Expression of antigen tf and galectin-3 in fibroadenoma.

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Gallegos, Itandehui Belem; Pérez-Campos, Eduardo; Martinez, Margarito; Mayoral, Miguel Ángel; Pérez, Laura; Aguilar, Sergio; Zenteno, Edgar; Pina, Maria del Socorro; Hernández, Pedro

2012-12-24

Fibroadenomas are benign human breast tumors, characterized by proliferation of epithelial and stromal components of the terminal ductal unit. They may grow, regress or remain unchanged, as the hormonal environment of the patient changes. Expression of antigen TF in mucin or mucin-type glycoproteins and of galectin-3 seems to contribute to proliferation and transformations events; their expression has been reported in ductal breast cancer and in aggressive tumors. Lectin histochemistry, immunohistochemistry, and immunofluorescence were used to examine the expression and distribution of antigen TF and galectin-3. We used lectins from Arachis hypogaea, Artocarpus integrifolia, and Amaranthus lecuocarpus to evaluate TF expression and a monoclonal antibody to evaluate galectin-3 expression. We used paraffin-embedded blocks from 10 breast tissues diagnosed with fibroadenoma and as control 10 healthy tissue samples. Histochemical and immunofluorescence analysis showed positive expression of galectin-3 in fibroadenoma tissue, mainly in stroma, weak interaction in ducts was observed; whereas, in healthy tissue samples the staining was also weak in ducts. Lectins from A. leucocarpus and A. integrifolia specificaly recognized ducts in healthy breast samples, whereas the lectin from A. hypogaea recognized ducts and stroma. In fibroadenoma tissue, the lectins from A. integrifolia, A. Hypogaea, and A. leucocarpus recognized mainly ducts. Our results suggest that expression of antigen TF and galectin-3 seems to participate in fibroadenoma development.

Expression of antigen tf and galectin-3 in fibroadenoma

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Gallegos Itandehui Belem

2012-12-01

Full Text Available Abstract Background Fibroadenomas are benign human breast tumors, characterized by proliferation of epithelial and stromal components of the terminal ductal unit. They may grow, regress or remain unchanged, as the hormonal environment of the patient changes. Expression of antigen TF in mucin or mucin-type glycoproteins and of galectin-3 seems to contribute to proliferation and transformations events; their expression has been reported in ductal breast cancer and in aggressive tumors. Findings Lectin histochemistry, immunohistochemistry, and immunofluorescence were used to examine the expression and distribution of antigen TF and galectin-3. We used lectins from Arachis hypogaea, Artocarpus integrifolia, and Amaranthus lecuocarpus to evaluate TF expression and a monoclonal antibody to evaluate galectin-3 expression. We used paraffin-embedded blocks from 10 breast tissues diagnosed with fibroadenoma and as control 10 healthy tissue samples. Histochemical and immunofluorescence analysis showed positive expression of galectin-3 in fibroadenoma tissue, mainly in stroma, weak interaction in ducts was observed; whereas, in healthy tissue samples the staining was also weak in ducts. Lectins from A. leucocarpus and A. integrifolia specificaly recognized ducts in healthy breast samples, whereas the lectin from A. hypogaea recognized ducts and stroma. In fibroadenoma tissue, the lectins from A. integrifolia, A. Hypogaea, and A. leucocarpus recognized mainly ducts. Conclusions Our results suggest that expression of antigen TF and galectin-3 seems to participate in fibroadenoma development.

Comparative immunological evaluation of recombinant Salmonella Typhimurium strains expressing model antigens as live oral vaccines.

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Zheng, Song-yue; Yu, Bin; Zhang, Ke; Chen, Min; Hua, Yan-Hong; Yuan, Shuofeng; Watt, Rory M; Zheng, Bo-Jian; Yuen, Kwok-Yung; Huang, Jian-Dong

2012-09-26

Despite the development of various systems to generate live recombinant Salmonella Typhimurium vaccine strains, little work has been performed to systematically evaluate and compare their relative immunogenicity. Such information would provide invaluable guidance for the future rational design of live recombinant Salmonella oral vaccines. To compare vaccine strains encoded with different antigen delivery and expression strategies, a series of recombinant Salmonella Typhimurium strains were constructed that expressed either the enhanced green fluorescent protein (EGFP) or a fragment of the hemagglutinin (HA) protein from the H5N1 influenza virus, as model antigens. The antigens were expressed from the chromosome, from high or low-copy plasmids, or encoded on a eukaryotic expression plasmid. Antigens were targeted for expression in either the cytoplasm or the outer membrane. Combinations of strategies were employed to evaluate the efficacy of combined delivery/expression approaches. After investigating in vitro and in vivo antigen expression, growth and infection abilities; the immunogenicity of the constructed recombinant Salmonella strains was evaluated in mice. Using the soluble model antigen EGFP, our results indicated that vaccine strains with high and stable antigen expression exhibited high B cell responses, whilst eukaryotic expression or colonization with good construct stability was critical for T cell responses. For the insoluble model antigen HA, an outer membrane expression strategy induced better B cell and T cell responses than a cytoplasmic strategy. Most notably, the combination of two different expression strategies did not increase the immune response elicited. Through systematically evaluating and comparing the immunogenicity of the constructed recombinant Salmonella strains in mice, we identified their respective advantages and deleterious or synergistic effects. Different construction strategies were optimally-required for soluble versus

Paired Expression Analysis of Tumor Cell Surface Antigens

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Rimas J. Orentas

2017-08-01

Full Text Available Adoptive immunotherapy with antibody-based therapy or with T cells transduced to express chimeric antigen receptors (CARs is useful to the extent that the cell surface membrane protein being targeted is not expressed on normal tissues. The most successful CAR-based (anti-CD19 or antibody-based therapy (anti-CD20 in hematologic malignancies has the side effect of eliminating the normal B cell compartment. Targeting solid tumors may not provide a similar expendable marker. Beyond antibody to Her2/NEU and EGFR, very few antibody-based and no CAR-based therapies have seen broad clinical application for solid tumors. To expand the way in which the surfaceome of solid tumors can be analyzed, we created an algorithm that defines the pairwise relative overexpression of surface antigens. This enables the development of specific immunotherapies that require the expression of two discrete antigens on the surface of the tumor target. This dyad analysis was facilitated by employing the Hotelling’s T-squared test (Hotelling–Lawley multivariate analysis of variance for two independent variables in comparison to a third constant entity (i.e., gene expression levels in normal tissues. We also present a unique consensus scoring mechanism for identifying transcripts that encode cell surface proteins. The unique application of our bioinformatics processing pipeline and statistical tools allowed us to compare the expression of two membrane protein targets as a pair, and to propose a new strategy based on implementing immunotherapies that require both antigens to be expressed on the tumor cell surface to trigger therapeutic effector mechanisms. Specifically, we found that, for MYCN amplified neuroblastoma, pairwise expression of ACVR2B or anaplastic lymphoma kinase (ALK with GFRA3, GFRA2, Cadherin 24, or with one another provided the strongest hits. For MYCN, non-amplified stage 4 neuroblastoma, neurotrophic tyrosine kinase 1, or ALK paired with GFRA2, GFRA3, SSK

Expression and immunogenicity of novel subunit enterovirus 71 VP1 antigens

International Nuclear Information System (INIS)

Xu, Juan; Wang, Shixia; Gan, Weihua; Zhang, Wenhong; Ju, Liwen; Huang, Zuhu; Lu, Shan

2012-01-01

Highlights: ► EV71 is a major emerging infectious disease in many Asian countries. ► Inactivated EV71 vaccines are in clinical studies but their safety and efficacy are unknown. ► Developing subunit based EV71 vaccines is significant and novel antigen design is needed. ► DNA immunization is an efficient tool to test the immunogenicity of VP1 based EV71 vaccines. ► Multiple VP1 antigens are developed showing immunogenic potential. -- Abstract: Hand, foot, and mouth disease (HFMD) is a common viral illness in young children. HFMD is caused by viruses belonging to the enterovirus genus of the picornavirus family. Recently, enterovirus 71 (EV71) has emerged as a virulent agent for HFMD with severe clinical outcomes. In the current report, we conducted a pilot antigen engineering study to optimize the expression and immunogenicity of subunit VP1 antigen for the design of EV71 vaccines. DNA immunization was adopted as a simple technical approach to test different designs of VP1 antigens without the need to express VP1 protein in vitro first. Our studies indicated that the expression and immunogenicity of VP1 protein can be improved with alternated VP1 antigen designs. Data presented in the current report revealed novel pathways to optimize the design of VP1 antigen-based EV71 vaccines.

Expression and immunogenicity of novel subunit enterovirus 71 VP1 antigens

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Xu, Juan [China-US Vaccine Research Center, The First Affiliated Hospital, Nanjing Medical University (China); Department of Microbiology and Immunology, Nanjing Medical University (China); Wang, Shixia [China-US Vaccine Research Center, The First Affiliated Hospital, Nanjing Medical University (China); Department of Medicine, University of Massachusetts Medical School (United States); Gan, Weihua [Department of Pediatrics, The Second Affiliated Hospital, Nanjing Medical University (China); Zhang, Wenhong [Department of Infectious Diseases, Huashan Hospital, Fudan University (China); Ju, Liwen [School of Public Health, Fudan University (China); Huang, Zuhu [Department of Infectious Diseases, The First Affiliated Hospital, Nanjing Medical University (China); China-US Vaccine Research Center, The First Affiliated Hospital, Nanjing Medical University (China); Lu, Shan, E-mail: shan.lu@umassmed.edu [Department of Infectious Diseases, The First Affiliated Hospital, Nanjing Medical University (China); China-US Vaccine Research Center, The First Affiliated Hospital, Nanjing Medical University (China); Department of Medicine, University of Massachusetts Medical School (United States)

2012-04-20

Highlights: Black-Right-Pointing-Pointer EV71 is a major emerging infectious disease in many Asian countries. Black-Right-Pointing-Pointer Inactivated EV71 vaccines are in clinical studies but their safety and efficacy are unknown. Black-Right-Pointing-Pointer Developing subunit based EV71 vaccines is significant and novel antigen design is needed. Black-Right-Pointing-Pointer DNA immunization is an efficient tool to test the immunogenicity of VP1 based EV71 vaccines. Black-Right-Pointing-Pointer Multiple VP1 antigens are developed showing immunogenic potential. -- Abstract: Hand, foot, and mouth disease (HFMD) is a common viral illness in young children. HFMD is caused by viruses belonging to the enterovirus genus of the picornavirus family. Recently, enterovirus 71 (EV71) has emerged as a virulent agent for HFMD with severe clinical outcomes. In the current report, we conducted a pilot antigen engineering study to optimize the expression and immunogenicity of subunit VP1 antigen for the design of EV71 vaccines. DNA immunization was adopted as a simple technical approach to test different designs of VP1 antigens without the need to express VP1 protein in vitro first. Our studies indicated that the expression and immunogenicity of VP1 protein can be improved with alternated VP1 antigen designs. Data presented in the current report revealed novel pathways to optimize the design of VP1 antigen-based EV71 vaccines.

Cloning and expression of the Legionella micdadei "common antigen" in Escherichia coli

DEFF Research Database (Denmark)

Bangsborg, Jette Marie; Collins, M T; Høiby, N

1989-01-01

To study individual Legionella antigens, a Legionella micdadei genomic library in Escherichia coli SC181 was established. Partially Sau3A digested L. micdadei DNA fragments (15-25 kilobase pairs (kb] were cloned into the tetracycline resistance gene of the cosmid vector pHC79. Four thousand...... ampicillin resistant recombinants were obtained; seven hundred were screened for expression of Legionella antigens in Western blot analysis with a polyspecific E. coli-absorbed anti-L. micdadei rabbit antibody. One of the positive clones expressed a 60 kilodalton (K) antigen, which reacted strongly...... will provide important information with respect to genetic vs. antigenic relatedness among Legionellae and other Gram-negative species, as well as to CA structure and possible function....

Isolation and characterization of NIH 3T3 cells expressing polyomavirus small T antigen

International Nuclear Information System (INIS)

Noda, T.; Satake, M.; Robins, T.; Ito, Y.

1986-01-01

The polyomavirus small T-antigen gene, together with the polyomavirus promoter, was inserted into retrovirus vector pGV16 which contains the Moloney sarcoma virus long terminal repeat and neomycin resistance gene driven by the simian virus 40 promoter. This expression vector, pGVST, was packaged into retrovirus particles by transfection of PSI2 cells which harbor packaging-defective murine retrovirus genome. NIH 3T3 cells were infected by this replication-defective retrovirus containing pGVST. Of the 15 G418-resistant cell clones, 8 express small T antigen at various levels as revealed by immunoprecipitation. A cellular protein with an apparent molecular weight of about 32,000 coprecipitates with small T antigen. Immunofluorescent staining shows that small T antigen is mainly present in the nuclei. Morphologically, cells expressing small T antigen are indistinguishable from parental NIH 3T3 cells and have a microfilament pattern similar to that in parental NIH 3T3 cells. Cells expressing small T antigen form a flat monolayer but continue to grow beyond the saturation density observed for parental NIH 3T3 cells and eventually come off the culture plate as a result of overconfluency. There is some correlation between the level of expression of small T antigen and the growth rate of the cells. Small T-antigen-expressing cells form small colonies in soft agar. However, the proportion of cells which form these small colonies is rather small. A clone of these cells tested did not form tumors in nude mice within 3 months after inoculation of 10 6 cells per animal. Thus, present studies establish that the small T antigen of polyomavirus is a second nucleus-localized transforming gene product of the virus (the first one being large T antigen) and by itself has a function which is to stimulate the growth of NIH 3T3 cells beyond their saturation density in monolayer culture

Expression of blood group antigens A and B in pancreas of vertebrates

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ELENKA GEORGIEVA

2012-01-01

Full Text Available The biological role of blood group antigens (BGA A and B in tissues of different vertebrates is still controversial. There are few investigations on vertebrate pancreas and no obvious explanation of their tissue expression. The aim of the present study is to follow and compare the pancreatic expression of BGA A and B in representatives of five vertebrate classes. The biotin-streptavidin-proxidase labeling system was used for immunohistochemical detection of BGA by monoclonal antibodies to human A and B antigens. The present study reveals specific immunoreactivity in acinar and epithelial cells of pancreatic efferent ducts in species free-living vertebrates. The immunoperoxidase staining shows antigenic heterogeneity in the cellular localization. The number of positive cells and the intensity of expression vary in different species. Endothelial cells are positive only in the pancreas of Emys orbicularis. The lack of BGA A and B in some species suggests that the expression of these antigens is dependent not only on the evolutionary level of the species, but mainly on some genetic control mechanisms. The production of BGA A and B and the variability in their cellular localization probably reflect the stage of cell differentiation and the mechanisms of pancreatic secretor function. The presence of histo BGA in endodermal acinar pancreatic cells confirms the assumption for the high antigenic stability and conservatism of these molecules in vertebrate histogenesis and evolution.

Enhanced specificity in immunoscreening of expression cDNA clones using radiolabeled antigen overlay

International Nuclear Information System (INIS)

Chao, S.; Chao, L.; Chao, J.

1989-01-01

A highly sensitive and specific method has been developed for immunoscreening clones from an expression cDNA library. The procedures utilize a radiolabeled antigen detection method described originally for the immunoblotting of plasma proteins. Screening of rat alpha 1-antitrypsin clones was used. Comparison between Western blots of alpha 1-antitrypsin using both labeled antigen and protein A detection methods showed that the former yielded lower background and greater sensitivity than the latter. Further, this technique was shown to have a lower detection limit of less than 20 ng through Western blot analysis of varying concentrations of alpha 1-antitrypsin. The procedures are based on the expression of the protein by cDNA clones containing the DNA inserts in the correct reading frame. Following the transfer of phage proteins to nitrocellulose membranes, the bivalent antibodies bind monovalently to both nitrocellulose-bound-antigen in the phage lysates and radiolabeled antigen. The radiolabeled antigen overlay method is superior to the protein A detection method in sensitivity, specificity and reproducibility. This improved method can be applied in general for screening expression cDNA libraries, provided that the specific antiserum and radiolabeled antigen are available

Liver cancer: expression features of hepatitis B antigens

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V. A. Tumanskiy

2013-12-01

Full Text Available Introduction. Hepatocellular carcinoma (HCC is currently the fifth most common malignancy in men and the eighth in women worldwide. According to the latest European Union countries’ statistics the incidence of HC cancer is about 8,29 per 100000 accidents, cholangiocellular (CC cancer – 0,9-1,3 per 100 thousand of population per year[10,14]. Hepatitis B virus (HBV is the major etiologic factor for the development of HCC [18]. People chronically infected with HBV are 20 times more likely to develop liver cancer than uninfected people [1,22,28]. Many studies have shown the association between Hepatitis B virus (HBV and hepatitis C virus (HCV infections and the development of cholangiocarcinoma (CCA [4,6,9,11,12]. At the same time, the expression features of HBsAg, HBcAg in HCC and CCA have not been studied clearly yet. Aim of investigation: to study the expression features of hepatitis B antigens in tumor tissue from patients with hepatocellular carcinoma and cholangiocarcinoma. Materials and methods. The complex pathomorphological research was performed using liver biopsies of 87 patients aged from 33 up to 83 years, where 50 (57,47% of them had HCC carcinoma and 37 (42,53% had cholangiocellular cancer. 15 patients among examined 87 ones were ill with chronic viral hepatitis (11 were ill with HCV, 3 – HBV B, 1 – HBV + HCV before, 72 cancer patients, corresponding to the clinical data, never had this one in their past medical history. The localization of hepatitis B surface antigen (HBsAg and core antigen (HBcAg was investigated by an indirect immunoperoxidase method in formalin-fixed, paraffin-embedded liver specimens obtained from 50 (57,47% patients with hepatocellular carcinoma and 37 (42,53% patients with cholangiocarcinoma. using antibodies Rb a-Hu Primary Hepatitis B Virus Core Antigen (HBcAg and Mo a-Hu Primary Hepatitis B Virus Surface Antigen (HBsAg, Сlone 3E7, and visualization system DAKO EnVision+ with diaminobenzidine. Liver

Antigenic characterisation of yeast-expressed lyssavirus nucleoproteins.

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Kucinskaite, Indre; Juozapaitis, Mindaugas; Serva, Andrius; Zvirbliene, Aurelija; Johnson, Nicholas; Staniulis, Juozas; Fooks, Anthony R; Müller, Thomas; Sasnauskas, Kestutis; Ulrich, Rainer G

2007-12-01

In Europe, three genotypes of the genus Lyssavirus, family Rhabdoviridae, are present, classical rabies virus (RABV, genotype 1), European bat lyssavirus type 1 (EBLV-1, genotype 5) and European bat lyssavirus type 2 (EBLV-2, genotype 6). The entire authentic nucleoprotein (N protein) encoding sequences of RABV (challenge virus standard, CVS, strain), EBLV-1 and EBLV-2 were expressed in yeast Saccharomyces cerevisiae at high level. Purification of recombinant N proteins by caesium chloride gradient centrifugation resulted in yields between 14-17, 25-29 and 18-20 mg/l of induced yeast culture for RABV-CVS, EBLV-1 and EBLV-2, respectively. The purified N proteins were evaluated by negative staining electron microscopy, which revealed the formation of nucleocapsid-like structures. The antigenic conformation of the N proteins was investigated for their reactivity with monoclonal antibodies (mAbs) directed against different lyssaviruses. The reactivity pattern of each mAb was virtually identical between immunofluorescence assay with virus-infected cells, and ELISA and dot blot assay using the corresponding recombinant N proteins. These observations lead us to conclude that yeast-expressed lyssavirus N proteins share antigenic properties with naturally expressed virus protein. These recombinant proteins have the potential for use as components of serological assays for lyssaviruses.

The relationship between oral Candida carriage and the secretor status of blood group antigens in saliva.

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Shin, Eun-Seop; Chung, Sung-Chang; Kim, Young-Ku; Lee, Sung-Woo; Kho, Hong-Seop

2003-07-01

The aim of the study was to investigate the relationship between oral Candida carriage and the secretor status of blood group antigens. Unstimulated whole saliva and oral rinse samples were obtained from 180 healthy subjects. These samples were plated on Sabouraud's dextrose agar media to determine oral Candida carriage. Sodium dodecylsulfate polyacrylamide gel electrophoresis and immunoblotting were performed on whole saliva samples to determine the secretor status of blood group antigens. The oral Candida carriage rate was found to be 45.0%. The sensitivity of the concentrated rinse culture proved to be superior. Oral Candida carriage was not significantly related to the blood group or secretor status of ABH or Lewis antigens. No significant relationship was found between oral Candida carriage and salivary flow rate. However, smoking affected oral Candida carriage. Oral Candida carriage in healthy individuals is not significantly related to blood group or secretor status.

Phase variable O antigen biosynthetic genes control expression of the major protective antigen and bacteriophage receptor in Vibrio cholerae O1.

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Kimberley D Seed

2012-09-01

Full Text Available The Vibrio cholerae lipopolysaccharide O1 antigen is a major target of bacteriophages and the human immune system and is of critical importance for vaccine design. We used an O1-specific lytic bacteriophage as a tool to probe the capacity of V. cholerae to alter its O1 antigen and identified a novel mechanism by which this organism can modulate O antigen expression and exhibit intra-strain heterogeneity. We identified two phase variable genes required for O1 antigen biosynthesis, manA and wbeL. manA resides outside of the previously recognized O1 antigen biosynthetic locus, and encodes for a phosphomannose isomerase critical for the initial step in O1 antigen biosynthesis. We determined that manA and wbeL phase variants are attenuated for virulence, providing functional evidence to further support the critical role of the O1 antigen for infectivity. We provide the first report of phase variation modulating O1 antigen expression in V. cholerae, and show that the maintenance of these phase variable loci is an important means by which this facultative pathogen can generate the diverse subpopulations of cells needed for infecting the host intestinal tract and for escaping predation by an O1-specific phage.

Cloning and Expressing Recombinant Protective Antigen Domains of B. anthracis

Science.gov (United States)

2011-09-01

future predictive modeling toolkits. 1 1. Introduction The use of Bacillus anthracis as a bio - weapon in the United States in 2001 affirmed the need...for improved sensing and detection of biological weapons of mass destruction (WMD). Protective Antigen (PA) protein of Bacillus anthracis is the...Cloning and Expressing Recombinant Protective Antigen Domains of B. anthracis by Deborah A. Sarkes, Joshua M. Kogot, Irene Val-Addo

Biosynthetic basis of incompatible histo-blood group A antigen expression

DEFF Research Database (Denmark)

David, L; Leitao, D; Sobrinho-Simoes, M

1993-01-01

The expression of incompatible A carbohydrate antigens in some adenocarcinomas may provide an explanation for the generally observed lower incidence of adenocarcinoma among types O and B versus type A individuals. The chemistry and genetic basis of incompatible A expression is largely unknown. He...

Axotomy induces MHC class I antigen expression on rat nerve cells

DEFF Research Database (Denmark)

Maehlen, J; Schröder, H D; Klareskog, L

1988-01-01

Immunomorphological staining demonstrates that class I major histocompatibility complex (MHC)-coded antigen expression can be selectively induced on otherwise class I-negative rat nerve cells by peripheral axotomy. Induction of class I as well as class II antigen expression was simultaneously seen...... on non-neural cells in the immediate vicinity of the injured nerve cells. As nerve regeneration after axotomy includes growth of new nerve cell processes and formation of new nerve cell contacts, the present findings raise the question of a role for MHC-coded molecules in cell-cell interactions during...... nerve cell growth....

Effect of radiation on the expression of carcinoembryonic antigen of human gastric adenocarcinoma cells

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Hareyama, M.; Imai, K.; Kubo, K.; Takahashi, H.; Koshiba, H.; Hinoda, Y.; Shidou, M.; Oouchi, A.; Yachi, A.; Morita, K. (Sapporo Medical College (Japan))

1991-05-01

The changes of antigenic expression of cultured human gastric adenocarcinoma MKN45 cells caused by irradiation were investigated to elucidate the immune responses to localized irradiation. The expression of carcinoembryonic antigen (CEA) showed remarkable increases in the culture supernatant and on the surface of the membrane of irradiated cells. The expression of major histocompatibility complex Class I antigen on the membrane also was enhanced by irradiation. In addition, the irradiated cell groups, when analyzed using a CEA-specific probe, showed remarkable increases in the CEA mRNA. These enhancements increased in the 10-Gy and 15-Gy irradiated populations compared with the 5-Gy irradiated population. These results suggest that the enhancement of expression of CEA by radiation takes place at the CEA gene expression (mRNA) level but not at the protein level.

Differential expression of the Escherichia coli autoaggregation factor antigen 43

DEFF Research Database (Denmark)

Schembri, Mark; Hjerrild, Louise; Gjermansen, Morten

2003-01-01

Antigen 43 (Ag43) is a self-recognizing surface adhesin found in most Escherichia coli strains. Due to its excellent cell-to-cell aggregation characteristics, Ag43 expression confers clumping and fluffing of cells and promotes biofilm formation. Ag43 expression is repressed by the cellular redox...

Effect of radiation on the expression of tumor-associated antigens of human lung adenocarcinoma cells. Immunological study using monoclonal antibodies

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Hareyama, Masato

1988-12-01

We studied the effects of irradiation on the expression of a tumor-associated antigen (YH206 antigen) of cultured human lung adenocarcinoma A549 cells by using enzyme-linked immunosorbent assay (ELISA) and flow cytometry. YH206 antigen is preferentially expressed on adenocarcinoma cells. Irradiation of A549 cells remarkably increased the expression of YH206 antigen on the cell surface and the level of the antigen in the culture supernatant as well as in the cell lysate, whereas it significantly affected the expression of HLA (MHC-class I) antigen on the same cells. The expression of HLA antigen on the cell was also increased after treatment of the cells with interferon-..gamma... In an additional experiment, cells were stained simultaneously for surface antigens (fluorescein coupled antibodies) and for DNA content (propidium iodide), and then dual parameter measurements were performed by flow cytometry to analyse the relationship between antigen levels and the cell cycle. YH206 antigen and HLA antigen increased more in the S and G/sub 2//M phases of the cell cycle than in G/sub 0//G/sub 1/. The expression of YH206 antigen was enhanced in the S and G/sub 2//M phases by irradiation, whereas the expression of HLA antigen was enhanced in each phase of the cell cycle with irradiation or IFN. These results suggest that irradiation plays a key role in the change of the expression of certain tumor-associated antigens.

Unpolarized release of vaccinia virus and HIV antigen by colchicine treatment enhances intranasal HIV antigen expression and mucosal humoral responses.

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Yan Zhang

Full Text Available The induction of a strong mucosal immune response is essential to building successful HIV vaccines. Highly attenuated recombinant HIV vaccinia virus can be administered mucosally, but even high doses of immunization have been found unable to induce strong mucosal antibody responses. In order to solve this problem, we studied the interactions of recombinant HIV vaccinia virus Tiantan strain (rVTT-gagpol in mucosal epithelial cells (specifically Caco-2 cell layers and in BALB/c mice. We evaluated the impact of this virus on HIV antigen delivery and specific immune responses. The results demonstrated that rVTT-gagpol was able to infect Caco-2 cell layers and both the nasal and lung epithelia in BALB/c mice. The progeny viruses and expressed p24 were released mainly from apical surfaces. In BALB/c mice, the infection was limited to the respiratory system and was not observed in the blood. This showed that polarized distribution limited antigen delivery into the whole body and thus limited immune response. To see if this could be improved upon, we stimulated unpolarized budding of the virus and HIV antigens by treating both Caco-2 cells and BALB/c mice with colchicine. We found that, in BALB/c mice, the degree of infection and antigen expression in the epithelia went up. As a result, specific immune responses increased correspondingly. Together, these data suggest that polarized budding limits antigen delivery and immune responses, but unpolarized distribution can increase antigen expression and delivery and thus enhance specific immune responses. This conclusion can be used to optimize mucosal HIV vaccine strategies.

Construction, expression, purification and biotin labeling of a single recombinant multi-epitope antigen for double-antigen sandwich ELISA to detect hepatitis C virus antibody.

Science.gov (United States)

He, Jing; Xiu, Bingshui; Wang, Guohua; Chen, Kun; Feng, Xiaoyan; Song, Xiaoguo; Zhu, Cuixia; Yang, Xiqin; Bai, Guanzhong; Ling, Shigan; Zhang, Heqiu

2011-08-01

Based on B cell epitope predictions, a recombinant antigen with multiple epitopes from four Hepatitis C Virus fragments (C, NS3, NS4 and NS5) were engineered. The recombinant gene was then highly expressed in E. coli. The non-modified and C-terminal-modified recombinant proteins were used for coating and biotin labeling, respectively, to establish the double-antigen sandwich ELISA. Ten positive reference samples confirmed by the CHIRON RIBA HCV 3.0 SIA kit were detected positive, Forty one plasma samples were positive among samples from 441 volunteers, which indicated that the recombinant antigen could readily react well with plasma HCV antibody. As critical reagents of double-antigen sandwich ELISA, the recombinant multi-epitope antigen and the C-terminal-modified and biotin-conjugated antigen show good antigenicity. In this study, we provide a simple approach to produce multiple epitopes within one recombinant protein in order to avoid the costly expression of less-effective pools of multiple proteins, which is the conventional strategy of diagnostic antigen production for HCV antibody detection.

Expression of Lewisa, Sialyl Lewisa, Lewisx, Sialyl Lewisx, Antigens as Prognostic Factors in Patients with Colorectal Cancer

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Tohru Nakagoe

2000-01-01

Full Text Available BACKGROUND: Altered expression of blood group-related carbohydrate antigens such as sialyl Lewis (Lex antigen in tumours is associated with tumour progression behaviour and subsequent prognosis. However, the prognostic value of the expression of Le-related antigens in colorectal tumours remains unclear.

Epstein-Barr virus nuclear antigen 2 specifically induces expression of the B-cell activation antigen CD23

International Nuclear Information System (INIS)

Wang, F.; Gregory, C.D.; Rowe, M.; Rickinson, A.B.; Wang, D.; Birkenbach, M.; Kikutani, H.; Kishimoto, T.; Kieff, E.

1987-01-01

Epstein-Barr virus (EBV) infection of EBV-negative Burkitt lymphoma (BL) cells includes some changes similar to those seen in normal B lymphocytes that have been growth transformed by EBV. The role of individual EBV genes in this process was evaluated by introducing each of the viral genes that are normally expressed in EBV growth-transformed and latently infected lymphoblasts into an EBV-negative BL cell line, using recombinant retrovirus-mediated transfer. Clones of cells were derived that stably express the EBV nuclear antigen 1 (EBNA-1), EBNA-2, EBNA-3, EBNA-leader protein, or EBV latent membrane protein (LMP). These were compared with control clones infected with the retrovirus vector. All 10 clones converted to EBNA-2 expression differed from control clones or clones expressing other EBV proteins by growth in tight clumps and by markedly increased expression of one particular surface marker of B-cell activation, CD23. Other activation antigens were unaffected by EBNA-2 expression, as were markers already expressed on the parent BL cell line. The results indicate that EBNA-2 is a specific direct or indirect trans-activator of CD23. This establishes a link between an EBV gene and cell gene expression. Since CD23 has been implicated in the transduction of B-cell growth signals, its specific induction by EBNA-2 could be important in EBV induction of B-lymphocyte transformation

Effects of radiation on the expression of antigens on the membranes of human adenocarcinoma cells

International Nuclear Information System (INIS)

Hareyama, Masato; Imai, Kozo; Oouchi, Atsushi; Shidou, Mitsuo; Takahashi, Hiroki; Koshiba, Hirohumi; Yachi, Akira; Morita, Kazuo

1992-01-01

We have investigated the effects of irradiation on the membranes of tumor cells. X-ray irradiation caused remarkable increases in the expression of tumor-associated antigens (YH206, CEA) and c-erbB-2 protein by flow cytometry, whereas IFN had no obvious effect on the expression of tumor-associated antigens and c-erbB-2 protein. On the other hand, the expression of MHC Class I and ICAM-1 antigen on the membrane by flow cytometry was enhanced by both irradiation and IFN. In addition, the irradiated cells, when analyzed using a CEA specific probe, showed remarkable increases in the CEA mRNA compared to IFN-treated cells. It is possible that enhancement of the expression of tumor-associated antigens and c-erbB-2 protein, together with the enhancement of that of MHC-Class I and ICAM-1, would help cytotoxic killer cells recognize the tumor cells. (author)

Neural Crest Cells Isolated from the Bone Marrow of Transgenic Mice Express JCV T-Antigen.

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Jennifer Gordon

Full Text Available JC virus (JCV, a common human polyomavirus, is the etiological agent of the demyelinating disease, progressive multifocal leukoencephalopathy (PML. In addition to its role in PML, studies have demonstrated the transforming ability of the JCV early protein, T-antigen, and its association with some human cancers. JCV infection occurs in childhood and latent virus is thought to be maintained within the bone marrow, which harbors cells of hematopoietic and non-hematopoietic lineages. Here we show that non-hematopoietic mesenchymal stem cells (MSCs isolated from the bone marrow of JCV T-antigen transgenic mice give rise to JCV T-antigen positive cells when cultured under neural conditions. JCV T-antigen positive cells exhibited neural crest characteristics and demonstrated p75, SOX-10 and nestin positivity. When cultured in conditions typical for mesenchymal cells, a population of T-antigen negative cells, which did not express neural crest markers arose from the MSCs. JCV T-antigen positive cells could be cultured long-term while maintaining their neural crest characteristics. When these cells were induced to differentiate into neural crest derivatives, JCV T-antigen was downregulated in cells differentiating into bone and maintained in glial cells expressing GFAP and S100. We conclude that JCV T-antigen can be stably expressed within a fraction of bone marrow cells differentiating along the neural crest/glial lineage when cultured in vitro. These findings identify a cell population within the bone marrow permissible for JCV early gene expression suggesting the possibility that these cells could support persistent viral infection and thus provide clues toward understanding the role of the bone marrow in JCV latency and reactivation. Further, our data provides an excellent experimental model system for studying the cell-type specificity of JCV T-antigen expression, the role of bone marrow-derived stem cells in the pathogenesis of JCV-related diseases

Synthesis and expression of CDw75 antigen in human colorectal cancer

International Nuclear Information System (INIS)

Costa-Nogueira, Clotilde; Villar-Portela, Susana; Cuevas, Elisa; Gil-Martín, Emilio; Fernández-Briera, Almudena

2009-01-01

Increased ST6Gal I activity has been associated with the α(2,6)sialylation enhancement of membrane glycoconjugates observed in metastatic colorectal carcinomas (CRC). Siaα(2,6)Galβ(1,4)GlcNAc sequence, known as CDw75, is a sialylated carbohydrate determinant generated by the ST6Gal I. This epitope has been reported to be associated with the progression of gastric and colorectal tumours, hence there are only a few conclusive studies to date. By radioisotopic techniques we evaluated the ST6Gal I activity in healthy, transitional and tumour tissues from 43 patients with CRC. By immunohistochemistry we assessed the CDw75 expression in 25 colorectal adenomas, 43 tumours, 13 transitional and 28 healthy tissues of CRC patients. ST6Gal I activity was likewise found to be statistically higher in tumour tissue respect to healthy tissue from CRC patients. CDw75 expression was positive in 20% of colorectal adenomas. Furthermore, 70% of tumour specimens and 8.3% of transitional specimens were positive for CDw75 expression, whereas none of the healthy ones showed the presence of the epitope. The major contribution of this study is the inclusion of data from transitional tissue and the analysis of CDw75 antigen expression in CRC and in colorectal adenomas, little known so far. ST6Gal I activity and CDw75 antigen expression were increased in CRC. Although their comparison did not reach the statistical significance, a great extent of patients showed both, an enhanced tumour ST6Gal I activity and an increased CDw75 expression in the tumour tissue. So, these two variables may play a role in malignant transformation. The expression of CDw75 in colorectal adenomas suggests that this antigen may be a tumour marker in CRC

Stabilization of Transfected Cells Expressing Low-Incidence Blood Group Antigens: Novel Methods Facilitating Their Use as Reagent-Cells.

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Cecilia González

Full Text Available The identification of erythrocyte antibodies in the serum of patients rely on panels of human red blood cells (RBCs, which coexpress many antigens and are not easily available for low-incidence blood group phenotypes. These problems have been addressed by generating cell lines expressing unique blood group antigens, which may be used as an alternative to human RBCs. However, the use of cell lines implies several drawbacks, like the requirement of cell culture facilities and the high cost of cryopreservation. The application of cell stabilization methods could facilitate their use as reagent cells in clinical laboratories.We generated stably-transfected cells expressing low-incidence blood group antigens (Dia and Lua. High-expresser clones were used to assess the effect of TransFix® treatment and lyophilization as cell preservation methods. Cells were kept at 4°C and cell morphology, membrane permeability and antigenic properties were evaluated at several time-points after treatment.TransFix® addition to cell suspensions allows cell stabilization and proper antigen detection for at least 120 days, despite an increase in membrane permeability and a reduction in antigen expression levels. Lyophilized cells showed minor morphological changes and antigen expression levels were rather conserved at days 1, 15 and 120, indicating a high stability of the freeze-dried product. These stabilized cells have been proved to react specifically with human sera containing alloantibodies.Both stabilization methods allow long-term preservation of the transfected cells antigenic properties and may facilitate their distribution and use as reagent-cells expressing low-incidence antigens, overcoming the limited availability of such rare RBCs.

Simple mucin-type carbohydrate antigens in pleomorphic adenomas

DEFF Research Database (Denmark)

Therkildsen, M H; Mandel, U; Christensen, M

1993-01-01

Simple mucin-type carbohydrate structures, T, Tn and sialosyl-Tn, are regarded as general markers of carcinomas in several epithelial tissues as a result of incomplete synthesis with precursor accumulation. The structures have a very limited distribution in normal tissues and secretions, including...... saliva and salivary glands. The expression of simple mucin-type carbohydrate structures and ABH(O) variants was studied in paraffin-embedded and frozen tissue sections from 37 pleomorphic adenomas with associated normal parotid tissue, using immunohistology and a panel of MAbs with well...

Blood group antigen A type 3 expression is a favorable prognostic factor in advanced NSCLC.

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Schmidt, L H; Kuemmel, A; Schliemann, C; Schulze, A; Humberg, J; Mohr, M; Görlich, D; Hartmann, W; Bröckling, S; Marra, A; Hillejan, L; Goletz, S; Karsten, U; Berdel, W E; Spieker, T; Wiewrodt, R

2016-02-01

Several blood group-related carbohydrate antigens are prognosis-relevant markers of tumor tissues. A type 3 (repetitive A) is a blood group antigen specific for A1 erythrocytes. Its potential expression in tumor tissues has so far not been examined. We have evaluated its expression in normal lung and in lung cancer using a novel antibody (A69-A/E8). For comparison an anti-A antibody specific to A types 1 and 2 was used, because its expression on lung cancer tissue has been previously reported to be of prognostic relevance. Resected tissue samples of 398 NSCLC patients were analyzed in immunohistochemistry using tissue microarrays. Expression of A type 3 was not observed in non-malignant lung tissues. A type 3 was expressed on tumor cells of around half of NSCLC patients of blood group A1 (ptype 1/2 antigen was observed (p=0.562), the expression of A type 3 by tumor cells indicated a highly significant favorable prognosis among advanced NSCLC patients (p=0.011) and in NSCLC patients with lymphatic spread (p=0.014). Univariate prognostic results were confirmed in a Cox proportional hazards model. In this study we present for the first time prognostic data for A type 3 antigen expression in lung cancer patients. Prospective studies should be performed to confirm the prognostic value of A type 3 expression for an improved risk stratification in NSCLC patients. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Expression cloning of camelid nanobodies specific for Xenopus embryonic antigens.

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Keiji Itoh

Full Text Available Developmental biology relies heavily on the use of conventional antibodies, but their production and maintenance involves significant effort. Here we use an expression cloning approach to identify variable regions of llama single domain antibodies (known as nanobodies, which recognize specific embryonic antigens. A nanobody cDNA library was prepared from lymphocytes of a llama immunized with Xenopus embryo lysates. Pools of bacterially expressed cDNAs were sib-selected for the ability to produce specific staining patterns in gastrula embryos. Three different nanobodies were isolated: NbP1 and NbP3 stained yolk granules, while the reactivity of NbP7 was predominantly restricted to the cytoplasm and the cortex. The isolated nanobodies recognized specific protein bands in immunoblot analysis. A reverse proteomic approach identified NbP1 target antigen as EP45/Seryp, a serine protease inhibitor. Given the unique stability of nanobodies and the ease of their expression in diverse systems, we propose that nanobody cDNA libraries represent a promising resource for molecular markers for developmental biology.

Age-Associated Decline in Thymic B Cell Expression of Aire and Aire-Dependent Self-Antigens

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Sergio Cepeda

2018-01-01

Full Text Available Although autoimmune disorders are a significant source of morbidity and mortality in older individuals, the mechanisms governing age-associated increases in susceptibility remain incompletely understood. Central T cell tolerance is mediated through presentation of self-antigens by cells constituting the thymic microenvironment, including epithelial cells, dendritic cells, and B cells. Medullary thymic epithelial cells (mTECs and B cells express distinct cohorts of self-antigens, including tissue-restricted self-antigens (TRAs, such that developing T cells are tolerized to antigens from peripheral tissues. We find that expression of the TRA transcriptional regulator Aire, as well as Aire-dependent genes, declines with age in thymic B cells in mice and humans and that cell-intrinsic and cell-extrinsic mechanisms contribute to the diminished capacity of peripheral B cells to express Aire within the thymus. Our findings indicate that aging may diminish the ability of thymic B cells to tolerize T cells, revealing a potential mechanistic link between aging and autoimmunity.

Expression of the Gastrin-Releasing Peptide Receptor, the Prostate Stem Cell Antigen and the Prostate-Specific Membrane Antigen in Lymph Node and Bone Metastases of Prostate Cancer

NARCIS (Netherlands)

Ananias, Hildo J. K.; van den Heuvel, Marius C.; Helfrich, Wijnand; de Jong, Igle J.

2009-01-01

OBJECTIVE. Cell membrane antigens like the gastrin-releasing peptide receptor (GRPR), the prostate stem cell antigen (PSCA), and the prostate-specific membrane antigen (PSMA), expressed in prostate cancer, are attractive targets for new therapeutic and diagnostic applications. Therefore, we

Heterologous expression of antigenic peptides in Bacillus subtilis biofilms.

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Vogt, Cédric M; Schraner, Elisabeth M; Aguilar, Claudio; Eichwald, Catherine

2016-08-11

Numerous strategies have been developed for the display of heterologous proteins in the surface of live bacterial carriers, which can be used as vaccines, immune-modulators, cancer therapy or bioremediation. Bacterial biofilms have emerged as an interesting approach for the expression of proteins of interest. Bacillus subtilis is a well-described, endospore-forming organism that is able to form biofilms and also used as a probiotic, thus making it a suitable candidate for the display of heterologous proteins within the biofilm. Here, we describe the use of TasA, an important structural component of the biofilms formed by B. subtilis, as a genetic tool for the display of heterologous proteins. We first engineered the fusion protein TasA-mCherry and showed that was widely deployed within the B. subtilis biofilms. A significant enhancement of the expression of TasA-mCherry within the biofilm was obtained when depleting both tasA and sinR genes. We subsequently engineered fusion proteins of TasA to antigenic peptides of the E. granulosus parasite, paramyosin and tropomyosin. Our results show that the antigens were well expressed within the biofilm as denoted by macrostructure complementation and by the detection of the fusion protein in both immunoblot and immunohistochemistry. In addition, we show that the recombinant endospores of B. subtilis preserve their biophysical and morphological properties. In this work we provide strong evidence pointing that TasA is a suitable candidate for the display of heterologous peptides, such as antigens, cytokines, enzymes or antibodies, in the B. subtilis biofilms. Finally, our data portray that the recombinant endospores preserve their morphological and biophysical properties and could be an excellent tool to facilitate the transport and the administration.

Limited transplantation of antigen-expressing hematopoietic stem cells induces long-lasting cytotoxic T cell responses.

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Warren L Denning

2011-02-01

Full Text Available Harnessing the ability of cytotoxic T lymphocytes (CTLs to recognize and eradicate tumor or pathogen-infected cells is a critical goal of modern immune-based therapies. Although multiple immunization strategies efficiently induce high levels of antigen-specific CTLs, the initial increase is typically followed by a rapid contraction phase resulting in a sharp decline in the frequency of functional CTLs. We describe a novel approach to immunotherapy based on a transplantation of low numbers of antigen-expressing hematopoietic stem cells (HSCs following nonmyeloablative or partially myeloablative conditioning. Continuous antigen presentation by a limited number of differentiated transgenic hematopoietic cells results in an induction and prolonged maintenance of fully functional effector T cell responses in a mouse model. Recipient animals display high levels of antigen-specific CTLs four months following transplantation in contrast to dendritic cell-immunized animals in which the response typically declines at 4-6 weeks post-immunization. Majority of HSC-induced antigen-specific CD8+ T cells display central memory phenotype, efficiently kill target cells in vivo, and protect recipients against tumor growth in a preventive setting. Furthermore, we confirm previously published observation that high level engraftment of antigen-expressing HSCs following myeloablative conditioning results in tolerance and an absence of specific cytotoxic activity in vivo. In conclusion, the data presented here supports potential application of immunization by limited transplantation of antigen-expressing HSCs for the prevention and treatment of cancer and therapeutic immunization of chronic infectious diseases such as HIV-1/AIDS.

Role of ABO secretor status in mucosal innate immunity and H. pylori infection.

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Sara Lindén

2008-01-01

Full Text Available The fucosylated ABH antigens, which constitute the molecular basis for the ABO blood group system, are also expressed in salivary secretions and gastrointestinal epithelia in individuals of positive secretor status; however, the biological function of the ABO blood group system is unknown. Gastric mucosa biopsies of 41 Rhesus monkeys originating from Southern Asia were analyzed by immunohistochemistry. A majority of these animals were found to be of blood group B and weak-secretor phenotype (i.e., expressing both Lewis a and Lewis b antigens, which are also common in South Asian human populations. A selected group of ten monkeys was inoculated with Helicobacter pylori and studied for changes in gastric mucosal glycosylation during a 10-month period. We observed a loss in mucosal fucosylation and concurrent induction and time-dependent dynamics in gastric mucosal sialylation (carbohydrate marker of inflammation, which affect H. pylori adhesion targets and thus modulate host-bacterial interactions. Of particular relevance, gastric mucosal density of H. pylori, gastritis, and sialylation were all higher in secretor individuals compared to weak-secretors, the latter being apparently "protected." These results demonstrate that the secretor status plays an intrinsic role in resistance to H. pylori infection and suggest that the fucosylated secretor ABH antigens constitute interactive members of the human and primate mucosal innate immune system.

Evaluation of Antigen-Conjugated Fluorescent Beads to Identify Antigen-Specific B Cells

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Isabel Correa

2018-03-01

Full Text Available Selection of single antigen-specific B cells to identify their expressed antibodies is of considerable interest for evaluating human immune responses. Here, we present a method to identify single antibody-expressing cells using antigen-conjugated fluorescent beads. To establish this, we selected Folate Receptor alpha (FRα as a model antigen and a mouse B cell line, expressing both the soluble and the membrane-bound forms of a human/mouse chimeric antibody (MOv18 IgG1 specific for FRα, as test antibody-expressing cells. Beads were conjugated to FRα using streptavidin/avidin-biotin bridges and used to select single cells expressing the membrane-bound form of anti-FRα. Bead-bound cells were single cell-sorted and processed for single cell RNA retrotranscription and PCR to isolate antibody heavy and light chain variable regions. Variable regions were then cloned and expressed as human IgG1/k antibodies. Like the original clone, engineered antibodies from single cells recognized native FRα. To evaluate whether antigen-coated beads could identify specific antibody-expressing cells in mixed immune cell populations, human peripheral blood mononuclear cells (PBMCs were spiked with test antibody-expressing cells. Antigen-specific cells could comprise up to 75% of cells selected with antigen-conjugated beads when the frequency of the antigen-positive cells was 1:100 or higher. In PBMC pools, beads conjugated to recombinant antigens FRα and HER2 bound antigen-specific anti-FRα MOv18 and anti-HER2 Trastuzumab antibody-expressing cells, respectively. From melanoma patient-derived B cells selected with melanoma cell line-derived protein-coated fluorescent beads, we generated a monoclonal antibody that recognized melanoma antigen-coated beads. This approach may be further developed to facilitate analysis of B cells and their antibody profiles at the single cell level and to help unravel humoral immune repertoires.

Evaluation of Antigen-Conjugated Fluorescent Beads to Identify Antigen-Specific B Cells.

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Correa, Isabel; Ilieva, Kristina M; Crescioli, Silvia; Lombardi, Sara; Figini, Mariangela; Cheung, Anthony; Spicer, James F; Tutt, Andrew N J; Nestle, Frank O; Karagiannis, Panagiotis; Lacy, Katie E; Karagiannis, Sophia N

2018-01-01

Selection of single antigen-specific B cells to identify their expressed antibodies is of considerable interest for evaluating human immune responses. Here, we present a method to identify single antibody-expressing cells using antigen-conjugated fluorescent beads. To establish this, we selected Folate Receptor alpha (FRα) as a model antigen and a mouse B cell line, expressing both the soluble and the membrane-bound forms of a human/mouse chimeric antibody (MOv18 IgG1) specific for FRα, as test antibody-expressing cells. Beads were conjugated to FRα using streptavidin/avidin-biotin bridges and used to select single cells expressing the membrane-bound form of anti-FRα. Bead-bound cells were single cell-sorted and processed for single cell RNA retrotranscription and PCR to isolate antibody heavy and light chain variable regions. Variable regions were then cloned and expressed as human IgG1/k antibodies. Like the original clone, engineered antibodies from single cells recognized native FRα. To evaluate whether antigen-coated beads could identify specific antibody-expressing cells in mixed immune cell populations, human peripheral blood mononuclear cells (PBMCs) were spiked with test antibody-expressing cells. Antigen-specific cells could comprise up to 75% of cells selected with antigen-conjugated beads when the frequency of the antigen-positive cells was 1:100 or higher. In PBMC pools, beads conjugated to recombinant antigens FRα and HER2 bound antigen-specific anti-FRα MOv18 and anti-HER2 Trastuzumab antibody-expressing cells, respectively. From melanoma patient-derived B cells selected with melanoma cell line-derived protein-coated fluorescent beads, we generated a monoclonal antibody that recognized melanoma antigen-coated beads. This approach may be further developed to facilitate analysis of B cells and their antibody profiles at the single cell level and to help unravel humoral immune repertoires.

Evaluation of Antigen-Conjugated Fluorescent Beads to Identify Antigen-Specific B Cells

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Correa, Isabel; Ilieva, Kristina M.; Crescioli, Silvia; Lombardi, Sara; Figini, Mariangela; Cheung, Anthony; Spicer, James F.; Tutt, Andrew N. J.; Nestle, Frank O.; Karagiannis, Panagiotis; Lacy, Katie E.; Karagiannis, Sophia N.

2018-01-01

Selection of single antigen-specific B cells to identify their expressed antibodies is of considerable interest for evaluating human immune responses. Here, we present a method to identify single antibody-expressing cells using antigen-conjugated fluorescent beads. To establish this, we selected Folate Receptor alpha (FRα) as a model antigen and a mouse B cell line, expressing both the soluble and the membrane-bound forms of a human/mouse chimeric antibody (MOv18 IgG1) specific for FRα, as test antibody-expressing cells. Beads were conjugated to FRα using streptavidin/avidin-biotin bridges and used to select single cells expressing the membrane-bound form of anti-FRα. Bead-bound cells were single cell-sorted and processed for single cell RNA retrotranscription and PCR to isolate antibody heavy and light chain variable regions. Variable regions were then cloned and expressed as human IgG1/k antibodies. Like the original clone, engineered antibodies from single cells recognized native FRα. To evaluate whether antigen-coated beads could identify specific antibody-expressing cells in mixed immune cell populations, human peripheral blood mononuclear cells (PBMCs) were spiked with test antibody-expressing cells. Antigen-specific cells could comprise up to 75% of cells selected with antigen-conjugated beads when the frequency of the antigen-positive cells was 1:100 or higher. In PBMC pools, beads conjugated to recombinant antigens FRα and HER2 bound antigen-specific anti-FRα MOv18 and anti-HER2 Trastuzumab antibody-expressing cells, respectively. From melanoma patient-derived B cells selected with melanoma cell line-derived protein-coated fluorescent beads, we generated a monoclonal antibody that recognized melanoma antigen-coated beads. This approach may be further developed to facilitate analysis of B cells and their antibody profiles at the single cell level and to help unravel humoral immune repertoires. PMID:29628923

Extracellular Expression in Aspergillus niger of an Antibody Fused to Leishmania sp. Antigens.

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Magaña-Ortíz, Denis; Fernández, Francisco; Loske, Achim M; Gómez-Lim, Miguel A

2018-01-01

Nucleoside hydrolase and sterol 24-c-methyltransferase, two antigenic proteins of Leishmania sp., were expressed in Aspergillus niger. Genetic transformation of conidia was achieved using underwater shock waves. scFv antibody addressed to DEC205, a receptor of dendritic cells, was fused to two proteins of Leishmania sp. Receptor 205 has a relevant role in the immune system in mammals; it can modulate T cell response to different antigens. Extracellular expression strategy of recombinant antibody was achieved using a fragment of native glucoamylase A (514 aa) as a carrier. Fermentations in shake flasks showed that the recombinant protein (104 kDa) was expressed and secreted only when maltose was used as carbon source; on the contrary, the expression was highly repressed in presence of xylose. Noteworthy, recombinant protein was secreted without glucoamylase-carrier and accumulation at intracellular level was not observed. The results presented here demonstrate the high value of Aspergillus niger as biotechnological platform for recombinant antibodies against Leishmania sp. at low cost. To the best of our knowledge, this is the first report about the recombinant expression of antigenic proteins of Leishmania sp. in filamentous fungi. The protein obtained can be used to explore novel strategies to induce immunity against Leishmania sp. or it can be employed in diagnostic kits to detect this neglected disease.

[Effect of gene optimization on the expression and purification of HDV small antigen produced by genetic engineering].

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Ding, Jun-Ying; Meng, Qing-Ling; Guo, Min-Zhuo; Yi, Yao; Su, Qiu-Dong; Lu, Xue-Xin; Qiu, Feng; Bi, Sheng-Li

2012-10-01

To study the effect of gene optimization on the expression and purification of HDV small antigen produced by genetic engineering. Based on the colon preference of E. coli, the HDV small antigen original gene from GenBank was optimized. Both the original gene and the optimized gene expressed in prokaryotic cells, SDS-PAGE was made to analyze the protein expression yield and to decide which protein expression style was more proportion than the other. Furthermore, two antigens were purified by chromatography in order to compare the purity by SDS-PAGE and Image Lab software. SDS-PAGE indicated that the molecular weight of target proteins from two groups were the same as we expected. Gene optimization resulted in the higher yield and it could make the product more soluble. After chromatography, the purity of target protein from optimized gene was up to 96.3%, obviously purer than that from original gene. Gene optimization could increase the protein expression yield and solubility of genetic engineering HDV small antigen. In addition, the product from the optimized gene group was easier to be purified for diagnosis usage.

Enhanced Expression of Interferon-γ-Induced Antigen-Processing Machinery Components in a Spontaneously Occurring Cancer

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Fulvia Cerruti

2007-11-01

Full Text Available In human tumors, changes in the surface expression and/or function of major histocompatibility complex (MHC class I antigens are frequently found and may provide malignant cells with a mechanism to escape control of the immune system. This altered human lymphocyte antigen (HLA class I phenotype can be caused by either structural alterations or dysregulation of genes encoding subunits of HLA class I antigens and/or components of the MHC class I antigen-processing machinery (APM. Herein we analyze the expression of several proteins involved in the generation of MHC class I epitopes in feline injection site sarcoma, a spontaneously occurring tumor in cats that is an informative model for the study of tumor biology in other species, including humans. Eighteen surgically removed primary fibrosarcoma lesions were analyzed, and an enhanced expression of two catalytic subunits of immunoproteasomes, PA28 and leucine aminopeptidase, was found in tumors compared to matched normal tissues. As a functional counterpart of these changes in protein levels, proteasomal activities were increased in tissue extracts from fibrosarcomas. Taken together, these results suggest that alterations in the APM system may account for reduced processing of selected tumor antigens and may potentially provide neoplastic fibroblasts with a mechanism for escape from T-cell recognition and destruction.

[Neoretinal antigen expression: a comparison of anatomical and clinical features of a murine uveoretinitis model].

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Terrada, C; Pâques, M; Fisson, S; De Kozak, Y; Klatzmann, D; Salomon, B; LeHoang, P; Bodaghi, B

2008-02-01

Uveitis is an inflammation involving the retina. The antigens targeted by the experimental models are located in the pigmentary epithelium-photoreceptor complex. To gain insights into the variations in topographic expression of the antigen in the retina, we studied a new mouse model. and methods: Stable retinal expression of the influenza virus hemagglutinin (HA) was obtained after intravitreal or subretinal injection of recombinant adeno-associated virus carrying HA (AAV-HA). One month later, we transferred HA-specific T cells, followed by a subcutaneous immunization of the cognate antigen emulsified in CFA. The animals were clinically examined with a slit lamp biomicroscope. Infiltration of donor cells was detected by immunostaining on retina flatmounts with anti-Thy-1.1 antibody, and infiltrating cells were studied using FACS analysis. Whatever the location of the HA expression, intraocular inflammation was clinically and histologically detected in all animals, between 10 and 15 days after immunization with HA. Lesions were identified with histopathological analysis. The ocular infiltrate was mostly composed of macrophages and HA-specific T cells in different proportions. The topographic variations of targeted ocular antigens do not seem to modify the development of inflammatory reactions in our model. By targeting different antigen-presenting cells, ocular infiltrating cells are different.

Infrequent and low expression of cancer-testis antigens located on the X chromosome in colorectal cancer: implications for immunotherapy in South African populations.

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Dakshinamurthy, Amirtha Ganesh; Ramesar, Rajkumar; Goldberg, Paul; Blackburn, Jonathan M

2008-11-01

Cancer-testis (CT) antigens are a group of tumor antigens that are expressed in the testis and aberrantly in cancerous tissue but not in somatic tissues. The testis is an immune-privileged site because of the presence of a blood-testis barrier; as a result, CT antigens are considered to be essentially tumor specific and are attractive targets for immunotherapy. CT antigens are classified as the CT-X and the non-X CT antigens depending on the chromosomal location to which the genes are mapped. CT-X antigens are typically highly immunogenic and hence the first step towards tailored immunotherapy is to elucidate the expression profile of CT-X antigens in the respective tumors. In this study we investigated the expression profile of 16 CT-X antigen genes in 34 colorectal cancer (CRC) patients using reverse transcription-polymerase chain reaction. We observed that 12 of the 16 CT-X antigen genes studied did not show expression in any of the CRC samples analyzed. The other 4 CT-X antigen genes showed low frequency of expression and exhibited a highly variable expression profile when compared to other populations. Thus, our study forms the first report on the expression profile of CT-X antigen genes among CRC patients in the genetically diverse South African population. The results of our study suggest that genetic and ethnic variations in population might have a role in the expression of the CT-X antigen genes. Thus our results have significant implications for anti-CT antigen-based immunotherapy trials in this population.

Analysis of GAGE, NY-ESO-1 and SP17 cancer/testis antigen expression in early stage non-small cell lung carcinoma

International Nuclear Information System (INIS)

Gjerstorff, Morten F; Pøhl, Mette; Olsen, Karen E; Ditzel, Henrik J

2013-01-01

The unique expression pattern and immunogenic properties of cancer/testis antigens make them ideal targets for immunotherapy of cancer. The MAGE-A3 cancer/testis antigen is frequently expressed in non-small cell lung cancer (NSCLC) and vaccination with MAGE-A3 in patients with MAGE-A3-positive NSCLC has shown promising results. However, little is known about the expression of other cancer/testis antigens in NSCLC. In the present study the expression of cancer/testis antigens GAGE, NY-ESO-1 and SP17 was investigated in patients with completely resected, early stage, primary NSCLC. Tumor biopsies from normal lung tissue and from a large cohort (n = 169) of NSCLC patients were examined for GAGE, NY-ESO-1 and SP17 protein expression by immunohistochemical analysis. The expression of these antigens was further matched to clinical and pathological features using univariate cox regression analysis. GAGE and NY-ESO-1 cancer/testis antigens were not expressed in normal lung tissue, while SP17 was expressed in ciliated lung epithelia. The frequency of GAGE, NY-ESO-1 and SP17 expression in NSCLC tumors were 26.0% (44/169), 11.8% (20/169) and 4.7% (8/169), respectively, and 33.1% (56/169) of the tumors expressed at least one of these antigens. In general, the expression of GAGE, NY-ESO-1 and SP17 was not significantly associated with a specific histotype (adenocarcinoma vs. squamous cell carcinoma), but high-level GAGE expression (>50%) was more frequent in squamous cell carcinoma (p = 0.02). Furthermore, the frequency of GAGE expression was demonstrated to be significantly higher in stage II-IIIa than stage I NSCLC (17.0% vs. 35.8%; p = 0.02). Analysis of the relation between tumor expression of GAGE and NY-ESO-1 and survival endpoints revealed no significant associations. Our study demonstrates that GAGE, NY-ESO-1 and SP17 cancer/testis antigens are candidate targets for immunotherapy of NSCLC and further suggest that multi-antigen vaccines may be beneficial

Expression of Tac antigen component of bovine interleukin-2 receptor in different leukocyte populations infected with Theileria parva or Theileria annulata.

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Dobbelaere, D A; Prospero, T D; Roditi, I J; Kelke, C; Baumann, I; Eichhorn, M; Williams, R O; Ahmed, J S; Baldwin, C L; Clevers, H

1990-01-01

The Tac antigen component of the bovine interleukin-2 receptor was expressed as a Cro-beta-galactosidase fusion protein in Escherichia coli and used to raise antibodies in rabbits. These antibodies were used for flow cytofluorimetric analysis to investigate the expression of Tac antigen in a variety of Theileria parva-infected cell lines and also in three Theileria annulata-infected cell lines. Cells expressing Tac antigen on their surface were found in all T. parva-infected cell lines tested whether these were of T- or B-cell origin. T cells expressing Tac antigen could be CD4- CD8-, CD4+ CD8-, CD4- CD8+, or CD4+ CD8+. Tac antigen expression was observed both in cultures which had been maintained in the laboratory for several years and in transformed cell lines which had recently been established by infection of lymphocytes in vitro with T. parva. Northern (RNA) blot analysis demonstrated Tac antigen transcripts in RNA isolated from all T. parva-infected cell lines. Three T. annulata-infected cell lines which were not of T-cell origin were also tested. Two of them expressed Tac antigen on their surface. Abundant Tac antigen mRNA was detected in these T. annulata-infected cell lines, but only trace amounts were demonstrated in the third cell line, which contained very few Tac antigen-expressing cells. In all cell lines tested, whether cloned or uncloned, a proportion of the cells did not express detectable levels of Tac antigen on their surface. This was also the case for a number of other leukocyte surface markers. In addition, we showed that the interleukin-2 receptors were biologically functional, because addition of recombinant interleukin-2 to cultures stimulated cell proliferation. Recombinant interleukin-2 treatment also resulted in increased amounts of steady-state Tac antigen mRNA. The relevance of interleukin-2 receptor expression on Theileria-infected cells is discussed. Images PMID:1979317

The effects of Ostertagia occidentalis somatic antigens on ovine TLR2 and TLR4 expression

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Hassan BORJI

2015-10-01

Full Text Available Background: Recognition of helminth-derived pathogen associated molecular patterns (PAMPs by pattern recognition receptors (PRRs, including toll like recep­tors (TLRs is the first step towards initiating anti–helminth immune re­sponses.Methods: Using somatic antigens of Ostertagia occidentalis, an important abomasal parasite of ruminants, the expression of ovine TLR2 and TLR4 in peripheral blood mononuclear cells (PBMCs was analyzed by real-time quatitative reverse-transcrip­tion polymerase chain reaction (qRT-PCR. Somatic antigens of O. occidentalis were prepared to stimulate ovine PBMCs in a time and dose dependent manner.Results: A high expression of TLR2 and TLR4 was observed in PBMCs cultured with somatic antigens of the parasites specially when PBMCs were cultured with 100 µg/ml of somatic antigens and incubated for 2h. Up-regulation of TLR2 expres­sion was more pronounced and evident in our study.Conclsusion: Somatic antigens of O. occidentalis have immunostimulatory and domi­nant role on peripheral immune cells. This study provide for the first time evidence of induction of TLRs in ovine PBMCs by somatic antigen of O. occidentalis

T cells expressing VHH-directed oligoclonal chimeric HER2 antigen receptors

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Jamnani, Fatemeh Rahimi; Rahbarizadeh, Fatemeh; Shokrgozar, Mohammad Ali

2014-01-01

Adoptive cell therapy with engineered T cells expressing chimeric antigen receptors (CARs) originated from antibodies is a promising strategy in cancer immunotherapy. Several unsuccessful trials, however, highlight the need for alternative conventional binding domains and the better combination...

[Cloning expression and serological evaluation on Mycobacterium tuberculosis four new antigens].

