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Sample records for vi antigen expression

  1. Cloning and expression of a Vi mimotope of Salmonella enterica ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-09-15

    Sep 15, 2009 ... A recombinant His-Vi protein of Salmonella enterica serovar Typhi was successfully constructed and cloned into an expression vector ... recombinant protein can be used to detect specific anti-Vi antibody produced by typhoid patients. Overall, the His-Vi ... E-mail: khchua@um.edu.my. Tel.:603-. 79676607.

  2. Expression and distribution of type VI collagen in gynecomastia.

    Science.gov (United States)

    Lanzafame, S; Magro, G; Colombatti, A

    1994-06-01

    We investigated the distribution of type VI collagen in 36 cases of routinely fixed and paraffin-embedded gynecomastia using an immunoperoxidase method for light microscopic visualization. Four samples of normal male mammary gland tissue were also included as controls. A protease predigestion was essential for the visualization of this extracellular matrix (ECM) glycoprotein. In normal male breast, no immunoreaction for type VI collagen was detected in the stroma surrounding the ducts. Gynecomastia was classified into three histological types: florid (type I), fibrous (type II), and intermediate (type III). Type VI collagen was differentially expressed in the periductal stroma of all types. This collagen was markedly expressed at the early disease stage (type I) when the periductal stroma is highly cellular and vascular. Its expression decreased when periductal stroma undergoing fibrotic transformation (type III) and completely disappeared from the dense periductal stroma of fibrous stage (type II). These findings suggest that type VI collagen is involved in the ECM remodelling occurring in gynecomastia.

  3. Cloning, expression, purification and antigenic evaluation of ...

    African Journals Online (AJOL)

    Streptococcus pyogenes produce an extracellular hyaluronidase which is associated with the spread of the organism during infection. Enzyme hyaluronidase is capable of degrading hyaluronic acid. The aim of the present study was to clone and express antigenic regions of the hylA of S.pyogenes in Escherichia coli.

  4. Cloning, expression, purification and antigenic evaluation of ...

    African Journals Online (AJOL)

    Yomi

    2012-01-31

    Jan 31, 2012 ... Key word: Hyaluronidase gene, cloning, expression of recombinant gene, antigenic region. INTRODUCTION. Group A streptococcus (Streptococcus pyogenes) is an important species of gram-positive pathogenic extra- cellular bacteria. This bacteria can produce wide range of infectious diseases like ...

  5. Expression of Treponema pallidum Antigens in Escherichia coli

    Science.gov (United States)

    Walfield, Alan M.; Hanff, Philip A.; Lovett, Michael A.

    1982-04-01

    Treponema pallidum DNA was cloned in a bacteriophage. Clones were screened for expression of Treponema pallidum antigens by an in situ radio-immunoassay on nitrocellulose, with the use of subsequent reactions with syphilitic serum and radioiodinated Staphylococcus aureus protein A. One clone, which gave a strong signal, codes for at least seven antigens that react specifically with human antibodies to Treponema pallidum.

  6. Constitutive type VI secretion system expression gives Vibrio cholerae intra- and interspecific competitive advantages.

    Directory of Open Access Journals (Sweden)

    Daniel Unterweger

    Full Text Available The type VI secretion system (T6SS mediates protein translocation across the cell membrane of Gram-negative bacteria, including Vibrio cholerae - the causative agent of cholera. All V. cholerae strains examined to date harbor gene clusters encoding a T6SS. Structural similarity and sequence homology between components of the T6SS and the T4 bacteriophage cell-puncturing device suggest that the T6SS functions as a contractile molecular syringe to inject effector molecules into prokaryotic and eukaryotic target cells. Regulation of the T6SS is critical. A subset of V. cholerae strains, including the clinical O37 serogroup strain V52, express T6SS constitutively. In contrast, pandemic strains impose tight control that can be genetically disrupted: mutations in the quorum sensing gene luxO and the newly described regulator gene tsrA lead to constitutive T6SS expression in the El Tor strain C6706. In this report, we examined environmental V. cholerae isolates from the Rio Grande with regard to T6SS regulation. Rough V. cholerae lacking O-antigen carried a nonsense mutation in the gene encoding the global T6SS regulator VasH and did not display virulent behavior towards Escherichia coli and other environmental bacteria. In contrast, smooth V. cholerae strains engaged constitutively in type VI-mediated secretion and displayed virulence towards prokaryotes (E. coli and other environmental bacteria and a eukaryote (the social amoeba Dictyostelium discoideum. Furthermore, smooth V. cholerae strains were able to outcompete each other in a T6SS-dependent manner. The work presented here suggests that constitutive T6SS expression provides V. cholerae with an advantage in intraspecific and interspecific competition.

  7. Identification of Enterococcus faecalis antigens specifically expressed in vivo

    OpenAIRE

    Seok-Woo Lee; Shet, Uttom K.; Sang-Won Park; Hyun-Pil Lim; Kwi-Dug Yun; Seong Soo Kang; Se Eun Kim

    2015-01-01

    Objectives Molecular mechanism of the pathogenicity of Enterococcus faecalis (E. faecalis), a suspected endodontic pathogen, has not yet been adequately elucidated due to limited information on its virulence factors. Here we report the identification of in vivo expressed antigens of E. faecalis by using a novel immunoscreening technique called change-mediated antigen technology (CMAT) and an experimental animal model of endodontic infection. Materials and Methods Among 4,500 E. coli...

  8. Transgenic expression of neuronal dystonin isoform 2 partially rescues the disease phenotype of the dystonia musculorum mouse model of hereditary sensory autonomic neuropathy VI

    Science.gov (United States)

    Ferrier, Andrew; Sato, Tadasu; De Repentigny, Yves; Gibeault, Sabrina; Bhanot, Kunal; O'Meara, Ryan W.; Lynch-Godrei, Anisha; Kornfeld, Samantha F.; Young, Kevin G.; Kothary, Rashmi

    2014-01-01

    A newly identified lethal form of hereditary sensory and autonomic neuropathy (HSAN), designated HSAN-VI, is caused by a homozygous mutation in the bullous pemphigoid antigen 1 (BPAG1)/dystonin gene (DST). The HSAN-VI mutation impacts all major neuronal BPAG1/dystonin protein isoforms: dystonin-a1, -a2 and -a3. Homozygous mutations in the murine Dst gene cause a severe sensory neuropathy termed dystonia musculorum (dt). Phenotypically, dt mice are similar to HSAN-VI patients, manifesting progressive limb contractures, dystonia, dysautonomia and early postnatal death. To obtain a better molecular understanding of disease pathogenesis in HSAN-VI patients and the dt disorder, we generated transgenic mice expressing a myc-tagged dystonin-a2 protein under the regulation of the neuronal prion protein promoter on the dtTg4/Tg4 background, which is devoid of endogenous dystonin-a1 and -a2, but does express dystonin-a3. Restoring dystonin-a2 expression in the nervous system, particularly within sensory neurons, prevented the disorganization of organelle membranes and microtubule networks, attenuated the degeneration of sensory neuron subtypes and ameliorated the phenotype and increased life span in these mice. Despite these improvements, complete rescue was not observed likely because of inadequate expression of the transgene. Taken together, this study provides needed insight into the molecular basis of the dt disorder and other peripheral neuropathies including HSAN-VI. PMID:24381311

  9. [Prokaryotic expression, purification and antigenicity identification of human renal cell carcinoma-associated antigen G250].

    Science.gov (United States)

    Xiao, Yi; Gao, Jiangping; Gao, Kun; Yan, Jinqi; Zhang, Liang; Wang, Yu; Xu, Yuanji; Wang, Wei; Wang, Xiaoxiong; Yu, Jiyun

    2013-03-01

    To amplify human renal cell carcinoma (RCC)-associated antigen G250 gene and construct a recombinant plasmid pET-42a-hG250, express and purify human G250 protein and identify its antigenicity. The gene of human G250 was amplified from pGEM-T-G250 by PCR. After sequencing, the PCR product (112-1242 bp) was cloned into pET-42a prokaryotic expression vector to construct the recombinant plasmid pET-42a-hG250. The plasmid was transformed into BL21 (DE3) and human G250 protein was expressed under the induction of IPTG. The fusion protein was purified and identified by SDS-PAGE, Western blotting and ELISA sequentially. The human G250 prokaryotic expression vector pET-42a-hG250 was successfully constructed as confirmed by enzyme digestion and DNA sequencing. After transformation into BL21 (DE3), the target protein was successfully induced to express and purified as expected. Western blotting and ELISA demonstrated that the purified human G250 protein had a desirable immunogenicity. The recombinant prokaryotic expression vector pET-42a-hG250 has been constructed successfully. The purified human G250 protein has a good antigenicity.

  10. Expression of hepatitis B surface antigen in transgenic banana plants.

    Science.gov (United States)

    Kumar, G B Sunil; Ganapathi, T R; Revathi, C J; Srinivas, L; Bapat, V A

    2005-10-01

    Embryogenic cells of bananan cv. Rasthali (AAB) have been transformed with the 's' gene of hepatitis B surface antigen (HBsAg) using Agrobacterium mediated transformation. Four different expression cassettes (pHBS, pHER, pEFEHBS and pEFEHER) were utilized to optimize the expression of HBsAg in banana. The transgenic nature of the plants and expression of the antigen was confirmed by PCR, Southern hybridization and reverse transcription (RT)-PCR. The expression levels of the antigen in the plants grown under in vitro conditions as well as the green house hardened plants were estimated by ELISA for all the four constructs. Maximum expression level of 38 ng/g F.W. of leaves was noted in plants transformed with pEFEHBS grown under in vitro conditions, whereas pHER transformed plants grown in the green house showed the maximum expression level of 19.92 ng/g F.W. of leaves. Higher monoclonal antibody binding of 67.87% of the antigen was observed when it was expressed with a C-terminal ER retention signal. The buoyant density in CsCl of HBsAg derived from transgenic banana leaves was determined and found to be 1.146 g/ml. HBsAg obtained from transgenic banana plants is similar to human serum derived one in buoyant density properties. The transgenic plants were grown up to maturity in the green house and the expression of HBsAg in the fruits was confirmed by RT-PCR. These transgenic plants were multiplied under in vitro using floral apex cultures. Attempts were also made to enhance the expression of HBsAg in the leaves of transgenic banana plants by wounding and/or treatment with plant growth regulators. This is the first report on the expression of HBsAg in transgenic banana fruits.

  11. Kinetics of Antigen Expression and Epitope Presentation during Virus Infection

    Science.gov (United States)

    Croft, Nathan P.; Smith, Stewart A.; Wong, Yik Chun; Tan, Chor Teck; Dudek, Nadine L.; Flesch, Inge E. A.; Lin, Leon C. W.; Tscharke, David C.; Purcell, Anthony W.

    2013-01-01

    Current knowledge about the dynamics of antigen presentation to T cells during viral infection is very poor despite being of fundamental importance to our understanding of anti-viral immunity. Here we use an advanced mass spectrometry method to simultaneously quantify the presentation of eight vaccinia virus peptide-MHC complexes (epitopes) on infected cells and the amounts of their source antigens at multiple times after infection. The results show a startling 1000-fold range in abundance as well as strikingly different kinetics across the epitopes monitored. The tight correlation between onset of protein expression and epitope display for most antigens provides the strongest support to date that antigen presentation is largely linked to translation and not later degradation of antigens. Finally, we show a complete disconnect between the epitope abundance and immunodominance hierarchy of these eight epitopes. This study highlights the complexity of viral antigen presentation by the host and demonstrates the weakness of simple models that assume total protein levels are directly linked to epitope presentation and immunogenicity. PMID:23382674

  12. Expression of antigen tf and galectin-3 in fibroadenoma

    Directory of Open Access Journals (Sweden)

    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.

  13. Cancer-testis antigen expression is shared between epithelial ovarian cancer tumors.

    Science.gov (United States)

    Garcia-Soto, Arlene E; Schreiber, Taylor; Strbo, Natasa; Ganjei-Azar, Parvin; Miao, Feng; Koru-Sengul, Tulay; Simpkins, Fiona; Nieves-Neira, Wilberto; Lucci, Joseph; Podack, Eckhard R

    2017-06-01

    Cancer-testis (CT) antigens have been proposed as potential targets for cancer immunotherapy. Our objective was to evaluate the expression of a panel of CT antigens in epithelial ovarian cancer (EOC) tumor specimens, and to determine if antigen sharing occurs between tumors. RNA was isolated from EOC tumor specimens, EOC cell lines and benign ovarian tissue specimens. Real time-PCR analysis was performed to determine the expression level of 20 CT antigens. A total of 62 EOC specimens, 8 ovarian cancer cell lines and 3 benign ovarian tissues were evaluated for CT antigen expression. The majority of the specimens were: high grade (62%), serous (68%) and advanced stage (74%). 58 (95%) of the EOC tumors analyzed expressed at least one of the CT antigens evaluated. The mean number of CT antigen expressed was 4.5 (0-17). The most frequently expressed CT antigen was MAGE A4 (65%). Antigen sharing analysis showed the following: 9 tumors shared only one antigen with 62% of the evaluated specimens, while 37 tumors shared 4 or more antigens with 82%. 5 tumors expressed over 10 CT antigens, which were shared with 90% of the tumor panel. CT antigens are expressed in 95% of EOC tumor specimens. However, not a single antigen was universally expressed across all samples. The degree of antigen sharing between tumors increased with the total number of antigens expressed. These data suggest a multi-epitope approach for development of immunotherapy for ovarian cancer treatment. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. 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......-forming potential of E. coli. Finally, we demonstrated that Ag43-mediated cell aggregation confers significant protection against hydrogen peroxide killing....

  15. Identification of Enterococcus faecalis antigens specifically expressed in vivo

    Science.gov (United States)

    Shet, Uttom K.; Park, Sang-Won; Lim, Hyun-Pil; Yun, Kwi-Dug; Kang, Seong Soo; Kim, Se Eun

    2015-01-01

    Objectives Molecular mechanism of the pathogenicity of Enterococcus faecalis (E. faecalis), a suspected endodontic pathogen, has not yet been adequately elucidated due to limited information on its virulence factors. Here we report the identification of in vivo expressed antigens of E. faecalis by using a novel immunoscreening technique called change-mediated antigen technology (CMAT) and an experimental animal model of endodontic infection. Materials and Methods Among 4,500 E. coli recombinant clones screened, 19 positive clones reacted reproducibly with hyperimmune sera obtained from rabbits immunized with E. faecalis cells isolated from an experimental endodontic infection. DNA sequences from 16 of these in vivo-induced (IVI) genes were determined. Results Identified protein antigens of E. faecalis included enzymes involved in housekeeping functions, copper resistance protein, putative outer membrane proteins, and proteins of unknown function. Conclusions In vivo expressed antigens of E. faecalis could be identified by using a novel immune-screening technique CMAT and an experimental animal model of endodontic infection. Detailed analysis of these IVI genes will lead to a better understanding of the molecular mechanisms involved in the endodontic infection of E. faecalis. PMID:26587417

  16. Flow cytometric assay detecting cytotoxicity against human endogenous retrovirus antigens expressed on cultured multiple sclerosis cells

    DEFF Research Database (Denmark)

    Møller-Larsen, A; Brudek, T; Petersen, T

    2013-01-01

    expressing increased amounts of human endogenous retrovirus antigens. MS patients also have increased antibody levels to these antigens. The target cells are spontaneously growing peripheral blood mononuclear cells (PBMCs) of B cell lineage, expressing human endogenous retrovirus HERV epitopes...

  17. Allogeneic H-2 antigen expression is insufficient for tumor rejection.

    Science.gov (United States)

    Cole, G A; Cole, G A; Clements, V K; Garcia, E P; Ostrand-Rosenberg, S

    1987-12-01

    Murine A strain (KkDdLd) sarcoma I (SaI) tumor cells have been transfected with a cloned H-2Kb gene. The resulting clones (SKB clones) stably express high levels of a molecule that is serologically and biochemically indistinguishable from the H-2Kb antigen. SKB clones are not susceptible to cytotoxic T lymphocyte-mediated lysis by H-2Kb-specific bulk, cloned, or H-2Kb-restricted lymphocytic choriomeningitis virus-specific effectors. Survival times of A/J and B10.A mice challenged i.p. with the H-2Kb-expressing transfectants and the parental SaI cells are similar, suggesting that the presence of an allogeneic major histocompatibility complex class I antigen on the surface of this tumor line is insufficient for tumor rejection.

  18. Heterologous expression of antigenic peptides in Bacillus subtilis biofilms.

    Science.gov (United States)

    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.

  19. Cloning, expression, and antigenicity of 14 proteins from Campylobacter jejuni.

    Science.gov (United States)

    Zhang, Maojun; Meng, Fanliang; Cao, Fangfang; Qiao, Bo; Liu, Guodong; Liu, Hongying; Zhou, Yizhuang; Dong, Haiyan; Gu, Yixin; Xiao, Di; Zhang, Yongchan; Zhang, Jianzhong

    2012-08-01

    Fourteen Campylobacter jejuni genes--porA, cadF, omp18, dnaK, flaC, peb1, peb2, peb3, peb4, ahpC, groEL, tuF, hipO, and Cj0069--were cloned and expressed in Escherichia coli BL21. The recombinant proteins were purified on histidine (His) and glutathione S-transferase (GST) trap columns using the ÄKTA Explorer 100 System. Recombinant proteins were visualized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The antigenicities of these recombinant proteins were assessed by Western blotting and enzyme-linked immunosorbent assays with anti-C. jejuni immune rabbit sera. Four recombinant proteins, including rGST-PorA, rHis-CadF, rGST-GroEL, and rGST-TuF, demonstrated reactions with both anti-serum and preimmune serum, while rHis-DnaK, rGST-FlaC, rGST-PEB2, rGST-PEB3, rGST-PEB4, and rGST-HipO showed variable antigenicity characteristics to the anti-sera derived from different C. jejuni strains. rHis-Omp18, rHis-PEB1, and rGST-AhpC demonstrated universal and specific antigenities with the entire anti-sera panel tested in this present study, while recombinant rGST-Cj0069 and rHis-DnaK did not react with any of the anti-C. jejuni sera tested. In conclusion, rGST-AhpC may be useful as a potential serodiagnostic antigen for C. jejuni infection.

  20. Serum protein expression profiling and bioinformatics analysis in workers occupationally exposed to chromium (VI).

    Science.gov (United States)

    Hu, Guiping; Wang, Tianjing; Liu, Jiaxing; Chen, Zhangjian; Zhong, Lijun; Yu, Shanfa; Zhao, Zuchang; Zhai, Min; Jia, Guang

    2017-08-05

    Cr(VI) is widely-recognized as occupational and environmental contaminant, but the precise underlying mechanisms of Cr(VI) induced carcinogenic toxicity remain to be elucidated. Among kinds of toxic mechanisms, alteration of protein profiling usually elaborate a key mechanism of Cr(VI) induced toxicity and carcinogenesis. Large-scale proteins changes can reflect the onset or progression of carcinogenic toxicity, and potential serum protein biomarkers of Cr(VI) exposure. To gain an insight into the serum proteins expression profiling in chromate workers and find potential novel serum proteins biomarkers of Cr(VI) exposure, 107 male participants from a chromate production plant were recruited into the study. Questionnaire was applied to collect personal information and occupational history. Chromium concentration in blood (CrB) was measured to evaluate the participants' internal exposure. Serum proteins profiling and bioinformatics analysis were performed to explore differentially expressed proteins, proteins-chemical interaction network, critical proteins nodes related to the signaling pathways among 16 controls and 25 exposure workers in the first stage. ELISA tests were applied to verify the critical interested proteins nodes in the remaining 41 exposure workers and 25 controls. The results showed that the CrB levels in the control group were significantly lower than that in the exposure group (P<0.05). 44 significantly differentially expressed serum proteins formed 16 significant signaling pathways and a complex proteins-chemical interaction network, which associated with the immune system and extracellular matrix organization. C reactive protein (CRP), sonic hedgehog protein (SHH) and calcium located at critical nodes in proteins-chemical interaction network. There was a significant negative correlation between serum CRP level and CrB (P<0.05), and a significant positive correlation between SHH concentrations and CrB (P<0.05), which indicated that CRP and SHH

  1. Expression in E. coli of the cloned cDNA for the major antigen of foot ...

    Indian Academy of Sciences (India)

    DNA produced proteins .which cross-reacted with the antibodies generated against the structural proteins of the virus in an enzyme linked immunosorbent assay, indicating that the. cDNA of the major antigen is expressed in the cloned cell. Keywords. Foot and mouth disease virus; cDNA; cloning; antigen; expression. 1.

  2. Nitrification inhibition by hexavalent chromium Cr(VI)--Microbial ecology, gene expression and off-gas emissions.

    Science.gov (United States)

    Kim, Young Mo; Park, Hongkeun; Chandran, Kartik

    2016-04-01

    The goal of this study was to investigate the responses in the physiology, microbial ecology and gene expression of nitrifying bacteria to imposition of and recovery from Cr(VI) loading in a lab-scale nitrification bioreactor. Exposure to Cr(VI) in the reactor strongly inhibited nitrification performance resulting in a parallel decrease in nitrate production and ammonia consumption. Cr(VI) exposure also led to an overall decrease in total bacterial concentrations in the reactor. However, the fraction of ammonia oxidizing bacteria (AOB) decreased to a greater extent than the fraction of nitrite oxidizing bacteria (NOB). In terms of functional gene expression, a rapid decrease in the transcript concentrations of amoA gene coding for ammonia oxidation in AOB was observed in response to the Cr(VI) shock. In contrast, transcript concentrations of the nxrA gene coding for nitrite oxidation in NOB were relatively unchanged compared to Cr(VI) pre-exposure levels. Therefore, Cr(VI) exposure selectively and directly inhibited activity of AOB, which indirectly resulted in substrate (nitrite) limitation to NOB. Significantly, trends in amoA expression preceded performance trends both during imposition of and recovery from inhibition. During recovery from the Cr(VI) shock, the high ammonia concentrations in the bioreactor resulted in an irreversible shift towards AOB populations, which are expected to be more competitive in high ammonia environments. An inadvertent impact during recovery was increased emission of nitrous oxide (N2O) and nitric oxide (NO), consistent with recent findings linking AOB activity and the production of these gases. Therefore, Cr(VI) exposure elicited multiple responses on the microbial ecology, gene expression and both aqueous and gaseous nitrogenous conversion in a nitrification process. A complementary interrogation of these multiple responses facilitated an understanding of both direct and indirect inhibitory impacts on nitrification. Copyright

  3. Antigenic assessment of a recombinant human CD90 protein expressed in prokaryotic expression system.

    Science.gov (United States)

    Yousefi-Rad, Narges; Shokrgozar, Mohammad Ali; Behdani, Mahdi; Moradi-Kalbolandi, Shima; Motamedi-Rad, Mahdieh; Habibi-Anbouhi, Mahdi

    2015-12-01

    Cluster of Differentiation 90 (CD90, Thy-1) has been proposed as one of the most important biomarkers in several cancer cells including cancer stem cells (CSCs). CD90 is considered as a potential normal stem cell and CSCs biomarker and also has been identified in lung cancer stem cells, hepatocellular carcinoma cells and high-grade gliomas. Using eukaryotic host systems involves complex procedures and frequently results in low protein yields. The expression of recombinant proteins in Escherichia coli is comparatively easier than eukaryotic host cells. The potential of large scale production of recombinant protein has made this system an economic production platform. In this study we expressed the extra-membrane domain of human CD90 (exCD90) antigen (Gln15-Cys130) in E. coli expression host cells. The epitope integrity of purified recombinant antigen was confirmed by antibody-antigen interaction using 5E10 anti-CD90 monoclonal antibody and binding study through ELISA and florescent staining of CD90(+) cells in a flow cytometry experiment. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Protective antigens against glanders identified by expression library immunization

    Directory of Open Access Journals (Sweden)

    Gregory C. Whitlock

    2011-11-01

    Full Text Available Burkholderia are highly evolved Gram-negative bacteria that primarily infect solipeds but are transmitted to humans by ingestion and cutaneous or aerosol exposures. Heightened concern over human infections of Burkholderia (B. mallei and the very closely related species B. pseudomallei is due to the pathogens’ proven effectiveness as bioweapons, and to the increased potential for natural opportunistic infections in the growing diabetic and immuno-compromised populations. These Burkholderia species are nearly impervious to antibiotic treatments and no vaccine exists. In this study, the genome of the highly virulent B. mallei ATCC23344 strain was examined by expression library immunization for gene-encoded protective antigens. This protocol for genomic-scale functional screening was customized to accommodate the unusually large complexity of Burkholderia, and yielded 12 new putative vaccine candidates. Five of the candidates were individually tested as protein immunogens and three were found to confer significant partial protection against a lethal pulmonary infection in a murine model of disease. Determinations of peripheral blood cytokine and chemokine profiles following individual protein immunizations show that IL-2 and IL-4 are elicited by the three confirmed candidates, but unexpectedly interferon-and tumor necrosis factor-are not. We suggest that these pathogen components, discovered using genetic immunization and confirmed in a conventional protein format, will be useful toward the development of a safe and effective glanders vaccine.

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

    Directory of Open Access Journals (Sweden)

    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.

  6. Expression of Tn, sialosyl-Tn and T antigens in human foetal large intestine

    Directory of Open Access Journals (Sweden)

    G Barresi

    2009-12-01

    Full Text Available Tn, sialosyl-Tn and T antigens are simple mucintype carbohydrate antigens that may be expressed in human neoplasies due to alteration of the glycoprotein biosynthetic pathway. Utilising specific monoclonal antibodies (HB-Tn1, HB-STn1 and HB-T1, we have investigated the expression of these simple mucin-type carbohydrate antigens in large intestine of 8 human foetuses at early gestational age (9-10 weeks, obtained after therapeutic abortion. In all cases the expression of Tn antigen was mainly localised as a thin rim at the cell membrane and occasionally in the supranuclear region of epithelial cells, while sialosyl-Tn antigen was documented in some goblet cell vacuoles and occasionally in the cytoplasm of columnar cells. T antigen was not expressed in any case. These results indicate that Tn and sialosyl-Tn antigens are expressed as early as nine weeks of gestation, further supporting the notion that they may be considered as oncodevelopmental cancerassociated antigens in the large intestine.

  7. HLA Class II Antigen Expression in Colorectal Carcinoma Tumors as a Favorable Prognostic Marker

    Directory of Open Access Journals (Sweden)

    Giuseppe Sconocchia

    2014-01-01

    Full Text Available The goal of this study was to determine the frequency of HLA class II antigen expression in colorectal carcinoma (CRC tumors, its association with the clinical course of the disease, and the underlying mechanism(s. Two tissue microarrays constructed with 220 and 778 CRC tumors were stained with HLA-DR, DQ, and DP antigen-specific monoclonal antibody LGII-612.14, using the immunoperoxidase staining technique. The immunohistochemical staining results were correlated with the clinical course of the disease. The functional role of HLA class II antigens expressed on CRC cells was analyzed by investigating their in vitro interactions with immune cells. HLA class II antigens were expressed in about 25% of the 220 and 21% of the 778 tumors analyzed with an overall frequency of 23%. HLA class II antigens were detected in 19% of colorectal adenomas. Importantly, the percentage of stained cells and the staining intensity were significantly lower than those detected in CRC tumors. However, HLA class II antigen staining was weakly detected only in 5.4% of 37 normal mucosa tissues. HLA class II antigen expression was associated with a favorable clinical course of the disease. In vitro stimulation with interferon gamma (IFNγ induced HLA class II antigen expression on two of the four CRC cell lines tested. HLA class II antigen expression on CRC cells triggered interleukin-1α (IL-1α production by resting monocytes. HLA class II antigen expression in CRC tumors is a favorable prognostic marker. This association may reflect stimulation of IL-1α production by monocytes.

  8. Expression of the Thomsen-Friedenreich (TF) tumor antigen in human abort placentas.

    Science.gov (United States)

    Richter, D U; Jeschke, U; Bergemann, C; Makovitzky, J; Lüthen, F; Karsten, U; Briese, V

    2005-01-01

    The Thomsen-Friedenreich antigen (TF), or more precisely epitope, has been known as a pancarcinoma antigen. It consists of galactose-beta1-3-N-acetylgalactose. We have already described the expression of TF in the normal placenta. TF is expressed by the syncytium and by extravillous trophoblast cells. In this study, we investigated the expression of TF in the abort placenta. Frozen samples of human abort placentas (12 placentas), obtained from the first and second trimesters of pregnancy and, for comparison, samples of normal placentas (17 placentas) from the first, second and third trimesters of pregnancy, were used. Expression of TF was investigated by immunohistochemical methods. For identification of TF-positive cells in abort placentas, immunofluorescence methods were used. Evaluation of simple and double immunofluorescence was performed on a laser scanning microscope. Furthermore, we isolated trophoblast cells from first and third trimester placentas and evaluated cytokeratin 7 and Muc1 expression by immunofluorescence methods. We observed expression of TF antigen in the syncytiotrophoblasts layer of the placenta in all three trimesters of pregnancy in normal and abort placentas evaluated by immunohistochemical methods. There was no expression of TF antigen in the decidua of abort placentas. Immunofluorescence double staining of TF antigen and cytokeratin 7 showed reduced expression of both antigens in the abort decidua and co-expression of both antigens in the syncytiotrophoblast layer of normal and abort placentas. TF expression in the syncytiotrophoblast was reduced in abort placentas. In the isolated trophoblast cells, no TF expression was found, however, Muc1 expression was visualized. Expression of TF antigen was reduced in the first and second trimester abort decidua compared to the normal decidua during the same time of pregnancy. TF antigen was restricted to the syncytiotrophoblast and extravillous trophoblast cells in the decidua. Abort placentas

  9. Microfluidic cell surface antigen expression analysis using a single antibody type.

    Science.gov (United States)

    Zhang, Ye; Pappas, Dimitri

    2016-02-21

    Antigen expression plays a significant role in clinical studies, pathology, biology and chemistry. The type and degree of antigen expression can provide information for disease diagnosis/monitoring and is used for phenotype analysis of cells. In this work, an affinity capture method was developed to capture cells based on antigen expression differences in a single microfluidic chip. Microfluidic chips with two affinity regions-at different antibody concentrations-captured two cell types based on differences in the expression of a single antigen. Using herringbone-modified capture channels, a separation purity of 95% and a capture efficiency of 15% were achieved under continuous-flow conditions. We observed that the capture ratio of Ramos B lymphocytes and HuT 78 T lymphocytes matched the expression ratio of CD71 for the two cell lines (R(2) = 0.94). To further validate our analytical method, Ramos B lymphocytes were spiked into blood samples to demonstrate performance with a complex sample. Expression ratios matched conventional flow cytometry measurements over a 40-fold difference, and the sample enrichment was 9.5×. This method has proven to be a robust system to measure the differences in antigen expression, and can be used to distinguish cells without having a unique surface antigen if the expression level is sufficiently high in one cell type.

  10. Mendelian and non-mendelian mutations affecting surface antigen expression in Paramecium tetraurelia

    Energy Technology Data Exchange (ETDEWEB)

    Epstein, L.M.; Forney, J.D.

    1984-08-01

    A screening procedure was devised for the isolation of X-ray-induced mutations affecting the expression of the A immobilization antigen (i-antigen) in Paramecium tetraurelia. Two of the mutations isolated by this procedure proved to be in modifier genes. The two genes are unlinked to each other and unlinked to the structural A i-antigen gene. These are the first modifier genes identified in a Paramecium sp. that affect surface antigen expression. Another mutation was found to be a deletion of sequences just downstream from the A i-antigen gene. In cells carrying this mutation, the A i-antigen gene lies in close proximity to the end of a macronuclear chromosome. The expression of the A i-antigen is not affected in these cells, demonstrating that downstream sequences are not important for the regulation and expression of the A i-antigen gene. A stable cell line was also recovered which shows non-Mendelian inheritance of a macronuclear deletion of the A i-antigen gene. This mutant does not contain the gene in its macronucleus, but contains a complete copy of the gene in its micronucleus. In the cytoplasm of wild-type animals, the micronuclear gene is included in the developing macronucleus; in the cytoplasm of the mutant, the incorporation of the A i-antigen gene into the macronucleus is inhibited. This is the first evidence that a mechanism is available in ciliates to control the expression of a gene by regulating its incorporation into developing macronuclei.

  11. Mendelian and non-mendelian mutations affecting surface antigen expression in Paramecium tetraurelia.

    Science.gov (United States)

    Epstein, L M; Forney, J D

    1984-08-01

    A screening procedure was devised for the isolation of X-ray-induced mutations affecting the expression of the A immobilization antigen (i-antigen) in Paramecium tetraurelia. Two of the mutations isolated by this procedure proved to be in modifier genes. The two genes are unlinked to each other and unlinked to the structural A i-antigen gene. These are the first modifier genes identified in a Paramecium sp. that affect surface antigen expression. Another mutation was found to be a deletion of sequences just downstream from the A i-antigen gene. In cells carrying this mutation, the A i-antigen gene lies in close proximity to the end of a macronuclear chromosome. The expression of the A i-antigen is not affected in these cells, demonstrating that downstream sequences are not important for the regulation and expression of the A i-antigen gene. A stable cell line was also recovered which shows non-Mendelian inheritance of a macronuclear deletion of the A i-antigen gene. This mutant does not contain the gene in its macronucleus, but contains a complete copy of the gene in its micronucleus. In the cytoplasm of wild-type animals, the micronuclear gene is included in the developing macronucleus; in the cytoplasm of the mutant, the incorporation of the A i-antigen gene into the macronucleus is inhibited. This is the first evidence that a mechanism is available in ciliates to control the expression of a gene by regulating its incorporation into developing macronuclei.

  12. T cells expressing VHH-directed oligoclonal chimeric HER2 antigen receptors

    DEFF Research Database (Denmark)

    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 o...

  13. Enhanced expression of beta2-microglobulin and HLA antigens on human lymphoid cells by interferon

    DEFF Research Database (Denmark)

    Heron, I; Hokland, M; Berg, K

    1979-01-01

    Mononuclear cells from the blood of healthy normal humans were kept in cultures under nonstimulating conditions for 16 hr in the presence or absence of human interferon. The relative quantities of HLA antigens and beta(2)-microglobulin on the cultured cells were determined by quantitative...... immunofluorescence (fluorescence-activated cell sorter) and by the capacity of cells to absorb out cytotoxic antibodies against the relevant antigens. Interferons of different origin and purities enhanced the expression of HLA antigens and beta(2)-microglobulins, whereas membrane immunoglobulins and antigens...... recognized by antiserum raised against human brain and T cells were the same on interferon-treated and control cells. Similar interferon effects were observed on an Epstein-Barrvirus-negative Burkitt lymphoma cell line. The enhanced expression of histocompatibility antigen subsequent to intereferon treatment...

  14. Antigen expression of metastasizing and non-metastasizing human melanoma cells xenografted into nude mice.

    Science.gov (United States)

    Van Muijen, G N; Cornelissen, L M; Jansen, C F; Figdor, C G; Johnson, J P; Bröcker, E B; Ruiter, D J

    1991-01-01

    In order to study differences in antigen expression related to the different stages of the process of metastasis of human melanoma cell lines, we determined the expression pattern of a series of well-characterized genes in a set of human melanoma cell lines with different metastatic behavior in nude mice. This set included non-metastatic (IF6, 530), sporadically metastatic (M14, Mel 57), and frequently metastatic (BLM, MV3) cell lines after subcutaneous inoculation. To study the phenotype of these cell lines both the cultured cells and representative samples of local tumors at the inoculation site and their metastases in the lungs were immunostained with a panel of monoclonal antibodies directed against melanocytic differentiation or progression antigens. Although most cell lines (IF6, 530, M14 and Mel 57) showed HLA-DR expression in vitro, these antigens were lacking in all xenografted lesions studied with exception of the 530 cell line. 530 Xenografts, however, showed a dramatic down-regulation of HLA-DR compared with the cell line in vitro. The same phenomenon was seen with respect to ICAM-1 expression. The expression of all other antigens studied in xenografts, both in subcutaneous tumors and in lung lesions, was in general comparable to that in the melanoma cell lines in vitro, with exception of the 530 cell line. In all melanoma cell lines except 530 the degree of intra- and interlesional heterogeneity regarding the expression of all antigens studied was limited. Remarkably, comparison of the immunophenotype of the frequently metastasizing (BLM, MV3) and the sporadically (M14, Mel 57) or non-metastasizing (IF6, 530) cell lines showed that the two frequently metastasizing cell lines had marked expression of the progression antigens VLA-2 and epidermal growth factor receptor, and lack of expression of the differentiation antigen NKI-beteb. These findings warrant further studies on the role of these antigens in the process of metastasis of human melanoma cells

  15. Expression of a cell surface immobilization antigen during serotype transformation in Tetrahymena thermophila.

    OpenAIRE

    Williams, N. E.; Doerder, F. P.; Ron, A

    1985-01-01

    A temperature shift from 40 to 28 degrees C rapidly induced expression of a specific immobilization antigen at the cell surface in Tetrahymena thermophila. This transformation was inhibited by actinomycin D and cycloheximide but not by colchicine or cytochalasin B. The major surface antigen expressed at 28 degrees C in cells homozygous for the SerH3 allele was partially purified, and an antiserum against this preparation was raised in rabbits. Electrophoresis, immunoblot, and [35S]methionine ...

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

    Directory of Open Access Journals (Sweden)

    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.

  17. P System antigenic determiners expression in Ascaris lumbricoides

    Directory of Open Access Journals (Sweden)

    Ponce De León Patricia

    2003-01-01

    Full Text Available The P System antigens have been detected in numerous parasites, bacterias and viruses, nevertheless the clinical significance is still unknown. The aim was to study the presence of P1 antigenic determiners in A. lumbricoides extracts by means of the use of 6 different monoclonal antibodies of well-known concentrations and Ig class. We worked with 14 A. lumbricoides extracts. Inhibition Agglutination Test was made in a bromelin enzymatic medium and 4 masculineC temperature. Titre, Score and Sensitivity Parameter were determined for each monoclonal antibody against red cells suspension used as revealing system. Ten extracts inhibited the agglutination of all anti P1 monoclonal antibodies. The 4 remaining extracts only inhibited the agglutination of some of them. It is demonstrated that the extracts have P1 activity. This activity is independent of titre, Score, Sensitivity Parameter, concentration and Ig class and it depends on the epitope at which the monoclonal antibody is directed.

  18. Expression and immunogenicity of novel subunit enterovirus 71 VP1 antigens

    Energy Technology Data Exchange (ETDEWEB)

    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.

  19. [MHC class I antigens, CD4 and CD8 expressions in polymyositis and dermatomyositis].

    Science.gov (United States)

    Graça, Carla Renata; Kouyoumdjian, João Aris

    2015-01-01

    To analyze the frequencies of the expression of major histocompatibility complex class I (MHC-I) antigens, and CD4 and CD8 cells in skeletal muscle in polymyositis (PM) and dermatomyositis (DM). This was a retrospective study of 34 PM cases, 8 DM cases, and 29 control patients with non-inflammatory myopathies. MHC-I antigens were expressed in the sarcolemma and/or sarcoplasm in 79.4% of PM cases, 62.5% of DM cases, and 27.6% of controls (CD4 expression was observed in 76.5%, 75%, and 13.8%, respectively). There was a high suspicion of PM/DM (mainly PM) in patients in whom MHC-I antigens and CD4 were co-expressed. In 14.3% of PM/DM cases, we observed MHC-I antigens expression alone, without inflammatory cells. MCH-I antigens expression and CD4 positivity might add to strong diagnostic suspicion of PM/DM. No cellular infiltration was observed in 14.3% of such cases. Copyright © 2014 Elsevier Editora Ltda. All rights reserved.

  20. 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....

  1. Expression of a cell surface immobilization antigen during serotype transformation in Tetrahymena thermophila.

    Science.gov (United States)

    Williams, N E; Doerder, F P; Ron, A

    1985-08-01

    A temperature shift from 40 to 28 degrees C rapidly induced expression of a specific immobilization antigen at the cell surface in Tetrahymena thermophila. This transformation was inhibited by actinomycin D and cycloheximide but not by colchicine or cytochalasin B. The major surface antigen expressed at 28 degrees C in cells homozygous for the SerH3 allele was partially purified, and an antiserum against this preparation was raised in rabbits. Electrophoresis, immunoblot, and [35S]methionine incorporation studies are reported which support the conclusion that the H3 antigen is an acidic protein with an Mr of approximately 52,000 daltons. An induced synthesis of the H3 immobilization antigen was detected within 30 min after a shift from 40 to 28 degrees C. This protein appeared to be synthesized in the microsomal fraction and transferred without cleavage to the cell surface, where it was inserted first into nonciliated regions.

  2. Expression of PCV2 antigen in the ovarian tissues of gilts

    Science.gov (United States)

    TUMMARUK, Padet; PEARODWONG, Pachara

    2015-01-01

    The present study was performed to determine the expression of porcine circovirus type 2 (PCV2) antigen in the ovarian tissue of naturally infected gilts. Ovarian tissues were obtained from 11 culled gilts. The ovarian tissues sections were divided into two groups according to PCV2 DNA detection using PCR. PCV2 antigen was assessed in the paraffin embedded ovarian tissue sections by immunohistochemistry. A total of 2,131 ovarian follicles (i.e., 1,437 primordial, 133 primary, 353 secondary and 208 antral follicles), 66 atretic follicles and 131 corpora lutea were evaluated. It was found that PCV2 antigen was detected in 280 ovarian follicles (i.e., 239 primordial follicles, 12 primary follicles, 10 secondary follicles and 19 antral follicles), 1 atretic follicles and 3 corpora lutea (P<0.05). PCV2 antigen was detected in primordial follicles more often than in secondary follicles, atretic follicles and corpora lutea (P<0.05). The detection of PCV2 antigen was found mainly in oocytes. PCV2 antigen was found in both PCV2 DNA positive and negative ovarian tissues. It can be concluded that PCV2 antigen is expressed in all types of the ovarian follicles and corpora lutea. Further studies should be carried out to determine the influence of PCV2 on porcine ovarian function and oocyte quality. PMID:26522687

  3. 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...

  4. Gene expression profiling using nanostring digital RNA counting to identify potential target antigens for melanoma immunotherapy.

    Science.gov (United States)

    Beard, Rachel E; Abate-Daga, Daniel; Rosati, Shannon F; Zheng, Zhili; Wunderlich, John R; Rosenberg, Steven A; Morgan, Richard A

    2013-09-15

    The success of immunotherapy for the treatment of metastatic cancer is contingent on the identification of appropriate target antigens. Potential targets must be expressed on tumors but show restricted expression on normal tissues. To maximize patient eligibility, ideal target antigens should be expressed on a high percentage of tumors within a histology and, potentially, in multiple different malignancies. A Nanostring probeset was designed containing 97 genes, 72 of which are considered potential candidate genes for immunotherapy. Five established melanoma cell lines, 59 resected metastatic melanoma tumors, and 31 normal tissue samples were profiled and analyzed using Nanostring technology. Of the 72 potential target genes, 33 were overexpressed in more than 20% of studied melanoma tumor samples. Twenty of those genes were identified as differentially expressed between normal tissues and tumor samples by ANOVA analysis. Analysis of normal tissue gene expression identified seven genes with limited normal tissue expression that warrant further consideration as potential immunotherapy target antigens: CSAG2, MAGEA3, MAGEC2, IL13RA2, PRAME, CSPG4, and SOX10. These genes were highly overexpressed on a large percentage of the studied tumor samples, with expression in a limited number of normal tissue samples at much lower levels. The application of Nanostring RNA counting technology was used to directly quantitate the gene expression levels of multiple potential tumor antigens. Analysis of cell lines, 59 tumors, and normal tissues identified seven potential immunotherapy targets for the treatment of melanoma that could increase the number of patients potentially eligible for adoptive immunotherapy. ©2013 AACR.

  5. Stage-specific embryonic antigen: determining expression in canine glioblastoma, melanoma, and mammary cancer cells.

    Science.gov (United States)

    Lin, Weiming; Modiano, Jaime F; Ito, Daisuke

    2017-03-30

    The expression of stage-specific embryonic antigens (SSEAs) was determined in several types of canine cancer cells. Flow cytometry showed SSEA-1 expression in glioblastoma, melanoma, and mammary cancer cells, although none expressed SSEA-3 or SSEA-4. Expression of SSEA-1 was not detected in lymphoma, osteosarcoma, or hemangiosarcoma cell lines. Relatively stable SSEA-1 expression was observed between 24 and 72 h of culture. After 8 days in culture, sorted SSEA-1(-) and SSEA-1(+) cells re-established SSEA-1 expression to levels comparable to those observed in unsorted cells. Our results document, for the first time, the expression of SSEA-1 in several canine cancer cell lines.

  6. Differential in vivo expression of mycobacterial antigens in Mycobacterium tuberculosis infected lungs and lymph node tissues.

    Science.gov (United States)

    Mustafa, Tehmina; Leversen, Nils Anders; Sviland, Lisbet; Wiker, Harald Gotten

    2014-10-03

    The clinical course of tuberculosis (TB) infection, bacterial load and the morphology of lesions vary between pulmonary and extrapulmonary TB. Antigens expressed in abundance during infection could represent relevant antigens in the development of diagnostic tools, but little is known about the in vivo expression of various M. tuberculosis antigens in different clinical manifestations. The aim of this study was to study the differences in the presence of major secreted as well as somatic mycobacterial antigens in host tissues during advanced rapidly progressing and fatal pulmonary disease with mainly pneumonic infiltrates and high bacterial load, and to compare this to the presence of the same antigens in TB lymphadenitis cases, which is mainly chronic and self-limiting disease with organised granulomas and lower bacterial load. Human pulmonary (n = 3) and lymph node (n = 17) TB biopsies, and non-TB controls (n = 12) were studied. Ziehl-Neelsen stain, nested PCR 1S6110 and immunohistochemistry were performed. Major secreted (MPT32, MPT44, MPT46, MPT51, MPT53, MPT59, MPT63, and MPT64) and somatic mycobacterial antigens (Mce1A, Hsp65, and MPT57) were detected by using rabbit polyclonal antibodies. Plenty of bacilli were detectable with Ziehl-Neelsen stain in the lung biopsies while no bacilli were detected in the lymph node biopsies. All the cases were shown to be positive by PCR. Both secretory and somatic antigens were expressed in abundance in pulmonary infiltrates, while primarily somatic antigens were detected in the lymphadenitis cases. Of the secreted antigens, only MPT64 was consistently detected in both cases, indicating a preferential accumulation of this antigen within the inflammatory cells, even if the cells of the granuloma can efficiently restrict bacterial growth and clear away the secreted antigens. This study shows that major secreted mycobacterial antigens were found in high amounts in advanced pulmonary lesions without proper granuloma

  7. Changes in monoclonal HLA-DR antigen expression in acute organophosphorus pesticide-poisoned patients.

    Science.gov (United States)

    Xia, Chenyun; Wang, Mi; Liang, Qi; Yun, Ling'an; Kang, Housheng; Fan, Lei; Wang, Dongsheng; Zhang, Guoyuan

    2014-01-01

    The aim of this study was to investigate changes in human leukocyte antigen (HLA)-DR expression of peripheral blood mononuclear cells (MNCs) in patients with acute organophosphorus pesticide poisoning (AOPP). HLA-DR antigen expression of peripheral blood MNCs was examined in 75 patients with AOPP, including 36 patients without multiple organ dysfunction syndrome (non-MODS) and 39 patients with multiple organ dysfunction syndrome (MODS), as well as in 30 healthy individuals using flow cytometry assay. The associations between HLA-DR antigen expression and certain parameters were analyzed, including acute physiology and chronic health evaluation II (APACHE II) score, serum cholinesterase (ChE) activity, cardiac troponin I (cTnI), cardiac enzymes, and liver and kidney function. The mean fluorescence intensity (MCF) of HLA-DR expression in the AOPP group (21.59±5.36) was significantly lower than that in the control group (27.85±4.86) (PDR expression in patients with AOPP was significantly decreased compared with that in healthy individuals; HLA-DR expression may therefore be a good indicator for evaluating AOPP, MODS disease severity, immune function, efficacy of prognosis and prognosis. Examination of HLA-DR antigen expression may be of crucial clinical value.

  8. Human leukocyte antigen DR surface expression on CD14+ monocytes during adverse events after hematopoietic stem cell transplantation.

    Science.gov (United States)

    Döring, Michaela; Rohrer, Karin Melanie; Erbacher, Annika; Gieseke, Friederike; Schwarze, Carl-Philipp; Bader, Peter; Handgretinger, Rupert; Hofbeck, Michael; Kerst, Gunter

    2015-02-01

    The human leukocyte antigen DR surface expression on CD14+ monocytes reflects the degree to which these cells have been activated. Given the central role monocytes and macrophages play in the immune system, a decreased human leukocyte antigen DR expression on CD14+ monocytes results in a hallmark of altered immune status during systemic inflammatory response syndrome. We hypothesize that human leukocyte antigen DR expression might be similarly altered after hematopoietic stem cell transplantation and during post-transplant complications. Using flow cytometry, this study investigates the human leukocyte antigen DR surface expression of CD14+ monocytes in 30 pediatric and young adult patients up to 1 year after hematopoietic stem cell transplantation. Normal values were derived from a control group of healthy children, adolescents, and young adults. Human leukocyte antigen DR expression decreased significantly prior and during bacterial infection or sepsis. By contrast, human leukocyte antigen DR expression levels were elevated before and at the time of viremia. Human leukocyte antigen DR expression was also elevated during acute graft-versus-host disease. In contrast, the expression was reduced when patients had hepatic veno-occlusive disease. A significant decrease of human leukocyte antigen DR expression was associated with a relapse of the underlying disease and before death. Human leukocyte antigen DR expression on CD14+ monocytes appears to be a promising parameter that might allow identification of patients at risk after hematopoietic stem cell transplantation.

  9. Temperature-related differential expression of antigens in the Lyme disease spirochete, Borrelia burgdorferi.

    Science.gov (United States)

    Stevenson, B; Schwan, T G; Rosa, P A

    1995-11-01

    Previous studies have demonstrated that Borrelia burgdorferi in the midguts of infected ticks shows increased expression of the antigenic outer surface protein OspC after the ticks have ingested a blood meal. This differential expression is at least partly due to a change in temperature, as an increase in OspC levels is also observed when cultures are shifted from 23 to 35 degrees C. Immunoblotting of bacterial lysates with sera from infected mice indicated that the levels of several additional antigens were also increased in bacterial cultures shifted to 35 degrees C; we have identified one antigen as OspE. We have also observed differential expression of OspF, which has been proposed to be coexpressed in an operon with the gene encoding OspE.

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

    Directory of Open Access Journals (Sweden)

    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.

  11. DNA rearrangements and antigenic variation in Trypanosoma equiperdum: multiple expression-linked sites in independent isolates of trypanosomes expressing the same antigen.

    Science.gov (United States)

    Longacre, S; Hibner, U; Raibaud, A; Eisen, H; Baltz, T; Giroud, C; Baltz, D

    1983-03-01

    African trypanosomes resist the immune response of their mammalian hosts by varying the surface glycoprotein which constitutes their antigenic identity. The molecular mechanism of this antigenic variation involves the successive activation of a series of genes which code for different variant surface glycoproteins (VSGs). We have studied the expression of two VSG genes (those of VSG-1 and VSG-28) in Trypanosoma equiperdum, and we report the following findings. (i) The expression of both VSG genes is associated with the duplication and transposition of corresponding basic copy genes. (ii) The duplicated transposed copy appears to be the expressed copy. (iii) Although there are multiple genes which cross-hybridize with the VSG-1 cDNA probe, only one of these appears to be used as a template for the expression-linked copy in four independent BoTat-1 clones. (iv) Analysis of the genomic environments of the expressed VSG-1 genes from each of four independently derived BoTat-1 trypanosome clones revealed that there are at least three different sites into which the expression-linked copy can be inserted.

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

    Directory of Open Access Journals (Sweden)

    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

  13. SMIM1 variants rs1175550 and rs143702418 independently modulate Vel blood group antigen expression

    Science.gov (United States)

    Christophersen, Mikael K.; Jöud, Magnus; Ajore, Ram; Vege, Sunitha; Ljungdahl, Klara W.; Westhoff, Connie M.; Olsson, Martin L.; Storry, Jill R.; Nilsson, Björn

    2017-01-01

    The Vel blood group antigen is expressed on the red blood cells of most individuals. Recently, we described that homozygosity for inactivating mutations in SMIM1 defines the rare Vel-negative phenotype. Still, Vel-positive individuals show great variability in Vel antigen expression, creating a risk for Vel blood typing errors and transfusion reactions. We fine-mapped the regulatory region located in SMIM1 intron 2 in Swedish blood donors, and observed a strong correlation between expression and rs1175550 as well as with a previously unreported tri-nucleotide insertion (rs143702418; C > CGCA). While the two variants are tightly linked in Caucasians, we separated their effects in African Americans, and found that rs1175550G and to a lesser extent rs143702418C independently increase SMIM1 and Vel antigen expression. Gel shift and luciferase assays indicate that both variants are transcriptionally active, and we identified binding of the transcription factor TAL1 as a potential mediator of the increased expression associated with rs1175550G. Our results provide insight into the regulatory logic of Vel antigen expression, and extend the set of markers for genetic Vel blood group typing. PMID:28084402

  14. 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.

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

    Directory of Open Access Journals (Sweden)

    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

  16. Expression of neuronal antigens by astrocytes derived from EGF-generated neuroprogenitor cells.

    Science.gov (United States)

    Schinstine, M; Iacovitti, L

    1996-09-01

    Previous studies have demonstrated that astrocytes reacting to CNS injury can express antigens normally associated with neurons. The origin of the reactive astrocytes, i.e., whether they are newly differentiated glial cells or preexisting astrocytes somehow triggered to express neuronal markers, remains difficult to determine using an in vivo model system. An in vitro model may prove more manageable. In the present study, primary brain cultures and EGF-generated neuroprogenitor cells were used to study the expression of neuronal antigens by established (primary) and nascent astrocytes, respectively. Astrocytes derived directly from dissociated mouse brains exhibited a flat morphology typical of type 1 astrocytes. These cells were nestin and GFAP positive and, in most cases, the antigens were colocalized. Primary astrocytes did not appear to express the putative neuronal markers GABA, Tau, or MAP2. Nascent astrocytes derived from EGF-generated progenitor cells showed a similar pattern of GFAP and nestin immunoreactivity. Contrary to primary astrocytes, many GFAP-intensive, stellate astrocytes exhibited Tau and MAP2. These cells also exhibited an intense nestin immunoreactivity. These data suggest that the reactive astrocytes expressing neuronal antigens in response to CNS trauma may be derived from neural progenitor cells rather than from previously differentiated astrocytes.

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

    Directory of Open Access Journals (Sweden)

    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.

  18. Inhibiting DNA methylation activates cancer testis antigens and expression of the antigen processing and presentation machinery in colon and ovarian cancer cells.

    Science.gov (United States)

    Siebenkäs, Cornelia; Chiappinelli, Katherine B; Guzzetta, Angela A; Sharma, Anup; Jeschke, Jana; Vatapalli, Rajita; Baylin, Stephen B; Ahuja, Nita

    2017-01-01

    Innovative therapies for solid tumors are urgently needed. Recently, therapies that harness the host immune system to fight cancer cells have successfully treated a subset of patients with solid tumors. These responses have been strong and durable but observed in subsets of patients. Work from our group and others has shown that epigenetic therapy, specifically inhibiting the silencing DNA methylation mark, activates immune signaling in tumor cells and can sensitize to immune therapy in murine models. Here we show that colon and ovarian cancer cell lines exhibit lower expression of transcripts involved in antigen processing and presentation to immune cells compared to normal tissues. In addition, treatment with clinically relevant low doses of DNMT inhibitors (that remove DNA methylation) increases expression of both antigen processing and presentation and Cancer Testis Antigens in these cell lines. We confirm that treatment with DNMT inhibitors upregulates expression of the antigen processing and presentation molecules B2M, CALR, CD58, PSMB8, PSMB9 at the RNA and protein level in a wider range of colon and ovarian cancer cell lines and treatment time points than had been described previously. In addition, we show that DNMTi treatment upregulates many Cancer Testis Antigens common to both colon and ovarian cancer. This increase of both antigens and antigen presentation by epigenetic therapy may be one mechanism to sensitize patients to immune therapies.

  19. Chemoresistance Is Associated with MUC1 and Lewis y Antigen Expression in Ovarian Epithelial Cancers

    Directory of Open Access Journals (Sweden)

    Danye Zhang

    2013-05-01

    Full Text Available Objective: The aim of this study was to analyze the correlation and clinical significance between the expression of Mucin-1 (MUC1 and the Lewis y antigen with chemoresistance in ovarian epithelial cancers. Methods: Ovarian cancer patients (n = 92 treated at our hospital from May 2005 to July 2009 were divided, according to their treatment and follow-up outcomes, into a resistant group (n = 37 or sensitive group (n = 55. The expression of MUC1 and Lewis y antigen in ovarian cancer tissues was detected using immunohistochemistry and correlated with chemoresistance. Results: The positive rates of MUC1 and Lewis y antigen in the resistant group were both 91.89%, significantly higher than their positive rates in the sensitive group (65.45% and 69.09%, respectively, and both p < 0.05. MUC1 or Lewis y expression and the pathological stage of the tissue were independent risk factors for chemoresistance (all p < 0.05. Conclusion: The increased expression of MUC1 and the Lewis y antigen is a significant risk factor for chemoresistance in patients with ovarian epithelial cancer.

  20. Trypanosoma cruzi: Maintenance in Culture Modify Gene and Antigenic Expression of Metacyclic Trypomastigotes

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    Víctor T Contreras

    1998-11-01

    Full Text Available In this study we examined whether the maintenance of Trypanosoma cruzi by long-time in axenic culture produces changes in gene expression and antigenic profiles. The studies were made with a Dm30L-clone from a low-virulent strain and a non-cloned virulent EP-strain of T. cruzi. Both parasites were maintained, for at least seven years, by successive alternate passage triatomine/mouse (triatomine condition, or by serial passage in axenic medium (culture condition. The comparison of the [35S]methionine metabolic labeling products of virulent and non-virulent parasites by 2D-SDS-PAGE, clearly indicates that the expression of metacyclic trypomastigotes (but not of epimastigotes proteins have been altered by laboratory maintenance conditions. Western blot analysis of EP and Dm30L-epimastigotes using a serum anti-epimastigotes revealed that although most of antigens are conserved, four antigens are characteristics of triatomine condition parasites and three other are characteristics of culture condition parasites. Anti-metacyclics serum revealed significative differences in EP- and Dm30L-metacyclic trypomastigotes from triatomine condition. However, avirulent metacyclic forms were antigenically very similar. These results suggest that besides a possible selection of avirulent subpopulation from T. cruzi strains genetically heterogeneous when maintained by long time in axenic culture, changes in virulence might be due to post-translational modifications of the antigens induced by the absence of the natural alternability (vertebrate-invertebrate in the life-cycle of T. cruzi

  1. Transgenic tomato plants expressing the antigen gene PfCP-2.9 of Plasmodium falciparum

    Directory of Open Access Journals (Sweden)

    Mihail Kantor

    2013-01-01

    Full Text Available The objective of this work was to obtain transgenic tomato plants expressing the PfCP-2.9 protein (a chimera of the antigens MSP1 and AMA1 of Plasmodium falciparum. Cotyledons of seven-day-old tomatoes, cultivar Summers, were transformed via Agrobacterium tumefaciens. Transgenic expression in the T0 plants was verified in the DNA extracted from fruits. PCR analysis was used to test the presence of the gene of interest in the T1 generation. Reverse transcriptase PCR provided evidence of gene expression at the RNA level, and Western blot analysis confirmed the presence of the protein of interest in the T1 plants. This is the first report of successful transformation with the expression of a malaria antigen (PfCP-2.9 in transgenic tomato plants from the T0 and T1 generations.

  2. 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

    NAc-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......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 ST6Gal......NAc-I as the major enzyme controlling the expression of cancer-associated sialyl-Tn antigen in gastrointestinal tissues....

  3. Cloning, expression and purification of Mycobacterium tuberculosis ESAT-6 and CFP-10 antigens

    OpenAIRE

    Shima Mahmoudi; Setareh Mamishi; Mona Ghazi; Reihaneh Hosseinpour Sadeghi; Babak Pourakbari

    2013-01-01

    Background and Objectives ESAT-6 (6-kDaearly secretory antigenic target) and CFP-10 (10-kDa culture filtrate protein) have been described as dominant antigens recognized by T-cells and considered as virulence factors in Mycobacterium tuberculosis. The aim of this study was to clone, express and purify recombinant ESAT-6 andCFP-10 proteins of M. tuberculosis in soluble form. Materials and Methods ESAT-6 andCFP-10 genes were amplified by PCR, cloned into pET32a (+) vector, and overexpress-ed us...

  4. Thyroid peroxidase (TPO) expressed in thyroid and breast tissues shows similar antigenic properties.

    Science.gov (United States)

    Godlewska, Marlena; Arczewska, Katarzyna D; Rudzińska, Magdalena; Łyczkowska, Anna; Krasuska, Wanda; Hanusek, Karolina; Ruf, Jean; Kiedrowski, Mirosław; Czarnocka, Barbara

    2017-01-01

    Thyroid peroxidase (TPO) is essential for physiological function of the thyroid gland. The high prevalence of thyroid peroxidase antibodies (TPOAbs) in patients with breast cancer and their protective role had previously been demonstrated, indicating a link between breast cancer and thyroid autoimmunity. Recently, TPO was shown to be present in breast cancer tissue samples but its antigenicity has not been analyzed. In this study, we investigated TPO expression levels in a series of fifty-six breast cancer samples paired with normal (peri-tumoral) tissue and its antigenic activity using a panel of well-characterized murine anti-human TPOAbs. We have shown that TPO transcripts were present in both normal and cancer tissue samples, although the amounts in the latter were reduced. Additionally, we observed that TPO levels are lower in more advanced cancers. TPO protein expression was confirmed in all tissue samples, both normal and cancerous. We also found that the antigenicity of the immunodominant regions (IDRs) in breast TPO resembles that of thyroid TPO, which is crucial for effective interactions with human TPOAbs. Expression of TPO in breast cancer together with its antigenic activity may have beneficial effects in TPOAb-positive breast cancer patients. However, further studies are needed to confirm the beneficial role of TPOAbs and to better understand the underlying mechanism.

  5. Limits of Flow-Cytometry Histogram Analysis Methods to Assess Bladder Tumour Antigen Expression

    Directory of Open Access Journals (Sweden)

    Agnès Chabanas

    1997-01-01

    Full Text Available Tumour‐associated antigens detected in cells obtained from bladder washings or tumours are useful markers in bladder cancer. Flow cytometry is commonly used to quantify immuno‐stained cells. A straightforward way to analyze data is to count the fluorescent cells above a threshold empirically determined on a control histogram representation. However, specific antigens expressed at highly variable rates give rise to wide range distributions in flow cytometry as illustrated when a mucin antigen for urinary bladder was titrated by M344 monoclonal antibody in urothelial cancer cells. We have evaluated several methods of background estimation and subtraction in order to determine the proportion of M344 Mab positive cells. These include threshold setting (Histogram Shape Dependent (HSD threshold developped in this study, 2% preset or 5% preset background, subtraction of the blank from the test histograms, and Kolmogorov–Smirnov statistical test. The HSD method appeared to be a more reliable method for background estimation; however, in the case of very low antigen expression, where specific fluorescence histograms could hardly be distinguished from that of the background, fluorescence microscopy remained the only valid method, since it allowed the distinction between specific and non‐specific fluorescence on the basis of structural differences between the two.

  6. Increased expression of natural-killer-associated and activation antigens in multiple myeloma.

    Science.gov (United States)

    Gonzalez, M; San Miguel, J F; Gascon, A; Moro, M J; Hernandez, J M; Ortega, F; Jimenez, R; Guerras, L; Romero, M; Casanova, F

    1992-02-01

    The expression of both natural-killer (NK)-associated and activation antigens was studied by flow cytometry in the peripheral blood of 47 untreated multiple myeloma (MM) patients. A significant increase in both absolute and relative numbers of CD57 positive cells as well as in the proportion of CD16 and CD11b cells was observed in patients with MM, specially in those in early stages of the disease (clinical stages I and II), suggesting a possible surveillance mechanism in response to an emerging malignant clone. Additional double stainings showed that strong CD16+ NK cells coexpress the CD56, CD11b, and CD2 antigens, while they lacked CD3, CD5, and WT31 antigens. Moreover, the previously reported increase in CD8 cells present in MM would be mainly due to a subset of CD8 cells that coexpress the CD57 Ags. The expression of activation antigens, especially CD38, was increased in peripheral blood lymphocytes of MM patients, the differences reaching statistical significance both in absolute and relative numbers in those cases with high numbers of CD16 NK cells and thus suggesting that these cells are functionally activated. These results reveal the existence of an increase in NK and activated cells in the peripheral blood of myeloma patients that may reflect a host's immunological mechanism in an attempt to modulate tumor cell growth.

