WorldWideScience

Sample records for variant antigens expressed

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

    Science.gov (United States)

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

    2017-01-01

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

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

    DEFF Research Database (Denmark)

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

    2006-01-01

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

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

    DEFF Research Database (Denmark)

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

    1999-01-01

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

  4. Antigenic variation of Plasmodium knowlesi malaria: identification of the variant antigen on infected erythrocytes.

    OpenAIRE

    Howard, R J; Barnwell, J W; Kao, V

    1983-01-01

    Erythrocytes infected with mature asexual stages of Plasmodium knowlesi express a new surface antigen such that rhesus monkey antisera specifically agglutinate these cells. Cloned parasites can express different antigenic variants of this antigen. The variant antigen has been identified by comparison of the surface membrane antigens of a clone and of an antigenic variant of that clone of different agglutination phenotype. After lactoperoxidase labeling, 125I-labeled proteins of Mrs 210,000 an...

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

    Directory of Open Access Journals (Sweden)

    Anna Bachmann

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

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

    DEFF Research Database (Denmark)

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

    2003-01-01

    Placenta-sequestered Plasmodium falciparum parasites that cause pregnancy-associated malaria (PAM) in otherwise clinically immune women express distinct variant surface antigens (VSA(PAM)) not expressed by parasites in nonpregnant individuals. We report here that parasites from the peripheral blo...

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

    Directory of Open Access Journals (Sweden)

    Kremsner Peter G

    2004-07-01

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

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

    Science.gov (United States)

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

    2017-07-01

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

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

    DEFF Research Database (Denmark)

    Khattab, Ayman; Reinhardt, Christina; Staalsoe, Trine

    2004-01-01

    BACKGROUND: Pregnancy-associated malaria (PAM) is caused by Plasmodium falciparum-infected erythrocytes that can sequester in placental intervillous space by expressing particular variant surface antigens (VSA) that can mediate adhesion to chondroitin sulfate A (CSA) in vitro. IgG antibodies......: In the study cohort, primiparous as well as secundiparous women had the greatest risk of infection at delivery as well as during pregnancy. Primiparous women with infected placentas at delivery showed higher levels of VSAPAM-specific IgG compared to women who had no malaria infections at delivery. Placental...

  10. Squamous cell carcinoma antigen 1 and 2 mRNA and a new variant expressed in hepatocellular carcinoma.

    Science.gov (United States)

    Li, S; Gao, Y; Yang, B; Liang, Z; Wang, Y; Zhai, D; Jing, L; Liu, T; Wang, F; DU, Z; Wang, Y

    2014-01-01

    New tools for diagnostic of HCC remain to further investigate. We have evaluated the expression of SCCA1, 2 mRNA and their prognostic value in hepatocellular carcinoma (HCC). Reverse transcription polymerase chain reaction (RT-PCR) and direct sequencing were performed to evaluate the mRNA expression of SCCA1 and SCCA2 in 93 HCCs, and 93 paired adjacent non-cancerous tissues (PNT), 16 cirrhosis livers and 9 normal livers. The correlation of SCCA variants expression with the clinical parameters and the factors affecting survival were analyzed statistically.Total SCCA was detected in 33.3% of HCCs (31/93), in 9.68% of PNT (9/93) and 22.2% of normal livers (2/9). No expression was found in cirrhosis livers (0/16). The frequencies of total SCCA expression were significantly higher in HCCs than that in PNT and liver cirrhosis (p = 0.000, 0.006). From mRNA sequencing of HCCs, a new SCCA1 variant (presenting a T357A mutation) was identified in 16 specimens, while wild type SCCA1 was identified in 11 specimens and SCCA2 in 27 specimens. Clinicopathological analysis showed that the frequency of SCCA1 was significantly higher in poorly differentiated HCC, compared with moderately and well differentiated tumors (p = 0.021). T357A variant has a significantly higher frequency in nonencapsulated tumors than wild type SCCA1 (p = 0.034).The SCCA1, 2 mRNA is effective for detecting HCC and could be potentially applied in HCC diagnosis.

  11. Plasmodium falciparum variant surface antigen expression varies between isolates causing severe and nonsevere malaria and is modified by acquired immunity

    DEFF Research Database (Denmark)

    Nielsen, Morten A; Staalsoe, Trine; Kurtzhals, Jørgen

    2002-01-01

    of immunity, as disease-causing parasites appear to be those not controlled by preexisting VSA-specific Abs. In this work we report that VSA expressed by parasites from young Ghanaian children with P. falciparum malaria were commonly and strongly recognized by plasma Abs from healthy children in the same area...... to limit the risk of severe disease by discriminating against the expression of VSA likely to cause life-threatening complications, such as cerebral malaria and severe anemia. Such VSA seem to be preferred by parasites infecting a nonimmune host, suggesting that VSA expression and switching are not random......In areas of endemic parasite transmission, protective immunity to Plasmodium falciparum malaria is acquired over several years with numerous disease episodes. Acquisition of Abs to parasite-encoded variant surface Ags (VSA) on the infected erythrocyte membrane is important in the development...

  12. DNA rearrangements and antigenic variation in Trypanosoma equiperdum: expression-independent DNA rearrangements in the basic copy of a variant surface glycoprotein gene.

    Science.gov (United States)

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

    1983-03-01

    Antigenic variation in Trypanosoma equiperdum is associated with the sequential expression of variant surface glycoprotein (VSG) genes in a process which involves gene duplication and transposition events. In this paper we present evidence that the genomic environment of the VSG-1 basic copy gene, the template for duplicated, expression-linked VSG-1 genes, differs in every trypanosome clone examined. This variation is thus independent of the expression of the VSG-1 gene, and it also appears to be restricted to the 3' genomic environment. It is also demonstrated that the DNA located 3' to the VSG-1 basic copy gene is moderately sensitive to digestion when the nuclei of either expressor or non-expressor trypanosomes are treated with DNase I.

  13. Genomic environment of variant surface antigen genes of Trypanosoma equiperdum.

    Science.gov (United States)

    Raibaud, A; Gaillard, C; Longacre, S; Hibner, U; Buck, G; Bernardi, G; Eisen, H

    1983-01-01

    Expression of variant antigen genes in Trypanosoma equiperdum is accompanied by the duplication of a silent basic copy gene and the transposition of the copy to an expression site elsewhere in the genome. We have analyzed the genomic locations of both the basic and expression-linked copies of the T. equiperdum gene for variable surface glycoprotein VSG-1. Both copies are situated proximal to termini in both extracted DNA and in chromatin. The regions between the VSG-1 genes and the termini have a very high buoyant density in CsCl and contain an unidentified nucleoside that replaces deoxycytidine. Images PMID:6308614

  14. Genomic environment of variant surface antigen genes of Trypanosoma equiperdum.

    OpenAIRE

    Raibaud, A; Gaillard, C; Longacre, S; Hibner, U; Buck, G; Bernardi, G; Eisen, H

    1983-01-01

    Expression of variant antigen genes in Trypanosoma equiperdum is accompanied by the duplication of a silent basic copy gene and the transposition of the copy to an expression site elsewhere in the genome. We have analyzed the genomic locations of both the basic and expression-linked copies of the T. equiperdum gene for variable surface glycoprotein VSG-1. Both copies are situated proximal to termini in both extracted DNA and in chromatin. The regions between the VSG-1 genes and the termini ha...

  15. Overexpression of squamous cell carcinoma antigen variants in hepatocellular carcinoma.

    Science.gov (United States)

    Pontisso, P; Calabrese, F; Benvegnù, L; Lise, M; Belluco, C; Ruvoletto, M G; Marino, M; Valente, M; Nitti, D; Gatta, A; Fassina, G

    2004-02-23

    Pathogenetic mechanisms of hepatocellular carcinoma (HCC) are still unclear and new tools for diagnostic and therapeutic purposes are ongoing. We have assessed whether squamous cell carcinoma antigen (SCCA), a serpin overexpressed in neoplastic cells of epithelial origin, is also expressed in liver cancer. Squamous cell carcinoma antigen was evaluated by immunohistochemistry in 65 HCCs of different aetiology and in 20 normal livers. Proliferative activity was assessed using MIB-1 antibody. In 18 surgical samples, tumour and nontumour liver tissue was available for SCCA cDNA amplification and sequencing. Squamous cell carcinoma antigen was detected in 55 out of 65 (85%) tumour specimens, but in none of the 20 controls. In the majority of the cases, the positive signal was found in the cytoplasm of more than 50% of the hepatocytes. Low or undetectable SCCA (scoreSCCA score >or=2 (mean+/-s.d.: 2%+/-2.4 vs 7.5%+/-10.3, PSCCA1 variant (G(351) to A) was identified in five cases, while SCCA1 was revealed in six cases and SCCA2 in three cases. In conclusion, SCCA variants are overexpressed in HCC, independently of tumour aetiology. A novel SCCA1 variant has been identified in one third of liver tumours.

  16. Isolation and characterization of human rhinovirus antigenic variants

    Energy Technology Data Exchange (ETDEWEB)

    Watson, D.G.

    1985-01-01

    Isolation of antigenic variants of human rhinovirus types 2, 14, and 17 was attempted by plaquing untreated virus (P-isolates), selecting variants in the presence of homologous antiserum (C-isolates), and by selecting variants in the presence of antibody following 5-fluorouracil mutagenesis (M-isolates). All viruses were triple-plaque purified and purity neutralization tested prior to isolate selection. Based on a fourfold reduction in neutralizing antibody titer to homologous antiserum, no antigenic variation was found in P-isolates from the three serotypes examined. Antigenic variants of all three serotypes could be isolated by the antiserum selection method (C-isolates). However, antigenic variants of RV17 were isolated at a much higher frequency and showed a larger degree of variation than those of RV2 and RV14. At least two of the variants selected, RV17 (C301) and RV2 (M803), failed to be neutralized by the known 89 rhinovirus antiserum. SDS-polyacrylamide gel electrophoresis of (/sup 35/S) methionine-labelled virion polypeptides revealed that each serotype had a characteristic pattern and that selected RV2 and RV17 isolates had patterns identical to those of the prototype strains. By isoelectric focusing an antigenic variant of RV2 was shown to contain altered virion polypeptides VP1 and VP2 whereas two RV17 antigenic variants demonstrated alterations only in the VP1 polypeptide.

  17. Geographical and temporal conservation of antibody recognition of Plasmodium falciparum variant surface antigens

    DEFF Research Database (Denmark)

    Nielsen, Morten A; Vestergaard, Lasse S; Lusingu, John

    2004-01-01

    The slow acquisition of protection against Plasmodium falciparum malaria probably reflects the extensive diversity of important antigens. The variant surface antigens (VSA) that mediate parasite adhesion to a range of host molecules are regarded as important targets of acquired protective immunity...... (VSASM) that were better recognized by plasma IgG than VSA expressed by other parasites, but importantly, VSASM-type antigens also appeared to show substantial antigenic homogeneity. Our finding that the repertoire of immunologically distinct VSA in general, and in particular that of VSASM...

  18. Selection of antigenically advanced variants of seasonal influenza viruses

    Science.gov (United States)

    Ozawa, Makoto; Taft, Andrew S.; Das, Subash C.; Hanson, Anthony P.; Song, Jiasheng; Imai, Masaki; Wilker, Peter R.; Watanabe, Tokiko; Watanabe, Shinji; Ito, Mutsumi; Iwatsuki-Horimoto, Kiyoko; Russell, Colin A.; James, Sarah L.; Skepner, Eugene; Maher, Eileen A.; Neumann, Gabriele; Kelso, Anne; McCauley, John; Wang, Dayan; Shu, Yuelong; Odagiri, Takato; Tashiro, Masato; Xu, Xiyan; Wentworth, David E.; Katz, Jacqueline M.; Cox, Nancy J.; Smith, Derek J.; Kawaoka, Yoshihiro

    2016-01-01

    Influenza viruses mutate frequently, necessitating constant updates of vaccine viruses. To establish experimental approaches that may complement the current vaccine strain selection process, we selected antigenic variants from human H1N1 and H3N2 influenza virus libraries possessing random mutations in the globular head of the haemagglutinin protein (which includes the antigenic sites) by incubating them with human and/or ferret convalescent sera to human H1N1 and H3N2 viruses. Further, we selected antigenic escape variants from human viruses treated with convalescent sera and from mice that had been previously immunized against human influenza viruses. Our pilot studies with past influenza viruses identified escape mutants that were antigenically similar to variants that emerged in nature, establishing the feasibility of our approach. Our studies with contemporary human influenza viruses identified escape mutants before they caused an epidemic in 2014–2015. This approach may aid in the prediction of potential antigenic escape variants and the selection of future vaccine candidates before they become widespread in nature. PMID:27572841

  19. Selection of antigenically advanced variants of seasonal influenza viruses

    NARCIS (Netherlands)

    Li, C. (Chengjun); Hatta, M. (Masato); D.F. Burke (David); Ping, J. (Jihui); Zhang, Y. (Ying); Ozawa, M. (Makoto); Taft, A.S. (Andrew S.); Das, S.C. (Subash C.); Hanson, A.P. (Anthony P.); Song, J. (Jiasheng); M. Imai; Wilker, P.R. (Peter R.); Watanabe, T. (Tokiko); Watanabe, S. (Shinji); Ito, M. (Mutsumi); Iwatsuki-Horimoto, K. (Kiyoko); C.A. Russell (Colin); S.L. James (Sarah ); E. Skepner (Eugene); E. Maher (Eileen); G. Neumann (Gabriele); A. Klimov (Alexander); A. Kelso; McCauley, J. (John); D. Wang (Dayan); Y.L. Shu (Yue-Long); T. Odagiri (Takato); Tashiro, M. (Masato); X. Xu (Xiyan); Wentworth, D.E. (David E.); J. Katz (Jacqueline); N.J. Cox (Nancy); D.J. Smith (Derek James); Y. Kawaoka (Yoshihiro)

    2016-01-01

    textabstractInfluenza viruses mutate frequently, necessitating constant updates of vaccine viruses. To establish experimental approaches that may complement the current vaccine strain selection process, we selected antigenic variants from human H1N1 and H3N2 influenza virus libraries possessing

  20. Canine parvovirus: the worldwide occurrence of antigenic variants.

    Science.gov (United States)

    Miranda, Carla; Thompson, Gertrude

    2016-09-01

    The most important enteric virus infecting canids is canine parvovirus type 2 (CPV-2). CPV is the aetiologic agent of a contagious disease, mainly characterized by clinical gastroenteritis signs in younger dogs. CPV-2 emerged as a new virus in the late 1970s, which could infect domestic dogs, and became distributed in the global dog population within 2 years. A few years later, the virus's original type was replaced by a new genetic and antigenic variant, called CPV-2a. Around 1984 and 2000, virus variants with the single change to Asp or Glu in the VP2 residue 426 were detected (sometimes termed CPV-2b and -2c). The genetic and antigenic changes in the variants have also been correlated with changes in their host range; in particular, in the ability to replicate in cats and also host range differences in canine and other tissue culture cells. CPV-2 variants have been circulating among wild carnivores and have been well-documented in several countries around the world. Here, we have reviewed and summarized the current information about the worldwide distribution and evolution of CPV-2 variants since they emerged, as well as the host ranges they are associated with.

