of a number of bands in purified gp96 preparations is due to heterogeneity within gp96 molecules rather than to contamination with unrelated proteins.
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cellular requirements for Tumor specific immunity elicited by heat shock proteins Tumor Rejection Antigen gp96 primes cd8 t cells in vivo
Proceedings of the National Academy of Sciences of the United States of America, 1994Co-Authors: Heiichiro Udono, Daniel L Levey, Pramod K SrivastavaAbstract:Abstract Purified preparations of 96-kDa heat shock proteins (gp96) have been previously shown to elicit Tumor-specific immunity to the Tumor from which gp96 is obtained but not to Antigenically distinct chemically induced Tumors. The cellular requirements of gp96-elicited immunity have been examined. It is observed that depletion of CD8+, but not CD4+, T cells in the priming phase abrogates the immunity elicited by gp96. The CD8+ T cells elicited by immunization with gp96 are active at least up to 5 weeks after immunization. Depletion of macrophages by treatment of mice with carrageenan during the priming phase also results in loss of gp96-elicited immunity. In the effector phase, all three compartments, CD4+ and CD8+ T cells and macrophages, are required. Immunity elicited by whole irradiated Tumor cells shows a different profile of cellular requirements. In contrast to immunization with gp96, depletion of CD4+, but not CD8+, T cells during priming with whole Tumor cells abrogates Tumor immunity. Further, ablation of macrophage function during priming or effector phases has no effect on Tumor immunity elicited by whole cells. Our results suggest the existence of a macrophage-dependent and a macrophage-independent pathway of Tumor immunity. Our observations also show that in spite of exogenous administration, vaccination with gp96 preparations elicits a CD8+ T-cell response in vivo, and it is therefore a useful method of vaccination against cancer and infectious diseases.
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Tumor Rejection Antigen gp96 grp94 is an atpase implications for protein folding and Antigen presentation
The EMBO Journal, 1993Co-Authors: Pramod K SrivastavaAbstract:Immunization of mice with gp96/grp94 heat shock proteins (HSPs) elicits Tumor-specific cellular immunity to the Tumors from which gp96 is isolated. However, the cDNA sequence of gp96 is identical among Tumors and normal tissues. This raises the question regarding the structural basis of the specific immunogenicity of gp96. As HSPs bind a wide array of molecules including peptides, we have proposed that gp96 may not be immunogenic per se, but may chaperone Antigenic peptides. Furthermore, gp96 is localized predominantly in the lumen of the endoplasmic reticulum (ER) suggesting that it may act as a peptide acceptor and as accessory to peptide loading of MHC class I molecules. We demonstrate here that gp96 molecules contain ATP-binding cassettes, bind ATP and possess an Mg(2+)-dependent ATPase activity. Gp96 preparations are also observed to contain tightly bound peptides, which can be eluted by acid extraction. These properties of gp96 are consistent with its proposed roles in chaperoning Antigenic peptides and in facilitating MHC class I--peptide assembly in the ER lumen. We present a model to explain how interaction of gp96 with MHC class I may result in transfer of peptides to the latter.
John R Aitken - One of the best experts on this subject based on the ideXlab platform.