  7. Immunohistochemical and genetic exploration of incompatible A blood group antigen expression in invasive micropapillary breast carcinoma: A case report.

    Science.gov (United States)

    Zouine, S; Orfi, Z; Kojok, K; Benayad, S; Zaid, Y; Marnissi, F; Habti, N

    Invasive Micropapillary Carcinoma (IMPC) of the breast is a relatively rare subtype of invasive ductal carcinoma and represents the most inherently aggressive form. Expression of incompatible blood group A antigen in cancer of type O patients has been reported in several types of cancer, however, the biosynthetic mechanism and the genetic basis remain unclear until today. The aim of the present case report study was to evaluate the expression of incompatible blood group A antigen and to identify the genetic basis of this expression in IMPC of the breast. One patient blood group O with Invasive Micropapillary Carcinoma was screened at Pathology Department of University Hospital CHU Ibn Rochd, Casablanca. ABH antigens expression was assessed by immunohistochemistry. ABO genotyping was performed by allele specific primers PCR-ASP and Exon 6 of ABO gene was sequenced with Sanger method. H antigen was expressed in endothelial and epithelial cells of normal tissue. However, H antigen expression was lost in both invasive micropapillary carcinomas. A antigen was expressed in IMPC with approximately 80% of positive cells. Tumor DNA was genotyped as heterozygous A/O. In normal DNA, we identified a single frameshift deletion c.320delA p.(Glu107Glyfs*12), which is removed from tumor DNA. Our findings suggest that incompatible A antigen expression in IMPC is due to glycosyltransferase A encoded by an A allele which is derived from O allele with a deletion at the position 320. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  8. [Expression characteristics of differentiation antigens on granulocytes in patients with megaloblastic anemia].

    Science.gov (United States)

    Zhou, Hai-Tao; Ke, Pei-Feng; Shen, Wen-Hong; Chen, Su-Ning; Wang, Guo-Zheng

    2013-08-01

    This study was aimed to explore the change characteristics of cell differentiation antigen (CD) on bone marrow (BM) granulocytes in patients,with megaloblastic anemia (MA). In combination with BM cell morphology, hemogram, level of blood serum folic acid, level of Vit B(12), cell genetics and biological examination data, the BM granulocytes differentiation antigens in 13 patients with MA were detected by flow cyto metry and analyzed retrospectively, in order to summarize the variation characteristics of CD13, CD33 and CD15 expressed on myeloid cells in patient with MA, including forward scatter light (FSC) and side scatter light (SSC) signal intensity, then these findings were compared with that in normal healthy persons. The results showed that the expression rates of CD13, CD15 and CD33 on granulocytic in patients with MA and normal healthy persons were (44.53 ± 16)%, (96.16 ± 2.67)%, (80.81 ± 14.71)% and (62.33 ± 11.02)%, (99.53 ± 0.46)%, (70.00 ± 7.81)% respectively, in which the expression rate of CD13 and CD15 in patients with MA decreased (P granulocytes in MA patients decreased (P granulocytes in patents with MA shows dysmaturity, but the expressing feature of differentiation antigens on BM granulocytes in MA patients also displays dysmaturity.These findings will contribute to the clinical diagnosis of MA patients.

  9. Epigenetic priming restores the HLA class-I antigen processing machinery expression in Merkel cell carcinoma

    DEFF Research Database (Denmark)

    Ritter, Cathrin; Fan, Kaiji; Paschen, Annette

    2017-01-01

    Merkel cell carcinoma (MCC) is a rare and aggressive, yet highly immunogenic skin cancer. The latter is due to its viral or UV-associated carcinogenesis. For tumor progression MCC has to escape the host's immuno-surveillance, e.g. by loss of HLA class-I expression. Indeed, a reduced HLA class......-I expression was observed in MCC tumor tissues and MCC cell lines. This reduced HLA class-I surface expression is caused by an impaired expression of key components of the antigen processing machinery (APM), including LMP2 and LMP7 as well as TAP1 and TAP2. Notably, experimental provisions of HLA class......-I binding peptides restored HLA class-I surface expression on MCC cells. Silencing of the HLA class-I APM is due to histone deacetylation as inhibition of histone deacetylases (HDACs) not only induced acetylation of histones in the respective promoter regions but also re-expression of APM components. Thus...

  10. Prognostic relevance of melanoma antigen D1 expression in colorectal carcinoma

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    Zeng Zhao-lei

    2012-08-01

    Full Text Available Abstract Background Melanoma antigen D1 (MAGED1 is a member of the type II melanoma antigen (MAGE family. The down-regulation of MAGED1 expression has been shown in breast carcinoma cell lines and in glioma stem cells and may play an important role in apoptosis and anti-tumorigenesis. However, there is no report on its clinical role in colorectal cancer (CRC. Methods We examined the expression of MAGED1 by qPCR in colorectal cancer tissues and their adjacent non-tumorous tissues taken from 6 cases and performed Western blotting and IHC analyses. In addition, we analyzed MAGED1 expression in 285 clinicopathologically characterized colorectal cancer patients. Results MAGED1 expression was significantly down-regulated in colorectal cancer tissues compared with adjacent non-tumorous tissues and was associated with clinical stage (p p = 0.001, N classification (p p p = 0.002. Patients with lower MAGED1 expression had a shorter survival time than those with higher MAGED1 expression. Univariate and multivariate analyses indicated that MAGED1 expression was an independent prognostic factors (p  Conclusions MAGED1 may serve as a novel prognostic biomarker of human colorectal cancer.

  11. S-Allyl cysteine alleviates inflammation by modulating the expression of NF-κB during chromium (VI)-induced hepatotoxicity in rats.

    Science.gov (United States)

    Anandasadagopan, S K; Sundaramoorthy, C; Pandurangan, A K; Nagarajan, V; Srinivasan, K; Ganapasam, S

    2017-11-01

    Hexavalent chromium (Cr (VI)) is a common environmental pollutant. Cr (VI) exposure can lead to severe damage to the liver, but the preventive measures to diminish Cr (VI)-induced hepatotoxicity need further study. S-allyl cysteine (SAC) is a constituent of garlic ( Allium sativum) and has many beneficial effects to humans and rodents. In this study, we intended to analyze the mechanistic role of SAC during Cr (VI)-induced hepatotoxicity. Male Wistar albino rats were induced with 17 mg/kg body weight to damage the liver. The Cr (VI)-induced rats were treated with 100 mg/kg body weight of SAC as an optimum dosage to treat hepatotoxicity. We observed that the levels of oxidants, lipid peroxidation and hydroxyl radical (OH(•)) were increased, and enzymatic antioxidants such as superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase were found to be decreased in Cr (VI)-induced rats. While treated with SAC, the levels of oxidants were decreased and enzymatic antioxidants were significantly ( p < 0.05) increased. Lysosomal enzyme activities were increased in Cr (VI)-induced rats and on treatment with SAC, the activities were significantly decreased. The expressions of nuclear factor-kappa B (p65-NF-κB), tumor necrosis factor α (TNF-α), and inducible nitric oxide synthase (iNOS) were increased during induction with Cr (VI). Subsequent administration of SAC to animals showed a decrease in the expressions of NF-κB, TNF-α, and iNOS. Results obtained from this study clearly demonstrated that SAC protects the liver cells from the Cr (VI)-induced free radical damage.

  12. Signal transduction by HLA class II antigens expressed on activated T cells

    DEFF Research Database (Denmark)

    Ødum, Niels; Martin, P J; Schieven, G L

    1991-01-01

    Human T cells express HLA class II antigens upon activation. Although activated, class II+ T cells can present alloantigens under certain circumstances, the functional role of class II antigens on activated T cells remains largely unknown. Here, we report that cross-linking of HLA-DR molecules...... after cross-linking CD4. Ligation of CD4 and class II molecules generated a synergistic effect of the intracellular free Ca2+ concentration response that required an interaction between the molecules on the cell surface. Since class II is the natural ligand for CD4, the present data suggest that class...... expressed on allospecific, CD4+ T clones and cell lines can function as transduction elements that trigger rapid cellular responses including tyrosine phosphorylation of cellular proteins and mobilization of Ca2+ from internal stores. The proteins phosphorylated on tyrosine were distinct from those observed...

  13. Capsular Polysaccharide Expression in Commensal Streptococcus Species: Genetic and Antigenic Similarities to Streptococcus pneumoniae

    OpenAIRE

    Skov Sørensen, Uffe B.; Kaihu Yao; Yonghong Yang; Hervé Tettelin; Mogens Kilian

    2016-01-01

    ABSTRACT Expression of a capsular polysaccharide is considered a hallmark of most invasive species of bacteria, including Streptococcus pneumoniae, in which the capsule is among the principal virulence factors and is the basis for successful vaccines. Consequently, it was previously assumed that capsule production distinguishes S.?pneumoniae from closely related commensals of the mitis group streptococci. Based on antigenic and genetic analyses of 187 mitis group streptococci, including 90 re...

  14. [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.

  15. Expression of proliferating cell nuclear antigen in cultured middle ear epithelial cells of the guinea pig.

    Science.gov (United States)

    Takeno, S; Hamamura, N; Hirakawa, K; Yajin, K

    1996-01-01

    Primary cultures of middle ear epithelium from the guinea pig were successfully established on type I collagen coated dishes. To characterize cellular outgrowth, antibodies to the proliferating cell nuclear antigen were used as a marker for spreading cells in the S phase of the cell cycle. A number of migrating epithelial cells positively stained for proliferating cell nuclear antigen after 7 and 14 days in culture. Confocal laser scanning microscopy was used to evaluate the localization pattern of this antigen, and the fluorescence intensity was quantified in different areas of the migrating epithelial sheet after various times in culture. Two distinct areas proved to be major sites of proliferating cell nuclear antigen expression. One was at the edge of the tissue explants from which multilayered epithelial cells had begun to migrate. The other was along the margin of the outgrowth, where the cells often had elongated shapes and were aligned in rows. The cells in both areas were identified as nonciliated cells; ciliated cells in the outgrowth showed little staining. We hypothesized that the outgrowth cells in this experiment might be identical to the migrating cells usually observed in renewing epithelia after injury. This model may provide a simple and reproducible method of evaluating the regenerative ability of the middle ear epithelium.

  16. Characterization of Citrobacter sp. line 328 as a source of Vi for a Vi-CRM(197) glycoconjugate vaccine against Salmonella Typhi.

    Science.gov (United States)

    Rondini, Simona; Micoli, Francesca; Lanzilao, Luisa; Pisoni, Ivan; Di Cioccio, Vito; Saul, Allan J; Martin, Laura B

    2012-11-26

    Salmonella enterica serovar Typhi is the causative agent of typhoid fever with over 22 million cases and over 200,000 deaths reported annually. A vaccine is much needed for use in young children and the Novartis Vaccines Institute for Global Health (NVGH) is developing a conjugate vaccine which targets S. Typhi Vi capsular polysaccharide. Here we describe a method suitable for industrial scale production of the Vi antigen based on expression by a Citrobacter line. We optimized the production of Vi by selecting a suitable Citrobacter strain (Citrobacter 328) that yields high and stable expression of Vi in chemically defined medium under industrial-scale fermentation conditions. Vi-CRM197 made using Vi from Citrobacter 328 elicited high anti-Vi antibody levels in mice and rabbits. Citrobacter 328 is a suitable strain for production of Vi for conjugate anti-Typhi vaccines. Being a BSL-1 organism, which grows in defined medium and stably produces high yields of Vi, it offers excellent potential for safe production of inexpensive vaccines for populations at risk of typhoid fever.

  17. Isolation and characterization of expression cDNA clones encoding antigens of Onchocerca volvulus infective larvae.

    Science.gov (United States)

    Unnasch, T R; Gallin, M Y; Soboslay, P T; Erttmann, K D; Greene, B M

    1988-01-01

    The isolation of recombinant cDNA clones expressing antigens found in Onchocerca volvulus infective larvae is described. To isolate such clones, an expression cDNA library constructed from adult O. volvulus RNA was screened with antiserum raised against infective larvae. One clone, designated lambda RAL-1 was characterized further. The recombinant antigen produced by lambda RAL-1 stimulates proliferation of peripheral blood mononuclear cells from O. volvulus infected humans. Lambda RAL-1 is derived from a 1450 bases message that encodes a protein with an apparent molecular weight of 42,000 in adult O. volvulus. The inserted DNA of lambda RAL-1 contains an open reading frame of 1008 bp. The amino acid sequence predicted by this open reading frame contains three repeats of the sequence KKPEDWD. The identification of clones such as lambda RAL-1 will provide quantities of purified antigens sufficient to begin to study the immune response to and explore the development of immunity against the infectious form of the parasite. Images PMID:2455736

  18. Potentiation of anthrax vaccines using protective antigen-expressing viral replicon vectors.

    Science.gov (United States)

    Wang, Hai-Chao; An, Huai-Jie; Yu, Yun-Zhou; Xu, Qing

    2015-02-01

    DNA vaccines require improvement for human use because they are generally weak stimulators of the immune system in humans. The efficacy of DNA vaccines can be improved using a viral replicon as vector to administer antigen of pathogen. In this study, we comprehensively evaluated the conventional non-viral DNA, viral replicon DNA or viral replicon particles (VRP) vaccines encoding different forms of anthrax protective antigen (PA) for specific immunity and protective potency against anthrax. Our current results clearly suggested that these viral replicon DNA or VRP vaccines derived from Semliki Forest virus (SFV) induced stronger PA-specific immune responses than the conventional non-viral DNA vaccines when encoding the same antigen forms, which resulted in potent protection against challenge with the Bacillus anthracis strain A16R. Additionally, the naked PA-expressing SFV replicon DNA or VRP vaccines without the need for high doses or demanding particular delivery regimens elicited robust immune responses and afforded completely protective potencies, which indicated the potential of the SFV replicon as vector of anthrax vaccines for use in clinical application. Therefore, our results suggest that these PA-expressing SFV replicon DNA or VRP vaccines may be suitable as candidate vaccines against anthrax. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Extinction of Tumor Antigen Expression by SF2/ASF in JCV-Transformed Cells.

    Science.gov (United States)

    Uleri, Elena; Beltrami, Sarah; Gordon, Jennifer; Dolei, Antonina; Sariyer, Ilker Kudret

    2011-07-01

    The human neurotropic polyomavirus JC (JCV) induces a broad range of neural-origin tumors in experimental animals and has been repeatedly detected in several human cancers, most notably neural crest-origin tumors including medulloblastomas and glioblastomas. The oncogenic activity of JCV is attributed to the viral early gene products, large T and small t antigens, as evident by results from in vitro cell culture and in vivo animal studies. Recently, we have shown that alternative splicing factor, SF2/ASF, has the capacity to exert a negative effect on transcription and splicing of JCV genes in glial cells through direct association with a specific DNA motif within the viral promoter region. Here, we demonstrate that SF2/ASF suppresses large T antigen expression in JCV-transformed tumor cell lines, and the expression of SF2/ASF in such tumor cells thereby inhibits the transforming capacity of the viral tumor antigens. Moreover, down-regulation of SF2/ASF in viral-transformed tumor cell lines induces growth and proliferation of the tumor cells. Mapping analysis of the minimal peptide domain of SF2/ASF responsible for JCV promoter silencing and tumor suppressor activity suggests that amino acid residues 76 to 100 of SF2/ASF are functionally sufficient to suppress the growth of the tumor cells. These observations demonstrate a role for SF2/ASF in JCV-mediated cellular transformation and provide a new avenue of research to pathogenic mechanisms of JCV-induced tumors.

  20. 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

    NAc-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......NAc-I as the major enzyme controlling the expression of cancer-associated sialyl-Tn antigen in gastrointestinal tissues....

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

    DEFF Research Database (Denmark)

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

    2015-01-01

    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...... inhibitors has been proposed as an attractive strategy to increase the expression of cancer/testis antigens in tumors before immunotargeting; however, neither ADAM2, CALR3 nor SAGE1 could be significantly induced in lung and breast cancer cell lines using this strategy. Our results suggest that ADAM2, CALR3...... 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...

  2. Intragastric immunization with recombinant Lactobacillus casei expressing flagellar antigen confers antibody-independent protective immunity against Salmonella enterica serovar Enteritidis

    NARCIS (Netherlands)

    Kajikawa, A.; Satoh, E.; Leer, R.J.; Yamamoto, S.; Igimi, S.

    2007-01-01

    A recombinant Lactobacillus casei expressing a flagellar antigen from Salmonella enterica serovar Enteritidis was constructed and evaluated as a mucosal vaccine. Intragastric immunization of the recombinant strain conferred protective immunity against Salmonella infection in mice. This immunization

  3. The C-terminal N-glycosylation sites of the human alpha1,3/4-fucosyltransferase III, -V, and -VI (hFucTIII, -V, adn -VI) are necessary for the expression of full enzyme activity.

    Science.gov (United States)

    Christensen, L L; Jensen, U B; Bross, P; Orntoft, T F

    2000-09-01

    The alpha1,3/4-fucosyltransferases are involved in the synthesis of fucosylated cell surface glycoconjugates. Human alpha1,3/4-fucosyltransferase III, -V, and -VI (hFucTIII, -V, and -VI) contain two conserved C-terminal N-glycosylation sites (hFucTIII: Asn154 and Asn185; hFucTV: Asn167 and Asn198; and hFucTVI: Asn153 and Asn184). In the present study, we have analyzed the functional role of these potential N-glycosylation sites, laying the main emphasis on the sites in hFucTIII. Tunicamycin treatment completely abolished hFucTIII enzyme activity while castanospermine treatment diminished hFucTIII enzyme activity to approximately 40% of the activity of the native enzyme. To further analyze the role of the conserved N-glycosylation sites in hFucTIII, -V, and -VI, we made a series of mutant genomic DNAs in which the asparagine residues in the potential C-terminal N-glycosylation sites were replaced by glutamine. Subsequently, the hFucTIII, -V, and -VI wild type and the mutants were expressed in COS-7 cells. All the mutants exhibited lower enzyme activity than the wild type and elimination of individual sites had different effects on the activity. The mutations did not affect the protein level of the mutants in the cells, but reduced the molecular mass as predicted. Kinetic analysis of hFucTIII revealed that lack of glycosylation at Asn185 did not change the Km values for the oligosaccharide acceptor and the nucleotide sugar donor. The present study demonstrates that hFucTIII, -V, and -VI require N-glycosylation at the two conserved C-terminal N-glycosylation sites for expression of full enzyme activity.

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

    Directory of Open Access Journals (Sweden)

    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.

  5. Multiple cancer/testis antigens are preferentially expressed in hormone-receptor negative and high-grade breast cancers.

    Directory of Open Access Journals (Sweden)

    Yao-Tseng Chen

    Full Text Available BACKGROUND: Cancer/testis (CT antigens are protein antigens normally expressed only in germ cells of testis, and yet are expressed in a proportion of a wide variety of human cancers. CT antigens can elicit spontaneous immune responses in cancer patients with CT-positive cancers, and CT antigen-based therapeutic cancer vaccine trials are ongoing for "CT-rich" tumors. Although some previous studies found breast cancer to be "CT-poor", our recent analysis identified increased CT mRNA transcripts in the ER-negative subset of breast cancer. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we performed a comprehensive immunohistochemical study to investigate the protein expression of eight CT genes in 454 invasive ductal carcinomas, including 225 ER/PR/HER2-negative (triple-negative carcinomas. We found significantly more frequent expression of all eight CT antigens in ER-negative cancers, and five of them--MAGEA, CT7, NY-ESO-1, CT10 and CT45, were expressed in 12-24% of ER-negative cancers, versus 2-6% of ER-positive cancers (p2 cm. CONCLUSIONS/SIGNIFICANCE: CT antigens are preferentially expressed in hormone receptor-negative and high-grade breast cancer. Considering the limited treatment options for ER/PR/HER2 triple-negative breast cancer, the potential of CT-based immunotherapy should be explored.

  6. Burkholderia mallei and Burkholderia pseudomallei cluster 1 type VI secretion system gene expression is negatively regulated by iron and zinc.

    Science.gov (United States)

    Burtnick, Mary N; Brett, Paul J

    2013-01-01

    Burkholderia mallei is a facultative intracellular pathogen that causes glanders in humans and animals. Previous studies have demonstrated that the cluster 1 type VI secretion system (T6SS-1) expressed by this organism is essential for virulence in hamsters and is positively regulated by the VirAG two-component system. Recently, we have shown that T6SS-1 gene expression is up-regulated following internalization of this pathogen into phagocytic cells and that this system promotes multinucleated giant cell formation in infected tissue culture monolayers. In the present study, we further investigated the complex regulation of this important virulence factor. To assess T6SS-1 expression, B. mallei strains were cultured in various media conditions and Hcp1 production was analyzed by Western immunoblotting. Transcript levels of several VirAG-regulated genes (bimA, tssA, hcp1 and tssM) were also determined using quantitative real time PCR. Consistent with previous observations, T6SS-1 was not expressed during growth of B. mallei in rich media. Curiously, growth of the organism in minimal media (M9G) or minimal media plus casamino acids (M9CG) facilitated robust expression of T6SS-1 genes whereas growth in minimal media plus tryptone (M9TG) did not. Investigation of this phenomenon confirmed a regulatory role for VirAG in this process. Additionally, T6SS-1 gene expression was significantly down-regulated by the addition of iron and zinc to M9CG. Other genes under the control of VirAG did not appear to be as tightly regulated by these divalent metals. Similar results were observed for B. pseudomallei, but not for B. thailandensis. Collectively, our findings indicate that in addition to being positively regulated by VirAG, B. mallei and B. pseudomallei T6SS-1 gene expression is negatively regulated by iron and zinc.

  7. Burkholderia mallei and Burkholderia pseudomallei cluster 1 type VI secretion system gene expression is negatively regulated by iron and zinc.

    Directory of Open Access Journals (Sweden)

    Mary N Burtnick

    Full Text Available Burkholderia mallei is a facultative intracellular pathogen that causes glanders in humans and animals. Previous studies have demonstrated that the cluster 1 type VI secretion system (T6SS-1 expressed by this organism is essential for virulence in hamsters and is positively regulated by the VirAG two-component system. Recently, we have shown that T6SS-1 gene expression is up-regulated following internalization of this pathogen into phagocytic cells and that this system promotes multinucleated giant cell formation in infected tissue culture monolayers. In the present study, we further investigated the complex regulation of this important virulence factor. To assess T6SS-1 expression, B. mallei strains were cultured in various media conditions and Hcp1 production was analyzed by Western immunoblotting. Transcript levels of several VirAG-regulated genes (bimA, tssA, hcp1 and tssM were also determined using quantitative real time PCR. Consistent with previous observations, T6SS-1 was not expressed during growth of B. mallei in rich media. Curiously, growth of the organism in minimal media (M9G or minimal media plus casamino acids (M9CG facilitated robust expression of T6SS-1 genes whereas growth in minimal media plus tryptone (M9TG did not. Investigation of this phenomenon confirmed a regulatory role for VirAG in this process. Additionally, T6SS-1 gene expression was significantly down-regulated by the addition of iron and zinc to M9CG. Other genes under the control of VirAG did not appear to be as tightly regulated by these divalent metals. Similar results were observed for B. pseudomallei, but not for B. thailandensis. Collectively, our findings indicate that in addition to being positively regulated by VirAG, B. mallei and B. pseudomallei T6SS-1 gene expression is negatively regulated by iron and zinc.

  8. Burkholderia mallei and Burkholderia pseudomallei Cluster 1 Type VI Secretion System Gene Expression Is Negatively Regulated by Iron and Zinc

    Science.gov (United States)

    Burtnick, Mary N.; Brett, Paul J.

    2013-01-01

    Burkholderia mallei is a facultative intracellular pathogen that causes glanders in humans and animals. Previous studies have demonstrated that the cluster 1 type VI secretion system (T6SS-1) expressed by this organism is essential for virulence in hamsters and is positively regulated by the VirAG two-component system. Recently, we have shown that T6SS-1 gene expression is up-regulated following internalization of this pathogen into phagocytic cells and that this system promotes multinucleated giant cell formation in infected tissue culture monolayers. In the present study, we further investigated the complex regulation of this important virulence factor. To assess T6SS-1 expression, B. mallei strains were cultured in various media conditions and Hcp1 production was analyzed by Western immunoblotting. Transcript levels of several VirAG-regulated genes (bimA, tssA, hcp1 and tssM) were also determined using quantitative real time PCR. Consistent with previous observations, T6SS-1 was not expressed during growth of B. mallei in rich media. Curiously, growth of the organism in minimal media (M9G) or minimal media plus casamino acids (M9CG) facilitated robust expression of T6SS-1 genes whereas growth in minimal media plus tryptone (M9TG) did not. Investigation of this phenomenon confirmed a regulatory role for VirAG in this process. Additionally, T6SS-1 gene expression was significantly down-regulated by the addition of iron and zinc to M9CG. Other genes under the control of VirAG did not appear to be as tightly regulated by these divalent metals. Similar results were observed for B. pseudomallei, but not for B. thailandensis. Collectively, our findings indicate that in addition to being positively regulated by VirAG, B. mallei and B. pseudomallei T6SS-1 gene expression is negatively regulated by iron and zinc. PMID:24146925

  9. Nogo-B receptor expression correlates negatively with malignancy grade and ki-67 antigen expression in invasive ductal breast carcinoma.

    Science.gov (United States)

    Pula, Bartosz; Olbromski, Mateusz; Owczarek, Tomasz; Ambicka, Aleksandra; Witkiewicz, Wojciech; Ugorski, Maciej; Rys, Janusz; Zabel, Maciej; Dziegiel, Piotr; Podhorska-Okolow, Marzena

    2014-09-01

    Nogo-B receptor (NgBR) has been shown to be involved in endothelial cell chemotaxis and morphogenesis. However, few studies analyzing its expression in cancer cells have been performed. We examined NgBR expression in 233 patients with invasive ductal breast carcinoma (IDC) and corresponding non-malignant breast tissues (NMBT) on mRNA (real-time polymerase chain reaction) and protein levels (immunohistochemistry; IHC and western-blot analysis). NgBR expression was found also analyzed in breast cancer cell lines of varying invasiveness. NgBR expression was increased in IDC compared to NMBT on the mRNA (p=0.0007) and protein level (p=0.018). NgBR expression decreased significantly with IDC malignancy grade and correlated negatively with the Ki-67 antigen expression (r=-0.18; p=0.0005). High NgBR mRNA expression was associated with estrogen receptor negativity (p=0.0023) and the triple-negative phenotype of the tumors (p=0.0129). NgBR may be involved in IDC development, however, its role in its progression requires further research. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  10. Antigen Binding and Site-Directed Labeling of Biosilica-Immobilized Fusion Proteins Expressed in Diatoms

    Energy Technology Data Exchange (ETDEWEB)

    Ford, Nicole R.; Hecht, Karen A.; Hu, Dehong; Orr, Galya; Xiong, Yijia; Squier, Thomas; Rorrer, Gregory L.; Roesijadi, Guritno

    2016-01-08

    The diatom Thalassiosira pseudonana was genetically modified to express biosilica-targeted fusion proteins incorporating a tetracysteine tag for site-directed labeling with biarsenical affinity probes and either EGFP or single chain antibody to test colocalization of probes with the EGFP-tagged recombinant protein or binding of biosilica-immobilized antibodies to large and small molecule antigens, respectively. Site-directed labeling with the biarsenical probes demonstrated colocalization with EGFP-encoded proteins in nascent and mature biosilica, supporting their use in studying biosilica maturation. Isolated biosilica transformed with a single chain antibody against either the Bacillus anthracis surface layer protein EA1 or small molecule explosive trinitrotoluene (TNT) effectively bound the respective antigens. A marked increase in fluorescence lifetime of the TNT surrogate Alexa Fluor 555-trinitrobenzene reflected the high binding specificity of the transformed isolated biosilica. These results demonstrated the potential use of biosilica-immobilized single chain antibodies as binders for large and small molecule antigens in sensing and therapeutics.

  11. Construction of two Listeria ivanovii attenuated strains expressing Mycobacterium tuberculosis antigens for TB vaccine purposes.

    Science.gov (United States)

    Lin, Qingqing; Zhou, Mengying; Xu, Zongkai; Khanniche, Asma; Shen, Hao; Wang, Chuan

    2015-02-20

    Bacillus Calmette-Guerin (BCG) has failed in complete control of tuberculosis (TB), thus, novel tuberculosis vaccines are urgently needed. We have constructed several TB vaccine candidates, which are characterized by the use of Listeria ivanovii (LI) strain as an antigen delivery vector. Two L. ivanovii attenuated recombinant strains L. ivanovii△actAplcB-Rv0129c and L. ivanovii△actAplcB-Rv3875 were successfully screened. Results from genome PCR and sequencing showed that the Mycobacterium tuberculosis antigen gene cassette coding for Ag85C or ESAT-6 protein respectively had been integrated into LI genome downstream of mpl gene. Western blot confirmed the secretion of Ag85C or ESAT-6 protein from the recombinant LI strains. These two recombinant strains showed similar growth curves as wide type strain in vitro. In vivo, they transiently propagated in mice spleen and liver, and induced specific CD8(+) IFN-γ secretion. Therefore, in this paper, two novel LI attenuated strains expressing specific TB antigens were successfully constructed. The promising growth characteristics in mice immune system and the capability of induction of IFN-γ secretion make them of potential interest for development of TB vaccines. Copyright © 2015 Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    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.

  13. Cloning, expression and purification of Mycobacterium tuberculosis ESAT-6 and CFP-10 antigens.

    Science.gov (United States)

    Mahmoudi, Shima; Mamishi, Setareh; Ghazi, Mona; Hosseinpour Sadeghi, Reihaneh; Pourakbari, Babak

    2013-12-01

    ESAT-6 (6-kDaearly secretory antigenic target) and CFP-10 (10-kDa culture filtrate protein) have been described as dominant antigens recognized by T-cells and considered as virulence factors in Mycobacterium tuberculosis. The aim of this study was to clone, express and purify recombinant ESAT-6 andCFP-10 proteins of M. tuberculosis in soluble form. ESAT-6 andCFP-10 genes were amplified by PCR, cloned into pET32a (+) vector, and overexpress-ed using isopropyl-beta-D-thiogalactopyranoside in E. coli BL21 (DE3). ESAT-6 andCFP-10 proteins were purified by Ni-NTA affinity chromatography and were detected by anti- ESAT-6 and anti -CFP-10 antibodies. ESAT-6 andCFP-10 genes were successfully expressed and purified. Anti- ESAT-6 and anti-CFP-10 antibodies were produced after induction of immunization against purified ESAT-6 andCFP-10 proteins in rabbit. In this study, we cloned, expressed and purified sufficient amounts of ESAT-6 andCFP-10 and it would be tested for the development of diagnostic kit for M. tuberculosis in future.

  14. Targeted expression of anthrax protective antigen by Lactobacillus gasseri as an anthrax vaccine.

    Science.gov (United States)

    Mohamadzadeh, Mansour; Durmaz, Evelyn; Zadeh, Mojgan; Pakanati, Krishna Chaitanya; Gramarossa, Matthew; Cohran, Valeria; Klaenhammer, Todd R

    2010-08-01

    Induction of protective immunity against pathogenic microbes, including Bacillus anthracis, requires efficient vaccines that potentiate antibody avidity and increase T-cell longevity. We recently reported that the delivery of targeted B. anthracis protective antigen (PA) genetically fused to a DC-binding peptide (DCpep) by Lactobacillus acidophilus induced mucosal and systemic immunity against B. anthracis challenge in mice. Improvement of this oral vaccine strategy was attempted by use of the high copy and genetically stable q-replicating vector, pTRKH2, for expression of the targeted PA fusion protein in Lactobacillus gasseri, a common human commensal microbe, to vaccinate animals against anthrax Sterne infection. Oral application of L. gasseri expressing the PA-DCpep fusion proteins elicited robust PA-neutralizing antibody and T-cell mediated immune responses against anthrax Sterne challenge, resulting in complete animal survival. Collectively, this improved expression vaccine strategy reduced the number of inoculations and length of the boosting period, leading to animal protection via efficacious bacterial adjuvanticity and safe oral delivery of this vaccine to mucosal immune cells, including dendritic cells. Lactobacillus-based delivery offers tremendous practical advantages. Recombinant antigens such as PA would not require chemical coupling agents, and the recombinant bacteria can be administered orally where upon both mucosal and systemic immune responses are elicited.

  15. Children with postsurgical capillary leak syndrome can be distinguished by antigen expression on neutrophils and monocytes

    Science.gov (United States)

    Tarnok, Attila; Pipek, Michal; Valet, Guenter; Richter, Jacqueline; Hambsch, Joerg; Schneider, Peter

    1999-04-01

    Our initial studies indicate that children who develop post- operative capillary leak syndrome (CLS) following cardiac surgery with cardiopulmonary bypass (CPB) can be distinguished based on their pre-operative level of circulating cytokines an adhesion molecules. We tested flow cytometric analysis of surface antigen expression as a potential assay for risk assessment of CLS. 24th preoperative blood samples were stained with monoclonal antibodies for the adhesion molecules ICAM-1, LFA1, MAC1, (beta) -integrin, activation markers CD25, CD54, CD69, HLA- DR, CD14 or CD4. Cells were measured on a dual-laser flow cytometer calibrated with microbeads. Antigen expression was detected as mean fluorescence intensity. The data indicate, that neutrophils of CLS patients express preoperatively higher levels of LFA1 and monocytes higher levels of HLA-DR and activation markers thus are in a state of activation. This could in combination with surgical trauma and CPB lead to their additional stimulation and migration into sites of inflammation and induce postoperative CLS. It is planned to set up a Flow-Classification program for individual risk assessment. By discriminate analysis over 80 percent of the patients were correctly classified. Our preliminary study indicates that flow cytometry with its low samples requirements and rapid access of the results could be a powerful tool to perform risk assessment prior to pediatric open heart surgery.

  16. Oral Vaccination Against Anthrax Using a Transgenic Plant Expressing Protective Antigen.

    Science.gov (United States)

    1996-09-01

    34 Anal. Biochem. 169:227-223. 10. Gould, J., M. Devey, 0. Hasegawa, E.C. Ulian, G. Peterson, and R.H. Smith (1991) "Transformation of Zea mays L. using...Against Anthrax Using a Transgenic Plant Expressing Protective Antigen PRINCIPAL INVESTIGATOR: Dr. Karen K. Oishi CONTRACTING ORGANIZATION: CropTech/Vet...COVERED I September 1996 Final - Phase I, 15 Auxg 95-14 Aug 96 4. TITLE AND 9UBTITLE 5. FUNDING NUMBERS Oral Vaccination Against Anthrax Using a Transgenic

  17. Aberrant expression of tumor-associated carbohydrate antigen Globo H in thyroid carcinoma.

    Science.gov (United States)

    Cheng, Shih-Ping; Yang, Po-Sheng; Chien, Ming-Nan; Chen, Ming-Jen; Lee, Jie-Jen; Liu, Chien-Liang

    2016-12-01

    The induction of tumor-associated carbohydrate antigen results from altered glycosylation in transformed cells. Globo H is a hexasaccharide glycosphingolipid overexpressed on malignancies of epithelial origin and has become an attractive vaccine target. We aimed to investigate the expression patterns and prognostic value of Globo H in thyroid neoplasms. Globo H expression was examined by immunohistochemical analysis using commercial and in-house tissue microarrays. The expression was correlated with clinicopathologic characteristics in papillary thyroid cancer. Normal or benign thyroid lesions were negative for Globo H expression. Globo H was positive in 33% medullary, 24% papillary, 11% undifferentiated, and 8% follicular thyroid cancer. Globo H expression in papillary thyroid cancer was associated with extrathyroidal invasion (P = 0.017), BRAF mutation (P = 0.010), AMES high risk (P = 0.045), and increased ATA risk of recurrence (P = 0.022). Globo H is specifically expressed in a subset of thyroid malignancies. In papillary thyroid cancer, Globo H expression is associated with invasiveness and BRAF mutation. Immunotherapy targeting Globo H may have potential applications in thyroid cancer. J. Surg. Oncol. 2016;114:853-858. © 2016 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  18. 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...

  19. Overlapping antigenic repertoires of variant antigens expressed on the surface of erythrocytes infected by Plasmodium falciparum

    DEFF Research Database (Denmark)

    Giha, H A; Staalsoe, T; Dodoo, D

    1999-01-01

    genes and undergoes clonal variation. Using agglutination and mixed agglutination tests and flow cytometry to analyse the recognition of variant antigens on parasitized erythrocytes by plasma antibodies from individuals living in Daraweesh in eastern Sudan, an area of seasonal and unstable malaria...

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

    Science.gov (United States)

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

    2008-01-01

    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. PMID:18479514

  1. 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.

  2. Soluble Expression and Characterization of Biologically Active Bacillus anthracis Protective Antigen in Escherichia coli

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    Nagendra Suryanarayana

    2016-01-01

    Full Text Available Bacillus anthracis secretory protein protective antigen (PA is primary candidate for subunit vaccine against anthrax. Attempts to obtain large quantity of PA from Escherichia coli expression system often result in the formation of insoluble inclusion bodies. Therefore, it is always better to produce recombinant proteins in a soluble form. In the present study, we have obtained biologically active recombinant PA in small scale E. coli shake culture system using three different expression constructs. The PA gene was cloned in expression vectors bearing trc, T5, and T7 promoters and transformed into their respective E. coli hosts. The growth conditions were optimized to obtain maximum expression of PA in soluble form. The expression construct PA-pET32c in DE3-pLysS E. coli host resulted in a maximum production of soluble PA (15 mg L−1 compared to other combinations. Purified PA was subjected to trypsin digestion and binding assay with lethal factor to confirm the protein’s functionality. Biological activity was confirmed by cytotoxicity assay on J774.1 cells. Balb/c mice were immunized with PA and the immunogenicity was tested by ELISA and toxin neutralization assay. This study highlights the expression of soluble and biologically active recombinant PA in larger quantity using simpler E. coli production platform.

  3. Antibody production by injection of living cells expressing non self antigens as cell surface type II transmembrane fusion protein.

    Science.gov (United States)

    Nizet, Yannick; Gillet, Laurent; Schroeder, Hélène; Lecuivre, Corinne; Louahed, J; Renauld, J-C; Gianello, Pierre; Vanderplasschen, Alain

    2011-03-31

    Antigen expression and purification are laborious, time consuming and frequently difficult steps in the process of antibody production. In the present study, we developed a method avoiding these two steps. This method relies on the injection of histocompatible living cells stably expressing the antigen as a cell surface type II transmembrane fusion protein. A vector, nicknamed pCD1-CD134L, was constructed to express the antigen fused at the carboxyterminal end of the human CD134 ligand (CD134L) type II transmembrane protein on the surface of eucaryotic cells. This vector was shown to induce cell surface expression of epitopes from human c-Myc (soluble protein), uterogloblin-related protein 1 (secreted protein) and CD94 (type II transmembrane protein). Using this vector, we developed a method to produce antibodies without antigen production. The flowchart of this method is as follows: (i) cloning of the antigen in the pCD1-CD134L vector; (ii) production of a histocompatible cell line stably expressing the CD134L-antigen fusion protein; (iii) testing for cell surface expression of the fusion protein by targeting the CD134L carrier; and (iv) prime-boost immunisation with living cells expressing the fusion protein. This method was successfully used for production of polyclonal antibodies raised against Ixodes ricinus calreticulin (secreted protein) in mice and for production of monoclonal antibodies raised against an epitope of Vaccinia virus A56 (type I transmembrane protein) protein in rat. The present study is the first to demonstrate the use of a type II transmembrane protein as a carrier for cell surface display of antigens. Copyright © 2011 Elsevier B.V. All rights reserved.

  4. Unique antigenic gene expression at different developmental stages of Trichinella pseudospiralis.

    Science.gov (United States)

    Wu, X P; Liu, X L; Wang, X L; Blaga, R; Fu, B Q; Liu, P; Bai, X; Wang, Z J; Rosenthal, B M; Shi, H N; Sandrine, L; Vallee, I; Boireau, P; Wang, F; Zhou, X N; Zhao, Y; Liu, M Y

    2013-05-20

    Parasite-induced and parasite-regulated larval capsule formation and host immunosuppression are two major characteristics that are unique in Trichinella spp. infections, but the molecule(s) and mechanism(s) that mediate these processes remain largely unknown. Trichinella pseudospiralis and Trichinella spiralis, are obviously different with respect to these two characteristics. A comparative study of these two species, in particular their antigen expression profiles at different developmental stages (the main molecules involved in the cross-talk or interaction between each parasite and its host), may help us better understand the parasite molecules and mechanisms involved. Here, we constructed cDNA libraries from T. pseudospiralis adults (Ad), newborn larvae (NBL) and muscle larvae (ML) mRNA and screened them with pig anti-T. pseudospiralis serum collected 26, 32 and 60 days post-infection (p.i.). The most abundant antigens were found to vary among life-cycle stages. Pyroglutamy peptidase 1-like and 6-phosphogluconolactonase-like genes predominated in the Ad stage and a serine protease (SS2-1-like gene) predominated in NBL similar to that observed in T. spiralis. Muscle larvae expressed proteasome activator complex subunit 3-like and 21 kDa excretory/secretory protein-like genes. This study indicated that parasites of two species may utilise different molecules and mechanisms for larvae capsule formation and host immunosuppression during their infections. Proteins of antigenic genes identified in this study may be also good candidates for diagnosis, treatment or vaccination for T. pseudospiralis infection, and also for the differential diagnosis of two species' infections. Copyright © 2013 Elsevier B.V. All rights reserved.

  5. Sarcoidosis Th17 Cells are ESAT-6 Antigen Specific but Demonstrate Reduced IFN-γ Expression

    Science.gov (United States)

    Richmond, Bradley W.; Ploetze, Kristen; Isom, Joan; Chambers-Harris, Isfahan; Braun, Nicole A.; Taylor, Thyneice; Abraham, Susamma; Mageto, Yolanda; Culver, Dan A.; Oswald-Richter, Kyra A.; Drake, Wonder P.

    2013-01-01

    Rationale Sarcoidosis is a granulomatous disease of unknown etiology. Many patients with sarcoidosis demonstrate antigen-specific immunity to mycobacterial virulence factors. Th-17 cells are crucial to the immune response in granulomatous inflammation, and have recently been shown to be present in greater numbers in the peripheral blood and bronchoalveolar lavage (BAL) fluid (BALF) of sarcoidosis patients than healthy controls. It is unclear whether Th-17 cells in sarcoidosis are specific for mycobacterial antigens, or whether they have similar functionality to control Th-17 cells. Methods Flow cytometry was used to determine the numbers of Th-17 cells present in the peripheral blood and BALF of patients with sarcoidosis, the percentage of Th-17 cells that were specific to the mycobacterial virulence factor ESAT-6, and as well as to assess IFN-γ expression in Th-17 cells following polyclonal stimulation. Results Patients with sarcoidosis had greater numbers of Th-17 cells in the peripheral blood and BALF than controls and produced significantly more extracellular IL-17A (p=0.03 and p=0.02, respectively). ESAT-6 specific Th-17 cells were present in both peripheral blood and BALF of sarcoidosis patients (psarcoidosis patients produced less IFN-γ than healthy controls. Conclusions Patients with sarcoidosis have mycobacterial antigen-specific Th-17 cells peripherally and in sites of active sarcoidosis involvement. Despite the Th1 immunophenotype of sarcoidosis immunology, the Th-17 cells have reduced IFN-γ expression, compared to healthy controls. This reduction in immunity may contribute to sarcoidosis pathogenesis. PMID:23073617

  6. Variability in expression of antigens responsible for serotype specificity in Candida albicans.

    Science.gov (United States)

    Barturen, B; Bikandi, J; San Millán, R; Moragues, M D; Regulez, P; Quindós, G; Pontón, J

    1995-07-01

    The monoclonal antibody (mAb) B9E, which reacts with a cell wall surface determinant of Candida albicans serotype A, and a polyclonal monospecific antiserum against the antigen 6 (IF6) were used to investigate the expression of the antigens responsible for the serotype specificity in C. albicans under different growth conditions. By indirect immunofluorescence, both antibodies reacted with the cell wall surface of serotype A yeast cells and germ tubes grown in vitro but no reactivity was observed with serotype B yeast cells. In some cases, only a weak reactivity restricted to a zone close to the parent yeast cell was observed in serotype B germ tubes stained with mAb B9E. Both antibodies reacted strongly with yeast cells and germ tubes present in kidney abscesses from rabbits infected with both serotypes, but only serotype A yeast cells and germ tubes present in smears from patients with vulvovaginal candidiasis reacted with B9E and IF6 antibodies. The expression of antigens reactive with both antibodies was modulated by the pH of the environment in which the fungus was grown. Both antibodies showed a similar pattern of reactivity when studied with a spectrofluorometer. Serotype A yeast cells showed maximum reactivity when cells were grown on Sabouraud dextrose broth supplemented with yeast extract at pH 4.6. The lowest reactivity was observed in cells grown at pH 2.0. Conversely, the reactivity of serotype B yeast cells increased at alkaline pH values, the highest being in cells grown at pH values of 7.2 and 9.5.(ABSTRACT TRUNCATED AT 250 WORDS)

  7. Association between CTL precursor frequency to HLA-C mismatches and HLA-C antigen cell surface expression

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    Moshe eIsraeli

    2014-10-01

    Full Text Available Previous studies showed the relevance of the cytotoxic T cell precursor frequency assay (CTLp for prediction of the outcome of HLA mismatched hematopoietic cell transplantation (HCT. Recently it has been shown that HLA-C cell surface expression is correlated with virus specific cytotoxic T cell responses and viremia control in HIV patients.The aim of the current study was to investigate the association between HLA-C antigen expression and the CTLp frequency to the mismatched HLA-C antigen.In total 115 recipient–donor pairs, for whom a successful CTLp assay was performed, were evaluated for this pilot study. All donor-recipient pairs were matched at 9/10 alleles with a single mismatch at the HLA-C locus. Antigen expression level of the mismatched HLA-C allele for each recipient and donor was based on the MFI values as described by Apps et al (Science, 2013.The cell surface expression of recipient’s mismatched HLA-C antigen was significantly lower among CTLp negative (n=59 compared to CTLp positive (n=56 pairs (154 and 193 MFI units, respectively; p=0.0031. This difference was more pronounced in donor-recipient pairs that were mismatched for amino-acid residue-116 located in the groove of the HLA-C antigen, suggesting the importance of peptide binding in the allo-recognition. Furthermore, in the particular case of low expression of the recipient mismatched HLA-C antigen (MFI<115, CTLp reactivity depended on HLA-C expression level in the donor; the median MFI of donor’s mismatched HLA-C antigen was 114 in CTLp negative cases (n=26, while in CTLp positive cases (n=15 the median MFI of donor’s HLA-C antigen was 193. (P=0.0093.We conclude that the expression level of the donor and recipient mismatched HLA-C antigens affect CTLp outcome. HLA-C antigen expression levels in combination with the CTLp assay may prove useful for the prediction of the clinical outcome of HLA-C mismatched HCT.

  8. Carbohydrate antigen expression in primary tumors, metastatic lesions, and serous effusions from patients diagnosed with epithelial ovarian carcinoma: evidence of up-regulated Tn and Sialyl Tn antigen expression in effusions.

    Science.gov (United States)

    Davidson, B; Berner, A; Nesland, J M; Risberg, B; Kristensen, G B; Tropé, C G; Bryne, M

    2000-09-01

    The object of this study was the investigation of carbohydrate antigen expression in malignant epithelial cells and benign mesothelial cells in serous effusions from patients diagnosed with epithelial ovarian carcinomas. In addition, to compare antigen expression in carcinoma cells in effusions with those of corresponding primary tumors and metastatic lesions. Sections from 63 malignant effusions from ovarian carcinoma patients and 15 reactive effusions were immunohistochemically stained, using 5 monoclonal antibodies for Lewis(y), Sialyl Lewis(x), Tn, and Sialyl Tn antigens. Tissue sections (n = 97) from corresponding primary ovarian carcinomas and metastatic lesions, as well as from 12 malignant mesotheliomas, were additionally stained using the above panel. Staining for the 4 antigens was seen in carcinoma cells in serous effusions in the majority of cases (range = 71% to 85%). In contrast, immunoreactivity was detected in mesothelial cells in only 6% to 23% of the specimens studied (P < .001 for all 5 markers). With the exception of B3 antibody against Lewis(y) antigen, malignant mesotheliomas stained negative, infrequently showing focal immunoreactivity. An up-regulation of Tn and Sialyl Tn expression was detected in carcinoma cells in effusions when compared with both primary tumors (P < .003 and P < .007, respectively) and metastatic lesions (P < .034 and .041, respectively). Cancer-associated carbohydrate antigens can thus be used as an adjunct in the differentiation between malignant epithelial and reactive mesothelial cells. Ovarian carcinoma cells in effusions show up-regulation of Tn and Sialyl Tn, possibly representing a transient phenotypic alteration facilitating metastasis.

  9. Systemic minor histocompatibility antigen expression in blood endothelial cells prevents T cell-mediated vascular immunopathology.

    Science.gov (United States)

    Caviezel-Firner, Sonja; Engeler, Daniel; Bolinger, Beatrice; Onder, Lucas; Scandella, Elke; Yu, Meimei; Kroczek, Richard A; Ludewig, Burkhard

    2013-12-01

    Attenuation of T cell-mediated damage of blood endothelial cells (BECs) in transplanted organs is important to prevent transplant vasculopathy (TV) and chronic rejection. Here, we assessed the importance of minor histocompatibility antigen (mHA) distribution and different coinhibitory molecules for T cell-BEC interaction. A transgenic mHA was directed specifically to BECs using the Tie2 promoter and cellular interactions were assessed in graft-versus-host disease-like and heterotopic heart transplantation settings. We found that cognate CD4(+) T-cell help was critical for the activation of BEC-specific CD8(+) T cells. However, systemic mHA expression on BECs efficiently attenuated adoptively transferred, BEC-specific CD4(+) and CD8(+) T cells and hence prevented tissue damage, whereas restriction of mHA expression to heart BECs precipitated the development of TV. Importantly, the lack of the coinhibitory molecules programmed death-1 (PD-1) and B and T lymphocyte attenuator fostered the initial activation of BEC-specific CD4(+) T cells, but did not affect development of TV. In contrast, TV was significantly augmented in the absence of PD-1 on BEC-specific CD8(+) T cells. Taken together, these results indicate that antigen distribution in the vascular bed determines the impact of coinhibition and, as a consequence, critically impinges on T cell-mediated vascular immunopathology. © 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Plant expressed coccidial antigens as potential vaccine candidates in protecting chicken against coccidiosis.

    Science.gov (United States)

    Sathish, Kota; Sriraman, Rajan; Subramanian, B Mohana; Rao, N Hanumantha; Kasa, Balaji; Donikeni, Jagan; Narasu, M Lakshmi; Srinivasan, V A

    2012-06-22

    Coccidiosis is a disease caused by intracellular parasites belonging to the genus Eimeria. In the present study, we transiently expressed two coccidial antigens EtMIC1 and EtMIC2 as poly histidine-tagged fusion proteins in tobacco. We have evaluated the protective efficacy of plant expressed EtMIC1 as monovalent and as well as bi-valent formulation where EtMIC1 and EtMIC2 were used in combination. The protective efficacy of these formulations was evaluated using homologous challenge in chickens. We observed better serum antibody response, weight gain and reduced oocyst shedding in birds immunized with EtMIC1 and EtMIC2 as bivalent formulation compared to monovalent formulation. However, IFN-γ response was not significant in birds immunized with EtMIC1 compared to the birds immunized with EtMIC2. Our results indicate the potential use of these antigens as vaccine candidates. Copyright © 2012 Elsevier Ltd. All rights reserved.

  11. Capsular Polysaccharide Expression in Commensal Streptococcus Species: Genetic and Antigenic Similarities to Streptococcus pneumoniae.

    Science.gov (United States)

    Skov Sørensen, Uffe B; Yao, Kaihu; Yang, Yonghong; Tettelin, Hervé; Kilian, Mogens

    2016-11-15

    Expression of a capsular polysaccharide is considered a hallmark of most invasive species of bacteria, including Streptococcus pneumoniae, in which the capsule is among the principal virulence factors and is the basis for successful vaccines. Consequently, it was previously assumed that capsule production distinguishes S. pneumoniae from closely related commensals of the mitis group streptococci. Based on antigenic and genetic analyses of 187 mitis group streptococci, including 90 recognized serotypes of S. pneumoniae, we demonstrated capsule production by the Wzy/Wzx pathway in 74% of 66 S. mitis strains and in virtually all tested strains of S. oralis (subspecies oralis, dentisani, and tigurinus) and S. infantis Additional analyses of genomes of S. cristatus, S. parasanguinis, S. australis, S. sanguinis, S. gordonii, S. anginosus, S. intermedius, and S. constellatus revealed complete capsular biosynthesis (cps) loci in all strains tested. Truncated cps loci were detected in three strains of S. pseudopneumoniae, in 26% of S. mitis strains, and in a single S. oralis strain. The level of sequence identities of cps locus genes confirmed that the structural polymorphism of capsular polysaccharides in S. pneumoniae evolved by import of cps fragments from commensal Streptococcus species, resulting in a mosaic of genes of different origins. The demonstrated antigenic identity of at least eight of the numerous capsular polysaccharide structures expressed by commensal streptococci with recognized serotypes of S. pneumoniae raises concerns about potential misidentifications in addition to important questions concerning the consequences for vaccination and host-parasite relationships both for the commensals and for the pathogen. Expression of a capsular polysaccharide is among the principal virulence factors of Streptococcus pneumoniae and is the basis for successful vaccines against infections caused by this important pathogen. Contrasting with previous

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

    Directory of Open Access Journals (Sweden)

    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.

  13. Variation of expression defects in cell surface 190-kDa protein antigen of Streptococcus mutans.

    Science.gov (United States)

    Lapirattanakul, Jinthana; Nomura, Ryota; Matsumoto-Nakano, Michiyo; Srisatjaluk, Ratchapin; Ooshima, Takashi; Nakano, Kazuhiko

    2015-05-01

    Streptococcus mutans, which consists of four serotypes, c, e, f, and k, possesses a 190-kDa cell surface protein antigen (PA) for initial tooth adhesion. We used Western blot analysis to determine PA expression in 750 S. mutans isolates from 150 subjects and found a significantly higher prevalence of the isolates with PA expression defects in serotypes f and k compared to serotypes c and e. Moreover, the defect patterns could be classified into three types; no PA expression on whole bacterial cells and in their supernatant samples (Type N1), PA expression mainly seen in supernatant samples (Type N2), and only low expression of PA in the samples of whole bacterial cells (Type W). The underlying reasons for the defects were mutations in the gene encoding PA as well as in the transcriptional processing of this gene for Type N1, defects in the sortase gene for Type N2, and low mRNA expression of PA for Type W. Since cellular hydrophobicity and phagocytosis susceptibility of the PA-defective isolates were significantly lower than those of the normal expression isolates, the potential implication of such defective isolates in systemic diseases involving bacteremia other than dental caries was suggested. Additionally, multilocus sequence typing was utilized to characterize S. mutans clones that represented a proportion of isolates with PA defects of 65-100%. Therefore, we described the molecular basis for variation defects in PA expression of S. mutans. Furthermore, we also emphasized the strong association between PA expression defects and serotypes f and k as well as the clonal relationships among these isolates. Copyright © 2015 Elsevier GmbH. All rights reserved.

  14. Cancer-testis antigen expression in synovial sarcoma: NY-ESO-1, PRAME, MAGEA4, and MAGEA1.

    Science.gov (United States)

    Iura, Kunio; Maekawa, Akira; Kohashi, Kenichi; Ishii, Takeaki; Bekki, Hirofumi; Otsuka, Hiroshi; Yamada, Yuichi; Yamamoto, Hidetaka; Harimaya, Katsumi; Iwamoto, Yukihide; Oda, Yoshinao

    2017-03-01

    Synovial sarcoma (SS) is regarded as a relatively chemosensitive sarcoma, but the prognosis of advanced SSs remains poor. Here we identified highly expressed cancer-testis antigens that could be promising immunotherapy targets for SS, using a previously conducted cDNA microarray, and we assessed the clinicopathological or prognostic relationships of these antigens in SS. We compared the gene expression profiles of 11 SSs with those of 3 normal adipose tissues. Among the up-regulated cancer-testis antigens, we analyzed PRAME, MAGEA1, and MAGEA4 and another cancer-testis antigen (NY-ESO-1) together, by immunohistochemistry and real-time polymerase chain reaction in 108 SSs. Immunohistochemically, NY-ESO-1, PRAME, MAGEA4, and MAGEA1 were positive in 66 (61%), 93 (86%), 89 (82%), and 16 (15%) of 108 SSs, respectively, and 104 (96%) of 108 SSs showed the immunohistochemical expression of at least 1 of NY-ESO-1, PRAME, and MAGEA4. Moreover, the high expression of at least 1 of these 3 antigens was observed in 83% of the SSs. High expression of NY-ESO-1 and MAGEA4 was significantly correlated with the presence of necrosis and advanced clinical stage. The immunohistochemical expression of these cancer-testis antigens was not correlated with prognosis, but the coexpression of NY-ESO-1, PRAME, and MAGEA4 was significantly associated with adverse prognosis. The real-time polymerase chain reaction results were closely related to the immunohistochemical results: NY-ESO-1 (P = .0019), PRAME (P = .039), MAGEA4 (P = .0149), and MAGEA1 (P = .0766). These data support the potential utility of NY-ESO-1, PRAME, and MAGEA4 as immunotherapy targets and ancillary prognostic parameters, suggesting the possible benefit of the combined use of these cancer-testis antigens as an SS immunotherapy target. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Co-expression of tumor antigen and interleukin-2 from an adenoviral vector augments the efficiency of therapeutic tumor vaccination

    DEFF Research Database (Denmark)

    Jensen, Benjamin Anderschou Holbech; Steffensen, Maria Abildgaard; Nørgaard Nielsen, Karen

    2014-01-01

    We have previously shown that for the majority of antigens, adenoviral vaccines expressing the target antigen fused to the MHC associated invariant chain (Ii) induce an accelerated, augmented, and prolonged transgene-specific CD8+ T-cell response. Here we describe a new adenoviral vaccine vector...... tailored to meet specific demands: in the context of therapeutic vaccination, the capacity to promote an augmented effector T-cell response.Molecular Therapy (2014); doi:10.1038/mt.2014.130....

  16. Análise da expressão do colágeno VI na distrofia muscular congênita Analysis of the expression of collagen VI in congenital muscular dystrophy

    Directory of Open Access Journals (Sweden)

    Regina Toni Loureiro de Freitas

    2005-06-01

    Full Text Available A distrofia muscular congênita (DMC compõe um grupo de miopatias caracterizadas por hipotonia e fraqueza muscular notadas já no primeiro ano de vida. A forma de Ullrich é caracterizada por retrações musculares proximais e hiperextensibilidade distal. Cerca de 40% destes pacientes apresentam mutações em um dos genes que codificam as três sub-unidades do colágeno VI (COL6, acarretando deficiência total ou parcial na marcação da proteína. Analisamos, através de imunofluorescência, a marcação do COL6 em fragmentos musculares de 50 pacientes com DMC, 20 deles com ausência da marcação para merosina. Identificamos 4 casos com deficiência total da marcação do COL6 (8% do total, representando 13% dos casos com marcação normal para merosina. As alterações histológicas musculares dos pacientes com COL6 deficiente eram indistinguíveis das outras formas de DMC, porém mais brandas que as observadas na DMC com deficiência de merosina. Em três dos pacientes com COL6 deficiente observou-se hipotonia e fraqueza muscular, notadas já no período neonatal, atraso do desenvolvimento motor, retrações musculares em joelhos e cotovelos, hiperextensibilidade distal e luxação congênita do quadril (dois pacientes. Um paciente perdeu a capacidade para a marcha, e outro faleceu por problemas respiratórios. A análise da marcação do COL6, assim como da merosina, no tecido muscular de pacientes com DMC pode auxiliar na identificação e caracterização fenotípica dos diversos subtipos de DMC.Congenital muscular dystrophy (CMD composes a group of disorders characterized by hypotonia and muscular weakness noticed in the first year of life. The Ullrich's form is characterized by proximal joint contractures and distal hiperextensibility. About 40% of these patients present mutations in one of the genes that codify the sub-units of the collagen VI protein (COL6, producing total or partial deficiency of the protein expression. We analyzed

  17. 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...

  18. Amount of H Antigen Expressed on Circulating von Willebrand Factor Is Modified by ABO Blood Group Genotype and Is a Major Determinant of Plasma von Willebrand Factor Antigen Levels

    National Research Council Canada - National Science Library

    O’Donnell, James; Boulton, Frank E; Manning, Richard A; Laffan, Michael A

    2002-01-01

    .... AvWF was correlated strongly with plasma levels of A transferase activity. Thus, we have clearly demonstrated a direct relationship between ABO genotype, A transferase expression, and the amount of A antigen expressed on circulating vWF...

  19. Prokaryotic Expression, Purification and Immunogenicity in Rabbits of the Small Antigen of Hepatitis Delta Virus

    Directory of Open Access Journals (Sweden)

    Vera L. Tunitskaya

    2016-10-01

    Full Text Available Hepatitis delta virus (HDV is a viroid-like blood-borne human pathogen that accompanies hepatitis B virus infection in 5% patients. HDV has been studied for four decades; however, the knowledge on its life-cycle and pathogenesis is still sparse. The studies are hampered by the absence of the commercially-available HDV-specific antibodies. Here, we describe a set of reproducible methods for the expression in E. coli of His-tagged small antigen of HDV (S-HDAg, its purification, and production of polyclonal anti-S-HDAg antibodies in rabbits. S-HDAg was cloned into a commercial vector guiding expression of the recombinant proteins with the C-terminal His-tag. We optimized S-HDAg protein purification procedure circumventing a low affinity of the His-tagged S-HDAg to the Ni-nitrilotriacetyl agarose (Ni-NTA-agarose resin. Optimization allowed us to obtain S-HDAg with >90% purity. S-HDAg was used to immunize Shinchilla grey rabbits which received 80 μg of S-HDAg in two subcutaneous primes in the complete, followed by four 40 μg boosts in incomplete Freunds adjuvant. Rabbits were bled two weeks post each boost. Antibody titers determined by indirect ELISA exceeded 107. Anti-S-HDAg antibodies detected the antigen on Western blots in the amounts of up-to 100 pg. They were also successfully used to characterize the expression of S-HDAg in the eukaryotic cells by immunofluorescent staining/confocal microscopy.

  20. The effect of bile acids and piroxicam on MHC antigen expression in rat colonocytes during colon cancer development.

    Science.gov (United States)

    Rigas, B; Tsioulias, G J; Allan, C; Wali, R K; Brasitus, T A

    1994-10-01

    The effect of bile acids and piroxicam on the expression of major histocompatibility complex (MHC) antigens in colonocytes was evaluated in rats treated with the colonic carcinogen azoxymethane (AOM). Male Fischer-344 rats were fed a basal diet (AIN-76) supplemented with 0.4% cholic acid, 0.4% ursodeoxycholic acid, 0.2% ursodeoxycholic acid plus 0.2% cholic acid, or 75 p.p.m. piroxicam. Rats were injected subcutaneously once a week for 2 weeks with AOM (15 mg/kg body weight/week) or vehicle, after being fed their respective diets for two weeks. The rats were killed at 16 weeks, while parallel identical groups of rats were killed at 28 weeks, and colon tumours were counted. None of the rats treated with AOM-vehicle developed tumours at 28 weeks, while in the AOM-treated rats the frequency of colonic tumours was as follows: AOM alone 50%, cholic acid 74%, ursodeoxycholic acid 17%, piroxicam 28%, ursodeoxycholic plus cholic acid 46%. The expression of RT1A, RT1B and RT1D was determined in isolated colonocytes by immune fluocytometry. Normal rat colonocytes express all three MHC antigens strongly. Neither the bile acids nor piroxicam affected MHC antigen expression in AOM-vehicle-treated rats. AOM did not effect MHC antigen expression compared to normal controls. Cholic acid had no significant effect on the expression of MHC antigens in AOM-treated rats. Ursodeoxycholic acid alone or in combination with cholic acid increased the expression of RT1A compared to normal controls, of RT1B compared to AOM-treated rats, and of RT1D compared to controls or AOM-treated rats. Piroxicam increased the expression of all three antigens compared to either control or AOM-treated rats. These findings indicate that (1) ursodeoxycholic acid and piroxicam up-regulate colonic MHC antigen expression in the AOM model of colonic carcinogenesis; (2) the colon of rats exposed to AOM responds differently than the normal colon with respect to MHC regulation; and (3) the protective effect of

  1. Frequent High Expression of Kita-Kyushu Lung Cancer Antigen-1 (KK-LC-1) in Gastric Cancer.

    Science.gov (United States)

    Shida, Akiko; Futawatari, Nobue; Fukuyama, Takashi; Ichiki, Yoshinobu; Takahashi, Yoshihito; Nishi, Yatsushi; Kobayashi, Noritada; Yamazaki, Hitoshi; Watanabe, Masahiko

    2015-06-01

    The tumor-associated antigen Kita-Kyushu lung cancer antigen-1 (KK-LC-1) has been reported as not being expressed in normal tissues, except for the testis, and in the setting of non-small cell lung cancer. The present study demonstrated that KK-LC-1 is expressed in gastric cancer. We analyzed the expression of KK-LC-1 and cancer/testis antigens (CTAs) in surgical specimens of 49 gastric carcinomas. The expression of KK-LC-1 and CTAs was assessed using reverse transcription-polymerase chain reaction. KK-LC-1 expression was observed in gastric carcinomas. The number of lesions with expression of KK-LC-1, Melanoma antigen gene encoding-A1 (MAGE-A1), MAGE-A3 and New York Esophageal squamous cell carcinoma-1 (NY-ESO-1) was 40 (81.6%), 17 (34.7%), 22 (44.9%) and 8 (16.3%) out of the 49 specimens, respectively. KK-LC-1 should be categorized as a CTA. The frequency of KK-LC-1 expression was higher than that of the other CTAs. KK-LC-1 might be a useful target for immunotherapy and in diagnosis of gastric cancer. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  2. Seasonal tracking of histo-blood group antigen expression and norovirus binding in oyster gastrointestinal cells.