  1. In vitro selection of Plasmodium falciparum 3D7 for expression of variant surface antigens associated with severe malaria in African children

    DEFF Research Database (Denmark)

    Staalsoe, Trine; Nielsen, Morten A; Vestergaard, Lasse S

    2003-01-01

    . Parasites isolated from young children with severe malaria (SM) tend to express a limited and conserved set of VSA (VSASM) that are both stronger and more commonly recognized by IgG in the plasma of malaria-exposed individuals than VSA (VSAUM) expressed by parasites causing uncomplicated malaria (UM...... and epidemiologically diverse areas of endemic parasite transmission. The described selection method appears a useful tool in the identification of genes encoding VSA involved in severe and life-threatening P. falciparum malaria....

  2. Malaria-induced acquisition of antibodies to Plasmodium falciparum variant surface antigens

    DEFF Research Database (Denmark)

    Ofori, Michael F; Dodoo, Daniel; Staalsoe, Trine

    2002-01-01

    In areas of intense Plasmodium falciparum transmission, protective immunity is acquired during childhood in parallel with acquisition of agglutinating antibodies to parasite-encoded variant surface antigens (VSA) expressed on parasitized red blood cells. In a semi-immune child in such an area......, clinical disease is caused mainly by parasites expressing VSA not recognized by preexisting VSA-specific antibodies in that child. Such malaria episodes are known to cause an increase in agglutinating antibodies specifically recognizing VSA expressed by the parasite isolate causing the illness, whereas...

  3. A novel category of antigens enabling CTL immunity to tumor escape variants: Cinderella antigens.

    Science.gov (United States)

    Seidel, Ursula J E; Oliveira, Claudia C; Lampen, Margit H; Hall, Thorbald van

    2012-01-01

    Deficiencies in MHC class I antigen presentation are a common feature of tumors and allows escape from cytotoxic T lymphocyte (CTL)-mediated killing. It is crucial to take this capacity of tumors into account for the development of T-cell-based immunotherapy, as it may strongly impair their effectiveness. A variety of escape mechanisms has been described thus far, but progress in counteracting them is poor. Here we review a novel strategy to target malignancies with defects in the antigenic processing machinery (APM). The concept is based on a unique category of CD8+ T-cell epitopes that is associated with impaired peptide processing, which we named TEIPP. We characterized this alternative peptide repertoire emerging in MHC-I on tumors lacking classical antigen processing due to defects in the peptide transporter TAP (transporter associated with peptide processing). These TEIPPs exemplify interesting parallels with the folktale figure Cinderella: they are oppressed and neglected by a stepmother (like functional TAP prevents TEIPP presentation), until the suppression is released and Cinderella/TEIPP achieves unexpected recognition. TEIPP-specific CTLs and their cognate peptide-epitopes provide a new strategy to counteract immune evasion by APM defects and bear potential to targeting escape variants observed in a wide range of cancers.

  4. Improvements and Variants of the Multiple Antigen Blot Assay-MABA: An Immunoenzymatic Technique for Simultaneous Antigen and Antibody Screening.

    Science.gov (United States)

    Noya, Oscar; Losada, Sandra; Toledo, Marilyan; Gauna, Adriana; Lorenzo, María Angelita; Bermúdez, Henry; de Noya, Belkisyolé Alarcón

    2015-01-01

    This simple, versatile, reliable, reproducible, multipurpose, and inexpensive technique is based on the adhesion of different antigens to a single nitrocellulose strip using, as template, an acrylic device containing 28 parallel channels. The inclusion of channels containing normal human serum improves the quality control of this assay. Antigen-sensitized nitrocellulose strips are cut perpendicularly to the antigen-rows, exposed to immune sera followed by the appropriate conjugate. Positive signals are recorded using chemiluminescent or precipitable colorimetric substrates. This assay allows the simultaneous qualitative demonstration of antigenicity and immunogenicity of antigens obtained as synthetic peptides, recombinant molecules, or crude preparations, with high sensitivity and specificity. Its major value is based on the rapid and simultaneous comparative evaluation of various antigenic preparations allowing the diagnosis of a variety of infectious, allergic, and autoimmune diseases. It can in general be used to detect any type of antibody or circulating antigen. Some improvements and variants of the original technique are included.

  5. Induction of endoplasmic reticulum-derived oxidative stress by an occult infection related S surface antigen variant.

    Science.gov (United States)

    Lee, In-Kyung; Lee, Seoung-Ae; Kim, Hong; Won, You-Sub; Kim, Bum-Joon

    2015-06-14

    To investigate the mechanism of endoplasmic reticulum (ER) stress induction by an occult infection related hepatitis B virus S surface antigen (HBsAg) variant. We used an HBsAg variant with lower secretion capacity, which was a KD variant from a Korean subject who was occultly infected with the genotype C. We compared the expression profiles of ER stress-related proteins between HuH-7 cells transfected with HBsAg plasmids of a wild-type and a KD variant using Western blot. Confocal microscopy indicated that the KD variant had higher levels of co-localization with ER than the wild-type HBsAg. The KD variant up-regulated ER stress-related proteins and induced reactive oxygen species (ROS) compared to the wild-type via an increase in calcium. The KD variant also down-regulated anti-oxidant proteins (HO-1, catalase and SOD) compared to the wild-type, which indicates positive amplification loops of the ER-ROS axis. The KD variant also induced apoptotic cell death via the up-regulation of caspase proteins (caspase 6, 9 and 12). Furthermore, the KD variant induced a higher level of nitric oxide than wild-type HBsAg via the up-regulation of the iNOS protein. Our data indicate that occult infection related HBsAg variants can lead to ER-derived oxidative stress and liver cell death in HuH-7 cells.

  6. 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.

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

    African Journals Online (AJOL)

    Yomi

    2012-01-31

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

  8. Absence of kinetoplast DNA in a late antigenic variant of Trypanosoma equiperdum.

    Science.gov (United States)

    Riou, G; Baltz, T; Gabillot, M; Pautrizel, R

    1980-04-01

    We have analysed by several biochemical techniques the DNA components of two antigenic variants isolated from Trypanosoma equiperdum. We did not observe any kinetoplast DNA (kDNA) structures or networks in the late antigen variant BoTat 28. Furthermore, the results of reassociation kinetics of in vitro labelled kDNA show that neither kDNA minicircle sequences nor kDNA maxicircle sequences of BoTat 1, the basic antigen type, can be detected in the total DNA of BoTat 28.

  9. Antibodies to variant antigens on the surfaces of infected erythrocytes are associated with protection from malaria in Ghanaian children

    DEFF Research Database (Denmark)

    Dodoo, D; Staalsoe, T; Giha, H

    2001-01-01

    Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a variant antigen expressed on the surface of infected erythrocytes. Each parasite genome contains about 40 PfEMP1 genes, but only 1 PfEMP1 gene is expressed at a given time. PfEMP1 serves as a parasite-sequestering ligand...... to endothelial cells and enables the parasites to avoid splenic passage. PfEMP1 antibodies may protect from disease by inhibiting sequestration, thus facilitating the destruction of infected erythrocytes in the spleen. In this study, we have measured antibodies in Ghanaian children to a conserved region of PfEMP...... during the season. The prevalences of antibodies to both the conserved PfEMP1 peptide and the variant epitopes were greater than 50%, and the levels of immunoglobulin G (IgG) correlated with age. The levels of antibodies to both the conserved peptide and the variant epitopes were higher in protected than...

  10. Characterization of antigenic variants of hepatitis C virus in immune evasion

    Directory of Open Access Journals (Sweden)

    Hershow Ronald

    2011-07-01

    Full Text Available Abstract Background Antigenic variation is an effective way by which viruses evade host immune defense leading to viral persistence. Little is known about the inhibitory mechanisms of viral variants on CD4 T cell functions. Results Using sythetic peptides of a HLA-DRB1*15-restricted CD4 epitope derived from the non-structural (NS 3 protein of hepatitis C virus (HCV and its antigenic variants and the peripheral blood mononuclear cells (PBMC from six HLA-DRB1*15-positive patients chronically infected with HCV and 3 healthy subjects, the in vitro immune responses and the phenotypes of CD4+CD25+ cells of chronic HCV infection were investigated. The variants resulting from single or double amino acid substitutions at the center of the core region of the Th1 peptide not only induce failed T cell activation but also simultaneously up-regulate inhibitory IL-10, CD25-TGF-β+ Th3 and CD4+IL-10+ Tr1 cells. In contrast, other variants promote differentiation of CD25+TGF-β+ Th3 suppressors that attenuate T cell proliferation. Conclusions Naturally occuring HCV antigenic mutants of a CD4 epitope can shift a protective peripheral Th1 immune response into an inhibitory Th3 and/or Tr1 response. The modulation of antigenic variants on CD4 response is efficient and extensive, and is likely critical in viral persistence in HCV infection.

  11. Structure-guided evolution of antigenically distinct adeno-associated virus variants for immune evasion.

    Science.gov (United States)

    Tse, Longping Victor; Klinc, Kelli A; Madigan, Victoria J; Castellanos Rivera, Ruth M; Wells, Lindsey F; Havlik, L Patrick; Smith, J Kennon; Agbandje-McKenna, Mavis; Asokan, Aravind

    2017-06-13

    Preexisting neutralizing antibodies (NAbs) against adeno-associated viruses (AAVs) pose a major, unresolved challenge that restricts patient enrollment in gene therapy clinical trials using recombinant AAV vectors. Structural studies suggest that despite a high degree of sequence variability, antibody recognition sites or antigenic hotspots on AAVs and other related parvoviruses might be evolutionarily conserved. To test this hypothesis, we developed a structure-guided evolution approach that does not require selective pressure exerted by NAbs. This strategy yielded highly divergent antigenic footprints that do not exist in natural AAV isolates. Specifically, synthetic variants obtained by evolving murine antigenic epitopes on an AAV serotype 1 capsid template can evade NAbs without compromising titer, transduction efficiency, or tissue tropism. One lead AAV variant generated by combining multiple evolved antigenic sites effectively evades polyclonal anti-AAV1 neutralizing sera from immunized mice and rhesus macaques. Furthermore, this variant displays robust immune evasion in nonhuman primate and human serum samples at dilution factors as high as 1:5, currently mandated by several clinical trials. Our results provide evidence that antibody recognition of AAV capsids is conserved across species. This approach can be applied to any AAV strain to evade NAbs in prospective patients for human gene therapy.

  12. Novel Non-Histocompatibility Antigen Mismatched Variants Improve the Ability to Predict Antibody-Mediated Rejection Risk in Kidney Transplant

    Directory of Open Access Journals (Sweden)

    Silvia Pineda

    2017-12-01

    Full Text Available Transplant rejection is the critical clinical end-point limiting indefinite survival after histocompatibility antigen (HLA mismatched organ transplantation. The predominant cause of late graft loss is antibody-mediated rejection (AMR, a process whereby injury to the organ is caused by donor-specific antibodies, which bind to HLA and non-HLA (nHLA antigens. AMR is incompletely diagnosed as donor/recipient (D/R matching is only limited to the HLA locus and critical nHLA immunogenic antigens remain to be identified. We have developed an integrative computational approach leveraging D/R exome sequencing and gene expression to predict clinical post-transplant outcome. We performed a rigorous statistical analysis of 28 highly annotated D/R kidney transplant pairs with biopsy-confirmed clinical outcomes of rejection [either AMR or T-cell-mediated rejection (CMR] and no-rejection (NoRej, identifying a significantly higher number of mismatched nHLA variants in AMR (ANOVA—p-value = 0.02. Using Fisher’s exact test, we identified 123 variants associated mainly with risk of AMR (p-value < 0.001. In addition, we applied a machine-learning technique to circumvent the issue of statistical power and we found a subset of 65 variants using random forest, that are predictive of post-tx AMR showing a very low error rate. These variants are functionally relevant to the rejection process in the kidney and AMR as they relate to genes and/or expression quantitative trait loci (eQTLs that are enriched in genes expressed in kidney and vascular endothelium and underlie the immunobiology of graft rejection. In addition to current D/R HLA mismatch evaluation, additional mismatch nHLA D/R variants will enhance the stratification of post-tx AMR risk even before engraftment of the organ. This innovative study design is applicable in all solid organ transplants, where the impact of mitigating AMR on graft survival may be greater, with considerable benefits on

  13. 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.

  14. 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.

  15. TYPE-SPECIFIC ANTIGENS, M AND T, OF MATT AND GLOSSY VARIANTS OF GROUP A HEMOLYTIC STREPTOCOCCI.

    Science.gov (United States)

    Lancefield, R C

    1940-03-31

    1. Two qualitatively different type-specific antigens, designated M and T, have been found present in matt variants of group A hemolytic streptococci, but only one of these, the T antigen, occurs in the degraded glossy variant. 2. The protein nature of the M antigen, present in matt variants only, has been demonstrated in previous work, but the chemical characteristics of the newly recognized antigenic factor, T, present in both variants, have not been determined. This T factor is identified only by its immunological reactions. It is unknown whether the two type-specific antigenic factors, M and T, occur as separate chemical entities in the matt variant or in conjugation. 3. Antibody to the type-specific protein, M, appears responsible for the M precipitin reaction, for type-specific protection, and, as a rule, for part of the type-specific agglutination of matt variants, but in type 1 it does not cause agglutination. 4. Antibody to the second type-specific antigen, T, seems to be solely responsible for type-specific agglutination of the glossy form and to play a large rôle in type-specific agglutination of the matt form, but apparently it is not involved in protection. This T antibody causes all of the type-specific agglutination of type 1. Consequently, type 1 matt and glossy variants agglutinate and absorb agglutinin alike, and antisera to both are identical in content of type-specific agglutinin though they differ in respect to M antibody. 5. Recognition of the principle underlying type-specific agglutination of glossy variants makes it possible to suggest, with certain reservations, the use of glossy variants for type classification by agglutination. These variants are suitable for preparing type-specific agglutinating antisera, and they form stable suspensions for use in the reaction. Improved methods are needed for deriving glossy from matt variants.

  16. 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.

  17. A LAIR-1 insertion generates broadly reactive antibodies against malaria variant antigens

    OpenAIRE

    Tan, Joshua; Pieper, Kathrin; Piccoli, Luca; Abdi, Abdirahman; Perez, Mathilde Foglierini; Geiger, Roger; Tully, Claire Maria; Jarrossay, David; Maina Ndungu, Francis; Wambua, Juliana; Bejon, Philip; Fregni, Chiara Silacci; Fernandez-Rodriguez, Blanca; Barbieri, Sonia; Bianchi, Siro

    2015-01-01

    Plasmodium falciparum antigens expressed on the surface of infected erythrocytes are important targets of naturally acquired immunity against malaria, but their high number and variability provide the pathogen with a powerful means of escape from host antibodies 1?4 . Although broadly reactive antibodies against these antigens could be useful as therapeutics and in vaccine design, their identification has proven elusive. Here, we report the isolation of human monoclonal antibodies that recogn...

