WorldWideScience

Sample records for antigen gene expression

  1. Expression of A, G and B melanoma antigen genes in human hepatocellular carcinoma.

    Science.gov (United States)

    Chen, Zhi; Shao, Jun-Bing; Wu, Wei

    2002-11-01

    To observe the expression of the A melanoma antigen (MAGE), G melanoma antigen (GAGE) and B melanoma antigen (BAGE) genes in human hepatocellular carcinoma cell lines. The MAGE-1,MAGE-3,GAGE1-8,GAGE1-2 and BAGE mRNA lever in hepatocellular carcinoma cell lines SMMC-7721, QQY-7701, BEL-7402 were studied by reverse transcription polymerase chain reaction and were compared with biopsied liver tissues. MAGE-1 and BAGE mRNA were expressed in SMMC-7721, MAGE-3 and BAGE in QGY-7701, MAGE-1 and GAGE1-2 in BEL-7402. None of these genes was expressed in biopsied liver tissues. MAGE-1, MAGE-3, GAGE1-8, GAGE1-2 and BAGE were expressed in hepatocellular carcinoma cell lines, respectively. These tumor-specific antigens can be used as molecular markers and possible targets of immunotherapy for patients with hepatocellular carcinoma.

  2. 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.

  3. The antigen processing machinery of class I human leukocyte antigens: linked patterns of gene expression in neoplastic cells.

    Science.gov (United States)

    Giorda, Ezio; Sibilio, Leonardo; Martayan, Aline; Moretti, Sara; Venturo, Irene; Mottolese, Marcella; Ferrara, Giovan Battista; Cappellacci, Sandra; Eibenschutz, Laura; Catricalà, Caterina; Grammatico, Paola; Giacomini, Patrizio

    2003-07-15

    The ultimate outcome of an immune response (escape or surveillance) depends on a delicate balance of opposing signals delivered by activating and inhibitory immune receptors expressed by cytotoxic T lymphocytes and natural killer cells. In this light, loss and down-regulation of human leukocyte antigens (HLA) class I molecules, while important for keeping tumors below the T-cell detection levels, may incite recognition of missing self. Conversely, the maintenance of normal levels of expression (or even up-regulation) may be favorable to tumors, at least in certain cases. In this study, we took advantage of a previously characterized panel of 15 early passage tumor cell lines (mainly from melanoma and lung carcinoma lesions) enriched with class I-low phenotypes. These cells were systematically characterized by Northern and/or Western blotting (e.g., mini-transcriptome/mini-proteome analysis) for the expression of HLA-A, -B, -C, beta(2)-microglobulin, and the members of the "antigen processing machinery" of class I molecules (LMP2, LMP7, TAP1, TAP2, tapasin, calreticulin, calnexin, and ERp57). In addition, we established four pairs of cultures, each comprising melanoma cells and normal melanocytes from the same patient. We found that approximately 97% of the 185 tested gene products are expressed (although often weakly), and in many cases coordinately regulated in 18 of 19 tumor cell lines. Linked expression patterns could be hierarchically arranged by statistical methods and graphically described as a class I HLA "coordinome." Deviations (both down- and up-regulation) from the coordinome expression pattern inherited from the normal, paired melanocyte counterpart, were allowed but limited in magnitude, as if melanoma cells were trying to keep a "low profile" HLA phenotype. We conclude that irreversible HLA loss is a rare event, and class I expression in tumor cells almost invariably results from reversible gene regulatory (rather than gene disruption) events.

  4. A gene family expressing a host-protective antigen of Echinococcus granulosus.

    Science.gov (United States)

    Chow, C; Gauci, C G; Cowman, A F; Lightowlers, M W

    2001-11-01

    Echinococcus granulosus causes cystic hydatidosis in humans. A recombinant antigen vaccine has been developed, for use in the parasite's natural animal intermediate hosts, that may provide a new tool for control of hydatid disease transmission. The antigen, designated EG95, is encoded by a cDNA the features of which indicate it to be an incomplete copy of the associated mRNA. Characterisation of the gene(s) encoding the antigen was undertaken in order to enable subsequent study of genetic variability in the gene and associated protein in different parasite isolates. Southern hybridisation studies of E. granulosus genomic DNA probed with the eg95 cDNA revealed that the gene belonged to a gene family. DNA sequence analysis of cloned genomic fragments indicated that the gene family consists of at least seven members, one of which is a pseudogene. The gene having identity with the eg95 cDNA was cloned and sequenced, and the full length mRNA characterised. Genomic sequence and structure of the eg95 gene family members are highly conserved with respect to the gene encoding EG95. Four eg95-related genes are predicted to express an identical EG95 protein and all four were shown to be expressed in the oncosphere life-cycle stage. The full length EG95 protein has a predicted molecular mass of 16.9 kDa, secretory signal sequence, carboxy-terminal glycosylphosphatidylinositol hydrophobic anchor motif and a fibronectin type III domain. PCR amplification conditions were established which allow gene-specific characterisation of the eg95 gene in E. granulosus isolates from different host species and geographical locations.

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

    Directory of Open Access Journals (Sweden)

    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

  6. The Echinococcus granulosus antigen B gene family comprises at least 10 unique genes in five subclasses which are differentially expressed.

    Science.gov (United States)

    Zhang, Wenbao; Li, Jun; Jones, Malcolm K; Zhang, Zhuangzhi; Zhao, Li; Blair, David; McManus, Donald Peter

    2010-08-10

    Antigen B (EgAgB) is a major protein produced by the metacestode cyst of Echinococcus granulosus, the causative agent of cystic hydatid disease. This protein has been shown to play an important role in modulating host immune responses, although its precise biological function still remains unknown. It is generally accepted that EgAgB is comprised of a gene family of five subfamilies which are highly polymorphic, but the actual number of genes present is unknown. Based on published sequences for the family, we designed specific primers for each subfamily and used PCR to amplify them from genomic DNA isolated from individual mature adult worms (MAW) taken from an experimentally infected dog in China and individual larval protoscoleces (PSC) excised from a single hydatid cyst taken from an Australian kangaroo. We then used real-time PCR to measure expression of each of the genes comprising the five EgAgB subfamilies in all life-cycle stages including the oncosphere (ONC). Based on sequence alignment analysis, we found that the EgAgB gene family comprises at least ten unique genes. Each of the genes was identical in both larval and adult E. granulosus isolates collected from two geographical areas (different continents). DNA alignment comparisons with EgAgB sequences deposited in GenBank databases showed that each gene in the gene family is highly conserved within E. granulosus, which contradicts previous studies claiming significant variation and polymorphism in EgAgB. Quantitative PCR analysis revealed that the genes were differentially expressed in different life-cycle stages of E. granulosus with EgAgB3 expressed predominantly in all stages. These findings are fundamental for determining the expression and the biological function of antigen B.

  7. [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.

  8. 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.

  9. Phase Variable O Antigen Biosynthetic Genes Control Expression of the Major Protective Antigen and Bacteriophage Receptor in Vibrio cholerae O1

    Science.gov (United States)

    Seed, Kimberley D.; Faruque, Shah M.; Mekalanos, John J.; Calderwood, Stephen B.; Qadri, Firdausi; Camilli, Andrew

    2012-01-01

    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. PMID:23028317

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

    Science.gov (United States)

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

    2016-08-01

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

  11. Gene cloning, expression, and localization of antigen 5 in the life cycle of Echinococcus granulosus.

    Science.gov (United States)

    Li, Yuzhe; Xu, Hongxu; Chen, Jiajia; Gan, Wenjia; Wu, Weihua; Wu, Weiping; Hu, Xuchu

    2012-06-01

    Antigen 5 (Ag5) has been identified as a dominant component of cyst fluid of Echinococcus granulosus and is considered as a member of serine proteases family, which in other helminth, plays an important role in the egg hatch and larva invasion. However, whether Ag5 is expressed and secreted in all life stages is unknown. In this study, according to the sequence in GenBank, we cloned and sequenced the open reading frame (ORF) of Ag5 gene from the protoscolices of E. granulosus isolated from the sheep in Qinhai Province of China, and found several substitutions and a base insert and deletion in a short region near the stop code, leading to a frameshift mutation which is conserved with the homologue of other cestode. The ORF is 1,455 bp in length, encoding 484 amino acids with a secretory signal peptide. Bioinformatics analysis predicted several phosphorylation and myristoylation sites and a N-glycosylation site and a species-specific linear B epitope in the protein. The ORF was cloned into the plasmid pET28a(+) vector and expressed in Escherichia coli . The recombinant protein was purified by affinity chromatography. Anti-rEgAg5 antiserum was prepared in rats and used to analyze the localization of Ag5 in protoscolex and adult worm by immunofluorescence technique. Results demonstrated that the Ag5 is strongly expressed in the tegument of protoscolex and the embryonic membrane of egg and surface of oncosphere; meanwhile, it is also weakly expressed in tegument of the adult. This study showed that Ag5 is expressed in all stages of life cycle, secreted from the surface of the worm and may be anchored in membrane by its myristoylation sites; these characteristics make it a candidate antigen for diagnosis and vaccine for both intermediate and definitive hosts.

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

    International Nuclear Information System (INIS)

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

    2005-01-01

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

  13. Cloning and Expression of Genes for Dengue Virus Type-2 Encoded Antigens for Rapid Diagnosis and Vaccine Development

    Science.gov (United States)

    1986-11-26

    SIE cop AD nCloning and Expression of Genes for Dengue Virus ,4. CJ Type 2 Encoded Antigens for Rapid Diagnosis and Vaccine Development 0ANNUAL...pVVI and pVVI7 cDNA clones, synthetic peptides homologous to NS5 and NSI regions were synthesized. These peptides are being used at Walter Reed Army...NO. Frederick, MD 21701-5012 63750A 63750 D808 A i 031 11. TITLE (Include Serurity Classification) Cloning and Expression of Genes for Dengue Virus

  14. Paraneoplastic antigen-like 5 gene (PNMA5) is preferentially expressed in the association areas in a primate specific manner.

    Science.gov (United States)

    Takaji, Masafumi; Komatsu, Yusuke; Watakabe, Akiya; Hashikawa, Tsutomu; Yamamori, Tetsuo

    2009-12-01

    To understand the relationship between the structure and function of primate neocortical areas at a molecular level, we have been screening for genes differentially expressed across macaque neocortical areas by restriction landmark cDNA scanning (RLCS). Here, we report enriched expression of the paraneoplastic antigen-like 5 gene (PNMA5) in association areas but not in primary sensory areas, with the lowest expression level in primary visual cortex. In situ hybridization in the primary sensory areas revealed PNMA5 mRNA expression restricted to layer II. Along the ventral visual pathway, the expression gradually increased in the excitatory neurons from the primary to higher visual areas. This differential expression pattern was very similar to that of retinol-binding protein (RBP) mRNA, another association-area-enriched gene that we reported previously. Additional expression analysis for comparison of other genes in the PNMA gene family, PNMA1, PNMA2, PNMA3, and MOAP1 (PNMA4), showed that they were widely expressed across areas and layers but without the differentiated pattern of PNMA5. In mouse brains, PNMA1 was only faintly expressed and PNMA5 was not detected. Sequence analysis showed divergence of PNMA5 sequences among mammals. These findings suggest that PNMA5 acquired a certain specialized role in the association areas of the neocortex during primate evolution.

  15. Preferentially Expressed Antigen of Melanoma (PRAME and Wilms’ Tumor 1 (WT 1 Genes Expression in Childhood Acute Lymphoblastic Leukemia, Prognostic Role and Correlation with Survival

    Directory of Open Access Journals (Sweden)

    Engy El Khateeb

    2015-03-01

    CONCLUSION: It is concluded that the expression of PRAME and WT1 genes are indicators of favorable prognosis and can be useful tools for monitoring minimal residual disease (MRD in acute leukemia especially in patients without known genetic markers. Differential expression between acute leukemia patients and healthy volunteers suggests that the immunogenic antigens (PRAME and WT1 are potential candidates for immunotherapy in childhood acute leukemia.

  16. [Cloning, expression and antigenic analysis of VP1-VP4 gene encoding the structural protein of Coxsackie virus A16].

    Science.gov (United States)

    Song, Yuanbin; He, Sijie; Yu, Nan; Chen, Xinxin; Wang, Bin; Che, Xiaoyan; Zeng, Qiyi

    2012-12-01

    To clone and express VP1-VP4 genes encoding the structural proteins of Coxsackie virus A16 and analyze the antigenicity of the expressed recombinant proteins. The VP1-VP4 cDNAs were amplified with RT-PCR from the extracted viral RNA and cloned into pMD19-T vectors. The VP1-VP4 genes were inserted to the multi-cloning sites of the plasmid pQE30a, and the protein expressions in E. coli M15 were induced by IPTG. After purification by washing with 8 mol/L urea under denaturing condition, the recombinant proteins were identified by Western blotting and ELISA for their immunogenicity against rabbit antisera of Coxsackie virus A16 and enterovirus 71, respectively. The recombinant VP1-VP4 proteins were highly expressed in E. coli M15. The purified proteins could be recognized by rabbit antiserum of Coxsackie virus A16 and showed cross reactivity with the rabbit antiserum of Enterovirus 71. The recombinant Coxsackie virus A16 VP1-VP4 proteins obtained possess good antigenicity.

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

    Science.gov (United States)

    Rama, Ana R; Hernandez, Rosa; Perazzoli, Gloria; Burgos, Miguel; Melguizo, Consolación; Vélez, Celia; Prados, Jose

    2015-06-04

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  19. Leishmania infantum: gene cloning of the GRP94 homologue, its expression as recombinant protein, and analysis of antigenicity.

    Science.gov (United States)

    Larreta, R; Soto, M; Alonso, C; Requena, J M

    2000-10-01

    The complete nucleotide sequence for the Leishmania infantum homologue to the glucose-regulated protein 94 (GRP94) gene was determined from the isolation and characterization of a genomic clone. Like the mammalian and plant GRP94s, the L. infantum GRP94 sequence possesses both an N-terminal signal peptide and a putative endoplasmic reticulum retention signal, consisting of the C-terminal tetrapeptide EDDL. Thus, L. infantum is the first protozoan organism in which GRP94 has been identified. Southern blot analysis has indicated that this protein is encoded by a single-copy gene. The L. infantum GRP94 gene was expressed in Escherichia coli and the recombinant protein used to evaluate its antigenicity and immunogenicity. Eighty-four percent of sera from dogs with visceral leishmaniasis reacted with the protein, indicating that GRP94 is a potent immunogen during Leishmania infection. Given the immunogenic and antigenic properties shown by the L. infantum GRP94, we think that this protein constitutes a valuable molecule for diagnostic purposes and a potential candidate for studies of protective immunogenicity. Copyright 2000 Academic Press.

  20. Upregulated Expression of a Unique Gene by Hepatitis B x Antigen Promotes Hepatocellular Growth and Tumorigenesis

    Directory of Open Access Journals (Sweden)

    Zhaorui Lian

    2003-05-01

    Full Text Available Hepatitis B x antigen (HBxAg is a trans-activating protein that may be involved in hepatocarcinogenesis, although few natural effectors of HBxAg that participate in this process have been identified. To identify additional effectors, whole cell RNA isolated from HBxAg-positive and HBxAg-negative HepG2 cells were compared by polymerase chain reaction select cDNA subtraction, and one clone, upregulated gene, clone 11 (URG11, was chosen for further characterization. Elevated levels of URG11 mRNA and protein were observed in HBxAg-positive compared to HBxAg-negative HepG2 cells. Costaining was observed in infected liver (P<.01. URG11 stimulated cell growth in culture (P<.01, anchorage-independent growth in soft agar (P<.001, and accelerated tumor formation (P<.01, and yielded larger tumors (P<.02 in SCID mice injected subcutaneously with HepG2 cells. These data suggest that URG11 is a natural effector of HBxAg that may promote the development of hepatocellular carcinoma.

  1. Oral Administration of Lactococcus lactis Expressing Synthetic Genes of Myelin Antigens in Decreasing Experimental Autoimmune Encephalomyelitis in Rats.

    Science.gov (United States)

    Kasarello, Kaja; Kwiatkowska-Patzer, Barbara; Lipkowski, Andrzej W; Bardowski, Jacek K; Szczepankowska, Agnieszka K

    2015-05-31

    Multiple sclerosis is a human autoimmunological disease that causes neurodegeneration. One of the potential ways to stop its development is induction of oral tolerance, whose effect lies in decreasing immune response to the fed antigen. It was shown in animal models that administration of specific epitopes of the three main myelin proteins - myelin oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP), and proteolipid protein (PLP) - results in induction of oral tolerance and suppression of disease symptoms. Use of bacterial cells to produce and deliver antigens to gut mucosa seems to be an attractive method for oral tolerance induction in treatment of diseases with autoimmune background. Synthetic genes of MOG35-55, MBP85-97, and PLP139-151 myelin epitopes were generated and cloned in Lactococcus lactis under a CcpA-regulated promoter. The tolerogenic effect of bacterial preparations was tested on experimental autoimmune encephalomyelitis, which is the animal model of MS. EAE was induced in rats by intradermal injection of guinea pig spinal cord homogenate into hind paws. Rats were administered preparations containing whole-cell lysates of L. lactis producing myelin antigens using different feeding schemes. Our study demonstrates that 20-fold, but not 4-fold, intragastric administration of autoantigen-expressing L. lactis cells under specific conditions reduces the clinical symptoms of EAE in rats. The present study evaluated the use of myelin antigens produced in L. lactis in inhibiting the onset of experimental autoimmune encephalomyelitis in rats. Obtained results indicate that application of such recombinant cells can be an attractive method of oral tolerance induction.

  2. Enhanced expression of HIV and SIV vaccine antigens in the structural gene region of live attenuated rubella viral vectors and their incorporation into virions.

    Science.gov (United States)

    Virnik, Konstantin; Ni, Yisheng; Berkower, Ira

    2013-04-19

    Despite the urgent need for an HIV vaccine, its development has been hindered by virus variability, weak immunogenicity of conserved epitopes, and limited durability of the immune response. For other viruses, difficulties with immunogenicity were overcome by developing live attenuated vaccine strains. However, there is no reliable method of attenuation for HIV, and an attenuated strain would risk reversion to wild type. We have developed rubella viral vectors, based on the live attenuated vaccine strain RA27/3, which are capable of expressing important HIV and SIV vaccine antigens. The rubella vaccine strain has demonstrated safety, immunogenicity, and long lasting protection in millions of children. Rubella vectors combine the growth and immunogenicity of live rubella vaccine with the antigenicity of HIV or SIV inserts. This is the first report showing that live attenuated rubella vectors can stably express HIV and SIV vaccine antigens at an insertion site located within the structural gene region. Unlike the Not I site described previously, the new site accommodates a broader range of vaccine antigens without interfering with essential viral functions. In addition, antigens expressed at the structural site were controlled by the strong subgenomic promoter, resulting in higher levels and longer duration of antigen expression. The inserts were expressed as part of the structural polyprotein, processed to free antigen, and incorporated into rubella virions. The rubella vaccine strain readily infects rhesus macaques, and these animals will be the model of choice for testing vector growth in vivo and immunogenicity. Published by Elsevier Ltd.

  3. Molecular interplay between T-Antigen and splicing factor, arginine/serine-rich 1 (SRSF1) controls JC virus gene expression in glial cells.

    Science.gov (United States)

    Craigie, Michael; Regan, Patrick; Otalora, Yolanda-Lopez; Sariyer, Ilker Kudret

    2015-11-24

    Human polyomavirus JCV is the etiologic agent of progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease characterized by lytic infection of glial cells in the central nervous system. PML is seen primarily in immunosuppressed patients and is mainly classified as an AIDS-defining disease. In addition to structural capsid proteins, JCV encodes multiple regulatory proteins, including T-antigen and agnoprotein, which are required for functional lytic infection. Previous studies have suggested that molecular interaction between viral proteins and host factors play an important role in reactivation of JCV and progression of the viral life cycle in glial cells. Recently, serine/arginine rich splicing factor 1 (SRSF1), a cellular alternative splicing factor, was identified as a strong negative regulator of JCV in glial cells. SRSF1 inhibits JCV gene expression and viral replication by directly interacting with viral promoter sequences. Here, we have investigated possible impact of JCV regulatory proteins, T-antigen and agnoprotein, on SRSF1-mediated suppression of JCV gene expression in glial cells. Reporter gene analysis has suggested that T-antigen rescues viral transcriptional suppression mediated by SRSF1. Further analyses have revealed that T-antigen promotes viral gene expression by suppressing SRSF1 gene transcription in glial cells. A subsequent ChIP analysis revealed that T-antigen associates with the promoter region of SRSF1 to induce the transcriptional suppression. These findings have revealed a molecular interplay between cellular SRSF1 and viral T-antigen in controlling JCV gene expression, and may suggest a novel mechanism of JCV reactivation in patients who are at risk of developing PML.

  4. 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.

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

    International Nuclear Information System (INIS)

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

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    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.

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

    DEFF Research Database (Denmark)

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

    2009-01-01

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

  8. New BAGE (B melanoma antigen) genes mapping to the juxtacentromeric regions of human chromosomes 13 and 21 have a cancer/testis expression profile.

    Science.gov (United States)

    Ruault, Myriam; van der Bruggen, Pierre; Brun, Marie-Elisabeth; Boyle, Shelagh; Roizès, Gérard; De Sario, Albertina

    2002-12-01

    A first BAGE (B melanoma antigen) gene, BAGE1, was identified because it encodes a human tumour antigen recognised by a cytolytic T lymphocyte. Here, we characterised five new BAGE genes mapping to the juxtacentromeric regions of human chromosomes 13 and 21 and nine BAGE gene fragments mapping to the juxtacentromeric regions of chromosomes 9, 13, 18, and 21. Genes and gene fragments share extensive regions of 90-99% nucleotide identity. We analysed the expression of BAGE genes on 215 tumours of various histological types and on nine normal tissues. Similar to BAGE1, the new BAGE genes are expressed in melanomas, bladder and lung carcinomas and in a few tumours of other histological types. All the normal tissues were negative, with the exception of testis. Our results show that human juxtacentromeric regions harbour genes, which are transcribed and translated, in addition to gene fragments that are generally not expressed. We suggest that the pattern of expression restricted to cancer/testis is a feature of the few genes mapping to juxtacentromeric regions.

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

    African Journals Online (AJOL)

    The transformed nature of the plants was confirmed by polymerase chain reaction (PCR) analysis and Southern blot hybridization. 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 ...

  10. The mechanism of tissue-restricted antigen gene expression by AIRE.

    Science.gov (United States)

    Zumer, Kristina; Saksela, Kalle; Peterlin, B Matija

    2013-03-15

    The autoimmune regulator is a critical transcription factor for generating central tolerance in the thymus. Recent studies have revealed how the autoimmune regulator targets many otherwise tissue-restricted Ag genes to enable negative selection of autoreactive T cells.

  11. 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

    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......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......-4 expression than healthy controls. Cell surface expression of CTLA-4 on CD4(+) CD25(high) T cells correlated with expression of FOXP3 mRNA in untreated patients and increased significantly with time from most recent injection in patients treated with IFN-β. FOXP3 mRNA expression correlated...

  12. 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.

  13. Expression of differentiation antigens and growth-related genes in normal kidney, autosomal dominant polycystic kidney disease, and renal cell carcinoma.

    Science.gov (United States)

    Klingel, R; Dippold, W; Störkel, S; Meyer zum Büschenfelde, K H; Köhler, H

    1992-01-01

    Cellular differentiation and mRNA levels of genes involved in kidney growth were investigated in normal kidney cells, cyst-lining epithelial cells of polycystic kidney disease, and renal carcinoma cells (RCC). All cells comparatively studied exhibited an antigenic phenotype of proximal tubular cells as shown by the expression of a panel of brush border membrane enzymes and kidney-associated cell surface antigens. The epithelial developmental antigen Exo-1 was expressed in 50% to 80% of cyst-lining epithelia in polycystic kidney tissue and in 20% to 30% of polycystic kidney cells cultured in vitro. Normal kidney cells and RCC were negative under identical culture conditions. The expression of antigen Exo-1 is associated with hyperproliferation in an epithelial tissue compartment composed of cells which have not yet reached their terminal differentiation state. Increased amounts of mRNA of the growth factor receptor system of epidermal growth factor (EGF) receptor and its ligand transforming growth factor (TGF)-alpha were associated with the malignant phenotype of RCC. Increased expression of EGF receptor and TGF-alpha, although less prominent, were also observed in polycystic kidney cells compared with normal kidney cells. In conclusion, the expression of Exo-1 in cyst-lining epithelial cells of autosomal dominant polycystic kidney disease (ADPKD) and the altered regulation of TGF-alpha and EGF receptor in these cells contribute to the hypothesis that hyperproliferation is an underlying pathogenic mechanism of ADPKD.

  14. Gene expression

    International Nuclear Information System (INIS)

    Hildebrand, C.E.; Crawford, B.D.; Walters, R.A.; Enger, M.D.

    1983-01-01

    We prepared probes for isolating functional pieces of the metallothionein locus. The probes enabled a variety of experiments, eventually revealing two mechanisms for metallothionein gene expression, the order of the DNA coding units at the locus, and the location of the gene site in its chromosome. Once the switch regulating metallothionein synthesis was located, it could be joined by recombinant DNA methods to other, unrelated genes, then reintroduced into cells by gene-transfer techniques. The expression of these recombinant genes could then be induced by exposing the cells to Zn 2+ or Cd 2+ . We would thus take advantage of the clearly defined switching properties of the metallothionein gene to manipulate the expression of other, perhaps normally constitutive, genes. Already, despite an incomplete understanding of how the regulatory switch of the metallothionein locus operates, such experiments have been performed successfully

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

    Science.gov (United States)

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

    2017-02-01

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

  16. Expression in bacteria of the gene encoding the gp43 antigen of Paracoccidioides brasiliensis: Immunological reactivity of the recombinant fusion proteins

    OpenAIRE

    Diniz, Susana N.; Carvalho, Katia C.; Cisalpino, Patricia S.; Silveira, Jose F.; Travassos, Luiz Rodolpho [UNIFESP; Puccia, Rosana [UNIFESP

    2002-01-01

    gp43 is the major diagnostic antigen of Paracoccidioides brasiliensis, the agent of paracoccidioidomycosis (PCM) in humans. In the present study, cDNA of the gp43 gene (PbGP43) was obtained by reverse transcriptase PCR, inserted into a pGEX vector in frame with the glutathione S-transferase (GST) gene, and expressed in Escherichia coli as inclusion bodies. Immunoblotting showed that all sera from patients with chronic pulmonary and acute lymphatic forms of PCM reacted with the recombinant fus...

  17. 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.

  18. 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.

  19. Modulation of MHC antigen expression by viruses and oncogenes.

    Science.gov (United States)

    Maudsley, D J; Pound, J D

    1991-12-01

    It is becoming increasingly clear that regulation of MHC antigen expression by viruses and oncogenes, leading to either immune evasion or autoimmunity, is widespread and important in disease. At a recent meeting*, which brought together workers interested in tumour immunology, viral infection and the MHC, a number of mechanisms for the regulation of MHC antigen expression were revealed and the importance of balanced expression of MHC gene products to effective immunity was underlined.

  20. Expression and Antigenic Evaluation of VacA Antigenic Fragment of Helicobacter Pylori

    Science.gov (United States)

    Hasanzadeh, Leila; Ghaznavi-Rad, Ehsanollah; Soufian, Safieh; Farjadi, Vahideh; Abtahi, Hamid

    2013-01-01

    Objective(s) : Helicobacter pylori, a human specific gastric pathogen is a causative agent of chronic active gastritis. The vacuolating cytotoxin (VacA) is an effective virulence factor involved in gastric injury. The aim of this study was to construct a recombinant protein containing antigenic region of VacA gene and determine its antigenicity. Materials and Methods: The antigenic region of VacA gene was detected by bioinformatics methods. The polymerase chain reaction method was used to amplify a highly antigenic region of VacA gene from chromosomal DNA of H. pylori. The eluted product was cloned into the prokaryotic expression vector pET32a. The target protein was expressed in the Escherichia coli BL21 (DE3) pLysS. The bacteria including pET32a-VacA plasmids were induced by IPTG. The antigenicity was finally studied by western blotting using sera of 15 H. pylori infected patients after purification. Results: Enzyme digestion analysis, PCR and DNA sequencing results showed that the target gene was inserted correctly into the recombinant vector. The expressed protein was purified successfully via affinity chromatography. Data indicated that antigenic region of VacA protein from Helicobacter pylori was recognized by all 15 patient’s sera. Conclusion : Our data showed that antigenic region of VacA protein can be expressed by in E. co.li. This protein was recognized by sera patients suffering from H. pylori infection. the recombinant protein has similar epitopes and close antigenic properties to the natural form of this antigen. Recombinant antigenic region of VacA protein also seems to be a promising antigen for protective and serologic diagnosis . PMID:23997913

  1. Expression and Antigenic Evaluation of VacA Antigenic Fragment of Helicobacter Pylori

    Directory of Open Access Journals (Sweden)

    Leila Hasanzadeh

    2013-07-01

    Full Text Available Objective(s: Helicobacter pylori, a human specific gastric pathogen is a causative agent of chronic active gastritis. The vacuolating cytotoxin (VacA is an effective virulence factor involved in gastric injury. The aim of this study was to construct a recombinant protein containing antigenic region of VacA gene and determine its antigenicity.   Materials and Methods: The antigenic region of VacA gene was detected by bioinformatics methods. The polymerase chain reaction method was used to amplify a highly antigenic region of VacA gene from chromosomal DNA of H. pylori. The eluted product was cloned into the prokaryotic expression vector pET32a. The target protein was expressed in the Escherichia coli BL21 (DE3 pLysS. The bacteria including pET32a-VacA plasmids were induced by IPTG. The antigenicity was finally studied by western blotting using sera of 15 H. pylori infected patients after purification. Results: Enzyme digestion analysis, PCR and DNA sequencing results showed that the target gene was inserted correctly into the recombinant vector. The expressed protein was purified successfully via affinity chromatography. Data indicated that antigenic region of VacA protein from Helicobacter pylori was recognized by all 15 patient’s sera. Conclusion : Our data showed that antigenic region of VacA protein can be expressed by in E. co.li. This protein was recognized by sera patients suffering from H. pylori infection. the recombinant protein has similar epitopes and close antigenic properties to the natural form of this antigen. Recombinant antigenic region of VacA protein also seems to be a promising antigen for protective and serologic diagnosis .

  2. In Vitro Variant Surface Antigen Expression in Plasmodium falciparum Parasites from a Semi-Immune Individual Is Not Correlated with Var Gene Transcription

    Science.gov (United States)

    Tschan, Serena; Flötenmeyer, Matthias; Koch, Iris; Berger, Jürgen; Kremsner, Peter; Frank, Matthias

    2016-01-01

    Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is considered to be the main variant surface antigen (VSA) of Plasmodium falciparum and is mainly localized on electron-dense knobs in the membrane of the infected erythrocyte. Switches in PfEMP1 expression provide the basis for antigenic variation and are thought to be critical for parasite persistence during chronic infections. Recently, strain transcending anti-PfEMP1 immunity has been shown to develop early in life, challenging the role of PfEMP1 in antigenic variation during chronic infections. In this work we investigate how P. falciparum achieves persistence during a chronic asymptomatic infection. The infected individual (MOA) was parasitemic for 42 days and multilocus var gene genotyping showed persistence of the same parasite population throughout the infection. Parasites from the beginning of the infection were adapted to tissue culture and cloned by limiting dilution. Flow cytometry using convalescent serum detected a variable surface recognition signal on isogenic clonal parasites. Quantitative real-time PCR with a field isolate specific var gene primer set showed that the surface recognition signal was not correlated with transcription of individual var genes. Strain transcending anti-PfEMP1 immunity of the convalescent serum was demonstrated with CD36 selected and PfEMP1 knock-down NF54 clones. In contrast, knock-down of PfEMP1 did not have an effect on the antibody recognition signal in MOA clones. Trypsinisation of the membrane surface proteins abolished the surface recognition signal and immune electron microscopy revealed that antibodies from the convalescent serum bound to membrane areas without knobs and with knobs. Together the data indicate that PfEMP1 is not the main variable surface antigen during a chronic infection and suggest a role for trypsin sensitive non-PfEMP1 VSAs for parasite persistence in chronic infections. PMID:27907004

  3. High homogeneity of MAGE, BAGE, GAGE, tyrosinase and Melan-A/MART-1 gene expression in clusters of multiple simultaneous metastases of human melanoma: implications for protocol design of therapeutic antigen-specific vaccination strategies.

    Science.gov (United States)

    Dalerba, P; Ricci, A; Russo, V; Rigatti, D; Nicotra, M R; Mottolese, M; Bordignon, C; Natali, P G; Traversari, C

    1998-07-17

    Human melanoma cells express several antigens which are recognized by autologous and specific CTL clones in association with HLA-class-I molecules. Many of these antigens represent suitable targets for tumor immunotherapy, since their expression in human melanoma cells is common and highly specific. In order to achieve real clinical success with therapeutic vaccination strategies, one important requirement is the expression of the target antigen by all the tumor lesions of a patient. We have studied this issue by assessing, through an RT-PCR approach, the expression of MAGE-1, MAGE-2, MAGE-3, BAGE, GAGE-1/2, Tyrosinase and Melan-A/MART-1 genes in 17 clusters of simultaneous in-transit or regional lymph-node metastases collected from 15 stage-III and 1 stage-IV (AJCC/UICC pTNM system) melanoma patients. In 14 out of 17 clusters of simultaneous metastatic lesions (82%), the homogeneity in the pattern of gene expression within the cluster was complete. Heterogeneity within the same cluster was observed in only 3 out of 17 clusters (18%) and represented only minor features. Our data reveal that, in AJCC-stage-III melanoma patients, different but simultaneous metastatic lesions express the same pattern of antigen-coding genes. These observations have 2 main clinical implications: (i) the antigenic characterization of one single and easily accessible lesion allows identification of optimal targets for an active antigen-specific immunotherapy treatment; (ii) almost all the metastatic lesions are expected to be hit by the immune response eventually induced against the tumor antigen. Moreover, these data suggest that active specific immunotherapy directed against MAGE-1, MAGE-3, BAGE, GAGE-1/2, Melan-A/MART-1 and Tyrosinase antigens could be exploited as an adjuvant treatment to surgery in high-risk AJCC-stage-III-melanoma patients.

  4. gene structure, gene expression

    Indian Academy of Sciences (India)

    and seedling leaves were sampled at 6 h after the treatment. For cold stress, the seedlings were transferred to 4◦C growth chamber for 30 min. Control seedlings were exposed to none of these treatments. To examine the expression patterns of these predicted genes in Poplar and to further confirm their stress responsive-.

  5. Genomic organization, splice variants and expression of CGM1, a CD66-related member of the carcinoembryonic antigen gene family

    NARCIS (Netherlands)

    Nagel, G.; Grunert, F.; Kuijpers, T. W.; Watt, S. M.; Thompson, J.; Zimmermann, W.

    1993-01-01

    The tumor marker carcinoembryonic antigen (CEA) belongs to a family of proteins which are composed of one immunoglobulin variable domain and a varying number of immunoglobulin constant-like domains. Most of the membrane-bound members, which are anchored either by a glycosylphosphatidylinositol

  6. Oral Administration of Lactococcus lactis Expressing Synthetic Genes of Myelin Antigens in Decreasing Experimental Autoimmune Encephalomyelitis in Rats

    OpenAIRE

    Kasarello, Kaja; Kwiatkowska-Patzer, Barbara; Lipkowski, Andrzej W.; Bardowski, Jacek K.; Szczepankowska, Agnieszka K.

    2015-01-01

    Background Multiple sclerosis is a human autoimmunological disease that causes neurodegeneration. One of the potential ways to stop its development is induction of oral tolerance, whose effect lies in decreasing immune response to the fed antigen. It was shown in animal models that administration of specific epitopes of the three main myelin proteins ? myelin oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP), and proteolipid protein (PLP) ? results in induction of oral tolerance ...

  7. Cloning and Expression of Genes for Dengue Virus Type-2 Encoded-Antigens for Rapid Diagnosis and Vaccine Development

    Science.gov (United States)

    1990-12-12

    problem associated with monovalent dengue vaccines is that individuals infected with one serotype are fully susceptible to infection with other...replication and virion assembly. Use of synthetic peptides encoding the epitopes of viral antigens recognized by host immune system has augmented our...Della-Porta and Westaway, 1977; Kitano et al., 1974; Heinz et al., 1981). In order to develop a subunit vaccine against dengue virus, it is important to

  8. Partial Characterisation of Salmonella gallinarum Clinical Isolate and Expression of Its Antigenic Outer Membrane Protein C (OmpC Gene In Planta

    Directory of Open Access Journals (Sweden)

    Ee Leen Pang

    2015-05-01

    Full Text Available Fowl typhoid’s epidemiology and disease intervention have been extensively studied since 1950's owing to its high mortality and morbidity rates. Even up-to-date, outbreaks are incessantly haunting poultry industries of major continents. Salmonella gallinarum, the etiologic agent of fowl typhoid, was used to develop a series of vaccination regime. However, treatments are gradually losing effectiveness due to residual virulence from mutated strains and rapid evolution of multi-drug resistance isolates. Hence, in planta subunit vaccine production is proposed to surpass current limitations. The homotrimeric osmoporin (outer membrane protein C is a potent candidate antigen that confers momentous stimulation of humoral and cell-mediated immune responses in broilers. This research signified the potential development of a plant-expressed OmpC immunogen. The project scope embarked on the identification of S. gallinarum clinical isolate, construction of expression cassette and delivery of constructs into Nicotiana benthamiana via agroinfiltration. The OmpC transcripts and proteins were detected successfully at the molecular weights of ~1.002 kbp and ~35 kDa, respectively. These preliminary findings pave the feasibility of biomanufacturing a safe and cost-effective fowl typhoid vaccine that would confer multi-protection against other significant Salmonella infections attributed to the high sequence homology of the OmpC gene. Speed improvement is demonstrated and transient expression appears to outperform conventional platforms in expediting vaccine production for an emerging pandemic strain.

  9. 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.

  10. Edible bird's nest impact on rats' uterine histomorphology, expressions of genes of growth factors and proliferating cell nuclear antigen, and oxidative stress level

    Directory of Open Access Journals (Sweden)

    Abdulla A. Albishtue

    2018-01-01

    Full Text Available Aim: This study aimed to evaluate the effect of edible bird's nest (EBN supplementation on the uteri of rats based on analyses of the morphological and histomorphometric changes, and expressions of epidermal growth factor (EGF and its receptor (REGF genes, vascular endothelial growth factor (VEGF, proliferating cell nuclear antigen (PCNA, and steroid receptors. Materials and Methods: Twenty-four Sprague Dawley rats were equally distributed into the following four groups: G1 (control, G2, G3, and G4 represented the groups treated with EBN at graded concentrations of 0, 30, 60, and 120 mg/kg body weight (BW per day for 8 weeks, respectively. During the experimental period, the BW of each rat was recorded weekly. At the proestrus stage of estrous cycle, blood samples were collected from the hearts of anesthetized rats that were later sacrificed. The uteri were removed for histological and immunohistochemical analyses. Results: The EBN-treated groups showed an increase in the weights and lengths of uteri as compared to the control. Results showed that relative to G1 and G2, G3 and G4 exhibited proliferation in their uterine luminal and glandular epithelia and uterine glands, and up-regulated expressions of EGF, REGF, VEGF, PCNA, and progesterone receptor, and estrogen receptor in their uteri. The EBN increased the antioxidant (AO and total AO capacities and reduced the oxidative stress (OS levels in non-pregnant rats. Conclusion: Findings of this study revealed that EBN promotes proliferation of the uterine structures as evidenced by the upregulation of the expressions of steroid receptors,EGF, REGF, VEGF, and PCNA in the uterus and increased in the plasma concentrations of AO and reduced levels of OS.

  11. Immunogenic profiling in mice of a HIV/AIDS vaccine candidate (MVA-B expressing four HIV-1 antigens and potentiation by specific gene deletions.

    Directory of Open Access Journals (Sweden)

    Juan García-Arriaza

    Full Text Available BACKGROUND: The immune parameters of HIV/AIDS vaccine candidates that might be relevant in protection against HIV-1 infection are still undefined. The highly attenuated poxvirus strain MVA is one of the most promising vectors to be use as HIV-1 vaccine. We have previously described a recombinant MVA expressing HIV-1 Env, Gag, Pol and Nef antigens from clade B (referred as MVA-B, that induced HIV-1-specific immune responses in different animal models and gene signatures in human dendritic cells (DCs with immunoregulatory function. METHODOLOGY/PRINCIPAL FINDINGS: In an effort to characterize in more detail the immunogenic profile of MVA-B and to improve its immunogenicity we have generated a new vector lacking two genes (A41L and B16R, known to counteract host immune responses by blocking the action of CC-chemokines and of interleukin 1beta, respectively (referred as MVA-B DeltaA41L/DeltaB16R. A DNA prime/MVA boost immunization protocol was used to compare the adaptive and memory HIV-1 specific immune responses induced in mice by the parental MVA-B and by the double deletion mutant MVA-B DeltaA41L/DeltaB16R. Flow cytometry analysis revealed that both vectors triggered HIV-1-specific CD4(+ and CD8(+ T cells, with the CD8(+ T-cell compartment responsible for >91.9% of the total HIV-1 responses in both immunization groups. However, MVA-B DeltaA41L/DeltaB16R enhanced the magnitude and polyfunctionality of the HIV-1-specific CD4(+ and CD8(+ T-cell immune responses. HIV-1-specific CD4(+ T-cell responses were polyfunctional and preferentially Env-specific in both immunization groups. Significantly, while MVA-B induced preferentially Env-specific CD8(+ T-cell responses, MVA-B DeltaA41L/DeltaB16R induced more GPN-specific CD8(+ T-cell responses, with an enhanced polyfunctional pattern. Both vectors were capable of producing similar levels of antibodies against Env. CONCLUSIONS/SIGNIFICANCE: These findings revealed that MVA-B and MVA-B DeltaA41L/DeltaB16R

  12. Transgenic tomato plants expressing the antigen gene PfCP-2.9 of Plasmodium falciparum Plantas transgênicas de tomate expressando o gene do antígeno PfCP-2.9 de 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.O objetivo deste trabalho foi obter plantas transgênicas de tomate que expressem a proteína PfCP-2.9 (uma quimera dos antígenos MSP1 e AMA1 de Plasmodium falciparum. Cotilédones de tomate, cultivar Summers, com sete dias de idade, foram transformados via Agrobacterium tumefaciens. A expressão transgênica nas plantas T0 foi verificada no DNA extraído dos frutos. A análise por PCR foi utilizada para testar a presença do gene de interesse na geração T1. A evidência da expressão do gene no RNA foi constatada por meio da PCR de transcriptase reversa, e a análise "Western blot" confirmou a presença da proteína de interesse nas plantas T1. Este é o primeiro relato de transformação bem sucedida com a expressão de um antígeno da malária (PfCP-2,9 em plantas transgênicas de tomate da geração T0 e T1.

  13. 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.

  14. 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.

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

    International Nuclear Information System (INIS)

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

    1984-01-01

    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

  16. Antigenic structures stably expressed by recombinant TGEV-derived vectors.

    Science.gov (United States)

    Becares, Martina; Sanchez, Carlos M; Sola, Isabel; Enjuanes, Luis; Zuñiga, Sonia

    2014-09-01

    Coronaviruses (CoVs) are positive-stranded RNA viruses with potential as immunization vectors, expressing high levels of heterologous genes and eliciting both secretory and systemic immune responses. Nevertheless, its high recombination rate may result in the loss of the full-length foreign gene, limiting their use as vectors. Transmissible gastroenteritis virus (TGEV) was engineered to express porcine reproductive and respiratory syndrome virus (PRRSV) small protein domains, as a strategy to improve heterologous gene stability. After serial passage in tissue cultures, stable expression of small PRRSV protein antigenic domains was achieved. Therefore, size reduction of the heterologous genes inserted in CoV-derived vectors led to the stable expression of antigenic domains. Immunization of piglets with these TGEV vectors led to partial protection against a challenge with a virulent PRRSV strain, as immunized animals showed reduced clinical signs and lung damage. Further improvement of TGEV-derived vectors will require the engineering of vectors with decreased recombination rate. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Enhance the anti-renca carcinoma effect of a DNA vaccine targeting G250 gene by co-expression with cytotoxic T-lymphocyte associated antigen-4(CTLA-4).

    Science.gov (United States)

    Zhao, Youguang; Wei, Zhitao; Yang, Hang; Li, Xiaowei; Wang, Qiwu; Wang, Liang; Li, Shadan

    2017-06-01

    Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is a negative regulator of T cell activation, which competes with CD28 for B7.1/B7.2 binding with a greater affinity. Co-expression specific antigens and extracellular domain of CTLA4 represents a promising approach to increase the immunogenicity of DNA vaccines. In this study, we evaluated this interesting approach for its enhancement on G250/MN/CA IX (G250)-specific immune responses and its anti-tumor effects in renal carcinoma mice model. Consequently, we constructed a DNA vaccine containing the G250 and the CTLA-4 gene. Vaccination with the co-expression DNA not only induced much higher level of anti-CTLA4 and anti-G250 antibody, but also increased G250-specific T cell response in mice. To evaluate the anti-tumor efficacy of the plasmids, murine models with G250-expressing tumors were generated. After injection into the tumor-bearing mouse model, the plasmid carrying the co-expression gene of CTLA4 and G250 showed stronger inhibition of tumor growth than the plasmid expressing CTLA4 or G250 alone. These observations emphasize the potential of the CTLA4 and G250 co-expression DNA vaccine, which could represent a promising approach for tumor immunotherapy. Copyright © 2017. Published by Elsevier Masson SAS.

  18. Development of gene expression assays measuring immune ...

    African Journals Online (AJOL)

    Using qPCR, the relative expression stability of the reference genes ACTB, GAPDH, YWHAZ and TBP in these samples was determined as well as the mean fold change in the expression of IFNG, CXCL8, CXCL9, CXCL10 and CXCL11 in M. bovis-antigen stimulated blood. The expression of YWHAZ and TBP showed ...

  19. 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

  20. Recombinant carcinoembryonic antigen as a reporter gene for molecular imaging

    Energy Technology Data Exchange (ETDEWEB)

    Kenanova, Vania; Barat, Bhaswati; Olafsen, Tove; Chatziioannou, Arion; Herschman, Harvey R.; Wu, Anna M. [David Geffen School of Medicine at the University of California Los Angeles, Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, Los Angeles, CA (United States); Braun, Jonathan [David Geffen School of Medicine at the University of California Los Angeles, Department of Pathology and Laboratory Medicine, Los Angeles, CA (United States)

    2009-01-15

    Reporter genes can provide a way of noninvasively assessing gene activity in vivo. However, current reporter gene strategies may be limited by the immunogenicity of foreign reporter proteins, endogenous expression, or unwanted biological activity. We have developed a reporter gene based on carcinoembryonic antigen (CEA), a human protein with limited normal tissue expression. To construct a CEA reporter gene for PET, a CEA minigene (N-A3) was fused to the extracellular and transmembrane domains of the human Fc{gamma}RIIb receptor. The NA3-Fc{gamma}RIIb recombinant gene, driven by a CMV promoter, was transfected in Jurkat (human T cell leukemia) cells. Expression was analyzed by flow cytometry, immunohistochemistry (IHC), and microPET imaging. Flow cytometry identified Jurkat clones stably expressing NA3-Fc{gamma}RIIb at low, medium, and high levels. High and medium NA3-Fc{gamma}RIIb expression could also be detected by Western blot. Reporter gene positive and negative Jurkat cells were used to establish xenografts in athymic mice. IHC showed staining of the tumor with high reporter gene expression; medium and low N-A3 expression was not detected. MicroPET imaging, using an anti-CEA {sup 124}I-labeled single-chain Fv-Fc antibody fragment, demonstrated that only high N-A3 expression could be detected. Specific accumulation of activity was visualized at the N-A3 positive tumor as early as 4 h. MicroPET image quantitation showed tumor activity of 1.8 {+-} 0.2, 15.2 {+-} 1.3, and 4.6 {+-} 1.2 percent injected dose per gram (%ID/g) at 4, 20, and 48 h, respectively. Biodistribution at 48 h demonstrated tumor uptake of 4.8 {+-} 0.8%ID/g. The CEA N-A3 minigene has the potential to be used as a reporter gene for imaging cells in vivo. (orig.)

  1. Genomic Methylation Inhibits Expression of Hepatitis B Virus Envelope Protein in Transgenic Mice: A Non-Infectious Mouse Model to Study Silencing of HBV Surface Antigen Genes.

    Science.gov (United States)

    Graumann, Franziska; Churin, Yuri; Tschuschner, Annette; Reifenberg, Kurt; Glebe, Dieter; Roderfeld, Martin; Roeb, Elke

    2015-01-01

    The Hepatitis B virus genome persists in the nucleus of virus infected hepatocytes where it serves as template for viral mRNA synthesis. Epigenetic modifications, including methylation of the CpG islands contribute to the regulation of viral gene expression. The present study investigates the effects of spontaneous age dependent loss of hepatitis B surface protein- (HBs) expression due to HBV-genome specific methylation as well as its proximate positive effects in HBs transgenic mice. Liver and serum of HBs transgenic mice aged 5-33 weeks were analyzed by Western blot, immunohistochemistry, serum analysis, PCR, and qRT-PCR. From the third month of age hepatic loss of HBs was observed in 20% of transgenic mice. The size of HBs-free area and the relative number of animals with these effects increased with age and struck about 55% of animals aged 33 weeks. Loss of HBs-expression was strongly correlated with amelioration of serum parameters ALT and AST. In addition lower HBs-expression went on with decreased ER-stress. The loss of surface protein expression started on transcriptional level and appeared to be regulated epigenetically by DNA methylation. The amount of the HBs-expression correlated negatively with methylation of HBV DNA in the mouse genome. Our data suggest that methylation of specific CpG sites controls gene expression even in HBs-transgenic mice with truncated HBV genome. More important, the loss of HBs expression and intracellular aggregation ameliorated cell stress and liver integrity. Thus, targeted modulation of HBs expression may offer new therapeutic approaches. Furthermore, HBs-transgenic mice depict a non-infectious mouse model to study one possible mechanism of HBs gene silencing by hypermethylation.

  2. Recombinant gene expression protocols

    National Research Council Canada - National Science Library

    Tuan, Rocky S

    1997-01-01

    .... A fundamental requirement for successful recombinant gene expression is the design of the cloning vector and the choice of the host organism for expression. Recombinant Gene Expression Protocols grows out of the need for a laboratory manual that provides the reader the background and rationale, as well as the practical protocols for the preparation of...

  3. Simultaneous expression of growth hormone releasing hormone (GHRH) and hepatitis B surface antigen/somatostatin (HBsAg/SS) fusion genes in a construct in the skeletal muscle enhances rabbit weight gain.

    Science.gov (United States)

    Dai, Jian-wei; Liu, Song-cai; Hao, Lin-lin; Zhang, Yong-liang; Zhang, Qianqian; Ren, Xiao-hui; Jiang, Qing-yan

    2008-01-01

    Somatostatin (SS) and growth hormone-releasing hormone (GHRH) are synthesized and secreted by the hypothalamus, which can control the synthesis and secretion of the growth hormone (GH) from the hypophysis as well as regulate the GH concentrations in animals and humans. In this article, we describe the regulation of animal growth using plasmid DNA encoding both the GHRH gene and the SS gene fused with the hepatitis B surface antigen (HBsAg) gene. We constructed a series of expression plasmids to express the GHRH and HBsAg-SS fusion genes individually as well as collectively. The fusion gene and GHRH were successfully expressed in Chinese hamster ovary (CHO) cells, as proven by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblotting tests. Poly D, L-lactide-co-glycolic acid (PLGA) plasmid-encapsulating microspheres were prepared and injected intramuscularly into the leg skeletal muscles of rabbits. Weight gain/day and the levels of insulinlike growth factor-I (IGF-I), SS, and hepatitis B surface antibody (HBsAb) were monitored. During days 30 postinjection, increase in weight gain/day and IGF- I concentration and decrease in SS were observed in treatment groups. From days 15 to 30 postinjection, the weight gain/day significantly increased (P gain/day (P gain/day in rabbits.

  4. Subcloning and Expression of Recombinant Echinococcus granulosus Antigen B, in Pqe-30 Expression Vector

    Directory of Open Access Journals (Sweden)

    A Haghighi

    2009-12-01

    Full Text Available "nBackground: Echinococcosis or hydatid disease is a zoonotic infection caused by larval (metacestode stages of cestodes belonging to the genus Echinococcus, family Taeniidae. We aimed to subclone antigen B gene in pQE-30 plasmid, its expression, and purification."nMethods: We subcloned HI gene into pQE-30 expression vector. The recombinant vector was transformed into E. coli, M15 and mass cultured. The subcloned gene was expressed by IPTG. Subcloning of gene was confirmed by both PCR and enzyme digestion."nResults: Production of recombinant protein was confirmed by SDS-PAGE. Western blot analysis was carried out by both His-Tag monoclonal Ab and human serum to estimate the expressed protein in E. coli cells. Recombinant protein was purified and its specificity was proved by Western blotting."nConclusion: Production of this recombinant protein can increase sensitivity and specificity in serological test (ELISA.