    Science.gov (United States)

    Tian, Peng; Engelbrektson, Anna L; Mandrell, Robert E

    2008-08-01

    Noroviruses (NORs) are the most common cause of viral gastroenteritis outbreaks. Outbreaks are often associated with the consumption of contaminated oysters and generally occur between the months of November and March, when oysters produce the highest levels of glycogen. Oyster glycogen has been proposed as playing a role in NOR accumulation. Recent research indicates that histo-blood group antigens (HBGAs) function as viral receptors on human gastrointestinal cells. In this study, oyster glycogen was tested to determine whether it contains HBGA-like molecules and whether it plays a role in NOR binding. The correlation between the amount of HBGA expression and NOR binding also was measured. We also tested whether seasonal changes affected HBGA expression and binding of recombinant NORs. The results indicate that recombinant NOR binding is highly correlated with HBGA expression in Virginica (Crassostrea virginica), Pacific (Crassostrea gigas), and Kumamato (Crassostrea sikamea) oysters, but the association does not have a seasonal pattern. No obvious trend in either HBGA expression or recombinant NOR binding by month was noted. A significant increase in recombinant NOR binding was observed in Virginica and Pacific oysters in a season not generally associated with NOR gastroenteritis outbreaks. A significant increase in HBGA expression also was observed for Pacific and Virginica oysters in the same season. Paradoxically, HBGA expression and NOR binding both were higher in oysters produced in the non-NOR gastroenteritis season (April through October) than in those produced in the NOR gastroenteritis season (November through March), suggesting that seasonal NOR gastroenteritis outbreaks are not associated with high levels of HBGA expression or NOR binding.

  3. Aire mediates thymic expression and tolerance of pancreatic antigens via an unconventional transcriptional mechanism.

    Science.gov (United States)

    Danso-Abeam, Dina; Staats, Kim A; Franckaert, Dean; Van Den Bosch, Ludo; Liston, Adrian; Gray, Daniel H D; Dooley, James

    2013-01-01

    The autoimmune regulator (Aire), mediates central tolerance of peripheral self. Its activity in thymic epithelial cells (TECs) directs the ectopic expression of thousands of tissue-restricted antigens (TRAs), causing the deletion of autoreactive thymocytes. The molecular mechanisms orchestrating the breadth of transcriptional regulation by Aire remain unknown. One prominent model capable of explaining both the uniquely high number of Aire-dependent targets and their specificity posits that tissue-specific transcription factors induced by Aire directly activate their canonical targets, exponentially adding to the total number of Aire-dependent TRAs. To test this "Hierarchical Transcription" model, we analysed mice deficient in the pancreatic master transcription factor pancreatic and duodenal homeobox 1 (Pdx1), specifically in TECs (Pdx1(ΔFoxn1) ), for the expression and tolerance of pancreatic TRAs. Surprisingly, we found that lack of Pdx1 in TECs did not reduce the transcription of insulin or somatostatin, or alter glucagon expression. Moreover, in a model of thymic deletion driven by a neo-TRA under the control of the insulin promoter, Pdx1 in TECs was not required to affect thymocyte deletion or the generation of regulatory T (Treg) cells. These findings suggest that the capacity of Aire to regulate expression of a huge array of TRAs relies solely on an unconventional transcriptional mechanism, without intermediary transcription factors. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Ectopic expression of HLA-DO in mouse dendritic cells diminishes MHC class II antigen presentation.

    Science.gov (United States)

    Fallas, Jennifer L; Tobin, Helen M; Lou, Olivia; Guo, Donglin; Sant'Angelo, Derek B; Denzin, Lisa K

    2004-08-01

    The MHC class II-like molecule HLA-DM (DM) (H-2M in mice) catalyzes the exchange of CLIP for antigenic peptides in the endosomes of APCs. HLA-DO (DO) (H-2O in mice) is another class II-like molecule that is expressed in B cells, but not in other APCs. Studies have shown that DO impairs or modifies the peptide exchange activity of DM. To further evaluate the role of DO in Ag processing and presentation, we generated transgenic mice that expressed the human HLA-DOA and HLA-DOB genes under the control of a dendritic cell (DC)-specific promoter. Our analyses of DCs from these mice showed that as DO levels increased, cell surface levels of A(b)-CLIP also increased while class II-peptide levels decreased. The presentation of some, but not all, exogenous Ags to T cells or T hybridomas was significantly inhibited by DO. Surprisingly, H-2M accumulated in DO-expressing DCs and B cells, suggesting that H-2O/DO prolongs the half-life of H-2M. Overall, our studies showed that DO expression impaired H-2M function, resulting in Ag-specific down-modulation of class II Ag processing and presentation.

  5. Antigenicity and Immunogenicity of Rotavirus VP6 Protein Expressed on the Surface of Lactococcus lactis

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    L. E. Esteban

    2013-01-01

    Full Text Available Group A rotaviruses are the major etiologic agents of acute gastroenteritis worldwide in children and young animals. Among its structural proteins, VP6 is the most immunogenic and is highly conserved within this group. Lactococcus lactis is a food-grade, Gram-positive, and nonpathogenic lactic acid bacteria that has already been explored as a mucosal delivery system of heterologous antigens. In this work, the nisin-controlled expression system was used to display the VP6 protein at the cell surface of L. lactis. Conditions for optimal gene expression were established by testing different nisin concentrations, cell density at induction, and incubation times after induction. Cytoplasmic and cell wall protein extracts were analyzed by Western blot and surface expression was confirmed by flow cytometry. Both analysis provided evidence that VP6 was efficiently expressed and displayed on the cell surface of L. lactis. Furthermore, the humoral response of mice immunized with recombinant L. lactis was evaluated and the displayed recombinant VP6 protein proved to be immunogenic. In conclusion, this is the first report of displaying VP6 protein on the surface of L. lactis to induce a specific immune response against rotavirus. These results provide the basis for further evaluation of this VP6-displaying L. lactis as a mucosal delivery vector in a mouse model of rotavirus infection.

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

    Directory of Open Access Journals (Sweden)

    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.

  7. Human leukocyte antigen-G (HLA-G polymorphism and expression in breast cancer patients.

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    Seri Jeong

    Full Text Available Human leukocyte antigen-G (HLA-G is known to be implicated in a tumor-driven immune escape mechanism in malignancies. The purpose of this study was to investigate HLA-G polymorphism and expression in breast cancer. HLA-G alleles were determined by direct DNA sequencing procedures from blood samples of 80 breast cancer patients and 80 healthy controls. Soluble HLA-G (sHLA-G was measured by enzyme-linked immunosorbent assay (ELISA from serum specimens. HLA-G expression in breast cancer lesions was also analyzed by immunohistochemistry staining. The presence of HLA-G 3' untranslated region (UTR 14-bp sequence was analyzed and found to be associated with reduced risk of breast cancer susceptibility based on HLA-G expression in tissues (P = 0.0407. Levels of sHLA-G were higher in the breast cancer group (median 117.2 U/mL compared to the control group (median 10.1 U/mL, P<0.001. The area under the receiver operating characteristic curve (AU-ROC values of sHLA-G for differentiating breast cancer from normal controls and for detecting metastasis from other stages of breast cancer were 0.89 and 0.79, respectively. HLA-G polymorphism and expression may be involved in breast carcinogenesis and sHLA-G concentrations could be used as a diagnostic marker for detecting breast cancer.

  8. Mode of Tolerance Induction and Requirement for Aire Are Governed by the Cell Types That Express Self-Antigen and Those That Present Antigen.

    Science.gov (United States)

    Mouri, Yasuhiro; Ueda, Yoshihiro; Yamano, Tomoyoshi; Matsumoto, Minoru; Tsuneyama, Koichi; Kinashi, Tatsuo; Matsumoto, Mitsuru

    2017-12-15

    Aire controls the fate of autoreactive thymocytes (i.e., clonal deletion or development into regulatory T cells [Tregs]) through transcriptional control of the expression of tissue-restricted self-antigens (TRAs) from medullary thymic epithelial cells (mTECs) and bone marrow (BM)-derived cells. Although TRAs expressed by mTECs and BM-derived cells are suggested to complement each other to generate a full spectrum of TRAs, little is known about the relative contribution of TRAs from each component for establishment of self-tolerance. Furthermore, the precise role of Aire in specific types of Aire-expressing APCs remains elusive. We have approached these issues by generating two different types of transgenic mouse (Tg) model, which express a prefixed model self-antigen driven by the insulin promoter or the Aire promoter. In the insulin-promoter Tg model, mTECs alone were insufficient for clonal deletion, and BM-derived APCs were required for this action by utilizing Ag transferred from mTECs. In contrast, mTECs alone were able to induce Tregs, although at a much lower efficiency in the absence of BM-derived APCs. Importantly, lack of Aire in mTECs, but not in BM-derived APCs, impaired both clonal deletion and production of Tregs. In the Aire-promoter Tg model, both mTECs and BM-derived APCs could independently induce clonal deletion without Aire, and production of Tregs was impaired by the lack of Aire in mTECs, but not in BM-derived APCs. These results suggest that the fate of autoreactive thymocytes together with the requirement for Aire depend on the cell types that express self-antigens and the types of APCs involved in tolerance induction. Copyright © 2017 by The American Association of Immunologists, Inc.

  9. 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.

  10. Genomic expression libraries for the identification of cross-reactive orthopoxvirus antigens.

    Science.gov (United States)

    Miller, Lilija; Richter, Marco; Hapke, Christoph; Stern, Daniel; Nitsche, Andreas

    2011-01-01

    Increasing numbers of human cowpox virus infections that are being observed and that particularly affect young non-vaccinated persons have renewed interest in this zoonotic disease. Usually causing a self-limiting local infection, human cowpox can in fact be fatal for immunocompromised individuals. Conventional smallpox vaccination presumably protects an individual from infections with other Orthopoxviruses, including cowpox virus. However, available live vaccines are causing severe adverse reactions especially in individuals with impaired immunity. Because of a decrease in protective immunity against Orthopoxviruses and a coincident increase in the proportion of immunodeficient individuals in today's population, safer vaccines need to be developed. Recombinant subunit vaccines containing cross-reactive antigens are promising candidates, which avoid the application of infectious virus. However, subunit vaccines should contain carefully selected antigens to confer a solid cross-protection against different Orthopoxvirus species. Little is known about the cross-reactivity of antibodies elicited to cowpox virus proteins. Here, we first identified 21 immunogenic proteins of cowpox and vaccinia virus by serological screenings of genomic Orthopoxvirus expression libraries. Screenings were performed using sera from vaccinated humans and animals as well as clinical sera from patients and animals with a naturally acquired cowpox virus infection. We further analyzed the cross-reactivity of the identified immunogenic proteins. Out of 21 identified proteins 16 were found to be cross-reactive between cowpox and vaccinia virus. The presented findings provide important indications for the design of new-generation recombinant subunit vaccines.

  11. Genomic expression libraries for the identification of cross-reactive orthopoxvirus antigens.

    Directory of Open Access Journals (Sweden)

    Lilija Miller

    Full Text Available Increasing numbers of human cowpox virus infections that are being observed and that particularly affect young non-vaccinated persons have renewed interest in this zoonotic disease. Usually causing a self-limiting local infection, human cowpox can in fact be fatal for immunocompromised individuals. Conventional smallpox vaccination presumably protects an individual from infections with other Orthopoxviruses, including cowpox virus. However, available live vaccines are causing severe adverse reactions especially in individuals with impaired immunity. Because of a decrease in protective immunity against Orthopoxviruses and a coincident increase in the proportion of immunodeficient individuals in today's population, safer vaccines need to be developed. Recombinant subunit vaccines containing cross-reactive antigens are promising candidates, which avoid the application of infectious virus. However, subunit vaccines should contain carefully selected antigens to confer a solid cross-protection against different Orthopoxvirus species. Little is known about the cross-reactivity of antibodies elicited to cowpox virus proteins. Here, we first identified 21 immunogenic proteins of cowpox and vaccinia virus by serological screenings of genomic Orthopoxvirus expression libraries. Screenings were performed using sera from vaccinated humans and animals as well as clinical sera from patients and animals with a naturally acquired cowpox virus infection. We further analyzed the cross-reactivity of the identified immunogenic proteins. Out of 21 identified proteins 16 were found to be cross-reactive between cowpox and vaccinia virus. The presented findings provide important indications for the design of new-generation recombinant subunit vaccines.

  12. Cowpea mosaic virus-based systems for the expression of antigens and antibodies in plants.

    Science.gov (United States)

    Sainsbury, Frank; Liu, Li; Lomonossoff, George P

    2009-01-01

    This chapter describes the use of Cowpea mosaic virus-based vectors for the production of foreign proteins such as antigens and antibodies in plants. The systems include vectors based on both full-length and deleted versions of RNA-2. In both cases, the modified RNA-2 is replicated by coinoculation with RNA-1. The constructs based on full-length RNA-2 retain the ability to spread systemically throughout an inoculated plant and the infection can be passaged. The vector based on a deleted version of RNA-2 can stably incorporate larger inserts but lacks the ability to move systemically. However, it has the added advantage of biocontainment. In both cases, vector constructs modified to contain a foreign gene of interest can be delivered by agroinfiltration to obtain transient expression of the foreign protein. If required, the same constructs can also be used for stable nuclear transformation. Both types of vector have proved effective for the production in plants of a diverse range of proteins including antigens and antibodies.

  13. Identification of cDNA clones expressing immunodiagnostic antigens from Trichinella spiralis

    Energy Technology Data Exchange (ETDEWEB)

    Zarlenga, D.; Gamble, H.R.

    1987-05-01

    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 TSP 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.

  14. Platelet receptor expression and shedding: glycoprotein Ib-IX-V and glycoprotein VI.

    Science.gov (United States)

    Gardiner, Elizabeth E; Andrews, Robert K

    2014-04-01

    Quantity, quality, and lifespan are 3 important factors in the physiology, pathology, and transfusion of human blood platelets. The aim of this review is to discuss the proteolytic regulation of key platelet-specific receptors, glycoprotein(GP)Ib and GPVI, involved in the function of platelets in hemostasis and thrombosis, and nonimmune or immune thrombocytopenia. The scope of the review encompasses the basic science of platelet receptor shedding, practical aspects related to laboratory analysis of platelet receptor expression/shedding, and clinical implications of using the proteolytic fragments as platelet-specific biomarkers in vivo in terms of platelet function and clearance. These topics can be relevant to platelet transfusion regarding both changes in platelet receptor expression occurring ex vivo during platelet storage and/or clinical use of platelets for transfusion. In this regard, quantitative analysis of platelet receptor profiles on blood samples from individuals could ultimately enable stratification of bleeding risk, discrimination between causes of thrombocytopenia due to impaired production vs enhanced clearance, and monitoring of response to treatment prior to change in platelet count. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. 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.

  16. Elevated expression of squamous cell carcinoma antigen (SCCA is associated with human breast carcinoma.

    Directory of Open Access Journals (Sweden)

    Joseph M Catanzaro

    Full Text Available Squamous cell carcinoma antigen (SCCA belongs to the serine protease inhibitor (Serpin family of proteins. Elevated expression of SCCA has been used as a biomarker for aggressive squamous cell carcinoma (SCC in cancers of the cervix, lung, head and neck, and liver. However, SCCA expression in breast cancer has not been investigated. Immunohistochemical analysis of SCCA expression was performed on tissue microarrays containing breast tumor tissues (n = 1,360 and normal breast epithelium (n = 124. SCCA expression was scored on a tiered scale (0-3 independently by two evaluators blind to the patient's clinical status. SCCA expression was observed in Grade I (0.3%, Grade II (2.5%, and Grade III (9.4% breast cancers (p<0.0001. Comparing tissues categorized into the three non-metastatic TNM stages, I-III, SCCA positivity was seen in 2.4% of Stage I cancers, 3.1% of Stage II cancers, and 8.6% of Stage III breast cancers (p = 0.0005. No positive staining was observed in normal/non-neoplastic breast tissue (0 out of 124. SCCA expression also correlated to estrogen receptor/progesterone receptor (ER/PR double-negative tumors (p = 0.0009. Compared to SCCA-negative patients, SCCA-positive patients had both a worse overall survival and recurrence-free survival (p<0.0001 and p<0.0001, respectively. This study shows that SCCA is associated with both advanced stage and high grade human breast carcinoma, and suggests the necessity to further explore the role of SCCA in breast cancer development and treatment.

  17. Differences in sialyl-Tn antigen expression between keratoacanthomas and cutaneous squamous cell carcinomas.

    Science.gov (United States)

    Jensen, P; Clausen, O P; Bryne, M

    1999-04-01

    Keratoacanthoma and squamous cell carcinoma are common skin tumors, especially in immunosuppressed transplant recipients, but the distinction between these two types of epidermal neoplasia may be difficult. Sialyl-Tn (Sia-GalNAc-O-Ser/Thr) is a cell surface carbohydrate associated with hyperplasia in squamous epithelium, and correlated with poor prognosis in several human adenocarcinomas. Twenty-seven keratoacanthomas and 29 cutaneous squamous cell carcinomas were examined for the expression of sialyl-Tn and of the Ki67 epitope, the latter a marker for cell proliferation. By immunohistochemistry, basaloid tumor cells at the periphery of tumor nests showed some degree of sialyl-Tn expression in 16 keratoacanthomas (59%), while only three squamous cell carcinomas (10%) showed sialyl-Tn-positive basaloid tumor cells (p<0.001). Keratinized, differentiated tumor cells were more often sialyl-Tn-positive in keratoacanthomas (89%) than in squamous cell carcinomas (31%) (p<0.001). A striking sialyl-Tn-positivity in the basal cell layer was found in a border zone directly adjacent to most tumors of both types (88 and 88%). By immunohistochemical examination of parallel sections and by double immunofluorescence, sialyl-Tn antigen expression was primarily seen in cells that did not express Ki67, although some overlap was present. Keratoacanthomas from transplant recipients did not differ in sialyl-Tn expression compared to those from non-immunosuppressed patients. The results indicate that sialyl-Tn expression is not directly related to cell proliferation, but rather to cellular features of post-mitotic cells, and that sialyl-Tn is not associated with a malignant phenotype. Sialyl-Tn may be linked to tumor regression, as seen in keratoacanthomas.

  18. Purification and characterization of fetal hematopoietic cells that express the common acute lymphoblastic leukemia antigen (CALLA)

    DEFF Research Database (Denmark)

    Hokland, P; Rosenthal, P; Griffin, J D

    1983-01-01

    Fetal hematopoietic cells that express the common acute lymphoblastic leukemia antigen (CALLA) were purified from both fetal liver and fetal bone marrow by immune rosetting with sheep erythrocytes coated with rabbit anti-mouse immunoglobulin and by fluorescence-activated cell sorting. Dual...... lymphoblastic leukemia cell with respect to surface marker phenotype. A population of CALLA- cells devoid of mature erythroid and myeloid surface markers was found to contain higher numbers of TdT+ cells but lower numbers of cyto-mu, B1, and Ia+ cells than the CALLA+ subset. In vitro analysis of normal...... that these cells are relatively immature lymphoid cells, CALLA+ cells do not appear to contain either myeloid precursor cells (CFU-G/M) or the earliest lymphoid stem cells. Udgivelsesdato: 1983-Jan-1...

  19. Expression, characterisation and antigenicity of a truncated Hendra virus attachment protein expressed in the protozoan host Leishmania tarentolae.

    Science.gov (United States)

    Fischer, Kerstin; dos Reis, Vinicius Pinho; Finke, Stefan; Sauerhering, Lucie; Stroh, Eileen; Karger, Axel; Maisner, Andrea; Groschup, Martin H; Diederich, Sandra; Balkema-Buschmann, Anne

    2016-02-01

    Hendra virus (HeV) is an emerging zoonotic paramyxovirus within the genus Henipavirus that has caused severe morbidity and mortality in humans and horses in Australia since 1994. HeV infection of host cells is mediated by the membrane bound attachment (G) and fusion (F) glycoproteins, that are essential for receptor binding and fusion of viral and cellular membranes. The eukaryotic unicellular parasite Leishmania tarentolae has recently been established as a powerful tool to express recombinant proteins with mammalian-like glycosylation patterns, but only few viral proteins have been expressed in this system so far. Here, we describe the purification of a truncated, Strep-tag labelled and soluble version of the HeV attachment protein (sHeV G) expressed in stably transfected L. tarentolae cells. After Strep-tag purification the identity of sHeV G was confirmed by immunoblotting and mass spectrometry. The functional binding of sHeV G to the HeV cell entry receptor ephrin-B2 was confirmed in several binding assays. Generated polyclonal rabbit antiserum against sHeV G reacted with both HeV and Nipah virus (NiV) G proteins in immunofluorescence assay and efficiently neutralised NiV infection, thus further supporting the preserved antigenicity of the purified protein. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  20. Expression of Thy1, TL and LYT antigens on spontaneous leukemias of BALB/MO strain of mice.

    Science.gov (United States)

    Strzadała, L; Opolski, A; Kisielow, P

    1981-01-01

    The expression of Thy1, TL and Tyt antigens on spontaneous BALB/Mo leukemias has been studied. The results obtained thus far indicate that a majority of leukemias express TL antigen and differentiated Lyt phenotype. This observation and other findings demonstrating the existence of TL+ thymocytes with different Lyt phenotypes in normal thymus of young mice lends support for the hypothesis that TL+Lyt2+ phenotype defines this stage in the T cell differentiation pathway, which is particularly sensitive to leukemic transformation.

  1. Transfer and expression of the gene encoding a human myeloid membrane antigen (gp150).

    Science.gov (United States)

    Look, A T; Peiper, S C; Rebentisch, M B; Ashmun, R A; Roussel, M F; Rettenmier, C W; Sherr, C J

    1985-02-01

    DNA from the human myeloid cell line HL-60 was cotransfected with the cloned thymidine kinase (tk) gene of herpes simplex virus into tk-deficient mouse L cells. tk-positive recipients expressing antigens detected on HL-60 cells were isolated with a fluorescence-activated cell sorter by use of a panel of monoclonal antibodies that detect epitopes on both normal and malignant myeloid cells. Independently sorted populations of transformed mouse cells showed concordant reactivities with four of the monoclonal antibodies in the panel (DU-HL60-4, MY7, MCS.2, and SJ-D1), which suggested that these antibodies reacted to products of a single human gene. A second round of DNA transfection and cell sorting was performed with donor DNA from primary transformants. Two different dominant selection systems were used to isolate secondary mouse L cell and NIH/3T3 cell transformants that coexpressed the same epitopes. Analysis of cellular DNA from secondary mouse cell subclones with a probe specific for human repetitive DNA sequences revealed a minimal human DNA complement containing a characteristic set of restriction fragments common to independently derived subclones. Two glycoproteins, of 130,000 (gp130) and 150,000 (gp150) mol wt, were specifically immunoprecipitated from metabolically labeled lysates of mouse cell transformants and were shown to contain [35S]methionine-labeled tryptic peptides identical to those of analogous glycoproteins expressed in the donor human myeloid cell line. Kinetic and biochemical analyses established that gp130 is a precursor that differs in its carbohydrate moiety from gp150, the mature form of the glycoprotein detected on the cell surface. The isolation of human gene sequences encoding gp150 in a mouse cell genetic background provides the possibility of molecularly cloning the gene and represents a general strategy for isolating human genes encoding differentiation-specific cell surface antigens.

  2. Capsular Polysaccharide Expression in Commensal Streptococcus Species: Genetic and Antigenic Similarities to Streptococcus pneumoniae

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    Uffe B. Skov Sørensen

    2016-11-01

    Full Text Available Expression of a capsular polysaccharide is considered a hallmark of most invasive species of bacteria, including Streptococcus pneumoniae, in which the capsule is among the principal virulence factors and is the basis for successful vaccines. Consequently, it was previously assumed that capsule production distinguishes S. pneumoniae from closely related commensals of the mitis group streptococci. Based on antigenic and genetic analyses of 187 mitis group streptococci, including 90 recognized serotypes of S. pneumoniae, we demonstrated capsule production by the Wzy/Wzx pathway in 74% of 66 S. mitis strains and in virtually all tested strains of S. oralis (subspecies oralis, dentisani, and tigurinus and S. infantis. Additional analyses of genomes of S. cristatus, S. parasanguinis, S. australis, S. sanguinis, S. gordonii, S. anginosus, S. intermedius, and S. constellatus revealed complete capsular biosynthesis (cps loci in all strains tested. Truncated cps loci were detected in three strains of S. pseudopneumoniae, in 26% of S. mitis strains, and in a single S. oralis strain. The level of sequence identities of cps locus genes confirmed that the structural polymorphism of capsular polysaccharides in S. pneumoniae evolved by import of cps fragments from commensal Streptococcus species, resulting in a mosaic of genes of different origins. The demonstrated antigenic identity of at least eight of the numerous capsular polysaccharide structures expressed by commensal streptococci with recognized serotypes of S. pneumoniae raises concerns about potential misidentifications in addition to important questions concerning the consequences for vaccination and host-parasite relationships both for the commensals and for the pathogen.

  3. Identification of a protective antigen of Coccidioides immitis by expression library immunization.

    Science.gov (United States)

    Ivey, F Douglas; Magee, D Mitchell; Woitaske, Melanie D; Johnston, Stephen Albert; Cox, Rebecca A

    2003-10-01

    Coccidioides immitis is a fungal pathogen of humans and is classified as a Select Agent. We have identified a new potential vaccine candidate for this pathogen using cDNA expression library immunization (ELI). A C. immitis spherule-phase cDNA library containing 800-1000 genes was divided into 10 pools and each was tested for its protective capacity in BALB/c mice against intraperitoneal challenge with 2500 arthroconidia of this dimorphic fungus. The most protective pool, designated Pool 7, was fractionated into five sublibraries, each containing 60 genes, and of these, only Pool 7-3 induced a significant level of protection in mice. Fractionation of Pool 7-3 into six sublibraries, each with 10 genes, yielded a protective fraction, designated Pool 7-3-5. Subsequent fraction of the latter pool into 10 sublibraries, each with one clone, yielded a clone designated 7-3-5-5 that was highly protective. Clone 7-3-5-5 was sequenced and found to contain a 672bp ORF encoding a 224 amino acid protein having a 19 amino acid signal sequence on the N-terminus and a 15 amino acid C-terminal GPI anchor site. The 7-3-5-5 clone, designated ELI-Antigen 1 (ELI-Ag1), showed partial homology with a hypothetical protein from Neurospora crassa. This is the first study to identify a protective antigen from a fungus using ELI, and it is also the first report in which sequential fractionation of an expression library successfully identified a single protective gene.

  4. Over-expression of superoxide dismutase ameliorates Cr(VI induced adverse effects via modulating cellular immune system of Drosophila melanogaster.

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    Prakash Pragya

    Full Text Available The evolutionarily conserved innate immune system plays critical role for maintaining the health of an organism. However, a number of environmental chemicals including metals are known to exert adverse effects on immune system. The present study assessed the in vivo effect of a major environmental chemical, Cr(VI, on cellular immune response using Drosophila melanogaster and subsequently the protective role of superoxide dismutase (SOD based on the comparable performance of the tested anti-oxidant enzymes. The immuno-modulatory potential of Cr(VI was demonstrated by observing a significant reduction in the total hemocyte count along with impaired phagocytic activity in exposed organism. Concurrently, a significant increase in the percentage of Annexin V-FITC positive cells, activation of DEVDase activity, generation of free radical species along with inhibition of anti-oxidant enzyme activities was observed in the hemocytes of exposed organism. In addition, we have shown that ONOO(- is primarily responsible for Cr(VI induced adverse effects on Drosophila hemocytes along with O2(-. While generation of O2(-/ONOO(- in Cr(VI exposed Drosophila hemocytes was found to be responsible for the suppression of Drosophila cellular immune response, Cr(VI induced alteration was significantly reduced by the over-expression of sod in Drosophila hemocytes. Overall, our results suggest that manipulation of one of the anti-oxidant genes, sod, benefits the organism from Cr(VI induced alteration in cellular immunity. Further, this study demonstrates the applicability of D. melanogaster to examine the possible effects of environmental chemicals on innate immunity which can be extrapolated to higher organisms due to evolutionary conservation of innate immune system between Drosophila and mammals.

  5. T cells expressing chimeric antigen receptors can cause anaphylaxis in humans

    Science.gov (United States)

    Maus, Marcela V.; Haas, Andrew R.; Beatty, Gregory L.; Albelda, Steven M.; Levine, Bruce L.; Liu, Xiaojun; Zhao, Yangbing; Kalos, Michael; June, Carl H.

    2013-01-01

    T cells can be redirected to overcome tolerance to cancer by engineering with integrating vectors to express a chimeric antigen receptor (CAR). In preclinical models, we have previously demonstrated that transfection of T cells with messenger RNA (mRNA) coding for a CAR is an alternative strategy that has antitumor efficacy and the potential to evaluate the on-target off-tumor toxicity of new CAR targets safely due to transient mRNA CAR expression. Here, we report the safety observed in four patients treated with autologous T cells that had been electroporated with mRNA coding for a CAR derived from a murine antibody to human mesothelin. Due to the transient nature of CAR expression on the T cells, subjects in the clinical study were given repeated infusions of the CAR-T cells in order to assess their safety. One subject developed anaphylaxis and cardiac arrest within minutes of completing the 3rd infusion. Although human anti-mouse IgG antibodies have been known to develop with CAR-transduced T cells, they have been thought to have no adverse clinical consequences. This is the first description of clinical anaphylaxis resulting from CAR-modified T cells, most likely through IgE antibodies specific to the CAR. These results indicate that the potential immunogenicity of CARs derived from murine antibodies may be a safety issue for mRNA CARs, especially when administered using an intermittent dosing schedule. PMID:24432303

  6. Mycobacteria Exploit p38 Signaling To Affect CD1 Expression and Lipid Antigen Presentation by Human Dendritic Cells▿

    Science.gov (United States)

    Gagliardi, Maria Cristina; Teloni, Raffaela; Giannoni, Federico; Mariotti, Sabrina; Remoli, Maria Elena; Sargentini, Valeria; Videtta, Melissa; Pardini, Manuela; De Libero, Gennaro; Coccia, Eliana Marina; Nisini, Roberto

    2009-01-01

    Group I CD1 proteins are specialized antigen-presenting molecules that present both microbial and self lipid antigens to CD1-restricted α/β T lymphocytes. The production of high levels of gamma interferon and lysis of infected macrophages by lipid-specific T lymphocytes are believed to play pivotal roles mainly in the defense against mycobacterial infections. We previously demonstrated that Mycobacterium tuberculosis and bacillus Calmette-Guérin (Mycobacterium bovis BCG) induce human monocytes to differentiate into CD1− dendritic cells (DC), which cannot present lipid antigens to specific T cells. Here, we show that in human monocytes mycobacteria trigger phosphorylation of p38 mitogen-activated protein kinase to inhibit CD1 expression in DC derived from infected monocytes. Pretreatment with a specific p38 inhibitor renders monocytes insensitive to mycobacterial subversion and allows them to differentiate into CD1+ DC, which are fully capable of presenting lipid antigens to specific T cells. We also report that one of the pathogen recognition receptors triggered by BCG to activate p38 is complement receptor 3 (CR3), as shown by reduced p38 phosphorylation and partial reestablishment of CD1 membrane expression obtained by CR3 blockade before infection. In conclusion, we propose that p38 signaling is a novel pathway exploited by mycobacteria to affect the expression of CD1 antigen-presenting cells and avoid immune recognition. PMID:19720761

  7. Prokaryotic expression and identification of B- and T-cell combined epitopes of Em95 antigen of Echinococcus multilocularis.

    Science.gov (United States)

    Wang, Hongying; Zhang, Fengbo; Ma, Xiumin; Ma, Haimei; Zhu, Yuejie; Liu, Xianfei; Zhu, Min; Wen, Hao; Ding, Jianbing

    2014-01-01

    This study is to clone and identify B- and T-cell combined epitopes from Em95 antigen. The B- and T-cell combined epitopes were predicted using bioinformatic software. Two DNA sequences of Em95-1 (which contained the coding region of one B- and T-cell combined epitope) and Em95-2 (which contained the coding regions of two B- and T-cell combined epitopes) were amplified by PCR. Em95-1 and Em95-2 were cloned into pET32a vector for protein expression. Rabbit was immunized with the expressed proteins of rEm95-1 and rEm95-2 to produce polyclonal antibodies. The immunogenicity and antigenicity of rEm95-1 and rEm95-2 were examined by Western blot analysis. The three B- and T-cell combined epitopes were successfully cloned and expressed in PET32a vector. The recombinant antigens of rEm95-1 and rEm95-2 could specifically bind the human serum from patients with alveolar echinococcosis and specifically bind the prepared polyclonal antibodies. Three B- and T-cell combined epitopes were successfully cloned with good immunogenicity and antigenicity. Our data suggest that B- and T-cell combined epitopes predicted from the Em95 antigen may be used for the construction of high-valence vaccines and as targets for prevention of echinococcosis.

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

    Directory of Open Access Journals (Sweden)

    Tanzer Fiona L

    2008-06-01

    Full Text Available Abstract Background Human immunodeficiency virus type 1 (HIV-1 has infected more than 40 million people worldwide, mainly in sub-Saharan Africa. The high prevalence of HIV-1 subtype C in southern Africa necessitates the development of cheap, effective vaccines. One means of production is the use of plants, for which a number of different techniques have been successfully developed. HIV-1 Pr55Gag is a promising HIV-1 vaccine candidate: we compared the expression of this and a truncated Gag (p17/p24 and the p24 capsid subunit in Nicotiana spp. using transgenic plants and transient expression via Agrobacterium tumefaciens and recombinant tobamovirus vectors. We also investigated the influence of subcellular localisation of recombinant protein to the chloroplast and the endoplasmic reticulum (ER on protein yield. We partially purified a selected vaccine candidate and tested its stimulation of a humoral and cellular immune response in mice. Results Both transient and transgenic expression of the HIV antigens were successful, although expression of Pr55Gag was low in all systems; however, the Agrobacterium-mediated transient expression of p24 and p17/p24 yielded best, to more than 1 mg p24/kg fresh weight. Chloroplast targeted protein levels were highest in transient and transgenic expression of p24 and p17/p24. The transiently-expressed p17/p24 was not immunogenic in mice as a homologous vaccine, but it significantly boosted a humoral and T cell immune response primed by a gag DNA vaccine, pTHGagC. Conclusion Transient agroinfiltration was best for expression of all of the recombinant proteins tested, and p24 and p17/p24 were expressed at much higher levels than Pr55Gag. Our results highlight the usefulness of plastid signal peptides in enhancing the production of recombinant proteins meant for use as vaccines. The p17/p24 protein effectively boosted T cell and humoral responses in mice primed by the DNA vaccine pTHGagC, showing that this plant

  9. An affinity matured minibody for PET imaging of prostate stem cell antigen (PSCA)-expressing tumors

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    Lepin, Eric J.; Leyton, Jeffrey V.; Olafsen, Tove; Salazar, Felix B.; McCabe, Katelyn E.; Wu, Anna M. [University of California, Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, Los Angeles, CA (United States); Zhou, Yu; Marks, James D. [University of California, Department of Anesthesia, San Francisco, CA (United States); Hahm, Scott; Reiter, Robert E. [University of California, Department of Urology, David Geffen School of Medicine, Los Angeles, CA (United States)

    2010-08-15

    Prostate stem cell antigen (PSCA), a cell surface glycoprotein expressed in normal human prostate and bladder, is over-expressed in the majority of localized prostate cancer and most bone metastases. We have previously shown that the hu1G8 minibody, a humanized anti-PSCA antibody fragment (single-chain Fv-C{sub H}3 dimer, 80 kDa), can localize specifically and image PSCA-expressing xenografts at 21 h post-injection. However, the humanization and antibody fragment reformatting decreased its apparent affinity. Here, we sought to evaluate PET imaging contrast with affinity matured minibodies. Yeast scFv display, involving four rounds of selection, was used to generate the three affinity matured antibody fragments (A2, A11, and C5) that were reformatted into minibodies. These three affinity matured anti-PSCA minibodies were characterized in vitro, and following radiolabeling with {sup 124}I were evaluated in vivo for microPET imaging of PSCA-expressing tumors. The A2, A11, and C5 minibody variants all demonstrated improved affinity compared to the parental (P) minibody and were ranked as follows: A2 > A11 > C5 > P. The {sup 124}I-labeled A11 minibody demonstrated higher immunoreactivity than the parental minibody and also achieved the best microPET imaging contrast in two xenograft models, LAPC-9 (prostate cancer) and Capan-1 (pancreatic cancer), when evaluated in vivo. Of the affinity variant minibodies tested, the A11 minibody that ranked second in affinity was selected as the best immunoPET tracer to image PSCA-expressing xenografts. This candidate is currently under development for evaluation in a pilot clinical imaging study. (orig.)

  10. Prostate-specific membrane antigen expression is a potential prognostic marker in endometrial adenocarcinoma.

    Science.gov (United States)

    Mhawech-Fauceglia, Paulette; Smiraglia, Dominic J; Bshara, Wiam; Andrews, Christopher; Schwaller, Juerg; South, Stacey; Higgs, Donald; Lele, Shashikant; Herrmann, Francois; Odunsi, Kunle

    2008-03-01

    The aim of this study was to determine the role of prostate-specific membrane antigen (PSMA) as a prognostic marker in endometrial adenocarcinoma (EAC) and to explore whether its down-regulation could be due to epigenetic mechanism. First, we examined the expression and the prognostic value of PSMA by semiquantitative reverse transcription-PCR and immunohistochemistry in EAC tissue samples. Second, to explore the role of CpG methylation in down-regulation PSMA in EAC, we evaluated PSMA CpG island methylation using methylation-specific PCR in cells lines and in a subset of patients' samples. Furthermore, association of the status of tumor methylation to the clinical and histologic variables was also evaluated. Higher PSMA mRNA levels were associated with stage I (P = 0.046) and PSMA protein intensity by immunohistochemistry (P = 0.032). In multivariate analysis, loss of PSMA expression was associated with a worse disease-free survival (P = 0.02). PSMA was methylated in prostate cell lines (DU145 and PC3) and endometrial cell lines. In addition, PSMA was methylated in 5 of 18 samples (all 5 had low PSMA mRNA value). There was a significant association between PSMA methylation and loss of protein expression by immunohistochemistry and PSMA-RNA level with P value of 0.036 and 0.011, respectively. In addition, there was an association between PSMA methylation and tumor size (P = 0.025). In summary, (a) PSMA is underexpressed in advanced stage EAC, (b) loss of PSMA expression can be considered as a prognostic marker in patients with EAC, and (c) loss of PSMA expression in a subset of EAC cases could be due to epigenetic silencing.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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.

  13. Tumor Expression of the Carcinoembryonic Antigen Correlates with High Mitotic Activity and Cell Pleomorphism Index in Lung Carcinoma

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    Rancés Blanco

    2013-01-01

    Full Text Available At present, some research efforts are focusing on the evaluation of a variety of tumor associated antigens (TAAs for a better understanding of tumor biology and genetics of lung tumors. For this reason, we evaluated the tissue expression of carcinoembryonic antigen (CEA and ior C2 (a cell surface O-linked glycoprotein carbohydrate chain TAA in lung carcinomas, as well as its correlation with a variety of clinicopathological features. The tissue expression of CEA was evidenced in 22/43 (51.16% lung carcinomas and it was correlated with mitotic activity, cell pleomorphism indexes, and age of patients. The expression of ior C2 was observed in 15/43 (34.88% tumors but no correlation with the clinicopathological features mentioned above was obtained. No correlation between both CEA and ior C2 antigens expression and the overall survival (OS of non-small-cell lung cancer patients was also observed. However, CEA-negative patients displayed higher OS rates as compared with positive ones (69.74 versus 58.26 months. Our results seem to be in agreement with the role of CEA expression in tumor cell proliferation, inhibition of cell polarizations and tissue architecture distortion. The significance of ior C2 antigen in these malignancies and it potential use in diagnosis, prognosis, and/or immunotherapy must be reevaluated.

  14. Coexpression of GM-CSF and antigen in DNA prime-adenoviral vector boost immunization enhances polyfunctional CD8+ T cell responses, whereas expression of GM-CSF antigen fusion protein induces autoimmunity

    OpenAIRE

    Tenbusch, Matthias; Kuate, Seraphin; Tippler, Bettina; Gerlach, Nicole; Schimmer, Simone; Dittmer, Ulf; Überla, Klaus

    2008-01-01

    Abstract Background Granulocyte-macrophage colony-stimulating factor (GM-CSF) has shown promising results as a cytokine adjuvant for antiviral vaccines and in various models of tumor gene therapy. To explore whether the targeting of antigens to GM-CSF receptors on antigen-presenting cells enhances antigen-specific CD8 T-cell responses, fusion proteins of GM-CSF and ovalbumin (OVA) were expressed by DNA and adenoviral vector vaccines. In addition, bicistronic vectors allowing independent expre...

  15. Immuno-fluorescence based Vi capsular polysaccharide detection for specific recognition of Salmonella enterica serovar Typhi in clinical samples.

    Science.gov (United States)

    Pandey, Satish K; Vinayaka, Aaydha C; Rishi, Dharam B; Rishi, Praveen; Suri, C Raman

    2014-09-02

    Typhoid fever is a life threatening bacterial infection that remains a major global health concern. This continued high burden associated with significant morbidity and mortality rate demands specific and rapid detection technique. This work reports a new sandwich type fluorescence immunoassay format using polymyxin B, a cationic receptor molecule, as a binder agent while anti-Vi antibody served as the capturing agent for specifically detecting Salmonella enterica serovar Typhi. Anti-Vi IgG antibody raised against Vi-BSA conjugate revealed affinity of 7.779nM(-1) signifying immunodominancy of O-acetyls groups in Vi polysaccharide. The detection limit of the developed assay was around 10(1) cellsmL(-1) of Vi expressing Salmonella enterica serovar Typhi with a correlation coefficient (R(2)) equal to 0.97. Positive response obtained for all the tested serovar Typhi clinical isolates as well as the pathogen spiked blood samples recommended specificity and accuracy of Vi antigen as a biomarker during typhoid fever. The intra- and inter-assay precision with Vi spiked samples were satisfactory revealing coefficient of variance (CV%) with a mean of 4.05% and 5.97% respectively. This may be the novel attempt and constructive report on the fluorescence based detection of Vi antigen of serovar Typhi in the epidemic as well as pandemic outbreaks. Copyright © 2014 Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    Singh, Harjeet; Figliola, Matthew J; Dawson, Margaret J; Olivares, Simon; Zhang, Ling; Yang, Ge; Maiti, Sourindra; Manuri, Pallavi; Senyukov, Vladimir; Jena, Bipulendu; Kebriaei, Partow; Champlin, Richard E; Huls, Helen; Cooper, Laurence J N

    2013-01-01

    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.

  18. Manufacture of Clinical-Grade CD19-Specific T Cells Stably Expressing Chimeric Antigen Receptor Using Sleeping Beauty System and Artificial Antigen Presenting Cells

    Science.gov (United States)

    Singh, Harjeet; Figliola, Matthew J.; Dawson, Margaret J.; Olivares, Simon; Zhang, Ling; Yang, Ge; Maiti, Sourindra; Manuri, Pallavi; Senyukov, Vladimir; Jena, Bipulendu; Kebriaei, Partow; Champlin, Richard E.; Huls, Helen; Cooper, Laurence J. N.

    2013-01-01

    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 ∼1010 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. PMID:23741305

  19. Granuloma cells in chronic inflammation express CD205 (DEC205) antigen and harbor proliferating T lymphocytes: similarity to antigen-presenting cells.

    Science.gov (United States)

    Ohtani, Haruo

    2013-02-01

    Granulomas are classified as immune or foreign body granulomas. Of these, the immune granulomas, a hallmark of granulomatous inflammation, are closely related to cell-mediated immune responses. The aim of the present study is to characterize immune granuloma cells in 33 patients with granulomatous inflammation focusing on the expression of CD205 (DEC205), a cell surface marker of antigen presenting cells, and their spatial relationship to T cells. CD205 was frequently expressed by immune granuloma cells, in contrast to foreign body granuloma cells that lacked CD205 expression. T cells were not only distributed in a lymphocyte collar around the granuloma, but also present among the granuloma cells (termed 'intra-granuloma T cells'). Intra-granuloma T cells stained positive for Ki-67 (median positivity = 9.4%) by double immunostaining for CD3 and Ki-67. This indicated the presence of proliferative stimuli within the granuloma that could activate the intra-granuloma T cells. The labeling index of Ki-67 in intra-granuloma T cells was significantly higher than that of T cells in the lymphocyte collar (P granulomas and antigen presenting cells. © 2013 The Author. Pathology International © 2013 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd.

  20. Expression of the cancer-testis antigen BORIS correlates with prostate cancer.

    Science.gov (United States)

    Cheema, Zubair; Hari-Gupta, Yukti; Kita, Georgia-Xanthi; Farrar, Dawn; Seddon, Ian; Corr, John; Klenova, Elena

    2014-02-01

    BORIS, a paralogue of the transcription factor CTCF, is a member of the cancer-testis antigen (CT) family. BORIS is normally present at high levels in the testis; however it is aberrantly expressed in various tumors and cancer cell lines. The main objectives of this study were to investigate BORIS expression together with sub-cellular localization in both prostate cell lines and tumor tissues, and assess correlations between BORIS and clinical/pathological characteristics. We examined BORIS mRNA expression, protein levels and cellular localization in a panel of human prostate tissues, cancer and benign, together with a panel prostate cell lines. We also compared BORIS levels and localization with clinical/pathological characteristics in prostate tumors. BORIS was detected in all inspected prostate cancer cell lines and tumors, but was absent in benign prostatic hyperplasia. Increased levels of BORIS protein positively correlated with Gleason score, T-stage and androgen receptor (AR) protein levels in prostate tumors. The relationship between BORIS and AR was further highlighted in prostate cell lines by the ability of ectopically expressed BORIS to activate the endogenous AR mRNA and protein. BORIS localization in the nucleus plus cytoplasm was also associated with higher BORIS levels and Gleason score. Detection of BORIS in prostate tumors suggests potential applications of BORIS as a biomarker for prostate cancer diagnosis, as an immunotherapy target and, potentially, a prognostic marker of more aggressive prostate cancer. The ability of BORIS to activate the AR gene indicates BORIS involvement in the growth and development of prostate tumors. © 2013 Wiley Periodicals, Inc.

  1. Addition of calmodulin antagonists to NRK cells during G1 inhibits proliferating cell nuclear antigen expression.

    Science.gov (United States)

    López-Girona, A; Bosch, M; Bachs, O; Agell, N

    1995-07-01

    The mRNAs of most proteins involved in DNA synthesis show an S phase correlated expression when mammalian cells are stimulated to proliferate from G0. This is the case for proliferating cell nuclear antigen (PCNA), a cofactor of DNA polymerase delta that is essential for the synthesis of the leading and lagging strands of DNA. Normal rat kidney cells re-entering the cell cycle from quiescence start DNA synthesis at 12 h and reach a maximum at 20 h. The expression of PCNA parallels the synthesis of DNA. Progression through the S phase was inhibited by addition of the anticalmodulin drug W13 to the cells during G1, 5 h after activation. W13 also inhibited the increase in both PCNA protein and mRNA indicating that calmodulin regulates its expression. Using TK-ts13 cells transfected with a plasmid containing the thymidine kinase gene under the control of the human 2.8 kb PCNA promoter, we demonstrated that this promoter is not regulated by calmodulin. The half-life of PCNA mRNA during G1/S transition was not modified by the treatment with W13, indicating that the decrease in the mRNA found when calmodulin was inhibited is not due to changes in its stability. Run-on assays revealed that control cells produced predominantly complete PCNA transcripts during S phase, while short incomplete transcripts were generated in W13-treated cells at the same time. These results indicate that calmodulin participates in a more direct or indirect way during G1 in the activation of PCNA expression. From data presented here it can be suggested that calmodulin activates the release of a transcriptional block leading to an increase in the amount of PCNA during S phase.

  2. Evaluation of Nicotiana tabacum plants transformed for the expression of verocytotoxic Escherichia coli antigens.

    Directory of Open Access Journals (Sweden)

    Angela Lombardi

    2015-07-01

    Full Text Available Two transgenic Nicotiana tabacum plants, carrying respectively the F18 adhesive fimbriae and the B subunit of verocytotoxin genes from O138 Verocytotoxic E.coli serotype were developed as a model of edible vaccine. Tobacco plants were transformed by agroinfection according to Rossi et al. (2013 stably.  The F18 adhesive fimbriae and VT2e B-subunit were placed under control of the GLOB promoter for the seed-specific protein expression. Agrobacterium tumefaciens binary vector system is an efficient tool to transform plant cells; however, the exogenous gene integrates at semi-random into the nuclear chromosome. PCR products, using specific oligonucleotides putatively encoding the B-subunit of VT2e-B and F18 fimbriae were identified on agarose gel (1.5% - 0.9% as bands with a length of 270 and 519 base pairs, respectively. We showed that the foreign VT2e-B and F18 fimbriae genes were stably integrated into the tobacco genome. Northern blot and Western blot analyses carried out respectively on total mRNA and total soluble protein extract obtained from seeds. For each line, the obtained amount of antigens is sufficient for subsequent oral immunization trials. Three lines of tobacco seeds (F18, VT2e-B, and WT were seeded in homogeneous conditions and were harvested simultaneously. Tobacco plants were analysed also by optical and electronic microscope in different phases of growth. Germination of transgenic seeds were delayed of three/five days compared to WT in two replicated experiments, suggesting that genetic manipulation may influenced mechanisms leading to germination. In conclusion the genes coding for VT2e-B and the F18 are stably maintained in the seeds and obtained tobacco seeds represent a valid strategy to ferry antigenic proteins to the gut and a promising non-invasive method of vaccination in pig industry.

  3. A macrophage phenotype for a constitutive, class II antigen-expressing, human dermal perivascular dendritic cell.

    Science.gov (United States)

    Sontheimer, R D; Matsubara, T; Seelig, L L

    1989-07-01

    A previously uncharacterized population of class II antigen-bearing dendritic cells that are intimately associated with the dermal microvasculature was identified in normal human skin using a double-label, indirect immunofluorescence technique. The only other major HLA-DR positive dermal cell type noted in these studies, the dermal microvascular endothelial cell (DMVEC), appeared to express lesser amounts of HLA-DR region gene product than did this dermal perivascular dendritic cell (DPDC). These DPDC were particularly common around small vessels in the superficial vascular plexus of the papillary dermis and were distinct from the mast cell, another cell type normally seen in a similar location. Phenotypic and ultrastructural studies have determined that the DPDC is more closely related to the monocyte/macrophage lineage than the dendritic cell lineage. The perivascular location and phenotype of this cell distinguishes it from other previously described constitutive dermal cell types such as the classic "histiocyte," veiled cell, and dendrocyte. The relatively rich expression of all three major HLA-D region gene products by this dermal perivascular dendritic macrophage would suggest that it could play a significant role in the immunobiology of the dermal microvascular unit.

  4. Trichohyalin expression in skin tumors: retrieval of trichohyalin antigenicity in tissues by microwave irradiation.

    Science.gov (United States)

    Manabe, M; Yaguchi, H; Iqbal Butt, K; O'Guin, W M; Loomis, C A; Sung, T T; Ogawa, H

    1996-05-01

    The antitrichohyalin antibody AE 15 is effective for identifying the cell lineage that undergoes the pathway of inner root sheath-type differentiation. Unfortunately, the AE 15 does not react with trichohyalin in tissue that is formalin-fixed and embedded in paraffin according to routine procedures. We attempted to retrieve the trichohyalin antigenicity in formalin-fixed, paraffin-embedded biopsy specimens that included normal skin as well as skin tumors such as trichofolliculoma and pilotricoma. We found that the use of a metal solution in combination with microwave oven heating improves the trichohyalin immunoreactivity substantially. Further, trichohyalin was found to be expressed not only in the secondary hair structure in trichofolliculoma but also in a certain cell lineage that differentiates to squamoid cells in pilomatricoma. Our findings established that surgical specimens processed under routine procedures can be successfully investigated with AE 15 using the microwave irradiation method. Studies of epidermal diseases expressing trichohyalin should provide valuable insights into our understanding the functional significance of trichohyalin during abnormal keratinization.

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

    Science.gov (United States)

    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.

  6. Corticosteroids decrease the expression of beta 2-microglobulin and histocompatibility antigens on human peripheral blood lymphocytes in vitro

    DEFF Research Database (Denmark)

    Hokland, M; Larsen, B; Heron, I

    1982-01-01

    The in vitro effect of two different glucocorticoids (prednisolone and dexamethasone) on the expression of beta 2-microglobulin and HLA-A-A, -B and -C-antigens on the surface of cultured lymphocytes was measured by quantitative immunofluorescence (flow cytofluorometry) and by a radioimmunoassay. ...

  7. Yeast expressing hepatitis B virus surface antigen determinants on its surface: Implications for a possible oral vaccine

    NARCIS (Netherlands)

    Schreuder, M.P.; Deen, C.; Boersma, W.J.A.; Pouwels, P.H.; Klis, F.M.

    1996-01-01

    The two major hydrophilic regions of the hepatitis B virus surface antigen (HBsAg) have been expressed in the outer mannoprotein layer of the cell wall of 'Bakers Yeast', Saccharomyces cerevisiae, by fusing them between the yeast invertase signal sequence and the yeast α-agglutinin carboxyterminal

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

    DEFF Research Database (Denmark)

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

    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 and the ov...

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  11. 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...

  12. Plant bioreactors for the antigenic hook-associated flgK protein expression

    Directory of Open Access Journals (Sweden)

    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.

  13. Tissue expression of Squamous Cellular Carcinoma Antigen (SCCA) is inversely correlated to tumor size in HCC.

    Science.gov (United States)

    Trerotoli, Paolo; Fransvea, Emilia; Angelotti, Umberto; Antonaci, Giovanni; Lupo, Luigi; Mazzocca, Antonio; Mangia, Anita; Antonaci, Salvatore; Giannelli, Gianluigi

    2009-05-27

    This study aimed to investigate squamous cellular carcinoma antigen (SCCA) in serum and in tumoral and paired peritumoral tissues. We studied 27 patients with liver cirrhosis (LC) and 55 with HCC: 20 with a single nodule HCC) and 35 with a single nodule > 3 cm or multifocal (l-HCC). Serum SCCA was measured by the ELISA kit, and in frozen tissues by immunohistochemistry, quantified with appropriate imaging analysis software and expressed in square microns. Continuous variables are reported as means and 95% confidence intervals. Comparisons between independent groups were performed with a generalized linear model and Tukey grouping. Pearson's correlation coefficients were determined to evaluate relations between markers. Qualitative variables were summarized as count and percentage. Statistical significance was set at p-value SCCA values in LC patients were 0.41 (0.31-0.55) ng/ml and statistically different from both HCC groups: 1.6 (1.0-2.6) ng/ml in s-HCC, 2.2 (1.28-2.74) ng/ml in l-HCC. SCCA in hepatic tissue was 263.8 (176.6-394.01) microm2 in LC patients, statistically different from values in s-HCC: 1163.2 (863.6-1566.8) microm2 and l-HCC: 625.8 (534.5-732.6). All pairwise comparisons between groups yielded statistically significant differences. Tumoral SCCA resulted linearly related with nodule size, showing a statistically significant inverse relation between the two variables (b = -0.099, p = 0.024). There was no statistically significant correlation between tissue and serum levels of SCCA. The significantly stronger expression of SCCA in smaller compared to larger HCC could be important for early HCC detection. However, the increased expression in peritumoral tissue could affect the significance of serological detection.

  14. Tissue expression of Squamous Cellular Carcinoma Antigen (SCCA is inversely correlated to tumor size in HCC

    Directory of Open Access Journals (Sweden)

    Mangia Anita

    2009-05-01

    Full Text Available Abstract Background This study aimed to investigate squamous cellular carcinoma antigen (SCCA in serum and in tumoral and paired peritumoral tissues. We studied 27 patients with liver cirrhosis (LC and 55 with HCC: 20 with a single nodule 3 cm or multifocal (l-HCC. Methods Serum SCCA was measured by the ELISA kit, and in frozen tissues by immunohistochemistry, quantified with appropriate imaging analysis software and expressed in square microns. Continuous variables are reported as means and 95% confidence intervals. Comparisons between independent groups were performed with a generalized linear model and Tukey grouping. Pearson's correlation coefficients were determined to evaluate relations between markers. Qualitative variables were summarized as count and percentage. Statistical significance was set at p-value Results Serum SCCA values in LC patients were 0.41 (0.31–0.55 ng/ml and statistically different from both HCC groups: 1.6 (1.0–2.6 ng/ml in s-HCC, 2.2 (1.28–2.74 ng/ml in l-HCC. SCCA in hepatic tissue was 263.8 (176.6–394.01 μm2 in LC patients, statistically different from values in s-HCC: 1163.2 (863.6–1566.8 μm2 and l-HCC: 625.8 (534.5–732.6. All pairwise comparisons between groups yielded statistically significant differences. Tumoral SCCA resulted linearly related with nodule size, showing a statistically significant inverse relation between the two variables (b = -0.099, p = 0.024. Conclusion There was no statistically significant correlation between tissue and serum levels of SCCA. The significantly stronger expression of SCCA in smaller compared to larger HCC could be important for early HCC detection. However, the increased expression in peritumoral tissue could affect the significance of serological detection.

  15. The thymus in "bare lymphocyte" syndrome : Significance of expression of major histocompatibility complex antigens on thymic epithelial cells in intrathymic T-cell maturation

    NARCIS (Netherlands)

    Schuurman, Henk-Jan; Wijngaert, Frank P. van de; Huber, Jonne; Schuurman, R.K.B.; Zegers, Ben J.M.; Roord, John J.; Kater, L.

    1985-01-01

    Thymic biopsies from two patients with combined immunodeficiency and defective expression of HLA class I and class II antigens on blood mononuclear cells (“bare lymphocyte” syndrome) were investigated. This made possible an evaluation of the significance of HLA antigen expression in a detailed

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

    DEFF Research Database (Denmark)

    Gjerstorff, Morten F; Pøhl, Mette; Olsen, Karen E

    2013-01-01

    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....

  17. 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

    in vitro is paralleled by differential kinetics in the expression of the T-cell adhesion and activation antigens leukocyte function-associated antigen 1 (LFA-1; CD11a/CD18), interleukin-2 receptor (IL-2R; CD25), and intercellular adhesion molecule 1 (ICAM-1; CD54). Furthermore, the changes in expression...... prestimulation levels, and CD25 expression was decreasing. This indicates that T-cell expression of all the 3 surface antigens examined is reversible. While this is in agreement with previous reports of the expression kinetics of IL-2R and ICAM-1, this is the first report indicating that the regulation of T...

  18. Identification of Protective Brucella Antigens and their Expressions in Vaccinia Virus to Prevent Disease in Animals and Humans.

    Science.gov (United States)

    1996-05-01

    selected antigens is through fractionation of Brucella strain RB51 or E.coli recombinants expressing the appropriateBrucella antigen. Briefly, the method...animal species infected with Brucella spp. It is also able to induce the in vitro production of INF-y with lymphocytes of RB51 vaccinated mice (Table...SOD RB51 1IkDa 20 15 x0 0- 10 E 0- Uve Acetone Buffer Void 0-0.1 0.1-0-25 0.25->0.5 0.5-0.75 0.75->1.0 Klled 14 Preparation of new vaccinia/ Brucella

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

    Directory of Open Access Journals (Sweden)

    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

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

    Science.gov (United States)

    Cepeda, Sergio; Cantu, Carolina; Orozco, Stephanie; Xiao, Yangming; Brown, Zoe; Semwal, Manpreet K; Venables, Thomas; Anderson, Mark S; Griffith, Ann V

    2018-01-30

    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. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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

  3. Low Cost Tuberculosis Vaccine Antigens in Capsules: Expression in Chloroplasts, Bio-Encapsulation, Stability and Functional Evaluation In Vitro

    Science.gov (United States)

    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 (6kDa 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

  4. Melanoma-associated antigen expression and the efficacy of tyrosine kinase inhibitors in head and neck cancer

    DEFF Research Database (Denmark)

    Hartmann, Stefan; Brands, Roman C; Küchler, Nora

    2015-01-01

    Melanoma-associated antigen (MAGE) has been identified in a variety of types of cancer. The expression of several MAGE subgroups is correlated with poor prognosis and chemotherapeutic resistance. One target of chemotherapeutic treatment in head and neck cancer is the epidermal growth factor...... receptor (EGFR). The efficacy of tyrosine kinase inhibitors (TKI) in the context of melanoma-associated antigens is discussed in the present study. Five human squamous cell carcinoma cell lines were treated with the EGFR TKIs, erlotinib and gefitinib. The efficacy of these agents was measured using...... a crystal violet assay. Furthermore, the expression levels of MAGE-A1, -A5, -A8, -A9, -A11 and -A12 were determined by reverse transcription-quantitative polymerase chain reaction. The association between TKI efficacy and MAGE-A expression was analyzed by linear regression. The cell lines revealed...

  5. Development of Recombinant Chimeric Antigen Expressing Immunodominant B Epitopes of Leishmania infantum for Serodiagnosis of Visceral Leishmaniasis

    Science.gov (United States)

    Boarino, A.; Scalone, A.; Gradoni, L.; Ferroglio, E.; Vitale, F.; Zanatta, R.; Giuffrida, M. G.; Rosati, S.

    2005-01-01

    Wild canids and domestic dogs are the main reservoir of zoonotic visceral leishmaniasis (VL) caused by Leishmania infantum (syn.: Leishmania chagasi). Serological diagnosis of VL is therefore important in both human and dog leishmaniasis from a clinical and epidemiological point of view. Routine diagnosis of VL is traditionally carried out by immunofluorescent antibody test (IFAT), which is laborious and difficult to standardize and to interpret. In the last decade, however, several specific antigens of Leishmania infantum have been characterized, allowing the development of a recombinant-based immunoassay. Among them, the whole open reading frame encoding K9 antigen, the gene fragment encoding the repetitive sequence of K26, and the 3′-terminal gene fragment of the kinesin-related protein (K39sub) were previously evaluated as diagnostic markers for canine leishmaniasis and proved to be independent in their antibody reactivity. Since sensitivity of serological test is usually higher in multiple-epitope format, in this study the relevant epitopes of K9, K26, and K39 antigens were joined by PCR strategy to produce the chimeric recombinant protein. The resulting mosaic antigen was found highly expressed in Escherichia coli and efficiently purified by affinity chromatography. Antigenic properties of this recombinant antigen were evaluated by indirect enzyme-linked immunosorbent assay (ELISA) using a panel of human and dog sera previously characterized by parasitological and/or serological techniques. Chimeric ELISA showed 99% specificity in both human (n = 180) and canine (n = 343) control groups, while sensitivity was higher in canine VL (96%, n = 213) than in human VL (82%, n = 185). Accordingly, concordance between IFAT and canine chimeric ELISA (k = 0.95, 95% confidence interval = 0.93 to 0.98) was higher than between IFAT and human chimeric ELISA (k = 0.81, 95% confidence interval = 0.76 to 0.87). Results suggest the potential use of this new antigen for routine

  6. Rhythmic expressed clock regulates the transcription of proliferating cellular nuclear antigen in teleost retina.

    Science.gov (United States)

    Song, Hang; Wang, Defeng; De Jesus Perez, Felipe; Xie, Rongrong; Liu, Zhipeng; Chen, Chun-Chun; Yu, Meijuan; Yuan, Liudi; Fernald, Russell D; Zhao, Sheng

    2017-07-01

    Teleost fish continues to grow their eyes throughout life with the body size. In Astatotilapia burtoni, the fish retina increases by adding new retinal cells at the ciliary marginal zone (CMZ) and in the outer nuclear layer (ONL). Cell proliferation at both sites exhibits a daily rhythm in number of dividing cells. To understand how this diurnal rhythm of new cell production is controlled in retinal progenitor cells, we studied the transcription pattern of clock genes in retina, including clock1a, clock1b, bmal1a (brain and muscle ARNT-Like), and per1b (period1b). We found that these genes have a strong diurnal rhythmic transcription during light-dark cycles but not in constant darkness. An oscillation in pcna transcription was also observed during light-dark cycles, but again not in constant darkness. Our results also indicate an association between Clock proteins and the upstream region of pcna (proliferating cellular nuclear antigen) gene. A luciferase reporter assay conducted in an inducible clock knockdown cell line further demonstrated that the mutation on predicted E-Boxes in pcna promoter region significantly attenuated the transcriptional activation induced by Clock protein. These results suggested that the diurnal rhythmic expression of clock genes in A. burtoni retina could be light dependent and might contribute to the daily regulation of the proliferation of the retina progenitors through key components of cell cycle machinery, for instance, pcna. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Expression and secretion of the protective antigen of Bacillus anthracis in Bacillus brevis.