Science.gov (United States)

Luo, Q; Li, S J; Xiao, T Y; Li, M C; Liu, H C; Lou, Y L; Wan, K L

2018-04-10

Objective: To evaluate the serological diagnostic value of Mycobacterium (M.) tuberculosis four new antigens Rv0432, Rv0674, Rv1566c and Rv1547. Methods: Rv0432, Rv0674, Rv1566c and Rv1547 were amplified from M. tuberculosis strain H37Rv genomic DNA by using PCR, among which Rv1547 was divided into two segments for amplification ( Rv1547-1 and Rv1547-2 ). The segments were cloned into expression vector PET-32a while the recombinant proteins were purified by affinity chromatography. Serums were incubated with BL21 (DE3) proteins. Antibodies IgG against M. tuberculosis were tested with 151 serum samples (41 healthy people and 110 TB patients) by using ELISA. The diagnostic efficiency of antigens was analyzed by means of receiver operating characteristic curve. Difference of the objective proteins in TB patients and healthy controls was compared by t -test. Results: Recombinant antigens Rv0432, Rv0674, Rv1566c, Rv1547-1 and Rv1547-2 were successfully expressed and purified. Results from ELISA showed that the sensitivity, specificity, positive predictive value, negative predictive value, Youden index and area under the curve of Rv0432, Rv0674, Rv1566c, Rv1547-1 and Rv1547-2, as 43.64%-92.73%, 80.49%-92.68%, 0.92-0.94, 0.38-0.80, 0.363-0.732 and 0.649-0.915. All the objective proteins showed significantly higher antibody levels in TB patients, when compared to the healthy controls ( P <0.000 1). Conclusion: The newly identified antigens Rv0432, Rv0674, Rv1566c, Rv1547-1 and Rv1547-2 all performed well when being used for TB serological diagnosis, thus were expected to be new candidate antigens used for TB diagnosis.

Lack of ADAM2, CALR3 and SAGE1 Cancer/Testis Antigen Expression in Lung and Breast Cancer

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Maheswaran, Emeaga; Pedersen, Christina B; Ditzel, Henrik J

2015-01-01

and antigenic properties, but the expression patterns of most of the more than 200 identified cancer/testis antigens in various cancers remain largely uncharacterized. In this study, we investigated the expression of the cancer/testis antigens ADAM2, CALR3 and SAGE1 in lung and breast cancer, the two most...... frequent human cancers, with the purpose of providing novel therapeutic targets for these diseases. We used a set of previously uncharacterized antibodies against the cancer/testis antigens ADAM2, CALR3 and SAGE1 to investigate their expression in a large panel of normal tissues as well as breast and lung...... cancers. Staining for the well-characterized MAGE-A proteins was included for comparison. Immunohistochemical staining confirmed previous mRNA analysis demonstrating that ADAM2, CALR3 and SAGE1 proteins are confined to testis in normal individuals. Negative tissues included plancenta, which express many...

In vivo and in vitro expression of myeloid antigens on B-lineage acute lymphoblastic leukemia cells.

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Hara, J; Kawa-Ha, K; Yumura-Yagi, K; Kurahashi, H; Tawa, A; Ishihara, S; Inoue, M; Murayama, N; Okada, S

1991-01-01

The expression of myeloid antigens has been extensively examined using two-color analysis in 43 children with B-lineage acute lymphoblastic leukemia (ALL). On pre-culture cells, CD33 expression was frequently observed in CD19+, CD10- B-precursor ALL, and CD14 was expressed only on the cells from B-precursor ALL expressing CD19, CD10 and CD20, and B-ALL. After 2 or 3 days of culture without TPA, CD13 emerged on the cells from 21 of 29 patients irrespective of the presence or the absence of fetal calf serum in the culture. Of four patients with CD10+ B-precursor ALL, which showed no expression of CD13 after culture, two had T-cell associated antigens. Whereas the addition of TPA to the culture enhanced the expression of CD13 on the cells from acute non-lymphocytic leukemia (ANLL), TPA reduced the expression of this antigen on B-precursor cells. These findings suggest that the regulatory mechanism of CD13 expression may be different between B-precursor ALL and ANLL. Co-culture with cycloheximide mostly abrogated the induction of CD13, suggesting that CD13 expression was mainly dependent on de novo protein synthesis.

SACCHAROTHRIX SP. ABH26, A NEW ACTINOBACTERIAL STRAIN FROM ALGERIAN SAHARAN SOIL: ISOLATION, IDENTIFICATION AND ANTIMICROBIAL ACTIVITY

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Abdelhadi Lahoum

2015-04-01

Full Text Available A new strain of actinobacteria, designated ABH26, was isolated from a Saharan soil in the Adrar region (Algeria, by the dilution agar plating method using a chitin-vitamins B medium supplemented with polymyxin and penicillin. The morphological studies showed that this strain represents a member of the Saccharothrix genus. Phylogenetic analysis showed that this strain had 16S rRNA gene sequence similarities ranging from 97.63% (with Saccharothrix violaceirubra NBRC 102064T to 99.86% (with Saccharothrix xinjiangensis NBRC 101911T. Furthermore, strain ABH26 presented a strong activity against mycotoxigenic and phytopathogenic fungi including Aspergillus carbonarius (M333, A. flavus (NRRL 3251, A. westerdijkiae (ATCC 3174, Fusarium oxysporum f. sp. lini (Fol and F. solani (Fsol. Additionally, the strain exhibited an important antimicrobial activity against many strains of the pathogenic yeast Candida albicans (M2, M3 and IPA200 and against methicillin resistant Staphylococcus aureus (MRSA 639c. Thus, four solvents (n-hexane, dichloromethane, ethyl acetate and n-butanol were used for the extraction of produced antibiotic compounds. The highest antimicrobial activities were obtained using the butanolic extract. The thin layer chromatography (TLC method showed two bioactive spots, named HAD1 and HAD2, which were reveled negatively by using chemical revelators (ninhydrin, naphtoresorcinol-sulfuric acid, ferrous iron chloride and formaldehyde-sulfuric. These results indicated the absence of amine group, sugar, hydroxamic acid, phenol and aromatic compound.

Characterization of a new Lactobacillus salivarius strain engineered to express IBV multi-epitope antigens by chromosomal integration.

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Ma, Bing-cun; Yang, Xin; Wang, Hong-ning; Cao, Hai-peng; Xu, Peng-wei; Ding, Meng-die; Liu, Hui

2016-01-01

To obtain adhesive and safe lactic acid bacteria (LAB) strains for expressing heterologous antigens, we screened LAB inhabitants in intestine of Tibetan chickens by analyzing their adhesion and safety properties and the selected LAB was engineered to express heterologous antigen (UTEpi C-A) based on chromosomal integration strategy. We demonstrated that a new Lactobacillu salivarius TCMM17 strain is strongly adhesive to chicken intestinal epithelial cells, contains no endogenous plasmids, is susceptible to tested antimicrobials, and shows no toxicities. In order to examine the potential of TCMM17 strain as heterogenous antigen delivering vehicle, we introduced a UTEpi C-A expression cassette in its chromosome by constructing a non-replicative plasmid (pORI280-UUTEpi C-AD). The recombinant TCMM17 strain (∆TCMM17) stably was found to keep the gene cassette through 50 generations, and successfully displayed EpiC encoded by the cassette on its surface. This work provides a universal platform for development of novel oral vaccines and expression of further antigens of avian pathogens.

Enhanced expression of codon optimized Mycobacterium avium subsp. paratuberculosis antigens in Lactobacillus salivarius

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Christopher D Johnston

2014-09-01

Full Text Available It is well documented that open reading frames containing high GC content show poor expression in A+T rich hosts. Specifically, G+C-rich codon usage is a limiting factor in heterologous expression of Mycobacterium avium subsp. paratuberculosis (MAP proteins using Lactobacillus salivarius. However, re-engineering opening reading frames through synonymous substitutions can offset codon bias and greatly enhance MAP protein production in this host. In this report, we demonstrate that codon-usage manipulation of two MAP genes (MAP2121c and MAP3733c can enhance the heterologous expression of two antigens (MMP and MptD respectively, analogous to the form to which they are produced natively by MAP bacilli. When heterologously over-expressed, antigenic determinants were preserved in synthetic MMP proteins as shown by monoclonal antibody mediated ELISA. Moreover, MMP is a membrane protein in MAP, which is also targeted to the cellular surface of recombinant L. salivarius at levels comparable to MAP. Additionally, codon optimised MptD displayed the tendency to associate with the cytoplasmic membrane boundary under confocal microscopy and the intracellularly accumulated protein selectively adhered with the MptD-specific bacteriophage fMptD. This work demonstrates there is potential for L. salivarius as a viable antigen delivery vehicle for MAP, which may provide an effective mucosal vaccine against Johne’s disease.

Ectopic Expression of O Antigen in Bordetella pertussis by a Novel Genomic Integration System.

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Ishigaki, Keisuke; Shinzawa, Naoaki; Nishikawa, Sayaka; Suzuki, Koichiro; Fukui-Miyazaki, Aya; Horiguchi, Yasuhiko

2018-01-01

We describe a novel genome integration system that enables the introduction of DNA fragments as large as 50 kbp into the chromosomes of recipient bacteria. This system, named BPI, comprises a bacterial artificial chromosome vector and phage-derived gene integration machinery. We introduced the wbm locus of Bordetella bronchiseptica , which is required for O antigen biosynthesis, into the chromosome of B. pertussis , which intrinsically lacks O antigen, using the BPI system. After the introduction of the wbm locus, B. pertussis presented an additional substance in the lipooligosaccharide fraction that was specifically recognized by the anti- B. bronchiseptica antibody but not the anti- B. pertussis antibody, indicating that B. pertussis expressed O antigen corresponding to that of B. bronchiseptica . O antigen-expressing B. pertussis was less sensitive to the bactericidal effects of serum and polymyxin B than the isogenic parental strain. In addition, an in vivo competitive infection assay showed that O antigen-expressing B. pertussis dominantly colonized the mouse respiratory tract over the parental strain. These results indicate that the BPI system provides a means to alter the phenotypes of bacteria by introducing large exogenous DNA fragments. IMPORTANCE Some bacterial phenotypes emerge through the cooperative functions of a number of genes residing within a large genetic locus. To transfer the phenotype of one bacterium to another, a means to introduce the large genetic locus into the recipient bacterium is needed. Therefore, we developed a novel system by combining the advantages of a bacterial artificial chromosome vector and phage-derived gene integration machinery. In this study, we succeeded for the first time in introducing a gene locus involved in O antigen biosynthesis of Bordetella bronchiseptica into the chromosome of B. pertussis , which intrinsically lacks O antigen, and using this system we analyzed phenotypic alterations in the resultant

Monoclonal antibodies to murine thrombospondin-1 and thrombospondin-2 reveal differential expression patterns in cancer and low antigen expression in normal tissues

International Nuclear Information System (INIS)

Bujak, Emil; Pretto, Francesca; Ritz, Danilo; Gualandi, Laura; Wulhfard, Sarah; Neri, Dario

2014-01-01

There is a considerable interest for the discovery and characterization of tumor-associated antigens, which may facilitate antibody-based pharmacodelivery strategies. Thrombospondin-1 and thrombospondin-2 are homologous secreted proteins, which have previously been reported to be overexpressed during remodeling typical for wound healing and tumor progression and to possibly play a functional role in cell proliferation, migration and apoptosis. To our knowledge, a complete immunohistochemical characterization of thrombospondins levels in normal rodent tissues has not been reported so far. Using antibody phage technology, we have generated and characterized monoclonal antibodies specific to murine thrombospondin-1 and thrombospondin-2, two antigens which share 62% aminoacid identity. An immunofluorescence analysis revealed that both antigens are virtually undetectable in normal mouse tissues, except for a weak staining of heart tissue by antibodies specific to thrombospondin-1. The analysis also showed that thrombospondin-1 was strongly expressed in 5/7 human tumors xenografted in nude mice, while it was only barely detectable in 3/8 murine tumors grafted in immunocompetent mice. By contrast, a high-affinity antibody to thrombospondin-2 revealed a much lower level of expression of this antigen in cancer specimens. Our analysis resolves ambiguities related to conflicting reports on thrombosponding expression in health and disease. Based on our findings, thrombospondin-1 (and not thrombospondin-2) may be considered as a target for antibody-based pharmacodelivery strategies, in consideration of its low expression in normal tissues and its upregulation in cancer. - Highlights: • High affinity monoclonal antibodies to murine and human TSP1 and 2 were raised. • Both antigens are virtually undetectable in normal mouse tissues. • Strong positivity of human tumor xenografts for TSP1 was detected. • Study revealed much lower level of TSP2 expression in cancer specimens

Monoclonal antibodies to murine thrombospondin-1 and thrombospondin-2 reveal differential expression patterns in cancer and low antigen expression in normal tissues

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Bujak, Emil [Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zürich), Vladimir-Prelog-Weg 2, CH-8093 Zurich (Switzerland); Pretto, Francesca; Ritz, Danilo; Gualandi, Laura; Wulhfard, Sarah [Philochem AG, Libernstrasse 3, CH-8112 Otelfingen (Switzerland); Neri, Dario, E-mail: neri@pharma.ethz.ch [Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zürich), Vladimir-Prelog-Weg 2, CH-8093 Zurich (Switzerland)

2014-09-10

There is a considerable interest for the discovery and characterization of tumor-associated antigens, which may facilitate antibody-based pharmacodelivery strategies. Thrombospondin-1 and thrombospondin-2 are homologous secreted proteins, which have previously been reported to be overexpressed during remodeling typical for wound healing and tumor progression and to possibly play a functional role in cell proliferation, migration and apoptosis. To our knowledge, a complete immunohistochemical characterization of thrombospondins levels in normal rodent tissues has not been reported so far. Using antibody phage technology, we have generated and characterized monoclonal antibodies specific to murine thrombospondin-1 and thrombospondin-2, two antigens which share 62% aminoacid identity. An immunofluorescence analysis revealed that both antigens are virtually undetectable in normal mouse tissues, except for a weak staining of heart tissue by antibodies specific to thrombospondin-1. The analysis also showed that thrombospondin-1 was strongly expressed in 5/7 human tumors xenografted in nude mice, while it was only barely detectable in 3/8 murine tumors grafted in immunocompetent mice. By contrast, a high-affinity antibody to thrombospondin-2 revealed a much lower level of expression of this antigen in cancer specimens. Our analysis resolves ambiguities related to conflicting reports on thrombosponding expression in health and disease. Based on our findings, thrombospondin-1 (and not thrombospondin-2) may be considered as a target for antibody-based pharmacodelivery strategies, in consideration of its low expression in normal tissues and its upregulation in cancer. - Highlights: • High affinity monoclonal antibodies to murine and human TSP1 and 2 were raised. • Both antigens are virtually undetectable in normal mouse tissues. • Strong positivity of human tumor xenografts for TSP1 was detected. • Study revealed much lower level of TSP2 expression in cancer specimens

ABH antigens as recognition sites for the activation of red blood cell anion exchange by the lectin ulex europaeus agglutinin I.

Science.gov (United States)

Engelmann, B

1993-11-01

The blood group antigen H (blood group O) and fucose-specific lectin Ulex europaeus agglutinin I (UEA1) (10 micrograms/ml) was found to increase the rate constant of Cl- efflux into 100 mM Na+ oxalate media by about 40% in erythrocytes taken from antigen H donors. In 100 mM K+ oxalate, 150 mM Na+ pyruvate and in 150 mM Na+ acetate media the lectin elevated the rate constant of Cl- efflux by 20-50%. The acceleration of Cl- efflux by UEA1 was completely blocked by 10 microM 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS) indicating that the effect of the lectin is mediated by the anion exchanger of human erythrocytes (band 3 protein). In antigen A1 erythrocytes no significant stimulation of anion exchange by UEA1 was seen. The activation of Cl- efflux was completely prevented by addition of 1 mM fucose to the medium. These results suggest that the effect of UEA1 is mediated through interaction with the fucose residues of H antigens. Increasing extracellular Ca++ from 0.5 to 5 mM in Na+ pyruvate or Na+ acetate media slightly reduced the acceleration of anion exchange by the lectin. On the other hand, replacing part of extracellular chloride by bicarbonate did not considerably alter the (previously reported) stimulatory effect of UEA1 on red blood cell Ca++ uptake. This suggests that the acceleration of anion exchange and of Ca++ uptake by UEA1, respectively, are mediated by different mechanisms. It is concluded that UEA1 activates anion exchange of human erythrocytes most probably by a direct interaction with H antigens present on extracellular domains of the band 3 protein.

An inducible transgenic mouse model for immune mediated hepatitis showing clearance of antigen expressing hepatocytes by CD8+ T cells.

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Marcin Cebula

Full Text Available The liver has the ability to prime immune responses against neo antigens provided upon infections. However, T cell immunity in liver is uniquely modulated by the complex tolerogenic property of this organ that has to also cope with foreign agents such as endotoxins or food antigens. In this respect, the nature of intrahepatic T cell responses remains to be fully characterized. To gain deeper insight into the mechanisms that regulate the CD8+ T cell responses in the liver, we established a novel OVA_X_CreER(T2 mouse model. Upon tamoxifen administration OVA antigen expression is observed in a fraction of hepatocytes, resulting in a mosaic expression pattern. To elucidate the cross-talk of CD8+ T cells with antigen-expressing hepatocytes, we adoptively transferred K(b/OVA257-264-specific OT-I T cells to OVA_X_CreER(T2 mice or generated triple transgenic OVA_X CreER(T2_X_OT-I mice. OT-I T cells become activated in OVA_X_CreER(T2 mice and induce an acute and transient hepatitis accompanied by liver damage. In OVA_X_CreER(T2_X_OT-I mice, OVA induction triggers an OT-I T cell mediated, fulminant hepatitis resulting in 50% mortality. Surviving mice manifest a long lasting hepatitis, and recover after 9 weeks. In these experimental settings, recovery from hepatitis correlates with a complete loss of OVA expression indicating efficient clearance of the antigen-expressing hepatocytes. Moreover, a relapse of hepatitis can be induced upon re-induction of cured OVA_X_CreER(T2_X_OT-I mice indicating absence of tolerogenic mechanisms. This pathogen-free, conditional mouse model has the advantage of tamoxifen inducible tissue specific antigen expression that reflects the heterogeneity of viral antigen expression and enables the study of intrahepatic immune responses to both de novo and persistent antigen. It allows following the course of intrahepatic immune responses: initiation, the acute phase and antigen clearance.

Early prostate cancer antigen expression in predicting presence of prostate cancer in men with histologically negative biopsies.

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Hansel, D E; DeMarzo, A M; Platz, E A; Jadallah, S; Hicks, J; Epstein, J I; Partin, A W; Netto, G J

2007-05-01

Early prostate cancer antigen is a nuclear matrix protein that was recently shown to be expressed in prostate adenocarcinoma and adjacent benign tissue. Previous studies have demonstrated early prostate cancer antigen expression in benign prostate tissue up to 5 years before a diagnosis of prostate carcinoma, suggesting that early prostate cancer antigen could be used as a potential predictive marker. We evaluated early prostate cancer antigen expression by immunohistochemistry using a polyclonal antibody (Onconome Inc., Seattle, Washington) on benign biopsies from 98 patients. Biopsies were obtained from 4 groups that included 39 patients with first time negative biopsy (group 1), 24 patients with persistently negative biopsies (group 2), 8 patients with initially negative biopsies who were subsequently diagnosed with prostate carcinoma (group 3) and negative biopsies obtained from 27 cases where other concurrent biopsies contained prostate carcinoma (group 4). Early prostate cancer antigen staining was assessed by 2 of the authors who were blind to the group of the examined sections. Staining intensity (range 0 to 3) and extent (range 1 to 3) scores were assigned. The presence of intensity 3 staining in any of the blocks of a biopsy specimen was considered as positive for early prostate cancer antigen for the primary outcome in the statistical analysis. In addition, as secondary outcomes we evaluated the data using the proportion of blocks with intensity 3 early prostate cancer antigen staining, the mean of the product of staining intensity and staining extent of all blocks within a biopsy, and the mean of the product of intensity 3 staining and extent. Primary outcome analysis revealed the proportion of early prostate cancer antigen positivity to be highest in group 3 (6 of 8, 75%) and lowest in group 2 (7 of 24, 29%, p=0.04 for differences among groups). A relatively higher than expected proportion of early prostate cancer antigen positivity was present in

[Expression, purification and protective antigen analysis of cell wall protein MRP of Streptococcus suis type 2].

Science.gov (United States)

Wang, Ping-ping; Pian, Ya-ya; Yuan, Yuan; Zheng, Yu-ling; Jiang, Yong-qiang; Xiong, Zheng-ying

2012-02-01

To amplify the mrp gene of Streptococcus suis type 2 05ZYH33, express it in E.coli BL21 in order to acquire high purity recombinant protein MRP, then evaluate the protective antigen of recombinant protein MRP. Using PCR technology to obtain the product of mrp gene of 05ZYH33, and then cloned it into the expression vector pET28a(+). The recombinant protein was purified by affinity chromatography, later immunized New Zealand rabbit to gain anti-serum, then test the anti-serum titer by ELISA. The opsonophagocytic killing test demonstrated the abilities of protective antigen of MRP. The truncated of MRP recombinant protein in E.coli BL21 expressed by inclusion bodies, and purified it in high purity. After immunoprotection, the survival condition of CD-1 was significantly elevated. The survival rate of wild-type strain 05ZYH33 in blood was apparently decreased after anti-serum opsonophagocyticed, but the mutant delta; MRP showed no differences. MRP represent an important protective antigen activity.

Spontaneous loss and alteration of antigen receptor expression in mature CD4+ T cells

International Nuclear Information System (INIS)

Kyoizumi, Seishi; Akiyama, Mitoshi; Hirai, Yuko; Kusunoki; Yoichiro; Tanabe, Kazumi; Umeki, Shigeko; Nakamura, Nori; Yamakido, Michio; Hamamoto, Kazuko.

1990-04-01

The T-cell receptor CD3 (TCR/CD3) complex plays a central role in antigen recognition and activation of mature T cells, and therefore abnormalities in the expression of the complex should induce unresponsiveness of T cells to antigen stimulus. Using flow cytometry, we detected and enumerated variant cells with loss or alteration of surface TCR/CD3 expression among human mature CD4 + T cells. The presence of variant CD4 + T cells was demonstrated by isolating and cloning them from peripheral blood, and their abnormalities can be accounted for by alterations in TCR expression such as defects of protein expression and partial protein deletion. The variant frequency in peripheral blood increased with aging in normal donors and was highly elevated in patients with ataxia telangiectasia, an autosomal recessive inherited disease with defective DNA repair and variable T-cell immunodeficiency. These findings suggest that such alterations in TCR expression are induced by somatic mutagenesis of TCR genes and can be important factors related to age-dependent and genetic disease-associated T-cell dysfunction. (author)

Antigen uptake and expression of antigen presentation-related immune genes in flounder (Paralichthys olivaceus) after vaccination with an inactivated Edwardsiella tarda immersion vaccine, following hyperosmotic treatment.

Science.gov (United States)

Gao, Yingli; Tang, Xiaoqian; Sheng, Xiuzhen; Xing, Jing; Zhan, Wenbin

2016-08-01

Antigen uptake is a critical process for activation of the immune system, and therefore the ability to enhance antigen uptake is a primary consideration in the development of an immersion vaccination of fish. In the present work, flounders (Paralichthys olivaceus) were immersed in three hyperosmotic solutions with 40, 50 and 60‰ salinities, then transferred into seawater of normal salinity (i.e. 30‰) containing formalin-inactivated Edwardsiella tarda for 30 min. The antigen uptake in vaccinated flounder was determined using an absolute quantitative PCR (qPCR). The results showed significantly higher antigen uptake in the tissues of flounders immersed in solutions with 50‰ and 60‰ salinity compared to the control group directly immersed in vaccine (DI) (P immersed in the 50‰ salinity solution, whereas there was no significant difference in antigen uptake between the 40‰ salinity group and the DI group (P > 0.05). A rapid and significant increase in antigen uptake was detected in the mucosal-associated tissues including the gill, skin and intestine (P immersion, which was significantly higher than the levels of uptake measured in the other tissues (P immersion (hpi). The expression profiles of four antigen presentation-related immune genes (MHC Iα, MHC IIα, CD4-1 and CD8α) were investigated after immersion. These four genes showed a significantly stronger response in the immersed flounders exposed to 50‰ salinity compared with the DI group (P immersion, notably 50‰ salinity significantly enhanced antigen uptake and the expression of selected genes associated with antigen presentation, providing evidence for an enhanced immune activation of the fish's immune response by the hyperosmotic immersion treatment prior to vaccination. Copyright © 2016 Elsevier Ltd. All rights reserved.

Low cost tuberculosis vaccine antigens in capsules: expression in chloroplasts, bio-encapsulation, stability and functional evaluation in vitro.

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Priya Saikumar Lakshmi

Full Text Available Tuberculosis (TB caused by Mycobacterium tuberculosis is one of the leading fatal infectious diseases. The development of TB vaccines has been recognized as a major public health priority by the World Health Organization. In this study, three candidate antigens, ESAT-6 (6 kDa early secretory antigenic target and Mtb72F (a fusion polyprotein from two TB antigens, Mtb32 and Mtb39 fused with cholera toxin B-subunit (CTB and LipY (a cell wall protein were expressed in tobacco and/or lettuce chloroplasts to facilitate bioencapsulation/oral delivery. Site-specific transgene integration into the chloroplast genome was confirmed by Southern blot analysis. In transplastomic leaves, CTB fusion proteins existed in soluble monomeric or multimeric forms of expected sizes and their expression levels varied depending upon the developmental stage and time of leaf harvest, with the highest-level of accumulation in mature leaves harvested at 6PM. The CTB-ESAT6 and CTB-Mtb72F expression levels reached up to 7.5% and 1.2% of total soluble protein respectively in mature tobacco leaves. Transplastomic CTB-ESAT6 lettuce plants accumulated up to 0.75% of total leaf protein. Western blot analysis of lyophilized lettuce leaves stored at room temperature for up to six months showed that the CTB-ESAT6 fusion protein was stable and preserved proper folding, disulfide bonds and assembly into pentamers for prolonged periods. Also, antigen concentration per gram of leaf tissue was increased 22 fold after lyophilization. Hemolysis assay with purified CTB-ESAT6 protein showed partial hemolysis of red blood cells and confirmed functionality of the ESAT-6 antigen. GM1-binding assay demonstrated that the CTB-ESAT6 fusion protein formed pentamers to bind with the GM1-ganglioside receptor. The expression of functional Mycobacterium tuberculosis antigens in transplastomic plants should facilitate development of a cost-effective and orally deliverable TB booster vaccine with potential

Low cost tuberculosis vaccine antigens in capsules: expression in chloroplasts, bio-encapsulation, stability and functional evaluation in vitro.

Science.gov (United States)

Lakshmi, Priya Saikumar; Verma, Dheeraj; Yang, Xiangdong; Lloyd, Bethany; Daniell, Henry

2013-01-01

Tuberculosis (TB) caused by Mycobacterium tuberculosis is one of the leading fatal infectious diseases. The development of TB vaccines has been recognized as a major public health priority by the World Health Organization. In this study, three candidate antigens, ESAT-6 (6 kDa early secretory antigenic target) and Mtb72F (a fusion polyprotein from two TB antigens, Mtb32 and Mtb39) fused with cholera toxin B-subunit (CTB) and LipY (a cell wall protein) were expressed in tobacco and/or lettuce chloroplasts to facilitate bioencapsulation/oral delivery. Site-specific transgene integration into the chloroplast genome was confirmed by Southern blot analysis. In transplastomic leaves, CTB fusion proteins existed in soluble monomeric or multimeric forms of expected sizes and their expression levels varied depending upon the developmental stage and time of leaf harvest, with the highest-level of accumulation in mature leaves harvested at 6PM. The CTB-ESAT6 and CTB-Mtb72F expression levels reached up to 7.5% and 1.2% of total soluble protein respectively in mature tobacco leaves. Transplastomic CTB-ESAT6 lettuce plants accumulated up to 0.75% of total leaf protein. Western blot analysis of lyophilized lettuce leaves stored at room temperature for up to six months showed that the CTB-ESAT6 fusion protein was stable and preserved proper folding, disulfide bonds and assembly into pentamers for prolonged periods. Also, antigen concentration per gram of leaf tissue was increased 22 fold after lyophilization. Hemolysis assay with purified CTB-ESAT6 protein showed partial hemolysis of red blood cells and confirmed functionality of the ESAT-6 antigen. GM1-binding assay demonstrated that the CTB-ESAT6 fusion protein formed pentamers to bind with the GM1-ganglioside receptor. The expression of functional Mycobacterium tuberculosis antigens in transplastomic plants should facilitate development of a cost-effective and orally deliverable TB booster vaccine with potential for long

Infrequent Expression of the Cancer-Testis Antigen, PASD1, in Ovarian Cancer

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Ghazala Khan

2015-01-01

Full Text Available Ovarian cancer is very treatable in the early stages of disease; however, it is usually detected in the later stages, at which time, treatment is no longer as effective. If discovered early (Stage I, there is a 90% chance of five-year survival. Therefore, it is imperative that early-stage biomarkers are identified to enhance the early detection of ovarian cancer. Cancer-testis antigens (CTAs, such as Per ARNT SIM (PAS domain containing 1 (PASD1, are unique in that their expression is restricted to immunologically restricted sites, such as the testis and placenta, which do not express MHC class I, and cancer, making them ideally positioned to act as targets for immunotherapy as well as potential biomarkers for cancer detection where expressed. We examined the expression of PASD1a and b in a number of cell lines, as well as eight healthy ovary samples, eight normal adjacent ovarian tissues, and 191 ovarian cancer tissues, which were predominantly stage I ( n = 164 and stage II ( n = 14 disease. We found that despite the positive staining of skin cancer, only one stage Ic ovarian cancer patient tissue expressed PASD1a and b at detectable levels. This may reflect the predominantly stage I ovarian cancer samples examined. To examine the restriction of PASD1 expression, we examined endometrial tissue arrays and found no expression in 30 malignant tumor tissues, 23 cases of hyperplasia, or 16 normal endometrial tissues. Our study suggests that the search for a single cancer-testes antigen/biomarker that can detect early ovarian cancer must continue.

Hepatitis B surface antigen (HBsAg) expression in plant cell culture: Kinetics of antigen accumulation in batch culture and its intracellular form.

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Smith, Mark L; Mason, Hugh S; Shuler, Michael L

2002-12-30

The production of edible vaccines in transgenic plants and plant cell culture may be improved through a better understanding of antigen processing and assembly. The hepatitis B surface antigen (HBsAg) was chosen for study because it undergoes substantial and complex post-translational modifications, which are necessary for its immunogenicity. This antigen was expressed in soybean (Glycine max L. Merr. cv Williams 82) and tobacco NT1 (Nicotiana tabacum L.) cell suspension cultures, and HBsAg production in batch culture was characterized. The plant-derived antigen consisted predominantly of disulfide cross-linked HBsAg protein (p24(s)) dimers, which were all membrane associated. Similar to yeast, the plant-expressed HBsAg was retained intracellularly. The maximal HBsAg titers were obtained with soybean suspension cultures (20-22 mg/L) with titers in tobacco cultures being approximately 10-fold lower. For soybean cells, electron microscopy and immunolocalization demonstrated that all the HBsAg was localized to the endoplasmic reticulum (ER) and provoked dilation and proliferation of the ER network. Sucrose gradient analysis of crude extracts showed that HBsAg had a complex size distribution uncharacteristic of the antigen's normal structure of uniform 22-nm virus-like particles. The extent of authentic epitope formation was assessed by comparing total p24(s) synthesized to that reactive by polyclonal and monoclonal immunoassays. Depending on culture age, between 40% and 100% of total p24(s) was polyclonal antibody reactive whereas between 6% and 37% was recognized by a commercial monoclonal antibody assay. Possible strategies to increase HBsAg production and improve post-translational processing are discussed. Copyright 2002 Wiley Periodicals, Inc.

ST6GalNAc-I controls expression of sialyl-Tn antigen in gastrointestinal tissues

DEFF Research Database (Denmark)

Marcos, Nuno T; Bennett, Eric Paul; Gomes, Joana

2011-01-01

-Tn biosynthesis. We developed novel monoclonal antibodies specific for ST6GalNAc-I and evaluated its expression in gastrointestinal tissues. ST6GalNAc-I was detected in normal colon mucosa co-localized with O-acetylated sialyl-Tn. Expression was largely unaltered in colorectal adenocarcinomas. In contrast, we......NAc-I as the major enzyme controlling the expression of cancer-associated sialyl-Tn antigen in gastrointestinal tissues....

Expression of endogenous ALV antigens and susceptibility to subgroup E ALV in three strains of chickens (Endogenous avian C. type virus)

International Nuclear Information System (INIS)

Robinson, H.L.; Lamoreux, W.F.

1976-01-01

Cells from three strains of Kimber Farms chickens, K16, K18, and K28, have been characterized for the expression of endogenous avian C-type virus (ALV) antigens and for susceptibility to subgroup E ALV. In K16 the coordinate dominant expression of chick helper factor (chf ), the type specific antigen of endogenous subgroup E ALV, and the group specific (gs) antigen of ALV was observed. The expression of chf and gs antigen in K16 chf + gs + cells was similar to that observed in SPAFAS and H and N gs + cells. In K18 chf but not gs antigen was expressed. The expression of chf in K18 chf + gs + cells was distinct from that observed for the chf + gs + pedigrees but similar to that found in SPAFAS chf + gs - helper extremely high (h/sub E/) cells. Cells from K16 and K18 chickens were uniformly resistant to subgroup E ALV. In cells from K28 chickens, susceptibility to subgroup B virus correlated with susceptibility to subgroup E virus. The efficiency of plating of subgroup E virus on susceptible K28 cells with 10 3 --10 4 -fold lower on chf + cells than on chf - cells

Epigenetic modulation of cancer-germline antigen gene expression in tumorigenic human mesenchymal stem cells: implications for cancer therapy

DEFF Research Database (Denmark)

Gjerstorff, Morten; Burns, Jorge S; Nielsen, Ole

2009-01-01

Cancer-germline antigens are promising targets for cancer immunotherapy, but whether such therapies will also eliminate the primary tumor stem cell population remains undetermined. We previously showed that long-term cultures of telomerized adult human bone marrow mesenchymal stem cells can...... spontaneously evolve into tumor-initiating, mesenchymal stem cells (hMSC-TERT20), which have characteristics of clinical sarcoma cells. In this study, we used the hMSC-TERT20 tumor stem cell model to investigate the potential of cancer-germline antigens to serve as tumor stem cell targets. We found...... of cancer-germline antigens in hMSC-TERT20 cells, while their expression levels in primary human mesenchymal stem cells remained unaffected. The expression pattern of cancer-germline antigens in tumorigenic mesenchymal stem cells and sarcomas, plus their susceptibility to enhancement by epigenetic...

A mouse model for chronic lymphocytic leukemia based on expression of the SV40 large T antigen

DEFF Research Database (Denmark)

ter Brugge, Petra J; Ta, Van B T; de Bruijn, Marjolein J W

2009-01-01

The simian virus 40 (SV40) T antigen is a potent oncogene able to transform many cell types and has been implicated in leukemia and lymphoma. In this report, we have achieved sporadic SV40 T-antigen expression in mature B cells in mice, by insertion of a SV40 T antigen gene in opposite...... transcriptional orientation in the immunoglobulin (Ig) heavy (H) chain locus between the D and J(H) segments. SV40 T-antigen expression appeared to result from retention of the targeted germline allele and concomitant antisense transcription of SV40 large T in mature B cells, leading to chronic lymphocytic...... leukemia (CLL). Although B-cell development was unperturbed in young mice, aging mice showed accumulation of a monoclonal B-cell population in which the targeted IgH allele was in germline configuration and the wild-type IgH allele had a productive V(D)J recombination. These leukemic B cells were Ig...

Purification and refolding of anti-T-antigen single chain antibodies (scFvs) expressed in Escherichia coli as inclusion bodies.

Science.gov (United States)

Yuasa, Noriyuki; Koyama, Tsubasa; Fujita-Yamaguchi, Yoko

2014-02-01

T-antigen (Galβ1-3GalNAcα-1-Ser/Thr) is an oncofetal antigen that is commonly expressed as a carbohydrate determinant in many adenocarcinomas. Since it is associated with tumor progression and metastasis, production of recombinant antibodies specific for T-antigen could lead to the development of cancer diagnostics and therapeutics. Previously, we isolated and characterized 11 anti-T-antigen phage clones from a phage library displaying human single-chain antibodies (scFvs) and purified one scFv protein, 1G11. More recently, we purified and characterized 1E8 scFv protein using a Drosophila S2 expression system. In the current study, four anti-T-antigen scFv genes belonging to Groups 1-4 were purified from inclusion bodies expressed in Escherichia coli cells. Inclusion bodies isolated from E. coli cells were denatured in 3.5 M Gdn-HCl. Solubilized His-tagged scFv proteins were purified using Ni(2+)-Sepharose column chromatography in the presence of 3.5 M Gdn-HCl. Purified scFv proteins were refolded according to a previously published method of step-wise dialysis. Two anti-T-antigen scFv proteins, 1E6 and 1E8 that belong to Groups 1 and 2, respectively, were produced in sufficient amounts, thus allowing further characterization of their binding activity with T-antigen. Specificity and affinity constants determined using enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR), respectively, provided evidence that both 1E8 and 1E6 scFv proteins are T-antigen specific and suggested that 1E8 scFv protein has a higher affinity for T-antigen than 1E6 scFv protein.

Detection of proliferating cell nuclear antigens and interleukin-2 beta receptor molecules on mitogen- and antigen-stimulated lymphocytes.

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Hesketh, J; Dobbelaere, D; Griffin, J F; Buchan, G

1993-01-01

The expression of interleukin-2 receptors (IL-2R) and proliferating cell nuclear antigens (PCNA) were compared for their usefulness as markers of lymphocyte activation. Heterologous polyclonal (anti-bovine IL-2R) and monoclonal (anti-human PCNA) antibodies were used to detect the expression of these molecules on activated deer lymphocytes. Both molecules were co-expressed on blast cells which had been activated with mitogen [concanavalin A (Con A)]. There was detectable up-regulation of IL-2R expression in response to antigen [Mycobacterium bovis-derived purified protein derivative (PPD)] stimulation while PCNA expression mimicked lymphocyte transformation (LT) reactivity. PCNA expression was found to more accurately reflect both antigen- and mitogen-activated lymphocyte activation, as estimated by LT activity. The expression of PCNA was used to identify antigen reactive cells from animals exposed to M. bovis. A very low percentage (1.1 +/- 0.4%) of peripheral blood lymphocytes from non-infected animals could be stimulated to express PCNA by in vitro culture with antigen (PPD). Within the infected group both diseased and healthy, 'in-contact', animals expressed significantly higher levels of PCNA upon antigen stimulation. PMID:8104884

Exosomes derived from tumor cells genetically modified to express Mycobacterium tuberculosis antigen: a novel vaccine for cancer therapy.

Science.gov (United States)

Koyama, Yoshiyuki; Ito, Tomoko; Hasegawa, Aya; Eriguchi, Masazumi; Inaba, Toshio; Ushigusa, Takahiro; Sugiura, Kikuya

2016-11-01

To examine the potential of exosomes derived from the tumor cells, which had been genetically modified to express a Mycobacterium tuberculosis antigen, as a cancer vaccine aimed at overcoming the weak immunogenicity of tumor antigens. We transfected B16 melanoma cells with a plasmid encoding the M. tuberculosis antigen, early secretory antigenic target-6 (ESAT-6). The secreted exosomes bearing both tumor-associated antigens and the pathogenic antigen (or their epitopes) were collected. When the exosomes were injected into foot pads of mice, they significantly (p exosomes significantly suppressed (p exosomes derived from the non-transfected B16 cells showed no effect on tumor growth, although both exosomes should have similar tumor antigens. Exosomes bearing both tumor antigens and the M. tuberculosis antigen (or their epitopes) have a high potential as a candidate for cancer vaccine to overcome the immune escape by tumor cells.

Vascular expression of endothelial antigen PAL-E indicates absence of blood-ocular barriers in the normal eye

NARCIS (Netherlands)

Schlingemann, R. O.; Hofman, P.; Anderson, L.; Troost, D.; van der Gaag, R.

1997-01-01

The endothelium-specific antigen PAL-E is expressed in capillaries and veins throughout the body with the exception of the brain, where the antigen is absent from anatomical sites with a patent blood-brain barrier. In this study we determined vascular endothelial staining for PAL-E in the normal eye

Expression of Hepatitis C Virus Core and E2 antigenic recombinant proteins and their use for development of diagnostic assays.

Science.gov (United States)

Ali, Amjad; Nisar, Muhammad; Idrees, Muhammad; Rafique, Shazia; Iqbal, Muhammad

2015-05-01

Early diagnosis of HCV infection is based on detection of antibodies against HCV proteins using recombinant viral antigens. The present study was designed to select, clone and express the antigenic regions of Core and E2 genes from local HCV-3a genotype and to utilize the antigenic recombinant proteins (Core & E2) to develop highly sensitive, specific and economical diagnostic assays for detection of HCV infection. The antigenic sites were determined within Core and E2 genes and were then cloned in pET-28a expression vector. The right orientation of the desired inserted fragments of Core and E2 were confirmed via sequencing prior to expression and were then transformed in BL21 (DE3) pLysS strains of E. coli and induced with 0.5mM Isopropyl-b-D-thiogalactopyranoside (IPTG) for the production of antigenic recombinant proteins. The produced truncated antigens were then purified by Nickel affinity chromatography and were confirmed by western blotting, immunoblotting and enzyme-linked immunosorbent assay (ELISA). The expressed Core and E2 recombinant antigens were used to develop immunoblotting assay for the detection of anti-HCV antibodies in sera. With immunoblotting, a total of 93-HCV infected sera and 35-HCV negative individuals were tested for the presence of anti-HCV antibodies to the Core and E2 antigens. Recombinant antigen showed 100% reactivity against HCV infected sera, with no cross reactivity against HCV-negative sera. The immunoblot assay mixture of recombinant antigens (Core+E2) showed a strong reaction intensity in the test area (TA) as compared to the individual truncated Core and E2 recombinant antigens. In the in-house ELISA assay, mixed Core and E2 recombinant antigens showed 100% reactivity against a standardized panel of 150-HCV-positive sera and non reactivity against a standardized panel of 150 HCV-negative sera while also being non reactive to sera positive for other viral infections. The antigenic recombinant antigens also were tested for the

Positive Selection of γδ CTL by TL Antigen Expressed in the Thymus

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Tsujimura, Kunio; Takahashi, Toshitada; Morita, Akimichi; Hasegawa-Nishiwaki, Hitomi; Iwase, Shigeru; Obata, Yuichi

1996-01-01

To elucidate the function of the mouse TL antigen in the thymus, we have derived two TL transgenic mouse strains by introducing Tla a -3 of A strain origin with its own promoter onto a C3H background with no expression of TL in the thymus. These transgenic mouse strains, both of which express high levels of Tlaa-3-TL antigen in their thymus, were analyzed for their T cell function with emphasis on cytotoxic T lymphocyte (CTL) generation. A T cell response against TL was induced in Tg.Tlaa-3-1, Tg.Tlaa-3-2, and control C3H mice by skin grafts from H-2K b/T3 b transgenic mice, Tg.Con.3-1, expressing T3b-TL ubiquitously. Spleen cells from mice that had rejected the T3b-TL positive skin grafts were restimulated in vitro with Tg.Con.3-1 irradiated spleen cells. In mixed lymphocyte cultures (MLC), approximately 20% and 15% of Thy-1+ T cells derived from Tg.Tlaa-3-1 and Tg.Tlaa-3-2, respectively, expressed TCRγδ, whereas almost all those from C3H expressed TCRαβ. The MLC from Tg.Tlaa-3-2 and C3H demonstrated high CTL activity against TL, while those from Tg.Tlaa-3-1 had little or none. The generation of γδ CTL recognizing TL in Tg.Tlaa-3-2, but not C3H mice, was confirmed by the establishment of CTL clones. A total of 14 γδ CTL clones were established from Tg.Tlaa-3-2, whereas none were obtained from C3H. Of the 14 γδ CTL clones, 8 were CD8+ and 6 were CD4−CD8− double negative. The CTL activity of all these clones was TL specific and inhibited by anti-TL, but not by anti-H-2 antibodies, demonstrating that they recognize TL directly without antigen presentation by H-2. The CTL activity was blocked by antibodies to TCRγδ and CD3, and also by antibodies to CD8α and CD8β in CD8+ clones, showing that the activity was mediated by TCRγδ and coreceptors. The thymic origin of these γδ CTL clones was indicated by the expression of Thy-1 and Ly-1 (CD5), and also CD8αβ heterodimers in CD8+ clones on their surfaces and by the usage of TCR Vγ4 chains in 12 of

Expression of cancer-testis antigens MAGE-A4 and MAGE-C1 in oral squamous cell carcinoma.

Science.gov (United States)

Montoro, José Raphael de Moura Campos; Mamede, Rui Celso Martins; Neder Serafini, Luciano; Saggioro, Fabiano Pinto; Figueiredo, David Livingstone Alves; Silva, Wilson Araújo da; Jungbluth, Achim A; Spagnoli, Giulio Cesare; Zago, Marco Antônio

2012-08-01

Tumor markers are genes or their products expressed exclusively or preferentially in tumor cells and cancer-testis antigens (CTAs) form a group of genes with a typical expression pattern expressed in a variety of malignant neoplasms. CTAs are considered potential targets for cancer vaccines. It is possible that the CTA MAGE-A4 (melanoma antigen) and MAGE-C1 are expressed in carcinoma of the oral cavity and are related with survival. This study involved immunohistochemical analysis of 23 patients with oral squamous cell carcinoma (SCC) and was carried out using antibodies for MAGE-A4 and MAGE-C1. Fisher's exact test and log-rank test were used to evaluate the results. The expression of the MAGE-A4 and MAGE-C1 were 56.5% and 47.8% without statistical difference in studied variables and survival. The expression of at least 1 CTA was present in 78.3% of the patients, however, without correlation with clinicopathologic variables and survival. Copyright © 2011 Wiley Periodicals, Inc.

Antigen presentation and MHC class II expression by human esophageal epithelial cells: role in eosinophilic esophagitis.

Science.gov (United States)

Mulder, Daniel J; Pooni, Aman; Mak, Nanette; Hurlbut, David J; Basta, Sameh; Justinich, Christopher J

2011-02-01

Professional antigen-presenting cells (APCs) play a crucial role in initiating immune responses. Under pathological conditions, epithelial cells at mucosal surfaces act as nonprofessional APCs, thereby regulating immune responses at the site of exposure. Epithelial cells in the esophagus may contribute to the pathogenesis of eosinophilic esophagitis (EoE) by presenting antigens on the major histocompatibility complex (MHC) class II. Our goal was to demonstrate the ability of esophageal epithelial cells to process and present antigens on the MHC class II system and to investigate the contribution of epithelial cell antigen presentation to EoE. Immunohistochemistry detected HLA-DR, CD80, and CD86 expression and enzyme-linked immunosorbent assay detected interferon-γ (IFNγ) in esophageal biopsies. Antigen presentation was studied using the human esophageal epithelial cell line HET-1A by reverse transcriptase-PCR, flow cytometry, and confocal microscopy. T helper cell lymphocyte proliferation was assessed by flow cytometry and IL-2 secretion. IFNγ and MHC class II were increased in mucosa of patients with EoE. IFNγ increased mRNA of HLA-DP, HLA-DQ, HLA-DR, and CIITA in HET-1A cells. HET-1A engulfed cell debris and processed ovalbumin. HET-1A cells expressed HLA-DR after IFNγ treatment. HET-1A stimulated T helper cell activation. In this study, we demonstrated the ability of esophageal epithelial cells to act as nonprofessional APCs in the presence of IFNγ. Esophageal epithelial cell antigen presentation may contribute to the pathophysiology of eosinophilic esophagitis. Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Immunophenotyping of Waldenstroms macroglobulinemia cell lines reveals distinct patterns of surface antigen expression: potential biological and therapeutic implications.

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Aneel Paulus

Full Text Available Waldenströms macroglobulinemia (WM is a subtype of Non-Hodgkin's lymphoma in which the tumor cell population is markedly heterogeneous, consisting of immunoglobulin-M secreting B-lymphocytes, plasmacytoid lymphocytes and plasma cells. Due to rarity of disease and scarcity of reliable preclinical models, many facets of WM molecular and phenotypic architecture remain incompletely understood. Currently, there are 3 human WM cell lines that are routinely used in experimental studies, namely, BCWM.1, MWCL-1 and RPCI-WM1. During establishment of RPCI-WM1, we observed loss of the CD19 and CD20 antigens, which are typically present on WM cells. Intrigued by this observation and in an effort to better define the immunophenotypic makeup of this cell line, we conducted a more comprehensive analysis for the presence or absence of other cell surface antigens that are present on the RPCI-WM1 model, as well as those on the two other WM cell lines, BCWM.1 and MWCL-1. We examined expression of 65 extracellular and 4 intracellular antigens, comprising B-cell, plasma cell, T-cell, NK-cell, myeloid and hematopoietic stem cell surface markers by flow cytometry analysis. RPCI-WM1 cells demonstrated decreased expression of CD19, CD20, and CD23 with enhanced expression of CD28, CD38 and CD184, antigens that were differentially expressed on BCWM.1 and MWCL-1 cells. Due to increased expression of CD184/CXCR4 and CD38, RPCI-WM1 represents a valuable model in which to study the effects anti-CXCR4 or anti-CD38 targeted therapies that are actively being developed for treatment of hematologic cancers. Overall, differences in surface antigen expression across the 3 cell lines may reflect the tumor clone population predominant in the index patients, from whom the cell lines were developed. Our analysis defines the utility of the most commonly employed WM cell lines as based on their immunophenotype profiles, highlighting unique differences that can be further studied for

[Expression changes of major outer membrane protein antigens in Leptospira interrogans during infection and its mechanism].

Science.gov (United States)

Zheng, Linli; Ge, Yumei; Hu, Weilin; Yan, Jie

2013-03-01

To determine expression changes of major outer membrane protein(OMP) antigens of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai during infection of human macrophages and its mechanism. OmpR encoding genes and OmpR-related histidine kinase (HK) encoding gene of L.interrogans strain Lai and their functional domains were predicted using bioinformatics technique. mRNA level changes of the leptospiral major OMP-encoding genes before and after infection of human THP-1 macrophages were detected by real-time fluorescence quantitative RT-PCR. Effects of the OmpR-encoding genes and HK-encoding gene on the expression of leptospiral OMPs during infection were determined by HK-peptide antiserum block assay and closantel inhibitive assays. The bioinformatics analysis indicated that LB015 and LB333 were referred to OmpR-encoding genes of the spirochete, while LB014 might act as a OmpR-related HK-encoding gene. After the spirochete infecting THP-1 cells, mRNA levels of leptospiral lipL21, lipL32 and lipL41 genes were rapidly and persistently down-regulated (P Expression levels of L.interrogans strain Lai major OMP antigens present notable changes during infection of human macrophages. There is a group of OmpR-and HK-encoding genes which may play a major role in down-regulation of expression levels of partial OMP antigens during infection.

Epithelium Expressing the E7 Oncoprotein of HPV16 Attracts Immune-Modulatory Dendritic Cells to the Skin and Suppresses Their Antigen-Processing Capacity.

Directory of Open Access Journals (Sweden)

Janin Chandra

Full Text Available Antigen presenting cells (APCs in skin can promote either antigen-specific effector functions or antigen tolerance, and thus determine clearance or persistence of cutaneous viral infections. Human papillomavirus (HPV infections can persist in squamous epithelium in immunocompetent individuals, and some persisting HPV infections, particularly with HPV16, promote malignant epithelial transformation. Here, we investigate whether local expression of the HPV16 protein most associated with malignant transformation, HPV16-E7, affects the phenotype and function of APC subsets in the skin. We demonstrate an expanded population of Langerhans cells in HPV16-E7 transgenic skin with distinct cell surface markers which express immune-modulatory enzymes and cytokines not expressed by cells from non transgenic skin. Furthermore, HPV16-E7 transgene expression in keratinocytes attracts new APC subsets to the epidermis. In vivo migration and transport of antigen to the draining lymph node by these APCs is markedly enhanced in HPV16-E7 expressing skin, whereas antigen-processing, as measured by proteolytic cleavage of DQ-OVA and activation of T cells in vivo by APCs, is significantly impaired. These data suggest that local expression of HPV16-E7 in keratinocytes can contribute to persisting infection with this oncogenic virus, by altering the phenotype and function of local APCs.

Codon optimisation to improve expression of a Mycobacterium avium ssp. paratuberculosis-specific membrane-associated antigen by Lactobacillus salivarius.

Science.gov (United States)

Johnston, Christopher; Douarre, Pierre E; Soulimane, Tewfik; Pletzer, Daniel; Weingart, Helge; MacSharry, John; Coffey, Aidan; Sleator, Roy D; O'Mahony, Jim

2013-06-01

Subunit and DNA-based vaccines against Mycobacterium avium ssp. paratuberculosis (MAP) attempt to overcome inherent issues associated with whole-cell formulations. However, these vaccines can be hampered by poor expression of recombinant antigens from a number of disparate hosts. The high G+C content of MAP invariably leads to a codon bias throughout gene expression. To investigate if the codon bias affects recombinant MAP antigen expression, the open reading frame of a MAP-specific antigen MptD (MAP3733c) was codon optimised for expression against a Lactobacillus salivarius host. Of the total 209 codons which constitute MAP3733c, 172 were modified resulting in a reduced G+C content from 61% for the native gene to 32.7% for the modified form. Both genes were placed under the transcriptional control of the PnisA promoter; allowing controlled heterologous expression in L. salivarius. Expression was monitored using fluorescence microscopy and microplate fluorometry via GFP tags translationally fused to the C-termini of the two MptD genes. A > 37-fold increase in expression was observed for the codon-optimised MAP3733synth variant over the native gene. Due to the low cost and improved expression achieved, codon optimisation significantly improves the potential of L. salivarius as an oral vaccine stratagem against Johne's disease. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Transgenic Expression of IL15 Improves Antiglioma Activity of IL13Rα2-CAR T Cells but Results in Antigen Loss Variants.

Science.gov (United States)

Krenciute, Giedre; Prinzing, Brooke L; Yi, Zhongzhen; Wu, Meng-Fen; Liu, Hao; Dotti, Gianpietro; Balyasnikova, Irina V; Gottschalk, Stephen

2017-07-01

Glioblastoma (GBM) is the most aggressive primary brain tumor in adults and is virtually incurable with conventional therapies. Immunotherapy with T cells expressing GBM-specific chimeric antigen receptors (CAR) is an attractive approach to improve outcomes. Although CAR T cells targeting GBM antigens, such as IL13 receptor subunit α2 (IL13Rα2), HER2, and EGFR variant III (EGFRvIII), have had antitumor activity in preclinical models, early-phase clinical testing has demonstrated limited antiglioma activity. Transgenic expression of IL15 is an appealing strategy to enhance CAR T-cell effector function. We tested this approach in our IL13Rα2-positive glioma model in which limited IL13Rα2-CAR T-cell persistence results in recurrence of antigen-positive gliomas. T cells were genetically modified with retroviral vectors encoding IL13Rα2-CARs or IL15 (IL13Rα2-CAR.IL15 T cells). IL13Rα2-CAR.IL15 T cells recognized glioma cells in an antigen-dependent fashion, had greater proliferative capacity, and produced more cytokines after repeated stimulations in comparison with IL13Rα2-CAR T cells. No autonomous IL13Rα2-CAR.IL15 T-cell proliferation was observed; however, IL15 expression increased IL13Rα2-CAR T-cell viability in the absence of exogenous cytokines or antigen. In vivo , IL13Rα2-CAR.IL15 T cells persisted longer and had greater antiglioma activity than IL13Rα2-CAR T cells, resulting in a survival advantage. Gliomas recurring after 40 days after T-cell injection had downregulated IL13Rα2 expression, indicating that antigen loss variants occur in the setting of improved T-cell persistence. Thus, CAR T cells for GBM should not only be genetically modified to improve their proliferation and persistence, but also to target multiple antigens. Summary: Glioblastoma responds imperfectly to immunotherapy. Transgenic expression of IL15 in T cells expressing CARs improved their proliferative capacity, persistence, and cytokine production. The emergence of antigen

A recombinant raccoon poxvirus vaccine expressing both Yersinia pestis F1 and truncated V antigens protects animals against lethal plague.

Science.gov (United States)

Rocke, Tonie E.; Kingstad-Bakke, B; Berlier, W; Osorio, J.E.

2014-01-01

In previous studies, we demonstrated in mice and prairie dogs that simultaneous administration of two recombinant raccoon poxviruses (rRCN) expressing Yersinia pestis antigens (F1 and V307-a truncated version of the V protein) provided superior protection against plague challenge compared to individual single antigen constructs. To reduce costs of vaccine production and facilitate implementation of a sylvatic plague vaccine (SPV) control program for prairie dogs, a dual antigen construct is more desirable. Here we report the construction and characterization of a novel RCN-vectored vaccine that simultaneously expresses both F1 and V307 antigens. This dual antigen vaccine provided similar levels of protection against plague in both mice and prairie dogs as compared to simultaneous administration of the two single antigen constructs and was also shown to protect mice against an F1 negative strain of Y. pestis.. The equivalent safety, immunogenicity and efficacy profile of the dual RCN-F1/V307 construct warrants further evaluation in field efficacy studies in sylvatic plague endemic areas.

Effects of irradiation on the expression of major histocompatibility complex class I antigen and adhesion costimulation molecules ICAM-1 in human cervical cancer

International Nuclear Information System (INIS)

Santin, Alessandro D.; Hermonat, Paul L.; Hiserodt, John C.; Chiriva-Internati, Maurizio; Woodliff, Jeff; Theus, John W.; Barclay, David; Pecorelli, Sergio; Parham, Groesbeck P.

1997-01-01

Purpose: We initiated studies to analyze the effects of high doses of gamma irradiation on the surface antigen expression of MHC Class I, Class II, and ICAM-1 on human cervical carcinoma cell lines. Methods and Materials: The expression of surface antigens (MHC Class I, Class II, and ICAM-1) was evaluated by FACS analysis on two cervical cell lines at different time points, following their exposure to high doses of gamma irradiation (i.e., 25.00, 50.00, and 100.00 Gy). Results: The CaSki and SiHa cervical cancer cells we analyzed in this study expressed variable levels of MHC Class I and ICAM-1 antigens, while Class II surface antigens were not detectable. Whereas irradiation doses of 25.00 Gy were not sufficient to totally block cell replication in both cell lines, exposure to 50.00 or 100.00 Gy was able to completely inhibit cell replication. Range doses from 25.00 to 100.00 Gy significantly and consistently increased the expression of all surface antigens present on the cells prior to irradiation but were unable to induce neoexpression of antigens previously not expressed by these cells (i.e., MHC Class II). Importantly, such upregulation was shown to be dose dependent, with higher radiation doses associated with increased antigen expression. Moreover, when the kinetic of this upregulation was studied after 2 and 6 days after irradiation, it was shown to be persistent and lasted until all the cells died. Conclusions: These findings may partially explain the increased immunogenicity of tumor cells following irradiation and may suggest enhanced immune recognition in tumor tissue in patients receiving radiation therapy

Expression of the VP40 antigen from the Zaire ebolavirus in tobacco plants.

Science.gov (United States)

Monreal-Escalante, Elizabeth; Ramos-Vega, Abel A; Salazar-González, Jorge A; Bañuelos-Hernández, Bernardo; Angulo, Carlos; Rosales-Mendoza, Sergio

2017-07-01

The plant cell is able to produce the VP40 antigen from the Zaire ebolavirus , retaining the antigenicity and the ability to induce immune responses in BALB/c mice. The recent Ebola outbreak evidenced the need for having vaccines approved for human use. Herein we report the expression of the VP40 antigen from the Ebola virus as an initial effort in the development of a plant-made vaccine that could offer the advantages of being cheap and scalable, which is proposed to overcome the rapid need for having vaccines to deal with future outbreaks. Tobacco plants were transformed by stable DNA integration into the nuclear genome using the CaMV35S promoter and a signal peptide to access the endoplasmic reticulum, reaching accumulation levels up to 2.6 µg g -1 FW leaf tissues. The antigenicity of the plant-made VP40 antigen was evidenced by Western blot and an initial immunogenicity assessment in test animals that revealed the induction of immune responses in BALB/c mice following three weekly oral or subcutaneous immunizations at very low doses (125 and 25 ng, respectively) without accessory adjuvants. Therefore, this plant-based vaccination prototype is proposed as an attractive platform for the production of vaccines in the fight against Ebola virus disease outbreaks.

A Case Report of Angina Bullosa Haemorrhagica (ABH in Patient with Churg Strauss Syndrome

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M. Koopaie

2015-11-01

Full Text Available Bullosa Haemorrhagica (ABH is a rare oral blood blisters disorder. This recurrent disorder is restricted to oral mucosa and its difference with other oral vesiculobullos disorders is that blood vesicles are formed after minor trauma and in the absence of any blood dyscrasia, vesiculobullos disease and systemic disorder. This blood blisters are without pain, with color of dark red and full of bloods that rapidly enlarge and rapidly burst. The incidence of Angina Bullosa Haemorrhagica is unknown. Diagnosis of this disorder and differentiation from vesiculobullos disease like pemphigus and pemphiguid is important for patient's health. In this report we introduce a 55 years old woman with a 4­years history of oral blood blisters. These vesicles were in lateral border and ventral surface of tongue and on the ferenum. In medical history the patient had churg strauss syndrome and using inhalation corticosteroid for control of asthmatic attacks (An etiologic factor for appearing or aggravation of the disease.

Expression of cathepsins B, L, S, and D by gastric epithelial cells implicates them as antigen presenting cells in local immune responses.

Science.gov (United States)

Barrera, C; Ye, G; Espejo, R; Gunasena, S; Almanza, R; Leary, J; Crowe, S; Ernst, P; Reyes, V E

2001-10-01

Helicobacter pylori infection is linked to chronic gastritis, peptic ulcer and gastric carcinoma. During H. pylori infection, class II MHC expression by the gastric epithelium increases, as does the number of local CD4(+) T cells, which appear to be important in the associated pathogenesis. These observations suggested that the epithelium might present antigens to T cells. Thus, we sought to determine whether gastric epithelial cells process antigens to establish their function as local antigen presenting cells (APC). We examined a panel of gastric epithelial cell lines for expression of the antigen processing cathepsins B (CB), L (CL), S (CS), and D (CD). The mRNA for these enzymes were detected by RT-PCR and the enzymes in the gastric epithelial cells were identified by various independent methods. We corroborated the expression of CB and CD on gastric epithelial cells from human biopsy samples. The functions of these proteases were confirmed by assessing their ability to digest ovalbumin, a conventional dietary antigen, and proteins from H. pylori. In summary, multiple lines of evidence suggest gastric epithelial cells process antigens for presentation to CD4(+) T cells. To our knowledge, these are the first studies to document the antigen processing capacity of human gastric epithelial cells.

Enhanced expression of codon optimized Mycobacterium avium subsp. paratuberculosis antigens in Lactobacillus salivarius.

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Johnston, Christopher D; Bannantine, John P; Govender, Rodney; Endersen, Lorraine; Pletzer, Daniel; Weingart, Helge; Coffey, Aidan; O'Mahony, Jim; Sleator, Roy D

2014-01-01

It is well documented that open reading frames containing high GC content show poor expression in A+T rich hosts. Specifically, G+C-rich codon usage is a limiting factor in heterologous expression of Mycobacterium avium subsp. paratuberculosis (MAP) proteins using Lactobacillus salivarius. However, re-engineering opening reading frames through synonymous substitutions can offset codon bias and greatly enhance MAP protein production in this host. In this report, we demonstrate that codon-usage manipulation of MAP2121c can enhance the heterologous expression of the major membrane protein (MMP), analogous to the form in which it is produced natively by MAP bacilli. When heterologously over-expressed, antigenic determinants were preserved in synthetic MMP proteins as shown by monoclonal antibody mediated ELISA. Moreover, MMP is a membrane protein in MAP, which is also targeted to the cellular surface of recombinant L. salivarius at levels comparable to MAP. Additionally, we previously engineered MAP3733c (encoding MptD) and show herein that MptD displays the tendency to associate with the cytoplasmic membrane boundary under confocal microscopy and the intracellularly accumulated protein selectively adheres to the MptD-specific bacteriophage fMptD. This work demonstrates there is potential for L. salivarius as a viable antigen delivery vehicle for MAP, which may provide an effective mucosal vaccine against Johne's disease.

[Optimization of prokaryotic expression conditions of Leptospira interrogans trigeminy genus-specific protein antigen based on surface response analysis].