  5. N-myc amplification causes down-modulation of MHC class I antigen expression in neuroblastoma

    NARCIS (Netherlands)

    Bernards, R.A.; Dessain, S.K.; Weinberg, R.A.

    1986-01-01

    Amplification of the N-myc gene is correlated with increased metastatic ability of human neuroblastomas. We show here that overexpression of the N-myc gene in a rat neuroblastoma cell line following gene transfer causes down-modulation of class I histocompatibility antigen expression and increases

  6. Protein antigen delivery by gene gun-mediated epidermal antigen incorporation (EAI).

    Science.gov (United States)

    Scheiblhofer, Sandra; Ritter, Uwe; Thalhamer, Josef; Weiss, Richard

    2013-01-01

    The gene gun technology can not only be employed for efficient transfer of gene vaccines into upper layers of the skin, but also for application of protein antigens. As a tissue rich in professional antigen presenting cells, the skin represents an attractive target for immunizations. In this chapter we present a method for delivery of the model antigen ovalbumin into the skin of mice termed epidermal antigen incorporation and describe in detail how antigen-specific proliferation in draining lymph nodes can be followed by flow cytometry.

  7. Genetical and functional investigation of fliC genes encoding flagellar serotype H4 in wildtype strains of Escherichia coli and in a laboratory E. coli K-12 strain expressing flagellar antigen type H48

    Directory of Open Access Journals (Sweden)

    Schaudinn Christoph

    2005-01-01

    Full Text Available Abstract Background Serotyping of O-(lipopolysaccharide and H-(flagellar antigens is a wideley used method for identification of pathogenic strains and clones of Escherichia coli. At present, 176 O- and 53 H-antigens are described for E. coli which occur in different combinations in the strains. The flagellar antigen H4 is widely present in E. coli strains of different O-serotypes and pathotypes and we have investigated the genetic relationship between H4 encoding fliC genes by PCR, nucleotide sequencing and expression studies. Results The complete nucleotide sequence of fliC genes present in E. coli reference strains U9-41 (O2:K1:H4 and P12b (O15:H17 was determined and both were found 99.3% (1043 of 1050 nucleotides identical in their coding sequence. A PCR/RFLP protocol was developed for typing of fliC-H4 strains and 88 E. coli strains reacting with H4 antiserum were investigated. Nucleotide sequencing of complete fliC genes of six E. coli strains which were selected based on serum agglutination titers, fliC-PCR genotyping and reference data revealed 96.6 to 100% identity on the amino acid level. The functional expression of flagellin encoded by fliC-H4 from strain U9-41 and from our strain P12b which is an H4 expressing variant type was investigated in the E. coli K-12 strain JM109 which encodes flagellar type H48. The fliC recombinant plasmid carrying JM109 strains reacted with both H4 and H48 specific antisera whereas JM109 reacted only with the H48 antiserum. By immunoelectron microscopy, we could show that the flagella made by the fliC-H4 recombinant plasmid carrying strain are constituted of H48 and H4 flagellins which are co-assembled into functional flagella. Conclusion The flagellar serotype H4 is encoded by closely related fliC genes present in serologically different types of E. coli strainswhich were isolated at different time periods and geographical locations. Our expression studies show for the first time, that flagellins of

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1986-10-01

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

  9. Cancer-testis antigen expression in bladder cancer.

    Science.gov (United States)

    Fradet, Yves; Picard, V; Bergeron, A; LaRue, H

    2005-12-01

    To evaluate the potential of cCancer-t/Testis antigens (CTAs) as targets for immunotherapy of bladder cancer, we evaluated the expression of 9 CTA genes or families of genes in normal urothelia, bladder tumours and bladder cancer human bladder tissuescell lines. As expression of most CTAs is controlled by epigenetic mechanisms, we also evaluated the effect of the DNA methylase inhibitor 5-aza-2'-deoxycytidine (5-AZA-DC), and/or theand histone deacetylase inhibitors Trichostatin A (TSA) on their expression in bladder cancer cell lines. Expression of NY-ESO-1/LAGE-1, MAGE-A, MAGE-C1, BAGE, HOM-TES-85, SCP-1, SSX-1, SSX-2 and SSX-4 was analyzed by semi-quantitative RT-PCR and Western blotting on 10 normal urothelia, 23 24 superficial and 223 invasive tumours and on 10 cell lines treated with 5-aza-2'-deoxycytidine (5-AZA-DC) and/or Trichostatin A (TSA). Expression of all CTA genes could be observed in at least 1 tumour except for HOM-TES-85 for which mRNA was never detected. MAGE-A, BAGE and NY-ESO-1/LAGE-1 mRNAs were the most frequently detected, respectively in 5677%, 212% and 89% of superficial and in 6461%, 4139% and 276% of invasive tumours. With the exception of MAGE-A, CTA transcripts were rarely detected in the cell lines. However, expression of all CTA genes, except SCP-1, could be induced at various levels by the drugs and 5-AZA-DC was a much more potent inducer than TSA. These data suggest that immunotherapy of bladder cancer could target CTAs, especially those expressed at higher frequency such as MAGE-A, BAGE and NY-ESO-1/LAGE-1. Moreover, their induction by chemotherapeutic agents such as 5-AZA-DC, provides a potential pretreatment aimed at inducing the immunogenicity of the tumours.

  10. Mature IgM-expressing plasma cells sense antigen and develop competence for cytokine production upon antigenic challenge

    Science.gov (United States)

    Blanc, Pascal; Moro-Sibilot, Ludovic; Barthly, Lucas; Jagot, Ferdinand; This, Sébastien; de Bernard, Simon; Buffat, Laurent; Dussurgey, Sébastien; Colisson, Renaud; Hobeika, Elias; Fest, Thierry; Taillardet, Morgan; Thaunat, Olivier; Sicard, Antoine; Mondière, Paul; Genestier, Laurent; Nutt, Stephen L.; Defrance, Thierry

    2016-01-01

    Dogma holds that plasma cells, as opposed to B cells, cannot bind antigen because they have switched from expression of membrane-bound immunoglobulins (Ig) that constitute the B-cell receptor (BCR) to production of the secreted form of immunoglobulins. Here we compare the phenotypical and functional attributes of plasma cells generated by the T-cell-dependent and T-cell-independent forms of the hapten NP. We show that the nature of the secreted Ig isotype, rather than the chemical structure of the immunizing antigen, defines two functionally distinct populations of plasma cells. Fully mature IgM-expressing plasma cells resident in the bone marrow retain expression of a functional BCR, whereas their IgG+ counterparts do not. Antigen boost modifies the gene expression profile of IgM+ plasma cells and initiates a cytokine production program, characterized by upregulation of CCL5 and IL-10. Our results demonstrate that IgM-expressing plasma cells can sense antigen and acquire competence for cytokine production upon antigenic challenge. PMID:27924814

  11. Paired Expression Analysis of Tumor Cell Surface Antigens

    Directory of Open Access Journals (Sweden)

    Rimas J. Orentas

    2017-08-01

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

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

    Science.gov (United States)

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

  13. Identification of cancer/testis-antigen genes by massively parallel signature sequencing.

    Science.gov (United States)

    Chen, Yao-Tseng; Scanlan, Matthew J; Venditti, Charis A; Chua, Ramon; Theiler, Gregory; Stevenson, Brian J; Iseli, Christian; Gure, Ali O; Vasicek, Tom; Strausberg, Robert L; Jongeneel, C Victor; Old, Lloyd J; Simpson, Andrew J G

    2005-05-31

    Massively parallel signature sequencing (MPSS) generates millions of short sequence tags corresponding to transcripts from a single RNA preparation. Most MPSS tags can be unambiguously assigned to genes, thereby generating a comprehensive expression profile of the tissue of origin. From the comparison of MPSS data from 32 normal human tissues, we identified 1,056 genes that are predominantly expressed in the testis. Further evaluation by using MPSS tags from cancer cell lines and EST data from a wide variety of tumors identified 202 of these genes as candidates for encoding cancer/testis (CT) antigens. Of these genes, the expression in normal tissues was assessed by RT-PCR in a subset of 166 intron-containing genes, and those with confirmed testis-predominant expression were further evaluated for their expression in 21 cancer cell lines. Thus, 20 CT or CT-like genes were identified, with several exhibiting expression in five or more of the cancer cell lines examined. One of these genes is a member of a CT gene family that we designated as CT45. The CT45 family comprises six highly similar (>98% cDNA identity) genes that are clustered in tandem within a 125-kb region on Xq26.3. CT45 was found to be frequently expressed in both cancer cell lines and lung cancer specimens. Thus, MPSS analysis has resulted in a significant extension of our knowledge of CT antigens, leading to the discovery of a distinctive X-linked CT-antigen gene family.

  14. 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.

  15. Gene Expression Omnibus (GEO)

    Data.gov (United States)

    U.S. Department of Health & Human Services — Gene Expression Omnibus is a public functional genomics data repository supporting MIAME-compliant submissions of array- and sequence-based data. Tools are provided...

  16. Frequency of mutant T lymphocytes defective in the expression of the T-cell antigen receptor gene among radiation-exposed people

    International Nuclear Information System (INIS)

    Kyoizumi, Seishi; Umeki, Shigeko; Akiyama, Mitoshi

    1991-06-01

    The frequency of mutant T lymphocytes defective in T-cell receptor gene (α or β) expression was measured using the two-color flow cytometric technique. Results for a total of 203 atomic bomb survivors, 78 of whom were proximally exposed (DS86 doses of ≥ 1.5 Gy) and 125 of whom were distally exposed (DS86 doses of 228 Th formerly used for radiodiagnosis. In addition, thyroid disease patients treated with 131 I showed a dose-related increase of mutant frequency. It was suggested that the present T-cell receptor mutation assay has a unique characteristic as a biological dosimeter for the measurement of recent exposures to genotoxic agents. (author)

  17. 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.

  18. A Survey of ABO, Rhesus (D) Antigen and Haemoglobin Genes ...

    African Journals Online (AJOL)

    Summary: A survey of ABO and Rhesus (Rh D) antigens and variants of haemoglobin genes (HbGen) in Oyo state was carried out. This longitudinal study involved the determination of ABO and Rh(D) antigens in 3241 and HbGen in 2622 male and female adults (aged 26-65years) respectively using standard methods.

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

    DEFF Research Database (Denmark)

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

    2009-01-01

    Tumor antigens are the primary target of therapeutic cancer vaccines. We set out to define and compare the expression pattern of tumor antigen genes in esophagus carcinoma biopsies and in an allogeneic tumor lysate-based cancer vaccine, MelCancerVac. Cells used for vaccine production were treated...... with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-CdR) to determine whether this treatment could improve the profile of tumor antigen genes expressed in these cells. In addition, the presence of MAGE-A tumor antigen protein was evaluated in the purified tumor cell lysate used...

  20. Evaluation of melanoma antigen gene A3 expression in drug resistance of epidermal growth factor receptor-tyrosine kinase inhibitors in advanced nonsmall cell lung cancer treatment

    Directory of Open Access Journals (Sweden)

    Ju Jin

    2015-01-01

    Conclusion: MAGE-A3 expression in EGFR-TKIs target therapy in NSCLC patient suggests that there might be EGFR-TKIs drug resistance, and the higher the level of expression, the shorter the time of acquired drug resistance.

  1. MAGE, BAGE and GAGE: tumour antigen expression in benign and malignant ovarian tissue.

    Science.gov (United States)

    Gillespie, A M; Rodgers, S; Wilson, A P; Tidy, J; Rees, R C; Coleman, R E; Murray, A K

    1998-09-01

    To determine if ovarian cancer patients would be suitable for MAGE-peptide vaccine-based immunotherapy, the frequency of expression of the MAGE-1-4 genes in ovarian tumours was assessed using reverse transcription polymerase chain reaction (RT-PCR) and product verification with digoxigenin-labelled oligonucleotide probes specific for each MAGE gene. In addition, the frequency of expression of more recently discovered tumour antigens (BAGE, GAGE -1, -2 and GAGE -3, -6) was established using RT-PCR and ethidium bromide staining. In this study 1/16 normal ovarian tissue specimens and 11/25 benign lesions expressed MAGE-1. In non-malignant tissue there was preferential expression of MAGE-1 in premenopausal women. A total of 15/27 malignant specimens expressed MAGE-1, including 10/14 serous cystadenocarcinomas. Expression of other tumour antigens was infrequent. The finding of MAGE-1 expression in both benign and malignant tissue questions previous assumptions regarding the role of MAGE genes in carcinogenesis. In addition, preferential MAGE-1 gene expression in non-malignant premenopausal tissue suggests that the MAGE genes may be involved in cellular proliferation as opposed to carcinogenesis or possibly that MAGE gene expression is under cyclical hormonal control. Finally, this study indicates that serous cystadenocarcinomas may be suitable tumours for MAGE-1 peptide immunotherapy.

  2. PRAME gene expression profile in medulloblastoma

    Directory of Open Access Journals (Sweden)

    Tânia Maria Vulcani-Freitas

    2011-02-01

    Full Text Available Medulloblastoma is the most common malignant tumors of central nervous system in the childhood. The treatment is severe, harmful and, thus, has a dismal prognosis. As PRAME is present in various cancers, including meduloblastoma, and has limited expression in normal tissues, this antigen can be an ideal vaccine target for tumor immunotherapy. In order to find a potential molecular target, we investigated PRAME expression in medulloblastoma fragments and we compare the results with the clinical features of each patient. Analysis of gene expression was performed by real-time quantitative PCR from 37 tumor samples. The Mann-Whitney test was used to analysis the relationship between gene expression and clinical characteristics. Kaplan-Meier curves were used to evaluate survival. PRAME was overexpressed in 84% samples. But no statistical association was found between clinical features and PRAME overexpression. Despite that PRAME gene could be a strong candidate for immunotherapy since it is highly expressed in medulloblastomas.

  3. Gene expression and gene therapy imaging

    International Nuclear Information System (INIS)

    Rome, Claire; Couillaud, Franck; Moonen, Chrit T.W.

    2007-01-01

    The fast growing field of molecular imaging has achieved major advances in imaging gene expression, an important element of gene therapy. Gene expression imaging is based on specific probes or contrast agents that allow either direct or indirect spatio-temporal evaluation of gene expression. Direct evaluation is possible with, for example, contrast agents that bind directly to a specific target (e.g., receptor). Indirect evaluation may be achieved by using specific substrate probes for a target enzyme. The use of marker genes, also called reporter genes, is an essential element of MI approaches for gene expression in gene therapy. The marker gene may not have a therapeutic role itself, but by coupling the marker gene to a therapeutic gene, expression of the marker gene reports on the expression of the therapeutic gene. Nuclear medicine and optical approaches are highly sensitive (detection of probes in the picomolar range), whereas MRI and ultrasound imaging are less sensitive and require amplification techniques and/or accumulation of contrast agents in enlarged contrast particles. Recently developed MI techniques are particularly relevant for gene therapy. Amongst these are the possibility to track gene therapy vectors such as stem cells, and the techniques that allow spatiotemporal control of gene expression by non-invasive heating (with MRI guided focused ultrasound) and the use of temperature sensitive promoters. (orig.)

  4. BAGE: a new gene encoding an antigen recognized on human melanomas by cytolytic T lymphocytes.

    Science.gov (United States)

    Boël, P; Wildmann, C; Sensi, M L; Brasseur, R; Renauld, J C; Coulie, P; Boon, T; van der Bruggen, P

    1995-02-01

    Several tumor antigens are recognized by autologous cytolytic T lymphocytes (CTL) on human melanoma MZ2-MEL. Some of them are encoded by genes MAGE-1 and MAGE-3, which are not expressed in normal tissues except in testis. Here, we report the identification of a new gene that codes for another of these antigens. This gene, named BAGE, codes for a putative protein of 43 aa and seems to belong to a family of several genes. The antigen recognized by the autologous CTL consists of BAGE-encoded peptide AARAVFLAL bound to an HLA-Cw 1601 molecule. Gene BAGE is expressed in 22% of melanomas, 30% of infiltrating bladder carcinomas, 10% of mammary carcinomas, 8% of head and neck squamous cell carcinomas, and 6% of non-small cell lung carcinomas. Like the MAGE genes, it is silent in normal tissues with the exception of testis. Because of its tumor-specific expression, the BAGE-encoded antigen may prove useful for cancer immunotherapy.

  5. Uterus human leucocyte antigen expression in the perspective of transplantation.

    Science.gov (United States)

    Gauthier, Tristan; Filloux, Matthieu; Guillaudeau, Angelique; Essig, Marie; Bibes, Romain; Pacha, Adam Fodil; Piver, Pascal; Aubard, Yves; Marquet, Pierre; Drouet, Mireille

    2016-12-01

    To describe class I and II human leucocyte antigen (HLA) expression using different uterine tissues in the perspective of uterus transplantation. Human uterine tissues were obtained from 12 women who had undergone hysterectomy for the treatment of benign disease. HLA class I and HLA-antigen D related (DR) expression were assessed via immunochemistry. HLA class I expression in the uterus was compared with expression in other organs and tissues, including kidney and myocardium samples. HLA class I expression was strong in the endometrial glands and mild in the myometrium. Staining of endometrial glands was similar to glomerular staining in the kidney. The myometrium seems to express HLA class I similarly to hepatocytes and myocardial cells. HLA class I expression in the uterus did not differ in younger or post-menopausal women. HLA-DR was expressed in the endometrial glands, but not in the myometrium. A lack of HLA-DR expression seemed to be correlated with cell proliferation. HLA expression in the endometrium and myometrium is different. The endometrium should be the major target of alloreactive response. As for other transplanted organs, assessment of HLA unacceptable antigens and multiple immunosuppressive treatments is necessary in uterus transplantation. © 2016 Japan Society of Obstetrics and Gynecology.

  6. Identification of cancer/testis-antigen genes by massively parallel signature sequencing

    Science.gov (United States)

    Chen, Yao-Tseng; Scanlan, Matthew J.; Venditti, Charis A.; Chua, Ramon; Theiler, Gregory; Stevenson, Brian J.; Iseli, Christian; Gure, Ali O.; Vasicek, Tom; Strausberg, Robert L.; Jongeneel, C. Victor; Old, Lloyd J.; Simpson, Andrew J. G.

    2005-01-01

    Massively parallel signature sequencing (MPSS) generates millions of short sequence tags corresponding to transcripts from a single RNA preparation. Most MPSS tags can be unambiguously assigned to genes, thereby generating a comprehensive expression profile of the tissue of origin. From the comparison of MPSS data from 32 normal human tissues, we identified 1,056 genes that are predominantly expressed in the testis. Further evaluation by using MPSS tags from cancer cell lines and EST data from a wide variety of tumors identified 202 of these genes as candidates for encoding cancer/testis (CT) antigens. Of these genes, the expression in normal tissues was assessed by RT-PCR in a subset of 166 intron-containing genes, and those with confirmed testis-predominant expression were further evaluated for their expression in 21 cancer cell lines. Thus, 20 CT or CT-like genes were identified, with several exhibiting expression in five or more of the cancer cell lines examined. One of these genes is a member of a CT gene family that we designated as CT45. The CT45 family comprises six highly similar (>98% cDNA identity) genes that are clustered in tandem within a 125-kb region on Xq26.3. CT45 was found to be frequently expressed in both cancer cell lines and lung cancer specimens. Thus, MPSS analysis has resulted in a significant extension of our knowledge of CT antigens, leading to the discovery of a distinctive X-linked CT-antigen gene family. PMID:15905330

  7. Quantitation of MHC antigen expression on colorectal tumours and its association with tumour progression.

    OpenAIRE

    Durrant, L. G.; Ballantyne, K. C.; Armitage, N. C.; Robins, R. A.; Marksman, R.; Hardcastle, J. D.; Baldwin, R. W.

    1987-01-01

    A flow cytometric technique has been established for accurately quantitating the cell surface density of MHC antigens and the percentage of cells expressing MHC antigens in 38 colorectal tumours. Thirty-four percent of tumours were partially or completely negative for HLA-ABC antigen expression. Although the quantity of HLA-ABC antigens varied widely, there was no correlation between the density of HLA-ABC antigens, or the percentage of cells expressing these antigens and clinicopathological ...

  8. Multivalent Chromosomal Expression of the Clostridium botulinum Serotype A Neurotoxin Heavy-Chain Antigen and the Bacillus anthracis Protective Antigen in Lactobacillus acidophilus.

    Science.gov (United States)

    O'Flaherty, Sarah; Klaenhammer, Todd R

    2016-10-15

    Clostridium botulinum and Bacillus anthracis produce potent toxins that cause severe disease in humans. New and improved vaccines are needed for both of these pathogens. For mucosal vaccine delivery using lactic acid bacteria, chromosomal expression of antigens is preferred over plasmid-based expression systems, as chromosomal expression circumvents plasmid instability and the need for antibiotic pressure. In this study, we constructed three strains of Lactobacillus acidophilus NCFM expressing from the chromosome (i) the nontoxic host receptor-binding domain of the heavy chain of Clostridium botulinum serotype A neurotoxin (BoNT/A-Hc), (ii) the anthrax protective antigen (PA), and (iii) both the BoNT/A-Hc and the PA. The BoNT/A-Hc vaccine cassette was engineered to contain the signal peptide from the S-layer protein A from L. acidophilus and a dendritic-cell-targeting peptide. A chromosomal region downstream of lba0889 carrying a highly expressed enolase gene was selected for insertion of the vaccine cassettes. Western blot analysis confirmed the heterologous expression of the two antigens from plasmid and chromosome locations. Stability assays demonstrated loss of the vaccine cassettes from expression plasmids without antibiotic maintenance. RNA sequencing showed high expression of each antigen and that insertion of the vaccine cassettes had little to no effect on the transcription of other genes in the chromosome. This study demonstrated that chromosomal integrative recombinant strains are promising vaccine delivery vehicles when targeted into high-expression chromosomal regions. Levels of expression match high-copy-number plasmids and eliminate the requirement for antibiotic selective maintenance of recombinant plasmids. Clostridium botulinum and Bacillus anthracis produce potent neurotoxins that pose a biochemical warfare concern; therefore, effective vaccines against these bacteria are required. Chromosomal expression of antigens is preferred over plasmid

  9. Human leukocyte antigen-E alleles and expression in patients with serous ovarian cancer

    OpenAIRE

    Zheng, Hui; Lu, Renquan; Xie, Suhong; Wen, Xuemei; Wang, Hongling; Gao, Xiang; Guo, Lin

    2015-01-01

    Human leukocyte antigen-E (HLA-E) is one of the most extensively studied non-classical MHC class I molecules that is almost non-polymorphic. Only two alleles (HLA-E*0101 and HLA-E*0103) are found in worldwide populations, and suggested to be functional differences between these variants. The HLA-E molecule can contribute to the escape of cancer cells from host immune surveillance. However, it is still unknown whether HLA-E gene polymorphisms might play a role in cancer immune escape. To explo...

  10. [cDNA library constructing and specific antigen expression of Streptomyces thermohydroscopicus].

    Science.gov (United States)

    Xu, Lei; Wang, Ling-ling; Liu, Shuo; Ling, Yuan; Ma, Lie; Wang, Qun; Zhang, Li-jiao; He, Xiao-yu; Zhao, Ming-jing; Wang, Xiao-ge

    2012-03-01

    To construct a cDNA library from Streptomyces thermohydroscopicus and screen genes with virulence, obtain the recombinant fusion virulence proteins by prokaryotic expression system. The Streptomyces thermohydroscopicus cDNA library was constructed by switching mechanism at 5'end of RNA transcript approach. A total of 1020 clones randomly selected from the cDNA library were sequenced and these expressed sequence tags (EST) were further analyzed for the screen of antigen-specific genes. The two candidate genes were subcloned into expression vector pET-28a. The recombinants were transformed into BL2 and proteins were expressed by the induction of isopropyl-β-D-1-thiogalactopyranoside (IPTG). A high-quality cDNA library from Streptomyces thermohydroscopicus was constructed and a set of 978 valid sequences were obtained. Clustering and assembly of these cDNA sequences resulted in 347 unique genes, among which 2 potential antigen-specific genes were highly allied with outer membrane lipoprotein (51%) and transferring-binding protein B (42%) from Actinobacillus pleuropneumoniae serotype (APP). The open reading frame (ORF) of the two candidate genes are 1554 bp and 726 bp, which coded two peptides with 517 and 241 amino acids, respectively. The molecular weights of the recombinant fusion proteins were 63 000 and 30 000. The cDNA library of Streptomyces thermohydroscopicus reached the quality requirement of gene library. EST database in the library would greatly facilitate further screening of virulence genes.

  11. Nucleotide Sequence of the Protective Antigen Gene of Bacillus Anthracis

    Science.gov (United States)

    1988-02-02

    the bands excised, and the DNA extracted with phenol for cloning in M13. 6 Nuclotida sequence analysis. The two fragments were each cloned into phages ...E.c. ToxA a £scherichia coli heat-labile enterotoxin A gene (45) V.c. CTxA - Vibrio ctolerae cholera toxin alfa subunlt gene (28) atotal nurber of specific amino acid residues deduced from p’otective antigen gene.

  12. Gene Expression in Bone

    Science.gov (United States)

    D'Ambrogio, A.

    Skeletal system has two main functions, to provide mechanical integrity for both locomotion and protection and to play an important role in mineral homeostasis. There is extensive evidence showing loss of bone mass during long-term Space-Flights. The loss is due to a break in the equilibrium between the activity of osteoblasts (the cells that forms bone) and the activity of osteoclasts (the cells that resorbs bone). Surprisingly, there is scanty information about the possible altered gene expression occurring in cells that form bone in microgravity.(Just 69 articles result from a "gene expression in microgravity" MedLine query.) Gene-chip or microarray technology allows to screen thousands of genes at the same time: the use of this technology on samples coming from cells exposed to microgravity could provide us with many important informations. For example, the identification of the molecules or structures which are the first sensors of the mechanical stress derived from lack of gravity, could help in understanding which is the first event leading to bone loss due to long-term exposure to microgravity. Consequently, this structure could become a target for a custom-designed drug. It is evident that bone mass loss, observed during long-time stay in Space, represents an accelerated model of what happens in aging osteoporosis. Therefore, the discovery and design of drugs able to interfere with the bone-loss process, could help also in preventing negative physiological processes normally observed on Earth. Considering the aims stated above, my research is designed to:

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

    Science.gov (United States)

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

    2012-12-24

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

  14. 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.

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

    Science.gov (United States)

    2012-01-01

    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. PMID:23265237

  16. 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.

  17. 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.

  18. Red Maca (Lepidium meyenii) did not affect cell viability despite increased androgen receptor and prostate-specific antigen gene expression in the human prostate cancer cell line LNCaP.

    Science.gov (United States)

    Díaz, P; Cardenas, H; Orihuela, P A

    2016-10-01

    We examined whether aqueous extract of Lepidium meyenii (red Maca) could inhibit growth, potentiate apoptotic activity of two anticancer drugs Taxol and 2-methoxyestradiol (2ME) or change mRNA expression for the androgen target genes, androgen receptor (Ar) and prostate-specific antigen (Psa) in the human prostate cancer cell line LNCaP. Red Maca aqueous extract at 0, 10, 20, 40 or 80 μg/ml was added to LNCaP cells, and viability was evaluated by the MTS assay at 24 or 48 hr after treatment. Furthermore, LNCaP cells were treated with 80 μg/ml of red Maca plus Taxol or 2ME 5 μM and viability was assessed 48 hr later. Finally, LNCaP cells were treated with red Maca 0, 20, 40 or 80 μg/ml, and 12 hr later, mRNA level for Ar or Psa was assessed by real-time PCR. Treatment with red Maca did not affect viability of LNCaP cells. Apoptotic activity induced by Taxol and 2ME in LNCaP cells was not altered with red Maca treatment. Relative expression of the mRNA for Ar and Psa increased with red Maca 20 and 40 μg/ml, but not at 80 μg/ml. We conclude that red Maca aqueous extract does not have toxic effects, but stimulates androgen signalling in LNCaP cells. © 2016 Blackwell Verlag GmbH.

  19. 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....

  20. Imaging gene expression in gene therapy

    International Nuclear Information System (INIS)

    Wiebe, Leonard I.

    1997-01-01

    Full text. Gene therapy can be used to introduce new genes, or to supplement the function of indigenous genes. At the present time, however, there is non-invasive test to demonstrate efficacy of the gene transfer and expression processes. It has been postulated that scintigraphic imaging can offer unique information on both the site at which the transferred gene is expressed, and the degree of expression, both of which are critical issue for safety and clinical efficacy. Many current studies are based on 'suicide gene therapy' of cancer. Cells modified to express these genes commit metabolic suicide in the presence of an enzyme encoded by the transferred gene and a specifically-convertible pro drug. Pro drug metabolism can lead to selective metabolic trapping, required for scintigraphy. Herpes simplex virus type-1 thymidine kinase (H S V-1 t k + ) has been use for 'suicide' in vivo tumor gene therapy. It has been proposed that radiolabelled nucleosides can be used as radiopharmaceuticals to detect H S V-1 t k + gene expression where the H S V-1 t k + gene serves a reporter or therapeutic function. Animal gene therapy models have been studied using purine-([ 18 F]F H P G; [ 18 F]-A C V), and pyrimidine- ([ 123 / 131 I]I V R F U; [ 124 / 131I ]) antiviral nucleosides. Principles of gene therapy and gene therapy imaging will be reviewed and experimental data for [ 123 / 131I ]I V R F U imaging with the H S V-1 t k + reporter gene will be presented

  1. Expression of MAGE and BAGE genes in Japanese breast cancers.

    Science.gov (United States)

    Fujie, T; Mori, M; Ueo, H; Sugimachi, K; Akiyoshi, T

    1997-04-01

    The MAGE and BAGE genes code for distinct antigens, which are recognized on melanoma cells as well as on other various tumor cells by autologous cytolytic T lymphocytes. These antigens may thus constitute useful targets for specific immunotherapy, since no expression of MAGE or BAGE genes has been recognized in normal tissue except for the testis. We studied the MAGE-1, MAGE-3, and BAGE gene expression observed in 49 Japanese breast cancers. Gene expression was evaluated by reverse transcription polymerase chain reaction. Out of 49 tumor tissue specimens of primary breast cancers, the expression of MAGE-1, -3 and BAGE was recognized in 15 (31%), 12 (24%), and 4 (8%) tumors, respectively. The expression of MAGE and BAGE genes is not recognized in normal breast tissue. The expression of the MAGE-3 gene was frequently recognized in tumors with lymphatic and/or vascular vessel permeations. Either MAGE-1 or -3 gene expressions were induced in 1 of 3 MAGE-1 negative breast cell lines or 1 of 3 MAGE-3 negative breast cell lines by the treatment with 5-aza-2'-deoxycytidine. These findings suggest that: 1) the identification of such antigens coded by MAGE or BAGE genes may thus offer the possibility of using specific immunotherapy, and 2) the use of a demethylating agent may increase the number of patients who might be candidates for MAGE specific immunotherapy.

  2. Suicide Gene Therapy to Increase the Safety of Chimeric Antigen Receptor-Redirected T Lymphocytes

    Directory of Open Access Journals (Sweden)

    Monica Casucci, Attilio Bondanza

    2011-01-01

    Full Text Available Chimeric antigen receptors (CARs are generated by fusing the antigen-binding motif of a monoclonal antibody (mAb with the signal transduction machinery of the T-cell receptor (TCR. The genetic modification of T lymphocytes with chimeric receptors specific for tumor-associated antigens (TAAs allows for the redirection towards tumor cells. Clinical experience with CAR-redirected T cells suggests that antitumor efficacy associates with some degree of toxicity, especially when TAA expression is shared with healthy tissues. This situation closely resembles the case of allogeneic hematopoietic stem cell transplantation (HSCT, wherein allorecognition causes both the graft-versus-leukemia (GVL effect and graft-versus-host disease (GVHD. Suicide gene therapy, i.e. the genetic induction of a conditional suicide phenotype into donor T cells, enables dissociating the GVL effect from GVHD. Applying suicide gene modification to CAR-redirected T cells may therefore greatly increase their safety profile and facilitate their clinical development.

  3. Evolution of gene expression after gene amplification.

    Science.gov (United States)

    Garcia, Nelson; Zhang, Wei; Wu, Yongrui; Messing, Joachim

    2015-04-24

    We took a rather unique approach to investigate the conservation of gene expression of prolamin storage protein genes across two different subfamilies of the Poaceae. We took advantage of oat plants carrying single maize chromosomes in different cultivars, called oat-maize addition (OMA) lines, which permitted us to determine whether regulation of gene expression was conserved between the two species. We found that γ-zeins are expressed in OMA7.06, which carries maize chromosome 7 even in the absence of the trans-acting maize prolamin-box-binding factor (PBF), which regulates their expression. This is likely because oat PBF can substitute for the function of maize PBF as shown in our transient expression data, using a γ-zein promoter fused to green fluorescent protein (GFP). Despite this conservation, the younger, recently amplified prolamin genes in maize, absent in oat, are not expressed in the corresponding OMAs. However, maize can express the oldest prolamin gene, the wheat high-molecular weight glutenin Dx5 gene, even when maize Pbf is knocked down (through PbfRNAi), and/or another maize transcription factor, Opaque-2 (O2) is knocked out (in maize o2 mutant). Therefore, older genes are conserved in their regulation, whereas younger ones diverged during evolution and eventually acquired a new repertoire of suitable transcriptional activators. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  4. Regulation of eucaryotic gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Brent, R.; Ptashne, M.S

    1989-05-23

    This patent describes a method of regulating the expression of a gene in a eucaryotic cell. The method consists of: providing in the eucaryotic cell, a peptide, derived from or substantially similar to a peptide of a procaryotic cell able to bind to DNA upstream from or within the gene, the amount of the peptide being sufficient to bind to the gene and thereby control expression of the gene.

  5. MAGE, BAGE and GAGE gene expression in human rhabdomyosarcomas.

    Science.gov (United States)

    Dalerba, P; Frascella, E; Macino, B; Mandruzzato, S; Zambon, A; Rosolen, A; Carli, M; Ninfo, V; Zanovello, P

    2001-07-01

    MAGE, BAGE and GAGE genes encode tumor-associated antigens that are presented by HLA class I molecules and recognized by CD8(+) cytolytic T lymphocytes. These antigens are currently regarded as promising targets for active, specific tumor immunotherapy because MAGE, BAGE and GAGE genes are expressed in many human cancers of different histotype and are silent in normal tissues, with the exception of spermatogonia and placental cells. MAGE, BAGE and GAGE gene expression has been extensively studied in different tumors of adults but is largely unknown in many forms of pediatric solid cancer. Using RT-PCR, we analyzed MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-6, BAGE, GAGE-1,-2 or -8 and GAGE-3,-4,-5,-6 or -7b gene expression in 31 samples of pediatric rhabdomyosarcoma, the most frequent form of malignant soft tissue tumor in children. MAGE genes were expressed in a substantial proportion of patients (MAGE-1, 38%; MAGE-2, 51%; MAGE-3, 35%; MAGE-4, 22%; MAGE-6, 35%), while expression of BAGE (6%); GAGE-1, GAGE-2 and GAGE-8 (9%); and GAGE-3, GAGE-4, GAGE-5, GAGE-6 and GAGE-7B (16%) was less frequent. Overall, 58% of tumors expressed at least 1 gene, and 35% expressed 3 or more genes simultaneously. Our data suggest that a subset of rhabdomyosarcoma patients could be eligible for active, specific immunotherapy directed against MAGE, BAGE and GAGE antigens. Copyright 2001 Wiley-Liss, Inc.

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

    DEFF Research Database (Denmark)

    Larsen, Margit Hørup; Hviid, Thomas

    2009-01-01

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

  7. Differential Gene Expression and Aging

    Directory of Open Access Journals (Sweden)

    Laurent Seroude

    2002-01-01

    Full Text Available It has been established that an intricate program of gene expression controls progression through the different stages in development. The equally complex biological phenomenon known as aging is genetically determined and environmentally modulated. This review focuses on the genetic component of aging, with a special emphasis on differential gene expression. At least two genetic pathways regulating organism longevity act by modifying gene expression. Many genes are also subjected to age-dependent transcriptional regulation. Some age-related gene expression changes are prevented by caloric restriction, the most robust intervention that slows down the aging process. Manipulating the expression of some age-regulated genes can extend an organism's life span. Remarkably, the activity of many transcription regulatory elements is linked to physiological age as opposed to chronological age, indicating that orderly and tightly controlled regulatory pathways are active during aging.

  8. 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

  9. Sequencing and Gene Expression Analysis of Leishmania tropica LACK Gene.

    Science.gov (United States)

    Hammoudeh, Nour; Kweider, Mahmoud; Abbady, Abdul-Qader; Soukkarieh, Chadi

    2014-01-01

    Leishmania Homologue of receptors for Activated C Kinase (LACK) antigen is a 36-kDa protein, which provokes a very early immune response against Leishmania infection. There are several reports on the expression of LACK through different life-cycle stages of genus Leishmania, but only a few of them have focused on L.tropica. The present study provides details of the cloning, DNA sequencing and gene expression of LACK in this parasite species. First, several local isolates of Leishmania parasites were typed in our laboratory using PCR technique to verify of Leishmania parasite species. After that, LACK gene was amplified and cloned into a vector for sequencing. Finally, the expression of this molecule in logarithmic and stationary growth phase promastigotes, as well as in amastigotes, was evaluated by Reverse Transcription-PCR (RT-PCR) technique. The typing result confirmed that all our local isolates belong to L.tropica. LACK gene sequence was determined and high similarity was observed with the sequences of other Leishmania species. Furthermore, the expression of LACK gene in both promastigotes and amastigotes forms was confirmed. Overall, the data set the stage for future studies of the properties and immune role of LACK gene products.

  10. Effect of radiation on the expression of tumor-associated antigens of human lung adenocarcinoma cells

    International Nuclear Information System (INIS)

    Hareyama, Masato

    1988-01-01

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

  11. Genes encoding homologous antigens in taeniid cestode parasites: Implications for development of recombinant vaccines produced in Escherichia coli.

    Science.gov (United States)

    Gauci, Charles; Lightowlers, Marshall W

    2013-01-01

    Recombinant vaccine antigens are being evaluated for their ability to protect livestock animals against cysticercosis and related parasitic infections. Practical use of some of these vaccines is expected to reduce parasite transmission, leading to a reduction in the incidence of neurocysticercosis and hydatid disease in humans. We recently showed that an antigen (TSOL16), expressed in Escherichia coli, confers high levels of protection against Taenia solium cysticercosis in pigs, which provides a strategy for control of T. solium parasite transmission. Here, we discuss the characteristics of this antigen that may affect the utility of TSOL16 and related antigens for development as recombinant vaccines. We also report that genes encoding antigens closely related to TSOL16 from T. solium also occur in other related species of parasites. These highly homologous antigens have the potential to be used as vaccines and may provide protection against related species of Taenia that cause infection in other hosts.

  12. [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.

  13. Transgenic Carrot Expressing Fusion Protein Comprising M. tuberculosis Antigens Induces Immune Response in Mice

    Directory of Open Access Journals (Sweden)

    Natalia V. Permyakova

    2015-01-01

    Full Text Available Tuberculosis remains one of the major infectious diseases, which continues to pose a major global health problem. Transgenic plants may serve as bioreactors to produce heterologous proteins including antibodies, antigens, and hormones. In the present study, a genetic construct has been designed that comprises the Mycobacterium tuberculosis genes cfp10, esat6 and dIFN gene, which encode deltaferon, a recombinant analog of the human γ-interferon designed for expression in plant tissues. This construct was transferred to the carrot (Daucus carota L. genome by Agrobacterium-mediated transformation. This study demonstrates that the fusion protein CFP10-ESAT6-dIFN is synthesized in the transgenic carrot storage roots. The protein is able to induce both humoral and cell-mediated immune responses in laboratory animals (mice when administered either orally or by injection. It should be emphasized that M. tuberculosis antigens contained in the fusion protein have no cytotoxic effect on peripheral blood mononuclear cells.

  14. 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.

  15. Biosynthetic basis of incompatible histo-blood group A antigen expression

    DEFF Research Database (Denmark)

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

    1993-01-01

    , we have screened 31 cases of gastric tumors of phenotype O for the expression of blood group A gene-defined glycosyltransferase by immunohistology on frozen sections using newly developed monoclonal antibodies to the transferases. Three cases were positive, and transferase expression was confirmed...... by enzyme analysis of extracts from the specimens. Blood group A carbohydrate antigens were also identified immunohistologically in these three cases as well as in five other cases. Thin-layer chromatography immunostaining analysis of glycolipid extracts from the three cases did not confirm the chemical...

  16. Prevalence, serologic and genetic studies of high expressers of the blood group A antigen on platelets*

    Science.gov (United States)

    Sant’Anna Gomes, B M; Estalote, A C; Palatnik, M; Pimenta, G; Pereira, B de B; do Nascimento, E M

    2010-01-01

    Objective/Aim: The aim of this study is to describe the distribution of the platelet blood group A antigenicity in Euro-Brazilians (EUBs) and Afro-Brazilians (AFBs). Background: A small but significant proportion of individuals express high levels of A or B antigen on their platelets corresponding to the erythrocyte ABO group. The mechanism of increased antigen expression has not been elucidated. Material/Methods: A cohort of 241 blood group A donors was analysed by flow cytometry. Although mean fluorescence intensity (MFI) is a typical continuous variable, platelets were screened and divided into two categories: low expressers (LEs) and high expressers (HEs). A three-generation family was investigated looking for an inheritance mechanism. Results: The prevalence of the HE platelet phenotype among group A1 donors was 2%. The mean of MFI on platelets of A1 subgroup of EUBs differs from that of AFBs (P = 0·0115), whereas the frequency of the HE phenotype was similar between them (P = 0·5251). A significant difference was found between sexes (P = 0·0039). Whereas the serum glycosyltransferase from HE family members converted significantly more H antigen on group O erythrocytes into A antigens compared with that in LE serum, their ABO, FUT1 and FUT2 genes were consensus. The theoretically favourable, transcriptionally four-repeat ABO enhancer was not observed. Conclusion: The occurrence of HE in several members suggests familial aggregation. Indeed, in repeated measures, stability of the MFI values is suggesting an inherited condition. Factors outside the ABO locus might be responsible for the HE phenotype. Whether the real mechanism of inheritance is either of a polygenic or of a discrete Mendelian nature remains to be elucidated. PMID:20553427

  17. Human antigen-specific regulatory T cells generated by T cell receptor gene transfer.

    Directory of Open Access Journals (Sweden)

    Todd M Brusko

    2010-07-01

    Full Text Available Therapies directed at augmenting regulatory T cell (Treg activities in vivo as a systemic treatment for autoimmune disorders and transplantation may be associated with significant off-target effects, including a generalized immunosuppression that may compromise beneficial immune responses to infections and cancer cells. Adoptive cellular therapies using purified expanded Tregs represents an attractive alternative to systemic treatments, with results from animal studies noting increased therapeutic potency of antigen-specific Tregs over polyclonal populations. However, current methodologies are limited in terms of the capacity to isolate and expand a sufficient quantity of endogenous antigen-specific Tregs for therapeutic intervention. Moreover, FOXP3+ Tregs fall largely within the CD4+ T cell subset and are thus routinely MHC class II-specific, whereas class I-specific Tregs may function optimally in vivo by facilitating direct tissue recognition.To overcome these limitations, we have developed a novel means for generating large numbers of antigen-specific Tregs involving lentiviral T cell receptor (TCR gene transfer into in vitro expanded polyclonal natural Treg populations. Tregs redirected with a high-avidity class I-specific TCR were capable of recognizing the melanoma antigen tyrosinase in the context of HLA-A*0201 and could be further enriched during the expansion process by antigen-specific reactivation with peptide loaded artificial antigen presenting cells. These in vitro expanded Tregs continued to express FOXP3 and functional TCRs, and maintained the capacity to suppress conventional T cell responses directed against tyrosinase, as well as bystander T cell responses. Using this methodology in a model tumor system, murine Tregs designed to express the tyrosinase TCR effectively blocked antigen-specific effector T cell (Teff activity as determined by tumor cell growth and luciferase reporter-based imaging.These results support the

  18. Gene expression in colorectal cancer

    DEFF Research Database (Denmark)

    Birkenkamp-Demtroder, Karin; Christensen, Lise Lotte; Olesen, Sanne Harder

    2002-01-01

    Understanding molecular alterations in colorectal cancer (CRC) is needed to define new biomarkers and treatment targets. We used oligonucleotide microarrays to monitor gene expression of about 6,800 known genes and 35,000 expressed sequence tags (ESTs) on five pools (four to six samples in each......' C, and clustered Dukes' D separately. Real-time PCR of 10 known genes and 5 ESTs demonstrated excellent reproducibility of the array-based findings. The most frequently altered genes belonged to functional categories of metabolism (22%), transcription and translation (11%), and cellular processes (9...

  19. Expression cloning of camelid nanobodies specific for Xenopus embryonic antigens.

    Directory of Open Access Journals (Sweden)

    Keiji Itoh

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

  20. Human Lacrimal Gland Gene Expression.

    Directory of Open Access Journals (Sweden)

    Vinay Kumar Aakalu

    Full Text Available The study of human lacrimal gland biology and development is limited. Lacrimal gland tissue is damaged or poorly functional in a number of disease states including dry eye disease. Development of cell based therapies for lacrimal gland diseases requires a better understanding of the gene expression and signaling pathways in lacrimal gland. Differential gene expression analysis between lacrimal gland and other embryologically similar tissues may be helpful in furthering our understanding of lacrimal gland development.We performed global gene expression analysis of human lacrimal gland tissue using Affymetrix ® gene expression arrays. Primary data from our laboratory was compared with datasets available in the NLM GEO database for other surface ectodermal tissues including salivary gland, skin, conjunctiva and corneal epithelium.The analysis revealed statistically significant difference in the gene expression of lacrimal gland tissue compared to other ectodermal tissues. The lacrimal gland specific, cell surface secretory protein encoding genes and critical signaling pathways which distinguish lacrimal gland from other ectodermal tissues are described.Differential gene expression in human lacrimal gland compared with other ectodermal tissue types revealed interesting patterns which may serve as the basis for future studies in directed differentiation among other areas.

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

    Directory of Open Access Journals (Sweden)

    Christopher D Johnston

    2014-09-01

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

  2. Erasing the Epigenetic Memory and Beginning to Switch—The Onset of Antigenic Switching of var Genes in Plasmodium falciparum

    Science.gov (United States)

    Fastman, Yair; Noble, Robert; Recker, Mario; Dzikowski, Ron

    2012-01-01

    Antigenic variation in Plasmodium falciparum is regulated by transcriptional switches among members of the var gene family, each expressed in a mutually exclusive manner and encoding a different variant of the surface antigens collectively named PfEMP1. Antigenic switching starts when the first merozoites egress from the liver and begin their asexual proliferation within red blood cells. By erasing the epigenetic memory we created parasites with no var background, similar to merozoites that egress from the liver where no var gene is expressed. Creating a null-var background enabled us to investigate the onset of antigenic switches at the early phase of infection. At the onset of switching, var transcription pattern is heterogeneous with numerous genes transcribed at low levels including upsA vars, a subtype that was implicated in severe malaria, which are rarely activated in growing cultures. Analysis of subsequent in vitro switches shows that the probability of a gene to turn on or off is not associated with its chromosomal position or promoter type per se but on intrinsic properties of each gene. We concluded that var switching is determined by gene specific associated switch rates rather than general promoter type or locus associated switch rates. In addition, we show that fine tuned reduction in var transcription increases their switch rate, indicating that transcriptional perturbation can alter antigenic switching. PMID:22461905

  3. Gene expression in colorectal cancer

    DEFF Research Database (Denmark)

    Birkenkamp-Demtroder, Karin; Christensen, Lise Lotte; Olesen, Sanne Harder

    2002-01-01

    Understanding molecular alterations in colorectal cancer (CRC) is needed to define new biomarkers and treatment targets. We used oligonucleotide microarrays to monitor gene expression of about 6,800 known genes and 35,000 expressed sequence tags (ESTs) on five pools (four to six samples in each...... pool) of total RNA from left-sided sporadic colorectal carcinomas. We compared normal tissue to carcinoma tissue from Dukes' stages A-D (noninvasive to distant metastasis) and identified 908 known genes and 4,155 ESTs that changed remarkably from normal to tumor tissue. Based on intensive filtering 226...

  4. Heat shock protein-90 inhibitors enhance antigen expression on melanomas and increase T cell recognition of tumor cells.

    Directory of Open Access Journals (Sweden)

    Timothy J Haggerty

    Full Text Available In an effort to enhance antigen-specific T cell recognition of cancer cells, we have examined numerous modulators of antigen-expression. In this report we demonstrate that twelve different Hsp90 inhibitors (iHsp90 share the ability to increase the expression of differentiation antigens and MHC Class I antigens. These iHsp90 are active in several molecular and cellular assays on a series of tumor cell lines, including eleven human melanomas, a murine B16 melanoma, and two human glioma-derived cell lines. Intra-cytoplasmic antibody staining showed that all of the tested iHsp90 increased expression of the melanocyte differentiation antigens Melan-A/MART-1, gp100, and TRP-2, as well as MHC Class I. The gliomas showed enhanced gp100 and MHC staining. Quantitative analysis of mRNA levels showed a parallel increase in message transcription, and a reporter assay shows induction of promoter activity for Melan-A/MART-1 gene. In addition, iHsp90 increased recognition of tumor cells by T cells specific for Melan-A/MART-1. In contrast to direct Hsp90 client proteins, the increased levels of full-length differentiation antigens that result from iHsp90 treatment are most likely the result of transcriptional activation of their encoding genes. In combination, these results suggest that iHsp90 improve recognition of tumor cells by T cells specific for a melanoma-associated antigen as a result of increasing the expressed intracellular antigen pool available for processing and presentation by MHC Class I, along with increased levels of MHC Class I itself. As these Hsp90 inhibitors do not interfere with T cell function, they could have potential for use in immunotherapy of cancer.

  5. Remote control of gene expression.

    Science.gov (United States)

    Long, Xiaochun; Miano, Joseph M

    2007-06-01

    The elucidation of a growing number of species' genomes heralds an unprecedented opportunity to ascertain functional attributes of non-coding sequences. In particular, cis regulatory modules (CRMs) controlling gene expression constitute a rich treasure trove of data to be defined and experimentally validated. Such information will provide insight into cell lineage determination and differentiation and the genetic basis of heritable diseases as well as the development of novel tools for restricting the inactivation of genes to specific cell types or conditions. Historically, the study of CRMs and their individual transcription factor binding sites has been limited to proximal regions around gene loci. Two important by-products of the genomics revolution, artificial chromosome vectors and comparative genomics, have fueled efforts to define an increasing number of CRMs acting remotely to control gene expression. Such regulation from a distance has challenged our perspectives of gene expression control and perhaps the very definition of a gene. This review summarizes current approaches to characterize remote control of gene expression in transgenic mice and inherent limitations for accurately interpreting the essential nature of CRM activity.

  6. Abnormal expression of blood group-related antigens in uterine endometrial cancers.

    Science.gov (United States)

    Tsukazaki, K; Sakayori, M; Arai, H; Yamaoka, K; Kurihara, S; Nozawa, S

    1991-08-01

    The expression of A, B, and H group antigens, Lewis group antigens (Lewis(a), Lewis(b), Lewis(x), and Lewis(y)), and Lc4 and nLc4 antigens, the precursor antigens of both groups, was examined immunohistochemically with monoclonal antibodies in 9 normal endometria, 6 endometrial hyperplasias, and 31 endometrial cancers. 1) A, B and/or H antigens were detected in endometrial cancers at an incidence of 51.6%, while no distinct localization of these antigens was observed in normal endometria. H antigen, the precursor of A and B antigens, was particularly frequently detected in endometrial cancers. 2) An increased rate of expression of Lewis group antigens, particularly Lewis(b) antigen, was observed in endometrial cancers compared with its expression in normal endometria. 3) Lc4 and nLc4 antigens were detected in endometrial cancers at rates of 41.9% and 38.7%, respectively, these expressions being increased compared with those in normal endometria. 4) These results suggest that a highly abnormal expression of blood group-related antigens in endometrial cancers occurs not only at the level of A, B, and H antigens and Lewis group antigens, but also at the level of their precursor Lc4 and nLc4 antigens. 5) Lewis(a), Lewis(b), and Lc4 antigens, built on the type-1 chain, are more specific to endometrial cancers than their respective positional isomers, Lewis(x), Lewis(y), and nLc4 antigens, built on the type-2 chain.

  7. Correlation between expression of major histocompatibility complex class I and that of antigen presenting machineries in carcinoma cell lines of the pancreas, biliary tract and colon

    OpenAIRE

    Imanishi, Tatsuya; Kamigaki, Takashi; Nakamura, Tetsu; Hayashi, Shun; Yasuda, Takashi; Kawasaki, Kentaro; Takase, Shiro; Ajiki, Tetsuo; Kuroda, Yoshikazu

    2006-01-01

    To elicit a tumor immune response, tumor antigens represented by majorhistocompatibility (MHC) class I complex on the cell surface is indispensable. Someinvestigators demonstrated that many cancer cells reduce expression ofβ2-microglobulin, a transporter of antigen presenting (TAP) or low molecular protein(LMP), due to the deletion mutant or point mutation. We investigated gene expressionlevels of antigen presenting machineries in 13 cell lines of the pancreas, biliary tractand colon cancer b...