    Science.gov (United States)

    Rhie, Gi-Eun; Park, Young-Mia; Chun, Jeong-Hoon; Yoo, Cheon-Kwon; Seong, Won-Keun; Oh, Hee-Bok

    2005-08-01

    We used the Bacillus brevis-pNU212 system to develop a mass production system for the protective antigen (PA) of Bacillus anthracis. A moderately efficient expression-secretion system for PA was constructed by fusing the PA gene from B. anthracis with the B. brevis cell-wall protein signal-peptide encoding region of pNU212, and by introducing the recombinant plasmid, pNU212-mPA, into B. brevis 47-5Q. The clone producing PA secreted about 300 microg of recombinant PA (rPA) per ml of 5PY-erythromycin medium after 4 days incubation at 30 degrees C. The rPA was fractionated from the culture supernatant of B. brevis 47-5Q carrying pNU212-mPA using ammonium sulfate at 70% saturation followed by anion exchange chromatography on a Hitrap Q, a Hiload 16/60 Superdex 200 gel filtration column and a phenyl sepharose hydrophobic interaction column, yielding 70 mg rPA per liter of culture. The N-terminal sequence of the purified rPA was identical to that of native PA from B. anthracis. The purified rPA exhibited cytotoxicity towards J774A.1 cells when combined with lethal factor. The rPA formulated in either Rehydragel HPA or MPL-TDM-CWS adjuvant (Ribi-Trimix) elicited the expression of a large amount of anti-PA and neutralizing antibodies in guinea pigs and completely protected them against a 100 LD50 challenge with fully virulent B. anthracis spores.

  8. Patterns of antigen expression in hepatoblastoma and hepatocellular carcinoma in childhood.

    Science.gov (United States)

    O'Brien, W J; Finlay, J L; Gilbert-Barness, E F

    1989-01-01

    Two hepatocellular carcinomas and six hepatoblastomas were examined for the presence of 13 antigens using immunoperoxidase, avidin-biotin, staining techniques. Primary antibodies were directed against alpha-fetoprotein (AFP), alpha-1-antitrypsin (AAT), lysozyme (LYS), carcinoembryonic antigen (CEA), human chorionic gonadotropin (HCG), glial fibrillary acidic protein (GFAP), neuron specific enolase (NSE), epithelial membrane antigen (EMA), hepatitis B surface antigen (HbSA), lactoferrin (LF), desmin (DES), vimentin (VIM), and keratin (KER). Except for HbSA, the antigen staining pattern was unable to differentiate between hepatoblastoma and hepatocellular carcinoma. Both neoplasms where positive for AFP, AAT, CEA, EMA, and KER; however, neither stained for GFAP, NSE, LYS, LF, HCG, or DES. Vimentin was weakly positive in those hepatoblastomas where mesenchymal tissue was present in the tumor. Only the tissue adjacent to hepatocellular carcinomas stained positively for HbSA and correlated with the elevated serum levels of HbSA.

  9. Development of Escherichia coli and Mycobacterium smegmatis recombinants expressing major Mycobacterium tuberculosis-specific antigenic proteins.

    Science.gov (United States)

    Amoudy, Hanady A; Safar, Hussain A; Mustafa, Abu S

    2016-12-01

    Mycobacterium tuberculosis is an obligate pathogenic bacterial species in the family Mycobacteriaceae and the causative agent of most tuberculosis (TB) cases. Until today, the only approved TB vaccine is Bacille Calmette Guerin (BCG), which has been used since 1921. While BCG provides fairly effective protection for infants and young children, its efficacy in adults is variable around the world. This could be due to several parameters including strains of the vaccine and exposure of individuals to different environmental bacterial infections. The situation is complicated by the emergence of multidrug resistant strains of M. tuberculosis. This urged the demand to develop new improved vaccines and immunotherapies against TB. Development of nonpathogenic recombinant constructs delivering M. tuberculosis-specific antigenic proteins provides the chance to evaluate candidates to be included in diagnostic tools and preventive vaccines. In our study, we are introducing some of the major M. tuberculosis genes in Escherichia coli and Mycobacterium smegmatis. DNA corresponding to the genes Rv3891, Rv3020, Rv0287, Rv3875, Rv3874, Rv3872, Rv2346c, and Rv3619 were PCR-amplified from M. tuberculosis genomic DNA and visualized on gel electrophoresis at the expected DNA size. Products were subsequently ligated to the plasmid pGEMTeasy and used to transform TOP10 E. coli. Transformed colonies were selected on appropriate media. At the second stage, genes-DNA were subcultured in expression vectors pDE22 and pGESTH1; the recombinant plasmids were finally used to transform. M. smegmatis and E. coli, respectively. Expression of proteins in E. coli was confirmed by Western blotting and in M. smegmatis by reverse transcriptase polymerase chain reaction (RT-PCR). Amplified genes were successfully cloned and transformed in E. coli and M. smegmatis. Colonies of recombinant bacteria were detected on appropriate media. Western blotting and RT-PCR confirmed the expression of our corresponding

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

    Energy Technology Data Exchange (ETDEWEB)

    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

  11. High yield expression and single-step purification of Toxoplasma gondii SAG1, GRA1, and GRA7 antigens in Escherichia coli

    DEFF Research Database (Denmark)

    Hiszczynska-Sawicka, E.; Brillowska-Dabrowska, A.; Dabrowski, Slawomir

    2003-01-01

    This report describes a simple, highly efficient and reproducible method for obtaining large quantities of highly pure recombinant Toxoplasma gondii antigens, which can be used for diagnostic application. The obtained T gondii SAG1, GRA1, and GRA7 antigens (as fusion proteins), expressed in Esche......This report describes a simple, highly efficient and reproducible method for obtaining large quantities of highly pure recombinant Toxoplasma gondii antigens, which can be used for diagnostic application. The obtained T gondii SAG1, GRA1, and GRA7 antigens (as fusion proteins), expressed...

  12. Intracellular synthesis of Epstein-Barr virus membrane antigen gp350/220. Inhibitory effect of monensin on its expression.

    Science.gov (United States)

    Bertoni, G; Nguyen, Q V; Humphreys, R E; Sairenji, T

    1989-01-01

    We have defined the intracellular expression and localization of gp350/220, one of the Epstein-Barr virus (EBV) induced membrane antigens, on 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and n-butyrate-treated P3HR-1 cells. 1B6 monoclonal antibody (mAb) immunoprecipitated gp350/220 from [35S]-methionine-labeled cells, as confirmed with other mAbs (2L10, 72A1, and C1), to the same membrane antigen. The appearance of gp350/220 was observed about 14 h after TPA and n-butyrate activation and reached a maximal level at about 48 h. 1B6 mAb membrane immunofluorescence-positive and cytoplasmic fluorescence-positive cells appeared progressively in cell populations at the same frequencies. Cytoplasmic immunofluorescent staining with 1B6 mAb demonstrated a paranuclear complex which was identical to a rhodamine-labeled wheat germ agglutinin-stained pattern which has been ascribed to the Golgi apparatus. We investigated the effect of monensin on gp350/220 expression and processing. Monensin at 10(-7) M significantly inhibited membrane antigen expression in the Golgi apparatus and on the cell surface, but had a negligible effect on synthesis of viral capsid antigen, early antigen, and viral DNA. The inhibition of gp350/220 with monensin was further characterized by the immunoprecipitation of gp350/220 with anti-MA-positive human sera and mAbs. Monensin treatment resulted in the accumulation of a 165-kD molecule which was judged to be a precursor of gp350/220. These results were consistent with the view that the Golgi apparatus plays an important role as a place of synthesis, processing, and maturation of gp350/220.

  13. Toxoplasma gondii antigen SAG2A differentially modulates IL-1β expression in resistant and susceptible murine peritoneal cells.

    Science.gov (United States)

    Leal-Sena, Jamilly Azevedo; Dos Santos, Jane Lima; Dos Santos, Thaise Anne Rocha; de Andrade, Edson Mário; de Oliveira Mendes, Tiago Antônio; Santana, Juliano Oliveira; Mineo, Tiago Wilson Patriarca; Mineo, José Roberto; da Cunha-Júnior, Jair Pereira; Pirovani, Carlos Priminho

    2018-03-01

    The cell surface of Toxoplasma gondii is covered by antigens (SAGs) from the SRS family anchored by glycosylphosphatidylinositol (GPI) and includes antigens from the SAG2 family. Among these, the SAG2A surface antigen shows great potential in activating humoral responses and has been used in characterizing the acute phase of infection and in the serological diagnosis of toxoplasmosis. In this study, we aimed to evaluate rSAG2A-induced proteins in BALB/c and C57BL/c mice macrophages and to evaluate the phenotypic polarization induced in the process. We treated the peritoneal macrophages from mouse strains that were resistant or susceptible to T. gondii with rSAG2A to analyze their proteomic profile by mass spectrometry and systems biology. We also examined the gene expression of these cells by RT-qPCR using the phenotypic markers of M1 and M2 macrophages. Differences were observed in the expression of proteins involved in the inflammatory process in both resistant and susceptible cells, and macrophages were preferentially induced to obtain a pro-inflammatory immune response (M1) via the overexpression of IL-1β in mice susceptible to this parasite. These data suggest that the SAG2A antigen induces phenotypic and classical activation of macrophages in both resistant and susceptible strains of mice during the acute phase of the disease.

  14. 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...... by single and double immunohistochemical staining. RESULTS: We found that GAGE was expressed in the primordial germ cells of the gonadal primordium, whereas MAGE-A1 and NY-ESO-1 were first detected in germ cells of both testis and ovary after sexual differentiation was initiated. The number of positive germ...... cells and the staining intensity of all three CTAs peaked during the second trimester and gradually decreased towards birth in both male and female germ cells. In oocytes, MAGE-A1 expression terminated around birth, whereas NY-ESO-1 expression persisted through the neonatal stage and GAGE expression...

  15. Prostate-Specific Antigen Modulates the Expression of Genes Involved in Prostate Tumor Growth

    Directory of Open Access Journals (Sweden)

    B. Bindukumar

    2005-03-01

    Full Text Available Prostate-specific antigen (PSA is a serine protease that is widely used as a surrogate marker in the early diagnosis and management of prostate cancer. The physiological relevance of tissue PSA levels and their role in prostate tumor growth and metastasis are not known. Free-PSA (f-PSA was purified to homogeneity from human seminal plasma by column chromatography, eliminating hk2 and all known PSA complexes and retaining its protease activity. Confluent monolayers of prostate cancer cell lines, PC-3M and LNCaP, were treated with f-PSA in a series of in vitro experiments to determine the changes in expression of various genes that are known to regulate tumor growth and metastasis. Gene array, quantitative polymerase chain reaction (QPCR, enzyme-linked immunosorbent assay (ELISA results show significant changes in the expression of various cancer-related genes in PC-3M and LNCaP cells treated with f-PSA. In a gene array analysis of PC-3M cells treated with 10 4tM f-PSA, 136 genes were upregulated and 137 genes were downregulated. In LNCaP cells treated with an identical concentration of f-PSA, a total of 793 genes was regulated. QPCR analysis reveals that the genes for urokinase-type plasminogen activator (uPA, VEGF, Pim-1 oncogene, known to promote tumor growth, were significantly downregulated, whereas IFN-γ, known to be a tumor-suppressor gene, was significantly upregulated in f-PSA-treated PC-3M cells. The effect of f-PSA on VEGF and IFN-γ gene expression and on protein release in PC-3M cells was distinctly dose-dependent. In vivo studies showed a significant reduction (P = .03 in tumor load when fPSA was administered in the tumor vicinity of PC-3M tumor-bearing BALB/c nude mice. Our data support the hypothesis that f-PSA plays a significant role in prostate tumor growth by regulating various proangiogenic and antiangiogenic growth factors.

  16. 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

    Numerous studies have examined kinetics of T-cell functional responses following non-specific and antigen-specific stimulation in vitro. However, while reports of phenotypic T-cell changes after non-specific stimulation are abundant, only little information on phenotypic effects of antigen......-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...... in vitro is paralleled by differential kinetics in the expression of the T-cell adhesion and activation antigens leukocyte function-associated antigen 1 (LFA-1; CD11a/CD18), interleukin-2 receptor (IL-2R; CD25), and intercellular adhesion molecule 1 (ICAM-1; CD54). Furthermore, the changes in expression...

  17. Human Platelet Antigen Genotyping and Expression of CD109 (Human Platelet Antigen 15 mRNA in Various Human Cell Types

    Directory of Open Access Journals (Sweden)

    Sang Mee Hwang

    2013-01-01

    Full Text Available CD109 gene encodes a glycosylphosphatidylinositol-linked glycoprotein found in a subset of platelets and endothelial cell, and human platelet antigen (HPA 15 is found on CD109. We evaluated the HPA genotype and/or the CD109 mRNA expression on two peripheral blood stem cells (PBSC, two peripheral bloods (PB, 12 granulocyte products, natural killer (NK-92, B-lymphocyte (CO88BV59-1, K-562 leukemia cell line, human embryonic stem cell (hESC, and human fibroblasts (HF. HPA genotyping was performed by SNaPshot assay and CD109 mRNA expression was evaluated by real-time PCR with SYBR green and melting curve analysis. Genotype HPA-15a/-15a was found in PBSC#1 and two granulocyte products, and HPA-15a/-15b was found in PBSC#2, eight granulocyte products, NK-92, K-562, hESC, and HF, and HPA-15b/-15b was found in two granulocyte products. CD109 mRNA expression was highly increased in HF and increased in CD34+ and CD34− PBSCs and some granulocyte products, compared to the PB. However, the increase of expression level varied among the PBSC and granulocyte products. The CD109 mRNA expression of NK-92, K-562, hESC, and CO 88BV59-1 was not detected. HPA genotype was evaluated in various cells and the expression of CD109, which contains HPA 15, was different among cell lines and high in HF and PBSCs.

  18. MHC class I expression in intestinal cells is reduced by rotavirus infection and increased in bystander cells lacking rotavirus antigen.

    Science.gov (United States)

    Holloway, Gavan; Fleming, Fiona E; Coulson, Barbara S

    2018-01-08

    Detection of viral infection by host cells leads to secretion of type I interferon, which induces antiviral gene expression. The class I major histocompatibility complex (MHCI) is required for viral antigen presentation and subsequent infected cell killing by cytotoxic T lymphocytes. STAT1 activation by interferon can induce NLRC5 expression, promoting MHCI expression. Rotavirus, an important pathogen, blocks interferon signalling through inhibition of STAT1 nuclear translocation. We assessed MHCI expression in HT-29 intestinal epithelial cells following rotavirus infection. MHCI levels were upregulated in a partially type I interferon-dependent manner in bystander cells lacking rotavirus antigen, but not in infected cells. MHCI and NLRC5 mRNA expression also was elevated in bystander, but not infected, cells, suggesting a transcriptional block in infected cells. STAT1 was activated in bystander and infected cells, but showed nuclear localisation in bystander cells only. Overall, the lack of MHCI upregulation in rotavirus-infected cells may be at least partially due to rotavirus blockade of interferon-induced STAT1 nuclear translocation. The reduced MHCI protein levels in infected cells support the existence of an additional, non-transcriptional mechanism that reduces MHCI expression. It is possible that rotavirus also may suppress MHCI expression in vivo, which might limit T cell-mediated killing of rotavirus-infected enterocytes.

  19. Escherichia coli–expressed near full length HIV-1 envelope glycoprotein is a highly sensitive and specific diagnostic antigen

    Directory of Open Access Journals (Sweden)

    Talha Sheikh M

    2012-11-01

    Full Text Available Abstract Background The Human Immunodeficiency Virus type 1 (HIV-1 envelope glycoprotein gp160, useful in detecting anti-HIV-1 antibodies, is difficult to express in heterologous hosts. The major hurdles are its signal sequence, strong hydrophobic regions and heavy glycosylation. While it has not been possible to express full length recombinant (r-gp160 in E. coli, it can be expressed in insect and mammalian cells, but at relatively higher cost. In this work, we report E. coli-based over-expression of r-gp160 variant and evaluate its performance in diagnostic immunoassays for the detection of anti-HIV-1 antibodies. Methods A deletion variant of r-gp160 lacking hydrophobic regions of the parent full length molecule was expressed in E. coli and purified to near homogeneity using single-step Ni(II-affinity chromatography. Biotinylated and europium(III chelate-labeled versions of this antigen were used to set up one- and two-step time-resolved fluorometric double antigen sandwich assays. The performance of these assays was evaluated against a collection of well-characterized human sera (n=131, that included an in-house panel and four commercially procured panels. Results In-frame deletion of three hydrophobic regions, spanning amino acid residues 1–43, 519–538 and 676–706, of full length HIV-1 gp160 resulted in its expression in E. coli. Both the one- and two-step assays manifested high sensitivity unambiguously identifying 75/77 and 77/77 HIV-1 positive sera, respectively. Both assays also identified all 52 HIV-seronegative sera correctly. Between the two assays, the mean signal-to-cutoff value of the two-step assay was an order of magnitude greater than that of the one-step assay. Both assays were highly specific manifesting no cross-reactivity towards antibodies specific to other viruses like hepatitis B, C, and human T cell leukemia viruses. Conclusions This study has demonstrated the expression of r-gp160 variant in E. coli, by deletion

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

    Directory of Open Access Journals (Sweden)

    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.

  1. Influence of different rol gene products on the chain length of Shigella dysenteriae type 1 lipopolysaccharide O antigen expressed by Shigella flexneri carrier strains.

    OpenAIRE

    Klee, S R; Tzschaschel, B D; Timmis, K. N.; Guzman, C.A.

    1997-01-01

    Introduction of the rol genes of Shigella dysenteriae 1 and Escherichia coli K-12 into Shigella flexneri carrier strains expressing the heterologous S. dysenteriae type 1 lipopolysaccharide resulted in the formation of longer chains of S. dysenteriae 1 O antigen. In bacteria producing both homologous and heterologous O antigen, this resulted in a reduction of the masking of heterologous O antigen by homologous lipopolysaccharide and an increased immune response induced by intraperitoneal immu...

  2. Plant-based strategies aimed at expressing HIV antigens and neutralizing antibodies at high levels. Nef as a case study

    OpenAIRE

    Marusic, Carla; Vitale, Allessandro; Pedrazzini, Emanuela; Donini, Marcello; Frigerio, Lorenzo; Bock, Ralph; Dix, Philip J.; McCabe, Matthew S.; Bellucci, Michele; Benvenuto, Eugenio

    2009-01-01

    The first evidence that plants represent a valid, safe and cost-effective alternative to traditional expression systems for large-scale production of antigens and antibodies was described more than 10?years ago. Since then, considerable improvements have been made to increase the yield of plant-produced proteins. These include the use of signal sequences to target proteins to different cellular compartments, plastid transformation to achieve high transgene dosage, codon usage optimization to ...

  3. Prostate stem cell antigen (PSCA) expression increases with high gleason score, advanced stage and bone metastasis in prostate cancer.

    Science.gov (United States)

    Gu, Z; Thomas, G; Yamashiro, J; Shintaku, I P; Dorey, F; Raitano, A; Witte, O N; Said, J W; Loda, M; Reiter, R E

    2000-03-02

    Prostate stem cell antigen (PSCA) is a recently defined homologue of the Thy-1/Ly-6 family of glycosylphosphatidylinositol (GPI)-anchored cell surface antigens. PSCA mRNA is expressed in the basal cells of normal prostate and in more than 80% of prostate cancers. The purpose of the present study was to examine PSCA protein expression in clinical specimens of human prostate cancer. Five monoclonal antibodies were raised against a PSCA-GST fusion protein and screened for their ability to recognize PSCA on the cell surface of human prostate cancer cells. Immunohistochemical analysis of PSCA expression was performed on paraffin-embedded sections from 25 normal tissues, 112 primary prostate cancers and nine prostate cancers metastatic to bone. The level of PSCA expression in prostate tumors was quantified and compared with expression in adjacent normal glands. The antibodies detect PSCA expression on the cell surface of normal and malignant prostate cells and distinguish three extracellular epitopes on PSCA. Prostate and transitional epithelium reacted strongly with PSCA. PSCA staining was also seen in placental trophoblasts, renal collecting ducts and neuroendocrine cells in the stomach and colon. All other normal tissues tested were negative. PSCA protein expression was identified in 105/112 (94%) primary prostate tumors and 9/9 (100%) bone metastases. The level of PSCA expression increased with higher Gleason score (P=0.016), higher tumor stage (P=0.010) and progression to androgen-independence (P=0. 021). Intense, homogeneous staining was seen in all nine bone metastases. PSCA is a cell surface protein with limited expression in extraprostatic normal tissues. PSCA expression correlates with tumor stage, grade and androgen independence and may have prognostic utility. Because expression on the surface of prostate cancer cells increases with tumor progression, PSCA may be a useful molecular target in advanced prostate cancer.

  4. Cell Culture Evaluation of the Semliki Forest Virus Expression System As a Novel Approach for Antigen Delivery and Expression in Fish.

    Science.gov (United States)

    Phenix; McKenna; Fitzpatrick; Vaughan; Atkins; Liljestrom; Todd

    2000-01-01

    Heterologous gene expression by Semliki Forest virus (SFV) expression vectors was investigated in fish cell culture. Experiments performed using an infectious strain of SFV, replication-defective SFV particles, and recombinant SFV RNA constructs encoding the Escherchia coli LacZ or firefly luciferase reporter genes indicated that levels of SFV-mediated expression in fish cells were dependent on cell type and temperature. Maximal expression levels were observed in the two salmonid-derived cell lines CHSE-214 and F95/9 at 25 degrees C and 20 degrees C. As the temperature was lowered to 15 degrees C or below, levels of reporter gene expression were reduced up to 1000-fold, indicating that the SFV replication complex functioned inefficiently at low temperatures. The ability of SFV expression systems to function in fish cells was further investigated by analyzing the expression of the protective VP2 antigen of infectious pancreatic necrosis virus (IPNV) from the various constructs, including a novel DNA-based SFV plasmid. The VP2 protein produced in CHSE-214 and F95/9 cells transfected or infected with the recombinant SFV-IPNV VP2 constructs appeared to be synthesized in an antigenically correct form, as evidenced by the ability to react with several conformation-dependent IPNV-specific monoclonal antibodies. Whether the temperature-restricted replication and expression displayed by SFV-based constructs in fish cell culture also occurs in vivo remains to be determined.

  5. Characterization of antigens expressed in normal baboon trophoblast and cross-reactive with HIV/SIV antibodies.

    Science.gov (United States)

    Langat, D K; Johnson, P M; Rote, N S; Wango, E O; Owiti, G O; Isahakia, M A; Mwenda, J M

    1999-01-01

    Electron microscopic studies have revealed the presence of endogenous retroviral (ERV) particles in normal primate placental tissues. These particles have ultrastructural similarities to type C retroviral particles and are mainly associated with the trophoblast. In normal human placental tissues, they have antigenic similarity with exogenous retroviruses, such as the human immunodeficiency virus (HIV), and may have a role to play in the regulation of cellular gene expression, syncytiotrophoblast formation or pregnancy-related immunosuppression. In this study, a panel of antibodies (polyclonal and monoclonal antibodies) against viral proteins (anti-HIV and anti-SIV) and endogenous retroviral (ERV) proteins were assessed by immunohistochemistry and immunoblotting, for their cross-reactivity with ERV particles isolated from normal baboon placental tissues. The antibodies (anti-HERV-K RT, anti-ERV3 env, anti-HIV-1 p17, anti-HIV-2 gp120) reacted positively with the syncytiotrophoblast and each antibody recognized one or two proteins of molecular weights (MW) 38, 58 or 64 kDa present in the baboon placental villous tissues and SIV-infected molt-4 Cl8 cells, but not in uninfected cells. The results of this study confirm the specific expression of retroviral cross-reactive antigens in normal baboon placental tissues and suggest placental cellular proteins may have antigenic similarity with those recognized by anti-HIV/SIV antibodies. The role of these retroviral-related proteins expressed at the maternal-fetal interface remain unclear.

  6. FOXP3, CBLB and ITCH gene expression and cytotoxic T lymphocyte antigen 4 expression on CD4(+) CD25(high) T cells in multiple sclerosis

    DEFF Research Database (Denmark)

    Sellebjerg, F; Krakauer, M; Khademi, M

    2012-01-01

    Expression of the forkhead box protein 3 (FoxP3) transcription factor is regulated by the E3 ubiquitin ligases Itch and Cbl-b and induces regulatory activity CD4(+) CD25(high) T cells. Treatment with interferon (IFN)-β enhances regulatory T cell activity in multiple sclerosis (MS). We studied...... the phenotype of CD4(+) CD25(high) T cells in MS by flow cytometry and its relationship with expression of the FOXP3, ITCH and CBLB genes. We found that untreated MS patients had lower cell surface expression of cytotoxic T lymphocyte antigen 4 (CTLA-4) on CD4(+) CD25(high) T cells and higher intracellular CTLA...

  7. Geometry VI

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 1; Issue 8. Geometry VI - Space-the Final Frontier. Kapil H Paranjape. Series Article Volume 1 Issue 8 August 1996 pp 28-33. Fulltext. Click here to view fulltext PDF. Permanent link: http://www.ias.ac.in/article/fulltext/reso/001/08/0028-0033 ...

  8. VI KA’

    DEFF Research Database (Denmark)

    Sprogøe, Jonas

    2012-01-01

    Artiklen handler om hvordan man kan bruge et spil til at udvikle og måle kompetencer. Artiklen diskuterer forskellige forståelser kompetencebegrebet og diskuterer hvordan Vi Ka'-spillet bidrager til at indfange den mere aktive forståelse af kompetence, som noget du gør i en bestemt kontekst....

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

    Directory of Open Access Journals (Sweden)

    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

  10. Effect of UV radiation on the surface of mammalian immunocompetent cells. 1. The change in expression of some antigens and receptors of murine spleen lymphocyte surface

    Energy Technology Data Exchange (ETDEWEB)

    Krylenkov, V.A.; Malygin, A.M. (AN SSSR, Leningrad. Inst. Tsitologii)

    1982-12-01

    Short-wave (254nm) and long-wave (365 nm) UV rays (ShUS and LUV rays) induce the increase in the expression of surface markers of T lymphocytes-THETA(Thy-1) antigens and B lymphocytes-MBLA-antigens and EAS receptors when affecting mouse spleen cells in nonlethal and small lethal doses. Total cell content with T and B lymphocyte characters in an irradiated suspension exceeds even the total cell quantity in non-irradiated suspension (100%) which points to the possibility of the expression of plasmatic membrane antigens and receptors not manifested on the surface of nonirradiated lymphocytes. In the isolethal dose range (LD/sup 15/-LD/sup 28/) ShUV rays suppress and LUV rays induce further increase of THETA and MBLA antigens expression. Among B lymphocytes surface markers the MBLA antigens are more resistant to ShUV an LUV radiation as compared with the EAC receptors.

  11. 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...

  12. Algae as protein factories: expression of a human antibody and the respective antigen in the diatom Phaeodactylum tricornutum.

    Directory of Open Access Journals (Sweden)

    Franziska Hempel

    Full Text Available Microalgae are thought to offer great potential as expression system for various industrial, therapeutic and diagnostic recombinant proteins as they combine high growth rates with all benefits of eukaryotic expression systems. Moreover, microalgae exhibit a phototrophic lifestyle like land plants, hence protein expression is fuelled by photosynthesis, which is CO(2-neutral and involves only low production costs. So far, however, research on algal bioreactors for recombinant protein expression is very rare calling for further investigations in this highly promising field. In this study, we present data on the expression of a monoclonal human IgG antibody against the Hepatitis B surface protein and the respective antigen in the diatom Phaeodactylum tricornutum. Antibodies are fully-assembled and functional and accumulate to 8.7% of total soluble protein, which complies with 21 mg antibody per gram algal dry weight. The Hepatitis B surface protein is functional as well and is recognized by algae-produced and commercial antibodies.

  13. Algae as Protein Factories: Expression of a Human Antibody and the Respective Antigen in the Diatom Phaeodactylum tricornutum

    Science.gov (United States)

    Hempel, Franziska; Lau, Julia; Klingl, Andreas; Maier, Uwe G.

    2011-01-01

    Microalgae are thought to offer great potential as expression system for various industrial, therapeutic and diagnostic recombinant proteins as they combine high growth rates with all benefits of eukaryotic expression systems. Moreover, microalgae exhibit a phototrophic lifestyle like land plants, hence protein expression is fuelled by photosynthesis, which is CO2-neutral and involves only low production costs. So far, however, research on algal bioreactors for recombinant protein expression is very rare calling for further investigations in this highly promising field. In this study, we present data on the expression of a monoclonal human IgG antibody against the Hepatitis B surface protein and the respective antigen in the diatom Phaeodactylum tricornutum. Antibodies are fully-assembled and functional and accumulate to 8.7% of total soluble protein, which complies with 21 mg antibody per gram algal dry weight. The Hepatitis B surface protein is functional as well and is recognized by algae-produced and commercial antibodies. PMID:22164289

  14. A false expression of CD8 antigens on CD4+ T cells in a routine flow cytometry analysis.

    Directory of Open Access Journals (Sweden)

    Danuta Kowalczyk

    2006-09-01

    Full Text Available The two-colour flow cytometry method applied in a routine enumeration of peripheral blood T lymphocyte subsets reveals that in some patients the entire population of CD4+ lymphocytes seems to express CD8 determinants as well. However, expression of the CD8 antigens on the cell surface is much lower in comparison with typical CD8+ cells. Moreover, in one-colour staining with an anti-CD8 antibody, cells with weak CD8 expression are not observed and only one typical population of CD8+ lymphocytes is seen. Investigating this phenomenon, we showed that after washing patient cells in RPMI before CD4/CD8 staining, the CD4+ T cell population did not show CD8 "co-expression". These results suggest that CD4+ lymphocytes, which seem to co-express CD8 antigen, in fact do not have this antigen on their surface. Moreover, after the addition of patient plasma to healthy donor cells prior to CD4/CD8 staining, a weak CD8 expression on normal CD4+ cells was noticed. Therefore we can assume that the agent(s causing this phenomenon is/are present in the plasma of some patients. Altogether, these observations suggest that this phenomenon is nonspecific and probably results from cross-linking of anti-CD8 mAbs with anti-CD4 mAbs caused by factor(s present in plasma of some patient. However, identification of that/these factor(s requires further research.

  15. A potential role for immunotherapy in thyroid cancer by enhancing NY-ESO-1 cancer antigen expression.

    Science.gov (United States)

    Gunda, Viswanath; Frederick, Dennie T; Bernasconi, Maria J; Wargo, Jennifer A; Parangi, Sareh

    2014-08-01

    NY-ESO-1 is one of the most immunogenic members of the cancer/testis antigen family and its levels can be increased after exposure to demethylating and deacetylating agents. This cytoplasmic antigen can serve as a potent target for cancer immunotherapy and yet has not been well studied in differentiated thyroid cancer cells. We studied the baseline expression of NY-ESO-1 messenger RNA and protein before and after exposure to 5-aza-2'-deoxycytidine (DAC) (72 hours) in a panel of thyroid cancer cell lines using quantitative polymerase chain reaction and Western blot. HLA-A2+, NY-ESO-1+ thyroid cell lines were then co-cultured with peripheral blood lymphocytes transduced with NY-ESO-1 specific T-cell receptor (TCR) and assayed for interferon-gamma and Granzyme-B release in the medium. SCID mice injected orthotopically with BCPAP cells were treated with DAC to evaluate for NY-ESO-1 gene expression in vivo. None of the thyroid cancer cell lines showed baseline expression of NY-ESO-1. Three cell lines, BCPAP, TPC-1, and 8505c, showed an increase in NY-ESO-1 gene expression with DAC treatment and were found to be HLA-A2 positive. DAC-treated target BCPAP and TPC-1 tumor cells with up-regulated NY-ESO-1 levels were able to mount an appropriate interferon-gamma and Granzyme-B response upon co-culture with the NY-ESO-1-TCR-transduced peripheral blood lymphocytes. In vivo DAC treatment was able to increase NY-ESO-1 expression in an orthotopic mouse model with BCPAP cells. Our data suggest that many differentiated thyroid cancer cells can be pressed to express immune antigens, which can then be utilized in TCR-based immunotherapeutic interventions.

  16. Binding to histo-blood group antigen-expressing bacteria protects human norovirus from acute heat stress

    Directory of Open Access Journals (Sweden)

    Dan eLi

    2015-07-01

    Full Text Available This study aims to investigate if histo-blood group antigen (HBGA expressing bacteria have any protective role on human norovirus (NoV from acute heat stress. Eleven bacterial strains were included, belonging to Escherichia coli, Enterobacter cloacae, Enterobacter aerogenes, Clostridium difficile, Bifidobacterium adolescentis, and Bifidobacterium longum. HBGA expression of the bacteria as well as binding of human NoV virus-like particles (VLPs, GI.1 and GII.4 strains to the bacteria were detected by flow cytometry. NoV VLPs pre-incubated with HBGA expressing or non-HBGA expressing bacteria were heated and detected by both direct ELISA and porcine gastric mucin-binding assay. The NoV-binding abilities of the bacteria correlated well with their HBGA expression profiles. Two HBGA expressing E.coli (LMG8223 and LFMFP861, both GI.1 and GII.4 binders and one non-HBGA expressing E.coli (ATCC8739, neither GI.1 nor GII.4 binder were selected for the heat treatment test with NoV VLPs. Compared with the same cell numbers of non-HBGA expressing E.coli, the presence of HBGA-expressing E.coli could always maintain higher antigen integrity, as well as mucin-binding ability of NoV VLPs of both GI.1 and GII.4 after heat-treatment at 90°C for 2 min. These results indicate that HBGA-expressing bacteria may protect NoVs during the food processing treatments, thereby facilitating their transmission.

  17. Live attenuated rubella vectors expressing SIV and HIV vaccine antigens replicate and elicit durable immune responses in rhesus macaques

    Science.gov (United States)

    2013-01-01

    Background Live attenuated viruses are among our most potent and effective vaccines. For human immunodeficiency virus, however, a live attenuated strain could present substantial safety concerns. We have used the live attenuated rubella vaccine strain RA27/3 as a vector to express SIV and HIV vaccine antigens because its safety and immunogenicity have been demonstrated in millions of children. One dose protects for life against rubella infection. In previous studies, rubella vectors replicated to high titers in cell culture while stably expressing SIV and HIV antigens. Their viability in vivo, however, as well as immunogenicity and antibody persistence, were unknown. Results This paper reports the first successful trial of rubella vectors in rhesus macaques, in combination with DNA vaccines in a prime and boost strategy. The vectors grew robustly in vivo, and the protein inserts were highly immunogenic. Antibody titers elicited by the SIV Gag vector were greater than or equal to those elicited by natural SIV infection. The antibodies were long lasting, and they were boosted by a second dose of replication-competent rubella vectors given six months later, indicating the induction of memory B cells. Conclusions Rubella vectors can serve as a vaccine platform for safe delivery and expression of SIV and HIV antigens. By presenting these antigens in the context of an acute infection, at a high level and for a prolonged duration, these vectors can stimulate a strong and persistent immune response, including maturation of memory B cells. Rhesus macaques will provide an ideal animal model for demonstrating immunogenicity of novel vectors and protection against SIV or SHIV challenge. PMID:24041113

  18. Expression of Trypanosoma brucei gambiense Antigens in Leishmania tarentolae. Potential for Use in Rapid Serodiagnostic Tests (RDTs.

    Directory of Open Access Journals (Sweden)

    Barrie Rooney

    2015-12-01

    Full Text Available The development of rapid serodiagnostic tests for sleeping sickness and other diseases caused by kinetoplastids relies on the affordable production of parasite-specific recombinant antigens. Here, we describe the production of recombinant antigens from Trypanosoma brucei gambiense (T.b. gambiense in the related species Leishmania tarentolae (L. tarentolae, and compare their diagnostic sensitivity and specificity to native antigens currently used in diagnostic kits against a panel of human sera. A number of T.b. gambiense protein antigen candidates were chosen for recombinant expression in L. tarentolae based on current diagnostics in field use and recent findings on immunodiagnostic antigens found by proteomic profiling. In particular, the extracellular domains of invariant surface glycoprotein 65 (ISG65, variant surface glycoproteins VSG LiTat 1.3 and VSG LiTat 1.5 were fused with C-terminal histidine tags and expressed as soluble proteins in the medium of cultured, recombinant L. tarentolae. Using affinity chromatography, on average 10 mg/L of recombinant protein was purified from cultures and subsequently tested against a panel of sera from sleeping sickness patients from controls, i.e. persons without sleeping sickness living in HAT endemic countries. The evaluation on sera from 172 T.b. gambiense human African trypanosomiasis (HAT patients and from 119 controls showed very high diagnostic potential of the two recombinant VSG and the rISG65 fragments with areas under the curve between 0.97 and 0.98 compared to 0.98 and 0.99 with native VSG LiTat 1.3 and VSG LiTat 1.5 (statistically not different. Evaluation on sera from 78 T.b. rhodesiense HAT patients and from 100 controls showed an acceptable diagnostic potential of rISG65 with an area under the curve of 0.83. These results indicate that a combination of these recombinant antigens has the potential to be used in next generation rapid serodiagnostic tests. In addition, the L. tarentolae

  19. 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.

  20. [Expression pattern and level of cytotoxic T lymphocyte-associated antigen-4 targeted anti-caries plasmids in eukaryotic cells].

    Science.gov (United States)

    Guo, Ji-hua; Fan, Ming-wen; Jia, Rong; Bian, Zhuan; Chen, Zhi; Yu, Fei

    2006-05-01

    To investigate and compare the expression pattern and level of targeted anti-caries plasmids encoding different-size antigens in eukaryotic cells. The A-P fragment of PAc (surface protein antigen) was removed from pGJA-P encoding the signal peptide, extracellular domains of human CTLA-4, human Ig hinge, CH2 and CH3 domains, A-P fragment of PAc and GLU (glucan binding domain) region of GTF-I of Streptococcus mutans, to obtain the plasmid pGJGLU. pCI vector skeleton of pGJA-P or pGJGLU was replaced by pVAX1 to construct plasmids pGJA-P/VAX and pGJGLU/VAX. CTLA4-Ig-GLU fragment was removed from pGJGLU and inserted into the vector pEGFP-N1 to obtain the recombinant plasmid pGJGLU/GFP. The CHO cells were transfected with those plasmids by using liposome and the expression of fusion protein was observed with fluorescence microscope. ELISA was used to detect the expression level of fusion proteins in cultured supernatants. Specific vesicles with green fluorescence could be observed in the CHO cells transfected with pGJGLU/GFP. The recombinant fusion protein could be detected in the cultured supernatants of CHO cells transfected with pGJA-P/VAX, pGJGLU/VAX and pGJGLU/GFP, of which the concentration was different. The highest concentration of recombinant fusion protein was observed in the supernatants of CHO cells transfected with pGJGLU/VAX. CTLA-4 targeted fusion protein could be expressed and secreted by eukaryotic cells. The size of antigen may affect the expression level of CTLA-4 targeted anti-caries DNA vaccine.

  1. Early gastric cancer frequently has high expression of KK-LC-1, a cancer-testis antigen.

    Science.gov (United States)

    Futawatari, Nobue; Fukuyama, Takashi; Yamamura, Rui; Shida, Akiko; Takahashi, Yoshihito; Nishi, Yatsushi; Ichiki, Yoshinobu; Kobayashi, Noritada; Yamazaki, Hitoshi; Watanabe, Masahiko

    2017-12-14

    To assess cancer-testis antigens (CTAs) expression in gastric cancer patients and examined their associations with clinicopathological factors. Eighty-three gastric cancer patients were evaluated in this study. Gastric cancer specimens were evaluated for the gene expression of CTAs, Kitakyushu lung cancer antigen-1 (KK-LC-1), melanoma antigen (MAGE)-A1, MAGE-A3 and New York esophageal cancer-1 (NY-ESO-1), by reverse transcription PCR. Clinicopathological background information, such as gender, age, tumor size, macroscopic type, tumor histology, depth of invasion, lymph node metastasis, lymphatic invasion, venous invasion, and pathological stage, was obtained. Statistical comparisons between the expression of each CTA and each clinicopathological background were performed using the χ2 test. The expression rates of KK-LC-1, MAGE-A1, MAGE-A3, and NY-ESO-1 were 79.5%, 32.5%, 39.8%, and 15.7%, respectively. In early stage gastric cancer specimens, the expression of KK-LC-1 was 79.4%, which is comparable to the 79.6% observed in advanced stage specimens. The expression of KK-LC-1 was not significantly associated with clinicopathological factors, while there were considerable differences in the expression rates of MAGE-A1 and MAGE-A3 with vs without lymphatic invasion (MAGE-A1, 39.3% vs 13.6%, P = 0.034; MAGE-A3, 47.5% vs 18.2%, P = 0.022) and/or vascular invasion (MAGE-A1, 41.5% vs 16.7%, P = 0.028; MAGE-A3, 49.1% vs 23.3%, P = 0.035) and, particularly, MAGE-A3, in patients with early vs advanced stage (36.5% vs 49.0%, P = 0.044), respectively. Patients expressing MAGE-A3 and NY-ESO-1 were older than those not expressing MAGE-A3 and NY-ESO-1 (MAGE-A3, 73.7 ± 7.1 vs 67.4 ± 12.3, P = 0.009; NY-ESO-1, 75.5 ± 7.2 vs 68.8 ± 11.2, P = 0.042). The KK-LC-1 expression rate was high even in patients with stage I cancer, suggesting that KK-LC-1 is a useful biomarker for early diagnosis of gastric cancer.

  2. A 6-kb promoter fragment mimics in transgenic mice the prostate-specific and androgen-regulated expression of the endogenous prostate-specific antigen gene in humans

    NARCIS (Netherlands)

    C.B.J.M. Cleutjens (Kitty); H.A.G.M. van der Korput (Hetty); C.C.E.M. Ehren-van Eekelen (Conny); R.A. Sikes; C. Fasciana (Claudia); L.W. Chung; J. Trapman (Jan)

    1997-01-01

    textabstractProstate-specific antigen (PSA) is a kallikrein-like serine protease, which is almost exclusively synthesized in the luminal epithelial cells of the human prostate. PSA expression is androgen regulated. Previously, we characterized in vitro the proximal

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

    DEFF Research Database (Denmark)

    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

  4. 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

    New strategies are needed to develop better tools to control TB, including identification of novel antigens for vaccination. Such Mtb antigens must be expressed during Mtb infection in the major target organ, the lung, and must be capable of eliciting human immune responses. Using genome......-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....... By further analysing high-level conservation among whole-genome sequenced Mtb-complex strains (n = 219) and algorithms predicting HLA-class-Ia and II presented epitopes, we selected the most promising IVE-TB candidate antigens. Several of these were recognized by T-cells from in-vitro Mtb-PPD and ESAT6/CFP10...

  5. [Novel therapy for malignant lymphoma: adoptive immuno-gene therapy using chimeric antigen receptor(CAR)-expressing T lymphocytes].

    Science.gov (United States)

    Ozawa, Keiya

    2014-03-01

    Adoptive T-cell therapy using chimeric antigen receptor (CAR) technology is a novel approach to cancer immuno-gene therapy. CARs are hybrid proteins consisting of target-antigen-specific single-chain antibody fragment fused to intracellular T-cell activation domains (CD28 or CD137/CD3 zeta receptor). CAR-expressing engineered T lymphocytes can directly recognize and kill tumor cells in an HLA independent manner. In the United States, promising results have been obtained in the clinical trials of adoptive immuno-gene therapy using CD19-CAR-T lymphocytes for the treatment of refractory B-cell malignancies, including chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL). In this review article, CD19-CAR-T gene therapy for refractory B-cell non-Hodgkin lymphoma is discussed.

  6. Cloning, expression and evaluation of the efficacy of a recombinant Baylisascaris schroederi Bs-Ag3 antigen in mice.

    Science.gov (United States)

    Wang, Tao; He, Guangzhi; Yang, Guangyou; Fei, Yuxiang; Zhang, Zhihe; Wang, Chengdong; Yang, Zhi; Lan, Jingchao; Luo, Li; Liu, Li

    2008-12-09

    The gene Bs-Ag3 enconding a antigen of 37kDa from Baylisascaris schroederi (giant panda isolates), as well as the recombinant Bs-Ag3, obtained by cloning and expression of the Bs-Ag3 gene in heterologous host Escherichia coli BL-21 (DE3), were used to evaluate their ability to induce immune protective responses in BALB/c mice against L3-challenge infection in a mouse-B. schroederi model. There was a significant reduction (62.91%) of mice vaccinated with rBs-Ag3 coupled with Freund's complete adjuvant (FCA) in recovery of challenged B. schroederi L3 compared with either controls or mice vaccinated with FCA alone. Our data indicate recombinant Bs-Ag3 may be a potential target as a vaccine antigen for giant panda ascariasis.

  7. Clinical significance of surface antigen expression in children with acute myeloid leukemia: results of study AML-BFM-87.

    Science.gov (United States)

    Creutzig, U; Harbott, J; Sperling, C; Ritter, J; Zimmermann, M; Löffler, H; Riehm, H; Schellong, G; Ludwig, W D

    1995-10-15

    Immunophenotyping using a panel of 15 antibodies was performed in 267 (87%) and cytogenetic analysis in 196 (64%) of 307 children under 17 years of age enrolled in the AML-BFM-87 study. Treatment consisted of cytosine arabinoside, daunorubicin, etoposide induction and a 6-week seven-drug consolidation chemotherapy, followed by two blocks of high-dose cytosine arabinoside with or without cranial irradiation and maintenance therapy for 1 year. Five-year event-free survival for patients with immunophenotypic data was .43 +/- .03 SE. The diagnostic value of the pan-myeloid reagents CD13, CD33, and CDw65 for the recognition of childhood acute myeloid leukemia (AML) was high with a sensitivity of 98% (positivity of at least one of these antigens), whereas, with the exception of CD41 for French American British (FAB) subtype M7, the expression of single cell-surface antigens showed no correlation with morphologic or cytogenetic subgroups. On the other hand, characteristic subgroups of AML defined by morphologic features and karyotypes could be described by low or high rates of surface antigen expression compared with those of other patients. These immunophenotypic features most probably associated with specific entities include expression of CD34 or CD13 and absence of CD14 or CD4 in M2 with Auer rods/t(8;21); absence of HLA-DR, CD34, and CD14, but expression of CD33 in M3/t(15;17); positivity of either CD34 or CD13 and either CD14 or CD2 for M4Eo/inv(16); and absence of either CD34 or CD13 and expression of either CD33 or CDw65 and either CD15 or CD4 for M5/t(9;11). In FAB M0, negativity of one or two of the three panmyeloid-associated markers (CD13/33/w65) was common; and cytogenetic results frequently showed random abnormalities. Expression of lymphoid-, progenitor- and most myeloid-associated antigens had no influence on the prognosis, whereas the outcome was significantly better for children with M2 with Auer rods, M3, or M4Eo or for those with the associated

  8. Human leucocyte antigen (HLA) expression of primary trophoblast cells and placental cell lines, determined using single antigen beads to characterize allotype specificities of anti-HLA antibodies.

    Science.gov (United States)

    Apps, Richard; Murphy, Shawn P; Fernando, Raymond; Gardner, Lucy; Ahad, Tashmeeta; Moffett, Ashley

    2009-05-01

    Human trophoblast cells express an unusual repertoire of human leucocyte antigen (HLA) molecules which has been difficult to define. Close homology between and extreme polymorphism at the classical HLA class-I (HLA-I) loci has made it difficult to generate locus-specific monoclonal antibodies (mAbs). The problem of defining an antibody's reactivity against the thousands of existing HLA-I allotypes has often made it impossible to determine the HLA bound by a mAb in biological samples from a normal outbred population. Here we have used commercially available beads coated with individual HLA-I to characterize experimentally the reactivity of nine mAb against 96 common HLA-I allotypes. In conjunction with donor HLA-I genotyping, we could then define the specific HLA molecules bound by these antibodies in normal individuals. We used this approach to analyse the HLA expression of primary trophoblast cells from normal pregnancies; the choriocarcinoma cells JEG-3 and JAR; and the placental cell lines HTR-8/SVneo, Swan-71 and TEV-1. We confirm that primary villous trophoblast cells are HLA null whereas extravillous trophoblast cells express HLA-C, HLA-G and HLA-E, but not HLA-A, HLA-B or HLA-DR molecules in normal pregnancy. Tumour-derived JEG-3 and JAR cells reflect extravillous and villous trophoblast HLA phenotypes, respectively, but the HLA repertoire of the in vitro derived placental cell lines is not representative of either in vivo trophoblast phenotype. This study raises questions regarding the validity of using the placental cell lines that are currently available as model systems for immunological interactions between fetal trophoblast and maternal leucocytes bearing receptors for HLA molecules.

  9. Interactive effects of sulfur and chromium on antioxidative defense systems and BnMP1 gene expression in canola (Brassica napus L.) cultivars differing in Cr(VI) tolerance.

    Science.gov (United States)

    Terzi, Hakan; Yıldız, Mustafa

    2015-07-01

    Plants suffer with combined stress of sulfur (S) deficiency and hexavalent chromium [Cr(VI)] in soils. There are a few reports on the interactive effects of S-deficiency and Cr(VI) stress. Therefore, the interactions between S nutrition and Cr(VI) stress were investigated in hydroponically grown canola (Brassica napus L.) cultivars differing in Cr(VI) tolerance. The relatively Cr(VI)-tolerant (NK Petrol) and Cr(VI)-susceptible (Sary) cultivars were grown in S-sufficient nutrient solution and then exposed to variable S concentrations [deficient (0 mM S, -S) and sufficient (1 mM S, +S)]. The seedlings were then exposed to 100 μM Cr(VI) for 3 days. S-deficiency (-S/-Cr) and combined stress (-S/+Cr) caused a significant decrease in growth parameters of Sary than NK Petrol (P < 0.05). In -S/+Cr treatment, Cr accumulation in Sary was significantly higher than NK Petrol. The higher level of Cr in Sary increased lipid peroxidation and decreased chlorophyll content. The activities of antioxidant enzymes and cysteine content were significantly higher in NK Petrol than in Sary under combined stress. The levels of ascorbate (AsA) and glutathione (GSH) were significantly decreased by S deficiency. The expression level of metallothionein gene (BnMP1) in the tolerant NK Petrol was increased by -S/+Cr treatment. However, expression level of BnMP1 gene in the susceptible Sary was enhanced by +S/+Cr treatment. This result suggests metallothionein (MT) may be involved in Cr(VI) tolerance under S-deficient condition. In conclusion, S nutrition influenced Cr accumulation and enhanced tolerance caused by a positive effect on defense systems and gene expression.

  10. Whole Pichia pastoris yeast expressing measles virus nucleoprotein as a production and delivery system to multimerize Plasmodium antigens.

    Directory of Open Access Journals (Sweden)

    Daria Jacob

    Full Text Available Yeasts are largely used as bioreactors for vaccine production. Usually, antigens are produced in yeast then purified and mixed with adjuvants before immunization. However, the purification costs and the safety concerns recently raised by the use of new adjuvants argue for alternative strategies. To this end, the use of whole yeast as both production and delivery system appears attractive. Here, we evaluated Pichia pastoris yeast as an alternative vaccine production and delivery system for the circumsporozoite protein (CS of Plasmodium, the etiologic agent of malaria. The CS protein from Plasmodium berghei (Pb was selected given the availability of the stringent C57Bl/6 mouse model of infection by Pb sporozoites, allowing the evaluation of vaccine efficacy in vivo. PbCS was multimerized by fusion to the measles virus (MV nucleoprotein (N known to auto-assemble in yeast in large-size ribonucleoprotein rods (RNPs. Expressed in P. pastoris, the N-PbCS protein generated highly multimeric and heterogenic RNPs bearing PbCS on their surface. Electron microscopy and immunofluorescence analyses revealed the shape of these RNPs and their localization in peripheral cytoplasmic inclusions. Subcutaneous immunization of C57Bl/6 mice with heat-inactivated whole P. pastoris expressing N-PbCS RNPs provided significant reduction of parasitemia after intradermal challenge with a high dose of parasites. Thus, in the absence of accessory adjuvants, a very low amount of PbCS expressed in whole yeast significantly decreased clinical damages associated with Pb infection in a highly stringent challenge model, providing a proof of concept of the intrinsic adjuvancy of this vaccine strategy. In addition to PbCS multimerization, the N protein contributed by itself to parasitemia delay and long-term mice survival. In the future, mixtures of whole recombinant yeasts expressing relevant Plasmodium antigens would provide a multivalent formulation applicable for antigen

  11. Cholera vaccine candidate 638: intranasal immunogenicity and expression of a foreign antigen from the pulmonary pathogen Coccidioides immitis.

    Science.gov (United States)

    Silva, Anisia J; Mohan, Archana; Benitez, Jorge A

    2003-12-01

    Vibrio cholerae strain 638 is a live genetically attenuated candidate cholera vaccine in which the CTXPhi prophage encoding cholera toxin has been deleted and hapA, encoding an extracellular Zn-dependent metalloprotease, was insertionally inactivated. Strain 638 was highly immunogenic when inoculated to adult Swiss mice by the intranasal route as judged by the induction of a strong serum vibriocidal antibody response. A side-by-side comparison of strain 638 with its isogenic hapA(+) precursor (strain 81) in the above model indicated that inactivation of hapA does not affect immunogenicity. The spherule-associated antigen 2/proline-rich antigen (Ag2/PRA) of Coccidioides immitis has been shown to protect mice against coccidioidomycosis to an extent dependent on the modes of antigen presentation and challenge with C. immitis arthrospores. In this work, we demonstrate the use of a live genetically attenuated V. cholerae strain to deliver Ag2/PRA. Ag2/PRA was expressed in 638 as a fusion protein with the Escherichia coli heat labile toxin B subunit leader peptide using the strong Tac promoter. The recombinant Ag2/PRA was efficiently expressed, processed and secreted to the periplasmic space. Intranasal immunizations of adult mice with strain 638 expressing Ag2/PRA induced serum vibriocidal antibody response to the vector strain and serum total IgG response to Ag2/PRA. Strain 638 expressing PRA could be recovered from trachea and lung up to 20h after immunization but was effectively cleared 72h post-inoculation.

  12. Expression of Helicobacter pylori TonB protein in transgenic Arabidopsis thaliana: toward production of vaccine antigens in plants.

    Science.gov (United States)

    Kalbina, Irina; Engstrand, Lars; Andersson, Sören; Strid, Ake

    2010-10-01

    The aim of this study was to produce a recombinant version of the highly antigenic Helicobacter pylori TonB (iron-dependent siderophore transporter protein HP1341) in transgenic plants as a candidate oral vaccine antigen. Using Agrobacterium-mediated gene transfer, we introduced three different constructs of the tonB gene into the genome of the model plant Arabidopsis thaliana. We investigated transgene insertion by PCR, produced TonB antibodies for analysis of the production of the recombinant protein in plants, verified the identity of the protein produced by mass spectrometry analysis, and analyzed the number of genetic inserts in the plants by Southern blotting. Three different constructs of the expression cassette (full-length tonB, tonB truncated in the 5' end removing the codons for a transmembrane helix, and the latter construct with codons for the endoplasmic reticulum SEKDEL retention signal added to the 3' end) were used to find the most effective way to express the TonB antigen. Production of TonB protein was detected in plants transformed with each of the constructs, confirmed by both Western blotting and mass spectrometry analysis. No considerable differences in protein expression from the three different constructs were observed. The protein concentration in the plants was at least 0.05% of the total soluble proteins. The Helicobacter pylori TonB protein can be produced in Arabidopsis thaliana plants in a form that is recognizable by rabbit anti-TonB antiserum. These TonB-expressing plants are highly suitable for animal studies of oral administration as a route for immunization against Helicobacter infections. © 2010 Blackwell Publishing Ltd.

  13. Antigen stimulation of peripheral blood mononuclear cells from Mycobacterium bovis infected cattle yields evidence for a novel gene expression program

    Directory of Open Access Journals (Sweden)

    Zhao Yingdong

    2008-09-01

    Full Text Available Abstract Background Bovine tuberculosis (BTB caused by Mycobacterium bovis continues to cause substantial losses to global agriculture and has significant repercussions for human health. The advent of high throughput genomics has facilitated large scale gene expression analyses that present a novel opportunity for revealing the molecular mechanisms underlying mycobacterial infection. Using this approach, we have previously shown that innate immune genes in peripheral blood mononuclear cells (PBMC from BTB-infected animals are repressed in vivo in the absence of exogenous antigen stimulation. In the present study, we hypothesized that the PBMC from BTB-infected cattle would display a distinct gene expression program resulting from exposure to M. bovis. A functional genomics approach was used to examine the immune response of BTB-infected (n = 6 and healthy control (n = 6 cattle to stimulation with bovine tuberculin (purified protein derivative – PPD-b in vitro. PBMC were harvested before, and at 3 h and 12 h post in vitro stimulation with bovine tuberculin. Gene expression changes were catalogued within each group using a reference hybridization design and a targeted immunospecific cDNA microarray platform (BOTL-5 with 4,800 spot features representing 1,391 genes. Results 250 gene spot features were significantly differentially expressed in BTB-infected animals at 3 h post-stimulation contrasting with only 88 gene spot features in the non-infected control animals (P ≤ 0.05. At 12 h post-stimulation, 56 and 80 gene spot features were differentially expressed in both groups respectively. The results provided evidence of a proinflammatory gene expression profile in PBMC from BTB-infected animals in response to antigen stimulation. Furthermore, a common panel of eighteen genes, including transcription factors were significantly expressed in opposite directions in both groups. Real-time quantitative reverse transcription PCR (qRT-PCR demonstrated

  14. 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...

  15. Increased expression of beta 2-microglobulin and histocompatibility antigens on human lymphoid cells induced by interferon

    DEFF Research Database (Denmark)

    Hokland, M; Heron, I; Berg, K

    1982-01-01

    Normal human peripheral blood lymphocytes were incubated in the presence of different concentrations of interferon for various incubation periods. Subsequently, the amount of beta 2-Microglobulin and HLA-A, B and C surface antigens was estimated by means of quantitative immunofluorescence (flow c...

  16. 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.

  17. Retinoic acid-induced expression of CD38 antigen in myeloid cells is mediated through retinoic acid receptor-alpha.

    Science.gov (United States)

    Drach, J; McQueen, T; Engel, H; Andreeff, M; Robertson, K A; Collins, S J; Malavasi, F; Mehta, K

    1994-04-01

    CD38 is a leukocyte differentiation antigen that has been thought to be a phenotypic marker of different subpopulations of T- and B-lymphocytes. In myeloid cells, CD38 is expressed during early stages of differentiation. Virtually no information is available on regulation and functions of CD38. Recently we reported that all-trans-retinoic acid (ATRA) is a potent and highly specific inducer of CD38 expression in human promyelocytic leukemia cells. Here we report that ATRA-induced expression of CD38 antigen in myeloid cells is mediated through retinoic acid-alpha receptor (RAR alpha). ATRA failed to induce CD38 expression in a mutant subclone of the HL-60 myeloid leukemia cell line (designated HL-60R) that is relatively resistant to ATRA-induced granulocytic differentiation. Retroviral vector-mediated transduction of RA receptor (RAR alpha) into this HL-60R subclone completely restored the sensitivity of these cells to ATRA in terms of their ability to express CD38. In contrast, CD38 expression was not inducible by ATRA in HL-60R cells, transfected with a functional RAR beta, RAR gamma, or RXR alpha receptor. Induction of CD38 in acute promyelocytic and acute myeloblastic leukemia cells was independent of ATRA-induced cytodifferentiation. Following culture with ATRA, increased CD38 protein levels were also observed in normal CD34+ bone marrow cells, but not on normal circulating granulocytes. From these results, we conclude that CD38 is ATRA inducible in myeloid leukemia cells and normal CD34+ bone marrow cells. This effect is independent of differentiation and is mediated by RAR alpha in HL-60 cells, suggesting a similar role for RAR alpha in CD38 expression in other hematopoietic cells.

  18. Transcriptome analysis of hepatopancreas from the Cr (VI)-stimulated mantis shrimp (Oratosquilla oratoria) by Illumina Paired-End Sequencing: Assembly, Annotation and Expression Analysis.

    Science.gov (United States)

    Zhang, Daizhen; Liu, Jun; Qi, Tingting; Ge, Baoming; Wang, Zhengfei; Jiang, Senhao; Liu, Qiuning; Zhang, Huabin; Ge, Ding; Tang, Boping

    2018-02-09

    Cr (VI), the pathogenicity factor, which is widely known to cause toxic effects in living organisms. Given the economic importance of the mantis shrimp (Oratosquilla oratoria), the understanding of impacts by Cr (VI) is considered important. In this study, transcriptome of mantis shrimp was characterized by a comparison between control and Cr (VI)-treated samples using RNA-seq approach. Totally, 88,234,826 bp and 13.24G clean reads were obtained. The total length and number of unigenes were 68,411,206 bp and 100,918, respectively. The maximal and average length of unigenes was 24,906 bp and 678 bp, respectively (N50, 798 bp). 7115 of these unigenes accounted for 7.05% of the total were annotated in all databases. After annotation of assembled unigenes, 35,619 of them were assigned into 3 functional categories and 56 subcategories using Gene Ontology; 18,580 of them were assigned into 26 functional categories using Clusters of Orthologous Groups of proteins; 16,864 of them were assigned into 5 major categories and 32 subclasses using KEGG. Finally, 1,730 genes were differentially expressed (DGEs) and 9 up-regulated pathways (Protein Digestion and Absorption, Neuroactive Ligand-receptor Interaction, Pancreatic Secretion, Tyrosine Metabolism, Amoebiasis, ECM-receptor Interaction, Riboflavin Metabolism, Amino Sugar and Nucleotide Sugar Metabolism and AGE-RAGE Signaling Pathway in Diabetic Complications) were significantly enriched (q<0.05), one down-regulated pathway (Staphylococcus Aureus Infection) was significantly enriched (q<0.05). Up-regulation of genes in pathways of Protein Digestion/Absorption (PepT1/SLC15A and ATP1B), Environment Information Processing (COL1AS, COL4A; LAMA3_5, LAMB3; FN1 and TN) may imply the potentially positive toxicity resistance mechanisms.

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

    Science.gov (United States)

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

    2013-10-08

    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.

  20. Enhanced protective efficacy of nonpathogenic recombinant leishmania tarentolae expressing cysteine proteinases combined with a sand fly salivary antigen.

    Science.gov (United States)

    Zahedifard, Farnaz; Gholami, Elham; Taheri, Tahereh; Taslimi, Yasaman; Doustdari, Fatemeh; Seyed, Negar; Torkashvand, Fatemeh; Meneses, Claudio; Papadopoulou, Barbara; Kamhawi, Shaden; Valenzuela, Jesus G; Rafati, Sima

    2014-03-01

    Novel vaccination approaches are needed to prevent leishmaniasis. Live attenuated vaccines are the gold standard for protection against intracellular pathogens such as Leishmania and there have been new developments in this field. The nonpathogenic to humans lizard protozoan parasite, Leishmania (L) tarentolae, has been used effectively as a vaccine platform against visceral leishmaniasis in experimental animal models. Correspondingly, pre-exposure to sand fly saliva or immunization with a salivary protein has been shown to protect mice against cutaneous leishmaniasis. Here, we tested the efficacy of a novel combination of established protective parasite antigens expressed by L. tarentolae together with a sand fly salivary antigen as a vaccine strategy against L. major infection. The immunogenicity and protective efficacy of different DNA/Live and Live/Live prime-boost vaccination modalities with live recombinant L. tarentolae stably expressing cysteine proteinases (type I and II, CPA/CPB) and PpSP15, an immunogenic salivary protein from Phlebotomus papatasi, a natural vector of L. major, were tested both in susceptible BALB/c and resistant C57BL/6 mice. Both humoral and cellular immune responses were assessed before challenge and at 3 and 10 weeks after Leishmania infection. In both strains of mice, the strongest protective effect was observed when priming with PpSP15 DNA and boosting with PpSP15 DNA and live recombinant L. tarentolae stably expressing cysteine proteinase genes. The present study is the first to use a combination of recombinant L. tarentolae with a sand fly salivary antigen (PpSP15) and represents a novel promising vaccination approach against leishmaniasis.