Science.gov (United States)

Wang, Jiang; Luo, Dongjiao; Sun, Aihua; Yan, Jie

2008-07-01

Lipoproteins LipL32 and LipL21 and transmembrane protein OMPL1 have been confirmed as the superficial genus-specific antigens of Leptospira interrogans, which can be used as antigens for developing a universal genetic engineering vaccine. In order to obtain high expression of an artificial fusion gene lipL32/1-lipL21-ompL1/2, we optimized prokaryotic expression conditions. We used surface response analysis based on the central composite design to optimize culture conditions of a new antigen protein by recombinant Escherichia coli DE3.The culture conditions included initial pH, induction start time, post-induction time, Isopropyl beta-D-thiogalactopyranoside (IPTG) concentration, and temperature. The maximal production of antigen protein was 37.78 mg/l. The optimal culture conditions for high recombinant fusion protein was determined: initial pH 7.9, induction start time 2.5 h, a post-induction time of 5.38 h, 0.20 mM IPTG, and a post-induction temperature of 31 degrees C. Surface response analysis based on CCD increased the target production. This statistical method reduced the number of experiments required for optimization and enabled rapid identification and integration of the key culture condition parameters for optimizing recombinant protein expression.

MAGE-A1, GAGE and NY-ESO-1 cancer/testis antigen expression during human gonadal development

DEFF Research Database (Denmark)

Gjerstorff, Morten F; Kock, Kirsten; Nielsen, Ole

2007-01-01

BACKGROUND: Cancer/testis antigens (CTAs) are expressed in several cancers and during normal adult male germ cell differentiation. Little is known about their role in fetal development of human germ cells. METHODS: We examined expression of the CTAs MAGE-A1, GAGE and NY-ESO-1 in fetal gonads...

Overview of Plant-Made Vaccine Antigens against Malaria

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Marina Clemente

2012-01-01

Full Text Available This paper is an overview of vaccine antigens against malaria produced in plants. Plant-based expression systems represent an interesting production platform due to their reduced manufacturing costs and high scalability. At present, different Plasmodium antigens and expression strategies have been optimized in plants. Furthermore, malaria antigens are one of the few examples of eukaryotic proteins with vaccine value expressed in plants, making plant-derived malaria antigens an interesting model to analyze. Up to now, malaria antigen expression in plants has allowed the complete synthesis of these vaccine antigens, which have been able to induce an active immune response in mice. Therefore, plant production platforms offer wonderful prospects for improving the access to malaria vaccines.

Recombinant Salmonella Expressing Burkholderia mallei LPS O Antigen Provides Protection in a Murine Model of Melioidosis and Glanders.

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Moustafa, Dina A; Scarff, Jennifer M; Garcia, Preston P; Cassidy, Sara K B; DiGiandomenico, Antonio; Waag, David M; Inzana, Thomas J; Goldberg, Joanna B

2015-01-01

Burkholderia pseudomallei and Burkholderia mallei are the etiologic agents of melioidosis and glanders, respectively. These bacteria are highly infectious via the respiratory route and can cause severe and often fatal diseases in humans and animals. Both species are considered potential agents of biological warfare; they are classified as category B priority pathogens. Currently there are no human or veterinary vaccines available against these pathogens. Consequently efforts are directed towards the development of an efficacious and safe vaccine. Lipopolysaccharide (LPS) is an immunodominant antigen and potent stimulator of host immune responses. B. mallei express LPS that is structurally similar to that expressed by B. pseudomallei, suggesting the possibility of constructing a single protective vaccine against melioidosis and glanders. Previous studies of others have shown that antibodies against B. mallei or B. pseudomallei LPS partially protect mice against subsequent lethal virulent Burkholderia challenge. In this study, we evaluated the protective efficacy of recombinant Salmonella enterica serovar Typhimurium SL3261 expressing B. mallei O antigen against lethal intranasal infection with Burkholderia thailandensis, a surrogate for biothreat Burkholderia spp. in a murine model that mimics melioidosis and glanders. All vaccine-immunized mice developed a specific antibody response to B. mallei and B. pseudomallei O antigen and to B. thailandensis and were significantly protected against challenge with a lethal dose of B. thailandensis. These results suggest that live-attenuated SL3261 expressing B. mallei O antigen is a promising platform for developing a safe and effective vaccine.

Recombinant Salmonella Expressing Burkholderia mallei LPS O Antigen Provides Protection in a Murine Model of Melioidosis and Glanders.

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Dina A Moustafa

Full Text Available Burkholderia pseudomallei and Burkholderia mallei are the etiologic agents of melioidosis and glanders, respectively. These bacteria are highly infectious via the respiratory route and can cause severe and often fatal diseases in humans and animals. Both species are considered potential agents of biological warfare; they are classified as category B priority pathogens. Currently there are no human or veterinary vaccines available against these pathogens. Consequently efforts are directed towards the development of an efficacious and safe vaccine. Lipopolysaccharide (LPS is an immunodominant antigen and potent stimulator of host immune responses. B. mallei express LPS that is structurally similar to that expressed by B. pseudomallei, suggesting the possibility of constructing a single protective vaccine against melioidosis and glanders. Previous studies of others have shown that antibodies against B. mallei or B. pseudomallei LPS partially protect mice against subsequent lethal virulent Burkholderia challenge. In this study, we evaluated the protective efficacy of recombinant Salmonella enterica serovar Typhimurium SL3261 expressing B. mallei O antigen against lethal intranasal infection with Burkholderia thailandensis, a surrogate for biothreat Burkholderia spp. in a murine model that mimics melioidosis and glanders. All vaccine-immunized mice developed a specific antibody response to B. mallei and B. pseudomallei O antigen and to B. thailandensis and were significantly protected against challenge with a lethal dose of B. thailandensis. These results suggest that live-attenuated SL3261 expressing B. mallei O antigen is a promising platform for developing a safe and effective vaccine.

Humoral immunity provides resident intestinal eosinophils access to luminal antigen via eosinophil-expressed low affinity Fc gamma receptors

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Smith, Kalmia M.; Rahman, Raiann S.; Spencer, Lisa A.

2016-01-01

Eosinophils are native to the healthy gastrointestinal tract, and are associated with inflammatory diseases likely triggered by exposure to food allergens (e.g. food allergies and eosinophilic gastrointestinal disorders). In models of allergic respiratory diseases and in vitro studies, direct antigen engagement elicits eosinophil effector functions including degranulation and antigen presentation. However, it was not known whether intestinal tissue eosinophils that are separated from luminal food antigens by a columnar epithelium might similarly engage food antigens. Using an intestinal ligated loop model in mice, here we determined that resident intestinal eosinophils acquire antigen from the lumen of antigen-sensitized but not naïve mice in vivo. Antigen acquisition was immunoglobulin-dependent; intestinal eosinophils were unable to acquire antigen in sensitized immunoglobulin-deficient mice, and passive immunization with immune serum or antigen-specific IgG was sufficient to enable intestinal eosinophils in otherwise naïve mice to acquire antigen in vivo. Intestinal eosinophils expressed low affinity IgG receptors, and the activating receptor FcγRIII was necessary for immunoglobulin-mediated acquisition of antigens by isolated intestinal eosinophils in vitro. Our combined data suggest that intestinal eosinophils acquire lumen-derived food antigens in sensitized mice via FcγRIII antigen focusing, and may therefore participate in antigen-driven secondary immune responses to oral antigens. PMID:27683752

Ultraviolet radiation (UVR) induces cell-surface Ro/SSA antigen expression by human keratinocytes in vitro: a possible mechanism for the UVR induction of cutaneous lupus lesions

International Nuclear Information System (INIS)

Jones, S.K.

1992-01-01

Antinuclear antibodies are useful markers of connective tissue disease. In this study, UVB but not UVA induced the expression of Ro/SSA antigen on keratinocyte surfaces in vitro. This expression was also found with the extractable nuclear antigens RnP and Sm, but not with single or double-stranded DNA. The expression was prevented by blocking protein synthesis, suggesting that it was an active process. The results suggest that UVB exposure may result in the expression of Ro/SSA antigen on the surfaces of basal keratinocytes in vivo. This antigen could then bind circulating antibody leading to the cutaneous lesions in neonatal and subacute cutaneous lupus erythematosus. (Author)

Chronic Hepatitis B Virus Infection: The Relation between Hepatitis B Antigen Expression, Telomere Length, Senescence, Inflammation and Fibrosis.

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Phaedra M Tachtatzis

Full Text Available Chronic Hepatitis B virus (HBV infection can lead to the development of chronic hepatitis, cirrhosis and hepatocellular carcinoma. We hypothesized that HBV might accelerate hepatocyte ageing and investigated the effect of HBV on hepatocyte cell cycle state and biological age. We also investigated the relation between inflammation, fibrosis and cell cycle phase.Liver samples from patients with chronic HBV (n = 91, normal liver (n = 55 and regenerating liver (n = 15 were studied. Immunohistochemistry for cell cycle phase markers and HBV antigens was used to determine host cell cycle phase. Hepatocyte-specific telomere length was evaluated by quantitative fluorescent in-situ hybridization (Q-FISH in conjunction with hepatocyte nuclear area and HBV antigen expression. The effects of induced cell cycle arrest and induced cellular senescence on HBV production were assessed in vitro.13.7% hepatocytes in chronic HBV had entered cell cycle, but expression of markers for S, G2 and M phase was low compared with regenerating liver. Hepatocyte p21 expression was increased (10.9% in chronic HBV and correlated with liver fibrosis. Mean telomere length was reduced in chronic HBV compared to normal. However, within HBV-affected livers, hepatocytes expressing HBV antigens had longer telomeres. Telomere length declined and hepatocyte nuclear size increased as HBV core antigen (HBcAg expression shifted from the nucleus to cytoplasm. Nuclear co-expression of HBcAg and p21 was not observed. Cell cycle arrest induced in vitro was associated with increased HBV production, in contrast to in vitro induction of cellular senescence, which had no effect.Chronic HBV infection was associated with hepatocyte G1 cell cycle arrest and accelerated hepatocyte ageing, implying that HBV induced cellular senescence. However, HBV replication was confined to biologically younger hepatocytes. Changes in the cellular location of HBcAg may be related to the onset of cellular senescence.

Biological and clinical meaning of myeloid antigen expression in the acute lymphocytic leukemia in children

International Nuclear Information System (INIS)

Marsan Suarez, Vianed; Sanchez Segura, Miriam; Socarras Ferrer, Bertha B; Valle Perez, Lazaro O del

2009-01-01

In 238 children presenting with acute lymphoid leukemia (ALL) authors studied the possible association between the myeloid antigens expression with determined biologic and clinic features at disease onset. The cellular immunophenotyping was performed by ultraimmunocytochemical method. From the total of diagnosed ALLs, the 21,8% were LLA-Mi+. There was a lymphadenopathies predominance (71,2%), splenomegaly (65,4%) and hepatomegaly (57,7%) in patients with LLA-Mi+ and very significant differences (p =0,003, p = 0,0068, and p = 0,000, respectively. There was also alight predominance of mediastinum adenopathies, CNS infiltration and hemorrahagic manifestations in patients with LLA-Mi+, no statistically significant. Results showed that in our patients the myeloid antigen expression on the lymphoid blasts influenced on appearance of determined presentation of morphologic and clinical features in children

Expression of androgen receptor and prostate-specific antigen in male breast carcinoma

International Nuclear Information System (INIS)

Kidwai, Noman; Gong, Yun; Sun, Xiaoping; Deshpande, Charuhas G; Yeldandi, Anjana V; Rao, M Sambasiva; Badve, Sunil

2004-01-01

The androgen-regulated proteins prostate-specific antigen (PSA) and prostate-specific acid phosphatase (PSAP) are present in high concentrations in normal prostate and prostatic cancer and are considered to be tissue-specific to prostate. These markers are commonly used to diagnose metastatic prostate carcinoma at various sites including the male breast. However, expression of these two proteins in tumors arising in tissues regulated by androgens such as male breast carcinoma has not been thoroughly evaluated. In this study we analyzed the expression of PSA, PSAP and androgen receptor (AR) by immunohistochemistry in 26 cases of male breast carcinomas and correlated these with the expression of other prognostic markers. AR, PSA and PSAP expression was observed in 81%, 23% and 0% of carcinomas, respectively. Combined expression of AR and PSA was observed in only four tumors. Although the biological significance of PSA expression in male breast carcinomas is not clear, caution should be exercised when it is used as a diagnostic marker of metastatic prostate carcinoma

A new adenovirus based vaccine vector expressing an Eimeria tenella derived TLR agonist improves cellular immune responses to an antigenic target.

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Daniel M Appledorn

2010-03-01

Full Text Available Adenoviral based vectors remain promising vaccine platforms for use against numerous pathogens, including HIV. Recent vaccine trials utilizing Adenovirus based vaccines expressing HIV antigens confirmed induction of cellular immune responses, but these responses failed to prevent HIV infections in vaccinees. This illustrates the need to develop vaccine formulations capable of generating more potent T-cell responses to HIV antigens, such as HIV-Gag, since robust immune responses to this antigen correlate with improved outcomes in long-term non-progressor HIV infected individuals.In this study we designed a novel vaccine strategy utilizing an Ad-based vector expressing a potent TLR agonist derived from Eimeria tenella as an adjuvant to improve immune responses from a [E1-]Ad-based HIV-Gag vaccine. Our results confirm that expression of rEA elicits significantly increased TLR mediated innate immune responses as measured by the influx of plasma cytokines and chemokines, and activation of innate immune responding cells. Furthermore, our data show that the quantity and quality of HIV-Gag specific CD8(+ and CD8(- T-cell responses were significantly improved when coupled with rEA expression. These responses also correlated with a significantly increased number of HIV-Gag derived epitopes being recognized by host T cells. Finally, functional assays confirmed that rEA expression significantly improved antigen specific CTL responses, in vivo. Moreover, we show that these improved responses were dependent upon improved TLR pathway interactions.The data presented in this study illustrate the potential utility of Ad-based vectors expressing TLR agonists to improve clinical outcomes dependent upon induction of robust, antigen specific immune responses.

Human leukocyte antigen-G polymorphism in relation to expression, function, and disease

DEFF Research Database (Denmark)

Larsen, Margit Hørup; Hviid, Thomas

2009-01-01

Human leukocyte antigen-G (HLA-G) is a nonclassical class Ib molecule belonging to the major histocompatibility complex. HLA-G appears to play a role in the suppression of immune responses and contribute to long-term immune escape or tolerance. The focus of this review is polymorphism in the HLA......-G gene and protein and its possible importance in expression, function, and disease associations....

Expression and the antigenicity of recombinant coat proteins of tungro viruses expressed in Escherichia coli.

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Yee, Siew Fung; Chu, Chia Huay; Poili, Evenni; Sum, Magdline Sia Henry

2017-02-01

Rice tungro disease (RTD) is a recurring disease affecting rice farming especially in the South and Southeast Asia. The disease is commonly diagnosed by visual observation of the symptoms on diseased plants in paddy fields and by polymerase chain reaction (PCR). However, visual observation is unreliable and PCR can be costly. High-throughput as well as relatively cheap detection methods are important for RTD management for screening large number of samples. Due to this, detection by serological assays such as immunoblotting assays and enzyme-linked immunosorbent assay are preferred. However, these serological assays are limited by lack of continuous supply of antibodies as reagents due to the difficulty in preparing sufficient purified virions as antigens. This study aimed to generate and evaluate the reactivity of the recombinant coat proteins of Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV) as alternative antigens to generate antibodies. The genes encoding the coat proteins of both viruses, RTBV (CP), and RTSV (CP1, CP2 and CP3) were cloned and expressed as recombinant fusion proteins in Escherichia coli. All of the recombinant fusion proteins, with the exception of the recombinant fusion protein of the CP2 of RTSV, were reactive against our in-house anti-tungro rabbit serum. In conclusion, our study showed the potential use of the recombinant fusion coat proteins of the tungro viruses as alternative antigens for production of antibodies for diagnostic purposes. Copyright © 2016 Elsevier B.V. All rights reserved.

Multiparameter flow cytometry reveals myelodysplasia-related aberrant antigen expression in myelodysplastic/myeloproliferative neoplasms.

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Kern, Wolfgang; Bacher, Ulrike; Schnittger, Susanne; Alpermann, Tamara; Haferlach, Claudia; Haferlach, Torsten

2013-05-01

Within the myelodysplastic/myeloproliferative neoplasm (MDS/MPN) category of the WHO (2008), only chronic myelomonocytic leukemia was so far evaluated by multiparameter flow cytometry (MFC). To investigate the potential of MFC for MDS/MPNs, unclassifiable (MDS/MPNu), and refractory anemia associated with ring sideroblasts and marked thrombocytosis (RARS-T), we studied 91 patients with these entities (60 males/31 females; 35.3-87.4 years) for MDS-related aberrant immunophenotypes (≥ 2 different cell lineages with ≥ 3 aberrantly expressed antigens). Data were correlated with cytomorphology and cytogenetics. MFC identified MDS-related immunophenotypes in 54/91 (59.3%) of patients. Patients with or without MDS-related immunophenotype did not differ significantly by demographic characteristics, blood values, or median overall survival. MDS-related immunophenotype cases showed a higher number of aberrantly expressed antigens (mean ± SD, 4.9 ± 2.4 vs. 2.0 ± 1.4; P MPNu and RARS-T. MFC therefore may be helpful to separate cases into more "MDS-like" or "MPN-like" subgroups. Copyright © 2012 International Clinical Cytometry Society.

Molecular cloning of cDNA for the human tumor-associated antigen CO-029 and identification of related transmembrane antigens

International Nuclear Information System (INIS)

Szala, S.; Kasai, Yasushi; Steplewski, Z.; Rodeck, U.; Koprowski, H.; Linnenbach, A.J.

1990-01-01

The human tumor-associated antigen CO-029 is a monoclonal antibody-defined cell surface glycoprotein of 27-34 kDa. By using the high-efficiency COS cell expression system, a full-length cDNA clone for CO-029 was isolated. When transiently expressed in COS cells, the cDNA clone directed the synthesis of an antigen reactive to monoclonal antibody CO-029 in mixed hemadsorption and immunoblot assays. Sequence analysis revealed that CO-029 belongs to a family of cell surface antigens that includes the melanoma-associated antigen ME491, the leukocyte cell surface antigen CD37, and the Sm23 antigen of the parasitic helminth Schistosoma mansoni. CO-029 and ME491 antigen expression and the effect of their corresponding monoclonal antibodies on cell growth were compared in human tumor cell lines of various histologic origins

Inhibitory activities of microalgal extracts against Epstein-Barr Virus (EBV antigen expression in lymphoblastoid cells

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Koh Yih Yih

2014-01-01

Full Text Available The inhibitory activities of microalgal extracts against the expression of three EBV antigens, latent membrane protein (LMP1, Epstein-Barr nuclear antigen (EBNA1 and Z Epstein-Barr reactivation activator (ZEBRA were assessed by immunocytochemistry. The observation that the methanol extracts and their fractions from Ankistrodesmus convolutus, Synechococcus elongatus and Spirulina platensis exhibited inhibitory activity against EBV proteins in three Burkitt’s lymphoma cell lines at concentrations as low as 20 μg/ml suggests that microalgae could be a potential source of antiviral compounds against EBV.

Plasmodium falciparum variant STEVOR antigens are expressed in merozoites and possibly associated with erythrocyte invasion

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Petter Michaela

2008-07-01

Full Text Available Abstract Background Plasmodium falciparum STEVOR proteins, encoded by the multicopy stevor gene family have no known biological functions. Their expression and unique locations in different parasite life cycle stages evoke multiple functionalities. Their abundance and hypervariability support a role in antigenic variation. Methods Immunoblotting of total parasite proteins with an anti-STEVOR antibody was used to identify variant antigens of this gene family and to follow changes in STEVOR expression in parasite populations panned on CSA or CD36 receptors. Immunofluorescence assays and immunoelectron microscopy were performed to study the subcellular localization of STEVOR proteins in different parasite stages. The capacity of the antibody to inhibit merozoite invasion of erythrocytes was assessed to determine whether STEVOR variants were involved in the invasion process. Results Antigenic variation of STEVORs at the protein level was observed in blood stage parasites. STEVOR variants were found to be present on the merozoite surface and in rhoptries. An insight into a participation in erythrocyte invasion was gained through an immunofluorescence analysis of a sequence of thin slides representing progressive steps in erythrocyte invasion. An interesting feature of the staining pattern was what appeared to be the release of STEVORs around the invading merozoites. Because the anti-STEVOR antibody did not inhibit invasion, the role of STEVORs in this process remains unknown. Conclusion The localization of STEVOR proteins to the merozoite surface and the rhoptries together with its prevalence as a released component in the invading merozoite suggest a role of these antigens in adhesion and/or immune evasion in the erythrocyte invasion process. These observations would also justify STEVORs for undergoing antigenic variation. Even though a role in erythrocyte invasion remains speculative, an association of members of the STEVOR protein family with

Cell-free translational screening of an expression sequence tag library of Clonorchis sinensis for novel antigen discovery.

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Kasi, Devi; Catherine, Christy; Lee, Seung-Won; Lee, Kyung-Ho; Kim, Yu Jung; Ro Lee, Myeong; Ju, Jung Won; Kim, Dong-Myung

2017-05-01

The rapidly evolving cloning and sequencing technologies have enabled understanding of genomic structure of parasite genomes, opening up new ways of combatting parasite-related diseases. To make the most of the exponentially accumulating genomic data, however, it is crucial to analyze the proteins encoded by these genomic sequences. In this study, we adopted an engineered cell-free protein synthesis system for large-scale expression screening of an expression sequence tag (EST) library of Clonorchis sinensis to identify potential antigens that can be used for diagnosis and treatment of clonorchiasis. To allow high-throughput expression and identification of individual genes comprising the library, a cell-free synthesis reaction was designed such that both the template DNA and the expressed proteins were co-immobilized on the same microbeads, leading to microbead-based linkage of the genotype and phenotype. This reaction configuration allowed streamlined expression, recovery, and analysis of proteins. This approach enabled us to identify 21 antigenic proteins. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:832-837, 2017. © 2017 American Institute of Chemical Engineers.

Heterologous expression of carcinoembryonic antigen in Lactococcus lactis via LcsB-mediated surface displaying system for oral vaccine development.

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Zhang, Xiaowei; Hu, Shumin; Du, Xue; Li, Tiejun; Han, Lanlan; Kong, Jian

2016-12-01

Carcinoembryonic antigen (CEA) is an attractive target for immunotherapy because it is expressed minimally in normal tissue, but is overexpressed in a wide variety of malignant epithelial tissues. Lactic acid bacteria (LABs), widely used in food processes, are attractive candidates for oral vaccination. Thus, we examined whether LABs could be used as a live vaccine vector to deliver CEA antigen. CEA was cloned into an Escherichia coli/Lactococcus lactis shuttle vector pSEC:LEISS under the control of a nisin promoter. For displaying the CEA on the cell surface of the L. lactis strain, the anchor motif LcsB from the S-layer protein of Lactobacillus crispatus was fused with CEA. Intracellular and cell surface expression of the CEA-LcsB fusion was confirmed by western blot analysis. Significantly higher levels of CEA-specific secretory immunoglobulin A in the sera of mice were observed upon oral administration of strain cultures containing the CEA-LcsB fused protein. In addition, the CEA-LcsB antigen group showed a higher spleen index compared to the CEA antigen alone or negative control, demonstrating that surface-displayed CEA antigen could induce a higher immune response. These results provided the first evidence for displaying CEA antigen on the cell surfaces of LABs as oral vaccines against cancer or infectious diseases. Copyright © 2014. Published by Elsevier B.V.

Plasmodium knowlesi Sporozoite Antigen: Expression by Infectious Recombinant Vaccinia Virus

Science.gov (United States)

Smith, Geoffrey L.; Godson, G. Nigel; Nussenzweig, Victor; Nussenzweig, Ruth S.; Barnwell, John; Moss, Bernard

1984-04-01

The gene coding for the circumsporozoite antigen of the malaria parasite Plasmodium knowlesi was inserted into the vaccinia virus genome under the control of a defined vaccinia virus promoter. Cells infected with the recombinant virus synthesized polypeptides of 53,000 to 56,000 daltons that reacted with monoclonal antibody against the repeating epitope of the malaria protein. Furthermore, rabbits vaccinated with the recombinant virus produced antibodies that bound specifically to sporozoites. These data provide evidence for expression of a cloned malaria gene in mammalian cells and illustrate the potential of vaccinia virus recombinants as live malaria vaccines.

Variation of prostate-specific antigen expression in different tumour growth patterns present in prostatectomy specimens

NARCIS (Netherlands)

M.P.W. Gallee; E. Visser-de Jong (E.); J.A.G.M. van der Korput (J. A G M); Th.H. van der Kwast (Theo); F.J.W. ten Kate (Fiebo); F.H. Schröder (Fritz); J. Trapman (Jan)

1990-01-01

textabstractA series of 55 randomly chosen radical prostatectomy specimens was analyzed for expression of prostate-specific antigen (PSA) by immunohistochemical techniques. Tissue sections were selected in such a manner that in addition to glandular benign prostatic hyperplasia (BPH), one or more

Analysis of a cDNA clone expressing a human autoimmune antigen: full-length sequence of the U2 small nuclear RNA-associated B antigen

International Nuclear Information System (INIS)

Habets, W.J.; Sillekens, P.T.G.; Hoet, M.H.; Schalken, J.A.; Roebroek, A.J.M.; Leunissen, J.A.M.; Van de Ven, W.J.M.; Van Venrooij, W.J.

1987-01-01

A U2 small nuclear RNA-associated protein, designated B'', was recently identified as the target antigen for autoimmune sera from certain patients with systemic lupus erythematosus and other rheumatic diseases. Such antibodies enabled them to isolate cDNA clone λHB''-1 from a phage λgt11 expression library. This clone appeared to code for the B'' protein as established by in vitro translation of hybrid-selected mRNA. The identity of clone λHB''-1 was further confirmed by partial peptide mapping and analysis of the reactivity of the recombinant antigen with monospecific and monoclonal antibodies. Analysis of the nucleotide sequence of the 1015-base-pair cDNA insert of clone λHB''-1 revealed a large open reading frame of 800 nucleotides containing the coding sequence for a polypeptide of 25,457 daltons. In vitro transcription of the λHB''-1 cDNA insert and subsequent translation resulted in a protein product with the molecular size of the B'' protein. These data demonstrate that clone λHB''-1 contains the complete coding sequence of this antigen. The deduced polypeptide sequence contains three very hydrophilic regions that might constitute RNA binding sites and/or antigenic determinants. These findings might have implications both for the understanding of the pathogenesis of rheumatic diseases as well as for the elucidation of the biological function of autoimmune antigens

Cell-Free Expression and In Situ Immobilization of Parasite Proteins from Clonorchis sinensis for Rapid Identification of Antigenic Candidates.

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Christy Catherine

Full Text Available Progress towards genetic sequencing of human parasites has provided the groundwork for a post-genomic approach to develop novel antigens for the diagnosis and treatment of parasite infections. To fully utilize the genomic data, however, high-throughput methodologies are required for functional analysis of the proteins encoded in the genomic sequences. In this study, we investigated cell-free expression and in situ immobilization of parasite proteins as a novel platform for the discovery of antigenic proteins. PCR-amplified parasite DNA was immobilized on microbeads that were also functionalized to capture synthesized proteins. When the microbeads were incubated in a reaction mixture for cell-free synthesis, proteins expressed from the microbead-immobilized DNA were instantly immobilized on the same microbeads, providing a physical linkage between the genetic information and encoded proteins. This approach of in situ expression and isolation enables streamlined recovery and analysis of cell-free synthesized proteins and also allows facile identification of the genes coding antigenic proteins through direct PCR of the microbead-bound DNA.

Spleen-dependent regulation of antigenic variation in malaria parasites: Plasmodium knowlesi SICAvar expression profiles in splenic and asplenic hosts.

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Stacey A Lapp

Full Text Available Antigenic variation by malaria parasites was first described in Plasmodium knowlesi, which infects humans and macaque monkeys, and subsequently in P. falciparum, the most virulent human parasite. The schizont-infected cell agglutination (SICA variant proteins encoded by the SICAvar multigene family in P. knowlesi, and Erythrocyte Membrane Protein-1 (EMP-1 antigens encoded by the var multigene family in P. falciparum, are expressed at the surface of infected erythrocytes, are associated with virulence, and serve as determinants of naturally acquired immunity. A parental P. knowlesi clone, Pk1(A+, and a related progeny clone, Pk1(B+1+, derived by an in vivo induced variant antigen switch, were defined by the expression of distinct SICA variant protein doublets of 210/190 and 205/200 kDa, respectively. Passage of SICA[+] infected erythrocytes through splenectomized rhesus monkeys results in the SICA[-] phenotype, defined by the lack of surface expression and agglutination with variant specific antisera.We have investigated SICAvar RNA and protein expression in Pk1(A+, Pk1(B+1+, and SICA[-] parasites. The Pk1(A+ and Pk1(B+1+ parasites express different distinct SICAvar transcript and protein repertoires. By comparison, SICA[-] parasites are characterized by a vast reduction in SICAvar RNA expression, the lack of full-length SICAvar transcript signals on northern blots, and correspondingly, the absence of any SICA protein detected by mass spectrometry.SICA protein expression may be under transcriptional as well as post-transcriptional control, and we show for the first time that the spleen, an organ central to blood-stage immunity in malaria, exerts an influence on these processes. Furthermore, proteomics has enabled the first in-depth characterization of SICA[+] protein phenotypes and we show that the in vivo switch from Pk1(A+ to Pk1(B+1+ parasites resulted in a complete change in SICA profiles. These results emphasize the importance of studying

Rh antigen expression during erythropoeisis: Comparison of cord and adult derived CD34 + cells

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Gupta Namita

2008-01-01

Full Text Available Objectives: Concentrations of O 2 and CO 2 in the fetal circulation differ to that in maternal blood. Previous studies done in algae demonstrate the functional role of Rh antigen as CO 2 transporter. As a preliminary study, it was the aim of this project to compare the expression of Rh polypeptides on cord and adult red blood cell progenitors during ex vivo proliferation and differentiation of CD34 + cells during erythropoeisis. Materials and Methods: CD34 positive hematopoeitic progenitor cells were isolated from umbilical cord blood and adult peripheral blood using an immunomagnetic system and cultured in serum free medium containing erythropoietin in order to compel them along the erythroid lineage. Cultured cells were analyzed for cell surface marker expression by flow cytometry, using monoclonal antibodies to RhAG, Glycophorin A, Rh polypeptides, CD47 and Band 3. Cytospin analysis was also done to study the morphology of cultured cells. Results: The appearance of cell surface markers analyzed on different days of culture varied slightly between samples. There was no evidence to suggest that RhAG, GPA, CD47 and Band 3 expression was any different between adult and cord derived cells. Nevertheless, the results of Rh antigenic expression suggest a reasonable difference between the two groups with adult sample derived cells showing higher and earlier expression than cord blood derived cells. These preliminary findings require further investigation. Conclusion: Comparing the expression of cell surface markers especially Rh polypeptides between adult and cord blood derived erythroid progenitors might assist in discerning their functions and could be valuable in the study of erythropoeisis.

Chloroplast-Derived Vaccine Antigens and Biopharmaceuticals: Expression, Folding, Assembly and Functionality

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Chebolu, S.; Daniell, H.

2009-01-01

Chloroplast genetic engineering offers several advantages, including high levels of transgene expression, transgene containment via maternal inheritance, and multi-gene expression in a single transformation event. Oral delivery is facilitated by hyperexpression of vaccine antigens against cholera, tetanus, anthrax, plague, or canine parvovirus (4%–31% of total soluble protein, TSP) in transgenic chloroplasts (leaves) or non-green plastids (carrots, tomato) as well as the availability of antibiotic free selectable markers or the ability to excise selectable marker genes. Hyperexpression of several therapeutic proteins, including human serum albumin (11.1% TSP), somatotropin (7% TSP), interferon-alpha (19% TSP), interferon-gamma (6% TSP), and antimicrobial peptide (21.5% TSP), facilitates efficient and economic purification. Also, the presence of chaperones and enzymes in chloroplasts facilitates assembly of complex multisubunit proteins and correct folding of human blood proteins with proper disulfide bonds. Functionality of chloroplast-derived vaccine antigens and therapeutic proteins has been demonstrated by several assays, including the macrophage lysis assay, GM1-ganglioside binding assay, protection of HeLA cells or human lung carcinoma cells against encephalomyocarditis virus, systemic immune response, protection against pathogen challenge, and growth or inhibition of cell cultures. Purification of human proinsulin has been achieved using novel purification strategies (inverse temperature transition property) that do not require expensive column chromatography techniques. Thus, transgenic chloroplasts are ideal bioreactors for production of functional human and animal therapeutic proteins in an environmentally friendly manner. PMID:19401820

In vitro stimulation of rabbit T lymphocytes by cells expressing herpes simplex antigens.

Science.gov (United States)

Kapoor, A K; Ling, N R; Nash, A A; Bachan, A; Wildy, P

1982-04-01

Lymphocyte stimulation responses to herpes antigens were studied using virus-infected X-irradiated cells. Rabbits were immunized with herpes simplex virus type 1 (strain HFEM) grown in RK 13 cells. For in vitro stimulation assay BHK21 cells were X-irradiated (15 000 rad) and infected with a high m.o.i. of a temperature-sensitive (ts) mutant (N102) of HFEM strain at the non-permissive temperature (38.5 degrees C) of virus. Virus antigens were expressed on the infected cells and there was no leakage of infectious virus into the medium at 38.5 degrees C. T lymphocytes from rabbits immunized with herpes simplex virus were specifically activated by herpesvirus-infected X-irradiated cells; lymph node cells from rabbits immunized with RK13 cells and from non-immune rabbits showed no proliferative response.

Adoptitive immunotherapy with genetically engineered T lymphocytes modified to express chimeric antigen receptors

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A. А. Pavlova

2017-01-01

Full Text Available Significant mortality due to oncological diseases as a whole, and oncohematological diseases in particular, motivates scientific and medical community to develop new treatment methods. One of the newest methods is adoptive cell therapy using patient’s own T-cells modified to express chimeric antigen receptors (CAR to tumor-specific antigens. Despite high cost and side effects of treatment, promising clinical trials even in patients with advanced disease allow to anticipate successful use of this method in clinical practice.The article includes a review of the main principles of this technique, published results of clinical studies of CAR T-cells with a focus on CD19 gene targeting, complications of this therapy, mechanisms of tumor resistance to CAR T-cells, and potential ways to overcome it.

Expression and Immunogenicity of the Mycobacterial Ag85B/ESAT-6 Antigens Produced in Transgenic Plants by Elastin-Like Peptide Fusion Strategy

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Doreen Manuela Floss

2010-01-01

Full Text Available This study explored a novel system combining plant-based production and the elastin-like peptide (ELP fusion strategy to produce vaccinal antigens against tuberculosis. Transgenic tobacco plants expressing the mycobacterial antigens Ag85B and ESAT-6 fused to ELP (TBAg-ELP were generated. Purified TBAg-ELP was obtained by the highly efficient, cost-effective, inverse transition cycling (ICT method and tested in mice. Furthermore, safety and immunogenicity of the crude tobacco leaf extracts were assessed in piglets. Antibodies recognizing mycobacterial antigens were produced in mice and piglets. A T-cell immune response able to recognize the native mycobacterial antigens was detected in mice. These findings showed that the native Ag85B and ESAT-6 mycobacterial B- and T-cell epitopes were conserved in the plant-expressed TBAg-ELP. This study presents the first results of an efficient plant-expression system, relying on the elastin-like peptide fusion strategy, to produce a safe and immunogenic mycobacterial Ag85B-ESAT-6 fusion protein as a potential vaccine candidate against tuberculosis.

Induction of carcinoembryonic antigen expression in a three-dimensional culture system

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Jessup, J. M.; Brown, D.; Fitzgerald, W.; Ford, R. D.; Nachman, A.; Goodwin, T. J.; Spaulding, G.

1994-01-01

MIP-101 is a poorly differentiated human colon carcinoma cell line established from ascites that produces minimal amounts of carcinoembryonic antigen (CEA), a 180 kDa glycoprotein tumor marker, and nonspecific cross-reacting antigen (NCA), a related protein that has 50 and 90 kDa isoforms, in vitro in monolayer culture. MIP-101 produces CEA when implanted into the peritoneum of nude mice but not when implanted into subcutaneous tissue. We tested whether MIP-101 cells may be induced to express CEA when cultured on microcarrier beads in three-dimensional cultures, either in static cultures as non-adherent aggregates or under dynamic conditions in a NASA-designed low shear stress bioreactor. MIP- 101 cells proliferated well under all three conditions and increased CEA and NCA production 3 - 4 fold when grown in three-dimensional cultures compared to MIP-101 cells growing logarithmically in monolayers. These results suggest that three-dimensional growth in vitro simulates tumor function in vivo and that three-dimensional growth by itself may enhance production of molecules that are associated with the metastatic process.

Alphavirus Replicon DNA Vectors Expressing Ebola GP and VP40 Antigens Induce Humoral and Cellular Immune Responses in Mice

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Shoufeng Ren

2018-01-01

Full Text Available Ebola virus (EBOV causes severe hemorrhagic fevers in humans, and no approved therapeutics or vaccine is currently available. Glycoprotein (GP is the major protective antigen of EBOV, and can generate virus-like particles (VLPs by co-expression with matrix protein (VP40. In this study, we constructed a recombinant Alphavirus Semliki Forest virus (SFV replicon vector DREP to express EBOV GP and matrix viral protein (VP40. EBOV VLPs were successfully generated and achieved budding from 293 cells after co-transfection with DREP-based GP and VP40 vectors (DREP-GP+DREP-VP40. Vaccination of BALB/c mice with DREP-GP, DREP-VP40, or DREP-GP+DREP-VP40 vectors, followed by immediate electroporation resulted in a mixed IgG subclass production, which recognized EBOV GP and/or VP40 proteins. This vaccination regimen also led to the generation of both Th1 and Th2 cellular immune responses in mice. Notably, vaccination with DREP-GP and DREP-VP40, which produces both GP and VP40 antigens, induced a significantly higher level of anti-GP IgG2a antibody and increased IFN-γ secreting CD8+ T-cell responses relative to vaccination with DREP-GP or DREP-VP40 vector alone. Our study indicates that co-expression of GP and VP40 antigens based on the SFV replicon vector generates EBOV VLPs in vitro, and vaccination with recombinant DREP vectors containing GP and VP40 antigens induces Ebola antigen-specific humoral and cellular immune responses in mice. This novel approach provides a simple and efficient vaccine platform for Ebola disease prevention.

Manufacture of clinical-grade CD19-specific T cells stably expressing chimeric antigen receptor using Sleeping Beauty system and artificial antigen presenting cells.

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Harjeet Singh

Full Text Available Adoptive transfer of T cells expressing a CD19-specific chimeric antigen receptor (CAR is being evaluated in multiple clinical trials. Our current approach to adoptive immunotherapy is based on a second generation CAR (designated CD19RCD28 that signals through a CD28 and CD3-ζ endodomain. T cells are electroporated with DNA plasmids from the Sleeping Beauty (SB transposon/transposase system to express this CAR. Stable integrants of genetically modified T cells can then be retrieved when co-cultured with designer artificial antigen presenting cells (aAPC in the presence of interleukin (IL-2 and 21. Here, we reveal how the platform technologies of SB-mediated transposition and CAR-dependent propagation on aAPC were adapted for human application. Indeed, we have initiated clinical trials in patients with high-risk B-lineage malignancies undergoing autologous and allogeneic hematopoietic stem-cell transplantation (HSCT. We describe the process to manufacture clinical grade CD19-specific T cells derived from healthy donors. Three validation runs were completed in compliance with current good manufacturing practice for Phase I/II trials demonstrating that by 28 days of co-culture on γ-irradiated aAPC ∼10(10 T cells were produced of which >95% expressed CAR. These genetically modified and propagated T cells met all quality control testing and release criteria in support of infusion.

Cloning, sequence analysis, and expression of the large subunit of the human lymphocyte activation antigen 4F2

International Nuclear Information System (INIS)

Lumadue, J.A.; Glick, A.B.; Ruddle, F.H.

1987-01-01

Among the earliest expressed antigens on the surface of activated human lymphocytes is the surface antigen 4F2. The authors have used DNA-mediated gene transfer and fluorescence-activated cell sorting to obtain cell lines that contain the gene encoding the large subunit of the human 4F2 antigen in a mouse L-cell background. Human DNAs cloned from these cell lines were subsequently used as hybridization probes to isolate a full-length cDNA clone expressing 4F2. Sequence analysis of the coding region has revealed an amino acid sequence of 529 residues. Hydrophobicity plotting has predicted a probable structure for the protein that includes an external carboxyl terminus, an internal leader sequence, a single hydrophobic transmembrane domain, and two possible membrane-associated domains. The 4F2 cDNA detects a single 1.8-kilobase mRNA in T-cell and B-cell lines. RNA gel blot analysis of RNA derived from quiescent and serum-stimulated Swiss 3T3 fibroblasts reveals a cell-cycle modulation of 4F2 gene expression: the mRNA is present in quiescent fibroblasts but increases 8-fold 24-36 hr after stimulation, at the time of maximal DNA synthesis

Cloning, sequence analysis, and expression of the large subunit of the human lymphocyte activation antigen 4F2

Energy Technology Data Exchange (ETDEWEB)

Lumadue, J.A.; Glick, A.B.; Ruddle, F.H.

1987-12-01

Among the earliest expressed antigens on the surface of activated human lymphocytes is the surface antigen 4F2. The authors have used DNA-mediated gene transfer and fluorescence-activated cell sorting to obtain cell lines that contain the gene encoding the large subunit of the human 4F2 antigen in a mouse L-cell background. Human DNAs cloned from these cell lines were subsequently used as hybridization probes to isolate a full-length cDNA clone expressing 4F2. Sequence analysis of the coding region has revealed an amino acid sequence of 529 residues. Hydrophobicity plotting has predicted a probable structure for the protein that includes an external carboxyl terminus, an internal leader sequence, a single hydrophobic transmembrane domain, and two possible membrane-associated domains. The 4F2 cDNA detects a single 1.8-kilobase mRNA in T-cell and B-cell lines. RNA gel blot analysis of RNA derived from quiescent and serum-stimulated Swiss 3T3 fibroblasts reveals a cell-cycle modulation of 4F2 gene expression: the mRNA is present in quiescent fibroblasts but increases 8-fold 24-36 hr after stimulation, at the time of maximal DNA synthesis.

STUDIES IN DYNAMICS OF APOPTOSIS-RELATED SURFACE ANTIGEN (CD95 EXPRESSION ON NEUTROPHILS FROM CERVICAL AND VAGINAL SECRETIONS IN WOMEN WITH CHLAMIDIA INFECTION

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O. A. Giesinger

2010-01-01

Full Text Available CD95 (Fas/APO-1 antigen expression was studied on the surface of neutrophil granulocytes from cervical secretions. Sixty-five female patients with established Chlamydia infection were found to have an increased CD95+ antigen expression following basic therapy. CD95+ receptors on neutrophils in the patients with Chlamydia infection have been shown to return to normal levels following a combined magnetic laser treatment.

Temporal expression and localization patterns of variant surface antigens in clinical Plasmodium falciparum isolates during erythrocyte schizogony.

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Anna Bachmann

Full Text Available Avoidance of antibody-mediated immune recognition allows parasites to establish chronic infections and enhances opportunities for transmission. The human malaria parasite Plasmodium falciparum possesses a number of multi-copy gene families, including var, rif, stevor and pfmc-2tm, which encode variant antigens believed to be expressed on the surfaces of infected erythrocytes. However, most studies of these antigens are based on in vitro analyses of culture-adapted isolates, most commonly the laboratory strain 3D7, and thus may not be representative of the unique challenges encountered by P. falciparum in the human host. To investigate the expression of the var, rif-A, rif-B, stevor and pfmc-2tm family genes under conditions that mimic more closely the natural course of infection, ex vivo clinical P. falciparum isolates were analyzed using a novel quantitative real-time PCR approach. Expression patterns in the clinical isolates at various time points during the first intraerythrocytic developmental cycle in vitro were compared to those of strain 3D7. In the clinical isolates, in contrast to strain 3D7, there was a peak of expression of the multi-copy gene families rif-A, stevor and pfmc-2tm at the young ring stage, in addition to the already known expression peak in trophozoites. Furthermore, most of the variant surface antigen families were overexpressed in the clinical isolates relative to 3D7, with the exception of the pfmc-2tm family, expression of which was higher in 3D7 parasites. Immunofluorescence analyses performed in parallel revealed two stage-dependent localization patterns of RIFIN, STEVOR and PfMC-2TM. Proteins were exported into the infected erythrocyte at the young trophozoite stage, whereas they remained inside the parasite membrane during schizont stage and were subsequently observed in different compartments in the merozoite. These results reveal a complex pattern of expression of P. falciparum multi-copy gene families during

Cancer-testis antigen expression and immunogenicity in AL amyloidosis

International Nuclear Information System (INIS)

Rosenzweig, M A; Landau, H; Seldin, D; O'Hara, C; Girnius, S; Hanson, N; Frosina, D; Sedrak, C; Arcila, M; Comenzo, R L; Giralt, S; Gnjatic, S; Jungbluth, A A; Koehne, G

2012-01-01

Light-chain amyloidosis (AL) is a plasma cell dyscrasia closely related to multiple myeloma. In multiple myeloma, the cancer-testis antigens (CTAs) CT7 (MAGE-C1), CT10 (MAGE-C2) and MAGE-A CTAs are expressed in up to 80% of cases. In this study, we investigated the expression and immunogenicity of several CTAs in patients with AL amyloidosis in a total of 38 bone marrow specimens by employing standard immunohistochemistry techniques on paraffin-embedded archival tissues. Plasma samples from 35 patients (27 with matched bone marrow samples) were also analyzed by ELISA for sero reactivity to a group of full-length CTA proteins. CT7 was present in 25/38 (66%) while CT10 was demonstrated in 3/38 and GAGE in 1/38 AL amyloid cases. The expression pattern was mostly focal. There were no significant differences with regard to organ involvement, response to treatment, or prognosis in CTA positive compared to negative cases. None of the specimens showed spontaneous humoral immunity to CT7, but sero reactivity was observed in individual patients to other CTAs. This study identifies CT7 as the prevalent CTA in plasma cells of patients with AL amyloidosis. Further analyses determining the biology of CTAs in AL amyloidosis and their value as potential targets for immunotherapy are warranted

NY-BR-1 Antigen Expression and anti-NY-BR-1 IgG in Egyptian Breast Cancer Patients: Clinicopathological and Prognostic Significance.

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Abu El-Nazar, Salma Y; Ghazy, Amany A; Ghoneim, Hossam E; Zoheir, Malak; Ahmed, Ahmed S; Sorour, Sally S; Abouelella, Amira M

2015-01-01

Breast cancer is the most common gynecological malignancy in the world. In Egypt, it ranks the first among female malignancies with incidence of 37.7%. Over the last decades, the integration of prognostic and predictive markers in treatment decisions has led to more individualized and optimized therapy. NY-BR-1 antigen has been shown to be frequently expressed in breast cancers. The study aimed to assess the tissue expression of NY-BR-1 antigen and serum IgG antibody to this antigen in Egyptian breast cancer females. The study was conducted on 60 females (10 healthy, 10 having benign breast lesions, 40 with malignant breast cancer). NY-BR-1 Ag expression was evaluated by immunohistochemistry and anti-NY-BR-1 IgG was assessed by ELISA. Results revealed a significant difference in NY-BR-1 Ag expression between benign and malignant breast cancer patients. There was a significant correlation between NY-BR-1 antigen expression and estrogen receptor's status (P = 0.019), stage of the disease (P = 0.008), menopausal status (P = 0.008), lymph node involvement (P = 0.022) and anti-NY-BR-1 IgG (P = 0.032) among the studied individuals. In addition, there was a statistically significant increase in anti-NY-BR-1 IgG O.D. results among malignant breast cancer group. It is correlated with tumor type (P < 0.001) and progesterone receptor status (P = 0.038). In conclusion, our work may represent a step towards identification of a new prognostic marker specific for breast cancer.

BRAF inhibition is associated with enhanced melanoma antigen expression and a more favorable tumor microenvironment in patients with metastatic melanoma

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Frederick, Dennie Tompers; Piris, Adriano; Cogdill, Alexandria P.; Cooper, Zachary A.; Lezcano, Cecilia; Ferrone, Cristina R.; Mitra, Devarati; Boni, Andrea; Newton, Lindsay P.; Liu, Chengwen; Peng, Weiyi; Sullivan, Ryan J; Lawrence, Donald P.; Hodi, F. Stephen; Overwijk, Willem W.; Lizée, Gregory; Murphy, George F.; Hwu, Patrick; Flaherty, Keith T.; Fisher, David E.; Wargo, Jennifer A.

2013-01-01

Purpose To evaluate the effects BRAF inhibition on the tumor microenvironment in patients with metastatic melanoma. Experimental Design Thirty-five biopsies were collected from 16 patients with metastatic melanoma pretreatment (day 0) and at 10-14 days after initiation of treatment with either BRAF inhibitor alone (vemurafenib) or BRAF + MEK inhibition (dabrafenib + trametinib), and were also taken at time of progression. Biopsies were analyzed for melanoma antigens, T cell markers, and immunomodulatory cytokines. Results Treatment with either BRAF inhibitor alone or BRAF + MEK inhibitor was associated with an increased expression of melanoma antigens and an increase in CD8+ T cell infiltrate. This was also associated with a decrease in immunosuppressive cytokines (IL-6 & IL-8) and an increase in markers of T cell cytotoxicity. Interestingly, expression of exhaustion markers TIM-3 and PD1 and the immunosuppressive ligand PDL1 were increased on treatment. A decrease in melanoma antigen expression and CD8 T cell infiltrate was noted at time of progression on BRAF inhibitor alone, and was reversed with combined BRAF and MEK inhibition. Conclusions Together, this data suggests that treatment with BRAF inhibition enhances melanoma antigen expression and facilitates T cell cytotoxicity and a more favorable tumor microenvironment, providing support for potential synergy of BRAF-targeted therapy and immunotherapy. Interestingly, markers of T cell exhaustion and the immunosuppressive ligand PDL1 are also increased with BRAF inhibition, further implying that immune checkpoint blockade may be critical in augmenting responses to BRAF-targeted therapy in patients with melanoma. PMID:23307859

Simultaneous cytoplasmic and nuclear protein expression of melanoma antigen-A family and NY-ESO-1 cancer-testis antigens represents an independent marker for poor survival in head and neck cancer.

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Laban, Simon; Atanackovic, Djordje; Luetkens, Tim; Knecht, Rainald; Busch, Chia-Jung; Freytag, Marcus; Spagnoli, Giulio; Ritter, Gerd; Hoffmann, Thomas K; Knuth, Alexander; Sauter, Guido; Wilczak, Waldemar; Blessmann, Marco; Borgmann, Kerstin; Muenscher, Adrian; Clauditz, Till S

2014-09-01

The prognosis of head and neck squamous cell carcinoma (HNSCC) patients remains poor. The identification of high-risk subgroups is needed for the development of custom-tailored therapies. The expression of cancer-testis antigens (CTAs) has been linked to a worse prognosis in other cancer types; however, their prognostic value in HNSCC is unclear because only few patients have been examined and data on CTA protein expression are sparse. A tissue microarray consisting of tumor samples from 453 HNSCC patients was evaluated for the expression of CTA proteins using immunohistochemistry. Frequency of expression and the subcellular expression pattern (nuclear, cytoplasmic, or both) was recorded. Protein expression of melanoma antigen (MAGE)-A family CTA, MAGE-C family CTA and NY-ESO-1 was found in approximately 30, 7 and 4% of tumors, respectively. The subcellular expression pattern in particular had a marked impact on the patients' prognosis. Median overall survival (OS) of patients with (i) simultaneous cytoplasmic and nuclear expression compared to (ii) either cytoplasmic or nuclear expression and (iii) negative patients was 23.0 versus 109.0 versus 102.5 months, for pan-MAGE (p family members or NY-ESO-1 represent a subgroup with an extraordinarily poor survival. The development of immunotherapeutic strategies targeting these CTA may, therefore, be a promising approach to improve the outcome of HNSCC patients. © 2014 UICC.

Expression of the small T antigen of Lymphotropic Papovavirus is sufficient to transform primary mouse embryo fibroblasts

Energy Technology Data Exchange (ETDEWEB)

Gupta, Tushar; Robles, Maria Teresa Sáenz [Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260 (United States); Schowalter, Rachel M.; Buck, Christopher B. [Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892-4263 (United States); Pipas, James M., E-mail: pipas@pitt.edu [Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260 (United States)

2016-01-15

Polyomaviruses induce cell proliferation and transformation through different oncoproteins encoded within the early region (ER): large T antigen (LT), small T antigen (sT) and, in some cases, additional components. Each virus utilizes different mechanisms to achieve transformation. For instance, the LTs of Simian virus 40 (SV40), BK and/or JC virus can induce transformation; but Merkel Cell Polyomavirus (MCPyV) requires expression of sT. Lymphotropic Papovavirus (LPV) is closely related to Human Polyomavirus 9 (HuPyV9) and, under similar conditions, mice expressing LPV.ER exhibit higher rates of tumor formation than mice expressing SV40.ER. We have investigated the contributions of individual LPV.ER components to cell transformation. In contrast to SV40, LPV.ER transforms mouse embryonic fibroblasts (MEFs), but expression of LPV LT is insufficient to transform MEFs. Furthermore, LPV sT induces immortalization and transformation of MEFs. Thus, in the case of LPV, sT is the main mediator of oncogenesis. - Highlights: • Characterization of early region products from the Lymphotropic Polyomavirus (LPV). • On its own, sT immortalizes and transforms mouse primary cells, and is able to block p53 activation. • Combined LT and sT expression induces a greater rate of proliferation than either LT or sT alone.

Cancer testis antigens and NY-BR-1 expression in primary breast cancer: prognostic and therapeutic implications.

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Balafoutas, Dimitrios; zur Hausen, Axel; Mayer, Sebastian; Hirschfeld, Marc; Jaeger, Markus; Denschlag, Dominik; Gitsch, Gerald; Jungbluth, Achim; Stickeler, Elmar

2013-06-03

Cancer-testis antigens (CTA) comprise a family of proteins, which are physiologically expressed in adult human tissues solely in testicular germ cells and occasionally placenta. However, CTA expression has been reported in various malignancies. CTAs have been identified by their ability to elicit autologous cellular and or serological immune responses, and are considered potential targets for cancer immunotherapy. The breast differentiation antigen NY-BR-1, expressed specifically in normal and malignant breast tissue, has also immunogenic properties. Here we evaluated the expression patterns of CTAs and NY-BR-1 in breast cancer in correlation to clinico-pathological parameters in order to determine their possible impact as prognostic factors. The reactivity pattern of various mAbs (6C1, MA454, M3H67, 57B, E978, GAGE #26 and NY-BR-1 #5) were assessed by immunohistochemistry in a tissue micro array series of 210 randomly selected primary invasive breast cancers in order to study the diversity of different CTAs (e.g. MAGE-A, NY-ESO-1, GAGE) and NY-BR-1. These expression data were correlated to clinico-pathological parameters and outcome data including disease-free and overall survival. Expression of at least one CTA was detectable in the cytoplasm of tumor cells in 37.2% of the cases. NY-BR-1 expression was found in 46.6% of tumors, respectively. Overall, CTA expression seemed to be linked to adverse prognosis and M3H67 immunoreactivity specifically was significantly correlated to shorter overall and disease-free survival (p=0.000 and 0.024, respectively). Our findings suggest that M3H67 immunoreactivity could serve as potential prognostic marker in primary breast cancer patients. The exclusive expression of CTAs in tumor tissues as well as the frequent expression of NY-BR-1 could define new targets for specific breast cancer therapies.

Plasmodium falciparum parasites expressing pregnancy-specific variant surface antigens adhere strongly to the choriocarcinoma cell line BeWo

DEFF Research Database (Denmark)

Haase, Rikke N; Megnekou, Rosette; Lundquist, Maja

2006-01-01

Placenta-sequestering Plasmodium falciparum parasites causing pregnancy-associated malaria express pregnancy-specific variant surface antigens (VSA(PAM)). We report here that VSA(PAM)-expressing patient isolates adhere strongly to the choriocarcinoma cell line BeWo and that the BeWo line can...... be used to efficiently select for VSA(PAM) expression in vitro....

Multiepitope fusion antigen induces broadly protective antibodies that prevent adherence of Escherichia coli strains expressing colonization factor antigen I (CFA/I), CFA/II, and CFA/IV.

Science.gov (United States)

Ruan, Xiaosai; Knudsen, David E; Wollenberg, Katie M; Sack, David A; Zhang, Weiping

2014-02-01

Diarrhea is the second leading cause of death in children younger than 5 years and continues to be a major threat to global health. Enterotoxigenic Escherichia coli (ETEC) strains are the most common bacteria causing diarrhea in developing countries. ETEC strains are able to attach to host small intestinal epithelial cells by using bacterial colonization factor antigen (CFA) adhesins. This attachment helps to initiate the diarrheal disease. Vaccines that induce antiadhesin immunity to block adherence of ETEC strains that express immunologically heterogeneous CFA adhesins are expected to protect against ETEC diarrhea. In this study, we created a CFA multiepitope fusion antigen (MEFA) carrying representative epitopes of CFA/I, CFA/II (CS1, CS2, and CS3), and CFA/IV (CS4, CS5, and CS6), examined its immunogenicity in mice, and assessed the potential of this MEFA as an antiadhesin vaccine against ETEC. Mice intraperitoneally immunized with this CFA MEFA exhibited no adverse effects and developed immune responses to CFA/I, CFA/II, and CFA/IV adhesins. Moreover, after incubation with serum of the immunized mice, ETEC or E. coli strains expressing CFA/I, CFA/II, or CFA/IV adhesins were significantly inhibited in adherence to Caco-2 cells. Our results indicated this CFA MEFA elicited antibodies that not only cross-reacted to CFA/I, CFA/II and CFA/IV adhesins but also broadly inhibited adherence of E. coli strains expressing these seven adhesins and suggested that this CFA MEFA could be a candidate to induce broad-spectrum antiadhesin protection against ETEC diarrhea. Additionally, this antigen construction approach (creating an MEFA) may be generally used in vaccine development against heterogenic pathogens.

MRl of prostate cancer antigen expression for diagnosis and immunotherapy.

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Jing Ren

Full Text Available BACKGROUND: Tumor antigen (TA-targeted monoclonal antibody (mAb immunotherapy can be effective for the treatment of a broad range of cancer etiologies; however, these approaches have demonstrated variable clinical efficacy for the treatment of patients with prostate cancer (PCa. An obstacle currently impeding translational progress has been the inability to quantify the mAb dose that reaches the tumor site and binds to the targeted TAs. The coupling of mAb to nanoparticle-based magnetic resonance imaging (MRI probes should permit in vivo measurement of patient-specific biodistributions; these measurements could facilitate future development of novel dosimetry paradigms wherein mAb dose is titrated to optimize outcomes for individual patients. METHODS: The prostate stem cell antigen (PSCA is broadly expressed on the surface of prostate cancer (PCa cells. Anti-human PSCA monoclonal antibodies (mAb 7F5 were bound to Au/Fe(3O(4 (GoldMag nanoparticles (mAb 7F5@GoldMag to serve as PSCA-specific theragnostic MRI probe permitting visualization of mAb biodistribution in vivo. First, the antibody immobilization efficiency of the GoldMag particles and the efficacy for PSCA-specific binding was assessed. Next, PC-3 (prostate cancer with PSCA over-expression and SMMC-7721 (hepatoma cells without PSCA expression tumor-bearing mice were injected with mAb 7F5@GoldMag for MRI. MRI probe biodistributions were assessed at increasing time intervals post-infusion; therapy response was evaluated with serial tumor volume measurements. RESULTS: Targeted binding of the mAb 7F5@GoldMag probes to PC-3 cells was verified using optical images and MRI; selective binding was not observed for SMMC-7721 tumors. The immunotherapeutic efficacy of the mAb 7F5@GoldMag in PC-3 tumor-bearing mice was verified with significant inhibition of tumor growth compared to untreated control animals. CONCLUSION: Our promising results suggest the feasibility of using mAb 7F5@GoldMag probes as a

Expression of tomato yellow leaf curl virus coat protein using baculovirus expression system and evaluation of its utility as a viral antigen.

Science.gov (United States)

Elgaied, Lamiaa; Salem, Reda; Elmenofy, Wael

2017-08-01

DNA encoding the coat protein (CP) of an Egyptian isolate of tomato yellow leaf curl virus (TYLCV) was inserted into the genome of Autographa californica nucleopolyhedrovirus (AcNPV) under the control of polyhedrin promoter. The generated recombinant baculovirus construct harboring the coat protein gene was characterized using PCR analysis. The recombinant coat protein expressed in infected insect cells was used as a coating antigen in an indirect Enzyme-linked immunosorbent assay (ELISA) and dot blot to test its utility for the detection of antibody generated against TYLCV virus particles. The results of ELISA and dot blot showed that the TYLCV-antibodies reacted positively with extracts of infected cells using the recombinant virus as a coating antigen with strong signals as well as the TYLCV infected tomato and beat plant extracts as positive samples. Scanning electron microscope examination showed that the expressed TYLCV coat protein was self-assembled into virus-like particles (VLPs) similar in size and morphology to TYLCV virus particles. These results concluded that, the expressed coat protein of TYLCV using baculovirus vector system is a reliable candidate for generation of anti-CP antibody for inexpensive detection of TYLCV-infected plants using indirect CP-ELISA or dot blot with high specificity.

Nuclear antigen expression by ultraviolet light irradiation - a contribution to the UV-induced autoimmunity

Energy Technology Data Exchange (ETDEWEB)

Wollina, U

1986-05-01

A review is given of nuclear antigen expression due to UVB, UVA, and PUVA. UVB alters DNA resulting in strong immunogenic UVDNA and complementary antibodies. Antibodies to UVDNA cross react with double-stranded DNA. UVDNA plays a (hypothetical) role in the induction of cutaneous lesions in lupus erythematosus (LE). Investigations of SS-A/Ro expression due to UVB seem to be more important under this view. Antibodies against SS-A/Ro are related to an increased photosensitivity in LE. PUVA and UVA are able to induce antinuclear antibodies of unknown specificity. It is likely that PUVA enhances SS-A/Ro expression in vitro. The results are discussed in sense of LE photobiology and unwanted side effects of photo(chemo)therapy in psoriasis. 54 references.

Nuclear antigen expression by ultraviolet light irradiation - a contribution to the UV-induced autoimmunity

International Nuclear Information System (INIS)

Wollina, U.

1986-01-01

A review is given about nuclear antigen expression due to UVB, UVA, and PUVA. UVB alters DNA resulting in strong immunogenic UVDNA and complementary antibodies. Antibodies to UVDNA cross react with double-stranded DNA. UVDNA plays a (hypothetical) role in the induction of cutaneous lesions in lupus erythematosus (LE). Investigations about SS-A/Ro expression due to UVB seem to be more important under this view. Antibodies against SS-A/Ro are related to an increased photosensitivity in LE. PUVA and UVA are able to induce antinuclear antibodies of unknown specificity. It is likely that PUVA enhances SS-A/Ro expression in vitro. The results are discussed in sense of LE photobiology and unwanted side effects of photo(chemo)therapy in psoriasis. (author)

ST6GalNAc-I controls expression of sialyl-Tn antigen in gastrointestinal tissues

DEFF Research Database (Denmark)

Marcos, Nuno T; Bennett, Eric P; Gomes, Joana

2011-01-01

Sialyl-Tn is a simple mucin-type carbohydrate antigen aberrantly expressed in gastrointestinal adenocarcinomas and in the precursor lesion intestinal metaplasia. Sialyl-Tn tumour expression is an independent indicator of poor prognosis. We have previously shown in vitro that ST6GalNAc-I and ST6GalNAc......-II sialyltransferases can synthesize sialyl-Tn. The aim of the present study was to establish whether ST6GalNAc-I is the major enzyme responsible for the expression of sialyl-Tn. We used a model of CHO-ldlD cells producing only MUC1-Tn glycoform and showed that ST6GalNAc-I is the key-enzyme leading to sialyl......-Tn biosynthesis. We developed novel monoclonal antibodies specific for ST6GalNAc-I and evaluated its expression in gastrointestinal tissues. ST6GalNAc-I was detected in normal colon mucosa co-localized with O-acetylated sialyl-Tn. Expression was largely unaltered in colorectal adenocarcinomas. In contrast, we...

Expression of HIV-1 antigens in plants as potential subunit vaccines

CSIR Research Space (South Africa)

Meyers, A

2008-06-23

Full Text Available Open AcceResearch article Expression of HIV-1 antigens in plants as potential subunit vaccines Ann Meyers1,2, Ereck Chakauya1,2,3, Enid Shephard1,4, Fiona L Tanzer1,2, James Maclean1,2, Alisson Lynch1,2, Anna-Lise Williamson1,5 and Edward P Rybicki...Figure 1 The HIV-1 Gag-derived proteins used in this study. Scale diagram showing (A) native Pr55Gag ORF organisation in the Page 2 of 15 (page number not for citation purposes) gag gene, (B) the p17/p24 fusion protein ORF, (C) p24 ORF. ORFs labelled p7...

The SnSAG merozoite surface antigens of Sarcocystis neurona are expressed differentially during the bradyzoite and sporozoite life cycle stages.