  8. Liver cancer: expression features of hepatitis B antigens

    Directory of Open Access Journals (Sweden)

    V. A. Tumanskiy

    2013-12-01

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

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

    Directory of Open Access Journals (Sweden)

    A. А. Pavlova

    2017-01-01

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

  10. Acute lymphoblastic leukemia-derived dendritic cells express tumor associated antigens: PNPT1, PMPCB, RHAMM, BSG and ERCC1.

    Science.gov (United States)

    Luczynski, W; Kowalczuk, O; Stasiak-Barmuta, A; Ilendo, E; Krawczuk-Rybak, M; Chyczewski, L

    2009-01-01

    In all types of leukemia both in children and adults there is a need for novel therapies that could reduce the risk of relapse after standard treatment. Acute lymphoblastic leukemia (ALL) cells are ineffective antigen presenting cells, but as shown by many authors including results from our laboratory, stimulation with CD40L restores their antigen expressing capacity. The development of T-cell therapies for leukemic patients can be based on discovery of leukemia-associated antigens (LAA) which could be recognized by the host immune system. The aim of our present study was to test the hypothesis that leukemia-derived dendritic cells maintain the expression of tumor associated antigens. Twenty five children with B-cell precursor ALL were prospectively enrolled into the study. The mononuclear cells from peripheral blood or bone marrow were cultured and stimulated (or not) with CD40L and IL-4. The assessment of costimulatory/adhesion molecules with the use of flow cytometry and real-time RT PCR were used to confirm the possibility of turning ALL cells into dendritic-like cells. Additionally 22 tumor associated antigens mRNA levels were determined by real-time PCR technique with the TaqMan chemistry using ready-to-use Low Density Arrays for Gene Expression. The results of the study showed maintained expression and even up-regulation of some (PNPT1, PMPCB, HMMR/RHAMM, BSG and ERCC1) tumor associated antigens in CD40-activated leukemic cells. CD40L stimulation leading to the differentiation of leukemic cells into DCs which combine both antigen presenting function and expression of tumor associated antigens represents an interesting approach in cancer immunotherapy.

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

    International Nuclear Information System (INIS)

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

    1990-04-01

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

  12. Transgenic Arabidopsis Gene Expression System

    Science.gov (United States)

    Ferl, Robert; Paul, Anna-Lisa

    2009-01-01

    The Transgenic Arabidopsis Gene Expression System (TAGES) investigation is one in a pair of investigations that use the Advanced Biological Research System (ABRS) facility. TAGES uses Arabidopsis thaliana, thale cress, with sensor promoter-reporter gene constructs that render the plants as biomonitors (an organism used to determine the quality of the surrounding environment) of their environment using real-time nondestructive Green Fluorescent Protein (GFP) imagery and traditional postflight analyses.

  13. 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.

  14. Human papillomavirus gene expression

    International Nuclear Information System (INIS)

    Chow, L.T.; Hirochika, H.; Nasseri, M.; Stoler, M.H.; Wolinsky, S.M.; Chin, M.T.; Hirochika, R.; Arvan, D.S.; Broker, T.R.

    1987-01-01

    To determine the role of tissue differentiation on expression of each of the papillomavirus mRNA species identified by electron microscopy, the authors prepared exon-specific RNA probes that could distinguish the alternatively spliced mRNA species. Radioactively labeled single-stranded RNA probes were generated from a dual promoter vector system and individually hybridized to adjacent serial sections of formalin-fixed, paraffin-embedded biopsies of condylomata. Autoradiography showed that each of the message species had a characteristic tissue distribution and relative abundance. The authors have characterized a portion of the regulatory network of the HPVs by showing that the E2 ORF encodes a trans-acting enhancer-stimulating protein, as it does in BPV-1 (Spalholz et al. 1985). The HPV-11 enhancer was mapped to a 150-bp tract near the 3' end of the URR. Portions of this region are duplicated in some aggressive strains of HPV-6 (Boshart and zur Hausen 1986; Rando et al. 1986). To test the possible biological relevance of these duplications, they cloned tandem arrays of the enhancer and demonstrated, using a chloramphenicol acetyltransferase (CAT) assay, that they led to dramatically increased transcription proportional to copy number. Using the CAT assays, the authors found that the E2 proteins of several papillomavirus types can cross-stimulate the enhancers of most other types. This suggests that prior infection of a tissue with one papillomavirus type may provide a helper effect for superinfection and might account fo the HPV-6/HPV-16 coinfections in condylomata that they have observed

  15. 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.

  16. Homeobox gene expression in Brachiopoda

    DEFF Research Database (Denmark)

    Altenburger, Andreas; Martinez, Pedro; Wanninger, Andreas

    2011-01-01

    The molecular control that underlies brachiopod ontogeny is largely unknown. In order to contribute to this issue we analyzed the expression pattern of two homeobox containing genes, Not and Cdx, during development of the rhynchonelliform (i.e., articulate) brachiopod Terebratalia transversa. Not...

  17. Expression of tumor-specific antigen MAGE, GAGE and BAGE in ovarian cancer tissues and cell lines

    Directory of Open Access Journals (Sweden)

    Yu Hao

    2010-04-01

    Full Text Available Abstract Background To observe mRNA expression of tumor-specific antigen MAGE, BAGE and GAGE in epithelial ovarian cancer tissues and cell lines, to explore the relationship between gene expression and diagnosis, treatment and prognosis of ovarian cancer, and to evaluate the feasibility of their gene products as markers, and an immunotherapy target for ovarian cancer. Methods mRNA expression of MAGE-1, MAGE-3, GAGE-1/2 and BAGE were determined by reverse transcription polymerase chain reaction (RT-PCR in 14 cases of normal ovarian tissue, 20 cases of ovarian benign tumor specimens, 41 cases of ovarian cancer specimens, and ovarian cancer cell lines SKOV3, A2780, and COC1. Results MAGE, GAGE and BAGE genes were not expressed in normal ovarian tissue. In benign tumors, only the MAGE gene was expressed; the expression rate of this gene in benign tumors was 15% (3/20. In ovarian cancer tissues, MAGE-1 and MAGE-3 was highly expressed, with expression rates of 53.7% (22/41 and 36.6% (15/41, while GAGE-1/2 and BAGE had relatively low expression, with rates of 26.8% (11/41 and 14.6% (6/41. In metastatic lesions of ovarian cancer, only MAGE-1 and BAGE were expressed, with expression rates of 28.6% (2/7 and 14.3% (1/7. The positive expression rates of MAGE-1 and MAGE-3 in serous cystadenocarcinoma were significantly higher than that in other types of ovarian cancer (P P Conclusion Tumor-specific antigen MAGE, BAGE and GAGE may play a role in the occurrence and development of ovarian cancer. These genes can be used as one of the important indicators for early diagnosis, efficacy evaluation and prognostic determination of ovarian cancer.

  18. Correction of gene expression data

    DEFF Research Database (Denmark)

    Darbani Shirvanehdeh, Behrooz; Stewart, C. Neal, Jr.; Noeparvar, Shahin

    2014-01-01

    an analytical approach to examine the suitability of correction methods by considering the inter-treatment bias as well as the inter-replicate variance, which allows use of the best correction method with minimum residual bias. Analyses of RNA sequencing and microarray data showed that the efficiencies......This report investigates for the first time the potential inter-treatment bias source of cell number for gene expression studies. Cell-number bias can affect gene expression analysis when comparing samples with unequal total cellular RNA content or with different RNA extraction efficiencies....... For maximal reliability of analysis, therefore, comparisons should be performed at the cellular level. This could be accomplished using an appropriate correction method that can detect and remove the inter-treatment bias for cell-number. Based on inter-treatment variations of reference genes, we introduce...

  19. MRl of prostate cancer antigen expression for diagnosis and immunotherapy.

    Directory of Open Access Journals (Sweden)

    Jing Ren

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

  20. Analysis of CD83 antigen expression in human breast fibroadenoma and adjacent tissue

    Directory of Open Access Journals (Sweden)

    Marcus Nascimento Borges

    Full Text Available CONTEXT AND OBJECTIVE: Dendritic cell maturation is considered essential for starting an immune response. The CD83 antigen is an important marker of dendritic cell maturation. The objectives here were to analyze CD83 antigen expression in human breast fibroadenoma and breast tissue adjacent to the lesion and to identify clinical factors that might influence this expression. DESIGN AND SETTING: This was a retrospective study at a public university hospital, in which 29 histopathological samples of breast fibroadenoma and adjacent breast tissue, from 28 women of reproductive age, were analyzed. METHODS: The immunohistochemistry method was used to analyze the cell expression of the antigen. The antigen expression in the cells was evaluated by means of random manual counting using an optical microscope. RESULTS: Positive expression of the CD83 antigen in the epithelial cells of the fibroadenoma (365.52; standard deviation ± 133.13 in relation to the adjacent breast tissue cells (189.59; standard deviation ± 140.75 was statistically larger (P < 0.001. Several clinical features were analyzed, but only parity was shown to influence CD83 antigen expression in the adjacent breast tissue, such that positive expression was more evident in nulliparous women (P = 0.042. CONCLUSIONS: The expression of the CD83 antigen in the fibroadenoma was positive and greater than in the adjacent breast tissue. Positive expression of the antigen in the adjacent breast tissue was influenced by parity, and was significantly more evident in nulliparous women.

  1. Vascular Gene Expression: A Hypothesis

    Directory of Open Access Journals (Sweden)

    Angélica Concepción eMartínez-Navarro

    2013-07-01

    Full Text Available The phloem is the conduit through which photoassimilates are distributed from autotrophic to heterotrophic tissues and is involved in the distribution of signaling molecules that coordinate plant growth and responses to the environment. Phloem function depends on the coordinate expression of a large array of genes. We have previously identified conserved motifs in upstream regions of the Arabidopsis genes, encoding the homologs of pumpkin phloem sap mRNAs, displaying expression in vascular tissues. This tissue-specific expression in Arabidopsis is predicted by the overrepresentation of GA/CT-rich motifs in gene promoters. In this work we have searched for common motifs in upstream regions of the homologous genes from plants considered to possess a primitive vascular tissue (a lycophyte, as well as from others that lack a true vascular tissue (a bryophyte, and finally from chlorophytes. Both lycophyte and bryophyte display motifs similar to those found in Arabidopsis with a significantly low E-value, while the chlorophytes showed either a different conserved motif or no conserved motif at all. These results suggest that these same genes are expressed coordinately in non- vascular plants; this coordinate expression may have been one of the prerequisites for the development of conducting tissues in plants. We have also analyzed the phylogeny of conserved proteins that may be involved in phloem function and development. The presence of CmPP16, APL, FT and YDA in chlorophytes suggests the recruitment of ancient regulatory networks for the development of the vascular tissue during evolution while OPS is a novel protein specific to vascular plants.

  2. [Immune response of melanoma antigen gene-3 modified dendritic cell vaccines in gastric carcinoma].

    Science.gov (United States)

    He, Song-bing; Wang, Liang; Zhang, Yan-yun

    2009-05-01

    To investigate the anti-gastric carcinoma immunological efficacy of dendritic cells (DC) precursors, that were mobilized into the peripheral blood by injection of macrophage inflammation protein-1 alpha (MIP-1 alpha), and induced by DC vaccine expressing melanoma antigen gene-3 (MAGE-3) ex vivo and in vivo. 615 mice were injected with MIP-1 alpha via the tail vein. Freshly isolated B220(-) CD11c+ cells were cultured with cytokines and assayed by phenotype analysis and mixed lymphocyte reaction (MLR). For adenoviral (Ad)-mediated gene transduction, cultured B220(-) CD11c+ cells were incubated with Ad-melanoma antigen gene-3. MIP-1 alpha-mobilized B220(-) CD11c+ cells pulsed MFC cells tumor lysate were used as positive control. The stimulated DC vaccination-induced T lymphocytes, and the killing effect of the T cells on gastric carcinoma cells were assayed by MTT. INF-gamma production was determined with the INF-gamma ELISA kit. To establish the solid tumor model, groups of 615 mice were injected with MFC cells subcutaneously into the abdominal wall. MIP-1 alpha-mobilized DC vaccines expressing MAGE-3 gene were used to immunize the mice after the challenge of MFC cells, then the tumor size and the survival of mice were examined to detect the therapeutic effect of DC vaccines. B220(-) CD11c+ cells increased obviously after MIP-1 alpha injection, and freshly isolated B220(-) CD11c+ cells cultured with mGM-CSF, IL-4, and mTNF-alpha were phenotypically identical to typical DC, gained the capacity to stimulate allogeneic T cells. These MIP-1 alpha-mobilized DCs were transduced with Ad-MAGE-3, which were prepared for DC vaccines expressing tumor antigen. T lymphocytes stimulated with DC-transduced with Ad-MAGE-3 showed specific killing effect on gastric carcinoma cells and produced high levels of INF-gamma [(1460.00 +/- 16.82) pg/ml]. Five days after the MFC cells challenge, the mice were subsequently injected with DC vaccines. The tumor size of the experimental group was

  3. Prevalence, serologic and genetic studies of high expressers of the blood group A antigen on platelets*

    OpenAIRE

    Sant?Anna Gomes, B M; Estalote, A C; Palatnik, M; Pimenta, G; Pereira, B de B; do Nascimento, E M

    2010-01-01

    Objective/Aim: The aim of this study is to describe the distribution of the platelet blood group A antigenicity in Euro-Brazilians (EUBs) and Afro-Brazilians (AFBs). Background: A small but significant proportion of individuals express high levels of A or B antigen on their platelets corresponding to the erythrocyte ABO group. The mechanism of increased antigen expression has not been elucidated. Material/Methods: A cohort of 241 blood group A donors was analysed by flow cytometry. Although m...

  4. Cytomegalovirus selectively blocks antigen processing and presentation of its immediate-early gene product.

    Science.gov (United States)

    Gilbert, M J; Riddell, S R; Plachter, B; Greenberg, P D

    1996-10-24

    Recognition of virus-infected cells by CD8+ cytotoxic T lymphocytes requires that the viral proteins be processed into peptides, the derived peptides transported into the endoplasmic reticulum and inserted into the binding groove of a major histocompatibility complex class I molecule, and the antigenic complex exported to the cell surface. However, viral pathogens can disrupt this process and interfere with immune recognition. These mechanisms may be vital to large viruses such as human cytomegalovirus (CMV), which causes persistent infection despite producing over 200 potentially antigenic proteins during the sequential immediate-early, early and late phases of viral gene expression. Products of CMV early-phase gene expression can globally block class I presentation and prevent recognition of infected cells by cytotoxic T lymphocytes, but an essential viral transcription factor, the 72K principal immediate-early protein, is abundantly expressed before this blockade. However, only a few host CD8+ cytotoxic T lymphocytes specific for immediate-early protein are present in seropositive individuals, and these lyse CMV-infected cells poorly. Here we demonstrate selective abrogation of immediate-early peptide presentation by a CMV matrix protein with associated kinase activity and suggest that modification of a viral protein can result in limiting access to the processing machinery and evasion of cytotoxic-T-cell recognition.

  5. Neighboring Genes Show Correlated Evolution in Gene Expression

    Science.gov (United States)

    Ghanbarian, Avazeh T.; Hurst, Laurence D.

    2015-01-01

    When considering the evolution of a gene’s expression profile, we commonly assume that this is unaffected by its genomic neighborhood. This is, however, in contrast to what we know about the lack of autonomy between neighboring genes in gene expression profiles in extant taxa. Indeed, in all eukaryotic genomes genes of similar expression-profile tend to cluster, reflecting chromatin level dynamics. Does it follow that if a gene increases expression in a particular lineage then the genomic neighbors will also increase in their expression or is gene expression evolution autonomous? To address this here we consider evolution of human gene expression since the human-chimp common ancestor, allowing for both variation in estimation of current expression level and error in Bayesian estimation of the ancestral state. We find that in all tissues and both sexes, the change in gene expression of a focal gene on average predicts the change in gene expression of neighbors. The effect is highly pronounced in the immediate vicinity (genes increasing their expression in humans tend to avoid nuclear lamina domains and be enriched for the gene activator 5-hydroxymethylcytosine, we conclude that, most probably owing to chromatin level control of gene expression, a change in gene expression of one gene likely affects the expression evolution of neighbors, what we term expression piggybacking, an analog of hitchhiking. PMID:25743543

  6. [Gene expression profile of spinal ventral horn in ALS].

    Science.gov (United States)

    Yamamoto, Masahiko; Tanaka, Fumiaki; Sobue, Gen

    2007-10-01

    The causative pathomechanism of sporadic amyotrophic lateral sclerosis (ALS) is not clearly understood. Using microarray technology combined with laser-captured microdissection, gene expression profiles of degenerating spinal motor neurons as well as spinal ventral horn from autopsied patients with sporadic ALS were examined. Spinal motor neurons showed a distinct gene expression profile from the whole spinal ventral horn. Three percent of genes examined were significantly downregulated, and 1% were upregulated in motor neurons. In contrast with motor neurons, the total spinal ventral horn homogenates demonstrated 0.7% and 0.2% significant upregulation and downregulation of gene expression, respectively. Downregulated genes in motor neurons included those associated with cytoskeleton/axonal transport, transcription and cell surface antigens/receptors, such as dynactin 1 (DCTN1) and early growth response 3 (EGR3). In particular, DCTN1 was markedly downregulated in most residual motor neurons prior to the accumulation of pNF-H and ubiquitylated protein. Promoters for cell death pathway, death receptor 5 (DR5), cyclins C (CCNC) and A1 (CCNA), and caspases were upregulated, whereas cell death inhibitors, acetyl-CoA transporter (ACATN) and NF-kappaB (NFKB) were also upregulated. In terms of spinal ventral horn, the expression of genes related to cell surface antigens/receptors, transcription and cell adhesion/ECM were increased. The gene expression resulting in neurodegenerative and neuroprotective changes were both present in spinal motor neurons and ventral horn. Moreover, Inflammation-related genes, such as belonging to the cytokine family were not, however, significantly upregulated in either motor neurons or ventral horn. The sequence of motor neuron-specific gene expression changes from early DCTN1 downregulation to late CCNC upregulation in sporadic ALS can provide direct information on the genes leading to neurodegeneration and neuronal death, and are helpful

  7. Biosynthetic basis of incompatible histo-blood group A antigen expression

    DEFF Research Database (Denmark)

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

    1993-01-01

    presence of A antigen. The ABO genotype of all patients was found to be OO, showing that all carried O alleles with a structural defect at nucleotide position 261 leading to a shift in the reading frame. The data suggest that incompatible A antigen expression is a result of transferase expression derived...

  8. Gene expression profile of pulpitis

    Science.gov (United States)

    Galicia, Johnah C.; Henson, Brett R.; Parker, Joel S.; Khan, Asma A.

    2016-01-01

    The cost, prevalence and pain associated with endodontic disease necessitate an understanding of the fundamental molecular aspects of its pathogenesis. This study was aimed to identify the genetic contributors to pulpal pain and inflammation. Inflamed pulps were collected from patients diagnosed with irreversible pulpitis (n=20). Normal pulps from teeth extracted for various reasons served as controls (n=20). Pain level was assessed using a visual analog scale (VAS). Genome-wide microarray analysis was performed using Affymetrix GeneTitan Multichannel Instrument. The difference in gene expression levels were determined by the Significance Analysis of Microarray program using a false discovery rate (q-value) of 5%. Genes involved in immune response, cytokine-cytokine receptor interaction and signaling, integrin cell surface interactions, and others were expressed at relatively higher levels in the in the pulpitis group. Moreover, several genes known to modulate pain and inflammation showed differential expression in asymptomatic and mild pain patients (≥30mm on VAS) compared to those with moderate to severe pain. This exploratory study provides a molecular basis for the clinical diagnosis of pulpitis. With an enhanced understanding of pulpal inflammation, future studies on treatment and management of pulpitis and on pain associated with it can have a biological reference to bridge treatment strategies with pulpal biology. PMID:27052691

  9. Gene expression profile of pulpitis.

    Science.gov (United States)

    Galicia, J C; Henson, B R; Parker, J S; Khan, A A

    2016-06-01

    The cost, prevalence and pain associated with endodontic disease necessitate an understanding of the fundamental molecular aspects of its pathogenesis. This study was aimed to identify the genetic contributors to pulpal pain and inflammation. Inflamed pulps were collected from patients diagnosed with irreversible pulpitis (n=20). Normal pulps from teeth extracted for various reasons served as controls (n=20). Pain level was assessed using a visual analog scale (VAS). Genome-wide microarray analysis was performed using Affymetrix GeneTitan Multichannel Instrument. The difference in gene expression levels were determined by the significance analysis of microarray program using a false discovery rate (q-value) of 5%. Genes involved in immune response, cytokine-cytokine receptor interaction and signaling, integrin cell surface interactions, and others were expressed at relatively higher levels in the pulpitis group. Moreover, several genes known to modulate pain and inflammation showed differential expression in asymptomatic and mild pain patients (⩾30 mm on VAS) compared with those with moderate to severe pain. This exploratory study provides a molecular basis for the clinical diagnosis of pulpitis. With an enhanced understanding of pulpal inflammation, future studies on treatment and management of pulpitis and on pain associated with it can have a biological reference to bridge treatment strategies with pulpal biology.

  10. Gene Expression in Trypanosomatid Parasites

    Directory of Open Access Journals (Sweden)

    Santiago Martínez-Calvillo

    2010-01-01

    Full Text Available The parasites Leishmania spp., Trypanosoma brucei, and Trypanosoma cruzi are the trypanosomatid protozoa that cause the deadly human diseases leishmaniasis, African sleeping sickness, and Chagas disease, respectively. These organisms possess unique mechanisms for gene expression such as constitutive polycistronic transcription of protein-coding genes and trans-splicing. Little is known about either the DNA sequences or the proteins that are involved in the initiation and termination of transcription in trypanosomatids. In silico analyses of the genome databases of these parasites led to the identification of a small number of proteins involved in gene expression. However, functional studies have revealed that trypanosomatids have more general transcription factors than originally estimated. Many posttranslational histone modifications, histone variants, and chromatin modifying enzymes have been identified in trypanosomatids, and recent genome-wide studies showed that epigenetic regulation might play a very important role in gene expression in this group of parasites. Here, we review and comment on the most recent findings related to transcription initiation and termination in trypanosomatid protozoa.

  11. Gene Therapy Induces Antigen-Specific Tolerance in Experimental Collagen-Induced Arthritis.

    Directory of Open Access Journals (Sweden)

    Sara Tengvall

    Full Text Available Here, we investigate induction of immunological tolerance by lentiviral based gene therapy in a mouse model of rheumatoid arthritis, collagen II-induced arthritis (CIA. Targeting the expression of the collagen type II (CII to antigen presenting cells (APCs induced antigen-specific tolerance, where only 5% of the mice developed arthritis as compared with 95% of the control mice. In the CII-tolerized mice, the proportion of Tregs as well as mRNA expression of SOCS1 (suppressors of cytokine signaling 1 increased at day 3 after CII immunization. Transfer of B cells or non-B cell APC, as well as T cells, from tolerized to naïve mice all mediated a certain degree of tolerance. Thus, sustainable tolerance is established very early during the course of arthritis and is mediated by both B and non-B cells as APCs. This novel approach for inducing tolerance to disease specific antigens can be used for studying tolerance mechanisms, not only in CIA but also in other autoimmune diseases.

  12. Surface expression of Helicobacter pylori HpaA adhesion antigen on Vibrio cholerae, enhanced by co-expressed enterotoxigenic Escherichia coli fimbrial antigens.

    Science.gov (United States)

    Tobias, Joshua; Lebens, Michael; Wai, Sun Nyunt; Holmgren, Jan; Svennerholm, Ann-Mari

    2017-04-01

    Helicobacter pylori infection can cause peptic ulceration and is associated with gastric adenocarcinoma. This study aimed to construct and characterize a non-virulent Vibrio cholerae O1 strain, which grows more rapidly than H. pylori, as vector for H. pylori antigens for possible use as a vaccine strain against H. pylori. This was done by recombinant expression of the H. pylori adhesion antigen HpaA alone or, as a proof of principle, together with different colonization factor (CF) antigens of enterotoxigenic Escherichia coli (ETEC) which may enhance immune responses against HpaA. A recombinant V. cholerae strain co-expressing HpaA and a fimbrial CF antigens CFA/I or CS5, but not the non-fimbrial CF protein CS6, was shown to express larger amounts of HpaA on the surface when compared with the same V. cholerae strain expressing HpaA alone. Mutations in the CFA/I operon showed that the chaperon, possibly together with the usher, was involved in enhancing the surface expression of HpaA. Oral immunization of mice with formaldehyde-inactivated recombinant V. cholerae expressing HpaA alone or together with CFA/I induced significantly higher serum antibody responses against HpaA than mice similarly immunized with inactivated HpaA-expressing H. pylori bacteria. Our results demonstrate that a non-virulent V. cholerae strain can be engineered to allow strong surface expression of HpaA, and that the expression can be further increased by co-expressing it with ETEC fimbrial antigens. Such recombinant V. cholerae strains expressing HpaA, and possibly also other H. pylori antigens, may have the potential as oral inactivated vaccine candidates against H. pylori. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Human embryonic stem cells hemangioblast express HLA-antigens

    Directory of Open Access Journals (Sweden)

    Min Wei-Ping

    2009-04-01

    Full Text Available Abstract Background It has been suggested that the initial differentiation of endothelial and hematopoietic cells during embryogenesis occurs from a common progenitor, called hemangioblast (hB. We hypothesized that these cells with dual hematopoietic/endothelial potential could be used in future regenerative medicine. Methods We used the two-step differentiation technology to generate bipotential blast cells from human embryonic stem cells (hES. This involved short differentiation in our in vitro EB system followed by differentiation in semisolid culture medium supplemented with mixture of cytokines. Results The occurrence of blast-colony-forming cells (BL-CFC during EB differentiation (day 0–6 was transient and peaked on day 3. The emergence of this event was associated with expression of mesoderm gene T, and inversely correlated with expression of endoderm gene FoxA2. Similarly, the highest BL-CFC number was associated with increase in expression of early hematopoietic/endothelial genes: CD34, CD31 and KDR. The derived colonies were composed of 30–50 blast cells on day 6 in culture. These cells had homogenous appearance in Wright-Giemsa stain, but to a different extent expressed markers of immature hematopoietic and endothelial cells (CD31, CD34, VE-cadherin, Flt-1 and mature differentiated cells (CD45, CD33, CD146. We found that some of them expressed fetal and embryonic globin genes. Interestingly, these cells expressed also HLA class I molecules, however at very low levels compared to endothelial and hematopoietic cells. The blast cells could be successfully differentiated to hematopoietic cells in a CFU assay. In these conditions, blast cells formed CFU-M colonies (63.4 ± 0.8% containing macrophages, BFU-E colonies (19.5 ± 3.5% containing nucleated red blood cells, and CFU-EM colonies (17.1 ± 2.7% composed of macrophages and nucleated erythrocytes. Cells of CFU-EM and BFU-E colonies expressed both ε – and γ- globin genes, but

  14. Expression of tumor-specific antigen MAGE, GAGE and BAGE in ovarian cancer tissues and cell lines

    International Nuclear Information System (INIS)

    Zhang, Shiqian; Zhou, Xiaoliang; Yu, Hao; Yu, Yunhai

    2010-01-01

    To observe mRNA expression of tumor-specific antigen MAGE, BAGE and GAGE in epithelial ovarian cancer tissues and cell lines, to explore the relationship between gene expression and diagnosis, treatment and prognosis of ovarian cancer, and to evaluate the feasibility of their gene products as markers, and an immunotherapy target for ovarian cancer. mRNA expression of MAGE-1, MAGE-3, GAGE-1/2 and BAGE were determined by reverse transcription polymerase chain reaction (RT-PCR) in 14 cases of normal ovarian tissue, 20 cases of ovarian benign tumor specimens, 41 cases of ovarian cancer specimens, and ovarian cancer cell lines SKOV3, A2780, and COC1. MAGE, GAGE and BAGE genes were not expressed in normal ovarian tissue. In benign tumors, only the MAGE gene was expressed; the expression rate of this gene in benign tumors was 15% (3/20). In ovarian cancer tissues, MAGE-1 and MAGE-3 was highly expressed, with expression rates of 53.7% (22/41) and 36.6% (15/41), while GAGE-1/2 and BAGE had relatively low expression, with rates of 26.8% (11/41) and 14.6% (6/41). In metastatic lesions of ovarian cancer, only MAGE-1 and BAGE were expressed, with expression rates of 28.6% (2/7) and 14.3% (1/7). The positive expression rates of MAGE-1 and MAGE-3 in serous cystadenocarcinoma were significantly higher than that in other types of ovarian cancer (P < 0.05). Gene expression rate was not correlated with menopause or lymph node metastasis. Positive expression of MAGE-1 and MAGE-3 was positively correlated with tumor differentiation and the clinical stage of the ovarian cancer. In addition, the positive expression rate of BAGE was significantly higher in ovarian cancer patients with ascites (P < 0.05). The mRNA expression profiles of MAGE, GAGE and BAGE in ovarian carcinoma cell lines SKOV3, A2780 and COC1 varied, but there was at least one gene expressed in each cell line. Tumor-specific antigen MAGE, BAGE and GAGE may play a role in the occurrence and development of ovarian cancer

  15. [Expressions of HLA class I antigen and CD8 and their clinical significance in cervical cancer].

    Science.gov (United States)

    Qi, Yue; Huang, Jin-Shuang; Wang, Dong-dong; Zhang, Fan; Zhang, Shu-lan

    2008-12-01

    To examine the expressions of HLA class I antigen and CD8 in various cervical diseases and investigate their association with cervical cancer. The expressions of HLA class I antigen and CD8 in cervical tissues sampled from patients with cervical cancer, cervical intraepithelial neoplasia (CIN), and chronic cervicitis were detected using SP immunohistochemistry. The association of the expressions of HLA class I antigen and CD8 with the clinicopathologic indices of the patients was analyzed. The positive expression rates of HLA class I antigen in cervical cancer, CIN, and chronic cervicitis were 22.6%, 100.0%, and 100.0%, and the positive expression rates of CD8 were 22.6%, 95.5%, and 100.0%, respectively. The positive rates of HLA class I antigen and CD8 were significantly lower in patients with cervical cancer (Pcancer had significantly higher positive rates of HLA class I antigen and CD8 than those with stage II cervical cancer (46.7% vs 0.0%, 46.7% vs 0.0%, both PHLA class I antigen and CD8 decreased with the progression of the clinicopathological stages, and may even become undetectable. The expressions of HLA class I antigen and CD8 were not related to the differentiation degree of the tumor or lymph node metastasis (P>0.05). A positive correlation was found between HLA class I antigen expression and CD8 expression. The expressions of HLA class I antigen and CD8 are down-regulated or deleted in CIN and cervical cancer, and they may play important roles in the development and progression of CIN and cervical cancer.

  16. Increased parasite surface antigen-2 expression in clinical isolates of Leishmania donovani augments antimony resistance.

    Science.gov (United States)

    Bhandari, Vasundhra; Kumar, Dhiraj; Verma, Sandeep; Srividya, Gurumurthy; Negi, Narendra Singh; Singh, Ruchi; Salotra, Poonam

    2013-11-01

    Resistance to sodium antimony gluconate (SAG) is a major cause of therapeutic failure in a large proportion of visceral leishmaniasis (VL) cases. Determinants of SAG resistance have been widely studied; however, the mechanism operating in clinical isolates is poorly understood. In the present study, expression of parasite surface antigen-2 (PSA-2) gene was studied in clinical isolates of Leishmania donovani comprising of antimony resistant (n=10) and sensitive (n=4) parasites. The expression of PSA-2 gene was found to be consistently high in SAG resistant clinical isolates (≥1.5-fold) at both transcript and protein level. Further, over-expression of PSA-2 in L. donovani isolates (LdPSA-2(++)) resulted in conversion of SAG sensitive phenotype to resistant. The LdPSA-2(++) parasites showed significantly decreased susceptibility towards SAG (>12-fold), amphotericin B (>4-fold) and miltefosine (>2.5-fold). Marked decrease in antimony accumulation and enhanced tolerance towards complement mediated lysis was evident in LdPSA-2(++) parasites. The study established the role of PSA-2 gene in SAG resistance and its potential as a biomarker to distinguish resistant and sensitive clinical isolates of L. donovani. Copyright © 2013 Elsevier Inc. All rights reserved.

  17. Unpolarized Release of Vaccinia Virus and HIV Antigen by Colchicine Treatment Enhances Intranasal HIV Antigen Expression and Mucosal Humoral Responses

    Science.gov (United States)

    Zhang, Yan; Yang, Jingyi; Bao, Rong; Chen, Yaoqing; Zhou, Dihan; He, Benxia; Zhong, Maohua; Li, Yaoming; Liu, Fang; Li, Qiaoli; Yang, Yi; Han, Chen; Sun, Ying; Cao, Yuan; Yan, Huimin

    2011-01-01

    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. PMID:21935396

  18. 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.

  19. [Expression of foreign gene by cysteine proteinase null recombinant baculovirus].

    Science.gov (United States)

    Likhoradova, O A; Ogaĭ, I D; Podpisnova, M M; Slack, J M; Azimova, Sh S

    2008-01-01

    The baculovirus expression vector systems (BEVS) are broadly used for producing foreign proteins in lepidopteran larvae. Most commercial BEVS are engineered to insert foreign genes into the polyhedrin (polh) locus and lack the polh gene. These viruses cannot produce occlusion bodies and are inconvenient for per os inoculation of larvae. Current knowledge in baculovirus genomics makes it possible to engineer BEVS into other parts of the virus genome. In our work, we have expressed recombinant M-HBsAg (middle surface antigen of human hepatitis B) in the baculovirus construct, rBmNPV-Deltav-cath-M-HBsAg, inserting foreign gene into the v-cath locus of the Bombyx mori nucleopolyhedrovirus (BmNPV) such that the v-cath gene is deleted and the native polh gene is retained. Silkworm larvae were infected per os and M-HBsAg was observed to be abundantly produced at a very late stage of infection.

  20. Gene expression of transcription factor NFATc1 in periodontal diseases

    OpenAIRE

    Belibasakis, G N; Emingil, G; Saygan, B; Turkoglu, O; Atilla, G; Bostanci, N

    2011-01-01

    Belibasakis GN, Emingil G, Saygan B, Turkoglu O, Atilla G, Bostanci N. Gene expression of transcription factor NFATc1 in periodontal diseases. APMIS 2011; 119: 167-172. Periodontitis is a disease of infectious aetiology that causes inflammatory destruction of the tooth-supporting tissues. Activated T cells are central to the pathogenesis of the disease, by producing receptor activator of nuclear factor κB (NF-κB) ligand (RANKL) that stimulates bone resorption. Antigenic activation of T cells ...

  1. Expression of a preproinsulin-beta-galactosidase gene fusion in mammalian cells.

    OpenAIRE

    Nielsen, D A; Chou, J; MacKrell, A J; Casadaban, M J; Steiner, D F

    1983-01-01

    As an approach to the study of mammalian gene expression, the promoters and translation initiation regions of the rat preproinsulin II and the simian virus 40 early genes were fused to the structural gene of Escherichia coli beta-galactosidase, a sensitive probe for gene expression. These fusions were introduced into COS-7 cells, a simian virus 40 large tumor-antigen-producing monkey kidney cell line, where they directed the synthesis of enzymatically active hybrid beta-galactosidase proteins...

  2. The evolutionary dynamics of variant antigen genes in Babesia reveal a history of genomic innovation underlying host-parasite interaction

    KAUST Repository

    Jackson, Andrew P.

    2014-05-05

    Babesia spp. are tick-borne, intraerythrocytic hemoparasites that use antigenic variation to resist host immunity, through sequential modification of the parasite-derived variant erythrocyte surface antigen (VESA) expressed on the infected red blood cell surface. We identified the genomic processes driving antigenic diversity in genes encoding VESA (ves1) through comparative analysis within and between three Babesia species, (B. bigemina, B. divergens and B. bovis). Ves1 structure diverges rapidly after speciation, notably through the evolution of shortened forms (ves2) from 5? ends of canonical ves1 genes. Phylogenetic analyses show that ves1 genes are transposed between loci routinely, whereas ves2 genes are not. Similarly, analysis of sequence mosaicism shows that recombination drives variation in ves1 sequences, but less so for ves2, indicating the adoption of different mechanisms for variation of the two families. Proteomic analysis of the B. bigemina PR isolate shows that two dominant VESA1 proteins are expressed in the population, whereas numerous VESA2 proteins are co-expressed, consistent with differential transcriptional regulation of each family. Hence, VESA2 proteins are abundant and previously unrecognized elements of Babesia biology, with evolutionary dynamics consistently different to those of VESA1, suggesting that their functions are distinct. 2014 The Author(s) 2014.

  3. Harnessing gene expression networks to prioritize candidate epileptic encephalopathy genes.

    Science.gov (United States)

    Oliver, Karen L; Lukic, Vesna; Thorne, Natalie P; Berkovic, Samuel F; Scheffer, Ingrid E; Bahlo, Melanie

    2014-01-01

    We apply a novel gene expression network analysis to a cohort of 182 recently reported candidate Epileptic Encephalopathy genes to identify those most likely to be true Epileptic Encephalopathy genes. These candidate genes were identified as having single variants of likely pathogenic significance discovered in a large-scale massively parallel sequencing study. Candidate Epileptic Encephalopathy genes were prioritized according to their co-expression with 29 known Epileptic Encephalopathy genes. We utilized developing brain and adult brain gene expression data from the Allen Human Brain Atlas (AHBA) and compared this to data from Celsius: a large, heterogeneous gene expression data warehouse. We show replicable prioritization results using these three independent gene expression resources, two of which are brain-specific, with small sample size, and the third derived from a heterogeneous collection of tissues with large sample size. Of the nineteen genes that we predicted with the highest likelihood to be true Epileptic Encephalopathy genes, two (GNAO1 and GRIN2B) have recently been independently reported and confirmed. We compare our results to those produced by an established in silico prioritization approach called Endeavour, and finally present gene expression networks for the known and candidate Epileptic Encephalopathy genes. This highlights sub-networks of gene expression, particularly in the network derived from the adult AHBA gene expression dataset. These networks give clues to the likely biological interactions between Epileptic Encephalopathy genes, potentially highlighting underlying mechanisms and avenues for therapeutic targets.

  4. Tolerance induction to cytoplasmic beta-galactosidase by hepatic AAV gene transfer: implications for antigen presentation and immunotoxicity.

    Directory of Open Access Journals (Sweden)

    Ashley T Martino

    2009-08-01

    Full Text Available Hepatic gene transfer, in particular using adeno-associated viral (AAV vectors, has been shown to induce immune tolerance to several protein antigens. This approach has been exploited in animal models of inherited protein deficiency for systemic delivery of therapeutic proteins. Adequate levels of transgene expression in hepatocytes induce a suppressive T cell response, thereby promoting immune tolerance. This study addresses the question of whether AAV gene transfer can induce tolerance to a cytoplasmic protein.AAV-2 vector-mediated hepatic gene transfer for expression of cytoplasmic beta-galactosidase (beta-gal was performed in immune competent mice, followed by a secondary beta-gal gene transfer with E1/E3-deleted adenoviral Ad-LacZ vector to provoke a severe immunotoxic response. Transgene expression from the AAV-2 vector in approximately 2% of hepatocytes almost completely protected from inflammatory T cell responses against beta-gal, eliminated antibody formation, and significantly reduced adenovirus-induced hepatotoxicity. Consequently, approximately 10% of hepatocytes continued to express beta-gal 45 days after secondary Ad-LacZ gene transfer, a time point when control mice had lost all Ad-LacZ derived expression. Suppression of inflammatory T cell infiltration in the liver and liver damage was linked to specific transgene expression and was not seen for secondary gene transfer with Ad-GFP. A combination of adoptive transfer studies and flow cytometric analyses demonstrated induction of Treg that actively suppressed CD8(+ T cell responses to beta-gal and that was amplified in liver and spleen upon secondary Ad-LacZ gene transfer.These data demonstrate that tolerance induction by hepatic AAV gene transfer does not require systemic delivery of the transgene product and that expression of a cytoplasmic neo-antigen in few hepatocytes can induce Treg and provide long-term suppression of inflammatory responses and immunotoxicity.

  5. 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.

  6. 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.

  7. 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...

  8. Modulation of gene expression made easy

    DEFF Research Database (Denmark)

    Solem, Christian; Jensen, Peter Ruhdal

    2002-01-01

    A new approach for modulating gene expression, based on randomization of promoter (spacer) sequences, was developed. The method was applied to chromosomal genes in Lactococcus lactis and shown to generate libraries of clones with broad ranges of expression levels of target genes. In one example...... that the method can be applied to modulating the expression of native genes on the chromosome. We constructed a series of strains in which the expression of the las operon, containing the genes pfk, pyk, and ldh, was modulated by integrating a truncated copy of the pfk gene. Importantly, the modulation affected...

  9. Cerebrovascular gene expression in spontaneously hypertensive rats

    DEFF Research Database (Denmark)

    Grell, Anne-Sofie; Frederiksen, Simona Denise; Edvinsson, Lars

    2017-01-01

    in the middle cerebral arteries from hypertensive compared to normotensive rats. The gene expression of 72 genes was decreased and the gene expression of 97 genes was increased. The following genes with a fold difference ≥1.40 were verified by quantitative PCR; Postn, Olr1, Fas, Vldlr, Mmp2, Timp1, Serpine1......, Mmp11, Cd34, Ptgs1 and Ptgs2. The gene expression of Postn, Olr1, Fas, Vldlr, Mmp2, Timp1 and Serpine1 and the protein expression of LOX1 (also known as OLR1) were significantly increased in the middle cerebral arteries from spontaneously hypertensive rats compared to Wistar-Kyoto rats. In conclusion...

  10. Stable expression of Mycobacterium bovis antigen 85B in auxotrophic M. bovis bacillus Calmette-Guérin

    Directory of Open Access Journals (Sweden)

    Caroline Rizzi

    Full Text Available BACKGROUND Bovine tuberculosis (TB is a zoonotic disease caused by Mycobacterium bovis, responsible for causing major losses in livestock. A cost effective alternative to control the disease could be herd vaccination. The bacillus Calmette-Guérin (BCG vaccine has a limited efficacy against bovine TB, but can improved by over-expression of protective antigens. The M. bovis antigen 85B demonstrates ability to induce protective immune response against bovine TB in animal models. However, current systems for the construction of recombinant BCG expressing multiple copies of the gene result in strains of low genetic stability that rapidly lose the plasmid in vivo. Employing antibiotic resistance as selective markers, these systems also compromise vaccine safety. We previously reported the construction of a stable BCG expression system using auxotrophic complementation as a selectable marker. OBJECTIVES The fundamental aim of this study was to construct strains of M. bovis BCG Pasteur and the auxotrophic M. bovis BCG ΔleuD expressing Ag85B and determine their stability in vivo. METHODS Employing the auxotrophic system, we constructed rBCG strains that expressed M. bovis Ag85B and compared their stability with a conventional BCG strain in mice. Stability was measured in terms of bacterial growth on the selective medium and retention of antigen expression. FINDINGS The auxotrophic complementation system was highly stable after 18 weeks, even during in vivo growth, as the selective pressure and expression of antigen were maintained comparing to the conventional vector. MAIN CONCLUSION The Ag85B continuous expression within the host may generate a stronger and long-lasting immune response compared to conventional systems.

  11. Human Tregs Made Antigen Specific by Gene Modification: The Power to Treat Autoimmunity and Antidrug Antibodies with Precision

    Directory of Open Access Journals (Sweden)

    Patrick R. Adair

    2017-09-01

    Full Text Available Human regulatory CD4+ T cells (Tregs are potent immunosuppressive lymphocytes responsible for immune tolerance and homeostasis. Since the seminal reports identifying Tregs, vast research has been channeled into understanding their genesis, signature molecular markers, mechanisms of suppression, and role in disease. This research has opened the doors for Tregs as a potential therapeutic for diseases and disorders such as multiple sclerosis, type I diabetes, transplantation, and immune responses to protein therapeutics, like factor VIII. Seminal clinical trials have used polyclonal Tregs, but the frequency of antigen-specific Tregs among polyclonal populations is low, and polyclonal Tregs may risk non-specific immunosuppression. Antigen-specific Treg therapy, which uses genetically modified Tregs expressing receptors specific for target antigens, greatly mitigates this risk. Building on the principles of T-cell receptor cloning, chimeric antigen receptors (CARs, and a novel CAR derivative, called B-cell antibody receptors, our lab has developed different types of antigen-specific Tregs. This review discusses the current research and optimization of gene-modified antigen-specific human Tregs in our lab in several disease models. The preparations and considerations for clinical use of such Tregs also are discussed.

  12. 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.

  13. Adaptive Evolution of Gene Expression in Drosophila

    Directory of Open Access Journals (Sweden)

    Armita Nourmohammad

    2017-08-01

    Full Text Available Gene expression levels are important quantitative traits that link genotypes to molecular functions and fitness. In Drosophila, population-genetic studies have revealed substantial adaptive evolution at the genomic level, but the evolutionary modes of gene expression remain controversial. Here, we present evidence that adaptation dominates the evolution of gene expression levels in flies. We show that 64% of the observed expression divergence across seven Drosophila species are adaptive changes driven by directional selection. Our results are derived from time-resolved data of gene expression divergence across a family of related species, using a probabilistic inference method for gene-specific selection. Adaptive gene expression is stronger in specific functional classes, including regulation, sensory perception, sexual behavior, and morphology. Moreover, we identify a large group of genes with sex-specific adaptation of expression, which predominantly occurs in males. Our analysis opens an avenue to map system-wide selection on molecular quantitative traits independently of their genetic basis.

  14. 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.

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

    International Nuclear Information System (INIS)

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

    1992-01-01

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

  16. Expression of tumor-specific antigen MAGE, GAGE and BAGE in ovarian cancer tissues and cell lines.

    Science.gov (United States)

    Zhang, Shiqian; Zhou, Xiaoliang; Yu, Hao; Yu, Yunhai

    2010-04-27

    To observe mRNA expression of tumor-specific antigen MAGE, BAGE and GAGE in epithelial ovarian cancer tissues and cell lines, to explore the relationship between gene expression and diagnosis, treatment and prognosis of ovarian cancer, and to evaluate the feasibility of their gene products as markers, and an immunotherapy target for ovarian cancer. mRNA expression of MAGE-1, MAGE-3, GAGE-1/2 and BAGE were determined by reverse transcription polymerase chain reaction (RT-PCR) in 14 cases of normal ovarian tissue, 20 cases of ovarian benign tumor specimens, 41 cases of ovarian cancer specimens, and ovarian cancer cell lines SKOV3, A2780, and COC1. MAGE, GAGE and BAGE genes were not expressed in normal ovarian tissue. In benign tumors, only the MAGE gene was expressed; the expression rate of this gene in benign tumors was 15% (3/20). In ovarian cancer tissues, MAGE-1 and MAGE-3 was highly expressed, with expression rates of 53.7% (22/41) and 36.6% (15/41), while GAGE-1/2 and BAGE had relatively low expression, with rates of 26.8% (11/41) and 14.6% (6/41). In metastatic lesions of ovarian cancer, only MAGE-1 and BAGE were expressed, with expression rates of 28.6% (2/7) and 14.3% (1/7). The positive expression rates of MAGE-1 and MAGE-3 in serous cystadenocarcinoma were significantly higher than that in other types of ovarian cancer (P BAGE was significantly higher in ovarian cancer patients with ascites (P BAGE in ovarian carcinoma cell lines SKOV3, A2780 and COC1 varied, but there was at least one gene expressed in each cell line. Tumor-specific antigen MAGE, BAGE and GAGE may play a role in the occurrence and development of ovarian cancer. These genes can be used as one of the important indicators for early diagnosis, efficacy evaluation and prognostic determination of ovarian cancer.

  17. Vaccine protection of chickens against antigenically diverse H5 highly pathogenic avian influenza isolates with a live HVT vector vaccine expressing the influenza hemagglutinin gene derived from a clade 2.2 avian influenza virus.

    Science.gov (United States)

    Kapczynski, Darrell R; Esaki, Motoyuki; Dorsey, Kristi M; Jiang, Haijun; Jackwood, Mark; Moraes, Mauro; Gardin, Yannick

    2015-02-25

    Vaccination is an important tool in the protection of poultry against avian influenza (AI). For field use, the overwhelming majority of AI vaccines produced are inactivated whole virus formulated into an oil emulsion. However, recombinant vectored vaccines are gaining use for their ability to induce protection against heterologous isolates and ability to overcome maternal antibody interference. In these studies, we compared protection of chickens provided by a turkey herpesvirus (HVT) vector vaccine expressing the hemagglutinin (HA) gene from a clade 2.2 H5N1 strain (A/swan/Hungary/4999/2006) against homologous H5N1 as well as heterologous H5N1 and H5N2 highly pathogenic (HP) AI challenge. The results demonstrated all vaccinated birds were protected from clinical signs of disease and mortality following homologous challenge. In addition, oral and cloacal swabs taken from challenged birds demonstrated that vaccinated birds had lower incidence and titers of viral shedding compared to sham-vaccinated birds. Following heterologous H5N1 or H5N2 HPAI challenge, 80-95% of birds receiving the HVT vector AI vaccine at day of age survived challenge with fewer birds shedding virus after challenge than sham vaccinated birds. In vitro cytotoxicity analysis demonstrated that splenic T lymphocytes from HVT-vector-AI vaccinated chickens recognized MHC-matched target cells infected with H5, as well as H6, H7, or H9 AI virus. Taken together, these studies provide support for the use of HVT vector vaccines expressing HA to protect poultry against multiple lineages of HPAI, and that both humoral and cellular immunity induced by live vaccines likely contributes to protection. Published by Elsevier Ltd.

  18. Nonlinear dimensionality reduction of gene expression data

    OpenAIRE

    Nilsson, Jens

    2006-01-01

    Using microarray measurements techniques, it is possible to measure the activity of genes simultaneously across the whole genome. Since genes influence each others activity levels through complex regulatory networks, such gene expression measurements are state samples of a dynamical system. Gene expression data has proven useful for diagnosis and definition of disease subgroups, for inference of the functional role of a given gene or for the deciphering of complex disease mechanisms. However,...

  19. Analysis of CD83 antigen expression in human breast fibroadenoma and adjacent tissue.

    Science.gov (United States)

    Borges, Marcus Nascimento; Facina, Gil; Silva, Ismael Dale Cotrin Guerreiro; Waitzberg, Angela Flávia Logullo; Nazario, Afonso Celso Pinto

    2011-12-01

    Dendritic cell maturation is considered essential for starting an immune response. The CD83 antigen is an important marker of dendritic cell maturation. The objectives here were to analyze CD83 antigen expression in human breast fibroadenoma and breast tissue adjacent to the lesion and to identify clinical factors that might influence this expression. This was a retrospective study at a public university hospital, in which 29 histopathological samples of breast fibroadenoma and adjacent breast tissue, from 28 women of reproductive age, were analyzed. The immunohistochemistry method was used to analyze the cell expression of the antigen. The antigen expression in the cells was evaluated by means of random manual counting using an optical microscope. Positive expression of the CD83 antigen in the epithelial cells of the fibroadenoma (365.52; standard deviation ± 133.13) in relation to the adjacent breast tissue cells (189.59; standard deviation ± 140.75) was statistically larger (P fibroadenoma was positive and greater than in the adjacent breast tissue. Positive expression of the antigen in the adjacent breast tissue was influenced by parity, and was significantly more evident in nulliparous women.

  20. Proliferating cell nuclear antigen (Pcna) as a direct downstream target gene of Hoxc8

    Energy Technology Data Exchange (ETDEWEB)

    Min, Hyehyun; Lee, Ji-Yeon; Bok, Jinwoong; Chung, Hyun Joo [Department of Anatomy, Embryology Laboratory, Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of); Kim, Myoung Hee, E-mail: mhkim1@yuhs.ac [Department of Anatomy, Embryology Laboratory, Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of)

    2010-02-19

    Hoxc8 is a member of Hox family transcription factors that play crucial roles in spatiotemporal body patterning during embryogenesis. Hox proteins contain a conserved 61 amino acid homeodomain, which is responsible for recognition and binding of the proteins onto Hox-specific DNA binding motifs and regulates expression of their target genes. Previously, using proteome analysis, we identified Proliferating cell nuclear antigen (Pcna) as one of the putative target genes of Hoxc8. Here, we asked whether Hoxc8 regulates Pcna expression by directly binding to the regulatory sequence of Pcna. In mouse embryos at embryonic day 11.5, the expression pattern of Pcna was similar to that of Hoxc8 along the anteroposterior body axis. Moreover, Pcna transcript levels as well as cell proliferation rate were increased by overexpression of Hoxc8 in C3H10T1/2 mouse embryonic fibroblast cells. Characterization of 2.3 kb genomic sequence upstream of Pcna coding region revealed that the upstream sequence contains several Hox core binding sequences and one Hox-Pbx binding sequence. Direct binding of Hoxc8 proteins to the Pcna regulatory sequence was verified by chromatin immunoprecipitation assay. Taken together, our data suggest that Pcna is a direct downstream target of Hoxc8.