  1. A bispecific antibody targeting CD47 and CD20 selectively binds and eliminates dual antigen expressing lymphoma cells.

    Science.gov (United States)

    Piccione, Emily C; Juarez, Silvia; Liu, Jie; Tseng, Serena; Ryan, Christine E; Narayanan, Cyndhavi; Wang, Lijuan; Weiskopf, Kipp; Majeti, Ravindra

    2015-01-01

    Agents that block the anti-phagocytic signal CD47 can synergize with pro-phagocytic anti-tumor antigen antibodies to potently eliminate tumors. While CD47 is overexpressed on cancer cells, its expression in many normal tissues may create an 'antigen sink' that could minimize the therapeutic efficacy of CD47 blocking agents. Here, we report development of bispecific antibodies (BsAbs) that co-target CD47 and CD20, a therapeutic target for non-Hodgkin lymphoma (NHL), that have reduced affinity for CD47 relative to the parental antibody, but retain strong binding to CD20. These characteristics facilitate selective binding of BsAbs to tumor cells, leading to phagocytosis. Treatment of human NHL-engrafted mice with BsAbs reduced lymphoma burden and extended survival while recapitulating the synergistic efficacy of anti-CD47 and anti-CD20 combination therapy. These findings serve as proof of principle for BsAb targeting of CD47 with tumor-associated antigens as a viable strategy to induce selective phagocytosis of tumor cells and recapitulate the synergy of combination antibody therapy. This approach may be broadly applied to cancer to add a CD47 blocking component to existing antibody therapies.

  2. Production and diagnostic application of a purified, E. coli-expressed, serological-specific chicken anaemia virus antigen VP3.

    Science.gov (United States)

    Lee, M-S; Chou, Y-M; Lien, Y-Y; Lin, M-K; Chang, W-T; Lee, H-Z; Lee, M-S; Lai, G-H; Chen, H-J; Huang, C-H; Lin, W-H

    2011-06-01

    The aim of this study was to evaluate the production of chicken anaemia virus VP3 protein in different Escherichia coli strains and to address the diagnostic application of purified E. coli-expressed VP3 protein for the detection of chicken anaemia virus (CAV) infection and the development of an ELISA kit. Three E. coli strains, BL21, BL21 codonplus RP and BL21 pLysS, each harbouring a VP3 protein expressing plasmid, were investigated after induction to produce recombinant VP3 protein. After isopropyl-β-D-thiogalactoside (IPTG) induction, VP3 protein was successfully expressed in all three E. coli strains. The BL21 pLysS strain gave the best performance in terms of protein productivity and growth profile. In addition, the optimal culture temperature and IPTG concentration were found to be 0.25 mM and 20 °C, respectively. Using Ni-NTA-purified VP3 protein as an ELISA coating antigen, the purified VP3 was shown to be highly antigenic and able to discriminate sera from chickens infected with CAV from those that were uninfected during an evaluation of CAV infection serodiagnosis. A VP3-based ELISA demonstrated 100% (6/6 x 100%) specificity and sensitivities of 91.3% (21/23 x 100%) and 82.6% (19/23 x 100%) using cut-off values of the mean plus 2 SD and the mean plus 3 SD, respectively. © 2011 Blackwell Verlag GmbH.

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

    Directory of Open Access Journals (Sweden)

    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.

  4. Expression of costimulatory molecules in antigen-activated peritoneal macrophages treated with either ovalbumin or palmitoyl-ova conjugates

    Directory of Open Access Journals (Sweden)

    Flávia Márcia Oliveira

    2013-12-01

    Full Text Available One of the mechanisms by which adjuvants are believed to promote T-cell activation and prevent induction of oral tolerance is by up-regulating the expression of co-stimulatory molecules on antigen presenting cells. Mice treated orally with palmitoyl-ovalbumin conjugates become immunized, while those treated with native ovalbumin (Ova become tolerant. Cells from the peritoneal cavity of B6D2F1 mice were cultured in the presence of 0.01, or 0.1 mg/100ml of either Ova, or palmitoyl-Ova and tested for the presence of cell markers. PE-conjugated anti-mouse CD80, CD86, and CD11b antibodies as well as biotin-PE were used to stain the antigen-activated peritoneal cells. A significant increase in the expression of CD86 and CD80 was observed following in vitro stimulation with palmitoyl-Ova; additionally, both Ova and palmitoyl-Ova induced the basal expression of CD11b. These findings could be related with the strong T-cell proliferative response induced by palmitoyl-Ova.

  5. Influence of ammonium availability on expression of nifD and amtB genes during biostimulation of a U(VI) contaminated aquifer: implications for U(VI) removal and monitoring the metabolic state of Geobacteraceae

    Energy Technology Data Exchange (ETDEWEB)

    Mouser, Paula J.; N' Guessan, A. Lucie; Elifantz, Hila; Holmes, Dawn E.; Williams, Kenneth H; Wilkins, Michael J.; Long, Philip E.; Lovley, Derek R.

    2009-03-25

    The influence of ammonium availability on bacterial community structure and the physiological status of Geobacter species during in situ bioremediation of uranium-contaminated groundwater was evaluated. Ammonium concentrations varied by 2 orders of magnitude (<4 to 400 ?M) across the study site. Analysis of 16S rRNA sequences suggested that ammonium may have been one factor influencing the community composition prior to acetate amendment with Rhodoferax species predominating over Geobacter species with higher ammonium and Dechloromonas species dominating at the site with lowest ammonium. However, once acetate was added and dissimilatory metal reduction was stimulated, Geobacter species became the predominant organisms at all locations. Rates of U(VI) reduction appeared to be more related to acetate concentrations rather than ammonium levels. In situ mRNA transcript abundance of the nitrogen fixation gene, nifD, and the ammonium transporter gene, amtB, in Geobacter species indicated that ammonium was the primary source of nitrogen during uranium reduction. The abundance of amtB was inversely correlated to ammonium levels, whereas nifD transcript levels were similar across all sites examined. These results suggest that nifD and amtB expression are closely regulated in response to ammonium availability to ensure an adequate supply of nitrogen while conserving cell resources. Thus, quantifying nifD and amtB transcript expression appears to be a useful approach for monitoring the nitrogen-related physiological status of subsurface Geobacter species. This study also emphasizes the need for more detailed analysis of geochemical and physiological interactions at the field scale in order to adequately model subsurface microbial processes during bioremediation.

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

    Directory of Open Access Journals (Sweden)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

  8. SSX cancer testis antigens are expressed in most multiple myeloma patients: co-expression of SSX1, 2, 4, and 5 correlates with adverse prognosis and high frequencies of SSX-positive PCs.

    NARCIS (Netherlands)

    Taylor, B.J.; Reiman, T.; Pittman, J.A.; Keats, J.J.; Bruijn, D.R.H. de; Mant, M.J.; Belch, A.R.; Pilarski, L.M.

    2005-01-01

    Cancer testis antigens (CTAs) are tumor-specific antigens that may be useful targets for cancer vaccines. Here, CTA expression was examined in multiple myeloma (MM), a B-cell cancer characterized by malignant plasma cells (PCs) in the bone marrow (BM), and monoclonal gammopathy of undetermined

  9. Mucopolysaccharidosis VI

    Science.gov (United States)

    2010-01-01

    Mucopolysaccharidosis VI (MPS VI) is a lysosomal storage disease with progressive multisystem involvement, associated with a deficiency of arylsulfatase B leading to the accumulation of dermatan sulfate. Birth prevalence is between 1 in 43,261 and 1 in 1,505,160 live births. The disorder shows a wide spectrum of symptoms from slowly to rapidly progressing forms. The characteristic skeletal dysplasia includes short stature, dysostosis multiplex and degenerative joint disease. Rapidly progressing forms may have onset from birth, elevated urinary glycosaminoglycans (generally >100 μg/mg creatinine), severe dysostosis multiplex, short stature, and death before the 2nd or 3rd decades. A more slowly progressing form has been described as having later onset, mildly elevated glycosaminoglycans (generally sialidosis and mucolipidosis. Before enzyme replacement therapy (ERT) with galsulfase (Naglazyme®), clinical management was limited to supportive care and hematopoietic stem cell transplantation. Galsulfase is now widely available and is a specific therapy providing improved endurance with an acceptable safety profile. Prognosis is variable depending on the age of onset, rate of disease progression, age at initiation of ERT and on the quality of the medical care provided. PMID:20385007

  10. Mucopolysaccharidosis VI

    Directory of Open Access Journals (Sweden)

    Harmatz Paul

    2010-04-01

    Full Text Available Abstract Mucopolysaccharidosis VI (MPS VI is a lysosomal storage disease with progressive multisystem involvement, associated with a deficiency of arylsulfatase B leading to the accumulation of dermatan sulfate. Birth prevalence is between 1 in 43,261 and 1 in 1,505,160 live births. The disorder shows a wide spectrum of symptoms from slowly to rapidly progressing forms. The characteristic skeletal dysplasia includes short stature, dysostosis multiplex and degenerative joint disease. Rapidly progressing forms may have onset from birth, elevated urinary glycosaminoglycans (generally >100 μg/mg creatinine, severe dysostosis multiplex, short stature, and death before the 2nd or 3rd decades. A more slowly progressing form has been described as having later onset, mildly elevated glycosaminoglycans (generally ARSB gene, located in chromosome 5 (5q13-5q14. Over 130 ARSB mutations have been reported, causing absent or reduced arylsulfatase B (N-acetylgalactosamine 4-sulfatase activity and interrupted dermatan sulfate and chondroitin sulfate degradation. Diagnosis generally requires evidence of clinical phenotype, arylsulfatase B enzyme activity ®, clinical management was limited to supportive care and hematopoietic stem cell transplantation. Galsulfase is now widely available and is a specific therapy providing improved endurance with an acceptable safety profile. Prognosis is variable depending on the age of onset, rate of disease progression, age at initiation of ERT and on the quality of the medical care provided.

  11. MAGE-A3/4 and NY-ESO-1 antigens expression in metastatic esophageal squamous cell carcinoma

    Directory of Open Access Journals (Sweden)

    T. Bujas

    2011-03-01

    Full Text Available In the present study we analyzed immunohistochemical expression of MAGE-A 3/4 and NY-ESO-1 in 55 samples of esophageal squamous cell carcinomas (ESCC and their respective lymph node metastases. To our knowledge this is the first study to assess and compare the expression of these antigens in ESCC lymph node metastases. Fifty (90.9% primary ESCC were positive for MAGE-A 3/4 and 53 (96.6% were positive for NY-ESO-1. MAGE-A 3/4 was expressed in all lymph node metastases and the intensity of expression was high in a majority of cases. NY-ESO-1 was negative in 2 (7.1% lymph nodes metastases, while the reaction was predominantly moderate in the positive group. In primary tumors MAGE-A 3/4 showed a significantly higher intensity of expression compared to NY-ESO-1 (P=0.047, while in lymph node metastases the intensity of expression was not significantly different (P=0.387. Primary tumors with and without lymph node metastases showed no significant differences in MAGE-A 3/4 (P=0.672 and NY-ESO-1 (P=0.444 expression. Intensity of MAGE-A 3/4 (P=0.461 and NY-ESO-1 (P=0.414 expression in primary tumors was not significantly different compared to the expression in their respective lymph nodes metastases. Expression of MAGE-A 3/4 in primary tumors showed significant positive correlation with primary tumor expression of NY-ESO-1 (P=0.021 but no significant correlation with the expression of MAGE-A 3/4 in lymph node metastases (P=0.056. Expression of NY-ESO-1 in primary tumors showed significant positive correlation with the expression of NY-ESO-1 in lymph node metastases (P=0.001 and significant negative correlation with patients’ age (P<0.001. Expression of MAGE-A 3/4 and NY-ESO-1 in primary tumors and lymph node metastases showed no significant correlation with prognostic parameters such as tumor grade and TNM stage (P>0.05. We have shown different levels of MAGE-A 3/4 and NY-ESO-1 expression in almost all specimens of primary tumor and lymph node

  12. MAGE-A3/4 and NY-ESO-1 antigens expression in metastatic esophageal squamous cell carcinoma.

    Science.gov (United States)

    Bujas, T; Marusic, Z; Peric Balja, M; Mijic, A; Kruslin, B; Tomas, D

    2011-03-21

    In the present study we analyzed immunohistochemical expression of MAGE-A 3/4 and NY-ESO-1 in 55 samples of esophageal squamous cell carcinomas (ESCC) and their respective lymph node metastases. To our knowledge this is the first study to assess and compare the expression of these antigens in ESCC lymph node metastases. Fifty (90.9%) primary ESCC were positive for MAGE-A 3/4 and 53 (96.6%) were positive for NY-ESO-1. MAGE-A 3/4 was expressed in all lymph node metastases and the intensity of expression was high in a majority of cases. NY-ESO-1 was negative in 2 (7.1%) lymph nodes metastases, while the reaction was predominantly moderate in the positive group. In primary tumors MAGE-A 3/4 showed a significantly higher intensity of expression compared to NY-ESO-1 (P=0.047), while in lymph node metastases the intensity of expression was not significantly different (P=0.387). Primary tumors with and without lymph node metastases showed no significant differences in MAGE-A 3/4 (P=0.672) and NY-ESO-1 (P=0.444) expression. Intensity of MAGE-A 3/4 (P=0.461) and NY-ESO-1 (P=0.414) expression in primary tumors was not significantly different compared to the expression in their respective lymph nodes metastases. Expression of MAGE-A 3/4 in primary tumors showed significant positive correlation with primary tumor expression of NY-ESO-1 (P=0.021) but no significant correlation with the expression of MAGE-A 3/4 in lymph node metastases (P=0.056). Expression of NY-ESO-1 in primary tumors showed significant positive correlation with the expression of NY-ESO-1 in lymph node metastases (P=0.001) and significant negative correlation with patients’ age (P<0.001). Expression of MAGE-A 3/4 and NY-ESO-1 in primary tumors and lymph node metastases showed no significant correlation with prognostic parameters such as tumor grade and TNM stage (P>0.05). We have shown different levels of MAGE-A 3/4 and NY-ESO-1 expression in almost all specimens of primary tumor and lymph node metastases

  13. Production and characterization of an orally immunogenic Plasmodium antigen in plants using a virus-based expression system.

    Science.gov (United States)

    Webster, Diane E; Wang, Lina; Mulcair, Mark; Ma, Charles; Santi, Luca; Mason, Hugh S; Wesselingh, Steve L; Coppel, Ross L

    2009-12-01

    Increasing numbers of plant-made vaccines and pharmaceuticals are entering the late stage of product development and commercialization. Despite the theoretical benefits of such production, expression of parasite antigens in plants, particularly those from Plasmodium, the causative parasites for malaria, have achieved only limited success. We have previously shown that stable transformation of tobacco plants with a plant-codon optimized form of the Plasmodium yoelii merozoite surface protein 4/5 (PyMSP4/5) gene resulted in PyMSP4/5 expression of up to approximately 0.25% of total soluble protein. In this report, we describe the rapid expression of PyMSP4/5 in Nicotiana benthamiana leaves using the deconstructed tobacco mosaic virus-based magnICON expression system. PyMSP4/5 yields of up to 10% TSP or 1-2 mg/g of fresh weight were consistently achieved. Characterization of the recombinant plant-made PyMSP4/5 indicates that it is structurally similar to PyMSP4/5 expressed by Escherichia coli. It is notable that the plant-made PyMSP4/5 protein retained its immunogenicity following long-term storage at ambient temperature within freeze-dried leaves. With assistance from a mucosal adjuvant the PyMSP4/5-containing leaves induced PyMSP4/5-specific antibodies when delivered orally to naïve mice or mice primed by a DNA vaccine. This study provides evidence that immunogenic Plasmodium antigens can be produced in large quantities in plants using the magnICON viral vector system.

  14. Expression of carbohydrate antigens in advanced-stage ovarian carcinomas and their metastases-A clinicopathologic study.

    Science.gov (United States)

    Davidson, B; Gotlieb, W H; Ben-Baruch, G; Kopolovic, J; Goldberg, I; Nesland, J M; Berner, A; Bjåmer, A; Bryne, M

    2000-04-01

    Up-regulated expression or loss of expression of various carbohydrate antigens on the surface of cancer cells has been associated with a metastatic phenotype and poor survival in epithelial malignancies of different origins. The object of this study was to investigate the expression of carbohydrate antigens in two groups of patients diagnosed with advanced-stage ovarian carcinoma-one with an extremely favorable outcome and the other with a uniformly poor survival. Sections from 76 paraffin-embedded blocks (primary ovarian carcinomas and metastatic lesions) from 45 patients diagnosed with advanced-stage ovarian carcinomas (FIGO stages III-IV) were immunohistochemically stained using five monoclonal antibodies for Lewis(y) (Le(y))(two antibodies), Sialyl Lewis(x) (Slex), Tn, and Sialyl Tn (STn) antigens. Patients were divided in two groups based on outcome. Long-term survivors (21 patients) and short-term survivors (24 patients) were defined using a double cut-off of 36 months for disease-free survival (DFS) and 60 months for overall survival (OS). Staining results for primary tumors and metastases were analyzed separately. Mean follow-up period was 70 months. The mean values for DFS and OS were 109 and 125 months for long-term survivors and 3 and 25 months for short-term survivors. Staining for all four antigens was seen in the majority of cases (range = 72-96%) and tended to be comparable in primary tumors and their metastases. However, absence of immunoreactivity for STn was seen in 9/38 (24%) metastatic lesions and only 1/38 (3%) primary tumors. This finding did not reach statistical significance (P > 0.05). A combined pattern of membranous and cytoplasmic staining was predominant in the majority of cases. Enhanced staining for Le(y) and STn was detected in the invasive front of some tumors, while Slex and Tn immunoreactivity did not relate to cell location. Primary tumors and metastatic lesions of long-term survivors displayed immunoreactivity patterns that were

  15. Pectinesterase Inhibitor from Jelly Fig (Ficus awkeotsang Makino Achene Inhibits Surface Antigen Expression by Human Hepatitis B Virus

    Directory of Open Access Journals (Sweden)

    Yu-Chuen Huang

    2013-01-01

    Full Text Available Pectinesterase inhibitor (PEI isolated from jelly fig (Ficus awkeotsang Makino is an edible component of a popular drink consumed in Asia. Hepatitis B virus (HBV infection is prevalent in Asia, and current treatments for HBV infection need improvement. This study aimed to evaluate the effect of PEI on the surface antigen expression by HBV (HBsAg. Human hepatoma cell lines Hep3B and Huh7 served as in vitro models for assessing the cytotoxicity and HBsAg expression. A culture of primary hepatocytes cultured from mice served as the normal counterpart. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT colorimetric assay. HBsAg expression was evaluated by measuring HBsAg secretion into the culture medium using an enzyme-linked immunosorbent assay. The results showed that PEI did not affect the viability of the human hepatoma cell lines or primary mouse hepatocytes. PEI inhibited the expression of HBsAg in hepatoma cell lines harboring endogenous (Hep3B and integrated (Huh7 HBV genomes in a concentration- and time-dependent manner, thus implicating a universal activity against HBV gene expression. In conclusion, it suggests that PEI from jelly fig inhibits the expression of human HBsAg in host cells without toxic effects on normal primary hepatocytes.

  16. Myosin VI and Associated Proteins Are Expressed in Human Macrophages but Do Not Play a Role in Foam Cell Formation in THP-1 Cells

    Directory of Open Access Journals (Sweden)

    Hayley J. Dawson

    2013-01-01

    Full Text Available Myosin VI (Myo6 functions in endocytosis in conjunction with binding partners including adaptor protein (AP-2, disabled 2 (Dab2, and GAIP interacting protein C terminus 1 (GIPC1. This study aimed to investigate the expression and function of Myo6 in macrophages and its possible role in the endocytosis of lipoproteins during the induction of foam cell formation. Expression of Myo6, AP-2 (α2 subunit, and Dab2 in THP-1 macrophages and primary human monocyte-derived macrophages was demonstrated at the mRNA and protein level, but GIPC1 was only detected at the mRNA level. Immunofluorescence showed that Myo6 was distributed similarly to F-actin in both macrophage types. AP-2α2 was found to have a similar subcellular distribution to Myo6 and Dab2 in THP-1 cells. Myo6 was located within membrane ruffles and protrusions of the plasma membrane. These results suggest that in macrophages Myo6 is required for several functions including cell adhesion, cell progression, and macropinocytosis. Low-density lipoprotein (LDL and oxidised LDL (oxLDL decreased Myo6 and GIPC1 mRNA expression in THP-1 cells, but uptake of the fluorescence-labelled lipoproteins was unaffected by knockdown of the expression of Myo6 or associated proteins with siRNA. Our findings, therefore, do not support the idea that Myo6 plays a major role in foam cell formation.

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

    Directory of Open Access Journals (Sweden)

    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.

  18. Recombinant Pvs48/45 antigen expressed in E. coli generates antibodies that block malaria transmission in Anopheles albimanus mosquitoes.

    Directory of Open Access Journals (Sweden)

    Myriam Arévalo-Herrera

    Full Text Available Transmission of malaria parasites from humans to Anopheles mosquitoes can be inhibited by specific antibodies elicited during malaria infection, which target surface Plasmodium gametocyte/gamete proteins. Some of these proteins may have potential for vaccine development. Pvs48/45 is a P. vivax gametocyte surface antigen orthologous to Pfs48/45, which may play a role during parasite fertilization and thus has potential for transmission blocking (TB activity. Here we describe the expression of a recombinant Pvs48/45 protein expressed in Escherichia coli as a ∼60kDa construct which we tested for antigenicity using human sera and for its immunogenicity and transmission blocking activity of specific anti-mouse and anti-monkey Pvs48/45 antibodies. The protein reacted with sera of individuals from malaria-endemic areas and in addition induced specific IgG antibody responses in BALB/c mice and Aotus l. griseimembra monkeys. Sera from both immunized animal species recognized native P. vivax protein in Western blot (WB and immunofluorescence assays. Moreover, sera from immunized mice and monkeys produced significant inhibition of parasite transmission to An. Albimanus mosquitoes as shown by membrane feeding assays. Results indicate the presence of reactive epitopes in the Pvs48/45 recombinant product that induce antibodies with TB activity. Further testing of this protein is ongoing to determine its vaccine potential.

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

    National Research Council Canada - National Science Library

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

    .... 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...

  20. 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.

  1. Expression of class II major histocompatibility complex antigens (HLA-DR) and lymphocyte subset immunotyping in chronic pulmonary transplant rejection.

    Science.gov (United States)

    Hasegawa, S; Ockner, D M; Ritter, J H; Patterson, G A; Trulock, E P; Cooper, J D; Wick, M R

    1995-05-01

    Currently, the bronchiolitis obliterans syndrome (BOS) of chronic airway rejection represents the most significant obstacle to the long-term function of isolated pulmonary allografts in humans. Between 20% and 30% of recipients are affected by this condition. To define the possible pathogenetic role of altered expression of class II major histocompatibility complex antigens (ie, HLA-DR) in BOS, the authors studied well-characterized examples of this process immunohistologically. Eleven BOS specimens were compared with seven controls, represented by allografts with no pathologic abnormalities taken from patients with normal posttransplant respiratory function, as well as 14 biopsies showing acute rejection. In addition, immunophenotypic subtyping of lymphocytes in all specimens was undertaken. Control tissues exhibited variable but weak expression of HLA-DR in bronchiolar epithelium and alveolar pneumocytes. In comparison, immunostaining for class II major histocompatibility complex antigens in BOS showed no statistically significant differences, whereas the 14 examples of acute rejection manifested intense HLA-DR expression in epithelia and endothelial cells. The numbers of intrabronchiolar and peribronchiolar lymphocytes were clearly higher in both acute rejection and BOS than in controls, but these cells differed in lineage in the two rejection states. Acute rejection showed an obvious preponderance of CD43-positive T lymphocytes, whereas lymphoid cells in BOS were a relatively equal mixture of CD20-positive B cells and CD43-positive T cells. Moreover, incipient peribronchiolar B-cell follicles were observed in BOS. Natural killer (CD57-positive) lymphocytes were rare in all specimens. These data suggest that alterations in HLA-DR expression probably do not play a central role in the genesis of BOS, as they do in acute rejection. In contrast, the results of lymphocyte immunophenotyping and correlative histologic findings in BOS suggest that both T cells and B

  2. Methimazole therapy in Graves' disease influences the abnormal expression of CD69 (early activation antigen) on T cells.

    Science.gov (United States)

    Corrales, J J; López, A; Ciudad, J; Mories, M T; Miralles, J M; Orfao, A

    1997-12-01

    At present, the in vivo response of T, B and natural killer (NK) cells to antithyroid drug therapy remains largely unknown. In the present study, we have prospectively analyzed the in vivo effects of methimazole treatment on a large number of circulating T and NK cell subsets, some of them expressing cell surface activation antigens involved in the very early phase of the immune response, in a group of 17 hyperthyroid, untreated patients with Graves' disease (GD). As one of the first events during T cell activation is the expression of interleukin (IL) receptors, we also studied the binding of IL-2 and IL-6 to T cells. Patients with Graves' disease were sequentially studied at diagnosis/before treatment (day 0) and 7, 14, 30, 90 and 180 days after methimazole therapy. The results were compared with both a group of 19 age- and sex-matched control volunteers and a group of 20 untreated/euthyroid patients with Graves' disease in long-term remission. The combination of flow cytometry and three-color immunofluorescence revealed a clear (P effect of the drug. Expression of the low-affinity receptor for IL-2 (CD25)--another early T cell activation marker--was not altered in Graves' disease, but the binding of IL-2 and IL-6 to T cells exhibited a progressive and parallel increase during the first 30 days of therapy, decreasing thereafter. Our results show that methimazole therapy downregulates the abnormally high expression of the CD69 early activation antigen on T cells, being less effective on inducing changes in other T cell activation markers and in NK cells.

  3. Programmed death-1 expression on HIV-1-specific CD8+ T cells is shaped by epitope specificity, T-cell receptor clonotype usage and antigen load

    DEFF Research Database (Denmark)

    Kløverpris, Henrik N; McGregor, Reuben; McLaren, James E

    2014-01-01

    ), CD244 and lymphocyte activation gene-3 (LAG-3), which modulate the functional capabilities of CD8+ T cells. DESIGN AND METHODS: Here, we used an array of different human leukocyte antigen(HLA)-B*15:03 and HLA-B*42:01 tetramers to characterize inhibitory receptor expression as a function...... by effector memory CD8+ T cells. CONCLUSION: Collectively, these data suggest that PD-1 expression on HIV-1-specific CD8+ T cells tracks antigen load at the level of epitope specificity and TCR clonotype usage. These findings are important because they provide evidence that PD-1 expression levels...

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

    Directory of Open Access Journals (Sweden)

    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.

  5. Immunohistochemical analysis of the expression of MAGE-A and NY-ESO-1 cancer/testis antigens in diffuse large B-cell testicular lymphoma

    OpenAIRE

    Hudolin, Tvrtko; Kaštelan, Željko; Ilić, Ivana; Levarda-Hudolin, Katarina; Bašić-Jukić, Nikolina; Rieken, Malte; Spagnoli, Giulio C; Juretić, Antonio; Mengus, Chantal

    2013-01-01

    BACKGROUND: Primary testicular lymphoma (PTL) is a rare and lethal disease. The most common histological subtype is diffuse large B-cell lymphoma (DLBCL). Standard treatments are frequently ineffective. Thus, the development of novel forms of therapy is urgently required. Specific immunotherapy generating immune responses directed against antigen predominantly expressed by cancer cells such as cancer-testis antigens (CTA) may provide a valid alternative treatment for patients bearing PTL...

  6. 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 blo...

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

    Directory of Open Access Journals (Sweden)

    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.

  8. Expression of cancer/testis antigens in salivary gland carcinomas with reference to MAGE-A and NY-ESO-1 expression in adenoid cystic carcinoma.

    Science.gov (United States)

    Beppu, Shintaro; Ito, Yohei; Fujii, Kana; Saida, Kosuke; Takino, Hisashi; Masaki, Ayako; Murase, Takayuki; Kusafuka, Kimihide; Iida, Yoshiyuki; Onitsuka, Tetsuro; Yatabe, Yasushi; Hanai, Nobuhiro; Hasegawa, Yasuhisa; Ijichi, Kei; Murakami, Shingo; Inagaki, Hiroshi

    2017-08-01

    Cancer/testis antigens (CTAs) are detected in cancer cells but not in healthy normal tissues, with the exception of gametogenic tissues. CTAs are highly immunogenic proteins, and thus represent ideal targets for cytotoxic T-lymphocyte-mediated specific immune therapy. The aim of this study was to screen CTA expression in various types of salivary gland carcinoma and to clarify clinicopathological significance of MAGE-A and NY-ESO-1 expression in adenoid cystic carcinomas (AdCCs) of the salivary gland, which is one of the most common salivary gland carcinomas, and usually has a fatal outcome. We used immunohistochemistry to examine the expression of four CTAs (MAGE-A, NY-ESO-1, CT7, and GAGE7) in various types of salivary gland carcinoma (n = 95). When carcinoma cases were divided into low-grade and intermediate/high-grade types, NY-ESO-1 and CT7 were expressed more frequently in intermediate/high-grade carcinomas. We then focused on MAGE-A and NY-ESO-1 expression in a large cohort of adenoid cystic carcinomas (AdCCs) (n = 46). MAGE-A and NY-ESO-1 were frequently expressed in AdCC; specifically, MAGE-A was expressed in >60% of the AdCC cases. MAGE-A expression and tumour site (minor salivary gland) were identified as independent risk factors for locoregional tumour recurrence. These findings suggest that CTAs may be expressed in a variety of salivary gland carcinomas, especially in those with higher histological grades. In addition, MAGE-A, which is frequently expressed in AdCC cases, may be a useful prognostic factor for poorer locoregional recurrence-free survival. © 2017 John Wiley & Sons Ltd.

  9. Capsular Polysaccharide Expression in Commensal Streptococcus Species: Genetic and Antigenic Similarities to Streptococcus pneumoniae

    National Research Council Canada - National Science Library

    Skov Sørensen, Uffe B; Yao, Kaihu; Yang, Yonghong; Tettelin, Hervé; Kilian, Mogens

    2016-01-01

    Expression of a capsular polysaccharide is considered a hallmark of most invasive species of bacteria, including Streptococcus pneumoniae, in which the capsule is among the principal virulence factors...

  10. A booster vaccine expressing a latency-associated antigen augments BCG induced immunity and confers enhanced protection against tuberculosis.

    Directory of Open Access Journals (Sweden)

    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.

  11. Expression of hepatitis B surface antigen (HBsAg) gene in ...

    African Journals Online (AJOL)

    Expression of HBsAg was confirmed by western blotting and levels of expression were assayed by enzyme-linked immunosorbent assay (ELISA). Southern blot hybridization confirmed the stable integration of the target genes into the genomes of cherry tomato, while western blotting showed high levels of biologically active ...

  12. Expression of the activation antigen CD97 and its ligand CD55 in rheumatoid synovial tissue

    NARCIS (Netherlands)

    Hamann, J. [=Jörg; Wishaupt, J. O.; van Lier, R. A.; Smeets, T. J.; Breedveld, F. C.; Tak, P. P.

    1999-01-01

    OBJECTIVE: Fibroblast-like synoviocytes (FLS) express decay-accelerating factor (CD55) at high levels. Recently, it was found that CD55 is a specific cellular ligand for the 7-span transmembrane receptor CD97. The objective of this study was to define the expression of this receptor-ligand pair in

  13. Suppression of MHC class I antigen expression by N-myc through enhancer inactivation

    NARCIS (Netherlands)

    Lenardo, M.; Rustgi, A.K.; Schievella, A.R.; Bernards, R.A.

    1989-01-01

    Amplification of the N-myc oncogene in human neuroblastoma is associated with increased metastatic ability. We previously found that over-expression of N-myc in rat neuroblastoma tumor cells causes a dramatic reduction in the expression of MHC class I mRNA. We show here that two distinct

  14. Expression of a highly antigenic and native-like folded extracellular domain of the human α1 subunit of muscle nicotinic acetylcholine receptor, suitable for use in antigen specific therapies for Myasthenia Gravis.

    Directory of Open Access Journals (Sweden)

    Athanasios Niarchos

    Full Text Available We describe the expression of the extracellular domain of the human α1 nicotinic acetylcholine receptor (nAChR in lepidopteran insect cells (i-α1-ECD and its suitability for use in antigen-specific therapies for Myasthenia Gravis (MG. Compared to the previously expressed protein in P. pastoris (y-α1-ECD, i-α1-ECD had a 2-fold increased expression yield, bound anti-nAChR monoclonal antibodies and autoantibodies from MG patients two to several-fold more efficiently and resulted in a secondary structure closer to that of the crystal structure of mouse α1-ECD. Our results indicate that i-α1-ECD is an improved protein for use in antigen-specific MG therapeutic strategies.

  15. Gene expression correlates with process rates quantified for sulfate- and Fe(III-reducing bacteria in U(VI-contaminated sediments

    Directory of Open Access Journals (Sweden)

    Denise M Akob

    2012-08-01

    Full Text Available Though iron- and sulfate-reducing bacteria are well known for mediating uranium(VI reduction in contaminated subsurface environments, quantifying the in situ activity of the microbial groups responsible remains a challenge. The objective of this study was to demonstrate the use of quantitative molecular tools that target mRNA transcripts of key genes related to Fe(III and sulfate reduction pathways in order to monitor these processes during in situ U(VI remediation in the subsurface. Expression of the Geobacteraceae-specific citrate synthase gene (gltA and the dissimilatory (bisulfite reductase gene (dsrA, were correlated with the activity of iron- or sulfate-reducing microorganisms, respectively, under stimulated bioremediation conditions in microcosms of sediments sampled from the U.S. Department of Energy’s Oak Ridge Integrated Field Research Challenge (OR-IFRC site at Oak Ridge, Tennessee. In addition, Geobacteraceae-specific gltA and dsrA transcript levels were determined in parallel with the predominant electron acceptors present in moderately and highly contaminated subsurface sediments from the OR-IFRC. Phylogenetic analysis of the cDNA generated from dsrA mRNA, sulfate-reducing bacteria-specific 16S rRNA, and gltA mRNA identified activity of specific microbial groups. Active sulfate reducers were members of the Desulfovibrio, Desulfobacterium, and Desulfotomaculum genera. Members of the subsurface Geobacter clade, closely related to uranium-reducing Geobacter uraniireducens and Geobacter daltonii, were the metabolically-active iron-reducers in biostimulated microcosms and in situ core samples. Direct correlation of transcripts and process rates demonstrated evidence of competition between the functional guilds in subsurface sediments. We further showed that active populations of Fe(III-reducing bacteria and sulfate-reducing bacteria are present in OR-IFRC sediments and are good potential targets for in situ bioremediation.

  16. Recombinant Mycobacterium bovis BCG expressing the Sm14 antigen of Schistosoma mansoni protects mice from cercarial challenge.

    Science.gov (United States)

    Varaldo, Paula B; Leite, Luciana C C; Dias, Waldely O; Miyaji, Eliane N; Torres, Fabio I G; Gebara, Vera C; Armôa, Geraldo R G; Campos, Adriano S; Matos, Denise C S; Winter, Nathalie; Gicquel, Brigitte; Vilar, Mônica M; McFadden, Johnjoe; Almeida, Marilia S; Tendler, Miriam; McIntosh, Douglas

    2004-06-01

    The Sm14 antigen of Schistosoma mansoni was cloned and expressed in Mycobacterium bovis BCG as a fusion with the Mycobacterium fortuitum beta-lactamase protein under the control of its promoter, pBlaF*; the protein was localized in the bacterial cell wall. The rBCG-Sm14 strain was shown to be relatively stable in cultured murine and bovine monocytes in terms of infectivity, bacterial persistence, and plasmid stability. The immunization of mice with rBCG-Sm14 showed no induction of anti-Sm14 antibodies; however, splenocytes of immunized mice released increased levels of gamma interferon upon stimulation with recombinant Sm14 (rSm14), indicating an induction of a Th1-predominant cellular response against Sm14. Mice immunized with one or two doses of rBCG-Sm14 and challenged with live S. mansoni cercaria showed a 48% reduction in worm burden, which was comparable to that obtained by immunization with three doses of rSm14 purified from Escherichia coli. The data presented here further enhance the status of Sm14 as a promising candidate antigen for the control of schistosomiasis and indicate that a one-dose regimen of rBCG-Sm14 could be considered a convenient means to overcome many of the practical problems associated with the successful implementation of a multiple-dose vaccine schedule in developing countries.

  17. 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

  18. Thymic Self-Antigen Expression for the Design of a Negative/Tolerogenic Self-Vaccine against Type 1 Diabetes

    Directory of Open Access Journals (Sweden)

    Aziz Alami Chentoufi

    2011-01-01

    Full Text Available Before being able to react against infectious non-self-antigens, the immune system has to be educated in the recognition and tolerance of neuroendocrine proteins, and this critical process essentially takes place in the thymus. The development of the autoimmune diabetogenic response results from a thymus dysfunction in programming central self-tolerance to pancreatic insulin-secreting islet β cells, leading to the breakdown of immune homeostasis with an enrichment of islet β cell reactive effector T cells and a deficiency of β cell-specific natural regulatory T cells (nTreg in the peripheral T-lymphocyte repertoire. Insulin-like growth factor 2 (IGF-2 is the dominant member of the insulin family expressed during fetal life by the thymic epithelium under the control of the autoimmune regulator (AIRE gene/protein. Based on the close homology and cross-tolerance between insulin, the primary T1D autoantigen, and IGF-2, the dominant self-antigen of the insulin family, a novel type of vaccination, so-called “negative/tolerogenic self-vaccination”, is currently developed for prevention and cure of T1D. If this approach were found to be effective for reprogramming immunological tolerance in T1D, it could pave the way for the design of negative self-vaccines against autoimmune endocrine diseases, as well as other organ-specific autoimmune diseases.

  19. 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...... gene is expressed per cell at a time. We measured var mRNA transcript levels by real-time Q-PCR, analysed var gene transcripts by single-cell FISH and directly compared these with PfEMP1 antigen surface expression and cytoadhesion in three different antibody-selected P. falciparum 3D7 sub-lines using......-line was found to bind both CD31/PECAM1 and CD54/ICAM1 and to adhere twice as efficiently to human endothelial cells, compared to infected cells having only one PfEMP1 variant on the surface. These new results on PfEMP1 antigen expression indicate that a re-evaluation of the molecular mechanisms involved in P...

  20. Expression of whole and hybrid genes in Trypanosoma equiperdum antigenic variation.

    Science.gov (United States)

    Longacre, S; Eisen, H

    1986-05-01

    A rapid technique involving the S1 nuclease resistance of RNA:DNA duplexes has been used to screen four Trypanosoma equiperdum variant surface glycoprotein (VSG) genes for evidence of hybrid gene structure in their transcribed regions. The results suggest that VSGs appearing early in a chronic infection each have a complete co-linear basic copy (BC) of their expressed gene while VSGs appearing later in infection are particularly associated with BC genes which are recombined before being expressed. The intensities of the S1-protected bands from hybrid VSGs indicate that the basic and expression linked copies are present in equivalent gene dosages. In addition, studies are presented on the expression of two additional VSG genes in T. equiperdum, VSG 4 and VSG 78, which (i) show that the basic copies are not located on telomeres even though one (VSG 4) is expressed early in infection and (ii) emphasize the role of a predominant expression site in T. equiperdum while nevertheless confirming the presence of multiple expression sites.

  1. Expression and Purification of Recombinant Mycobacterium Tuberculosis (TB) Antigens, ESAT-6, CFP-10 and ESAT- 6/CFP-10 and Their Diagnosis Potential for Detection of TB Patients.

    Science.gov (United States)

    Hemmati, M; Seghatoleslam, A; Rasti, M; Ebadat, S; Mosavari, N; Habibagahi, M; Taheri, M; Sardarian, A R; Mostafavi-Pour, Z

    2011-08-01

    One of the most widely used methods to detect tuberculosis (TB) infection is the tuberculin skin test (TST). The completion of Mycobacterium tuberculosis (M. tuberculosis) genome sequence has led to identification of several antigens that can be utilized for accurate diagnosis and control of TB. The aim of this study was to purify the recombinant M. tuberculosis antigens for the evaluation of their potential in TB diagnosis. The recombinant secretory antigens, ESAT-6, CFP-10 and ESAT-6/CFP-10 were produced by PCR and cloning methods. To investigate antigen specific responses of these recombinant antigens in detection of TB, ex vivo enzyme linked immunospot (ELISPOT) test in 30 clinically diagnosed TB patients was evaluated. The selected M. tuberculosis antigens were cloned, expressed and purified in Escherichia coli (BL21). ELISPOT assay for detection of TB showed the sensitivity of 93, 90 and 100% for recombinant ESAT-6, CFP-10 and ESAT-6/CFP-10 proteins respectively, which is significantly higher than conventional TST. The recombinant antigens of ESAT-6, CFP-10 and ESAT-6/CFP-10 can be used as an accurate means of detecting TB in Iran.

  2. Antigen-Specific Monoclonal Antibodies Isolated from B Cells Expressing Constitutively Active STAT5

    NARCIS (Netherlands)

    Scheeren, F.A.; van Geelen, C.M.M.; Yasuda, E.; Spits, H.; Beaumont, T.

    2011-01-01

    Background: Fully human monoclonal antibodies directed against specific pathogens have a high therapeutic potential, but are difficult to generate. Methodology/Principal Findings: Memory B cells were immortalized by expressing an inducible active mutant of the transcription factor Signal Transducer

  3. Tissue expression of Squamous Cellular Carcinoma Antigen (SCCA) is inversely correlated to tumor size in HCC

    National Research Council Canada - National Science Library

    Trerotoli, Paolo; Fransvea, Emilia; Angelotti, Umberto; Antonaci, Giovanni; Lupo, Luigi; Mazzocca, Antonio; Mangia, Anita; Antonaci, Salvatore; Giannelli, Gianluigi

    2009-01-01

    ...) and 35 with a single nodule > 3 cm or multifocal (l-HCC). Serum SCCA was measured by the ELISA kit, and in frozen tissues by immunohistochemistry, quantified with appropriate imaging analysis software and expressed in square microns...

  4. Pretargeted 177Lu radioimmunotherapy of carcinoembryonic antigen-expressing human colonic tumors in mice

    National Research Council Canada - National Science Library

    Schoffelen, R; Graaf, W.T.A. van der; Franssen, G.M; Sharkey, R.M; Goldenberg, D.M; McBride, W.J; Rossi, E.A; Eek, A; Oyen, W.J.G; Boerman, O.C

    2010-01-01

    ... (CEA)-expressing human tumors. METHODS: To obtain the optimal therapeutic efficacy, several strategies were evaluated to increase the total amount of radioactivity targeted to subcutaneous LS174T colon cancer tumors in BALB/c nude mice...

  5. EXPRESSION PROFILING OF FIVE RAT STRAINS REVEAL TRANSCRIPTIONAL MODES IN THE ANTIGEN PROCESSING PATHWAY

    Science.gov (United States)

    Comparative gene expression profiling of rat strains with genetic predisposition to diverse cardiovascular diseases can help decode the transcriptional program that governs cellular behavior. We hypothesized that co-transcribed, intra-pathway, functionally coherent genes can be r...

  6. Development of Escherichia coli and Mycobacterium smegmatis recombinants expressing major Mycobacterium tuberculosis-specific antigenic proteins

    Directory of Open Access Journals (Sweden)

    Hanady A Amoudy

    2016-01-01

    Conclusion: Positive results of cloning and expression suggest that the constructed clones are ready tools for further assessment of their immunogenicity and can be included in improved diagnostic tools and vaccines against TB.

  7. Constitutive intracellular expression of human leukocyte antigen (HLA)-DO and HLA-DR but not HLA-DM in trophoblast cells.

    Science.gov (United States)

    Ranella, Anthi; Vassiliadis, Simon; Mastora, Chrisa; Valentina, Michailidou; Dionyssopoulou, Eva; Athanassakis, Irene

    2005-01-01

    The nonclassic human leukocyte antigen (HLA)-DM molecules have been proved to positively regulate antigen presentation in classic antigen-presenting cells, whereas in B lymphocytes HLA-DO have been identified as negative regulators of the process. The present report examines whether the negative expression of classic class II molecules in trophoblasts implies negative regulation by HLA-DO. It was revealed by immunofluorescence, confocal microscopy, and subcellular fractionation techniques that human trophoblasts, although not expressing any surface HLA-DR antigens, constitutively express intracellular HLA-DR, HLA-DO, and CD74, but not HLA-DM. Administration of interferon-gamma to the cell culture increased HLA-DR and CD74, induced HLA-DM, but did not alter the expression of HLA-DO and induced HLA-DR release from the cells. These results were confirmed by reverse transcriptase-polymerase chain reaction analysis except that HLA-DM mRNA was detected in control cells, indicating a posttranscriptional regulation. Under the same experimental conditions, human monocytes/macrophages were not expressing intracellular HLA-DO while exhibiting significant levels of HLA-DR, HLA-DM, and CD74. The results presented here reveal for the first time expression of HLA-DO in trophoblasts, which can be of great importance in maintaining the class II-negative state in these cells and consequently protecting the fetus from maternal immune attack.

  8. Early Secretory Antigenic Target-6 Drives Matrix Metalloproteinase-10 Gene Expression and Secretion in Tuberculosis.

    Science.gov (United States)

    Brilha, Sara; Sathyamoorthy, Tarangini; Stuttaford, Laura H; Walker, Naomi F; Wilkinson, Robert J; Singh, Shivani; Moores, Rachel C; Elkington, Paul T; Friedland, Jon S

    2017-02-01

    Tuberculosis (TB) causes disease worldwide, and multidrug resistance is an increasing problem. Matrix metalloproteinases (MMPs), particularly the collagenase MMP-1, cause lung extracellular matrix destruction, which drives disease transmission and morbidity. The role in such tissue damage of the stromelysin MMP-10, a key activator of the collagenase MMP-1, was investigated in direct Mycobacterium tuberculosis (Mtb)-infected macrophages and in conditioned medium from Mtb-infected monocyte-stimulated cells. Mtb infection increased MMP-10 secretion from primary human macrophages 29-fold, whereas Mtb-infected monocytes increased secretion by 4.5-fold from pulmonary epithelial cells and 10.5-fold from fibroblasts. Inhibition of MMP-10 activity decreased collagen breakdown. In two independent cohorts of patients with TB from different continents, MMP-10 was increased in both induced sputum and bronchoalveolar lavage fluid compared with control subjects and patients with other respiratory diseases (both P < 0.05). Mtb drove 3.5-fold greater MMP-10 secretion from human macrophages than the vaccine strain bacillus Calmette-Guerin (P < 0.001), whereas both mycobacteria up-regulated TNF-α secretion equally. Using overlapping, short, linear peptides covering the sequence of early secretory antigenic target-6, a virulence factor secreted by Mtb, but not bacillus Calmette-Guerin, we found that stimulation of human macrophages with a single specific 15-amino acid peptide sequence drove threefold greater MMP-10 secretion than any other peptide (P < 0.001). Mtb-driven MMP-10 secretion was inhibited in a dose-dependent manner by p38 and extracellular signal-related kinase mitogen-activated protein kinase blockade (P < 0.001 and P < 0.01 respectively), but it was not affected by inhibition of NF-κB. In summary, Mtb activates inflammatory and stromal cells to secrete MMP-10, and this is partly driven by the virulence factor early secretory antigenic target-6

  9. Characterization of Fetal Antigen 1/Delta-Like 1 Homologue Expressing Cells in the Rat Nigrostriatal System

    DEFF Research Database (Denmark)

    Liechti, Rémy; Ducray, Angélique D; Jensen, Pia

    2015-01-01

    suggested as a potential supplementary marker of dopaminergic neurons. The present study aimed at investigating the distribution of FA1/dlk1-immunoreactive (-ir) cells in the early postnatal and adult midbrain as well as in the nigrostriatal system of 6-hydroxydopamine (6-OHDA)-lesioned hemiparkinsonian......Fetal antigen 1/delta-like 1 homologue (FA1/dlk1) belongs to the epidermal growth factor superfamily and is considered to be a non-canonical ligand for the Notch receptor. Interactions between Notch and its ligands are crucial for the development of various tissues. Moreover, FA1/dlk1 has been...... adult rats. FA1/dlk1-ir cells were predominantly distributed in the substantia nigra (SN) pars compacta (SNc) and in the ventral tegmental area. Interestingly, the expression of FA1/dlk1 significantly increased in tyrosine hydroxylase (TH)-ir cells during early postnatal development. Co...

  10. Expression, Purification and Characterization of Ricin vectors used for exogenous antigen delivery into the MHC Class I presentation pathway

    Directory of Open Access Journals (Sweden)

    Smith Daniel C.

    2003-01-01

    Full Text Available Disarmed versions of the cytotoxin ricin can deliver fused peptides into target cells leading to MHC class I-restricted antigen presentation [Smith et al. J Immunol 2002; 169:99-107]. The ricin delivery vector must contain an attenuated catalytic domain to prevent target cell death, and the fused peptide epitope must remain intact for delivery and functional loading to MHC class I molecules. Expression in E. coli and purification by cation exchange chromatography of the fusion protein is described. Before used for delivery, the activity of the vector must be characterized in vitro, via an N-glycosidase assay, and in vivo, by a cytotoxicity assay. The presence of an intact epitope must be confirmed using mass spectrometry by comparing the actual mass with the predicted mass.

  11. Small-scale expressed sequence tag analysis of Theileria uilenbergi: identification of a gene family encoding potential antigenic proteins.

    Science.gov (United States)

    Liu, Zhijie; Dang, Zhisheng; Luo, Jianxun; Yin, Hong; Ahmed, Jabbar S; Seitzer, Ulrike

    2008-12-01

    Recently, Theileria sp. (China) has been designated as T. luwenshuni[formerly Theileria sp. (China 1)] and T. uilenbergi[formerly Theileria sp. (China 2)]. A cDNA library of T. uilenbergi merozoites was constructed and subjected to random sequencing. Among the obtained sequences were three highly identical cDNA clones, indicating a gene family. Bioinformatic analyses indicated these genes contain signal peptides and encode potential immunogenic proteins. The presence of tandemly arranged and additional variants of these genes was shown. Analysis of one recombinantly expressed clone revealed immunoreactivity for serum from Theileria-infected animals. No cross-reaction with serum of T. lestoquardi-, Babesia motasi-, or Anaplasma ovis-infected animals was observed, indicating a potential antigen for development of serological diagnostic tools.

  12. Human leukocyte antigen-G expression in differentiated human airway epithelial cells: lack of modulation by Th2-associated cytokines

    Directory of Open Access Journals (Sweden)

    White Steven R

    2013-01-01

    Full Text Available Abstract Background Human leukocyte antigen (HLA-G is a nonclassical class I antigen with immunomodulatory roles including up-regulation of suppressor T regulatory lymphocytes. HLA-G was recently identified as an asthma susceptibility gene, and expression of a soluble isoform, HLA-G5, has been demonstrated in human airway epithelium. Increased presence of HLA-G5 has been demonstrated in bronchoalveolar lavage fluid recovered from patients with mild asthma; this suggests a role for this isoform in modulating airway inflammation though the mechanisms by which this occurs is unclear. Airway inflammation associated with Th2 cytokines such as IL-4 and IL-13 is a principal feature of asthma, but whether these cytokines elicit expression of HLA-G is not known. Methods We examined gene and protein expression of both soluble (G5 and membrane-bound (G1 HLA-G isoforms in primary differentiated human airway epithelial cells collected from normal lungs and grown in air-liquid interface culture. Cells were treated with up to 10 ng/ml of either IL-4, IL-5, or IL-13, or 100 ng/ml of the immunomodulatory cytokine IL-10, or 10,000 U/ml of the Th1-associated cytokine interferon-beta, for 24 hr, after which RNA was isolated for evaluation by quantitative PCR and protein was collected for Western blot analysis. Results HLA-G5 but not G1 was present in dAEC as demonstrated by quantitative PCR, western blot and confocal microscopy. Neither G5 nor G1 expression was increased by the Th2-associated cytokines IL-4, IL-5 or IL-13 over 24 hr, nor after treatment with IL-10, but was increased 4.5 ± 1.4 fold after treatment with 10,000 U/ml interferon-beta. Conclusions These data demonstrate the constitutive expression of a T lymphocyte regulatory molecule in differentiated human airway epithelial cells that is not modulated by Th2-associated cytokines.

  13. 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 loss

  14. γ-Radiation promotes immunological recognition of cancer cells through increased expression of cancer-testis antigens in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Anu Sharma

    Full Text Available BACKGROUND: γ-radiation is an effective treatment for cancer. There is evidence that radiotherapy supports tumor-specific immunity. It was described that irradiation induces de novo protein synthesis and enhances antigen presentation, we therefore investigated whether γ-radiation results in increased expression of cancer-testis (CT antigens and MHC-I, thus allowing efficient immunological control. This is relevant because the expression of CT-antigens and MHC-I on tumor cells is often heterogeneous. We found that the changes induced by γ-radiation promote the immunological recognition of the tumor, which is illustrated by the increased infiltration by lymphocytes after radiotherapy. METHODS/FINDINGS: We compared the expression of CT-antigens and MHC-I in various cancer cell lines and fresh biopsies before and after in vitro irradiation (20 Gy. Furthermore, we compared paired biopsies that were taken before and after radiotherapy from sarcoma patients. To investigate whether the changed expression of CT-antigens and MHC-I is specific for γ-radiation or is part of a generalized stress response, we analyzed the effect of hypoxia, hyperthermia and genotoxic stress on the expression of CT-antigens and MHC-I. In vitro irradiation of cancer cell lines and of fresh tumor biopsies induced a higher or de novo expression of different CT-antigens and a higher expression of MHC-I in a time- and dose-dependent fashion. Importantly, we show that irradiation of cancer cells enhances their recognition by tumor-specific CD8+ T cells. The analysis of paired biopsies taken from a cohort of sarcoma patients before and after radiotherapy confirmed our findings and, in addition showed that irradiation resulted in higher infiltration by lymphocytes. Other forms of stress did not have an impact on the expression of CT-antigens or MHC-I. CONCLUSIONS: Our findings suggest that γ-radiation promotes the immunological recognition of the tumor. We therefore propose that

  15. The O-Antigen Capsule of Salmonella enterica Serovar Typhimurium Facilitates Serum Resistance and Surface Expression of FliC.

    Science.gov (United States)

    Marshall, Joanna M; Gunn, John S

    2015-10-01

    Group IV polysaccharide capsules are common in enteric bacteria and have more recently been described in nontyphoidal Salmonella species. Such capsules are known as O-antigen (O-Ag) capsules, due to their high degree of similarity to the O-Ag of the lipopolysaccharide (LPSO-Ag). Capsular polysaccharides are known virulence factors of many bacterial pathogens, facilitating evasion of immune recognition and systemic dissemination within the host. Previous studies on the O-Ag capsule of salmonellae have focused primarily on its role in bacterial surface attachment and chronic infection; however, the potential effects of the O-Ag capsule on acute pathogenesis have yet to be investigated. While much of the in vivo innate immune resistance of Salmonella enterica serovar Typhimurium is attributed to the high-molecular-weight LPS, we hypothesized that the O-Ag capsule may enhance this resistance by diminishing surface expression of pathogen-associated molecular patterns, such as flagella, and increasing resistance to host immune molecules. To test this hypothesis, O-Ag capsule-deficient mutants were constructed, and the loss of O-Ag capsular surface expression was confirmed through microscopy and immunoblotting. Loss of O-Ag capsule production did not alter bacterial growth or production of LPS. Western blot analysis and confocal microscopy revealed that O-Ag capsule-deficient mutants demonstrate reduced resistance to killing by human serum. Furthermore, O-Ag capsule-deficient mutants produced exclusively phase I flagellin (FliC). Although O-Ag capsule-deficient mutants did not exhibit reduced virulence in a murine model of acute infection, in vitro results indicate that the O-Ag capsule may function to modify the antigenic nature of the bacterial surface, warranting additional investigation of a potential role of the structure in pathogenesis. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  16. Changes in expression of putative antigens encoded by pigment genes in mouse melanomas at different stages of malignant progression.

    Science.gov (United States)

    Orlow, S J; Hearing, V J; Sakai, C; Urabe, K; Zhou, B K; Silvers, W K; Mintz, B

    1995-10-24

    Cutaneous melanomas of Tyr-SV40E transgenic mice (mice whose transgene consists of the tyrosinase promoter fused to the coding regions of simian virus 40 early genes) strikingly resemble human melanomas in their development and progression. Unlike human melanomas, the mouse tumors all arise in genetically identical individuals, thereby better enabling expression of specific genes to be characterized in relation to advancing malignancy. The products of pigment genes are of particular interest because peptides derived from these proteins have been reported to function as autoantigens with immunotherapeutic potential in some melanoma patients. However, the diminished pigmentation characteristic of many advanced melanomas raises the possibility that some of the relevant products may no longer be expressed in the most malignant cells. We have therefore investigated the contributions of several pigment genes in melanotic vs. relatively amelanotic components of primary and metastatic mouse melanomas. The analyses reveal marked differences within and among tumors in levels of mRNAs and proteins encoded by the wild-type alleles at the albino, brown, slaty, and silver loci. Tyrosinase (the protein encoded by the albino locus) was most often either absent or undetectable as melanization declined. The protein encoded by the slaty locus (tyrosinase-related protein 2) was the only one of those tested that was clearly present in all the tumor samples. These results suggest that sole reliance on targeting tyrosinase-based antigens might selectively favor survival of more malignant cells, whereas targeting the ensemble of the antigens tested might contribute toward a more inclusive and effective antimelanoma strategy.

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

    Directory of Open Access Journals (Sweden)

    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

  18. The expression and antigenicity of a truncated spike-nucleocapsid fusion protein of severe acute respiratory syndrome-associated coronavirus

    Directory of Open Access Journals (Sweden)

    Qiu Yan

    2008-11-01

    Full Text Available Abstract Background In the absence of effective drugs, controlling SARS relies on the rapid identification of cases and appropriate management of the close contacts, or effective vaccines for SARS. Therefore, developing specific and sensitive laboratory tests for SARS as well as effective vaccines are necessary for national authorities. Results Genes encoding truncated nucleocapsid (N and spike (S proteins of SARSCoV were cloned into the expression vector pQE30 and fusionally expressed in Escherichia coli M15. The fusion protein was analyzed for reactivity with SARS patients' sera and with anti-sera against the two human coronaviruses HCoV 229E and HCoV OC43 by ELISA, IFA and immunoblot assays. Furthermore, to evaluate the antigen-specific humoral antibody and T-cell responses in mice, the fusion protein was injected into 6-week-old BALB/c mice and a neutralization test as well as a T-cell analysis was performed. To evaluate the antiviral efficacy of immunization, BALB/c mice were challenged intranasally with SARSCoV at day 33 post injection and viral loads were determined by fluorescent quantitative RT-PCR. Serological results showed that the diagnostic sensitivity and specificity of the truncated S-N fusion protein derived the SARS virus were > 99% (457/460 and 100.00% (650/650, respectively. Furthermore there was no cross-reactivity with other two human coronaviruses. High titers of antibodies to SRASCoV appeared in the immunized mice and the neutralization test showed that antibodies to the fusion protein could inhibit SARSCoV. The T cell proliferation showed that the fusion protein could induce an antigen-specific T-cell response. Fluorescent quantitative RT-PCR showed that BALB/c mice challenged intranasally with SARSCoV at day 33 post injection were completely protected from virus replication. Conclusion The truncated S-N fusion protein is a suitable immunodiagnostic antigen and vaccine candidate.

  19. Specific Colon Cancer Cell Cytotoxicity Induced by Bacteriophage E Gene Expression under Transcriptional Control of Carcinoembryonic Antigen Promoter

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    Ana R. Rama

    2015-06-01

    Full Text Available Colorectal cancer is one of the most prevalent cancers in the world. Patients in advanced stages often develop metastases that require chemotherapy and usually show a poor response, have a low survival rate and develop considerable toxicity with adverse symptoms. Gene therapy may act as an adjuvant therapy in attempts to destroy the tumor without affecting normal host tissue. The bacteriophage E gene has demonstrated significant antitumor activity in several cancers, but without any tumor-specific activity. The use of tumor-specific promoters may help to direct the expression of therapeutic genes so they act against specific cancer cells. We used the carcinoembryonic antigen promoter (CEA to direct E gene expression (pCEA-E towards colon cancer cells. pCEA-E induced a high cell growth inhibition of human HTC-116 colon adenocarcinoma and mouse MC-38 colon cancer cells in comparison to normal human CCD18co colon cells, which have practically undetectable levels of CEA. In addition, in vivo analyses of mice bearing tumors induced using MC-38 cells showed a significant decrease in tumor volume after pCEA-E treatment and a low level of Ki-67 in relation to untreated tumors. These results suggest that the CEA promoter is an excellent candidate for directing E gene expression specifically toward colon cancer cells.

  20. Constitutive transgene expression of Stem Cell Antigen-1 in the hair follicle alters the sensitivity to tumor formation and progression

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    Rikke Christensen

    2017-08-01

    Full Text Available The cell surface protein Stem Cell Antigen-1 (Sca-1 marks stem or progenitor cells in several murine tissues and is normally upregulated during cancer development. Although the specific function of Sca-1 remains unknown, Sca-1 seems to play a role in proliferation, differentiation and cell migration in a number of tissues. In the skin epithelium, Sca-1 is highly expressed in the interfollicular epidermis but is absent in most compartments of the hair follicle; however, the function of Sca-1 in the skin has not been investigated. To explore the role of Sca-1 in normal and malignant skin development we generated transgenic mice that express Sca-1 in the hair follicle stem cells that are normally Sca-1 negative. Development of hair follicles and interfollicular epidermis appeared normal in Sca-1 mutant mice; however, follicular induction of Sca-1 expression in bulge region and isthmus stem cells reduced the overall yield of papillomas in a chemical carcinogenesis protocol. Despite that fewer papillomas developed in transgenic mice a higher proportion of the papillomas underwent malignant conversion. These findings suggest that overexpression of Sca-1 in the hair follicle stem cells contributes at different stages of tumour development. In early stages, overexpression of Sca-1 decreases tumour formation while at later stages overexpression of Sca-1 seems to drive tumours towards malignant progression.

  1. The porcine circovirus type 1 capsid gene promoter improves antigen expression and immunogenicity in a HIV-1 plasmid vaccine

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    Burger Marieta

    2011-02-01

    Full Text Available Abstract Background One of the promising avenues for development of vaccines against Human immunodeficiency virus type 1 (HIV-1 and other human pathogens is the use of plasmid-based DNA vaccines. However, relatively large doses of plasmid must be injected for a relatively weak response. We investigated whether genome elements from Porcine circovirus type 1 (PCV-1, an apathogenic small ssDNA-containing virus, had useful expression-enhancing properties that could allow dose-sparing in a plasmid vaccine. Results The linearised PCV-1 genome inserted 5' of the CMV promoter in the well-characterised HIV-1 plasmid vaccine pTHgrttnC increased expression of the polyantigen up to 2-fold, and elicited 3-fold higher CTL responses in mice at 10-fold lower doses than unmodified pTHgrttnC. The PCV-1 capsid gene promoter (Pcap alone was equally effective. Enhancing activity was traced to a putative composite host transcription factor binding site and a "Conserved Late Element" transcription-enhancing sequence previously unidentified in circoviruses. Conclusions We identified a novel PCV-1 genome-derived enhancer sequence that significantly increased antigen expression from plasmids in in vitro assays, and improved immunogenicity in mice of the HIV-1 subtype C vaccine plasmid, pTHgrttnC. This should allow significant dose sparing of, or increased responses to, this and other plasmid-based vaccines. We also report investigations of the potential of other circovirus-derived sequences to be similarly used.

  2. Zinc oxide nanoparticle reduced biofilm formation and antigen 43 expressions in uropathogenic Escherichia coli

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    Ali Shakerimoghaddam

    2017-04-01

    Full Text Available Objective(s: This study aimed to investigate the effect of zinc oxide nanoparticles (ZnO-np on biofilm formation and expression of the flu gene in uropathogenic Escherichia coli (UPEC strains. Materials and Methods: Minimum inhibitory concentration (MIC of ZnO-np was determined by agar dilution method. The effect of MIC and sub-MIC concentrations of ZnO-np on biofilm formation were determined by microtiter plate assay. The expression level of the flu gene was assessed by Real-Time PCR assay. Results: MIC and sub-MIC ZnO-np concentrations reduced biofilm formation by 50% and 33.4%, respectively. Sub-MIC ZnO-np concentration significantly reduced the flu gene expression in the UPEC isolates (P

  3. Ectopic expression of cancer/testis antigen SSX2 induces DNA damage and promotes genomic instability

    DEFF Research Database (Denmark)

    Greve, Katrine Buch Vidén; Lindgreen, Jonas; Terp, Mikkel Green

    2015-01-01

    of senescence (i.e. an irregular and enlarged cell shape, enhanced β-galactosidase activity and DNA double-strand breaks). Since replication defects, DNA damage and senescence are interconnected and well-documented effects of oncogene expression, we tested the oncogenic potential of SSX2. Importantly, knockdown...... in an increased DNA content and enlargement of cell nuclei, suggestive of replication aberrations. The cells further displayed signs of DNA damage and genomic instability, associated with p53-mediated G1 cell cycle arrest and a late apoptotic response. These results suggest a model wherein SSX2-mediated...... replication stress translates into mitotic defects and genomic instability. Arrest of cell growth and induction of DNA double-strand breaks was also observed in MCF7 breast cancer cells in response to SSX2 expression. Additionally, MCF7 cells with ectopic SSX2 expression demonstrated typical signs...