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

    Science.gov (United States)

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

    1983-03-01

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

  19. The role of Plasmodium falciparum variant surface antigens in protective immunity and vaccine development

    DEFF Research Database (Denmark)

    Hviid, Lars

    2010-01-01

    that development of PfEMP1-based vaccines to protect specifically against severe malaria syndromes-in particular PAM-is feasible. This review summarizes the evidence that VSAs are important targets of NAI, discusses why VSA-based vaccines might be feasible despite the extensive intra- and interclonal variation...... of VSAs, and how vaccines based on this type of antigens fit into the current global strategy to reduce, eliminate and eventually eradicate the burden of malaria.......There is substantial immuno-epidemiological evidence that the parasite-encoded, so-called variant surface antigens (VSAs), such as PfEMP1 on the surface of infected erythrocytes (IEs) are important-in some cases probably decisive determinants of clinical outcome of P. falciparum malaria...

  20. The role of Plasmodium falciparum variant surface antigens in protective immunity and vaccine development

    DEFF Research Database (Denmark)

    Hviid, Lars

    2010-01-01

    that development of PfEMP1-based vaccines to protect specifically against severe malaria syndromes-in particular PAM-is feasible. This review summarizes the evidence that VSAs are important targets of NAI, discusses why VSA-based vaccines might be feasible despite the extensive intra- and interclonal variation...... of VSAs, and how vaccines based on this type of antigens fit into the current global strategy to reduce, eliminate and eventually eradicate the burden of malaria.......There is substantial immuno-epidemiological evidence that the parasite-encoded, so-called variant surface antigens (VSAs) such as PfEMP1 on the surface of infected erythrocytes (IEs) are important-in some cases probably decisive-determinants of clinical outcome of P. falciparum malaria...

  1. Identification of Enterococcus faecalis antigens specifically expressed in vivo

    OpenAIRE

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

    2015-01-01

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

  2. Neutralizing monoclonal antibodies recognize antigenic variants among isolates of infectious hematopoietic necrosis virus

    Science.gov (United States)

    Winton, J.R.; Arakawa, C.N.; Lannan, C.N.; Fryer, J.L.

    1988-01-01

    eutralizing monoclonal antibodies were developed against strains of infectious hematopoietic necrosis virus (IHNV) from steelhead trout Salmo gairdneri in the Deschutes River of Oregon, chinook salmon Oncorhynchus tshawytscha in the Sacramento River of California, and rainbow trout Salmo gairdneri reared in the Hagerman Valley of Idaho, USA. These antibodies were tested for neutralization of 12 IHNV isolates obtained from salmonids in Japan, Alaska, Washington, Oregon, California, and Idaho. The antibodies recognized antigenic variants among the isolates and could be used to separate the viruses into 4 groups. The members of each group tended to be related by geographic area rather than by source host species, virulence, or date of isolation.

  3. Variant surface antigen-specific IgG and protection against clinical consequences of pregnancy-associated Plasmodium falciparum malaria

    DEFF Research Database (Denmark)

    Staalsoe, Trine; Shulman, Caroline E; Bulmer, Judith N

    2004-01-01

    BACKGROUND: Pregnancy-associated malaria caused by Plasmodium falciparum adherence to chondroitin sulfate A in the placental intervillous space is a major cause of low birthweight and maternal anaemia in areas of endemic P falciparum transmission. Adhesion-blocking antibodies that specifically...... recognise parasite-encoded variant surface antigens (VSA) are associated with resistance to pregnancy-associated malaria. We looked for a possible relation between VSA-specific antibody concentrations, placental infection, and protection from low birthweight and maternal anaemia. METHODS: We used flow...... selected or not selected for chondroitin sulfate A adhesiveness in-vitro. FINDINGS: Concentrations of plasma IgG specific for VSA, expressed by chondroitin sulfate A-adhering parasites (VSA in pregnancy-associated malaria or vsa-pam), increased with gravidity and were associated with placental histological...

  4. Recruitment of PfSET2 by RNA polymerase II to variant antigen encoding loci contributes to antigenic variation in P. falciparum.

    Directory of Open Access Journals (Sweden)

    Uchechi E Ukaegbu

    2014-01-01

    Full Text Available Histone modifications are important regulators of gene expression in all eukaryotes. In Plasmodium falciparum, these epigenetic marks regulate expression of genes involved in several aspects of host-parasite interactions, including antigenic variation. While the identities and genomic positions of many histone modifications have now been cataloged, how they are targeted to defined genomic regions remains poorly understood. For example, how variant antigen encoding loci (var are targeted for deposition of unique histone marks is a mystery that continues to perplex the field. Here we describe the recruitment of an ortholog of the histone modifier SET2 to var genes through direct interactions with the C-terminal domain (CTD of RNA polymerase II. In higher eukaryotes, SET2 is a histone methyltransferase recruited by RNA pol II during mRNA transcription; however, the ortholog in P. falciparum (PfSET2 has an atypical architecture and its role in regulating transcription is unknown. Here we show that PfSET2 binds to the unphosphorylated form of the CTD, a property inconsistent with its recruitment during mRNA synthesis. Further, we show that H3K36me3, the epigenetic mark deposited by PfSET2, is enriched at both active and silent var gene loci, providing additional evidence that its recruitment is not associated with mRNA production. Over-expression of a dominant negative form of PfSET2 designed to disrupt binding to RNA pol II induced rapid var gene expression switching, confirming both the importance of PfSET2 in var gene regulation and a role for RNA pol II in its recruitment. RNA pol II is known to transcribe non-coding RNAs from both active and silent var genes, providing a possible mechanism by which it could recruit PfSET2 to var loci. This work unifies previous reports of histone modifications, the production of ncRNAs, and the promoter activity of var introns into a mechanism that contributes to antigenic variation by malaria parasites.

  5. Increased expression of the type IV secretion system in piliated Neisseria gonorrhoeae variants.

    Science.gov (United States)

    Salgado-Pabón, Wilmara; Du, Ying; Hackett, Kathleen T; Lyons, Katelynn M; Arvidson, Cindy Grove; Dillard, Joseph P

    2010-04-01

    Neisseria gonorrhoeae produces a type IV secretion system that secretes chromosomal DNA. The secreted DNA is active in the transformation of other gonococci in the population and may act to transfer antibiotic resistance genes and variant alleles for surface antigens, as well as other genes. We observed that gonococcal variants that produced type IV pili secreted more DNA than variants that were nonpiliated, suggesting that the process may be regulated. Using microarray analysis, we found that a piliated strain showed increased expression of the gene for the putative type IV secretion coupling protein TraD, whereas a nonpiliated variant showed increased expression of genes for transcriptional and translational machinery, consistent with its higher growth rate compared to that of the piliated strain. These results suggested that type IV secretion might be controlled by either traD expression or growth rate. A mutant with a deletion in traD was found to be deficient in DNA secretion. Further mutation and complementation analysis indicated that traD is transcriptionally and translationally coupled to traI, which encodes the type IV secretion relaxase. We were able to increase DNA secretion in a nonpiliated strain by inserting a gene cassette with a strong promoter to drive the expression of the putative operon containing traI and traD. Together, these data suggest a model in which the type IV secretion system apparatus is made constitutively, while its activity is controlled through regulation of traD and traI.

  6. Rational development and characterization of humanized anti–EGFR variant III chimeric antigen receptor T cells for glioblastoma

    Science.gov (United States)

    Johnson, Laura A.; Scholler, John; Ohkuri, Takayuki; Kosaka, Akemi; Patel, Prachi R.; McGettigan, Shannon E.; Nace, Arben K.; Dentchev, Tzvete; Thekkat, Pramod; Loew, Andreas; Boesteanu, Alina C.; Cogdill, Alexandria P.; Chen, Taylor; Fraietta, Joseph A.; Kloss, Christopher C.; Posey, Avery D.; Engels, Boris; Singh, Reshma; Ezell, Tucker; Idamakanti, Neeraja; Ramones, Melissa H.; Li, Na; Zhou, Li; Plesa, Gabriela; Seykora, John T.; Okada, Hideho; June, Carl H.; Brogdon, Jennifer L.; Maus, Marcela V.

    2015-01-01

    Chimeric antigen receptors (CARs) are synthetic molecules designed to redirect T cells to specific antigens. CAR-modified T cells can mediate long-term durable remissions in B cell malignancies, but expanding this platform to solid tumors requires the discovery of surface targets with limited expression in normal tissues. The variant III mutation of the epidermal growth factor receptor (EGFRvIII) results from an in-frame deletion of a portion of the extracellular domain, creating a neoepitope. We chose a vector backbone encoding a second-generation CAR based on efficacy of a murine scFv–based CAR in a xenograft model of glioblastoma. Next, we generated a panel of humanized scFvs and tested their specificity and function as soluble proteins and in the form of CAR-transduced T cells; a low-affinity scFv was selected on the basis of its specificity for EGFRvIII over wild-type EGFR. The lead candidate scFv was tested in vitro for its ability to direct CAR-transduced T cells to specifically lyse, proliferate, and secrete cytokines in response to antigen-bearing targets. We further evaluated the specificity of the lead CAR candidate in vitro against EGFR-expressing keratinocytes and in vivo in a model of mice grafted with normal human skin. EGFRvIII-directed CAR T cells were also able to control tumor growth in xenogeneic subcutaneous and orthotopic models of human EGFRvIII+ glioblastoma. On the basis of these results, we have designed a phase 1 clinical study of CAR T cells transduced with humanized scFv directed to EGFRvIII in patients with either residual or recurrent glioblastoma (NCT02209376). PMID:25696001

  7. Rational development and characterization of humanized anti-EGFR variant III chimeric antigen receptor T cells for glioblastoma.

    Science.gov (United States)

    Johnson, Laura A; Scholler, John; Ohkuri, Takayuki; Kosaka, Akemi; Patel, Prachi R; McGettigan, Shannon E; Nace, Arben K; Dentchev, Tzvete; Thekkat, Pramod; Loew, Andreas; Boesteanu, Alina C; Cogdill, Alexandria P; Chen, Taylor; Fraietta, Joseph A; Kloss, Christopher C; Posey, Avery D; Engels, Boris; Singh, Reshma; Ezell, Tucker; Idamakanti, Neeraja; Ramones, Melissa H; Li, Na; Zhou, Li; Plesa, Gabriela; Seykora, John T; Okada, Hideho; June, Carl H; Brogdon, Jennifer L; Maus, Marcela V

    2015-02-18

    Chimeric antigen receptors (CARs) are synthetic molecules designed to redirect T cells to specific antigens. CAR-modified T cells can mediate long-term durable remissions in B cell malignancies, but expanding this platform to solid tumors requires the discovery of surface targets with limited expression in normal tissues. The variant III mutation of the epidermal growth factor receptor (EGFRvIII) results from an in-frame deletion of a portion of the extracellular domain, creating a neoepitope. We chose a vector backbone encoding a second-generation CAR based on efficacy of a murine scFv-based CAR in a xenograft model of glioblastoma. Next, we generated a panel of humanized scFvs and tested their specificity and function as soluble proteins and in the form of CAR-transduced T cells; a low-affinity scFv was selected on the basis of its specificity for EGFRvIII over wild-type EGFR. The lead candidate scFv was tested in vitro for its ability to direct CAR-transduced T cells to specifically lyse, proliferate, and secrete cytokines in response to antigen-bearing targets. We further evaluated the specificity of the lead CAR candidate in vitro against EGFR-expressing keratinocytes and in vivo in a model of mice grafted with normal human skin. EGFRvIII-directed CAR T cells were also able to control tumor growth in xenogeneic subcutaneous and orthotopic models of human EGFRvIII(+) glioblastoma. On the basis of these results, we have designed a phase 1 clinical study of CAR T cells transduced with humanized scFv directed to EGFRvIII in patients with either residual or recurrent glioblastoma (NCT02209376). Copyright © 2015, American Association for the Advancement of Science.

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

    Science.gov (United States)

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

    2013-03-01

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

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

    Science.gov (United States)

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

    2005-10-01

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

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

    Directory of Open Access Journals (Sweden)

    Stacey A Lapp

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

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Gallegos Itandehui Belem

    2012-12-01

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

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

    Science.gov (United States)

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

    2017-06-01

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

  14. Differential expression of the Escherichia coli autoaggregation factor antigen 43

    DEFF Research Database (Denmark)

    Schembri, Mark; Hjerrild, Louise; Gjermansen, Morten

    2003-01-01

    Antigen 43 (Ag43) is a self-recognizing surface adhesin found in most Escherichia coli strains. Due to its excellent cell-to-cell aggregation characteristics, Ag43 expression confers clumping and fluffing of cells and promotes biofilm formation. Ag43 expression is repressed by the cellular redox......-forming potential of E. coli. Finally, we demonstrated that Ag43-mediated cell aggregation confers significant protection against hydrogen peroxide killing....

  15. High affinity antigen recognition of the dual specific variants of herceptin is entropy-driven in spite of structural plasticity.

    Directory of Open Access Journals (Sweden)

    Jenny Bostrom

    Full Text Available The antigen-binding site of Herceptin, an anti-human Epidermal Growth Factor Receptor 2 (HER2 antibody, was engineered to add a second specificity toward Vascular Endothelial Growth Factor (VEGF to create a high affinity two-in-one antibody bH1. Crystal structures of bH1 in complex with either antigen showed that, in comparison to Herceptin, this antibody exhibited greater conformational variability, also called "structural plasticity". Here, we analyzed the biophysical and thermodynamic properties of the dual specific variants of Herceptin to understand how a single antibody binds two unrelated protein antigens. We showed that while bH1 and the affinity-improved bH1-44, in particular, maintained many properties of Herceptin including binding affinity, kinetics and the use of residues for antigen recognition, they differed in the binding thermodynamics. The interactions of bH1 and its variants with both antigens were characterized by large favorable entropy changes whereas the Herceptin/HER2 interaction involved a large favorable enthalpy change. By dissecting the total entropy change and the energy barrier for dual interaction, we determined that the significant structural plasticity of the bH1 antibodies demanded by the dual specificity did not translate into the expected increase of entropic penalty relative to Herceptin. Clearly, dual antigen recognition of the Herceptin variants involves divergent antibody conformations of nearly equivalent energetic states. Hence, increasing the structural plasticity of an antigen-binding site without increasing the entropic cost may play a role for antibodies to evolve multi-specificity. Our report represents the first comprehensive biophysical analysis of a high affinity dual specific antibody binding two unrelated protein antigens, furthering our understanding of the thermodynamics that drive the vast antigen recognition capacity of the antibody repertoire.

  16. Identification of Enterococcus faecalis antigens specifically expressed in vivo

    Science.gov (United States)

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

    2015-01-01

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

  17. In vivo switching between variant surface antigens in human Plasmodium falciparum infection

    DEFF Research Database (Denmark)

    Staalsoe, Trine; Hamad, Amel A; Hviid, Lars

    2002-01-01

    A semi-immune individual was retrospectively found to have maintained an apparently monoclonal and genotypically stable asymptomatic infection for months after clinical cure of a Plasmodium falciparum malaria episode. Before the attack, the individual had no antibodies to variant surface antigens...

  18. The sickle cell trait is associated with enhanced immunoglobulin G antibody responses to Plasmodium falciparum variant surface antigens.