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Localization and Significance of Molecular Chaperones, Heat Shock Protein 1, and Tumor Rejection Antigen gp96 in the Male Reproductive Tract and During
2016Co-Authors: Acrosome Reaction, Kelly L Asquith, Eileen A Mclaughlin, Brett Nixon, A J. Harman, John R AitkenAbstract:Although the molecular basis of sperm-oocyte interaction is unclear, recent studies have implicated two chaperone proteins, heat shock protein 1 (HSPD1; previously known as heat shock protein 60) and Tumor Rejection Antigen gp96 (TRA1; previously known as endoplasmin), in the formation of a functional zona-receptor complex on the surface of mammalian spermatozoa. The current study was undertaken to investigate the expression of these chaperones during the ontogeny of male germ cells through spermatogenesis, epididymal sperm maturation, capac-itation, and acrosomal exocytosis. In testicular sections, both HSPD1 and TRA1 were closely associated with the mitochondria of spermatogonia and primary spermatocytes. However, this la-beling pattern disappeared from the male germ line during sper-miogenesis to become undetectable in testicular spermatozoa
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localization and significance of molecular chaperones heat shock protein 1 and Tumor Rejection Antigen gp96 in the male reproductive tract and during capacitation and acrosome reaction
Biology of Reproduction, 2005Co-Authors: Kelly L Asquith, Amanda Harman, Eileen A Mclaughlin, Brett Nixon, John R AitkenAbstract:Although the molecular basis of sperm-oocyte interaction is unclear, recent studies have implicated two chaperone proteins, heat shock protein 1 (HSPD1; previously known as heat shock protein 60) and Tumor Rejection Antigen gp96 (TRA1; previously known as endoplasmin), in the formation of a functional zonareceptor complex on the surface of mammalian spermatozoa. The current study was undertaken to investigate the expression of these chaperones during the ontogeny of male germ cells through spermatogenesis, epididymal sperm maturation, capacitation, and acrosomal exocytosis. In testicular sections, both HSPD1 and TRA1 were closely associated with the mitochondria of spermatogonia and primary spermatocytes. However, this labeling pattern disappeared from the male germ line during spermiogenesis to become undetectable in testicular spermatozoa. Subsequently, these chaperones could be detected in epididymal spermatozoa and in previously unreported ‘‘dense bodies’’ in the epididymal lumen. The latter appeared in the precise region of the epididymis (proximal corpus), where spermatozoa acquire the capacity to recognize and bind to the zona pellucida, implicating these structures in the functional remodeling of the sperm surface during epididymal maturation. Both HSPD1 and TRA1 were subsequently found to become coexpressed on the surface of live mouse spermatozoa following capacitation in vitro and were lost once these cells had undergone the acrosome reaction, as would be expected of cell surface molecules involved in sperm-egg interaction. These data reinforce the notion that these chaperones are intimately involved in the mechanisms by which mammalian spermatozoa both acquire and express their ability to recognize the zona pellucida.
Steven A Rosenberg - One of the best experts on this subject based on the ideXlab platform.
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gp100 pmel 17 is a murine Tumor Rejection Antigen induction of self reactive Tumoricidal t cells using high affinity altered peptide ligand
Journal of Experimental Medicine, 1998Co-Authors: Willem W Overwijk, Maria R Parkhurst, Allan Tsung, Kari R Irvine, Theresa J Goletz, Kangla Tsung, Miles W Carroll, Chunlei Liu, Bernard Moss, Steven A RosenbergAbstract:Many Tumor-associated Antigens are nonmutated, poorly immunogenic tissue differentiation Antigens. Their weak immunogenicity may be due to “self”-tolerance. To induce autoreactive T cells, we studied immune responses to gp100/pmel 17, an Antigen naturally expressed by both normal melanocytes and melanoma cells. Although a recombinant vaccinia virus (rVV) encoding the mouse homologue of gp100 was nonimmunogenic, immunization of normal C57BL/6 mice with the rVV encoding the human gp100 elicited a specific CD8+ T cell response. These lymphocytes were cross-reactive with mgp100 in vitro and treated established B16 melanoma upon adoptive transfer. To understand the mechanism of the greater immunogenicity of the human version of gp100, we characterized a 9-amino acid (AA) epitope, restricted by H-2Db, that was recognized by the T cells. The ability to induce specific T cells with human but not mouse gp100 resulted from differences within the major histocompatibility complex (MHC) class I–restricted epitope and not from differences elsewhere in the molecule, as was evidenced by experiments in which mice were immunized with rVV containing minigenes encoding these epitopes. Although the human (hgp10025–33) and mouse (mgp10025–33) epitopes were homologous, differences in the three NH2-terminal AAs resulted in a 2-log increase in the ability of the human peptide to stabilize “empty” Db on RMA-S cells and a 3-log increase in its ability to trigger interferon γ release by T cells. Thus, the fortuitous existence of a peptide homologue with significantly greater avidity for MHC class I resulted in the generation of self-reactive T cells. High-affinity, altered peptide ligands might be useful in the rational design of recombinant and synthetic vaccines that target tissue differentiation Antigens expressed by Tumors.