  4. Expression of RoTat 1.2 cross-reactive variable antigen type in Trypanosoma evansi and T. equiperdum.

    Science.gov (United States)

    Claes, Filip; Verloo, D; De Waal, D T; Urakawa, T; Majiwa, P; Goddeeris, B M; Buscher, P

    2002-10-01

    The variable antigen type (VAT) RoTat 1.2 has been cloned from a T. evansi strain, isolated in 1982 from a water buffalo in Indonesia. All T. evansi isolates hitherto tested express this VAT. In a study on the differential diagnosis of T. equiperdum and T. evansi in horses, we investigated serological evidence for the expression of RoTat 1.2 in 11 T. evansi and six T. equiperdum populations originating from Asia, Europe, Africa, and the Americas. Preinfection sera and sera of days 7, 14, 25, and 35 post-infection (p.i.) were analyzed for the presence of antibodies reactive with RoTat 1.2 in immune trypanolysis, ELISA/T. evansi and CATT/T. evansi. Within the duration of the experiment, all rabbits infected with T. evansi became positive in the three serological tests. Five out of six rabbits infected with T. equiperdum also became positive in the three tests. Only one T. equiperdum strain (the OVI strain from South Africa) did not induce the production of antibodies reactive with RoTat 1.2 and thus might not contain or express a VSG that shares epitopes similar to those on the RoTat 1.2 VSG. The data lead to the conclusion that T. equiperdum can express VSGs containing epitopes serologically similar to those in the T. evansi RoTat 1.2 VAT. This explains, in part, why the antibody detection tests based on Ro Tat 1.2 VSG cannot reliably distinguish between the infections caused by T. evansi and those caused by T. equiperdum. There are no data that contradict the possibility that the putative T. equiperdum strains, which express VSGs with epitopes similar to those on RoTat 1.2, are actually T. evansi.

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

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    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.

  6. Study of Prostate Specific Antigen Gene Expression and Telomerase in Breast Cancer Patients: Relationship to Steroid Hormone Receptors

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    N. Zarghami

    2007-10-01

    Full Text Available Introduction & Objective: Breast cancer is the most common disease in women. In the expansion and progression of breast tumors combination of tumor markers including prostate specific antigen (PSA and telomerase are engaged. The aim of this study was to evaluate relationship between telomerase activity and prostate specific antigen gene expression with steroid hormone receptors in breast cancer patients. Materials & Methods: This study was a case-control and consisted of 50 women diagnosed with breast benign tumors as control and 50 women having malignant tumors as cases. Telomerase activity was measured in tumor cytosol of samples by telomeric repeat amplification protocol (TRAP assay. PSA protein was measured using ultra sensitive immunoflourometric assay and PSA mRNA expression was carried out using RT-PCR technique in all tumor tissues. Estrogen and progesterone receptors were stained using immunohistochemistry technique in tumor tissues. Data analysis was carried out by using SPSS software version 11.6 and paired t-student test. Results: Using TRAP assay, presence of the telomerase activity was positive in all of the breast cancer patients. The difference of relative telomerase activity (RTA values between stages and also all grades were more statistically significant (p<0.05. The mRNA of PSA was detected only in benign tumors and stage I and grade I malignant tumor cytosols. Difference of tumor cytosol PSA levels between the cases and control groups and also between all grades and stages of diseases were significant (p <0.05. In all, there was an inverse significant correlation between the RTA and PSA protein levels in the case groups. (r=-0.42, p<0.05.There was a statistically difference between steroid hormone receptors (ER and PR positive and negative on PSA and telomerase gene expression in breast tumor tissues (p<0.05. Conclusion: It is speculated that differential expression of PSA and telomerase genes in breast tumors are under

  7. Comparison of cancer stem cell antigen expression by tumor cell lines and by tumor biopsies from dogs with melanoma and osteosarcoma.

    Science.gov (United States)

    Guth, Amanda M; Deogracias, Mike; Dow, Steven W

    2014-10-15

    Cancer stem cells (CSCs) represent a small subpopulation of tumor cells that play a critical role in initiating and sustaining tumor growth. However, we currently have an incomplete understanding of the expression patterns of CSC antigens in tumors of dogs, nor do we understand how expression of these antigens vary between tumor cell lines and tumor biopsy specimens. Therefore, we used flow cytometry and commonly reported CSC surface and intracellular markers to evaluate the phenotype and overall frequency of CSC subpopulations in tumor cell lines and primary tumor biopsy samples from dogs with melanoma and osteosarcoma. We found that cells expressing common CSC antigens were rare in tumor cell lines, with the exception of tumor cells expressing CD44 and CD90. In contrast, tumor cells expressing conventional CSC antigens such as CD133, CD34, CD44, CD24 and Oct3/4 were much more common in tumor biopsy samples. Notably, the frequency and types of putative CSC subpopulations were very similar in biopsy samples from dogs with either melanoma or osteosarcoma. Our results suggest that the tumor microenvironment significantly influences CSC subpopulations within tumors and that tumor cell lines may not accurately reflect the actual frequency or types of CSC subpopulations present in tumor tissues in vivo. Copyright © 2014 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    We have previously identified the mycobacterial high G+C codon usage bias as a limiting factor in heterologous expression of MAP proteins from Lb.salivarius, and demonstrated that codon optimisation of a synthetic coding gene greatly enhances MAP protein production. Here, we effectively demonstrate ...

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

    CSIR Research Space (South Africa)

    Meyers, A

    2008-06-23

    Full Text Available to the chloroplast and the endoplasmic reticulum (ER) on protein yield. The authors have partially purified a selected vaccine candidate and tested its stimulation of a humoral and cellular immune response in mice. Both transient and transgenic expression of the HIV...

  10. Change in antigen specificity of cytotoxic T lymphocytes is associated with the rearrangement and expression of a T-cell receptor beta-chain gene.

    Science.gov (United States)

    Epplen, J T; Bartels, F; Becker, A; Nerz, G; Prester, M; Rinaldy, A; Simon, M M

    1986-06-01

    Cloned H-Y-specific murine cytotoxic T lymphocytes, which alter antigen specificity in vitro ("aging"), simultaneously exhibit changes in the T-cell antigen receptor beta-chain rearrangements and respective mRNAs expressed. beta-chain cDNA clones were isolated from a library prepared from mRNA of aged killer T cells. The sequence of the beta-chain variable region element (VAK) was found to be identical with germ-line DNA. Four bases at the beta-chain diversity-joining region (D beta--J beta) junction cannot be explained by known germ-line D beta and J beta elements. These results illustrate that in T-cell clones altered antigen specificity correlates with a switch in productive beta-chain rearrangements of the T-cell receptor. When tested for its expression under physiological conditions, significant levels of VAK mRNA were found in normal lymphocyte populations.

  11. Immunohistochemical localization of retroviral-related antigens expressed in normal baboon placental villous tissue.

    Science.gov (United States)

    Langat, D K; Johnson, P M; Rote, N S; Wango, E O; Owiti, G O; Mwenda, J M

    1998-12-01

    Endogenous retroviral particles (ERVs) have been detected in the genome of all eukaryotes. They are generally non-pathogenic except in mice where they have been found to induce tumors and immunological disorders. The ERVs have morphological features consistent with type-C retroviral particles and are commonly expressed in normal placental villous tissues. ERVs may have a role in the regulation of placental gene expression, syncytiotrophoblast formation, or pregnancy-related immunosuppression. In this study, well-characterized antibodies (monoclonal and polyclonal antibodies) raised against retroviral proteins (anti-HIV and anti-SIV) and endogenous retroviral (ERV) particles were assessed for their cross-reactivity (by using immunohistochemistry) with normal baboon placental and other adult tissues. The monoclonal antibodies to exogenous retroviral proteins (anti-HIV-2 gp120, anti-HIV-1 gp41, anti-SIVmac p27, anti-HIV-1 RT, and anti-HIV-2 core protein) showed specific immunohistochemical reactivity with the syncytiotrophoblast. Antibodies to endogenous retroviral gene products (anti-ERV3 env, anti-HERV-K RT, and anti-HERV-K env) also reacted in a similar manner and did not cross-react with other adult tissues. These studies have shown that retroviral-cross-reactive proteins are expressed in baboon placental syncytiotrophoblast and may have a role to play at the feto-maternal interface.

  12. Molecular characterisation and expression analysis of SEREX-defined antigen NUCB2 in gastric epithelium, gastritis and gastric cancer

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    Z Kalnina

    2009-08-01

    Full Text Available NUCB2 is an EF-hand Ca2+ binding protein that has been implicated in various physiological processes like calcium homeostasis, hypothalamic regulation of feeding and TNF receptor shedding. In our previous study we identified NUCB2 as a potential tumour antigen eliciting autoantibody responses in 5.4% of gastric cancer patients but not in the healthy individuals. The current study aimed to elucidate the molecular mechanism underlying NUCB2 immunogenicity and to gain an insight into the physiological functions of NUCB2 in the stomach. mRNA expression analysis demonstrated that NUCB2 is ubiquitously expressed in normal tissues, including lymphoid tissues, and downregulated in gastric tumours when compared with the adjacent relatively normal stomach tissues. The search for molecular alterations resulted in the identification of novel mRNA variants transcribed from an alternative promoter and expressed predominantly in gastric cancers. Western blot analysis demonstrated that the protein levels correspond to mRNA levels and revealed that NUCB2 is phosphorylated in gastric mucosa. Furthermore, a 55 kDa isoform, generated presumably by yet an unidentified post-translational modification was detected in gastric tumours and AGS gastric cancer cells but was absent in the relatively normal gastric mucosa and thereby might have served as a trigger for the immune response against NUCB2. Staining of stomach tissue microarray with anti-NUCB2 antibody revealed that it is expressed in the secretory granules of chief cells and in the cytoplasm of parietal cells in the functioning gastric glands which are lost in atrophic glands and tumour cells. Hence we propose that NUCB2 may be implicated in gastric secretion by establishing an agonist-releasable Ca2+ store in ER or Golgi apparatus, signalling via heterotrimeric Ga proteins and/or mediating the exocytosis of the secretory granules.

  13. Expression and refolding of the protective antigen of Bacillus anthracis: A model for high-throughput screening of antigenic recombinant protein refolding.

    Science.gov (United States)

    Pavan, María Elisa; Pavan, Esteban Enrique; Cairó, Fabián Martín; Pettinari, María Julia

    2016-01-01

    Bacillus anthracis protective antigen (PA) is a well known and relevant immunogenic protein that is the basis for both anthrax vaccines and diagnostic methods. Properly folded antigenic PA is necessary for these applications. In this study a high level of PA was obtained in recombinant Escherichia coli. The protein was initially accumulated in inclusion bodies, which facilitated its efficient purification by simple washing steps; however, it could not be recognized by specific antibodies. Refolding conditions were subsequently analyzed in a high-throughput manner that enabled nearly a hundred different conditions to be tested simultaneously. The recovery of the ability of PA to be recognized by antibodies was screened by dot blot using a coefficient that provided a measure of properly refolded protein levels with a high degree of discrimination. The best refolding conditions resulted in a tenfold increase in the intensity of the dot blot compared to the control. The only refolding additive that consistently yielded good results was L-arginine. The statistical analysis identified both cooperative and negative interactions between the different refolding additives. The high-throughput approach described in this study that enabled overproduction, purification and refolding of PA in a simple and straightforward manner, can be potentially useful for the rapid screening of adequate refolding conditions for other overexpressed antigenic proteins. Copyright © 2015 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  14. 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.

  15. Molecular modeling and expression of the Litopenaeus vannamei proliferating cell nuclear antigen (PCNA) after white spot syndrome virus shrimp infection.

    Science.gov (United States)

    de-la-Re-Vega, Enrique; Muhlia-Almazan, Adriana; Arvizu-Flores, Aldo A; Islas-Osuna, Maria A; Yepiz-Plascencia, Gloria; Brieba, Luis G; Sotelo-Mundo, Rogerio R

    2011-01-01

    Proliferating cell nuclear antigen (PCNA) is the eukaryotic sliding clamp that tethers DNA polymerase to DNA during replication. The full-length cDNA of the Pacific white shrimp Litopenaeus vannamei PCNA (LvPCNA) was cloned and encoded a protein of 260 amino acids that is highly similar to other Crustacean PCNAs. The theoretical shrimp PCNA structure has all the domains that are necessary for its interaction with template DNA and DNA polymerase. RT-PCR analysis showed that LvPCNA is expressed mainly in muscle and hemocytes and much less in hepatopancreas and gills. LvPCNA mRNA levels are not statistically different in muscle from healthy and challenged shrimp with the white spot syndrome virus (WSSV). In contrast, the mRNA levels of the viral DNA polymerase show a biphasic pattern with expression at 6 h post-infection and later at 24 and 48 h. These results suggest that in shrimp muscle LvPCNA levels are steadily kept to allow viral replication and that WSSV DNA polymerase (WSSV-DNApol) is more responsive towards later stages of infection. More knowledge of the DNA replication machinery would result in a better understanding of the mechanism and components of viral replication, since the WSSV genome does not have all the components required for assembly of a fully functional replisome.

  16. Mechanisms and effects of loss of human leukocyte antigen class II expression in immune-privileged site-associated B-cell lymphoma

    NARCIS (Netherlands)

    Booman, M; Douwes, J; Glas, AM; Riemersma, SA; Jordanova, ES; Kok, K; Rosenwald, A; de Jong, D; Schuuring, E; Kluin, PM

    2006-01-01

    Purpose and Experimental Design: Loss of human leukocyte antigen (HLA) expression on tumor cells is frequent in diffuse large B-cell lymphoma (DLBCL) arising in immune-privileged sites, such as the testis and central nervous system, and is associated with small homozygous deletions of HLA-DQ/HLA-DR

  17. 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 and the ov...

  18. Coexpression of GM-CSF and antigen in DNA prime-adenoviral vector boost immunization enhances polyfunctional CD8+ T cell responses, whereas expression of GM-CSF antigen fusion protein induces autoimmunity.

    Science.gov (United States)

    Tenbusch, Matthias; Kuate, Seraphin; Tippler, Bettina; Gerlach, Nicole; Schimmer, Simone; Dittmer, Ulf; Uberla, Klaus

    2008-04-11

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) has shown promising results as a cytokine adjuvant for antiviral vaccines and in various models of tumor gene therapy. To explore whether the targeting of antigens to GM-CSF receptors on antigen-presenting cells enhances antigen-specific CD8 T-cell responses, fusion proteins of GM-CSF and ovalbumin (OVA) were expressed by DNA and adenoviral vector vaccines. In addition, bicistronic vectors allowing independent expression of the antigen and the cytokine were tested in parallel. In vitro, the GM-CSF ovalbumin fusion protein (GM-OVA) led to the better stimulation of OVA-specific CD8+ T cells by antigen-presenting cells than OVA and GM-CSF given as two separate proteins. However, prime-boost immunizations of mice with DNA and adenoviral vector vaccines encoding GM-OVA suppressed CD8+ T-cell responses to OVA. OVA-specific IgG2a antibody levels were also reduced, while the IgG1 antibody response was enhanced. Suppression of CD8+ T cell responses by GM-OVA vaccines was associated with the induction of neutralizing antibodies to GM-CSF. In contrast, the coexpression of GM-CSF and antigens in DNA prime adenoviral boost immunizations led to a striking expansion of polyfunctional OVA-specific CD8+ T cells without the induction of autoantibodies. The induction of autoantibodies suggests a general note of caution regarding the use of highly immunogenic viral vector vaccines encoding fusion proteins between antigens and host proteins. In contrast, the expansion of polyfunctional OVA-specific CD8+ T cells after immunizations with bicistronic vectors further support a potential application of GM-CSF as an adjuvant for heterologous prime-boost regimens with genetic vaccines. Since DNA prime adenoviral vector boost regimenes are presently considered as one of the most efficient ways to induce CD8+ T cell responses in mice, non-human primates and humans, further enhancement of this response by GM-CSF is a striking observation.

  19. Coexpression of GM-CSF and antigen in DNA prime-adenoviral vector boost immunization enhances polyfunctional CD8+ T cell responses, whereas expression of GM-CSF antigen fusion protein induces autoimmunity

    Directory of Open Access Journals (Sweden)

    Gerlach Nicole

    2008-04-01

    Full Text Available Abstract Background Granulocyte-macrophage colony-stimulating factor (GM-CSF has shown promising results as a cytokine adjuvant for antiviral vaccines and in various models of tumor gene therapy. To explore whether the targeting of antigens to GM-CSF receptors on antigen-presenting cells enhances antigen-specific CD8 T-cell responses, fusion proteins of GM-CSF and ovalbumin (OVA were expressed by DNA and adenoviral vector vaccines. In addition, bicistronic vectors allowing independent expression of the antigen and the cytokine were tested in parallel. Results In vitro, the GM-CSF ovalbumin fusion protein (GM-OVA led to the better stimulation of OVA-specific CD8+ T cells by antigen-presenting cells than OVA and GM-CSF given as two separate proteins. However, prime-boost immunizations of mice with DNA and adenoviral vector vaccines encoding GM-OVA suppressed CD8+ T-cell responses to OVA. OVA-specific IgG2a antibody levels were also reduced, while the IgG1 antibody response was enhanced. Suppression of CD8+ T cell responses by GM-OVA vaccines was associated with the induction of neutralizing antibodies to GM-CSF. In contrast, the coexpression of GM-CSF and antigens in DNA prime adenoviral boost immunizations led to a striking expansion of polyfunctional OVA-specific CD8+ T cells without the induction of autoantibodies. Conclusion The induction of autoantibodies suggests a general note of caution regarding the use of highly immunogenic viral vector vaccines encoding fusion proteins between antigens and host proteins. In contrast, the expansion of polyfunctional OVA-specific CD8+ T cells after immunizations with bicistronic vectors further support a potential application of GM-CSF as an adjuvant for heterologous prime-boost regimens with genetic vaccines. Since DNA prime adenoviral vector boost regimenes are presently considered as one of the most efficient ways to induce CD8+ T cell responses in mice, non-human primates and humans, further

  20. 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.

    Science.gov (United States)

    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 < 0.0001), 46.6 versus 50.0 versus 109.0 for MAGE-A3/A4 (p = 0.0074) and 13.3 versus 50.0 versus 100.2 months for NY-ESO-1 (p = 0.0019). By multivariate analysis, these factors were confirmed as independent markers for poor survival. HNSCC patients showing protein expression of MAGE-A 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.

  1. 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.

  2. Oral Delivery of Probiotics Expressing Dendritic Cell-Targeting Peptide Fused with Porcine Epidemic Diarrhea Virus COE Antigen: A Promising Vaccine Strategy against PEDV

    Directory of Open Access Journals (Sweden)

    Xiaona Wang

    2017-10-01

    Full Text Available Porcine epidemic diarrhea virus (PEDV, an enteric coronavirus, is the causative agent of porcine epidemic diarrhea (PED that damages intestinal epithelial cells and results in severe diarrhea and dehydration in neonatal suckling pigs with up to 100% mortality. The oral vaccine route is reported as a promising approach for inducing protective immunity against PEDV invasion. Furthermore, dendritic cells (DCs, professional antigen-presenting cells, link humoral and cellular immune responses for homeostasis of the intestinal immune environment. In this study, in order to explore an efficient oral vaccine against PEDV infection, a mucosal DC-targeting oral vaccine was developed using Lactobacillus casei to deliver the DC-targeting peptide (DCpep fused with the PEDV core neutralizing epitope (COE antigen. This probiotic vaccine could efficiently elicit secretory immunoglobulin A (SIgA-based mucosal and immunoglobulin G (IgG-based humoral immune responses via oral vaccination in vivo. Significant differences (p < 0.05 in the immune response levels were observed between probiotics expressing the COE-DCpep fusion protein and COE antigen alone, suggesting better immune efficiency of the probiotics vaccine expressing the DC-targeting peptide fused with PEDV COE antigen. This mucosal DC-targeting oral vaccine delivery effectively enhances vaccine antigen delivery efficiency, providing a useful strategy to induce efficient immune responses against PEDV infection.

  3. 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.

  4. Tumor-associated antigens identified by mRNA expression profiling as tumor rejection epitopes

    DEFF Research Database (Denmark)

    Andersen, Marie Louise; Ruhwald, Morten; Thorn, Mette

    2003-01-01

    Thirteen H-2b-binding peptides derived from six potentially overexpressed proteins in p53-/- thymoma (SM7) cells were studied for immunogenecity and vaccine-induced prevention of tumor growth in mice inoculated with SM7 tumor cells. Six of the peptides generated specific CTL responses after immun...... derived from potentially overexpressed tumor proteins, as identified by mRNA expression profiling of p53-/- thymoma cells, at best results in a weak tumor protection thus questioning this way of detection of new tumor rejection epitopes....

  5. Bacterially expressed human papillomavirus type 6 and 11 bivalent vaccine: Characterization, antigenicity and immunogenicity.

    Science.gov (United States)

    Pan, Huirong; Li, Zhihai; Wang, Jin; Song, Shuo; Wang, Daning; Wei, Minxi; Gu, Ying; Zhang, Jun; Li, Shaowei; Xia, Ningshao

    2017-05-31

    Human papillomavirus (HPV)-6 and HPV11 are the major etiological causes of condylomata acuminate. HPV neutralization by vaccine-elicited neutralizing antibodies can block viral infection and prevent subsequent disease. Currently, two commercially available HPV vaccines cover these two genotypes, expressed by Saccharomyces cerevisiae. Here we describe another HPV6/11 bivalent vaccine candidate derived from Escherichia coli. The soluble expression of N-terminally truncated L1 proteins was optimized to generate HPV6- and HPV11 L1-only virus-like particles (VLPs) as a scalable process. In a pilot scale, we used various biochemical, biophysical and immunochemical approaches to comprehensively characterize the scale and lot consistency of the vaccine candidate at 30L and 100L. Cryo-EM structure analysis showed that these VLPs form a T=7 icosahedral lattice, imitating the L1 capsid of the authentic HPV virion. This HPV6/11 bivalent vaccine confers a neutralization titer and antibody production profile in monkey that is comparable with the quadrivalent vaccine, Gardasil. This study demonstrates the robustness and scalability of a potential HPV6/11 bivalent vaccine using a prokaryotic system for vaccine production. Copyright © 2017. Published by Elsevier Ltd.

  6. Retinoic acid receptor antagonist inhibits CD38 antigen expression on human hematopoietic cells in vitro.

    Science.gov (United States)

    Prus, Eugenia; Chandraratna, Roshantha A S; Fibach, Eitan

    2004-05-01

    The CD34+ CD38- subset of human hematopoietic stem cells are crucial for long-term ex-vivo expansion; conditions that decreased this specific sub-population reduced the self-renewal capacity and shortened the duration of the proliferative phase of the culture. Retinoids, such as all-trans retinoic acid (ATRA), have been shown to induce CD38 expression. ATRA present in serum may be responsible for the high CD38 of cells grown in serum-containing medium. In the present study we analyzed the effects of AGN 194310, a retinoic acid receptor pan-antagonist, on CD38 expression of human hematopoietic cells. Normal cells (cord blood derived CD34+ cells) and abnormal cells (myeloid leukemic lines) were studied when grown in either serum-containing or serum-free media. The results showed that both serum and ATRA enhanced differentiation and, thereby, reduced the proportion of CD34+ CD38- cells and total CD34+ cell expansion. AGN reversed these effects of serum and ATRA: it delayed differentiation and increased CD34+ CD38- cells. These results suggest that physiological ATRA levels in serum may prevent efficient cell expansion. AGN, by neutralizing ATRA, improves cell expansion in serum-containing cultures, thus making AGN a useful agent for ex vivo expansion of stem cells and other specific sub-populations for research and clinical use.

  7. The cancer-testis antigen NY-ESO-1 is highly expressed in myxoid and round cell subset of liposarcomas.

    Science.gov (United States)

    Hemminger, Jessica A; Ewart Toland, Amanda; Scharschmidt, Thomas J; Mayerson, Joel L; Kraybill, William G; Guttridge, Denis C; Iwenofu, O Hans

    2013-02-01

    Liposarcomas are a heterogenous group of fat-derived sarcomas, and surgery with or without chemoradiation therapy remains the main stay of treatment. NY-ESO-1 is a cancer-testis antigen expressed in various cancers where it can induce both cellular and humoral immunity. Immunotherapy has shown promise in clinical trials involving NY-ESO-1-expressing tumors. Gene expression studies have shown upregulation of the gene for NY-ESO-1, CTAG1B, in myxoid and round cell liposarcomas. Herein, we evaluated the expression of NY-ESO-1 among liposarcoma subtypes by quantitative real-time PCR, western blot analysis, and immunohistochemistry. Frozen tissue for quantitative real-time PCR and western blot analysis was obtained for the following liposarcoma subtypes (n=15): myxoid and round cell (n=8); well-differentiated (n=4), and dedifferentiated (n=3). Formalin-fixed paraffin-embedded blocks were obtained for the following liposarcoma subtypes (n=44): myxoid and round cell (n=18); well-differentiated (n=10); dedifferentiated (n=10); and pleomorphic (n=6). Full sections were stained with monoclonal antibody NY-ESO-1, and staining was assessed for intensity (1-3+), percentage of tumor positivity, and location. In all, 7/8 (88%) and 16/18 (89%) myxoid and round cell expressed CTAG1B and NY-ESO-1 by quantitative real-time PCR and immunohistochemistry, respectively. Western blot correlated with mRNA expression levels. By immunohistochemistry, 94% (15/16) of positive cases stained homogenously with 2-3+ intensity. Also, 3/6 (50%) pleomorphic liposarcomas demonstrated a range of staining: 1+ intensity in 50% of cells; 2+ intensity in 5% of cells; and 3+ intensity in 90% of cells. One case of dedifferentiated liposarcoma showed strong, diffuse staining (3+ intensity in 75% of cells). Our study shows that both CTAG1B mRNA and protein are overexpressed with high frequency in myxoid and round cell liposarcoma, enabling the potential use of targeted immunotherapy in the treatment of this

  8. [Effect of hydrogen peroxide on human leukocyte antigen-G expression in placental trophoblasts in pre-eclampsia].

    Science.gov (United States)

    Zhou, Xue; Sun, Li-zhou; Yin, Yin

    2010-05-01

    To explore the effect of hydrogen peroxide (H2O2) on the expression of human leukocyte antigen-G (HLA-G) in placental trophoblasts in pregnant women with pre-eclampsia. Forty pregnant women, delivered through cesarean section in the Department of Obstetrics of and Gynecology, the First Affiliated Hospital of Nanjing Medical University from October 2008 to October 2009, were enrolled, including 20 women with pre-eclampsia and 20 healthy gravidas (control group). Colorimetry and western blot were applied, respectively, to determine the level of H2O2 and the expression of HLA-G protein in placental tissues and the correlation between them were analyzed. After 24 hours of seeding, JEG-3 cells (the HLA-G positive cell line of choriocarcinoma) were divided into two groups: intervention group (exposure to 175 micromol/L H2O2) and control group (without H2O2). Immunofluorescence and western blot were used to investigate the expression of HLA-G protein in JEG-3 cells at 24 hours and 48 hours after incubation. (1) The level of H2O2 in placenta in the pre-eclampsia group was significantly higher than that in control group [(105+/-13) nmolxmg(-1)xprot(-1) vs (62+/-18) nmol.mg(-1)xprot(-1), PHLA-G protein in placenta of the pre-eclampsia group was reduced by 88% compared with that of the control (0.20+/-0.08 vs 1.67+/-0.65, PHLA-G level and H2O2 expression in the placenta in both groups (r=-0.895, P=0.000). (4) Compared with the control group, the expression of HLA-G protein in JEG-3 cells, after 24 hours and 48 hours exposure to H2O2, reduced by 39% and 80%, respectively, (3.21+/-0.33 vs 1.95+/-0.25 and 0.65+/-0.08, PHLA-G in JEG-E cells in the intervention group at 48 hours compared to the control group (PHLA-G expression in the placental trophoblasts in pre-eclampsia which may be involved in the pathogenesis of pre-eclampsia.

  9. Immunohistochemical expression of tumor antigens MAGE-A3/4 and NY-ESO-1 in renal oncocytoma and chromophobe renal cell carcinoma.

    Science.gov (United States)

    Demirović, Alma; Džombeta, Tihana; Tomas, Davor; Spajić, Borislav; Pavić, Ivana; Hudolin, Tvrtko; Milošević, Milan; Cupić, Hrvoje; Krušlin, Božo

    2010-10-15

    The distinction between renal oncocytoma (RO) and chromophobe renal cell carcinoma (ChRCC), especially the eosinophilic variant, can often be difficult. Our study has documented for the first time the expression of MAGE-A3/4 and NY-ESO-1 cancer testis antigens (CTAs) in these tumors. A total of 35 patients (17 ROs and 18 ChRCCs) were included in the study. Two antibodies were used for immunohistochemical staining: 57B recognizing multiple MAGE-A and D8.38 recognizing NY-ESO-1 CTAs. Fifteen (88.2%) samples of RO stained positively for both MAGE-A3/4 and NY-ESO-1 antigens. Regarding ChRCC, seven (38.9%) stained positively for MAGE-A3/4 and six (33.3%) for NY-ESO-1 antigens. Median MAGE-A3/4 expression was moderately positive in RO and negative in ChRCC. The difference in MAGE-A3/4 expression between two tumor groups was significant (P=0.0013). Median NY-ESO-1 expression was strongly positive in RO and negative in ChRCC. The difference in NY-ESO-1 expression between two tumor groups was also significant (P=0.0008). Our study has shown that RO had a significantly higher expression of both CTAs. However, additional research is needed to clarify their potential diagnostic implications. Copyright © 2010 Elsevier GmbH. All rights reserved.

  10. Construction, expression, purification and antigenicity of recombinant Campylobacter jejuni flagellar proteins.

    Science.gov (United States)

    Yeh, Hung-Yueh; Hiett, Kelli L; Line, John E; Oakley, Brian B; Seal, Bruce S

    2013-05-06

    Campylobacter jejuni, a flagellated, spiral-rod Gram-negative bacterium, is the leading etiologic agent of human acute bacterial gastroenteritis worldwide. The source of this microorganism for human infection has been implicated as consumption and handling of poultry meat where this microorganism is a commensal in the gut. Because the genomes of many C. jejuni isolates have been sequenced, our ultimate goal is to develop protein arrays for exploring this microorganism and host interactions. In this communication, we report cloning, expression and purification of C. jejuni flagellar proteins in a bacterial expression system. Twelve recombinant proteins were purified, which were confirmed by SDS-PAGE analysis and a His tag detection kit. The FlgE1, FlgG, FlgK, FliE, FlgH/FliH and FlaA recombinant proteins were further confirmed by LC-ESI-MS/MS. The purified recombinant proteins were tested whether they were immunogenic using antibodies from several sources. BacTrace anti-Campylobacter species antibody reacted to the FlaA recombinant protein, but not others. Rabbit anti-MOMP1 peptide antibody reacted strongly to FliE and weakly to FlaA, but not others. Rabbit anti-MOMP2 peptide antibody reacted strongly to the FlaA, FliG, FliE, FlhF, FlgG, FlgE1 and FliD recombinant proteins, less to FlgK and FlgH/FliH, and did not react to the FliY, FliS and FliH recombinant proteins. These antibody studies suggest that these recombinant flagellar proteins have potential for novel targets for vaccine development. It is also anticipated that these recombinant proteins provide us a very useful tool for investigating host immune response to C. jejuni. Published by Elsevier GmbH.

  11. Lymphoid associated antigen expression in new cases of Acute Myeloid Leukemia

    Directory of Open Access Journals (Sweden)

    R Jha

    2013-10-01

    Full Text Available Background: Occurrence of aberrant phenotype has been reported in acute leukemias with varying frequency though its prognostic importance remains controversial. In acute myeloid leukemias, aberrant phenotype, as high as 88 %, has been reported. To evaluate the occurrence of aberrant lymphoid phenotypes and to correlate their presence with various French American British classification, 100 cases of fresh acute myeloid leukemias were analyzed for lymphoid markers CD 4,7,8,10 and 19. Materials and Methods: Whole blood or bone marrow aspirate collected in EDTA were processed by standard method and subjected to immunophenotyping for B Cells marker CD 19 and 10 and T cell marker CD 4, 7 and 8. Results: Aberrant lymphoid markers were seen in 35(35% cases. All FAB subtypes except M7 showed aberrancy for the markers studied. However it was the most common in M0 (100%, followed by M2 (51.9%. T cell aberrancy was the most common, comprising 62.8% (22/35 of total aberrancy. CD 7 was the most common aberrantly expressed marker, seen in 20% AML, followed by CD 4(14% and CD 19 (8%. Conclusion: Occurrence of lymphoid phenotypes is frequent in pediatric as well adult AML. Though T cell markers are more common, only B cell as well as both B and T cell markers may be co expressed. DOI: http://dx.doi.org/10.3126/jpn.v3i6.8999   Journal of Pathology of Nepal (2013 Vol. 3, 487-490

  12. Adaptive resistance to immunotherapy directed against p53 can be overcome by global expression of tumor antigens in dendritic cells

    Directory of Open Access Journals (Sweden)

    Matjaz eHumar

    2014-10-01

    Full Text Available Immunotherapy of cancer utilizes dendritic cells (DC for antigen presentation and the induction of tumor-specific immune responses. However, the therapeutic induction of anti-tumor immunity is limited by tumor escape mechanisms. In this study immortalized dendritic D2SC/1 cells were transduced with a mutated version of the p53 tumor suppressor gene, p53M234I or p53C132F/E164G, which are overexpressed in MethA fibrosarcoma tumor cells. In addition, D2SC/1 cells were fused with MethA tumor cells to generate a vaccine that potentially expresses a large repertoire of tumor antigens. Cellular vaccines were transplanted onto Balb/c mice and MethA tumor growth and anti-tumor immune responses were examined in vaccinated animals. D2SC/1-p53M234I and D2SC/1-p53C132F/E168G cells induced strong therapeutic and protective MethA tumor immunity upon transplantation in Balb/c mice. However, in a fraction of immunized mice MethA tumor growth resumed after an extended latency period. Analysis of these tumors indicated loss of p53 expression. Mice, pre-treated with fusion hybrids generated from D2SC/1 and MethA tumor cells, suppressed MethA tumor growth and averted adaptive immune escape. Polyclonal B-cell responses directed against various MethA tumor proteins could be detected in the sera of D2SC/1-MethA inoculated mice. Athymic nude mice and Balb/c mice depleted of CD4+ or CD8+ T-cells were not protected against MethA tumor cell growth after immunization with D2SC/1-MethA hybrids. Our results highlight a potential drawback of cancer immunotherapy by demonstrating that the induction of a specific anti-tumor response favours the acquisition of tumor phenotypes promoting immune evasion. In contrast, the application of DC/tumor cell fusion hybrids prevents adaptive immune escape by a T-cell dependent mechanism and provides a simple strategy for personalized anti-cancer treatment without the need of selectively priming the host immune system.

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

    Directory of Open Access Journals (Sweden)

    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.

  14. Long-term expression of Fos-related antigen and transient expression of delta FosB associated with seizures in the rat hippocampus and striatum.

    Science.gov (United States)

    Bing, G; Wang, W; Qi, Q; Feng, Z; Hudson, P; Jin, L; Zhang, W; Bing, R; Hong, J S

    1997-01-01

    Systemic administration of kainic acid (KA), an analogue of glutamic acid, causes limbic seizures and pathophysiological changes in adult rats that are very similar to human temporal lobe epilepsy. One of the earliest changes in gene expression after treatment with KA is the induction of immediate-early genes. The fos and jun families are frequently studied immediate-early genes that are induced by KA. Several groups, including ours, have recently reported that a 35-kDa Fos-related antigen (FRA) is induced for a protracted time by various stimuli. It has been suggested that this FRA is delta FosB, which has a molecular mass of approximately 35 kDa. The present study characterizes the long-term expression of FRA and delta FosB after systemic treatment with KA. Immunocytochemistry and western blot analysis using an antibody that cross-reacts with all known FRAs showed that a 35-kDa FRA was induced at high levels in both the hippocampus and striatum for up to 1 month by KA. A semiquantitative PCR analysis showed that delta FosB was induced by KA, but its expression lasted for only 6 h. This result was also verified by northern blot analysis. These results suggested that the 35-kDa FRA with long-term elevated levels seen with western blot analysis and immunocytochemistry is a new species of the FRA and not delta FosB. The long-term expression of FRA in both the hippocampus and striatum may be associated with the pathophysiological changes after KA administration.

  15. Oral immunogenicity of porcine reproductive and respiratory syndrome virus antigen expressed in transgenic banana.

    Science.gov (United States)

    Chan, Hui-Ting; Chia, Min-Yuan; Pang, Victor Fei; Jeng, Chian-Ren; Do, Yi-Yin; Huang, Pung-Ling

    2013-04-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is a persistent threat of economically significant influence to the swine industry worldwide. Recombinant DNA technology coupled with tissue culture technology is a viable alternative for the inexpensive production of heterologous proteins in planta. Embryogenic cells of banana cv. 'Pei chiao' (AAA) have been transformed with the ORF5 gene of PRRSV envelope glycoprotein (GP5) using Agrobacterium-mediated transformation and have been confirmed. Recombinant GP5 protein levels in the transgenic banana leaves were detected and ranged from 0.021%-0.037% of total soluble protein. Pigs were immunized with recombinant GP5 protein by orally feeding transgenic banana leaves for three consecutive doses at a 2-week interval and challenged with PRRSV at 7 weeks postinitial immunization. A vaccination-dependent gradational increase in the elicitation of serum and saliva anti-PRRSV IgG and IgA was observed. Furthermore, significantly lower viraemia and tissue viral load were recorded when compared with the pigs fed with untransformed banana leaves. The results suggest that transgenic banana leaves expressing recombinant GP5 protein can be an effective strategy for oral delivery of recombinant subunit vaccines in pigs and can open new avenues for the production of vaccines against PRRSV. © 2012 The Authors Plant Biotechnology Journal © 2012 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

  16. Cranial mononeuropathy VI

    Science.gov (United States)

    ... Abducens palsy; Lateral rectus palsy; VIth nerve palsy; Cranial nerve VI palsy; Sixth nerve palsy; Neuropathy - sixth nerve ... Cranial mononeuropathy VI is damage to the sixth cranial nerve. This nerve is also called the abducens nerve. ...

  17. Antibodies produced by mice immunized with recombinant vaccinia viruses expressing two different types of a major Theileria sergenti surface antigen (p32) react with the native surface antigen.

    Science.gov (United States)

    Takasima, Y; Xuan, X; Matsumoto, Y; Onuma, M; Otsuka, H

    1999-07-01

    A 32 kDa major surface antigen, p32, of Theileria sergenti at the piroplasm stage is the main target of the host immune response. The immunogenic property of the p32 varies in some strains among the population of Theileria sergenti in Japan where the Chitose type and the Ikeda type are the most common varieties. We have constructed vaccinia virus recombinants vv/p32C and vv/p32I which harbor the Chitose and Ikeda types of p32 gene, respectively. It was found that vv/p32C and vv/p32I produced type-specific p32 which did not cross react with the monoclonal antibodies (mAbs) against the other type of p32. When mice were immunized with vv/p32C and vv/p32I, antibodies against p32 were detectable 2 weeks after the immunization, and these antibodies reacted with the native surface antigen in purified T. sergenti merozoite.

  18. Stable expression of Shigella dysenteriae serotype 1 O-antigen genes integrated into the chromosome of live Salmonella oral vaccine vector Ty21a.

    Science.gov (United States)

    Dharmasena, Madushini N; Osorio, Manuel; Filipova, Svetlana; Marsh, Christina; Stibitz, Scott; Kopecko, Dennis J

    2016-09-20

    Typhoid fever and shigellosis cause high morbidity and mortality worldwide, yet no anti-Shigella vaccine is currently available. However, to protect against typhoid fever, an approved vaccine, based on the attenuated Salmonella enterica serovar Typhi strain Ty21a is available. We have investigated Ty21a as a live oral vaccine vector for expression of heterologous foreign antigens to protect against other diseases (e.g. shigellosis, anthrax, and plague). Shigella LPS is a potent vaccine antigen for serotype-specific protection against Shigellae. We previously reported the construction of a Ty21a derivative expressing S. sonnei O-antigen by insertion of a large (∼12.5 kb) operon comprising the S. sonnei O-antigen biosynthetic genes into a targeted site within the Ty21a chromosome using modified λ red recombineering methods. In the current study, S. dysenteriae 1 O-antigen biosynthetic genes from 2 separate genetic loci, rfp and rfb were assembled and inserted into the Ty21a chromosome by λ red-mediated recombineering to construct strain Ty21a-Sd. To obtain a high level of heterologous LPS expression, the native upstream promoter was replaced with the constitutive lpp promoter, which resulted in Ty21a-Sdl with enhanced heterologous LPS expression. Both Ty21a-Sd and Ty21a-Sdl elicited significant serum antibody responses in mice against both Ty21a and this heterologous Shigella LPS, and conferred protection against virulent S. dysenteriae 1 challenge. This work represents progress toward the goal of a safe and effective vaccine against Shigella. Published by Oxford University Press on behalf of FEMS 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  19. Alpha-interferon induces enhanced expression of HLA-ABC antigens and beta-2-microglobulin in vivo and in vitro in various subsets of human lymphoid cells

    DEFF Research Database (Denmark)

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

    1987-01-01

    The effect of cloned alpha-interferon (alpha-IFN) on the in vitro and in vivo expression of HLA-ABC antigens and beta-2-microglobulin (beta-2-m) on subpopulations of human lymphoid cells was studied by flow cytometry. Mononuclear cells isolated from patients and cell cultures were labelled...... with saturating amounts of FITC conjugated monoclonal anti-HLA-ABC or anti-beta-2-m. Phycoerythrin conjugated monoclonal antibodies were simultaneously used for the selection of T lymphocytes. T helper lymphocytes, T suppressor lymphocytes, B lymphocytes and monocytes. In vitro, alpha-IFN induced a significant......, except for T suppressor lymphocytes. The increase in beta-2-m only reached significance on T lymphocytes. T helper lymphocytes and monocytes (P less than 0.02). At 48 h after administration of alpha-IFN, expression of HLA-ABC antigens and beta-2-m approached pretreatment levels. Enhanced expression...

  20. Mouse thymic epithelial cell lines expressing "Aire" and peripheral tissue-specific antigens reproduce in vitro negative selection of T cells.

    Science.gov (United States)

    Yamaguchi, Yoshitaka; Takayanagi, Atsushi; Chen, Jiabing; Sakai, Kosuke; Kudoh, Jun; Shimizu, Nobuyoshi

    2011-08-15

    In the human thymus, AIRE (autoimmune regulator) gene is expressed in a very limited type of medullary thymic epithelial cells (mTECs) and no cognate cell lines are available, hence the molecular analysis of AIRE gene function has been difficult. To improve this situation, we attempted to isolate Aire-expressing cells and established three cell lines (Aire⁺TEC1, Aire⁺TEC2, Aire⁺DC) from the abnormally enlarged thymus, which was developed in the transgenic mice expressing SV40 T-antigen driven by the mouse Aire gene promoter. When these Aire⁺ cell lines were co-cultured with fresh thymocytes, they adhered to the majority of thymocytes and induced apoptosis as if negative selection of T-cells in the thymus is occurring in vitro. Further analysis revealed that these Aire⁺ cell lines are derived from mTECs and exhibit characteristic natures of "antigen presenting cells" including several distinct abilities: to express a variety of peripheral tissue-specific antigens, to produce immunoproteasome and immunological synapse, and to express some of TNFSFs (tumor necrosis factor super families). Thus, the newly established Aire⁺ cell lines will be invaluable for the further detailed analysis of AIRE gene function in the central tolerance of immunity and autoimmune disease. Copyright © 2011 Elsevier Inc. All rights reserved.

  1. Ganglioside Metabolism in a Transgenic Mouse Model of Alzheimer's Disease: Expression of Chol-1α Antigens in the Brain

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    Toshio Ariga

    2010-08-01

    Full Text Available The accumulation of Aβ (amyloid β-protein is one of the major pathological hallmarks in AD (Alzheimer's disease. Gangliosides, sialic acid-containing glycosphingolipids enriched in the nervous system and frequently used as biomarkers associated with the biochemical pathology of neurological disorders, have been suggested to be involved in the initial aggregation of Aβ. In the present study, we have examined ganglioside metabolism in the brain of a double-Tg (transgenic mouse model of AD that co-expresses mouse/human chimaeric APP (amyloid precursor protein with the Swedish mutation and human presenilin-1 with a deletion of exon 9. Although accumulation of Aβ was confirmed in the double-Tg mouse brains and sera, no statistically significant change was detected in the concentration and composition of major ganglio-N-tetraosyl-series gangliosides in the double-Tg brain. Most interestingly, Chol-1α antigens (cholinergic neuron-specific gangliosides, such as GTlaα and GQlbα, which are minor species in the brain, were found to be increased in the double-Tg mouse brain. We interpret that the occurrence of these gangliosides may represent evidence for generation of cholinergic neurons in the AD brain, as a result of compensatory neurogenesis activated by the presence of Aβ.

  2. Mass Spectrometry Reveals Changes in MHC I Antigen Presentation After Lentivector Expression of a Gene Regulation System

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    Roland Vogel

    2013-01-01

    Full Text Available The rapamycin-inducible gene regulation system was designed to minimize immune reactions in man and may thus be suited for gene therapy. We assessed whether this system indeed induces no immune responses. The protein components of the regulation system were produced in the human cell lines HEK 293T, D407, and HER 911 following lentiviral transfer of the corresponding genes. Stable cell lines were established, and the peptides presented by major histocompatibility complex class I (MHC I molecules on transduced and wild-type (wt cells were compared by differential mass spectrometry. In all cell lines examined, expression of the transgenes resulted in prominent changes in the repertoire of MHC I-presented self-peptides. No MHC I ligands originating from the transgenic proteins were detected. In vitro analysis of immunogenicity revealed that transduced D407 cells displayed slightly higher capacity than wt controls to promote proliferation of cytotoxic T cells. These results indicate that therapeutic manipulations within the genome of target cells may affect pathways involved in the processing of peptide antigens and their presentation by MHC I. This makes the genomic modifications visible to the immune system which may recognize these events and respond. Ultimately, the findings call attention to a possible immune risk.

  3. Expression and immunolocalisation of TpFABP as a candidate antigen for the serodiagnosis of rabbit Taenia pisiformis cysticercosis.

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    Yang, Deying; Chen, Lin; Xie, Yue; Wu, Xuhang; Nong, Xiang; Peng, Xi; Lai, Weimin; Gu, Xiaobin; Wang, Shuxian; Peng, Xuerong; Yang, Guangyou

    2013-01-01

    The larval stage of Taenia pisiformis, also known as Cysticercus pisiformis, is the causative agent of cysticercosis and the cause of severe health problems in rabbits that negatively impacts on husbandry production. To date, there is no fast detection method to identify early infections in rabbits. In the present study, a new dot-ELISA-based on an endogenous antigen fatty acid-binding protein (FABP) was developed for the detection of cysticercosis, and its potential was then evaluated using test serum samples. Immunolocalisation showed that T. pisiformis FABP (TpFABP) localised to the parenchyma of the bladder wall of the cysticercus and perinuclear cytoplasm of parenchyma of the adult parasite. After cloning and expression, recombinant TpFABP (rTpFABP) protein was used for serodiagnosis of T. pisiformis infection in rabbits by dot-ELISA. The antibody was detected 14 days post-infection in rabbits experimentally infected with T. pisiformis. Based on the necropsy results, the sensitivity and specificity of 169 serum samples tested by rTpFABP dot-ELISA were found to be 98.2% (54/55) and 92.1% (105/114), respectively. These data suggest that the dot-ELISA developed in this study has potential for detection of T. pisiformis infection in rabbits. © D. Yang et al., published by EDP Sciences, 2013.

  4. Antigenic expression and spontaneous immune responses support the use of a selected peptide set from the IMA950 glioblastoma vaccine for immunotherapy of grade II and III glioma

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    Dutoit, Valérie; Migliorini, Denis; Ranzanici, Giulia; Marinari, Eliana; Widmer, Valérie; Lobrinus, Johannes Alexander; Momjian, Shahan; Costello, Joseph; Walker, Paul R.; Okada, Hideho; Weinschenk, Toni; Herold-Mende, Christel; Dietrich, Pierre-Yves

    2018-01-01

    ABSTRACT Gliomas are lethal brain tumors that resist standard therapeutic approaches. Immunotherapy is a promising alternative strategy mostly developed in the context of glioblastoma. However, there is a need for implementing immunotherapy for grade II/III gliomas, as these are the most common CNS tumors in young adults with a high propensity for recurrence, making them lethal despite current treatments. We recently identified HLA-A2-restricted tumor-associated antigens by peptide elution from glioblastoma and formulated a multipeptide vaccine (IMA950) evaluated in phase I/II clinical trials with promising results. Here, we investigated expression of the IMA950 antigens in patients with grade II/III astrocytoma, oligodendroglioma or ependymoma, at the mRNA, protein and peptide levels. We report that the BCAN, CSPG4, IGF2BP3, PTPRZ1 and TNC proteins are significantly over-expressed at the mRNA (n = 159) and protein (n = 36) levels in grade II/III glioma patients as compared to non-tumor samples (IGF2BP3 being absent from oligodendroglioma). Most importantly, we detected spontaneous antigen-specific T cell responses to one or more of the IMA950 antigens in 100% and 71% of grade II and grade III patients, respectively (27 patients tested). These patients displayed T cell responses of better quality (higher frequency, broader epitope targeting) than patients with glioblastoma. Detection of spontaneous T cell responses to the IMA950 antigens shows that these antigens are relevant for tumor targeting, which will be best achieved by combination with CD4 epitopes such as the IDH1R132H peptide. Altogether, we provide the rationale for using a selective set of IMA950 peptides for vaccination of patients with grade II/III glioma. PMID:29308320

  5. Immunohistochemical expression of tumor antigens MAGE-A1, MAGE-A3/4, and NY-ESO-1 in cancerous and benign prostatic tissue.

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    Hudolin, Tvrtko; Juretic, Antonio; Spagnoli, Giulio Cesare; Pasini, Josip; Bandic, Daniela; Heberer, Michael; Kosicek, Miljenko; Cacic, Mirjana

    2006-01-01

    To investigate immunohistochemical expression of MAGE-A and NY-ESO-1/LAGE-1, cancer testis antigens in prostate tissues showing evidence of malignant transformation or benign hyperplasia. 112 prostate samples from patients undergoing surgery at the Urology Clinic at the Zagreb Clinical Hospital Center from 1995 to 2003 were investigated in this study. Of these, 92 carcinoma samples were obtained by radical prostatectomy, and 20 benign prostatic hyperplasia samples by transvesical prostatectomy. Three monoclonal antibodies were used for immunohistochemical staining: 77B for MAGE-A1, 57B for multi-MAGE-A and D8.38 for NY-ESO-1 expression. Expression of MAGE-A1 was observed in 10.8% of carcinoma samples, whereas multi-MAGE-A and NY-ESO-1/LAGE-1 stained 85.9% and 84.8% of samples. Immunohistochemical staining was only detectable in the cytoplasm. A significant heterogeneity could be observed within a same tissue sample where areas with strong positivities coexisted with cancer testis antigens negative areas. Interestingly, a majority of 57B positive cases were also found to be D8.38 positive (correlation coefficient r=0.727 (P<0.01)). Cancer testis antigens expression was neither significantly correlated with PSA values nor with Gleason score. In benign prostatic hyperplasia tissues MAGE-A1 expression was detected in 5%, while 57B and D8.38 staining was observed in 15% samples, and in all cases percentages of positive cells were always <10%. Our data underline the peculiar relevance of cancer testis antigens expression in prostate cancers, with potential implications regarding both diagnosis and therapy. Copyright (c) 2005 Wiley-Liss, Inc.

  6. Programmed death-1 expression on HIV-1-specific CD8+ T cells is shaped by epitope specificity, T-cell receptor clonotype usage and antigen load

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    Kløverpris, Henrik N.; McGregor, Reuben; McLaren, James E.; Ladell, Kristin; Stryhn, Anette; Koofhethile, Catherine; Brener, Jacqui; Chen, Fabian; Riddell, Lynn; Graziano, Luzzi; Klenerman, Paul; Leslie, Alasdair; Buus, Søren; Price, David A.; Goulder, Philip

    2014-01-01

    Objectives: Although CD8+ T cells play a critical role in the control of HIV-1 infection, their antiviral efficacy can be limited by antigenic variation and immune exhaustion. The latter phenomenon is characterized by the upregulation of multiple inhibitory receptors, such as programmed death-1 (PD-1), CD244 and lymphocyte activation gene-3 (LAG-3), which modulate the functional capabilities of CD8+ T cells. Design and methods: Here, we used an array of different human leukocyte antigen (HLA)-B∗15 : 03 and HLA-B∗42 : 01 tetramers to characterize inhibitory receptor expression as a function of differentiation on HIV-1-specific CD8+ T-cell populations (n = 128) spanning 11 different epitope targets. Results: Expression levels of PD-1, but not CD244 or LAG-3, varied substantially across epitope specificities both within and between individuals. Differential expression of PD-1 on T-cell receptor (TCR) clonotypes within individual HIV-1-specific CD8+ T-cell populations was also apparent, independent of clonal dominance hierarchies. Positive correlations were detected between PD-1 expression and plasma viral load, which were reinforced by stratification for epitope sequence stability and dictated by effector memory CD8+ T cells. Conclusion: Collectively, these data suggest that PD-1 expression on HIV-1-specific CD8+ T cells tracks antigen load at the level of epitope specificity and TCR clonotype usage. These findings are important because they provide evidence that PD-1 expression levels are influenced by peptide/HLA class I antigen exposure. PMID:24906112

  7. Self-Amplifying mRNA Vaccines Expressing Multiple Conserved Influenza Antigens Confer Protection against Homologous and Heterosubtypic Viral Challenge

    Science.gov (United States)

    Magini, Diletta; Giovani, Cinzia; Mangiavacchi, Simona; Maccari, Silvia; Cecchi, Raffaella; Ulmer, Jeffrey B.; De Gregorio, Ennio; Geall, Andrew J.; Brazzoli, Michela; Bertholet, Sylvie

    2016-01-01

    Current hemagglutinin (HA)-based seasonal influenza vaccines induce vaccine strain-specific neutralizing antibodies that usually fail to provide protection against mismatched circulating viruses. Inclusion in the vaccine of highly conserved internal proteins such as the nucleoprotein (NP) and the matrix protein 1 (M1) was shown previously to increase vaccine efficacy by eliciting cross-reactive T-cells. However, appropriate delivery systems are required for efficient priming of T-cell responses. In this study, we demonstrated that administration of novel self-amplifying mRNA (SAM®) vectors expressing influenza NP (SAM(NP)), M1 (SAM(M1)), and NP and M1 (SAM(M1-NP)) delivered with lipid nanoparticles (LNP) induced robust polyfunctional CD4 T helper 1 cells, while NP-containing SAM also induced cytotoxic CD8 T cells. Robust expansions of central memory (TCM) and effector memory (TEM) CD4 and CD8 T cells were also measured. An enhanced recruitment of NP-specific cytotoxic CD8 T cells was observed in the lungs of SAM(NP)-immunized mice after influenza infection that paralleled with reduced lung viral titers and pathology, and increased survival after homologous and heterosubtypic influenza challenge. Finally, we demonstrated for the first time that the co-administration of RNA (SAM(M1-NP)) and protein (monovalent inactivated influenza vaccine (MIIV)) was feasible, induced simultaneously NP-, M1- and HA-specific T cells and HA-specific neutralizing antibodies, and enhanced MIIV efficacy against a heterologous challenge. In conclusion, systemic administration of SAM vectors expressing conserved internal influenza antigens induced protective immune responses in mice, supporting the SAM® platform as another promising strategy for the development of broad-spectrum universal influenza vaccines. PMID:27525409

  8. Self-Amplifying mRNA Vaccines Expressing Multiple Conserved Influenza Antigens Confer Protection against Homologous and Heterosubtypic Viral Challenge.

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    Diletta Magini

    Full Text Available Current hemagglutinin (HA-based seasonal influenza vaccines induce vaccine strain-specific neutralizing antibodies that usually fail to provide protection against mismatched circulating viruses. Inclusion in the vaccine of highly conserved internal proteins such as the nucleoprotein (NP and the matrix protein 1 (M1 was shown previously to increase vaccine efficacy by eliciting cross-reactive T-cells. However, appropriate delivery systems are required for efficient priming of T-cell responses. In this study, we demonstrated that administration of novel self-amplifying mRNA (SAM® vectors expressing influenza NP (SAM(NP, M1 (SAM(M1, and NP and M1 (SAM(M1-NP delivered with lipid nanoparticles (LNP induced robust polyfunctional CD4 T helper 1 cells, while NP-containing SAM also induced cytotoxic CD8 T cells. Robust expansions of central memory (TCM and effector memory (TEM CD4 and CD8 T cells were also measured. An enhanced recruitment of NP-specific cytotoxic CD8 T cells was observed in the lungs of SAM(NP-immunized mice after influenza infection that paralleled with reduced lung viral titers and pathology, and increased survival after homologous and heterosubtypic influenza challenge. Finally, we demonstrated for the first time that the co-administration of RNA (SAM(M1-NP and protein (monovalent inactivated influenza vaccine (MIIV was feasible, induced simultaneously NP-, M1- and HA-specific T cells and HA-specific neutralizing antibodies, and enhanced MIIV efficacy against a heterologous challenge. In conclusion, systemic administration of SAM vectors expressing conserved internal influenza antigens induced protective immune responses in mice, supporting the SAM® platform as another promising strategy for the development of broad-spectrum universal influenza vaccines.

  9. Gene expression profiles in hypoxic preconditioning using cDNA microarray analysis: altered expression of an angiogenic factor, carcinoembryonic antigen-related cell adhesion molecule 1.

    Science.gov (United States)

    Chen, Wen-Jone; Chen, Huei-Wen; Yu, Sung-Liang; Huang, Chien-Hua; Wang, Tzung-Dau; Chen, Jeremy J W; Chien, Chiang-Ting; Chen, Hsuan-Yu; Yang, Pan-Chyr; Lee, Yuan-Teh

    2005-08-01

    Hypoxic preconditioning has been shown to exhibit cardioprotective effects on myocardium from ischemic or reperfusion injury. The specific regulated gene involved in the hypoxia-induced cardioprotective effects is profiled in this study. Young male Wistar rats and ICR mice were exposed to sea level (as normal control) or simulated high altitude for 15 h/day for 2, 4, or 8 weeks, or for 4 weeks at high altitude after 2 weeks at sea level. The left ventricles of the animals were isolated for mRNA isolation and cDNA microarray analysis. Our data demonstrated that hypoxic preconditioning significantly ameliorated cardiac ischemic injury by minimizing the infarct size. After cluster analysis of expression profiles after different courses of hypoxic preconditioning (0, 2, 4, and 8 weeks), 386 genes showed an ascending pattern, whereas 301 genes showed a descending pattern. The ascending genes include several angiogenic factors: FGF receptor 4, vascular endothelial growth factor (vEGF), and carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM-1). The microvessel density was also significantly increased in hypoxic hearts. Using Western blotting and immunohistochemical analysis, the protein expression level and localization of CEACAM-1 were observed in hypoxic myocardium. The results also indicated that CEACAM-1 was upregulated as with other hypoxic angiogenic factors, heme oxygenase 1 (HO-1) and hypoxia inducible factor-1alpha (HIF-1alpha), in in vitro cultured cardiomyocytes (H9c2) after hypoxia treatment and in vivo hypoxic preconditioning. Furthermore, incubation with recombinant vEGF could also increase the expression level of CEACAM-1 in H9c2 cells. These results demonstrated that hypoxic preconditioning resulted in transcriptional changes, and some of these genes have been correlated with angiogenesis. The HIF-1/vEGF/CEACAM-1 pathway might be important for hypoxia-induced angiogenesis in the heart during hypoxic preconditioning.

  10. Alphavirus replicon DNA expressing HIV antigens is an excellent prime for boosting with recombinant modified vaccinia Ankara (MVA or with HIV gp140 protein antigen.

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    Maria L Knudsen

    Full Text Available Vaccination with DNA is an attractive strategy for induction of pathogen-specific T cells and antibodies. Studies in humans have shown that DNA vaccines are safe, but their immunogenicity needs further improvement. As a step towards this goal, we have previously demonstrated that immunogenicity is increased with the use of an alphavirus DNA-launched replicon (DREP vector compared to conventional DNA vaccines. In this study, we investigated the effect of varying the dose and number of administrations of DREP when given as a prime prior to a heterologous boost with poxvirus vector (MVA and/or HIV gp140 protein formulated in glucopyranosyl lipid A (GLA-AF adjuvant. The DREP and MVA vaccine constructs encoded Env and a Gag-Pol-Nef fusion protein from HIV clade C. One to three administrations of 0.2 μg DREP induced lower HIV-specific T cell and IgG responses than the equivalent number of immunizations with 10 μg DREP. However, the two doses were equally efficient as a priming component in a heterologous prime-boost regimen. The magnitude of immune responses depended on the number of priming immunizations rather than the dose. A single low dose of DREP prior to a heterologous boost resulted in greatly increased immune responses compared to MVA or protein antigen alone, demonstrating that a mere 0.2 μg DREP was sufficient for priming immune responses. Following a DREP prime, T cell responses were expanded greatly by an MVA boost, and IgG responses were also expanded when boosted with protein antigen. When MVA and protein were administered simultaneously following multiple DREP primes, responses were slightly compromised compared to administering them sequentially. In conclusion, we have demonstrated efficient priming of HIV-specific T cell and IgG responses with a low dose of DREP, and shown that the priming effect depends on number of primes administered rather than dose.

  11. Induction of antigen-positive cell death by the expression of perforin, but not DTa, from a DNA vaccine enhances the immune response.

    Science.gov (United States)

    Gargett, Tessa; Grubor-Bauk, Branka; Garrod, Tamsin J; Yu, Wenbo; Miller, Darren; Major, Lee; Wesselingh, Steve; Suhrbier, Andreas; Gowans, Eric J

    2014-04-01

    The failure of traditional protein-based vaccines to prevent infection by viruses such as HIV or hepatitis C highlights the need for novel vaccine strategies. DNA vaccines have shown promise in small animal models, and are effective at generating anti-viral T cell-mediated immune responses; however, they have proved to be poorly immunogenic in clinical trials. We propose that the induction of necrosis will enhance the immune response to vaccine antigens encoded by DNA vaccines, as necrotic cells are known to release a range of intracellular factors that lead to dendritic cell (DC) activation and enhanced cross-presentation of antigen. Here we provide evidence that induction of cell death in DNA vaccine-targeted cells provides an adjuvant effect following intradermal vaccination of mice; however, this enhancement of the immune response is dependent on both the mechanism and timing of cell death after antigen expression. We report that a DNA vaccine encoding the cytolytic protein, perforin, resulted in DC activation, enhanced broad and multifunctional CD8 T-cell responses to the HIV-1 antigen GAG and reduced viral load following challenge with a chimeric virus, EcoHIV, compared with the canonical GAG DNA vaccine. This effect was not observed for a DNA vaccine encoding an apoptosis-inducing toxin, DTa, or when the level of perforin expression was increased to induce cell death sooner after vaccination. Thus, inducing lytic cell death following a threshold level of expression of a viral antigen can improve the immunogenicity of DNA vaccines, whereas apoptotic cell death has an inhibitory effect on the immune response.

  12. Over-expression of homologous antigens in a leucine auxotroph of Brucella abortus strain RB51 protects mice against a virulent B. suis challenge.

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    Rajasekaran, Parthiban; Surendran, Naveen; Seleem, Mohamed N; Sriranganathan, Nammalwar; Schurig, Gerhardt G; Boyle, Stephen M

    2011-04-12

    Infection by members of the Gram-negative bacterial genus Brucella causes brucellosis in a variety of mammals. Brucellosis in swine remains a challenge, as there is no vaccine in the USA approved for use in swine against brucellosis. Here, we developed an improved recombinant Brucella abortus vaccine strain RB51 that could afford protection against Brucella suis infection by over-expressing genes encoding homologous proteins: L7/L12 ribosomal protein, Cu/Zn superoxide dismutase [SOD] and glycosyl-transferase [WboA]. Using strain RB51leuB as a platform and an antibiotic-resistance marker free plasmid, strains RB51leuB/SOD, RB51leuB/SOD/L7/L12 and RB51leuB/SOD/WboA were constructed to over-express the antigens: SOD alone, SOD and ribosomal protein L7/L12 or SOD and glycosyl-transferase, respectively. The ability of these vaccine candidates to protect against a virulent B. suis challenge were evaluated in a mouse model. All vaccine groups protected mice significantly (PBrucella antigens can be over-expressed in strain RB51leuB and elicit protective immune responses against brucellosis. Since the plasmid over-expressing homologous antigens does not carry an antibiotic resistance gene, it complies with federal regulations and therefore could be used to develop safer multi-species vaccines for prevention of brucellosis caused by other species of Brucella. Copyright © 2011 Elsevier Ltd. All rights reserved.