  1. The Expression of HCG Epitope Fused to Hepatits B Virus Core Antigen.

    Science.gov (United States)

    Li, Guang-Di; Wang, Bin; Chen, Zuo-Yi; Wang, Yuan; Gong, Yue-Ting

    1996-01-01

    The DNA fragments encoding HGC-beta-37-CTP was amplified by PCR and fused to the core gene of HBV at the position of amino Acid 1(N-terminal fusion, pCn-HCG), 154(C-terminal fusion, pCc-HCG), 75-83(internal fusion,pCm-HCG), and both 75-83 and 154 (pC-HCG2) respectively. The fused genes were expressed in E. coli, and the antigenicity of both HBcAg and HCG as well as the expression level were analyzed. In addition, the chimeric particular characteristics of the proteins and their immunogenicity were identified. It revealed that the fusion proteins pCm-HCG and pCc-HCG were able to form particles, and that the fusion protein pCm-HCG could induce antibody of anti-HCG of high titers in mice, suggesting that the position at 75-83 amino acid residue should be a relatively promising fusion site for HCG.

  2. In silico Analysis of Human Telomerase Reverse Transcriptase (hTERT Gene: Identification of a Distant Homolog of Melanoma Antigen Family Gene (MAGE

    Directory of Open Access Journals (Sweden)

    Ruhul Amin

    2009-11-01

    Full Text Available Melanoma antigen family (MAGE genes are widely expressed in various tumor types but silent in normal cells except germ-line cells lacking human leukocyte antigen (HLA expression. Over 25 MAGE genes have been identified in different tissues, mostly located in Xq28 of human chromosome and some of them in chromosome 3 and 15, containing either single or multiple-exons. This in silico study predicted the genes on hTERT location and identified a distant relative of MAGE gene located on chromosome 5. The study identified a single exon coding ∼850 residues polypeptide sharing ∼30% homology with Macfa-MAGE E1 and hMAGE-E1. dbEST search of the predicted transcript matches 5' and 3' flanking ESTs. The predicted protein showed sequence homology within the MAGE homology domain 2 (MHD2. UCSC genome annotation of CpG Island around the coding region reveals that this gene could be silent by methylation. Affymetrix all-exon track indicates the gene could be expressed in different tissues particularly in cancer cells as they widely undergo a genome wide demethylation process.

  3. 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.

  4. An evolutionarily conserved mutual interdependence between Aire and microRNAs in promiscuous gene expression

    OpenAIRE

    Ucar, Olga; Tykocinski, Lars-Oliver; Dooley, James; Liston, Adrian; Kyewski, Bruno

    2013-01-01

    The establishment and maintenance of central tolerance depends to a large extent on the ability of medullary thymic epithelial cells to express a variety of tissue-restricted antigens, the so-called promiscuous gene expression (pGE). Autoimmune regulator (Aire) is to date the best characterised transcriptional regulator known to at least partially coordinate pGE. There is accruing evidence that the expression of Aire-dependent and -independent genes is modulated by higher order chromatin conf...

  5. Biochemical activities of T-antigen proteins encoded by simian virus 40 A gene deletion mutants.

    OpenAIRE

    Clark, R; Peden, K; Pipas, J M; Nathans, D; Tjian, R

    1983-01-01

    We have analyzed T antigens produced by a set of simian virus 40 (SV40) A gene deletion mutants for ATPase activity and for binding to the SV40 origin of DNA replication. Virus stocks of nonviable SV40 A gene deletion mutants were established in SV40-transformed monkey COS cells. Mutant T antigens were produced in mutant virus-infected CV1 cells. The structures of the mutant T antigens were characterized by immunoprecipitation with monoclonal antibodies directed against distinct regions of th...

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

    International Nuclear Information System (INIS)

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

    2012-01-01

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

  7. 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.

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

    Directory of Open Access Journals (Sweden)

    L.T.M. Figueiredo

    2008-07-01

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

  9. Prediction of the gene expression in normal lung tissue by the gene expression in blood.

    Science.gov (United States)

    Halloran, Justin W; Zhu, Dakai; Qian, David C; Byun, Jinyoung; Gorlova, Olga Y; Amos, Christopher I; Gorlov, Ivan P

    2015-11-17

    Comparative analysis of gene expression in human tissues is important for understanding the molecular mechanisms underlying tissue-specific control of gene expression. It can also open an avenue for using gene expression in blood (which is the most easily accessible human tissue) to predict gene expression in other (less accessible) tissues, which would facilitate the development of novel gene expression based models for assessing disease risk and progression. Until recently, direct comparative analysis across different tissues was not possible due to the scarcity of paired tissue samples from the same individuals. In this study we used paired whole blood/lung gene expression data from the Genotype-Tissue Expression (GTEx) project. We built a generalized linear regression model for each gene using gene expression in lung as the outcome and gene expression in blood, age and gender as predictors. For ~18 % of the genes, gene expression in blood was a significant predictor of gene expression in lung. We found that the number of single nucleotide polymorphisms (SNPs) influencing expression of a given gene in either blood or lung, also known as the number of quantitative trait loci (eQTLs), was positively associated with efficacy of blood-based prediction of that gene's expression in lung. This association was strongest for shared eQTLs: those influencing gene expression in both blood and lung. In conclusion, for a considerable number of human genes, their expression levels in lung can be predicted using observable gene expression in blood. An abundance of shared eQTLs may explain the strong blood/lung correlations in the gene expression.

  10. 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.

  11. Synthetic promoter libraries- tuning of gene expression

    DEFF Research Database (Denmark)

    Hammer, Karin; Mijakovic, Ivan; Jensen, Peter Ruhdal

    2006-01-01

    The study of gene function often requires changing the expression of a gene and evaluating the consequences. In principle, the expression of any given gene can be modulated in a quasi-continuum of discrete expression levels but the traditional approaches are usually limited to two extremes: gene...... be met by using promoter libraries. This approach generally consists of inserting a library of promoters in front of the gene to be studied, whereby the individual promoters might deviate either in their spacer sequences or bear slight deviations from the consensus sequence of a vegetative promoter. Here......, we describe the two different methods for obtaining promoter libraries and compare their applicability....

  12. Expression of sperm-associated antigen 6 in liver cancer tissue and its clinical significance

    Directory of Open Access Journals (Sweden)

    GU Junming

    2017-10-01

    Full Text Available ObjectiveTo investigate the expression of sperm-associated antigen 6 (Spag6 in liver cancer tissue and its association with the clinicopathological features and prognosis of liver cancer patients, as well as the effect of Spag6 on the proliferation and migration of HCCLM3 hepatoma cells. MethodsClinical samples were collected from 102 liver cancer patients who were treated in Xiangya Hospital of Central South University from August 2006 to November 2009, and Western blot was used to measure the expression of Spag6 in hepatoma cells, normal liver tissue, tumor tissue, and corresponding adjacent tissue. Immunohistochemistry was used to measure the expression of Spag6 in 102 liver cancer tissue samples, and according to the immunohistochemical scoring criteria, the patients were divided into high Spag6 expression group and low Spag6 expression group. Lentivirus-mediated RNA interference technique was used to silence Spag6 expression in HCCLM3 cells; Western blot was used to analyze silencing effect, wound-healing assay was used to investigate the effect of Spag6 gene silencing on the migration of HCCLM3 cells, and colony formation assay was performed to observe the effect of Spag6 gene silencing on the proliferation of HCCLM3 cells. The chi-square test was used to investigate the association between Spag6 expression and clinicopathological features of liver cancer patients, and the Kaplan-Meier survival analysis and log-rank test were used to analyze the association between Spag6 expression and the prognosis of liver cancer patients. ResultsHepatoma cells and liver cancer tissue had significantly higher expression of Spag6 than the normal LO2 cells and normal liver tissue. Immunohistochemistry showed that the expression rate of Spag6 was 58.8% (60/102 in liver cancer tissue samples and 12.7% (13/102 in adjacent tissue samples (χ2=47123,P<0.001. According to the results of the chi-square test, Spag6 expression was associated with the number of

  13. [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.

  14. Abnormal gene expression and gene fusion in lung adenocarcinoma with high-throughput RNA sequencing.

    Science.gov (United States)

    Yang, Z-H; Zheng, R; Gao, Y; Zhang, Q; Zhang, H

    2014-02-01

    To explore the universal law of the abnormal gene expression and the structural variation of genes related to lung adenocarcinoma, the gene expression profile of GSE37765 were downloaded from Gene Expression Omnibus database. The differentially expressed genes (DEGs) were analyzed with t-test and NOISeq tool, and the core DEGs were screened out by combining with another RNA-seq data containing totally 77 pairs of samples in 77 patients with lung adenocarcinoma. Moreover, the functional annotation of the core DEGs was performed by using the Database for Annotation Visualization and Integrated Discovery following selection of oncogene and tumor suppressor by combining with tumor suppressor genes and Cancer Genes database, and motif-finding of core DEGs was performed with motif-finding algorithm Seqpos. We also used Tophat-fusion tool to further explore the fusion genes. In total, 850 downregulated DEGs and 206 upregulated DEGs were screened out in lung adenocarcinoma tissues. Next, we selected 543 core DEGs, including 401 downregulated and 142 upregulated genes, and vasculature development (P=1.89E-06) was significantly enriched among downregulated core genes, as well as mitosis (P=6.26E-04) enriched among upregulated core genes. On the basis of the cellular localization analysis of core genes, wnt-1-induced secreted protein 1 (WISP1) and receptor (G protein-coupled) activity modifying protein 1 (RAMP1) identified mainly located in extracellular region and extracellular space. We also screened one oncogene, v-myb avian myeloblastosis viral oncogene homolog-like 2 (MYBL2). Moreover, transcription factor GATA2 was mined by motif-finding analysis. Finally, four fusion genes belonged to the human leukocyte antigen (HLA) family. WISP1, RAMP1, MYBL2 and GATA2 could be potential targets of treatment for lung adenocarcinoma and the fusion of HLA family genes might have important roles in lung adenocarcinoma.

  15. The effects of a partitioned var gene repertoire of Plasmodium falciparum on antigenic diversity and the acquisition of clinical immunity

    Directory of Open Access Journals (Sweden)

    Arinaminpathy Nimalan

    2008-01-01

    Full Text Available Abstract Background The human malaria parasite Plasmodium falciparum exploits antigenic diversity and within-host antigenic variation to evade the host's immune system. Of particular importance are the highly polymorphic var genes that encode the family of cell surface antigens PfEMP1 (Plasmodium falciparum Erythrocyte Membrane Protein 1. It has recently been shown that in spite of their extreme diversity, however, these genes fall into distinct groups according to chromosomal location or sequence similarity, and that recombination may be confined within these groups. Methods This study presents a mathematical analysis of how recombination hierarchies affect diversity, and, by using simple stochastic simulations, investigates how intra- and inter-genic diversity influence the rate at which individuals acquire clinical immunity. Results The analysis demonstrates that the partitioning of the var gene repertoire has a limiting effect on the total diversity attainable through recombination and that the limiting effect is strongly influenced by the respective sizes of each of the partitions. Furthermore, by associating expression of one of the groups with severe malaria it is demonstrated how a small number of infections can be sufficient to protect against disease despite a seemingly limitless number of possible non-identical repertoires. Conclusion Recombination hierarchies within the var gene repertoire of P. falciparum have a severe effect on strain diversity and the process of acquiring immunity against clinical malaria. Future studies will show how the existence of these recombining groups can offer an evolutionary advantage in spite of their restriction on diversity.

  16. 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

  17. 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...... on their surface. Polyclonal antibodies against defined peptides in the Env- and Gag-regions of the HERVs were raised in rabbits and used in antibody-dependent cell-mediated cytotoxicity (ADCC) -assays. Rituximab® (Roche), a chimeric monoclonal antibody against CD20 expressed primarily on B cells, was used...

  18. Profiling Gene Expression in Germinating Brassica Roots.

    Science.gov (United States)

    Park, Myoung Ryoul; Wang, Yi-Hong; Hasenstein, Karl H

    2014-01-01

    Based on previously developed solid-phase gene extraction (SPGE) we examined the mRNA profile in primary roots of Brassica rapa seedlings for highly expressed genes like ACT7 (actin7), TUB (tubulin1), UBQ (ubiquitin), and low expressed GLK (glucokinase) during the first day post-germination. The assessment was based on the mRNA load of the SPGE probe of about 2.1 ng. The number of copies of the investigated genes changed spatially along the length of primary roots. The expression level of all genes differed significantly at each sample position. Among the examined genes ACT7 expression was most even along the root. UBQ was highest at the tip and root-shoot junction (RS). TUB and GLK showed a basipetal gradient. The temporal expression of UBQ was highest in the MZ 9 h after primary root emergence and higher than at any other sample position. Expressions of GLK in EZ and RS increased gradually over time. SPGE extraction is the result of oligo-dT and oligo-dA hybridization and the results illustrate that SPGE can be used for gene expression profiling at high spatial and temporal resolution. SPGE needles can be used within two weeks when stored at 4 °C. Our data indicate that gene expression studies that are based on the entire root miss important differences in gene expression that SPGE is able to resolve for example growth adjustments during gravitropism.

  19. Polycistronic gene expression in Aspergillus niger.

    Science.gov (United States)

    Schuetze, Tabea; Meyer, Vera

    2017-09-25

    Genome mining approaches predict dozens of biosynthetic gene clusters in each of the filamentous fungal genomes sequenced so far. However, the majority of these gene clusters still remain cryptic because they are not expressed in their natural host. Simultaneous expression of all genes belonging to a biosynthetic pathway in a heterologous host is one approach to activate biosynthetic gene clusters and to screen the metabolites produced for bioactivities. Polycistronic expression of all pathway genes under control of a single and tunable promoter would be the method of choice, as this does not only simplify cloning procedures, but also offers control on timing and strength of expression. However, polycistronic gene expression is a feature not commonly found in eukaryotic host systems, such as Aspergillus niger. In this study, we tested the suitability of the viral P2A peptide for co-expression of three genes in A. niger. Two genes descend from Fusarium oxysporum and are essential to produce the secondary metabolite enniatin (esyn1, ekivR). The third gene (luc) encodes the reporter luciferase which was included to study position effects. Expression of the polycistronic gene cassette was put under control of the Tet-On system to ensure tunable gene expression in A. niger. In total, three polycistronic expression cassettes which differed in the position of luc were constructed and targeted to the pyrG locus in A. niger. This allowed direct comparison of the luciferase activity based on the position of the luciferase gene. Doxycycline-mediated induction of the Tet-On expression cassettes resulted in the production of one long polycistronic mRNA as proven by Northern analyses, and ensured comparable production of enniatin in all three strains. Notably, gene position within the polycistronic expression cassette matters, as, luciferase activity was lowest at position one and had a comparable activity at positions two and three. The P2A peptide can be used to express at

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

    Science.gov (United States)

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

    2016-02-01

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

  1. Tolerance associated gene expression following allogeneic hematopoietic cell transplantation.

    Directory of Open Access Journals (Sweden)

    Joseph Pidala

    Full Text Available Biologic markers of immune tolerance may facilitate tailoring of immune suppression duration after allogeneic hematopoietic cell transplantation (HCT. In a cross-sectional study, peripheral blood samples were obtained from tolerant (n = 15, median 38.5 months post-HCT and non-tolerant (n = 17, median 39.5 post-HCT HCT recipients and healthy control subjects (n = 10 for analysis of immune cell subsets and differential gene expression. There were no significant differences in immune subsets across groups. We identified 281 probe sets unique to the tolerant (TOL group and 122 for non-tolerant (non-TOL. These were enriched for process networks including NK cell cytotoxicity, antigen presentation, lymphocyte proliferation, and cell cycle and apoptosis. Differential gene expression was enriched for CD56, CD66, and CD14 human lineage-specific gene expression. Differential expression of 20 probe sets between groups was sufficient to develop a classifier with > 90% accuracy, correctly classifying 14/15 TOL cases and 15/17 non-TOL cases. These data suggest that differential gene expression can be utilized to accurately classify tolerant patients following HCT. Prospective investigation of immune tolerance biologic markers is warranted.

  2. MHC class I antigen expression in patients with IDDM and their siblings.

    Science.gov (United States)

    Anal, O; Akkoç, N; Sen, A; Yesil, S; Yüksel, F; Büyükgebiz, A

    1997-01-01

    MHC class I antigen expression was found to be low on the lymphocytes of patients with insulin dependent diabetes mellitus (IDDM). Thus, it has been proposed that the defective expression of MHC antigens could lead to faulty immunological responses with the eventual destruction of the pancreatic beta cells. The objective in this study was to compare MHC antigen expression in IDDM patients and their presently healthy siblings. Nineteen children (mean age 10.8 +/- 3.9 years) with diabetes and their 25 siblings (mean age 10.7 +/- 4.6 years) were enrolled in the study. Peripheral blood lymphocytes isolated from venous blood samples were incubated with FITC conjugated monoclonal antibody W6/32. The amount of antibody binding by cell surface MHC class I antigens was assessed by flow cytometry. MHC class I molecule expression did not differ significantly among IDDM patients and their siblings. It was concluded that MHC class I antigen expression did not appear to be indicative of a susceptibility to develop autoimmune diabetes.

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

    International Nuclear Information System (INIS)

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

    1989-01-01

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

  4. Fas antigen (CD95) expression and apoptosis in hepatocytes of patients with chronic viral hepatitis.

    Science.gov (United States)

    Kiyici, Murat; Gurel, Selim; Budak, Ferah; Dolar, Enver; Gulten, Macit; Nak, Selim Giray; Memik, Faruk

    2003-10-01

    Apoptosis may be defined as programmed cell death. It is involved in the normal development and homeostasis of tissues in multicellular organisms. An increased or decreased rate of apoptosis may lead to a range of diseases. Fas antigen is a cell-surface receptor that induces apoptotic pathways when treated with Fas ligand or anti-Fas antibody. There is increasing evidence that apoptosis plays an important role in the immunopathogenesis of chronic viral hepatitis, in which the Fas antigen-Fas ligand pathway is particularly involved. Fas antigen expression and apoptosis (apoptotic index) were assayed using flow cytometry in the hepatocytes of 27 patients with chronic viral hepatitis. Histopathological activity, scored by Knodell's histological activity index, other histopathological parameters, serum transaminase values and patient age were then compared with apoptotic index and Fas antigen expression. Apoptosis and Fas antigen expression in hepatocytes were correlated closely with histological activity (grade) of chronic viral hepatitis, but there were no correlations with histological stage, patient age or serum transaminase levels. Apoptosis and its triggering molecule, Fas antigen, induce mechanisms that appear to be associated with the pathogenesis of chronic viral hepatitis.

  5. Phylogenetic analysis of the swine leukocyte antigen - 2 gene for Korean native pigs

    Science.gov (United States)

    The objective of this study was to investigate genetic distances of the SLA-2 gene, to characterize SLA-2 alleles, and to provide basic genetic information of Korean pigs. The swine leukocyte antigen - 2 (SLA-2) gene in the MHC classical region was cloned with spleen tissues from Korean native pigs ...

  6. Gene Expression and Microarray Investigation of Dendrobium ...

    African Journals Online (AJOL)

    Result: Between the diabetic rat group and the wild-type group, 1339 functional genes showed differences in expression levels (p < 0.05). ... Genes whose expression normalized were mainly those affected by the disease state and associated with glucose and lipid metabolism, cell growth, apoptosis, biosynthesis, olfactory ...

  7. Expression of conserved signalling pathway genes during

    Indian Academy of Sciences (India)

    Hence, we analysed the expression of Notch, Wnt and Sonic Hedgehog (Shh) pathway genes during differentiation of R1 cells into early vascular lineages. Notch-, Wnt-and Shh-mediated signalling is important during embryonic development. Regulation of gene expression through these signalling molecules is a frequently ...

  8. Serial analysis of gene expression (SAGE)

    NARCIS (Netherlands)

    van Ruissen, Fred; Baas, Frank

    2007-01-01

    In 1995, serial analysis of gene expression (SAGE) was developed as a versatile tool for gene expression studies. SAGE technology does not require pre-existing knowledge of the genome that is being examined and therefore SAGE can be applied to many different model systems. In this chapter, the SAGE

  9. Arabidopsis gene expression patterns during spaceflight

    Science.gov (United States)

    Paul, A.-L.; Ferl, R. J.

    The exposure of Arabidopsis thaliana (Arabidopsis) plants to spaceflight environments resulted in the differential expression of hundreds of genes. A 5 day mission on orbiter Columbia in 1999 (STS-93) carried transgenic Arabidopsis plants engineered with a transgene composed of the alcohol dehydrogenase (Adh) gene promoter linked to the β -Glucuronidase (GUS) reporter gene. The plants were used to evaluate the effects of spaceflight on two fronts. First, expression patterns visualized with the Adh/GUS transgene were used to address specifically the possibility that spaceflight induces a hypoxic stress response, and to assess whether any spaceflight response was similar to control terrestrial hypoxia-induced gene expression patterns. (Paul et al., Plant Physiol. 2001, 126:613). Second, genome-wide patterns of native gene expression were evaluated utilizing the Affymetrix ATH1 GeneChip? array of 8,000 Arabidopsis genes. As a control for the veracity of the array analyses, a selection of genes identified with the arrays was further characterized with quantitative Real-Time RT PCR (ABI - TaqmanTM). Comparison of the patterns of expression for arrays of hybridized with RNA isolated from plants exposed to spaceflight compared to the control arrays revealed hundreds of genes that were differentially expressed in response to spaceflight, yet most genes that are hallmarks of hypoxic stress were unaffected. These results will be discussed in light of current models for plant responses to the spaceflight environment, and with regard to potential future flight opportunities.

  10. Mapping of the antigenic and allergenic epitopes of Lol p VB using gene fragmentation.

    Science.gov (United States)

    Ong, E K; Knox, R B; Singh, M B

    1995-03-01

    The recombinant proteins of Lol p VA and Lol p VB expressed in E. coli reacted with IgE antibodies from sera of allergic patients and mAbs FMC A7 and PpV1. Cross-absorption analyses using these recombinant proteins showed that Lol p VA and Lol p VB possess both similar and unique IgE binding determinants. Gene fragmentation was utilized to localize the antigenic and allergenic determinants of Lol p VB. When full-length cDNA of Lol p VB was digested into three fragments and expressed as the fusions from the glutathione transferase of pGEX vectors, fragments Met1-Val196 and Asp197-Val339 bound IgE while fragment Met1-Pro96 did not. The data suggest that there are at least two IgE binding determinants in Lol p VB. In addition, only fragment Met1-Val196 reacted with mAb PpV1. The localization of these determinants was further resolved using random fragment expression libraries. The mAb PpV1 determinant was near the N-terminal region of Lol p VB molecule. The IgE binding determinants were distributed in the central region: region I (amino acids 111-195) and II (199-254). These IgE binding determinants are conserved in Lol p VA.

  11. 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

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

    International Nuclear Information System (INIS)

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

    1987-01-01

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

  13. Expression of Sox genes in tooth development.

    Science.gov (United States)

    Kawasaki, Katsushige; Kawasaki, Maiko; Watanabe, Momoko; Idrus, Erik; Nagai, Takahiro; Oommen, Shelly; Maeda, Takeyasu; Hagiwara, Nobuko; Que, Jianwen; Sharpe, Paul T; Ohazama, Atsushi

    2015-01-01

    Members of the Sox gene family play roles in many biological processes including organogenesis. We carried out comparative in situ hybridization analysis of seventeen sox genes (Sox1-14, 17, 18, 21) during murine odontogenesis from the epithelial thickening to the cytodifferentiation stages. Localized expression of five Sox genes (Sox6, 9, 13, 14 and 21) was observed in tooth bud epithelium. Sox13 showed restricted expression in the primary enamel knots. At the early bell stage, three Sox genes (Sox8, 11, 17 and 21) were expressed in pre-ameloblasts, whereas two others (Sox5 and 18) showed expression in odontoblasts. Sox genes thus showed a dynamic spatio-temporal expression during tooth development.

  14. Determinants of human adipose tissue gene expression

    DEFF Research Database (Denmark)

    Viguerie, Nathalie; Montastier, Emilie; Maoret, Jean-José

    2012-01-01

    of environmental and individual factors controlling AT adaptation is therefore essential. Here, expression of 271 transcripts, selected for regulation according to obesity and weight changes, was determined in 515 individuals before, after 8-week low-calorie diet-induced weight loss, and after 26-week ad libitum...... interconnection between expression of genes involved in de novo lipogenesis and components of the metabolic syndrome. Sex had a marked influence on AT expression of 88 transcripts, which persisted during the entire dietary intervention and after control for fat mass. In women, the influence of body mass index...... on expression of a subset of genes persisted during the dietary intervention. Twenty-two genes revealed a metabolic syndrome signature common to men and women. Genetic control of AT gene expression by cis signals was observed for 46 genes. Dietary intervention, sex, and cis genetic variants independently...

  15. A Survey of ABO, Rhesus (D) Antigen and Haemoglobin Genes ...

    African Journals Online (AJOL)

    olayemitoyin

    while 5.5% were Rh(D) negative respectively based on the detection (Positive) or absence (Negative) of Rh(D) antigen. 22.8% of the subjects had ABO blood group A, 26.4% were group B, 4.1% were group AB while 46.7% were group O. Further analysis revealed that 695 (21.4%) of the group A were Apositive while 44 ...

  16. Gene set analysis for longitudinal gene expression data

    Directory of Open Access Journals (Sweden)

    Piepho Hans-Peter

    2011-07-01

    Full Text Available Abstract Background Gene set analysis (GSA has become a successful tool to interpret gene expression profiles in terms of biological functions, molecular pathways, or genomic locations. GSA performs statistical tests for independent microarray samples at the level of gene sets rather than individual genes. Nowadays, an increasing number of microarray studies are conducted to explore the dynamic changes of gene expression in a variety of species and biological scenarios. In these longitudinal studies, gene expression is repeatedly measured over time such that a GSA needs to take into account the within-gene correlations in addition to possible between-gene correlations. Results We provide a robust nonparametric approach to compare the expressions of longitudinally measured sets of genes under multiple treatments or experimental conditions. The limiting distributions of our statistics are derived when the number of genes goes to infinity while the number of replications can be small. When the number of genes in a gene set is small, we recommend permutation tests based on our nonparametric test statistics to achieve reliable type I error and better power while incorporating unknown correlations between and within-genes. Simulation results demonstrate that the proposed method has a greater power than other methods for various data distributions and heteroscedastic correlation structures. This method was used for an IL-2 stimulation study and significantly altered gene sets were identified. Conclusions The simulation study and the real data application showed that the proposed gene set analysis provides a promising tool for longitudinal microarray analysis. R scripts for simulating longitudinal data and calculating the nonparametric statistics are posted on the North Dakota INBRE website http://ndinbre.org/programs/bioinformatics.php. Raw microarray data is available in Gene Expression Omnibus (National Center for Biotechnology Information with

  17. Positron emission tomography imaging of gene expression

    International Nuclear Information System (INIS)

    Tang Ganghua

    2001-01-01

    The merging of molecular biology and nuclear medicine is developed into molecular nuclear medicine. Positron emission tomography (PET) of gene expression in molecular nuclear medicine has become an attractive area. Positron emission tomography imaging gene expression includes the antisense PET imaging and the reporter gene PET imaging. It is likely that the antisense PET imaging will lag behind the reporter gene PET imaging because of the numerous issues that have not yet to be resolved with this approach. The reporter gene PET imaging has wide application into animal experimental research and human applications of this approach will likely be reported soon

  18. Multilocus Sequence Analysis of Housekeeping Genes and Antigenic Determinant Genes in Bordetella pertussis Strains Isolated in Korea.

    Science.gov (United States)

    Jung, Sang-Oun; Moon, Yu Mi; Kim, So-Hyeon; Sung, Hwa Young; Kwon, Seung-Jik; Kang, Yeon Ho; Yu, Jae Yon

    2011-09-01

    To confirm genotype diversities of clinical isolates of Bordetella pertussis and to evaluate the risk of pertussis outbreak in Korea. Seven housekeeping genes and 10 antigenic determinant genes from clinical B. pertussis isolates were analyzed by Multilocus sequence typing (MLST). More variant pattern was observed in antigenic determinant genes. Especially, PtxS1 gene was the most variant gene; five genotypes were observed from eight global genotypes. In the bacterial type, the number of observed sequence types in the isolates was seven and the most frequent form was type 1 (79.6%). This major sequence type also showed a time-dependent transition pattern. Older isolates (1968 and 1975) showed type 1 and 6 in housekeeping genes and antigenic determinant genes, respectively. However, these were changed to type 2 and 1 in isolates 1999-2008. This transition was mainly attributed to genotype change of PtxS1 and Fim3 gene; the tendency of genotype change was to avoid vaccine-derived genotype. In addition, there was second transition in 2009. In this period, only the sequence type of antigenic determinant genes was changed to type 2. Based Upon Related Sequence Types (BURST) analysis confirmed that there were two clonal complexes (ACCI and ACCII) in the Korean isolates. Moreover, the recently increased sequence type was revealed as AST2 derived from AST 3 in ACCI. Genotype changes in Korean distributing strains are still progressing and there was a specific driving force in antigenic determinant genes. Therefore continuous surveillance of genotype change of the distributing strains should be performed to confirm interrelationship of genotype change with vaccine immunity.

  19. 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.

  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. Photosynthetic gene expression in higher plants.

    Science.gov (United States)

    Berry, James O; Yerramsetty, Pradeep; Zielinski, Amy M; Mure, Christopher M

    2013-11-01

    Within the chloroplasts of higher plants and algae, photosynthesis converts light into biological energy, fueling the assimilation of atmospheric carbon dioxide into biologically useful molecules. Two major steps, photosynthetic electron transport and the Calvin-Benson cycle, require many gene products encoded from chloroplast as well as nuclear genomes. The expression of genes in both cellular compartments is highly dynamic and influenced by a diverse range of factors. Light is the primary environmental determinant of photosynthetic gene expression. Working through photoreceptors such as phytochrome, light regulates photosynthetic genes at transcriptional and posttranscriptional levels. Other processes that affect photosynthetic gene expression include photosynthetic activity, development, and biotic and abiotic stress. Anterograde (from nucleus to chloroplast) and retrograde (from chloroplast to nucleus) signaling insures the highly coordinated expression of the many photosynthetic genes between these different compartments. Anterograde signaling incorporates nuclear-encoded transcriptional and posttranscriptional regulators, such as sigma factors and RNA-binding proteins, respectively. Retrograde signaling utilizes photosynthetic processes such as photosynthetic electron transport and redox signaling to influence the expression of photosynthetic genes in the nucleus. The basic C3 photosynthetic pathway serves as the default form used by most of the plant species on earth. High temperature and water stress associated with arid environments have led to the development of specialized C4 and CAM photosynthesis, which evolved as modifications of the basic default expression program. The goal of this article is to explain and summarize the many gene expression and regulatory processes that work together to support photosynthetic function in plants.

  2. Generation of multi-functional antigen-specific human T-cells by lentiviral TCR gene transfer.

    Science.gov (United States)

    Perro, M; Tsang, J; Xue, S-A; Escors, D; Cesco-Gaspere, M; Pospori, C; Gao, L; Hart, D; Collins, M; Stauss, H; Morris, E C

    2010-06-01

    T-cell receptor (TCR) gene transfer is an attractive strategy to generate antigen-specific T-cells for adoptive immunotherapy of cancer and chronic viral infection. However, current TCR gene transfer protocols trigger T-cell differentiation into terminally differentiated effector cells, which likely have reduced ability to mediate disease protection in vivo. We have developed a lentiviral gene transfer strategy to generate TCR-transduced human T-cells without promoting T-cell differentiation. We found that a combination of interleukin-15 (IL15) and IL21 facilitated lentiviral TCR gene transfer into non-proliferating T-cells. The transduced T-cells showed redirection of antigen specificity and produced IL2, IFNgamma and TNFalpha in a peptide-dependent manner. A significantly higher proportion of the IL15/IL21-stimulated T-cells were multi-functional and able to simultaneously produce all three cytokines (P<0.01), compared with TCR-transduced T-cells generated by conventional anti-CD3 plus IL2 stimulation, which primarily secreted only one cytokine. Similarly, IL15/IL21 maintained high levels of CD62L and CD28 expression in transduced T-cells, whereas anti-CD3 plus IL2 accelerated the loss of CD62L/CD28 expression. The data demonstrate that the combination of lentiviral TCR gene transfer together with IL15/IL21 stimulation can efficiently redirect the antigen specificity of resting primary human T-cells and generate multi-functional T-cells.

  3. 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

  4. 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.

  5. Expression of factor VIII-related antigen in human aqueous drainage channels.

    Science.gov (United States)

    Krohn, J

    1999-02-01

    Previous studies have indicated that there are preformed channels at the inner aspect of the anterior sclera, capable of draining fluid from the suprachoroidal space into scleral veins. The aim of this study was to characterize the cellular lining of these channels, and to compare it with the endothelium of Schlemm's canal, the collector channels and the scleral blood vessels. Histological sections from the angular region of human eyes were prepared by an immunoperoxidase method to evaluate the expression of factor VIII-related antigen in different aqueous drainage channels. The cellular lining of the scleral channels showed a weak immunostaining to factor VIII-related antigen. Factor VIII-related antigen was also detected in the endothelium of the collector channels and Schlemm's canal. The positive immunoreaction to factor VIII-related antigen indicates that the previously described scleral channels, the collector channels and Schlemm's canal are all lined by an endothelium derived from a vascular origin.

  6. 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.

  7. Caleydo: connecting pathways and gene expression.

    Science.gov (United States)

    Streit, Marc; Lex, Alexander; Kalkusch, Michael; Zatloukal, Kurt; Schmalstieg, Dieter

    2009-10-15

    Understanding the relationships between pathways and the altered expression of their components in disease conditions can be addressed in a visual data analysis process. Caleydo uses novel visualization techniques to support life science experts in their analysis of gene expression data in the context of pathways and functions of individual genes. Pathways and gene expression visualizations are placed in a 3D scene where selected entities (i.e. genes) are visually connected. This allows Caleydo to seamlessly integrate interactive gene expression visualization with cross-database pathway exploration. The Caleydo visualization framework is freely available on www.caleydo.org for non-commercial use. It runs on Windows and Linux and requires a 3D capable graphics card.

  8. Adaptive Evolution of Gene Expression in Drosophila.

    Science.gov (United States)

    Nourmohammad, Armita; Rambeau, Joachim; Held, Torsten; Kovacova, Viera; Berg, Johannes; Lässig, Michael

    2017-08-08

    Gene expression levels are important quantitative traits that link genotypes to molecular functions and fitness. In Drosophila, population-genetic studies have revealed substantial adaptive evolution at the genomic level, but the evolutionary modes of gene expression remain controversial. Here, we present evidence that adaptation dominates the evolution of gene expression levels in flies. We show that 64% of the observed expression divergence across seven Drosophila species are adaptive changes driven by directional selection. Our results are derived from time-resolved data of gene expression divergence across a family of related species, using a probabilistic inference method for gene-specific selection. Adaptive gene expression is stronger in specific functional classes, including regulation, sensory perception, sexual behavior, and morphology. Moreover, we identify a large group of genes with sex-specific adaptation of expression, which predominantly occurs in males. Our analysis opens an avenue to map system-wide selection on molecular quantitative traits independently of their genetic basis. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  9. Differential gene expression during Trypanosoma cruzi metacyclogenesis

    Directory of Open Access Journals (Sweden)

    Marco Aurelio Krieger

    1999-09-01

    Full Text Available The transformation of epimastigotes into metacyclic trypomastigotes involves changes in the pattern of expressed genes, resulting in important morphological and functional differences between these developmental forms of Trypanosoma cruzi. In order to identify and characterize genes involved in triggering the metacyclogenesis process and in conferring to metacyclic trypomastigotes their stage specific biological properties, we have developed a method allowing the isolation of genes specifically expressed when comparing two close related cell populations (representation of differential expression or RDE. The method is based on the PCR amplification of gene sequences selected by hybridizing and subtracting the populations in such a way that after some cycles of hybridization-amplification genes specific to a given population are highly enriched. The use of this method in the analysis of differential gene expression during T. cruzi metacyclogenesis (6 hr and 24 hr of differentiation and metacyclic trypomastigotes resulted in the isolation of several clones from each time point. Northern blot analysis showed that some genes are transiently expressed (6 hr and 24 hr differentiating cells, while others are present in differentiating cells and in metacyclic trypomastigotes. Nucleotide sequencing of six clones characterized so far showed that they do not display any homology to gene sequences available in the GeneBank.

  10. Differentiation and major histocompatibility complex antigen expression in human liver-derived stem cells.

    Science.gov (United States)

    Lee, J-H; Park, H-J; Kim, Y-A; Lee, D-H; Noh, J-K; Kwon, C H D; Jung, S-M; Lee, S-K

    2012-05-01

    Stem cells are a promising source for liver repopulation after cell transplantation, but whether the adult liver contains hepatic stem cells is controversial. The purpose of this study was to characterize the properties and expression profile of major histocompatibility complex (MHC) antigens on the surface of human-derived stem cells. Human liver-derived stem cells (HLSC7) were isolated from the nontumorous tissue of a patient who underwent a resection of an hepatic hemangioendothelioma. We characterized HLSC7 using a fluorescence-activated cell sorter, polymerase chain reactions, and immunofluorescence assays. HLSC7 expressed mesenchymal but not hematopoietic stem cell markers. HLSC7 underwent osteogenic, chondrogenic, and hepatogenic differentiation when cultured in appropriate differentiation media. However, HLSC7 did not differentiate into adipocytes. In addition, HLSC7 did not express MHC class II (HLA-DP, -DQ, and -DR) antigens. However, they did express MHC class I antigens. These results suggest that human liver-derived stem cells express MHC class I antigens and thus may be rejected on transplantation. Therefore, in addition to studies on stem cell differentiation, one must overcome immunologic barriers for successful clinical application of this therapy. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. Mosquito Passage Dramatically Changes var Gene Expression in Controlled Human Plasmodium falciparum Infections.

    Science.gov (United States)

    Bachmann, Anna; Petter, Michaela; Krumkamp, Ralf; Esen, Meral; Held, Jana; Scholz, Judith A M; Li, Tao; Sim, B Kim Lee; Hoffman, Stephen L; Kremsner, Peter G; Mordmüller, Benjamin; Duffy, Michael F; Tannich, Egbert

    2016-04-01

    Virulence of the most deadly malaria parasite Plasmodium falciparum is linked to the variant surface antigen PfEMP1, which is encoded by about 60 var genes per parasite genome. Although the expression of particular variants has been associated with different clinical outcomes, little is known about var gene expression at the onset of infection. By analyzing controlled human malaria infections via quantitative real-time PCR, we show that parasite populations from 18 volunteers expressed virtually identical transcript patterns that were dominated by the subtelomeric var gene group B and, to a lesser extent, group A. Furthermore, major changes in composition and frequency of var gene transcripts were detected between the parental parasite culture that was used to infect mosquitoes and Plasmodia recovered from infected volunteers, suggesting that P. falciparum resets its var gene expression during mosquito passage and starts with the broad expression of a specific subset of var genes when entering the human blood phase.

  12. EXPRESSION OF BACTERIOOPSIN GENES IN ESCHERICHIA COLI

    OpenAIRE

    TSUJIUCHI, Yutaka; IWASA, Tatsuo; TOKUNAGA, Fumio

    1994-01-01

    An inducible expression vector pUBO was constructed with native codons in order to express the gene of Bacteriorhodopsin (BOP) in Escherichia coli (E. coli). Vector pUBO contains lac-promoter followed by the partial structural gene of lacZ and the structural gene of BOP. The expression of this fusion protein was detected by ELISA with anti-BOP antiserum. The fusion protein obtained from E. coli trnsformed with pUBO formed approximately 0.1% of the total protein of the E. coli membrane fraction.

  13. Differential expression of cell adhesion genes

    DEFF Research Database (Denmark)

    Stein, Wilfred D; Litman, Thomas; Fojo, Tito

    2005-01-01

    that compare cells grown in suspension to similar cells grown attached to one another as aggregates have suggested that it is adhesion to the extracellular matrix of the basal membrane that confers resistance to apoptosis and, hence, resistance to cytotoxins. The genes whose expression correlates with poor...... survival might, therefore, act through such a matrix-to-cell suppression of apoptosis. Indeed, correlative mining of gene expression and patient survival databases suggests that poor survival in patients with metastatic cancer correlates highly with tumor expression of a common theme: the genes involved...

  14. Efficacy of a DNA vaccine carrying Eimeria maxima Gam56 antigen gene against coccidiosis in chickens.

    Science.gov (United States)

    Xu, Jinjun; Zhang, Yan; Tao, Jianping

    2013-04-01

    To control coccidiosis without using prophylactic medications, a DNA vaccine targeting the gametophyte antigen Gam56 from Eimeria maxima in chickens was constructed, and the immunogenicity and protective effects were evaluated. The ORF of Gam56 gene was cloned into an eukaryotic expression vector pcDNA3.1(zeo)+. Expression of Gam56 protein in COS-7 cells transfected with recombinant plasmid pcDNA-Gam56 was confirmed by indirect immunofluorescence assay. The DNA vaccine was injected intramuscularly to yellow feathered broilers of 1-week old at 3 dosages (25, 50, and 100 µg/chick). Injection was repeated once 1 week later. One week after the second injection, birds were challenged orally with 5×10(4) sporulated oocysts of E. maxima, then weighed and killed at day 8 post challenge. Blood samples were collected and examined for specific peripheral blood lymphocyte proliferation activity and serum antibody levels. Compared with control groups, the administration of pcDNA-Gam56 vaccine markedly increased the lymphocyte proliferation activity (Pcoccidiosis control.

  15. Expression characteristics of the human CD97 antigen

    NARCIS (Netherlands)

    Eichler, W.; Hamann, J. [=Jörg; Aust, G.

    1997-01-01

    Molecules whose expression is increased upon stimulation on leukocyte populations are of considerable interest because insights into their structure and function extend knowledge of the intracellular and intercellular events that accompany cellular activation. One such molecule is CD97, a cell

  16. Microarray Gene Expression Analysis to Evaluate Cell Type Specific Expression of Targets Relevant for Immunotherapy of Hematological Malignancies.

    Directory of Open Access Journals (Sweden)

    M J Pont

    Full Text Available Cellular immunotherapy has proven to be effective in the treatment of hematological cancers by donor lymphocyte infusion after allogeneic hematopoietic stem cell transplantation and more recently by targeted therapy with chimeric antigen or T-cell receptor-engineered T cells. However, dependent on the tissue distribution of the antigens that are targeted, anti-tumor responses can be accompanied by undesired side effects. Therefore, detailed tissue distribution analysis is essential to estimate potential efficacy and toxicity of candidate targets for immunotherapy of hematological malignancies. We performed microarray gene expression analysis of hematological malignancies of different origins, healthy hematopoietic cells and various non-hematopoietic cell types from organs that are often targeted in detrimental immune responses after allogeneic stem cell transplantation leading to graft-versus-host disease. Non-hematopoietic cells were also cultured in the presence of IFN-γ to analyze gene expression under inflammatory circumstances. Gene expression was investigated by Illumina HT12.0 microarrays and quality control analysis was performed to confirm the cell-type origin and exclude contamination of non-hematopoietic cell samples with peripheral blood cells. Microarray data were validated by quantitative RT-PCR showing strong correlations between both platforms. Detailed gene expression profiles were generated for various minor histocompatibility antigens and B-cell surface antigens to illustrate the value of the microarray dataset to estimate efficacy and toxicity of candidate targets for immunotherapy. In conclusion, our microarray database provides a relevant platform to analyze and select candidate antigens with hematopoietic (lineage-restricted expression as potential targets for immunotherapy of hematological cancers.

  17. MHC-restricted antigen presentation and recognition: constraints on gene, recombinant and peptide vaccines in humans

    Directory of Open Access Journals (Sweden)

    Cunha-Neto E.

    1999-01-01

    Full Text Available The target of any immunization is to activate and expand lymphocyte clones with the desired recognition specificity and the necessary effector functions. In gene, recombinant and peptide vaccines, the immunogen is a single protein or a small assembly of epitopes from antigenic proteins. Since most immune responses against protein and peptide antigens are T-cell dependent, the molecular target of such vaccines is to generate at least 50-100 complexes between MHC molecule and the antigenic peptide per antigen-presenting cell, sensitizing a T cell population of appropriate clonal size and effector characteristics. Thus, the immunobiology of antigen recognition by T cells must be taken into account when designing new generation peptide- or gene-based vaccines. Since T cell recognition is MHC-restricted, and given the wide polymorphism of the different MHC molecules, distinct epitopes may be recognized by different individuals in the population. Therefore, the issue of whether immunization will be effective in inducing a protective immune response, covering the entire target population, becomes an important question. Many pathogens have evolved molecular mechanisms to escape recognition by the immune system by variation of antigenic protein sequences. In this short review, we will discuss the several concepts related to selection of amino acid sequences to be included in DNA and peptide vaccines.

  18. Cloning of synthetic gene including antigens against Urinary Tract Infections in pET28a+ vector

    Directory of Open Access Journals (Sweden)

    Zohreh Haghri

    2017-12-01

    Full Text Available There are many different bacterial infections in the world that patients are suffering from and research teams are trying to find suitable ways to prevent and treat them. Urinary Tract Infections (UTIs are most important infections in the world , and they are more common among women because vaginal cavity is near to urethral opening. The aim of this study is cloning of synthetic gene include antigens against UTIs in pET28a+ vector. Antibiotic resistant has been increasing because of antibiotic overuse recently, so It shows the necessity of developing a vaccine against these infections. There for, it will be imperative to develop a vaccine instead of antibiotics. This infection causes by many organisms, most important of which are Uropathogenic Escherichia coli (UPEC, Proteus mirabilis and Klebsiella pneumoniae Uropathogenic Escherichia .coli is the most important microorganism that causes these infections more than other bacteria, so in developing a vaccine it is the most important one, that have to be considered. The synthetic Gene which was designed against these three bacteria including antigens which are important and common to cause these infections. This gene has involved 1293bp. It was ordered to Gene Ray Biotechnology. Primers were designed by Gene Runner. Gene and pET28a+ vector was checked by SnappGene. Synthetic gene was multiplied by PCR and cloned in pET28a+ vector. Construct was transformed into E. coli TOP10.The clone was confirmed by PCR, Digestion. This data indicates that this gene can be expressed and it might be a vaccine candidate to protect people from these infections in the future.

  19. Immunoglobulin (Ig) D in Labeo rohita is widely expressed and differentially modulated in viral, bacterial and parasitic antigenic challenges.

    Science.gov (United States)

    Basu, Madhubanti; Lenka, Saswati S; Paichha, Mahismita; Swain, Banikalyan; Patel, Bhakti; Banerjee, Rajanya; Jayasankar, Pallipuram; Das, Surajit; Samanta, Mrinal

    2016-10-15

    Immunoglobulins (Igs) play critical roles in protecting host against diverse pathogenic invasion and diseases. Among all Ig isotypes, IgD is the most recently-evolved and enigmatic molecule detected in all vertebrates species except birds. In South-East Asia, Labeo rohita (rohu) is the leading candidate fish species for freshwater aquaculture, and this article describes about IgD gene expression in rohu following viral, bacterial and parasitic antigenic challenges. The partial cDNA (761bp) of Labeo rohita-IgD (LrIgD) was cloned and submitted in the GenBank with the accession no KT883581. Phylogenetically, LrIgD was closely related to grass carp IgD. Analysis of LrIgD gene expression in juveniles by quantitative real-time PCR (qRT-PCR) assay revealed gradual increase in IgD expression with the advancement of time. In the healthy rohu fingerlings, LrIgD expression occurred predominantly in kidney followed by liver and spleen. In response to rhabdoviral antigenic stimulation, LrIgD expression was significantly enhanced in all tested tissues. In bacterial (Aeromonas hydrophila) infection, transcripts of LrIgD increased more dramatically in liver followed by kidney and gill. In parasitic (Argulus) infection, most significant expression of IgD was noted in the skin, followed by kidney, liver, spleen and gill. These results collectively suggest the key role of IgD in the immune response of rohu during viral, bacterial and parasitic infections. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Selvaraju Kanagarajan

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

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

    Science.gov (United States)

    Kanagarajan, Selvaraju; Tolf, Conny; Lundgren, Anneli; Waldenström, Jonas; Brodelius, Peter E

    2012-01-01

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

  2. A rapid and potent DNA vaccination strategy defined by in vivo monitoring of antigen expression

    NARCIS (Netherlands)

    Bins, Adriaan D.; Jorritsma, Annelies; Wolkers, Monika C.; Hung, Chien-Fu; Wu, T.-C.; Schumacher, Ton N. M.; Haanen, John B. A. G.

    2005-01-01

    Induction of immunity after DNA vaccination is generally considered a slow process. Here we show that DNA delivery to the skin results in a highly transient pulse of antigen expression. Based on this information, we developed a new rapid and potent intradermal DNA vaccination method. By

  3. Drosophila melanogaster gene expression changes after spaceflight.

    Data.gov (United States)

    National Aeronautics and Space Administration — Gene expression levels were determined in 3rd instar and adult Drosophila melanogaster reared during spaceflight to elucidate the genetic and molecular mechanisms...

  4. Homeobox genes expressed during echinoderm arm regeneration.

    Science.gov (United States)

    Ben Khadra, Yousra; Said, Khaled; Thorndyke, Michael; Martinez, Pedro

    2014-04-01

    Regeneration in echinoderms has proved to be more amenable to study in the laboratory than the more classical vertebrate models, since the smaller genome size and the absence of multiple orthologs for different genes in echinoderms simplify the analysis of gene function during regeneration. In order to understand the role of homeobox-containing genes during arm regeneration in echinoderms, we isolated the complement of genes belonging to the Hox class that are expressed during this process in two major echinoderm groups: asteroids (Echinaster sepositus and Asterias rubens) and ophiuroids (Amphiura filiformis), both of which show an extraordinary capacity for regeneration. By exploiting the sequence conservation of the homeobox, putative orthologs of several Hox genes belonging to the anterior, medial, and posterior groups were isolated. We also report the isolation of a few Hox-like genes expressed in the same systems.

  5. Evaluation of suitable reference genes for gene expression studies ...

    Indian Academy of Sciences (India)

    2011-12-14

    Dec 14, 2011 ... MADS family of TFs control floral organ identity within each whorl of the flower by activating downstream genes. Measuring gene expression in different tissue types and developmental stages is of fundamental importance in TFs functional research. In last few years, quantitative real-time. PCR (qRT-PCR) ...

  6. 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...

  7. Optimal Reference Genes for Gene Expression Normalization in Trichomonas vaginalis.

    Science.gov (United States)

    dos Santos, Odelta; de Vargas Rigo, Graziela; Frasson, Amanda Piccoli; Macedo, Alexandre José; Tasca, Tiana

    2015-01-01

    Trichomonas vaginalis is the etiologic agent of trichomonosis, the most common non-viral sexually transmitted disease worldwide. This infection is associated with several health consequences, including cervical and prostate cancers and HIV acquisition. Gene expression analysis has been facilitated because of available genome sequences and large-scale transcriptomes in T. vaginalis, particularly using quantitative real-time polymerase chain reaction (qRT-PCR), one of the most used methods for molecular studies. Reference genes for normalization are crucial to ensure the accuracy of this method. However, to the best of our knowledge, a systematic validation of reference genes has not been performed for T. vaginalis. In this study, the transcripts of nine candidate reference genes were quantified using qRT-PCR under different cultivation conditions, and the stability of these genes was compared using the geNorm and NormFinder algorithms. The most stable reference genes were α-tubulin, actin and DNATopII, and, conversely, the widely used T. vaginalis reference genes GAPDH and β-tubulin were less stable. The PFOR gene was used to validate the reliability of the use of these candidate reference genes. As expected, the PFOR gene was upregulated when the trophozoites were cultivated with ferrous ammonium sulfate when the DNATopII, α-tubulin and actin genes were used as normalizing gene. By contrast, the PFOR gene was downregulated when the GAPDH gene was used as an internal control, leading to misinterpretation of the data. These results provide an important starting point for reference gene selection and gene expression analysis with qRT-PCR studies of T. vaginalis.

  8. Optimal Reference Genes for Gene Expression Normalization in Trichomonas vaginalis.

    Directory of Open Access Journals (Sweden)

    Odelta dos Santos

    Full Text Available Trichomonas vaginalis is the etiologic agent of trichomonosis, the most common non-viral sexually transmitted disease worldwide. This infection is associated with several health consequences, including cervical and prostate cancers and HIV acquisition. Gene expression analysis has been facilitated because of available genome sequences and large-scale transcriptomes in T. vaginalis, particularly using quantitative real-time polymerase chain reaction (qRT-PCR, one of the most used methods for molecular studies. Reference genes for normalization are crucial to ensure the accuracy of this method. However, to the best of our knowledge, a systematic validation of reference genes has not been performed for T. vaginalis. In this study, the transcripts of nine candidate reference genes were quantified using qRT-PCR under different cultivation conditions, and the stability of these genes was compared using the geNorm and NormFinder algorithms. The most stable reference genes were α-tubulin, actin and DNATopII, and, conversely, the widely used T. vaginalis reference genes GAPDH and β-tubulin were less stable. The PFOR gene was used to validate the reliability of the use of these candidate reference genes. As expected, the PFOR gene was upregulated when the trophozoites were cultivated with ferrous ammonium sulfate when the DNATopII, α-tubulin and actin genes were used as normalizing gene. By contrast, the PFOR gene was downregulated when the GAPDH gene was used as an internal control, leading to misinterpretation of the data. These results provide an important starting point for reference gene selection and gene expression analysis with qRT-PCR studies of T. vaginalis.