    NARCIS (Netherlands)

    Cabrera, G.; Cot, M.; Migot-Nabias, F.; Kremsner, P.G.; Deloron, P.; Luty, A.J.F.

    2005-01-01

    The sickle cell trait (HbAS) protects against severe Plasmodium falciparum malaria in young African children. We investigated the extent of the association between HbAS and antibodies directed to parasite-derived variant surface antigens (VSAs) on the membrane of infected erythrocytes. We measured

  19. 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...

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

    Science.gov (United States)

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

    1987-12-01

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

  1. Insecticide-treated bed nets reduce plasma antibody levels and limit the repertoire of antibodies to Plasmodium falciparum variant surface antigens

    DEFF Research Database (Denmark)

    Askjaer, N; Maxwell, C; Chambo, W

    2001-01-01

    variant surface antigens (VSA) are important in the development of naturally acquired immunity to Plasmodium falciparum malaria and may thus be good indicators of immune status. We have compared the levels of VSA antibodies in plasma from children who have used ITN for 4 years to levels in plasma from...... children from a nearby village not using ITN. A total of 97 plasma samples were analyzed using 13 different P. falciparum isolates. We found that the children using ITN had significantly lower VSA antibody levels and recognized a smaller proportion of the VSA expressed by the tested parasite isolates than...... children not using ITN....

  2. Utility of immunohistochemical analysis of KAI1, epithelial-specific antigen, and epithelial-related antigen for distinction of chromophobe renal cell carcinoma, an eosinophilic variant from renal oncocytoma.

    Science.gov (United States)

    Ohe, Chisato; Kuroda, Naoto; Takasu, Kosho; Senzaki, Hideto; Shikata, Nobuaki; Yamaguchi, Tadanori; Miyasaka, Chika; Nakano, Yorika; Sakaida, Noriko; Uemura, Yoshiko

    2012-06-01

    Distinction of renal oncocytoma (RO) from chromophobe renal cell carcinoma (ChRCC) is important because their clinical behavior is different. As part of a search for the best available immunohistochemical markers to distinguish ChRCC from RO, we investigated the immunohistochemical profiles of these tumors. We selected 30 renal tumors consisting of ChRCC, typical variant (n = 14), ChRCC, eosinophilic variant (n = 6), and RO (n = 10). Their expression of cytokeratin (CK) 7, KAI1, epithelial-specific antigen (ESA), epithelial-related antigen (ERA), Claudin- 7, and Claudin-8 was studied using an autostainer. Immunoreactivity was assessed based on a combined score of the extent and intensity of staining. Compared to RO, a significantly higher percentage of the total ChRCCs stained positive for CK7 (85% vs. 10%, respectively), KAI1 (90% vs. 10%), ESA (95% vs. 10%), ERA (95% vs. 10%), and Claudin-7 (95% vs. 20%) (P < 0.001). Additionally, there was a significant difference between the percentage of ChRCC eosinophilic variant (ChRCC-E) and RO that stained positive for KAI1 (100% vs. 10%, respectively), ESA (83% vs. 10%), and ERA (83% vs. 10%) (P < 0.001). We recommend immunohistochemical analysis of KAI1, ESA, and ERA to distinguish ChRCC-E from RO.

  3. Immune selection and within-host competition can structure the repertoire of variant surface antigens in Plasmodium falciparum -a mathematical model

    DEFF Research Database (Denmark)

    van Noort, Sander P; Nunes, Marta C; Weedall, Gareth D

    2010-01-01

    BACKGROUND: The evolutionary mechanisms structuring the expression pattern of variant surface antigen (VSA) families that allow pathogens to evade immune responses and establish chronic and repeated infections pose major challenges to theoretical research. In Plasmodium falciparum, the best......-studied VSA family is erythrocyte membrane protein 1 (PfEMP1). Each parasite genome encodes about 60 PfEMP1 variants, which are important virulence factors and major targets of host antibody responses. Transcriptional switching is the basis of clonal PfEMP1 variation and immune evasion. A relatively conserved......-host and diverse blocks that are favoured by immune selection at the population level. CONCLUSIONS/SIGNIFICANCE: The application of a monotonic dominance profile to VSAs encoded by a gene family generates two opposing selective forces and, consequently, two distinct clusters of genes emerge in adaptation to naïve...

  4. 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.

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

    Science.gov (United States)

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

    2012-08-01

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

  6. Expression of CD44 variants in human inflammatory synovitis

    Energy Technology Data Exchange (ETDEWEB)

    Hale, L.P. [Duke Univ. Medical Center, Durham, NC (United States); Haynes, B.F.; McCachren, S. [Duke Univ. Arthritis Center, Durham, NC (United States)

    1995-11-01

    The cell surface hyaluronate receptor CD44 has previously been shown to have immunomodulatory activity and to be upregulated in inflammatory synovitis. Since these findings were reported, the genomic structure of CD44 has been delineated, and multiple splice variants have been described. Therefore, we determined which CD44 variant exons are present during inflammatory synovitis by a combination of Northern blot analysis and reverse transcription followed by polymerase chain reaction amplification of synovial RNA. Immunohistochemical staining was used to define the sites of expression of individual v6 and v9 exons in synovial tissue. The standard (S) or hematopoietic isoform, CD44S, was the predominant form of CD44 expressed in synovium and was expressed by most cell types. Other isoforms, containing alternatively spliced exons in the proximal extracellular domain, were found by RT-PCR, but at lower levels than CD44S. The second most prevalent form was CD44E, which has an insertion of three exons (v8-v10) in the proximal extracellular domain. Immunohistochemical studies showed that reactivity with v9-specific antibodies was primarily in macrophages, particularly those in the synovial lining layer. CD44 exon v6, previously reported to be important in immune activation and in epithelial tumor metastasis, was also expressed in synovial lining cells and in occasional synovial interstitial cells. The presence of CD44 variants containing v9 in rheumatoid synovial macrophages may be important in the adhesion and activation of mononuclear phagocytes in the synovium and, thus, may be a target for novel antiinflammatory therapies in the future. The role of CD44 isoforms in cellular adhesion, immune activation, and joint erosion in inflammatory synovitis deserves further study. 7 figs., 2 tabs., 56 refs.

  7. Hepatitis B surface antigen variants in voluntary blood donors in Nanjing, China

    Directory of Open Access Journals (Sweden)

    Yong-lin Yang

    2012-04-01

    Full Text Available Abstract Background Hepatitis B virus (HBV is still one of the serious infectious risks for the blood transfusion safety in China. One plausible reason is the emergence of the variants in the major antigenic alpha determinant within the major hydrophilic region (MHR of hepatitis B surface antigen (HBsAg, which have been assumed to evade the immune surveillance and pose a challenge to the disease diagnosis. It is well documented that some commercial ELISA kits could detect the wild-type but not the mutant viruses. The high prevalence of HBV in China also impaired the application of nucleic acid testing (NAT in the improvement of blood security. Molecular epidemiological study of HBsAg variations in China is still limited. This study was designed to identify the prevalence of mutations in the HBsAg in voluntary blood donors in Nanjing, China. Methods A total of 20,326 blood units were enrolled in this study, 39 donors were positive for HBV S gene in the nested-PCR. Mutations in the major hydrophilic region (MHR; aa 99-169 were identified by direct sequencing of S region. Results Among of 20,326 blood units in the Red Cross Transfusion Center of Nanjing from October 2008 to April 2009, 296 samples (1.46%, 296/20,326 were HBsAg positive in the 2 successive rounds of the ELISA test. In these HBsAg positive units, HBV S gene could be successfully amplified from 39 donors (13.18%, 39/296 in the nested-PCR. Sequence analysis revealed that 32 strains (82.1%, 32/39 belong to genotype B, 7 strains (17.9%, 7/39 to genotype C. Besides well known G145R, widely dispersed variations in the MHR of S region, were observed in 20 samples of all the strains sequenced. Conclusions HBV/B and HBV/C are dominant in Nanjing, China. The mutations in the MHR of HBsAg associated with disease diagnosis are common.

  8. Nine-year longitudinal study of antibodies to variant antigens on the surface of Plasmodium falciparum-infected erythrocytes

    DEFF Research Database (Denmark)

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

    1999-01-01

    PfEMP1 is an antigenically variable molecule which mediates the adhesion of parasitized erythrocytes to a variety of cell types and which is believed to constitute an important target for naturally acquired protective immune responses in malaria. For 9 years we have monitored individuals living...... in an area of low-intensity, seasonal, and unstable malaria transmission in eastern Sudan, and we have used this database to study the acquisition, specificity, and duration of the antibody response to variant parasitized erythrocyte surface antigens. Both the levels and the spectrum of reactivity...... of these antibodies varied considerably among individuals, ranging from low levels of antibodies recognizing only few parasitized erythrocyte surface antigens to high levels of broad-specificity antibodies. In general, episodes of clinical malaria were associated with increases in the levels of parasitized...

  9. Expression of androgen receptor splice variants in clinical breast cancers.

    Science.gov (United States)

    Hickey, Theresa E; Irvine, Connie M; Dvinge, Heidi; Tarulli, Gerard A; Hanson, Adrienne R; Ryan, Natalie K; Pickering, Marie A; Birrell, Stephen N; Hu, Dong Gui; Mackenzie, Peter I; Russell, Roslin; Caldas, Carlos; Raj, Ganesh V; Dehm, Scott M; Plymate, Stephen R; Bradley, Robert K; Tilley, Wayne D; Selth, Luke A

    2015-12-29

    The importance of androgen receptor (AR) signaling is increasingly being recognized in breast cancer, which has elicited clinical trials aimed at assessing the efficacy of androgen deprivation therapy (ADT) for metastatic disease. In prostate cancer, resistance to ADT is frequently associated with the emergence of androgen-independent splice variants of the AR (AR variants, AR-Vs) that lack the LBD and are constitutively active. Women with breast cancer may be prone to a similar phenomenon. Herein, we show that in addition to the prototypical transcript, the AR gene produces a diverse range of AR-V transcripts in primary breast tumors. The most frequently and highly expressed variant was AR-V7 (exons 1/2/3/CE3), which was detectable at the mRNA level in > 50% of all breast cancers and at the protein level in a subset of ERα-negative tumors. Functionally, AR-V7 is a constitutively active and ADT-resistant transcription factor that promotes growth and regulates a transcriptional program distinct from AR in ERα-negative breast cancer cells. Importantly, we provide ex vivo evidence that AR-V7 is upregulated by the AR antagonist enzalutamide in primary breast tumors. These findings have implications for treatment response in the ongoing clinical trials of ADT in breast cancer.

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

    Directory of Open Access Journals (Sweden)

    Talha Sheikh M

    2012-11-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-08-15

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

  12. Characterisation of centromere (kinetochore) antigen reactive with sera of patients with a scleroderma variant (CREST syndrome).

    Science.gov (United States)

    Nishikai, M; Okano, Y; Yamashita, H; Watanabe, M

    1984-01-01

    Anticentromere (kinetochore) antibody is the marker antibody in CREST syndrome, but the precise molecular composition of the partner antigen has been poorly defined. This report describes for the first time a procedure for the successful extraction and biochemical characterisation of the centromere antigen molecule. The centromere antigen was extracted with 4M NaCl solution. The molecular weight of the partner antigen of the centromere antibody was determined to be 70 000 daltons by the SDS-PAGE and immunoblotting methods. A Sephacryl S-300 column experiment confirmed these results. Centromere antigenic activity was preserved at pHs between 3 and 11 and was resistant to three enzymes, trypsin, RNase, and DNase. Images PMID:6524984

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

    DEFF Research Database (Denmark)

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

    2010-01-01

    The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) antigens play a major role in cytoadhesion of infected erythrocytes (IE), antigenic variation, and immunity to malaria. The current consensus on control of variant surface antigen expression is that only one PfEMP1 encoded by one var...... gene is expressed per cell at a time. We measured var mRNA transcript levels by real-time Q-PCR, analysed var gene transcripts by single-cell FISH and directly compared these with PfEMP1 antigen surface expression and cytoadhesion in three different antibody-selected P. falciparum 3D7 sub-lines using......-line was found to bind both CD31/PECAM1 and CD54/ICAM1 and to adhere twice as efficiently to human endothelial cells, compared to infected cells having only one PfEMP1 variant on the surface. These new results on PfEMP1 antigen expression indicate that a re-evaluation of the molecular mechanisms involved in P...

  14. Molecular characterization of atypical antigenic variants of canine rabies virus reveals its reintroduction by wildlife vectors in southeastern Mexico.

    Science.gov (United States)

    Garcés-Ayala, Fabiola; Aréchiga-Ceballos, Nidia; Ortiz-Alcántara, Joanna M; González-Durán, Elizabeth; Pérez-Agüeros, Sandra I; Méndez-Tenorio, Alfonso; Torres-Longoria, Belem; López-Martínez, Irma; Hernández-Rivas, Lucía; Díaz-Quiñonez, José Alberto; Ramírez-González, José Ernesto

    2017-12-01

    Rabies is an infectious viral disease that is practically always fatal following the onset of clinical signs. In Mexico, the last case of human rabies transmitted by dogs was reported in 2006 and canine rabies has declined significantly due to vaccination campaigns implemented in the country. Here we report on the molecular characterization of six rabies virus strains found in Yucatan and Chiapas, remarkably, four of them showed an atypical reaction pattern when antigenic characterization with a reduced panel of eight monoclonal antibodies was performed. Phylogenetic analyses on the RNA sequences unveiled that the three atypical strains from Yucatan are associated with skunks. Analysis using the virus entire genome showed that they belong to a different lineage distinct from the variants described for this animal species in Mexico. The Chiapas atypical strain was grouped in a lineage that was considered extinct, while the others are clustered within classic dog variants.

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

    Indian Academy of Sciences (India)

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

  16. Novel Plasmodium falciparum malaria vaccines: evidence-based searching for variant surface antigens as candidates for vaccination against pregnancy-associated malaria

    DEFF Research Database (Denmark)

    Staalsoe, Trine; Jensen, Anja T R; Theander, Thor G

    2002-01-01

    Malaria vaccine development has traditionally concentrated on careful molecular, biochemical, and immunological characterisation of candidate antigens. In contrast, evidence of the importance of identified antigens in immunity to human infection and disease has generally been limited......) in particular, to provide robust evidence of a causal link between the two in order to allow efficient and evidence-based identification of candidate antigens for malaria vaccine development....... to statistically significant co-variation with protection rather than on demonstration of causal relationships. We have studied the relationship between variant surface antigen-specific antibodies and clinical protection from Plasmodium falciparum malaria in general, and from pregnancy-associated malaria (PAM...

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

    Science.gov (United States)

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

    2015-12-01

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

  18. 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.

  19. Prevalence of naturally occurring surface gene variants of hepatitis B virus in nonimmunized surface antigen-negative Chinese carriers.