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identification of tyrosinase related protein 2 as a Tumor Rejection Antigen for the b16 melanoma
Journal of Experimental Medicine, 1997Co-Authors: Matthew B Bloom, Paul F Robbins, Steven A Rosenberg, Donna Perrylalley, Yong Li, Mona Elgamil, James Chihhsin YangAbstract:Recently, major advances have been made in the identification of Antigens from human melanoma which are recognized by T cells. In spite of this, little is known about the optimal ways to use these Antigens to treat patients with cancer. Progress in this area is likely to require accurate preclinical animal models, but the availability of such models has lagged behind developments in human Tumor immunology. Whereas many of the identified human melanoma Antigens are normal tissue differentiation proteins, analogous murine Tumor Antigens have not yet been identified. In this paper we identify a normal tissue differentiation Antigen, tyrosinaserelated protein 2 (TRP-2), expressed by the murine B16 melanoma which was found by screening a cDNA library from B16 with Tumor-reactive cytotoxic T lymphocytes (CTL). A peptide conforming to the predicted MHC class I H2-Kb binding motif, TRP-2181-188, was identified as the major reactive epitope within TRP-2 recognized by these anti-B16 CTLs. By site-directed mutagenesis, it was shown that alteration of this epitope eliminated recognition of TRP-2. It was further demonstrated that a CTL line raised from splenocytes by repeated stimulation in vitro with this peptide could recognize B16 Tumor and was therapeutic against 3-d-old established pulmonary metastases. The use of TRP-2 in a preclinical model of Tumor immunotherapy may be helpful in suggesting optimal vaccination strategies for cancer therapy in patients.
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utilization of an alternative open reading frame of a normal gene in generating a novel human cancer Antigen
Journal of Experimental Medicine, 1996Co-Authors: Rongfu Wang, Yutaka Kawakami, Paul F Robbins, Maria R Parkhurst, Steven A RosenbergAbstract:Tumor infiltrating lymphocytes (TILs) derived from Tumor-bearing patients recognize Tumor-associated Antigens presented by major histocompatibility complex (MHC) class I molecules. The infusion of TIL586 along with interleukin (IL) 2 into an autologous patient with metastatic melanoma resulted in the objective regression of Tumor. A gene encoding a Tumor Antigen recognized by TIL586 was recently isolated and shown to encode gp75. Here we report that an Antigenic peptide, MSLQRQFLR, recognized by TIL586 was not derived from the normal gp75 protein. Instead, this nonamer peptide resulted from translation of an alternative open reading frame of the same gene. Thus, the gp75 gene encodes two completely different polypeptides, gp75 as an Antigen recognized by immunoglobulin G antibodies in sera from a patient with cancer, and a 24-amino acid product as a Tumor Rejection Antigen recognized by T cells. This represents the first demonstration that a human Tumor Rejection Antigen can be generated from a normal cellular gene using an open reading frame other than that used to encode the normal protein. These findings revealed a novel mechanism for generating Tumor Antigens, which may be useful as vaccines to induce Tumor-specific cell-mediated immunity against cancer.
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identification of a human melanoma Antigen recognized by Tumor infiltrating lymphocytes associated with in vivo Tumor Rejection
Proceedings of the National Academy of Sciences of the United States of America, 1994Co-Authors: Yutaka Kawakami, Siona Eliyahu, Cynthia H Delgado, John R Yannelli, Gosse Jan Adema, Paul F Robbins, Ettore Appella, Kazuyasu Sakaguchi, Toru Miki, Steven A RosenbergAbstract:Abstract The cultured T-cell line TIL1200, established from the Tumor-infiltrating lymphocytes (TILs) of a patient with advanced metastatic melanoma, recognized an Antigen on most HLA-A2+ melanomas and on all HLA-A2+ cultured neonatal melanocytes in an HLA-A2 restricted manner but not on other types of tissues or cell lines tested. A cDNA encoding an Antigen recognized by TIL1200 was isolated by screening an HLA-A2+ breast cancer cell line transfected with an expression cDNA library prepared from an HLA-A2+ melanoma cell line. The nucleotide and amino acid sequences of this cDNA were almost identical to the genes encoding glycoprotein gp100 or Pmel17 previously registered in the GenBank. Expression of this gene was restricted to melanoma and melanocyte cell lines and retina but was not expressed on other fresh or cultured normal tissues or other types of Tumor tested. The cell line transfected with this cDNA also expressed Antigen recognized by the melanoma-specific antibody HMB45 that bound to gp100. A synthetic 10-amino acid peptide derived from gp100 was recognized by TIL1200 in the context of HLA-A2.1. Since the administration of TIL1200 plus interleukin 2 resulted in regression of metastatic cancer in the autologous patient, gp100 is a possible Tumor Rejection Antigen and may be useful for the development of immunotherapies for patients with melanoma.
Lance A Liotta - One of the best experts on this subject based on the ideXlab platform.