  9. Inferring gene networks from discrete expression data

    KAUST Repository

    Zhang, L.

    2013-07-18

    The modeling of gene networks from transcriptional expression data is an important tool in biomedical research to reveal signaling pathways and to identify treatment targets. Current gene network modeling is primarily based on the use of Gaussian graphical models applied to continuous data, which give a closedformmarginal likelihood. In this paper,we extend network modeling to discrete data, specifically data from serial analysis of gene expression, and RNA-sequencing experiments, both of which generate counts of mRNAtranscripts in cell samples.We propose a generalized linear model to fit the discrete gene expression data and assume that the log ratios of the mean expression levels follow a Gaussian distribution.We restrict the gene network structures to decomposable graphs and derive the graphs by selecting the covariance matrix of the Gaussian distribution with the hyper-inverse Wishart priors. Furthermore, we incorporate prior network models based on gene ontology information, which avails existing biological information on the genes of interest. We conduct simulation studies to examine the performance of our discrete graphical model and apply the method to two real datasets for gene network inference. © The Author 2013. Published by Oxford University Press. All rights reserved.

  10. Perspectives: Gene Expression in Fisheries Management

    Science.gov (United States)

    Nielsen, Jennifer L.; Pavey, Scott A.

    2010-01-01

    Functional genes and gene expression have been connected to physiological traits linked to effective production and broodstock selection in aquaculture, selective implications of commercial fish harvest, and adaptive changes reflected in non-commercial fish populations subject to human disturbance and climate change. Gene mapping using single nucleotide polymorphisms (SNPs) to identify functional genes, gene expression (analogue microarrays and real-time PCR), and digital sequencing technologies looking at RNA transcripts present new concepts and opportunities in support of effective and sustainable fisheries. Genomic tools have been rapidly growing in aquaculture research addressing aspects of fish health, toxicology, and early development. Genomic technologies linking effects in functional genes involved in growth, maturation and life history development have been tied to selection resulting from harvest practices. Incorporating new and ever-increasing knowledge of fish genomes is opening a different perspective on local adaptation that will prove invaluable in wild fish conservation and management. Conservation of fish stocks is rapidly incorporating research on critical adaptive responses directed at the effects of human disturbance and climate change through gene expression studies. Genomic studies of fish populations can be generally grouped into three broad categories: 1) evolutionary genomics and biodiversity; 2) adaptive physiological responses to a changing environment; and 3) adaptive behavioral genomics and life history diversity. We review current genomic research in fisheries focusing on those that use microarrays to explore differences in gene expression among phenotypes and within or across populations, information that is critically important to the conservation of fish and their relationship to humans.

  11. Expression of Deinococcus geothermalis trehalose synthase gene ...

    African Journals Online (AJOL)

    A novel trehalose synthase gene from Deinococcus geothermalis (DSMZ 11300) containing 1692 bp reading-frame encoding 564 amino acids was amplified using polymerase chain reaction (PCR). The gene was ligated into pET30Ek/LIC vector and expressed after isopropyl β-D-thiogalactopyranoside induction in ...

  12. Bayesian assignment of gene ontology terms to gene expression experiments.

    Science.gov (United States)

    Sykacek, P

    2012-09-15

    Gene expression assays allow for genome scale analyses of molecular biological mechanisms. State-of-the-art data analysis provides lists of involved genes, either by calculating significance levels of mRNA abundance or by Bayesian assessments of gene activity. A common problem of such approaches is the difficulty of interpreting the biological implication of the resulting gene lists. This lead to an increased interest in methods for inferring high-level biological information. A common approach for representing high level information is by inferring gene ontology (GO) terms which may be attributed to the expression data experiment. This article proposes a probabilistic model for GO term inference. Modelling assumes that gene annotations to GO terms are available and gene involvement in an experiment is represented by a posterior probabilities over gene-specific indicator variables. Such probability measures result from many Bayesian approaches for expression data analysis. The proposed model combines these indicator probabilities in a probabilistic fashion and provides a probabilistic GO term assignment as a result. Experiments on synthetic and microarray data suggest that advantages of the proposed probabilistic GO term inference over statistical test-based approaches are in particular evident for sparsely annotated GO terms and in situations of large uncertainty about gene activity. Provided that appropriate annotations exist, the proposed approach is easily applied to inferring other high level assignments like pathways. Source code under GPL license is available from the author. peter.sykacek@boku.ac.at.

  13. Bayesian assignment of gene ontology terms to gene expression experiments

    Science.gov (United States)

    Sykacek, P.

    2012-01-01

    Motivation: Gene expression assays allow for genome scale analyses of molecular biological mechanisms. State-of-the-art data analysis provides lists of involved genes, either by calculating significance levels of mRNA abundance or by Bayesian assessments of gene activity. A common problem of such approaches is the difficulty of interpreting the biological implication of the resulting gene lists. This lead to an increased interest in methods for inferring high-level biological information. A common approach for representing high level information is by inferring gene ontology (GO) terms which may be attributed to the expression data experiment. Results: This article proposes a probabilistic model for GO term inference. Modelling assumes that gene annotations to GO terms are available and gene involvement in an experiment is represented by a posterior probabilities over gene-specific indicator variables. Such probability measures result from many Bayesian approaches for expression data analysis. The proposed model combines these indicator probabilities in a probabilistic fashion and provides a probabilistic GO term assignment as a result. Experiments on synthetic and microarray data suggest that advantages of the proposed probabilistic GO term inference over statistical test-based approaches are in particular evident for sparsely annotated GO terms and in situations of large uncertainty about gene activity. Provided that appropriate annotations exist, the proposed approach is easily applied to inferring other high level assignments like pathways. Availability: Source code under GPL license is available from the author. Contact: peter.sykacek@boku.ac.at PMID:22962488

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

    Directory of Open Access Journals (Sweden)

    Koh Yih Yih

    2014-01-01

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

  15. Mismatch repair gene expression in gastroesophageal cancers.

    Science.gov (United States)

    Dracea, Amelia; Angelescu, Cristina; Danciulescu, Mihaela; Ciurea, Marius; Ioana, Mihai; Burada, Florin

    2015-09-01

    Mismatch repair (MMR) genes play a critical role in maintaining genomic stability, and the impairment of MMR machinery is associated with different human cancers, mainly colorectal cancer. The purpose of our study was to analyze gene expression patterns of three MMR genes (MSH2, MHS6, and EXO1) in gastroesophageal cancers, a pathology in which the contribution of DNA repair genes remains essentially unclear. A total of 45 Romanian patients diagnosed with sporadic gastroesophageal cancers were included in this study. For each patient, MMR mRNA levels were measured in biopsied tumoral (T) and peritumoral (PT) tissues obtained by upper endoscopy. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) with specific TaqMan probes was used to measure gene expression levels for MSH2, MSH6, and EXO1 genes. A significant association was observed for the investigated MMR genes, all of which were detected to be upregulated in gastroesophageal tumor samples when compared with paired normal samples. In the stratified analysis, the association was limited to gastric adenocarcinoma samples. We found no statistically significant associations between MMR gene expression and tumor site or histological grade. In our study, MSH2, MSH6, and EXO1 genes were overexpressed in gastroesophageal cancers. Further investigations based on more samples are necessary to validate our findings.

  16. Identification of genes showing differential expression profile ...

    Indian Academy of Sciences (India)

    in pig with genetic propensity for higher growth rate were identified by sequence analysis of 12 differentially expressed clones selected by differential screening following the generation of the subtracted cDNA population. Real-time PCR analysis con- firmed difference in expression profiles of the identified genes in ...

  17. 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.

  18. Nonstructural Protein 4 of Porcine Reproductive and Respiratory Syndrome Virus Modulates Cell Surface Swine Leukocyte Antigen Class I Expression by Downregulating β2-Microglobulin Transcription.

    Science.gov (United States)

    Qi, Pengfei; Liu, Ke; Wei, Jianchao; Li, Yuming; Li, Beibei; Shao, Donghua; Wu, Zhuanchang; Shi, Yuanyuan; Tong, Guangzhi; Qiu, Yafeng; Ma, Zhiyong

    2017-03-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of PRRS, which has important impacts on the pig industry. PRRSV infection results in disruption of the swine leukocyte antigen class I (SLA-I) antigen presentation pathway. In this study, highly pathogenic PRRSV (HP-PRRSV) infection inhibited transcription of the β2-microglobulin (β2M) gene ( B2M ) and reduced cellular levels of β2M, which forms a heterotrimeric complex with the SLA-I heavy chain and a variable peptide and plays a critical role in SLA-I antigen presentation. HP-PRRSV nonstructural protein 4 (Nsp4) was involved in the downregulation of β2M expression. Exogenous expression of Nsp4 downregulated β2M expression at both the mRNA and the protein level and reduced SLA-I expression on the cell surface. Nsp4 bound to the porcine B2M promoter and inhibited its transcriptional activity. Domain III of Nsp4 and the enhancer PAM element of the porcine B2M promoter were identified as essential for the interaction between Nsp4 and B2M These findings demonstrate a novel mechanism whereby HP-PRRSV may modulate the SLA-I antigen presentation pathway and provide new insights into the functions of HP-PRRSV Nsp4. IMPORTANCE PRRSV modulates the host response by disrupting the SLA-I antigen presentation pathway. We show that HP-PRRSV downregulates SLA-I expression on the cell surface via transcriptional inhibition of B2M expression by viral Nsp4. The interaction between domain III of Nsp4 and the enhancer PAM element of the porcine B2M promoter is essential for inhibiting B2M transcription. These observations reveal a novel mechanism whereby HP-PRRSV may modulate SLA-I antigen presentation and provide new insights into the functions of viral Nsp4. Copyright © 2017 American Society for Microbiology.

  19. Expression of vascular endothelial growth factor and proliferation cell nuclear antigen in esophageal carcinoma treated by radiotherapy

    International Nuclear Information System (INIS)

    Wang Yang; He Shaoqin; Shi Xuehui; Li Xiaoqiu; Lu Hongfen; Shi Daren

    2002-01-01

    Objective: To assess the correlation between the expression of vascular endothelial growth factor (VEGF) and proliferation cell nuclear antigen (PCNA) and the outcome of esophageal carcinoma patients treated by radiotherapy. Methods: In 59 esophageal cancer patients, expressions of VEGF and PCNA gene proteins in the biopsy samples obtained by endoscopic examination were assessed by immunohistochemical stain. Correlation between the expression of VEGF and PCNA gene and prognosis and clinical or pathological characters such as gender, stage and histological grade were analyzed. Results: Preliminary study demonstrated that the expression of VEGF and PCNA was not correlated with clinical or pathological characters such as gender, stage and histological grade, while VEGF was an independent prognostic factor for overall survival rate by multivariate analysis. Neither correlation between VEGF and local control nor that between PCNA and local control or overall survival rates was found significant. The correlation between VEGF and PCNA was significantly positive. Conclusions: Expression of VEGF, which can reflect the cell proliferation, may have significant impact on overall survival rate in esophageal carcinoma treated by radiotherapy and is worthy of further study. Although no correlation between PCNA and local control or overall survival rate is found, further study is still needed because of the limited cases alloted

  20. Use of serial analysis of gene expression to reveal the specific regulation of gene expression profile in asthmatic rats treated by acupuncture

    Directory of Open Access Journals (Sweden)

    Xu Yu-Dong

    2009-05-01

    Full Text Available Abstract Background Asthma has become an important public health issue and approximately 300 million people have suffered from the disease worldwide. Nowadays, the use of acupuncture in asthma is increasing. This study intended to systematically analyze and compare the gene expression profiles between the asthmatic and acupuncture-treated asthmatic rat lung, and tried to gain insight into the molecular mechanism underlying the early airway response (EAR phase of asthma treated by acupuncture. Methods Four tag libraries of serial analysis of gene expression (SAGE were established from lung tissues of control rats (CK, asthmatic rats (AS, asthmatic rats treated by acupuncture (ASAC, and control rats treated by acupuncture (CKAC. Bioinformatic analyses were carried out by using the methods including unsupervised hierarchical clustering, functional annotation tool of the database for annotation, visualization, and integrated discovery (DAVID, gene ontology (GO tree machine, and Kyoto encyclopedia of genes and genomes (KEGG pathway analysis. Results There were totally 186 differentially expressed tags (P CK/AS between the libraries of CK and AS, 130 differentially expressed tags between libraries of AS/ASAC (P AS/ASAC, and 144 differentially expressed tags between libraries of CK/CKAC (P CK/CKAC. The gene expression profiles of AS and ASAC were more similar than other libraries via unsupervised SAGE clustering. By comparison of PCK/AS and PAS/ASAC, the DAVID genes functional classification was found to be changed from "immune response" to "response to steroid hormone stimulus", and the GO term "antigen processing and presentation of peptide antigen" disappeared in PAS/ASAC. Totally 3 same KEGG pathways were found among the three groups. Moreover, 21 specific tags of the acupuncture in treating asthma were detected using Venn diagrams. Conclusion Our SAGE research indicates that the gene expression profile of the EAR phase of asthma could be

  1. Aberrant lymphoid antigen expression in acute myeloid leukemia in Saudi Arabia.

    Science.gov (United States)

    El-Sissy, Azza H; El-Mashari, May A; Bassuni, Wafaa Y; El-Swaayed, Aziza F

    2006-09-01

    Immunophenotyping improves both accuracy and reproducibility of acute leukemia classification and is considered particularly useful for identifying aberrant lineage association of acute leukemia, biphenotypic and bilineal acute leukemia, as well as monitoring minimal residual disease. Some immunophenotypes correlate with cytogenetic abnormalities and prognosis. Is to determine aberrant lymphoid antigen expression in Saudi acute myeloid leukemia (AML), correlate them with FAB subtypes, evaluate early surface markers CD7 and CD56, and to investigate the role of cytoplasmic CD79a (a B cell marker that is assigned a high score of 2.0 in the WHO classification). Thirty four newly diagnosed AML cases were included in this study, 47% showed aberrant lymphoid antigen expression. CD9 was the most frequently expressed lymphoid antigen (29.4%) followed by CD7 & CD19 (11.8%), CD4 (8.8%) and CD22 (2.9%). CD9 was expressed in 3/6 (50%) of M3 cases, CD7 was expressed in 11.8% and was mostly confined to FAB M1 and M2 and associated with immature antigens CD34, HLA-DR and TdT. CD56 was expressed in 7/34 (20.6%) cases, three of these cases (42.9%) belonged to the monocytic group. CD56 was also detected in 2 cases with 11q23 rearrangement. CD56 was expressed in 2/7 (28.6%) M2 cases, and was associated with t (8;21) (q22;q22) together with CD19. Co-expression of CD56 and CD7 was detected in 2.9% of the cases. CD79a was expressed in one case together with CD19, diagnosed as acute biphenotypic leukemia, and was associated with t(8;21) (q22;q22). Minimal residual disease in AML is very difficult to trace, detection of aberrant expression of lymphoid antigens will make it easier. The high score given to CD79a by EGIL is questionable based on cytogenetic classification.

  2. Nerve/glial antigen (NG) 2 is a crucial regulator of intercellular adhesion molecule (ICAM)-1 expression.

    Science.gov (United States)

    Schmitt, Beate M; Laschke, Matthias W; Rössler, Oliver G; Huang, Wenhui; Scheller, Anja; Menger, Michael D; Ampofo, Emmanuel

    2018-01-01

    The proteoglycan nerve/glial antigen (NG) 2 is expressed on multiple cell types and mediates cell proliferation and migration. However, little is known about its function in gene regulation. In this study, we demonstrate that in pericytes and glioblastoma cells intercellular adhesion molecule (ICAM)-1, an essential protein for leukocyte adhesion and transmigration, underlies a NG2-dependent expression. As shown by flow cytometry, Western blot analysis and quantitative real-time polymerase chain reaction (qRT-PCR), silencing of NG2 in human placenta-derived pericytes increased the expression of ICAM-1. Pathway analyses revealed that this is mediated by extracellular-regulated-kinases (ERK) 1/2 signaling. Moreover, leukocyte adhesion to NG2 siRNA-treated pericytes was significantly enhanced when compared to scrambled (scr) siRNA-treated control cells. In vivo, we detected increased ICAM-1 protein levels in the retina of mice lacking NG2 expression. To exclude that this novel mechanism is pericyte-specific, we additionally analyzed the expression of ICAM-1 in dependency of NG2 in two glioblastoma cell lines. We found that A1207 and M059K cells exhibit an inverse expression pattern of NG2 and ICAM-1. Finally, downregulation of NG2 in A1207 cells significantly increased ICAM-1 expression. Taken together, these findings indicate that NG2 may represent a promising target for the modulation of ICAM-1-mediated immune responses. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2018-01-01

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

  4. [Study of testicular cancer gene expression in samples of oral leukoplakia and squamous cell carcinoma of the mouth].

    Science.gov (United States)

    Skorodumova, L O; Muraev, A A; Zakharova, E S; Shepelev, M V; Korobko, I V; Zaderenko, I A; Ivanov, S Iu; Gnuchev, N V; Georgiev, G P; Larin, S S

    2012-01-01

    Cancer-testis (CT) antigens are normally expressed mostly in human germ cells, there is also an aberrant expression in some tumor cells. This expression profile makes them potential tumor growth biomarkers and a promising target for tumor immunotherapy. Specificity of CT genes expression in oral malignant and potentially malignant diseases, e.g. oral leukoplakia, is not yet studied. Data on CT genes expression profile in leukoplakia would allow developing new diagnostic methods with potential value for immunotherapy and prophylaxis of leukoplakia malignization. In our study we compared CT genes expression in normal oral mucosa, oral leukoplakia and oral squamous cell carcinoma. We are the first to describe CT genes expression in oral leukoplakia without dysplasia. This findings make impossible differential diagnosis of oral leukoplakia and squamous cell carcinoma on the basis of CT genes expression. The prognostic value of CT genes expression is still unclear, therefore the longitudinal studies are necessary.

  5. Genes involved in nonpermissive temperature-induced cell differentiation in Sertoli TTE3 cells bearing temperature-sensitive simian virus 40 large T-antigen

    International Nuclear Information System (INIS)

    Tabuchi, Yoshiaki; Kondo, Takashi; Suzuki, Yoshihisa; Obinata, Masuo

    2005-01-01

    Sertoli TTE3 cells, derived from transgenic mice bearing temperature-sensitive simian virus 40 large T (tsSV40LT)-antigen, proliferated continuously at a permissive temperature (33 deg C) whereas inactivation of the large T-antigen by a nonpermissive temperature (39 deg C) led to differentiation as judged by elevation of transferrin. To clarify the detailed mechanisms of differentiation, we investigated the time course of changes in gene expression using cDNA microarrays. Of the 865 genes analyzed, 14 genes showed increased levels of expression. Real-time quantitative PCR revealed that the mRNA levels of p21 waf1 , milk fat globule membrane protein E8, heat-responsive protein 12, and selenoprotein P were markedly elevated. Moreover, the differentiated condition induced by the nonpermissive temperature significantly increased mRNA levels of these four genes in several cell lines from the transgenic mice bearing the oncogene. The present results regarding changes in gene expression will provide a basis for a further understanding of molecular mechanisms of differentiation in both Sertoli cells and cell lines transformed by tsSV40LT-antigen

  6. Regulation of gene expression in human tendinopathy

    Directory of Open Access Journals (Sweden)

    Archambault Joanne M

    2011-05-01

    Full Text Available Abstract Background Chronic tendon injuries, also known as tendinopathies, are common among professional and recreational athletes. These injuries result in a significant amount of morbidity and health care expenditure, yet little is known about the molecular mechanisms leading to tendinopathy. Methods We have used histological evaluation and molecular profiling to determine gene expression changes in 23 human patients undergoing surgical procedures for the treatment of chronic tendinopathy. Results Diseased tendons exhibit altered extracellular matrix, fiber disorientation, increased cellular content and vasculature, and the absence of inflammatory cells. Global gene expression profiling identified 983 transcripts with significantly different expression patterns in the diseased tendons. Global pathway analysis further suggested altered expression of extracellular matrix proteins and the lack of an appreciable inflammatory response. Conclusions Identification of the pathways and genes that are differentially regulated in tendinopathy samples will contribute to our understanding of the disease and the development of novel therapeutics.

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

    Science.gov (United States)

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

    2016-12-01

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

  8. Noise minimization in eukaryotic gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Fraser, Hunter B.; Hirsh, Aaron E.; Giaever, Guri; Kumm, Jochen; Eisen, Michael B.

    2004-01-15

    All organisms have elaborate mechanisms to control rates of protein production. However, protein production is also subject to stochastic fluctuations, or noise. Several recent studies in Saccharomyces cerevisiae and Escherichia coli have investigated the relationship between transcription and translation rates and stochastic fluctuations in protein levels, or more generally, how such randomness is a function of intrinsic and extrinsic factors. However, the fundamental question of whether stochasticity in protein expression is generally biologically relevant has not been addressed, and it remains unknown whether random noise in the protein production rate of most genes significantly affects the fitness of any organism. We propose that organisms should be particularly sensitive to variation in the protein levels of two classes of genes: genes whose deletion is lethal to the organism and genes that encode subunits of multiprotein complexes. Using an experimentally verified model of stochastic gene expression in S. cerevisiae, we estimate the noise in protein production for nearly every yeast gene, and confirm our prediction that the production of essential and complex-forming proteins involves lower levels of noise than does the production of most other genes. Our results support the hypothesis that noise in gene expression is a biologically important variable, is generally detrimental to organismal fitness, and is subject to natural selection.

  9. Global gene expression in Escherichia coli biofilms

    DEFF Research Database (Denmark)

    Schembri, Mark; Kjærgaard, K.; Klemm, Per

    2003-01-01

    It is now apparent that microorganisms undergo significant changes during the transition from planktonic to biofilm growth. These changes result in phenotypic adaptations that allow the formation of highly organized and structured sessile communities, which possess enhanced resistance...... to antimicrobial treatments and host immune defence responses. Escherichia coli has been used as a model organism to study the mechanisms of growth within adhered communities. In this study, we use DNA microarray technology to examine the global gene expression profile of E. coli during sessile growth compared...... the transition to biofilm growth, and these included genes expressed under oxygen-limiting conditions, genes encoding (putative) transport proteins, putative oxidoreductases and genes associated with enhanced heavy metal resistance. Of particular interest was the observation that many of the genes altered...

  10. Profiling helper T cell subset gene expression in deer mice

    Directory of Open Access Journals (Sweden)

    Hjelle Brian

    2006-08-01

    Full Text Available Abstract Background Deer mice (Peromyscus maniculatus are the most common mammals in North America and are reservoirs for several zoonotic agents, including Sin Nombre virus (SNV, the principal etiologic agent of hantavirus cardiopulmonary syndrome (HCPS in North America. Unlike human HCPS patients, SNV-infected deer mice show no overt pathological symptoms, despite the presence of virus in the lungs. A neutralizing IgG antibody response occurs, but the virus establishes a persistent infection. Limitations of detailed analysis of deer mouse immune responses to SNV are the lack of reagents and methods for evaluating such responses. Results We developed real-time PCR-based detection assays for several immune-related transcription factor and cytokine genes from deer mice that permit the profiling of CD4+ helper T cells, including markers of Th1 cells (T-bet, STAT4, IFNγ, TNF, LT, Th2 cells (GATA-3, STAT6, IL-4, IL-5 and regulatory T cells (Fox-p3, IL-10, TGFβ1. These assays compare the expression of in vitro antigen-stimulated and unstimulated T cells from individual deer mice. Conclusion We developed molecular methods for profiling immune gene expression in deer mice, including a multiplexed real-time PCR assay for assessing expression of several cytokine and transcription factor genes. These assays should be useful for characterizing the immune responses of experimentally- and naturally-infected deer mice.

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

    Science.gov (United States)

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

    2011-02-01

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

  12. 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

    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...... 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...... live confocal microscopy, flow cytometry and in vitro adhesion assays. We found that one selected parasite sub-line simultaneously expressed two different var genes as surface antigens, on single IE. Importantly, and of physiological relevance to adhesion and malaria pathogenesis, this parasite sub-line...

  13. The detection of K88, K99 fimbrial antigen and enterotoxin genes of Escherichia coli isolated from piglets and calves with diarrhoea in Indonesia

    Directory of Open Access Journals (Sweden)

    Supar

    1996-03-01

    Full Text Available Enterotoxigenic Escherichia coli (ETEC strains cause diarrhoeal disease in piglets and calves in Indonesia. These strains possess two virulence factors namely attachment and enterotoxin antigens . These factors could be detected phenotypically and genetically. Haemolytic Escherichia coli (E coli isolates possessing K88 fimbrial antigen associated with 0-group 108 and 149. They were positive for K88 gene and demonstrated their ability to produce heat labile enterotoxin (LT and genetically were all positive for LT gene . Seventeen isolates ofE coli K88 which associated with 0-group 149 were positive forSTb gene, other O-serotypes were negative . Ten isolates of Ecoli K88 which associated with 0-group 108 possessed K88, K99, LT and STa genes, but negative for STb gene . However, phenotypically the K99 antigen and STa toxin were not expressed under laboratory conditions, the reason was not well understood . E. coli K99 strains isolated from calves wit h diarrhoea were all associated with 0-group 9 and produced STa toxin when tested by suckling mousse bioassay. The E. coli K99 calf isolates were all hybridized with K99 and STa gene only . It is likely that K99 gene is associated with STa gene . The DNA hybridization technique is more convenience to be used for confirmation diagnosis of colibacillosis, however, not all veterinary laboratories could perform these tests .

  14. 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...... prolonged tumor control in vaccinated wild type (WT) mice. The improved tumor control required antigen-specific cells, since no tumor control was observed, unless the melanoma cells expressed the vaccine targeted antigen. We also tested our new vaccine in immunodeficient (CD80/86 deficient) mice. Following...... approach where the target antigen fused to Ii is expressed from the adenoviral E1 region and IL-2 is expressed from the E3 region. Immunization of mice with this new vector construct resulted in an augmented primary effector CD8+ T-cell response. Furthermore, in a melanoma model we observed significantly...

  15. 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

    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...... frequent human cancers, with the purpose of providing novel therapeutic targets for these diseases. We used a set of previously uncharacterized antibodies against the cancer/testis antigens ADAM2, CALR3 and SAGE1 to investigate their expression in a large panel of normal tissues as well as breast and lung...

  16. Phasevarions mediate random switching of gene expression in pathogenic Neisseria.

    Directory of Open Access Journals (Sweden)

    Yogitha N Srikhanta

    2009-04-01

    Full Text Available Many host-adapted bacterial pathogens contain DNA methyltransferases (mod genes that are subject to phase-variable expression (high-frequency reversible ON/OFF switching of gene expression. In Haemophilus influenzae, the random switching of the modA gene controls expression of a phase-variable regulon of genes (a "phasevarion", via differential methylation of the genome in the modA ON and OFF states. Phase-variable mod genes are also present in Neisseria meningitidis and Neisseria gonorrhoeae, suggesting that phasevarions may occur in these important human pathogens. Phylogenetic studies on phase-variable mod genes associated with type III restriction modification (R-M systems revealed that these organisms have two distinct mod genes--modA and modB. There are also distinct alleles of modA (abundant: modA11, 12, 13; minor: modA4, 15, 18 and modB (modB1, 2. These alleles differ only in their DNA recognition domain. ModA11 was only found in N. meningitidis and modA13 only in N. gonorrhoeae. The recognition site for the modA13 methyltransferase in N. gonorrhoeae strain FA1090 was identified as 5'-AGAAA-3'. Mutant strains lacking the modA11, 12 or 13 genes were made in N. meningitidis and N. gonorrhoeae and their phenotype analyzed in comparison to a corresponding mod ON wild-type strain. Microarray analysis revealed that in all three modA alleles multiple genes were either upregulated or downregulated, some of which were virulence-associated. For example, in N. meningitidis MC58 (modA11, differentially expressed genes included those encoding the candidate vaccine antigens lactoferrin binding proteins A and B. Functional studies using N. gonorrhoeae FA1090 and the clinical isolate O1G1370 confirmed that modA13 ON and OFF strains have distinct phenotypes in antimicrobial resistance, in a primary human cervical epithelial cell model of infection, and in biofilm formation. This study, in conjunction with our previous work in H. influenzae, indicates

  17. Phasevarions mediate random switching of gene expression in pathogenic Neisseria.

    Science.gov (United States)

    Srikhanta, Yogitha N; Dowideit, Stefanie J; Edwards, Jennifer L; Falsetta, Megan L; Wu, Hsing-Ju; Harrison, Odile B; Fox, Kate L; Seib, Kate L; Maguire, Tina L; Wang, Andrew H-J; Maiden, Martin C; Grimmond, Sean M; Apicella, Michael A; Jennings, Michael P

    2009-04-01

    Many host-adapted bacterial pathogens contain DNA methyltransferases (mod genes) that are subject to phase-variable expression (high-frequency reversible ON/OFF switching of gene expression). In Haemophilus influenzae, the random switching of the modA gene controls expression of a phase-variable regulon of genes (a "phasevarion"), via differential methylation of the genome in the modA ON and OFF states. Phase-variable mod genes are also present in Neisseria meningitidis and Neisseria gonorrhoeae, suggesting that phasevarions may occur in these important human pathogens. Phylogenetic studies on phase-variable mod genes associated with type III restriction modification (R-M) systems revealed that these organisms have two distinct mod genes--modA and modB. There are also distinct alleles of modA (abundant: modA11, 12, 13; minor: modA4, 15, 18) and modB (modB1, 2). These alleles differ only in their DNA recognition domain. ModA11 was only found in N. meningitidis and modA13 only in N. gonorrhoeae. The recognition site for the modA13 methyltransferase in N. gonorrhoeae strain FA1090 was identified as 5'-AGAAA-3'. Mutant strains lacking the modA11, 12 or 13 genes were made in N. meningitidis and N. gonorrhoeae and their phenotype analyzed in comparison to a corresponding mod ON wild-type strain. Microarray analysis revealed that in all three modA alleles multiple genes were either upregulated or downregulated, some of which were virulence-associated. For example, in N. meningitidis MC58 (modA11), differentially expressed genes included those encoding the candidate vaccine antigens lactoferrin binding proteins A and B. Functional studies using N. gonorrhoeae FA1090 and the clinical isolate O1G1370 confirmed that modA13 ON and OFF strains have distinct phenotypes in antimicrobial resistance, in a primary human cervical epithelial cell model of infection, and in biofilm formation. This study, in conjunction with our previous work in H. influenzae, indicates that

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

    Science.gov (United States)

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

    2017-12-01

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

  19. High-throughput identification of antigen-specific TCRs by TCR gene capture

    DEFF Research Database (Denmark)

    Linnemann, Carsten; Heemskerk, Bianca; Kvistborg, Pia

    2013-01-01

    have developed a high-throughput DNA-based strategy to identify TCR sequences by the capture and sequencing of genomic DNA fragments encoding the TCR genes. We establish the value of this approach by assembling a large library of cancer germline tumor antigen-reactive TCRs. Furthermore, by exploiting......The transfer of T cell receptor (TCR) genes into patient T cells is a promising approach for the treatment of both viral infections and cancer. Although efficient methods exist to identify antibodies for the treatment of these diseases, comparable strategies to identify TCRs have been lacking. We...... of antigen specificities, which may be the first step toward the development of autologous TCR gene therapy to target patient-specific neoantigens in human cancer....

  20. Human AZU-1 gene, variants thereof and expressed gene products

    Science.gov (United States)

    Chen, Huei-Mei; Bissell, Mina

    2004-06-22

    A human AZU-1 gene, mutants, variants and fragments thereof. Protein products encoded by the AZU-1 gene and homologs encoded by the variants of AZU-1 gene acting as tumor suppressors or markers of malignancy progression and tumorigenicity reversion. Identification, isolation and characterization of AZU-1 and AZU-2 genes localized to a tumor suppressive locus at chromosome 10q26, highly expressed in nonmalignant and premalignant cells derived from a human breast tumor progression model. A recombinant full length protein sequences encoded by the AZU-1 gene and nucleotide sequences of AZU-1 and AZU-2 genes and variant and fragments thereof. Monoclonal or polyclonal antibodies specific to AZU-1, AZU-2 encoded protein and to AZU-1, or AZU-2 encoded protein homologs.

  1. Expression Study of Banana Pathogenic Resistance Genes

    Directory of Open Access Journals (Sweden)

    Fenny M. Dwivany

    2016-10-01

    Full Text Available Banana is one of the world's most important trade commodities. However, infection of banana pathogenic fungi (Fusarium oxysporum race 4 is one of the major causes of decreasing production in Indonesia. Genetic engineering has become an alternative way to control this problem by isolating genes that involved in plant defense mechanism against pathogens. Two of the important genes are API5 and ChiI1, each gene encodes apoptosis inhibitory protein and chitinase enzymes. The purpose of this study was to study the expression of API5 and ChiI1 genes as candidate pathogenic resistance genes. The amplified fragments were then cloned, sequenced, and confirmed with in silico studies. Based on sequence analysis, it is showed that partial API5 gene has putative transactivation domain and ChiI1 has 9 chitinase family GH19 protein motifs. Data obtained from this study will contribute in banana genetic improvement.

  2. Mapping of antigenic sites in human neuron-specific enolase by expression subcloning.

    Science.gov (United States)

    Quinn, G B; Reeves, I G; Day, I N

    1994-05-01

    Human serum neuron-specific enolase (NSE) is a marker of neurons and of small-cell carcinoma of the lung; improved immunoassays of NSE remain an important goal. Here, we used overlapping complementary DNA (cDNA) clones for reconstruction to express full-length recombinant NSE, and also to express a set of cloned subfragments through the prokaryotic expression vectors pUEX and pUBEX. Subfragments expressed as fusion proteins were used to characterize immunogenic and antigenic regions and epitopes and, expressed as affinity matrices, to derive purified, fractionated polyclonal antibodies. NSE epitope data can be visualized with yeast enolase-1 crystal structure coordinates: The two protein sequences align almost perfectly and are 61% identical. This approach demonstrates the complementarity of cDNA expression with techniques of polyclonal antiserum and monoclonal antibody production and with chemical peptide synthesis in the refinement of immunodiagnostic reagents.

  3. Epigenetic aspects of lymphocyte antigen receptor gene rearrangement or ‘when stochasticity completes randomness’

    Science.gov (United States)

    Jaeger, Sébastien; Fernandez, Bastien; Ferrier, Pierre

    2013-01-01

    To perform their specific functional role, B and T lymphocytes, cells of the adaptive immune system of jawed vertebrates, need to express one (and, preferably, only one) form of antigen receptor, i.e. the immunoglobulin or T-cell receptor (TCR), respectively. This end goal depends initially on a series of DNA cis-rearrangement events between randomly chosen units from separate clusters of V, D (at some immunoglobulin and TCR loci) and J gene segments, a biomolecular process collectively referred to as V(D)J recombination. V(D)J recombination takes place in immature T and B cells and relies on the so-called RAG nuclease, a site-specific DNA cleavage apparatus that corresponds to the lymphoid-specific moiety of the VDJ recombinase. At the genome level, this recombinase's mission presents substantial biochemical challenges. These relate to the huge distance between (some of) the gene segments that it eventually rearranges and the need to achieve cell-lineage-restricted and developmentally ordered routines with at times, mono-allelic versus bi-allelic discrimination. The entire process must be completed without any recombination errors, instigators of chromosome instability, translocation and, potentially, tumorigenesis. As expected, such a precisely choreographed and yet potentially risky process demands sophisticated controls; epigenetics demonstrates what is possible when calling upon its many facets. In this vignette, we will recall the evidence that almost from the start appeared to link the two topics, V(D)J recombination and epigenetics, before reviewing the latest advances in our knowledge of this joint venture. PMID:23278765

  4. 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.

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

    Science.gov (United States)

    Huang, Yu-Chuen; Jiang, Chii-Ming; Chen, Yu-Jen; Chen, Yu-Yawn

    2013-01-01

    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.

  6. Gene expression analysis of flax seed development

    Directory of Open Access Journals (Sweden)

    Sharpe Andrew

    2011-04-01

    Full Text Available Abstract Background Flax, Linum usitatissimum L., is an important crop whose seed oil and stem fiber have multiple industrial applications. Flax seeds are also well-known for their nutritional attributes, viz., omega-3 fatty acids in the oil and lignans and mucilage from the seed coat. In spite of the importance of this crop, there are few molecular resources that can be utilized toward improving seed traits. Here, we describe flax embryo and seed development and generation of comprehensive genomic resources for the flax seed. Results We describe a large-scale generation and analysis of expressed sequences in various tissues. Collectively, the 13 libraries we have used provide a broad representation of genes active in developing embryos (globular, heart, torpedo, cotyledon and mature stages seed coats (globular and torpedo stages and endosperm (pooled globular to torpedo stages and genes expressed in flowers, etiolated seedlings, leaves, and stem tissue. A total of 261,272 expressed sequence tags (EST (GenBank accessions LIBEST_026995 to LIBEST_027011 were generated. These EST libraries included transcription factor genes that are typically expressed at low levels, indicating that the depth is adequate for in silico expression analysis. Assembly of the ESTs resulted in 30,640 unigenes and 82% of these could be identified on the basis of homology to known and hypothetical genes from other plants. When compared with fully sequenced plant genomes, the flax unigenes resembled poplar and castor bean more than grape, sorghum, rice or Arabidopsis. Nearly one-fifth of these (5,152 had no homologs in sequences reported for any organism, suggesting that this category represents genes that are likely unique to flax. Digital analyses revealed gene expression dynamics for the biosynthesis of a number of important seed constituents during seed development. Conclusions We have developed a foundational database of expressed sequences and collection of plasmid

  7. Gene expression analysis of flax seed development.

    Science.gov (United States)

    Venglat, Prakash; Xiang, Daoquan; Qiu, Shuqing; Stone, Sandra L; Tibiche, Chabane; Cram, Dustin; Alting-Mees, Michelle; Nowak, Jacek; Cloutier, Sylvie; Deyholos, Michael; Bekkaoui, Faouzi; Sharpe, Andrew; Wang, Edwin; Rowland, Gordon; Selvaraj, Gopalan; Datla, Raju

    2011-04-29

    Flax, Linum usitatissimum L., is an important crop whose seed oil and stem fiber have multiple industrial applications. Flax seeds are also well-known for their nutritional attributes, viz., omega-3 fatty acids in the oil and lignans and mucilage from the seed coat. In spite of the importance of this crop, there are few molecular resources that can be utilized toward improving seed traits. Here, we describe flax embryo and seed development and generation of comprehensive genomic resources for the flax seed. We describe a large-scale generation and analysis of expressed sequences in various tissues. Collectively, the 13 libraries we have used provide a broad representation of genes active in developing embryos (globular, heart, torpedo, cotyledon and mature stages) seed coats (globular and torpedo stages) and endosperm (pooled globular to torpedo stages) and genes expressed in flowers, etiolated seedlings, leaves, and stem tissue. A total of 261,272 expressed sequence tags (EST) (GenBank accessions LIBEST_026995 to LIBEST_027011) were generated. These EST libraries included transcription factor genes that are typically expressed at low levels, indicating that the depth is adequate for in silico expression analysis. Assembly of the ESTs resulted in 30,640 unigenes and 82% of these could be identified on the basis of homology to known and hypothetical genes from other plants. When compared with fully sequenced plant genomes, the flax unigenes resembled poplar and castor bean more than grape, sorghum, rice or Arabidopsis. Nearly one-fifth of these (5,152) had no homologs in sequences reported for any organism, suggesting that this category represents genes that are likely unique to flax. Digital analyses revealed gene expression dynamics for the biosynthesis of a number of important seed constituents during seed development. We have developed a foundational database of expressed sequences and collection of plasmid clones that comprise even low-expressed genes such as

  8. Human leukocyte antigen (HLA)-E and HLA-F expression in gastric cancer.

    Science.gov (United States)

    Ishigami, Sumiya; Arigami, Takaaki; Okumura, Hiroshi; Uchikado, Yasuto; Kita, Yoshiaki; Kurahara, Hiroshi; Maemura, Kosei; Kijima, Yuko; Ishihara, Yuka; Sasaki, Ken; Uenosono, Yoshikazu; Natsugoe, Shoji

    2015-04-01

    Human leukocyte antigen (HLA)-E and HLA-F are classified as non-classical HLA class Ib antigens. Ectopic HLA-E and HLA-F expression was recently detected in cancer cells; however, the clinical implication of their expression remains unknown. A total of 209 patients with gastric cancer were enrolled in this study. Immunohistochemistry was used to evaluate the expression of HLA-E and HLA-F in gastric cancer specimens. HLA-E and HLA-F expression were seen in the cell membrane. HLA-E and HLA-F expression significantly correlated with depth of invasion, nodal involvement, lymphatic invasion, and venous invasion. No significant correlation between HLA-E and HLA-F expression was found (pHLA-E-positive group and HLA-F-positive group were significantly poorer than that of their respective negative groups. Combination of HLA-E and HLA-F made the p-value smaller than single analysis (pHLA-E and HLA-F expression simultaneously in gastric cancer. We identified that the HLA-E and HLA-F in gastric cancer independently affected clinical factors, including postoperative outcome. For HLA-E- or HLA-F-positive gastric cancer, we should settle on a treatment strategy that reinforces the host immune response. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  9. Gene expression profiles in skeletal muscle after gene electrotransfer

    DEFF Research Database (Denmark)

    Hojman, Pernille; Zibert, John R; Gissel, Hanne

    2007-01-01

    ) followed by a long low voltage pulse (LV, 100 V/cm, 400 ms); a pulse combination optimised for efficient and safe gene transfer. Muscles were transfected with green fluorescent protein (GFP) and excised at 4 hours, 48 hours or 3 weeks after treatment. RESULTS: Differentially expressed genes were...... with the control muscles. Most interestingly, no changes in the expression of proteins involved in inflammatory responses or muscle regeneration was detected, indicating limited muscle damage and regeneration. Histological analysis revealed structural changes with loss of cell integrity and striation pattern......BACKGROUND: Gene transfer by electroporation (DNA electrotransfer) to muscle results in high level long term transgenic expression, showing great promise for treatment of e.g. protein deficiency syndromes. However little is known about the effects of DNA electrotransfer on muscle fibres. We have...

  10. Visualizing Gene Expression In Situ

    Energy Technology Data Exchange (ETDEWEB)

    Burlage, R.S.

    1998-11-02

    Visualizing bacterial cells and describing their responses to the environment are difficult tasks. Their small size is the chief reason for the difficulty, which means that we must often use many millions of cells in a sample in order to determine what the average response of the bacteria is. However, an average response can sometimes mask important events in bacterial physiology, which means that our understanding of these organisms will suffer. We have used a variety of instruments to visualize bacterial cells, all of which tell us something different about the sample. We use a fluorescence activated cell sorter to sort cells based on the fluorescence provided by bioreporter genes, and these can be used to select for particular genetic mutations. Cells can be visualized by epifluorescent microscopy, and sensitive photodetectors can be added that allow us to find a single bacterial cell that is fluorescent or bioluminescent. We have also used standard photomultipliers to examine cell aggregates as field bioreporter microorganisms. Examples of each of these instruments show how our understanding of bacterial physiology has changed with the technology.

  11. The prognostic implications of macrophages expressing proliferating cell nuclear antigen in breast cancer depend on immune context.

    Directory of Open Access Journals (Sweden)

    Michael J Campbell

    Full Text Available Tumor associated macrophages (TAMs are recruited from the circulation to the tumor site, and can undergo a spectrum of phenotypic changes, with two contrasting activation states described in the literature: the M1 and M2 phenotypes. We previously identified a population of TAMs that express proliferating cell nuclear antigen (PCNA and are associated with high grade, hormone receptor negative breast cancers and poor outcomes. In the present exploratory study we again found that high PCNA(+ TAM counts in pre-treatment tumor biopsies (102 invasive breast cancer cases from the I-SPY 1 Trial, a prospective neoadjuvant trial with serial core biopsies and gene array data were associated with high grade, hormone receptor negativity, and decreased recurrence free survival. We explored the association of these PCNA(+ TAMs with the expression of M1 and M2 related genes and, contrary to expectation, observed that high PCNA(+ TAM levels were associated with more M1- than M2-related genes. An immune gene signature, derived from cytotoxic T cell and MHC Class II genes (Tc/ClassII, was developed and we found that high PCNA(+ TAM counts, in the context of a low Tc/ClassII signature score, were associated with significantly worse recurrence free survival in all cases and in hormone receptor negative only cases. We observed similar results using a gene signature-proxy for PCNA(+ TAMs in a larger independent set of 425 neoadjuvant-treated breast cancer cases. The results of this exploratory study indicate that high numbers of PCNA(+ TAMs, in the absence of an anti-tumor immune microenvironment (as indicated by a low Tc/ClassII signature score, are associated with poor outcomes in breast cancer patients treated with neoadjuvant chemotherapy. This, along with the observation that PCNA(+ TAMs were associated predominantly with M1-related genes, may provide new insights into the role of the immune microenvironment in breast cancer.

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

    International Nuclear Information System (INIS)

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

    2009-01-01

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

  13. Renal Gene Expression Database (RGED): a relational database of gene expression profiles in kidney disease

    Science.gov (United States)

    Zhang, Qingzhou; Yang, Bo; Chen, Xujiao; Xu, Jing; Mei, Changlin; Mao, Zhiguo

    2014-01-01

    We present a bioinformatics database named Renal Gene Expression Database (RGED), which contains comprehensive gene expression data sets from renal disease research. The web-based interface of RGED allows users to query the gene expression profiles in various kidney-related samples, including renal cell lines, human kidney tissues and murine model kidneys. Researchers can explore certain gene profiles, the relationships between genes of interests and identify biomarkers or even drug targets in kidney diseases. The aim of this work is to provide a user-friendly utility for the renal disease research community to query expression profiles of genes of their own interest without the requirement of advanced computational skills. Availability and implementation: Website is implemented in PHP, R, MySQL and Nginx and freely available from http://rged.wall-eva.net. Database URL: http://rged.wall-eva.net PMID:25252782

  14. Synthesis and expression of CDw75 antigen in human colorectal cancer

    International Nuclear Information System (INIS)

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

    2009-01-01

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

  15. Expression of BAGE, GAGE, and MAGE genes in human gastric carcinoma.

    Science.gov (United States)

    Li, J; Yang, Y; Fujie, T; Baba, K; Ueo, H; Mori, M; Akiyoshi, T

    1996-09-01

    The MAGE, BAGE, and GAGE genes code for distinct antigens that are recognized by autologous cytolytic T lymphocytes. We investigated the expression of these genes in both cell lines and surgical samples of gastric carcinoma, using reverse transcription-PCR. Furthermore, the induction of these genes by 5-aza-2'-deoxycytidine (DAC), a demethylating agent, was also examined in several cell lines. Of 11 cell lines, BAGE, GAGE1-6, GAGE1-2, MAGE-1, and MAGE-3 were detected in 7 (64%), 4 (36%), 3 (27%), 8 (73%), and 8 (73%) cell lines, respectively. After the in vitro treatment of the negative cell lines with DAC, the expression of these genes became positive in 46 to 91% of these cell lines. No expression of these genes was seen in any of the 57 samples of normal gastric tissue. In contrast, the tumor tissue samples expressed BAGE, GAGE1-6, GAGE1-2, MAGE-1, and MAGE-3 in 13 (23%), 9 (16%), 6 (11%), 25 (44%), and 23 (40%) tissue samples, respectively. Thus, at least one of these genes was expressed in 35 (61%) of 57 carcinomas. An analysis of the relationship between clinicopathological factors and the expression of these genes revealed that either BAGE or one of these genes was more frequently expressed in histologically intestinal-type than in diffuse-type carcinomas. Our results suggest that, because of the higher expression of these genes and the possible induction of these genes by DAC, patients with gastric carcinoma may, therefore, be potential candidates for tumor-specific immunotherapy directed against these antigens.

  16. Reduced experimental autoimmune encephalomyelitis after intranasal and oral administration of recombinant lactobacilli expressing myelin antigens

    OpenAIRE

    Maassen, Kitty; Laman, Jon; Holten-Neelen, C.; Hoogteijling, L.; Groenewegen, Lizet; Visser, Lizette; Schellekens, M.; Boersma, Wim; Claassen, Eric

    2003-01-01

    textabstractOral administration of autoantigens is a safe and convenient way to induce peripheral T-cell tolerance in autoimmune diseases like multiple sclerosis (MS). To increase the efficacy of oral tolerance induction and obviate the need for large-scale purification of human myelin proteins, we use genetically modified lactobacilli expressing myelin antigens. A panel of recombinant lactobacilli was constructed producing myelin proteins and peptides, including human and guinea pig myelin b...

  17. 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...... of costimulatory endodomains for CAR construction to improve the effector functions of the engineered T cells. Camelid single-domain antibodies (VHHs), which are the smallest single domain antibodies, can endow great targeting ability to CAR-engineered T cells....

  18. Limitations of the Echinococcus granulosus genome sequence assemblies for analysis of the gene family encoding the EG95 vaccine antigen.

    Science.gov (United States)

    Gauci, Charles G; Alvarez Rojas, Cristian A; Chow, Conan; Lightowlers, Marshall W

    2017-11-27

    Echinococcus granulosus is an important zoonotic parasite that is distributed worldwide. The EG95 vaccine was developed to assist with control of E. granulosus transmission through the parasite's livestock intermediate hosts. The vaccine is based on a recombinant antigen encoded by a gene which is a member of a multi-gene family. With the recent availability of two E. granulosus draft genomes, we sought to map the eg95 gene family to the genomes. We were unable to map unequivocally any of the eg95 gene family members which had previously been characterized by cloning and sequencing both strands of genomic DNA fragments. Our inability to map EG95-related genes to the genomes has revealed limitations in the assembled sequence data when utilized for gene family analyses. This study contrasts with the expectations expressed in often high-profile publications describing draft genomes of parasitic organisms, highlighting deficiencies in currently available genomic resources for E. granulosus and provides a cautionary note for research which seeks to utilize these genome datasets.

  19. [Imprinting genes and it's expression in Arabidopsis].

    Science.gov (United States)

    Zhang, Hong-Yu; Xu, Pei-Zhou; Yang, Hua; Wu, Xian-Jun

    2010-07-01

    Genomic imprinting refers to the phenomenon that the expression of a gene copy depends on its parent of origin. The Arabidopsis imprinted FIS (Fertilisation-independent seed) genes, mea, fis2, and fie, play essential roles in the repression of central cell and the regulation of early endosperm development. fis mutants display two phenotypes: autonomous diploid endosperm development when fertilization is absent and un-cellularised endosperm formation when fertilization occurs. The FIS Polycomb protein complex including the above three FIS proteins catalyzes histone H3 K27 tri-methylation on target loci. DME (DEMETER), a DNA glycosylase, and AtMET1 (Methyltransferase1), a DNA methyltransferase, are involved in the regulation of imprinted expression of both mea and fis2. This review summarizes the studies on the Arabidopsis imprinted FIS genes and other related genes. Recent works have shown that the insertion of transposons may affect nearby gene expression, which may be the main driving force behind the evolution of genomic imprinting. This summary covers the achievements on Arabidopsis imprinted genes will provide important information for studies on genomic imprinting in the important crops such as rice and maize.

  20. Gene expression profiling for pharmaceutical toxicology screening.

    Science.gov (United States)

    Bugelski, Peter J

    2002-01-01

    Advances in medicinal chemistry and high-throughput pharmacological screening are creating a multitude of potential lead compounds. There is also heightened concern about drug-induced toxicity, which is all too often uncovered late in development or at the post marketing stage. Together, these factors have created a need for novel approaches to screen for toxicity. There have been technological advances that enable study of changes in the gene expression profile caused by toxic insults and important steps made toward unraveling target organ toxicity at the molecular level. Thus, gene expression profile-based screens hold the promise to revolutionize the way in which compounds are selected for development. For screens focused on specific mechanisms of toxicity, reporter gene systems have proven utility, albeit modest because of our limited knowledge of which genes are true surrogate markers for toxicity. For broader forecasts of toxicity, DNA microarrays hold great promise for delivering practical gene expression profile screens (GEPS). For this promise to be realized, however, a number of technological hurdles must be cleared: (i) cost; (ii) reproducibility; (iii) throughput; and (iv) data analysis. Of equal if not greater importance, issues relating to the test systems used, the requisite number of genes to be studied and the size and scope of the database upon which forecasts will be based must be addressed. At present, the proof-of-concept for GEPS for toxicity is in hand, and we are poised to realize the goal of creating practical GEPS for application in compound prioritization.

  1. Human leukocyte antigen class II genes and Helicobacter pylori infection: does genotype overwhelm environmental exposure?