Science.gov (United States)

Gautam, A; Dubey, J P; Saville, W J; Howe, D K

2011-12-29

Sarcocystis neurona is a two-host coccidian parasite whose complex life cycle progresses through multiple developmental stages differing at morphological and molecular levels. The S. neurona merozoite surface is covered by multiple, related glycosylphosphatidylinositol-linked proteins, which are orthologous to the surface antigen (SAG)/SAG1-related sequence (SRS) gene family of Toxoplasma gondii. Expression of the SAG/SRS proteins in T. gondii and another related parasite Neospora caninum is life-cycle stage specific and seems necessary for parasite transmission and persistence of infection. In the present study, the expression of S. neurona merozoite surface antigens (SnSAGs) was evaluated in the sporozoite and bradyzoite stages. Western blot analysis was used to compare SnSAG expression in merozoites versus sporozoites, while immunocytochemistry was performed to examine expression of the SnSAGs in merozoites versus bradyzoites. These analyses revealed that SnSAG2, SnSAG3 and SnSAG4 are expressed in sporozoites, while SnSAG5 was appeared to be downregulated in this life cycle stage. In S. neurona bradyzoites, it was found that SnSAG2, SnSAG3, SnSAG4 and SnSAG5 were either absent or expression was greatly reduced. As shown for T. gondii, stage-specific expression of the SnSAGs may be important for the parasite to progress through its developmental stages and complete its life cycle successfully. Thus, it is possible that the SAG switching mechanism by these parasites could be exploited as a point of intervention. As well, the alterations in surface antigen expression during different life cycle stages may need to be considered when designing prospective approaches for protective vaccination. Copyright © 2011 Elsevier B.V. All rights reserved.

Chimeric antigen receptors with human scFvs preferentially induce T cell anti-tumor activity against tumors with high B7H6 expression.

Science.gov (United States)

Gacerez, Albert T; Hua, Casey K; Ackerman, Margaret E; Sentman, Charles L

2018-05-01

B7H6 is emerging as a promising tumor antigen that is known to be expressed on a wide array of tumors and is reported to stimulate anti-tumor responses from the immune system. As such, B7H6 presents a good target for tumor-specific immunotherapies. B7H6-specific chimeric antigen receptors (CAR) based on a murine antibody showed successful targeting and elimination of tumors expressing B7H6. However, mouse single chain variable fragments (scFvs) have the potential to induce host anti-CAR responses that may limit efficacy, so human scFvs specific for B7H6 were selected by yeast surface display. In this study, we validate the functionality of these human scFvs when formatted into chimeric antigen receptors. The data indicate that T cells expressing these B7H6-specific human scFvs as CARs induced potent anti-tumor activity in vitro and in vivo against tumors expressing high amounts of B7H6. Importantly, these human scFv-based CARs are sensitive to changes in B7H6 expression which may potentially spare non-tumor cells that express B7H6 and provides the foundation for future clinical development.

A Novel Vaccine Delivery Model of the Apicomplexan Eimeria tenella Expressing Eimeria maxima Antigen Protects Chickens against Infection of the Two Parasites.

Science.gov (United States)

Tang, Xinming; Liu, Xianyong; Yin, Guangwen; Suo, Jingxia; Tao, Geru; Zhang, Sixin; Suo, Xun

2017-01-01

Vaccine delivery is critical in antigen discovery and vaccine efficacy and safety. The diversity of infectious diseases in humans and livestock has required the development of varied delivery vehicles to target different pathogens. In livestock animals, previous strategies for the development of coccidiosis vaccines have encountered several hurdles, limiting the development of multiple species vaccine formulations. Here, we describe a novel vaccine delivery system using transgenic Eimeria tenella expressing immunodominant antigens of Eimeria maxima . In this delivery system, the immune mapped protein 1 of E. maxima (EmIMP1) was delivered by the closely related species of E. tenella to the host immune system during the whole endogenous life cycle. The overexpression of the exogenous antigen did not interfere with the reproduction and immunogenicity of transgenic Eimeria . After immunization with the transgenic parasite, we detected EmIMP1's and E. maxima oocyst antigens' specific humoral and cellular immune responses. In particular, we observed partial protection of chickens immunized with transgenic E. tenella against subsequent E. maxima infections. Our results demonstrate that the transgenic Eimeria parasite is an ideal coccidia antigen delivery vehicle and represents a new type of coccidiosis vaccines. In addition, this model could potentially be used in the development of malaria live sporozoite vaccines, in which antigens from different strains can be expressed in the vaccine strain.

Identification of immunogenic Salmonella enterica serotype Typhi antigens expressed in chronic biliary carriers of S. Typhi in Kathmandu, Nepal.

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Richelle C Charles

Full Text Available Salmonella enterica serotype Typhi can colonize and persist in the biliary tract of infected individuals, resulting in a state of asymptomatic chronic carriage. Chronic carriers may act as persistent reservoirs of infection within a community and may introduce infection to susceptible individuals and new communities. Little is known about the interaction between the host and pathogen in the biliary tract of chronic carriers, and there is currently no reliable diagnostic assay to identify asymptomatic S. Typhi carriage.To study host-pathogen interactions in the biliary tract during S. Typhi carriage, we applied an immunoscreening technique called in vivo-induced antigen technology (IVIAT, to identify potential biomarkers unique to carriers. IVIAT identifies humorally immunogenic bacterial antigens expressed uniquely in the in vivo environment, and we hypothesized that S. Typhi surviving in the biliary tract of humans may express a distinct antigenic profile. Thirteen S. Typhi antigens that were immunoreactive in carriers, but not in healthy individuals from a typhoid endemic area, were identified. The identified antigens included a number of putative membrane proteins, lipoproteins, and hemolysin-related proteins. YncE (STY1479, an uncharacterized protein with an ATP-binding motif, gave prominent responses in our screen. The response to YncE in patients whose biliary tract contained S. Typhi was compared to responses in patients whose biliary tract did not contain S. Typhi, patients with acute typhoid fever, and healthy controls residing in a typhoid endemic area. Seven of 10 (70% chronic carriers, 0 of 8 bile culture-negative controls (0%, 0 of 8 healthy Bangladeshis (0%, and 1 of 8 (12.5% Bangladeshis with acute typhoid fever had detectable anti-YncE IgG in blood. IgA responses were also present.Further evaluation of YncE and other antigens identified by IVIAT could lead to the development of improved diagnostic assays to identify asymptomatic

Surface co-expression of two different PfEMP1 antigens on single Plasmodium falciparum-infected erythrocytes facilitates binding to ICAM1 and PECAM1

DEFF Research Database (Denmark)

Joergensen, Louise; Bengtsson, Dominique C; Bengtsson, Anja

2010-01-01

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) antigens play a major role in cytoadhesion of infected erythrocytes (IE), antigenic variation, and immunity to malaria. The current consensus on control of variant surface antigen expression is that only one PfEMP1 encoded by one var...

Potential radioimmunoassay system for detection of Hanganutziu-Deicher type heterophile antigen(s) and antibodies in tissues and fluids

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Mukuria, J C; Naiki, Masaharu; Hashimoto, Masato; Nishiura, Katsumi; Okabe, Masahiro; Kato, Shiro

1985-06-12

A relatively simple, specific and sensitive radioimmunoassay system has been developed for the detection of heterophile Hanganutziu-Deicher (H-D) antigen(s) and antibodies. The SVI-labeled H-D antigen-active molecule used for the assay is a bovine erythrocyte major glycoprotein previously found to have a strong H-D antigen potency. Different H-D antigen-active molecules were compared for heterophile H-D antigen potency. Eight different lung cancer tissues were assayed for H-D antigen. The sera from the 8 lung cancer patients were also screened by ELISA and RIA in an attmept to correlate expression of H-D antigen on tissues with elevation of H-D antibodies.

Sarcocystis neurona merozoites express a family of immunogenic surface antigens that are orthologues of the Toxoplasma gondii surface antigens (SAGs) and SAG-related sequences.

Science.gov (United States)

Howe, Daniel K; Gaji, Rajshekhar Y; Mroz-Barrett, Meaghan; Gubbels, Marc-Jan; Striepen, Boris; Stamper, Shelby

2005-02-01

Sarcocystis neurona is a member of the Apicomplexa that causes myelitis and encephalitis in horses but normally cycles between the opossum and small mammals. Analysis of an S. neurona expressed sequence tag (EST) database revealed four paralogous proteins that exhibit clear homology to the family of surface antigens (SAGs) and SAG-related sequences of Toxoplasma gondii. The primary peptide sequences of the S. neurona proteins are consistent with the two-domain structure that has been described for the T. gondii SAGs, and each was predicted to have an amino-terminal signal peptide and a carboxyl-terminal glycolipid anchor addition site, suggesting surface localization. All four proteins were confirmed to be membrane associated and displayed on the surface of S. neurona merozoites. Due to their surface localization and homology to T. gondii surface antigens, these S. neurona proteins were designated SnSAG1, SnSAG2, SnSAG3, and SnSAG4. Consistent with their homology, the SnSAGs elicited a robust immune response in infected and immunized animals, and their conserved structure further suggests that the SnSAGs similarly serve as adhesins for attachment to host cells. Whether the S. neurona SAG family is as extensive as the T. gondii SAG family remains unresolved, but it is probable that additional SnSAGs will be revealed as more S. neurona ESTs are generated. The existence of an SnSAG family in S. neurona indicates that expression of multiple related surface antigens is not unique to the ubiquitous organism T. gondii. Instead, the SAG gene family is a common trait that presumably has an essential, conserved function(s).

Sarcocystis neurona Merozoites Express a Family of Immunogenic Surface Antigens That Are Orthologues of the Toxoplasma gondii Surface Antigens (SAGs) and SAG-Related Sequences†

Science.gov (United States)

Howe, Daniel K.; Gaji, Rajshekhar Y.; Mroz-Barrett, Meaghan; Gubbels, Marc-Jan; Striepen, Boris; Stamper, Shelby

2005-01-01

Sarcocystis neurona is a member of the Apicomplexa that causes myelitis and encephalitis in horses but normally cycles between the opossum and small mammals. Analysis of an S. neurona expressed sequence tag (EST) database revealed four paralogous proteins that exhibit clear homology to the family of surface antigens (SAGs) and SAG-related sequences of Toxoplasma gondii. The primary peptide sequences of the S. neurona proteins are consistent with the two-domain structure that has been described for the T. gondii SAGs, and each was predicted to have an amino-terminal signal peptide and a carboxyl-terminal glycolipid anchor addition site, suggesting surface localization. All four proteins were confirmed to be membrane associated and displayed on the surface of S. neurona merozoites. Due to their surface localization and homology to T. gondii surface antigens, these S. neurona proteins were designated SnSAG1, SnSAG2, SnSAG3, and SnSAG4. Consistent with their homology, the SnSAGs elicited a robust immune response in infected and immunized animals, and their conserved structure further suggests that the SnSAGs similarly serve as adhesins for attachment to host cells. Whether the S. neurona SAG family is as extensive as the T. gondii SAG family remains unresolved, but it is probable that additional SnSAGs will be revealed as more S. neurona ESTs are generated. The existence of an SnSAG family in S. neurona indicates that expression of multiple related surface antigens is not unique to the ubiquitous organism T. gondii. Instead, the SAG gene family is a common trait that presumably has an essential, conserved function(s). PMID:15664946

A Lactococcus lactis BFE920 feed vaccine expressing a fusion protein composed of the OmpA and FlgD antigens from Edwardsiella tarda was significantly better at protecting olive flounder (Paralichthys olivaceus) from edwardsiellosis than single antigen vaccines.

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Beck, Bo Ram; Lee, Soon Ho; Kim, Daniel; Park, Ji Hye; Lee, Hyun Kyung; Kwon, San-Sung; Lee, Kwan Hee; Lee, Jae Il; Song, Seong Kyu

2017-09-01

Edwardsiellosis is a major fish disease that causes a significant economic damage in the aquaculture industry. Here, we assessed vaccine efficacy after feeding oral vaccines to olive flounder (Paralichthys olivaceus), either L. lactis BFE920 expressing Edwardsiella tarda outer membrane protein A (OmpA), flagellar hook protein D (FlgD), or a fusion antigen of the two. Feed vaccination was done twice with a one-week interval. Fish were fed regular feed adsorbed with the vaccines. Feed vaccination was given over the course of one week to maximize the interaction between the feed vaccines and the fish intestine. Flounder fed the vaccine containing the fusion antigen had significantly elevated levels T cell genes (CD4-1, CD4-2, and CD8α), type 1 helper T cell (Th1) subset indicator genes (T-bet and IFN-γ), and antigen-specific antibodies compared to the groups fed the single antigen-expressing vaccines. Furthermore, the superiority of the fusion vaccine was also observed in survival rates when fish were challenged with E. tarda: OmpA-FlgD-expressing vaccine (82.5% survival); FlgD-vaccine (55.0%); OmpA-vaccine (50%); WT L. lactis BFE920 (37.5%); Ctrl (10%). In addition, vaccine-fed fish exhibited increased weight gain (∼20%) and a decreased feed conversion ratio (∼20%) during the four week vaccination period. Flounder fed the FlgD-expressing vaccine, either the single or the fusion form, had significantly increased expression of TLR5M, IL-1β, and IL-12p40, suggesting that the FlgD may be a ligand of olive flounder TLR5M receptor or closely related to the TLR5M pathway. In conclusion, the present study demonstrated that olive flounder fed L. lactis BFE920 expressing a fusion antigen composed of E. tarda OmpA and FlgD showed a strong protective effect against edwardsiellosis indicating this may be developed as an E. tarda feed vaccine. Copyright © 2017 Elsevier Ltd. All rights reserved.

Establishment of a common acute lymphoblastic leukemia cell line (LC4-1) and effects of phorbol myristate acetate (PMA) on the surface antigen expression of the cell line.

Science.gov (United States)

Yoshimura, T; Mayumi, M; Yorifuji, T; Kim, K M; Heike, T; Miyanomae, T; Shinomiya, K; Mikawa, H

1987-09-01

A common acute lymphoblastic leukemia (ALL) cell line, designated LC4-1, was established from peripheral blood mononuclear cells of a patient with acute non-T-cell ALL. LC4-1 cells were characteristically positive for Ia, B4, and common ALL antigens (CALLA), but negative for B2, Tac, T3, T4, T8, T11, and M1 antigens and E-rosette formation. Approximately 30% of LC4-1 cells expressed detectable amounts of B1 antigens. LC4-1 cells expressed neither Epstein-Barr-virus-associated nuclear antigen (EBNA), cytoplasmic immunoglobulins (cIg) nor surface immunoglobulins (sIg). Gene rearrangements had already occurred in LC4-1 in the D-J region of immunoglobulin heavy chain genes, but not in T-cell receptor (beta-chain) genes, suggesting that LC4-1 is a progenitor cell line of B-cell lineage earlier than pre-B-cells. The expression of cell surface antigens of LC4-1 was changed by treatment with 4-phorbol 12-myristate 13-acetate (PMA) (0.1 ng/ml) for 2 days. Before treatment with PMA, about 98% of LC4-1 cells were positive for B4, CALLA, and Ia. However, following treatment they lost CALLA expression without any change in expression of Ia and B4. There was no change in B1-positive population. The change in surface antigens on LC4-1 cells seems to be due to differentiation induced in the cells by PMA. These results support the hypothesis that CALLA is a differentiation antigen and suggest one possible differentiation pathway for pre-B-cells.

Expression analysis of carbohydrate antigens in ductal carcinoma in situ of the breast by lectin histochemistry

International Nuclear Information System (INIS)

Korourian, Soheila; Siegel, Eric; Kieber-Emmons, Thomas; Monzavi-Karbassi, Behjatolah

2008-01-01

The number of breast cancer patients diagnosed with ductal carcinoma in situ (DCIS) continues to grow. Laboratory and clinical data indicate that DCIS can progress to invasive disease. Carbohydrate-mediated cell-cell adhesion and tumor-stroma interaction play crucial roles in tumorigenesis and tumor aggressive behavior. Breast carcinogenesis may reflect quantitative as well as qualitative changes in oligosaccharide expression, which may provide a useful tool for early detection of breast cancer. Because tumor-associated carbohydrate antigens (TACA) are implicated in tumor invasion and metastasis, the purpose of this study was to assess the expression of selected TACA by lectin histochemistry on DCIS specimens from the archival breast cancer tissue array bank of the University of Arkansas for Medical Sciences. For detection of TACA expression, specimens were stained with Griffonia simplicifolia lectin-I (GS-I) and Vicia vilosa agglutinin (VVA). We studied associations of lectin reactivity with established prognostic factors, such as tumor size, tumor nuclear grade, and expression of Her-2/neu, p53 mutant and estrogen and progesterone receptors. We observed that both lectins showed significant associations with nuclear grade of DCIS. DCIS specimens with nuclear grades II and III showed significantly more intense reactivity than DCIS cases with nuclear grade I to GS-1 (Mean-score chi-square = 17.60, DF = 2; P = 0.0002) and VVA (Mean-score chi-square = 15.72, DF = 2; P = 0.0004). The results suggest that the expression of VVA- and GS-I-reactive carbohydrate antigens may contribute to forming higher grade DCIS and increase the recurrence risk

Characterization of a transcriptional promoter of human papillomavirus 18 and modulation of its expression by simian virus 40 and adenovirus early antigens

International Nuclear Information System (INIS)

Thierry, F.; Heard, J.M.; Dartmann, K.; Yaniv, M.

1987-01-01

RNA present in cells derived from cervical carcinoma that contained human papillomavirus 18 genomes was initiated in the 1.053-kilobase BamHI fragment that covered the complete noncoding region of this virus. When cloned upstream of the chloramphenicol acetyltransferase gene, this viral fragment directed the expression of the bacterial enzyme only in the sense orientation. Initiation sites were mapped around the ATG of open reading frame E6. This promoter was active in some human and simian cell lines, and its expression was modulated positively by simian virus 40 large T antigen and negatively by adenovirus type 5 E1a antigen

Changing of expression level of fas-antigen (CD95), cytokines synthesis and production after irradiation in low doses

International Nuclear Information System (INIS)

Kalinina, N.M.; Solntceva, O.S.; Bytchkova, N.V.; Nikiforov, A.M.

1997-01-01

It is known that bone marrow progenitor (CD34+), tymocytes and peripheral blood lymphocytes (PBL) are most radiosensitive than other cell types. Even low doses of radiation induce apoptosis. The investigators suggest that it is possible relationship between synthesis and production of cytokines and apoptotic process. With the purpose to determine correlation between expression of Fas-antigen and synthesis of cytokines after low doses irradiation the experiments by irradiation PBL of healthy persons in vitro were held. Cells were X-irradiated by 12,5, 25 and 50 cGy. In consequence of the experiments increasing of Fas-antigen was revealed. This increasing correlated with changing in synthesis and production of cytokines. Also the Chernobyl's accident liquidators (CAL) were investigated. After comparison data in the group CAL (I) with data in the control group (II) increasing of Fas-antigen expression was revealed. Also in I group was discovered increasing of the cell number sinthesied interleukine-4 (IL-4) and interleukine-6 (IL-6). Interleukine-lβ (IL-1 β) producing pell were decreased. These changes have been correlated with degree of immunodeficiency at CAL. These data allow to consider the apoptosis as cell mechanism included in pathogenesis of diseases, which can be showed later long time after irradiation. (author)

Kinetics of human T-cell expression of LFA-1, IL-2 receptor, and ICAM-1 following antigenic stimulation in vitro

DEFF Research Database (Denmark)

Hviid, L; Felsing, A; Theander, T G

1993-01-01

-specific stimulation is available. In the present study we have examined phenotypic T-cell changes after in vitro stimulation by the antigens purified derivative of tuberculin (PPD) and tetanus toxoid (TT). We show that the well-established differences in kinetics of mitogen- and antigen-induced T-cell proliferation...... of all 3 surface antigens showed similar kinetics, and correlated with the magnitude of the lymphoproliferative response. By day 8 (PHA-stimulation) or day 12 (PPD or TT stimulation), the lymphoproliferative response was essentially completed, the expression of CD11a and CD54 had approached...

Oncogenic cancer/testis antigens

DEFF Research Database (Denmark)

Gjerstorff, Morten F; Andersen, Mads H; Ditzel, Henrik J

2015-01-01

Recent developments have set the stage for immunotherapy as a supplement to conventional cancer treatment. Consequently, a significant effort is required to further improve efficacy and specificity, particularly the identification of optimal therapeutic targets for clinical testing. Cancer....../testis antigens are immunogenic, highly cancer-specific, and frequently expressed in various types of cancer, which make them promising candidate targets for cancer immunotherapy, including cancer vaccination and adoptive T-cell transfer with chimeric T-cell receptors. Our current understanding of tumor...... immunology and immune escape suggests that targeting oncogenic antigens may be beneficial, meaning that identification of cancer/testis antigens with oncogenic properties is of high priority. Recent work from our lab and others provide evidence that many cancer/testis antigens, in fact, have oncogenic...

Expression analysis of carbohydrate antigens in ductal carcinoma in situ of the breast by lectin histochemistry

Directory of Open Access Journals (Sweden)

Kieber-Emmons Thomas

2008-05-01

Full Text Available Abstract Background The number of breast cancer patients diagnosed with ductal carcinoma in situ (DCIS continues to grow. Laboratory and clinical data indicate that DCIS can progress to invasive disease. Carbohydrate-mediated cell-cell adhesion and tumor-stroma interaction play crucial roles in tumorigenesis and tumor aggressive behavior. Breast carcinogenesis may reflect quantitative as well as qualitative changes in oligosaccharide expression, which may provide a useful tool for early detection of breast cancer. Because tumor-associated carbohydrate antigens (TACA are implicated in tumor invasion and metastasis, the purpose of this study was to assess the expression of selected TACA by lectin histochemistry on DCIS specimens from the archival breast cancer tissue array bank of the University of Arkansas for Medical Sciences. Methods For detection of TACA expression, specimens were stained with Griffonia simplicifolia lectin-I (GS-I and Vicia vilosa agglutinin (VVA. We studied associations of lectin reactivity with established prognostic factors, such as tumor size, tumor nuclear grade, and expression of Her-2/neu, p53 mutant and estrogen and progesterone receptors. Results We observed that both lectins showed significant associations with nuclear grade of DCIS. DCIS specimens with nuclear grades II and III showed significantly more intense reactivity than DCIS cases with nuclear grade I to GS-1 (Mean-score chi-square = 17.60, DF = 2; P = 0.0002 and VVA (Mean-score chi-square = 15.72, DF = 2; P = 0.0004. Conclusion The results suggest that the expression of VVA- and GS-I-reactive carbohydrate antigens may contribute to forming higher grade DCIS and increase the recurrence risk.

Differentiation of EL4 lymphoma cells by tumoral environment is associated with inappropriate expression of the large chondroitin sulfate proteoglycan PG-M and the tumor-associated antigen HTgp-175.

Science.gov (United States)

Rottiers, P; Verfaillie, T; Contreras, R; Revets, H; Desmedt, M; Dooms, H; Fiers, W; Grooten, J

1998-11-09

Progression to malignancy of transformed cells involves complex genetic alterations and aberrant gene expression patterns. While aberrant gene expression is often caused by alterations in individual genes, the contribution of the tumoral environment to the triggering of this gene expression is less well established. The stable but heterogeneous expression in cultured EL4/13 cells of a novel tumor-associated antigen, designated as HTgp-175, was chosen for the investigation of gene expression during tumor formation. Homogeneously HTgp-175-negative EL4/13 cells, isolated by cell sorting or obtained by subcloning, acquired HTgp-175 expression as a result of tumor formation. The tumorigenicity of HTgp-175-negative vs. HTgp-175-positive EL4 variants was identical, indicating that induction but not selection accounted for the phenotypic switch from HTgp-175-negative to HTgp-175-positive. Although mutagenesis experiments showed that the protein was not essential for tumor establishment, tumor-derived cells showed increased malignancy, linking HTgp-175 expression with genetic changes accompanying tumor progression. This novel gene expression was not an isolated event, since it was accompanied by ectopic expression of the large chondroitin sulfate proteoglycan PG-M and of normal differentiation antigens. We conclude that signals derived from the tumoral microenvironment contribute significantly to the aberrant gene expression pattern of malignant cells, apparently by fortuitous activation of differentiation processes and cause expression of novel differentiation antigens as well as of inappropriate tumor-associated and ectopic antigens.

Highly efficient gene transfer using a retroviral vector into murine T cells for preclinical chimeric antigen receptor-expressing T cell therapy

International Nuclear Information System (INIS)

Kusabuka, Hotaka; Fujiwara, Kento; Tokunaga, Yusuke; Hirobe, Sachiko; Nakagawa, Shinsaku; Okada, Naoki

2016-01-01

Adoptive immunotherapy using chimeric antigen receptor-expressing T (CAR-T) cells has attracted attention as an efficacious strategy for cancer treatment. To prove the efficacy and safety of CAR-T cell therapy, the elucidation of immunological mechanisms underlying it in mice is required. Although a retroviral vector (Rv) is mainly used for the introduction of CAR to murine T cells, gene transduction efficiency is generally less than 50%. The low transduction efficiency causes poor precision in the functional analysis of CAR-T cells. We attempted to improve the Rv gene transduction protocol to more efficiently generate functional CAR-T cells by optimizing the period of pre-cultivation and antibody stimulation. In the improved protocol, gene transduction efficiency to murine T cells was more than 90%. In addition, almost all of the prepared murine T cells expressed CAR after puromycin selection. These CAR-T cells had antigen-specific cytotoxic activity and secreted multiple cytokines by antigen stimulation. We believe that our optimized gene transduction protocol for murine T cells contributes to the advancement of T cell biology and development of immunotherapy using genetically engineered T cells. - Highlights: • We established highly efficient gene transduction protocols for murine T cells. • CD8"+ CAR-T cells had antigen-specific cytotoxic activity. • CD4"+ CAR-T cells secreted multiple cytokines by antigen stimulation. • This finding can contribute to the development of T-cell biology and immunotherapy.

Highly efficient gene transfer using a retroviral vector into murine T cells for preclinical chimeric antigen receptor-expressing T cell therapy

Energy Technology Data Exchange (ETDEWEB)

Kusabuka, Hotaka; Fujiwara, Kento; Tokunaga, Yusuke; Hirobe, Sachiko; Nakagawa, Shinsaku, E-mail: nakagawa@phs.osaka-u.ac.jp; Okada, Naoki, E-mail: okada@phs.osaka-u.ac.jp

2016-04-22

Adoptive immunotherapy using chimeric antigen receptor-expressing T (CAR-T) cells has attracted attention as an efficacious strategy for cancer treatment. To prove the efficacy and safety of CAR-T cell therapy, the elucidation of immunological mechanisms underlying it in mice is required. Although a retroviral vector (Rv) is mainly used for the introduction of CAR to murine T cells, gene transduction efficiency is generally less than 50%. The low transduction efficiency causes poor precision in the functional analysis of CAR-T cells. We attempted to improve the Rv gene transduction protocol to more efficiently generate functional CAR-T cells by optimizing the period of pre-cultivation and antibody stimulation. In the improved protocol, gene transduction efficiency to murine T cells was more than 90%. In addition, almost all of the prepared murine T cells expressed CAR after puromycin selection. These CAR-T cells had antigen-specific cytotoxic activity and secreted multiple cytokines by antigen stimulation. We believe that our optimized gene transduction protocol for murine T cells contributes to the advancement of T cell biology and development of immunotherapy using genetically engineered T cells. - Highlights: • We established highly efficient gene transduction protocols for murine T cells. • CD8{sup +} CAR-T cells had antigen-specific cytotoxic activity. • CD4{sup +} CAR-T cells secreted multiple cytokines by antigen stimulation. • This finding can contribute to the development of T-cell biology and immunotherapy.

Transient expression of hemagglutinin antigen from low pathogenic avian influenza A (H7N7 in Nicotiana benthamiana.

Directory of Open Access Journals (Sweden)

Selvaraju Kanagarajan

Full Text Available The influenza A virus is of global concern for the poultry industry, especially the H5 and H7 subtypes as they have the potential to become highly pathogenic for poultry. In this study, the hemagglutinin (HA of a low pathogenic avian influenza virus of the H7N7 subtype isolated from a Swedish mallard Anas platyrhynchos was sequenced, characterized and transiently expressed in Nicotiana benthamiana. Recently, plant expression systems have gained interest as an alternative for the production of vaccine antigens. To examine the possibility of expressing the HA protein in N. benthamiana, a cDNA fragment encoding the HA gene was synthesized de novo, modified with a Kozak sequence, a PR1a signal peptide, a C-terminal hexahistidine (6×His tag, and an endoplasmic retention signal (SEKDEL. The construct was cloned into a Cowpea mosaic virus (CPMV-based vector (pEAQ-HT and the resulting pEAQ-HT-HA plasmid, along with a vector (pJL3:p19 containing the viral gene-silencing suppressor p19 from Tomato bushy stunt virus, was agro-infiltrated into N. benthamiana. The highest gene expression of recombinant plant-produced, uncleaved HA (rHA0, as measured by quantitative real-time PCR was detected at 6 days post infiltration (dpi. Guided by the gene expression profile, rHA0 protein was extracted at 6 dpi and subsequently purified utilizing the 6×His tag and immobilized metal ion adsorption chromatography. The yield was 0.2 g purified protein per kg fresh weight of leaves. Further molecular characterizations showed that the purified rHA0 protein was N-glycosylated and its identity confirmed by liquid chromatography-tandem mass spectrometry. In addition, the purified rHA0 exhibited hemagglutination and hemagglutination inhibition activity indicating that the rHA0 shares structural and functional properties with native HA protein of H7 influenza virus. Our results indicate that rHA0 maintained its native antigenicity and specificity, providing a good source of

A Novel Vaccine Delivery Model of the Apicomplexan Eimeria tenella Expressing Eimeria maxima Antigen Protects Chickens against Infection of the Two Parasites

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Tang, Xinming; Liu, Xianyong; Yin, Guangwen; Suo, Jingxia; Tao, Geru; Zhang, Sixin; Suo, Xun

2018-01-01

Vaccine delivery is critical in antigen discovery and vaccine efficacy and safety. The diversity of infectious diseases in humans and livestock has required the development of varied delivery vehicles to target different pathogens. In livestock animals, previous strategies for the development of coccidiosis vaccines have encountered several hurdles, limiting the development of multiple species vaccine formulations. Here, we describe a novel vaccine delivery system using transgenic Eimeria tenella expressing immunodominant antigens of Eimeria maxima. In this delivery system, the immune mapped protein 1 of E. maxima (EmIMP1) was delivered by the closely related species of E. tenella to the host immune system during the whole endogenous life cycle. The overexpression of the exogenous antigen did not interfere with the reproduction and immunogenicity of transgenic Eimeria. After immunization with the transgenic parasite, we detected EmIMP1’s and E. maxima oocyst antigens’ specific humoral and cellular immune responses. In particular, we observed partial protection of chickens immunized with transgenic E. tenella against subsequent E. maxima infections. Our results demonstrate that the transgenic Eimeria parasite is an ideal coccidia antigen delivery vehicle and represents a new type of coccidiosis vaccines. In addition, this model could potentially be used in the development of malaria live sporozoite vaccines, in which antigens from different strains can be expressed in the vaccine strain. PMID:29375584

Detection of Burkholderia pseudomallei O-antigen serotypes in near-neighbor species

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Stone Joshua K

2012-11-01

Full Text Available Abstract Background Burkholderia pseudomallei is the etiological agent of melioidosis and a CDC category B select agent with no available effective vaccine. Previous immunizations in mice have utilized the lipopolysaccharide (LPS as a potential vaccine target because it is known as one of the most important antigenic epitopes in B. pseudomallei. Complicating this strategy are the four different B. pseudomallei LPS O-antigen types: A, B, B2, and rough. Sero-crossreactivity is common among O-antigens of Burkholderia species. Here, we identified the presence of multiple B. pseudomallei O-antigen types and sero-crossreactivity in its near-neighbor species. Results PCR screening of O-antigen biosynthesis genes, phenotypic characterization using SDS-PAGE, and immunoblot analysis showed that majority of B. mallei and B. thailandensis strains contained the typical O-antigen type A. In contrast, most of B. ubonensis and B. thailandensis-like strains expressed the atypical O-antigen types B and B2, respectively. Most B. oklahomensis strains expressed a distinct and non-seroreactive O-antigen type, except strain E0147 which expressed O-antigen type A. O-antigen type B2 was also detected in B. thailandensis 82172, B. ubonensis MSMB108, and Burkholderia sp. MSMB175. Interestingly, B. thailandensis-like MSMB43 contained a novel serotype B positive O-antigen. Conclusions This study expands the number of species which express B. pseudomallei O-antigen types. Further work is required to elucidate the full structures and how closely these are to the B. pseudomallei O-antigens, which will ultimately determine the efficacy of the near-neighbor B serotypes for vaccine development.

Insect-cell expression, crystallization and X-ray data collection of the bradyzoite-specific antigen BSR4 from Toxoplasma gondii

International Nuclear Information System (INIS)

Grujic, Ognjen; Grigg, Michael E.; Boulanger, Martin J.

2008-01-01

Preliminary X-ray diffraction studies of the bradyzoite-specific surface antigen BSR4 from T. gondii are described. Toxoplasma gondii is an important global pathogen that infects nearly one third of the world’s adult population. A family of developmentally expressed structurally related surface-glycoprotein adhesins (SRSs) mediate attachment to and are utilized for entry into host cells. The latent bradyzoite form of T. gondii persists for the life of the host and expresses a distinct family of SRS proteins, of which the bradyzoite-specific antigen BSR4 is a prototypical member. Structural studies of BSR4 were initiated by first recombinantly expressing BSR4 in insect cells, which was followed by crystallization and preliminary X-ray data collection to 1.95 Å resolution. Data processing showed that BSR4 crystallized with one molecule in the asymmetric unit of the P4 1 2 1 2 or P4 3 2 1 2 space group, with a solvent content of 60% and a corresponding Matthews coefficient of 2.98 Å 3 Da −1

New Genome-Wide Algorithm Identifies Novel In-Vivo Expressed Mycobacterium Tuberculosis Antigens Inducing Human T-Cell Responses with Classical and Unconventional Cytokine Profiles

DEFF Research Database (Denmark)

Coppola, Mariateresa; van Meijgaarden, Krista E.; Franken, Kees L. M. C.

2016-01-01

-wide transcriptomics of Mtb infected lungs we developed data sets and methods to identify IVE-TB (in-vivo expressed Mtb) antigens expressed in the lung. Quantitative expression analysis of 2,068 Mtb genes from the predicted first operons identified the most upregulated IVE-TB genes during in-vivo pulmonary infection...

Peripheral nerve injury causes transient expression of MHC class I antigens in rat motor neurons and skeletal muscles

DEFF Research Database (Denmark)

Maehlen, J; Nennesmo, I; Olsson, A B

1989-01-01

After a peripheral nerve lesion (rat facial and sciatic) an induction of major histocompatibility complex (MHC) antigens class I was detected immunohistochemically in skeletal muscle fibers and motor neurons. This MHC expression was transient after a nerve crush, when regeneration occurred......, but persisted after a nerve cut, when regeneration was prevented. Since the time course of MHC class I expression correlates to that of regeneration a role for this cell surface molecule in regeneration may be considered....

Impaired cell surface expression of HLA-B antigens on mesenchymal stem cells and muscle cell progenitors

DEFF Research Database (Denmark)

Isa, Adiba; Nehlin, Jan; Sabir, Hardee Jawad

2010-01-01

HLA class-I expression is weak in embryonic stem cells but increases rapidly during lineage progression. It is unknown whether all three classical HLA class-I antigens follow the same developmental program. In the present study, we investigated allele-specific expression of HLA-A, -B, and -C...... at the mRNA and protein levels on human mesenchymal stem cells from bone marrow and adipose tissue as well as striated muscle satellite cells and lymphocytes. Using multicolour flow cytometry, we found high cell surface expression of HLA-A on all stem cells and PBMC examined. Surprisingly, HLA-B was either...... undetectable or very weakly expressed on all stem cells protecting them from complement-dependent cytotoxicity (CDC) using relevant human anti-B and anti-Cw sera. IFNgamma stimulation for 48-72 h was required to induce full HLA-B protein expression. Quantitative real-time RT-PCR showed that IFNgamma induced...

ABO blood group antigens in oral mucosa. What is new?

DEFF Research Database (Denmark)

Dabelsteen, Erik

2002-01-01

which represent secondary gene products. They are synthesized in a stepwise fashion from a precursor by the action of different glycosyltransferases. In non-keratinized oral mucosa, a sequential elongation of the carbohydrates is associated with differentiation of epithelial cells, resulting...... in expression of precursors on basal cells and A/B antigens on spinous cells. Reduction or complete deletion of A/B antigen expression in oral carcinomas has been reported, a phenotypic change that is correlated with invasive and metastatic potential of the tumours and with the mortality rates of the patients....... Disappearance of the antigens is ascribed to the absence of A or B transferase gene expression. Several studies have shown that loss of A and B antigen expression is associated with increased cell motility, invasion in matrigel, and tumourigenecity in syngenic animals. In vivo studies of human oral wound...

Expression, biosynthesis and release of preadipocyte factor-1/ delta-like protein/fetal antigen-1 in pancreatic beta-cells

DEFF Research Database (Denmark)

Friedrichsen, B N; Carlsson, C; Møldrup, Annette

2003-01-01

Preadipocyte factor-1 (Pref-1)/delta-like protein/fetal antigen-1 (FA1) is a member of the epidermal growth factor-like family. It is widely expressed in embryonic tissues, whereas in adults it is confined to the adrenal gland, the anterior pituitary, the endocrine pancreas, the testis...

Natural selection promotes antigenic evolvability.

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Graves, Christopher J; Ros, Vera I D; Stevenson, Brian; Sniegowski, Paul D; Brisson, Dustin

2013-01-01

The hypothesis that evolvability - the capacity to evolve by natural selection - is itself the object of natural selection is highly intriguing but remains controversial due in large part to a paucity of direct experimental evidence. The antigenic variation mechanisms of microbial pathogens provide an experimentally tractable system to test whether natural selection has favored mechanisms that increase evolvability. Many antigenic variation systems consist of paralogous unexpressed 'cassettes' that recombine into an expression site to rapidly alter the expressed protein. Importantly, the magnitude of antigenic change is a function of the genetic diversity among the unexpressed cassettes. Thus, evidence that selection favors among-cassette diversity is direct evidence that natural selection promotes antigenic evolvability. We used the Lyme disease bacterium, Borrelia burgdorferi, as a model to test the prediction that natural selection favors amino acid diversity among unexpressed vls cassettes and thereby promotes evolvability in a primary surface antigen, VlsE. The hypothesis that diversity among vls cassettes is favored by natural selection was supported in each B. burgdorferi strain analyzed using both classical (dN/dS ratios) and Bayesian population genetic analyses of genetic sequence data. This hypothesis was also supported by the conservation of highly mutable tandem-repeat structures across B. burgdorferi strains despite a near complete absence of sequence conservation. Diversification among vls cassettes due to natural selection and mutable repeat structures promotes long-term antigenic evolvability of VlsE. These findings provide a direct demonstration that molecular mechanisms that enhance evolvability of surface antigens are an evolutionary adaptation. The molecular evolutionary processes identified here can serve as a model for the evolution of antigenic evolvability in many pathogens which utilize similar strategies to establish chronic infections.

Natural selection promotes antigenic evolvability.

Directory of Open Access Journals (Sweden)

Christopher J Graves

Full Text Available The hypothesis that evolvability - the capacity to evolve by natural selection - is itself the object of natural selection is highly intriguing but remains controversial due in large part to a paucity of direct experimental evidence. The antigenic variation mechanisms of microbial pathogens provide an experimentally tractable system to test whether natural selection has favored mechanisms that increase evolvability. Many antigenic variation systems consist of paralogous unexpressed 'cassettes' that recombine into an expression site to rapidly alter the expressed protein. Importantly, the magnitude of antigenic change is a function of the genetic diversity among the unexpressed cassettes. Thus, evidence that selection favors among-cassette diversity is direct evidence that natural selection promotes antigenic evolvability. We used the Lyme disease bacterium, Borrelia burgdorferi, as a model to test the prediction that natural selection favors amino acid diversity among unexpressed vls cassettes and thereby promotes evolvability in a primary surface antigen, VlsE. The hypothesis that diversity among vls cassettes is favored by natural selection was supported in each B. burgdorferi strain analyzed using both classical (dN/dS ratios and Bayesian population genetic analyses of genetic sequence data. This hypothesis was also supported by the conservation of highly mutable tandem-repeat structures across B. burgdorferi strains despite a near complete absence of sequence conservation. Diversification among vls cassettes due to natural selection and mutable repeat structures promotes long-term antigenic evolvability of VlsE. These findings provide a direct demonstration that molecular mechanisms that enhance evolvability of surface antigens are an evolutionary adaptation. The molecular evolutionary processes identified here can serve as a model for the evolution of antigenic evolvability in many pathogens which utilize similar strategies to establish

Induction of Oral Tolerance with Transgenic Plants Expressing Antigens for Prevention/Treatment of Autoimmune, Allergic and Inflammatory Diseases.

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Ma, Shengwu; Liao, Yu-Cai; Jevnikar, Anthony M

2015-01-01

The prevalence and incidence of autoimmune and allergic diseases have increased dramatically over the last several decades, especially in the developed world. The treatment of autoimmune and allergic diseases is typically with the use of non-specific immunosuppressive agents that compromise the integrity of the host immune system and therefore, increase the risk of infections. Antigenspecific immunotherapy by reinstating immunological tolerance towards self antigens without compromising immune functions is a much desired goal for the treatment of autoimmune and allergic diseases. Mucosal administration of antigen is a long-recognized method of inducing antigen-specific immune tolerance known as oral tolerance, which is viewed as having promising potential in the treatment of autoimmune and allergic diseases. Plant-based expression and delivery of recombinant antigens provide a promising new platform to induce oral tolerance, having considerable advantages including reduced cost and increased safety. Indeed, in recent years the use of tolerogenic plants for oral tolerance induction has attracted increasing attention, and considerable progress has been made. This review summarizes recent advances in using plants to deliver tolerogens for induction of oral tolerance in the treatment of autoimmune, allergic and inflammatory diseases.

Expression of ras oncogene and major histocompatibility complex (MHC) antigen in carcinomas of the uterine cervix

International Nuclear Information System (INIS)

Cho, Kyung Ja; Jang, Ja June; Kim, Yong Dae; Ha, Chang Won; Koh, Jae Soo

1993-01-01

Consecutive 50 cases of squamous cell carcinomas of the uterine cervix diagnosed in 1992 were subjected to immunohistochemical study for ras oncogene product (p21) and MHC class II (DR) antigen using a microprobe immunostainer. Activated ras and aberrant DR expression were noted in 26 cases (52%) and 11 cases (22%) of cervical squamous cell carcinomas, respectively, without difference among histologic types. The reaction was mainly intracytoplasmic, with granular staining pattern and diffuse distribution. No direct histologic correlation between ras and DR expression was found. Four cases with HPV 16/18 DNA in superficial koilocytotic cells, revealed by in situ hybridization, showed various expression of ras and DR, and these 3 factors histologically did not seem to be affected one another. (Author)

Immune response to Lactobacillus plantarum expressing Borrelia burgdorferi OspA is modulated by the lipid modification of the antigen.

Directory of Open Access Journals (Sweden)

Beatriz del Rio

2010-06-01

Full Text Available Over the past decade there has been increasing interest in the use of lactic acid bacteria as mucosal delivery vehicles for vaccine antigens, microbicides and therapeutics. We investigated the mechanism by which a mucosal vaccine based in recombinant lactic acid bacteria breaks the immunological tolerance of the gut in order to elicit a protective immune response.We analyzed how the lipid modification of OspA affects the localization of the antigen in our delivery vehicle using a number of biochemistry techniques. Furthermore, we examined how OspA-expressing L. plantarum breaks the oral tolerance of the gut by stimulating human intestinal epithelial cells, peripheral blood mononuclear cells and monocyte derived dendritic cells and measuring cytokine production. We show that the leader peptide of OspA targets the protein to the cell envelope of L. plantarum, and it is responsible for protein export across the membrane. Mutation of the lipidation site in OspA redirects protein localization within the cell envelope. Further, we show that lipidated-OspA-expressing L. plantarum does not induce secretion of the pro-inflammatory cytokine IL-8 by intestinal epithelial cells. In addition, it breaks oral tolerance of the gut via Th1/Th2 cell mediated immunity, as shown by the production of pro- and anti-inflammatory cytokines by human dendritic cells, and by the production of IgG2a and IgG1 antibodies, respectively.Lipid modification of OspA expressed in L. plantarum modulates the immune response to this antigen through a Th1/Th2 immune response.

Identification of cDNA clones expressing immunodiagnostic antigens from Trichinella spiralis

International Nuclear Information System (INIS)

Zarlenga, D.; Gamble, H.R.

1987-01-01

A cDNA expression library was built in lambda gt11 phage using poly A mRNA isolated from Trichinella spiralis muscle stage larvae. This library was screened with rabbit antibodies to parasite excretory-secretory (ES) products and greater than 180 clones were isolated. Thirteen clones producing highly immunogenic protein antigens were plaque purified and rescreened with pig antisera to T.spiralis, Trichuris suis or Ascaris suum to identify clones producing epitopes specific to T.spiralis ES products, only. Two clones, TsAc-2 and TsAc-8, which displayed strong interactions with pig antisera to T. spiralis were lysogenized in E. coli Y1089 and the protein extracted. Western blots of the crude fusion proteins revealed molecular weights of 133 kD and 129 kD, respectively. Northern blot analysis of total RNA with 32 P labelled cDNA:lambda gt11 probes indicated single RNA transcripts for each clone with molecular sizes corresponding to 800-850 nucleotides. dscDNA inserts were estimated by southern blot analysis to be 500 bp and 340 bp, respectively, with no cross-hybridization observed between the cloned sequences. Dot blots using pig sera to screen crude fusion protein preparations, total bacterial protein (negative controls) and crude worm extract or ES products from T.spiralis, T.suis and A.suum (positive controls) corroborated the specificity and sensitivity of these clones as potential diagnostic antigens for swine trichinellosis

Tumor-associated antigens identified by mRNA expression profiling induce protective anti-tumor immunity

DEFF Research Database (Denmark)

Mathiassen, S; Lauemøller, S L; Ruhwald, M

2001-01-01

Defined tumor-associated antigens (TAA) are attractive targets for anti-tumor immunotherapy. Here, we describe a novel genome-wide approach to identify multiple TAA from any given tumor. A panel of transplantable thymomas was established from an inbred p53-/- mouse strain. The resulting tumors were...... of autoimmune reactions were observed. Thus, it appears possible to evaluate the entire metabolism of any given tumor and use this information rationally to identify multiple epitopes of value in the generation of tumor-specific immunotherapy. We expect that human tumors express similar tumor-specific metabolic...

Expression of the major histocompatibility antigens HLA-A2 and HLA-B7 by DNA-mediated gene transfer

NARCIS (Netherlands)

Bernabeu, C.; Finlay, D.; van de Rijn, M.; Maziarz, R. T.; Biro, P. A.; Spits, H.; de Vries, J.; Terhorst, C. P.

1983-01-01

Genes coding for the heavy chain of the class I antigens HLA-A2 or HLA-B7 of the human major histocompatibility complex have been introduced into mouse LtK- cells by cotransfection with the herpes simplex virus thymidine kinase gene. HAT-resistant colonies were isolated expressing either HLA-A2 or

Prostate-specific antigen and hormone receptor expression in male and female breast carcinoma

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Cohen Cynthia

2010-09-01

Full Text Available Abstract Background Prostate carcinoma is among the most common solid tumors to secondarily involve the male breast. Prostate specific antigen (PSA and prostate-specific acid phosphatase (PSAP are expressed in benign and malignant prostatic tissue, and immunohistochemical staining for these markers is often used to confirm the prostatic origin of metastatic carcinoma. PSA expression has been reported in male and female breast carcinoma and in gynecomastia, raising concerns about the utility of PSA for differentiating prostate carcinoma metastasis to the male breast from primary breast carcinoma. This study examined the frequency of PSA, PSAP, and hormone receptor expression in male breast carcinoma (MBC, female breast carcinoma (FBC, and gynecomastia. Methods Immunohistochemical staining for PSA, PSAP, AR, ER, and PR was performed on tissue microarrays representing six cases of gynecomastia, thirty MBC, and fifty-six FBC. Results PSA was positive in two of fifty-six FBC (3.7%, focally positive in one of thirty MBC (3.3%, and negative in the five examined cases of gynecomastia. PSAP expression was absent in MBC, FBC, and gynecomastia. Hormone receptor expression was similar in males and females (AR 74.1% in MBC vs. 67.9% in FBC, p = 0.62; ER 85.2% vs. 68.5%, p = 0.18; and PR 51.9% vs. 48.2%, p = 0.82. Conclusions PSA and PSAP are useful markers to distinguish primary breast carcinoma from prostate carcinoma metastatic to the male breast. Although PSA expression appeared to correlate with hormone receptor expression, the incidence of PSA expression in our population was too low to draw significant conclusions about an association between PSA expression and hormone receptor status in breast lesions.

The distribution of blood group antigens in experimentally produced carcinomas of rat palate

DEFF Research Database (Denmark)

Reibel, J; Philipsen, H P; Fisker, A V

1986-01-01

palate induced by a chemical carcinogen (4NQO). The H antigen, normally expressed on spinous cells in rats, was absent in malignant epithelium, whereas staining for the B antigen, normally expressed on basal cells, was variable. These changes are equivalent to those seen in human squamous cell carcinomas....... The blood group antigen staining pattern in experimentally produced verrucous carcinomas showed an almost normal blood group antigen expression. This may have diagnostic significance. Localized areas of hyperplastic palatal epithelium with slight dysplasia revealed loss of H antigen and the presence of B...... antigen in suprabasal strata equivalent to the pattern seen in human premalignant epithelium. We conclude from these findings, that the rat model is well suited to study changes in cell surface carbohydrates during chemical carcinogenesis....

The androgen receptor controls expression of the cancer-associated sTn antigen and cell adhesion through induction of ST6GalNAc1 in prostate cancer

Science.gov (United States)

Munkley, Jennifer; Oltean, Sebastian; Vodák, Daniel; Wilson, Brian T.; Livermore, Karen E.; Zhou, Yan; Star, Eleanor; Floros, Vasileios I.; Johannessen, Bjarne; Knight, Bridget; McCullagh, Paul; McGrath, John; Crundwell, Malcolm; Skotheim, Rolf I.; Robson, Craig N.; Leung, Hing Y.; Harries, Lorna W.; Rajan, Prabhakar; Mills, Ian G.; Elliott, David J.

2015-01-01

Patterns of glycosylation are important in cancer, but the molecular mechanisms that drive changes are often poorly understood. The androgen receptor drives prostate cancer (PCa) development and progression to lethal metastatic castration-resistant disease. Here we used RNA-Seq coupled with bioinformatic analyses of androgen-receptor (AR) binding sites and clinical PCa expression array data to identify ST6GalNAc1 as a direct and rapidly activated target gene of the AR in PCa cells. ST6GalNAc1 encodes a sialytransferase that catalyses formation of the cancer-associated sialyl-Tn antigen (sTn), which we find is also induced by androgen exposure. Androgens induce expression of a novel splice variant of the ST6GalNAc1 protein in PCa cells. This splice variant encodes a shorter protein isoform that is still fully functional as a sialyltransferase and able to induce expression of the sTn-antigen. Surprisingly, given its high expression in tumours, stable expression of ST6GalNAc1 in PCa cells reduced formation of stable tumours in mice, reduced cell adhesion and induced a switch towards a more mesenchymal-like cell phenotype in vitro. ST6GalNAc1 has a dynamic expression pattern in clinical datasets, being significantly up-regulated in primary prostate carcinoma but relatively down-regulated in established metastatic tissue. ST6GalNAc1 is frequently upregulated concurrently with another important glycosylation enzyme GCNT1 previously associated with prostate cancer progression and implicated in Sialyl Lewis X antigen synthesis. Together our data establishes an androgen-dependent mechanism for sTn antigen expression in PCa, and are consistent with a general role for the androgen receptor in driving important coordinate changes to the glycoproteome during PCa progression. PMID:26452038

Antigenic determinants and functional domains in core antigen and e antigen from hepatitis B virus

International Nuclear Information System (INIS)

Salfeld, J.; Pfaff, E.; Noah, M.; Schaller, H.

1989-01-01

The precore/core gene of hepatitis B virus directs the synthesis of two polypeptides, the 21-kilodalton subunit (p21c) forming the viral nucleocapsid (serologically defined as core antigen [HBcAg]) and a secreted processed protein (p17e, serologically defined as HBe antigen [HBeAg]). Although most of their primary amino acid sequences are identical, HBcAg and HBeAg display different antigenic properties that are widely used in hepatitis B virus diagnosis. To locate and to characterize the corresponding determinants, segments of the core gene were expressed in Escherichia coli and probed with a panel of polyclonal or monoclonal antibodies in radioimmunoassays or enzyme-linked immunosorbent assays, Western blots, and competition assays. Three distinct major determinants were characterized. It is postulated that HBcAg and HBeAg share common basic three-dimensional structure exposing the common linear determinant HBe1 but that they differ in the presentation of two conformational determinants that are either introduced (HBc) or masked (HBe2) in the assembled core. The simultaneous presentation of HBe1 and HBc, two distinctly different antigenic determinants with overlapping amino acid sequences, is interpreted to indicate the presence of slightly differently folded, stable conformational states of p21c in the hepatitis virus nucleocapsid

Comparison of altered expression of histocompatibility antigens with altered immune function in murine spleen cells treated with ultraviolet radiation and/or TPA

International Nuclear Information System (INIS)

Pretell, J.O.; Cone, R.E.

1985-01-01

Previous studies in our laboratory demonstrated that several treatments that inhibited the ability of cells to stimulate the mixed lymphocyte reaction (MLR) also blocked the shedding of histocompatibility antigens and Ia antigens from murine spleen cells. In the present studies, one of these treatments, ultraviolet radiation (UV), was shown to cause an initial loss in the density of H-2K, IA, and IE antigens prior to the block in shedding observed after culture of these cells. Further analysis revealed that the UV-induced loss of antigens could be prevented by the presence of colchicine during irradiation. Biosynthetic analyses revealed the IA antigen synthesis was also inhibited in the UV-irradiated cells. Examination of the effects of a second agent, 12-0-tetradecanoylphorbol-13-acetate (TPA) on the turnover of histocompatibility antigens revealed that the biosynthesis and shedding of these antigens were accelerated by this agent. However, addition of TPA to UV-irradiated cells did not result in a reversal of the UV-induced block in biosynthesis of IA antigens. Results of immune function assays correlated with the biochemical studies: UV-irradiation inhibited the generation of the MLR, but TPA enhanced this reaction, and addition of TPA to mixed lymphocyte cultures with UV-irradiated stimulators did not reverse the UV-induced inhibition. These results suggest that, although the turnover of histocompatibility antigens may be affected by TPA and UV in an antagonistic fashion, additional factors other than the expression of histocompatibility antigens are operating in the inhibition of stimulation of an MLR by UV radiation or its enhancement by TPA

A single point in protein trafficking by Plasmodium falciparum determines the expression of major antigens on the surface of infected erythrocytes targeted by human antibodies.

Science.gov (United States)

Chan, Jo-Anne; Howell, Katherine B; Langer, Christine; Maier, Alexander G; Hasang, Wina; Rogerson, Stephen J; Petter, Michaela; Chesson, Joanne; Stanisic, Danielle I; Duffy, Michael F; Cooke, Brian M; Siba, Peter M; Mueller, Ivo; Bull, Peter C; Marsh, Kevin; Fowkes, Freya J I; Beeson, James G

2016-11-01

Antibodies to blood-stage antigens of Plasmodium falciparum play a pivotal role in human immunity to malaria. During parasite development, multiple proteins are trafficked from the intracellular parasite to the surface of P. falciparum-infected erythrocytes (IEs). However, the relative importance of different proteins as targets of acquired antibodies, and key pathways involved in trafficking major antigens remain to be clearly defined. We quantified antibodies to surface antigens among children, adults, and pregnant women from different malaria-exposed regions. We quantified the importance of antigens as antibody targets using genetically engineered P. falciparum with modified surface antigen expression. Genetic deletion of the trafficking protein skeleton-binding protein-1 (SBP1), which is involved in trafficking the surface antigen PfEMP1, led to a dramatic reduction in antibody recognition of IEs and the ability of human antibodies to promote opsonic phagocytosis of IEs, a key mechanism of parasite clearance. The great majority of antibody epitopes on the IE surface were SBP1-dependent. This was demonstrated using parasite isolates with different genetic or phenotypic backgrounds, and among antibodies from children, adults, and pregnant women in different populations. Comparisons of antibody reactivity to parasite isolates with SBP1 deletion or inhibited PfEMP1 expression suggest that PfEMP1 is the dominant target of acquired human antibodies, and that other P. falciparum IE surface proteins are minor targets. These results establish SBP1 as part of a critical pathway for the trafficking of major surface antigens targeted by human immunity, and have key implications for vaccine development, and quantifying immunity in populations.

Host genetic resistance to symptomatic norovirus (GGII.4) infections in Denmark

DEFF Research Database (Denmark)

Kindberg, Elin; Akerlind, Britt; Johnsen, Christina

2007-01-01

A total of 61 individuals involved in five norovirus outbreaks in Denmark were genotyped at nucleotides 428 and 571 of the FUT2 gene, determining secretor status, i.e., the presence of ABH antigens in secretions and on mucosa. A strong correlation (P = 0.003) was found between the secretor phenot...

Production of dengue virus envelope protein domain III-based antigens in tobacco chloroplasts using inducible and constitutive expression systems.

Science.gov (United States)

Gottschamel, Johanna; Lössl, Andreas; Ruf, Stephanie; Wang, Yanliang; Skaugen, Morten; Bock, Ralph; Clarke, Jihong Liu

2016-07-01

Dengue fever is a disease in many parts of the tropics and subtropics and about half the world's population is at risk of infection according to the World Health Organization. Dengue is caused by any of the four related dengue virus serotypes DEN-1, -2, -3 and -4, which are transmitted to people by Aedes aegypti mosquitoes. Currently there is only one vaccine (Dengvaxia(®)) available (limited to a few countries) on the market since 2015 after half a century's intensive efforts. Affordable and accessible vaccines against dengue are hence still urgently needed. The dengue envelop protein domain III (EDIII), which is capable of eliciting serotype-specific neutralizing antibodies, has become the focus for subunit vaccine development. To contribute to the development of an accessible and affordable dengue vaccine, in the current study we have used plant-based vaccine production systems to generate a dengue subunit vaccine candidate in tobacco. Chloroplast genome engineering was applied to express serotype-specific recombinant EDIII proteins in tobacco chloroplasts using both constitutive and ethanol-inducible expression systems. Expression of a tetravalent antigen fusion construct combining EDIII polypeptides from all four serotypes was also attempted. Transplastomic EDIII-expressing tobacco lines were obtained and homoplasmy was verified by Southern blot analysis. Northern blot analyses showed expression of EDIII antigen-encoding genes. EDIII protein accumulation levels varied for the different recombinant EDIII proteins and the different expression systems, and reached between 0.8 and 1.6 % of total cellular protein. Our study demonstrates the suitability of the chloroplast compartment as a production site for an EDIII-based vaccine candidate against dengue fever and presents a Gateway(®) plastid transformation vector for inducible transgene expression.

Effective plague vaccination via oral delivery of plant cells expressing F1-V antigens in chloroplasts.

Science.gov (United States)

Arlen, Philip A; Singleton, Michael; Adamovicz, Jeffrey J; Ding, Yi; Davoodi-Semiromi, Abdolreza; Daniell, Henry

2008-08-01

The chloroplast bioreactor is an alternative to fermentation-based systems for production of vaccine antigens and biopharmaceuticals. We report here expression of the plague F1-V fusion antigen in chloroplasts. Site-specific transgene integration and homoplasmy were confirmed by PCR and Southern blotting. Mature leaves showed the highest level of transgene expression on the third day of continuous illumination, with a maximum level of 14.8% of the total soluble protein. Swiss Webster mice were primed with adjuvant-containing subcutaneous (s.c.) doses of F1-V and then boosted with either adjuvanted s.c. doses (s.c. F1-V mice) or unadjuvanted oral doses (oral F1-V mice). Oral F1-V mice had higher prechallenge serum immunoglobulin G1 (IgG1) titers than s.c. F1-V mice. The corresponding serum levels of antigen-specific IgG2a and IgA were 2 and 3 orders of magnitude lower, respectively. After vaccination, mice were exposed to an inhaled dose of 1.02 x 10(6) CFU of aerosolized Yersinia pestis CO92 (50% lethal dose, 6.8 x 10(4) CFU). All control animals died within 3 days. F1-V given s.c. (with adjuvant) protected 33% of the immunized mice, while 88% of the oral F1-V mice survived aerosolized Y. pestis challenge. A comparison of splenic Y. pestis CFU counts showed that there was a 7- to 10-log reduction in the mean bacterial burden in survivors. Taken together, these data indicate that oral booster doses effectively elicit protective immune responses in vivo. In addition, this is the first report of a plant-derived oral vaccine that protected animals from live Y. pestis challenge, bringing the likelihood of lower-cost vaccines closer to reality.

The MASP family of Trypanosoma cruzi: changes in gene expression and antigenic profile during the acute phase of experimental infection.

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Sara Lopes dos Santos

Full Text Available Trypanosoma cruzi is the etiological agent of Chagas disease, a debilitating illness that affects millions of people in the Americas. A major finding of the T. cruzi genome project was the discovery of a novel multigene family composed of approximately 1,300 genes that encode mucin-associated surface proteins (MASPs. The high level of polymorphism of the MASP family associated with its localization at the surface of infective forms of the parasite suggests that MASP participates in host-parasite interactions. We speculate that the large repertoire of MASP sequences may contribute to the ability of T. cruzi to infect several host cell types and/or participate in host immune evasion mechanisms.By sequencing seven cDNA libraries, we analyzed the MASP expression profile in trypomastigotes derived from distinct host cells and after sequential passages in acutely infected mice. Additionally, to investigate the MASP antigenic profile, we performed B-cell epitope prediction on MASP proteins and designed a MASP-specific peptide array with 110 putative epitopes, which was screened with sera from acutely infected mice.We observed differential expression of a few MASP genes between trypomastigotes derived from epithelial and myoblast cell lines. The more pronounced MASP expression changes were observed between bloodstream and tissue-culture trypomastigotes and between bloodstream forms from sequential passages in acutely infected mice. Moreover, we demonstrated that different MASP members were expressed during the acute T. cruzi infection and constitute parasite antigens that are recognized by IgG and IgM antibodies. We also found that distinct MASP peptides could trigger different antibody responses and that the antibody level against a given peptide may vary after sequential passages in mice. We speculate that changes in the large repertoire of MASP antigenic peptides during an infection may contribute to the evasion of host immune responses during the

Ultraviolet light-induced suppression of antigen presentation

International Nuclear Information System (INIS)

Spellman, C.W.; Tomasi, T.B.

1983-01-01

Ultraviolet (UV) light irradiation of animals results in the development of specific T suppressor cells that inhibit antitumor immune responses. It is thought that suppression may arise as a consequence of altered antigen presentation by UV-irradiated epidermal cells. This hypothesis is based on evidence demonstrating that specific lymphoid tissues from UV-irradiated hosts exhibit impaired antigen-presenting function and that animals cannot be contact sensitized when antigens are applied to a UV-irradiated skin site. Langerhans cells of the skin are likely candidates as targets of UV-induced defects in antigen presentation as they bear Fc and C3b receptors, express Ia antigens, are of bone marrow origin, and are capable of presenting antigen in vitro. We speculate on the possible clinical usefulness of UV-induced tolerance to specific antigens such as those encountered in monoclonal antibody therapy and tissue transplantation

Tumor-Targeted Human T Cells Expressing CD28-Based Chimeric Antigen Receptors Circumvent CTLA-4 Inhibition.

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Maud Condomines

Full Text Available Adoptive T cell therapy represents a promising treatment for cancer. Human T cells engineered to express a chimeric antigen receptor (CAR recognize and kill tumor cells in a MHC-unrestricted manner and persist in vivo when the CAR includes a CD28 costimulatory domain. However, the intensity of the CAR-mediated CD28 activation signal and its regulation by the CTLA-4 checkpoint are unknown. We investigated whether T cells expressing an anti-CD19, CD3 zeta and CD28-based CAR (19-28z displayed the same proliferation and anti-tumor abilities than T cells expressing a CD3 zeta-based CAR (19z1 costimulated through the CD80/CD28, ligand/receptor pathway. Repeated in vitro antigen-specific stimulations indicated that 19-28z+ T cells secreted higher levels of Th1 cytokines and showed enhanced proliferation compared to those of 19z1+ or 19z1-CD80+ T cells. In an aggressive pre-B cell leukemia model, mice treated with 19-28z+ T cells had 10-fold reduced tumor progression compared to those treated with 19z1+ or 19z1-CD80+ T cells. shRNA-mediated CTLA-4 down-regulation in 19z1-CD80+ T cells significantly increased their in vivo expansion and anti-tumor properties, but had no effect in 19-28z+ T cells. Our results establish that CTLA-4 down-regulation may benefit human adoptive T cell therapy and demonstrate that CAR design can elude negative checkpoints to better sustain T cell function.