  13. A bivalent typhoid live vector vaccine expressing both chromosome- and plasmid-encoded Yersinia pestis antigens fully protects against murine lethal pulmonary plague infection.

    Science.gov (United States)

    Galen, James E; Wang, Jin Yuan; Carrasco, Jose A; Lloyd, Scott A; Mellado-Sanchez, Gabriela; Diaz-McNair, Jovita; Franco, Olga; Buskirk, Amanda D; Nataro, James P; Pasetti, Marcela F

    2015-01-01

    Live attenuated bacteria hold great promise as multivalent mucosal vaccines against a variety of pathogens. A major challenge of this approach has been the successful delivery of sufficient amounts of vaccine antigens to adequately prime the immune system without overattenuating the live vaccine. Here we used a live attenuated Salmonella enterica serovar Typhi strain to create a bivalent mucosal plague vaccine that produces both the protective F1 capsular antigen of Yersinia pestis and the LcrV protein required for secretion of virulence effector proteins. To reduce the metabolic burden associated with the coexpression of F1 and LcrV within the live vector, we balanced expression of both antigens by combining plasmid-based expression of F1 with chromosomal expression of LcrV from three independent loci. The immunogenicity and protective efficacy of this novel vaccine were assessed in mice by using a heterologous prime-boost immunization strategy and compared to those of a conventional strain in which F1 and LcrV were expressed from a single low-copy-number plasmid. The serum antibody responses to lipopolysaccharide (LPS) induced by the optimized bivalent vaccine were indistinguishable from those elicited by the parent strain, suggesting an adequate immunogenic capacity maintained through preservation of bacterial fitness; in contrast, LPS titers were 10-fold lower in mice immunized with the conventional vaccine strain. Importantly, mice receiving the optimized bivalent vaccine were fully protected against lethal pulmonary challenge. These results demonstrate the feasibility of distributing foreign antigen expression across both chromosomal and plasmid locations within a single vaccine organism for induction of protective immunity. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  14. A Bivalent Typhoid Live Vector Vaccine Expressing both Chromosome- and Plasmid-Encoded Yersinia pestis Antigens Fully Protects against Murine Lethal Pulmonary Plague Infection

    Science.gov (United States)

    Wang, Jin Yuan; Carrasco, Jose A.; Lloyd, Scott A.; Mellado-Sanchez, Gabriela; Diaz-McNair, Jovita; Franco, Olga; Buskirk, Amanda D.; Nataro, James P.; Pasetti, Marcela F.

    2014-01-01

    Live attenuated bacteria hold great promise as multivalent mucosal vaccines against a variety of pathogens. A major challenge of this approach has been the successful delivery of sufficient amounts of vaccine antigens to adequately prime the immune system without overattenuating the live vaccine. Here we used a live attenuated Salmonella enterica serovar Typhi strain to create a bivalent mucosal plague vaccine that produces both the protective F1 capsular antigen of Yersinia pestis and the LcrV protein required for secretion of virulence effector proteins. To reduce the metabolic burden associated with the coexpression of F1 and LcrV within the live vector, we balanced expression of both antigens by combining plasmid-based expression of F1 with chromosomal expression of LcrV from three independent loci. The immunogenicity and protective efficacy of this novel vaccine were assessed in mice by using a heterologous prime-boost immunization strategy and compared to those of a conventional strain in which F1 and LcrV were expressed from a single low-copy-number plasmid. The serum antibody responses to lipopolysaccharide (LPS) induced by the optimized bivalent vaccine were indistinguishable from those elicited by the parent strain, suggesting an adequate immunogenic capacity maintained through preservation of bacterial fitness; in contrast, LPS titers were 10-fold lower in mice immunized with the conventional vaccine strain. Importantly, mice receiving the optimized bivalent vaccine were fully protected against lethal pulmonary challenge. These results demonstrate the feasibility of distributing foreign antigen expression across both chromosomal and plasmid locations within a single vaccine organism for induction of protective immunity. PMID:25332120

  15. Prognostic value of antigen expression in multiple myeloma: a PETHEMA/GEM study on 1265 patients enrolled in four consecutive clinical trials.

    Science.gov (United States)

    Arana, P; Paiva, B; Cedena, M-T; Puig, N; Cordon, L; Vidriales, M-B; Gutierrez, N C; Chiodi, F; Burgos, L; Anglada, L-L; Martinez-Lopez, J; Hernandez, M-T; Teruel, A-I; Gironella, M; Echeveste, M-A; Rosiñol, L; Martinez, R; Oriol, A; De la Rubia, J; Orfao, A; Blade, J; Lahuerta, J-J; Mateos, M-V; San Miguel, J-F

    2017-11-03

    Persistence of minimal residual disease (MRD) after treatment for myeloma predicts inferior outcomes, but within MRD-positive patients there is great heterogeneity with both early and very late relapses. Among different MRD techniques, flow cytometry provides additional information about antigen expression on tumor cells, which could potentially contribute to stratify MRD-positive patients. We investigated the prognostic value of those antigens required to monitor MRD in 1265 newly diagnosed patients enrolled in the GEM2000, GEM2005MENOS65, GEM2005MAS65 and GEM2010MAS65 protocols. Overall, CD19pos, CD27neg, CD38lo, CD45pos, CD81pos, CD117neg and CD138lo expression predicted inferior outcomes. Through principal component analysis, we found that simultaneous CD38lowCD81posCD117neg expression emerged as the most powerful combination with independent prognostic value for progression-free survival (HR:1.69; P=0.002). This unique phenotypic profile retained prognostic value among MRD-positive patients. We then used next-generation flow to determine antigen stability throughout the course of the disease, and found that the expression of antigens required to monitor MRD is mostly stable from diagnosis to MRD stages, except for CD81 whose expression progressively increased from baseline to chemoresistant tumor cells (14 vs 28%). Altogether, we showed that the phenotypic profile of tumor cells provides additional prognostic information, and could be used to further predict risk of relapse among MRD-positive patients.Leukemia advance online publication, 19 December 2017; doi:10.1038/leu.2017.320.

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

    Science.gov (United States)

    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 1) 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.

  17. 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.

  18. 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.

    2014-01-01

    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. PMID:24351757

  19. Expression and characterization of an anti-(hepatitis B surface antigen) glycosylated mouse antibody in transgenic tobacco (Nicotiana tabacum) plants and its use in the immunopurification of its target antigen.

    Science.gov (United States)

    Ramírez, Nadia; Rodríguez, Meilyn; Ayala, Marta; Cremata, José; Pérez, Marlene; Martínez, Anazuria; Linares, Marbelis; Hevia, Yasser; Páez, Rolando; Valdés, Rodolfo; Gavilondo, Jorge V; Selman-Housein, Guillermo

    2003-12-01

    Transgenic plants expressing recombinant immunoglobulins have arisen as an alternative technology for the large-scale production of antibodies useful in therapeutics and in industrial processes. In the present paper we report the expression in transgenic tobacco ( Nicotiana tabacum ) of an anti-HBsAg [anti-(hepatitis B virus surface antigen)] mouse IgG1 mAb (monoclonal antibody), currently used for the industrial purification of the recombinant vaccine antigen. Using the sweet potato sporamin signal peptide, a KDEL (Lys-Asp-Glu-Leu) ER (endoplasmic reticulum) anchorage domain, and a heavy- and light-chain gene tandem construction, we generated F1 plants in which the expression of the antibody accounted for 0.5% of the total soluble proteins. The 'plantibody' (functional IgG antibody produced in plants) was easily purified by Protein A-Sepharose chromatography with a yield of approximately 35 microg/g of fresh leaf material, and its glycosylation indicated that, irrespective of the KDEL signal, the molecule is modified in both the ER and Golgi. Finally, a successful comparison of the plantibody with the ascites-derived mAb in the immunoaffinity purification of the vaccine recombinant HBsAg was performed. Taken as a whole, our results show that the large-scale production of this antibody of industrial relevance in transgenic tobacco is feasible.

  20. Prevention of vertical transmission of Neospora caninum in C57BL/6 mice vaccinated with Brucella abortus strain RB51 expressing N. caninum protective antigens.

    Science.gov (United States)

    Ramamoorthy, Sheela; Sanakkayala, Neelima; Vemulapalli, Ramesh; Jain, Neeta; Lindsay, David S; Schurig, Gerhardt S; Boyle, Stephen M; Sriranganathan, Nammalwar

    2007-11-01

    Bovine abortions caused by the apicomplexan parasite Neospora caninum have been responsible for severe economic losses to the cattle industry. Infected cows either experience abortion or transmit the parasite transplacentally at a rate of up to 95%. Neospora caninum vaccines that can prevent vertical transmission and ensure disruption in the life cycle of the parasite greatly aid in the management of neosporosis in the cattle industry. Brucella abortus strain RB51, a commercially available vaccine for bovine brucellosis, can also be used as a vector to express plasmid-encoded proteins from other pathogens. Neospora caninum protective antigens MIC1, MIC3, GRA2, GRA6 and SRS2 were expressed in strain RB51. Female C57BL/6 mice were vaccinated with a recombinant strain RB51 expressing N. caninum antigen or irradiated tachyzoites, boosted 4 weeks later and then bred. Antigen-specific IgG, IFN-gamma and IL-10 were detected in vaccinated pregnant mice. Vaccinated mice were challenged with 5 x 10(6)N. caninum tachyzoites between days 11-13 of pregnancy. Brain tissue was collected from pups 3 weeks after birth and examined for the presence of N. caninum by real-time PCR. The RB51-MIC3, RB51-GRA6, irradiated tachyzoite vaccine, pooled strain RB51-Neospora vaccine, RB51-MIC1 and RB51-SRS2 vaccines elicited approximately 6-38% protection against vertical transmission. However, the differences in parasite burden in brain tissue of pups from the control and vaccinated groups were highly significant for all groups. Thus, B. abortus strain RB51 expressing the specific N. caninum antigens induced substantial protection against vertical transmission of N. caninum in mice.

  1. The CD3-gamma and CD3-delta subunits of the T cell antigen receptor can be expressed within distinct functional TCR/CD3 complexes.

    OpenAIRE

    Alarcón, B; Ley, S C; Sánchez-Madrid, F.; Blumberg, R. S.; Ju, S T; Fresno, M; Terhorst, C

    1991-01-01

    The T cell receptor for antigen (TCR) consists of two glycoproteins containing variable regions (TCR-alpha/beta or TCR-gamma/delta) which are expressed on the cell surface in association with at least four invariant proteins (CD3-gamma, -delta, -epsilon and -zeta). CD3-gamma and CD3-delta chains are highly homologous, especially in the cytoplasmic domain. The similarity observed in their genomic organization and their proximity in the chromosome indicate that both genes arose from duplication...

  2. Rapid de novo generation of antigen specific human B cells with expression of Blimp-1 and AID by in vitro immunization.

    Science.gov (United States)

    Fang, Xu; Tong, Yue; Tian, Hong; Ning, Hongyu; Gao, Xiangdong; Yao, Wenbing

    2017-03-01

    In vitro immunization with antigens and cytokines triggers specific human B-cell response in short periods and is therefore superior to conventional in vivo immunization for antibody development. However, this new technology is limited by low efficiency, poor reproducibility, and requirement of pre-immunized lymphocytes. In this study, we demonstrate a novel method for de novo inducing antigen-specific human B cells in vitro. Unlike previous in vitro immunization of unfractionated PBMCs, we firstly optimized the conditions for inducing monocyte-derived dendritic cells (DCs) to efficiently capture, process, and present antigens. Instead of using the conventional method to activate Th2 cells for in vitro immunization, we succeeded to differentiate naïve CD4+ T cells into T follicular helper (Tfh) cells using antigen-sensitized DCs and cytokine cocktail. We discovered the differentiated T cells expressed ICOS, PD-1, BCL-6, and IL-21 at high levels. After 12 days of T-B co-culture, we observed induced T cells efficiently promoted naïve B cells to differentiate into plasmablasts secreting antigen-specific antibodies, with expression of Blimp-1 and AID related to affinity maturation and class switching. Thus, we established a new co-culture system with naïve lymphocyte populations for de novo acquisition of specifically in vitro immunized B cells potentially for development of therapeutic antibodies, which also provides novel insights into understanding the complex interactions among immune cells in lymph nodes. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. LIGHT induces distinct signals to clear an AAV-expressed persistent antigen in the mouse liver and to induce liver inflammation.

    Directory of Open Access Journals (Sweden)

    Michael L Washburn

    2010-05-01

    Full Text Available Infection with adeno-associated virus (AAV vector with liver tropism leads to persistent expression of foreign antigens in the mouse liver, with no significant liver inflammation or pathology. This provides a model to investigate antigen persistence in the liver and strategies to modulate host immunity to reduce or clear the foreign antigen expressed from AAV vector in the liver.We showed that expressing LIGHT with an adenovirus vector (Ad in mice with established AAV in the liver led to clearance of the AAV. Ad-LIGHT enhanced CD8 effector T cells in the liver, correlated with liver inflammation. LTbetaR-Ig proteins blocked Ad-LIGHT in clearing AAV. Interestingly, in LTbetaR-null mice, Ad-LIGHT still cleared AAV but caused no significant liver inflammation.Our data suggest that LIGHT interaction with the LTbetaR plays a critical role in liver inflammation but is not required for LIGHT-mediated AAV clearance. These findings will shed light on developing novel immuno-therapeutics in treating people chronically infected with hepato-tropic viruses.

  4. Effective adoptive immunotherapy of triple-negative breast cancer by folate receptor-alpha redirected CAR T cells is influenced by surface antigen expression level

    Directory of Open Access Journals (Sweden)

    De-Gang Song

    2016-07-01

    Full Text Available Abstract Background The poor prognosis and the limited efficacy of targeted therapy in patients with triple-negative breast cancer (TNBC have raised the need for alternative therapies. Recent studies have demonstrated that folate receptor-alpha (FRα may represent an ideal tumor-associated marker for immunotherapy for TNBC. Methods The aim of the present study was to apply a chimeric antigen receptor (CAR approach for the targeting of FRα expressed on TNBC cells and evaluate the antitumor activity of CAR-engineered T cells in vitro and in vivo. Results We found that human T cells expressing a FRα-specific CAR were potent and specific killers of TNBC cells that express moderate levels of FRα in vitro and significantly inhibited tumor outgrowth following infusion into immunodeficient mice bearing an MDA-MB-231 tumor xenograft. However, the antitumor activity of the FRα CAR T cells was modest when compared to the same CAR T cells applied in an ovarian tumor xenograft model where FRα expression is more abundant. Notably, FRα CAR T cells induced superior tumor regression in vivo against MDA-MB-231 that was engineered for overexpression of FRα. Conclusions Taken together, our results show that FRα CAR T cells can mediate antitumor activity against established TNBC tumor, particularly when FRα is expressed at higher levels. These results have significant implications for the pre-selection of patients with high antigen expression levels when utilizing CAR-based adoptive T cell therapies of cancer in future clinical trials.

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

    Science.gov (United States)

    Paulus, Aneel; Chitta, Kasyapa S; Wallace, Paul K; Advani, Pooja P; Akhtar, Sharoon; Kuranz-Blake, Maja; Ailawadhi, Sikander; Chanan-Khan, Asher A

    2015-01-01

    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 therapeutic exploit.

  6. Expression of MAGE-A1, MAGE-A3/4 and NY-ESO-1 cancer-testis antigens in fetal testis.

    Science.gov (United States)

    Hudolin, Tvrtko; Kastelan, Zeljko; Derezić, Danijel; Basić-Jukić, Nikolina; Cesare Spagnoli, Giulio; Juretić, Antonio; Jezek, Davor

    2009-01-01

    Immunohistochemical expression of MAGE-A1, MAGE-A3/4 and NY-ESO-1/LAGE-1 cancer testis antigens (CTA) was assessed in 24 fetal testes from 15th to 36th week of gestation. Three monoclonal antibodies were used for immunohistochemical staining: 77B recognizing MAGE-A1, 57B recognizing multiple MAGE-A CTA, and D8.38 recognizing NY-ESO-1/LAGE-1. Expression of MAGE-A1 was not observed in fetal testis samples, whereas multi-MAGE-A and NY-ESO-1/LAGE-1 specific reagents stained pro-spermatogonia in all samples with different expression levels during the period of fetal development observed. Significant expression of MAGE-A3/4 and almost continuous expression of NY-ESO-1 in fetal testes after 22nd week of gestation suggested their important role in the development of sex cords and pro-spermatogonia in particular.

  7. MAGE-A10 cancer/testis antigen is highly expressed in high-grade non-muscle-invasive bladder carcinomas.

    Science.gov (United States)

    Mengus, Chantal; Schultz-Thater, Elke; Coulot, Julie; Kastelan, Zeljko; Goluza, Eleonora; Coric, Marijana; Spagnoli, Giulio C; Hudolin, Tvrtko

    2013-05-15

    Bladder cancer is a common urinary malignancy and a prevalent cause of cancer-related death. Current therapies of early stage non-muscle-invasive bladder cancer (NMIBC) are frequently associated with undesirable toxicities and recurrence. Active antigen-specific immunotherapy may provide a valid therapeutic option for patients with NMIBC. Cancer-testis antigens (CTA) expressed in various tumour types and in a limited range of healthy tissues may represent potential targets for specific immunotherapy. MAGE-A10 is probably the most immunogenic antigen of the MAGE-A family. We evaluated the expression of MAGE-A10 in NMIBC. Seventy-nine patients undergoing surgical treatment for NMIBC were enrolled in the study. MAGE-A10 gene expression was assessed by quantitative real-time polymerase chain reaction. Immunohistochemistry was performed on paraffin-embedded sections. MAGE-A10 gene was specifically expressed in one-third of NMIBC (n = 24: 32.43%). Gene expression was correlated with high tumour grade. MAGE-A10 protein was exclusively detectable in nuclei of tumour cells. More importantly, MAGE-A10 protein was also more frequently detectable in high-grade tumours (p = 0.0001) and in stage T1 tumours invading subepithelial tissue or lamina propria (p = 0.01). A strong correlation between MAGE-A10 staining score and tumour grade and stage could accordingly be observed. These data indicate that MAGE-A10 expression is a feature of aggressive NMIBC and might be used as a novel target for specific immunotherapy of these cancers. Copyright © 2012 UICC.

  8. Dendritic cells engineered to express defined allo-HLA peptide complexes induce antigen-specific cytotoxic T cells efficiently killing tumour cells

    DEFF Research Database (Denmark)

    Stronen, E; Abrahamsen, I W; Gaudernack, G

    2009-01-01

    presented by a non-self human leucocyte antigen (HLA) molecule and transferred to cancer patients expressing that HLA molecule. Obtaining allo-restricted CTL of high-avidity and low cross-reactivity has, however, proven difficult. Here, we show that dendritic cells transfected with mRNA encoding HLA-A*0201......, efficiently present externally loaded peptides from the antigen, Melan-A/MART-1 to T cells from HLA-A*0201-negative donors. CD8(+) T cells binding HLA-A*0201/MART-1 pentamers were detected already after 12 days of co-culture in 11/11 donors. The majority of cells from pentamer(+) cell lines were CTL......Most tumour-associated antigens (TAA) are non-mutated self-antigens. The peripheral T cell repertoire is devoid of high-avidity TAA-specific cytotoxic T lymphocytes (CTL) due to self-tolerance. As tolerance is major histocompatibility complex-restricted, T cells may be immunized against TAA...

  9. Vaccination with recombinant adenoviruses and dendritic cells expressing prostate-specific antigens is effective in eliciting CTL and suppresses tumor growth in the experimental prostate cancer.

    Science.gov (United States)

    Kim, Sol; Lee, Jee-Boong; Lee, Geon Kook; Chang, Jun

    2009-06-15

    Prostate cancer is currently the most commonly diagnosed cancer in men and the second leading cause of cancer-related death in men in the US. Immunological approaches may provide an alternative option for prevention and treatment of prostate cancer. To develop vaccine against prostate cancer using mouse model, we constructed three recombinant adenoviruses expressing prostate-specific membrane antigen (rAd/PSMA), prostate stem cell antigen (rAd/PSCA) and six-transmembrane epithelial antigen of the prostate (rAd/STEAP), that were specifically up-regulated in the transgenic murine prostate cancer. Male C57BL/6 mice were immunized by intravenous injection of these recombinant adenoviruses and subsequently by subcutaneous injection of dendritic cells pulsed with TRAMP-C1 tumor lysate. After subcutaneous challenge with TRAMP-C1 cells, tumor growth was significantly delayed in the immunized mice compared to the control group. Surprisingly, significant numbers of STEAP-specific CD8 T cells were detected in the peripheral blood and the spleen of immune mice using MHC I tetramers, and injection of rAd/STEAP alone followed by pulsed DC was sufficient to inhibit tumor growth. Therapeutic vaccination also significantly delayed the growth of pre-established tumors. Our results suggest that STEAP is a good immunologic target antigen against prostate cancer and our vaccination regimen successfully elicits anti-tumor CTL responses and suppresses tumor growth. More studies will expedite the development of this vaccine toward clinical application.

  10. Population and single-cell genomics reveal the Aire dependency, relief from Polycomb silencing, and distribution of self-antigen expression in thymic epithelia.

    Science.gov (United States)

    Sansom, Stephen N; Shikama-Dorn, Noriko; Zhanybekova, Saule; Nusspaumer, Gretel; Macaulay, Iain C; Deadman, Mary E; Heger, Andreas; Ponting, Chris P; Holländer, Georg A

    2014-12-01

    Promiscuous gene expression (PGE) by thymic epithelial cells (TEC) is essential for generating a diverse T cell antigen receptor repertoire tolerant to self-antigens, and thus for avoiding autoimmunity. Nevertheless, the extent and nature of this unusual expression program within TEC populations and single cells are unknown. Using deep transcriptome sequencing of carefully identified mouse TEC subpopulations, we discovered a program of PGE that is common between medullary (m) and cortical TEC, further elaborated in mTEC, and completed in mature mTEC expressing the autoimmune regulator gene (Aire). TEC populations are capable of expressing up to 19,293 protein-coding genes, the highest number of genes known to be expressed in any cell type. Remarkably, in mouse mTEC, Aire expression alone positively regulates 3980 tissue-restricted genes. Notably, the tissue specificities of these genes include known targets of autoimmunity in human AIRE deficiency. Led by the observation that genes induced by Aire expression are generally characterized by a repressive chromatin state in somatic tissues, we found these genes to be strongly associated with H3K27me3 marks in mTEC. Our findings are consistent with AIRE targeting and inducing the promiscuous expression of genes previously epigenetically silenced by Polycomb group proteins. Comparison of the transcriptomes of 174 single mTEC indicates that genes induced by Aire expression are transcribed stochastically at low cell frequency. Furthermore, when present, Aire expression-dependent transcript levels were 16-fold higher, on average, in individual TEC than in the mTEC population. © 2014 Sansom et al.; Published by Cold Spring Harbor Laboratory Press.

  11. File list: ALL.Brs.20.AllAg.MDA-MB-134-VI [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Brs.20.AllAg.MDA-MB-134-VI hg19 All antigens Breast MDA-MB-134-VI SRX355612,SRX...355611,SRX355609,SRX355610 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.Brs.20.AllAg.MDA-MB-134-VI.bed ...

  12. File list: ALL.Brs.05.AllAg.MDA-MB-134-VI [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Brs.05.AllAg.MDA-MB-134-VI hg19 All antigens Breast MDA-MB-134-VI SRX355612,SRX...355609,SRX355610,SRX355611 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.Brs.05.AllAg.MDA-MB-134-VI.bed ...

  13. File list: ALL.Brs.50.AllAg.MDA-MB-134-VI [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Brs.50.AllAg.MDA-MB-134-VI hg19 All antigens Breast MDA-MB-134-VI SRX355612,SRX...355611,SRX355609,SRX355610 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.Brs.50.AllAg.MDA-MB-134-VI.bed ...

  14. Anti-gene peptide nucleic acid specifically inhibits MYCN expression in human neuroblastoma cells leading to cell growth inhibition and apoptosis.

    Science.gov (United States)

    Tonelli, Roberto; Purgato, Stefania; Camerin, Consuelo; Fronza, Raffaele; Bologna, Fabrizio; Alboresi, Simone; Franzoni, Monica; Corradini, Roberto; Sforza, Stefano; Faccini, Andrea; Shohet, Jason M; Marchelli, Rosangela; Pession, Andrea

    2005-05-01

    We developed an anti-gene peptide nucleic acid (PNA) for selective inhibition of MYCN transcription in neuroblastoma cells, targeted against a unique sequence in the antisense DNA strand of exon 2 of MYCN and linked at its NH(2) terminus to a nuclear localization signal peptide. Fluorescence microscopy showed specific nuclear delivery of the PNA in six human neuroblastoma cell lines: GI-LI-N and IMR-32 (MYCN-amplified/overexpressed); SJ-N-KP and NB-100 (MYCN-unamplified/low-expressed); and GI-CA-N and GI-ME-N (MYCN-unamplified/unexpressed). Antiproliferative effects were observable at 24 hours (GI-LI-N, 60%; IMR-32, 70%) and peaked at 72 hours (GI-LI-N, 80%; IMR-32, 90%; SK-N-KP, 60%; NB-100, 50%); no reduction was recorded for GI-CA-N and GI-ME-N (controls). In MYCN-amplified/overexpressed IMR-32 cells and MYCN-unamplified/low-expressed SJ-N-KP cells, inhibition was recorded of MYCN mRNA (by real-time PCR) and N-Myc (Western blotting); these inhibitory effects increased over 3 days after single treatment in IMR-32. Anti-gene PNA induced G(1)-phase accumulation (39-53%) in IMR-32 and apoptosis (56% annexin V-positive cells at 24 hours in IMR-32 and 22% annexin V-positive cells at 48 hours in SJ-N-KP). Selective activity of the PNA was shown by altering three point mutations, and by the observation that an anti-gene PNA targeted against the noncoding DNA strand did not exert any effect. These findings could encourage research into development of an anti-gene PNA-based tumor-specific agent for neuroblastoma (and other neoplasms) with MYCN expression.

  15. Antigenic variation of Plasmodium knowlesi malaria: identification of the variant antigen on infected erythrocytes.

    OpenAIRE

    Howard, R J; Barnwell, J W; Kao, V

    1983-01-01

    Erythrocytes infected with mature asexual stages of Plasmodium knowlesi express a new surface antigen such that rhesus monkey antisera specifically agglutinate these cells. Cloned parasites can express different antigenic variants of this antigen. The variant antigen has been identified by comparison of the surface membrane antigens of a clone and of an antigenic variant of that clone of different agglutination phenotype. After lactoperoxidase labeling, 125I-labeled proteins of Mrs 210,000 an...

  16. Expression of E-cadherin, beta-catenin and Ki-67 antigen and their reciprocal correlations in fibrosarcomas of soft tissues in dogs.

    Science.gov (United States)

    Nowak, Marcin; Madej, Janusz A; Dziegiel, Piotr

    2007-01-01

    This study aimed at immunocytochemical demonstration of E-cadherin, beta-catenin and Ki-67 expressions and the examination of correlation between these markers in primary fibrosarcomas in dogs. Material for the study was sampled in the course of surgery from 24 mongrel dogs aged 5 to 16 years. The neoplastic tumors were subjected to histopathological verification and immunohistochemical reactions were performed to detect the studied markers. Microphotographs of the preparations were subjected to computer-assisted image analysis using the MultiScaneBase V 14.02 software. Expression of beta-catenin was detected in all tumours examined while E-cadherin was expressed in only 8.2%. Expression of the Ki-67 proliferation-associated antigen was noted in over 33% of the tumours. The lack of correlation (r=-0.1035) between expression of Ki-67 and that of beta-catenin detected here in contrast to the high values of Ki-67 antigen found as a prognostic factor in many other studies allowed us to conclude that the presence of beta-catenin in cells of soft tissue fibrosarcoma in dogs manifested no unequivocal relationship to augmented proliferative potential of neoplastic cells, although it did not exclude participation of the protein in the development of this neoplasia in dogs.

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

    Science.gov (United States)

    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.

  18. Cloning, expression and characterization of SeM protein of Streptococcus equi subsp. equi and evaluation of its use as antigen in an indirect ELISA

    Directory of Open Access Journals (Sweden)

    C.M. Moraes

    2014-08-01

    Full Text Available Strangles is an economically important horse disease caused by Streptococcus equi subsp. equi. The diagnosis can be confirmed either directly by bacterial isolation and PCR or by ELISA, which is an indirect method based on the detection of serum antibodies. The aim of this study was to clone, express and characterize the SeM protein of Streptococcus equi subsp. equi, evaluate its use as antigen in indirect ELISA and determine its performance to distinguish sera of negative, vaccinated and positive animals. This was initially performed by cloning the gene encoding the SeM protein and its expression in Escherichia coli. Subsequently, the protein produced was characterized and used as antigen in ELISA. Serum samples for evaluation were taken from 40 negative foals, 46 horses vaccinated with a commercial vaccine against strangles and 46 horses diagnosed with the disease. The test showed high specificity and sensitivity, allowing discrimination between negative and positive, positive and vaccinated animals, and vaccinated animals and negative sera. Thus, it was concluded that the protein produced rSeM, which can be used as antigen for disease diagnosis, and the described ELISA might be helpful to evaluate the immune status of the herd.

  19. Expression of a Gene Encoding 34.9 kDa PPE Antigen of Mycobacterium avium subsp. paratuberculosis in E. coli

    Directory of Open Access Journals (Sweden)

    Rajib Deb

    2010-01-01

    Full Text Available Mycobacterium avium subsp. paratuberculosis (Map contains PPE family antigens which are Proline and glutamic acid rich and may play important role as T cell antigens. Hence the identification and generation of antigens are necessary for immunological characterization. In the present study, the epitopic region of a unique PPE gene encoding 34.9 kDa protein from Map was amplified by polymerase chain reaction. The gene was cloned into Escherichia coli vector pQE30 UA. The recombinant plasmid designated as pQPPE was transformed into E. coli M15 and induced with IPTG revealed the high level expression of 37.1 kDa His-fusion protein (34.9 kDa PPE and 2.2 kDa His-tag, which was confirmed by immunoblotting. Recombinant PPE protein was then purified by Ni-NTA agarose chromatography. The polyclonal antiserum raised against purified recombinant PPE protein reacted with expressed 37.1 kDa His-fusion protein as well as with Map sonicate. The protein elicited significant delayed type hypersensitivity (DTH skin reaction in mice sensitized with Map. The results indicated that the recombinant PPE protein of Map was associated with cellular immune response.

  20. Expression of proliferating cell nuclear antigen in pulp cells of extracted immature teeth preserved in two different storage media.

    Science.gov (United States)

    Tekin, Uğur; Filippi, Andreas; Pohl, Yango; Kirschner, Horst

    2008-02-01

    A specially composed medium for storing avulsed teeth has been developed. In experimental and clinical studies it could be shown that PDL cells could be kept viable during storage in the medium for up to 53 h. In the present study the medium was tested on pulp cells. A total of 40 immature unerupted third molars with open apices were removed surgically and the teeth were stored in a special cell culture medium (SCCM) or in Hank's balanced salt solution (HBSS) at room temperature for 6, 12, 18 or 24 h. Five teeth were assigned to each group. A total of seven consecutive pulp cross-sections per tooth were examined, resulting in a total of 280 specimens. Viable cells were marked using proliferating cell nuclear antigen (PCNA). The pulp was divided in three regions: apical region (0-0.5 mm), middle region (>0.5-1.5 mm) and coronal region (>1.5 mm). The labelling index (LI) was calculated for the whole cut (regions 1, 2 and 3) and for each region separately. The statistical evaluation was made using the One-way anova and Mann-Whitney Test. Pulp cells of all teeth expressed PCNA. About 110 of 140 specimens in the SCCM and 101 of 140 specimens in the HBSS group showed PCNA-positive cells. The highest LI was observed within the apical region and decreased with increased distance from the medium. No marked cells were observed at a distance of more than 1.5 mm. The LI for both media showed a significant increase with storage intervals (P < 0.05). The pulp cells of teeth stored in SCCM showed a LI nearly twice as high compared to pulp cells of teeth stored in HBSS for the apical and middle region (time interval 6, 18 and 24 h: P < 0.05). The LI for the apical region was found to be 8.43% for the SCCM and 4.50% for the HBSS after 24 h. For the middle region the LI was found to be 2.02% for the SCCM and 0.81% for the HBSS after 24 h. Within the parameters of this study, it appears that the SCCM is able to maintain pulp cell viability better than HBSS. The use of special cell

  1. Entamoeba histolytica: cloning, expression and evaluation of the efficacy of a recombinant amebiasis cysteine proteinase gene (ACP1) antigen in minipig.

    Science.gov (United States)

    He, Guang-Zhi

    2012-02-01

    The amebiasis cysteine proteinase gene (ACP1) encoding an antigen from Entamoeba histolytica, as well as the recombinant ACP1, obtained by cloning and expression of the ACP1 gene in heterologous host Escherichia coli BL-21 (DE3), were used to evaluate their ability to induce immune protective responses in minipig against challenge infection in a minipig -E. histolytica model. There was a 64.52% reduction (P<0.001) in the group of recovery of challenged E. histolytica compared with that in the control group. Specific anti-ACP1 antibodies from immune protected minipig had significantly higher levels of immunoglobulin G (IgG) (P<0.001). Our data indicate recombinant ACP1 may be a potential target as a vaccine antigen. Copyright © 2011 Elsevier Inc. All rights reserved.

  2. [Expression in Escherichia coli of hepatitis B virus genes from minority nationality patients in Yunnan province with chronic hepatitis B and their antigenicity].

    Science.gov (United States)

    Bian, Zhong-qi; Hua, Zhan-lou; Yan, Wei-yao; Liu, Ming-qiu; Zheng, Zhao-xin

    2006-04-11

    To investigate the expression in Escherichia coli (E. coli) of hepatitis B virus (HBV) genes from minority nationality patients in Yunnan province with chronic hepatitis B (CHB) and their antigenicity. Peripheral blood samples were collected from 25 minority nationality patients with CHB in Yunnan province. Twenty-five CHB patients of Han nationality in Yunnan were used as controls. The full length HBV preS2/S and C genes were amplified by PCR, cloned, sequenced, and inserted into the prokaryotic expression vector p lambda PR. The recombinant plasmids p lambda PR-S2S and p lambda PR-C were transfected into E. coli of the line TOP10. The expression of the non-fusion proteins encoded by the HBV preS2S and C genes was determined by sodium dodecyl sulphate polyacrlamide gel electrophoresis (SDS-PAGE) and Western blotting. The antigenicity of the non-fusion proteins was examined by ELISA. Fifty samples of serum of patients with hepatitis A, 50 samples of serum of patients with hepatitis C, and 50 samples of serum of healthy blood donors were used as controls in the study of the antigenicity of non-fusion proteins. SDS-PAGE showed that the recombinant plasmids p lambda PR-S2S and p lambda PR-C were both stably and highly expressed in the E. coli for all 50 CHB patients. The molecular weights of the expressed non-fusion proteins, with a concentration of 16% and a purity of 50%, were between 31,000 and 21,000. Western blotting and ELISA showed that the purified recombinant non-fusion proteins reacted strongly with the antibodies HBpreS2S/SAb and HBcAb and the serum of CHB patients, but there was no cross-activity between the non-fusion proteins and all the serum samples of controls with HA and HC, and normal controls. The HBV preS2S and C genes from the minority nationality patients with CHB can be stably and highly expression in E. coli. The non-fusion proteins encoded by the HBV preS2S and C genes have high antigenicity. These findings have potential values in

  3. MUC1 and the simple mucin-type antigens: Tn and Sialyl-Tn are differently expressed in salivary gland acini and ducts from the submandibular gland, the vestibular folds, and the soft palate

    DEFF Research Database (Denmark)

    Kirkeby, Svend; Moe, Dennis; Jensen, Allan Bardow

    2010-01-01

    Autopsies of the submandibular gland, the vestibular folds and the soft palate from 65-87 old humans were examined to record the immunohistochemical expression of MUC1 and the simple mucin-type antigens Tn and Sialyl-Tn.......Autopsies of the submandibular gland, the vestibular folds and the soft palate from 65-87 old humans were examined to record the immunohistochemical expression of MUC1 and the simple mucin-type antigens Tn and Sialyl-Tn....

  4. Immunohistochemical expression of tumor antigens MAGE-A1, MAGE-A3/4, and NY-ESO-1 in squamous cell carcinoma of the penis.

    Science.gov (United States)

    Hudolin, Tvrtko; Juretic, Antonio; Pasini, Josip; Tomas, Davor; Spagnoli, Giulio Cesare; Heberer, Michael; Dimanovski, Jordan; Kruslin, Bozo

    2006-07-01

    To investigate by immunohistochemistry the expression of MAGE-A and NY-ESO-1/LAGE-1, cancer testis antigens (CTAs), in squamous cell carcinoma of the penis. A total of 30 penile carcinoma samples from patients undergoing penile amputation at the Urology Clinics at the Zagreb Clinical Hospital Center and University Hospital "Sestre milosrdnice" from 1997 to 2004 were investigated in this study. Three monoclonal antibodies were used for immunohistochemical staining: 77B specific for MAGE-A1, 57B recognizing multiple MAGE-A CTAs, and D8.38, specific for NY-ESO-1 antigen. The expression of MAGE-A1 was not observed in the carcinoma samples, but both multi-MAGE-A and NY-ESO-1-specific reagents stained 29 (97%) of 30 samples. Immunohistochemical staining was prevailingly detected in the cytoplasm. A significant heterogeneity was observed within the same specimen, in which areas with strong positivity coexisted with CTA-negative areas. The extent of CTA expression did not correlate significantly with tumor grade. The results of this study have documented for the first time the expression of CTAs in squamous cell carcinoma of the penis. Additional research is warranted to explore the potential implications regarding both diagnosis and therapy.

  5. Prevention of lethal experimental infection of C57BL/6 mice by vaccination with Brucella abortus strain RB51 expressing Neospora caninum antigens.

    Science.gov (United States)

    Ramamoorthy, Sheela; Sanakkayala, Neelima; Vemulapalli, Ramesh; Duncan, Robert B; Lindsay, David S; Schurig, Gerhart S; Boyle, Stephen M; Kasimanickam, Ramanathan; Sriranganathan, Nammalwar

    2007-11-01

    Bovine abortions caused by the intracellular protozoal parasite Neospora caninum are a major concern to cattle industries worldwide. A strong Th1 immune response is required for protection against N. caninum. Brucella abortus strain RB51 is currently used as a live, attenuated vaccine against bovine brucellosis. Strain RB51 can also be used as an expression vector for heterologous protein expression. In this study, putative protective antigens of N. caninum MIC1, MIC3, GRA2, GRA6 and SRS2, were expressed individually in B. abortus strain RB51. The ability of each of the recombinant RB51 strains to induce N. caninum-specific immunity was assessed in C57BL/6 mice. Mice were immunised by two i.p. inoculations, 4 weeks apart. Five weeks after the second immunisation, spleen cells from the vaccinated mice secreted high levels of IFN-gamma and IL-10 upon in vitro stimulation with N. caninum whole cell lysate antigens. N. caninum-specific antibodies of both IgG1 and IgG2a subtypes were detected in the serum of the vaccinated mice. Mice in the vaccinated and control groups were challenged with 2 x 10(7)N. caninum tachyzoites i.p. and observed for 28 days after vaccination. All unvaccinated control mice died within 7 days. Mice in the MIC1 and GRA6 vaccine groups were completely protected while the mice in the SRS2, GRA2 and MIC3 vaccinated groups were partially protected and experienced 10-50% mortality. The non-recombinant RB51 vector control group experienced an average protection of 69%. These results suggest that expression of protective antigens of N. caninum in B. abortus strain RB51 is a novel approach towards the development of a multivalent vaccine against brucellosis and neosporosis.

  6. Adoptive Immunotherapy for Hematological Malignancies Using T Cells Gene-Modified to Express Tumor Antigen-Specific Receptors

    Directory of Open Access Journals (Sweden)

    Hiroshi Fujiwara

    2014-12-01

    Full Text Available Accumulating clinical evidence suggests that adoptive T-cell immunotherapy could be a promising option for control of cancer; evident examples include the graft-vs-leukemia effect mediated by donor lymphocyte infusion (DLI and therapeutic infusion of ex vivo-expanded tumor-infiltrating lymphocytes (TIL for melanoma. Currently, along with advances in synthetic immunology, gene-modified T cells retargeted to defined tumor antigens have been introduced as “cellular drugs”. As the functional properties of the adoptive immune response mediated by T lymphocytes are decisively regulated by their T-cell receptors (TCRs, transfer of genes encoding target antigen-specific receptors should enable polyclonal T cells to be uniformly redirected toward cancer cells. Clinically, anticancer adoptive immunotherapy using genetically engineered T cells has an impressive track record. Notable examples include the dramatic benefit of chimeric antigen receptor (CAR gene-modified T cells redirected towards CD19 in patients with B-cell malignancy, and the encouraging results obtained with TCR gene-modified T cells redirected towards NY-ESO-1, a cancer-testis antigen, in patients with advanced melanoma and synovial cell sarcoma. This article overviews the current status of this treatment option, and discusses challenging issues that still restrain the full effectiveness of this strategy, especially in the context of hematological malignancy.

  7. High expression of carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 6 and 8 in primary myelofibrosis

    DEFF Research Database (Denmark)

    Riley, Caroline Hasselbalch; Skov, Vibe; Larsen, Thomas Stauffer

    2011-01-01

    for the egress of CD34+ cells from the bone marrow. Carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 6 has been implicated in cell adhesion, cellular invasiveness, angiogenesis, and inflammation, which are all key processes in the pathophysiology of PMF. Accordingly, CEACAMs may play an important...

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

    DEFF Research Database (Denmark)

    Mathiassen, Søren; Lauemøller, S L; Ruhwald, Morten

    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...

  9. Expression of intercellular and vascular cell adhesion molecules and class II major histocompatibility antigens in human lungs: lack of influence by conditions of organ preservation.

    Science.gov (United States)

    Hasegawa, S; Ritter, J H; Patterson, A; Ockner, D M; Sawa, H; Mohanakumar, T; Cooper, J D; Wick, M R

    1995-01-01

    The expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and class II major histocompatibility complex antigens was studied in control lung tissue and preserved human donor lungs. The three controls were represented by wedge biopsy specimens taken from non-neoplastic lung surrounding bronchogenic carcinomas. Nine lungs were harvested from six brain-dead donors, flushed with Euro-Collins solution or low potassium-dextran-glucose solution, and stored at 1 degree C or 10 degrees C. Samples of the latter organs were taken at the time of surgical harvest (baseline) and after 2, 12, 24, and 48 hours of preservation time. Immunostains with monoclonal antibodies against intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and class II major histocompatibility complex molecules were performed on all samples, and the relative presence of these determinants was evaluated. In both the controls and preserved lungs, intercellular adhesion molecule-1 expression was intense in the septal capillary endothelium and alveolar pneumocytes, but essentially absent in bronchial epithelium. Vascular cell adhesion molecule-1 was moderately to strongly labeled in the endothelia of large and small blood vessels of all types, and it was not seen in other cell types. Class II major histocompatibility complex antigens were variably observed in pulmonary epithelial cells, but they were not expressed by endothelia. There appeared to be no significant difference in the immunohistologic density of intercellular adhesion molecule-1 or vascular cell adhesion molecule-1 immunostaining in allografts at the specified time points of preservation; this conclusion was confirmed by Western blot analysis. Similar findings pertained to staining results for human leukocyte DR antigens. There was likewise no significant difference in the expression of the three analytes when donor lungs perfused with Euro-Collins solution versus low potassium

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

    DEFF Research Database (Denmark)

    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 ... of invasion of skin or deep fascia (= T1N0M0 and T2N0M0). The median follow-up time was 104 months (range 5-143 months). Immunohistochemical analysis of OA-519 expression was performed on formalin-fixed, paraffin-embedded tissue. The proliferative activity was estimated using a Ki-67 equivalent monoclonal...... analysis, both the MIB-1 index and OA-519 expression were of independent prognostic value (2p cancer who might benefit from adjuvant therapy....

  11. DNA rearrangements and antigenic variation in Trypanosoma equiperdum: expression-independent DNA rearrangements in the basic copy of a variant surface glycoprotein gene.

    Science.gov (United States)

    Longacre, S; Raibaud, A; Hibner, U; Buck, G; Eisen, H; Baltz, T; Giroud, C; Baltz, D

    1983-03-01

    Antigenic variation in Trypanosoma equiperdum is associated with the sequential expression of variant surface glycoprotein (VSG) genes in a process which involves gene duplication and transposition events. In this paper we present evidence that the genomic environment of the VSG-1 basic copy gene, the template for duplicated, expression-linked VSG-1 genes, differs in every trypanosome clone examined. This variation is thus independent of the expression of the VSG-1 gene, and it also appears to be restricted to the 3' genomic environment. It is also demonstrated that the DNA located 3' to the VSG-1 basic copy gene is moderately sensitive to digestion when the nuclei of either expressor or non-expressor trypanosomes are treated with DNase I.

  12. An evaluation of purified Salmonella Typhi protein antigens for the serological diagnosis of acute typhoid fever

    NARCIS (Netherlands)

    Tran Vu Thieu, Nga; Trinh van, Tan; Tran Tuan, Anh; Klemm, Elizabeth J.; Nguyen Ngoc Minh, Chau; Voong Vinh, Phat; Pham Thanh, Duy; Ho Ngoc Dan, Thanh; Pham Duc, Trung; Langat, Pinky; Martin, Laura B.; Galan, Jorge; Liang, Li; Felgner, Philip L.; Davies, D. Huw; de Jong, Hanna K.; Maude, Rapeephan R.; Fukushima, Masako; Wijedoru, Lalith; Ghose, Aniruddha; Samad, Rasheda; Dondorp, Arjen M.; Faiz, Abul; Darton, Thomas C.; Pollard, Andrew J.; Thwaites, Guy E.; Dougan, Gordon; Parry, Christopher M.; Baker, Stephen

    2017-01-01

    The diagnosis of typhoid fever is a challenge. Aiming to develop a typhoid diagnostic we measured antibody responses against Salmonella Typhi (S. Typhi) protein antigens and the Vi polysaccharide in a cohort of Bangladeshi febrile patients. IgM against 12 purified antigens and the Vi polysaccharide

  13. 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.

  14. The antigen presentation function of bone marrow-derived mast cells is spatiotemporally restricted to a subset expressing high levels of cell surface FcεRI and MHC II

    Directory of Open Access Journals (Sweden)

    Sun Liangwu

    2010-06-01

    Full Text Available Abstract Background At present, it is highly controversial whether pure mast cells can serve as antigen presenting cells, and it is not known whether the capacity of antigen presenting function is temporally restricted to a particular subset of differentiated mast cells. Evidence is presented for a novel surface FcεRIhi , MHC II +, and c-kit + pure mast cell subset, temporally restricted as antigen-presenting cells in the immune axis of T-cell activation. Results Bone marrow-derived mast cells (BMMC cultured in the presence of IL-3 for three weeks are pure mast cells based on surface expression of lineage-specific marker, c-kit and FcεRI. Herein we present the first demonstration that approximately 98.7% c-kit + and FcεRI expressing BMMC, further depleted of any contaminated professional antigen-presenting cells, are still fully capable of presenting antigens, i.e., OVA protein, OVA peptide, and IgE-TNP-OVA, to OVA peptide-specific T-cell hybridomas. Notably, IgE-dependent antigen presentation is more efficient compared to that resulting from direct antigen uptake. Importantly, we present the novel finding that only surface FcεRIhi mast cells, also expressing surface MHC II exhibited antigen-presenting function. In contrast, surface FcεRIlo mast cells without expressing surface MHC II were not capable of antigen presentation. Interestingly, the antigen-presenting function of BMMC was irrevocably lost during the third and fourth week in IL-3 or SCF containing cultures. Conclusions This is the first observation to attribute a spatiotemporally restricted antigen-presenting function to a subset of three-week old pure BMMC expressing both high levels of surface FcεRI and surface MHC II. We propose that mast cells play an important role in immune deviating and/or sustaining the activation of infiltrating CD4 T-cells, and modulating T-cell mediated allergic inflammation via its flexibility to present antigens and antigen-IgE complexes.

  15. ClearColi BL21(DE3)-based expression of Zika virus antigens illustrates a rapid method of antibody production against emerging pathogens.

    Science.gov (United States)

    Viranaicken, Wildriss; Nativel, Brice; Krejbich-Trotot, Pascale; Harrabi, Wissal; Bos, Sandra; El Kalamouni, Chaker; Roche, Marjolaine; Gadea, Gilles; Desprès, Philippe

    2017-11-01

    Available rapid, simple and accurate methods for detection and diagnosis of emerging viral diseases are required. Recently, there was an urgent need for specific antibodies against mosquito-borne Zika virus (ZIKV), which is an emerging zoonotic disease of medical concern in different regions of the world. Here, we showed that overexpression of ZIKV antigens in ClearColi BL21(DE3), a bacteria strain expressing a non-endotoxic form of LPS, is suitable for the production of specific ZIKV antisera. Two major ZIKV antigenic domains, the domain III from envelope E glycoprotein, which brings the virus-specific epitopes, and the N-terminal region of nonstructural NS1 glycoprotein, which is responsible for pathophysiological conditions, were overexpressed in ClearColi BL21(DE3). Immunization of adult rat with insoluble recombinant ZIKV antigens in inclusion bodies resulted in the production of specific antibodies in a few weeks. Anti-E and anti-NS1 antibodies are efficient as biological tools for ZIKV detection by indirect ELISA and immunoblot assay. This method could successfully be applied to any emerging viruses. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  16. Detection of specific antibodies directed against a consistently expressed surface antigen of Mycoplasma gallisepticum using a monoclonal blocking enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Czifra, G; Kleven, S H; Engström, B; Stipkovits, L

    1995-01-01

    Sera from 14 groups of chickens inoculated with different laboratory and field strains of Mycoplasma gallisepticum (MG) were used to compare the diagnostic potential of the hemagglutination-inhibition (HI) test and a recently developed monoclonal blocking enzyme-linked immunosorbent assay (ELISA). HI was performed with strain A5969, commonly used as hemagglutinating antigen, and it could detect 62.7% of the inoculated chickens as positive. Of all sera, 83% proved to be positive when examined with the blocking ELISA. The difference between the sensitivities of the two methods was due to group-specific insensitivity of the HI test. None of the sera from groups inoculated with strains K 1501, K 1503, K 503, or K 703 and only half of the sera from groups inoculated with K 1453 or 236C could inhibit the activity of the A5969 hemagglutinating antigen, indicating antigenic differences between these challenge strains and the diagnostic strain. ELISA detected MG-specific antibodies in every group of sera, although inoculation with variant strains K 503 or K 703 resulted in lower level of antibody production than inoculation with other strains. The monoclonal blocking ELISA can be useful in the serological diagnosis of MG infections, because it is based on a consistently expressed, specific region of MG.

  17. Natural killer T cells constitutively expressing the interleukin-2 receptor α chain early in life are primed to respond to lower antigenic stimulation.

    Science.gov (United States)

    Ladd, Mihoko; Sharma, Ashish; Huang, Qing; Wang, Adele Y; Xu, Lixin; Genowati, Indira; Levings, Megan K; Lavoie, Pascal M

    2010-10-01

    Invariant natural killer T (iNKT) cells are known to constitutively express the high affinity interleukin-2 receptor α chain (CD25) in neonates, but the functional consequence of this phenotype is unknown. Here, we show that high numbers of CD25-expressing iNKT cells are present early in gestation and represent a significant proportion of the developing immune system. Despite their activated phenotype, neonatal iNKT cells express high levels of the Krüppel-like factor-2, a transcription factor associated with quiescent T cells, and require de novo T-cell receptor and CD28 co-stimulation to proliferate. In contrast to bona fide CD4/CD25-expressing regulatory T cells, neonatal iNKT cells do not suppress T-cell responses, indicating that they do not represent an immunosuppressive cell subset. Evidence that neonatal iNKT cells respond to dramatically reduced amounts of CD1d-restricted antigen compared with adult iNKT cells or T cells, and that their proliferation can be induced in the absence of early interleukin-2 suggest that constitutive expression of CD25 'primes' neonatal iNKT cells to respond rapidly to low amounts of antigen. This unique phenotype, which is distinct from adult iNKT cells, as well as other CD25-expressing activated T or regulatory T cells, may be important to ensure stability of a structurally limited peripheral iNKT-cell repertoire early in life. © 2010 The Authors. Immunology © 2010 Blackwell Publishing Ltd.

  18. The hyperactive Sleeping Beauty transposase SB100X improves the genetic modification of T cells to express a chimeric antigen receptor1

    Science.gov (United States)

    Jin, Zeming; Maiti, Sourindra; Huls, Helen; Singh, Harjeet; Olivares, Simon; Mátés, Lajos; Izsvák, Zsuzsanna; Ivics, Zoltán; Lee, Dean A.; Champlin, Richard E.; Cooper, Laurence J.N.

    2014-01-01

    Sleeping Beauty (SB3) transposon and transposase constitute a DNA plasmid system used for therapeutic human cell genetic engineering. Here we report a comparison of SB100X, a newly developed hyperactive SB transposase, to a previous generation SB11 transposase to achieve stable expression of a CD19-specific chimeric antigen receptor (CAR3) in primary human T cells. The electro-transfer of SB100X expressed from a DNA plasmid or as an introduced mRNA species had superior transposase activity in T cells based on measurement of excision circles released after transposition and emergence of CAR expression on T cells selectively propagated upon CD19+ artificial antigen presenting cells. Given that T cells modified with SB100X and SB11 integrate on average one copy of the CAR transposon in each T-cell genome, the improved transposition mediated by SB100X apparently leads to an augmented founder effect of electroporated T cells with durable integration of CAR. In aggregate, SB100X improves SB transposition in primary human T cells and can be titrated with a SB transposon plasmid to improve the generation of CD19-specific CAR+ T cells. PMID:21451576

  19. The hyperactive Sleeping Beauty transposase SB100X improves the genetic modification of T cells to express a chimeric antigen receptor.

    Science.gov (United States)

    Jin, Z; Maiti, S; Huls, H; Singh, H; Olivares, S; Mátés, L; Izsvák, Z; Ivics, Z; Lee, D A; Champlin, R E; Cooper, L J N

    2011-09-01

    Sleeping Beauty (SB3) transposon and transposase constitute a DNA plasmid system used for therapeutic human cell genetic engineering. Here we report a comparison of SB100X, a newly developed hyperactive SB transposase, to a previous generation SB11 transposase to achieve stable expression of a CD19-specific chimeric antigen receptor (CAR3) in primary human T cells. The electro-transfer of SB100X expressed from a DNA plasmid or as an introduced mRNA species had superior transposase activity in T cells based on the measurement of excision circles released after transposition and emergence of CAR expression on T cells selectively propagated upon CD19+ artificial antigen-presenting cells. Given that T cells modified with SB100X and SB11 integrate on average one copy of the CAR transposon in each T-cell genome, the improved transposition mediated by SB100X apparently leads to an augmented founder effect of electroporated T cells with durable integration of CAR. In aggregate, SB100X improves SB transposition in primary human T cells and can be titrated with an SB transposon plasmid to improve the generation of CD19-specific CAR+ T cells.

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

    Energy Technology Data Exchange (ETDEWEB)

    Grujic, Ognjen [Biochemistry and Microbiology, University of Victoria, PO Box 3055 STN CSC, Victoria, BC, V8W 3P6 (Canada); Grigg, Michael E. [Molecular Parasitology Unit, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 4 Center Drive, Bethesda, MD 20892 (United States); Boulanger, Martin J., E-mail: mboulang@uvic.ca [Biochemistry and Microbiology, University of Victoria, PO Box 3055 STN CSC, Victoria, BC, V8W 3P6 (Canada)

    2008-05-01

    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{sub 1}2{sub 1}2 or P4{sub 3}2{sub 1}2 space group, with a solvent content of 60% and a corresponding Matthews coefficient of 2.98 Å{sup 3} Da{sup −1}.

  1. Expression and clinical significance of MAGE and NY-ESO-1 cancer-testis antigens in adenoid cystic carcinoma of the head and neck.

    Science.gov (United States)

    Veit, Johannes A; Heine, Daniela; Thierauf, Julia; Lennerz, Jochen; Shetty, Subasch; Schuler, Patrick J; Whiteside, Theresa; Beutner, Dirk; Meyer, Moritz; Grünewald, Inga; Ritter, Gerd; Gnjatic, Sacha; Sikora, Andrew G; Hoffmann, Thomas K; Laban, Simon

    2016-07-01

    Adenoid cystic carcinoma (ACC) of the head and neck is a rare but highly malignant tumor. Cancer-testis antigens (CTAs) represent an immunogenic family of cancer-specific proteins and thus represent an attractive target for immunotherapy. Eighty-four cases of ACC were identified, the CTAs pan-Melanoma antigen (pan-MAGE; M3H67) and New York esophageal squamous cell carcinoma (NY-ESO-1; E978) were detected immunohistochemically (IHC) and correlated with clinical data. Expression of NY-ESO-1 was found in 48 of 84 patients (57.1%) and of pan-MAGE in 28 of 84 patients (31.2%). Median overall survival (OS) in NY-ESO-1 positive versus negative patients was 130.8 and 282.0 months (p = .223), respectively. OS in pan-MAGE positive versus negative patients was 105.3 and 190.5 months, respectively (p = .096). Patients expressing both NY-ESO-1 and pan-MAGE simultaneously had significantly reduced OS with a median of 90.5 months compared with 282.0 months in negative patients (p = .047). A significant fraction of patients with ACC show expression of the CTAs NY-ESO-1 and/or pan-MAGE with promising immunotherapeutic implications. © 2016 Wiley Periodicals, Inc. Head Neck 38: 1008-1016, 2016. © 2016 Wiley Periodicals, Inc.

  2. Immunohistochemical analysis of the expression of MAGE-A and NY-ESO-1 cancer/testis antigens in diffuse large B-cell testicular lymphoma.

    Science.gov (United States)

    Hudolin, Tvrtko; Kastelan, Zeljko; Ilic, Ivana; Levarda-Hudolin, Katarina; Basic-Jukic, Nikolina; Rieken, Malte; Spagnoli, Giulio C; Juretic, Antonio; Mengus, Chantal

    2013-05-16

    Primary testicular lymphoma (PTL) is a rare and lethal disease. The most common histological subtype is diffuse large B-cell lymphoma (DLBCL). Standard treatments are frequently ineffective. Thus, the development of novel forms of therapy is urgently required. Specific immunotherapy generating immune responses directed against antigen predominantly expressed by cancer cells such as cancer-testis antigens (CTA) may provide a valid alternative treatment for patients bearing PTL, alone or in combination with current therapies. Three monoclonal antibodies (mAbs), 77B recognizing MAGE-A1, 57B recognizing an epitope shared by multiple MAGE-A CTA (multi-MAGE-A specific) and D8.38 recognizing NY-ESO-1/LAGE-1 were used for immunohistochemical staining of 27 PTL, including 24 DLBCL. Expression of MAGE-A1 was infrequently detectable in DLBCL specimens (12.50%), whereas multi-MAGE-A and NY-ESO-1/LAGE-1 specific reagents stained the cytoplasms of tumor cells in DLBCL specimens with higher frequencies (54.17% and 37.50%, respectively) with different expression levels. These results suggest that MAGE-A and NY-ESO-1/LAGE-1, possibly in combination with other CTA, might be used as targets for specific immunotherapy in DLBCL.

  3. Escherichia coli Nissle 1917 bacterial ghosts retain crucial surface properties and express chlamydial antigen: an imaging study of a delivery system for the ocular surface

    Directory of Open Access Journals (Sweden)

    Montanaro J

    2015-07-01

    Full Text Available Jacqueline Montanaro,1 Aleksandra Inic-Kanada,1 Angela Ladurner,1 Elisabeth Stein,1 Sandra Belij,1 Nora Bintner,1 Simone Schlacher,1 Nadine Schuerer,1 Ulrike Beate Mayr,2 Werner Lubitz,2 Nikolaus Leisch,3 Talin Barisani-Asenbauer11Laura Bassi Centres of Expertise, OCUVAC – Centre of Ocular Inflammation and Infection, Centre for Pathophysiology, Infectiology, and Immunology, Medical University of Vienna, Vienna, Austria; 2BIRD-C GmbH & Co KG, Kritzendorf, Austria; 3Department of Ecogenomics and Systems Biology, University of Vienna, Vienna, AustriaAbstract: To target chronic inflammatory ocular surface diseases, a drug delivery platform is needed that is safe, possesses immunomodulatory properties, and can be used either for drug delivery, or as a foreign antigen carrier. A new therapeutic approach that we have previously proposed uses nonliving bacterial ghosts (BGs as a carrier-delivery system which can be engineered to carry foreign antigens and/or be loaded with therapeutic drugs. The parent strain chosen for development of our BG delivery system is the probiotic Escherichia coli strain Nissle 1917 (EcN, whose intrinsic properties trigger the innate immune system with the flagella and fimbriae used to attach and stimulate epithelial cells. In previous studies, we have shown that EcN BGs are safe for the ocular surface route, but evidence that EcN BGs retain flagella and fimbriae after transformation, has never been visually confirmed. In this study, we used different visualization techniques to determine whether flagella and fimbriae are retained on EcN BGs engineered either for drug delivery or as a foreign antigen carrier. We have also shown by immunoelectron microscopy that EcN retains two foreign antigens after processing to become EcN BGs. Furthermore, we demonstrated that BGs derived from EcN and expressing a foreign antigen attachment to conjunctival epithelial cells in vitro without causing reduced cell viability. These results

  4. 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.

    Directory of Open Access Journals (Sweden)

    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.

  5. Interleukin 10 (IL-10) and viral IL-10 strongly reduce antigen-specific human T cell proliferation by diminishing the antigen-presenting capacity of monocytes via downregulation of class II major histocompatibility complex expression

    NARCIS (Netherlands)

    de Waal Malefyt, R.; Haanen, J.; Spits, H.; Roncarolo, M. G.; te Velde, Anje; Figdor, C.; Johnson, K.; Kastelein, R.; Yssel, H.; de Vries, J. E.

    1991-01-01

    Interleukin 10 (IL-10) and viral IL-10 (v-IL-10) strongly reduced antigen-specific proliferation of human T cells and CD4+ T cell clones when monocytes were used as antigen-presenting cells. In contrast, IL-10 and v-IL-10 did not affect the proliferative responses to antigens presented by autologous

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

    DEFF Research Database (Denmark)

    Khattab, Ayman; Reinhardt, Christina; Staalsoe, Trine

    2004-01-01

    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......: 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...

  7. Proliferating cell nuclear antigen in gonad and associated storage tissue of the Pacific oyster Crassostrea gigas: seasonal immunodetection and expression in laser microdissected tissues.

    Science.gov (United States)

    Franco, Alban; Jouaux, Aude; Mathieu, Michel; Sourdaine, Pascal; Lelong, Christophe; Kellner, Kristell; Heude Berthelin, Clothilde

    2010-04-01

    To understand the processes involved in tissue remodeling associated with the seasonal reproductive cycle of the oyster Crassostrea gigas, we used immunodetection and expression measurements of proliferating cell nuclear antigen (PCNA). The expression of the PCNA gene was measured by real-time polymerase chain reaction in the whole gonadal area compared with laser microdissected gonad and storage tissue. Results underlined the advantage of the laser microdissection approach to detect expression, mainly for early stages of spermatogenesis. In the storage tissue, PCNA expression was reduced in the gonadal tubules, but immunolabeled hemocytes and vesicular cells were detected when the storage tissue was being restored. In the gonadal tubules, the PCNA gene was more highly expressed in males than in females. As soon as spermatogenesis was initiated, PCNA expression showed a high and constant level. In females, the expression level increased gradually until the ripe stage. The immunological approach established the involvement of peritubular cells in gonadal tubule expansion during early gametogenesis. In both sexes, gonial mitosis was immunodetected throughout the reproductive cycle. In males, the occurrence of two types of spermatogonia was ascertained by differential immunolabeling, and intragonadal somatic cell proliferation was noted. As expected, immunolabeling was never observed from stage II spermatocytes to spermatozoa. In females, positively stained cells were detected from oogonia to growing oocytes with various labeled intracellular locations.