    Science.gov (United States)

    Russo, Antonio; Maconi, Giovanni; Lombardo, Claudia; Settesoldi, Daniela; Ferrari, Daniela; Ravagnani, Fernando; Andreola, Salvatore; Pizzetti, Paolo; Spinelli, Pasquale; Bertario, Lucio

    2003-09-01

    We investigated associations between human leukocyte antigen class II genes, environmental exposures, and Helicobacter pylori infection. Sixty-eight subjects with histologically confirmed H. pylori and intestinal metaplasia (cases) and 70 healthy subjects without H. pylori (controls) matched for age, sex, and year of birth were included in this study. All patients answered a detailed questionnaire designed to collect sociodemographic characteristics, smoking, alcohol drinking, and dietary habits. Human leukocyte antigen class II genes were typed with genomic DNA. The cytotoxins CagA and VacA were investigated with serology. Odds ratios and corresponding 95% confidence intervals were estimated from multivariate conditional logistic regression. Multiple correspondence analysis was used to represent the interrelationships of a multiple contingency table. Human leukocyte antigen DRB1, DQA1, and DQB1 genotypes were not significantly associated with H. pylori infection and intestinal metaplasia. No significant association with blood group or Lewis antigen system was found. However, multiple correspondence analysis clearly associated H. pylori with environmental exposure: the control group largely consumed olive oil, fresh fruits, and vegetables and histories of never or formerly smoking and the case group (those positive for H. pylori and metaplasia) largely consumed eggs, meat and butter and had histories of smoking cigarettes. These findings suggested that H. pylori infection is not influenced by a genetic compound and confirmed the relevance of environmental exposure.

  2. Regulation of major histocompatibility complex class II gene expression in trophoblast cells

    Directory of Open Access Journals (Sweden)

    Choi Jason C

    2004-07-01

    Full Text Available Abstract Trophoblast cells are unique because they are one of the few mammalian cell types that do not express major histocompatibility complex (MHC class II antigens, either constitutively or after exposure to IFN-γ. The absence of MHC class II antigen expression on trophoblast cells has been postulated to be one of the essential mechanisms by which the semi-allogeneic fetus evades immune rejection reactions by the maternal immune system. Consistent with this hypothesis, trophoblast cells from the placentas of women suffering from chronic inflammation of unknown etiology and spontaneous recurrent miscarriages have been reported to aberrantly express MHC class II antigens. The lack of MHC class II antigen expression on trophoblast cells is due to silencing of expression of the class II transactivator (CIITA, a transacting factor that is essential for constitutive and IFN-γ-inducible MHC class II gene transcription. Transfection of trophoblast cells with CIITA expression vectors activates both MHC class II and class Ia antigen expression, which confers on trophoblast cells both the ability to activate helper T cells, and sensitivity to lysis by cytotoxic T lymphocytes. Collectively, these studies strongly suggest that stringent silencing of CIITA (and therefore MHC class II gene expression in trophoblast cells is critical for the prevention of immune rejection responses against the fetus by the maternal immune system. The focus of this review is to summarize studies examining the novel mechanisms by which CIITA is silenced in trophoblast cells. The elucidation of the silencing of CIITA in trophoblast cells may shed light on how the semi-allogeneic fetus evades immune rejection by the maternal immune system during pregnancy.

  3. The expression of histocompatibility antigen HLA-DR in cervical squamous epithelium infected with human papilloma virus.

    Science.gov (United States)

    Warhol, M J; Gee, B

    1989-03-01

    Uterine cervices with histologic changes suggestive of human papilloma virus (HPV) infection were examined for the presence of papilloma virus capsid antigens and the Class II histocompatibility antigen HLA-DR. The purpose of this study was to determine whether papilloma virus infection could induce HLA-DR expression by squamous cells. This expression would allow squamous epithelium to function as antigen-presenting cells and perhaps initiate the immune response. In 20 cases in which HPV capsid antigens were identified, no HLA-DR expression was noted. HLA-DR expression was noted on Langerhans cells within the squamous epithelium and on mononuclear cells in the underlying lamina propria. HLA-DR-positive cells were also noted between columnar epithelial cells of the endocervix. We conclude that HPV infection does not induce HLA-DR expression in the cells it infects.

  4. Differential testicular gene expression in seasonal fertility

    Science.gov (United States)

    Maywood, Elizabeth S.; Chahad-Ehlers, Samira; Garabette, Martine L.; Pritchard, Claire; Underhill, Phillip; Greenfield, Andrew; Ebling, Francis J. P.; Kyriacou, Charalambos P.; Hastings, Michael H.; Reddy, Akhilesh B.

    2012-01-01

    Spermatogenesis is an essential precursor for successful sexual reproduction. Recently, there has been an expansion in our knowledge of the genes associated with particular stages of normal, physiological testicular development and pubertal activation. What has been lacking, however, is an understanding of those genes that are involved in specifically regulating sperm production, rather than in maturation and elaboration of the testis as an organ. By utilising the reversible (seasonal) fertility of the Syrian hamster as a model system, we sought to discover genes which are specifically involved in turning off sperm production and not in tissue specification and/or maturation. Using gene expression microarrays and in situ hybridisation in hamsters and genetically infertile mice, we have identified a variety of known and novel factors involved in reversible, transcriptional, translational and post-translational control of testicular function, as well those involved in cell division and macromolecular metabolism. The novel genes uncovered could be potential targets for therapies against fertility disorders. PMID:19346449

  5. AIRE: From promiscuous molecular partnerships to promiscuous gene expression.

    Science.gov (United States)

    Abramson, Jakub; Goldfarb, Yael

    2016-01-01

    Autoimmune regulator (AIRE) is a unique transcriptional regulator that induces promiscuous expression of thousands of tissue-restricted antigens (TRAs) in medullary thymic epithelial cells (mTECs), a step critical for the induction of immunological self-tolerance. The past 15 years have seen dramatic progress in our understanding of how AIRE induces immunological self-tolerance on a molecular level. This major advancement can be greatly attributed to the identification of a large variety of proteins that physically associate with AIRE, supporting and regulating its transcription-transactivation capacity. These diverse molecular partnerships have been shown to play roles in shuttling AIRE to the nucleus, securing AIRE's interaction with nuclear matrix and chromatin, releasing RNA polymerase-II from its stalled state and potentiating AIRE-mediated gene expression, among others. In this review we discuss the relationship of AIRE with its vast and rather diverse repertoire of partners and highlight how such "promiscuous partnerships" contribute to the phenomenon of "promiscuous gene expression" in the thymus. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Characterization and gene expression analysis of the cir multi-gene family of plasmodium chabaudi chabaudi (AS

    Directory of Open Access Journals (Sweden)

    Lawton Jennifer

    2012-03-01

    Full Text Available Abstract Background The pir genes comprise the largest multi-gene family in Plasmodium, with members found in P. vivax, P. knowlesi and the rodent malaria species. Despite comprising up to 5% of the genome, little is known about the functions of the proteins encoded by pir genes. P. chabaudi causes chronic infection in mice, which may be due to antigenic variation. In this model, pir genes are called cirs and may be involved in this mechanism, allowing evasion of host immune responses. In order to fully understand the role(s of CIR proteins during P. chabaudi infection, a detailed characterization of the cir gene family was required. Results The cir repertoire was annotated and a detailed bioinformatic characterization of the encoded CIR proteins was performed. Two major sub-families were identified, which have been named A and B. Members of each sub-family displayed different amino acid motifs, and were thus predicted to have undergone functional divergence. In addition, the expression of the entire cir repertoire was analyzed via RNA sequencing and microarray. Up to 40% of the cir gene repertoire was expressed in the parasite population during infection, and dominant cir transcripts could be identified. In addition, some differences were observed in the pattern of expression between the cir subgroups at the peak of P. chabaudi infection. Finally, specific cir genes were expressed at different time points during asexual blood stages. Conclusions In conclusion, the large number of cir genes and their expression throughout the intraerythrocytic cycle of development indicates that CIR proteins are likely to be important for parasite survival. In particular, the detection of dominant cir transcripts at the peak of P. chabaudi infection supports the idea that CIR proteins are expressed, and could perform important functions in the biology of this parasite. Further application of the methodologies described here may allow the elucidation of CIR sub

  7. Gene expression during normal and FSHD myogenesis

    Directory of Open Access Journals (Sweden)

    Sowden Janet

    2011-09-01

    Full Text Available Abstract Background Facioscapulohumeral muscular dystrophy (FSHD is a dominant disease linked to contraction of an array of tandem 3.3-kb repeats (D4Z4 at 4q35. Within each repeat unit is a gene, DUX4, that can encode a protein containing two homeodomains. A DUX4 transcript derived from the last repeat unit in a contracted array is associated with pathogenesis but it is unclear how. Methods Using exon-based microarrays, the expression profiles of myogenic precursor cells were determined. Both undifferentiated myoblasts and myoblasts differentiated to myotubes derived from FSHD patients and controls were studied after immunocytochemical verification of the quality of the cultures. To further our understanding of FSHD and normal myogenesis, the expression profiles obtained were compared to those of 19 non-muscle cell types analyzed by identical methods. Results Many of the ~17,000 examined genes were differentially expressed (> 2-fold, p DUX4 RNA isoform was detected by RT-PCR in FSHD myoblast and myotube preparations only at extremely low levels. Unique insights into myogenesis-specific gene expression were also obtained. For example, all four Argonaute genes involved in RNA-silencing were significantly upregulated during normal (but not FSHD myogenesis relative to non-muscle cell types. Conclusions DUX4's pathogenic effect in FSHD may occur transiently at or before the stage of myoblast formation to establish a cascade of gene dysregulation. This contrasts with the current emphasis on toxic effects of experimentally upregulated DUX4 expression at the myoblast or myotube stages. Our model could explain why DUX4's inappropriate expression was barely detectable in myoblasts and myotubes but nonetheless linked to FSHD.

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

    NARCIS (Netherlands)

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

    1997-01-01

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

  9. MHC class II expression and potential antigen-presenting cells in the retina during experimental autoimmune uveitis.

    Science.gov (United States)

    Lipski, Deborah A; Dewispelaere, Rémi; Foucart, Vincent; Caspers, Laure E; Defrance, Matthieu; Bruyns, Catherine; Willermain, François

    2017-07-18

    II-associated antigen presentation and in T cell activation than non-hematopoietic cells. Our results highlight the potential of cells of hematopoietic origin in local antigen presentation, whatever their Ly6C expression. Our work further provides a first transcriptomic study of MHC class II-expressing retinal cells during EAU and delivers a series of new candidate genes possibly implicated in the pathogenesis of retinal autoimmunity.

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

    Science.gov (United States)

    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.

  11. Human Leukocyte Antigen-G (HLA-G) Polymorphism and Expression in Breast Cancer Patients

    OpenAIRE

    Jeong, Seri; Park, Seho; Park, Byeong-Woo; Park, Younhee; Kwon, Oh-Joong; Kim, Hyon-Suk

    2014-01-01

    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 lesio...

  12. Development of stealth transgenes for gene therapy : evaluation of cis-acting inhibitors of antigen presentation

    NARCIS (Netherlands)

    Raamsman-Ossevoort, Martine

    2006-01-01

    In gene therapy, expression of a corrected gene leads to synthesis of proteins foreign to the immune system. Cells expressing these will therefore be recognized as aberrant and destructed. We used a known immune evasion mechanism to “stealth” transgene products. We fused the coding sequence of the

  13. Gene expression analysis identifies global gene dosage sensitivity in cancer

    DEFF Research Database (Denmark)

    Fehrmann, Rudolf S. N.; Karjalainen, Juha M.; Krajewska, Malgorzata

    2015-01-01

    Many cancer-associated somatic copy number alterations (SCNAs) are known. Currently, one of the challenges is to identify the molecular downstream effects of these variants. Although several SCNAs are known to change gene expression levels, it is not clear whether each individual SCNA affects gen...

  14. Expression and purification of neurotoxin-associated protein HA-33/A from Clostridium botulinum and evaluation of its antigenicity.

    Science.gov (United States)

    Sayadmanesh, Ali; Ebrahimi, Firouz; Hajizade, Abbas; Rostamian, Mosayeb; Keshavarz, Hani

    2013-01-01

    Botulinum neurotoxin (BoNT) complexes consist of neurotoxin and neurotoxin-associated proteins. Hemagglutinin-33 (HA-33) is a member of BoNT type A (BoNT/A) complex. Considering the protective role of HA-33 in preservation of BoNT/A in gastrointestinal harsh conditions and also its adjuvant role, recombinant production of this protein is favorable. Thus in this study, HA-33 was expressed and purified, and subsequently its antigenicity in mice was studied. Initially, ha-33 gene sequence of Clostridium botulinum serotype A was adopted from GenBank. The gene sequence was optimized and synthesized in pET28a (+) vector. E. coli BL21 (DE3) strain was transformed by the recombinant vector and the expression of HA-33 was optimized at 37°C and 5 h induction time. The recombinant protein was purified by nickel nitrilotriacetic acid agarose affinity chromatography and confirmed by immunoblotting. Enzyme Linked Immunoassay showed a high titer antibody production in mice. The results indicated a highly expressed and purified recombinant protein, which is able to evoke high antibody titers in mice.

  15. Gene expression analysis of zebrafish heart regeneration.

    Directory of Open Access Journals (Sweden)

    Ching-Ling Lien

    2006-08-01

    Full Text Available Mammalian hearts cannot regenerate. In contrast, zebrafish hearts regenerate even when up to 20% of the ventricle is amputated. The mechanism of zebrafish heart regeneration is not understood. To systematically characterize this process at the molecular level, we generated transcriptional profiles of zebrafish cardiac regeneration by microarray analyses. Distinct gene clusters were identified based on temporal expression patterns. Genes coding for wound response/inflammatory factors, secreted molecules, and matrix metalloproteinases are expressed in regenerating heart in sequential patterns. Comparisons of gene expression profiles between heart and fin regeneration revealed a set of regeneration core molecules as well as tissue-specific factors. The expression patterns of several secreted molecules around the wound suggest that they play important roles in heart regeneration. We found that both platelet-derived growth factor-a and -b (pdgf-a and pdgf-b are upregulated in regenerating zebrafish hearts. PDGF-B homodimers induce DNA synthesis in adult zebrafish cardiomyocytes. In addition, we demonstrate that a chemical inhibitor of PDGF receptor decreases DNA synthesis of cardiomyocytes both in vitro and in vivo during regeneration. Our data indicate that zebrafish heart regeneration is associated with sequentially upregulated wound healing genes and growth factors and suggest that PDGF signaling is required.

  16. Gene expression in early stage cervical cancer

    NARCIS (Netherlands)

    Biewenga, Petra; Buist, Marrije R.; Moerland, Perry D.; van Thernaat, Emiel Ver Loren; van Kampen, Antoine H. C.; ten Kate, Fiebo J. W.; Baas, Frank

    2008-01-01

    Objective. Pelvic lymph node metastases are the main prognostic factor for survival in early stage cervical cancer, yet accurate detection methods before surgery are lacking. In this study, we examined whether gene expression profiling can predict the presence of lymph node metastasis in early stage

  17. Identification of genes showing differential expression profile

    Indian Academy of Sciences (India)

    Suppression subtractive hybridization was used to identify genes showing differential expression profile associated withgrowth rate in skeletal muscle tissue of Landrace weanling pig. Two subtracted cDNA populations were generated from mus-culus longissimus muscle tissues of selected pigs with extreme expected ...

  18. Identification of genes showing differential expression profile ...

    Indian Academy of Sciences (India)

    Abstract. Suppression subtractive hybridization was used to identify genes showing differential expression profile associated with growth rate in skeletal muscle tissue of Landrace weanling pig. Two subtracted cDNA populations were generated from mus- culus longissimus muscle tissues of selected pigs with extreme ...

  19. Gene Expression and Microarray Investigation of Dendrobium ...

    African Journals Online (AJOL)

    diet. The rats were continuously fed for 16 months, and blood glucose monitored by a glucose meter. One wild-type rat and 4 high- fat/high-glucose rats died during ..... therapy not only changed gene expression patterns in type 2 diabetes but also improved immune activity and reduced the likelihood of cancer development.

  20. Genomics analysis of genes expressed reveals differential ...

    African Journals Online (AJOL)

    Genomics analysis of genes expressed reveals differential responses to low chronic nitrogen stress in maize. ... Most induced clones were largely involved in various metabolism processes including physiological process, organelle regulation of biological process, nutrient reservoir activity, transcription regulator activity and ...

  1. Global gene expression in Escherichia coli biofilms

    DEFF Research Database (Denmark)

    Schembri, Mark; Kjærgaard, K.; Klemm, Per

    2003-01-01

    to antimicrobial treatments and host immune defence responses. Escherichia coli has been used as a model organism to study the mechanisms of growth within adhered communities. In this study, we use DNA microarray technology to examine the global gene expression profile of E. coli during sessile growth compared...

  2. Identification of genes showing differential expression profile ...

    Indian Academy of Sciences (India)

    Suppression subtractive hybridization was used to identify genes showing differential expression profile associated withgrowth rate in skeletal muscle tissue of Landrace weanling pig. Two subtracted cDNA populations were generated from mus-culus longissimus muscle tissues of selected pigs with extreme expected ...

  3. Differentially expressed genes in pancreatic ductal adenocarcinomas identified through serial analysis of gene expression

    DEFF Research Database (Denmark)

    Hustinx, Steven R; Cao, Dengfeng; Maitra, Anirban

    2004-01-01

    genome and better biocomputational techniques have substantially improved the assignment of differentially expressed SAGE "tags" to human genes. These improvements have provided us with an opportunity to re-evaluate global gene expression in pancreatic cancer using existing SAGE libraries. SAGE libraries...... generated from six pancreatic cancers were compared to SAGE libraries generated from 11 non-neoplastic tissues. Compared to normal tissue libraries, we identified 453 SAGE tags as differentially expressed in pancreatic cancer, including 395 that mapped to known genes and 58 "uncharacterized" tags....... Of the 395 SAGE tags assigned to known genes, 223 were overexpressed in pancreatic cancer, and 172 were underexpressed. In order to map the 58 uncharacterized differentially expressed SAGE tags to genes, we used a newly developed resource called TAGmapper (http://tagmapper.ibioinformatics.org), to identify...

  4. [Prokaryotic expression and histological localization of the Taenia solium CDC37 gene].

    Science.gov (United States)

    Huang, Jiang; Li, Bo; Dai, Jia-Lin; Zhang, Ai-Hua

    2013-02-01

    To express Taenia solium gene encoding cell division cycle 37 protein (TsCDC37) and investigate its antigenicity and localization in adults of Taenia solium. The complete coding sequence of TsCDC37 was amplified by PCR based on the recombinant plasmid clone from the cDNA library of adult Taenia solium. The PCR product was cloned into a prokaryotic expression vector pET-28a (+). The recombinant expression plasmid was identified by PCR, double endonuclease digestion and sequencing. The recombinant plasmid was transformed into E. coli BL21/DE3 and followed by expression of the protein induced by IPTG. The mice were immunized subcutaneously with purified recombinant TsCDC37 formulated in Freund's adjuvant. The antigenicity of the recombinant protein was examined by Western blotting. The localization of TsCDC37 in adult worms was demonstrated by immunofluorescent technique. The recombinant expression vector was constructed successfully. The recombinant protein was about M(r) 52 000, it was then purified and specifically recognized by immuno sera of SD rats and sera from patients infected with Taenia solium, Taenia saginata or Taenia asiatica. The immunofluorescence assay revealed that TsCDC37 located at the tegument of T. solium adult and the eggs. TsCDC37 gene has been expressed with immunoreactivity. The recombinant protein is mainly expressed in tegument and egg, and is a common antigen of the three human taenia cestodes.

  5. Regulation of methane genes and genome expression

    Energy Technology Data Exchange (ETDEWEB)

    John N. Reeve

    2009-09-09

    At the start of this project, it was known that methanogens were Archaeabacteria (now Archaea) and were therefore predicted to have gene expression and regulatory systems different from Bacteria, but few of the molecular biology details were established. The goals were then to establish the structures and organizations of genes in methanogens, and to develop the genetic technologies needed to investigate and dissect methanogen gene expression and regulation in vivo. By cloning and sequencing, we established the gene and operon structures of all of the “methane” genes that encode the enzymes that catalyze methane biosynthesis from carbon dioxide and hydrogen. This work identified unique sequences in the methane gene that we designated mcrA, that encodes the largest subunit of methyl-coenzyme M reductase, that could be used to identify methanogen DNA and establish methanogen phylogenetic relationships. McrA sequences are now the accepted standard and used extensively as hybridization probes to identify and quantify methanogens in environmental research. With the methane genes in hand, we used northern blot and then later whole-genome microarray hybridization analyses to establish how growth phase and substrate availability regulated methane gene expression in Methanobacterium thermautotrophicus ΔH (now Methanothermobacter thermautotrophicus). Isoenzymes or pairs of functionally equivalent enzymes catalyze several steps in the hydrogen-dependent reduction of carbon dioxide to methane. We established that hydrogen availability determine which of these pairs of methane genes is expressed and therefore which of the alternative enzymes is employed to catalyze methane biosynthesis under different environmental conditions. As were unable to establish a reliable genetic system for M. thermautotrophicus, we developed in vitro transcription as an alternative system to investigate methanogen gene expression and regulation. This led to the discovery that an archaeal protein

  6. Prostate stem cell antigen is expressed in normal and malignant human brain tissues.

    Science.gov (United States)

    Ono, Hiroe; Sakamoto, Hiromi; Yoshida, Teruhiko; Saeki, Norihisa

    2018-03-01

    Prostate stem cell antigen (PSCA) is a glycosylphosphatidylinositol (GPI)-anchored cell surface protein and exhibits an organ-dependent expression pattern in cancer. PSCA is upregulated in prostate cancer and downregulated in gastric cancer. PSCA is expressed in a variety of human organs. Although certain studies previously demonstrated its expression in the mammalian and avian brain, its expression in the human brain has not been thoroughly elucidated. Additionally, it was previously reported that PSCA is weakly expressed in the astrocytes of the normal human brain but aberrantly expressed in glioma, suggesting that PSCA is a promising target of glioma therapy and prostate cancer therapy. The current study identified PSCA expression in the neural and choroid plexus cells of the normal human brain by immunohistochemistry. In brain tumors, PSCA was expressed in medulloblastoma and glioma, and its expression was also observed in papilloma and papillary carcinoma of the choroid plexus, ependymoma and meningioma. The results suggest that PSCA may have a tumor-promoting function in brain tumors and is a potential target for their therapy. However, its expression in normal neuronal and choroid plexus cells implies that a PSCA-targeted therapy may lead to certain adverse phenomena.

  7. Aberrant Gene Expression in Acute Myeloid Leukaemia

    DEFF Research Database (Denmark)

    Bagger, Frederik Otzen

    genes and genetic signatures and for reducing dimensionally of gene expression data. Next, we have used machine-learning methods to predict survival and to assess important predictors based on these results. General application of a number of these methods has been implemented into two public query......Summary Acute Myeloid Leukaemia (AML) is an aggressive cancer of the bone marrow, affecting formation of blood cells during haematopoiesis. This thesis presents investigation of AML using mRNA gene expression profiles (GEP) of samples extracted from the bone marrow of healthy and diseased subjects....... Here GEPs from purified healthy haematopoietic populations, with different levels of differentiation, form the basis for comparison with diseased samples. We present a mathematical transformation of mRNA microarray data to make it possible to compare AML samples, carrying expanded aberrant...

  8. Gene Expression Commons: an open platform for absolute gene expression profiling.

    Directory of Open Access Journals (Sweden)

    Jun Seita

    Full Text Available Gene expression profiling using microarrays has been limited to comparisons of gene expression between small numbers of samples within individual experiments. However, the unknown and variable sensitivities of each probeset have rendered the absolute expression of any given gene nearly impossible to estimate. We have overcome this limitation by using a very large number (>10,000 of varied microarray data as a common reference, so that statistical attributes of each probeset, such as the dynamic range and threshold between low and high expression, can be reliably discovered through meta-analysis. This strategy is implemented in a web-based platform named "Gene Expression Commons" (https://gexc.stanford.edu/ which contains data of 39 distinct highly purified mouse hematopoietic stem/progenitor/differentiated cell populations covering almost the entire hematopoietic system. Since the Gene Expression Commons is designed as an open platform, investigators can explore the expression level of any gene, search by expression patterns of interest, submit their own microarray data, and design their own working models representing biological relationship among samples.

  9. Comparative gene expression of intestinal metabolizing enzymes.

    Science.gov (United States)

    Shin, Ho-Chul; Kim, Hye-Ryoung; Cho, Hee-Jung; Yi, Hee; Cho, Soo-Min; Lee, Dong-Goo; Abd El-Aty, A M; Kim, Jin-Suk; Sun, Duxin; Amidon, Gordon L

    2009-11-01

    The purpose of this study was to compare the expression profiles of drug-metabolizing enzymes in the intestine of mouse, rat and human. Total RNA was isolated from the duodenum and the mRNA expression was measured using Affymetrix GeneChip oligonucleotide arrays. Detected genes from the intestine of mouse, rat and human were ca. 60% of 22690 sequences, 40% of 8739 and 47% of 12559, respectively. Total genes of metabolizing enzymes subjected in this study were 95, 33 and 68 genes in mouse, rat and human, respectively. Of phase I enzymes, the mouse exhibited abundant gene expressions for Cyp3a25, Cyp4v3, Cyp2d26, followed by Cyp2b20, Cyp2c65 and Cyp4f14, whereas, the rat showed higher expression profiles of Cyp3a9, Cyp2b19, Cyp4f1, Cyp17a1, Cyp2d18, Cyp27a1 and Cyp4f6. However, the highly expressed P450 enzymes were CYP3A4, CYP3A5, CYP4F3, CYP2C18, CYP2C9, CYP2D6, CYP3A7, CYP11B1 and CYP2B6 in the human. For phase II enzymes, glucuronosyltransferase Ugt1a6, glutathione S-transferases Gstp1, Gstm3 and Gsta2, sulfotransferase Sult1b1 and acyltransferase Dgat1 were highly expressed in the mouse. The rat revealed predominant expression of glucuronosyltransferases Ugt1a1 and Ugt1a7, sulfotransferase Sult1b1, acetyltransferase Dlat and acyltransferase Dgat1. On the other hand, in human, glucuronosyltransferases UGT2B15 and UGT2B17, glutathione S-transferases MGST3, GSTP1, GSTA2 and GSTM4, sulfotransferases ST1A3 and SULT1A2, acetyltransferases SAT1 and CRAT, and acyltransferase AGPAT2 were dominantly detected. Therefore, current data indicated substantial interspecies differences in the pattern of intestinal gene expression both for P450 enzymes and phase II drug-metabolizing enzymes. This genomic database is expected to improve our understanding of interspecies variations in estimating intestinal prehepatic clearance of oral drugs.

  10. Genetic analysis of the capsule polysaccharide (K antigen and exopolysaccharide genes in pandemic Vibrio parahaemolyticus O3:K6

    Directory of Open Access Journals (Sweden)

    Morris J Glenn

    2010-11-01

    Full Text Available Abstract Background Pandemic Vibrio parahaemolyticus has undergone rapid changes in both K- and O-antigens, making detection of outbreaks more difficult. In order to understand these rapid changes, the genetic regions encoding these antigens must be examined. In Vibrio cholerae and Vibrio vulnificus, both O-antigen and capsular polysaccharides are encoded in a single region on the large chromosome; a similar arrangement in pandemic V. parahaemolyticus would help explain the rapid serotype changes. However, previous reports on "capsule" genes are controversial. Therefore, we set out to clarify and characterize these regions in pandemic V. parahaemolyticus O3:K6 by gene deletion using a chitin based transformation strategy. Results We generated different deletion mutants of putative polysaccharide genes and examined the mutants by immuno-blots with O and K specific antisera. Our results showed that O- and K-antigen genes are separated in V. parahaemolyticus O3:K6; the region encoding both O-antigen and capsule biosynthesis in other vibrios, i.e. genes between gmhD and rjg, determines the K6-antigen but not the O3-antigen in V. parahaemolyticus. The previously identified "capsule genes" on the smaller chromosome were related to exopolysaccharide synthesis, not K-antigen. Conclusion Understanding of the genetic basis of O- and K-antigens is critical to understanding the rapid changes in these polysaccharides seen in pandemic V. parahaemolyticus. This report confirms the genetic location of K-antigen synthesis in V. parahaemolyticus O3:K6 allowing us to focus future studies of the evolution of serotypes to this region.

  11. Epigenetic regulation of inducible gene expression in the immune system.

    Science.gov (United States)

    Lim, Pek Siew; Li, Jasmine; Holloway, Adele F; Rao, Sudha

    2013-07-01

    T cells are exquisitely poised to respond rapidly to pathogens and have proved an instructive model for exploring the regulation of inducible genes. Individual genes respond to antigenic stimulation in different ways, and it has become clear that the interplay between transcription factors and the chromatin platform of individual genes governs these responses. Our understanding of the complexity of the chromatin platform and the epigenetic mechanisms that contribute to transcriptional control has expanded dramatically in recent years. These mechanisms include the presence/absence of histone modification marks, which form an epigenetic signature to mark active or inactive genes. These signatures are dynamically added or removed by epigenetic enzymes, comprising an array of histone-modifying enzymes, including the more recently recognized chromatin-associated signalling kinases. In addition, chromatin-remodelling complexes physically alter the chromatin structure to regulate chromatin accessibility to transcriptional regulatory factors. The advent of genome-wide technologies has enabled characterization of the chromatin landscape of T cells in terms of histone occupancy, histone modification patterns and transcription factor association with specific genomic regulatory regions, generating a picture of the T-cell epigenome. Here, we discuss the multi-layered regulation of inducible gene expression in the immune system, focusing on the interplay between transcription factors, and the T-cell epigenome, including the role played by chromatin remodellers and epigenetic enzymes. We will also use IL2, a key inducible cytokine gene in T cells, as an example of how the different layers of epigenetic mechanisms regulate immune responsive genes during T-cell activation. © 2013 John Wiley & Sons Ltd.

  12. [Expression of MAGE, GAGE, and BAGE genes in human hepatocellular carcinoma].

    Science.gov (United States)

    Shao, Jun-bin; Chen, Zhi

    2003-03-01

    To observe the expression of MAGE, GAGE and BAGE genes in human hepatocellular carcinoma (HCC) cell lines. The expression levels of MAGE-1, MAGE-3, GAGE1-8, GAGE1-2 and BAGE mRNAs in HCC cell lines SMMC-7721, QQY-7701, BEL-7402 were studied by reverse transcription polymerase chain reaction and were compared with those in biopsied liver tissues. MAGE-1 and BAGE mRNAs were expressed in SMMC-7721 cells. MAGE-3 and BAGE mRNAs were expressed in QQY-7701 cells. MAGE-1 and GAGE1-2 mRNAs were expressed in BEL-7402 cells. None of these genes was expressed in biopsied liver tissues. MAGE-1, MAGE-3, GAGE1-8, GAGE1-2 and BAGE are expressed in hepatocellular carcinoma cell lines. These tumor-specific antigens can be used as molecular markers for early diagnosis and possible targets for immunotherapy of human HCC.

  13. 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

  14. A novel tumor-associated antigen expressed in human uterine and ovarian carcinomas.

    Science.gov (United States)

    Sonoda, K; Nakashima, M; Kaku, T; Kamura, T; Nakano, H; Watanabe, T

    1996-04-15

    A large number of monoclonal antibodies (MoAbs) against human tumor cells have been generated and it has been shown that these MoAbs are useful tools in the diagnosis and treatment of cancer patients, as well as in the basic investigation of the oncogenesis and characterization of cancer cells. The 22-1-1 MoAb was established by cell fusion between mouse myeloma cells and spleen cells derived from mice immunized with the human uterine cervical adenocarcinoma cell line, SiSo. The tissue distribution and biologic characteristics of the 22-1-1 antigen (Ag) were examined. The 22-1-1 Ag was distinct from the known tumor-associated antigens such as YH 206, GA 733, CA 125, carcinoembryonic antigen, and sialyl Le(x) molecules in an expression pattern in human tumor cell lines. An immunohistochemical study revealed that 22-1-1 Ag was expressed in 87.5% of uterine cervical adenocarcinomas, 66% of uterine endometrial adenocarcinomas, and 58.8% of ovarian carcinomas. Moreover, 22-1-1 Ag was detected in 87.7% of uterine cervical squamous cell carcinomas; however, it was not detected in 87.7% of uterine cervical or ovarian tissues, except in uterine endometrial glands, in which its expression was observed at low levels. The 22-1-1 Ag was secreted into cell culture supernatant fluids and was also detected in the vaginal discharges of uterine cervical carcinoma patients. The antigenic epitope of 22-1-1 Ag was shown to be a protein with a molecular weight of 78 kilodaltons using sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The 22-1-1 MoAb reactive to a novel tumor-associated antigen was generated. This Ag was expressed in cancer cells derived mainly from the uterus and ovary. Moreover, 22-1-1 Ag was associated in the vaginal discharges of uterine cervical carcinoma patients. 22-1-1 MoAb is a potential tool for the study of oncogenesis and the management of cancer patients.

  15. Gene expression profiling of laterally spreading tumors.

    Science.gov (United States)

    Minemura, Shoko; Tanaka, Takeshi; Arai, Makoto; Okimoto, Kenichiro; Oyamada, Arata; Saito, Keiko; Maruoka, Daisuke; Matsumura, Tomoaki; Nakagawa, Tomoo; Katsuno, Tatsuro; Kishimoto, Takashi; Yokosuka, Osamu

    2015-06-06

    Laterally spreading tumors (LSTs) are generally defined as lesions >10 mm in diameter, are characterized by lateral expansion along the luminal wall with a low vertical axis. In contrast to other forms of tumor, LSTs are generally considered to have a superficial growth pattern and the potential for malignancy. We focused on this morphological character of LSTs, and analyzed the gene expression profile of LSTs. The expression of 168 genes in 41 colorectal tumor samples (17 LST-adenoma, 12 LST-carcinoma, 12 Ip [pedunculated type of the Paris classification)-adenoma, all of which were 10 mm or more in diameter] was analyzed by PCR array. Based on the results, we investigated the expression levels of genes up-regulated in LST-adenoma, compared to Ip-adenoma, by hierarchical and K-means clustering. To confirm the results of the array analysis, using an additional 60 samples (38 LST-adenoma, 22 Ip-adenoma), we determined the localization of the gene product by immunohistochemical staining. The expression of 129 genes differed in colorectal tumors from normal mucosa by PCR array analysis. As a result of K-means clustering, the expression levels of five genes, AKT1, BCL2L1, ERBB2, MTA2 and TNFRSF25, were found to be significantly up-regulated (p < 0.05) in LST-adenoma, compared to Ip-adenoma. Immunohistochemical analysis showed that the BCL2L1 protein was significantly and meaningfully up-regulated in LST-adenoma compared to Ip-adenoma (p = 0.010). With respect to apoptosis status in LST-Adenoma, it assumes that BCL2L1 is anti-apoptotic protein, the samples such as BCL2L1 positive and TUNEL negative, or BCL2L1 negative and TUNEL positive are consistent with the assumption. 63.2 % LST-adenoma samples were consistent with the assumption. LSTs have an unusual profile of gene expression compared to other tumors and BCL2L1 might be concerned in the organization of LSTs.

  16. Differential gene expression profiling in blood from patients with digestive system cancers.

    Science.gov (United States)

    Honda, Masao; Sakai, Yoshio; Yamashita, Taro; Yamashita, Tatsuya; Sakai, Akito; Mizukoshi, Eishiro; Nakamoto, Yasunari; Tatsumi, Isamu; Miyazaki, Yoshitaka; Tanno, Hiroshi; Kaneko, Shuichi

    2010-09-10

    To develop a non-invasive and sensitive diagnostic test for cancer using peripheral blood, we evaluated gene expression profiling of blood obtained from patients with cancer of the digestive system and normal subjects. The expression profiles of blood-derived total RNA obtained from 39 cancer patients (11 colon cancer, 14 gastric cancer, and 14 pancreatic cancer) was clearly different from those obtained from 15 normal subjects. By comparing the gene expression profiles of cancer patients and normal subjects, 25 cancer-differentiating genes (p3) were identified and an "expression index" deduced from the expression values of these genes differentiated the validation cohort (11 colon cancer, 8 gastric cancer, 18 pancreatic cancer, and 15 normal subjects) into cancer patients and normal subjects with 100% (37/37) and 87% (13/15) accuracy, respectively. Although, the expression profiles were not clearly different between the cancer patients, some characteristic genes were identified according to the stage and species of the cancer. Interestingly, many immune-related genes such as antigen presenting, cell cycle accelerating, and apoptosis- and stress-inducing genes were up-regulated in cancer patients, reflecting the active turnover of immune regulatory cells in cancer patients. These results showed the potential relevance of peripheral blood gene expression profiling for the development of new diagnostic examination tools for cancer patients. Copyright © 2010 Elsevier Inc. All rights reserved.

  17. Gene expression profiling in autoimmune diseases

    DEFF Research Database (Denmark)

    Bovin, Lone Frier; Brynskov, Jørn; Hegedüs, Laszlo

    2007-01-01

    ) patients and healthy individuals were specific for the arthritic process or likewise altered in other chronic inflammatory diseases such as chronic autoimmune thyroiditis (Hashimoto's thyroiditis, HT) and inflammatory bowel disease (IBD). Using qPCR for 18 RA-discriminative genes, there were no significant......A central issue in autoimmune disease is whether the underlying inflammation is a repeated stereotypical process or whether disease specific gene expression is involved. To shed light on this, we analysed whether genes previously found to be differentially regulated in rheumatoid arthritis (RA...... immunoinflammatory diseases, but only if accompanied by pronounced systemic manifestations. This suggests that at least some of the genes activated in RA are predominantly or solely related to general and disease-nonspecific autoimmune processes...

  18. [The role of miR-492 in the regulation of OK blood group antigen expression on red blood cells].

    Science.gov (United States)

    Ye, Luyi; Wang, Chen; Yang, Qixiu; Zhu, Ziyan

    2017-10-10

    To investigate whether miR-492 is involved in the post-transcriptional regulation of OK blood group antigen expression on red blood cells. Two 3'-UTR fragments of the BSG gene were synthesized with a chemical method, which respectively encompassed the BSG rs8259 TT or BSG rs8259 AA sites. The fragments were added with Xho I and Not I restriction enzyme cutting sites at both ends and cloned into a pUC57 vector, which in turn was constructed into a psiCHECK-2 vector and verified by sequencing. K562 cells were transfected with various combinations of miR-492 mimic and constructed psiCHECK2-BSG-T or psiCHECK2-BSG-A recombinant plasmid. A blank control group was set up. Each transfection experiment was repeated three times. The activity of Renilla reniformis luciferase was determined and normalized with that of firefly luciferase, and detected with a dual-luciferase reporter assay system. The data were subjected to statistical analysis. The sequencing results confirmed that the recombinant psiCHECK2 plasmids containing the BSG rs8259 TT or rs8259 AA sites were constructed successfully. The results of dual-luciferase report gene detection showed that the miR-492 mimic could significantly inhibit psiCHECK2-BSG-T at a concentration over 100 nmol/L. However, it could not inhibit psiCHECK-BSG-A. miR-492 may be involved in the regulation of OK antigen expression on red blood cells with the BSG rs8259 TT genotype.

  19. Coevolution of gene expression among interacting proteins

    Energy Technology Data Exchange (ETDEWEB)

    Fraser, Hunter B.; Hirsh, Aaron E.; Wall, Dennis P.; Eisen,Michael B.

    2004-03-01

    Physically interacting proteins or parts of proteins are expected to evolve in a coordinated manner that preserves proper interactions. Such coevolution at the amino acid-sequence level is well documented and has been used to predict interacting proteins, domains, and amino acids. Interacting proteins are also often precisely coexpressed with one another, presumably to maintain proper stoichiometry among interacting components. Here, we show that the expression levels of physically interacting proteins coevolve. We estimate average expression levels of genes from four closely related fungi of the genus Saccharomyces using the codon adaptation index and show that expression levels of interacting proteins exhibit coordinated changes in these different species. We find that this coevolution of expression is a more powerful predictor of physical interaction than is coevolution of amino acid sequence. These results demonstrate previously uncharacterized coevolution of gene expression, adding a different dimension to the study of the coevolution of interacting proteins and underscoring the importance of maintaining coexpression of interacting proteins over evolutionary time. Our results also suggest that expression coevolution can be used for computational prediction of protein protein interactions.

  20. Immunohistochemical antigenic expression and in vivo tumor uptake of monoclonal antibodies with specificity for tumors of the gastrointestinal tract

    International Nuclear Information System (INIS)

    Douillard, J.Y.; Lehur, P.A.; Aillet, G.; Kremer, M.; Bianco-Arco, A.; Peltier, P.; Chatal, J.F.

    1986-01-01

    Two monoclonal antibodies with specificity for carcinoembryonic antigen and Ca 19-9 gastrointestinal tract tumor associated antigens were infused after iodination with 125 I and 131 I, respectively, in six patients 3 days and in one patient 4 days before radical surgery for colon or rectal carcinoma. Biopsy specimens from tumor, normal colon, fat, muscle, and skin along with a blood sample were excised at surgery and counting was performed for gamma emission. Fragments were then studied by two independent pathologists for immunohistochemical expression of corresponding antigens using the avidin-biotin peroxidase complex. A correlation study was thereafter performed between the amount of antibody bound in vivo, expressed as the percentage of injected dose per gram of tissue and the quantitative expression of tumor associated antigens, taking into account both the percentage of cells expressing the antigen and intensity of staining. For this limited number of patients a good correlation was found between amount of targeted antibodies and amount of expressed antigens. For carcinoembryonic antigen, r values were 0.69 and 0.90 for each pathologist (with an r value of interobserver correlation of 0.74); for Ca 19-9, values of 0.78 and 0.84 were obtained for each observer, with an interobserver r value of 0.97. Based on this limited study, it may be assumed that the possibility of imaging a given tumor is in part correlated to intensity of antigenic expression at the tumor site; other parameters, like tumor vascularization and blood flow for instance, are, however, to be considered for accessibility of antibodies to corresponding antigens

  1. 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.

  2. Digital gene expression analysis of gene expression differences within Brassica diploids and allopolyploids.

    Science.gov (United States)

    Jiang, Jinjin; Wang, Yue; Zhu, Bao; Fang, Tingting; Fang, Yujie; Wang, Youping

    2015-01-27

    Brassica includes many successfully cultivated crop species of polyploid origin, either by ancestral genome triplication or by hybridization between two diploid progenitors, displaying complex repetitive sequences and transposons. The U's triangle, which consists of three diploids and three amphidiploids, is optimal for the analysis of complicated genomes after polyploidization. Next-generation sequencing enables the transcriptome profiling of polyploids on a global scale. We examined the gene expression patterns of three diploids (Brassica rapa, B. nigra, and B. oleracea) and three amphidiploids (B. napus, B. juncea, and B. carinata) via digital gene expression analysis. In total, the libraries generated between 5.7 and 6.1 million raw reads, and the clean tags of each library were mapped to 18547-21995 genes of B. rapa genome. The unambiguous tag-mapped genes in the libraries were compared. Moreover, the majority of differentially expressed genes (DEGs) were explored among diploids as well as between diploids and amphidiploids. Gene ontological analysis was performed to functionally categorize these DEGs into different classes. The Kyoto Encyclopedia of Genes and Genomes analysis was performed to assign these DEGs into approximately 120 pathways, among which the metabolic pathway, biosynthesis of secondary metabolites, and peroxisomal pathway were enriched. The non-additive genes in Brassica amphidiploids were analyzed, and the results indicated that orthologous genes in polyploids are frequently expressed in a non-additive pattern. Methyltransferase genes showed differential expression pattern in Brassica species. Our results provided an understanding of the transcriptome complexity of natural Brassica species. The gene expression changes in diploids and allopolyploids may help elucidate the morphological and physiological differences among Brassica species.

  3. Predicting gene expression from sequence: a reexamination.

    Directory of Open Access Journals (Sweden)

    Yuan Yuan

    2007-11-01

    Full Text Available Although much of the information regarding genes' expressions is encoded in the genome, deciphering such information has been very challenging. We reexamined Beer and Tavazoie's (BT approach to predict mRNA expression patterns of 2,587 genes in Saccharomyces cerevisiae from the information in their respective promoter sequences. Instead of fitting complex Bayesian network models, we trained naïve Bayes classifiers using only the sequence-motif matching scores provided by BT. Our simple models correctly predict expression patterns for 79% of the genes, based on the same criterion and the same cross-validation (CV procedure as BT, which compares favorably to the 73% accuracy of BT. The fact that our approach did not use position and orientation information of the predicted binding sites but achieved a higher prediction accuracy, motivated us to investigate a few biological predictions made by BT. We found that some of their predictions, especially those related to motif orientations and positions, are at best circumstantial. For example, the combinatorial rules suggested by BT for the PAC and RRPE motifs are not unique to the cluster of genes from which the predictive model was inferred, and there are simpler rules that are statistically more significant than BT's ones. We also show that CV procedure used by BT to estimate their method's prediction accuracy is inappropriate and may have overestimated the prediction accuracy by about 10%.

  4. Gene expression regulation in roots under drought.

    Science.gov (United States)

    Janiak, Agnieszka; Kwaśniewski, Mirosław; Szarejko, Iwona

    2016-02-01

    Stress signalling and regulatory networks controlling expression of target genes are the basis of plant response to drought. Roots are the first organs exposed to water deficiency in the soil and are the place of drought sensing. Signalling cascades transfer chemical signals toward the shoot and initiate molecular responses that lead to the biochemical and morphological changes that allow plants to be protected against water loss and to tolerate stress conditions. Here, we present an overview of signalling network and gene expression regulation pathways that are actively induced in roots under drought stress. In particular, the role of several transcription factor (TF) families, including DREB, AP2/ERF, NAC, bZIP, MYC, CAMTA, Alfin-like and Q-type ZFP, in the regulation of root response to drought are highlighted. The information provided includes available data on mutual interactions between these TFs together with their regulation by plant hormones and other signalling molecules. The most significant downstream target genes and molecular processes that are controlled by the regulatory factors are given. These data are also coupled with information about the influence of the described regulatory networks on root traits and root development which may translate to enhanced drought tolerance. This is the first literature survey demonstrating the gene expression regulatory machinery that is induced by drought stress, presented from the perspective of roots. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  5. PiggyBac-mediated cancer immunotherapy using EBV-specific cytotoxic T-cells expressing HER2-specific chimeric antigen receptor.

    Science.gov (United States)

    Nakazawa, Yozo; Huye, Leslie E; Salsman, Vita S; Leen, Ann M; Ahmed, Nabil; Rollins, Lisa; Dotti, Gianpietro; Gottschalk, Stephen M; Wilson, Matthew H; Rooney, Cliona M

    2011-12-01

    Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTLs) can be modified to function as heterologous tumor directed effector cells that survive longer in vivo than tumor directed T cells without virus specificity, due to chronic stimulation by viral antigens expressed during persistent infection in seropositive individuals. We evaluated the nonviral piggyBac (PB) transposon system as a platform for modifying EBV-CTLs to express a functional human epidermal growth factor receptor 2-specific chimeric antigen receptor (HER2-CAR) thereby directing virus-specific, gene modified CTLs towards HER2-positive cancer cells. Peripheral blood mononuclear cells (PBMCs) were nucleofected with transposons encoding a HER2-CAR and a truncated CD19 molecule for selection followed by specific activation and expansion of EBV-CTLs. HER2-CAR was expressed in ~40% of T cells after CD19 selection with retention of immunophenotype, polyclonality, and function. HER2-CAR-modified EBV-CTLs (HER2-CTLs) killed HER2-positive brain tumor cell lines in vitro, exhibited transient and reversible increases in HER2-CAR expression following antigen-specific stimulation, and stably expressed HER2-CAR beyond 120 days. Adoptive transfer of PB-modified HER2-CTLs resulted in tumor regression in a murine xenograft model. Our results demonstrate that PB can be used to redirect virus-specific CTLs to tumor targets, which should prolong tumor-specific T cell survival in vivo producing more efficacious immunotherapy.

  6. Use of serial analysis of gene expression to reveal the specific regulation of gene expression profile in asthmatic rats treated by acupuncture.

    Science.gov (United States)

    Yin, Lei-Miao; Jiang, Gong-Hao; Wang, Yu; Wang, Yan; Liu, Yan-Yan; Jin, Wei-Rong; Xu, Yu-Dong; Zhang, Qing-Hua; Yang, Yong-Qing

    2009-05-06

    Asthma has become an important public health issue and approximately 300 million people have suffered from the disease worldwide. Nowadays, the use of acupuncture in asthma is increasing. This study intended to systematically analyze and compare the gene expression profiles between the asthmatic and acupuncture-treated asthmatic rat lung, and tried to gain insight into the molecular mechanism underlying the early airway response (EAR) phase of asthma treated by acupuncture. Four tag libraries of serial analysis of gene expression (SAGE) were established from lung tissues of control rats (CK), asthmatic rats (AS), asthmatic rats treated by acupuncture (ASAC), and control rats treated by acupuncture (CKAC). Bioinformatic analyses were carried out by using the methods including unsupervised hierarchical clustering, functional annotation tool of the database for annotation, visualization, and integrated discovery (DAVID), gene ontology (GO) tree machine, and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis. There were totally 186 differentially expressed tags (P SAGE clustering. By comparison of P(CK/AS) and P(AS/ASAC), the DAVID genes functional classification was found to be changed from "immune response" to "response to steroid hormone stimulus", and the GO term "antigen processing and presentation of peptide antigen" disappeared in P(AS/ASAC). Totally 3 same KEGG pathways were found among the three groups. Moreover, 21 specific tags of the acupuncture in treating asthma were detected using Venn diagrams. Our SAGE research indicates that the gene expression profile of the EAR phase of asthma could be effectively and specifically regulated by acupuncture, which suggests that the gene expression of immune response and steroid hormone may play an important role in the treatment.

  7. [Transformation activity and antigenicity of the human papillomavirus type 58 E6E7 fusion gene mutant].

    Science.gov (United States)

    Wang, He; Yu, Ji-yun; Li, Li

    2013-07-01

    To develop a prophylactic and therapeutic vaccine against human papillomavirus (HPV) type 58-associated cervical carcinoma, and explore its transformation activity and antigenicity. The E6 and E7 three amino acid codons in the HPV 58 virus were modified respectively and fused. The modified and fused gene was named HPV58 mE6E7. The recombinant HPV58 mE6E7 gene was inserted into pIRES-neo vector to generate plasmid pIRES-neo-HPV58 mE6E7. Then NIH/3T3 cell line was transfected with plasmid pIRES-neo-HPV58 mE6E7. The pIRES-neo-HPV58 mE6E7-transfected cells were the experimental group, pIRES-neo-HPV58 E6E7-transfected cells were the positive control group, and pIRES-neo empty vector-transfected cells were the negative control group. The expression of HPV58 mE6E7 protein in the experimental cells was detected by flow cytometry, immunofluorescence and Western blot. The transformation activity of HPV58 mE6E7 was tested by soft agar colony formation assay and subcutaneously tumors in nude mice. Finally, DNA vaccine was constructed with HPV58 mE6E7 fusion antigen and used to immunize C57BL/6 mice with the vaccine plasmids. The specific serum antibodies were detected by EIISA, and the number of splenic specific CD8(+) T cells secreting IFN-γ of the immunized mice was detected by ELISPOT assay. Sequencing confirmed the expected mutation and a 100% homogeneity of the HPV58 E6E7 fusion gene. Stable transfected NIH/3T3 cells expressing HPV58 mE6E7 and HPV58 E6E7 gene were 70.3% and 84.1%, respectively. The relative expressions of HPV58 mE6E7 and HPV58 E6E7 fusion protein in 3T3-HPV58 mE6E7 experimental cells and 3T3-HPV58 E6E7 positive control cells were 2.1 ± 1.7 and 3.8 ± 1.4, respectively, and were negative in the negative control group. No colony formation was found in the experimental and 3T3-neo negative control cell groups, and 31 colonies were found in the positive control cell group, among them 10 colonies were consisted of more than 50 cells. No tumor mass was formed

  8. Gene expression profiles in skeletal muscle after gene electrotransfer

    Directory of Open Access Journals (Sweden)

    Eriksen Jens

    2007-06-01

    Full Text Available Abstract Background Gene transfer by electroporation (DNA electrotransfer to muscle results in high level long term transgenic expression, showing great promise for treatment of e.g. protein deficiency syndromes. However little is known about the effects of DNA electrotransfer on muscle fibres. We have therefore investigated transcriptional changes through gene expression profile analyses, morphological changes by histological analysis, and physiological changes by force generation measurements. DNA electrotransfer was obtained using a combination of a short high voltage pulse (HV, 1000 V/cm, 100 μs followed by a long low voltage pulse (LV, 100 V/cm, 400 ms; a pulse combination optimised for efficient and safe gene transfer. Muscles were transfected with green fluorescent protein (GFP and excised at 4 hours, 48 hours or 3 weeks after treatment. Results Differentially expressed genes were investigated by microarray analysis, and descriptive statistics were performed to evaluate the effects of 1 electroporation, 2 DNA injection, and 3 time after treatment. The biological significance of the results was assessed by gene annotation and supervised cluster analysis. Generally, electroporation caused down-regulation of structural proteins e.g. sarcospan and catalytic enzymes. Injection of DNA induced down-regulation of intracellular transport proteins e.g. sentrin. The effects on muscle fibres were transient as the expression profiles 3 weeks after treatment were closely related with the control muscles. Most interestingly, no changes in the expression of proteins involved in inflammatory responses or muscle regeneration was detected, indicating limited muscle damage and regeneration. Histological analysis revealed structural changes with loss of cell integrity and striation pattern in some fibres after DNA+HV+LV treatment, while HV+LV pulses alone showed preservation of cell integrity. No difference in the force generation capacity was observed in

  9. Expression of 5'-nucleotidase (CD73) related to other differentiation antigens in leukemias of B-cell lineage

    NARCIS (Netherlands)

    Pieters, R.; Thompson, L. F.; Broekema, G. J.; Huismans, D. R.; Peters, G. J.; Pals, S. T.; Horst, E.; Hählen, K.; Veerman, A. J.