Correlation Between Preoperative Serum Carcinoembryonic Antigen Levels and Expression on Pancreatic and Rectal Cancer Tissue

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LSF Boogerd

2017-05-01

Full Text Available Carcinoembryonic antigen (CEA–targeted imaging and therapeutic agents are being tested in clinical trials. If CEA overexpression in malignant tissue corresponds with elevated serum CEA, serum CEA could assist in selecting patients who may benefit from CEA-targeted agents. This study aims to assess the relationship between serum CEA and CEA expression in pancreatic (n = 20 and rectal cancer tissues (n = 35 using histopathology. According to local laboratory standards, a serum CEA >3 ng/mL was considered elevated. In pancreatic cancer patients a significant correlation between serum CEA and percentage of CEA-expressing tumor cells was observed ( P  = .04, ρ = .47. All 6 patients with homogeneous CEA expression in the tumor had a serum CEA >3 ng/mL. Most rectal cancer tissues (32/35 showed homogeneous CEA expression, independent of serum CEA levels. This study suggests that selection of pancreatic cancer patients for CEA-targeted agents via serum CEA appears adequate. For selection of rectal cancer patients, serum CEA levels are not informative.

The evaluaton of prevalence rate of p53 antigen expression in cutaneous malignant melanoma and it′s relation to tumor thickness

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Faghihi Gita

2005-01-01

Full Text Available P53 tumor suppressor gene mutation is one of the most common genetic alteration in human malignancies. This study was done to determine the prevalence of the P53 antigen expression by sex, age, type of melanoma, thickness of the lesion and site of the antigen expression either cytoplasmic or nuclear. Paraffin embeded block of 50 patients (45 primary and metastaticwith documented diagnosis of melanoma deparaffinized and immuno stained with DO-7 monoclonal antibody. The lesions were divided depending on the degree of the staining as follow: 1. no staining, 2. mild (less than 10%, 3. moderate (10%-50% staining, 4. severe (more than 50%. Fifty four percent of evaluated patients were female and 46% were male. Forty percent of lesions were graded as no staining, 36% of lesions showed mild staining, 14% moderate and 10% severe staining site of expression was excusively in the cytoplasm. There was no meaningful statistical deference between severity of staining and the age group, sex, type and thickness of melanoma. (p value was 0.532, 0.488, 0.626, 0.954 respectively.

Antigen-specific cytotoxic T cell and antigen-specific proliferating T cell clones can be induced to cytolytic activity by monoclonal antibodies against T3

NARCIS (Netherlands)

Spits, H.; Yssel, H.; Leeuwenberg, J.; de Vries, J. E.

1985-01-01

T3 is a human differentiation antigen expressed exclusively on mature T cells. In this study it is shown that anti-T3 monoclonal antibodies, in addition to their capacity to induce T cells to proliferate, are able to induce antigen-specific cytotoxic T lymphocyte clones to mediate antigen

Matrix Metalloproteinase-2, Squamous Cell Carcinoma Antigen, and Tissue Polypeptide-Specific Antigen Expression in Egyptian Patients with Cervical Carcinoma: Relationship with Prognosis

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Maha Imam Ahmed

2004-01-01

Full Text Available Matrix metalloproteinases (MMPs, a family of proteolytic enzymes produced by both stromal and tumor cells, appear to have a key role in the events leading to local invasion and metastasis by malignant neoplasms. In the present study, we evaluated the role of MMP-2, squamous cell carcinoma antigen (SCCA, and tissue polypeptide – specific antigen (TPS in cervical neoplasia. Using Western blotting and enzyme immunoassay (EIA, we analyzed 50 patients with cervical carcinoma (CC and 25 normal controls for expression of MMP-2 in tissue cell lysates. We also quantified SCCA and TPS with microparticle immunoassay and EIA, respectively. The results were correlated with human papilloma virus (HPV infection, clinicopathological findings, and disease outcome. The cutoff point for each marker was estimated from receiver operating characteristic curves. Logistic regression analysis was performed to estimate the odds ratio (OR and 95% confidence interval (CI for each marker. MMP-2, SCCA, and TPS protein expression were significantly higher in patients with CC than in normal controls. While TPS was the best marker for discriminating between patients and controls, MMP-2 was associated with an advanced tumor stage (OR, 13.9 [95% CI, 1.4-133.9] and poor histological grade (OR, 10.2 [95% CI, 1.7-60.5]. Moreover, independent of the effect of an advanced CC stage and grade, the patients' age, and the presence of HPV infection, MMP-2 was considered a strong predictor for CC recurrence (OR, 8.1 [95% CI, 1.3- 49.1]. Tissue markers may be used to select high-risk patients for early detection of and adjuvant therapy for recurrence. Our MMP-2 findings are particularly relevant to the development of protease inhibitors as a new cancer therapy approach.

Increasing a Robust Antigen-Specific Cytotoxic T Lymphocyte Response by FMDV DNA Vaccination with IL-9 Expressing Construct

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Qiang Zou

2010-01-01

Full Text Available Various chemokines and cytokines as adjuvants can be used to improve efficacy of DNA vaccination. In this study, we sought to investigate if a DNA construct expressing IL-9 (designed as proV-IL9 as a molecular adjuvant enhance antigen specific immune responses elicited by the pcD-VP1 DNA vaccination. Mice immunized with pcD-VP1 combined with proV-IL9 developed a strong humoral response. In addition, the coinoculation induced significant higher level of antigen-specific cell proliferation and cytotoxic response. This agreed well with higher expression level of IFN-γ and perforin in CD8+ T cells, but not with IL-17 in these T cells. The results indicate that IL-9 induces the development of IFN-γ-producing CD8+ T cells (Tc1, but not the IL-17-producing CD8+ T cells (Tc17. Up-regulated expressions of BCL-2 and BCL-XL were exhibited in these Tc1 cells, suggesting that IL-9 may trigger antiapoptosis mechanism in these cells. Together, these results demonstrated that IL-9 used as molecular adjuvant could enhance the immunogenicity of DNA vaccination, in augmenting humoral and cellular responses and particularly promoting Tc1 activations. Thus, the IL-9 may be utilized as a potent Tc1 adjuvant for DNA vaccines.

Milk-induced eczema is associated with the expansion of T cells expressing cutaneous lymphocyte antigen.

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Abernathy-Carver, K J; Sampson, H A; Picker, L J; Leung, D Y

1995-02-01

The extravasation of T cells at sites of inflammation is critically dependent on the activity of homing receptors (HR) involved in endothelial cell recognition and binding. Two such HR (the cutaneous lymphocyte antigen [CLA] and L-selectin) have been shown to be selectively involved in T cell migration to skin and peripheral lymph nodes, respectively. This study was designed to assess the relationship between the organ specificity of an allergic reaction to food and the expression of HR on T cells activated in vitro by the relevant food allergen. Peripheral blood mononuclear cells were isolated from seven milk allergic children with a history of eczema when exposed to milk. All patients had a positive prick skin test and double-blind placebo-controlled food challenge to milk. 10 children with either allergic eosinophilic gastroenteritis or milk-induced enterocolitis and 8 nonatopic adults served as controls. Five-parameter flow cytometry using monoclonal antibodies was used for detection of the specific HR on freshly isolated T cells versus T cell blasts induced by a 6-d incubation with casein, as compared with Candida albicans. After in vitro stimulation with casein, but not C. albicans, patients with milk allergy and atopic dermatitis had a significantly greater percentage of CLA+ T cells (P < 0.01) than controls with milk-induced enterocolitis, allergic eosinophilic gastroenteritis, or nonatopic healthy controls. In contrast, the percentage of L-selectin-expressing T cells did not differ significantly between these groups. These data suggest that after casein stimulation allergic patients with milk-induced skin disease have an expanded population of CLA+ T cells, as compared with nonatopics or allergic patients without skin involvement. We postulate that heterogeneity in the regulation of HR expression on antigen-specific T cells may play a role in determining sites of involvement in tissue-directed allergic responses.

Expression of variant surface antigens by Plasmodium falciparum parasites in the peripheral blood of clinically immune pregnant women indicates ongoing placental infection

DEFF Research Database (Denmark)

Ofori, Michael F; Staalsoe, Trine; Bam, Victoria

2003-01-01

Placenta-sequestered Plasmodium falciparum parasites that cause pregnancy-associated malaria (PAM) in otherwise clinically immune women express distinct variant surface antigens (VSA(PAM)) not expressed by parasites in nonpregnant individuals. We report here that parasites from the peripheral blood...... of clinically immune pregnant women also express VSA(PAM), making them a convenient source of VSA(PAM) expressors for PAM vaccine research....

The reliability of cytoplasmic CD3 and CD22 antigen expression in the immunodiagnosis of acute leukemia: a study of 500 cases.

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Janossy, G; Coustan-Smith, E; Campana, D

1989-03-01

Current views about the origin of acute lymphoid leukemia (ALL) emphasize the importance of maturation arrest at a precursor cell level. Recently, the CD22 antigen has been identified in the cytoplasm of normal bone marrow-borne immature B lineage cells, while the CD3 antigen (epsilon chain) has been detected within normal immature thymic blasts. In the first part our study performed on 100 cases of known acute leukemias, the expression of such cytoplasmic molecules, referred to as cCD22 and cCD3, was analyzed together with their appearance in the leukemic cells' membrane (mCD22 and mCD3). The presence of cCD22 in B-lineage ALL and that of cCD3 in T-ALL has indeed fully confirmed the diagnosis reached by other markers, and mCD22 and mCD3 were expressed on only a few cases of B- and T-lineage ALL, also revealing a degree of developmental asynchrony within leukemic blasts. In the subsequent analysis both cCD22 and cCD3 have been included in a standard panel of diagnostic reagents applied on 500 consecutive cases of acute leukemia. Here the aim was to analyze both the diagnostic precision of individual markers and the heterogeneity of various leukemic types in terms of the expression of membrane and intracellular antigens and their cytochemical features (Sudan Black B and esterases). It has been found that cCD22 and cCD3 are exquisitely specific for B-precursor ALL (TdT+, CD19+) and T-ALL (TdT+, CD7+), respectively, while both markers are absent in acute myeloblastic leukemia (AML) and acute myelomonocytic and monocytic leukemia (AMML/AMoL). These observations contrast the findings which demonstrate that 31% of cases among nonlymphoid acute leukemia (including AML and AMML) express CD7 and/or TdT. The study of myeloid antigens detected by CD13, CD33, and CD14 is also informative and complementary, both in diagnosing and subdividing the AML and AMML/AMoL groups. The peculiar main observation of this study is that only with the combined use of these markers in a

Multiple injections of electroporated autologous T cells expressing a chimeric antigen receptor mediate regression of human disseminated tumor.

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Zhao, Yangbing; Moon, Edmund; Carpenito, Carmine; Paulos, Chrystal M; Liu, Xiaojun; Brennan, Andrea L; Chew, Anne; Carroll, Richard G; Scholler, John; Levine, Bruce L; Albelda, Steven M; June, Carl H

2010-11-15

Redirecting T lymphocyte antigen specificity by gene transfer can provide large numbers of tumor-reactive T lymphocytes for adoptive immunotherapy. However, safety concerns associated with viral vector production have limited clinical application of T cells expressing chimeric antigen receptors (CAR). T lymphocytes can be gene modified by RNA electroporation without integration-associated safety concerns. To establish a safe platform for adoptive immunotherapy, we first optimized the vector backbone for RNA in vitro transcription to achieve high-level transgene expression. CAR expression and function of RNA-electroporated T cells could be detected up to a week after electroporation. Multiple injections of RNA CAR-electroporated T cells mediated regression of large vascularized flank mesothelioma tumors in NOD/scid/γc(-/-) mice. Dramatic tumor reduction also occurred when the preexisting intraperitoneal human-derived tumors, which had been growing in vivo for >50 days, were treated by multiple injections of autologous human T cells electroporated with anti-mesothelin CAR mRNA. This is the first report using matched patient tumor and lymphocytes showing that autologous T cells from cancer patients can be engineered to provide an effective therapy for a disseminated tumor in a robust preclinical model. Multiple injections of RNA-engineered T cells are a novel approach for adoptive cell transfer, providing flexible platform for the treatment of cancer that may complement the use of retroviral and lentiviral engineered T cells. This approach may increase the therapeutic index of T cells engineered to express powerful activation domains without the associated safety concerns of integrating viral vectors. Copyright © 2010 AACR.

Expression and immunological characterisation of Eimeria tenella glycosylphosphatidylinositol-anchored surface antigen-5

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Ho, Sue-Kim; Nathan, Sheila; Wan, Kiew-Lian

2016-11-01

Eimeria tenella is the most pathogenic of the Eimeria species that infect chickens and causes huge economic losses to the poultry industry. The glycosylphosphatidylinositol-anchored surface antigen-5 (SAG5) found on the surface of the parasite has been shown to activate the chicken's immune system. In this study, recombinant SAG5 was expressed, purified and used to investigate the immune-inducing characteristics of the molecule. Chickens were immunized with purified recombinant SAG5 and sera were subjected to Enzyme-linked Immunosorbant Assay (ELISA). Results indicated that specific antibodies against rSAG5 were produced, with IgG detected at a higher level compared to IgA and IgM. Information on the immunological responses elicited by SAG5 provides essential knowledge that will contribute towards the effort to develop more effective strategies against coccidiosis.

Leukemia-associated antigens in man.

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Brown, G; Capellaro, D; Greaves, M

1975-12-01

Rabbit antisera raised against acute lymphoblastic leukemia (ALL) cells were used to distinguish ALL from other leukemias, to identify rare leukemia cells in the bone marrow of patients in remission, and to define human leukemia-associated antigens. Antibody binding was studied with the use of immunofluorescence reagents and the analytic capacity of the Fluorescence Activated Cell Sorter-1 (FACS-1). The results indicated that most non-T-cell ALL have three leukemia-associated antigens on their surface which are absent from normal lymphoid cells: 1) an antigen shared with myelocytes, myeloblastic leukemia cells, and fetal liver (hematopoietic) cells; 2) an antigen shared with a subset of intermediate normoblasts in normal bone marrow and fetal liver; and 3) an antigen found thus far only on non-T-cell ALL and in some acute undifferentiated leukemias, which we therefore regard as a strong candidate for a leukemia-specific antigen. These antigens are absent from a subgroup of ALL patients in which the lymphoblasta express T-cell surface markers. Preliminary studies on the bone marrow samples of patients in remission indicated that rare leukemia cells were present in some samples. The implications of these findings with respect to the heterogeneity and cell origin(s) of ALL, its diagnosis, and its potential monitoring during treatment were discussed.

Cloning of a Recombinant Plasmid Encoding Thiol-Specific Antioxidant Antigen (TSA) Gene of Leishmania majorand Expression in the Chinese Hamster Ovary Cell Line.

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Fatemeh, Ghaffarifar; Fatemeh, Tabatabaie; Zohreh, Sharifi; Abdolhosein, Dalimiasl; Mohammad Zahir, Hassan; Mehdi, Mahdavi

2012-01-01

TSA (thiol-specific antioxidant antigen) is the immune-dominant antigen of Leishmania major and is considered to be the most promising candidate molecule for a recombinant or DNA vaccine against leishmaniasis. The aim of the present work was to express a plasmid containing the TSA gene in eukaryotic cells. Genomic DNA was extracted, and the TSA gene was amplified by polymerase chain reaction (PCR). The PCR product was cloned into the pTZ57R/T vector, followed by subcloning into the eukaryotic expression vector pcDNA3 (EcoRI and HindIII sites). The recombinant plasmid was characterised by restriction digest and PCR. Eukaryotic Chinese hamster ovary cells were transfected with the plasmid containing the TSA gene. Expression of the L. major TSA gene was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting. The plasmid containing the TSA gene was successfully expressed, as demonstrated by a band of 22.1 kDa on Western blots. The plasmid containing the TSA gene can be expressed in a eukaryotic cell line. Thus, the recombinant plasmid may potentially be used as a DNA vaccine in animal models.

Development of stable Vibrio cholerae O1 Hikojima type vaccine strains co-expressing the Inaba and Ogawa lipopolysaccharide antigens.

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Stefan L Karlsson

Full Text Available We describe here the development of stable classical and El Tor V. cholerae O1 strains of the Hikojima serotype that co-express the Inaba and Ogawa antigens of O1 lipopolysaccharide (LPS. Mutation of the wbeT gene reduced LPS perosamine methylation and thereby gave only partial transformation into Ogawa LPS on the cell surface. The strains express approximately equal amounts of Inaba- and Ogawa-LPS antigens which are preserved after formalin-inactivation of the bacteria. Oral immunizations of both inbred and outbred mice with formalin-inactivated whole-cell vaccine preparations of these strains elicited strong intestinal IgA anti-LPS as well as serum vibriocidal antibody responses against both Inaba and Ogawa that were fully comparable to the responses induced by the licensed Dukoral vaccine. Passive protection studies in infant mice showed that immune sera raised against either of the novel Hikojima vaccine strains protected baby mice against infection with virulent strains of both serotypes. This study illustrates the power of using genetic manipulation to improve the properties of bacteria strains for use in killed whole-cell vaccines.

Investigations on flexural wave propagation and attenuation in a modified one-dimensional acoustic black hole using a laser excitation technique

Science.gov (United States)

Ji, Hongli; Luo, Jing; Qiu, Jinhao; Cheng, Li

2018-05-01

Acoustic Black Holes (ABHs), as a new type of passive structure for vibration damping enhancement and noise attenuation, have been drawing increasing attentions of many researchers. Due to the difficulty in manufacturing the sharp edges required by the ABH structures, it is important to understand the wave propagation and attenuation process in the presence of damping layers in non-ideal ABHs with a truncated edge. In this paper, an analytical expression of the wave reflection coefficient in a modified one-dimensional ABH is derived and a time-domain experimental method based on a laser excitation technique is used to visualize the wave propagation. In the experimental studies, the flexural waves in the ABH were excited by a scanning pulse laser and measured by a Laser Doppler Vibrometer (LDV). The incident wave and reflected wave were separated from the measured original wave field and the decrease of the wave velocity in the ABH was exhibited. The reflection coefficient was calculated from the ratio of the amplitude of the reflected wave to that of the incident wave for different ABH parameters and different thicknesses of the damping layer. The measured reflection coefficients were used to identify the unknown coefficients in the theoretical formula. The results confirm that there exists an optimal thickness for the damping layer, which leads to the minimum wave reflection. Based on the laser-induced visualization technique and various signal processing and feature extraction methods, the entire process of the wave propagation in a non-ideal one-dimensional ABH structure can be visualized and scrutinized.

Cloning and Expression of Fusion Genes of Domain A-1 Protective Antigen of Bacillus Anthracis and Shigella Enterotoxin B Subunit (Stxb In E. Coil

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AH ahmadi

2015-02-01

Conclusion: The findings of the current study revealed that this antigen can be raised as an anti-cancer and recombinant vaccine candidate against types of Shigella, Escherichia coli and Bacillus anthracis which can be due to such factors as identification of antigen(PA by antibody PA20, its apoptosis induction properties, property of immunogenicity, adjuvant and delivery of STxB protein and high expression levels of Gb3 in human cancer cells.

Co-ordinate expression of the pre-T-cell receptor complex and a novel immature thymocyte-specific antigen, IMT-1, during thymocyte development.

Science.gov (United States)

Tong, J J; Kishi, H; Nagata, T; Muraguchi, A

1999-01-01

Previously we described a monoclonal antibody (mAb) that reacted with a cell-surface antigen, immature thymocyte antigen-1 (IMT-1), which is expressed on thymocytes of late CD4- CD8- (double negative) to early CD4+ CD8+ (double positive) differentiation stages. In this study, we investigated the expression of IMT-1 on various cell lineages in thymus as well as in peripheral lymphoid organs. We found that IMT-1 is expressed on T-cell receptor (TCR)-betalo and TCR-deltalo thymocytes, but not on TCR-betahi, TCR-deltahi or natural killer (NK)1.1+ thymocytes, or on peripheral alpha beta or gamma delta T cells. We also investigated the kinetics of expression of IMT-1 during fetal thymocyte development and compared it with the expression of the pre-TCR complex, comprising CD3, pre-TCR-alpha (pTalpha) and TCR-beta. We found that expression of both was similar, starting at day 14.5 of gestation, peaking on day 16.5 and gradually decreasing thereafter. Furthermore, the expression of both IMT-1 and pTalpha was drastically reduced when DN thymocytes in recombination activating gene (RAG)-2-/- mice were challenged in vivo with anti-CD3 mAb. These results indicate that IMT-1 is expressed on not only immature thymocytes of alpha beta T-cell lineage but also on those of gamma delta T-cell lineage, and that the expression of IMT-1 and the pre-TCR complex is co-ordinately regulated during the alpha beta lineage thymocyte development.

Expressed var gene repertoire and variant surface antigen diversity in a shrinking Plasmodium falciparum population.

Science.gov (United States)

Carlos, Bianca C; Fotoran, Wesley L; Menezes, Maria J; Cabral, Fernanda J; Bastos, Marcele F; Costa, Fabio T M; Sousa-Neto, Jayme A; Ribolla, Paulo E M; Wunderlich, Gerhard; Ferreira, Marcelo U

2016-11-01

The var gene-encoded erythrocyte membrane protein-1 of Plasmodium falciparum (PfEMP-1) is the main variant surface antigen (VSA) expressed on infected erythrocytes. The rate at which antibody responses to VSA expressed by circulating parasites are acquired depends on the size of the local VSA repertoire and the frequency of exposure to new VSA. Because parasites from areas with declining malaria endemicity, such as the Amazon, typically express a restricted PfEMP-1 repertoire, we hypothesized that Amazonians would rapidly acquire antibodies to most locally circulating VSA. Consistent with our expectations, the analysis of 5878 sequence tags expressed by 10 local P. falciparum samples revealed little PfEMP-1 DBL1α domain diversity. Among the most commonly expressed DBL1α types, 45% were shared by two or more independent parasite lines. Nevertheless, Amazonians displayed major gaps in their repertoire of anti-VSA antibodies, although the breadth of anti-VSA antibody responses correlated positively with their cumulative exposure to malaria. We found little antibody cross-reactivity even when testing VSA from related parasites expressing the same dominant DBL1α types. We conclude that variant-specific immunity to P. falciparum VSAs develops slowly despite the relatively restricted PfEMP-1 repertoire found in low-endemicity settings. Copyright © 2016 Elsevier Inc. All rights reserved.

Expression of cancer-associated simple mucin-type O-glycosylated antigens in parasites.

Science.gov (United States)

Osinaga, Eduardo

2007-01-01

Simple mucin-type O-glycan structures, such as Tn, TF, sialyl-Tn and Tk antigens, are among of the most specific human cancer-associated structures. These antigens are involved in several types of receptor-ligand interactions, and they are potential targets for immunotherapy. In the last few years several simple mucin-type O-glycan antigens were identified in different species belonging to the main two helminth parasite phyla, and sialyl-Tn bearing glycoproteins were detected in Trypanosoma cruzi. These results are of interest to understand new aspects in parasite glycoimmunology and may help identify new biological characteristics of parasites as well of the host-parasite relationship. Considering that different groups reported a negative correlation between certain parasite infections and cancer development, we could hypothesize that simple mucin-type O-glycosylated antigens obtained from parasites could be good potential targets for cancer immunotherapy.

Differential expression of carbohydrate antigen 19-9 in human colorectal cancer: A comparison with colon and rectal cancers

Science.gov (United States)

ZHANG, SHUAI; CHEN, YIJUN; ZHU, ZHANMENG; DING, YUNLONG; REN, SHUANGYI; ZUO, YUNFEI

2013-01-01

Colorectal cancer is one of the leading causes of cancer-related mortality, being the third most commonly diagnosed cancer among men and the second among women. Accumulating evidence regarding carbohydrate antigen (CA) demonstrated that tumor-associated antigens are clinically useful for the diagnosis, staging and monitoring of human gastrointestinal cancers, particularly colorectal cancer. There has been an extensive investigation for sensitive and specific markers of this disease. Currently, the gastrointestinal cancer-associated carbohydrate antigen 19-9 (CA19-9) is the most widely applied tumor marker in cancer diagnosis. Despite a similar etiology and cancer incidence rates, there are anatomical and clinical differences between colon and rectal cancer, as well as differences regarding tumor progression and adjuvant treatments. To investigate whether CA19-9 is differentially expressed between colon and rectal cancer, we conducted a differential analysis of serum CA19-9 levels among 227 cases of colorectal cancer, analyzing gender, age, Dukes’ stage and distant metastasis for human colon and rectal cancer as a single entity, separately and as matched pairs. We demonstrated that the serum CA19-9 levels in colorectal cancer were upregulated in advanced stages with distant metastasis. By contrast, the serum CA19-9 levels in colon cancer displayed a differential and upregulated behavior in advanced stages with distant metastasis. By analyzing as matched pairs, the upregulated serum CA19-9 levels in rectal cancer during the early stages without distant metastasis further supported our hypothesis that the expression of CA19-9 displays a site-specific differential behavior. The integrative analysis suggested a significant difference between human colon and rectal cancer, justifying individualized therapy for these two types of cancer. PMID:24649295

Analysis of IgG with specificity for variant surface antigens expressed by placental Plasmodium falciparum isolates

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Kremsner Peter G

2004-07-01

Full Text Available Abstract Background Pregnancy-associated malaria (PAM is caused by Plasmodium falciparum-infected erythrocytes that can sequester in placental intervillous space by expressing particular variant surface antigens (VSA that can mediate adhesion to chondroitin sulfate A (CSA in vitro. IgG antibodies with specificity for the VSA expressed by these parasites (VSAPAM are associated with protection from maternal anaemia, prematurity and low birth weight, which is the greatest risk factor for death in the first month of life. Methods In this study, the development of anti-VSAPAM antibodies in a group of 151 women who presented to the maternity ward of Albert Schweitzer Hospital in Lambaréné, Gabon for delivery was analysed using flow cytometry assays. Plasma samples from placenta infected primiparous women were also investigated for their capacity to inhibit parasite binding to CSA in vitro. Results In the study cohort, primiparous as well as secundiparous women had the greatest risk of infection at delivery as well as during pregnancy. Primiparous women with infected placentas at delivery showed higher levels of VSAPAM-specific IgG compared to women who had no malaria infections at delivery. Placental isolates of Gabonese and Senegalese origin tested on plasma samples from Gabon showed parity dependency and gender specificity patterns. There was a significant correlation of plasma reactivity as measured by flow cytometry between different placental isolates. In the plasma of infected primiparous women, VSAPAM-specific IgG measured by flow cytometry could be correlated with anti-adhesion antibodies measured by the inhibition of CSA binding. Conclusion Recognition of placental parasites shows a parity- and sex- dependent pattern, like that previously observed in laboratory strains selected to bind to CSA. Placental infections at delivery in primiparous women appear to be sufficient to induce functional antibodies which can both recognize the surface of

Novel Antitumor Strategy Utilizing a Plasmid Expressing a Mycobacterium tuberculosis Antigen as a “Danger Signal” to Block Immune Escape of Tumor Cells

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Yoshiyuki Koyama

2015-07-01

Full Text Available Immune escape of tumor cells is one of the main obstacles hindering the effectiveness of cancer immunotherapy. We developed a novel strategy to block immune escape by transfecting tumor cells in vivo with genes of pathogenic antigens from Mycobacterium tuberculosis (TB. This induces presentation of the TB antigen on tumor cell surfaces, which can be recognized by antigen presenting cells (APCs as a “danger signal” to stimulate antitumor immune response. This strategy is also expected to amplify the immune response against tumor-associated antigens, and block immune escape of the tumor. DNA/PEI/chondroitin sulfate ternary complex is a highly effective non-viral gene vector system for in vivo transfection. A therapeutic complex was prepared using a plasmid encoding the TB antigen, early secretory antigenic target-6 (ESAT-6. This was injected intratumorally into syngeneic tumor-bearing mice, and induced significant tumor growth suppression comparable to or higher than similar complexes expressing cytokines such as interleukin-2 (IL-2 and interleukin-12 (IL-12. Co-transfection of the cytokine-genes and the ESAT-6-gene enhanced the antitumor efficacy of either treatment alone. In addition, complete tumor regression was achieved with the combination of ESAT-6 and IL-2 genes.

Duffy blood group antigens: structure, serological properties and function

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Ewa Łukasik

2016-03-01

Full Text Available Duffy (Fy blood group antigens are located on seven-transmembrane glycoprotein expressed on erythrocytes and endothelial cells, which acts as atypical chemokine receptor (ACKR1 and malarial receptor. The biological role of the Duffy glycoprotein has not been explained yet. It is suggested that Duffy protein modulate the intensity of the inflammatory response. The Duffy blood group system consists of two major antigens, Fya and Fyb, encoded by two codominant alleles designated FY*A and FY*B which differ by a single nucleotide polymorphism (SNP at position 125G>A of the FY gene that results in Gly42Asp amino acid change in the Fya and Fyb antigens, respectively. The presence of antigen Fya and/or Fyb on the erythrocytes determine three Duffy-positive phenotypes: Fy(a+b-, Fy(a-b+ and Fy(a+b+, identified in Caucasian population. The Duffy-negative phenotype Fy(a-b-, frequent in Africans, but very rare in Caucasians, is defined by the homozygous state of FY*B-33 alleles. The FY*B-33 allele is associated with a SNP -33T>C in the promoter region of the FY gene, which suppresses erythroid expression of this gene without affecting its expression in other tissues. The FY*X allele, found in Caucasians, is correlated with weak expression of Fyb antigen. Fyx antigen differs from the native Fyb by the Arg89Cys and Ala100Thr amino acid substitutions due to SNPs: 265C>T and 298G>A in FY*B allele. The frequency of the FY alleles shows marked geographic disparities, the FY*B-33 allele is predominant in Africans, the FY*B in Caucasians, while the FY*A allele is dominant in Asians and it is the most prevalent allele globally.

Effective antigen presentation to helper T cells by human eosinophils.

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Farhan, Ruhaifah K; Vickers, Mark A; Ghaemmaghami, Amir M; Hall, Andrew M; Barker, Robert N; Walsh, Garry M

2016-12-01

Although eosinophils are inflammatory cells, there is increasing attention on their immunomodulatory roles. For example, murine eosinophils can present antigen to CD4 + T helper (Th) cells, but it remains unclear whether human eosinophils also have this ability. This study determined whether human eosinophils present a range of antigens, including allergens, to activate Th cells, and characterized their expression of MHC class II and co-stimulatory molecules required for effective presentation. Human peripheral blood eosinophils purified from non-allergic donors were pulsed with the antigens house dust mite extract (HDM), Timothy Grass extract (TG) or Mycobacterium tuberculosis purified protein derivative (PPD), before co-culture with autologous CD4 + Th cells. Proliferative and cytokine responses were measured, with eosinophil expression of HLA-DR/DP/DQ and the co-stimulatory molecules CD40, CD80 and CD86 determined by flow cytometry. Eosinophils pulsed with HDM, TG or PPD drove Th cell proliferation, with the response strength dependent on antigen concentration. The cytokine responses varied with donor and antigen, and were not biased towards any particular Th subset, often including combinations of pro- and anti-inflammatory cytokines. Eosinophils up-regulated surface expression of HLA-DR/DP/DQ, CD80, CD86 and CD40 in culture, increases that were sustained over 5 days when incubated with antigens, including HDM, or the major allergens it contains, Der p I or Der p II. Human eosinophils can, therefore, act as effective antigen-presenting cells to stimulate varied Th cell responses against a panel of antigens including HDM, TG or PPD, an ability that may help to determine the development of allergic disease. © 2016 John Wiley & Sons Ltd.

Plant bioreactors for the antigenic hook-associated flgK protein expression

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Luciana Rossi

2014-01-01

Full Text Available Plants engineered with genes encoding for the antigenic proteins of various microorganisms have shown to correctly express the proteins that elicit the production of antibodies in mammalian hosts. In livestock, plant-based vaccines could represent an innovative strategy for oral vaccination, especially to prevent infection by enteric pathogens. The aim of this study was to evaluate tobacco plants as a seedspecific expression system for the production of the flgK flagellar hook-associated protein from a wild type Salmonella typhimurium strain, as a model of an edible vaccine. The flgK gene is the principal component of bacterial flagella and is recognised as virulence factor by the innate immune system. It was isolated from the Salmonella typhimurium strain by PCR. The encoding sequence of flgK was transferred into a pBI binary vector, under control of soybean basic 7S globulin promoter for the seed-specific. Plant transformation was carried out using recombinant EHA 105 Agrobacterium tumefaciens. A transgenic population was obtained made up of independently kanamycin-resistant transgenic plants, which had a similar morphological appearance to the wild-type plants. Molecular analyses of seeds confirmed the integration of the gene and the average expression level of flgK was estimated to be about 0.6 mg per gram of seeds, corresponding to 0.33% of the total amount of soluble protein in tobacco seeds. This study showed that the foreign flgK gene could be stably incorporated into the tobacco plant genome by transcription through the nuclear apparatus of the plant, and that these genes are inherited by the next generation.

Enhanced expression in vivo of HLA-ABC antigens and beta 2-microglobulin on human lymphoid cells induced by human interferon-alpha in patients with lung cancer. Enhanced expression of class I major histocompatibility antigens prior to treatment

DEFF Research Database (Denmark)

Nissen, Mogens Holst; Plesner, T; Larsen, J K

1985-01-01

than 0.5, respectively) by day-to-day analysis of an untreated healthy control group. An increased expression of both HLA-ABC (mean 55%, P less than 0.0005) and beta 2m (mean 23%, P less than 0.01) was also observed prior to treatment in the lung cancer patients when compared to a group of age matched......The effect of cloned human interferon-alpha (IFN-alpha) on the expression of HLA-ABC antigens (HLA-ABC) and beta 2-microglobulin (beta 2m) on human peripheral lymphoid cells in vivo was studied by cytofluorometry using monoclonal antibodies and fluorescein-labelled rabbit anti-mouse immunoglobulin....... A significant increase in the mean fluorescence intensity of HLA-ABC (median 59%, P less than 0.001) and beta 2m (median 57%, P less than 0.001) on small lymphoid cells was observed 24 h after initiation of IFN-alpha treatment (50 X 10(6) units IFN-alpha/m2 three times a week). The enhanced expression...

Effector CD4+ T cells recognize intravascular antigen presented by patrolling monocytes.

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Westhorpe, Clare L V; Norman, M Ursula; Hall, Pam; Snelgrove, Sarah L; Finsterbusch, Michaela; Li, Anqi; Lo, Camden; Tan, Zhe Hao; Li, Songhui; Nilsson, Susan K; Kitching, A Richard; Hickey, Michael J

2018-02-21

Although effector CD4 + T cells readily respond to antigen outside the vasculature, how they respond to intravascular antigens is unknown. Here we show the process of intravascular antigen recognition using intravital multiphoton microscopy of glomeruli. CD4 + T cells undergo intravascular migration within uninflamed glomeruli. Similarly, while MHCII is not expressed by intrinsic glomerular cells, intravascular MHCII-expressing immune cells patrol glomerular capillaries, interacting with CD4 + T cells. Following intravascular deposition of antigen in glomeruli, effector CD4 + T-cell responses, including NFAT1 nuclear translocation and decreased migration, are consistent with antigen recognition. Of the MHCII + immune cells adherent in glomerular capillaries, only monocytes are retained for prolonged durations. These cells can also induce T-cell proliferation in vitro. Moreover, monocyte depletion reduces CD4 + T-cell-dependent glomerular inflammation. These findings indicate that MHCII + monocytes patrolling the glomerular microvasculature can present intravascular antigen to CD4 + T cells within glomerular capillaries, leading to antigen-dependent inflammation.

Selective Inhibition of Tumor Growth by Clonal NK Cells Expressing an ErbB2/HER2-Specific Chimeric Antigen Receptor

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Schönfeld, Kurt; Sahm, Christiane; Zhang, Congcong; Naundorf, Sonja; Brendel, Christian; Odendahl, Marcus; Nowakowska, Paulina; Bönig, Halvard; Köhl, Ulrike; Kloess, Stephan; Köhler, Sylvia; Holtgreve-Grez, Heidi; Jauch, Anna; Schmidt, Manfred; Schubert, Ralf; Kühlcke, Klaus; Seifried, Erhard; Klingemann, Hans G; Rieger, Michael A; Tonn, Torsten; Grez, Manuel; Wels, Winfried S

2015-01-01

Natural killer (NK) cells are an important effector cell type for adoptive cancer immunotherapy. Similar to T cells, NK cells can be modified to express chimeric antigen receptors (CARs) to enhance antitumor activity, but experience with CAR-engineered NK cells and their clinical development is still limited. Here, we redirected continuously expanding and clinically usable established human NK-92 cells to the tumor-associated ErbB2 (HER2) antigen. Following GMP-compliant procedures, we generated a stable clonal cell line expressing a humanized CAR based on ErbB2-specific antibody FRP5 harboring CD28 and CD3ζ signaling domains (CAR 5.28.z). These NK-92/5.28.z cells efficiently lysed ErbB2-expressing tumor cells in vitro and exhibited serial target cell killing. Specific recognition of tumor cells and antitumor activity were retained in vivo, resulting in selective enrichment of NK-92/5.28.z cells in orthotopic breast carcinoma xenografts, and reduction of pulmonary metastasis in a renal cell carcinoma model, respectively. γ-irradiation as a potential safety measure for clinical application prevented NK cell replication, while antitumor activity was preserved. Our data demonstrate that it is feasible to engineer CAR-expressing NK cells as a clonal, molecularly and functionally well-defined and continuously expandable cell therapeutic agent, and suggest NK-92/5.28.z cells as a promising candidate for use in adoptive cancer immunotherapy. PMID:25373520

Arabidopsis thaliana plants expressing Rift Valley fever virus antigens: Mice exhibit systemic immune responses as the result of oral administration of the transgenic plants.

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Kalbina, Irina; Lagerqvist, Nina; Moiane, Bélisario; Ahlm, Clas; Andersson, Sören; Strid, Åke; Falk, Kerstin I

2016-11-01

The zoonotic Rift Valley fever virus affects livestock and humans in Africa and on the Arabian Peninsula. The economic impact of this pathogen due to livestock losses, as well as its relevance to public health, underscores the importance of developing effective and easily distributed vaccines. Vaccines that can be delivered orally are of particular interest. Here, we report the expression in transformed plants (Arabidopsis thaliana) of Rift Valley fever virus antigens. The antigens used in this study were the N protein and a deletion mutant of the Gn glycoprotein. Transformed lines were analysed for specific mRNA and protein content by RT-PCR and Western blotting, respectively. Furthermore, the plant-expressed antigens were evaluated for their immunogenicity in mice fed the transgenic plants. After oral intake of fresh transgenic plant material, a proportion of the mice elicited specific IgG antibody responses, as compared to the control animals that were fed wild-type plants and of which none sero-converted. Thus, we show that transgenic plants can be readily used to express and produce Rift Valley Fever virus proteins, and that the plants are immunogenic when given orally to mice. These are promising findings and provide a basis for further studies on edible plant vaccines against the Rift Valley fever virus. Copyright © 2016 Elsevier Inc. All rights reserved.

The prognostic value of oncogenic antigen 519 (OA-519) expression and proliferative activity detected by antibody MIB-1 in node-negative breast cancer

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Jensen, V; Ladekarl, M; Holm-Nielsen, P

1995-01-01

The prognostic value of oncogenic antigen 519 (OA-519) expression and tumour proliferative activity was evaluated in a retrospective series of 118 patients with low-risk breast cancer. Low risk was defined as negative axillary nodes, tumour diameter histological evidence...... analysis, both the MIB-1 index and OA-519 expression were of independent prognostic value (2p breast cancer who might benefit from adjuvant therapy....

Natural Mutations in Streptococcus agalactiae Resulting in Abrogation of β Antigen Production.

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Vasilyeva, Anastasia; Santos Sanches, Ilda; Florindo, Carlos; Dmitriev, Alexander

2015-01-01

Streptococcus agalactiae genome encodes 21 two-component systems (TCS) and a variety of regulatory proteins in order to control gene expression. One of the TCS, BgrRS, comprising the BgrR DNA-binding regulatory protein and BgrS sensor histidine kinase, was discovered within a putative virulence island. BgrRS influences cell metabolism and positively control the expression of bac gene, coding for β antigen at transcriptional level. Inactivation of bgrR abrogated bac gene expression and increased virulence properties of S. agalactiae. In this study, a total of 140 strains were screened for the presence of bac gene, and the TCS bgrR and bgrS genes. A total of 53 strains carried the bac, bgrR and bgrS genes. Most of them (48 strains) expressed β antigen, while five strains did not express β antigen. Three strains, in which bac gene sequence was intact, while bgrR and/or bgrS genes had mutations, and expression of β antigen was absent, were complemented with a constructed plasmid pBgrRS(P) encoding functionally active bgrR and bgrS gene alleles. This procedure restored expression of β antigen indicating the crucial regulatory role of TCS BgrRS. The complemented strain A49V/BgrRS demonstrated attenuated virulence in intraperitoneal mice model of S. agalactiae infection compared to parental strain A49V. In conclusion we showed that disruption of β antigen expression is associated with: i) insertion of ISSa4 upstream the bac gene just after the ribosomal binding site; ii) point mutation G342A resulting a stop codon TGA within the bac gene and a truncated form of β antigen; iii) single deletion (G) in position 439 of the bgrR gene resulting in a frameshift and the loss of DNA-binding domain of the BgrR protein, and iv) single base substitutions in bgrR and bgrS genes causing single amino acid substitutions in BgrR (Arg187Lys) and BgrS (Arg252Gln). The fact that BgrRS negatively controls virulent properties of S. agalactiae gives a novel clue for understanding of S

Quantitative radioimmunoassay for membranous and soluble H-Y antigen typing

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Casanova-Bettane, M.; Latron, F.; Jakob, H.; Fellous, M.

1981-01-01

Two sensitive and quantitative methods for membranous or soluble H-Y antigen typing using rat anti-H-Y immune sera and /sup 125/I labelled protein A were carried out. These techniques were used to study H-Y antigen expression in human cell lines, and to refine the hypothesis that ..beta../sub 2/m serves as an anchorage point for H-Y antigen.

Antigenic and Genotypic Similarity between Primary Glioblastomas and Their Derived Neurospheres

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Valentina Caldera

2011-01-01

Full Text Available Formation of neurospheres (NS in cultures of glioblastomas (GBMs, with self-renewal, clonogenic capacities, and tumorigenicity following transplantation into immunodeficient mice, may denounce the existence of brain tumor stem cells (BTSCs in vivo. In sixteen cell lines from resected primary glioblastomas, NS showed the same genetic alterations as primary tumors and the expression of stemness antigens. Adherent cells (AC, after adding 10% of fetal bovine serum (FBS to the culture, were genetically different from NS and prevailingly expressed differentiation antigens. NS developed from a highly malignant tumor phenotype with proliferation, circumscribed necrosis, and high vessel density. Beside originating from transformed neural stem cells (NSCs, BTSCs may be contained within or correspond to dedifferentiated cells after mutation accumulation, which reacquire the expression of stemness antigens.

Cancer-germline antigen vaccines and epigenetic enhancers

DEFF Research Database (Denmark)

Gjerstorff, Morten Frier; Burns, Jorge; Ditzel, Henrik Jorn

2010-01-01

IMPORTANCE OF THE FIELD: Immunotherapy holds great potential for disseminated cancer, and cancer-germline (CG) antigens are among the most promising tumor targets. They are widely expressed in different cancer types and are essentially tumor-specific, since their expression in normal tissues is l...

Cell surface expression level variation between two common Human Leukocyte Antigen alleles, HLA-A2 and HLA-B8, is dependent on the structure of the C terminal part of the alpha 2 and the alpha 3 domains

DEFF Research Database (Denmark)

Dellgren, Christoffer; Nehlin, Jan O; Barington, Torben

2015-01-01

Constitutive cell surface expression of Human Leukocyte Antigen (HLA) class I antigens vary extremely from tissue to tissue and individual antigens may differ widely in expression levels. Down-regulation of class I expression is a known immune evasive mechanism used by cancer cells and viruses....... Moreover, recent observations suggest that even minor differences in expression levels may influence the course of viral infections and the frequency of complications to stem cell transplantation. We have shown that some human multipotent stem cells have high expression of HLA-A while HLA-B is only weakly...... expressed, and demonstrate here that this is also the case for the human embryonic kidney cell line HEK293T. Using quantitative flow cytometry and quantitative polymerase chain reaction we found expression levels of endogenous HLA-A3 (median 71,204 molecules per cell) 9.2-fold higher than the expression of...

Expression of senescent antigen on erythrocytes infected with a knobby variant of the human malaria parasite Plasmodium falciparum

International Nuclear Information System (INIS)

Winograd, E.; Greenan, J.R.T.; Sherman, I.W.

1987-01-01

Erythrocytes infected with a knobby variant of Plasmodium falciparum selectively bind IgG autoantibodies in normal human serum. Quantification of membrane-bound IgG, by use of 125 I-labeled protein A, revealed that erythrocytes infected with the knobby variant bound 30 times more protein A than did noninfected erythrocytes; infection with a knobless variant resulted in less than a 2-fold difference compared with noninfected erythrocytes. IgG binding to knobby erythrocytes appeared to be related to parasite development, since binding of 125 I-labeled protein A to cells bearing young trophozoites (less than 20 hr after parasite invasion) was similar to binding to uninfected erythrocytes. By immunoelectron microscopy, the membrane-bound IgG on erythrocytes infected with the knobby variant was found to be preferentially associated with the protuberances (knobs) of the plasma membrane. The removal of aged or senescent erythrocytes from the peripheral circulation is reported to involve the binding of specific antibodies to an antigen (senescent antigen) related to the major erythrocyte membrane protein band 3. Since affinity-purified autoantibodies against band 3 specifically bound to the plasma membrane of erythrocytes infected with the knobby variant of P. falciparum, it is clear that the malaria parasite induces expression of senescent antigen

Advances in alfalfa mosaic virus-mediated expression of anthrax antigen in planta

International Nuclear Information System (INIS)

Brodzik, R.; Bandurska, K.; Deka, D.; Golovkin, M.; Koprowski, H.

2005-01-01

Plant viruses show great potential for production of pharmaceuticals in plants. Such viruses can harbor a small antigenic peptide(s) as a part of their coat proteins (CP) and elicit an antigen-specific immune response. Here, we report the high yield and consistency in production of recombinant alfalfa mosaic virus (AlMV) particles for specific presentation of the small loop 15 amino acid epitope from domain-4 of the Bacillus anthracis protective antigen (PA-D4s). The epitope was inserted immediately after the first 25 N-terminal amino acids of AlMV CP to retain genome activation and binding of CP to viral RNAs. Recombinant AlMV particles were efficiently produced in tobacco, easily purified for immunological analysis, and exhibited extended stability and systemic proliferation in planta. Intraperitional injections of mice with recombinant plant virus particles harboring the PA-D4s epitope elicited a distinct immune response. Western blotting and ELISA analysis showed that sera from immunized mice recognized both native PA antigen and the AlMV CP

The Many Faces of Human Leukocyte Antigen-G

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Dahl, Mette; Djurisic, Snezana; Hviid, Thomas Vauvert F

2014-01-01

Pregnancy is an immunological paradox, where fetal antigens encoded by polymorphic genes inherited from the father do not provoke a maternal immune response. The fetus is not rejected as it would be theorized according to principles of tissue transplantation. A major contribution to fetal tolerance...... is the human leukocyte antigen (HLA)-G, a nonclassical HLA protein displaying limited polymorphism, restricted tissue distribution, and a unique alternative splice pattern. HLA-G is primarily expressed in placenta and plays multifaceted roles during pregnancy, both as a soluble and a membrane-bound molecule......, differences in HLA-G isoform expression, and possible differences in functional activity. Furthermore, we highlight important observations regarding HLA-G genetics and expression in preeclampsia that future research should address....

Human antibody and antigen response to IncA antibody of Chlamydia trachomatis.

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Tsai, P Y; Hsu, M C; Huang, C T; Li, S Y

2007-01-01

The high prevalence of C. trachomatis worldwide has underscored the importance of identifying specific immunogenic antigens in facilitating diagnosis as well as vaccine development. The aim of this study is to evaluate IncA antibody and antigen production in natural human infections. Our temporal expression study showed that IncA transcription and protein expression could be detected as early as 4 hours after the start of infection. Antibody responses could be detected in urine and genital swab samples from C. trachomatis-positive patients. It is especially interesting to note that the IncA antigen could be detected in urine. In conclusion, we have identified IncA as an important antigen in human. The potential applicability of the IncA antibody or antigen in the diagnosis as well as to vaccine development for C. trachomatis is also discussed.

Antigen Availability Shapes T Cell Differentiation and Function during Tuberculosis.

Science.gov (United States)

Moguche, Albanus O; Musvosvi, Munyaradzi; Penn-Nicholson, Adam; Plumlee, Courtney R; Mearns, Helen; Geldenhuys, Hennie; Smit, Erica; Abrahams, Deborah; Rozot, Virginie; Dintwe, One; Hoff, Søren T; Kromann, Ingrid; Ruhwald, Morten; Bang, Peter; Larson, Ryan P; Shafiani, Shahin; Ma, Shuyi; Sherman, David R; Sette, Alessandro; Lindestam Arlehamn, Cecilia S; McKinney, Denise M; Maecker, Holden; Hanekom, Willem A; Hatherill, Mark; Andersen, Peter; Scriba, Thomas J; Urdahl, Kevin B

2017-06-14

CD4 T cells are critical for protective immunity against Mycobacterium tuberculosis (Mtb), the cause of tuberculosis (TB). Yet to date, TB vaccine candidates that boost antigen-specific CD4 T cells have conferred little or no protection. Here we examined CD4 T cell responses to two leading TB vaccine antigens, ESAT-6 and Ag85B, in Mtb-infected mice and in vaccinated humans with and without underlying Mtb infection. In both species, Mtb infection drove ESAT-6-specific T cells to be more differentiated than Ag85B-specific T cells. The ability of each T cell population to control Mtb in the lungs of mice was restricted for opposite reasons: Ag85B-specific T cells were limited by reduced antigen expression during persistent infection, whereas ESAT-6-specific T cells became functionally exhausted due to chronic antigenic stimulation. Our findings suggest that different vaccination strategies will be required to optimize protection mediated by T cells recognizing antigens expressed at distinct stages of Mtb infection. Copyright © 2017 Elsevier Inc. All rights reserved.

The Thomsen-Friedenreich (T) simple mucin-type carbohydrate antigen in salivary gland carcinomas

DEFF Research Database (Denmark)

Therkildsen, M H; Mandel, U; Christensen, M

1995-01-01

acinic cell carcinomas and CinPA. A antigen was expressed in all tumour types from blood group A patients, except in CinPA. The expression of T, sialosyl-T, H and A antigens in relation to differentiation grade varied with tumour type in poorly differentiated areas. High and moderate differentiated areas...

Expression of class 5 antigens by meningococcal strains obtained from patients in Brazil and evaluation of two new monoclonal antibodies

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Elizabeth N. De Gaspari

Full Text Available Determining the profile of antigen expression among meningococci is important for epidemiologic surveillance and vaccine development. To this end, two new mouse monoclonal antibodies (MAbs have been derived against Neisseria meningitidis proteins (class 5. The MAbs were reactive against outer membrane antigens and were bactericidal. Selected anti-class 5 MAbs [(5.1-3E6-2; (5.3-3BH4-C7; (5.4-1BG11-C7; (5.5-3DH-F5G9 also 5F1F4-T3(5.c], and the two new monoclonal antibodies C14F10Br2 (5.8 and 7F11B5Br3 (5.9, were then tested against different meningococcal strains, (63 strains of serogroup A, 60 strains of serogroup C (from 1972 to 1974; and 136 strains of serogroup B (from 1992 meningococci. Our results demonstrated that the expression of class 5 proteins in the N. meningitidis B Brazilian strains studied is highly heterogeneous. The serotypes and subtypes of B:4:P1.15, B:4:P1.9, B:4:P1.7, B:4:P1.3, B:4:P1.14, B:4:P1.16, B:4:NT, and B:NT:NT were detected in N. meningitidis B serogroups.The strains C:2a:P1.2 and A:4.21:P1.9 were dominant in the C and A serogroups, respectively. Serogroup B organisms expressed the class 5 epitopes 5.4 (18%, 5.5 (22%, 5.8 (3.6%, 5.9 (8.0% and 5c (38%. Serogroup C expressed class 5 epitopes 5.1 (81%, 5.4 (35%, 5.5 (33% and 5.9 (5.0%; and serogroup A showed reactivity directed at the class 5 protein 5c (47%; and reactivity was present with the new monoclonal antibody, 5.9 (5.5%. We conclude that the two new MAbs are useful in detecting important group B, class 5 antigens, and that a broad selection of serogroup B, class 5 proteins would be required for an effective vaccine based on the class 5 proteins.

Cholera Toxin Promotes Th17 Cell Differentiation by Modulating Expression of Polarizing Cytokines and the Antigen-Presenting Potential of Dendritic Cells.

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Kang, Jung-Ok; Lee, Jee-Boong; Chang, Jun

2016-01-01

Cholera toxin (CT), an exotoxin produced by Vibrio cholera, acts as a mucosal adjuvant. In a previous study, we showed that CT skews differentiation of CD4 T cells to IL-17-producing Th17 cells. Here, we found that intranasal administration of CT induced migration of migratory dendritic cell (DC) populations, CD103+ DCs and CD11bhi DCs, to the lung draining mediastinal lymph nodes (medLN). Among those DC subsets, CD11bhi DCs that were relatively immature had a major role in Th17 cell differentiation after administration of CT. CT-treated BMDCs showed reduced expression of MHC class II and CD86, similar to CD11bhi DCs in medLN, and these BMDCs promoted Th17 cell differentiation more potently than other BMDCs expressing higher levels of MHC class II and CD86. By analyzing the expression of activation markers such as CD25 and CD69, proliferation and IL-2 production, we determined that CT-treated BMDCs showed diminished antigen-presenting potential to CD4+ T cells compared with normal BMDCs. We also found that CT-stimulated BMDCs promote activin A expression as well as IL-6 and IL-1β, and activin A had a synergic role with TGF-β1 in CT-mediated Th17 cell differentiation. Taken together, our results suggest that CT-stimulated DCs promote Th17 cell differentiation by not only modulating antigen-presenting potential but also inducing Th polarizing cytokines.

Interleukin-2 (rIL-2)-induced lymphokine-activated killer (LAK) cells and their precursors express the VGO1 antigen

International Nuclear Information System (INIS)

Denegri, J.F.; Peterson, J.; Tilley, P.

1989-01-01

Precursor and effector cells of recombinant interleukin-2 (r-IL-2)-induced lymphokine-activated killer (LAK) activity were investigated for their expression of VGO1. Peripheral blood lymphocytes (PBL) from normal donors were purified and separated in a FACS 420 into VGO1+- and VGO1- cell fractions before and after culture for 96 hr with 100 U/ml of r-IL-2. Their lytic activity against K 562 and Daudi cells was measured in a 51Cr release assay. The majority, if not all, of the LAK effector and precursor cells was VGO1+ lymphocytes. The expression of VGO1 by LAK precursor cells remained stable under the culture conditions used in our experiments. VGO1- lymphocytes cultured with r-IL-2 demonstrated neither LAK-induced activity nor expression of VGO1 antigen

Cancer/testis antigen SPATA19 is frequently expressed in benign prostatic hyperplasia and prostate cancer.

Science.gov (United States)

Wong, Kah Keng; Hussain, Faezahtul Arbaeyah; Loo, Suet Kee; López, José I

2017-12-01

Spermatogenesis-associated 19 (SPATA19) is a cancer/testis antigen overexpressed in various cancers. However, its protein expression profile in malignant or non-malignant tissues remains unknown. Thus, in this study, we investigated SPATA19 protein expression patterns in a panel of non-malignant human samples and primary prostate cancer (PCa) with or without benign prostatic hyperplasia (BPH) tissues. SPATA19 was absent in all non-malignant tissues investigated (n=14) except testis and prostate tissues. In terms of malignancies, all PCa cases were positive for SPATA19 exhibiting frequency between 20 and 100% (median 85%) with 63 (52.5%) and 57 (47.5%) cases demonstrating weak/moderate and strong intensities, respectively. Thirty-nine PCa cases (32.5%) contained BPH, and all BPH glands were SPATA19 positive (frequency between 20 and 100%; median 90%) with 13 (33.3%) demonstrating strong SPATA19 expression. Higher SPATA19 expression (higher frequency, intensity, or H-score) was not associated with overall survival or disease-specific survival (DFS) in all PCa cases. However, biochemical recurrence (BR) was associated with worse DFS (p = 0.005) in this cohort of 120 patients, and cases with strong SPATA19 intensity were associated with BR (p = 0.020). In conclusion, we showed that SPATA19 protein was frequently expressed in both BPH and PCa glands, and this warrants future investigations on its pathogenic roles in the disease. © 2017 APMIS. Published by John Wiley & Sons Ltd.

T cells targeting a neuronal paraneoplastic antigen mediate tumor rejection and trigger CNS autoimmunity with humoral activation.

Science.gov (United States)

Blachère, Nathalie E; Orange, Dana E; Santomasso, Bianca D; Doerner, Jessica; Foo, Patricia K; Herre, Margaret; Fak, John; Monette, Sébastien; Gantman, Emily C; Frank, Mayu O; Darnell, Robert B

2014-11-01

Paraneoplastic neurologic diseases (PND) involving immune responses directed toward intracellular antigens are poorly understood. Here, we examine immunity to the PND antigen Nova2, which is expressed exclusively in central nervous system (CNS) neurons. We hypothesized that ectopic expression of neuronal antigen in the periphery could incite PND. In our C57BL/6 mouse model, CNS antigen expression limits antigen-specific CD4+ and CD8+ T-cell expansion. Chimera experiments demonstrate that this tolerance is mediated by antigen expression in nonhematopoietic cells. CNS antigen expression does not limit tumor rejection by adoptively transferred transgenic T cells but does limit the generation of a memory population that can be expanded upon secondary challenge in vivo. Despite mediating cancer rejection, adoptively transferred transgenic T cells do not lead to paraneoplastic neuronal targeting. Preliminary experiments suggest an additional requirement for humoral activation to induce CNS autoimmunity. This work provides evidence that the requirements for cancer immunity and neuronal autoimmunity are uncoupled. Since humoral immunity was not required for tumor rejection, B-cell targeting therapy, such as rituximab, may be a rational treatment option for PND that does not hamper tumor immunity. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

An MHC-restricted antibody-based chimeric antigen receptor requires TCR-like affinity to maintain antigen specificity

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Marcela V Maus

2016-01-01

Full Text Available Chimeric antigen receptors (CARs are synthetic receptors that usually redirect T cells to surface antigens independent of human leukocyte antigen (HLA. Here, we investigated a T cell receptor-like CAR based on an antibody that recognizes HLA-A*0201 presenting a peptide epitope derived from the cancer-testis antigen NY-ESO-1. We hypothesized that this CAR would efficiently redirect transduced T cells in an HLA-restricted, antigen-specific manner. However, we found that despite the specificity of the soluble Fab, the same antibody in the form of a CAR caused moderate lysis of HLA-A2 expressing targets independent of antigen owing to T cell avidity. We hypothesized that lowering the affinity of the CAR for HLA-A2 would improve its specificity. We undertook a rational approach of mutating residues that, in the crystal structure, were predicted to stabilize binding to HLA-A2. We found that one mutation (DN lowered the affinity of the Fab to T cell receptor-range and restored the epitope specificity of the CAR. DN CAR T cells lysed native tumor targets in vitro, and, in a xenogeneic mouse model implanted with two human melanoma lines (A2+/NYESO+ and A2+/NYESO−, DN CAR T cells specifically migrated to, and delayed progression of, only the HLA-A2+/NY-ESO-1+ melanoma. Thus, although maintaining MHC-restricted antigen specificity required T cell receptor-like affinity that decreased potency, there is exciting potential for CARs to expand their repertoire to include a broad range of intracellular antigens.

Affinity Maturation of an Anti-V Antigen IgG Expressed In Situ Via Adenovirus Gene Delivery Confers Enhanced Protection Against Yersinia pestis Challenge

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Van Blarcom, Thomas J.; Sofer-Podesta, Carolina; Ang, John; Boyer, Julie L.; Crystal, Ronald G.; Georgiou, George

2013-01-01

Genetic transfer of neutralizing antibodies has been shown to confer strong and persistent protection against bacterial and viral infectious agents. While it is well established that for many exogenous neutralizing antibodies increased antigen affinity correlates with protection, the effect of antigen affinity on antibodies produced in situ following adenoviral gene transfer has not been examined. The mouse IgG2b monoclonal antibody 2C12.4 recognizes the Yersinia pestis Type III secretion apparatus protein LcrV (V antigen) and confers protection in mice when administered as an IgG intraperitoneally or, following genetic immunization with engineered, replication-defective serotype 5 human adenovirus (Ad) 1. 2C12.4 was expressed as a scFv fragment in E. coli and was shown to display a KD=3.5 nM by surface plasmon resonance (SPR) analysis. The 2C12.4 scFv was subjected to random mutagenesis and variants with increased affinity were isolated by flow cytometry using the Anchored Periplasmic Expression (APEx) bacterial display system. After a single round of mutagenesis, variants displaying up to 35-fold lower KD values (H8, KD=100 pM) were isolated. The variable domains of the H8 scFv were used to replace those of the parental 2C12.4 IgG encoded in the Ad vector, AdαV giving rise to AdαV.H8. The two adenoviral vectors resulted in similar titers of anti-V antigen antibodies 3 days post-immunization with 109, 1010 or 1011 particle units. Following intranasal challenge with 363 LD50Y. pestis CO92, 54% of the mice immunized with 1010 pu of AdαV.H8 survived at the 14 day end point compared to only 15% survivors for the group immunized with AdαV expressing the lower affinity 2C12.4 (Pgenetic transfer may confer increased protection not only for Y. pestis challenge but possibly for other pathogens. PMID:20393511

Mutational analysis of polyomavirus small-T-antigen functions in productive infection and in transformation.

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Martens, I; Nilsson, S A; Linder, S; Magnusson, G

1989-05-01

The function of polyomavirus small T antigen in productive infection and in transformation was studied. Transfection of permissive mouse cells with mixtures of mutants that express only one type of T antigen showed that small T antigen increased large-T-antigen-dependent viral DNA synthesis approximately 10-fold. Under the same conditions, small T antigen was also essential for the formation of infectious virus particles. To analyze these activities of small T antigen, mutants producing protein with single amino acid replacements were constructed. Two mutants, bc1073 and bc1075, were characterized. Although both mutations led to the substitution of amino acid residues of more than one T antigen, the phenotype of both mutants was associated with alterations of the small T antigen. Both mutant proteins had lost their activity in the maturation of infectious virus particles. The bc1075 but not the bc1073 small T antigen had also lost its ability to stimulate viral DNA synthesis in mouse 3T6 cells. Finally, both mutants retained a third activity of small T antigen: to confer on rat cells also expressing middle T antigen the ability to grow efficiently in semisolid medium. The phenotypes of the mutants in these three assays suggest that small T antigen has at least three separate functions.

Organic extract of diesel exhaust particles stimulates expression of Ia and costimulatory molecules associated with antigen presentation in rat peripheral blood monocytes but not in alveolar macrophages

International Nuclear Information System (INIS)

Koike, Eiko; Kobayashi, Takahiro

2005-01-01

We hypothesized that diesel exhaust particles (DEP) induce the activation of antigen-presenting cells (APC) in lung. The present study was designed to clarify the following about DEP: (1) whether it affects the expression of Ia and B7 molecules in alveolar macrophages (AM) as a mature cell or in peripheral blood monocytes (PBM) as an immature cell (2) if it affects the antigen-presenting (AP) activity of PBM (3) what component of DEP is responsible for the effects, and (4) whether the effect of DEP is related to oxidative stress. DEP was extracted with methylene chloride. Cells were exposed to whole DEP, organic extract, or residual particles for 24 h. Cell-surface molecules were measured by flow cytometry. AP activity was assessed by antigen-specific T cell proliferation. Whole DEP or organic extract significantly increased the expression of Ia and B7 molecules on PBM but not on AM. No significant effect of residual particles was observed. A low concentration of organic extract also increased the AP activity of PBM. When the induction of an antioxidative enzyme was assessed, heme oxygenase-1 protein was found to be significantly increased by exposure to whole DEP, and the organic extract was more effective than the residual particles. Furthermore, the organic extract-induced expression of Ia antigen on PBM was reduced by the addition of an antioxidative agent. These results suggest that DEP may act on immature APC and enhance their AP activity and that the action contributing to oxidative stress may be mediated by organic compounds of DEP

Generation and characterization of antigenic variants induced by exposure of tumor cells to UV radiation in vitro

International Nuclear Information System (INIS)

Hostetler, L.L.W.