    Science.gov (United States)

    Hou, J; Wang, Z; Cheng, J; Lin, Y; Lau, G K; Sun, J; Zhou, F; Waters, J; Karayiannis, P; Luo, K

    2001-11-01

    Previous studies have suggested that hepatitis B virus (HBV) variants may account for the presence of HBV DNA in hepatitis B surface antigen (HBsAg)-negative patients (occult HBV infection). However, it is not known how widespread these variants are and how they influence the course of liver disease. To determine the prevalence of variants within the major hydrophilic region (MHR) of HBsAg, we investigated 2,565 subjects, including subjects with chronic hepatitis, cryptogenic cirrhosis, hemodialysis patients, and blood donors. Fifty-one of them had occult HBV infection. The entire S gene from 46 of these patients was sequenced from amplified serum HBV DNA. Forty-three percent (20 of 46) had mutations in the MHR of HBsAg. Thirty-two amino acid substitutions between positions 100-160 of the MHR of HBsAg were detected in 18 patients, and these ranged from 1 to 4 per patient. These changes involved 11 positions inside and 5 outside of the historical first and second loops of the "a" determinant, and included the following: Q101K, T115A, K122N, T123A, T126N, Q129N, G130R, T131I, M133T, F134L, C138Y, K141E, P142S, G145R, N146S, and C147F/R. Combinations of mutations were detected in 9 patients, and 7 of these have not been described before. Two further patients had insertion mutations immediately before the "a" determinant. Monoclonal antibody binding tests with the Royal Free hepatitis B surface (RFHBs) panel of antibodies revealed decreased immunoreactivity in 6 novel variants of HBsAg. The existence of patients with occult HBV infection caused by HBsAg variants, therefore, has implications for their possible transmission through sexual contact and by blood transfusion.

  20. 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.

  1. A unique variant of streptococcal group O-antigen (C-polysaccharide) that lacks phosphocholine

    DEFF Research Database (Denmark)

    Bergström, N; Jansson, P.-E.; Kilian, Mogens

    2003-01-01

    Streptococcus mitis strain SK598, which represents a subgroup of biovar 1, possesses a unique variant of the C-polysaccharide found in the cell wall of all strains of Streptococcus pneumoniae and in some strains of S. mitis. This new variant lacks the choline methyl groups in contrast to the prev......Streptococcus mitis strain SK598, which represents a subgroup of biovar 1, possesses a unique variant of the C-polysaccharide found in the cell wall of all strains of Streptococcus pneumoniae and in some strains of S. mitis. This new variant lacks the choline methyl groups in contrast...... to the previously characterized forms of C-polysaccharide, which all contain one or two choline residues per repeat. The following structure of the repeating unit of the SK598 polysaccharide was established: where AAT is 2-acetamido-4-amino-2,4,6-trideoxy-d-galactose. This structure is identical to the double...... choline-substituted form of C-polysaccharide, except that it is substituted with ethanolamine instead of choline. This extends the number of recognized C-polysaccharide variants to four....

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

    Directory of Open Access Journals (Sweden)

    G Barresi

    2009-12-01

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

  3. Using multiple linear regression and physicochemical changes of amino acid mutations to predict antigenic variants of influenza A/H3N2 viruses.

    Science.gov (United States)

    Cui, Haibo; Wei, Xiaomei; Huang, Yu; Hu, Bin; Fang, Yaping; Wang, Jia

    2014-01-01

    Among human influenza viruses, strain A/H3N2 accounts for over a quarter of a million deaths annually. Antigenic variants of these viruses often render current vaccinations ineffective and lead to repeated infections. In this study, a computational model was developed to predict antigenic variants of the A/H3N2 strain. First, 18 critical antigenic amino acids in the hemagglutinin (HA) protein were recognized using a scoring method combining phi (ϕ) coefficient and information entropy. Next, a prediction model was developed by integrating multiple linear regression method with eight types of physicochemical changes in critical amino acid positions. When compared to other three known models, our prediction model achieved the best performance not only on the training dataset but also on the commonly-used testing dataset composed of 31878 antigenic relationships of the H3N2 influenza virus.

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

    Directory of Open Access Journals (Sweden)

    Giuseppe Sconocchia

    2014-01-01

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

  5. Determinants of variant surface antigen antibody response in severe Plasmodium falciparum malaria in an area of low and unstable malaria transmission

    DEFF Research Database (Denmark)

    A-Elgadir, T M E; Theander, T G; Elghazali, G

    2006-01-01

    The variant surface antigens (VSA) of infected erythrocytes are important pathogenic markers, a set of variants (VSA(SM)), were assumed to be associated with severe malaria (SM), while SM constitutes clinically diverse forms, such as, severe malarial anemia (SMA) and cerebral malaria (CM). This s......The variant surface antigens (VSA) of infected erythrocytes are important pathogenic markers, a set of variants (VSA(SM)), were assumed to be associated with severe malaria (SM), while SM constitutes clinically diverse forms, such as, severe malarial anemia (SMA) and cerebral malaria (CM...... range of isolates had a higher level of VSA Ab against the recognized isolates (correlation coefficient, 0.727, PSMA (P....001). Parasites obtained from patients with SMA or from children were better recognized than isolates obtained from patients with uncomplicated malaria or from adults, P

  6. Expression and secretion of defined cutinase variants by Aspergillus awamori

    NARCIS (Netherlands)

    Gemeren, I.A. van; Beijersbergen, A.; Hondel, C.A.M.J.J. van den; Verrips, C.T.

    1998-01-01

    Several cutinase variants derived by molecular modelling and site- directed mutagenesis of a cutinase gene from Fusarium solani pisi are poorly secreted by Saccharomyces cerevisiae. The majority of these variants are successfully produced by the filamentous fungus Aspergillus awamori. However, the

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

    Science.gov (United States)

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

    2005-01-01

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

  8. JC Polyomavirus Infection Is Strongly Controlled by Human Leucocyte Antigen Class II Variants

    DEFF Research Database (Denmark)

    Sundqvist, Emilie; Buck, Dorothea; Warnke, Clemens

    2014-01-01

    mark infection occur only in 50-60% of infected individuals, and high JCV-antibody titers seem to increase the risk of developing PML. We here investigated the role of human leukocyte antigen (HLA), instrumental in immune defense in JCV antibody response. Anti-JCV antibody status, as a surrogate...... antibody response and human leucocyte antigens supports the notion that CD4+ T cells are crucial in the immune defence to JCV and lays the ground for risk stratification for PML and development of therapy and prevention.......JC polyomavirus (JCV) carriers with a compromised immune system, such as in HIV, or subjects on immune-modulating therapies, such as anti VLA-4 therapy may develop progressive multifocal leukoencephalopathy (PML) which is a lytic infection of oligodendrocytes in the brain. Serum antibodies to JCV...

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

    Science.gov (United States)

    Zhang, Ye; Pappas, Dimitri

    2016-02-21

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1984-08-01

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

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

    Science.gov (United States)

    Epstein, L M; Forney, J D

    1984-08-01

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

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

    DEFF Research Database (Denmark)

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

    2014-01-01

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

  13. Genetic variant as a marker for bladder cancer therapy

    Science.gov (United States)

    Patients who have inherited a specific common genetic variant develop bladder cancer tumors that strongly express a protein known as prostate stem cell antigen (PSCA), which is also expressed in many pancreatic and prostate tumors, according to research a

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

    DEFF Research Database (Denmark)

    Heron, I; Hokland, M; Berg, K

    1979-01-01

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

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

    Science.gov (United States)

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

    1991-01-01

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

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

    OpenAIRE

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

    1985-01-01

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

  17. Lenalidomide enhances the function of chimeric antigen receptor T cells against the epidermal growth factor receptor variant III by enhancing immune synapses.

    Science.gov (United States)

    Kuramitsu, S; Ohno, M; Ohka, F; Shiina, S; Yamamichi, A; Kato, A; Tanahashi, K; Motomura, K; Kondo, G; Kurimoto, M; Senga, T; Wakabayashi, T; Natsume, A

    2015-10-01

    The epidermal growth factor receptor variant III (EGFRvIII) is exclusively expressed on the cell surface in ~50% of glioblastoma multiforme (GBM). This variant strongly and persistently activates the phosphatidylinositol 3-kinase-Akt signaling pathway in a ligand-independent manner resulting in enhanced tumorigenicity, cellular motility and resistance to chemoradiotherapy. Our group generated a recombinant single-chain variable fragment (scFv) antibody specific to the EGFRvIII, referred to as 3C10-scFv. In the current study, we constructed a lentiviral vector transducing the chimeric antigen receptor (CAR) that consisted of 3C10-scFv, CD3ζ, CD28 and 4-1BB (3C10-CAR). The 3C10-CAR-transduced peripheral blood mononuclear cells (PBMCs) and CD3(+) T cells specifically lysed the glioma cells that express EGFRvIII. Moreover, we demonstrated that CAR CD3(+) T cells migrated to the intracranial xenograft of GBM in the mice treated with 3C10-CAR PBMCs. An important and novel finding of our study was that a thalidomide derivative lenalidomide induced 3C10-CAR PBMC proliferation and enhanced the persistent antitumor effect of the cells in vivo. Lenalidomide also exhibited enhanced immunological synapses between the effector cells and the target cells as determined by CD11a and F-actin polymerization. Collectively, lentiviral-mediated transduction of CAR effectors targeting the EGFRvIII showed specific efficacy, and lenalidomide even intensified CAR cell therapy by enhanced formation of immunological synapses.

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

    Directory of Open Access Journals (Sweden)

    Barrie Rooney

    2015-12-01

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

  19. Expression of 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.

  20. P System antigenic determiners expression in Ascaris lumbricoides

    Directory of Open Access Journals (Sweden)

    Ponce De León Patricia

    2003-01-01

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

  1. 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.

  2. Multiple common variants for celiac disease influencing immune gene expression

    NARCIS (Netherlands)

    Dubois, Patrick C. A.; Trynka, Gosia; Franke, Lude; Hunt, Karen A.; Romanos, Jihane; Curtotti, Alessandra; Zhernakova, Alexandra; Heap, Graham A. R.; Adany, Roza; Aromaa, Arpo; Bardella, Maria Teresa; van den Berg, Leonard H.; Bockett, Nicholas A.; de la Concha, Emilio G.; Dema, Barbara; Fehrmann, Rudolf S. N.; Fernandez-Arquero, Miguel; Fiatal, Szilvia; Grandone, Elvira; Green, Peter M.; Groen, Harry J. M.; Gwilliam, Rhian; Houwen, Roderick H. J.; Hunt, Sarah E.; Kaukinen, Katri; Kelleher, Dermot; Korponay-Szabo, Ilma; Kurppa, Kalle; MacMathuna, Padraic; Maki, Markku; Mazzilli, Maria Cristina; McCann, Owen T.; Mearin, M. Luisa; Mein, Charles A.; Mirza, Muddassar M.; Mistry, Vanisha; Mora, Barbara; Morley, Katherine I.; Mulder, Chris J.; Murray, Joseph A.; Nunez, Concepcion; Oosterom, Elvira; Ophoff, Roel A.; Polanco, Isabel; Peltonen, Leena; Platteel, Mathieu; Rybak, Anna; Salomaa, Veikko; Schweizer, Joachim J.; Sperandeo, Maria Pia; Tack, Greetje J.; Turner, Graham; Veldink, Jan H.; Verbeek, Wieke H. M.; Weersma, Rinse K.; Wolters, Victorien M.; Urcelay, Elena; Cukrowska, Bozena; Greco, Luigi; Neuhausen, Susan L.; McManus, Ross; Barisani, Donatella; Deloukas, Panos; Barrett, Jeffrey C.; Saavalainen, Paivi; Wijmenga, Cisca; van Heel, David A.

    We performed a second-generation genome-wide association study of 4,533 individuals with celiac disease (cases) and 10,750 control subjects. We genotyped 113 selected SNPs with P(GWAS) <10(-4) and 18 SNPs from 14 known loci in a further 4,918 cases and 5,684 controls. Variants from 13 new regions

  3. JC polyomavirus infection is strongly controlled by human leucocyte antigen class II variants.

    Directory of Open Access Journals (Sweden)

    Emilie Sundqvist

    2014-04-01

    Full Text Available JC polyomavirus (JCV carriers with a compromised immune system, such as in HIV, or subjects on immune-modulating therapies, such as anti VLA-4 therapy may develop progressive multifocal leukoencephalopathy (PML which is a lytic infection of oligodendrocytes in the brain. Serum antibodies to JCV mark infection occur only in 50-60% of infected individuals, and high JCV-antibody titers seem to increase the risk of developing PML. We here investigated the role of human leukocyte antigen (HLA, instrumental in immune defense in JCV antibody response. Anti-JCV antibody status, as a surrogate for JCV infection, were compared to HLA class I and II alleles in 1621 Scandinavian persons with MS and 1064 population-based Swedish controls and associations were replicated in 718 German persons with MS. HLA-alleles were determined by SNP imputation, sequence specific (SSP kits and a reverse PCR sequence-specific oligonucleotide (PCR-SSO method. An initial GWAS screen displayed a strong HLA class II region signal. The HLA-DRB1*15 haplotype was strongly negatively associated to JCV sero-status in Scandinavian MS cases (OR = 0.42, p = 7×10(-15 and controls (OR = 0.53, p = 2×10(-5. In contrast, the DQB1*06:03 haplotype was positively associated with JCV sero-status, in Scandinavian MS cases (OR = 1.63, p = 0.006, and controls (OR = 2.69, p = 1×10(-5. The German dataset confirmed these findings (OR = 0.54, p = 1×10(-4 and OR = 1.58, p = 0.03 respectively for these haplotypes. HLA class II restricted immune responses, and hence CD4+ T cell immunity is pivotal for JCV infection control. Alleles within the HLA-DR1*15 haplotype are associated with a protective effect on JCV infection. Alleles within the DQB1*06:03 haplotype show an opposite association. These associations between JC virus antibody response and human leucocyte antigens supports the notion that CD4+ T cells are crucial in the immune defence to JCV and

  4. Expression of CD44 splice variants in human primary brain tumors

    NARCIS (Netherlands)

    Kaaijk, P.; Troost, D.; Morsink, F.; Keehnen, R. M.; Leenstra, S.; Bosch, D. A.; Pals, S. T.

    1995-01-01

    Expression of CD44, particularly of certain splice variants, has been linked to tumor progression and metastatic potential in a number of different animal and human cancers. Although differential expression of CD44 standard epitopes (CD44s) in human brain tumors has been reported, the expression of

  5. Association of breast cancer risk with genetic variants showing differential allelic expression

    DEFF Research Database (Denmark)

    Hamdi, Yosr; Soucy, Penny; Adoue, Véronique

    2016-01-01

    There are significant inter-individual differences in the levels of gene expression. Through modulation of gene expression, cis-acting variants represent an important source of phenotypic variation. Consequently, cis-regulatory SNPs associated with differential allelic expression are functional...

  6. [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.