    1991-01-01

    Ecto-5'nucleotidase (5'NT; CD73) expression was studied with a monoclonal antibody (7G2) and a radiochemical assay and compared with the expression of other antigens in B-cell-lineage leukemias on cells from 100 leukemic patients and two cell lines. A B-cell origin was confirmed by the expression of

  10. Prognostic impact of programmed death-ligand 1 expression in correlation with human leukocyte antigen class I expression status in stage I adenocarcinoma of the lung.

    Science.gov (United States)

    Hirai, Ayako; Yoneda, Kazue; Shimajiri, Shohei; Kuroda, Koji; Hanagiri, Takeshi; Fujino, Yoshihisa; Tanaka, Fumihiro

    2018-01-01

    The study objective was to investigate the prognostic impact of programmed death-ligand 1 expression in correlation with human leukocyte antigen class I expression on tumor cells in early-stage adenocarcinoma of the lung, because both programmed death-ligand 1 and human leukocyte antigen class I molecules play important roles in cancer immunity. Ninety-four patients with completely resected pathologic stage I lung adenocarcinoma were retrospectively reviewed. Programmed death-ligand 1 expression on tumor cells was evaluated with immunohistochemistry in correlation with several clinicopathologic and molecular features, including human leukocyte antigen class I expression on tumor cells. Fifteen patients (16.0%) had tumor with positive programmed death-ligand 1 expression (percentage of tumor cells expressing programmed death-ligand 1, ≥5%), and the incidence was significantly higher in poorly differentiated tumors. There was no significant correlation between human leukocyte antigen class I expression and programmed death-ligand 1 expression. Programmed death-ligand 1 positivity was a significant factor to predict a poor survival (5-year survival, 66.7% vs 85.9%; P = .049), which was enhanced in tumors with normal human leukocyte antigen class I expression (P = .029) but was not evident in tumors with reduced human leukocyte antigen class I expression (P = .552). The prognostic impact of programmed death-ligand 1 expression on tumor cells in early-stage lung adenocarcinoma may be distinct according to concurrent human leukocyte antigen class I expression. Copyright © 2017 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.

  11. Monitoring the Efficacy of Oncolytic Viruses via Gene Expression

    Directory of Open Access Journals (Sweden)

    Ashley Ansel

    2017-11-01

    Full Text Available With the recent success of oncolytic viruses in clinical trials, efforts toward improved monitoring of the viruses and their mechanism have intensified. Four main gene expression strategies have been employed to date including: analyzing overall gene expression in tumor cells, looking at gene expression of a few specific genes in the tumor cells, focusing on gene expression of specific transgenes introduced into the virus, and following gene expression of certain viral genes. Each strategy presents certain advantages and disadvantages over the others. Various methods to organize the dysregulated genes into clusters have provided a window into the mechanism of action for these viruses. Methodologically, the combined approach of looking at both overall gene expression, the tumor cells and gene expression of viral genes, enables researchers to assess correlation between the introduction of the virus and the changes in the tumor. This would seem to be the most productive approach for future studies, providing much information on mechanism and timing.

  12. 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.

  13. DNA vaccine constructs expressing Mycobacterium tuberculosis-specific genes induce immune responses.

    Science.gov (United States)

    Hanif, S N M; Al-Attiyah, R; Mustafa, A S

    2010-11-01

    RD1 PE35, PPE68, EsxA, EsxB and RD9 EsxV genes are present in Mycobacterium tuberculosis genome but deleted in Mycobacterium bovis BCG. The aim of this study was to clone these genes into DNA vaccine vectors capable of expressing them in eukaryotic cells as fusion proteins, fused with immunostimulatory signal peptides of human interleukin-2 (hIL-2) and tissue plasminogen activator (tPA), and evaluate the recombinant DNA vaccine constructs for induction of antigen-specific cellular immune responses in mice. DNA corresponding to the aforementioned RD1 and RD9 genes was cloned into DNA vaccine plasmid vectors pUMVC6 and pUMVC7 (with hIL-2 and tPA signal peptides, respectively), and a total of 10 recombinant DNA vaccine constructs were obtained. BALB/c mice were immunized with the parent and recombinant plasmids and their spleen cells were tested for antigen-induced proliferation with antigens of M. tuberculosis and pure proteins corresponding to the cloned genes. The results showed that antigen-specific proliferation responses were observed for a given antigen only with spleen cells of mice immunized with the homologous recombinant DNA vaccine construct. The mice immunized with the parent plasmids did not show positive immune responses to any of the antigens of the cloned genes. The ability of the DNA vaccine constructs to elicit cellular immune responses makes them an attractive weapon as a safer vaccine candidate for preventive and therapeutic applications against tuberculosis. © 2010 The Authors. Scandinavian Journal of Immunology © 2010 Blackwell Publishing Ltd.

  14. Regulation of antigenic variation in Plasmodium falciparum: censoring freedom of expression?

    Science.gov (United States)

    Duffy, Michael F; Reeder, John C; Brown, Graham V

    2003-03-01

    Plasmodium falciparum employs a strategy of clonal antigenic variation to evade the host immune response during the intraerythrocytic stage of its life cycle. The major variant parasite molecule is the P. falciparum erythrocyte membrane protein (PfEMP)1, which is encoded by the var multigene family. The parasite switches between different PfEMP1 molecules through regulation of var transcription. Recent studies have shed considerable light on this process, but much remains unknown. However, striking parallels between transcriptional control of var and genes in other organisms provide direction for future studies.

  15. Enhanced gene expression from retroviral vectors

    Directory of Open Access Journals (Sweden)

    Micklem David R

    2008-02-01

    Full Text Available Abstract Background Retroviruses are widely used to transfer genes to mammalian cells efficiently and stably. However, genetic elements required for high-level gene expression are incompatible with standard systems. The retroviral RNA genome is produced by cellular transcription and post-transcriptional processing within packaging cells: Introns present in the retroviral genomic transcript are removed by splicing, while polyadenylation signals lead to the production of ineffective truncated genomes. Furthermore strong enhancer/promoters within the retroviral payload lead to detrimental competition with the retroviral enhancer/promoter. Results By exploiting a new method of producing the retroviral genome in vitro it is possible to produce infectious retroviral particles carrying a high-level expression cassette that completely prohibits production of infectious retroviral particles by conventional methods. We produced an expression cassette comprising a strong enhancer/promoter, an optimised intron, the GFP open reading frame and a strong polyadenylation signal. This cassette was cloned into both a conventional MMLV retroviral vector and a vector designed to allow in vitro transcription of the retroviral genome by T7 RNA polymerase. When the conventional retroviral vector was transfected into packaging cells, the expression cassette drove strong GFP expression, but no infectious retrovirus was produced. Introduction of the in vitro produced uncapped retroviral genomic transcript into the packaging cells did not lead to any detectable GFP expression. However, infectious retrovirus was easily recovered, and when used to infect target primary human cells led to very high GFP expression – up to 3.5 times greater than conventional retroviral LTR-driven expression. Conclusion Retroviral vectors carrying an optimized high-level expression cassette do not produce infectious virions when introduced into packaging cells by transfection of DNA

  16. Gene expression in Pseudomonas aeruginosa swarming motility

    Directory of Open Access Journals (Sweden)

    Déziel Eric

    2010-10-01

    Full Text Available Abstract Background The bacterium Pseudomonas aeruginosa is capable of three types of motilities: swimming, twitching and swarming. The latter is characterized by a fast and coordinated group movement over a semi-solid surface resulting from intercellular interactions and morphological differentiation. A striking feature of swarming motility is the complex fractal-like patterns displayed by migrating bacteria while they move away from their inoculation point. This type of group behaviour is still poorly understood and its characterization provides important information on bacterial structured communities such as biofilms. Using GeneChip® Affymetrix microarrays, we obtained the transcriptomic profiles of both bacterial populations located at the tip of migrating tendrils and swarm center of swarming colonies and compared these profiles to that of a bacterial control population grown on the same media but solidified to not allow swarming motility. Results Microarray raw data were corrected for background noise with the RMA algorithm and quantile normalized. Differentially expressed genes between the three conditions were selected using a threshold of 1.5 log2-fold, which gave a total of 378 selected genes (6.3% of the predicted open reading frames of strain PA14. Major shifts in gene expression patterns are observed in each growth conditions, highlighting the presence of distinct bacterial subpopulations within a swarming colony (tendril tips vs. swarm center. Unexpectedly, microarrays expression data reveal that a minority of genes are up-regulated in tendril tip populations. Among them, we found energy metabolism, ribosomal protein and transport of small molecules related genes. On the other hand, many well-known virulence factors genes were globally repressed in tendril tip cells. Swarm center cells are distinct and appear to be under oxidative and copper stress responses. Conclusions Results reported in this study show that, as opposed to

  17. 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.

  18. Defining the expression of marker genes in equine mesenchymal stromal cells

    Directory of Open Access Journals (Sweden)

    Deborah J Guest

    2008-11-01

    Full Text Available Deborah J Guest1, Jennifer C Ousey1, Matthew RW Smith21Animal Health Trust, Lanwades Park, Kentford, Newmarket, Suffolk, CB8 7UU; 2Reynolds House Referrals, Greenwood Ellis and Partners, 166 High Street, Newmarket, Suffolk, CB8 9WS, UKAbstract: Mesenchymal stromal (MS cells have been derived from multiple sources in the horse including bone marrow, adipose tissue and umbilical cord blood. To date these cells have been investigated for their differentiation potential and are currently being used to treat damage to horse musculoskeletal tissues. However, no work has been done in horse MS cells to examine the expression profile of proteins and cell surface antigens that are expressed in human MS cells. The identification of such profiles in the horse will allow the comparison of putative MS cells isolated from different laboratories and different tissues. At present it is difficult to ascertain whether equivalent cells are being used in different reports. Here, we report on the expression of a range of markers used to define human MS cells. Using immunocytochemistry we show that horse MS cells homogenously express collagens, alkaline phosphatase activity, CD44, CD90 and CD29. In contrast, CD14, CD79α and the embryonic stem cell markers Oct-4, SSEA (stage specific embryonic antigen -1, -3, -4, TRA (tumor rejection antigen -1–60 and -1–81 are not expressed. The MS cells also express MHC class I antigens but do not express class II antigens, although they are inducible by treatment with interferon gamma (IFN-γ.Keywords: mesenchymal stem cells, equine, gene expression

  19. 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.

  20. Identification of novel genes expressed during host infection in Streptococcus equi ssp. zooepidemicus ATCC35246.

    Science.gov (United States)

    Ma, Zhe; Yu, Lei; Zhou, Hong; Liu, Tingting; Xu, Bin; Ma, Fang; Peng, Jie; Fan, Hongjie

    2015-02-01

    Infection with Streptococcus equi ssp. zooepidemicus (Streptococcus zooepidemicus, SEZ) can cause septicemia, meningitis, and mastitis in domesticated species. Identification of this organism's virulence factors is an effective way of clarifying its pathogenic mechanism. We employed in vivo-induced antigen technology (IVIAT) to find bacterial genes that were only expressed or upregulated in an infected host (IVI genes). Convalescent-phase sera from pigs infected with SEZ were pooled, adsorbed against in vitro antigens, and used to screen SEZ genomic expression libraries. This analysis identified 43 genes as IVI genes. Six of these 43 genes were verified via real-time PCR. Following the analysis, we were able to assign a putative function to 36 of the 43 proteins. These proteins included those involved in virulence and adaptation; formation of intermediary products; gene replication, transcription and expression; energy metabolism; transport and also various proteins of unknown function. The relationship between sagD gene and bacterial virulence was confirmed. This study provides new molecular data for the study of streptococcal disease in swine and is important for identifying the pathogenic mechanisms of SEZ. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

    International Nuclear Information System (INIS)

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

    1993-01-01

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

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

    International Nuclear Information System (INIS)

    Wollina, U.

    1986-01-01

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

  3. Inactivation of the alpha C protein antigen gene, bca, by a novel shuttle/suicide vector results in attenuation of virulence and immunity in group B Streptococcus.

    Science.gov (United States)

    Li, J; Kasper, D L; Ausubel, F M; Rosner, B; Michel, J L

    1997-11-25

    The alpha C protein of group B Streptococcus (GBS) is a major surface-associated antigen. Although its role in the biology and virulence of GBS has not been defined, it is opsonic and capable of eliciting protective immunity. The alpha C protein is widely distributed among clinical isolates and is a potential protein carrier and antigen in conjugate vaccines to prevent GBS infections. The structural gene for the alpha C protein, bca, has been cloned and sequenced. The protein encoded by bca is related to a class of surface-associated proteins of gram-positive cocci involved in virulence and immunity. To investigate the potential roles of the alpha C protein, bca null mutants were generated in which the bca gene was replaced with a kanamycin resistance cassette via homologous recombination using a novel shuttle/suicide vector. Studies of lethality in neonatal mice showed that the virulence of the bca null mutants was attenuated 5- to 7-fold when compared with the isogenic wild-type strain A909. Significant differences in mortality occurred in the first 24 h, suggesting that the role of the alpha antigen is important in the initial stages of the infection. In contrast to A909, bca mutants were no longer killed by polymorphonuclear leukocytes in the presence of alpha-specific antibodies in an in vitro opsonophagocytic assay. In contrast to previous studies, alpha antigen expression does not appear to play a role in resistance to opsonophagocytosis in the absence of alpha-specific antibodies. In addition, antibodies to the alpha C protein did not passively protect neonatal mice from lethal challenge with bca mutants, suggesting that these epitopes are uniquely present within the alpha antigen as expressed from the bca gene. Therefore, the alpha C protein is important in the pathogenesis of GBS infection and is a target for protective immunity in the development of GBS vaccines.

  4. Human Leukocyte Antigen-DR Expression is Significantly Related to an Increased Disease-Free and Disease-Specific Survival in Patients With Cervical Adenocarcinoma

    NARCIS (Netherlands)

    Samuels, Sanne; Spaans, Vivian M.; Osse, Michelle; Peters, Lex A. W.; Kenter, Gemma G.; Fleuren, Gertjan J.; Jordanova, Ekaterina S.

    2016-01-01

    Human leukocyte antigen (HLA) class II antigens are expressed on antigen-presenting cells, that is, macrophages, dendritic cells, and B lymphocytes. Under the influence of IFN-γ, HLA class II molecules can also be expressed on T lymphocytes, epithelial and endothelial cells. In addition, HLA class

  5. 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.

  6. Genetic diversity of K-antigen gene clusters of Escherichia coli and their molecular typing using a suspension array.

    Science.gov (United States)

    Yang, Shuang; Xi, Daoyi; Jing, Fuyi; Kong, Deju; Wu, Junli; Feng, Lu; Cao, Boyang; Wang, Lei

    2018-04-01

    Capsular polysaccharides (CPSs), or K-antigens, are the major surface antigens of Escherichia coli. More than 80 serologically unique K-antigens are classified into 4 groups (Groups 1-4) of capsules. Groups 1 and 4 contain the Wzy-dependent polymerization pathway and the gene clusters are in the order galF to gnd; Groups 2 and 3 contain the ABC-transporter-dependent pathway and the gene clusters consist of 3 regions, regions 1, 2 and 3. Little is known about the variations among the gene clusters. In this study, 9 serotypes of K-antigen gene clusters (K2ab, K11, K20, K24, K38, K84, K92, K96, and K102) were sequenced and correlated with their CPS chemical structures. On the basis of sequence data, a K-antigen-specific suspension array that detects 10 distinct CPSs, including the above 9 CPSs plus K30, was developed. This is the first report to catalog the genetic features of E. coli K-antigen variations and to develop a suspension array for their molecular typing. The method has a number of advantages over traditional bacteriophage and serum agglutination methods and lays the foundation for straightforward identification and detection of additional K-antigens in the future.

  7. The multiple immune-evasion genes of murine cytomegalovirus are not redundant: m4 and m152 inhibit antigen presentation in a complementary and cooperative fashion.

    Science.gov (United States)

    Kavanagh, D G; Gold, M C; Wagner, M; Koszinowski, U H; Hill, A B

    2001-10-01

    Both human cytomegaloviruses (HCMVs) and murine cytomegaloviruses (MCMVs) encode multiple genes that interfere with antigen presentation by major histocompatibility complex (MHC) class I, and thus protect infected targets from lysis by virus-specific cytotoxic T lymphocytes (CTLs). HCMV has been shown to encode four such genes and MCMV to encode two. MCMV m152 blocks the export of class I from a pre-Golgi compartment, and MCMV m6 directs class I to the lysosome for degradation. A third MCMV gene, m4, encodes a glycoprotein which is expressed at the cell surface in association with class I. Here we here show that m4 is a CTL-evasion gene which, unlike previously described immune-evasion genes, inhibited CTLs without blocking class I surface expression. m152 was necessary to block antigen presentation to both K(b)- and D(b)-restricted CTL clones, while m4 was necessary to block presentation only to K(b)-restricted clones. m152 caused complete retention of D(b), but only partial retention of K(b), in a pre-Golgi compartment. Thus, while m152 effectively inhibited D(b)-restricted CTLs, m4 was required to completely inhibit K(b)-restricted CTLs. We propose that cytomegaloviruses encode multiple immune-evasion genes in order to cope with the diversity of class I molecules in outbred host populations.

  8. Gene expression in Streptococcus mutans biofilms

    OpenAIRE

    Banu, L D

    2010-01-01

    Streptococcus mutans is considered the major aetiological agent of human dental caries. It is an obligate biofilm-forming bacterium, which resides on teeth and forms, together with other species, an oral biofilm that is often designated as supragingival plaque. This thesis consists of three distinct parts. The first part describes, using microarray analysis, how S. mutans modulates gene expression when grown under different conditions in biofilms. The goal of this analysis was to identify gen...

  9. Gene expression: RNA interference in adult mice

    Science.gov (United States)

    McCaffrey, Anton P.; Meuse, Leonard; Pham, Thu-Thao T.; Conklin, Douglas S.; Hannon, Gregory J.; Kay, Mark A.

    2002-07-01

    RNA interference is an evolutionarily conserved surveillance mechanism that responds to double-stranded RNA by sequence-specific silencing of homologous genes. Here we show that transgene expression can be suppressed in adult mice by synthetic small interfering RNAs and by small-hairpin RNAs transcribed in vivo from DNA templates. We also show the therapeutic potential of this technique by demonstrating effective targeting of a sequence from hepatitis C virus by RNA interference in vivo.

  10. Rat monocytes in a model of combined injury express the OX8 antigen

    Energy Technology Data Exchange (ETDEWEB)

    Kaffenberger, W.; Gruber, D.F.; MacVittie, T.J.

    1987-09-01

    We have analyzed peripheral blood mononuclear cell preparations from a rat model of combined injury (CI) (whole-body irradiation (500 cGy /sup 60/Co) followed by a thermal injury (20% body surface area, dorsal, scald burn)) for the expression of OX8 antigens. Ficoll-separated mononuclear fractions were labeled with monoclonal antibodies MRC OX8, MRC OX19, W3/13 HLK, or W3/25 for flow cytometric analysis. Combined-injury trauma resulted in decreased mononuclear cells to 6% of normal. This effect was due to the rapid decrease in radiosensitive lymphocytes from 83% to 10%. The relative numbers of monocytes increased from a normal 13% to 70% at day 4 after CI. Labeling of cells with OX8 after CI shifted to a population which was significantly larger in volume than normal lymphocytes. At the same time the mean fluorescence intensity of OX8-positive cells was considerably reduced. With the use of a F(ab) fragment of OX8 as a probe, these results could be partially explained as unspecific binding of the whole molecule of OX8 to Fc receptors expressed by activated monocytes. But, double-labeling and cell-sorting experiments also revealed the expression of OX8 antigens by a subset of OX8+/OX19- monocytes after CI.

  11. 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.

  12. Blood Gene Expression Predicts Bronchiolitis Obliterans Syndrome

    Directory of Open Access Journals (Sweden)

    Richard Danger

    2018-01-01

    Full Text Available Bronchiolitis obliterans syndrome (BOS, the main manifestation of chronic lung allograft dysfunction, leads to poor long-term survival after lung transplantation. Identifying predictors of BOS is essential to prevent the progression of dysfunction before irreversible damage occurs. By using a large set of 107 samples from lung recipients, we performed microarray gene expression profiling of whole blood to identify early biomarkers of BOS, including samples from 49 patients with stable function for at least 3 years, 32 samples collected at least 6 months before BOS diagnosis (prediction group, and 26 samples at or after BOS diagnosis (diagnosis group. An independent set from 25 lung recipients was used for validation by quantitative PCR (13 stables, 11 in the prediction group, and 8 in the diagnosis group. We identified 50 transcripts differentially expressed between stable and BOS recipients. Three genes, namely POU class 2 associating factor 1 (POU2AF1, T-cell leukemia/lymphoma protein 1A (TCL1A, and B cell lymphocyte kinase, were validated as predictive biomarkers of BOS more than 6 months before diagnosis, with areas under the curve of 0.83, 0.77, and 0.78 respectively. These genes allow stratification based on BOS risk (log-rank test p < 0.01 and are not associated with time posttransplantation. This is the first published large-scale gene expression analysis of blood after lung transplantation. The three-gene blood signature could provide clinicians with new tools to improve follow-up and adapt treatment of patients likely to develop BOS.

  13. 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.

  14. Design and development of therapies using chimeric antigen receptor-expressing T cells.

    Science.gov (United States)

    Dotti, Gianpietro; Gottschalk, Stephen; Savoldo, Barbara; Brenner, Malcolm K

    2014-01-01

    Investigators developed chimeric antigen receptors (CARs) for expression on T cells more than 25 years ago. When the CAR is derived from an antibody, the resultant cell should combine the desirable targeting features of an antibody (e.g. lack of requirement for major histocompatibility complex recognition, ability to recognize non-protein antigens) with the persistence, trafficking, and effector functions of a T cell. This article describes how the past two decades have seen a crescendo of research which has now begun to translate these potential benefits into effective treatments for patients with cancer. We describe the basic design of CARs, describe how antigenic targets are selected, and the initial clinical experience with CAR-T cells. Our review then describes our own and other investigators' work aimed at improving the function of CARs and reviews the clinical studies in hematological and solid malignancies that are beginning to exploit these approaches. Finally, we show the value of adding additional engineering features to CAR-T cells, irrespective of their target, to render them better suited to function in the tumor environment, and discuss how the safety of these heavily modified cells may be maintained. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  15. Immunohistochemical study of p53 and proliferating cell nuclear antigen expression in odontogenic keratocyst and periapical cyst

    Directory of Open Access Journals (Sweden)

    Thara Purath Sajeevan

    2014-01-01

    Full Text Available Introduction: p53 protein is a product of p53 gene, which is now classified as a tumor suppressor gene. The gene is a frequent target for mutation, being seen as a common step in the pathogenesis of many human cancers. Proliferating cell nuclear antigen (PCNA is an auxiliary protein of DNA polymerase delta and plays a critical role in initiation of cell proliferation. Aim: The aim of this study is to assess and compare the expression of p53 and PCNA in lining epithelium of odontogenic keratocyst (OKC and periapical cyst (PA. Materials and Methods: A total of 20 cases comprising 10 OKC and 10 PA were included in retrospective study. Three paraffin section of 4 μm were cut, one was used for routine hematoxylin and eosin stain, while the other two were used for immunohistochemistry. Statistical analysis was performed using Chi-square test. Results: The level of staining and intensity were assessed in all these cases. OKC showed PCNA expression in all cases (100%, whereas in perapical cyst only 60% of cases exhibited PCNA staining. (1 OKC showed p53 expression in 6 cases (60% whereas in PA only 10% of the cases exhibited p53 staining. Chi-square test showed PCNA staining intensity was more significant than p53 in OKC. (2 The staining intensity of PA using p53, PCNA revealed that PCNA stating intensity was more significant than p53. Conclusion: OKC shows significant proliferative activity than PA using PCNA and p53. PCNA staining was more intense when compared with p53 in both OKC and PA.

  16. Gene expression profile in obesity and type 2 diabetes mellitus

    Directory of Open Access Journals (Sweden)

    Rao Allam A

    2007-12-01

    Full Text Available Abstract Obesity is an important component of metabolic syndrome X and predisposes to the development of type 2 diabetes mellitus. The incidence of obesity, type 2 diabetes mellitus and metabolic syndrome X is increasing, and the cause(s for this increasing incidence is not clear. Although genetics could play an important role in the higher prevalence of these diseases, it is not clear how genetic factors interact with environmental and dietary factors to increase their incidence. We performed gene expression profile in subjects with obesity and type 2 diabetes mellitus with and without family history of these diseases. It was noted that genes involved in carbohydrate, lipid and amino acid metabolism pathways, glycan of biosynthesis, metabolism of cofactors and vitamin pathways, ubiquitin mediated proteolysis, signal transduction pathways, neuroactive ligand-receptor interaction, nervous system pathways, neurodegenerative disorders pathways are upregulated in obesity compared to healthy subjects. In contrast genes involved in cell adhesion molecules, cytokine-cytokine receptor interaction, insulin signaling and immune system pathways are downregulated in obese. Genes involved in signal transduction, regulation of actin cytoskeleton, antigen processing and presentation, complement and coagulation cascades, axon guidance and neurodegenerative disorders pathways are upregulated in subjects with type 2 diabetes with family history of diabetes compared to those who are diabetic but with no family history. Genes involved in oxidative phosphorylation, immune, nervous system, and metabolic disorders pathways are upregulated in those with diabetes with family history of diabetes compared to those with diabetes but with no family history. In contrast, genes involved in lipid and amino acid pathways, ubiquitin mediated proteolysis, signal transduction, insulin signaling and PPAR signaling pathways are downregulated in subjects with diabetes with family

  17. Mucoepidermoid carcinoma-associated expression of MUC5AC, MUC5B and mucin-type carbohydrate antigen sialyl-Tn in the parotid gland.

    Science.gov (United States)

    Matse, Johannes H; Bharos, Wiresh K; Veerman, Enno C I; Bloemena, Elisabeth; Bolscher, Jan G M

    2017-10-01

    The aberrant expression of mucins and mucin-type carbohydrates has been described in many types of cancer, including mucoepidermoid carcinoma (MEC), a malignant salivary gland tumor. In this study, we examined the aberrant expression patterns of mucins (MUC1, MUC4, MUC5AC and MUC5B), simple mucin-type carbohydrate antigens (Tn, sialyl-Tn and T) and mature carbohydrate antigens (Lewis a and sulfo-Lewis a antigens) in MEC originating from the parotid gland, which normally does not secrete mucins. We conducted an immunohistochemical study to investigate the presence of mucins and carbohydrates in 24 MEC samples originating from the parotid gland and in surrounding normal tissue of the same gland in comparison 6 samples of normal salivary glands. The expression levels were compared with respect to the histological grading. Furthermore, 24 MEC samples from non-parotid salivary glands were included. We observed loss of topology of membrane-bound MUC1 and MUC4, and de novo expression of MUC5AC, MUC5B and sialyl-Tn in MEC that originated in the parotid gland. Furthermore, mucins MUC1, MUC4 and carbohydrate antigens Tn, sialyl-Tn, T, Lewis a and sulfo-Lewis a were overexpressed in MEC samples compared to surrounding normal salivary gland tissues. MUC1 was expressed in both low- and high grade MECs, whereas MUC4 was not expressed in high grade MECs of the parotid gland. During the development of MEC in the parotid gland, the genes for gel-forming secretory mucins are switched on. Besides these MEC tissues overexpress short oligosaccharides, suggesting that the glycosylation machinery is altered. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Proteomic and gene expression patterns of keratoconus

    Directory of Open Access Journals (Sweden)

    Arkasubhra Ghosh

    2013-01-01

    Full Text Available Keratoconus is a progressive corneal thinning disease associated with significant tissue remodeling activities and activation of a variety of signaling networks. However, it is not understood how differential gene and protein expression direct function in keratoconus corneas to drive the underlying pathology, ectasia. Research in the field has focused on discovering differentially expressed genes and proteins and quantifying their levels and activities in keratoconus patient samples. In this study, both microarray analysis of total ribonucleic acid (RNA and whole proteome analyses are carried out using corneal epithelium and tears from keratoconus patients and compared to healthy controls. A number of structural proteins, signaling molecules, cytokines, proteases, and enzymes have been found to be deregulated in keratoconus corneas. Together, the data provide clues to the complex process of corneal degradation which suggest novel ways to clinically diagnose and manage the disease. This review will focus on discussing these recent advances in the knowledge of keratoconus biology from a gene expression and function point-of-view.

  19. Analysis of gene expression in rabbit muscle

    Directory of Open Access Journals (Sweden)

    Alena Gálová

    2014-02-01

    Full Text Available Increasing consumer knowledge of the link between diet and health has raised the demand for high quality food. Meat and meat products may be considered as irreplaceable in human nutrition. Breeding livestock to higher content of lean meat and the use of modern hybrids entails problems with the quality of meat. Analysing of livestock genomes could get us a great deal of important information, which may significantly affect the improvement process. Domestic animals are invaluable resources for study of the molecular architecture of complex traits. Although the mapping of quantitative trait loci (QTL responsible for economically important traits in domestic animals has achieved remarkable results in recent decades, not all of the genetic variation in the complex traits has been captured because of the low density of markers used in QTL mapping studies. The genome wide association study (GWAS, which utilizes high-density single-nucleotide polymorphism (SNP, provides a new way to tackle this issue. New technologies now allow producing microarrays containing thousands of hybridization probes on a single membrane or other solid support. We used microarray analysis to study gene expression in rabbit muscle during different developmental age stages. The outputs from GeneSpring GX sotware are presented in this work. After the evaluation of gene expression in rabbits, will be selected genes of interest in relation to meat quality parameters and will be further analyzed by the available methods of molecular biology and genetics.

  20. Moving Toward Integrating Gene Expression Profiling into ...

    Science.gov (United States)

    Microarray profiling of chemical-induced effects is being increasingly used in medium and high-throughput formats. In this study, we describe computational methods to identify molecular targets from whole-genome microarray data using as an example the estrogen receptor α (ERα), often modulated by potential endocrine disrupting chemicals. ERα biomarker genes were identified by their consistent expression after exposure to 7 structurally-diverse ERα agonists and 3 ERα antagonists in ERα-positive MCF-7 cells. Most of the biomarker genes were shown to be directly regulated by ERα as determined by ESR1 gene knockdown using siRNA as well as through ChIP-Seq analysis of ERα-DNA interactions. The biomarker was evaluated as a predictive tool using the fold-change rank-based Running Fisher algorithm by comparison to annotated gene expression data sets from experiments using MCF-7 cells, including those evaluating the transcriptional effects of hormones and chemicals. Using 141 comparisons from chemical- and hormone-treated cells, the biomarker gave a balanced accuracy for prediction of ERα activation or suppression of 94% and 93%, respectively. The biomarker was able to correctly classify 18 out of 21 (86%) ER reference chemicals including “very weak” agonists. Importantly, the biomarker predictions accurately replicated predictions based on 18 in vitro high-throughput screening assays that queried different steps in ERα signaling. For 114 chemicals,

  1. Differentially expressed genes in pancreatic ductal adenocarcinomas identified through serial analysis of gene expression

    DEFF Research Database (Denmark)

    Hustinx, Steven R; Cao, Dengfeng; Maitra, Anirban

    2004-01-01

    generated from six pancreatic cancers were compared to SAGE libraries generated from 11 non-neoplastic tissues. Compared to normal tissue libraries, we identified 453 SAGE tags as differentially expressed in pancreatic cancer, including 395 that mapped to known genes and 58 "uncharacterized" tags....... Of the 395 SAGE tags assigned to known genes, 223 were overexpressed in pancreatic cancer, and 172 were underexpressed. In order to map the 58 uncharacterized differentially expressed SAGE tags to genes, we used a newly developed resource called TAGmapper (http://tagmapper.ibioinformatics.org), to identify...

  2. Genome-wide gene expression profiling of homeodomain-interacting protein kinase 2 deficient medullary thymic epithelial cells

    Directory of Open Access Journals (Sweden)

    Kristin Rattay

    2015-12-01

    Full Text Available The establishment of central tolerance essentially depends on the promiscuous gene expression (pGE of a plethora of tissue restricted antigens by the medullary thymic epithelial cells. The antigens are presented to developing thymocytes in the thymus to select for non-self reactive T-cell receptors in order to prevent autoimmune reactions in the periphery. However the molecular regulation of tissue-restricted antigen expression is still poorly understood. The only regulator known to play a role in the transcriptional regulation so far is the autoimmune regulator (AIRE. AIRE is thought to act in a multi-protein complex, promoting transcription, elongation and splicing of target genes. Yet the full composition of this Aire-associated multi-protein complex and its mode of action remain to be elucidated. Here we describe the experimental details and controls of the gene array analysis on the impact of the homeodomain-interacting protein kinase 2 (Hipk2 on promiscuous gene expression in medullary thymic epithelial cells based on the analysis of newly generated TEC-specific Hipk2 conditional knockout mice. The changes in gene expression are presumably mediated through a regulatory effect of Hipk2 on AIRE as published in the study by Rattay and colleagues in the Journal of Immunology [1]. The gene array data reported in this paper have been deposited in the Gene Expression Omnibus (GEO database, www.ncbi.nlm.nih.gov/geo (accession no. GSE63432.

  3. Evaluation of variants of melanoma-associated antigen genes and mRNA transcripts in melanomas of dogs.

    Science.gov (United States)

    Stell, Anneliese J; Dobson, Jane M; Scase, Timothy J; Catchpole, Brian

    2009-12-01

    OBJECTIVE-To characterize variability in melanoma-associated antigen (MAA) genes and gene expression in melanomas of dogs. ANIMALS-18 dogs with malignant melanomas and 8 healthy control dogs. PROCEDURES-cDNA was prepared from malignant melanoma biopsy specimens and from pigmented oral mucocutaneous tissues of healthy control dogs. Genomic DNA was extracted from poorly pigmented melanomas. A PCR assay was performed by use of Melan-A, SILV, or tyrosinase-specific primers. RESULTS-Splice variants of Melan-A and SILV were identified in malignant melanomas and also in healthy pigmented tissues, whereas a tyrosinase splice variant was detected in melanoma tissues only. A short interspersed nuclear element (SINE) insertion mutation was identified in the SILV gene in 1 of 10 poorly pigmented melanomas. Six novel exonic single nucleotide polymorphisms (SNPs; 3 synonymous and 3 nonsynonymous) were detected in the tyrosinase gene, and 1 nonsynonymous exonic SNP was detected in the SILV gene. CONCLUSIONS AND CLINICAL RELEVANCE-Variants of MAA mRNA were detected in malignant melanoma tissues of dogs. The importance of MAA alternative transcripts expressed in melanomas and normal pigmented tissues was unclear, but they may have represented a means of regulating melanin synthesis. The tyrosinase splice variant was detected only in melanomas and could potentially be a tumor-specific target for immunotherapy. A SILV SINE insertion mutation was identified in a melanoma from a Great Dane, a breed known to carry this mutation (associated with merle coat color). The nonsynonymous SNPs detected in tyrosinase and SILV transcripts did not appear to affect tumor pigmentation.

  4. 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...

  5. Reduced expression of Autographa californica nucleopolyhedrovirus ORF34, an essential gene, enhances heterologous gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Salem, Tamer Z. [Department of Entomology, Michigan State University, East Lansing, MI 48824 (United States); Department of Microbial Molecular Biology, AGERI, Agricultural Research Center, Giza 12619 (Egypt); Division of Biomedical Sciences, Zewail University, Zewail City of Science and Technology, Giza 12588 (Egypt); Zhang, Fengrui [Department of Entomology, Michigan State University, East Lansing, MI 48824 (United States); Thiem, Suzanne M., E-mail: smthiem@msu.edu [Department of Entomology, Michigan State University, East Lansing, MI 48824 (United States); Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824 (United States)

    2013-01-20

    Autographa californica multiple nucleopolyhedrovirus ORF34 is part of a transcriptional unit that includes ORF32, encoding a viral fibroblast growth factor (FGF) and ORF33. We identified ORF34 as a candidate for deletion to improve protein expression in the baculovirus expression system based on enhanced reporter gene expression in an RNAi screen of virus genes. However, ORF34 was shown to be an essential gene. To explore ORF34 function, deletion (KO34) and rescue bacmids were constructed and characterized. Infection did not spread from primary KO34 transfected cells and supernatants from KO34 transfected cells could not infect fresh Sf21 cells whereas the supernatant from the rescue bacmids transfection could recover the infection. In addition, budded viruses were not observed in KO34 transfected cells by electron microscopy, nor were viral proteins detected from the transfection supernatants by western blots. These demonstrate that ORF34 is an essential gene with a possible role in infectious virus production.

  6. Three gene expression vector sets for concurrently expressing multiple genes in Saccharomyces cerevisiae.

    Science.gov (United States)

    Ishii, Jun; Kondo, Takashi; Makino, Harumi; Ogura, Akira; Matsuda, Fumio; Kondo, Akihiko

    2014-05-01

    Yeast has the potential to be used in bulk-scale fermentative production of fuels and chemicals due to its tolerance for low pH and robustness for autolysis. However, expression of multiple external genes in one host yeast strain is considerably labor-intensive due to the lack of polycistronic transcription. To promote the metabolic engineering of yeast, we generated systematic and convenient genetic engineering tools to express multiple genes in Saccharomyces cerevisiae. We constructed a series of multi-copy and integration vector sets for concurrently expressing two or three genes in S. cerevisiae by embedding three classical promoters. The comparative expression capabilities of the constructed vectors were monitored with green fluorescent protein, and the concurrent expression of genes was monitored with three different fluorescent proteins. Our multiple gene expression tool will be helpful to the advanced construction of genetically engineered yeast strains in a variety of research fields other than metabolic engineering. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

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

    Science.gov (United States)

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

    1987-09-01

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

  8. 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.

  9. Elevated Hu-Antigen Receptor (HuR) Expression is Associated with Tumor Aggressiveness and Poor Prognosis but not with COX-2 Expression in Invasive Breast Carcinoma Patients.

    Science.gov (United States)

    Giaginis, Constantinos; Sampani, Anastasia; Kotta-Loizou, Iolly; Giannopoulou, Ioanna; Danas, Eugene; Politi, Ekaterini; Tsourouflis, Gerasimos; Kouraklis, Gregorios; Patsouris, Efstratios; Keramopoulos, Antonios; Nakopoulou, Lydia; Theocharis, Stamatios

    2017-08-14

    Hu-antigen R (HuR), a RNA-binding protein, is considered to play a crucial role in tumor development and progression by stabilizing or regulating a group of cellular mRNAs of cancer-related genes, such as cyclooxygenase-2 (COX-2). The present study aimed to evaluate the clinical significance of HuR and COX-2 expression in invasive breast carcinoma. HuR and COX-2 protein expression was assessed immunohistochemically on paraffin-embedded breast cancer tissue sections obtained from 121 patients and was statistically analyzed with clinicopathological parameters, estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2), as well as with tumor cells' proliferative capacity and overall and disease-free patients' survival. High HuR expression was positively associated with larger tumor size and advanced disease stage (p = 0.0234 and p = 0.0361, respectively), being more frequently observed in ER negative cases (p = 0.0208). High COX-2 expression was negatively associated with histological (p Cox-regression analysis, p = 0.0223 and p = 0.0004, respectively) level. On the other hand, high COX-2 expression was associated with favorable overall and disease-free patients' survival merely at univariate level (log-rank test, p = 0.0389 and p = 0.0154, respectively). HuR expression was not associated with COX-2 expression (Spearman R = 0.1489, p = 0.1032). The present data support evidence that HuR is associated with tumor aggressiveness and poor prognosis in breast carcinoma, reinforcing its potential as promising therapeutic target in this type of neoplasia.

  10. Identification of cancer/testis-antigen genes by massively parallel signature sequencing

    OpenAIRE

    Chen, Yao-Tseng; Scanlan, Matthew J.; Venditti, Charis A.; Chua, Ramon; Theiler, Gregory; Stevenson, Brian J.; Iseli, Christian; Gure, Ali O.; Vasicek, Tom; Strausberg, Robert L.; Jongeneel, C. Victor; Old, Lloyd J.; Simpson, Andrew J. G.

    2005-01-01

    Massively parallel signature sequencing (MPSS) generates millions of short sequence tags corresponding to transcripts from a single RNA preparation. Most MPSS tags can be unambiguously assigned to genes, thereby generating a comprehensive expression profile of the tissue of origin. From the comparison of MPSS data from 32 normal human tissues, we identified 1,056 genes that are predominantly expressed in the testis. Further evaluation by using MPSS tags from cancer cell lines and EST data fro...

  11. HMME-based PDT restores expression and function of transporter associated with antigen processing 1 (TAP1) and surface presentation of MHC class I antigen in human glioma.

    Science.gov (United States)

    Zhang, Shan-Yi; Li, Jun-Liang; Xu, Xin-Ke; Zheng, Mei-Guang; Wen, Cheng-Cai; Li, Fang-Cheng

    2011-11-01

    Numerous studies have established that photodynamic therapy (PDT) can trigger tumor-specific immunity and cancer cell immunogenicity, both of which play a critical role in the long-term control of oncogenesis; however, the underlying mechanisms are largely unexplained. Deficiency of the transporter associated with antigen processing 1 (TAP1) has been observed in a variety of tumors, and the question has been raised whether the restoration of TAP1 could facilitate the activation of antitumor immunity. To elucidate the mechanisms underlying PDT-induced immunopotentiation, we examined the hypothesis that upregulating TAP1 via PDT may contribute to enhancement of antitumor immunity and cancer cell immunogenicity. In this study, we investigated the effects of PDT on the expression and function of TAP1 in glioma cells. We found that HMME-based PDT restored TAP1 expression in a rapid and transient manner. Furthermore, the newly synthesized TAP1 protein was capable of potentiating the activity of transporting antigen peptides. As a result, restoration of the expression and function of TAP1 translated into augmenting the presentation of surface MHC class I molecules. Overall, our data indicate that PDT enables glioma cells to recover both the expression of functional TAP1 and the presentation of surface MHC class I antigens, which are processes that may enhance antitumor immunity after PDT. These findings may have implications for PDT and provide new insights into the mechanisms underlying PDT-induced immunopotentiation.

  12. B-cell subpopulations from normal human secondary lymphoid tissues with specific gene expression profiles and phenotypes

    DEFF Research Database (Denmark)

    Johnsen, Hans Erik; Schmitz, Alexander; Perez Andres, Martin

    In order to improve insights into the B-cell biology and thereby B-cell myelomagenesis we have established a MSCNET standard for multiparametric flow cytometry (MFC) and cell sorting (FACS) for subsequent genetic analysis. The material analysed was fresh tonsils, blood and bone marrow. The method...... and single gene expression analysis (qRT-PCR) for transcription factors as well as global gene expression profiling (GEP; GeneChip Human Exon 1.0 ST Array). For example for tonsils, based on the immunophenotypic presentation (including CD3/44/CXCR4 in the panel), B-cell subsets were identified and sorted......-cell subpopulations identified have distinct gene expression profiles reflecting their functions but also revealing genes with subpopulation specific exon splicing. In conclusion a combination of surface markers expressed antigens and gene expression analysis of B cell subsets confirm a strong methodology to be used...

  13. Gene Expression Profiling of Xeroderma Pigmentosum

    Directory of Open Access Journals (Sweden)

    Bowden Nikola A

    2006-05-01

    Full Text Available Abstract Xeroderma pigmentosum (XP is a rare recessive disorder that is characterized by extreme sensitivity to UV light. UV light exposure results in the formation of DNA damage such as cyclobutane dimers and (6-4 photoproducts. Nucleotide excision repair (NER orchestrates the removal of cyclobutane dimers and (6-4 photoproducts as well as some forms of bulky chemical DNA adducts. The disease XP is comprised of 7 complementation groups (XP-A to XP-G, which represent functional deficiencies in seven different genes, all of which are believed to be involved in NER. The main clinical feature of XP is various forms of skin cancers; however, neurological degeneration is present in XPA, XPB, XPD and XPG complementation groups. The relationship between NER and other types of DNA repair processes is now becoming evident but the exact relationships between the different complementation groups remains to be precisely determined. Using gene expression analysis we have identified similarities and differences after UV light exposure between the complementation groups XP-A, XP-C, XP-D, XP-E, XP-F, XP-G and an unaffected control. The results reveal that there is a graded change in gene expression patterns between the mildest, most similar to the control response (XP-E and the severest form (XP-A of the disease, with the exception of XP-D. Distinct differences between the complementation groups with neurological symptoms (XP-A, XP-D and XP-G and without (XP-C, XP-E and XP-F were also identified. Therefore, this analysis has revealed distinct gene expression profiles for the XP complementation groups and the first step towards understanding the neurological symptoms of XP.

  14. Cholinergic regulation of VIP gene expression in human neuroblastoma cells

    DEFF Research Database (Denmark)

    Kristensen, Bo; Georg, Birgitte; Fahrenkrug, Jan

    1997-01-01

    Vasoactive intestinal polypeptide, muscarinic receptor, neuroblastoma cell, mRNA, gene expression, peptide processing......Vasoactive intestinal polypeptide, muscarinic receptor, neuroblastoma cell, mRNA, gene expression, peptide processing...

  15. 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.

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

    Science.gov (United States)

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

    2008-08-01

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

  17. Expression Analysis of ARMC3, a Testis-Specific Gene, in Breast Cancer Patients

    Directory of Open Access Journals (Sweden)

    Zare

    2016-02-01

    Full Text Available Background Breast cancer is the most prevalent cancer among women. Biomarkers that are expressed in tumors play a pivotal role in diagnosis and treatment. Cancer-testis (CT antigens are predominantly expressed in the testis and also have inappropriate expression in various tumor types. In the case of expression in tumors, they will be used as immunotherapy targets. Objectives Expression of ARMC3, a CT antigen, was analyzed to determine its potential as a tumor marker for breast cancer. Materials and Methods Eighty samples including 40 tumor samples and 40 normal adjacent tissue samples, were gathered from the ICBC biobank. RNA extraction was carried out on all samples. The extracted RNA was treated by DNaseI, after which cDNA was synthesized. Expression of ARMC3 with ACTB (internal control was studied using Real-Time PCR (polymerase chain reaction. Results Overall, 43.6% of tumors and 25.6% of normal adjacent tissues expressed ARMC3. ARMC3 was overexpressed in 41% of tumor samples (P = 0.00 and showed decreased expression in 46.2% (P = 0.00. Also, the expression of this gene in 12.8% of tumors was unchanged, which was statistically significant. It should be noted that all samples expressed ACTB gene. Conclusions Expression of ARMC3 in tumor samples and normal adjacent tissue is very important. The expression of this gene in tumor-adjacent tissue may be associated with the stage of cancer; it may be that these tissues are affected by epigenetic and oncogenic changes of breast cancer. Accordingly, aberrant expression of ARMC3 in tumor samples may be an attractive candidate for use as a tumor marker.

  18. Quantitative RT-PCR analysis of estrogen receptor gene expression in laser microdissected prostate cancer tissue.

    Science.gov (United States)

    Walton, Thomas J; Li, Geng; McCulloch, Thomas A; Seth, Rashmi; Powe, Desmond G; Bishop, Michael C; Rees, Robert C

    2009-06-01

    Real-time quantitative RT-PCR analysis of laser microdissected tissue is considered the most accurate technique for determining tissue gene expression. The discovery of estrogen receptor beta (ERbeta) has focussed renewed interest on the role of estrogen receptors in prostate cancer, yet few studies have utilized the technique to analyze estrogen receptor gene expression in prostate cancer. Fresh tissue was obtained from 11 radical prostatectomy specimens and from 6 patients with benign prostate hyperplasia. Pure populations of benign and malignant prostate epithelium were laser microdissected, followed by RNA isolation and electrophoresis. Quantitative RT-PCR was performed using primers for androgen receptor (AR), estrogen receptor beta (ERbeta), estrogen receptor alpha (ERalpha), progesterone receptor (PGR) and prostate specific antigen (PSA), with normalization to two housekeeping genes. Differences in gene expression were analyzed using the Mann-Whitney U-test. Correlation coefficients were analyzed using Spearman's test. Significant positive correlations were seen when AR and AR-dependent PSA, and ERalpha and ERalpha-dependent PGR were compared, indicating a representative population of RNA transcripts. ERbeta gene expression was significantly over-expressed in the cancer group compared with benign controls (P AR, ERalpha or PSA expression between the groups. This study represents the first to show an upregulation of ERbeta gene expression in laser microdissected prostate cancer specimens. In concert with recent studies the findings suggest differential production of ERbeta splice variants, which may play important roles in the genesis of prostate cancer. (c) 2009 Wiley-Liss, Inc.

  19. IDO, PTEN-expressing Tregs and control of antigen-presentation in the murine tumor microenvironment.

    Science.gov (United States)

    Munn, David H; Sharma, Madhav D; Johnson, Theodore S; Rodriguez, Paulo

    2017-08-01

    The tumor microenvironment is profoundly immunosuppressive. This creates a major barrier for attempts to combine immunotherapy with conventional chemotherapy or radiation, because the tumor antigens released by these cytotoxic agents are not cross-presented in an immunogenic fashion. In this Focused Research Review, we focus on mouse preclinical studies exploring the role of immunosuppressive Tregs expressing the PTEN lipid phosphatase, and the links between PTEN+ Tregs and the immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO). IDO has received attention because it can be expressed by a variety of human tumor types in vivo, but IDO can also be induced in host immune cells of both humans and mice in response to inflammation, infection or dying (apoptotic) cells. Mechanistically, IDO and PTEN+ Tregs are closely connected, with IDO causing activation of the PTEN pathway in Tregs. Genetic ablation or pharmacologic inhibition of PTEN in mouse Tregs destabilizes their suppressive phenotype, and this prevents transplantable and autochthonous tumors from creating their normal immunosuppressive microenvironment. Genetic ablation of either IDO or PTEN+ Tregs in mice results in a fundamental defect in the ability to maintain tolerance to antigens associated with apoptotic cells, including dying tumor cells. Consistent with this, pharmacologic inhibitors of either pathway show synergy when combined with cytotoxic agents such as chemotherapy or radiation. Thus, we propose that IDO and PTEN+ Tregs represent closely linked checkpoints that can influence the choice between immune activation versus tolerance to dying tumor cells.

  20. 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.

  1. Dynamic gene expression in the human cerebral cortex distinguishes children from adults.

    Directory of Open Access Journals (Sweden)

    Kirstin N Sterner

    Full Text Available In comparison with other primate species, humans have an extended juvenile period during which the brain is more plastic. In the current study we sought to examine gene expression in the cerebral cortex during development in the context of this adaptive plasticity. We introduce an approach designed to discriminate genes with variable as opposed to uniform patterns of gene expression and found that greater inter-individual variance is observed among children than among adults. For the 337 transcripts that show this pattern, we found a significant overrepresentation of genes annotated to the immune system process (pFDR ~/= 0. Moreover, genes known to be important in neuronal function, such as brain-derived neurotrophic factor (BDNF, are included among the genes more variably expressed in childhood. We propose that the developmental period of heightened childhood neuronal plasticity is characterized by more dynamic patterns of gene expression in the cerebral cortex compared to adulthood when the brain is less plastic. That an overabundance of these genes are annotated to the immune system suggests that the functions of these genes can be thought of not only in the context of antigen processing and presentation, but also in the context of nervous system development.

  2. 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.

  3. Gene expression in developing watermelon fruit

    Directory of Open Access Journals (Sweden)

    Hernandez Alvaro

    2008-06-01

    Full Text Available Abstract Background Cultivated watermelon form large fruits that are highly variable in size, shape, color, and content, yet have extremely narrow genetic diversity. Whereas a plethora of genes involved in cell wall metabolism, ethylene biosynthesis, fruit softening, and secondary metabolism during fruit development and ripening have been identified in other plant species, little is known of the genes involved in these processes in watermelon. A microarray and quantitative Real-Time PCR-based study was conducted in watermelon [Citrullus lanatus (Thunb. Matsum. & Nakai var. lanatus] in order to elucidate the flow of events associated with fruit development and ripening in this species. RNA from three different maturation stages of watermelon fruits, as well as leaf, were collected from field grown plants during three consecutive years, and analyzed for gene expression using high-density photolithography microarrays and quantitative PCR. Results High-density photolithography arrays, composed of probes of 832 EST-unigenes from a subtracted, fruit development, cDNA library of watermelon were utilized to examine gene expression at three distinct time-points in watermelon fruit development. Analysis was performed with field-grown fruits over three consecutive growing seasons. Microarray analysis identified three hundred and thirty-five unique ESTs that are differentially regulated by at least two-fold in watermelon fruits during the early, ripening, or mature stage when compared to leaf. Of the 335 ESTs identified, 211 share significant homology with known gene products and 96 had no significant matches with any database accession. Of the modulated watermelon ESTs related to annotated genes, a significant number were found to be associated with or involved in the vascular system, carotenoid biosynthesis, transcriptional regulation, pathogen and stress response, and ethylene biosynthesis. Ethylene bioassays, performed with a closely related watermelon

  4. Gene expression in first trimester preeclampsia placenta.

    Science.gov (United States)

    Founds, Sandra A; Terhorst, Lauren A; Conrad, Kirk P; Hogge, W Allen; Jeyabalan, Arun; Conley, Yvette P

    2011-04-01

    The goal of this study was to further validate eight candidate genes identified in a microarray analysis of first trimester placentas in preeclampsia. Surplus chorionic villus sampling (CVS) specimens of 4 women subsequently diagnosed with preeclampsia (PE) and 8 control women (C) without preeclampsia analyzed previously by microarray and 24 independent additional control samples (AS) were submitted for confirmatory studies by quantitative real-time polymerase chain reaction (qRT-PCR). Downregulation was significant in FSTL3 in PE as compared to C and AS (p = .04). PAEP was downregulated, but the difference was only significant between C and AS (p = .002) rather than between PE and either of the control groups. Expression levels for CFH, EPAS1, IGFBP1, MMP12, and SEMA3C were not statistically different among groups, but trends were consistent with microarray results; there was no anti-correlation. S100A8 was not measurable in all samples, probably because different probes and primers were needed. This study corroborates reduced FSTL3 expression in the first trimester of preeclampsia. Nonsignificant trends in the other genes may require follow-up in studies powered for medium or medium/large effect sizes. qRT-PCR verification of the prior microarray of CVS may support the placental origins of preeclampsia hypothesis. Replication is needed for the candidate genes as potential biomarkers of susceptibility, early detection, and/or individualized care of maternal-infant preeclampsia.