1988-01-01

Antigenic changes present in nonantigenic tumor cells exposed to UV radiation (UV) in vitro were investigated by addressing the following questions: (1) Are antigenic variants (AV) produced that are rejected in normal but not immunosuppressed mice? (2) Does generation of AV depend upon intrinsic properties of the cells exposed or result from the action of UV? (3) Is antigenic modification induced by UV due to increased histocompatibility antigen expression? (4) Do AV crossreact immunologically with parental tumor or with other AV? and (5) Is the UV-associated common antigen expressed on UV-induced tumors present on UV-irradiated tumor cells? AV were generated at different frequencies following in vitro UV irradiation of a spontaneous murine fibrosarcoma, a murine melanoma, and two melanoma clones. Immunological experiments demonstrated that the AV and parental cells shared a determinant that was susceptible to immune recognition, but incapable of inducing immunity. In contrast, the AV were noncrossreactive, suggesting that variant-specific antigens were also expressed. Finally, the AV were recognized by UV-induced suppressor cells, indicating that the UV-associated common antigen expressed by UV-induced tumors was also present

Expression of a hantavirus N protein and its efficacy as antigen in immune assays

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L.T.M. Figueiredo

2008-07-01

Full Text Available Hantavirus cardiopulmonary syndrome (HCPS has been recognized as an important public heath problem. Five hantaviruses associated with HCPS are currently known in Brazil: Juquitiba, Araraquara, Laguna Negra-like, Castelo dos Sonhos, and Anajatuba viruses. The laboratory diagnosis of HCPS is routinely carried out by the detection of anti-hantavirus IgM and/or IgG antibodies. The present study describes the expression of the N protein of a hantavirus detected in the blood sample of an HCPS patient. The entire S segment of the virus was amplified and found to be 1858 nucleotides long, with an open reading frame of 1287 nucleotides that encodes a protein of 429 amino acids. The nucleotide sequence described here showed a high identity with the N protein gene of Araraquara virus. The entire N protein was expressed using the vector pET200D and the Escherichia coli BL21 strain. The expression of the recombinant protein was confirmed by the detection of a 52-kDa protein by Western blot using a pool of human sera obtained from HCPS patients, and by specific IgG detection in five serum samples of HCPS patients tested by ELISA. These results suggest that the recombinant N protein could be used as an antigen for the serological screening of hantavirus infection.

Cancer antigens are expressed in a carcinogen-transformed Bloom syndrome B-lymphoblastoid cell line

International Nuclear Information System (INIS)

Shiraishi, Yukimasa; Soma, Hiroaki

1988-01-01

The authors have cloned malignant cells carrying specific antigens associated with ovarian cancer (OVC) and malignant lymphoma (ML) from BS-SHI-4M cells, a line derived from a 1-methyl-3-nitro-1-nitrosoguanidine-treated B-lymphoblastoid cell line isolated from a patient with Bloom syndrome. Since BS-SHI-4M cells react with sera from various individual cancer patients at relatively low frequencies (2-9%), as detected by an indirect immunofluorescence technique, cell clones that specifically react with sera from patients with OVC and ML were separated by the panning method in which polystyrene dishes were coated with sera from OVC and ML patients and cells with the corresponding antigens bound to the dishes. Subsequent cloning by limiting dilution provided cell clones highly enriched for OVC- and ML-associated antigens. Karyotype analyses revealed that cell clones with OVC and ML antigens had common marker chromosomes. Interestingly, in cell clones with a strong OVC antigen response, the distal part of the Y chromosome (Yq11) was missing in 100% of the cells. Therefore the cell line BS-SHI-4M appears to be a reservoir of cell clones each of which carries a specific tumor antigen and thus provides a potential tool for rapid serological diagnosis of cancer

Ionizing radiation modulates the surface expression of human leukocyte antigen-G in a human melanoma cell line

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Michelin, S.; Gallegos, C.E.; Dubner, D. [Radiopathology Laboratory, Nuclear Regulatory Authority, Buenos Aires (Argentina); Favier, B.; Carosella, E.D. [CEA, I2BM, Hopital Saint-Louis, IUH, Service de Recherches en Hemato-Immunologie, Paris (France)

2009-07-01

Human leukocyte antigen G (HLA-G) is a nonclassical HLA class I molecule involved in fetus protection from the maternal immune system, transplant tolerance, and viral and tumoral immune escape. Tumor-specific HLA-G expression has been described for a wide variety of malignancies, including melanomas. The aim of this study was to evaluate whether ionizing radiation (IR) could modulate the surface expression of HLA-G1 in a human melanoma cell line that expresses endogenously membrane-bound HLA-G1. For this purpose, cells were exposed to increasing doses of {gamma}-irradiation (0-20 Gy) and HLA-G1 levels at the plasma membrane were analyzed at different times postirradiation by flow cytometry. HLA-G total expression and the presence of the soluble form of HLA-G1 (sHLA-G1) in the culture medium of irradiated cells were also evaluated. IR was capable of down regulating cell surface and total HLA-G levels, with a concomitant increase of sHLA-G1 in the medium. These results could indicate that {gamma}-irradiation decreases HLA-G1 surface levels by enhancing the proteolytic cleavage of this molecule. (authors)

Ionizing radiation modulates the surface expression of human leukocyte antigen-G in a human melanoma cell line

International Nuclear Information System (INIS)

Michelin, S.; Gallegos, C.E.; Dubner, D.; Favier, B.; Carosella, E.D.

2009-01-01

Human leukocyte antigen G (HLA-G) is a nonclassical HLA class I molecule involved in fetus protection from the maternal immune system, transplant tolerance, and viral and tumoral immune escape. Tumor-specific HLA-G expression has been described for a wide variety of malignancies, including melanomas. The aim of this study was to evaluate whether ionizing radiation (IR) could modulate the surface expression of HLA-G1 in a human melanoma cell line that expresses endogenously membrane-bound HLA-G1. For this purpose, cells were exposed to increasing doses of γ-irradiation (0-20 Gy) and HLA-G1 levels at the plasma membrane were analyzed at different times postirradiation by flow cytometry. HLA-G total expression and the presence of the soluble form of HLA-G1 (sHLA-G1) in the culture medium of irradiated cells were also evaluated. IR was capable of down regulating cell surface and total HLA-G levels, with a concomitant increase of sHLA-G1 in the medium. These results could indicate that γ-irradiation decreases HLA-G1 surface levels by enhancing the proteolytic cleavage of this molecule. (authors)

Hepatitis Bx Antigen Stimulates Expression of a Novel Cellular Gene, URG4, that Promotes Hepatocellular Growth and Survival

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N. Lale Satiroglu Tufan

2002-01-01

Full Text Available Hepatitis B virus encoded X antigen (HBxAg may contribute to the development of hepatocellular carcinoma (HCC by up-or downregulating the expression of cellular genes that promote cell growth and survival. To test this hypothesis, HBxAg-positive and-negative HepG2 cells were constructed, and the patterns of cellular gene expression compared by polymerase chain reaction select cDNA subtraction. The full-length clone of one of these upregulated genes (URG, URG4, encoded a protein of about 104 kDa. URG4 was strongly expressed in hepatitis 13-infected liver and in HCC cells, where it costained with HBxAg, and was weakly expressed in uninfected liver, suggesting URG4 was an effector of HBxAg in vivo. Overexpression of URG4 in HepG2 cells promoted hepatocellular growth and survival in tissue culture and in soft agar, and accelerated tumor development in nude mice. Hence, URG4 may be a natural effector of HBxAg that contributes importantly to multistep hepatocarcinogenesis.

Expression, purification and characterization of the cancer-germline antigen GAGE12I: a candidate for cancer immunotherapy

DEFF Research Database (Denmark)

Gjerstorff, Morten F; Besir, Hüseyin; Larsen, Martin R

2010-01-01

GAGE cancer-germline antigens are frequently expressed in a broad range of different cancers, while their expression in normal tissues is limited to the germ cells of the immune privileged organs, testis and ovary. GAGE proteins are immunogenic in humans, which make them promising targets...... for immunotherapy and candidates for cancer vaccines. Recombinant proteins may be superior to peptides as immunogens, since they have the potential to prime both CD4(+) and CD8(+) T cells and are not dependent on patient HLA-type. We have developed a method for production of highly pure recombinant GAGE12I...... filtration and formaldehyde cross-linking indicated that GAGE12I forms tetramers. The purified recombinant GAGE12I represents a candidate molecule for vaccination of cancer patients and will form the basis for further structural analysis of GAGE proteins....

Expression pattern of immunosurveillance-related antigen in adult T cell leukaemia/lymphoma.

Science.gov (United States)

Asano, Naoko; Miyoshi, Hiroaki; Kato, Takeharu; Shimono, Joji; Yoshida, Noriaki; Kurita, Daisuke; Sasaki, Yuya; Kawamoto, Keisuke; Ohshima, Koichi; Seto, Masao

2018-05-01

Adult T cell leukaemia/lymphoma (ATLL) is an aggressive malignancy with a poor prognosis. Human leucocyte antigen (HLA) and β2 microglobulin (β2M) serve as key molecules in tumour immunity, and their expression is reduced frequently in tumour cells. Programmed cell death (PD)-1/PD-ligand1 (PD-L1) interactions play a role in escape of tumour cells from T cell immunity. Therefore, this study aimed to determine the clinicopathological relevance of HLA and β2M expressions in ATLL cells and PD-L1 expression in lymphoma or stromal cells and predict the overall survival of patients with ATLL. We analysed a total of 123 biopsy samples from patients newly diagnosed with ATLL by using immunohistochemical analysis. Of the patients enrolled, 91 (74%) were positive for HLA (in cell membrane, 60 patients), 89 (72%) were positive for β2M (in cell membrane, 54 patients) and 48 (39%) were positive for both HLA and β2M in the cell membrane (HLA m+ β2M m+ ). No significant clinical differences other than prognosis were found between the HLA m+ β2M m+ group and the other groups. Immunophenotypical evaluation revealed significantly higher rates of CD30-positive lymphoma cells (P = 0.003) and PD-L1-positive stromal cells in microenvironments (miPD-L1 high ) (P = 0.011) of the HLA m+ β2M m+ group than in the other groups. The HLA m+ β2M m+ group had a significantly better prognosis that the other groups (P = 0.0096), and patients showing HLA m+ β2M m+ with miPD-L1 high had the most favourable prognosis among all groups. The membranous expression of HLA and β2M is likely to reflect the immune response and would be useful to predict prognosis before starting ATLL therapy. © 2018 John Wiley & Sons Ltd.

Magnitude of Alloresponses to MHC Class I/II Expressing Human Cardiac Myocytes is Limited by their Intrinsic Ability to Process and Present Antigenic Peptides

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Aftab A. Ansari

2003-01-01

Full Text Available In this investigation we have explored the relationship between the weak allogenicity of cardiac myocytes and their capacity to present allo-antigens by examining the ability of a human cardiac myocyte cell line (W-1 to process and present nominal antigens. W-1 cells (HLA-A*0201 and HLA-DR β1*0301 pulsed with the influenza A matrix 1 (58-66 peptide (M1 were able to serve as targets for the HLA-A*0201 restricted CTL line PG, specific for M1-peptide. However, PG-CTLs were unable to lyse W-1 target cells infected with a recombinant vaccinia virus expressing the M1 protein (M1-VAC. Pretreatment of these M1-VAC targets with IFN-γ partially restored their ability to process and present the M1 peptide. However, parallel studies demonstrated that IFN-γ pretreated W-1's could not process tetanus toxin (TT or present the TT(830-843 peptide to HLA-DR3 restricted TT-primed T cells. Semi-quantitative RT-PCR measurements revealed significantly lower constitutive levels of expression for MHC class I, TAP-1/2, and LMP-2/7 genes in W-1s that could be elevated by pretreatment with IFN-γ to values equal to or greater than those expressed in EBV-PBLs. However, mRNA levels for the genes encoding MHC class II, Ii, CIITA, and DMA/B were markedly lower in both untreated and IFN-γ pretreated W-1s relative to EBV-PBLs. Furthermore, pulse-chase analysis of the corresponding genes revealed significantly lower protein levels and longer half-life expression in W-1s relative to EBV-PBLs. These results suggest that weak allogenicity of cardiac myocytes may be governed by their limited expression of MHC genes and gene products critical for antigen processing and presentation.

Nuclear localization of Merkel cell polyomavirus large T antigen in Merkel cell carcinoma

International Nuclear Information System (INIS)

Nakamura, Tomoyuki; Sato, Yuko; Watanabe, Daisuke; Ito, Hideki; Shimonohara, Nozomi; Tsuji, Takahiro; Nakajima, Noriko; Suzuki, Yoshio; Matsuo, Koma; Nakagawa, Hidemi; Sata, Tetsutaro; Katano, Harutaka

2010-01-01

To clarify whether mutations in the large T gene encoded by Merkel cell polyomavirus affect the expression and function of large T antigen in Merkel cell carcinoma cases, we investigated the expression of large T antigen in vitro and in vivo. Immunohistochemistry using a rabbit polyclonal antibody revealed that large T antigen was expressed in the nuclei of Merkel cell carcinoma cells with Merkel cell polyomavirus infection. Deletion mutant analyses identified an Arg-Lys-Arg-Lys sequence (amino acids 277-280) as a nuclear localization signal in large T antigen. Sequence analyses revealed that there were no mutations in the nuclear localization signal in any of the eleven Merkel cell polyomavirus strains examined. Furthermore, stop codons were not observed in the upstream of the nuclear localization signal in any of the Merkel cell carcinoma cases examined. These data suggest that the nuclear localization signal is highly conserved and functional in Merkel cell carcinoma cases.

Antigen Loss Variants: Catching Hold of Escaping Foes.

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Vyas, Maulik; Müller, Rolf; Pogge von Strandmann, Elke

2017-01-01

Since mid-1990s, the field of cancer immunotherapy has seen steady growth and selected immunotherapies are now a routine and preferred therapeutic option of certain malignancies. Both active and passive cancer immunotherapies exploit the fact that tumor cells express specific antigens on the cell surface, thereby mounting an immune response specifically against malignant cells. It is well established that cancer cells typically lose surface antigens following natural or therapy-induced selective pressure and these antigen-loss variants are often the population that causes therapy-resistant relapse. CD19 and CD20 antigen loss in acute lymphocytic leukemia and chronic lymphocytic leukemia, respectively, and lineage switching in leukemia associated with mixed lineage leukemia (MLL) gene rearrangements are well-documented evidences in this regard. Although increasing number of novel immunotherapies are being developed, majority of these do not address the control of antigen loss variants. Here, we review the occurrence of antigen loss variants in leukemia and discuss the therapeutic strategies to tackle the same. We also present an approach of dual-targeting immunoligand effectively retargeting NK cells against antigen loss variants in MLL-associated leukemia. Novel immunotherapies simultaneously targeting more than one tumor antigen certainly hold promise to completely eradicate tumor and prevent therapy-resistant relapses.

The expression of prostate-specific antigen in invasive breast carcinoma and its relationship with routine clinicopathologic parameters

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Fereshteh Mohammadizadeh

2012-01-01

Full Text Available Background: Invasive breast carcinoma is one of the most common cancers of women. Parameters such as lymph node status, tumor grade, and the status of hormone receptors are among the main prognostic determinants of this cancer. Immunohistochemical detection of prostate-specific antigen (PSA is widely used to identify metastatic prostatic adenocarcinoma. However, its immunoreactivity has been found in some non-prostatic tissues. This study was conducted to assess PSA expression in invasive breast carcinoma and its relationship with routine clinicopathologic parameters. Materials and Methods: 100 formalin-fixed and paraffin-embedded invasive breast carcinoma tissue specimens from the pathology archive of Alzahra hospital (Isfahan, Iran were studied for the expression of estrogen receptor (ER, progesterone receptor (PR, HER2/neu, and PSA by immunohistochemistry. Stained sections were classified according to the intensity of staining and the percentage of cells showing PSA staining. The relationship between PSA expression and other markers, age, lymph node status, tumor subtype, and tumor grade was then studied. Results: No association was found between PSA expression on one hand and PR, Her2/neu, age, lymph node status, tumor grade, and tumor subtype on the other. PSA score was reversely correlated with ER expression (P = 0.015. Conclusion: Despite the reverse relationship between PSA expression and the immunoreactivity of ER, PSA expression was not correlated with other prognostic factors. Therefore, the detection of PSA by immunohistochemistry does not seem to be a significant prognostic parameter in patients with invasive breast carcinoma.

Targeting tumor antigens to secreted membrane vesicles in vivo induces efficient antitumor immune responses.

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Zeelenberg, Ingrid S; Ostrowski, Matias; Krumeich, Sophie; Bobrie, Angélique; Jancic, Carolina; Boissonnas, Alexandre; Delcayre, Alain; Le Pecq, Jean-Bernard; Combadière, Béhazine; Amigorena, Sebastian; Théry, Clotilde

2008-02-15

Expression of non-self antigens by tumors can induce activation of T cells in vivo, although this activation can lead to either immunity or tolerance. CD8+ T-cell activation can be direct (if the tumor expresses MHC class I molecules) or indirect (after the capture and cross-presentation of tumor antigens by dendritic cells). The modes of tumor antigen capture by dendritic cells in vivo remain unclear. Here we examine the immunogenicity of the same model antigen secreted by live tumors either in association with membrane vesicles (exosomes) or as a soluble protein. We have artificially addressed the antigen to secreted vesicles by coupling it to the factor VIII-like C1C2 domain of milk fat globule epidermal growth factor-factor VIII (MFG-E8)/lactadherin. We show that murine fibrosarcoma tumor cells that secrete vesicle-bound antigen grow slower than tumors that secrete soluble antigen in immunocompetent, but not in immunodeficient, host mice. This growth difference is due to the induction of a more potent antigen-specific antitumor immune response in vivo by the vesicle-bound than by the soluble antigen. Finally, in vivo secretion of the vesicle-bound antigen either by tumors or by vaccination with naked DNA protects against soluble antigen-secreting tumors. We conclude that the mode of secretion can determine the immunogenicity of tumor antigens and that manipulation of the mode of antigen secretion may be used to optimize antitumor vaccination protocols.

Expression and characterization of highly antigenic domains of chicken anemia virus viral VP2 and VP3 subunit proteins in a recombinant E. coli for sero-diagnostic applications

Science.gov (United States)

2013-01-01

Background Chicken anemia virus (CAV) is an important viral pathogen that causes anemia and severe immunodeficiency syndrome in chickens worldwide. Generally, CAV infection occurs via vertical transmission in young chicks that are less than two weeks old, which are very susceptible to the disease. Therefore, epidemiological investigations of CAV infection and/or the evaluation of the immunization status of chickens is necessary for disease control. Up to the present, systematically assessing viral protein antigenicity and/or determining the immunorelevant domain(s) of viral proteins during serological testing for CAV infection has never been performed. The expression, production and antigenic characterization of CAV viral proteins such as VP1, VP2 and VP3, and their use in the development of diagnostic kit would be useful for CAV infection prevention. Results Three CAV viral proteins VP1, VP2 and VP3 was separately cloned and expressed in recombinant E. coli. The purified recombinant CAV VP1, VP2 and VP3 proteins were then used as antigens in order to evaluate their reactivity against chicken sera using indirect ELISA. The results indicated that VP2 and VP3 show good immunoreactivity with CAV-positive chicken sera, whereas VP1 was found to show less immunoreactivity than VP2 and VP3. To carry out the further antigenic characterization of the immunorelevant domains of the VP2 and VP3 proteins, five recombinant VP2 subunit proteins (VP2-435N, VP2-396N, VP2-345N, VP2-171C and VP2-318C) and three recombinant VP3 subunit proteins (VP3-123N, VP3-246M, VP3-366C), spanning the defined regions of VP2 and VP3 were separately produced by an E. coli expression system. These peptides were then used as antigens in indirect ELISAs against chicken sera. The results of these ELISAs using truncated recombinant VP2 and VP3 subunit proteins as coating antigen showed that VP2-345N, VP2-396N and VP3-246M gave good immunoreactivity with CAV-positive chicken sera compared to the other

Immunogenicity of 60 novel latency-related antigens of Mycobacterium tuberculosis.

Science.gov (United States)

Serra-Vidal, Mᵃdel Mar; Latorre, Irene; Franken, Kees L C M; Díaz, Jéssica; de Souza-Galvão, Maria Luiza; Casas, Irma; Maldonado, José; Milà, Cèlia; Solsona, Jordi; Jimenez-Fuentes, M Ángeles; Altet, Neus; Lacoma, Alícia; Ruiz-Manzano, Juan; Ausina, Vicente; Prat, Cristina; Ottenhoff, Tom H M; Domínguez, José

2014-01-01

The aim of our work here was to evaluate the immunogenicity of 60 mycobacterial antigens, some of which have not been previously assessed, notably a novel series of in vivo-expressed Mycobacterium tuberculosis (IVE-TB) antigens. We enrolled 505 subjects and separated them in individuals with and without latent tuberculosis infection (LTBI) vs. patients with active tuberculosis (TB). Following an overnight and 7 days stimulation of whole blood with purified recombinant M. tuberculosis antigens, interferon-γ (IFN-γ) levels were determined by ELISA. Several antigens could statistically significantly differentiate the groups of individuals. We obtained promising antigens from all studied antigen groups [dormancy survival regulon (DosR regulon) encoded antigens; resuscitation-promoting factors (Rpf) antigens; IVE-TB antigens; reactivation associated antigens]. Rv1733, which is a probable conserved transmembrane protein encoded in DosR regulon, turned out to be very immunogenic and able to discriminate between the three defined TB status, thus considered a candidate biomarker. Rv2389 and Rv2435n, belonging to Rpf family and IVE-TB group of antigens, respectively, also stood out as LTBI biomarkers. Although more studies are needed to support our findings, the combined use of these antigens would be an interesting approach to TB immunodiagnosis candidates.

Immunogenicity of 60 novel latency-related antigens of Mycobacterium tuberculosis

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Mªdel Mar eSerra Vidal

2014-10-01

Full Text Available The aim of our work here was to evaluate the immunogenicity of 60 mycobacterial antigens, some of which have not been previously assessed, notably a novel series of in vivo-expressed M.tuberculosis (IVE-TB antigens. We enrolled 505 subjects and separated them in individuals with and without latent tuberculosis infection (LTBI versus patients with active tuberculosis. Following an overnight and 7 day stimulation of whole blood with purified recombinant M.tb antigens, interferon-γ (IFN-γ levels were determined by ELISA. Several antigens could statistically significantly differentiate the groups of individuals. We obtained promising antigens from all studied antigen groups (DosR regulon encoded antigens; resuscitation-promoting factors (Rpf antigens; IVE-TB antigens; reactivation asociated antigens. Rv1733, which is a probable conserved transmembrane protein encoded in DosR regulon, turned out to be very immunogenic and able to discriminate between the three defined TB status, thus considered a candidate biomarker. Rv2389 and Rv2435n, belonging to Rpf family and IVE-TB group of antigens, respectively, also stood out as LTBI biomarkers. Although more studies are needed to support our findings, the combined use of these antigens would be an interesting approach to tuberculosis immunodiagnosis candidates.

Expression of verocytotoxic Escherichia coli antigens in tobacco seeds and evaluation of gut immunity after oral administration in mouse model

OpenAIRE

Rossi, Luciana; Di Giancamillo, Alessia; Reggi, Serena; Domeneghini, Cinzia; Baldi, Antonella; Sala, Vittorio; Dell'Orto, Vittorio; Coddens, Annelies; Cox, Eric; Fogher, Corrado

2013-01-01

Verocytotoxic Escherichia (E.) coli strains are responsible for swine oedema disease, which is an enterotoxaemia that causes economic losses in the pig industry. The production of a vaccine for oral administration in transgenic seeds could be an efficient system to stimulate local immunity. This study was conducted to transform tobacco plants for the seed-specific expression of antigenic proteins from a porcine verocytotoxic E. coli strain. Parameters related to an immunological response and ...

Endothelial cells present antigens in vivo

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Tellides George

2004-03-01

Full Text Available Abstract Background Immune recognition of vascular endothelial cells (EC has been implicated in allograft rejection, protection against pathogens, and lymphocyte recruitment. However, EC pervade nearly all tissues and predominate in none, complicating any direct test of immune recognition. Here, we examined antigen presentation by EC in vivo by testing immune responses against E. coli β-galactosidase (β-gal in two lines of transgenic mice that express β-gal exclusively in their EC. TIE2-lacZ mice express β-gal in all EC and VWF-lacZ mice express β-gal in heart and brain microvascular EC. Results Transgenic and congenic wild type FVB mice immunized with β-gal expression vector DNA or β-gal protein generated high titer, high affinity antisera containing comparable levels of antigen-specific IgG1 and IgG2a isotypes, suggesting equivalent activation of T helper cell subsets. The immunized transgenic mice remained healthy, their EC continued to express β-gal, and their blood vessels showed no histological abnormalities. In response to β-gal in vitro, CD4+ and CD8+ T cells from immunized transgenic and FVB mice proliferated, expressed CD25, and secreted IFN-γ. Infection with recombinant vaccinia virus encoding β-gal raised equivalent responses in transgenic and FVB mice. Hearts transplanted from transgenic mice into FVB mice continued to beat and the graft EC continued to express β-gal. These results suggested immunological ignorance of the transgene encoded EC protein. However, skin transplanted from TIE2-lacZ onto FVB mice lost β-gal+ EC and the hosts developed β-gal-specific antisera, demonstrating activation of host immune effector mechanisms. In contrast, skin grafted from TIE2-lacZ onto VWF-lacZ mice retained β-gal+ EC and no antisera developed, suggesting a tolerant host immune system. Conclusion Resting, β-gal+ EC in transgenic mice tolerize specific lymphocytes that would otherwise respond against β-gal expressed by EC within

Increasing vaccine potency through exosome antigen targeting.

Science.gov (United States)

Hartman, Zachary C; Wei, Junping; Glass, Oliver K; Guo, Hongtao; Lei, Gangjun; Yang, Xiao-Yi; Osada, Takuya; Hobeika, Amy; Delcayre, Alain; Le Pecq, Jean-Bernard; Morse, Michael A; Clay, Timothy M; Lyerly, Herbert K

2011-11-21

While many tumor associated antigens (TAAs) have been identified in human cancers, efforts to develop efficient TAA "cancer vaccines" using classical vaccine approaches have been largely ineffective. Recently, a process to specifically target proteins to exosomes has been established which takes advantage of the ability of the factor V like C1C2 domain of lactadherin to specifically address proteins to exosomes. Using this approach, we hypothesized that TAAs could be targeted to exosomes to potentially increase their immunogenicity, as exosomes have been demonstrated to traffic to antigen presenting cells (APC). To investigate this possibility, we created adenoviral vectors expressing the extracellular domain (ECD) of two non-mutated TAAs often found in tumors of cancer patients, carcinoembryonic antigen (CEA) and HER2, and coupled them to the C1C2 domain of lactadherin. We found that these C1C2 fusion proteins had enhanced expression in exosomes in vitro. We saw significant improvement in antigen specific immune responses to each of these antigens in naïve and tolerant transgenic animal models and could further demonstrate significantly enhanced therapeutic anti-tumor effects in a human HER2+ transgenic animal model. These findings demonstrate that the mode of secretion and trafficking can influence the immunogenicity of different human TAAs, and may explain the lack of immunogenicity of non-mutated TAAs found in cancer patients. They suggest that exosomal targeting could enhance future anti-tumor vaccination protocols. This targeting exosome process could also be adapted for the development of more potent vaccines in some viral and parasitic diseases where the classical vaccine approach has demonstrated limitations. Copyright © 2011 Elsevier Ltd. All rights reserved.

Identification of Schistosoma mansoni candidate antigens for diagnosis of schistosomiasis

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Gardenia Braz Figueiredo Carvalho

2011-11-01

Full Text Available The development of a more sensitive diagnostic test for schistosomiasis is needed to overcome the limitations of the use of stool examination in low endemic areas. Using parasite antigens in enzyme linked immunosorbent assay is a promising strategy, however a more rational selection of parasite antigens is necessary. In this study we performed in silico analysis of the Schistosoma mansoni genome, using SchistoDB database and bioinformatic tools for screening immunogenic antigens. Based on evidence of expression in all parasite life stage within the definitive host, extracellular or plasmatic membrane localization, low similarity to human and other helminthic proteins and presence of predicted B cell epitopes, six candidates were selected: a glycosylphosphatidylinositol-anchored 200 kDa protein, two putative cytochrome oxidase subunits, two expressed proteins and one hypothetical protein. The recognition in unidimensional and bidimensional Western blot of protein with similar molecular weight and isoelectric point to the selected antigens by sera from S. mansoni infected mice indicate a good correlation between these two approaches in selecting immunogenic proteins.

Study of CD69 antigen expression and integrity of leukocyte cellular membrane in stored platelet concentrates following irradiation and treatment with Mirasol® PRT System.

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Lachert, Elżbieta; Woźniak, Jolanta; Antoniewicz-Papis, Jolanta; Krzywdzińska, Agnieszka; Kubis, Jolanta; Mikołowska, Agata; Letowska, Magdalena

2017-01-01

Leukocytes in transfused blood components, particularly residual lymphocytes, have been shown to contribute to the occurrence of various adverse reactions. One of the most severe is transfusionassociated graft versus host disease (TA-GvHD) following transfusion of blood components contaminated with immunocompetent T lymphocytes. Irradiation is a routine method for protection against TA-GvHD. According to the literature, some pathogen reduction methods have also been proven effective for the inactivation of T lymphocytes, and so they may be considered as an alternative to irradiation. Comparison of CD69 antigen expression and the integrity of the leukocyte cellular membrane in stored platelet concentrates (PCs) following irradiation with the Gammacell 3000 Elan (Nordion Inc., Ottawa, Canada) and treatment with the Mirasol® Pathogen Reduction Technology (PRT) System (Terumo BCT, Lakewood, USA). The study included seven experiments. For each experiment we used 3 PCs, for Mirasol® PRT System treatment (M), for Gammacell 3000 Elan irradiation (R), and for the control (C). 7-amino-actinomycin D (7-AAD, Becton Dickinson, Franklin Lakes, USA) permeability was used to determine lymphocyte viability while CD69 antigen expression was the marker of lymphocyte activation. Analyses of 7-AAD and CD69 antigen expression were performed in a FACS Canto I flow cytometer (Becton Dickinson, USA). During 6 storage days, viable lymphocyte count decreased to 28% (p = 0.001) in the Mirasol® PRT System treated PCs and to 65% (p = 0.004) in the irradiated PCs. A statistically significant increase in CD69 expression in the irradiated PCs was observed; 1.3-fold on day 3 and 1.5-fold on day 6. In the Mirasol ® PRT System treated PCs, no statistically significant increase was observed. The in vitro results suggest that the Mirasol® PRT System is as effective as irradiation due to donor leukocyte inactivation capacity.

Count of splenic stromal precursor cells in mice and expression of cytokine genes in these cells in primary cultures during different periods after immunization of animals with S. typhimurium antigens.

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Gorskaya, Yu F; Danilova, T A; Mezentseva, M V; Shapoval, I M; Narovlyanskii, A N; Nesterenko, V G

2011-06-01

Injection of S. typhimurium antigens significantly (9-fold) increased cloning efficiency and, hence, the content of stromal precursor cells in the spleen as soon as after 24 h. These parameters returned to normal by days 6-15 after immunization. Cultured splenocytes collected from immune (but not intact) animals expressed the genes of proinflammatory cytokines IL-1β (on days 1, 6, 15) and IL-6 (on days 1 and 6), TNF-α (on days 6 and 15), and of IFN-α and IL-18 (on days 6 and 15). The expression of IL-4 gene was suppressed on day 6 after immunization, of IL-10 gene on days 1 and 6, of IL-6 gene on day 15. Hence, no signs of immune response suppression by stromal cells were found in this system. The spectrum and dynamics of the expression of pro- and anti-inflammatory cytokine genes in stromal cell cultures from the spleen of immunized mice seemed to correspond to those needed for support of the immune response to S. typhimurium antigens, observed in immunized animals. The results indicate possible involvement of stromal cells in the realization of immune response in vivo. The increase of stromal precursor cells cloning efficiency in response to antigen injection could not be reproduced in vitro: the presence of S. typhimurium antigens in primary cultures of intact mouse bone marrow and spleen throughout the entire period of culturing ≈ 20-fold reduced cloning efficiency in cultures.

Local CD4 and CD8 T-cell reactivity to HSV-1 antigens documents broad viral protein expression and immune competence in latently infected human trigeminal ganglia.

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Monique van Velzen

2013-08-01

Full Text Available Herpes simplex virus type 1 (HSV-1 infection results in lifelong chronic infection of trigeminal ganglion (TG neurons, also referred to as neuronal HSV-1 latency, with periodic reactivation leading to recrudescent herpetic disease in some persons. HSV-1 proteins are expressed in a temporally coordinated fashion during lytic infection, but their expression pattern during latent infection is largely unknown. Selective retention of HSV-1 reactive T-cells in human TG suggests their role in controlling reactivation by recognizing locally expressed HSV-1 proteins. We characterized the HSV-1 proteins recognized by virus-specific CD4 and CD8 T-cells recovered from human HSV-1-infected TG. T-cell clusters, consisting of both CD4 and CD8 T-cells, surrounded neurons and expressed mRNAs and proteins consistent with in situ antigen recognition and antiviral function. HSV-1 proteome-wide scans revealed that intra-TG T-cell responses included both CD4 and CD8 T-cells directed to one to three HSV-1 proteins per person. HSV-1 protein ICP6 was targeted by CD8 T-cells in 4 of 8 HLA-discordant donors. In situ tetramer staining demonstrated HSV-1-specific CD8 T-cells juxtaposed to TG neurons. Intra-TG retention of virus-specific CD4 T-cells, validated to the HSV-1 peptide level, implies trafficking of viral proteins from neurons to HLA class II-expressing non-neuronal cells for antigen presentation. The diversity of viral proteins targeted by TG T-cells across all kinetic and functional classes of viral proteins suggests broad HSV-1 protein expression, and viral antigen processing and presentation, in latently infected human TG. Collectively, the human TG represents an immunocompetent environment for both CD4 and CD8 T-cell recognition of HSV-1 proteins expressed during latent infection. HSV-1 proteins recognized by TG-resident T-cells, particularly ICP6 and VP16, are potential HSV-1 vaccine candidates.

Microglial MHC antigen expression after ischemic and kainic acid lesions of the adult rat hippocampus

DEFF Research Database (Denmark)

Finsen, B.R.; Jørgensen, Martin Balslev; Diemer, Nils Henrik

1993-01-01

Leukocyte common antigen, macrophages, blood-brain barrier, neural degeneration, fascia dentata, neuropathology......Leukocyte common antigen, macrophages, blood-brain barrier, neural degeneration, fascia dentata, neuropathology...

Use of mammary epithelial antigens as markers in mammary neoplasia

International Nuclear Information System (INIS)

Ceriani, R.L.; Peterson, J.A.; Blank, E.W.

1979-01-01

Cell-type specific antigens of the mammary epithelial cells can be used as markers of breast neoplasia. Methods are proposed for the detection of metastatic mammary tissue in vivo by injection of [ 125 I]-labeled antibodies against the mammary epithelial antigens. In addition, the reduced expression of mammary epithelial cell antigens in neoplastic breast cells, quantitated here on a cell per cell basis by flow cytofluorimetry, is a marker of neoplasia and an indication of a deletion accompanying the neoplastic transformation of these cells. (Auth.)

Cancer Patient T Cells Genetically Targeted to Prostate-Specific Membrane Antigen Specifically Lyse Prostate Cancer Cells and Release Cytokines in Response to Prostate-Specific Membrane Antigen

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Michael C. Gong

1999-06-01

Full Text Available The expression of immunoglobulin-based artificial receptors in normal T lymphocytes provides a means to target lymphocytes to cell surface antigens independently of major histocompatibility complex restriction. Such artificial receptors have been previously shown to confer antigen-specific tumoricidal properties in murine T cells. We constructed a novel ζ chain fusion receptor specific for prostate-specific membrane antigen (PSMA termed Pz-1. PSMA is a cell-surface glycoprotein expressed on prostate cancer cells and the neovascular endothelium of multiple carcinomas. We show that primary T cells harvested from five of five patients with different stages of prostate cancer and transduced with the Pz-1 receptor readily lyse prostate cancer cells. Having established a culture system using fibroblasts that express PSMA, we next show that T cells expressing the Pz-1 receptor release cytokines in response to cell-bound PSMA. Furthermore, we show that the cytokine release is greatly augmented by B7.1-mediated costimulation. Thus, our findings support the feasibility of adoptive cell therapy by using genetically engineered T cells in prostate cancer patients and suggest that both CD4+ and CD8+ T lymphocyte functions can be synergistically targeted against tumor cells.

Leukemia Associated Antigens: Their Dual Role as Biomarkers and Immunotherapeutic Targets for Acute Myeloid Leukemia

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Michael Schmitt

2007-01-01

Full Text Available Leukemia associated antigens (LAAs are being increasingly identified by methods such as cytotoxic T-lymphocyte (CTL cloning, serological analysis of recombinant cDNA expression libraries (SEREX and mass spectrometry (MS. In additional, large scale screening techniques such as microarray, single nucleotide polymorphisms (SNPs, serial analysis of gene expression (SAGE and 2-dimensional gel electrophoresis (2-DE have expanded our understanding of the role that tumor antigens play in the biological processes which are perturbed in acute myeloid leukemia (AML. It has become increasingly apparent that these antigens play a dual role, not only as targets for immunotherapy, but also as biomarkers of disease state, stage, response to treatment and survival. We need biomarkers to enable the identification of the patients who are most likely to benefit from specific treatments (conventional and/or novel and to help clinicians and scientists improve clinical end points and treatment design. Here we describe the LAAs identified in AML, to date, which have already been shown to play a dual role as biomarkers of AML disease.Abbreviations: AML: acute myeloid leukemia; APL: acute promyelocytic leukemia; ATRA: all-trans-retinoic acid; B-CLL: B-cell chronic lymphocytic leukemia; CT: cancer-testis; CTL: cytotoxic T-lymphocyte; FAB: French-American-British; HI: hypusination inhibitors; HSP: heat shock protein; ITD: internal tandem duplication; LAA: leukemia associated antigen; MDS: myelodysplastic syndrome; MGEA6: meningioma antigen 6; MPD: myeloproliferative disease; MS: mass spectrometry; NK: natural killer; PRAME: preferentially expressed antigen of melanoma; PRTN3: proteinase 3; RAGE-1: renal antigen 1; RHAMM: receptor for hyaluronic acid-mediated motility; RQ-PCR: real-time PCR; SAGE: serial analysis of gene expression; SCT: stem cell transplant; SEREX: serological analysis of recombinant cDNA expression libraries; SNPs: single nucleotide polymorphisms; UPD

Expression of T cell antigen receptor genes in the thymus of irradiated mice after bone marrow transplantation

International Nuclear Information System (INIS)

Matsuzaki, G.; Yoshikai, Y.; Kishihara, K.; Nomoto, K.

1988-01-01

Sequential appearance of the expression of T cell antigen receptor genes was investigated in the thymus of irradiated mice at the early stage after transplantation of Thy-1 congeneic H-2 compatible allogeneic bone marrow cells. The first cells to repopulate the thymus on day 7 after bone marrow transplantation were intrathymic radioresistant T cell precursors, which expanded mainly to CD4+CD8+ host-type thymocytes by day 14. A high level of gamma gene expression but a much reduced level of alpha and beta gene expression were detected in the host-type thymocytes on day 7. During regeneration of these cells, gamma-chain messages fell to low level and alpha and beta mRNA levels increased. The thymus of the recipients began to be repopulated by donor-derived T cells about 2 wk after bone marrow transplantation and was almost completely replaced by the third week. An ordered expression of gamma then beta and alpha-chain gene transcript was also observed in the donor-type thymocytes at the early stage after bone marrow transplantation. The use of thymocytes at early stage in whole-body irradiated bone marrow chimera provides a pertinent source for investigating the molecular mechanism of T cell differentiation in adult thymus

Comparison of glycoprotein expression between ovarian and colon adenocarcinomas

DEFF Research Database (Denmark)

Multhaupt, H A; Arenas-Elliott, C P; Warhol, M J

1999-01-01

, carcinoembryonic antigen, and cytokeratins 7 and 20 to detect tumor-associated glycoproteins and keratin proteins in ovarian and colonic carcinomas. RESULTS: CA125, carcinoembryonic antigen, and cytokeratins 7 and 20 can distinguish between colonic and serous or endometrioid adenocarcinomas of the ovary in both...... primary and metastatic lesions. Mucinous ovarian adenocarcinomas differed in that they express carcinoembryonic antigen and cytokeratins 7 and 20 and weakly express CA125. The other glycoprotein antigens were equally expressed by ovarian and colonic adenocarcinomas and therefore were of no use...... in distinguishing between these 2 entities. CONCLUSION: A panel of monoclonal antibodies against cytokeratins 7 and 20 antigens, CA125, and carcinoembryonic antigen is useful in differentiating serous and endometrioid adenocarcinomas of the ovary from colonic adenocarcinomas. Mucinous ovarian adenocarcinomas cannot...

Non-MHC Antigenic Targets of the Humoral Immune Response in Transplantation

OpenAIRE

Zhang, Qiuheng; Reed, Elaine F.

2010-01-01

There is a growing body of data supporting a role for non-HLA antibodies in acute and chronic rejection of solid organ transplants. While many of these non-HLA antigens remain poorly defined, the principle antigenic targets are expressed on cells of the allograft including endothelium and epithelium. These non-HLA antigens are classified as either alloantigens, such as the major histocompatibility complex class I chain-related gene A (MICA) or MICB, or tissue-specific autoantigens such as vim...

Phenotypic H-Antigen Typing by Mass Spectrometry Combined with Genetic Typing of H Antigens, O Antigens, and Toxins by Whole-Genome Sequencing Enhances Identification of Escherichia coli Isolates.

Science.gov (United States)

Cheng, Keding; Chui, Huixia; Domish, Larissa; Sloan, Angela; Hernandez, Drexler; McCorrister, Stuart; Robinson, Alyssia; Walker, Matthew; Peterson, Lorea A M; Majcher, Miles; Ratnam, Sam; Haldane, David J M; Bekal, Sadjia; Wylie, John; Chui, Linda; Tyler, Shaun; Xu, Bianli; Reimer, Aleisha; Nadon, Celine; Knox, J David; Wang, Gehua

2016-08-01

Mass spectrometry-based phenotypic H-antigen typing (MS-H) combined with whole-genome-sequencing-based genetic identification of H antigens, O antigens, and toxins (WGS-HOT) was used to type 60 clinical Escherichia coli isolates, 43 of which were previously identified as nonmotile, H type undetermined, or O rough by serotyping or having shown discordant MS-H and serotyping results. Whole-genome sequencing confirmed that MS-H was able to provide more accurate data regarding H antigen expression than serotyping. Further, enhanced and more confident O antigen identification resulted from gene cluster based typing in combination with conventional typing based on the gene pair comprising wzx and wzy and that comprising wzm and wzt The O antigen was identified in 94.6% of the isolates when the two genetic O typing approaches (gene pair and gene cluster) were used in conjunction, in comparison to 78.6% when the gene pair database was used alone. In addition, 98.2% of the isolates showed the existence of genes for various toxins and/or virulence factors, among which verotoxins (Shiga toxin 1 and/or Shiga toxin 2) were 100% concordant with conventional PCR based testing results. With more applications of mass spectrometry and whole-genome sequencing in clinical microbiology laboratories, this combined phenotypic and genetic typing platform (MS-H plus WGS-HOT) should be ideal for pathogenic E. coli typing. Copyright © 2016 Cheng et al.

Antigen Expression on Blast Cells and Hematological Parameters at Presentation in Acute Lymphoblastic Leukemia Patients

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Naeem, S.; Bukhari, M. H.

2015-01-01

Objective: To analyze the expression of various antigens on the leukemic blasts and to determine the hematological parameters, in Acute Lymphoblastic Leukemia (ALL) patients at presentation. Study Design: Observational study. Place and Duration of Study: King Edward Medical University, Lahore and Hameed Latif Hospital, Lahore, from February 2013 to March 2014. Methodology: A total of 50 newly diagnosed and untreated patients of ALL were selected from Mayo Hospital and Hameed Latif Hospital. These patients included both genders and all age groups. Hemoglobin, total leukocyte count and platelet count were determined on hematology analyser-Sysmex-Kx-2I. Blast cell percentage was estimated on Giemsa stained blood smears. Immuno phenotyping was done on bone marrow samples by 5 colour flow cytometery on Beckman Counter Navious Flow cytometer. An acute leukemia panel of 23 antibodies was used. The data was entered and analyzed in SPSS version 22. Results: Of the 50 ALL patients, 36 (72 percentage) were B-ALL and 14 (28 percentage) T-ALL. There were 18 (36 percentage) children and 32 (64 percentage) adults. T-ALL included 22 percentage of the childhood and 31 percentage of the adult cases. Immuno phenotypic analysis showed that CD19, CD79a and CD20 were B-lineage specific markers whereas cCD3, CD3 and CD5 were T-lineage specific. CD10 was the most sensitive marker for B-ALL and CD7 was the most sensitive marker of T-ALL. TdT was expressed in 92 percentage B-ALL and 71 percentage T-ALL cases, CD34 in 58 percentage and 43 percentage cases and CD45 in 83 percentage and 100 percentage respectively. High leukocyte count (> 50 x 109/L) was present in 58 percentage cases. Hemoglobin was < 10 g/dl in 74 percentage patients and platelet count was below 20 x 109/Lin 12 percentage patients. Leukocyte count, hemoglobin, platelet count and blast cell percentage did not show a significant difference in the two ALL immuno types. Conclusion: The frequency of T-ALL is higher in childhood

Deciphering the genetic control of gene expression following Mycobacterium leprae antigen stimulation.

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Manry, Jérémy; Nédélec, Yohann; Fava, Vinicius M; Cobat, Aurélie; Orlova, Marianna; Thuc, Nguyen Van; Thai, Vu Hong; Laval, Guillaume; Barreiro, Luis B; Schurr, Erwin

2017-08-01

Leprosy is a human infectious disease caused by Mycobacterium leprae. A strong host genetic contribution to leprosy susceptibility is well established. However, the modulation of the transcriptional response to infection and the mechanism(s) of disease control are poorly understood. To address this gap in knowledge of leprosy pathogenicity, we conducted a genome-wide search for expression quantitative trait loci (eQTL) that are associated with transcript variation before and after stimulation with M. leprae sonicate in whole blood cells. We show that M. leprae antigen stimulation mainly triggered the upregulation of immune related genes and that a substantial proportion of the differential gene expression is genetically controlled. Indeed, using stringent criteria, we identified 318 genes displaying cis-eQTL at an FDR of 0.01, including 66 genes displaying response-eQTL (reQTL), i.e. cis-eQTL that showed significant evidence for interaction with the M. leprae stimulus. Such reQTL correspond to regulatory variations that affect the interaction between human whole blood cells and M. leprae sonicate and, thus, likely between the human host and M. leprae bacilli. We found that reQTL were significantly enriched among binding sites of transcription factors that are activated in response to infection, and that they were enriched among single nucleotide polymorphisms (SNPs) associated with susceptibility to leprosy per se and Type-I Reaction, and seven of them have been targeted by recent positive selection. Our study suggested that natural selection shaped our genomic diversity to face pathogen exposure including M. leprae infection.

Novel Functions for Glycosyltransferases Jhp0562 and GalT in Lewis Antigen Synthesis and Variation in Helicobacter pylori

OpenAIRE

Pohl, Mary Ann; Kienesberger, Sabine; Blaser, Martin J.

2012-01-01

Lewis (Le) antigens are fucosylated oligosaccharides present in the Helicobacter pylori lipopolysaccharide. Expression of these antigens is believed to be important for H. pylori colonization, since Le antigens also are expressed on the gastric epithelia in humans. A galactosyltransferase encoded by β-(1,3)galT is essential for production of type 1 (Lea and Leb) antigens. The upstream gene jhp0562, which is present in many but not all H. pylori strains, is homologous to β-(1,3)galT but is of ...

Plasma membrane associated, virus-specific polypeptides required for the formation of target antigen complexes recognized by virus-specific cytotoxic T lymphocytes

International Nuclear Information System (INIS)

Domber, E.A.

1986-01-01

These studies were undertaken to define some of the poxvirus-specific target antigens which are synthesized in infected cells and recognized by vaccinia virus-specific CTLs (VV-CTLs). Since vaccinia virus infected, unmanipulated target cells express numerous virus-specific antigens on the plasma membrane, attempts were made to manipulate expression of the poxvirus genome after infection so that one or a few defined virus-specified antigens were expressed on the surface of infected cells. In vitro [ 51 Cr]-release assays determined that viral DNA synthesis and expression of late viral proteins were not necessary to form a target cell which was fully competent for lysis by VV-CTLs. Under the conditions employed in these experiments, 90-120 minutes of viral protein synthesis were necessary to produce a competent cell for lysis by VV-CTLs. In order to further inhibit the expression of early viral proteins in infected cells, partially UV-inactivated vaccinia virus was employed to infect target cells. It was determined that L-cells infected with virus preparations which had been UV-irradiated for 90 seconds were fully competent for lysis by VV-CTLs. Cells infected with 90 second UV-irr virus expressed 3 predominant, plasma membrane associated antigens of 36-37K, 27-28K, and 19-17K. These 3 viral antigens represent the predominant membrane-associated viral antigens available for interaction with class I, major histocompatibility antigens and hence are potential target antigens for VV-CTLs

Transgene Expression and Repression in Transgenic Rats Bearing the Phosphoenolpyruvate Carboxykinase-Simian Virus 40 T Antigen or the Phosphoenolpyruvate Carboxykinase-Transforming Growth Factor-α Constructs

Science.gov (United States)

Haas, Michael J.; Dragan, Yvonne P.; Hikita, Hiroshi; Shimel, Randee; Takimoto, Koichi; Heath, Susan; Vaughan, Jennifer; Pitot, Henry C.

1999-01-01

Transgenic Sprague-Dawley rats expressing either human transforming growth factor-α (TGFα) or simian virus 40 large and small T antigen (TAg), each under the control of the phosphoenolpyruvate carboxykinase (PEPCK) promoter, were developed as an approach to the study of the promotion of hepatocarcinogenesis in the presence of a transgene regulatable by diet and/or hormones. Five lines of PEPCK-TGFα transgenic rats were established, each genetic line containing from one to several copies of the transgene per haploid genome. Two PEPCK-TAg transgenic founder rats were obtained, each with multiple copies of the transgene. Expression of the transgene was undetectable in the TGFα transgenic rats and could not be induced when the animals were placed on a high-protein, low-carbohydrate diet. The transgene was found to be highly methylated in all of these lines. No pathological alterations in the liver and intestine were observed at any time (up to 2 years) during the lives of these rats. One line of transgenic rats expressing the PEPCK-TAg transgene developed pancreatic islet cell hyperplasias and carcinomas, with few normal islets evident in the pancreas. This transgene is integrated as a hypomethylated tandem array of 10 to 12 copies on chromosome 8q11. Expression of large T antigen is highest in pancreatic neoplasms, but is also detectable in the normal brain, kidney, and liver. Mortality is most rapid in males, starting at 5 months of age and reaching 100% by 8 months. Morphologically, islet cell differentiation in the tumors ranges from poor to well differentiated, with regions of necrosis and fibrosis. Spontaneous metastasis of TAg-positive tumor cells to regional lymph nodes was observed. These studies indicate the importance of DNA methylation in the repression of specific transgenes in the rat. However, the expression of the PEPCK-TAg induces neoplastic transformation in islet cells, probably late in neuroendocrine cell differentiation. T antigen expression

Adoptive cancer immunotherapy using DNA-demethylated T helper cells as antigen-presenting cells

DEFF Research Database (Denmark)

Kirkin, Alexei F.; Dzhandzhugazyan, Karine N.; Guldberg, Per

2018-01-01

In cancer cells, cancer/testis (CT) antigens become epigenetically derepressed through DNA demethylation and constitute attractive targets for cancer immunotherapy. Here we report that activated CD4+ T helper cells treated with a DNA-demethylating agent express a broad repertoire of endogenous CT...... antigens and can be used as antigen-presenting cells to generate autologous cytotoxic T lymphocytes (CTLs) and natural killer cells. In vitro, activated CTLs induce HLA-restricted lysis of tumor cells of different histological types, as well as cells expressing single CT antigens. In a phase 1 trial of 25...... patients with recurrent glioblastoma multiforme, cytotoxic lymphocytes homed to the tumor, with tumor regression ongoing in three patients for 14, 22, and 27 months, respectively. No treatment-related adverse effects were observed. This proof-of-principle study shows that tumor-reactive effector cells can...

Immunotoxicity of aflatoxin B1: Impairment of the cell-mediated response to vaccine antigen and modulation of cytokine expression

International Nuclear Information System (INIS)

Meissonnier, Guylaine M.; Pinton, Philippe; Laffitte, Joelle; Cossalter, Anne-Marie; Gong, Yun Yun; Wild, Christopher P.; Bertin, Gerard; Galtier, Pierre; Oswald, Isabelle P.

2008-01-01

Aflatoxin B1 (AFB1), a mycotoxin produced by Aspergillus flavus or A. parasiticus, is a frequent contaminant of food and feed. This toxin is hepatotoxic and immunotoxic. The present study analyzed in pigs the influence of AFB1 on humoral and cellular responses, and investigated whether the immunomodulation observed is produced through interference with cytokine expression. For 28 days, pigs were fed a control diet or a diet contaminated with 385, 867 or 1807 μg pure AFB1/kg feed. At days 4 and 15, pigs were vaccinated with ovalbumin. AFB1 exposure, confirmed by an observed dose-response in blood aflatoxin-albumin adduct, had no major effect on humoral immunity as measured by plasma concentrations of total IgA, IgG and IgM and of anti-ovalbumin IgG. Toxin exposure did not impair the mitogenic response of lymphocytes but delayed and decreased their specific proliferation in response to the vaccine antigen, suggesting impaired lymphocyte activation in pigs exposed to AFB1. The expression level of pro-inflammatory (TNF-α, IL-1β, IL-6, IFN-γ) and regulatory (IL-10) cytokines was assessed by real-time PCR in spleen. A significant up-regulation of all 5 cytokines was observed in spleen from pigs exposed to the highest dose of AFB1. In pigs exposed to the medium dose, IL-6 expression was increased and a trend towards increased IFN-γ and IL-10 was observed. In addition we demonstrate that IL-6 impaired in vitro the antigenic- but not the mitogenic-induced proliferation of lymphocytes from control pigs vaccinated with ovalbumin. These results indicate that AFB1 dietary exposure decreases cell-mediated immunity while inducing an inflammatory response. These impairments in the immune response could participate in failure of vaccination protocols and increased susceptibility to infections described in pigs exposed to AFB1

Real-time PCR analysis of genes encoding tumor antigens in esophageal tumors and a cancer vaccine

DEFF Research Database (Denmark)

Weinert, Brian T; Krishnadath, Kausilia K; Milano, Francesca

2009-01-01

Tumor antigens are the primary target of therapeutic cancer vaccines. We set out to define and compare the expression pattern of tumor antigen genes in esophagus carcinoma biopsies and in an allogeneic tumor lysate-based cancer vaccine, MelCancerVac. Cells used for vaccine production were treated...... in the production of the vaccine. Quantitative PCR was used to assay 74 tumor antigen genes in patients with squamous cell carcinoma of the esophagus. 81% (13/16) of tumors expressed more than five cancer/testis (CT) antigens. A total of 96 genes were assayed in the tumor cell clone (DDM1.7) used to make tumor cell...

Strains of Sarcocystis neurona exhibit differences in their surface antigens, including the absence of the major surface antigen SnSAG1.

Science.gov (United States)

Howe, Daniel K; Gaji, Rajshekhar Y; Marsh, Antoinette E; Patil, Bhagyashree A; Saville, William J; Lindsay, David S; Dubey, J P; Granstrom, David E

2008-05-01

A gene family of surface antigens is expressed by merozoites of Sarcocystis neurona, the primary cause of equine protozoal myeloencephalitis (EPM). These surface proteins, designated SnSAGs, are immunodominant and therefore excellent candidates for development of EPM diagnostics or vaccines. Prior work had identified an EPM isolate lacking the major surface antigen SnSAG1, thus suggesting there may be some diversity in the SnSAGs expressed by different S. neurona isolates. Therefore, a bioinformatic, molecular and immunological study was conducted to assess conservation of the SnSAGs. Examination of an expressed sequence tag (EST) database revealed several notable SnSAG polymorphisms. In particular, the EST information implied that the EPM strain SN4 lacked the major surface antigen SnSAG1. The absence of this surface antigen from the SN4 strain was confirmed by both Western blot and Southern blot. To evaluate SnSAG polymorphisms in the S. neurona population, 14 strains were examined by Western blots using monospecific polyclonal antibodies against the four described SnSAGs. The results of these analyses demonstrated that SnSAG2, SnSAG3, and SnSAG4 are present in all 14 S. neurona strains tested, although some variance in SnSAG4 was observed. Importantly, SnSAG1 was not detected in seven of the strains, which included isolates from four cases of EPM and a case of fatal meningoencephalitis in a sea otter. Genetic analyses by PCR using gene-specific primers confirmed the absence of the SnSAG1 locus in six of these seven strains. Collectively, the data indicated that there is heterogeneity in the surface antigen composition of different S. neurona isolates, which is an important consideration for development of serological tests and prospective vaccines for EPM. Furthermore, the diversity reported herein likely extends to other phenotypes, such as strain virulence, and may have implications for the phylogeny of the various Sarcocystis spp. that undergo sexual stages

Evaluation of a Salmonella vectored vaccine expressing Mycobacterium avium subsp. paratuberculosis antigens against challenge in a goat model.

Directory of Open Access Journals (Sweden)

Syed M Faisal

Full Text Available Johnes disease (JD, caused by Mycobacterium avium subsp paratuberculosis (MAP, occurs worldwide as chronic granulomatous enteritis of domestic and wild ruminants. To develop a cost effective vaccine, in a previous study we constructed an attenuated Salmonella strain that expressed a fusion product made up of partial fragments of MAP antigens (Ag85A, Ag85B and SOD that imparted protection against challenge in a mouse model. In the current study we evaluated the differential immune response and protective efficacy of the Sal-Ag vaccine against challenge in a goat model as compared to the live attenuated vaccine MAP316F. PBMCs from goats vaccinated with Sal-Ag and challenged with MAP generated significantly lower levels of IFN-γ, following in vitro stimulation with either Antigen-mix or PPD jhonin, than PBMC from MAP316F vaccinated animals. Flow cytometric analysis showed the increase in IFN-γ correlated with a significantly higher level of proliferation of CD4, CD8 and γδT cells and an increased expression of CD25 and CD45R0 in MAP316F vaccinated animals as compared to control animals. Evaluation of a range of cytokines involved in Th1, Th2, Treg, and Th17 immune responses by quantitative PCR showed low levels of expression of Th1 (IFN-γ, IL-2, IL-12 and proinflammatory cytokines (IL-6, IL-8, IL-18, TNF-α in the Sal-Ag immunized group. Significant levels of Th2 and anti-inflammatory cytokines transcripts (IL-4, IL-10, IL-13, TGF-β were expressed but their level was low and with a pattern similar to the control group. Over all, Sal-Ag vaccine imparted partial protection that limited colonization in tissues of some animals upon challenge with wild type MAP but not to the level achieved with MAP316F. In conclusion, the data indicates that Sal-Ag vaccine induced only a low level of protective immunity that failed to limit the colonization of MAP in infected animals. Hence the Sal-Ag vaccine needs further refinement to increase its efficacy.

Recombinant expression systems: the obstacle to helminth vaccines?

Science.gov (United States)

Geldhof, Peter; De Maere, Veerle; Vercruysse, Jozef; Claerebout, Edwin

2007-11-01

The need for alternative ways to control helminth parasites has in recent years led to a boost in vaccination experiments with recombinant antigens. Despite the use of different expression systems, only a few recombinants induced high levels of protection against helminths. This is often attributed to the limitations of the current expression systems. Therefore, the need for new systems that can modify and glycosylate the expressed antigens has been advocated. However, analysis of over 100 published vaccine trials with recombinant helminth antigens indicates that it is often not known whether the native parasite antigen itself can induce protection or, if it does, which epitopes are important. This information is vital for a well-thought-out strategy for recombinant production. So, in addition to testing more expression systems, it should be considered that prior evaluation and characterization of the native antigens might help the development of recombinant vaccines against helminths in the long term.

Molecular mimics of the tumour antigen MUC1.

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Tharappel C James

Full Text Available A key requirement for the development of cancer immunotherapy is the identification of tumour-associated antigens that are differentially or exclusively expressed on the tumour and recognized by the host immune system. However, immune responses to such antigens are often muted or lacking due to the antigens being recognized as "self", and further complicated by the tumour environment and regulation of immune cells within. In an effort to circumvent the lack of immune responses to tumour antigens, we have devised a strategy to develop potential synthetic immunogens. The strategy, termed mirror image phage display, is based on the concept of molecular mimicry as demonstrated by the idiotype/anti-idiotype paradigm in the immune system. Here as 'proof of principle' we have selected molecular mimics of the well-characterised tumour associated antigen, the human mucin1 protein (MUC1 from two different peptide phage display libraries. The putative mimics were compared in structure and function to that of the native antigen. Our results demonstrate that several of the mimic peptides display T-cell stimulation activity in vitro when presented by matured dendritic cells. The mimic peptides and the native MUC1 antigenic epitopes can cross-stimulate T-cells. The data also indicate that sequence homology and/or chemical properties to the original epitope are not the sole determining factors for the observed immunostimulatory activity of the mimic peptides.

A molecular approach to immunoscintigraphy: A study of the T-antigen conformation on the surface of tumors

International Nuclear Information System (INIS)

Noujaim, A.; Selvaraj, S.; Suresh, M.R.; Turner, C.; McLean, G.; Willans, D.; Longenecker, B.M.; Haines, D.M.

1987-01-01

The role of glycoconjugates in tumor cell differentiation has been well documented. We have examined the expression of the two anomers of the Thomsen-Friedenreich antigen on the surface of human, canine and murine tumor cell membranes both in vitro and in vivo. This has been accomplished through the synthesis of the disaccharide terminal residues in both α and β configuration. Both entities were used to generate murine monoclonal antibodies which recognized the carbohydrate determinants. The determination of fine specificities of these antibodies was effected by means of cellular uptake, immunohistopathology and immunoscintigraphy. Examination of pathological specimens of human and canine tumor tissue indicated that the expressed antigen was in the β configuration. More than 89% of all human carcinomas tested expressed the antigen in the above anomeric form. The combination of synthetic antigens and monoclonal antibodies raised specifically against them provide us with invaluable tools for the study of tumor marker expression in humans and their respective animal tumor models. (orig.) [de

Characterization of the Apa antigen from M. avium subsp. paratuberculosis: a conserved Mycobacterium antigen that elicits a strong humoral response in cattle.

Science.gov (United States)

Gioffré, A; Echeverría-Valencia, G; Arese, A; Morsella, C; Garbaccio, S; Delgado, F; Zumárraga, M; Paolicchi, F; Cataldi, A; Romano, M I

2009-12-15

Johne's disease or paratuberculosis is widespread in almost all countries and remains difficult to eradicate. Nowadays, diagnosis of Mycobacterium avium subsp. paratuberculosis (MPTB) infection is one of the main concerns. In this work, we evaluated the expression, biochemical properties and antigenicity of the Apa antigen, encoded by the gene annotated as MAP1569, in the MPTB genome. We confirmed its expression in MPTB and its glycosylation by the ConA binding assay. Although the MPTB-Apa is not an immunodominant antigen, MPTB-infected cattle showed a strong humoral response to recombinant Apa by Western blot and ELISA. Milk was also a suitable sample to be tested by ELISA. We comparatively analysed the humoral cross-reactivity to the Apa from MPTB (MPTB-Apa) and the orthologue from Mycobacterium tuberculosis (MT-Apa, identical to that from Mycobacterium bovis) in both infected and control cows. Response of M. bovis- and MPTB-infected animals against MT-Apa was similar (P=0.6985) but the response of the M. bovis-infected ones to MPTB-Apa was differential, being significantly diminished (PApa stimulation in the IFNgamma release assay, we found no significant differences when compared infected herds with non-infected ones (P=0.34). This antigen, in contrast to bovine Purified Protein Derivative (PPDb), was strongly represented in avian PPD (PPDa), as shown by the recognition of BALB/c mice hyperimmune sera against MPTB-Apa by Dot-blot immunoassay. We therefore demonstrated the antigenicity of Apa in MPTB-infected animals and a differential response to the recombinant antigen when compared to M. bovis-infected animals. These traits herein described, added to the usefulness of milk samples to detect IgG anti-Apa, could be important for routine screening in dairy cattle, considering a multiantigenic approach to overcome the lack of immunodominance.

Limited SP17 expression within tumors diminishes its therapeutic potential

DEFF Research Database (Denmark)

Gjerstorff, M F; Ditzel, H J

2012-01-01

In this study, we have investigated the expression of the tumor antigen sperm protein 17 (SP17) in a large panel of human cancers and compared it with the expression of two well-characterized families of tumor antigens, melanoma-associated antigen-A (MAGE-A) and G antigen (GAGE). We found that SP17...

The influence of concentration of inactivated Edwardsiella tarda bacterin and immersion time on antigen uptake and expression of immune-related genes in Japanese flounder (Paralichthys olivaceus).