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2d differential in gel electrophoresis for the identification of esophageal scans cell cancer specific protein markers
Molecular & Cellular Proteomics, 2002Co-Authors: Ge Zhou, Dianne Decamp, She Chen, Hongjun Shu, Yi Gong, Michael J Flaig, John W Gillespie, Philip R Taylor, Michael R Emmertbuck, Lance A LiottaAbstract:The reproducibility of conventional two-dimensional (2D) gel electrophoresis can be improved using differential in-gel electrophoresis (DIGE), a new emerging technology for proteomic analysis. In DIGE, two pools of proteins are labeled with 1-(5-carboxypentyl)-1'-propylindocarbocyanine halide (Cy3) N-hydroxy-succinimidyl ester and 1-(5-carboxypentyl)-1'-methylindodi-carbocyanine halide (Cy5) N-hydroxysuccinimidyl ester fluorescent dyes, respectively. The labeled proteins are mixed and separated in the same 2D gel. 2D DIGE was applied to quantify the differences in protein expression between laser capture microdissection-procured esophageal carcinoma cells and normal epithelial cells and to define cancer-specific and normal-specific protein markers. Analysis of the 2D images from protein lysates of approximately 250,000 cancer cells and normal cells identified 1038 protein spots in cancer cell lysates and 1088 protein spots in normal cell lysates. Of the detected proteins, 58 spots were up-regulated by >3-fold and 107 were down-regulated by >3-fold in cancer cells. In addition to previously identified down-regulated protein annexin I, Tumor Rejection Antigen (gp96) was found up-regulated in esophageal squamous cell cancer. Global quantification of protein expression between laser capture-microdissected patient-matched cancer cells and normal cells using 2D DIGE in combination with mass spectrometry is a powerful tool for the molecular characterization of cancer progression and identification of cancer-specific protein markers.
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2d differential in gel electrophoresis for the identification of esophageal scans cell cancer specific protein markers
Molecular & Cellular Proteomics, 2002Co-Authors: Ge Zhou, Dianne Decamp, She Chen, Hongjun Shu, Yi Gong, Michael J Flaig, John W Gillespie, Philip R Taylor, Michael R Emmertbuck, Lance A LiottaAbstract:The reproducibility of conventional two-dimensional (2D) gel electrophoresis can be improved using differential in-gel electrophoresis (DIGE), a new emerging technology for proteomic analysis. In DIGE, two pools of proteins are labeled with 1-(5-carboxypentyl)-1′-propylindocarbocyanine halide (Cy3) N-hydroxy-succinimidyl ester and 1-(5-carboxypentyl)-1′-methylindodi-carbocyanine halide (Cy5) N-hydroxysuccinimidyl ester fluorescent dyes, respectively. The labeled proteins are mixed and separated in the same 2D gel. 2D DIGE was applied to quantify the differences in protein expression between laser capture microdissection-procured esophageal carcinoma cells and normal epithelial cells and to define cancer-specific and normal-specific protein markers. Analysis of the 2D images from protein lysates of ∼ 250,000 cancer cells and normal cells identified 1038 protein spots in cancer cell lysates and 1088 protein spots in normal cell lysates. Of the detected proteins, 58 spots were up-regulated by >3-fold and 107 were down-regulated by >3-fold in cancer cells. In addition to previously identified down-regulated protein annexin I, Tumor Rejection Antigen (gp96) was found up-regulated in esophageal squamous cell cancer. Global quantification of protein expression between laser capture-microdissected patient-matched cancer cells and normal cells using 2D DIGE in combination with mass spectrometry is a powerful tool for the molecular characterization of cancer progression and identification of cancer-specific protein markers.
Eli Gilboa - One of the best experts on this subject based on the ideXlab platform.