  8. Uterine Natural Killer Cell and Human Leukocyte Antigen-G1 and Human Leukocyte Antigen-G5 Expression in Vaginal Discharge of Threatened-Abortion Women: A Case-Control Study

    Directory of Open Access Journals (Sweden)

    Saeideh Sadat Shobeiri

    2015-01-01

    Full Text Available The immunotolerant human leukocyte antigen-G (HLA-G molecules have a major role in fetal-maternal tolerance during pregnancy. Interaction between these molecules and uterine natural killer (uNK cells inhibitory receptors prevents NK cell invasion against fetus trophoblast cells. The aim of this study was to evaluate the percentages of uNK cells and HLA-G1 and HLA-G5 isoforms expression in vaginal discharge of threatened-abortion women in comparison with control. In a case-control study, we investigated 30 threatened-abortion women with bleeding or spotting less than 20 weeks of pregnancy as compared to 30 normal pregnant women. uNK cells percentage was assessed by flow cytometry. Furthermore, we evaluated HLA-G1 and HLA-G5 isoforms expression by Real-Time PCR in these groups. The results of this study showed that threatened-abortion women had increased uNK cells and decreased T cells percentage in vaginal discharge in comparison with normal pregnant women (p=0.01, p=0.003, resp.. In addition, HLA-G1 isoform had lower expression in threatened-abortion women in comparison with control group p=0.0001. The increase of uNK cells level with the decrease of HLA-G expression in vaginal discharge of threatened-abortion pregnant women is an indicator of mother’s immune dysregulation. It is concluded that HLA-G expression level with uNK cells percentage can be determined as a diagnostic marker for threatened-abortion women.

  9. The O-Antigen Capsule of Salmonella enterica Serovar Typhimurium Facilitates Serum Resistance and Surface Expression of FliC

    OpenAIRE

    Marshall, Joanna M.; Gunn, John S.

    2015-01-01

    Group IV polysaccharide capsules are common in enteric bacteria and have more recently been described in nontyphoidal Salmonella species. Such capsules are known as O-antigen (O-Ag) capsules, due to their high degree of similarity to the O-Ag of the lipopolysaccharide (LPSO-Ag). Capsular polysaccharides are known virulence factors of many bacterial pathogens, facilitating evasion of immune recognition and systemic dissemination within the host. Previous studies on the O-Ag capsule of salmonel...

  10. Expression and immunolocalisation of TpFABP as a candidate antigen for the serodiagnosis of rabbit Taenia pisiformis cysticercosis

    OpenAIRE

    Yang, Deying; Chen, Lin; Xie, Yue; Wu, Xuhang; Nong, Xiang; Peng, Xi; Lai, Weimin; Gu, Xiaobin; Wang, Shuxian; Peng, Xuerong; Yang, Guangyou

    2013-01-01

    The larval stage of Taenia pisiformis, also known as Cysticercus pisiformis, is the causative agent of cysticercosis and the cause of severe health problems in rabbits that negatively impacts on husbandry production. To date, there is no fast detection method to identify early infections in rabbits. In the present study, a new dot-ELISA-based on an endogenous antigen fatty acid-binding protein (FABP) was developed for the detection of cysticercosis, and its potential was then evaluated using ...

  11. Evaluation of Selected Borrelia burgdorferi lp54 Plasmid-Encoded Gene Products Expressed during Mammalian Infection as Antigens To Improve Serodiagnostic Testing for Early Lyme Disease.

    Science.gov (United States)

    Weiner, Zachary P; Crew, Rebecca M; Brandt, Kevin S; Ullmann, Amy J; Schriefer, Martin E; Molins, Claudia R; Gilmore, Robert D

    2015-11-01

    Laboratory testing for the diagnosis of Lyme disease is performed primarily by serologic assays and is accurate for detection beyond the acute stage of the infection. Serodiagnostic assays to detect the early stages of infection, however, are limited in their sensitivity, and improvement is warranted. We analyzed a series of Borrelia burgdorferi proteins known to be induced within feeding ticks and/or during mammalian infection for their utility as serodiagnostic markers against a comprehensive panel of Lyme disease patient serum samples. The antigens were assayed for IgM and IgG reactivity in line immunoblots and separately by enzyme-linked immunosorbent assay (ELISA), with a focus on reactivity against early Lyme disease with erythema migrans (EM), early disseminated Lyme neuroborreliosis, and early Lyme carditis patient serum samples. By IgM immunoblotting, we found that recombinant proteins BBA65, BBA70, and BBA73 reacted with early Lyme EM samples at levels comparable to those of the OspC antigen used in the current IgM blotting criteria. Additionally, these proteins reacted with serum samples from patients with early neuroborreliosis and early carditis, suggesting value in detecting early stages of this disease progression. We also found serological reactivity against recombinant proteins BBA69 and BBA73 with early-Lyme-disease samples using IgG immunoblotting and ELISA. Significantly, some samples that had been scored negative by the Centers for Disease Control and Prevention-recommended 2-tiered testing algorithm demonstrated positive reactivity to one or more of the antigens by IgM/IgG immunoblot and ELISA. These results suggest that incorporating additional in vivo-expressed antigens into the current IgM/IgG immunoblotting tier in a recombinant protein platform assay may improve the performance of early-Lyme-disease serologic testing. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  12. Expression of VP7, a Bluetongue Virus Group Specific Antigen by Viral Vectors: Analysis of the Induced Immune Responses and Evaluation of Protective Potential in Sheep

    Science.gov (United States)

    Bouet-Cararo, Coraline; Contreras, Vanessa; Caruso, Agathe; Top, Sokunthea; Szelechowski, Marion; Bergeron, Corinne; Viarouge, Cyril; Desprat, Alexandra; Relmy, Anthony; Guibert, Jean-Michel; Dubois, Eric; Thiery, Richard; Bréard, Emmanuel; Bertagnoli, Stephane; Richardson, Jennifer; Foucras, Gilles; Meyer, Gilles; Schwartz-Cornil, Isabelle; Zientara, Stephan; Klonjkowski, Bernard

    2014-01-01

    Bluetongue virus (BTV) is an economically important Orbivirus transmitted by biting midges to domestic and wild ruminants. The need for new vaccines has been highlighted by the occurrence of repeated outbreaks caused by different BTV serotypes since 1998. The major group-reactive antigen of BTV, VP7, is conserved in the 26 serotypes described so far, and its role in the induction of protective immunity has been proposed. Viral-based vectors as antigen delivery systems display considerable promise as veterinary vaccine candidates. In this paper we have evaluated the capacity of the BTV-2 serotype VP7 core protein expressed by either a non-replicative canine adenovirus type 2 (Cav-VP7 R0) or a leporipoxvirus (SG33-VP7), to induce immune responses in sheep. Humoral responses were elicited against VP7 in almost all animals that received the recombinant vectors. Both Cav-VP7 R0 and SG33-VP7 stimulated an antigen-specific CD4+ response and Cav-VP7 R0 stimulated substantial proliferation of antigen-specific CD8+ lymphocytes. Encouraged by the results obtained with the Cav-VP7 R0 vaccine vector, immunized animals were challenged with either the homologous BTV-2 or the heterologous BTV-8 serotype and viral burden in plasma was followed by real-time RT-PCR. The immune responses triggered by Cav-VP7 R0 were insufficient to afford protective immunity against BTV infection, despite partial protection obtained against homologous challenge. This work underscores the need to further characterize the role of BTV proteins in cross-protective immunity. PMID:25364822

  13. Evaluation of Selected Borrelia burgdorferi lp54 Plasmid-Encoded Gene Products Expressed during Mammalian Infection as Antigens To Improve Serodiagnostic Testing for Early Lyme Disease

    Science.gov (United States)

    Weiner, Zachary P.; Crew, Rebecca M.; Brandt, Kevin S.; Ullmann, Amy J.; Schriefer, Martin E.; Molins, Claudia R.

    2015-01-01

    Laboratory testing for the diagnosis of Lyme disease is performed primarily by serologic assays and is accurate for detection beyond the acute stage of the infection. Serodiagnostic assays to detect the early stages of infection, however, are limited in their sensitivity, and improvement is warranted. We analyzed a series of Borrelia burgdorferi proteins known to be induced within feeding ticks and/or during mammalian infection for their utility as serodiagnostic markers against a comprehensive panel of Lyme disease patient serum samples. The antigens were assayed for IgM and IgG reactivity in line immunoblots and separately by enzyme-linked immunosorbent assay (ELISA), with a focus on reactivity against early Lyme disease with erythema migrans (EM), early disseminated Lyme neuroborreliosis, and early Lyme carditis patient serum samples. By IgM immunoblotting, we found that recombinant proteins BBA65, BBA70, and BBA73 reacted with early Lyme EM samples at levels comparable to those of the OspC antigen used in the current IgM blotting criteria. Additionally, these proteins reacted with serum samples from patients with early neuroborreliosis and early carditis, suggesting value in detecting early stages of this disease progression. We also found serological reactivity against recombinant proteins BBA69 and BBA73 with early-Lyme-disease samples using IgG immunoblotting and ELISA. Significantly, some samples that had been scored negative by the Centers for Disease Control and Prevention-recommended 2-tiered testing algorithm demonstrated positive reactivity to one or more of the antigens by IgM/IgG immunoblot and ELISA. These results suggest that incorporating additional in vivo-expressed antigens into the current IgM/IgG immunoblotting tier in a recombinant protein platform assay may improve the performance of early-Lyme-disease serologic testing. PMID:26376927

  14. Expression of VP7, a Bluetongue virus group specific antigen by viral vectors: analysis of the induced immune responses and evaluation of protective potential in sheep.

    Directory of Open Access Journals (Sweden)

    Coraline Bouet-Cararo

    Full Text Available Bluetongue virus (BTV is an economically important Orbivirus transmitted by biting midges to domestic and wild ruminants. The need for new vaccines has been highlighted by the occurrence of repeated outbreaks caused by different BTV serotypes since 1998. The major group-reactive antigen of BTV, VP7, is conserved in the 26 serotypes described so far, and its role in the induction of protective immunity has been proposed. Viral-based vectors as antigen delivery systems display considerable promise as veterinary vaccine candidates. In this paper we have evaluated the capacity of the BTV-2 serotype VP7 core protein expressed by either a non-replicative canine adenovirus type 2 (Cav-VP7 R0 or a leporipoxvirus (SG33-VP7, to induce immune responses in sheep. Humoral responses were elicited against VP7 in almost all animals that received the recombinant vectors. Both Cav-VP7 R0 and SG33-VP7 stimulated an antigen-specific CD4+ response and Cav-VP7 R0 stimulated substantial proliferation of antigen-specific CD8+ lymphocytes. Encouraged by the results obtained with the Cav-VP7 R0 vaccine vector, immunized animals were challenged with either the homologous BTV-2 or the heterologous BTV-8 serotype and viral burden in plasma was followed by real-time RT-PCR. The immune responses triggered by Cav-VP7 R0 were insufficient to afford protective immunity against BTV infection, despite partial protection obtained against homologous challenge. This work underscores the need to further characterize the role of BTV proteins in cross-protective immunity.

  15. Engineering the chloroplast of Chlamydomonas reinhardtii to express the recombinant PfCelTOS-Il2 antigen-adjuvant fusion protein.

    Science.gov (United States)

    Shamriz, Shabnam; Ofoghi, Hamideh

    2018-01-20

    Malaria is an infectious disease having a large negative impact on economic growth. Vaccines are considered as a novel strategy to reduce the burden of malaria. Malaria parasite has a complex life cycle and attempts are being made to develop vaccines that target each stage of the life cycle. Oral vaccines seem to be more feasible to implement in poor countries, since they are relatively inexpensive, needle-free administrated, mostly stable at non-refrigerated conditions and painless. By using recombinant technology, suitable oral hosts could serve as antigen delivering vehicles in developing oral vaccines. Chlamydomonas reinhardtii offers beneficial attributes as oral recombinant protein expression platform. Moreover, C. reinhardtii chloroplast is an attractive platform for expressing malaria antigens because it is capable of folding complex proteins, including those requiring disulfide bond formation, while lacking the ability to glycosylate proteins; a valuable quality of any malaria protein expression system, since the Plasmodium parasite lacks N-linked glycosylation machinery. As a first step towards developing an oral vaccine candidate against malaria, here, we expressed a fusion protein consisting of PfCelTOS, a candidate for pre-erythrocytic and transmission-blocking vaccines, fused to human interleukin-2 (IL-2) as vaccine adjuvant in the chloroplast of C. reinhardtii. The effect of light and media on recombinant protein production and cell growth was then studied. Results demonstrated that expressed recombinant proteins accumulate as a soluble, properly folded and functional protein within algal chloroplasts. Moreover, results showed that the highest cell density can be achieved using mixotrophy mode. However, protein accumulation appears to be favored by cultivating in TAP medium in low light. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Epigenetic modulation of MAGE-A3 antigen expression in multiple myeloma following treatment with the demethylation agent 5-azacitidine and the histone deacetlyase inhibitor MGCD0103.

    Science.gov (United States)

    Moreno-Bost, Amberly; Szmania, Susann; Stone, Katie; Garg, Tarun; Hoerring, Antje; Szymonifka, Jackie; Shaughnessy, John; Barlogie, Bart; Prentice, H Grant; van Rhee, Frits

    2011-05-01

    Immunotherapy targeting MAGE-A3 in multiple myeloma (MM) could eradicate highly aggressive and proliferative clonal cell populations responsible for relapse. However, expression of many cancer-testis antigens, including MAGE-A3, can be heterogeneous, leading to the potential for tumor escape despite MAGE-A3-induced immunity. We hypothesized that a combination of the hypomethylating agent 5-azacitidine (5AC) and the histone deacetylase inhibitor (HDACi) MGCD0103 (MGC) could induce MAGE-A3 expression in MAGE-A3-negative MM, resulting in recognition and killing of MM cells by MAGE-A3-specific cytotoxic T lymphocytes (CTL). Gene expression analyses of MAGE-A3 expression in primary MM patient samples at diagnosis and relapse were completed to identify populations that would benefit from MAGE-A3 immunotherapy. MM cell lines were treated with 5AC and MGC. Real-time polymerase chain reaction (PCR) and Western blotting were performed to assess MAGE-A3 RNA and protein levels, respectively. Chromium-release assays and interferon (IFN) secretion assays were employed to ascertain MAGE-A3 CTL specificity against treated targets. Gene expression analysis revealed that MAGE-A3 is expressed in MM patients at diagnosis (25%) and at relapse (49%). We observed de novo expression of MAGE-A3 RNA and protein in MAGE-A3-negative cell lines treated with 5AC. MGC treatment alone did not induce expression but sequential 5AC/MGC treatment led to enhanced expression and augmented recognition by MAGE-A3-specific CTL, as assessed by (51)Cr-release assays (P = 0.047) and enzyme-linked immunosorbent assay (ELISA) for IFN-γ secretion (P = 0.004). MAGE-A3 is an attractive target for immunotherapy of MM and epigenetic modulation by 5AC, and MGC can induce MAGE-A3 expression and facilitate killing by MAGE-A3-specific CTL.

  17. Efficient expression of nuclear transgenes in the green alga Chlamydomonas: synthesis of an HIV antigen and development of a new selectable marker.

    Science.gov (United States)

    Barahimipour, Rouhollah; Neupert, Juliane; Bock, Ralph

    2016-03-01

    The unicellular green alga Chlamydomonas reinhardtii has become an invaluable model system in plant biology. There is also considerable interest in developing this microalga into an efficient production platform for biofuels, pharmaceuticals, green chemicals and industrial enzymes. However, the production of foreign proteins in the nucleocytosolic compartment of Chlamydomonas is greatly hampered by the inefficiency of transgene expression from the nuclear genome. We have recently addressed this limitation by isolating mutant algal strains that permit high-level transgene expression and by determining the contributions of GC content and codon usage to gene expression efficiency. Here we have applied these new tools and explored the potential of Chlamydomonas to produce a recombinant biopharmaceutical, the HIV antigen P24. We show that a codon-optimized P24 gene variant introduced into our algal expression strains give rise to recombinant protein accumulation levels of up to 0.25% of the total cellular protein. Moreover, in combination with an expression strain, a resynthesized nptII gene becomes a highly efficient selectable marker gene that facilitates the selection of transgenic algal clones at high frequency. By establishing simple principles of successful transgene expression, our data open up new possibilities for biotechnological research in Chlamydomonas.

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

    Directory of Open Access Journals (Sweden)

    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.

  19. Vi, de civiliserede

    DEFF Research Database (Denmark)

    Nyemann, Dorthe

    2016-01-01

    Vi har i årtier troet på, at mennesker under de rette omstændigheder kan lykkes med at leve i fred og fordragelighed med hinanden. Skal vi til at erkende, at også vores samfundsstrukturer kun er en tynd fernis ovenpå et utæmmeligt voldspotentiale og egoisme?......Vi har i årtier troet på, at mennesker under de rette omstændigheder kan lykkes med at leve i fred og fordragelighed med hinanden. Skal vi til at erkende, at også vores samfundsstrukturer kun er en tynd fernis ovenpå et utæmmeligt voldspotentiale og egoisme?...

  20. Enhanced therapeutic effect using sequential administration of antigenically distinct oncolytic viruses expressing oncostatin M in a Syrian hamster orthotopic pancreatic cancer model.

    Science.gov (United States)

    Nistal-Villan, Estanislao; Bunuales, Maria; Poutou, Joanna; Gonzalez-Aparicio, Manuela; Bravo-Perez, Carlos; Quetglas, Jose I; Carte, Beatriz; Gonzalez-Aseguinolaza, Gloria; Prieto, Jesus; Larrea, Esther; Hernandez-Alcoceba, Ruben

    2015-12-16

    The limited efficacy of current treatments against pancreatic cancer has prompted the search of new alternatives such as virotherapy. Activation of the immune response against cancer cells is emerging as one of the main mechanisms of action of oncolytic viruses (OV). Direct oncolysis releases tumor antigens, and viral replication within the tumor microenvironment is a potent danger signal. Arming OV with immunostimulatory transgenes further enhances their therapeutic effect. However, standard virotherapy protocols do not take full advantage of OV as cancer vaccines because repeated viral administrations may polarize immune responses against strong viral antigens, and the rapid onset of neutralizing antibodies limits the efficacy of redosing. An alternative paradigm based on sequential combination of antigenically distinct OV has been recently proposed. We have developed a protocol consisting of sequential intratumor administrations of new Adenovirus (Ad) and Newcastle Disease Virus (NDV)-based OV encoding the immunostimulatory cytokine oncostatin M (OSM). Transgene expression, toxicity and antitumor effect were evaluated using an aggressive orthotopic pancreatic cancer model in Syrian hamsters, which are sensitive to OSM and permissive for replication of both OVs. NDV-OSM was more cytolytic, whereas Ad-OSM caused higher OSM expression in vivo. Both viruses achieved only a marginal antitumor effect in monotherapy. In addition, strong secretion of OSM in serum limited the maximal tolerated dose of Ad-OSM. In contrast, moderate doses of Ad-OSM followed one week later by NDV-OSM were safe, showed a significant antitumor effect and stimulated immune responses against cancer cells. Similar efficacy was observed when the order of virus administrations was reversed. Sequential administration of oncolytic Ad and NDV encoding OSM is a promising approach against pancreatic cancer.

  1. Conformational states of vitronectin: preferential expression of an antigenic epitope when vitronectin is covalently and noncovalently complexed with thrombin-antithrombin III or treated with urea.

    Science.gov (United States)

    Tomasini, B R; Mosher, D F

    1988-09-01

    A difference in recognition of the adhesive glycoprotein vitronectin (also called S-protein, serum spreading factor, and epibolin) by monoclonal antibody 8E6 (Hayman EG, et al, Proc Natl Acad Sci USA 80:4003, 1983) was investigated using a competitive enzyme-immunosorbent assay and immunoaffinity chromatography. Recognition of vitronectin in serum was approximately 50-fold greater than recognition of vitronectin in plasma. Recognition of vitronectin incubated with heparin, thrombin-antithrombin III complex, or heparin and thrombin-antithrombin III complex together was 2.5-, 7-, or 32-fold greater, respectively, than recognition of vitronectin alone. Thrombin or antithrombin III by itself did not induce the antigenic change. Factor Xa-antithrombin III was less effective than thrombin-antithrombin III in induction of the change. Dextran sulfate and fucoidan were more potent than heparin in induction of the antigenic change, whereas dermatan sulfate, hyaluronic acid, heparan sulfate, chondroitin sulfate, or keratan sulfate were less effective. Immunoblotting analysis of serum and of vitronectin incubated with thrombin and antithrombin III demonstrated the presence of complexes composed of vitronectin and thrombin-antithrombin III that could only be dissociated with reducing agent. N-ethylmaleimide completely blocked the formation of the presumably disulfide-bonded complexes and partially blocked the antigenic change. Both non-disulfide-bonded and disulfide-bonded vitronectin bound to antibody-Sepharose from a mixture of vitronectin and thrombin-antithrombin III. Treatment of vitronectin with 8 mol/L urea resulted in enhanced recognition by the monoclonal antibody. Thus, the 8E6 antibody reacts with an epitope that is preferentially expressed by noncovalently and covalently linked vitronectin/thrombin-antithrombin III complexes and by urea-treated vitronectin. The change in vitronectin induced by thrombin-antithrombin III, therefore, is a physiological correlate of

  2. Artificial antigen-presenting cells expressing AFP(158-166) peptide and interleukin-15 activate AFP-specific cytotoxic T lymphocytes.

    Science.gov (United States)

    Sun, Longhao; Guo, Hao; Jiang, Ruoyu; Lu, Li; Liu, Tong; Zhang, Zhixiang; He, Xianghui

    2016-04-05

    Professional antigen-presenting cells (APCs) are potent generators of tumor antigen-specific cytotoxic T lymphocytes (CTLs) for adoptive immunotherapy; however, generation of APCs is cumbersome, expensive, and subject to the tumor microenvironment. Artificial APCs (aAPCs) have been developed as a cost-effective alternative to APCs. We developed a cellular aAPC that efficiently generated alpha-fetoprotein (AFP)-specific CTLs. We genetically modified the human B cell lymphoma cell line BJAB with a lentiviral vector to establish an aAPC called BA15. The expression of AFP(158-166)-HLA-A*02:01 complex, CD80, CD86, and interleukin (IL)-15 in BA15 cells was assessed. The efficiency of BA15 at generating AFP-specific CTLs and the specific cytotoxicity of CTLs against AFP+ cells were also determined. BA15 cells expressed high levels of AFP(158-166) peptide, HLA-A2, CD80, CD86, and IL-15. BA15 cells also exhibited higher efficiency in generating AFP-specific CTLs than did dendritic cells. These CTLs had greater cytotoxicity against AFP+ hepatocellular carcinoma cells than did CTLs obtained from dendritic cells in vitro and in vivo. Our novel aAPC system could provide a robust platform for the generation of functional AFP-specific CTLs for adoptive immunotherapy of hepatocellular carcinoma.

  3. Regression of established renal cell carcinoma in nude mice using lentivirus-transduced human T cells expressing a human anti-CAIX chimeric antigen receptor

    Directory of Open Access Journals (Sweden)

    Agnes Shuk-Yee Lo

    2014-01-01

    Full Text Available Carbonic anhydrase IX (CAIX is a tumor-associated antigen and marker of hypoxia that is overexpressed on > 90% of clear-cell type renal cell carcinoma (RCC but not on neighboring normal kidney tissue. Here, we report on the construction of two chimeric antigen receptors (CARs that utilize a carbonic anhydrase (CA domain mapped, human single chain antibody (scFv G36 as a targeting moiety but differ in their capacity to provide costimulatory signaling for optimal T cell proliferation and tumor cell killing. The resulting anti-CAIX CARs were expressed on human primary T cells via lentivirus transduction. CAR-transduced T cells (CART cells expressing second-generation G36-CD28-TCRζ exhibited more potent in vitro antitumor effects on CAIX+ RCC cells than first-generation G36-CD8-TCRζ including cytotoxicity, cytokine secretion, proliferation, and clonal expansion. Adoptive G36-CD28-TCRζ CART cell therapy combined with high-dose interleukin (IL-2 injection also lead to superior regression of established RCC in nude mice with evidence of tumor cell apoptosis and tissue necrosis. These results suggest that the fully human G36-CD28-TCRζ CARs should provide substantial improvements over first-generation mouse anti-CAIX CARs in clinical use through reduced human anti-mouse antibody responses against the targeting scFv and administration of lower doses of T cells during CART cell therapy of CAIX+ RCC.

  4. Expressing Redundancy among Linear-Epitope Sequence Data Based on Residue-Level Physicochemical Similarity in the Context of Antigenic Cross-Reaction

    Directory of Open Access Journals (Sweden)

    Salvador Eugenio C. Caoili

    2016-01-01

    Full Text Available Epitope-based design of vaccines, immunotherapeutics, and immunodiagnostics is complicated by structural changes that radically alter immunological outcomes. This is obscured by expressing redundancy among linear-epitope data as fractional sequence-alignment identity, which fails to account for potentially drastic loss of binding affinity due to single-residue substitutions even where these might be considered conservative in the context of classical sequence analysis. From the perspective of immune function based on molecular recognition of epitopes, functional redundancy of epitope data (FRED thus may be defined in a biologically more meaningful way based on residue-level physicochemical similarity in the context of antigenic cross-reaction, with functional similarity between epitopes expressed as the Shannon information entropy for differential epitope binding. Such similarity may be estimated in terms of structural differences between an immunogen epitope and an antigen epitope with reference to an idealized binding site of high complementarity to the immunogen epitope, by analogy between protein folding and ligand-receptor binding; but this underestimates potential for cross-reactivity, suggesting that epitope-binding site complementarity is typically suboptimal as regards immunologic specificity. The apparently suboptimal complementarity may reflect a tradeoff to attain optimal immune function that favors generation of immune-system components each having potential for cross-reactivity with a variety of epitopes.

  5. Supraphysiologic control over HIV-1 replication mediated by CD8 T cells expressing a re-engineered CD4-based chimeric antigen receptor.

    Directory of Open Access Journals (Sweden)

    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.

  6. Cross reactivity of serum antibody responses elicited by DNA vaccines expressing HA antigens from H1N1 subtype influenza vaccines in the past 30 years.

    Science.gov (United States)

    Almansour, Iman; Chen, Huaiqing; Wang, Shixia; Lu, Shan

    2013-10-01

    In the past three decades, ten H1 subtype influenza vaccines have been recommended for global seasonal flu vaccination. Some of them were used only for one year before being replaced by another H1 flu vaccine while others may be used for up to seven years. While the selection of a new seasonal flu vaccine was based on the escape of a new emerging virus that was not effectively protected by the existing flu formulation, there is limited information on the magnitude and breadth of cross reactivity among H1 subtype virus circulation over a long period. In the current study, HA-expressing DNA vaccines were constructed to express individual HA antigens from H1 subtype vaccines used in the past 30 y. Rabbits naïve to HA antibody responses were immunized with these HA DNA vaccines and the cross reactivity of these sera against HA antigen and related H1 viruses in the same period was studied. Our data indicate that the level of cross reactivity was different for different viral isolates and the key mutations responsible for the cross reactivity may involve only a limited number of residues. Our results provide useful information for the development of improved seasonal vaccines than can achieve broad protection against viruses within the same H1 subtype.

  7. Human microRNA hsa-miR-125a-5p interferes with expression of hepatitis B virus surface antigen

    Science.gov (United States)

    Potenza, Nicoletta; Papa, Umberto; Mosca, Nicola; Zerbini, Francesca; Nobile, Valentina; Russo, Aniello

    2011-01-01

    MicroRNAs are small non-coding RNAs that modulate gene expression at post-transcriptional level, playing a crucial role in cell differentiation and development. Recently, some reports have shown that a limited number of mammalian microRNAs are also involved in anti-viral defense. In this study, the analysis of the hepatitis B virus (HBV) genome by the computer program MiRanda led to the identification of seven sites that are potential targets for human liver microRNAs. These sites were found to be clustered in a 995-bp segment within the viral polymerase ORF and the overlapping surface antigen ORF, and conserved among the most common HBV subtypes. The HBV genomic targets were then subjected to a validation test based on cultured hepatic cells (HepG2, HuH-7 and PLC/PRF/5) and luciferase reporter genes. In this test, one of the selected microRNAs, hsa-miR-125a-5p, was found to interact with the viral sequence and to suppress the reporter activity markedly. The microRNA was then shown to interfere with the viral translation, down-regulating the expression of the surface antigen. Overall, these results support the emerging concept that some mammalian microRNAs play a role in virus-host interaction. Furthermore, they provide the basis for the development of new strategies for anti-HBV intervention. PMID:21317190

  8. 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

    tumorigenic hMSC-TERT20 single cell subclones exhibited heterogeneous expression of both GAGE and MAGE-A proteins, and similar patterns of expression were observed in clinical sarcomas. Importantly, histone deacetylase and DNA methyltransferase inhibitors were able to induce more ubiquitous expression levels...

  9. MetQ of Neisseria gonorrhoeae Is a Surface-Expressed Antigen That Elicits Bactericidal and Functional Blocking Antibodies.

    Science.gov (United States)

    Semchenko, Evgeny A; Day, Christopher J; Seib, Kate L

    2017-02-01

    Neisseria gonorrhoeae, the causative agent of the sexually transmitted infection (STI) gonorrhea, is a growing public health threat for which a vaccine is urgently needed. We characterized the functional role of the gonococcal MetQ protein, which is the methionine binding component of an ABC transporter system, and assessed its potential as a candidate antigen for inclusion in a gonococcal vaccine. MetQ has been found to be highly conserved in all strains investigated to date, it is localized on the bacterial surface, and it binds l-methionine with a high affinity. MetQ is also involved in gonococcal adherence to cervical epithelial cells. Mutants lacking MetQ have impaired survival in human monocytes, macrophages, and serum. Furthermore, antibodies raised against MetQ are bactericidal and are able to block gonococcal adherence to epithelial cells. These data suggest that MetQ elicits both bactericidal and functional blocking antibodies and is a valid candidate antigen for additional investigation and possible inclusion in a vaccine for prevention of gonorrhea. Copyright © 2017 Semchenko et al.

  10. Influence of hepatitis B virus genotypes on the intra- and extracellular expression of viral DNA and antigens.

    Science.gov (United States)

    Sugiyama, Masaya; Tanaka, Yasuhito; Kato, Takanobu; Orito, Etsuro; Ito, Kiyoaki; Acharya, Subrat K; Gish, Robert G; Kramvis, Anna; Shimada, Takashi; Izumi, Namiki; Kaito, Masahiko; Miyakawa, Yuzo; Mizokami, Masashi

    2006-10-01

    Various genotypes of the hepatitis B virus (HBV) induce liver disease of distinct severity, but the underlying virological differences are not well defined. Huh7 cells were transfected with plasmids carrying 1.24-fold the HBV genome of different genotypes/subgenotypes (2 strains each for Aa/A1, Ae/A2, Ba/B2 and D; 3 each for Bj/B1 and C). HBV DNA levels in cell lysates, determined by Southern hybridization, were the highest for C followed by Bj/Ba and D/Ae (P B e antigen (HBeAg) was excreted in a trend similar to that of HBV DNA with smaller differences. Secretion of hepatitis B surface antigen (HBsAg) was most abundant for Ae followed by Aa, Ba, Bj/C and remotely by D, which was consistent with mRNA levels. Cellular stress determined by the reporter assay for Grp78 promoter was higher for C and Ba than the other genotypes/subgenotypes (P type plasminogen activator (uPA/SCID), with the liver replaced for human hepatocytes, were inoculated with virions passed in mouse and recovered from culture supernatants. HBV DNA levels in their sera were higher for C than Ae by 2 logs during 4-7 weeks after inoculation. In conclusion, virological differences among HBV genotypes were demonstrated both in vitro and in vivo. These differences may influence HBV infections with distinct genotypes in clinical and epidemiological settings.

  11. Distinct domains of the CD3-gamma chain are involved in surface expression and function of the T cell antigen receptor

    DEFF Research Database (Denmark)

    Wegener, A M; Hou, X; Dietrich, J

    1995-01-01

    The T cell antigen receptor (TcR) is a multisubunit complex that consists of at least six different polypeptides. We have recently demonstrated that the CD3-delta subunit cannot substitute for the CD3-gamma subunit in TcR cell surface expression, in spite of significant amino acid homology between...... these two subunits. To identify CD3-gamma-specific domains that are required for assembly of the complete TcR and for surface expression and function of the TcR, chimeric CD3-gamma/CD3-delta molecules were constructed and expressed in T cells devoid of endogenous CD3-gamma. Substitution of the extracellular...... domain of CD3-gamma with that of CD3-delta did not allow cell surface expression of the TcR. In contrast, substitution of the transmembrane and/or the intracellular domains of CD3-gamma with those of CD3-delta did allow TcR cell surface expression. These results conclusively demonstrate...

  12. B-cell maturation antigen (BCMA) activation exerts specific proinflammatory effects in normal human keratinocytes and is preferentially expressed in inflammatory skin pathologies.

    Science.gov (United States)

    Alexaki, Vassilia-Ismini; Pelekanou, Vassiliki; Notas, George; Venihaki, Maria; Kampa, Marilena; Dessirier, Valérie; Sabour-Alaoui, Sanaa; Stathopoulos, Efstathios N; Tsapis, Andreas; Castanas, Elias

    2012-02-01

    TNFα is known to be expressed in human skin, regulating immune-related responses. Here we report that human normal skin keratinocytes express the members of the TNF superfamily members A proliferation-inducing ligand (APRIL; TNFSF13), B cell-activating factor (BAFF; TNFSF13B), and their receptors, B cell maturation antigen (BCMA; TNFRSF17) and transmembrane activator, calcium-modulator, and cyclophilin ligand interactor (TACI; TNFRSF13B), in a distinct spatial pattern. Our data show a differential expression of these molecules within epidermal layers and skin appendages, whereas the BAFF-specific receptor BAFFR (TNFRSF13C) is absent. Importantly, APRIL and BCMA but not BAFF or TACI are up-regulated in inflammatory skin lesions of psoriasis and squamous cell carcinomas. To explore the functional significance of this system in the skin, we assayed these receptors and ligands in cultured primary keratinocytes and HaCaT cells. We show that both cell types express BAFF, APRIL, BCMA, and TACI. Furthermore, APRIL and/or BAFF trigger nuclear factor-κB activation and IL-6 and granulocyte macrophage colony-stimulating factor (GM-CSF) expression through functional BCMA receptors, an activation inhibited by anti-BCMA short hairpin RNA. However, BAFF and/or APRIL do not induce IL-8 or TNFα production. Our data advance BCMA as an inflammation-related TNFSFR member in keratinocytes, of potential importance in the management of inflammatory skin conditions.

  13. Associations of Prostate-Specific Antigen, Prostate Carcinoma Tissue Gleason Score, and Androgen Receptor Expression with Bone Metastasis in Patients with Prostate Carcinoma.

    Science.gov (United States)

    Chen, Yehui; Lin, Yun; Nie, Pin; Jiang, Wen; Liu, Yanqing; Yuan, Runqiang; Li, Miaoyuan; Zhao, Shijia; Lin, Huaxin; Li, Penghui; Zhang, Jinxiang; Hu, Zhiwen; Xu, Jin; Zhu, Xusheng

    2017-04-12

    BACKGROUND Prostate carcinoma (PCa) is often not diagnosed until advanced disease with bone metastasis. Predictive factors for bone metastasis are required to improve patient outcomes. The study aimed to analyze the factors associated with bone metastases in newly diagnosed patients with PCa. MATERIAL AND METHODS This was a retrospective study of 80 patients newly diagnosed with PCa by pathological examination between January 2012 and December 2014. Bone metastases were diagnosed by positron emission computed tomography. Clinical data, serological laboratory results, and pathological examination results were collected. RESULTS Among the 80 patients, 45 (56%) had bone metastases. Age, serum alkaline phosphatase, prostate-specific antigen (PSA), erythrocyte sedimentation rate, PCa tissue Gleason score, androgen receptor (AR) expression, and Ki-67 expression were higher in patients with bone metastasis compared with those without (all PGleason score (OR: 4.095; 95%CI: 1.592-10.529; P=0.003), and AR expression (OR: 14.023; 95%CI: 3.531-55.6981; P=0.005) were independently associated with bone metastases. Cut-off values for PSA, Gleason score, and AR expression were 67.1 ng/ml (sensitivity: 55.6%; specificity: 97.1%), 7.5 (sensitivity: 75.6%; specificity: 82.9%), and 2.5 (sensitivity: 84.0%; specificity: 91.4%), respectively. CONCLUSIONS PSA, Gleason score, and AR expression in PCa tissues were independently associated with PCa bone metastases. These results could help identifying patients with PCa at high risk of bone metastases.

  14. Prolonged suppression of monocytic human leukocyte antigen-DR expression correlates with mortality in pediatric septic patients in a pediatric tertiary Intensive Care Unit.

    Science.gov (United States)

    Manzoli, Talita Freitas; Troster, Eduardo Juan; Ferranti, Juliana Ferreira; Sales, Maria Mirtes

    2016-06-01

    Immunoparalysis is a syndrome with no clinical symptoms that occurs in some septic patients. Monocytic human leukocyte antigen-DR (mHLA-DR) expression has been used to identify patients in immunoparalysis and prolonged periods of reduced mHLA-DR expression have been correlated with a poor prognosis in sepsis. However, there is a lack of studies investigating mHLA-DR expression in pediatric septic patients. To determine if mHLA-DR expression correlates with mortality in pediatric septic patients using the QuantiBRITE Anti HLA-DR/Anti-Monocyte,a Bechton Dickinson novel reagent that standardizes flow cytometry values. We determined mHLA-DR expression in 30 patients with severe sepsis or septic shock admitted to the pediatric intensive care unit at Hospital das Clinicas da Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil, between January 2013 and February 2015. mHLA-DR expression was quantified between days 3 to 5 and 5 to 7 after the onset of sepsis and the ΔmHLA-DR (mHLA-DR2 - mHLA-DR1) was calculated. We also measured mHLA-DR levels in 21 healthy control patients. Mean mHLA-DR expression was significantly lower in septic patients than in controls (P = .0001). Mortality was 46% in patients with negative ΔHLA-DR or 1000 mAb/cell. Mean ΔmHLA-DR levels were significantly different between survivors and non-survivors (P = .023). ΔHLA-DR correlates with mortality in pediatric patients with septic shock or severe sepsis. This is the first study to have used the QuantiBRITE Anti HLA-DR/Anti-Monocyte reagent to quantify monocyte HLA-DR expression in pediatric septic patients. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Expression of MAGE-A and NY-ESO-1 cancer-testis antigens is enriched in triple-negative invasive breast cancers.

    Science.gov (United States)

    Raghavendra, Ashwini; Kalita-de Croft, Priyakshi; Vargas, Ana Cristina; Smart, Chanel E; Simpson, Peter T; Saunus, Jodi M; Lakhani, Sunil R

    2018-02-21

    A better understanding of the expression of cancer testis antigens (CTA) in breast cancer might identify new immunotherapy options, especially for triple-negative (TN) tumours, which lack expression of conventional therapeutic targets ER, PR and HER2 (receptors for Oestrogen, Progesterone and Human epidermal growth factor). The aim of this study was to quantify the expression of MAGE-A and NY-ESO-1 CTAs in breast cancer, and relate this to known clinicopathologic parameters. We surveyed MAGE-A and NY-ESO-1 protein expression in an unselected cohort of 367 breast tumours (out of which 65 tumours were TN), with accompanying clinical follow-up data, using immunohistochemistry (IHC) analysis of tissue microarrays. Relevant to their potential as vaccine targets in breast cancer, MAGE-A was expressed in 13% of cases, and NY-ESO-1 in 3.8%, with the majority of tumours exhibiting fairly homogeneous staining within individual tissue cores (~85% of cases with staining in >75% tumour cells). Most NY-ESO-1 positive cases also expressed MAGE-A (p=2.06x10 -9 ), and both were strongly associated with the TN phenotype (pESO-1 are frequently expressed in TNBC (~47% and 17% of TN cases, respectively), suggesting that targeting them could be feasible in this patient group. Expression is reasonably homogeneous in positive cases suggesting that IHC analysis of tissue biopsies would be a reliable companion biomarker. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  16. The expression of hematopoietic progenitor cell antigen CD34 is regulated by DNA methylation in a site-dependent manner in gastrointestinal stromal tumours.

    Science.gov (United States)

    Bure, Irina; Braun, Alexander; Kayser, Claudia; Geddert, Helene; Schaefer, Inga-Marie; Cameron, Silke; Ghadimi, Michael B; Ströbel, Philipp; Werner, Martin; Hartmann, Arndt; Wiemann, Stefan; Agaimy, Abbas; Haller, Florian; Moskalev, Evgeny A

    2017-12-01

    The anatomic site-dependent expression of hematopoietic progenitor cell antigen CD34 is a feature of gastrointestinal stromal tumours (GISTs). The basis for the differential CD34 expression is only incompletely understood. This study aimed at understanding the regulation of CD34 in GISTs and clarification of its site-dependent expression. Two sample sets of primary GISTs were interrogated including 52 fresh-frozen and 134 paraffin-embedded and formalin-fixed specimens. DNA methylation analysis was performed by HumanMethylation450 BeadChip array in three cell lines derived from gastric and intestinal GISTs, and differentially methylated CpG sites were established upstream of CD34. The methylation degree was further quantified by pyrosequencing, and inverse correlation with CD34 mRNA and protein abundance was revealed. The gene's expression could be activated upon induction of DNA hypomethylation with 5-aza-2'-deoxycytidine in GIST-T1 cells. In patient samples, a strong inverse correlation of DNA methylation degree with immunohistochemically evaluated CD34 expression was documented. Both CD34 expression and DNA methylation levels were specific to the tumours' anatomic location and mutation status. A constant decrease in methylation levels was observed ranging from almost 100% hypermethylation in intestinal GISTs from duodenum to hypomethylation in rectum. CD34 was heavily methylated in gastric PDGFRA-mutant GISTs in comparison to hypomethylated KIT-mutant counterparts. Next to CD34 hypermethylation, miR-665 was predicted and experimentally confirmed to target CD34 mRNA in GIST-T1 cells. Our results suggest that CD34 expression in GISTs may undergo a complex control by DNA methylation and miR-665. Differential methylation and expression of CD34 in GISTs along the gastrointestinal tract axis and in tumours that harbour different gain-of-function mutations suggest the origin from different cell populations in the gastrointestinal tract. © 2017 UICC.

  17. Structure and expression of the human Lysyl hydroxylase gene (PLOD): Introns 9 and 16 contain Alu sequences at the sites of recombination in Ehlers-Danlos syndrome type VI patients

    Energy Technology Data Exchange (ETDEWEB)

    Heikkinen, J.; Hautala, T.; Kivirikko, K.I. [Univ. of Oulu (Finland)] [and others

    1994-12-01

    Lysyl hydroxylase (EC 1.14.11.4) catalyzes the formation of hydroxylysine in collagens by the hydroxylation of lysine residues in peptide linkages. This enzyme activity is known to be reduced in patients with the type VI variant of the Ehlers-Danlos syndrome, and the first mutations in the lysyl hydroxylase gene (PLOD) have recently been identified. We have now isolated genomic clones for human lysyl hydroxylase and determined the complete structure of the gene, which contains 19 exons and a 5{prime} flanking region with characteristics shared by housekeeping genes. The constitutive expression of the gene in different tissues further suggests that lysyl hydroxylase has an essential function. We have sequenced the introns of the gene in the region where many mutations and rearrangements analyzed to date are concentrated. Intron 9 and intron 16 show extensive homology resulting from the many Alu sequences found in these introns. Intron 9 contains five and intron 16 eight Alu sequences. The high homology and many short identical or complementary sequences in these introns generate many potential recombination sites with the gene. The delineation of the structure of the lysyl hydroxylase gene contributes significantly to our understanding of the rearrangements in the genome of Ehlers-Danlos type VI patients. 21 refs., 2 figs., 2 tabs.

  18. Expression and Possible Prognostic Role of MAGE-A4, NY-ESO-1, and HER-2 Antigens in Women with Relapsing Invasive Ductal Beast Cancer: Retrospective Immunohistochemical Study

    Science.gov (United States)

    Bandić, Daniela; Juretić, Antonio; Šarčević, Božena; Šeparović, Viktor; Kujundžić Tiljak, Mirjana; Hudolin, Tvrtko; Spagnoli, Giulio C.; Čović, Dinko; Šamija, Mirko

    2006-01-01

    Aim To evaluate the possible prognostic role of the expression of MAGE-A4 and NY-ESO-1 cancer/testis antigens in women diagnosed with invasive ductal breast cancer and determine the expression of HER-2 antigen. Methods The expression of MAGE-A4, NY-ESO-1, and HER-2 antigens was evaluated immunohistochemically on archival paraffin-embedded samples of breast cancer tissue from 81 patients. All patients had T1 to T3, N0 to N1, M0 tumors and underwent postoperative radiotherapy and, if indicated, systemic therapy (chemotherapy and hormonal therapy). The antigen expression in women who were disease-free for 5 years of follow up (n = 23) was compared with that in women with either locoregional relapse (n = 30) or bone metastases (n = 28). Patient survival after 10 years of follow up was assessed. Results The three groups of women were comparable in terms of age, type of operation, tumor size, tumor grade, number of metastatically involved axillary lymph nodes, Nottingham prognostic index (NPI), progesterone receptor (PR) status, and adjuvant hormonal therapy. Estrogen receptors (ER) were positive in 13 women in the 5-year relapse-free group vs 8 in locoregional relapse and 7 in bone metastases group (P = 0.032). There were significantly fewer women who received adjuvant chemotherapy in the 5-year relapse-free group than in other two groups (7 vs 23 with locoregional relapse and 25 with bone metastases; P<0.001). This group also had a significantly better 10-year survival (14 women vs 1 with locoregional relapse and 1 with bone metastases; P<0.001). The three groups did not differ in the NY-ESO-1 or HER-2 expression, but the number of patients expressing MAGE-A4 antigen was significantly lower in the group with locoregional relapse (P = 0.014). In all groups, MAGE-A4 antigen expression was associated with the NY-ESO-1 antigen expression (P = 0.006), but not with tumor size and grade, number of metastatically involved axillary lymph nodes, or the ER

  19. Expression and possible prognostic role of MAGE-A4, NY-ESO-1, and HER-2 antigens in women with relapsing invasive ductal breast cancer: retrospective immunohistochemical study.

    Science.gov (United States)

    Bandić, Daniela; Juretić, Antonio; Sarcević, Bozena; Separović, Viktor; Kujundzić-Tiljak, Mirjana; Hudolin, Tvrtko; Spagnoli, Giulio C; Cović, Dinko; Samija, Mirko

    2006-02-01

    To evaluate the possible prognostic role of the expression of MAGE-A4 and NY-ESO-1 cancer/testis antigens in women diagnosed with invasive ductal breast cancer and determine the expression of HER-2 antigen. The expression of MAGE-A4, NY-ESO-1, and HER-2 antigens was evaluated immunohistochemically on archival paraffin-embedded samples of breast cancer tissue from 81 patients. All patients had T1 to T3, N0 to N1, M0 tumors and underwent postoperative radiotherapy and, if indicated, systemic therapy (chemotherapy and hormonal therapy). The antigen expression in women who were disease-free for 5 years of follow up (n=23) was compared with that in women with either locoregional relapse (n=30) or bone metastases (n=28). Patient survival after 10 years of follow up was assessed. The three groups of women were comparable in terms of age, type of operation, tumor size, tumor grade, number of metastatically involved axillary lymph nodes, Nottingham prognostic index (NPI), progesterone receptor (PR) status, and adjuvant hormonal therapy. Estrogen receptors (ER) were positive in 13 women in the 5-year relapse-free group vs 8 in locoregional relapse and 7 in bone metastases group (P=0.032). There were significantly fewer women who received adjuvant chemotherapy in the 5-year relapse-free group than in other two groups (7 vs 23 with locoregional relapse and 25 with bone metastases; P<0.001). This group also had a significantly better 10-year survival (14 women vs 1 with locoregional relapse and 1 with bone metastases; P<0.001). The three groups did not differ in the NY-ESO-1 or HER-2 expression, but the number of patients expressing MAGE-A4 antigen was significantly lower in the group with locoregional relapse (P=0.014). In all groups, MAGE-A4 antigen expression was associated with the NY-ESO-1 antigen expression (P=0.006), but not with tumor size and grade, number of metastatically involved axillary lymph nodes, or the ER and PR status. MAGE-A4-positive patients had a

  20. Swine adipose stromal cells loaded with recombinant bovine herpesvirus 4 virions expressing a foreign antigen induce potent humoral immune responses in pigs.

    Science.gov (United States)

    Donofrio, Gaetano; Taddei, Simone; Franceschi, Valentina; Capocefalo, Antonio; Cavirani, Sandro; Martinelli, Nicola; Ottonello, Simone; Ferrari, Maura

    2011-01-29

    Increasingly effective vaccination strategies are needed to counteract the high incidence of contagious diseases associated with intensive swine breeding. Recombinant viral vaccines are a promising new avenue in this direction. Key features of viral vectors suitable for immunoprophylaxis are safety, ease of manipulation and the ability to replicate in a variety of hosts. Most of the above requirements are met by bovine herpesvirus 4 (BoHV-4), a non-pathogenic dsDNA virus capable of infecting a broad range of cell types in vitro. Here we report the results of an exploratory study using an engineered BoHV-4 virus (eBoHV-4) expressing two unrelated glycoprotein antigens from bovine viral diarrhea virus (BVDV) and bovine herpesvirus 1 (BoHV-1), to assess the potential of recombinant BoHV-4 as a self-adjuvanted immunogen in pigs. Free eBoHV-4 virions and virions preloaded into homologous swine adipose-derived stromal cells (SADSC) were tested. Neither virus formulation elicited neutralizing anti-BoHV-4 antibodies, nor any disease symptom, yet both induced specific immune responses against the heterologous antigens. However, a much earlier (18 vs 28 days post-infection) and more robust neutralizing response against BVDV and BoHV-1 viruses was elicited by eBoHV-4-preinfected SADSCs compared to free virions. The data validate BoHV-4 as a safe and effective heterologous antigen carrier/producer and identify SADSCs as helpful tools for the formulation of increasingly efficacious recombinant immunogens for pig vaccination. Copyright © 2010 Elsevier Ltd. All rights reserved.

  1. Bispecific T-cells expressing polyclonal repertoire of endogenous γδ T-cell receptors and introduced CD19-specific chimeric antigen receptor.

    Science.gov (United States)

    Deniger, Drew C; Switzer, Kirsten; Mi, Tiejuan; Maiti, Sourindra; Hurton, Lenka; Singh, Harjeet; Huls, Helen; Olivares, Simon; Lee, Dean A; Champlin, Richard E; Cooper, Laurence J N

    2013-03-01

    Even though other γδ T-cell subsets exhibit antitumor activity, adoptive transfer of γδ Tcells is currently limited to one subset (expressing Vγ9Vδ2 T-cell receptor (TCR)) due to dependence on aminobisphosphonates as the only clinically appealing reagent for propagating γδ T cells. Therefore, we developed an approach to propagate polyclonal γδ T cells and rendered them bispecific through expression of a CD19-specific chimeric antigen receptor (CAR). Peripheral blood mononuclear cells (PBMC) were electroporated with Sleeping Beauty (SB) transposon and transposase to enforce expression of CAR in multiple γδ T-cell subsets. CAR(+)γδ T cells were expanded on CD19(+) artificial antigen-presenting cells (aAPC), which resulted in >10(9) CAR(+)γδ T cells from <10(6) total cells. Digital multiplex assay detected TCR mRNA coding for Vδ1, Vδ2, and Vδ3 with Vγ2, Vγ7, Vγ8, Vγ9, and Vγ10 alleles. Polyclonal CAR(+)γδ T cells were functional when TCRγδ and CAR were stimulated and displayed enhanced killing of CD19(+) tumor cell lines compared with CAR(neg)γδ T cells. CD19(+) leukemia xenografts in mice were reduced with CAR(+)γδ T cells compared with control mice. Since CAR, SB, and aAPC have been adapted for human application, clinical trials can now focus on the therapeutic potential of polyclonal γδ T cells.

  2. Antibodies against In Vivo-Expressed Antigens Are Sufficient To Protect against Lethal Aerosol Infection with Burkholderia mallei and Burkholderia pseudomallei.

    Science.gov (United States)

    Zimmerman, Shawn M; Dyke, Jeremy S; Jelesijevic, Tomislav P; Michel, Frank; Lafontaine, Eric R; Hogan, Robert J

    2017-08-01

    Burkholderia mallei, a facultative intracellular bacterium and tier 1 biothreat, causes the fatal zoonotic disease glanders. The organism possesses multiple genes encoding autotransporter proteins, which represent important virulence factors and targets for developing countermeasures in pathogenic Gram-negative bacteria. In the present study, we investigated one of these autotransporters, BatA, and demonstrate that it displays lipolytic activity, aids in intracellular survival, is expressed in vivo, elicits production of antibodies during infection, and contributes to pathogenicity in a mouse aerosol challenge model. A mutation in the batA gene of wild-type strain ATCC 23344 was found to be particularly attenuating, as BALB/c mice infected with the equivalent of 80 median lethal doses cleared the organism. This finding prompted us to test the hypothesis that vaccination with the batA mutant strain elicits protective immunity against subsequent infection with wild-type bacteria. We discovered that not only does vaccination provide high levels of protection against lethal aerosol challenge with B. mallei ATCC 23344, it also protects against infection with multiple isolates of the closely related organism and causative agent of melioidosis, Burkholderia pseudomallei Passive-transfer experiments also revealed that the protective immunity afforded by vaccination with the batA mutant strain is predominantly mediated by IgG antibodies binding to antigens expressed exclusively in vivo Collectively, our data demonstrate that BatA is a target for developing medical countermeasures and that vaccination with a mutant lacking expression of the protein provides a platform to gain insights regarding mechanisms of protective immunity against B. mallei and B. pseudomallei, including antigen discovery. Copyright © 2017 American Society for Microbiology.

  3. Expression of sialyl-Tn sugar antigen in bladder cancer cells affects response toBacillus Calmette Guérin(BCG) and to oxidative damage.

    Science.gov (United States)

    Severino, Paulo F; Silva, Mariana; Carrascal, Mylene; Malagolini, Nadia; Chiricolo, Mariella; Venturi, Giulia; Astolfi, Annalisa; Catera, Mariangela; Videira, Paula A; Dall'Olio, Fabio

    2017-08-15

    The sialyl-Tn (sTn) antigen is an O -linked carbohydrate chain aberrantly expressed in bladder cancer (BC), whose biosynthesis is mainly controlled by the sialyltransferase ST6GALNAC1. Treatment with Bacillus Calmette-Guérin (BCG) is the most effective adjuvant immunotherapy for superficial BC but one third of the patients fail to respond. A poorly understood correlation between the expression of sTn and BC patient's response to BCG was previously observed. By analyzing tumor tissues, we showed that patients with high ST6GALNAC1 and IL-6 mRNA expression were BCG responders. To investigate the role of sTn in BC cell biology and BCG response, we established the cell lines MCR sTn and MCR Nc by retroviral transduction of the BC cell line MCR with the ST6GALNAC1 cDNA or with an empty vector, respectively. Compared with MCR Nc , BCG-stimulated MCR sTn secreted higher levels of IL-6 and IL-8 and their secretome induced a stronger IL-6, IL-1β, and TNFα secretion by macrophages, suggesting the induction of a stronger inflammatory response. Transcriptomic analysis of MCR Nc and MCR sTn revealed that ST6GALNAC1 /sTn expression modulates hundreds of genes towards a putative more malignant phenotype and down-regulates several genes maintaining genomic stability. Consistently, MCR sTn cells displayed higher H 2 O 2 sensitivity. In MCR sTn ,, BCG challenge induced an increased expression of several regulatory non coding RNA genes. These results indicate that the expression of ST6GALNAC1 /sTn improves the response to BCG therapy by inducing a stronger macrophage response and alters gene expression towards malignancy and genomic instability, increasing the sensitivity of BC cells to the oxidizing agents released by BCG.

  4. A Genetically Modified attenuated Listeria Vaccine Expressing HPV16 E7 Kill Tumor Cells in Direct and Antigen-Specific Manner

    Directory of Open Access Journals (Sweden)

    Yan Yan Jia

    2017-06-01

    Full Text Available Attenuated Listeria monocytogenes (L. monocytogenes, LM induces specific CD8+ and CD4+ T cell responses, and has been identified as a promising cancer vaccine vector. Cervical cancer is the third most common cancer in women worldwide, with human papillomavirus (HPV, particularly type 16, being the main etiological factor. The therapeutic HPV vaccines are urgently needed. The E7 protein of HPV is necessary for maintaining malignancy in tumor cells. Here, a genetically modified attenuated LM expressing HPV16 E7 protein was constructed. Intraperitoneal vaccination of LM4Δhly::E7 significantly reduced tumor size and even resulted in complete regression of established tumors in a murine model of cervical cancer. We provided evidence that recombinant LM strains could enter the tumor tissue and induce non-specific tumor cell death, probably via activation of reactive oxygen species and increased intracellular Ca2+ levels. LM4Δhly::E7 effectively triggered a strong antigen-specific cellular immunity in tumor-bearing mice, and elicited significant infiltration of T cells in the intratumoral milieu. In summary, these data showed LM4Δhly::E7 to be effective in a cervical cancer model and LM4Δhly::E7 induced an antitumor effect by antigen-specific cellular immune responses and direct killing of tumor cells, indicating a potential application against cervical cancer.

  5. Protein expression levels of carcinoembryonic antigen (CEA) in Danish ovarian cancer patients: from the Danish 'MALOVA'ovarian cancer study

    DEFF Research Database (Denmark)

    Hogdall, E.V.; Christensen, L.; Blaakaer, J.

    2008-01-01

    from 189 women diagnosed with low malignant potential ovarian tumours (LMP, borderline ovarian tumours) and 571 women diagnosed with ovarian cancer (OC). RESULTS: Using 30% as the cut-off level for CEA over-expression, 18% of LMPs and 4% of OCs were positive. A higher proportion of mucinous tumours...... (I to IV), the highest CEA expression compared with no expression was found to be a prognostic factor (level 3 versus negative: HR = 2.12, 95%CI 1.11-4.05). FIGO stage, residual tumour after primary surgery, age at diagnosis, other histological types versus serous adenocarcinoma and low versus high...

  6. Effect of codon optimization and subcellular targeting on Toxoplasma gondii antigen SAG1 expression in tobacco leaves to use in subcutaneous and oral immunization in mice.

    Science.gov (United States)

    Laguía-Becher, Melina; Martín, Valentina; Kraemer, Mauricio; Corigliano, Mariana; Yacono, María L; Goldman, Alejandra; Clemente, Marina

    2010-07-15

    Codon optimization and subcellular targeting were studied with the aim to increase the expression levels of the SAG178-322 antigen of Toxoplasma gondii in tobacco leaves. The expression of the tobacco-optimized and native versions of the SAG1 gene was explored by transient expression from the Agrobacterium tumefaciens binary expression vector, which allows targeting the recombinant protein to the endoplasmic reticulum (ER) and the apoplast. Finally, mice were subcutaneously and orally immunized with leaf extracts-SAG1 and the strategy of prime boost with rSAG1 expressed in Escherichia coli was used to optimize the oral immunization with leaf extracts-SAG1. Leaves agroinfiltrated with an unmodified SAG1 gene accumulated 5- to 10-fold more than leaves agroinfiltrated with a codon-optimized SAG1 gene. ER localization allowed the accumulation of higher levels of native SAG1. However, no significant differences were observed between the mRNA accumulations of the different versions of SAG1. Subcutaneous immunization with leaf extracts-SAG1 (SAG1) protected mice against an oral challenge with a non-lethal cyst dose, and this effect could be associated with the secretion of significant levels of IFN-gamma. The protection was increased when mice were ID boosted with rSAG1 (SAG1+boost). This group elicited a significant Th1 humoral and cellular immune response characterized by high levels of IFN-gamma. In an oral immunization assay, the SAG1+boost group showed a significantly lower brain cyst burden compared to the rest of the groups. Transient agroinfiltration was useful for the expression of all of the recombinant proteins tested. Our results support the usefulness of endoplasmic reticulum signal peptides in enhancing the production of recombinant proteins meant for use as vaccines. The results showed that this plant-produced protein has potential for use as vaccine and provides a potential means for protecting humans and animals against toxoplasmosis.

  7. Swine leukocyte antigen-DQ expression and its regulation by interferon-gamma at the maternal-fetal interface in pigs.

    Science.gov (United States)

    Kim, Mingoo; Seo, Heewon; Choi, Yohan; Shim, Jangsoo; Bazer, Fuller W; Ka, Hakhyun

    2012-02-01

    Successful pregnancy requires an appropriate intrauterine immune response to the conceptus, which is a semiallograft within the uterus. We reported that swine leukocyte antigen-DQA (SLA-DQA), a major histocompatibility complex (MHC) class II gene, is expressed in the uterine endometrium at the time of conceptus implantation in pigs. Because MHC molecules play critical roles in the immune system, SLA-DQ was hypothesized to be involved in immune regulation during pregnancy. Therefore, we examined expression of SLA-DQ in uterine endometrial tissues obtained during the estrous cycle and pregnancy. SLA-DQA and SLA-DQB mRNAs were detected as 1.3-kb and 1.2-kb bands, respectively. Real-time RT-PCR analysis indicated that SLA-DQA and SLA-DQB mRNA expression was affected by day and pregnancy status, with the highest expression on Day 15 of pregnancy. SLA-DQ was localized primarily to subepithelial stromal cells and endothelial cells of the uterus. Using endometrial explant cultures from Day 12 of the estrous cycle, we determined that expression of SLA-DQA and SLA-DQB mRNAs increased in response to interferon-gamma (IFNG), which is produced by pig conceptus trophectoderm between Days 14 and 18 of pregnancy. The abundance of SLA-DQ protein was less in endometria from gilts with conceptuses resulting from somatic cell nuclear transfer compared with endometria from gilts with conceptuses resulting from natural mating. These results support our hypothesis that SLA-DQ is expressed in response to IFNG from the conceptus, and likely regulates immune response at the maternal-fetal interface to support the maintenance of pregnancy in pigs.

  8. Brucella abortus Inhibits Major Histocompatibility Complex Class II Expression and Antigen Processing through Interleukin-6 Secretion via Toll-Like Receptor 2▿

    Science.gov (United States)

    Barrionuevo, Paula; Cassataro, Juliana; Delpino, M. Victoria; Zwerdling, Astrid; Pasquevich, Karina A.; Samartino, Clara García; Wallach, Jorge C.; Fossati, Carlos A.; Giambartolomei, Guillermo H.

    2008-01-01

    The strategies that allow Brucella abortus to survive inside macrophages for prolonged periods and to avoid the immunological surveillance of major histocompatibility complex class II (MHC-II)-restricted gamma interferon (IFN-γ)-producing CD4+ T lymphocytes are poorly understood. We report here that infection of THP-1 cells with B. abortus inhibited expression of MHC-II molecules and antigen (Ag) processing. Heat-killed B. abortus (HKBA) also induced both these phenomena, indicating the independence of bacterial viability and involvement of a structural component of the bacterium. Accordingly, outer membrane protein 19 (Omp19), a prototypical B. abortus lipoprotein, inhibited both MHC-II expression and Ag processing to the same extent as HKBA. Moreover, a synthetic lipohexapeptide that mimics the structure of the protein lipid moiety also inhibited MHC-II expression, indicating that any Brucella lipoprotein could down-modulate MHC-II expression and Ag processing. Inhibition of MHC-II expression and Ag processing by either HKBA or lipidated Omp19 (L-Omp19) depended on Toll-like receptor 2 and was mediated by interleukin-6. HKBA or L-Omp19 also inhibited MHC-II expression and Ag processing of human monocytes. In addition, exposure to the synthetic lipohexapeptide inhibited Ag-specific T-cell proliferation and IFN-γ production of peripheral blood mononuclear cells from Brucella-infected patients. Together, these results indicate that there is a mechanism by which B. abortus may prevent recognition by T cells to evade host immunity and establish a chronic infection. PMID:17984211

  9. GM-CSF/IL-3/IL-5 receptor common β chain (CD131 expression as a biomarker of antigen-stimulated CD8+ T cells

    Directory of Open Access Journals (Sweden)

    Maric Dragan

    2008-04-01

    Full Text Available Abstract Background Upon Ag-activation cytotoxic T cells (CTLs produce IFN-γ GM-CSF and TNF-α, which deliver simultaneously pro-apoptotic and pro-inflammatory signals to the surrounding microenvironment. Whether this secretion affects in an autocrine loop the CTLs themselves is unknown. Methods Here, we compared the transcriptional profile of Ag-activated, Flu-specific CTL stimulated with the FLU M1:58-66 peptide to that of convivial CTLs expanded in vitro in the same culture. PBMCs from 6 HLA-A*0201 expressing donors were expanded for 7 days in culture following Flu M1:58-66 stimulation in the presence of 300 IU/ml of interleukin-2 and than sorted by high speed sorting to high purity CD8+ expressing T cells gated according to FluM1:58-66 tetrameric human leukocyte antigen complexes expression. Results Ag-activated CTLs displayed higher levels of IFN-γ, GM-CSF (CSF2 and GM-CSF/IL-3/IL-5 r