Science.gov (United States)

Du, Yang; Tang, Xiaoqian; Sheng, Xiuzhen; Xing, Jing; Zhan, Wenbin

2017-02-01

Our previous work has demonstrated that the immune response of Japanese flounder was associated with the concentration of formalin-inactivated Edwardsiella tarda and immersion time. In order to further investigate the influence of immersion vaccine dose and bath time on the antigen uptake, formalin-killed Edwardsiella tarda bacterin was prepared and adjusted to four concentrations (10 9 , 10 8 , 10 7 , 10 6  cfu ml -1 ) for 30, 60 and 90 min immersion in Japanese flounder model, respectively. Absolute quantitative real-time PCR was employed to examine the bacterin uptake in gill, skin, spleen and kidney at 3 and 6 h post vaccination. The results showed that the antigen uptaken in gills and skin were significant higher than spleen and kidney, and the antigen amounts in gill and skin both declined from 3 to 6 h, whereas the antigen amounts in spleen and kidney gradually increased. Significant higher antigen amounts were detected in 10 9 -30, 10 9 -60, 10 8 -60, 10 8 -90 and 10 8 -90 groups than other groups (P immersion with formalin-inactivated E. tarda, especially under 10 8 -60 min condition could efficiently enhance the antigen uptake and the expression of immune-related genes, which provided evidences for an enhanced vaccination effects under an optimized combination of vaccine dose and immersion time. Copyright © 2016 Elsevier Ltd. All rights reserved.

Detection and quantification of Duffy antigen on bovine red blood cell membranes using a polyclonal antibody

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Ana Teresa B.F. Antonangelo

2012-09-01

Full Text Available Babesiosis is one of the most important diseases affecting livestock agriculture worldwide. Animals from the subspecies Bos taurus indicus are more resistant to babesiosis than those from Bos taurus taurus. The genera Babesia and Plasmodium are Apicomplexa hemoparasites and share features such as invasion of red blood cells (RBC. The glycoprotein Duffy is the only human erythrocyte receptor for Pasmodium vivax and a mutation which abolishes expression of this glycoprotein on erythrocyte surfaces is responsible for making the majority of people originating from the indigenous populations of West Africa resistant to P. vivax. The current work detected and quantified the Duffy antigen on Bos taurus indicus and Bos taurus taurus erythrocyte surfaces using a polyclonal antibody in order to investigate if differences in susceptibility to Babesia are due to different levels of Duffy antigen expression on the RBCs of these animals, as is known to be the case in human beings for interactions of Plasmodium vivax-Duffy antigen. ELISA tests showed that the antibody that was raised against Duffy antigens detected the presence of Duffy antigen in both subspecies and that the amount of this antigen on those erythrocyte membranes was similar. These results indicate that the greater resistance of B. taurus indicus to babesiosis cannot be explained by the absence or lower expression of Duffy antigen on RBC surfaces.

Increased seroreactivity to glioma-expressed antigen 2 in brain tumor patients under radiation.

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Sabrina M Heisel

Full Text Available BACKGROUND: Surgery and radiation are the mainstays of therapy for human gliomas that are the most common primary brain tumors. Most recently, cell culture and animal studies provided the first convincing evidence that radiation not only eliminates tumor cells, but also modulates the immune response and likely improves anti-tumor immunotherapy. METHODOLOGY/PRINCIPAL FINDINGS: We present an in vivo study that analyzes the effects of radiation on the immune response in tumor patients. As readout system, we utilized the reactivity of glioma patients' sera against antigen GLEA2 as the most frequent antigen immunogenic in glioblastoma patients. We established an ELISA assay to analyze reactivity of 24 glioblastoma patients over a period of several months. As control we used 30 sera from healthy donors as well as 30 sera from lung cancer patients. We compared the course of GLEA2 seroreactivity at different times prior, during and after radiation. The GLEA2 seroreactivity was increased by the time of surgery, decreased after surgery, increased again under radiation, and slightly decreased after radiation. CONCLUSIONS/SIGNIFICANCE: Our results provide in vivo evidence for an increased antibody response against tumor antigens under radiation. Antigens that become immunogenic with an increased antibody response as result of radiation can serve as ideal targets for immunotherapy of human tumors.

Purification of a polyclonal antibody against CD147 for ELISA using antigen-immunoaffinity chromatography

Science.gov (United States)

Liu, Shuangshuang; Li, Shasha; Zhang, Yang; Wang, Ye; Zhu, Yumeng; Wang, Bin; Chen, Zhi-Nan

2017-01-01

The immunoglobulin superfamily member CD147 is a widely expressed glycoprotein that occurs in both a membrane-spanning and soluble form. Sandwich ELISA is a powerful tool for analyzing soluble antigens. The aim of the present study was to obtain a highly specific polyclonal antibody against human CD147 that can be used for sandwich ELISA analysis. Expression of recombinant CD147 by a eukaryotic expression system was used to immunize rabbits to obtain antiserum. A highly specific polyclonal antibody that was able to detect soluble CD147 in sandwich ELISA was obtained by antigen-immunoaffinity chromatography purification. The purity of rabbit anti-CD147 polyclonal antibodies was ~99%, and ELISA analysis was able to determine the titer of the rabbit anti-CD147 polyclonal antibodies at 1:512,000. The lowest concentration of the standard CD147 antigen that the sandwich ELISA was able to detect was 31.25 pg/ml. The sandwich ELISA system was composed of anti-hepatoma HAb18 monoclonal antibodies and purified rabbit anti-CD147 polyclonal antibodies. The present study demonstrated that antigen-immunoaffinity chromatography may be a good technique for the purification of polyclonal antibodies, which may be used to detect antigen in sandwich ELISAs. PMID:28487989

Ubiquitinated Proteins Isolated From Tumor Cells Are Efficient Substrates for Antigen Cross-Presentation.

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Yu, Guangjie; Moudgil, Tarsem; Cui, Zhihua; Mou, Yongbin; Wang, Lixin; Fox, Bernard A; Hu, Hong-Ming

2017-06-01

We have previously shown that inhibition of the proteasome causes defective ribosomal products to be shunted into autophagosomes and subsequently released from tumor cells as defective ribosomal products in Blebs (DRibbles). These DRibbles serve as an excellent source of antigens for cross-priming of tumor-specific T cells. Here, we examine the role of ubiquitinated proteins (Ub-proteins) in this pathway. Using purified Ub-proteins from tumor cells that express endogenous tumor-associated antigen or exogenous viral antigen, we tested the ability of these proteins to stimulate antigen-specific T-cell responses, by activation of monocyte-derived dendritic cells generated from human peripheral blood mononuclear cells. Compared with total cell lysates, we found that purified Ub-proteins from both a gp100-specific melanoma cell line and from a lung cancer cell line expressing cytomegalovirus pp65 antigen produced a significantly higher level of IFN-γ in gp100- or pp65-specific T cells, respectively. In addition, Ub-proteins from an allogeneic tumor cell line could be used to stimulate tumor-infiltrating lymphocytes isolated and expanded from non-small cell lung cancer patients. These results establish that Ub-proteins provide a relevant source of antigens for cross-priming of antitumor immune responses in a variety of settings, including endogenous melanoma and exogenous viral antigen presentation, as well as antigen-specific tumor-infiltrating lymphocytes. Thus, ubiquitin can be used as an affinity tag to enrich for unknown tumor-specific antigens from tumor cell lysates to stimulate tumor-specific T cells ex vivo or to be used as vaccines to target short-lived proteins.

Cytotoxic T-Lymphocyte Antigen-2 alpha participates in axial ...

African Journals Online (AJOL)

Cytotoxic T-lymphocyte antigen-2 alpha (CTLA-2α) has been discovered and expressed in mouse activated T-cells and mast cells. Structurally, it is homologous to the proregion of mouse cathepsin L, a lysosomal cystein proteinase. Expressed recombinant CTLA-2α is shown to exhibit selective inhibition to cathepsin L and ...

Variability in surface antigen expression on neuroblastoma cells as revealed by monoclonal antibodies

International Nuclear Information System (INIS)

Malpas, J.S.; Kemshead, J.T.; Pritchard, J.; Greaves, M.F.

1982-01-01

In treatment programmes for neuroblastoma involving autologous bone marrow transplantation, a problem exists in the identification of small numbers of metastatic tumour cells present in the marrow aspirates. Reinfusion of tumour cells along with normal bone marrow may reseed the tumour within a patient who has received high dose chemotherapy. Formalin-induced fluorescence in neuroblastoma is a possible diagnostic aid, but this method has no therapeutic potential. Other methods of detecting tumour relying on gross physiological changes in the patient are not suitable for diagnosis of minimal metastatic disease. As an immunological approach to the problem, rabbit antisera to neuroblastoma have been raised but these reagents suffer from low titre after absorption to make them specific. The authors have used the technique of somatic cell hybridisation to raise monoclonal antibodies which bind to neuroblastoma cells and not to normal haemopoietic progenitors. A panel of such reagents to demonstrate heterogeneity in antigen expression amongst metastatic neuroblastoma cells was employed in a radioimmunoassay as diagnostic aid for this problem. (Auth.)

Rabies virus glycoprotein as a carrier for anthrax protective antigen

International Nuclear Information System (INIS)

Smith, Mary Ellen; Koser, Martin; Xiao Sa; Siler, Catherine; McGettigan, James P.; Calkins, Catherine; Pomerantz, Roger J.; Dietzschold, Bernhard; Schnell, Matthias J.

2006-01-01

Live viral vectors expressing foreign antigens have shown great promise as vaccines against viral diseases. However, safety concerns remain a major problem regarding the use of even highly attenuated viral vectors. Using the rabies virus (RV) envelope protein as a carrier molecule, we show here that inactivated RV particles can be utilized to present Bacillus anthracis protective antigen (PA) domain-4 in the viral membrane. In addition to the RV glycoprotein (G) transmembrane and cytoplasmic domains, a portion of the RV G ectodomain was required to express the chimeric RV G anthrax PA on the cell surface. The novel antigen was also efficiently incorporated into RV virions. Mice immunized with the inactivated recombinant RV virions exhibited seroconversion against both RV G and anthrax PA, and a second inoculation greatly increased these responses. These data demonstrate that a viral envelope protein can carry a bacterial protein and that a viral carrier can display whole polypeptides compared to the limited epitope presentation of previous viral systems

Human Tumor Antigens Yesterday, Today, and Tomorrow.

Science.gov (United States)

Finn, Olivera J

2017-05-01

The question of whether human tumors express antigens that can be recognized by the immune system has been answered with a resounding YES. Most were identified through spontaneous antitumor humoral and cellular immune responses found in cancer patients and include peptides, glycopeptides, phosphopeptides, viral peptides, and peptides resulting from common mutations in oncogenes and tumor-suppressor genes, or common gene fusion events. Many have been extensively tested as candidates for anticancer vaccines. More recently, attention has been focused on the potentially large number of unique tumor antigens, mutated neoantigens, that are the predicted products of the numerous mutations revealed by exome sequencing of primary tumors. Only a few have been confirmed as targets of spontaneous immunity and immunosurveillance, and even fewer have been tested in preclinical and clinical settings. The field has been divided for a long time on the relative importance of shared versus mutated antigens in tumor surveillance and as candidates for vaccines. This question will eventually need to be answered in a head to head comparison in well-designed clinical trials. One advantage that shared antigens have over mutated antigens is their potential to be used in vaccines for primary cancer prevention. Cancer Immunol Res; 5(5); 347-54. ©2017 AACR . ©2017 American Association for Cancer Research.

The expressions of P53 protein and proliferating cell nuclear antigen in specimens by CT-guidance percutaneous lung biopsy

International Nuclear Information System (INIS)

Zhuang Yiping; Shen Zongli; Zhang Jin; Kang Zheng; Zhu Yueqing; Feng Yong; Shen Wenrong; Wang Yaping

2004-01-01

Objective: To evaluate relations between lung cancer and the expressions of P53 protein together with proliferating cell nuclear antigen (PCNA) in specimens of lung lesions by needle biopsy. Methods: CT-guidance percutaneous biopsy of lung lesions were performed in 66 patients with the determination of expressions of p53 protein and PCNA by flow cytometer (FCM). Results: 1. The sensitivity of CT-guidance percutaneous biopsy was 94.3% in 53 cases of lung cancer with the diagnostic accuracy of 90.9% totally. The complication rate of pneumothorax was 4.6%. 2. The expression of P53 protein was (29.9 ± 2.7)% in lung cancer (53 cases), while (17.9 ± 2.8)% in benign lesions (13 cases) (t=2.0, P 2 =6.10, P 2 =9.71, P 0.05). Conclusions: FCM plays and valuable role in determining the expression of P53 protein and PCNA in the specimen of lung cancer by CT-guided percutaneous biopsy. The expression of p53 and PCNA may be useful in the diagnosis of lung cancer by providing the relation between imaging of lung cancer and the molecular mechanism, and furthermore revealing the characteristics of molecular biology of lung cancer at protein level. (authors)

Influence of radiotherapy on expression of the proliferating cell nuclear antigen (PCNA) and c-fos in human cervical cancer

International Nuclear Information System (INIS)

Shi Mei; Wei Lichun; Sun Chaoyang; Ma Haixin; Guo Yan

2001-01-01

Objective: To investigate changes of proliferating cell nuclear antigen (PCNA) expression in human cervical cancer following irradiation. Methods: Immunohistochemical staining for PCNA was performed in frozen sections of formalin-fixed cervical cancer biopsy tissues. Results: The majority of the cancer cells showed PCNA-immunoreactivity before irradiation. Following irradiation (30-40 Gy/15-20 f) PCNA-immuno-positive staining was hardly detectable in most of the cancer cells. The PCNA-immunoreactivity, however, increased after radiotherapy, and moderate or heavy immuno-positive staining for PCNA was seen in irradiated mesenchymal tissue cells. On the other hand, after irradiation Fos-immunoreactivity decreased remarkably, and Fos-immuno-positive staining was hardly detectable in most of cancer cells. No obvious change in Fos-immuno-reactivity, however, was seen in mesenchymal connective tissue following irradiation. Conclusion: Irradiation inhibits PCNA and c-fos expression in cervical cancer cells whereas it induces the expression of PCNA in mesenchymal tissue cells. The present results suggest that expression of PCNA and c-fos may be regarded as a molecular marker for evaluating the cancer cell proliferation and mesenchymal tissue repair during radiotherapy of human cervical cancer

Evidence against suppressor cell involvement in naturally acquired tolerance of a minor histocompatibility antigen

International Nuclear Information System (INIS)

Johnson, L.L.

1991-01-01

The hypothesis was investigated that suppressor cells may be responsible for maintenance of immunologic tolerance of a minor H3 antigen in mice that express the antigen naturally. Lymphoid cell populations from B6.C-H-24c (HW54) mice, a congenic-resistant strain histoincompatible with H-24b-expressing C57BL/6 (B6) mice only with respect to the H-24 locus, were examined in cell-transfer experiments to see if they contained naturally arising H-24c-specific suppressor cells. The H-24 antigen was chosen for these studies because, unlike most other minor and major histocompatibility (H) antigens, it is not detectable on mature lymphoid cells by any of several functional criteria. Thus transfer of HW54 lymphoid cells to B6 hosts could be done without the complication of inducing hyporesponsiveness de novo in the host, as occurs with other minor H antigens that are expressed on lymphocytes. B6 hosts were given HW54 skin grafts along with HW54 lymphoid cells to assess their tolerance of the H-24c-encoded antigen. The hosts were either (1) normal, nonimmune B6 mice; (2) B6 mice rendered immunodeficient by thymectomy and irradiation (TxB) and repopulated with H-24c-immune B6 lymphocytes; or (3) TxB B6 hosts repopulated with nonimmune B6 lymphocytes. In each case it was found that the additionally infused HW54 lymphoid cells did not suppress the ability of these hosts to reject HW54 skin grafts. In other words, HW54 lymphoid cells appear not to possess suppressive activity specific for the H-24c antigen that might maintain antigen-specific natural tolerance. Additional experiments were performed to determine whether HW54 lymphoid cells can inhibit the ability of sublethally irradiated B6 mice to regain the capacity to reject HW54 skin

Expression of neuronal antigens and related ventral and dorsal proteins in the normal spinal cord and a surgically induced open neural tube defect of the spine in chick embryos: an immunohistochemical study.

Science.gov (United States)

Lee, Do-Hun; Phi, Ji Hoon; Chung, You-Nam; Lee, Yun-Jin; Kim, Seung-Ki; Cho, Byung-Kyu; Kim, Dong Won; Park, Moon-Sik; Wang, Kyu-Chang

2010-05-01

The aims of this study were to elucidate the processes of neuronal differentiation and ventrodorsal patterning in the spinal cord of the chick embryo from embryonic day (E) 3 to E17 and to study the effect of a prenatal spinal open neural tube defect (ONTD) on these processes. Expression patterns of neuronal antigens (neuronal nuclear antigen, neurofilament-associated protein (NAP), and synaptophysin) and related ventral markers [sonic hedgehog, paired box gene (PAX)6, and islet-1], and dorsal markers (bone morphogenetic protein, Notch homolog 1, and PAX7) were investigated in the normal spinal cord and in a surgically induced spinal ONTD in chick embryos. Four normal and ONTD chick embryos were used for each antigen group. There were no differences in the expression of neuronal and ventrodorsal markers between the control and ONTD groups. NAP and synaptophysin were useful for identifying dorsal structures in the distorted anatomy of the ONTD chicks.

MYCN: from oncoprotein to tumor-associated antigen

International Nuclear Information System (INIS)

Pistoia, Vito; Morandi, Fabio; Pezzolo, Annalisa; Raffaghello, Lizzia; Prigione, Ignazia

2012-01-01

MYCN is a well-known oncogene over-expressed in different human malignancies including neuroblastoma (NB), rhabdomyosarcoma, medulloblastoma, astrocytoma, Wilms’ tumor, and small cell lung cancer. In the case of NB, MYCN amplification is an established biomarker of poor-prognosis. MYCN belongs to a family of transcription factors (the most important of which is C-MYC) that show a high degree of homology. Down-regulation of MYC protein expression leads to tumor regression in animal models, indicating that MYC proteins represent interesting therapeutic targets. Pre-requisites for a candidate tumor-associated antigen (TAA) to be targeted by immunotherapeutic approaches are the following, (i) expression should be tumor-restricted, (ii) the putative TAA should be up-regulated in cancer cells, and (iii) protein should be processed into immunogenic peptides capable of associating to major histocompatibility complex molecules with high affinity. Indeed, the MYCN protein is not expressed in human adult tissues and up-regulated variably in NB cells, and MYCN peptides capable of associating to HLA-A1 or HLA-A2 molecules with high affinity have been identified. Thus the MYCN protein qualifies as putative TAA in NB. Additional issues that determine the feasibility of targeting a putative TAA with cytotoxic T lymphocytes (CTLs) and will be here discussed are the following, (i) the inadequacy of tumor cells per se to act as antigen-presenting cells witnessed, in the case of NB cells, by the low to absent expression of HLA class I molecules, the lack of co-stimulatory molecules and multiple defects in the HLA class I related antigen processing machinery, and (ii) the immune evasion mechanisms operated by cancer cells to fool the host immune system, such as up-regulation of soluble immunosuppressive molecules (e.g., soluble MICA and HLA-G in the case of NB) or generation of immunosuppressive cells in the tumor microenvironment. A final issue that deserves consideration is the

MYCN: from oncoprotein to tumor-associated antigen

Energy Technology Data Exchange (ETDEWEB)

Pistoia, Vito; Morandi, Fabio; Pezzolo, Annalisa; Raffaghello, Lizzia; Prigione, Ignazia, E-mail: vitopistoia@ospedale-gaslini.ge.it [Laboratory of Oncology, Translational Research and Laboratory Medicine, G. Gaslini Institute, Genoa (Italy)

2012-11-16

MYCN is a well-known oncogene over-expressed in different human malignancies including neuroblastoma (NB), rhabdomyosarcoma, medulloblastoma, astrocytoma, Wilms’ tumor, and small cell lung cancer. In the case of NB, MYCN amplification is an established biomarker of poor-prognosis. MYCN belongs to a family of transcription factors (the most important of which is C-MYC) that show a high degree of homology. Down-regulation of MYC protein expression leads to tumor regression in animal models, indicating that MYC proteins represent interesting therapeutic targets. Pre-requisites for a candidate tumor-associated antigen (TAA) to be targeted by immunotherapeutic approaches are the following, (i) expression should be tumor-restricted, (ii) the putative TAA should be up-regulated in cancer cells, and (iii) protein should be processed into immunogenic peptides capable of associating to major histocompatibility complex molecules with high affinity. Indeed, the MYCN protein is not expressed in human adult tissues and up-regulated variably in NB cells, and MYCN peptides capable of associating to HLA-A1 or HLA-A2 molecules with high affinity have been identified. Thus the MYCN protein qualifies as putative TAA in NB. Additional issues that determine the feasibility of targeting a putative TAA with cytotoxic T lymphocytes (CTLs) and will be here discussed are the following, (i) the inadequacy of tumor cells per se to act as antigen-presenting cells witnessed, in the case of NB cells, by the low to absent expression of HLA class I molecules, the lack of co-stimulatory molecules and multiple defects in the HLA class I related antigen processing machinery, and (ii) the immune evasion mechanisms operated by cancer cells to fool the host immune system, such as up-regulation of soluble immunosuppressive molecules (e.g., soluble MICA and HLA-G in the case of NB) or generation of immunosuppressive cells in the tumor microenvironment. A final issue that deserves consideration is the

Molecular characteristics of an immobilization antigen gene of the fish-parasitic protozoan Ichthyophthirius multifiliis strain ARS-6

Science.gov (United States)

Ichthyophthirius multifiliis, a ciliated protozoan parasite of fish, expresses surface antigens (i-antigens), which react with host antibodies that render them immobile. The nucleotide sequence of an i-antigen gene of Ichthyophthirius multifiliis strain ARS-6 was deduced. The predicted protein of 47...

CD107a Expression and IFN-γ Production as Markers for Evaluation of Cytotoxic CD3+ CD8+ T Cell Response to CMV Antigen in Women with Recurrent Spontaneous Abortion.

Science.gov (United States)

Tarokhian, Batoul; Sherkat, Roya; Nasr Esfahani, Mohamma Hossein; Adib, Minoo; Kiani Esfahani, Abbas; Ataei, Behrooz

2014-01-01

Some evidence has shown a relationship between primary human cytomegalovirus (CMV) infection and pregnancy loss. The impact of CMV infection reactivation during pregnancy on adverse pregnancy outcomes is not completely understood. It is proposed that altered immune response, and therefore, recurrence or reactivation of latent CMV infection may relate to recurrent spontaneous abortion (RSA); however, few data are available in this regard. To find out about any cell mediated defect and reactivation of latent CMV infection in women with RPL, cellular immunity to the virus has been evaluated by specific cytotoxic T lymphocyte (CTL) response to CMV. In a case control study, CTL CD107a expression and in- tercellular IFN-γ production in response to CMV pp65 antigen and staphylococcus enterotoxin B (SEB) in women with RSA were assessed by flow cytometric analysis. Forty-four cases with history of recurrent pregnancy and forty-four controls with history of successful pregnancies were included. The FACSCaliber flow cytometer were used for analysis. No significant difference was observed between CD107a expression and IFN-γ production in response to CMV PP65 antigen in RPL patients and control group. How- ever, the cytotoxic response to SEB antigen in patients with RPL was significantly lower than control group (p=0.042). The results of this study show that impaired CD107a expression and IFN-γ production as CTL response to CMV does not appear to be a major contrib- uting and immune incompetence factor in patients with RPL, but cytotoxic T cell response defect to other antigens requires to be assessed further in these patients.

T-lymphocyte cytokine mRNA expression in cystic echinococcosis.

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Fauser, S; Kern, P

1997-04-01

In the present study we investigated cytokine mRNA expression by peripheral blood mononuclear cells (PBMC) from patients with cystic echinococcosis (CE) after stimulation with different antigens. By using reverse transcriptase polymerase chain reaction (RT-PCR) we could demonstrate that restimulation with crude Echinococcus granulosus antigen (Eg-Ag) induced or enhanced Th2 cytokine mRNA expression, especially IL-5 (by using antigen from sheep cyst fluid) in 23 out of 26 investigated CE patients and IL-10 (by using antigen from camel cyst fluid) in 10 out of 10 investigated CE patients. In contrast, IL-5 mRNA expression was absent in PBMC of healthy controls after Eg-Ag stimulation. To determine the specificity of this reaction we stimulated PBMC from 11 CE patients with crude Echinococcus multilocularis antigen (Em-Ag) and PBMC from 8 CE patients with Toxocara canis antigen (Tc-Ag). We found that the PBMC of patients showed a similar mRNA cytokine pattern on stimulation with Em-Ag when compared with Eg-Ag stimulation. The cytokine mRNA pattern on stimulation with Tc-Ag, however, resembled the cytokine mRNA pattern of unstimulated PBMC. Furthermore, the stimulation of PBMC with crude Mycobacterium tuberculosis antigen (H37Ra) and purified protein derivative (PPD) of M. tuberculosis revealed distinct IL-5 mRNA expression in all investigated CE patients, whereas in healthy controls IL-5 mRNA expression was very weak or totally absent. Thus, our results indicate an induction of Th2 cytokine mRNA expression in CE patients, which is frequently observed in parasite infections. Interestingly, this response persists after stimulation with tuberculosis antigens, which normally induce Th1 response.

Anti-GBM disease after nephrectomy for xanthogranulomatous pyelonephritis in a patient expressing HLA DR15 major histocompatibility antigens: a case report.

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O'Hagan, Emma; Mallett, Tamara; Convery, Mairead; McKeever, Karl

2015-01-01

Antiglomerular basement membrane (anti-GBM) antibody disease is uncommon in the pediatric population. There are no cases in the literature describing the development of anti-GBM disease following XGP or nephrectomy. We report the case of a 7-year-old boy with no past history of urological illness, treated with antimicrobials and nephrectomy for diffuse, unilateral xanthogranulomatous pyelonephritis (XGP). Renal function and ultrasound scan of the contralateral kidney postoperatively were normal. Three months later, the child represented in acute renal failure with rapidly progressive glomerulonephritis requiring hemodialysis. Renal biopsy showed severe crescentic glomerulonephritis with 95% of glomeruli demonstrating circumferential cellular crescents. Strong linear IgG staining of the glomerular basement membranes was present, in keeping with anti-GBM disease. Circulating anti-GBM antibodies were positive. Treatment with plasma exchange, methylprednisolone, and cyclophosphamide led to normalization of anti-GBM antibody titers. Frequency of hemodialysis was reduced as renal function improved, and he is currently independent of dialysis with estimated glomerular filtration rate 20.7 mls/min/1.73 m 2 . Case studies in the adult literature have reported the development of a rapidly progressive anti-GBM antibody-induced glomerulonephritis following renal surgery where patients expressed HLA DR2/HLA DR15 major histocompatibility (MHC) antigens. Of note, our patient also expresses the HLA DR15 MHC antigen.

Generation of human induced pluripotent stem cells from a Bombay individual: Moving towards 'universal-donor' red blood cells

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Seifinejad, Ali; Taei, Adeleh [Department of Stem Cells and Developmental Biology, Royan Institute for Stem Cell Biology and Technology, P.O. Box 19395-4644, ACECR, Tehran (Iran, Islamic Republic of); Totonchi, Mehdi; Vazirinasab, Hamed [Department of Genetics, Royan Institute for Reproductive Biomedicine, ACECR, Tehran (Iran, Islamic Republic of); Hassani, Seideh Nafiseh [Department of Stem Cells and Developmental Biology, Royan Institute for Stem Cell Biology and Technology, P.O. Box 19395-4644, ACECR, Tehran (Iran, Islamic Republic of); Aghdami, Nasser [Department of Stem Cells and Developmental Biology, Royan Institute for Stem Cell Biology and Technology, P.O. Box 19395-4644, ACECR, Tehran (Iran, Islamic Republic of); Department of Regenerative Biomedicine, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran (Iran, Islamic Republic of); Shahbazi, Ebrahim [Department of Stem Cells and Developmental Biology, Royan Institute for Stem Cell Biology and Technology, P.O. Box 19395-4644, ACECR, Tehran (Iran, Islamic Republic of); Yazdi, Reza Salman [Department of Genetics, Royan Institute for Reproductive Biomedicine, ACECR, Tehran (Iran, Islamic Republic of); Salekdeh, Ghasem Hosseini, E-mail: Salekdeh@royaninstitute.org [Department of Molecular Systems Biology, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran (Iran, Islamic Republic of); Department of Systems Biology, Agricultural Biotechnology Research Institute of Iran, Karaj (Iran, Islamic Republic of); Baharvand, Hossein, E-mail: Baharvand@royaninstitute.org [Department of Stem Cells and Developmental Biology, Royan Institute for Stem Cell Biology and Technology, P.O. Box 19395-4644, ACECR, Tehran (Iran, Islamic Republic of); Department of Regenerative Biomedicine, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran (Iran, Islamic Republic of); Department of Developmental Biology, University of Science and Culture, ACECR, Tehran (Iran, Islamic Republic of)

2010-01-01

Bombay phenotype is one of the rare phenotypes in the ABO blood group system that fails to express ABH antigens on red blood cells. Nonsense or missense mutations in fucosyltransfrase1 (FUT1) and fucosyltransfrase2 (FUT2) genes are known to create this phenotype. This blood group is compatible with all other blood groups as a donor, as it does not express the H antigen on the red blood cells. In this study, we describe the establishment of human induced pluripotent stem cells (iPSCs) from the dermal fibroblasts of a Bombay blood-type individual by the ectopic expression of established transcription factors Klf4, Oct4, Sox2, and c-Myc. Sequence analyses of fibroblasts and iPSCs revealed a nonsense mutation 826C to T (276 Gln to Ter) in the FUT1 gene and a missense mutation 739G to A (247 Gly to Ser) in the FUT2 gene in the Bombay phenotype under study. The established iPSCs resemble human embryonic stem cells in morphology, passaging, surface and pluripotency markers, normal karyotype, gene expression, DNA methylation of critical pluripotency genes, and in-vitro differentiation. The directed differentiation of the iPSCs into hematopoietic lineage cells displayed increased expression of the hematopoietic lineage markers such as CD34, CD133, RUNX1, KDR, {alpha}-globulin, and {gamma}-globulin. Such specific stem cells provide an unprecedented opportunity to produce a universal blood group donor, in-vitro, thus enabling cellular replacement therapies, once the safety issue is resolved.

Generation of human induced pluripotent stem cells from a Bombay individual: Moving towards 'universal-donor' red blood cells

International Nuclear Information System (INIS)

Seifinejad, Ali; Taei, Adeleh; Totonchi, Mehdi; Vazirinasab, Hamed; Hassani, Seideh Nafiseh; Aghdami, Nasser; Shahbazi, Ebrahim; Yazdi, Reza Salman; Salekdeh, Ghasem Hosseini; Baharvand, Hossein

2010-01-01

Bombay phenotype is one of the rare phenotypes in the ABO blood group system that fails to express ABH antigens on red blood cells. Nonsense or missense mutations in fucosyltransfrase1 (FUT1) and fucosyltransfrase2 (FUT2) genes are known to create this phenotype. This blood group is compatible with all other blood groups as a donor, as it does not express the H antigen on the red blood cells. In this study, we describe the establishment of human induced pluripotent stem cells (iPSCs) from the dermal fibroblasts of a Bombay blood-type individual by the ectopic expression of established transcription factors Klf4, Oct4, Sox2, and c-Myc. Sequence analyses of fibroblasts and iPSCs revealed a nonsense mutation 826C to T (276 Gln to Ter) in the FUT1 gene and a missense mutation 739G to A (247 Gly to Ser) in the FUT2 gene in the Bombay phenotype under study. The established iPSCs resemble human embryonic stem cells in morphology, passaging, surface and pluripotency markers, normal karyotype, gene expression, DNA methylation of critical pluripotency genes, and in-vitro differentiation. The directed differentiation of the iPSCs into hematopoietic lineage cells displayed increased expression of the hematopoietic lineage markers such as CD34, CD133, RUNX1, KDR, α-globulin, and γ-globulin. Such specific stem cells provide an unprecedented opportunity to produce a universal blood group donor, in-vitro, thus enabling cellular replacement therapies, once the safety issue is resolved.

An engineered Lactococcus lactis strain exerts significant immune responses through efficient expression and delivery of Helicobacter pylori Lpp20 antigen.

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Zhang, Rongguang; Peng, Xiaoyan; Duan, Guangcai; Shi, Qingfeng; Chen, Shuaiyin; Wang, Chen; Fan, Qingtang; Xi, Yuanlin

2016-12-01

To produce and deliver Helicobacter pylori lipoprotein Lpp20 via using Lactococcus lactis with aim of developing an efficient way to use this protective antigen in vaccine formulation. An engineered L. lactis strain carrying the lpp20 gene from H. pylori was constructed. The inducible expression of Lpp20 in L. lactis was detected as a 20 kDa intracellular protein by SDS-PAGE. Lpp20 constituted 10 % of the L. lactis cellular proteins. The expression product was highly immunoreactive, as demonstrated by western blot assays using mouse anti-H. pylori sera. Animal experimentation showed that oral vaccination with the engineered strain excited significantly elevated levels of serum Lpp20-specific IgG antibodies in BALB/c mice (P lactis, demonstrating an efficient utilization mode of Lpp20 in anti-H. pylori vaccination.

Sperm associated antigen 9 (SPAG9) expression and humoral response in benign and malignant salivary gland tumors

Science.gov (United States)

Agarwal, Sumit; Parashar, Deepak; Gupta, Namita; Jagadish, Nirmala; Thakar, Alok; Suri, Vaishali; Kumar, Rajive; Gupta, Anju; Ansari, Abdul S; Lohiya, Nirmal Kumar; Suri, Anil

2015-01-01

Salivary gland cancers are highly aggressive epithelial tumor associated with metastatic potential and high mortality. The tumors are biologically diverse and are of various histotypes. Besides, the detection and diagnosis is a major problem of salivary gland cancer for available treatment modalities. In the present study, we have investigated the association of sperm associated antigen 9 (SPAG9) expression with salivary gland tumor (SGT). Clinical specimens of benign (n = 16) and malignant tumors (n = 86) were examined for the SPAG9 expression. In addition, the sera and adjacent non-cancerous tissues (n = 72) from available patients were obtained. Our in situ RNA hybridization and immunohistochemistry (IHC) analysis revealed significant difference (p = 0.0001) in SPAG9 gene and protein expression in benign (63%) and malignant tumor (84%) specimens. Further, significant association was also observed between SPAG9 expression and malignant tumors (P = 0.05). A cut-off value of >10% cells expressing SPAG9 protein designated as positive in IHC, predicted presence of malignant SGT with 83.72% sensitivity, 100% specificity, 100% PPV and 83.72% NPV. Humoral response against SPAG9 protein was generated in 68% of SGT patients. A cut-off value of 0.212 OD for anti-SPAG9 antibodies in ELISA predicted presence of malignant SGT with 69.23% sensitivity, 100% specificity, 100% PPV and 78.94% NPV. Collectively, our data suggests that the majority of SGT show significant difference and association among benign and malignant tumors for SPAG9 gene and protein expression and also exhibit humoral response against SPAG9 protein. Hence, SPAG9 may be developed as a biomarker for detection and diagnosis of salivary gland tumors. PMID:25941602

Highly specific expression of luciferase gene in lungs of naive nude mice directed by prostate-specific antigen promoter

International Nuclear Information System (INIS)

Li Hongwei; Li Jinzhong; Helm, Gregory A.; Pan Dongfeng

2005-01-01

PSA promoter has been demonstrated the utility for tissue-specific toxic gene therapy in prostate cancer models. Characterization of foreign gene overexpression in normal animals elicited by PSA promoter should help evaluate therapy safety. Here we constructed an adenovirus vector (AdPSA-Luc), containing firefly luciferase gene under the control of the 5837 bp long prostate-specific antigen promoter. A charge coupled device video camera was used to non-invasively image expression of firefly luciferase in nude mice on days 3, 7, 11 after injection of 2 x 10 9 PFU of AdPSA-Luc virus via tail vein. The result showed highly specific expression of the luciferase gene in lungs of mice from day 7. The finding indicates the potential limitations of the suicide gene therapy of prostate cancer based on selectivity of PSA promoter. By contrary, it has encouraging implications for further development of vectors via PSA promoter to enable gene therapy for pulmonary diseases

Women's attitude towards routine human platelet antigen-screening in pregnancy.

Science.gov (United States)

Winkelhorst, Dian; Loeff, Rosanne M; van den Akker-Van Marle, M Elske; de Haas, Masja; Oepkes, Dick

2017-08-01

Fetal and neonatal alloimmune thrombocytopenia is a potentially life-threatening disease with excellent preventative treatment available for subsequent pregnancies. To prevent index cases, the effectiveness of a population-based screening program has been suggested repeatedly. Therefore, we aimed to evaluate women's attitude towards possible future human platelet antigen-screening in pregnancy. We performed a cross-sectional questionnaire study among healthy pregnant women receiving prenatal care in one of seven participating midwifery practices. Attitude was assessed using a questionnaire based on the validated Multidimensional Measurement of Informed Choice model, containing questions assessing knowledge, attitude and intention to participate. A total of 143 of the 220 women (65%) completed and returned the questionnaire. A positive attitude towards human platelet antigen-screening was expressed by 91% of participants, of which 94% was based on sufficient knowledge. Attitude was more likely to be negatively influenced by the opinion that screening can be frightening. Informed choices were made in 87% and occurred significantly less in women from non-European origin, 89% in European women vs. 60% in non-European women (p = 0.03). Pregnant women in the Netherlands expressed a positive attitude towards human platelet antigen-screening in pregnancy. We therefore expect a high rate of informed uptake when human platelet antigen-screening is implemented. In future counseling on human platelet antigen-screening, ethnicity and possible anxiety associated with a screening test need to be specifically addressed. © 2017 Nordic Federation of Societies of Obstetrics and Gynecology.

Carcinoma-associated antigens

International Nuclear Information System (INIS)

Bartorelli, A.; Accinni, R.

1981-01-01

This invention relates to novel antigens associated with breast carcinoma, anti-sera specific to said antigens, 125 I-labeled forms of said antigens and methods of detecting said antigens in serum or plasma. The invention also relates to a diagnostic kit containing standardised antigens or antisera or marked forms thereof for the detection of said antigens in human blood, serum or plasma. (author)

Cytotoxic T cells in chronic idiopathic neutropenia express restricted antigen receptors.

Science.gov (United States)

Mastrodemou, Semeli; Stalika, Evangelia; Vardi, Anna; Gemenetzi, Katerina; Spanoudakis, Michalis; Karypidou, Maria; Mavroudi, Irene; Hadzidimitriou, Anastasia; Stavropoulos-Giokas, Catherine; Papadaki, Helen A; Stamatopoulos, Kostas

2017-12-01

Chronic idiopathic neutropenia (CIN) is an acquired disorder of granulopoiesis characterized by female predominance and mostly uncomplicated course. Crucial to CIN pathophysiology is the presence of activated T lymphocytes with myelosuppressive properties in both peripheral blood (PB) and bone marrow (BM). We systematically profiled the T cell receptor beta chain (TRB) gene repertoire in CD8 + cells of 34 CIN patients through subcloning/Sanger sequencing analysis of TRBV-TRBD-TRBJ gene rearrangements. Remarkable repertoire skewing and oligoclonality were observed, along with shared clonotypes between different patients, alluding to antigen selection. Cross-comparison of our sequence dataset with public TRB sequence databases revealed that CIN may rarely share common immunogenetic features with other entities, however, the CIN TRB repertoire is largely disease-biased. Overall, these findings suggest that CIN may be driven by long-term exposure to a restricted set of specific CIN-associated antigens.

Identification of a peptide binding protein that plays a role in antigen presentation

International Nuclear Information System (INIS)

Lakey, E.K.; Margoliash, E.; Pierce, S.K.

1987-01-01

The helper T-cell response to globular proteins appears, in general, to require intracellular processing of the antigen, such that a peptide fragment containing the T-cell antigenic determinant is released and transported to and held on the surface of an Ia-expressing, antigen-presenting cell. However, the molecular details underlying these phenomena are largely unknown. The means by which antigenic peptides are anchored on the antigen-presenting cell surface was investigated. A cell surface protein is identified that was isolated by it ability to bind to a 24-amino acid peptide fragment of pigeon cytochrome c, residues 81-104, containing the major antigenic determinant for B10.A mouse T cells. This peptide binding protein, purified from [ 35 S]methionine-labeled cells, appears as two discrete bands of ≅72 and 74 kDa after NaDodSO 4 /PAGE. The protein can be eluted from the peptide affinity column with equivalent concentrations of either the antigenic pigeon cytochrome c peptide or the corresponding nonantigenic peptide of mouse cytochrome c. However, it does not bind to the native cytochromes c, either of pigeon or mouse, and thus the protein appears to recognize some structure available only in the free peptides. This protein plays a role in antigen presentation. Its expression is not major histocompatibility complex-restricted in that the blocking activity of the antisera can be absorbed on spleen cells from mice of different haplotypes. This peptide binding protein can be isolated from a variety of cell types, including B cells, T cells, and fibroblasts. The anchoring of processed peptides on the cell surface by such a protein may play a role in antigen presentation

Quantitative detection of the tumor-associated antigen large external antigen in colorectal cancer tissues and cells using quantum dot probe

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Wang S

2016-01-01

Full Text Available Shuo Wang, Wanming Li, Dezheng Yuan, Jindan Song, Jin Fang Department of Cell Biology, Key Laboratory of Cell Biology, Ministry of Public Health, and Key Laboratory of Medical Cell Biology, Ministry of Education, China Medical University, Shenyang, People’s Republic of China Abstract: The large external antigen (LEA is a cell surface glycoprotein that has been proven to be highly expressed in colorectal cancer (CRC as a tumor-associated antigen. To evaluate and validate the relationship between LEA expression and clinical characteristics of CRC with high efficiency, LEA expression levels were detected in 85 tissue blocks from CRC patients by quantum dot-based immunohistochemistry (QD-IHC combined with imaging quantitative analysis using quantum dots with a 605 nm emission wavelength (QD605 conjugated to an ND-1 monoclonal antibody against LEA as a probe. Conventional IHC was performed in parallel for comparison. Both QD-IHC and conventional IHC showed that LEA was specifically expressed in CRC, but not in non-CRC tissues, and high LEA expression was significantly associated with a more advanced T-stage (P<0.05, indicating that LEA is likely to serve as a CRC prognostic marker. Compared with conventional IHC, receiver operating characteristic analysis revealed that QD-IHC possessed higher sensitivity, resulting in an increased positive detection rate of CRC, from 70.1% to 89.6%. In addition, a simpler operation, objective analysis of results, and excellent repeatability make QD-IHC an attractive alternative to conventional IHC in clinical practice. Furthermore, to explore whether the QD probes can be utilized to quantitatively detect living cells or single cells, quantum dot-based immunocytochemistry (QD-ICC combined with imaging quantitative analysis was developed to evaluate LEA expression in several CRC cell lines. It was demonstrated that QD-ICC could also predict the correlation between LEA expression and the T-stage characteristics of

Mosaic VSGs and the scale of Trypanosoma brucei antigenic variation.

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James P J Hall

Full Text Available A main determinant of prolonged Trypanosoma brucei infection and transmission and success of the parasite is the interplay between host acquired immunity and antigenic variation of the parasite variant surface glycoprotein (VSG coat. About 0.1% of trypanosome divisions produce a switch to a different VSG through differential expression of an archive of hundreds of silent VSG genes and pseudogenes, but the patterns and extent of the trypanosome diversity phenotype, particularly in chronic infection, are unclear. We applied longitudinal VSG cDNA sequencing to estimate variant richness and test whether pseudogenes contribute to antigenic variation. We show that individual growth peaks can contain at least 15 distinct variants, are estimated computationally to comprise many more, and that antigenically distinct 'mosaic' VSGs arise from segmental gene conversion between donor VSG genes or pseudogenes. The potential for trypanosome antigenic variation is probably much greater than VSG archive size; mosaic VSGs are core to antigenic variation and chronic infection.

Chloroplast-derived vaccine antigens and biopharmaceuticals: protocols for expression, purification, or oral delivery and functional evaluation.

Science.gov (United States)

Singh, N Dolendro; Ding, Yi; Daniell, Henry

2009-01-01

Many vaccine antigens and biopharmaceutical proteins have been expressed at high levels via the chloroplast genome and their functionality has been evaluated using in vitro assays in cell cultures (i.e., macrophage lysis assay, inhibition of vesicular stomatitis virus-induced cytopathicity in baby hamster kidney cells, or inhibition of human HIV infection in TZM-BL cells) as well as protection after challenge with bacterial or viral pathogens or antitumor assays or delay the onset of insulitis in suitable animal models. Production of therapeutic proteins in chloroplasts eliminates the expensive fermentation technology. Moreover, oral delivery of chloroplast-derived therapeutic proteins eliminates expensive purification steps, cold storage, cold transportation, and delivery via sterile needles, thereby further decreasing their cost. In this chapter, we describe detailed protocols for chloroplast transformation including the construction of chloroplast transformation vectors, delivery of DNA into plant cells using particle bombardment, selection and regeneration of transformants by tissue culture, confirmation of transgene integration into the chloroplast genome and homoplasmy, evaluation of foreign gene expression, purification of foreign protein, or oral delivery via bioencapsulation, functional evaluation using in vitro and in vivo assays, and evaluation of immunity after challenge with pathogens in suitable animal models.

Genetic diversity and antigenicity variation of Babesia bovis merozoite surface antigen-1 (MSA-1) in Thailand.

Science.gov (United States)

Tattiyapong, Muncharee; Sivakumar, Thillaiampalam; Takemae, Hitoshi; Simking, Pacharathon; Jittapalapong, Sathaporn; Igarashi, Ikuo; Yokoyama, Naoaki

2016-07-01

Babesia bovis, an intraerythrocytic protozoan parasite, causes severe clinical disease in cattle worldwide. The genetic diversity of parasite antigens often results in different immune profiles in infected animals, hindering efforts to develop immune control methodologies against the B. bovis infection. In this study, we analyzed the genetic diversity of the merozoite surface antigen-1 (msa-1) gene using 162 B. bovis-positive blood DNA samples sourced from cattle populations reared in different geographical regions of Thailand. The identity scores shared among 93 msa-1 gene sequences isolated by PCR amplification were 43.5-100%, and the similarity values among the translated amino acid sequences were 42.8-100%. Of 23 total clades detected in our phylogenetic analysis, Thai msa-1 gene sequences occurred in 18 clades; seven among them were composed of sequences exclusively from Thailand. To investigate differential antigenicity of isolated MSA-1 proteins, we expressed and purified eight recombinant MSA-1 (rMSA-1) proteins, including an rMSA-1 from B. bovis Texas (T2Bo) strain and seven rMSA-1 proteins based on the Thai msa-1 sequences. When these antigens were analyzed in a western blot assay, anti-T2Bo cattle serum strongly reacted with the rMSA-1 from T2Bo, as well as with three other rMSA-1 proteins that shared 54.9-68.4% sequence similarity with T2Bo MSA-1. In contrast, no or weak reactivity was observed for the remaining rMSA-1 proteins, which shared low sequence similarity (35.0-39.7%) with T2Bo MSA-1. While demonstrating the high genetic diversity of the B. bovis msa-1 gene in Thailand, the present findings suggest that the genetic diversity results in antigenicity variations among the MSA-1 antigens of B. bovis in Thailand. Copyright © 2016 Elsevier B.V. All rights reserved.

Immunity to tumour antigens.

Science.gov (United States)

Li, Geng; Ali, Selman A; McArdle, Stephanie E B; Mian, Shahid; Ahmad, Murrium; Miles, Amanda; Rees, Robert C

2005-01-01

During the last decade, a large number of human tumour antigens have been identified. These antigens are classified as tumour-specific shared antigens, tissue-specific differentiation antigens, overexpressed antigens, tumour antigens resulting from mutations, viral antigens and fusion proteins. Antigens recognised by effectors of immune system are potential targets for antigen-specific cancer immunotherapy. However, most tumour antigens are self-proteins and are generally of low immunogenicity and the immune response elicited towards these tumour antigens is not always effective. Strategies to induce and enhance the tumour antigen-specific response are needed. This review will summarise the approaches to discovery of tumour antigens, the current status of tumour antigens, and their potential application to cancer treatment.

Transformation and radiosensitivity of human diploid skin fibroblasts transfected with activated ras oncogene and SV40 T-antigen.

Science.gov (United States)

Su, L N; Little, J B

1992-08-01

Three normal human diploid cell strains were transfected with an activated Ha-ras oncogene (EJ ras) or SV40 T-antigen. Multiple clones were examined for morphological alterations, growth requirements, ability to grow under anchorage independent conditions, immortality and tumorigenicity in nude mice. Clones expressing SV40 T-antigen alone or in combination with ras protein p21 were significantly radioresistant as compared with their parent cells or clones transfected with the neo gene only. This radioresistant phenotype persisted in post-crisis, immortalized cell lines. Cells transfected with EJ ras alone showed no morphological alterations nor significant changes in radiosensitivity. Cell clones expressing ras and/or SV40 T-antigen showed a reduced requirement for serum supplements, an increase in aneuploidy and chromosomal aberrations, and enhanced growth in soft agar as an early cellular response to SV40 T-antigen expression. The sequential order of transfection with SV40 T-antigen and ras influenced radio-sensitivity but not the induction of morphological changes. These data suggest that expression of the SV40 T-antigen but not activated Ha-ras plays an important role in the radiosensitivity of human diploid cells. The radioresistant phenotype in SV40 T transfected cells was not related to the enhanced level of genetic instability seen in pre-crisis and newly immortalized cells, nor to the process of immortalization itself.

Blood group antigen studies using CdTe quantum dots and flow cytometry

Directory of Open Access Journals (Sweden)

Cabral Filho PE

2015-07-01

Full Text Available Paulo E Cabral Filho,1 Maria IA Pereira,1 Heloise P Fernandes,2 Andre A de Thomaz,3 Carlos L Cesar,3 Beate S Santos,4 Maria L Barjas-Castro,2 Adriana Fontes1 1Departamento de Biofísica e Radiobiologia, Universidade Federal de Pernambuco, Recife, Pernambuco, 2Centro de Hematologia e Hemoterapia, Universidade Estadual de Campinas, Instituto Nacional de Ciência e Tecnologia do Sangue, Campinas, São Paulo, 3Departamento de Eletrônica Quântica, Instituto de Física Gleb Wataghin, Universidade Estadual de Campinas, Campinas, São Paulo, 4Departamento de Ciências Farmacêuticas, Universidade Federal de Pernambuco, Recife, PE, Brazil Abstract: New methods of analysis involving semiconductor nanocrystals (quantum dots [QDs] as fluorescent probes have been highlighted in life science. QDs present some advantages when compared to organic dyes, such as size-tunable emission spectra, broad absorption bands, and principally exceptional resistance to photobleaching. Methods applying QDs can be simple, not laborious, and can present high sensibility, allowing biomolecule identification and quantification with high specificity. In this context, the aim of this work was to apply dual-color CdTe QDs to quantify red blood cell (RBC antigen expression on cell surface by flow cytometric analysis. QDs were conjugated to anti-A or anti-B monoclonal antibodies, as well as to the anti-H (Ulex europaeus I lectin, to investigate RBCs of A1, B, A1B, O, A2, and Aweak donors. Bioconjugates were capable of distinguishing the different expressions of RBC antigens, both by labeling efficiency and by flow cytometry histogram profile. Furthermore, results showed that RBCs from Aweak donors present fewer amounts of A antigens and higher amounts of H, when compared to A1 RBCs. In the A group, the amount of A antigens decreased as A1 > A3 > AX = Ael, while H antigens were AX = Ael > A1. Bioconjugates presented stability and remained active for at least 6 months. In conclusion

Production of prostate-specific antigen by a breast cancer cell line, Sk-Br-3

International Nuclear Information System (INIS)

Kamali Sarvestani, E.; Ghaderi, A.

2002-01-01

Prostate-specific antigen is a 33-KDa serine protease that is produced predominantly by prostate epithelium. However, it has been shown that about 30-40% of female breast tumors produce prostate-specific antigen and its production is associated with the presence of estrogen and progesterone receptors. We have now developed a new tissue culture system to study prostate-specific antigen production in breast cancer and its association with prognostic factors such as progesterone receptor and c-erbB-2. For this purpose we investigated the ability of prostate-specific antigen production in five different cell lines, including two breast cancer cell lines, Sk-Br-3 and MDA-MB-453. The prostate-specific antigen in tissue culture supernatant and cytoplasm of the Sk-Br-3 cell line was detected by western blotting and immunoperoxidase, respectively. Furthermore, we found lower expression of c-erbB-2 in Sk-Br-3 than non-prostate-specific antigen producer breast cancer cell line, MDA-MB-453. Progesterone receptor was expressed by both prostate-specific antigen-positive and -negative cell lines and only the intensity of staining and the number of positive cells in Sk-Br-3 population was higher than MDA-MB-453. According to our findings prostate-specific antigen can be considered as a good prognostic factor in breast cancer and we suggest that these two cell lines are a good in vitro model to study the relationship of different breast cancer prognostic factors and their regulations

Cell surface antigens of radiation leukemia virus-induced BALB/c leukemias defined by syngeneic cytotoxic T lymphocytes

International Nuclear Information System (INIS)

Kaneko, Yukio; Oettgen, H.F.; Obata, Yuichi; Nakayama, Eiichi.

1989-01-01

Two cell surface antigens of mouse leukemias were defined by BALB/c cytotoxic T lymphocytes (CTL) generated against syngeneic radiation leukemia virus (RadLV)-induced leukemia, BALBRV1 or BALBRVD. Hyperimmunization of BALB/c mice with irradiated leukemias followed by in vitro sensitization of primed spleen cells resulted in the generation of CTL with high killing activity. The specificity of CTL was examined by direct cytotoxicity assays and competitive inhibition assays. A shared cell surface antigen, designated as BALBRV1 antigen, was detected by BALB/c anti-BALBRV1 CTL. BALBRV1 antigen was expressed not only on RadLV-induced BALB/c leukemias except for BALBRVD, but also on spontaneous or X-ray-induced BALB/c leukemias, chemically-induced leukemias with the H-2 d haplotype and some chemically-induced BALB/c sarcomas. In contrast, a unique cell surface antigen, designated as BALBRVD antigen, was detected by BALB/c anti-BALBRVD CTL. BALBRVD antigen was expressed only on BALBRVD, but not on thirty-nine normal lymphoid or tumor cells. These two antigens could be distinguished from those previously defined on Friend, Moloney, Rauscher or Gross murine leukemia virus (MuLV) leukemias, or MuLV-related antigens. Both cytotoxic responses were blocked by antisera against H-2K d , but not H-2D d . The relationship of BALBRV1 antigen and BALBRVD antigen to endogenous MuLV is discussed with regard to the antigenic distribution on tumor cell lines. (author)

Chimeric antigen receptor T cells: a novel therapy for solid tumors

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Shengnan Yu

2017-03-01

Full Text Available Abstract The chimeric antigen receptor T (CAR-T cell therapy is a newly developed adoptive antitumor treatment. Theoretically, CAR-T cells can specifically localize and eliminate tumor cells by interacting with the tumor-associated antigens (TAAs expressing on tumor cell surface. Current studies demonstrated that various TAAs could act as target antigens for CAR-T cells, for instance, the type III variant epidermal growth factor receptor (EGFRvIII was considered as an ideal target for its aberrant expression on the cell surface of several tumor types. CAR-T cell therapy has achieved gratifying breakthrough in hematological malignancies and promising outcome in solid tumor as showed in various clinical trials. The third generation of CAR-T demonstrates increased antitumor cytotoxicity and persistence through modification of CAR structure. In this review, we summarized the preclinical and clinical progress of CAR-T cells targeting EGFR, human epidermal growth factor receptor 2 (HER2, and mesothelin (MSLN, as well as the challenges for CAR-T cell therapy.

Correlation of mRNA and protein levels: Cell type-specific gene expression of cluster designation antigens in the prostate

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Deutsch Eric W

2008-05-01

Full Text Available Abstract Background: Expression levels of mRNA and protein by cell types exhibit a range of correlations for different genes. In this study, we compared levels of mRNA abundance for several cluster designation (CD genes determined by gene arrays using magnetic sorted and laser-capture microdissected human prostate cells with levels of expression of the respective CD proteins determined by immunohistochemical staining in the major cell types of the prostate – basal epithelial, luminal epithelial, stromal fibromuscular, and endothelial – and for prostate precursor/stem cells and prostate carcinoma cells. Immunohistochemical stains of prostate tissues from more than 50 patients were scored for informative CD antigen expression and compared with cell-type specific transcriptomes. Results: Concordance between gene and protein expression findings based on 'present' vs. 'absent' calls ranged from 46 to 68%. Correlation of expression levels was poor to moderate (Pearson correlations ranged from 0 to 0.63. Divergence between the two data types was most frequently seen for genes whose array signals exceeded background (> 50 but lacked immunoreactivity by immunostaining. This could be due to multiple factors, e.g. low levels of protein expression, technological sensitivities, sample processing, probe set definition or anatomical origin of tissue and actual biological differences between transcript and protein abundance. Conclusion: Agreement between these two very different methodologies has great implications for their respective use in both molecular studies and clinical trials employing molecular biomarkers.

Simple mucin-type carbohydrate antigens in major salivary glands

DEFF Research Database (Denmark)

Therkildsen, M H; Mandel, U; Thorn, J

1994-01-01

Simple mucin-type carbohydrate antigens Tn, sialosyl-Tn and T are often markers of neoplastic transformation and have very limited expression in normal tissues. We performed an immunohistological study of simple mucin-type carbohydrate antigens, including H and A variants, with well......-defined monoclonal antibodies (MAb) on frozen and paraffin-embedded normal salivary gland tissue from 22 parotid, 14 submandibular, six sublingual, and 13 labial glands to elucidate the simple mucin-type glycosylation pattern in relation to cyto- and histodifferentiation. The investigated carbohydrate structures...

CARbodies: Human Antibodies Against Cell Surface Tumor Antigens Selected From Repertoires Displayed on T Cell Chimeric Antigen Receptors

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Vanesa Alonso-Camino

2013-01-01

Full Text Available A human single-chain variable fragment (scFv antibody library was expressed on the surface of human T cells after transduction with lentiviral vectors (LVs. The repertoire was fused to a first-generation T cell receptor ζ (TCRζ-based chimeric antigen receptor (CAR. We used this library to isolate antibodies termed CARbodies that recognize antigens expressed on the tumor cell surface in a proof-of-principle system. After three rounds of activation-selection there was a clear repertoire restriction, with the emergence dominant clones. The CARbodies were purified from bacterial cultures as soluble and active proteins. Furthermore, to validate its potential application for adoptive cell therapy, human T cells were transduced with a LV encoding a second-generation costimulatory CAR (CARv2 bearing the selected CARbodies. Transduced human primary T cells expressed significant levels of the CARbodies-based CARv2 fusion protein on the cell surface, and importantly could be specifically activated, after stimulation with tumor cells. This approach is a promising tool for the generation of antibodies fully adapted to the display format (CAR and the selection context (cell synapse, which could extend the scope of current adoptive cell therapy strategies with CAR-redirected T cells.

Distribution of bovine viral diarrhoea virus antigen in persistently infected white-tailed deer (Odocoileus virginianus).

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Passler, T; Walz, H L; Ditchkoff, S S; van Santen, E; Brock, K V; Walz, P H

2012-11-01

Infection with bovine viral diarrhoea virus (BVDV), analogous to that occurring in cattle, is reported rarely in white-tailed deer (Odocoileus virginianus). This study evaluated the distribution of BVDV antigen in persistently infected (PI) white-tailed deer and compared the findings with those from PI cattle. Six PI fawns (four live-born and two stillborn) from does exposed experimentally to either BVDV-1 or BVDV-2 were evaluated. Distribution and intensity of antigen expression in tissues was evaluated by immunohistochemistry. Data were analyzed in binary fashion with a proportional odds model. Viral antigen was distributed widely and was present in all 11 organ systems. Hepatobiliary, integumentary and reproductive systems were respectively 11.8, 15.4 and 21.6 times more likely to have higher antigen scores than the musculoskeletal system. Pronounced labelling occurred in epithelial tissues, which were 1.9-3.0 times likelier than other tissues to contain BVDV antigen. Antigen was present in >90% of samples of liver and skin, suggesting that skin biopsy samples are appropriate for BVDV diagnosis. Moderate to severe lymphoid depletion was detected and may hamper reliable detection of BVDV in lymphoid organs. Muscle tissue contained little antigen, except for in the cardiovascular system. Antigen was present infrequently in connective tissues. In nervous tissues, antigen expression frequency was 0.3-0.67. In the central nervous system (CNS), antigen was present in neurons and non-neuronal cells, including microglia, emphasizing that the CNS is a primary target for fetal BVDV infection. BVDV antigen distribution in PI white-tailed deer is similar to that in PI cattle. Copyright © 2012 Elsevier Ltd. All rights reserved.

Autologous monoclonal antibodies recognize tumour-associated antigens in X-irradiated C57BL/6 mice

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Artus, A; Guillemain, B; Legrand, E; Astier-Gin, T; Mamoun, R; Duplan, J -F

1986-09-01

X-irradiation of C57BL/6 mice induces thymic lymphosarcomas which sometimes contain retroviruses which upon injection into normal mice mimic the effect of the irradiation. We examined whether specific antigenicities, viral or cellular, were expressed by tumour cells that could be recognized by antibodies from the irradiated animals. We developed monoclonal antibodies (MAbs) using splenocytes of the diseased animal. The reactivity of such MAbs towards thymoma cell lines established in vitro was investigated by means of an ELISA. At least 10 antibody specificities were detected on the 13 tumours investigated, allowing separation of the MAbs into three classes: (i) those recognizing the autologous tumour, heterologous tumours as well as normal thymic tissue, (ii) those specific for the autologous tumour, and (iii) those specific for one tumour, but not ones of autologous origin. The last two classes corresponded to specific tumour-associated antigens. Our panel of MAbs defined each tumour by the particular pattern of antigens harboured. It is striking that most of the antigens were present in the normal thymus and that only two tumours had additional antigenicities. Additionally, quantitative variations were observed in the levels of expression of these antigens.

Antigen delivery by α2-macroglobulin enhances the cytotoxic T lymphocyte response

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Bowers, Edith V.; Horvath, Jeffrey J.; Bond, Jennifer E.; Cianciolo, George J.; Pizzo, Salvatore V.

2009-01-01

α2M* targets antigens to APCs for rapid internalization, processing, and presentation. When used as an antigen-delivery vehicle, α2M* amplifies MHC class II presentation, as demonstrated by increased antibody titers. Recent evidence, however, suggests that α2M* encapsulation may also enhance antigen-specific CTL immunity. In this study, we demonstrate that α2M*-delivered antigen (OVA) enhances the production of specific in vitro and in vivo CTL responses. Murine splenocytes expressing a transgenic TCR specific for CTL peptide OVA257–264 (SIINFEKL) demonstrated up to 25-fold greater IFN-γ and IL-2 secretion when treated in vitro with α2M*-OVA compared with soluble OVA. The frequency of IFN-γ-producing cells was increased ∼15-fold, as measured by ELISPOT. Expansion of the OVA-specific CD8+ T cell population, as assayed by tetramer binding and [3H]thymidine incorporation, and OVA-specific cell-mediated cytotoxicity, as determined by a flow cytometric assay, were also enhanced significantly by α2M*-OVA. Furthermore, significant CTL responses were observed at antigen doses tenfold lower than those required with OVA alone. Finally, we also observed enhanced humoral and CTL responses by naïve mice following intradermal immunization with α2M*-OVA. These α2M*-OVA-immunized mice demonstrated increased protection against a s.c.-implanted, OVA-expressing tumor, as demonstrated by delayed tumor growth and prolonged animal survival. The observation that α2M*-mediated antigen delivery elicits specific CTL responses suggests the cross-presentation of antigen onto MHC class I. These results support α2M* as an effective antigen-delivery system that may be particularly useful for vaccines based on weakly immunogenic subunits or requiring dose sparing. PMID:19652028

A booster vaccine expressing a latency-associated antigen augments BCG induced immunity and confers enhanced protection against tuberculosis.

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Bappaditya Dey

Full Text Available BACKGROUND: In spite of a consistent protection against tuberculosis (TB in children, Mycobacterium bovis Bacille Calmette-Guerin (BCG fails to provide adequate protection against the disease in adults as well as against reactivation of latent infections or exogenous reinfections. It has been speculated that failure to generate adequate memory T cell response, elicitation of inadequate immune response against latency-associated antigens and inability to impart long-term immunity against M. tuberculosis infections are some of the key factors responsible for the limited efficiency of BCG in controlling TB. METHODS/PRINCIPAL FINDINGS: In this study, we evaluated the ability of a DNA vaccine expressing α-crystallin--a key latency antigen of M. tuberculosis to boost the BCG induced immunity. 'BCG prime-DNA boost' regimen (B/D confers robust protection in guinea pigs along with a reduced pathology in comparison to BCG vaccination (1.37 log(10 and 1.96 log(10 fewer bacilli in lungs and spleen, respectively; p<0.01. In addition, B/D regimen also confers enhanced protection in mice. Further, we show that B/D immunization in mice results in a heightened frequency of PPD and antigen specific multi-functional CD4 T cells (3(+ simultaneously producing interferon (IFNγ, tumor necrosis factor (TNFα and interleukin (IL2. CONCLUSIONS/SIGNIFICANCE: These results clearly indicate the superiority of α-crystallin based B/D regimen over BCG. Our study, also demonstrates that protection against TB is predictable by an increased frequency of 3(+ Th1 cells with superior effector functions. We anticipate that this study would significantly contribute towards the development of superior booster vaccines for BCG vaccinated individuals. In addition, this regimen can also be expected to reduce the risk of developing active TB due to reactivation of latent infection.