  7. The monoclonal antibody SM5-1 recognizes a fibronectin variant which is widely expressed in melanoma

    Directory of Open Access Journals (Sweden)

    Guo Yajun

    2006-01-01

    Full Text Available Abstract Background Previously we have generated the monoclonal antibody SM5-1 by using a subtractive immunization protocol of human melanoma. This antibody exhibits a high sensitivity for primary melanomas of 99% (248/250 tested and for metastatic melanoma of 96% (146/151 tested in paraffin embedded sections. This reactivity is superior to the one obtained by HMB-45, anti-MelanA or anti-Tyrosinase and is comparable to anti-S100. However, as compared to anti-S100, the antibody SM5-1 is highly specific for melanocytic lesions since 40 different neoplasms were found to be negative for SM5-1 by immunohistochemistry. The antigen recognized by SM5-1 is unknown. Methods In order to characterize the antigen recognized by mAb SM5-1, a cDNA library was constructed from the metastatic human melanoma cell line SMMUpos in the Uni-ZAP lambda phage and screened by mAb SM5-1. The cDNA clones identified by this approach were then sequenced and subsequently analyzed. Results Sequence analysis of nine independent overlapping clones (length 3100–5600 bp represent fibronectin cDNA including the ED-A, but not the ED-B region which are produced by alternative splicing. The 89aa splicing variant of the IIICS region was found in 8/9 clones and the 120aa splicing variant in 1/9 clones, both of which are included in the CS1 region of fibronectin being involved in melanoma cell adhesion and spreading. Conclusion The molecule recognized by SM5-1 is a melanoma associated FN variant expressed by virtually all primary and metastatic melanomas and may play an important role in melanoma formation and progression. This antibody is therefore not only of value in immunohistochemistry, but potentially also for diagnostic imaging and immunotherapy.

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

    DEFF Research Database (Denmark)

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

    1989-01-01

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

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

    Science.gov (United States)

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

    1985-08-01

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

  10. 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

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

    Science.gov (United States)

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

    2008-12-01

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

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

    DEFF Research Database (Denmark)

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

    1993-01-01

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

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

    Science.gov (United States)

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

    2013-09-15

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

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

    Science.gov (United States)

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

    2017-03-30

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

  15. Successful antiviral therapy determines a significant decrease in squamous cell carcinoma antigen-associated (SCCA) variants' serum levels in anti-HCV positive cirrhotic patients.

    Science.gov (United States)

    Giannini, E G; Basso, M; Bazzica, M; Contini, P; Marenco, S; Savarino, V; Picciotto, A

    2010-08-01

    Aberrant squamous cell carcinoma antigen (SCCA) expression is an early event in hepatocarcinogenesis, and increasing serum levels of SCCA variants IgM immune complexes (SCCA-IgM IC) have been found in cirrhotic patients developing hepatocellular carcinoma (HCC). We longitudinally evaluated a cohort of cirrhotic patients with hepatitis C virus infection (HCV) who underwent pegylated interferon (PEG-IFN) and ribavirin treatment. SCCA-IgM IC levels were assessed in the sera of 33 cirrhotic patients with HCV (21 males, median age 57 years) before, at the end and at 6-month and 1-year follow-up after treatment with PEG-IFN and ribavirin. SCCA-IgM IC serum levels (arbitrary units/mL, AU/mL) were evaluated according to treatment outcome: sustained virological response (SVR) vs nonresponse (NR). Overall, 15 patients obtained a SVR to antiviral therapy (45%). There was no significant difference in baseline SCCA-IgM IC serum levels between SVR and NR patients. When compared to baseline (451.2 AU/mL), SVR patients showed a significant decrease in median SCCA-IgM IC serum levels at the end of treatment (186.8 AU/mL, P = 0.013) and at both 6-month (96.8 AU/mL, P SCCA-IC serum levels. Because of the pathophysiological correlation between SCCA and liver carcinogenesis, it is hypothesized that in patients with liver cirrhosis, SVR may be accompanied by a decreased proliferative stimulation.

  16. Correlation of presentation of insulin with surface I-Ad and A alpha and A beta mRNA expression by cloned B lymphoma hybridoma variants.

    Science.gov (United States)

    Bernard, N F; Reid, P C; Phillips, M L; Delovitch, T L

    1988-10-01

    We investigated the relationship between Ia expression and antigen presentation in cloned B cells, using variants of TA3 antigen presenting cells. Two TA3 subclones were selected as high presenters and 5 as low presenters of insulin to pork insulin/I-Ad restricted T cells. All TA3 subclones express the surface I-Ak, I-Ek, I-Ad and I-Ed Ia antigens characteristic of the parental cell line. However, surface I-Ad levels correlated best with the ability to present insulin, since high presenters express 2- to 4-fold more I-Ad than low presenters. High presenters possess 2- to 4-times more A alpha and A beta Ia mRNA than low presenters and also transcribe these mRNAs 2- to 5-fold faster than most low presenters. Thus, the correlation noted between I-Ad surface density and capacity to present insulin by our panel of TA3 variants is regulated at the level of transcription and not translation of I-Ad specific mRNAs.

  17. 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

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Science.gov (United States)

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

    2010-10-15

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

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

    Science.gov (United States)

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

    2015-02-01

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

  1. Multiple common variants for celiac disease influencing immune gene expression

    OpenAIRE

    MCMANUS, ROSS; KELLEHER, DERMOT

    2010-01-01

    PUBLISHED We performed a second-generation genome-wide association study of 4,533 individuals with celiac disease (cases) and 10,750 control subjects. We genotyped 113 selected SNPs with P(GWAS) < 10(-4) and 18 SNPs from 14 known loci in a further 4,918 cases and 5,684 controls. Variants from 13 new regions reached genome-wide significance (P(combined) < 5 x 10(-8)); most contain genes with immune functions (BACH2, CCR4, CD80, CIITA-SOCS1-CLEC16A, ICOSLG and ZMIZ1), with ETS1, RUNX3, THEMI...

  2. Cloning, expression, purification and characterization of tryptophan hydroxylase variants

    DEFF Research Database (Denmark)

    Boesen, Jane

    such as depression and obsessive-compulsive disorder (OCD). Characterization of TPH and elucidation of the enzymes regulation and catalytic mechanism is therefore vital to our understanding of the serotonin balance. This study concerns variants of both human TPH isoform 1 (hTPH1) and human TPH isoform 2 (h PH2......Tryptophan hydroxylase (TPH) catalyzes the first and rate-limiting step in the biosynthesis of the neurotransmitter and hormone serotonin (5-hydroxytryptamine). Serotonin is involved in many physiological functions, such as appetite and sleep rhythm, as well as a wide range of psychiatric disorders...

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

    Science.gov (United States)

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

    1995-11-01

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

  4. 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.

  5. Multiple Sclerosis Risk Variant HLA-DRB1*1501 Associates with High Expression of DRB1 Gene in Different Human Populations

    Science.gov (United States)

    Abad-Grau, María del Mar; Fedetz, María; Izquierdo, Guillermo; Lucas, Miguel; Fernández, Óscar; Ndagire, Dorothy; Catalá-Rabasa, Antonio; Ruiz, Agustín; Gayán, Javier; Delgado, Concepción; Arnal, Carmen

    2012-01-01

    The human leukocyte antigen (HLA) DRB1*1501 has been consistently associated with multiple sclerosis (MS) in nearly all populations tested. This points to a specific antigen presentation as the pathogenic mechanism though this does not fully explain the disease association. The identification of expression quantitative trait loci (eQTL) for genes in the HLA locus poses the question of the role of gene expression in MS susceptibility. We analyzed the eQTLs in the HLA region with respect to MS-associated HLA-variants obtained from genome-wide association studies (GWAS). We found that the Tag of DRB1*1501, rs3135388 A allele, correlated with high expression of DRB1, DRB5 and DQB1 genes in a Caucasian population. In quantitative terms, the MS-risk AA genotype carriers of rs3135388 were associated with 15.7-, 5.2- and 8.3-fold higher expression of DQB1, DRB5 and DRB1, respectively, than the non-risk GG carriers. The haplotype analysis of expression-associated variants in a Spanish MS cohort revealed that high expression of DRB1 and DQB1 alone did not contribute to the disease. However, in Caucasian, Asian and African American populations, the DRB1*1501 allele was always highly expressed. In other immune related diseases such as type 1 diabetes, inflammatory bowel disease, ulcerative colitis, asthma and IgA deficiency, the best GWAS-associated HLA SNPs were also eQTLs for different HLA Class II genes. Our data suggest that the DR/DQ expression levels, together with specific structural properties of alleles, seem to be the causal effect in MS and in other immunopathologies rather than specific antigen presentation alone. PMID:22253788

  6. 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

  7. The promoter for a variant surface glycoprotein gene expression site in Trypanosoma brucei

    NARCIS (Netherlands)

    Zomerdijk, J. C.; Ouellette, M.; ten Asbroek, A. L.; Kieft, R.; Bommer, A. M.; Clayton, C. E.; Borst, P.

    1990-01-01

    The variant-specific surface glycoprotein (VSG) gene 221 of Trypanosoma brucei is transcribed as part of a 60 kb expression site (ES). We have identified the promoter controlling this multigene transcription unit by the use of 221 chromosome-enriched DNA libraries and VSG gene 221 expression site

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

    Science.gov (United States)

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

    2016-11-01

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

  9. Structural Analysis of Histo-Blood Group Antigen Binding Specificity in a Norovirus GII.4 Epidemic Variant: Implications for Epochal Evolution

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    Shanker, Sreejesh; Choi, Jae-Mun; Sankaran, Banumathi; Atmar, Robert L.; Estes, Mary K.; Prasad, B.V. Venkataram (Baylor); (LBNL)

    2012-03-23

    Susceptibility to norovirus (NoV), a major pathogen of epidemic gastroenteritis, is associated with histo-blood group antigens (HBGAs), which are also cell attachment factors for this virus. GII.4 NoV strains are predominantly associated with worldwide NoV epidemics with a periodic emergence of new variants. The sequence variations in the surface-exposed P domain of the capsid protein resulting in differential HBGA binding patterns and antigenicity are suggested to drive GII.4 epochal evolution. To understand how temporal sequence variations affect the P domain structure and contribute to epochal evolution, we determined the P domain structure of a 2004 variant with ABH and secretor Lewis HBGAs and compared it with the previously determined structure of a 1996 variant. We show that temporal sequence variations do not affect the binding of monofucosyl ABH HBGAs but that they can modulate the binding strength of difucosyl Lewis HBGAs and thus could contribute to epochal evolution by the potentiated targeting of new variants to Lewis-positive, secretor-positive individuals. The temporal variations also result in significant differences in the electrostatic landscapes, likely reflecting antigenic variations. The proximity of some of these changes to the HBGA binding sites suggests the possibility of a coordinated interplay between antigenicity and HBGA binding in epochal evolution. From the observation that the regions involved in the formation of the HBGA binding sites can be conformationally flexible, we suggest a plausible mechanism for how norovirus disassociates from salivary mucin-linked HBGA before reassociating with HBGAs linked to intestinal epithelial cells during its passage through the gastrointestinal tract.

  10. Genome-Wide Identification, Evolutionary, and Expression Analyses of Histone H3 Variants in Plants

    Directory of Open Access Journals (Sweden)

    Jinteng Cui

    2015-01-01

    Full Text Available Histone variants alter the nucleosome structure and play important roles in chromosome segregation, transcription, DNA repair, and sperm compaction. Histone H3 is encoded by many genes in most eukaryotic species and is the histone that contains the largest variety of posttranslational modifications. Compared with the metazoan H3 variants, little is known about the complex evolutionary history of H3 variants proteins in plants. Here, we study the identification, evolutionary, and expression analyses of histone H3 variants from genomes in major branches in the plant tree of life. Firstly we identified all the histone three related (HTR genes from the examined genomes, then we classified the four groups variants: centromeric H3, H3.1, H3.3 and H3-like, by phylogenetic analysis, intron information, and alignment. We further demonstrated that the H3 variants have evolved under strong purifying selection, indicating the conservation of HTR proteins. Expression analysis revealed that the HTR has a wide expression profile in maize and rice development and plays important roles in development.

  11. Expression of activation-induced cytidine deaminase splicing variants in patients with ankylosing spondylitis.

    Science.gov (United States)

    Kim, Ji-Young; Yoon, Hee-Kyung; Song, Seung Taek; Park, Seok-Rae; Shim, Seung-Cheol

    2017-12-01

    To investigate the expression patterns of activation-induced cytidine deaminase (AID) variants in peripheral blood mononuclear cells (PBMCs) of patients with ankylosing spondylitis (AS) and examine their clinical implications, we isolated PBMCs from healthy controls (HC, n = 33) and patients with AS (n = 62), and measured mRNA expression of AID variants and translesion synthesis (TLS) polymerases using quantitative real-time polymerase chain reaction. The proportion of patients with AS in whom AID splicing variant (sv) 2 was expressed was significantly higher than that of HC (p = .031). 80.7% of AS patients were treated with tumour necrosis factor inhibitor (TNFi). Significantly higher proportion of the TNFi-treated group expressed sv2 compared to the TNF-naïve group (p = .037). And we compared the level of AID variants expression between the TNFi-treated group and the TNF-naïve group. The expression levels of AID full-length (FL) and sv1 were significantly lower in the TNFi-treated group than the TNF-naïve group (FL: p = .002, sv1: p = .045). In addition, we investigated mRNA expression levels of translesion synthesis (TLS) polymerases in PBMCs from patients with AS and HC. The expression level of TLS pol ι was significantly lower in patients with AS than in HC (p = .007). In conclusion, AS patients expressed significantly higher levels of sv2 than HC. TNFi treatment restored the gene expression of the AID variants (FL, sv1, and sv2) in patients with AS. A clear understanding of the underlying cellular and molecular mechanisms will help to identify the pathogenesis of AS better and to develop novel therapeutic targets.

  12. 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

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

    Science.gov (United States)

    Schinstine, M; Iacovitti, L

    1996-09-01

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

  14. Codon-Optimized Luciola Italica Luciferase Variants for Mammalian Gene Expression in Culture and in Vivo

    Directory of Open Access Journals (Sweden)

    Casey A. Maguire

    2012-01-01

    Full Text Available Luciferases have proven to be useful tools in advancing our understanding of biologic processes. Having a multitude of bioluminescent reporters with different properties is highly desirable. We characterized codon-optimized thermostable green- and red-emitting luciferase variants from the Italian firefly Luciola italica for mammalian gene expression in culture and in vivo. Using lentivirus vectors to deliver and stably express these luciferases in mammalian cells, we showed that both variants displayed similar levels of activity and protein half-lives as well as similar light emission kinetics and higher stability compared to the North American firefly luciferase. Further, we characterized the red-shifted variant for in vivo bioluminescence imaging. Intramuscular injection of tumor cells stably expressing this variant into nude mice yielded a robust luciferase activity. Light emission peaked at 10 minutes post-D-luciferin injection and retained > 60% of signal at 1 hour. Similarly, luciferase activity from intracranially injected glioma cells expressing the red-shifted variant was readily detected and used as a marker to monitor tumor growth over time. Overall, our characterization of these codon-optimized luciferases lays the groundwork for their further use as bioluminescent reporters in mammalian cells.

  15. Recombination-driven generation of the largest pathogen repository of antigen variants in the protozoan Trypanosoma cruzi.