  5. Nuclear AXIN2 represses MYC gene expression

    International Nuclear Information System (INIS)

    Rennoll, Sherri A.; Konsavage, Wesley M.; Yochum, Gregory S.

    2014-01-01

    Highlights: •AXIN2 localizes to cytoplasmic and nuclear compartments in colorectal cancer cells. •Nuclear AXIN2 represses the activity of Wnt-responsive luciferase reporters. •β-Catenin bridges AXIN2 to TCF transcription factors. •AXIN2 binds the MYC promoter and represses MYC gene expression. -- Abstract: The β-catenin transcriptional coactivator is the key mediator of the canonical Wnt signaling pathway. In the absence of Wnt, β-catenin associates with a cytosolic and multi-protein destruction complex where it is phosphorylated and targeted for proteasomal degradation. In the presence of Wnt, the destruction complex is inactivated and β-catenin translocates into the nucleus. In the nucleus, β-catenin binds T-cell factor (TCF) transcription factors to activate expression of c-MYC (MYC) and Axis inhibition protein 2 (AXIN2). AXIN2 is a member of the destruction complex and, thus, serves in a negative feedback loop to control Wnt/β-catenin signaling. AXIN2 is also present in the nucleus, but its function within this compartment is unknown. Here, we demonstrate that AXIN2 localizes to the nuclei of epithelial cells within normal and colonic tumor tissues as well as colorectal cancer cell lines. In the nucleus, AXIN2 represses expression of Wnt/β-catenin-responsive luciferase reporters and forms a complex with β-catenin and TCF. We demonstrate that AXIN2 co-occupies β-catenin/TCF complexes at the MYC promoter region. When constitutively localized to the nucleus, AXIN2 alters the chromatin structure at the MYC promoter and directly represses MYC gene expression. These findings suggest that nuclear AXIN2 functions as a rheostat to control MYC expression in response to Wnt/β-catenin signaling

  6. Nuclear AXIN2 represses MYC gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Rennoll, Sherri A.; Konsavage, Wesley M.; Yochum, Gregory S., E-mail: gsy3@psu.edu

    2014-01-03

    Highlights: •AXIN2 localizes to cytoplasmic and nuclear compartments in colorectal cancer cells. •Nuclear AXIN2 represses the activity of Wnt-responsive luciferase reporters. •β-Catenin bridges AXIN2 to TCF transcription factors. •AXIN2 binds the MYC promoter and represses MYC gene expression. -- Abstract: The β-catenin transcriptional coactivator is the key mediator of the canonical Wnt signaling pathway. In the absence of Wnt, β-catenin associates with a cytosolic and multi-protein destruction complex where it is phosphorylated and targeted for proteasomal degradation. In the presence of Wnt, the destruction complex is inactivated and β-catenin translocates into the nucleus. In the nucleus, β-catenin binds T-cell factor (TCF) transcription factors to activate expression of c-MYC (MYC) and Axis inhibition protein 2 (AXIN2). AXIN2 is a member of the destruction complex and, thus, serves in a negative feedback loop to control Wnt/β-catenin signaling. AXIN2 is also present in the nucleus, but its function within this compartment is unknown. Here, we demonstrate that AXIN2 localizes to the nuclei of epithelial cells within normal and colonic tumor tissues as well as colorectal cancer cell lines. In the nucleus, AXIN2 represses expression of Wnt/β-catenin-responsive luciferase reporters and forms a complex with β-catenin and TCF. We demonstrate that AXIN2 co-occupies β-catenin/TCF complexes at the MYC promoter region. When constitutively localized to the nucleus, AXIN2 alters the chromatin structure at the MYC promoter and directly represses MYC gene expression. These findings suggest that nuclear AXIN2 functions as a rheostat to control MYC expression in response to Wnt/β-catenin signaling.

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

    Directory of Open Access Journals (Sweden)

    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

  8. Reduced experimental autoimmune encephalomyelitis after intranasal and oral administration of recombinant lactobacilli expressing myelin antigens.

    Science.gov (United States)

    Maassen, Catharina B M; Laman, Jon D; van Holten-Neelen, Conny; Hoogteijling, Linsy; Groenewegen, Lizet; Visser, Lizette; Schellekens, Marc M; Boersma, Wim J A; Claassen, Eric

    2003-12-01

    Oral administration of autoantigens is a safe and convenient way to induce peripheral T-cell tolerance in autoimmune diseases like multiple sclerosis (MS). To increase the efficacy of oral tolerance induction and obviate the need for large-scale purification of human myelin proteins, we use genetically modified lactobacilli expressing myelin antigens. A panel of recombinant lactobacilli was constructed producing myelin proteins and peptides, including human and guinea pig myelin basic protein (MBP) and proteolipid protein peptide 139-151 (PLP(139-151)). In this study we examined whether these Lactobacillus recombinants are able to induce oral and intranasal tolerance in an animal model for multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). Lewis rats received soluble cell extracts of Lactobacillus transformants intranasally three times prior to induction of EAE. For the induction of oral tolerance, rats were fed live transformed lactobacilli for 20 days. Ten days after the first oral administration EAE was induced. Intranasal administration of extracts containing guinea pig MBP (gpMBP) or MBP(72-85) significantly inhibited EAE in Lewis rats. Extracts of control transformants did not reduce EAE. Live lactobacilli expressing guinea pig MBP(72-85) fused to the marker enzyme beta-glucuronidase (beta-gluc) were also able to significantly reduce disease when administered orally. In conclusion, these experiments provide proof of principle that lactobacilli expressing myelin antigens reduce EAE after mucosal (intranasal and oral) administration. This novel method of mucosal tolerance induction by mucosal administration of recombinant lactobacilli expressing relevant autoantigens could find applications in autoimmune disease in general, such as multiple sclerosis, rheumatoid arthritis and uveitis.

  9. Molecular mechanisms of curcumin action: gene expression.

    Science.gov (United States)

    Shishodia, Shishir

    2013-01-01

    Curcumin derived from the tropical plant Curcuma longa has a long history of use as a dietary agent, food preservative, and in traditional Asian medicine. It has been used for centuries to treat biliary disorders, anorexia, cough, diabetic wounds, hepatic disorders, rheumatism, and sinusitis. The preventive and therapeutic properties of curcumin are associated with its antioxidant, anti-inflammatory, and anticancer properties. Extensive research over several decades has attempted to identify the molecular mechanisms of curcumin action. Curcumin modulates numerous molecular targets by altering their gene expression, signaling pathways, or through direct interaction. Curcumin regulates the expression of inflammatory cytokines (e.g., TNF, IL-1), growth factors (e.g., VEGF, EGF, FGF), growth factor receptors (e.g., EGFR, HER-2, AR), enzymes (e.g., COX-2, LOX, MMP9, MAPK, mTOR, Akt), adhesion molecules (e.g., ELAM-1, ICAM-1, VCAM-1), apoptosis related proteins (e.g., Bcl-2, caspases, DR, Fas), and cell cycle proteins (e.g., cyclin D1). Curcumin modulates the activity of several transcription factors (e.g., NF-κB, AP-1, STAT) and their signaling pathways. Based on its ability to affect multiple targets, curcumin has the potential for the prevention and treatment of various diseases including cancers, arthritis, allergies, atherosclerosis, aging, neurodegenerative disease, hepatic disorders, obesity, diabetes, psoriasis, and autoimmune diseases. This review summarizes the molecular mechanisms of modulation of gene expression by curcumin. Copyright © 2012 International Union of Biochemistry and Molecular Biology, Inc.

  10. Studying the Complex Expression Dependences between Sets of Coexpressed Genes

    Directory of Open Access Journals (Sweden)

    Mario Huerta

    2014-01-01

    Full Text Available Organisms simplify the orchestration of gene expression by coregulating genes whose products function together in the cell. The use of clustering methods to obtain sets of coexpressed genes from expression arrays is very common; nevertheless there are no appropriate tools to study the expression networks among these sets of coexpressed genes. The aim of the developed tools is to allow studying the complex expression dependences that exist between sets of coexpressed genes. For this purpose, we start detecting the nonlinear expression relationships between pairs of genes, plus the coexpressed genes. Next, we form networks among sets of coexpressed genes that maintain nonlinear expression dependences between all of them. The expression relationship between the sets of coexpressed genes is defined by the expression relationship between the skeletons of these sets, where this skeleton represents the coexpressed genes with a well-defined nonlinear expression relationship with the skeleton of the other sets. As a result, we can study the nonlinear expression relationships between a target gene and other sets of coexpressed genes, or start the study from the skeleton of the sets, to study the complex relationships of activation and deactivation between the sets of coexpressed genes that carry out the different cellular processes present in the expression experiments.

  11. Changes in gene expression following androgen receptor blockade ...

    Indian Academy of Sciences (India)

    Madhu urs

    Involution of the rat ventral prostate and concomitant modulation of gene expression post-castration is a well- documented phenomenon. While the rat castration model has been extensively used to study androgen regulation of gene expression in the ventral prostate, it is not clear whether all the gene expression changes ...

  12. Generation of antigenic diversity in Plasmodium falciparum by structured rearrangement of Var genes during mitosis.

    Directory of Open Access Journals (Sweden)

    Antoine Claessens

    2014-12-01

    Full Text Available The most polymorphic gene family in P. falciparum is the ∼60 var genes distributed across parasite chromosomes, both in the subtelomeres and in internal regions. They encode hypervariable surface proteins known as P. falciparum erythrocyte membrane protein 1 (PfEMP1 that are critical for pathogenesis and immune evasion in Plasmodium falciparum. How var gene sequence diversity is generated is not currently completely understood. To address this, we constructed large clone trees and performed whole genome sequence analysis to study the generation of novel var gene sequences in asexually replicating parasites. While single nucleotide polymorphisms (SNPs were scattered across the genome, structural variants (deletions, duplications, translocations were focused in and around var genes, with considerable variation in frequency between strains. Analysis of more than 100 recombination events involving var exon 1 revealed that the average nucleotide sequence identity of two recombining exons was only 63% (range: 52.7-72.4% yet the crossovers were error-free and occurred in such a way that the resulting sequence was in frame and domain architecture was preserved. Var exon 1, which encodes the immunologically exposed part of the protein, recombined in up to 0.2% of infected erythrocytes in vitro per life cycle. The high rate of var exon 1 recombination indicates that millions of new antigenic structures could potentially be generated each day in a single infected individual. We propose a model whereby var gene sequence polymorphism is mainly generated during the asexual part of the life cycle.

  13. Generation of antigenic diversity in Plasmodium falciparum by structured rearrangement of Var genes during mitosis.

    Science.gov (United States)

    Claessens, Antoine; Hamilton, William L; Kekre, Mihir; Otto, Thomas D; Faizullabhoy, Adnan; Rayner, Julian C; Kwiatkowski, Dominic

    2014-12-01

    The most polymorphic gene family in P. falciparum is the ∼60 var genes distributed across parasite chromosomes, both in the subtelomeres and in internal regions. They encode hypervariable surface proteins known as P. falciparum erythrocyte membrane protein 1 (PfEMP1) that are critical for pathogenesis and immune evasion in Plasmodium falciparum. How var gene sequence diversity is generated is not currently completely understood. To address this, we constructed large clone trees and performed whole genome sequence analysis to study the generation of novel var gene sequences in asexually replicating parasites. While single nucleotide polymorphisms (SNPs) were scattered across the genome, structural variants (deletions, duplications, translocations) were focused in and around var genes, with considerable variation in frequency between strains. Analysis of more than 100 recombination events involving var exon 1 revealed that the average nucleotide sequence identity of two recombining exons was only 63% (range: 52.7-72.4%) yet the crossovers were error-free and occurred in such a way that the resulting sequence was in frame and domain architecture was preserved. Var exon 1, which encodes the immunologically exposed part of the protein, recombined in up to 0.2% of infected erythrocytes in vitro per life cycle. The high rate of var exon 1 recombination indicates that millions of new antigenic structures could potentially be generated each day in a single infected individual. We propose a model whereby var gene sequence polymorphism is mainly generated during the asexual part of the life cycle.

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

    Science.gov (United States)

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

    2017-12-01

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

  15. Asthenoteratozoospermia in mice lacking testis expressed gene 18 (Tex18)

    NARCIS (Netherlands)

    Jaroszynski, L.; dev, A.; Li, M.; Meinhardt, A.; de rooij, D. G.; Mueller, Christian; Böhm, Detlef; Wolf, S.; Adham, I. M.; Wulf, G.; Engel, W.; Nayernia, K.

    2007-01-01

    Testis expressed gene 18 (Tex18) is a small gene with one exon of 240 bp, which is specifically expressed in male germ cells. The gene encodes for a protein of 80 amino acids with unknown domain. To investigate the function of (Tex18) gene, we generated mice with targeted disruption of the (Tex18)

  16. Elimination of contaminating cap genes in AAV vector virions reduces immune responses and improves transgene expression in a canine gene therapy model.

    Science.gov (United States)

    Wang, Z; Halbert, C L; Lee, D; Butts, T; Tapscott, S J; Storb, R; Miller, A D

    2014-04-01

    Animal and human gene therapy studies utilizing AAV vectors have shown that immune responses to AAV capsid proteins can severely limit transgene expression. The main source of capsid antigen is that associated with the AAV vectors, which can be reduced by stringent vector purification. A second source of AAV capsid proteins is that expressed from cap genes aberrantly packaged into AAV virions during vector production. This antigen source can be eliminated by the use of a cap gene that is too large to be incorporated into an AAV capsid, such as a cap gene containing a large intron (captron gene). Here, we investigated the effects of elimination of cap gene transfer and of vector purification by CsCl gradient centrifugation on AAV vector immunogenicity and expression following intramuscular injection in dogs. We found that both approaches reduced vector immunogenicity and that combining the two produced the lowest immune responses and highest transgene expression. This combined approach enabled the use of a relatively mild immunosuppressive regimen to promote robust micro-dystrophin gene expression in Duchenne muscular dystrophy-affected dogs. Our study shows the importance of minimizing AAV cap gene impurities and indicates that this improvement in AAV vector production may benefit human applications.

  17. Effects of Emdogain on osteoblast gene expression.

    Science.gov (United States)

    Carinci, F; Piattelli, A; Guida, L; Perrotti, V; Laino, G; Oliva, A; Annunziata, M; Palmieri, A; Pezzetti, F

    2006-05-01

    Emdogain (EMD) is a protein extract purified from porcine enamel and has been introduced in clinical practice to obtain periodontal regeneration. EMD is composed mainly of amelogenins (90%), while the remaining 10% is composed of non-amelogenin enamel matrix proteins such as enamelins, tuftelin, amelin and ameloblastin. Enamel matrix proteins seem to be involved in root formation. EMD has been reported to promote proliferation, migration, adhesion and differentiation of cells associated with healing periodontal tissues in vivo. How this protein acts on osteoblasts is poorly understood. We therefore attempted to address this question by using a microarray technique to identify genes that are differently regulated in osteoblasts exposed to enamel matrix proteins. By using DNA microarrays containing 20,000 genes, we identified several upregulated and downregulated genes in the osteoblast-like cell line (MG-63) cultured with enamel matrix proteins (Emd). The differentially expressed genes cover a broad range of functional activities: (i) signaling transduction, (ii) transcription, (iii) translation, (iv) cell cycle regulation, proliferation and apoptosis, (v) immune system, (vi) vesicular transport and lysosome activity, and (vii) cytoskeleton, cell adhesion and extracellular matrix production. The data reported are the first genome-wide scan of the effect of enamel matrix proteins on osteoblast-like cells. These results can contribute to our understanding of the molecular mechanisms of bone regeneration and as a model for comparing other materials with similar clinical effects.

  18. The relationship among gene expression, the evolution of gene dosage, and the rate of protein evolution.

    Directory of Open Access Journals (Sweden)

    Jean-François Gout

    2010-05-01

    Full Text Available The understanding of selective constraints affecting genes is a major issue in biology. It is well established that gene expression level is a major determinant of the rate of protein evolution, but the reasons for this relationship remain highly debated. Here we demonstrate that gene expression is also a major determinant of the evolution of gene dosage: the rate of gene losses after whole genome duplications in the Paramecium lineage is negatively correlated to the level of gene expression, and this relationship is not a byproduct of other factors known to affect the fate of gene duplicates. This indicates that changes in gene dosage are generally more deleterious for highly expressed genes. This rule also holds for other taxa: in yeast, we find a clear relationship between gene expression level and the fitness impact of reduction in gene dosage. To explain these observations, we propose a model based on the fact that the optimal expression level of a gene corresponds to a trade-off between the benefit and cost of its expression. This COSTEX model predicts that selective pressure against mutations changing gene expression level or affecting the encoded protein should on average be stronger in highly expressed genes and hence that both the frequency of gene loss and the rate of protein evolution should correlate negatively with gene expression. Thus, the COSTEX model provides a simple and common explanation for the general relationship observed between the level of gene expression and the different facets of gene evolution.

  19. [Expression, structure and antigenicity analysis of N51 derived from the N-terminal heptad repeat domain in gp41 of HIV-1 CRF07_BC strain].

    Science.gov (United States)

    Shao, Jiping; Jiang, Shibo; Liu, Shuwen

    2012-12-01

    To express N51 derived from the N-terminal heptad repeat (NHR) domain in gp41 of the HIV-1 CRF07_BC strain and analyze its molecular structure and antigenicity. Overlapping PCR was used to amplify the DNA fragment encoding N51Fd gene, which was then subcloned into the vector pFUSE-hIgG1-Fc2. The construct was confirmed by DNA sequencing. The structure and antigenicity of the recombinant protein N51FdFc-BC were analyzed using bioinformatic software, circular dichroism, and Western blotting. A recombinant expression vector pFUSE/N51Fd-BC was successfully constructed. N51FdFc-BC recombinant protein with a relative molecular mass of about 35 000 was effectively expressed in mammalian 293T cells and could be recognized by rabbit antibodies against HIV-1 gp41 N/C peptides as shown by Western blotting. Bioinformatic analysis showed that the recombinant protein N51FdFc-BC, with a relative molecular mass of 34 315.1 and a PI of 7.59, formed a secondary structure of random coil to allow its interactions as an antigen with antibodies. Circular dichroism measurement confirmed the random coil structure of N51FdFc-BC protein. The recombinant protein N51FdFc-BC has a random coil structure and can be used as an immunogen for development of HIV-1 subunit vaccine.

  20. Gene expression profiling of cutaneous wound healing

    Directory of Open Access Journals (Sweden)

    Wang Ena

    2007-02-01

    Full Text Available Abstract Background Although the sequence of events leading to wound repair has been described at the cellular and, to a limited extent, at the protein level this process has yet to be fully elucidated. Genome wide transcriptional analysis tools promise to further define the global picture of this complex progression of events. Study Design This study was part of a placebo-controlled double-blind clinical trial in which basal cell carcinomas were treated topically with an immunomodifier – toll-like receptor 7 agonist: imiquimod. The fourteen patients with basal cell carcinoma in the placebo arm of the trial received placebo treatment consisting solely of vehicle cream. A skin punch biopsy was obtained immediately before treatment and at the end of the placebo treatment (after 2, 4 or 8 days. 17.5K cDNA microarrays were utilized to profile the biopsy material. Results Four gene signatures whose expression changed relative to baseline (before wound induction by the pre-treatment biopsy were identified. The largest group was comprised predominantly of inflammatory genes whose expression was increased throughout the study. Two additional signatures were observed which included preferentially pro-inflammatory genes in the early post-treatment biopsies (2 days after pre-treatment biopsies and repair and angiogenesis genes in the later (4 to 8 days biopsies. The fourth and smallest set of genes was down-regulated throughout the study. Early in wound healing the expression of markers of both M1 and M2 macrophages were increased, but later M2 markers predominated. Conclusion The initial response to a cutaneous wound induces powerful transcriptional activation of pro-inflammatory stimuli which may alert the host defense. Subsequently and in the absence of infection, inflammation subsides and it is replaced by angiogenesis and remodeling. Understanding this transition which may be driven by a change from a mixed macrophage population to predominately M2

  1. Network Completion for Static Gene Expression Data

    Directory of Open Access Journals (Sweden)

    Natsu Nakajima

    2014-01-01

    Full Text Available We tackle the problem of completing and inferring genetic networks under stationary conditions from static data, where network completion is to make the minimum amount of modifications to an initial network so that the completed network is most consistent with the expression data in which addition of edges and deletion of edges are basic modification operations. For this problem, we present a new method for network completion using dynamic programming and least-squares fitting. This method can find an optimal solution in polynomial time if the maximum indegree of the network is bounded by a constant. We evaluate the effectiveness of our method through computational experiments using synthetic data. Furthermore, we demonstrate that our proposed method can distinguish the differences between two types of genetic networks under stationary conditions from lung cancer and normal gene expression data.

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

    Science.gov (United States)

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

    2013-01-01

    Verocytotoxic Escherichia (E.) coli strains are responsible for swine oedema disease, which is an enterotoxaemia that causes economic losses in the pig industry. The production of a vaccine for oral administration in transgenic seeds could be an efficient system to stimulate local immunity. This study was conducted to transform tobacco plants for the seed-specific expression of antigenic proteins from a porcine verocytotoxic E. coli strain. Parameters related to an immunological response and possible adverse effects on the oral administration of obtained tobacco seeds were evaluated in a mouse model. Tobacco was transformed via Agrobacteium tumefaciens with chimeric constructs containing structural parts of the major subunit FedA of the F18 adhesive fimbriae and VT2e B-subunit genes under control of a seed specific GLOB promoter. We showed that the foreign Vt2e-B and F18 genes were stably accumulated in storage tissue by the immunostaining method. In addition, Balb-C mice receiving transgenic tobacco seeds via the oral route showed a significant increase in IgA-positive plasma cell presence in tunica propria when compared to the control group with no observed adverse effects. Our findings encourage future studies focusing on swine for evaluation of the protective effects of transformed tobacco seeds against E. coli infection.

  3. Inferring gene expression dynamics via functional regression analysis

    Directory of Open Access Journals (Sweden)

    Leng Xiaoyan

    2008-01-01

    Full Text Available Abstract Background Temporal gene expression profiles characterize the time-dynamics of expression of specific genes and are increasingly collected in current gene expression experiments. In the analysis of experiments where gene expression is obtained over the life cycle, it is of interest to relate temporal patterns of gene expression associated with different developmental stages to each other to study patterns of long-term developmental gene regulation. We use tools from functional data analysis to study dynamic changes by relating temporal gene expression profiles of different developmental stages to each other. Results We demonstrate that functional regression methodology can pinpoint relationships that exist between temporary gene expression profiles for different life cycle phases and incorporates dimension reduction as needed for these high-dimensional data. By applying these tools, gene expression profiles for pupa and adult phases are found to be strongly related to the profiles of the same genes obtained during the embryo phase. Moreover, one can distinguish between gene groups that exhibit relationships with positive and others with negative associations between later life and embryonal expression profiles. Specifically, we find a positive relationship in expression for muscle development related genes, and a negative relationship for strictly maternal genes for Drosophila, using temporal gene expression profiles. Conclusion Our findings point to specific reactivation patterns of gene expression during the Drosophila life cycle which differ in characteristic ways between various gene groups. Functional regression emerges as a useful tool for relating gene expression patterns from different developmental stages, and avoids the problems with large numbers of parameters and multiple testing that affect alternative approaches.

  4. Expression regulation of design process gene in product design

    DEFF Research Database (Denmark)

    Li, Bo; Fang, Lusheng; Li, Bo

    2011-01-01

    is proposed and analyzed, as well as its three categories i.e., the operator gene, the structural gene and the regulator gene. Second, the trigger mechanism that design objectives and constraints trigger the operator gene is constructed. Third, the expression principle of structural gene is analyzed...... with the example of design management gene. Last, the regulation mode that the regulator gene regulates the expression of the structural gene is established and it is illustrated by taking the design process management gene as an example. © (2011) Trans Tech Publications....

  5. Intravaginal immunization of mice with recombinant Salmonella enterica serovar Typhimurium expressing human papillomavirus type 16 antigens as a potential route of vaccination against cervical cancer.

    Science.gov (United States)

    Echchannaoui, Hakim; Bianchi, Matteo; Baud, David; Bobst, Martine; Stehle, Jean-Christophe; Nardelli-Haefliger, Denise

    2008-05-01

    Cervical cancer, the second leading cause of cancer deaths in women, is the consequence of high-risk human papillomavirus (HPV) infections. Toward the development of therapeutic vaccines that can induce both innate and adaptive mucosal immune responses, we analyzed intravaginal (ivag) vaccine delivery of live attenuated Salmonella enterica serovar Typhimurium expressing HPV16L1 as a model antigen. Innate immune responses were examined in cervicovaginal tissues by determining gene expression patterns by microarray analysis using nylon membranes imprinted with cDNA fragments coding for inflammation-associated genes. At 24 h, a wide range of genes, including those for chemokines and Th1- and Th2-type cytokine and chemokine receptors were up-regulated in mice ivag immunized with Salmonella compared to control mice. However, the majority of transcripts returned to their steady-state levels 1 week after immunization, suggesting a transient inflammatory response. Indeed, cervicovaginal histology of immunized mice showed a massive, but transient, infiltration of macrophages and neutrophils, while T cells were still increased after 7 days. Ivag immunization also induced humoral and antitumor immune responses, i.e., serum and vaginal anti-HPV16VLP antibody titers similar to those induced by oral immunization, and significant protection in tumor protection experiments using HPV16-expressing C3 tumor cells. These results show that ivag immunization with live attenuated Salmonella expressing HPV16 antigens modulates the local mucosal gene expression pattern into a transient proinflammatory profile, elicits strong systemic and mucosal immunity against HPV16, and confers protection against HPV16 tumor cells subcutaneously implanted in mice. Examination of the efficacy with which ivag HPV16E7E6 Salmonella induces regression of tumors located in cervicovaginal tissue is warranted.

  6. Global expression differences and tissue specific expression differences in rice evolution result in two contrasting types of differentially expressed genes

    KAUST Repository

    Horiuchi, Youko

    2015-12-23

    Background Since the development of transcriptome analysis systems, many expression evolution studies characterized evolutionary forces acting on gene expression, without explicit discrimination between global expression differences and tissue specific expression differences. However, different types of gene expression alteration should have different effects on an organism, the evolutionary forces that act on them might be different, and different types of genes might show different types of differential expression between species. To confirm this, we studied differentially expressed (DE) genes among closely related groups that have extensive gene expression atlases, and clarified characteristics of different types of DE genes including the identification of regulating loci for differential expression using expression quantitative loci (eQTL) analysis data. Results We detected differentially expressed (DE) genes between rice subspecies in five homologous tissues that were verified using japonica and indica transcriptome atlases in public databases. Using the transcriptome atlases, we classified DE genes into two types, global DE genes and changed-tissues DE genes. Global type DE genes were not expressed in any tissues in the atlas of one subspecies, however changed-tissues type DE genes were expressed in both subspecies with different tissue specificity. For the five tissues in the two japonica-indica combinations, 4.6 ± 0.8 and 5.9 ± 1.5 % of highly expressed genes were global and changed-tissues DE genes, respectively. Changed-tissues DE genes varied in number between tissues, increasing linearly with the abundance of tissue specifically expressed genes in the tissue. Molecular evolution of global DE genes was rapid, unlike that of changed-tissues DE genes. Based on gene ontology, global and changed-tissues DE genes were different, having no common GO terms. Expression differences of most global DE genes were regulated by cis-eQTLs. Expression

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

    Science.gov (United States)

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

    2009-01-01

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

  8. Regulation and gene expression profiling of NKG2D positive human cytomegalovirus-primed CD4+ T-cells

    DEFF Research Database (Denmark)

    Jensen, Helle; Folkersen, Lasse; Skov, Søren

    2012-01-01

    we used genome-wide analysis of individual genes and gene set enrichment analysis (GSEA) to investigate the gene expression profile of NKG2D(+) CD4(+) T-cells, generated from HCMV-primed CD4(+) T-cells. We show that the HCMV-primed NKG2D(+) CD4(+) T-cells possess a higher differentiated phenotype...... than the NKG2D(-) CD4(+) T-cells, both at the gene expression profile and cytokine profile. The ability to express NKG2D at the cell surface was primarily determined by the activation or differentiation status of the CD4(+) T-cells and not by the antigen presenting cells. We observed a correlation...... CD4(+) T-cells. These findings provide novel information about the gene expression profile of HCMV-primed NKG2D(+) CD4(+) T-cells, as well as the mechanisms regulating NKG2D cell surface expression....

  9. Immune evasion by muscle-specific gene expression in dystrophic muscle.

    Science.gov (United States)

    Hartigan-O'Connor, D; Kirk, C J; Crawford, R; Mulé, J J; Chamberlain, J S

    2001-12-01

    Muscle tissue from Duchenne muscular dystrophy patients and the Dmd(mdx/mdx) (hereafter referred to as mdx) mouse is characterized by an abundance of necrotic myofibers and infiltrating macrophages. Both features may provide additional stimulus to the immune response directed against novel antigens, such as those delivered by gene therapy vectors. It has previously been shown that the immune evasion achieved by adeno-associated virus in healthy muscle fails in one model of muscular dystrophy. Here, we examined the immune response to adenoviral vectors and their transgenes in normal and mdx mice. We found that mdx mouse muscles contain 20 times more macrophages and 7 times more dendritic cells than healthy muscles. This higher professional antigen-presenting cell content results in a stronger immune response to antigens that can be directly presented by those cells, including viral antigens and constitutively expressed transgene products. However, we did not detect a significant immune response to beta-galactosidase expressed specifically in muscle, even at high expression levels. This result suggests that cross-presentation is not more effective in mdx mouse muscle, and that targeted vectors and tissue-specific promoters may be useful tools for evasion of the immune response in dystrophic muscle.

  10. Candidate genes expressed in human islets and their role in the pathogenesis of type 1 diabetes

    DEFF Research Database (Denmark)

    Storling, Joachim; Brorsson, Caroline Anna

    2013-01-01

    In type 1 diabetes (T1D), the insulin-producing β cells are destroyed by an immune-mediated process leading to complete insulin deficiency. There is a strong genetic component in T1D. Genes located in the human leukocyte antigen (HLA) region are the most important genetic determinants of disease......, but more than 40 additional loci are known to significantly affect T1D risk. Since most of the currently known genetic candidates have annotated immune cell functions, it is generally considered that most of the genetic susceptibility in T1D is caused by variation in genes affecting immune cell function....... Recent studies, however, indicate that most T1D candidate genes are expressed in human islets suggesting that the functions of the genes are not restricted to immune cells, but also play roles in the islets and possibly the β cells. Several candidates change expression levels within the islets following...

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

    Science.gov (United States)

    Jeong, Seri; Park, Seho; Park, Byeong-Woo; Park, Younhee; Kwon, Oh-Joong; Kim, Hyon-Suk

    2014-01-01

    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, Pcancer 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  13. 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.

  14. Expression, purification, and functional analysis of an antigen-targeting fusion protein composed of CD40 ligand and the C-terminal fragment of ovalbumin.

    Science.gov (United States)

    Shi, Yunnuo; Halperin, Scott A; Lee, Song F

    2018-02-01

    Delivering antigen via molecules specifically targeting receptors on the surface of antigen-presenting cells is a strategy to improve immune responses. In this study, an antigen-targeting fusion protein (OVA-CD40LS) composed of the C-terminal fragment of ovalbumin and the extracellular domain of mouse CD40 ligand was constructed by genetic fusion. The OVA-CD40LS and the control OVA (rOVA) genes were cloned in Escherichia coli and over-expressed as insoluble proteins. The rOVA protein was purified from the insoluble fraction of E. coli cell lysate by nickel affinity chromatography and refolded by step-wise dialysis to give a yield of 11.8 mg/L of culture. The OVA-CD40LS was purified by a 'two-round' nickel affinity and on-column protein-refolding chromatography. The yield was 528 μg/L of culture. The purified OVA-CD40LS, but not the rOVA, was able to simulate the production of pro-inflammatory cytokines and up-regulate cell surface marker proteins in mouse bone marrow-derived dendritic cells. The purified OVA-CD40LS elicited a robust immune response when injected submucosally in the oral cavity of mice. Collectively, the results indicate that the OVA-CD40LS fusion protein was biologically active, functioning as an antigen-targeting protein. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Prognostic Gene Expression Profiles in Breast Cancer

    DEFF Research Database (Denmark)

    Sørensen, Kristina Pilekær

    Each year approximately 4,800 Danish women are diagnosed with breast cancer. Several clinical and pathological factors are used as prognostic and predictive markers to categorize the patients into groups of high or low risk. Around 90% of all patients are allocated to the high risk group and offe......Each year approximately 4,800 Danish women are diagnosed with breast cancer. Several clinical and pathological factors are used as prognostic and predictive markers to categorize the patients into groups of high or low risk. Around 90% of all patients are allocated to the high risk group...... clinical courses, and they may be useful as novel prognostic biomarkers in breast cancer. The aim of the present project was to predict the development of metastasis in lymph node negative breast cancer patients by RNA profiling. We collected and analyzed 82 primary breast tumors from patients who...... developed metastasis and 82 primary breast tumors from patients who remained metastasis-free, by microarray gene expression profiling. We employed a nested case-control design, where samples were matched, in this study one-to-one, to exclude differences in gene expression based on tumor type, tumor size...

  16. The Effects of Hallucinogens on Gene Expression.

    Science.gov (United States)

    Martin, David A; Nichols, Charles D

    2018-01-01

    The classic serotonergic hallucinogens, or psychedelics, have the ability to profoundly alter perception and behavior. These can include visual distortions, hallucinations, detachment from reality, and mystical experiences. Some psychedelics, like LSD, are able to produce these effects with remarkably low doses of drug. Others, like psilocybin, have recently been demonstrated to have significant clinical efficacy in the treatment of depression, anxiety, and addiction that persist for at least several months after only a single therapeutic session. How does this occur? Much work has recently been published from imaging studies showing that psychedelics alter brain network connectivity. They facilitate a disintegration of the default mode network, producing a hyperconnectivity between brain regions that allow centers that do not normally communicate with each other to do so. The immediate and acute effects on both behaviors and network connectivity are likely mediated by effector pathways downstream of serotonin 5-HT2A receptor activation. These acute molecular processes also influence gene expression changes, which likely influence synaptic plasticity and facilitate more long-term changes in brain neurochemistry ultimately underlying the therapeutic efficacy of a single administration to achieve long-lasting effects. In this review, we summarize what is currently known about the molecular genetic responses to psychedelics within the brain and discuss how gene expression changes may contribute to altered cellular physiology and behaviors.

  17. Interactive visualization of gene regulatory networks with associated gene expression time series data

    NARCIS (Netherlands)

    Westenberg, Michel A.; Hijum, Sacha A.F.T. van; Lulko, Andrzej T.; Kuipers, Oscar P.; Roerdink, Jos B.T.M.; Linsen, L; Hagen, H; Hamann, B

    2008-01-01

    We present GENeVis, an application to visualize gene expression time series data in a gene regulatory network context. This is a network of regulator proteins that regulate the expression of their respective target genes. The networks are represented as graphs, in which the nodes represent genes,

  18. 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.

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

    Science.gov (United States)

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

    1994-01-01

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

  20. Analysis of Structures and Epitopes of Surface Antigen Glycoproteins Expressed in Bradyzoites of Toxoplasma gondii

    Directory of Open Access Journals (Sweden)

    Hua Cong

    2013-01-01

    Full Text Available Toxoplasma gondii is a protozoan parasite capable of infecting humans and animals. Surface antigen glycoproteins, SAG2C, -2D, -2X, and -2Y, are expressed on the surface of bradyzoites. These antigens have been shown to protect bradyzoites against immune responses during chronic infections. We studied structures of SAG2C, -2D, -2X, and -2Y proteins using bioinformatics methods. The protein sequence alignment was performed by T-Coffee method. Secondary structural and functional domains were predicted using software PSIPRED v3.0 and SMART software, and 3D models of proteins were constructed and compared using the I-TASSER server, VMD, and SWISS-spdbv. Our results showed that SAG2C, -2D, -2X, and -2Y are highly homologous proteins. They share the same conserved peptides and HLA-I restricted epitopes. The similarity in structure and domains indicated putative common functions that might stimulate similar immune response in hosts. The conserved peptides and HLA-restricted epitopes could provide important insights on vaccine study and the diagnosis of this disease.

  1. 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 ...

  2. Positive selection on gene expression in the human brain

    DEFF Research Database (Denmark)

    Khaitovich, Philipp; Tang, Kun; Franz, Henriette

    2006-01-01

    Recent work has shown that the expression levels of genes transcribed in the brains of humans and chimpanzees have changed less than those of genes transcribed in other tissues [1] . However, when gene expression changes are mapped onto the evolutionary lineage in which they occurred, the brain...... shows more changes than other tissues in the human lineage compared to the chimpanzee lineage [1] , [2] and [3] . There are two possible explanations for this: either positive selection drove more gene expression changes to fixation in the human brain than in the chimpanzee brain, or genes expressed...... in the brain experienced less purifying selection in humans than in chimpanzees, i.e. gene expression in the human brain is functionally less constrained. The first scenario would be supported if genes that changed their expression in the brain in the human lineage showed more selective sweeps than other genes...

  3. 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

  4. Expression of antigens coded in murine leukemia viruses on thymocytes of allogeneic donor origin in AKR mice following syngeneic or allogeneic bone marrow transplantation

    International Nuclear Information System (INIS)

    Wustrow, T.P.; Good, R.A.

    1985-01-01

    Removal of T-lymphocytes from marrow inoculum with monoclonal antibody plus complement permitted establishment of long-lived allogeneic chimeras between C57BL/6 and AKR/J mice. Development of leukemia was prevented for 15 mo. Protection from leukemia occurred with both young (4 wk) and older (4 mo) recipients. AKR mice reconstituted with syngeneic marrow or control AKR mice all developed leukemia-lymphoma before 1 yr of age. During spontaneous lymphomagenesis in AKR mice, amplified expression of gag or env gene-coded virus antigens on the surface of thymocytes preceded leukemia development and evidence for amplification of other virus genes. These changes generally appeared before 6 mo. Similar viral gene expression and viral gene amplification occurred in the thymus and spleen cells of leukemia-resistant chimeric mice. Using monoclonal antibodies to Mr 70,000 glycoprotein epitopes characteristic of ecotropic, xenotropic, or dualtropic viruses, antigens marking each virus form were found on thymocytes of allogeneic 4-wk and 4-mo chimeras as well as on the cells of AKR mice and of AKR mice reconstituted with syngeneic marrow. Flow cytometric analysis showed amplification of the virus genes in mice protected from leukemia-lymphoma by allogeneic bone marrow transplantation from leukemia-resistant mice. Allogeneic chimeras and syngeneically transplanted mice both showed evidence of accelerated viremia and of recombinant virus formation. The findings suggest that an event essential to leukemogenesis which occurs within the AKR lymphoid cells or their environment is lacking in the allogeneic chimeras. The nature of this influence of a resistance gene or genes introduced into AKR mice by allogeneic bone marrow transplantation deserves further study

  5. Gene expression profiling reveals the defining features of monocytes from septic patients with compensatory anti-inflammatory response syndrome.

    Science.gov (United States)

    Xu, Ping-bo; Lou, Jing-Sheng; Ren, Yu; Miao, Chang-Hong; Deng, Xiao-ming

    2012-11-01

    To characterize the expression profiles of genes in purified monocytes from septic patients during systemic inflammatory response syndrome (SIRS) and compensatory anti-inflammatory response syndrome (CARS), and then to investigate the potential mechanism of monocyte deactivation. Lipopolysaccharides (LPS)-induced cytokine responses, phagocytosis assay and migration assay were performed in monocytes from SIRS patients, CARS patients and healthy volunteers (n = 8). After functional assays, each pair of samples from the same group was pooled into one for gene expression analysis. All new samples (n = 4) were hybridized on NimbleGen human gene expression 12 × 135 K microarrays, and selected genes were validated by real-time polymerase chain reaction. Pathway analysis and Gene Ontology analysis were performed on differentially expressed genes using Agilent GeneSpring (version 11.0). A set of genes related to pro-inflammation, phagocytosis, chemotaxis, antigen presentation, and anti-apoptosis were significantly down-regulated, while some genes associated with pro-apoptosis and anti-inflammation were up-regulated instead on monocytes from CARS patients compared with SIRS patients and healthy volunteers. Monocytes from CARS patients showed impaired production of TNF-α and IL-6, and increased release of IL-10 when stimulated by LPS. Functional analysis confirmed reduced phagocytosis and migratory activity of monocytes from CARS patients. Human leukocyte antigen-DR (HLA-DR) measurements demonstrated decreased expression of HLA-DR on monocytes from CARS patients. Monocytes from CARS patients exhibited significant changes in mRNA expression of genes associated with phagocytosis, antigen presentation, inflammatory response, cell migration, and apoptosis, which might cause deactivation of monocytes during CARS. Copyright © 2012 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

  6. Identification of immune protective genes of Eimeria maxima through cDNA expression library screening.

    Science.gov (United States)

    Yang, XinChao; Li, MengHui; Liu, JianHua; Ji, YiHong; Li, XiangRui; Xu, LiXin; Yan, RuoFeng; Song, XiaoKai

    2017-02-16

    Eimeria maxima is one of the most prevalent Eimeria species causing avian coccidiosis, and results in huge economic loss to the global poultry industry. Current control strategies, such as anti-coccidial medication and live vaccines have been limited because of their drawbacks. The third generation anticoccidial vaccines including the recombinant vaccines as well as DNA vaccines have been suggested as a promising alternative strategy. To date, only a few protective antigens of E. maxima have been reported. Hence, there is an urgent need to identify novel protective antigens of E. maxima for the development of neotype anticoccidial vaccines. With the aim of identifying novel protective genes of E. maxima, a cDNA expression library of E. maxima sporozoites was constructed using Gateway technology. Subsequently, the cDNA expression library was divided into 15 sub-libraries for cDNA expression library immunization (cDELI) using parasite challenged model in chickens. Protective sub-libraries were selected for the next round of screening until individual protective clones were obtained, which were further sequenced and analyzed. Adopting the Gateway technology, a high-quality entry library was constructed, containing 9.2 × 10 6 clones with an average inserted fragments length of 1.63 kb. The expression library capacity was 2.32 × 10 7 colony-forming units (cfu) with an average inserted fragments length of 1.64 Kb. The expression library was screened using parasite challenged model in chickens. The screening yielded 6 immune protective genes including four novel protective genes of EmJS-1, EmRP, EmHP-1 and EmHP-2, and two known protective genes of EmSAG and EmCKRS. EmJS-1 is the selR domain-containing protein of E. maxima whose function is unknown. EmHP-1 and EmHP-2 are the hypothetical proteins of E. maxima. EmRP and EmSAG are rhomboid-like protein and surface antigen glycoproteins of E. maxima respectively, and involved in invasion of the parasite. Our

  7. Identification of Human HK Genes and Gene Expression Regulation Study in Cancer from Transcriptomics Data Analysis

    Science.gov (United States)

    Zhang, Zhang; Liu, Jingxing; Wu, Jiayan; Yu, Jun

    2013-01-01

    The regulation of gene expression is essential for eukaryotes, as it drives the processes of cellular differentiation and morphogenesis, leading to the creation of different cell types in multicellular organisms. RNA-Sequencing (RNA-Seq) provides researchers with a powerful toolbox for characterization and quantification of transcriptome. Many different human tissue/cell transcriptome datasets coming from RNA-Seq technology are available on public data resource. The fundamental issue here is how to develop an effective analysis method to estimate expression pattern similarities between different tumor tissues and their corresponding normal tissues. We define the gene expression pattern from three directions: 1) expression breadth, which reflects gene expression on/off status, and mainly concerns ubiquitously expressed genes; 2) low/high or constant/variable expression genes, based on gene expression level and variation; and 3) the regulation of gene expression at the gene structure level. The cluster analysis indicates that gene expression pattern is higher related to physiological condition rather than tissue spatial distance. Two sets of human housekeeping (HK) genes are defined according to cell/tissue types, respectively. To characterize the gene expression pattern in gene expression level and variation, we firstly apply improved K-means algorithm and a gene expression variance model. We find that cancer-associated HK genes (a HK gene is specific in cancer group, while not in normal group) are expressed higher and more variable in cancer condition than in normal condition. Cancer-associated HK genes prefer to AT-rich genes, and they are enriched in cell cycle regulation related functions and constitute some cancer signatures. The expression of large genes is also avoided in cancer group. These studies will help us understand which cell type-specific patterns of gene expression differ among different cell types, and particularly for cancer. PMID:23382867

  8. Expression, immunolocalization, and serological reactivity of a novel sphingomyelin phosphodiesterase-like protein, an excretory/secretory antigen from Clonorchis sinensis.

    Science.gov (United States)

    Huang, Yanwei; Zheng, Youwei; Li, Yuzhe; Yang, Mei; Li, Ting; Zeng, Suxiang; Yu, Xinbing; Huang, Huaiqiu; Hu, Xuchu

    2013-06-01

    Clonorchiasis, caused by Clonorchis sinensis infection, is a zoonotic parasitic disease of hepatobiliary system in which the proteins released by adult are major pathogenetic factors. In this study, we first characterized a putative sphingomyelin phosphodiesterase (CsSMPase) A-like secretory protein, which was highly expressed in the adult worm. The full-length gene was cloned. The putative protein is of relatively low homology comparing with SMPase from other species, and of rich T cell and B cell epitopes, suggesting that it is an antigen of strong antigenicity. The complete coding sequence of the gene was expressed in the Escherichia coli. The recombinant CsSMPase (rCsSMPase) can be recognized by C. sinensis-infected serum, and the protein immunoserum can recognize a specific band in excretory/secretory products (ESPs) of C. sinensis adult by western blotting. Immunolocalization revealed that CsSMPase was not only localized on tegument, ventral sucker of metacercaria, and the intestine of adult but also on the nearby epithelium of bile duct of the infected Sprague-Dawley rats, implying that CsSMPase was mainly secreted and excreted through adult intestine and directly interacted with bile duct epithelium. Although immunized rats evoked high level antibody response, the antigen level was low in clonorchiasis patients. And the sensitivity and specificity of rCsSMPase were 50.0 % (12/24) and 88.4 % (61/69), in sera IgG-ELISA, respectively. It is likely due to the fact that CsSMPase binding to the plasma membrane of biliary epithelium decreases the antigen immune stimulation.

  9. Transcriptional programs that control expression of the autoimmune regulator gene Aire.

    Science.gov (United States)

    Herzig, Yonatan; Nevo, Shir; Bornstein, Chamutal; Brezis, Miriam R; Ben-Hur, Sharon; Shkedy, Aya; Eisenberg-Bord, Michal; Levi, Ben; Delacher, Michael; Goldfarb, Yael; David, Eyal; Weinberger, Leehee; Viukov, Sergey; Ben-Dor, Shifra; Giraud, Matthieu; Hanna, Jacob H; Breiling, Achim; Lyko, Frank; Amit, Ido; Feuerer, Markus; Abramson, Jakub

    2017-02-01

    Aire is a transcriptional regulator that induces promiscuous expression of thousands of genes encoding tissue-restricted antigens (TRAs) in medullary thymic epithelial cells (mTECs). While the target genes of Aire are well characterized, the transcriptional programs that regulate its own expression have remained elusive. Here we comprehensively analyzed both cis-acting and trans-acting regulatory mechanisms and found that the Aire locus was insulated by the global chromatin organizer CTCF and was hypermethylated in cells and tissues that did not express Aire. In mTECs, however, Aire expression was facilitated by concurrent eviction of CTCF, specific demethylation of exon 2 and the proximal promoter, and the coordinated action of several transcription activators, including Irf4, Irf8, Tbx21, Tcf7 and Ctcfl, which acted on mTEC-specific accessible regions in the Aire locus.

  10. FlyTED: the Drosophila Testis Gene Expression Database

    OpenAIRE

    Zhao, Jun; Klyne, Graham; Benson, Elizabeth; Gudmannsdottir, Elin; White-Cooper, Helen; Shotton, David

    2009-01-01

    FlyTED, the Drosophila Testis Gene Expression Database, is a biological research database for gene expression images from the testis of the fruit fly Drosophila melanogaster. It currently contains 2762 mRNA in situ hybridization images and ancillary metadata revealing the patterns of gene expression of 817 Drosophila genes in testes of wild type flies and of seven meiotic arrest mutant strains in which spermatogenesis is defective. This database has been built by adapting a widely used digita...

  11. Sequence biases in large scale gene expression profiling data

    OpenAIRE

    Siddiqui, Asim S.; Delaney, Allen D.; Schnerch, Angelique; Griffith, Obi L.; Jones, Steven J. M.; Marra, Marco A.

    2006-01-01

    We present the results of a simple, statistical assay that measures the G+C content sensitivity bias of gene expression experiments without the requirement of a duplicate experiment. We analyse five gene expression profiling methods: Affymetrix GeneChip, Long Serial Analysis of Gene Expression (LongSAGE), LongSAGELite, ‘Classic’ Massively Parallel Signature Sequencing (MPSS) and ‘Signature’ MPSS. We demonstrate the methods have systematic and random errors leading to a different G+C content s...

  12. 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.

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

    Directory of Open Access Journals (Sweden)

    Joseph M Catanzaro

    2011-04-01

    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.

  14. The putative natural killer decoy early gene m04 (gp34) of murine cytomegalovirus encodes an antigenic peptide recognized by protective antiviral CD8 T cells.

    Science.gov (United States)

    Holtappels, R; Thomas, D; Podlech, J; Geginat, G; Steffens, H P; Reddehase, M J

    2000-02-01

    Several early genes of murine cytomegalovirus (MCMV) encode proteins that mediate immune evasion by interference with the major histocompatibility complex class I (MHC-I) pathway of antigen presentation to cytolytic T lymphocytes (CTL). Specifically, the m152 gene product gp37/40 causes retention of MHC-I molecules in the endoplasmic reticulum (ER)-Golgi intermediate compartment. Lack of MHC-I on the cell surface should activate natural killer (NK) cells recognizing the "missing self." The retention, however, is counteracted by the m04 early gene product gp34, which binds to folded MHC-I molecules in the ER and directs the complex to the cell surface. It was thus speculated that gp34 might serve to silence NK cells and thereby complete the immune evasion of MCMV. In light of these current views, we provide here results demonstrating an in vivo role for gp34 in protective antiviral immunity. We have identified an antigenic nonapeptide derived from gp34 and presented by the MHC-I molecule D(d). Besides the immunodominant immediate-early nonapeptide consisting of IE1 amino acids 168-176 (IE1(168-176)), the early nonapeptide m04(243-251) is the second antigenic peptide described for MCMV. The primary immune response to MCMV generates significant m04-specific CD8 T-cell memory. Upon adoptive transfer into immunodeficient recipients, an m04-specific CTL line controls MCMV infection with an efficacy comparable to that of an IE1-specific CTL line. Thus, gp34 is the first noted early protein of MCMV that escapes viral immune evasion mechanisms. These data document that MCMV is held in check by a redundance of protective CD8 T cells recognizing antigenic peptides in different phases of viral gene expression.

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

    Science.gov (United States)

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

    2016-12-01

    Carcinoembryonic antigen (CEA) is an attractive target for immunotherapy because it is expressed minimally in normal tissue, but is overexpressed in a wide variety of malignant epithelial tissues. Lactic acid bacteria (LABs), widely used in food processes, are attractive candidates for oral vaccination. Thus, we examined whether LABs could be used as a live vaccine vector to deliver CEA antigen. CEA was cloned into an Escherichia coli/Lactococcus lactis shuttle vector pSEC:LEISS under the control of a nisin promoter. For displaying the CEA on the cell surface of the L. lactis strain, the anchor motif LcsB from the S-layer protein of Lactobacillus crispatus was fused with CEA. Intracellular and cell surface expression of the CEA-LcsB fusion was confirmed by western blot analysis. Significantly higher levels of CEA-specific secretory immunoglobulin A in the sera of mice were observed upon oral administration of strain cultures containing th