Co-stimulatory signaling determines tumor antigen sensitivity and persistence of CAR T cells targeting PSCA+ metastatic prostate cancer.

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Priceman, Saul J; Gerdts, Ethan A; Tilakawardane, Dileshni; Kennewick, Kelly T; Murad, John P; Park, Anthony K; Jeang, Brook; Yamaguchi, Yukiko; Yang, Xin; Urak, Ryan; Weng, Lihong; Chang, Wen-Chung; Wright, Sarah; Pal, Sumanta; Reiter, Robert E; Wu, Anna M; Brown, Christine E; Forman, Stephen J

2018-01-01

Advancing chimeric antigen receptor (CAR)-engineered adoptive T cells for the treatment of solid cancers is a major focus in the field of immunotherapy, given impressive recent clinical responses in hematological malignancies. Prostate cancer may be amenable to T cell-based immunotherapy since several tumor antigens, including prostate stem-cell antigen (PSCA), are widely over-expressed in metastatic disease. While antigen selectivity of CARs for solid cancers is crucial, it is problematic due to the absence of truly restricted tumor antigen expression and potential safety concerns with "on-target off-tumor" activity. Here, we show that the intracellular co-stimulatory signaling domain can determine a CAR's sensitivity for tumor antigen expression. A 4-1BB intracellular co-stimulatory signaling domain in PSCA-CARs confers improved selectivity for higher tumor antigen density, reduced T cell exhaustion phenotype, and equivalent tumor killing ability compared to PSCA-CARs containing the CD28 co-stimulatory signaling domain. PSCA-CARs exhibit robust in vivo anti-tumor activity in patient-derived bone-metastatic prostate cancer xenograft models, and 4-1BB-containing CARs show superior T cell persistence and control of disease compared with CD28-containing CARs. Our study demonstrates the importance of co-stimulation in defining an optimal CAR T cell, and also highlights the significance of clinically relevant models in developing solid cancer CAR T cell therapies.

Co-stimulatory signaling determines tumor antigen sensitivity and persistence of CAR T cells targeting PSCA+ metastatic prostate cancer

Science.gov (United States)

Priceman, Saul J.; Gerdts, Ethan A.; Tilakawardane, Dileshni; Kennewick, Kelly T.; Murad, John P.; Park, Anthony K.; Jeang, Brook; Yamaguchi, Yukiko; Urak, Ryan; Weng, Lihong; Chang, Wen-Chung; Wright, Sarah; Pal, Sumanta; Reiter, Robert E.; Brown, Christine E.; Forman, Stephen J.

2018-01-01

ABSTRACT Advancing chimeric antigen receptor (CAR)-engineered adoptive T cells for the treatment of solid cancers is a major focus in the field of immunotherapy, given impressive recent clinical responses in hematological malignancies. Prostate cancer may be amenable to T cell-based immunotherapy since several tumor antigens, including prostate stem-cell antigen (PSCA), are widely over-expressed in metastatic disease. While antigen selectivity of CARs for solid cancers is crucial, it is problematic due to the absence of truly restricted tumor antigen expression and potential safety concerns with “on-target off-tumor” activity. Here, we show that the intracellular co-stimulatory signaling domain can determine a CAR's sensitivity for tumor antigen expression. A 4-1BB intracellular co-stimulatory signaling domain in PSCA-CARs confers improved selectivity for higher tumor antigen density, reduced T cell exhaustion phenotype, and equivalent tumor killing ability compared to PSCA-CARs containing the CD28 co-stimulatory signaling domain. PSCA-CARs exhibit robust in vivo anti-tumor activity in patient-derived bone-metastatic prostate cancer xenograft models, and 4-1BB-containing CARs show superior T cell persistence and control of disease compared with CD28-containing CARs. Our study demonstrates the importance of co-stimulation in defining an optimal CAR T cell, and also highlights the significance of clinically relevant models in developing solid cancer CAR T cell therapies. PMID:29308300

Chimeric antigen receptor-modified T cells for acute lymphoid leukemia.

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Grupp, Stephan A; Kalos, Michael; Barrett, David; Aplenc, Richard; Porter, David L; Rheingold, Susan R; Teachey, David T; Chew, Anne; Hauck, Bernd; Wright, J Fraser; Milone, Michael C; Levine, Bruce L; June, Carl H

2013-04-18

Chimeric antigen receptor-modified T cells with specificity for CD19 have shown promise in the treatment of chronic lymphocytic leukemia (CLL). It remains to be established whether chimeric antigen receptor T cells have clinical activity in acute lymphoblastic leukemia (ALL). Two children with relapsed and refractory pre-B-cell ALL received infusions of T cells transduced with anti-CD19 antibody and a T-cell signaling molecule (CTL019 chimeric antigen receptor T cells), at a dose of 1.4×10(6) to 1.2×10(7) CTL019 cells per kilogram of body weight. In both patients, CTL019 T cells expanded to a level that was more than 1000 times as high as the initial engraftment level, and the cells were identified in bone marrow. In addition, the chimeric antigen receptor T cells were observed in the cerebrospinal fluid (CSF), where they persisted at high levels for at least 6 months. Eight grade 3 or 4 adverse events were noted. The cytokine-release syndrome and B-cell aplasia developed in both patients. In one child, the cytokine-release syndrome was severe; cytokine blockade with etanercept and tocilizumab was effective in reversing the syndrome and did not prevent expansion of chimeric antigen receptor T cells or reduce antileukemic efficacy. Complete remission was observed in both patients and is ongoing in one patient at 11 months after treatment. The other patient had a relapse, with blast cells that no longer expressed CD19, approximately 2 months after treatment. Chimeric antigen receptor-modified T cells are capable of killing even aggressive, treatment-refractory acute leukemia cells in vivo. The emergence of tumor cells that no longer express the target indicates a need to target other molecules in addition to CD19 in some patients with ALL.

Supraphysiologic control over HIV-1 replication mediated by CD8 T cells expressing a re-engineered CD4-based chimeric antigen receptor.

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Rachel S Leibman

2017-10-01

Full Text Available HIV is adept at avoiding naturally generated T cell responses; therefore, there is a need to develop HIV-specific T cells with greater potency for use in HIV cure strategies. Starting with a CD4-based chimeric antigen receptor (CAR that was previously used without toxicity in clinical trials, we optimized the vector backbone, promoter, HIV targeting moiety, and transmembrane and signaling domains to determine which components augmented the ability of T cells to control HIV replication. This re-engineered CAR was at least 50-fold more potent in vitro at controlling HIV replication than the original CD4 CAR, or a TCR-based approach, and substantially better than broadly neutralizing antibody-based CARs. A humanized mouse model of HIV infection demonstrated that T cells expressing optimized CARs were superior at expanding in response to antigen, protecting CD4 T cells from infection, and reducing viral loads compared to T cells expressing the original, clinical trial CAR. Moreover, in a humanized mouse model of HIV treatment, CD4 CAR T cells containing the 4-1BB costimulatory domain controlled HIV spread after ART removal better than analogous CAR T cells containing the CD28 costimulatory domain. Together, these data indicate that potent HIV-specific T cells can be generated using improved CAR design and that CAR T cells could be important components of an HIV cure strategy.

Cancer vaccines: an update with special focus on ganglioside antigens.

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Bitton, Roberto J; Guthmann, Marcel D; Gabri, Mariano R; Carnero, Ariel J L; Alonso, Daniel F; Fainboim, Leonardo; Gomez, Daniel E

2002-01-01

Vaccine development is one of the most promising and exciting fields in cancer research; numerous approaches are being studied to developed effective cancer vaccines. The aim of this form of therapy is to teach the patient's immune system to recognize the antigens expressed in tumor cells, but not in normal tissue, to be able to destroy these abnormal cells leaving the normal cells intact. In other words, is an attempt to teach the immune system to recognize antigens that escaped the immunologic surveillance and are by it, therefore able to survive and, in time, disseminate. However each research group developing a cancer vaccine, uses a different technology, targeting different antigens, combining different carriers and adjuvants, and using different immunization schedules. Most of the vaccines are still experimental and not approved by the US or European Regulatory Agencies. In this work, we will offer an update in the knowledge in cancer immunology and all the anticancer vaccine approaches, with special emphasis in ganglioside based vaccines. It has been demonstrated that quantitative and qualitative changes occur in ganglioside expression during the oncogenic transformation. Malignant transformation appears to activate enzymes associated with ganglioside glycosylation, resulting in altered patterns of ganglioside expression in tumors. Direct evidence of the importance of gangliosides as potential targets for active immunotherapy has been suggested by the observation that human monoclonal antibodies against these glycolipids induce shrinkage of human cutaneous melanoma metastasis. Thus, the cellular over-expression and shedding of gangliosides into the interstitial space may play a central role in cell growth regulation, immune tolerance and tumor-angiogenesis, therefore representing a new target for anticancer therapy. Since 1993 researchers at the University of Buenos Aires and the University of Quilmes (Argentina), have taken part in a project carried out by

Interleukin-6, C-reactive protein, and expression of human leukocyte antigen-DR on peripheral blood mononuclear cells in patients after laparoscopic vs. conventional bowel resection - A randomized study

NARCIS (Netherlands)

Dunker, M. S.; ten Hove, T.; Bemelman, W. A.; Slors, J. F. M.; Gouma, D. J.; van Deventer, S. J. H.

2003-01-01

PURPOSE: The aim of the study was to investigate the effect of surgical trauma in terms of approach (laparoscopic vs. conventional surgery) and extent of bowel resection (ileocolic resection vs. colectomy) on interleukin-6 level, C-reactive protein level, and expression of human leukocyte antigen-DR

MYCN: From Oncoprotein To Tumor-Associated Antigen

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Vito ePistoia

2012-11-01

Full Text Available MYCN is a well known oncogene overexpressed in different human malignancies including neuroblastoma, rhabdomyosarcoma, medulloblastoma, astrocytoma, Wilms’ tumor and small cell lung cancer. In the case of neuroblastoma (NB, MYCN amplification is an established biomarker of poor prognosis. MYCN belongs to a family of transcription factors (the most important of which is CMYC that show a high degree of homology. Downregulation of MYC protein expression leads to tumor regression in animal models, indicating that MYC proteins represent interesting therapeutic targets.Pre-requisites for a candidate tumor-associated antigen (TAA to be targeted by immunotherapeutic approaches are the following, i expression should be tumor-restricted, ii the putative TAA should be up-regulated in cancer cells and iii protein should be processed into immunogenic peptides capable of associating to MHC molecules with high affinity. Indeed, the MYCN protein is not expressed in human adult tissues and upregulated variably in NB cells, and MYCN peptides capable of associating to HLA-A1 or –A2 molecules with high affinity have been identified. Thus the MYCN protein qualifies as putative TAA in NB.Additional issues that determine the feasibility of targeting a putative TAA with cytotoxic T lymphocytes (CTL and will be here discussed are the following, i the inadequacy of tumor cells per se to act as antigen-presenting cells witnessed, in the case of NB cells, by the low to absent expression of HLA- class I molecules, the lack of costimulatory molecules and multiple defects in the HLA class I related antigen processing machinery, and ii the immune evasion mechanisms operated by cancer cells to fool the host immune system, such as up-regulation of soluble immunosuppressive molecules (e.g. soluble MICA and HLA-G in the case of NB or generation of immunosuppressive cells in the tumor microenvironment. A final issue that deserves consideration is the strategy used to generate

Monoclonal antibodies reactive with common tumor antigens on UV-induced tumors also react with hyperplastic UV-irradiated skin

International Nuclear Information System (INIS)

Spellman, C.W.; Beauchamp, D.A.

1986-01-01

Most murine skin tumors induced by ultraviolet light (UVB, 280-340 nm) can be successfully transplanted only into syngeneic hosts that have received subcarcinogenic doses of UVB. The tumor susceptible state is long-lived and mediated by T suppressor cells that control effector responses against common antigens on UV-induced tumors. Because antigen specific suppression arises prior to the appearance of a tumor, questions arise about the source of the original antigen. They have previously reported transplantation studies indicating that UV-irradiated skin is antigenically cross-reactive with UV-induced tumors. They now report on flow cytometry analyses showing that a series of MoAb reactive with common antigens expressed by UV-induced tumors are also reactive on cells from UV-irradiated skin. Various antigens appear at different times in the UV irradiation scheme, and some persist while others are transient. They speculate that the common antigens detected may be the ones to which functional suppression is directed. If true, these results suggest that successful tumors need not escape host defenses to emerge. Rather, tumors may arise and grow progressively if they express antigens that cross-react with specificities to which the host has previously mounted a suppressive response

Antigen-specific T cell activation independently of the MHC: chimeric antigen receptor (CAR-redirected T cells.

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Hinrich eAbken

2013-11-01

Full Text Available Adoptive T cell therapy has recently shown powerful in initiating a lasting anti-tumor response with spectacular therapeutic success in some cases. Specific T cell therapy, however, is limited since a number of cancer cells are not recognized by T cells due to various mechanisms including the limited availability of tumor-specific T cells and deficiencies in antigen processing or major histocompatibility complex (MHC expression of cancer cells. To make adoptive cell therapy applicable for the broad variety of cancer entities, patient's T cells are engineered ex vivo with pre-defined specificity by a recombinant chimeric antigen receptor (CAR which consists in the extracellular part of an antibody-derived domain for binding with a tumor-associated antigen and in the intracellular part of a TCR-derived signaling moiety for T cell activation. The specificity of CAR mediated T cell recognition is defined by the antibody domain, is independent of MHC presentation and can be extended to any target for which an antibody is available. We discuss the advantages and limitations of MHC-independent T cell targeting by an engineered CAR and review most significant progress recently made in early stage clinical trials to treat cancer.

Augmentation of antigen-specific immune responses using DNA-fusogenic liposome vaccine

International Nuclear Information System (INIS)

Yoshikawa, Tomoaki; Imazu, Susumu; Gao Jianqing; Hayashi, Kazuyuki; Tsuda, Yasuhiro; Shimokawa, Mariko; Sugita, Toshiki; Niwa, Takako; Oda, Atushi; Akashi, Mitsuru; Tsutsumi, Yasuo; Mayumi, Tadanori; Nakagawa, Shinsaku

2004-01-01

In an attempt to enhance the immunological efficacy of genetic immunization, we investigated a new biological means for delivering antigen gene directly to the cytoplasm via membrane fusion. In this context, we investigated fusogenic liposome (FL) encapsulating DNA as a possible genetic immunization vehicle. RT-PCR analysis indicated that a FL could introduce and express encapsulating OVA gene efficiently and rapidly in vitro. Consistent with this observation, an in vitro assay showed that FL-mediated antigen-gene delivery can induce potent presentation of antigen via the MHC class I-dependent pathway. Accordingly, immunization with FL containing the OVA-gene induced potent OVA-specific Th1 and Th2 cytokine production. Additionally, OVA-specific CTL responses and antibody production were also observed in systemic compartments including the spleen, upon immunization with the OVA-gene encapsulating FL. These findings suggest that FL is an effective genetic immunization carrier system for the stimulation of antigen-specific immune responses against its encoding antigen

Blockade of CD7 expression in T cells for effective chimeric antigen receptor targeting of T-cell malignancies.

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Png, Yi Tian; Vinanica, Natasha; Kamiya, Takahiro; Shimasaki, Noriko; Coustan-Smith, Elaine; Campana, Dario

2017-11-28

Effective immunotherapies for T-cell malignancies are lacking. We devised a novel approach based on chimeric antigen receptor (CAR)-redirected T lymphocytes. We selected CD7 as a target because of its consistent expression in T-cell acute lymphoblastic leukemia (T-ALL), including the most aggressive subtype, early T-cell precursor (ETP)-ALL. In 49 diagnostic T-ALL samples (including 14 ETP-ALL samples), median CD7 expression was >99%; CD7 expression remained high at relapse (n = 14), and during chemotherapy (n = 54). We targeted CD7 with a second-generation CAR (anti-CD7-41BB-CD3ζ), but CAR expression in T lymphocytes caused fratricide due to the presence of CD7 in the T cells themselves. To downregulate CD7 and control fratricide, we applied a new method (protein expression blocker [PEBL]), based on an anti-CD7 single-chain variable fragment coupled with an intracellular retention domain. Transduction of anti-CD7 PEBL resulted in virtually instantaneous abrogation of surface CD7 expression in all transduced T cells; 2.0% ± 1.7% were CD7 + vs 98.1% ± 1.5% of mock-transduced T cells (n = 5; P < .0001). PEBL expression did not impair T-cell proliferation, interferon-γ and tumor necrosis factor-α secretion, or cytotoxicity, and eliminated CAR-mediated fratricide. PEBL-CAR T cells were highly cytotoxic against CD7 + leukemic cells in vitro and were consistently more potent than CD7 + T cells spared by fratricide. They also showed strong anti-leukemic activity in cell line- and patient-derived T-ALL xenografts. The strategy described in this study fits well with existing clinical-grade cell manufacturing processes and can be rapidly implemented for the treatment of patients with high-risk T-cell malignancies.

Myeloid antigens in childhood lymphoblastic leukemia:clinical data point to regulation of CD66c distinct from other myeloid antigens

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Madzo Jozef

2005-04-01

Full Text Available Abstract Background Aberrant expression of myeloid antigens (MyAgs on acute lymphoblastic leukemia (ALL cells is a well-documented phenomenon, although its regulating mechanisms are unclear. MyAgs in ALL are interpreted e.g. as hallmarks of early differentiation stage and/or lineage indecisiveness. Granulocytic marker CD66c – Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6 is aberrantly expressed on ALL with strong correlation to genotype (negative in TEL/AML1 and MLL/AF4, positive in BCR/ABL and hyperdiploid cases. Methods In a cohort of 365 consecutively diagnosed Czech B-precursor ALL patients, we analyze distribution of MyAg+ cases and mutual relationship among CD13, CD15, CD33, CD65 and CD66c. The most frequent MyAg (CD66c is studied further regarding its stability from diagnosis to relapse, prognostic significance and regulation of surface expression. For the latter, flow cytometry, Western blot and quantitative RT-PCR on sorted cells is used. Results We show CD66c is expressed in 43% patients, which is more frequent than other MyAgs studied. In addition, CD66c expression negatively correlates with CD13 (p Conclusion In contrast to general notion we show that different MyAgs in lymphoblastic leukemia represent different biological circumstances. We chose the most frequent and tightly genotype-associated MyAg CD66c to show its stabile expression in patients from diagnosis to relapse, which differs from what is known on the other MyAgs. Surface expression of CD66c is regulated at the gene transcription level, in contrast to previous reports.

Oral delivery of the Sj23LHD-GST antigen by Salmonella typhimurium type III secretion system protects against Schistosoma japonicum infection in mice.

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Guo Chen

2011-09-01

Full Text Available BACKGROUND: Schistosomiasis japonica is a zoonotic parasitic disease and oral vaccine delivery system would be benefit for prevention of this disease. Although attenuated salmonella has been used as an antigen expression vector for oral vaccine development, the membrane-bound vacuoles in which bacteria reside hinders the presentation of expressed heterologous antigens to the major histocompatibility complex (MHC molecules. The present work used an attenuated Salmonella typhimurium strain VNP20009 to secretory expression of Sj23LHDGST bivalent antigen from Schistosoma japonicum and tested the protective efficacy against S. japonicum infection in orally immunized mice. METHODOLOGY/PRINCIPAL FINDINGS: Promoters (nirB or pagC were used to express the antigen (Sj23LHDGST and the Salmonella type III or α-hemolysin secretion system was employed to secrete it. The immunoblotting analysis and fluorescent microscopy revealed that the antigen was effectively expressed and delivered to the cytosol of macrophages in vitro. Among recombinant vaccine strains, an engineered VNP20009 which expressed the antigen by nirB promoter and secreted it through type III secretion system (nirB-sopE(1-104-Sj23LHD-GST efficiently protected against S. japonicum infection in a mouse model. This strain elicited a predominantly IgG(2a antibody response and a markedly increase in the production of IL-12 and IFN-γ. The flow cytometric analysis demonstrated that this strain caused T cell activation as evidenced by significantly increased expression of CD44 and CD69. CONCLUSION/SIGNIFICANCE: Oral delivery of antigen by nirB-driven Salmonella typhimurium type III secretion system is a novel, safe, inexpensive, efficient and convenient approach for schistosome vaccine development.

Doxorubicin-anti-carcinoembryonic antigen immunoconjugate activity in vitro.

Science.gov (United States)

Richardson, V J; Ford, C H; Tsaltas, G; Gallant, M E

1989-04-01

An in vitro model consisting of a series of 11 human cancer cell lines with varying density of expression of membrane carcinoembryonic antigen (CEA) has been used to evaluate conjugates of doxorubicin (Adriamycin) covalently linked by a carbodiimide method to goat polyclonal antibodies and mouse monoclonal antibodies to CEA. Conjugates were produced which retained both antigen binding and drug cytotoxicity. IC50 values were determined for free drug, free drug mixed with unconjugated antibodies and for the immunoconjugates. Cell lines that were very sensitive to free drug (IC50 less than 100 ng/ml) were also found to be highly sensitive to conjugated drug and similarly cell lines resistant to drug (IC50 greater than 1,000 ng/ml) were also resistant to conjugated drug. Although there was no correlation between CEA expression and conjugates efficacy, competitive inhibition studies using autologous antibody to block conjugate binding to cells indicated immunoconjugates specificity for the CEA target.

Blood feeding by the Rocky Mountain spotted fever vector, Dermacentor andersoni, induces interleukin-4 expression by cognate antigen responding CD4+ T cells

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Wikel Stephen K

2009-10-01

Full Text Available Abstract Background Tick modulation of host defenses facilitates both blood feeding and pathogen transmission. Several tick species deviate host T cell responses toward a Th2 cytokine profile. The majority of studies of modulation of T cell cytokine expression by ticks were performed with lymphocytes from infested mice stimulated in vitro with polyclonal T cell activators. Those reports did not examine tick modulation of antigen specific responses. We report use of a transgenic T cell receptor (TCR adoptive transfer model reactive with influenza hemagglutinin peptide (110-120 to examine CD4+ T cell intracellular cytokine responses during infestation with the metastriate tick, Dermacentor andersoni, or exposure to salivary gland extracts. Results Infestation with pathogen-free D. andersoni nymphs or administration of an intradermal injection of female or male tick salivary gland extract induced significant increases of IL-4 transcripts in skin and draining lymph nodes of BALB/c mice as measured by quantitative real-time RT-PCR. Furthermore, IL-10 transcripts were significantly increased in skin while IL-2 and IFN-γ transcripts were not significantly changed by tick feeding or intradermal injection of salivary gland proteins, suggesting a superimposed Th2 response. Infestation induced TCR transgenic CD4+ T cells to divide more frequently as measured by CFSE dilution, but more notably these CD4+ T cells also gained the capacity to express IL-4. Intracellular levels of IL-4 were significantly increased. A second infestation administered 14 days after a primary exposure to ticks resulted in partially reduced CFSE dilution with no change in IL-4 expression when compared to one exposure to ticks. Intradermal inoculation of salivary gland extracts from both male and female ticks also induced IL-4 expression. Conclusion This is the first report of the influence of a metastriate tick on the cytokine profile of antigen specific CD4+ T cells. Blood feeding

Diagnostic value of soluble receptor-binding cancer antigen expressed on SiSo cells and carcinoembryonic antigen in differentiating malignant from benign pleural effusion.

Science.gov (United States)

Dong, Jiahui; Sun, Gengyun; Zhu, Hongbin

2016-03-01

Diagnosis of malignant pleural effusion (MPE) remains a major clinical challenge. The aim of this study was to evaluate the diagnostic value of combined detection of receptor-binding cancer antigen expressed on SiSo cells (RCAS1) and carcinoembryonic antigen (CEA) in patients with MPE and benign pleural effusion (BPE). The serum and pleural fluid samples were collected from 53 patients diagnosed with MPE and 49 patients with BPE. Enzyme-linked immunosorbent assay was used to detect the concentration of RCAS1 in serum and pleural effusion. The clinical data and laboratory information, including CEA levels, were gathered from these cases. The concentration of RCAS1 in MPE was significantly higher than that of BPE (P < 0.001). There was no significant difference between the two serum groups. The diagnostic sensitivity and specificity of pleural fluid RCAS1 were 67.92 and 81.63 %, respectively, at the optimized cutoff value of 7.326 U/mL; meanwhile, the sensitivity and specificity of pleural fluid CEA were 83.02 and 91.84 % at the cutoff value of 3.93 ng/mL. The specificity could be elevated to 98.50 % in serial detection, while the sensitivity may be improved to 94.55 % in parallel detection. Serum RCAS1 concentration was only detected in 53 serum samples out of the 102 samples, indicating that serum RCAS1 may not be a better option in differential diagnosis of malignancies compared with serum CEA, of which the diagnostic sensitivity and specificity were 64.15 and 83.67 % at the cutoff value of 3.90 ng/mL. No significant differences were found in pleural fluid RCAS1 concentration in MPE patients with different ages, gender, and pathological types of lung cancers. The detection of RCAS1 concentration in pleural fluid is informative for the diagnosis of MPE. Joint detection of RCAS1 and CEA can improve the diagnostic sensitivity and specificity. However, the diagnostic value of RCAS1 is not higher than that of CEA.

Stem Cell Antigen-1 in Skeletal Muscle Function

OpenAIRE

Bernstein, Harold S.; Samad, Tahmina; Cholsiripunlert, Sompob; Khalifian, Saami; Gong, Wenhui; Ritner, Carissa; Aurigui, Julian; Ling, Vivian; Wilschut, Karlijn J.; Bennett, Stephen; Hoffman, Julien; Oishi, Peter

2013-01-01

Stem cell antigen-1 (Sca-1) is a member of the Ly-6 multigene family encoding highly homologous, glycosyl-phosphatidylinositol-anchored membrane proteins. Sca-1 is expressed on muscle-derived stem cells and myogenic precursors recruited to sites of muscle injury. We previously reported that inhibition of Sca-1 expression stimulated myoblast proliferation in vitro and regulated the tempo of muscle repair in vivo. Despite its function in myoblast expansion during muscle repair, a role for Sca-1...

Expression of Immunoglobulin Receptors with Distinctive Features Indicating Antigen Selection by Marginal Zone B Cells from Human Spleen

Science.gov (United States)

Colombo, Monica; Cutrona, Giovanna; Reverberi, Daniele; Bruno, Silvia; Ghiotto, Fabio; Tenca, Claudya; Stamatopoulos, Kostas; Hadzidimitriou, Anastasia; Ceccarelli, Jenny; Salvi, Sandra; Boccardo, Simona; Calevo, Maria Grazia; De Santanna, Amleto; Truini, Mauro; Fais, Franco; Ferrarini, Manlio

2013-01-01

Marginal zone (MZ) B cells, identified as surface (s)IgMhighsIgDlowCD23low/−CD21+CD38− B cells, were purified from human spleens, and the features of their V(D)J gene rearrangements were investigated and compared with those of germinal center (GC), follicular mantle (FM) and switched memory (SM) B cells. Most MZ B cells were CD27+ and exhibited somatic hypermutations (SHM), although to a lower extent than SM B cells. Moreover, among MZ B-cell rearrangements, recurrent sequences were observed, some of which displayed intraclonal diversification. The same diversifying sequences were detected in very low numbers in GC and FM B cells and only when a highly sensitive, gene-specific polymerase chain reaction was used. This result indicates that MZ B cells could expand and diversify in situ and also suggested the presence of a number of activation-induced cytidine deaminase (AID)-expressing B cells in the MZ. The notion of antigen-driven expansion/selection in situ is further supported by the VH CDR3 features of MZ B cells with highly conserved amino acids at specific positions and by the finding of shared (“stereotyped”) sequences in two different spleens. Collectively, the data are consistent with the notion that MZ B cells are a special subset selected by in situ antigenic stimuli. PMID:23877718

Kinetics of T cell-activation molecules in response to Mycobacterium tuberculosis antigens

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Antas Paulo RZ

2002-01-01

Full Text Available The phenotypic features acquired subsequent to antigen-specific stimulation in vitro were evaluated by means of the kinetic expressions of CD69 and CD25 activation molecules on T lymphocytes and assayed by flow cytometry in response to PPD, Ag85B, and ferritin in PPD-positive healthy control individuals. In response to PHA, CD69 staining on both CD4+ and CD8+ T cells became initially marked after 4 h, peaked at 24 h, and quickly decreased after 120 h. For CD25, a latter expression was detected around 8 h, having increased after 96 h. As expected, the response rate to the mycobacterial antigens was much lower than that to the mitogen. Positive staining was high after 96 h for CD25 and after 24 h for CD69. CD69 expression was significantly enhanced (p < 0.05 on CD8+ as compared to CD4+ T cells. High levels were also found between 96-120 h. Regarding Ag85B, CD25+ cells were mostly CD4+ instead of CD8+ T cells. Moreover, in response to ferritin, a lower CD25 expression was noted. The present data will allow further characterization of the immune response to new mycobacterial-specific antigens and their evaluation for possible inclusion in developing new diagnostic techniques for tuberculosis as well in a new vaccine to prevent the disease.

Microarray Gene Expression Analysis to Evaluate Cell Type Specific Expression of Targets Relevant for Immunotherapy of Hematological Malignancies.

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M J Pont

Full Text Available Cellular immunotherapy has proven to be effective in the treatment of hematological cancers by donor lymphocyte infusion after allogeneic hematopoietic stem cell transplantation and more recently by targeted therapy with chimeric antigen or T-cell receptor-engineered T cells. However, dependent on the tissue distribution of the antigens that are targeted, anti-tumor responses can be accompanied by undesired side effects. Therefore, detailed tissue distribution analysis is essential to estimate potential efficacy and toxicity of candidate targets for immunotherapy of hematological malignancies. We performed microarray gene expression analysis of hematological malignancies of different origins, healthy hematopoietic cells and various non-hematopoietic cell types from organs that are often targeted in detrimental immune responses after allogeneic stem cell transplantation leading to graft-versus-host disease. Non-hematopoietic cells were also cultured in the presence of IFN-γ to analyze gene expression under inflammatory circumstances. Gene expression was investigated by Illumina HT12.0 microarrays and quality control analysis was performed to confirm the cell-type origin and exclude contamination of non-hematopoietic cell samples with peripheral blood cells. Microarray data were validated by quantitative RT-PCR showing strong correlations between both platforms. Detailed gene expression profiles were generated for various minor histocompatibility antigens and B-cell surface antigens to illustrate the value of the microarray dataset to estimate efficacy and toxicity of candidate targets for immunotherapy. In conclusion, our microarray database provides a relevant platform to analyze and select candidate antigens with hematopoietic (lineage-restricted expression as potential targets for immunotherapy of hematological cancers.

Invasive breast cancer in Argentine women: association between risk and prognostic factors with antigens of a peptidic and carbohydrate nature

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Croce MV

2011-12-01

Full Text Available Sandra O Demichelis, Marina T Isla-Larrain, Luciano Cermignani, Cecilio G Alberdi, Amada Segal-Eiras, María Virginia CroceCentre of Basic and Applied Immunological Research, Faculty of Medical Sciences, National University of La Plata, La Plata, ArgentinaObjective: In breast cancer, several tumor markers have been identified. The marker most extensively associated with breast cancer is MUC1. The objective of the study was to analyze prognostic and risk factors in relation to tumor markers in order to clarify breast cancer biology. A total of 349 primary tumor samples and lymph nodes from breast cancer patients were studied. Risk and prognostic factors were considered. An immunohistochemical approach was applied and an extensive statistical analysis was performed, including frequency analysis and analysis of variance. Correlation among variables was performed with principal component analysis.Results: All the antigens showed an increased expression according to tumor size increment; moreover, sialyl Lewis x expression showed a significant increase in relation to disease stage, whereas Tn and TF presented a positive tendency. Vascular invasion was related to sialyl Lewis x expression and number of metastatic lymph nodes. Taking into account risk factors, when a patient had at least one child, Lewis antigens diminished their expression. In relation to breastfeeding, sialyl Lewis x expression diminished, although its apical expression increased.Conclusion: Associations between MUC1 and carbohydrate antigens and risk and prognostic factors show the complexity of the cellular biological behavior that these antigens modulate in breast cancer.Keywords: breast cancer, Argentine women, risk factors, prognostic factors, antigenic expression

RM2 antigen (beta1,4-GalNAc-disialyl-Lc4) as a new marker for prostate cancer.

Science.gov (United States)

Saito, Seiichi; Egawa, Shin; Endoh, Mareyuki; Ueno, Seiji; Ito, Akihiro; Numahata, Kenji; Satoh, Makoto; Kuwao, Sadahito; Baba, Shiro; Hakomori, Senitiroh; Arai, Yoichi

2005-05-20

Although prostate-specific antigen (PSA) has been widely used for early detection of prostate cancer, PSA has problems with specificity and prediction of pathological stage. Therefore, a new marker for prostate cancer is urgently required. We examined expression of a novel carbohydrate antigen, beta1,4-GalNAc-disialyl-Lc(4), defined by the monoclonal antibody RM2, in prostate cancer using 75 cases of radical prostatectomy specimens. RM2 immunoreactivity was negative to weak in all benign glands, and weak to moderate in high-grade prostatic intraepithelial neoplasia. In prostatic adenocarcinoma, RM2 immunoreactivity was negative to weak (lower expression) in 20 cases, and moderate to strong (higher expression) in 55 cases. A clear difference of RM2 expression level was observed between Gleason patterns 3 and >/=4. Higher expression of RM2 antigen was significantly associated with primary Gleason pattern >/=4, high Gleason score (>/=8), larger tumor volume and advanced tumor stage. Furthermore, 5-year PSA failure-free survival was significantly lower in the higher expression group. However, no significant relationship was observed between RM2 expression level and preoperative serum PSA. Western blot analysis in prostate cancer cell lines PC3 and LNCap revealed that major 49-kDa and minor 39-kDa glycoproteins were common to both cells, but there was an increase of 59- and 125-kDa glycoproteins unique to LNCap and an increase of 88- and 98-kDa glycoproteins unique to PC3. RM2 antigen is a new histological marker for prostate cancer that may reflect the Gleason grading system. Identification of the glycoproteins carrying the RM2 antigen will provide new insights into the properties of prostate cancer. (c) 2005 Wiley-Liss, Inc.

Cancer/testis antigens: A prospective reagent as diagnostic and immunotherapeutic targets for squamous cell carcinoma of the head and neck

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Shohei Domae

2014-11-01

Full Text Available Numerous tumor antigens have so far been identified from various tumors using the serological identification of antigens by recombinant expression cloning (SEREX method. Among them, cancer/testis (CT antigens are considered promising target molecules for immunotherapy for patients with various cancers. We performed several SEREX analyses of various cancers to identify CT antigens, including gastric adenocarcinoma, lung adenocarcinoma, and colon cancer, and consequently identified additional CT antigens, such as XAGE-1, CCDC62-2, GKAP1, and TEKT5. However, although SEREX analysis of squamous cell carcinoma of the head and neck (HNSCC has been performed several times, only a few CT or HNSCC specific antigens have yet been isolated. Compared with other tumors, a small number of studies have been reported on the antigen proteins specific to HNSCC. We here reported the expression of selected CT antigens and their immunogenicity in patients with HNSCC. The results obtained suggested that CCDC62-2, GKAP1, and TEKT5 are immunogenic in HNSCC and also demonstrated their potencies as diagnostic markers for patients with HNSCC in combination with other CT antigens such as NY-ESO-1, MAGE-A3, and MAGE-A4.

Identification of a novel dendritic cell surface antigen defined by carbohydrate specific CD24 antibody cross-reactivity.

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Williams, L A; McLellan, A D; Summers, K L; Sorg, R V; Fearnley, D B; Hart, D N

1996-01-01

Dendritic cells (DC) are characterized as leucocytes that lack mature lineage specific markers and stimulate naive T-lymphocyte proliferation in vitro and in vivo. The mouse heat stable antigen (HSA) participates in T lymphocyte co-stimulation and is expressed by DC isolated from thymus, skin and spleen. The human HSA homologue, CD24, is predominantly expressed by B lymphocytes and granulocytes, but its expression on DC has not been studied in detail. CD24 clearly participates in B-lymphocyte signalling but co-stimulatory activity for T lymphocytes has not yet been described. We have examined the expression of CD24 on human peripheral blood DC populations isolated directly or following in vitro culture. The CD24 antigen was absent from blood DC however, cross-reactive sialylated carbohydrate epitopes were detected on DC with some CD24 monoclonal antibodies (mAb). These CD24 mAb define a protein surface antigen, which is expressed by an immature or resting subpopulation of peripheral blood DC and is down-regulated following activation differentiation in vitro. PMID:8911149

The systems biology of MHC class II antigen presentation

NARCIS (Netherlands)

Paul, Petra

2012-01-01

Major histocompatibility class II molecules (MHC class II) are one of the key regulators of adaptive immunity because of their specific expression by professional antigen presenting cells (APC). They present peptides derived from endocytosed material to T helper lymphocytes. Consequently, MHC class

Characterization of plant plasma membrane antigens: [Annual] progress report

International Nuclear Information System (INIS)

Galbraith, D.W.; Afonso, C.L.; Meyer, D.; Harkins, K.R.

1987-01-01

Protoplast plasma membranes were used to raise antibodies in mice to cell surface antigens. Monoclonal antibodies were selected from those produced and used for indirect immunofluorescence microscopic analysis of N. tabacum cells. In parallel studies cDNA expression libraries were prepared. (DT)

In Vitro Variant Surface Antigen Expression in Plasmodium falciparum Parasites from a Semi-Immune Individual Is Not Correlated with Var Gene Transcription

Science.gov (United States)

Tschan, Serena; Flötenmeyer, Matthias; Koch, Iris; Berger, Jürgen; Kremsner, Peter; Frank, Matthias

2016-01-01

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is considered to be the main variant surface antigen (VSA) of Plasmodium falciparum and is mainly localized on electron-dense knobs in the membrane of the infected erythrocyte. Switches in PfEMP1 expression provide the basis for antigenic variation and are thought to be critical for parasite persistence during chronic infections. Recently, strain transcending anti-PfEMP1 immunity has been shown to develop early in life, challenging the role of PfEMP1 in antigenic variation during chronic infections. In this work we investigate how P. falciparum achieves persistence during a chronic asymptomatic infection. The infected individual (MOA) was parasitemic for 42 days and multilocus var gene genotyping showed persistence of the same parasite population throughout the infection. Parasites from the beginning of the infection were adapted to tissue culture and cloned by limiting dilution. Flow cytometry using convalescent serum detected a variable surface recognition signal on isogenic clonal parasites. Quantitative real-time PCR with a field isolate specific var gene primer set showed that the surface recognition signal was not correlated with transcription of individual var genes. Strain transcending anti-PfEMP1 immunity of the convalescent serum was demonstrated with CD36 selected and PfEMP1 knock-down NF54 clones. In contrast, knock-down of PfEMP1 did not have an effect on the antibody recognition signal in MOA clones. Trypsinisation of the membrane surface proteins abolished the surface recognition signal and immune electron microscopy revealed that antibodies from the convalescent serum bound to membrane areas without knobs and with knobs. Together the data indicate that PfEMP1 is not the main variable surface antigen during a chronic infection and suggest a role for trypsin sensitive non-PfEMP1 VSAs for parasite persistence in chronic infections. PMID:27907004

Immunogenicity in pig-tailed macaques of poliovirus replicons expressing HIV-1 and SIV antigens and protection against SHIV-89.6P disease

International Nuclear Information System (INIS)

Fultz, Patricia N.; Stallworth, Jackie; Porter, Donna; Novak, Miroslav; Anderson, Marie J.; Morrow, Casey D.

2003-01-01

In the search for an effective vaccine against the human immunodeficiency virus (HIV), novel ways to deliver viral antigens are being evaluated. One such approach is the use of nonreplicating viral vectors encoding HIV and/or SIV genes that are expressed after infection of host cells. Nonreplicating poliovirus vectors, termed replicons, that expressed HIV-1/HXB2 and SIVmac239 gag and various HIV-1 env genes from different clades were tested for immunogenicity and protective efficacy against intravenous challenge of pig-tailed macaques with SHIV-89.6P. To maximize both cellular and humoral immune responses, a prime-boost regimen was used. Initially, macaques were immunized four times over 35 weeks by either the intranasal and intrarectal or the intramuscular (im) route with mixtures of poliovirus replicons expressing HIV-1 gag and multiple env genes. Immunization with replicons alone induced both serum antibodies and lymphocyte proliferative responses. After boosting with purified Env protein, neutralizing antibodies to SHIV-89.6P were induced in four of five immunized animals. In a second experiment, four macaques were immunized im three times over 27 weeks with replicons expressing the SIVmac239 gag and HIV-1/HXB2 env genes. All immunized animals were then boosted twice with purified HIV-1-89.6 rgp140-Env and SIVmac239 p55-Gag proteins. Four control animals received only the two protein inoculations. Immunized and control animals were then challenged intravenously with the pathogenic SHIV-89.6P. After challenge the animals were monitored for virus isolation from peripheral blood mononuclear cells and plasma viremia and for changes in virus-specific antibody titers. Naieve pig-tailed macaques experienced rapid loss of CD4 + T cells and died between 38 and 62 weeks after infection. In contrast, macaques immunized with replicons and proteins rapidly cleared plasma virus and did not experience sustained loss of CD4 + lymphocytes. Furthermore, two of the four macaques

In vitro selection of Plasmodium falciparum 3D7 for expression of variant surface antigens associated with severe malaria in African children

DEFF Research Database (Denmark)

Staalsoe, Trine; Nielsen, Morten A; Vestergaard, Lasse S

2003-01-01

) in older semi-immune children. Establishment of the genetic mechanism underlying changes in VSA expression in response to in vitro selective pressure is now possible because of the availability of the entire genomic sequence of the P. falciparum clone 3D7. As a first step towards direct molecular...... identification of VSASM-encoding genes in 3D7, we report here a method of enforcing expression of VSASM-like antigens in this parasite clone by a novel selection method using plasma from semi-immune children with low VSAUM-specific, but high VSASM-specific, IgG reactivity. In addition to the resulting increase...... and epidemiologically diverse areas of endemic parasite transmission. The described selection method appears a useful tool in the identification of genes encoding VSA involved in severe and life-threatening P. falciparum malaria....

Improved protection conferred by vaccination with a recombinant vaccinia virus that incorporates a foreign antigen into the extracellular enveloped virion

International Nuclear Information System (INIS)

Kwak, Heesun; Mustafa, Waleed; Speirs, Kendra; Abdool, Asha J.; Paterson, Yvonne; Isaacs, Stuart N.

2004-01-01

Recombinant poxviruses have shown promise as vaccine vectors. We hypothesized that improved cellular immune responses could be developed to a foreign antigen by incorporating it as part of the extracellular enveloped virion (EEV). We therefore constructed a recombinant vaccinia virus that replaced the cytoplasmic domain of the B5R protein with a test antigen, HIV-1 Gag. Mice immunized with the virus expressing Gag fused to B5R had significantly better primary CD4 T-cell responses than recombinant virus expressing HIV-Gag from the TK-locus. The CD8 T-cell responses were less different between the two groups. Importantly, although we saw differences in the immune response to the test antigen, the vaccinia virus-specific immune responses were similar with both constructs. When groups of vaccinated mice were challenged 30 days later with a recombinant Listeria monocytogenes that expresses HIV-Gag, mice inoculated with the virus that expresses the B5R-Gag fusion protein had lower colony counts of Listeria in the liver and spleen than mice vaccinated with the standard recombinant. Thus, vaccinia virus expressing foreign antigen incorporated into EEV may be a better vaccine strategy than standard recombinant vaccinia virus

HLA antigens in juvenile onset diabetes.

Science.gov (United States)

Kikuchi, T; Toyota, T; Ouchi, E

1980-11-01

To study association between juvenile onset diabetes (JOD) and major histocompatibility gene complex, 40 patients with childhood onset diabetes and 120 healthy subjects were typed for HLA. Bw54 was present in 33 percent of the patients with JOD, while it appeared in 8 percent of the controls. Expressed as a relative risk, the antigen Bw54 confers a susceptibility to the development of JOD which is 5.3 times that in the controls. JOD shows a little high degree of association with A9 (78%). However, the A9-antigen is common in the Japanese and appears in 58 percent. Though less striking, the decreased frequency of B12 was 3 percent of JOD, less than 15 percent of the controls (p less than 0.05). There was no association between Bw54 and JOD with family history of diabetes.

78 FR 13691 - Prospective Grant of Exclusive License: The Development of m971 and m972 Chimeric Antigen...

Science.gov (United States)

2013-02-28

... Exclusive License: The Development of m971 and m972 Chimeric Antigen Receptors (CARs) for the Treatment of B... ``M971 Chimeric Antigen Receptors'' [HHS Ref. E-291-2012/0-US-01], and (b) U.S. Patent Application 61/042... malignancies that express CD22 on their cell surface using chimeric antigen receptors which contain the m971 or...

Evaluation of Vitronectin Expression in Prostate Cancer and the Clinical Significance of the Association of Vitronectin Expression with Prostate Specific Antigen in Detecting Prostate Cancer.

Science.gov (United States)

Niu, Yue; Zhang, Ling; Bi, Xing; Yuan, Shuai; Chen, Peng

2016-03-05

To detect the expression of vitronectin (VTN) in the tissues and blood serum of prostate cancer (PCa) patients, and evaluate its clinical significance and to evaluate the significance of the combined assay of VTN and prostate specific antigens (PSA) in PCa diagnosis. To detect the expression of VTN as a potential marker for PCa diagnosis and prognosis, immunohistochemistry was performed on the tissues of 32 patients with metastatic PCa (PCaM), 34 patients with PCa without metastasis (PCa), and 41 patients with benign prostatic hyperplasia (BPH). The sera were then subjected to Western blot analysis. All cases were subsequently examined to determine the concentrations of PSA and VTN in the sera. The collected data were collated and analyzed. The positive expression rates of VTN in the tissues of the BPH and PCa groups (including PCa and PCaM groups) were 75.61% and 45.45%, respectively (P = .005). VTN was more highly expressed in the sera of the BPH patients (0.83 ± 0.07) than in the sera of the PCa patients (0.65 ± 0.06) (P < .05). It was also more highly expressed in the sera of the PCa patients than in the sera of the PCaM patients (0.35 ± 0.08) (P < .05). In the diagnosis of BPH and PCa, the Youden indexes of PSA detection, VTN detection, and combined detection were 0.2620, 0.3468, and 0.5635; the kappa values were 0.338, 0.304, and 0.448, respectively, and the areas under the receiver operating characteristic curve were 0.625, 0.673, and 0.703 (P < .05), respectively. VTN levels in sera may be used as a potential marker of PCa for the diagnosis and assessment of disease progression and metastasis. The combined detection of VTN and PSA in sera can be clinically applied in PCa diagnosis. .

Recombinant antigen-based immuno-slot blot method for serodiagnosis of syphilis

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N.S. Sato

2004-07-01

Full Text Available Three recombinant antigens of Treponema pallidum Nichols strain were fused with GST, cloned and expressed in Escherichia coli, resulting in high levels of GST-rTp47 and GST-rTp17 expression, and supplementation with arginine tRNA for the AGR codon was needed to obtain GST-rTp15 overexpression. Purified fusion protein yields were 1.9, 1.7 and 5.3 mg/l of cell culture for GST-rTp47, GST-rTp17 and GST-rTp15, respectively. The identities of the antigens obtained were confirmed by automated DNA sequencing using ABI Prism 310 and peptide mapping by Finningan LC/MS. These recombinant antigens were evaluated by immuno-slot blot techniques applied to 137 serum samples from patients with a clinical and laboratory diagnosis of syphilis (61 samples, from healthy blood donors (50 samples, individuals with sexually transmitted disease other than syphilis (3 samples, and from individuals with other spirochetal diseases such as Lyme disease (20 samples and leptospirosis (3 samples. The assay had sensitivity of 95.1% (95% CI, 86.1 to 98.7% and a specificity of 94.7% (95% CI, 87.0 to 98.7%; a stronger reactivity was observed with fraction rTp17. The immunoreactivity results showed that fusion recombinant antigens based-immuno-slot blot techniques are suitable for use in diagnostic assays for syphilis.

Skin-Resident T Cells Drive Dermal Dendritic Cell Migration in Response to Tissue Self-Antigen.

Science.gov (United States)

Ali, Niwa; Zirak, Bahar; Truong, Hong-An; Maurano, Megan M; Gratz, Iris K; Abbas, Abul K; Rosenblum, Michael D

2018-05-01

Migratory dendritic cell (DC) subsets deliver tissue Ags to draining lymph nodes (DLNs) to either initiate or inhibit T cell-mediated immune responses. The signals mediating DC migration in response to tissue self-antigen are largely unknown. Using a mouse model of inducible skin-specific self-antigen expression, we demonstrate that CD103 + dermal DCs (DDCs) rapidly migrate from skin to skin DLN (SDLNs) within the first 48 h after Ag expression. This window of time was characterized by the preferential activation of tissue-resident Ag-specific effector T cells (Teffs), with no concurrent activation of Ag-specific Teffs in SDLNs. Using genetic deletion and adoptive transfer approaches, we show that activation of skin-resident Teffs is required to drive CD103 + DDC migration in response to tissue self-antigen and this Batf3-dependent DC population is necessary to mount a fulminant autoimmune response in skin. Conversely, activation of Ag-specific Teffs in SDLNs played no role in DDC migration. Our studies reveal a crucial role for skin-resident T cell-derived signals, originating at the site of self-antigen expression, to drive DDC migration during the elicitation phase of an autoimmune response. Copyright © 2018 by The American Association of Immunologists, Inc.

Analysis of antigen-specific B-cell memory directly ex vivo.

Science.gov (United States)

McHeyzer-Williams, Louise J; McHeyzer-Williams, Michael G

2004-01-01

Helper T-cell-regulated B-cell memory develops in response to initial antigen priming as a cellular product of the germinal center (GC) reaction. On antigen recall, memory response precursors expand rapidly with exaggerated differentiation into plasma cells to produce the high-titer, high-affinity antibody(Ab) that typifies the memory B-cell response in vivo. We have devised a high-resolution flow cytometric strategy to quantify the emergence and maintenance of antigen-specific memory B cells directly ex vivo. Extended cell surface phenotype establishes a level of cellular diversity not previously appreciated for the memory B-cell compartment. Using an "exclusion transfer" strategy, we ascertain the capacity of two distinct memory B-cell populations to transfer antigen-specific memory into naive adoptive hosts. Finally, we sequence expressed messenger ribonucleic acid (mRNA) from single cells within the population to estimate the level of somatic hypermutation as the best molecular indicator of B-cell memory. In this chapter, we describe the methods used in each of these four sections that serve to provide high-resolution quantification of antigen-specific B-cell memory responses directly ex vivo.

A compound chimeric antigen receptor strategy for targeting multiple myeloma.

Science.gov (United States)

Chen, K H; Wada, M; Pinz, K G; Liu, H; Shuai, X; Chen, X; Yan, L E; Petrov, J C; Salman, H; Senzel, L; Leung, E L H; Jiang, X; Ma, Y

2018-02-01

Current clinical outcomes using chimeric-antigen receptors (CARs) against multiple myeloma show promise in the eradication of bulk disease. However, these anti-BCMA (CD269) CARs observe relapse as a common phenomenon after treatment due to the reemergence of either antigen-positive or -negative cells. Hence, the development of improvements in CAR design to target antigen loss and increase effector cell persistency represents a critical need. Here, we report on the anti-tumor activity of a CAR T-cell possessing two complete and independent CAR receptors against the multiple myeloma antigens BCMA and CS1. We determined that the resulting compound CAR (cCAR) T-cell possesses consistent, potent and directed cytotoxicity against each target antigen population. Using multiple mouse models of myeloma and mixed cell populations, we are further able to show superior in vivo survival by directed cytotoxicity against multiple populations compared to a single-expressing CAR T-cell. These findings indicate that compound targeting of BCMA and CS1 on myeloma cells can potentially be an effective strategy for augmenting the response against myeloma bulk disease and for initiation of broader coverage CAR therapy.

Survival and antigenic profile of irradiated malarial sporozoites in infected liver cells

International Nuclear Information System (INIS)

Suhrbier, A.; Winger, L.A.; Castellano, E.; Sinden, R.E.

1990-01-01

Exoerythrocytic (EE) stages of Plasmodium berghei derived from irradiated sporozoites were cultured in vitro in HepG2 cells. They synthesized several antigens, predominantly but not exclusively those expressed by normal early erythrocytic schizonts. After invasion, over half the intracellular sporozoites, both normal and irradiated, appeared to die. After 24 h, in marked contrast to the normal parasites, EE parasites derived from irradiated sporozoites continued to break open, shedding their antigens into the cytoplasm of the infected host cells. Increasing radiation dosage, which has previously been shown to reduce the ability of irradiated sporozoites to protect animals, correlated with reduced de novo antigen synthesis by EE parasites derived from irradiated sporozoites

Antigen-specific murine T cell clones produce soluble interleukin 2 receptor on stimulation with specific antigens

International Nuclear Information System (INIS)

Wagner, D.K.; York-Jolley, J.; Malek, T.R.; Berzofsky, J.A.; Nelson, D.L.

1986-01-01

In this study, monoclonal antibodies were used to the murine IL 2 receptor (IL 2R) termed 3C7 and 7D4, which bind to different epitopes on the murine IL 2R, to develop an ELISA to measure soluble murine IL 2R. Surprisingly, stimulated murine spleen cells not only expressed cell-associated IL 2R, but also produced a considerable level of cellfree IL 2R in the culture supernatant fluid. To assess the fine specificity of this response, myoglobin-immune murine T cell clones were stimulated with appropriate or inappropriate antigen and syngeneic or allogeneic presenting cells. Proliferation, measured by [ 3 H] thymidine incorporation, and levels of soluble IL 2R were determined at day 4. The production of soluble IL2R displayed the same epitope fine specificity, genetic restriction, and antigen dose-response as the proliferative response. Indeed, in some cases there was sharper discrimination of epitope specificity and genetic restriction with the soluble IL 2R levels. There was also reproducible clone-to-clone variation in the amount of soluble receptor produced in response to antigen among 12 T cell clones and lines tested. In time course experiments, proliferation was greatest at day 3, whereas soluble IL 2R levels continued to rise in subsequent days. To the authors' knowledge, this is the first demonstration of release of secretion of soluble IL 2R by murine T cells, and the first demonstration of the fine specificity and genetic restriction of the induction of soluble IL 2R by specific antigen

Nestin expression in neuroepithelial tumors.

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Schiffer, Davide; Manazza, Andrea; Tamagno, Ilaria

2006-05-29

Nestin is a marker of early stages of neurocytogenesis. It has been studied in 50 neuroepithelial tumors, mostly gliomas of different malignancy grades, by immunohistochemistry, immunofluorescence, immunoblotting, and confocal microscopy and compared with GFAP and Vimentin. As an early marker of differentiation, Nestin is almost not expressed in diffuse astrocytomas, variably expressed in anaplastic astrocytomas and strongly and irregularly expressed in glioblastomas. Negative in oligodendrogliomas, it stains ependymomas and shows a gradient of expression in pilocytic astrocytomas. In glioblastomas, Nestin distribution does not completely correspond to that of GFAP and Vimentin with which its expression varies in tumor cells in a complementary way, as confirmed by confocal microscopy. Tumor cells can thus either derive from or differentiate toward the neurocytogenetic stages. Hypothetically, they could be put in relation with radial glia where during embriogenesis the three antigens are successively expressed. Completely negative cells of invasive or recurrent glioblastomas may represent malignant selected clones after accumulation of mutations or early stem cells not expressing antigens.

Prevalence of Weak D Antigen In Western Indian Population

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Tanvi Sadaria

2015-12-01

Full Text Available Introduction: Discovery of Rh antigens in 1939 by Landsteiner and Weiner was the revolutionary stage in blood banking. Of these antigens, D, which decides Rh positivity or negativity, is the most antigenic. A problem is encountered when an individual has a weakened expression of D (Du, i.e., fewer numbers of D antigens on red cell membrane. Aims and Objectives: To know the prevalence of weak D in Indian population because incidence varies in different population. To determine the risk of alloimmunization among Rh D negative patients who receives the blood of weak D positive donors. Material and Methods: Rh grouping of 38,962 donors who came to The Department of Immunohematology and Blood Transfusion of Civil Hospital, Ahmedabad from 1st January 2013 to 30th September 2014 was done using the DIAGAST (Automated Grouping. The samples that tested negative for D antigen were further analysed for weak D (Du by indirect antiglobulin test using blend of Ig G and Ig M Anti D. This was done using Column agglutination method in ID card (gel card. Results: The total number of donors studied was 38,962. Out of these 3360(8.6% were tested Rh D negative. All Rh D negative donors were tested for weak D (Du. 22 (0.056% of total donors and 0.65% of Rh negative donors turned out to be weak D (Du positive. Conclusion: The prevalence of weak D (Du in Western Indian population is 0.056 %, So the risk of alloimmunization in our setting due to weak D (Du antigen is marginal. But, testing of weak D antigen is necessary in blood bank because weak D antigen is immunogenic and can produce alloimmunization if transfused to Rh D negative subjects.

Real-time PCR analysis of genes encoding tumor antigens in esophageal tumors and a cancer vaccine

NARCIS (Netherlands)

Weinert, Brian T.; Krishnadath, Kausilia K.; Milano, Francesca; Pedersen, Ayako W.; Claesson, Mogens H.; Zocca, Mai-Britt

2009-01-01

Tumor antigens are the primary target of therapeutic cancer vaccines. We set out to define and compare the expression pattern of tumor antigen genes in esophagus carcinoma biopsies and in an allogeneic tumor lysate-based cancer vaccine, MelCancerVac. Cells used for vaccine production were treated

Prostate Stem Cell Antigen: A Prospective Therapeutic and Diagnostic Target

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Raff, Adam B.; Gray, Andrew; Kast, W. Martin

2009-01-01

The development of novel clinical tools to combat cancer is an intense field of research and recent efforts have been directed at the identification of proteins that may provide diagnostic, prognostic and/or therapeutic applications due to their restricted expression. To date, a number of protein candidates have emerged as potential clinical tools in the treatment of prostate cancer. Discovered over ten year ago, prostate stem cell antigen (PSCA) is a cell surface antigen that belongs to the Ly-6/Thy-1 family of glycosylphosphatidylinositol-anchored proteins. PSCA is highly overexpressed in human prostate cancer, with limited expression in normal tissues, making it an ideal target for both diagnosis and therapy. Several studies have now clearly correlated the expression of PSCA with relevant clinical benchmarks, such as Gleason score and metastasis, while others have demonstrated the efficacy of PSCA targeting in treatment through various modalities. The purpose of this review is to present the current body of knowledge about PSCA and its potential role in the treatment of human prostate cancer. PMID:18838214

Recruitment of PfSET2 by RNA polymerase II to variant antigen encoding loci contributes to antigenic variation in P. falciparum.

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Uchechi E Ukaegbu

2014-01-01

Full Text Available Histone modifications are important regulators of gene expression in all eukaryotes. In Plasmodium falciparum, these epigenetic marks regulate expression of genes involved in several aspects of host-parasite interactions, including antigenic variation. While the identities and genomic positions of many histone modifications have now been cataloged, how they are targeted to defined genomic regions remains poorly understood. For example, how variant antigen encoding loci (var are targeted for deposition of unique histone marks is a mystery that continues to perplex the field. Here we describe the recruitment of an ortholog of the histone modifier SET2 to var genes through direct interactions with the C-terminal domain (CTD of RNA polymerase II. In higher eukaryotes, SET2 is a histone methyltransferase recruited by RNA pol II during mRNA transcription; however, the ortholog in P. falciparum (PfSET2 has an atypical architecture and its role in regulating transcription is unknown. Here we show that PfSET2 binds to the unphosphorylated form of the CTD, a property inconsistent with its recruitment during mRNA synthesis. Further, we show that H3K36me3, the epigenetic mark deposited by PfSET2, is enriched at both active and silent var gene loci, providing additional evidence that its recruitment is not associated with mRNA production. Over-expression of a dominant negative form of PfSET2 designed to disrupt binding to RNA pol II induced rapid var gene expression switching, confirming both the importance of PfSET2 in var gene regulation and a role for RNA pol II in its recruitment. RNA pol II is known to transcribe non-coding RNAs from both active and silent var genes, providing a possible mechanism by which it could recruit PfSET2 to var loci. This work unifies previous reports of histone modifications, the production of ncRNAs, and the promoter activity of var introns into a mechanism that contributes to antigenic variation by malaria parasites.

The relationship between apoptosis and the expression of proliferating cell nuclear antigen and the clinical stages in gastric carcinoma.

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Tao, K; Chen, D; Tian, Y; Lu, X; Yang, X

2000-01-01

The relationship between the apoptosis and the expression of proliferating cell nuclear antigen (PCNA) and the clinical stages in gastric cancers was studied. By using terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) technique and PCNA immunohistochemical staining, the apoptosis and the expression of PCNA in tissue of gastric carcinoma were assayed in situ, the index of apoptosis (AI), index of PCNA (PI) and the rate of AI/PI were calculated. AI and PI in gastric cancer tissues were (6.5 +/- 3.7)% and (49.8 +/- 15.9)% respectively, and the rate of AI/PI was 0.13 +/- 0.05, which were obviously different from those of normal gastric mucosa in paragastric cancer (P stages of gastric carcinoma, the AI was decreased, PI was increased and the rate of AI/PI decreased in gastric carcinoma. There was significant difference in them between the gastric cancer tissues and normal gastric mucosa in pericarcinoma in TNM stage II to IV (P gastric carcinoma. The AI, PI and the rate of AI/PI would become the prognostic factors in advanced gastric carcinoma.

THE THEORETICAL AND PRACTICAL ASPECTS OF THE RECOMBINANT ANTIGENS FOR THE DIAGNOSIS OF BOVINE LEUKEMIA PRODUCTION

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Shapovalova OV

2016-12-01

sequence construction. The efficiency of BLV gp51 and p24 encoding regions fusion-sequence integration was confirmed by the screening with the specially designed oligonucleotides. The recombinant antigen expression was induced by addition of IPTG. To isolate the antigen bacterial mass was destroyed by defrostation and ultrasonic disintegration in the experimentally selected modes. The activity and specificity of the antigen was determined by AGID with the use of the bovine fetal serum, positive and negative reference diagnostic serum by unified method in comparison with the standardized cultural BLV antigen in AGID. The antigen specificity was increased by adsorption with commercial anticolibacillosis serum. The antigen activity was confirmed by AGID. Conclusions. Nowadays the most promising BLV antigens expressing genetic constructions with E. coli and baculovirus. E. coli recombinant strains are the most available and effective using expressing system, which allows to get an active and specific antigenic product if an optimal vector constructions and commercially available systems of metal affinity chromatography purification and control with appropriate Mab are used. As an cultural and recombinant antigens alternative the mimicking critical BLV antigenic epitopes synthetic peptides were tested. In recent times many scientific works formed the basis for the bovine leukemia diagnostic test systems’ creation, which are now widely available on the biotechnological products market. Although the majority of manufacturers prefer the recombinant antigens of the pathogen, pilot studies on more improved and cheaper ways to obtain different diagnostic antigens preparations shall not lose relevance.

Fluorescence-based endoscopic imaging of Thomsen-Friedenreich antigen to improve early detection of colorectal cancer.

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Sakuma, Shinji; Yu, James Y H; Quang, Timothy; Hiwatari, Ken-Ichiro; Kumagai, Hironori; Kao, Stephanie; Holt, Alex; Erskind, Jalysa; McClure, Richard; Siuta, Michael; Kitamura, Tokio; Tobita, Etsuo; Koike, Seiji; Wilson, Kevin; Richards-Kortum, Rebecca; Liu, Eric; Washington, Kay; Omary, Reed; Gore, John C; Pham, Wellington

2015-03-01

Thomsen-Friedenreich (TF) antigen belongs to the mucin-type tumor-associated carbohydrate antigen. Notably, TF antigen is overexpressed in colorectal cancer (CRC) but is rarely expressed in normal colonic tissue. Increased TF antigen expression is associated with tumor invasion and metastasis. In this study, we sought to validate a novel nanobeacon for imaging TF-associated CRC in a preclinical animal model. We developed and characterized the nanobeacon for use with fluorescence colonoscopy. In vivo imaging was performed on an orthotopic rat model of CRC. Both white light and fluorescence colonoscopy methods were utilized to establish the ratio-imaging index for the probe. The nanobeacon exhibited specificity for TF-associated cancer. Fluorescence colonoscopy using the probe can detect lesions at the stage which is not readily confirmed by conventional visualization methods. Further, the probe can report the dynamic change of TF expression as tumor regresses during chemotherapy. Data from this study suggests that fluorescence colonoscopy can improve early CRC detection. Supplemented by the established ratio-imaging index, the probe can be used not only for early detection, but also for reporting tumor response during chemotherapy. Furthermore, since the data obtained through in vivo imaging confirmed that the probe was not absorbed by the colonic mucosa, no registered toxicity is associated with this nanobeacon. Taken together, these data demonstrate the potential of this novel probe for imaging TF antigen as a biomarker for the early detection and prediction of the progression of CRC at the molecular level. © 2014 UICC.

Numerical analysis of the vibroacoustic properties of plates with embedded grids of acoustic black holes.

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