    Science.gov (United States)

    Weatherly, D Brent; Peng, Duo; Tarleton, Rick L

    2016-09-13

    The protozoan parasite Trypanosoma cruzi, causative agent of Chagas disease, depends upon a cell surface-expressed trans-sialidase (ts) to avoid activation of complement-mediated lysis and to enhance intracellular invasion. However these functions alone fail to account for the size of this gene family in T. cruzi, especially considering that most of these genes encode proteins lacking ts enzyme activity. Previous whole genome sequencing of the CL Brener clone of T. cruzi identified ~1400 ts variants, but left many partially assembled sequences unannotated. In the current study we reevaluated the trans-sialidase-like sequences in this reference strain, identifying an additional 1779 full-length and partial ts genes with their important features annotated, and confirming the expression of previously annotated "pseudogenes" and newly annotated ts family members. Multiple EM for Motif Elicitation (MEME) analysis allowed us to generate a model T. cruzi ts (TcTS) based upon the most conserved motif patterns and demonstrated that a common motif order is highly conserved among ts family members. Using a newly developed pipeline for the analysis of recombination within large gene families, we further demonstrate that TcTS family members are undergoing frequent recombination, generating new variants from the thousands of functional and non-functional ts gene segments but retaining the overall structure of the core TcTS family members. The number and variety as well as high recombination frequency of TcTS family members supports strong evolutionary pressure, probably exerted by immune selection, for continued variation in ts sequences in T. cruzi, and thus for a unique immune evasion mechanism for the large ts gene family.

  16. Genetic variants regulating expression levels and isoform diversity during embryogenesis.

    Science.gov (United States)

    Cannavò, Enrico; Koelling, Nils; Harnett, Dermot; Garfield, David; Casale, Francesco P; Ciglar, Lucia; Gustafson, Hilary E; Viales, Rebecca R; Marco-Ferreres, Raquel; Degner, Jacob F; Zhao, Bingqing; Stegle, Oliver; Birney, Ewan; Furlong, Eileen E M

    2017-01-19

    Embryonic development is driven by tightly regulated patterns of gene expression, despite extensive genetic variation among individuals. Studies of expression quantitative trait loci (eQTL) indicate that genetic variation frequently alters gene expression in cell-culture models and differentiated tissues. However, the extent and types of genetic variation impacting embryonic gene expression, and their interactions with developmental programs, remain largely unknown. Here we assessed the effect of genetic variation on transcriptional (expression levels) and post-transcriptional (3' RNA processing) regulation across multiple stages of metazoan development, using 80 inbred Drosophila wild isolates, identifying thousands of developmental-stage-specific and shared QTL. Given the small blocks of linkage disequilibrium in Drosophila, we obtain near base-pair resolution, resolving causal mutations in developmental enhancers, validated transcription-factor-binding sites and RNA motifs. This fine-grain mapping uncovered extensive allelic interactions within enhancers that have opposite effects, thereby buffering their impact on enhancer activity. QTL affecting 3' RNA processing identify new functional motifs leading to transcript isoform diversity and changes in the lengths of 3' untranslated regions. These results highlight how developmental stage influences the effects of genetic variation and uncover multiple mechanisms that regulate and buffer expression variation during embryogenesis.

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

    Science.gov (United States)

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

    2017-01-01

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

  18. The BDNF Val66Met Variant Affects Gene Expression through miR-146b

    OpenAIRE

    Hsu, Pei-Ken; Xu, Bin; Mukai, Jun; Karayiorgou, Maria; Gogos, Joseph A.

    2015-01-01

    Variation in gene expression is an important mechanism underlying susceptibility to complex disease and traits. Single nucleotide polymorphisms (SNPs) account for a substantial portion of the total detected genetic variation in gene expression but how exactly variants acting in trans modulate gene expression and disease susceptibility remains largely unknown. The BDNF Val66Met SNP has been associated with a number of psychiatric disorders such as depression, anxiety disorders, schizophrenia a...

  19. Single Nucleotide Variants in Transcription Factors Associate More Tightly with Phenotype than with Gene Expression

    Science.gov (United States)

    Sudarsanam, Priya; Cohen, Barak A.

    2014-01-01

    Mapping the polymorphisms responsible for variation in gene expression, known as Expression Quantitative Trait Loci (eQTL), is a common strategy for investigating the molecular basis of disease. Despite numerous eQTL studies, the relationship between the explanatory power of variants on gene expression versus their power to explain ultimate phenotypes remains to be clarified. We addressed this question using four naturally occurring Quantitative Trait Nucleotides (QTN) in three transcription factors that affect sporulation efficiency in wild strains of the yeast, Saccharomyces cerevisiae. We compared the ability of these QTN to explain the variation in both gene expression and sporulation efficiency. We find that the amount of gene expression variation explained by the sporulation QTN is not predictive of the amount of phenotypic variation explained. The QTN are responsible for 98% of the phenotypic variation in our strains but the median gene expression variation explained is only 49%. The alleles that are responsible for most of the variation in sporulation efficiency do not explain most of the variation in gene expression. The balance between the main effects and gene-gene interactions on gene expression variation is not the same as on sporulation efficiency. Finally, we show that nucleotide variants in the same transcription factor explain the expression variation of different sets of target genes depending on whether the variant alters the level or activity of the transcription factor. Our results suggest that a subset of gene expression changes may be more predictive of ultimate phenotypes than the number of genes affected or the total fraction of variation in gene expression variation explained by causative variants, and that the downstream phenotype is buffered against variation in the gene expression network. PMID:24784239

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

    Directory of Open Access Journals (Sweden)

    Danye Zhang

    2013-05-01

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

  1. 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

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

    DEFF Research Database (Denmark)

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

    2011-01-01

    NAc-II sialyltransferases can synthesize sialyl-Tn. The aim of the present study was to establish whether ST6GalNAc-I is the major enzyme responsible for the expression of sialyl-Tn. We used a model of CHO-ldlD cells producing only MUC1-Tn glycoform and showed that ST6GalNAc-I is the key-enzyme leading to sialyl......Sialyl-Tn is a simple mucin-type carbohydrate antigen aberrantly expressed in gastrointestinal adenocarcinomas and in the precursor lesion intestinal metaplasia. Sialyl-Tn tumour expression is an independent indicator of poor prognosis. We have previously shown in vitro that ST6GalNAc-I and ST6Gal......NAc-I as the major enzyme controlling the expression of cancer-associated sialyl-Tn antigen in gastrointestinal tissues....

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

    Science.gov (United States)

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

    1998-11-09

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

  5. Antigenic variants of influenza A virus, PR8 strain. I. Their development during serial passage in the lungs of partially immune mice.

    Science.gov (United States)

    GERBER, P; LOOSLI, C G; HAMBRE, D

    1955-06-01

    Antigenically different strains of mouse-adapted PR8 influenza A virus have been produced by 17 serial passages of the virus in the lungs of mice immunized with the homologous agent. Comparative serological tests show that the variant strains share antigenic components with the parent strain but the dominant antigen is different. By means of antibody absorption it was shown that the "new" antigenic component of the variant was already present in minor amounts up to the eighth passage and thereafter gained prominence with continued passage in vaccinated mice. Groups of mice vaccinated with either the PR8-S or T(21) virus and having comparable antibody titers showed no growth of virus in the lungs following aid-borne challenge with homologous strains. On the other hand, following heterologous air-borne challenge no deaths occurred, but virus grew in the lungs of both groups of vaccinated mice. Almost unrestricted virus multiplication took place in the lungs of mice vaccinated with the parent strain and challenged with the PR8-T(21) virus which resulted in extensive consolidation. Less virus grew in the lungs of the mice vaccinated with the variant strains and challenged with the PR8-S virus. In these animals only microscopic evidence of changes due to virus growth in the lungs was observed. The successful serial passage of PR8 influenza A virus in immunized animals was dependent on the initial selection of mice with uniformly low H.I. antibody titers as determined on tail blood, and the intranasal instillation of sufficient virus to favor the survival of those virus particles least related to the antibodies present. The epidemiological implications of these observations are discussed briefly.

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

    OpenAIRE

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

    2013-01-01

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

  7. 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.

  8. Liver myofibroblasts of murine origins express mesothelin: Identification of novel rat mesothelin splice variants.

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    Michel Fausther

    Full Text Available Liver myofibroblasts are specialized effector cells that drive hepatic fibrosis, a hallmark process of chronic liver diseases, leading to progressive scar formation and organ failure. Liver myofibroblasts are increasingly recognized as heterogeneous with regards to their origin, phenotype, and functions. For instance, liver myofibroblasts express cell markers that are universally represented such as, ItgαV and Pdgfrβ, or restricted to a given subpopulation such as, Lrat exclusively expressed in hepatic stellate cells, and Gpm6a in mesothelial cells. To study liver myofibroblasts in vitro, we have previously generated and characterized a SV40-immortalized polyclonal rat activated portal fibroblast cell line called RGF-N2 expressing multiple mesothelin mRNA transcripts. Mesothelin, a cell-surface molecule expressed in normal mesothelial cells and overexpressed in several cancers such as, mesothelioma and cholangiocarcinoma, was recently identified as a key regulator of portal myofibroblast proliferation, and fibrosis progression in the setting of chronic cholestatic liver disease. Here, we identify novel mesothelin splice variants expressed in rat activated portal fibroblasts. RGF-N2 portal fibroblast cDNA was used as template for insertion of hemagglutinin tag consensus sequence into the complete open reading frame of rat mesothelin variant coding sequences by extension PCR. Purified amplicons were subsequently cloned into an expression vector for in vitro translation and transfection in monkey COS7 fibroblasts, before characterization of fusion proteins by immunoblot and immunofluorescence. We show that rat activated portal fibroblasts, hepatic stellate cells, and cholangiocarcinoma cells express wild-type mesothelin and additional splice variants, while mouse activated hepatic stellate cells appear to only express wild-type mesothelin. Notably, rat mesothelin splice variants differ from the wild-type isoform by their protein properties and

  9. Cloning and expression of a novel human profilin variant, profilin II

    DEFF Research Database (Denmark)

    Honoré, B; Madsen, Peder; Andersen, A H

    1993-01-01

    We have isolated a 1.7 kbp cDNA encoding a 140 amino acid protein (15.1 kDa, pI 5.91) with a high sequence similarity (62%) to human profilin (profilin I). We have termed this variant profilin II. Northern blot analysis showed that profilin II is highly expressed in brain, skeletal muscle...

  10. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria

    DEFF Research Database (Denmark)

    Andersen, Jens Bo; Sternberg, Claus; Poulsen, Lars K.

    1998-01-01

    Use of the green fluorescent protein (Gfp) from the jellyfish Aequorea victoria ia is a powerful method for nondestructive in situ monitoring, since expression of green fluorescence does not require any substrate addition. To expand the use of Gfp as a reporter protein, new variants have been con...

  11. Construction of Nef-positive doxycycline-dependent HIV-1 variants using bicistronic expression elements

    Energy Technology Data Exchange (ETDEWEB)

    Velden, Yme U. van der; Kleibeuker, Wendy; Harwig, Alex; Klaver, Bep; Siteur-van Rijnstra, Esther; Frankin, Esmay; Berkhout, Ben; Das, Atze T., E-mail: a.t.das@amc.uva.nl

    2016-01-15

    Conditionally replicating HIV-1 variants that can be switched on and off at will are attractive tools for HIV research. We previously developed a genetically modified HIV-1 variant that replicates exclusively when doxycycline (dox) is administered. The nef gene in this HIV-rtTA variant was replaced with the gene encoding the dox-dependent rtTA transcriptional activator. Because loss of Nef expression compromises virus replication in primary cells and precludes studies on Nef function, we tested different approaches to restore Nef production in HIV-rtTA. Strategies that involved translation via an EMCV or synthetic internal ribosome entry site (IRES) failed because these elements were incompatible with efficient virus replication. Fusion protein approaches with the FMDV 2A peptide and human ubiquitin were successful and resulted in genetically-stable Nef-expressing HIV-rtTA strains that replicate more efficiently in primary T-cells and human immune system (HIS) mice than Nef-deficient variants, thus confirming the positive effect of Nef on in vivo virus replication. - Highlights: • Different approaches to encode additional proteins in the HIV-1 genome were tested. • IRES translation elements are incompatible with efficient HIV-1 replication. • Ubiquitin and 2A fusion protein approaches allow efficient HIV-1 replication. • Doxycycline-controlled HIV-1 variants that encode all viral proteins were developed. • Nef stimulates HIV-rtTA replication in primary cells and human immune system mice.

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

    Directory of Open Access Journals (Sweden)

    Agnès Chabanas

    1997-01-01

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

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

    Science.gov (United States)

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

    1992-02-01

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

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

    Science.gov (United States)

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

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

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

    Science.gov (United States)

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

    2013-08-01

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

  16. Association of Cytotoxic T-Lymphocyte Antigen 4 (CTLA4) and Thyroglobulin (TG) Genetic Variants with Autoimmune Hypothyroidism

    Science.gov (United States)

    Patel, Hinal; Mansuri, Mohmmad Shoab; Singh, Mala; Begum, Rasheedunnisa; Shastri, Minal; Misra, Ambikanandan

    2016-01-01

    Autoimmune hypothyroidism is known to be caused by immune responses related to the thyroid gland and its immunological feature includes presence of autoimmune antibodies. Therefore the aim was to analyze presence of anti-TPO antibodies in hypothyroidism patients in Gujarat. Cytotoxic T-Lymphocyte Antigen 4 (CTLA4) is one of the susceptibility genes for various autoimmune diseases. Hence, exon1 +49A/G and 3’UTR CT60A/G single nucleotide polymorphisms (SNPs) in CTLA4 and its mRNA expression levels were investigated in autoimmune hypothyroidism patients. Thyroglobulin (TG) is known to be associated with autoimmune thyroid disorders and thus exon 33 (E33) SNP in TG was investigated. We analyzed the presence of anti-TPO antibodies in the plasma samples of 84 hypothyroidism patients and 62 controls by ELISA. PCR-RFLP technique was used for genotyping of polymorphisms. sCTLA4 and flCTLA4 mRNA expression levels were assessed by real time PCR. 59.52% of hypothyroid patients had anti-TPO antibodies in their circulation. The genotype and allele frequencies differed significantly for +49A/G (p = 0.0004 for +49AG, p = 0.0019 for +49GG & p = 0.0004 for allele), CT60 (p = 0.0110 for CT60AG, p = 0.0005 for CT60GG & phypothyroidism when adjusted for age and gender. Our results suggest +49A/G and CT60 polymorphism of CTLA4 and E33 polymorphism of TG may be genetic risk factors for autoimmune hypothyroidism susceptibility and down regulation of both forms of CTLA4 advocates the crucial role of CTLA4 in pathogenesis of autoimmune hypothyroidism. PMID:26963610

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

    DEFF Research Database (Denmark)

    Ritter, Cathrin; Fan, Kaiji; Paschen, Annette

    2017-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Zeng Zhao-lei

    2012-08-01

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

  19. Increased expression of Interleukin-6 related to nephritis in chickens challenged with an Avian infectious bronchitis virus variant

    Directory of Open Access Journals (Sweden)

    Filipe S. Fernando

    2015-03-01

    Full Text Available A Brazilian field isolate (IBV/Brazil/PR05 of avian infectious bronchitis virus (IBV, associated with development of nephritis in chickens, was previously genotyped as IBV variant after S1 gene sequencing. The aim of this study was to evaluate the levels of IL-6 in kidneys and trachea of birds vaccinated and challenged with IBV/Brazil/PR05 strain, correlating these results with scores of microscopic lesions, specific IBV antigen detection and viral load. The up-regulation of IL-6 and the increased levels of viral load on renal and tracheal samples were significantly correlated with scores of microscopic lesions. Reduced levels of viral load were detected in kidneys of birds previously vaccinated and challenged, compared to non-vaccinated challenged group, although markedly microscopic lesions were observed for both groups. The expression of IL-6, present both in the kidney and in the tracheas, was dependent on the load of the virus present in the tissue, and the development of lesions was related with IL-6 present in the tissues. These data suggest that variant IBV/Brazil/PR05 can induce the expression of proinflammatory cytokines in a manner correlated with viral load and increased IL-6 is involved in the tissue with the influx of inflammatory cells and subsequent nephritis. This may contribute with a model to the development of immunosuppressive agents of IL-6 to prevent acute inflammatory processes against infection with IBV and perhaps other coronaviruses, as well as contribute to the understanding of the immunopathogenesis of IBV nephropatogenic strains.