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Tumor immunotherapy targeting fibroblast activation protein a product expressed in Tumor associated fibroblasts
Cancer Research, 2005Co-Authors: Jaewoo Lee, Martin Fassnacht, Smita K Nair, David Boczkowski, Eli GilboaAbstract:Murine studies have shown that immunologic targeting of the Tumor vasculature, a key element of the Tumor stroma, can lead to protective immunity in the absence of significant pathology. In the current study, we expand the scope of stroma-targeted immunotherapy to Antigens expressed in Tumor-associated fibroblasts, the predominant component of the stroma in most types of cancer. Mice were immunized against fibroblast activation protein (FAP), a product up-regulated in Tumor-associated fibroblasts, using dendritic cells transfected with FAP mRNA. Using melanoma, carcinoma, and lymphoma models, we show that Tumor growth was inhibited in Tumor-bearing mice vaccinated against FAP and that the magnitude of the antiTumor response was comparable to that of vaccination against Tumor cell-expressed Antigens. Both s.c. implanted Tumors and lung metastases were susceptible to anti-FAP immunotherapy. The antiTumor response could be further enhanced by augmenting the CD4+ T-cell arm of the anti-FAP immune response, achieved by using a lysosomal targeting sequence to redirect the translated FAP product into the class II presentation pathway, or by covaccination against FAP and a Tumor cell-expressed Antigen, tyrosinase-related protein 2. No morbidity or mortality was associated with anti-FAP vaccination except for a small delay in wound healing. The study suggests that FAP, a product which is preferentially expressed in Tumor-associated fibroblasts, could function as a Tumor Rejection Antigen in a broad range of cancers.
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induction of cytotoxic t cell responses and Tumor immunity against unrelated Tumors using telomerase reverse transcriptase rna transfected dendritic cells
Nature Medicine, 2000Co-Authors: Smita K Nair, David Boczkowski, A Heiser, Anish Sen Majumdar, Michio Naoe, Jane S Lebkowski, Johannes Vieweg, Eli GilboaAbstract:The polypeptide component of telomerase (TERT) is an attractive candidate for a broadly expressed Tumor Rejection Antigen because telomerase is silent in normal tissues but is reactivated in more than 85% of cancers. Here we show that immunization against TERT induces immunity against Tumors of unrelated origin. Immunization of mice with TERT RNA-transfected dendritic cells (DC) stimulated cytotoxic T lymphocytes (CTL), which lysed melanoma and thymoma Tumor cells and inhibited the growth of three unrelated Tumors in mice of distinct genetic backgrounds. TERT RNA-transfected human DC stimulated TERT-specific CTL in vitro that lysed human Tumor cells, including Epstein Barr virus (EBV)-transformed B cells as well as autologous Tumor targets from patients with renal and prostate cancer. Tumor RNA-transfected DC were used as surrogate targets in the CTL assays, obviating the difficulties in obtaining Tumor cells from cancer patients. In one instance, where a Tumor cell line was successfully established in culture from a patient with renal cancer, the patient's Tumor cells were efficiently lysed by the CTL. Immunization with Tumor RNA was generally more effective than immunization with TERT RNA, suggesting that an optimal immunization protocol may have to include TERT as well as additional Tumor Antigens.
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the makings of a Tumor Rejection Antigen
Immunity, 1999Co-Authors: Eli GilboaAbstract:If isolation of unique group I–type Antigens from each cancer patients is not practical, and the use of shared nonmutated Antigens must await the development of appropriate Antigen isolation methodologies, what can one do in the meantime? One attractive option is to vaccinate with autologous Tumor-derived Antigenic mixtures. The Tumor-derived Antigenic mixtures will include the complete Antigenic repertoire of the Tumor, including the potent group I patient-specific Antigens, yet will obviate the need to identify the relevant Tumor Antigens in each patient. In animal Tumor models, vaccination with genetically modified irradiated Tumor cells (Gilboa and Lyerly 1994xSpecific active immunotherapy of cancer using genetically modified Tumor vaccines. Gilboa, E and Lyerly, H.K. See all ReferencesGilboa and Lyerly 1994), Tumor-derived heat shock proteins (Tamura et al. 1997xImmunotherapy of Tumors with autologous Tumor-derived heat shock protein preparations. Tamura, Y, Peng, P, Liu, K, Daou, M, and Srivastava, P.K. Science. 1997; 278: 117–120Crossref | PubMed | Scopus (578)See all ReferencesTamura et al. 1997), or with dendritic cells loaded with Tumor-derived peptides or proteins (Gilboa et al. 1998xImmunotherapy of cancer with dendritic-cell based vaccines. Gilboa, E, Nair, S.K, and Lyerly, H.K. Cancer Immunol. Immunother. 1998; 46: 82–87Crossref | PubMed | Scopus (247)See all ReferencesGilboa et al. 1998) is very potent. A common limitation of these strategies is that sufficient Tumor tissue for Antigen preparation cannot be obtained or generated from many cancer patients. In such instances, use of mRNA amplified from small amounts of available Tumor tissue could provide unlimited amounts of Antigen for vacccination protocols.*E-mail: e.gilboa@cgct.duke.edu.