  20. RBM20 and RBM24 cooperatively promote the expression of short enh splice variants.

    Science.gov (United States)

    Ito, Jumpei; Iijima, Masumi; Yoshimoto, Nobuo; Niimi, Tomoaki; Kuroda, Shun'ichi; Maturana, Andrés D

    2016-07-01

    PDZ-LIM protein ENH1 is a scaffold protein for protein kinases and transcriptional regulators. While ENH1 promotes the hypertrophic growth of cardiomyocytes, its short splice variant (ENH3) prevents the hypertrophic growth. The mechanism underlying the alternative splicing of enh mRNA between ENH short and long isoforms has remained unknown. Here, we found that two splicing factors, RNA-binding motif 20 (RBM20) and RNA-binding motif 24 (RBM24) together promoted the expression of short enh splice variants and bound the 5' intronic region of exon 11 containing an in-phase stop codon. In addition, expression of both RBMs is repressed by hypertrophic stimulations. Collectively, our results suggest that, in healthy conditions, RBM20 and RBM24 cooperate to promote the expression of short ENH isoforms. © 2016 Federation of European Biochemical Societies.

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

    DEFF Research Database (Denmark)

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

    1991-01-01

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

  2. The BDNF Val66Met variant affects gene expression through miR-146b.

    Science.gov (United States)

    Hsu, Pei-Ken; Xu, Bin; Mukai, Jun; Karayiorgou, Maria; Gogos, Joseph A

    2015-05-01

    Variation in gene expression is an important mechanism underlying susceptibility to complex disease and traits. Single nucleotide polymorphisms (SNPs) account for a substantial portion of the total detected genetic variation in gene expression but how exactly variants acting in trans modulate gene expression and disease susceptibility remains largely unknown. The BDNF Val66Met SNP has been associated with a number of psychiatric disorders such as depression, anxiety disorders, schizophrenia and related traits. Using global microRNA expression profiling in hippocampus of humanized BDNF Val66Met knock-in mice we showed that this variant results in dysregulation of at least one microRNA, which in turn affects downstream target genes. Specifically, we show that reduced levels of miR-146b (mir146b), lead to increased Per1 and Npas4 mRNA levels and increased Irak1 protein levels in vitro and are associated with similar changes in the hippocampus of hBDNF(Met/Met) mice. Our findings highlight trans effects of common variants on microRNA-mediated gene expression as an integral part of the genetic architecture of complex disorders and traits. Copyright © 2015 Elsevier Inc. All rights reserved.

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

    OpenAIRE

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

    2016-01-01

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

  4. [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.

  5. Cloning and characterization of a variant surface glycoprotein expression site from Trypanosoma equiperdum.

    Science.gov (United States)

    Raibaud, A; Buck, G; Baltz, T; Eisen, H

    1986-08-01

    Variant surface glycoprotein (VSG) genes of African trypanosomes are expressed when they are inserted into one of several telomere-linked expression sites. We cloned and characterized an 11-kilobase (kb) DNA fragment located upstream of an expressed VSG gene. A DNA sequence of 1.8 kb that is located immediately upstream of the inserted VSG gene contains sequences homologous to the 76-base-pair repeats described as being upstream of VSG genes in Trypanosoma brucei (D. A. Campbell, M. P. Van Bree, and J. C. Boothroyd, Nucleic Acids Res. 12:2759-2774). There are no such sequences elsewhere in the 11-kb cloned region. Southern blot analysis using probes from the cloned region revealed multiple unlinked copies of the same or very similar regions. At least three of these are located near telomeres, and two have been shown to be used for the expression of known Trypanosoma equiperdum VSG genes. Like VSG genes, the upstream sequences themselves can be duplicated and deleted. The choice of expression site to be used by a duplicated VSG gene is nonrandom; the site used for expression of the parental VSG gene is strongly favored for use in the daughter variant. Furthermore, even when the parental expression site is not used, the VSG gene occupying it is replaced. Thus, an active expression site is a preferential target for gene conversion in the next variation event.

  6. Dominant Negative Effects of a Non-conducting TREK1 Splice Variant Expressed in Brain*

    OpenAIRE

    Veale, Emma L; Rees, Kathryn A.; Mathie, Alistair; Trapp, Stefan

    2010-01-01

    Two-pore domain potassium (K2P) channels modulate neuronal excitability throughout the entire CNS. The stretch-activated channel TREK1 (K2P2.1) is expressed widely in brain and has been linked to depression, neuroprotection, pain perception, and epilepsy. Little, however, is known about the regulation of TREK1 expression on the transcriptional and translational level or about its trafficking to the plasma membrane. Here we have used PCR techniques to identify a splice variant of TREK1 express...

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

    Directory of Open Access Journals (Sweden)

    Z Kalnina

    2009-08-01

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

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

    Science.gov (United States)

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

    1996-01-01

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

  9. Detection of antibodies to variant antigens on Plasmodium falciparum-infected erythrocytes by flow cytometry

    DEFF Research Database (Denmark)

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

    1999-01-01

    BACKGROUND: Naturally induced antibodies binding to surface antigens of Plasmodium falciparum-infected erythrocytes can be detected by direct agglutination of infected erythrocytes or by indirect immunofluorescence on intact, unfixed, infected erythrocytes. Agglutinating antibodies have previously...

  10. Identification of Differentially Expressed K-Ras Transcript Variants in Patients With Leiomyoma.

    Science.gov (United States)

    Zolfaghari, Nooshin; Shahbazi, Shirin; Torfeh, Mahnaz; Khorasani, Maryam; Hashemi, Mehrdad; Mahdian, Reza

    2017-10-01

    Molecular studies have demonstrated a wide range of gene expression variations in uterine leiomyoma. The rat sarcoma virus/rapidly accelerated fibrosarcoma/mitogen-activated protein kinase (RAS/RAF/MAPK) is the crucial cellular pathway in transmitting external signals into nucleus. Deregulation of this pathway contributes to excessive cell proliferation and tumorigenesis. The present study aims to investigate the expression profile of the K-Ras transcripts in tissue samples from patients with leiomyoma. The patients were leiomyoma cases who had no mutation in mediator complex subunit 12 ( MED12) gene. A quantitative approach has been applied to determine the difference in the expression of the 2 main K-Ras messenger RNA (mRNA) variants. The comparison between gene expression levels in leiomyoma and normal myometrium group was performed using relative expression software tool. The expression of K-Ras4B gene was upregulated in leiomyoma group ( P = .016), suggesting the involvement of K-Ras4B in the disease pathogenesis. Pairwise comparison of the K-Ras4B expression between each leiomyoma tissue and its matched adjacent normal myometrium revealed gene upregulation in 68% of the cases. The expression of K-Ras4A mRNA was relatively upregulated in leiomyoma group ( P = .030). In addition, the mean expression of K-Ras4A gene in leiomyoma tissues relative to normal samples was 4.475 (95% confidence interval: 0.10-20.42; standard error: 0.53-12.67). In total, 58% of the cases showed more than 2-fold increase in K-Ras4A gene expression. Our results demonstrated increased expression of both K-Ras mRNA splicing variants in leiomyoma tissue. However, the ultimate result of KRAS expression on leiomyoma development depends on the overall KRAS isoform balance and, consequently, on activated signaling pathways.

  11. FTO Obesity Risk Variants Are Linked to Adipocyte IRX3 Expression and BMI of Children - Relevance of FTO Variants to Defend Body Weight in Lean Children?

    Science.gov (United States)

    Landgraf, Kathrin; Scholz, Markus; Kovacs, Peter; Kiess, Wieland; Körner, Antje

    2016-01-01

    Genome-wide association studies have identified variants within the FTO (fat mass and obesity associated) locus as the strongest predictors of obesity amongst all obesity-associated gene loci. Recent evidence suggests that variants in FTO directly affect human adipocyte function through targeting IRX3 and IRX5 and thermogenesis regulation. We addressed the relevance of this proposed FTO-IRX pathway in adipose tissue (AT) of children. Expression of IRX3 was higher in adipocytes compared to SVF. We found increased adipocyte-specific expression of IRX3 and IRX5 with the presence of the FTO risk haplotype in lean children, whereas it was unaffected by risk variants in obese peers. We further show that IRX3 expression was elevated in isolated adipocytes and AT of lean compared to obese children, particularly in UCP1-negative adipocytes, and inversely correlated with BMI SDS. Independent of BMI, IRX3 expression in adipocytes was significantly related to adipocyte hypertrophy, and subsequent associations with AT inflammation and HOMA-IR in the children. One interpretation of our observation of FTO risk variants linked to IRX3 expression and adipocyte size restricted to lean children, along with the decreased IRX3 expression in obese compared to lean peers, may reflect a defense mechanism for protecting body-weight, which is pertinent for lean children.

  12. Brain expression genome-wide association study (eGWAS identifies human disease-associated variants.

    Directory of Open Access Journals (Sweden)

    Fanggeng Zou

    Full Text Available Genetic variants that modify brain gene expression may also influence risk for human diseases. We measured expression levels of 24,526 transcripts in brain samples from the cerebellum and temporal cortex of autopsied subjects with Alzheimer's disease (AD, cerebellar n=197, temporal cortex n=202 and with other brain pathologies (non-AD, cerebellar n=177, temporal cortex n=197. We conducted an expression genome-wide association study (eGWAS using 213,528 cisSNPs within ± 100 kb of the tested transcripts. We identified 2,980 cerebellar cisSNP/transcript level associations (2,596 unique cisSNPs significant in both ADs and non-ADs (q<0.05, p=7.70 × 10(-5-1.67 × 10(-82. Of these, 2,089 were also significant in the temporal cortex (p=1.85 × 10(-5-1.70 × 10(-141. The top cerebellar cisSNPs had 2.4-fold enrichment for human disease-associated variants (p<10(-6. We identified novel cisSNP/transcript associations for human disease-associated variants, including progressive supranuclear palsy SLCO1A2/rs11568563, Parkinson's disease (PD MMRN1/rs6532197, Paget's disease OPTN/rs1561570; and we confirmed others, including PD MAPT/rs242557, systemic lupus erythematosus and ulcerative colitis IRF5/rs4728142, and type 1 diabetes mellitus RPS26/rs1701704. In our eGWAS, there was 2.9-3.3 fold enrichment (p<10(-6 of significant cisSNPs with suggestive AD-risk association (p<10(-3 in the Alzheimer's Disease Genetics Consortium GWAS. These results demonstrate the significant contributions of genetic factors to human brain gene expression, which are reliably detected across different brain regions and pathologies. The significant enrichment of brain cisSNPs among disease-associated variants advocates gene expression changes as a mechanism for many central nervous system (CNS and non-CNS diseases. Combined assessment of expression and disease GWAS may provide complementary information in discovery of human disease variants with functional implications. Our findings

  13. Dominant negative effects of a non-conducting TREK1 splice variant expressed in brain.

    Science.gov (United States)

    Veale, Emma L; Rees, Kathryn A; Mathie, Alistair; Trapp, Stefan

    2010-09-17

    Two-pore domain potassium (K(2P)) channels modulate neuronal excitability throughout the entire CNS. The stretch-activated channel TREK1 (K(2P)2.1) is expressed widely in brain and has been linked to depression, neuroprotection, pain perception, and epilepsy. Little, however, is known about the regulation of TREK1 expression on the transcriptional and translational level or about its trafficking to the plasma membrane. Here we have used PCR techniques to identify a splice variant of TREK1 expressed in the brain, which encodes a heavily truncated TREK1 protein retaining a single transmembrane domain. Functional expression of this splice variant TREK1ΔEx4 in tsA201 cells in the presence or absence of wild type TREK1 revealed that TREK1ΔEx4 has no channel activity itself but reduced TREK1 whole cell current amplitude. Confocal analysis of the expression of fluorescently tagged TREK1 variants revealed that TREK1ΔEx4 is translated, but it is retained in the intracellular compartment. Additionally, TREK1ΔEx4 reduced the level of TREK1 expression in the plasma membrane. Long and short forms of TREK1 derived from alternative translation initiation are differentially affected by TREK1ΔEx4, with the short form (lacking the first 41 amino acids at its N terminus) unaffected. This differential regulatory role of TREK1ΔEx4 will alter the functional profile of TREK1 current in neurons where they are expressed. These results indicate that the N-terminal domain and first transmembrane domain of TREK1 are likely to be important for channel dimerization and trafficking to the plasma membrane.

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

    Science.gov (United States)

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

    1988-01-01

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

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

    Science.gov (United States)

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

    2015-02-01

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

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

    Science.gov (United States)

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

    2011-07-01

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

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

    DEFF Research Database (Denmark)

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

    2011-01-01

    NAc-II sialyltransferases can synthesize sialyl-Tn. The aim of the present study was to establish whether ST6GalNAc-I is the major enzyme responsible for the expression of sialyl-Tn. We used a model of CHO-ldlD cells producing only MUC1-Tn glycoform and showed that ST6GalNAc-I is the key-enzyme leading to sialyl......-Tn biosynthesis. We developed novel monoclonal antibodies specific for ST6GalNAc-I and evaluated its expression in gastrointestinal tissues. ST6GalNAc-I was detected in normal colon mucosa co-localized with O-acetylated sialyl-Tn. Expression was largely unaltered in colorectal adenocarcinomas. In contrast, we......NAc-I as the major enzyme controlling the expression of cancer-associated sialyl-Tn antigen in gastrointestinal tissues....

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

    DEFF Research Database (Denmark)

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

    2015-01-01

    cancers. Staining for the well-characterized MAGE-A proteins was included for comparison. Immunohistochemical staining confirmed previous mRNA analysis demonstrating that ADAM2, CALR3 and SAGE1 proteins are confined to testis in normal individuals. Negative tissues included plancenta, which express many...... inhibitors has been proposed as an attractive strategy to increase the expression of cancer/testis antigens in tumors before immunotargeting; however, neither ADAM2, CALR3 nor SAGE1 could be significantly induced in lung and breast cancer cell lines using this strategy. Our results suggest that ADAM2, CALR3...... and antigenic properties, but the expression patterns of most of the more than 200 identified cancer/testis antigens in various cancers remain largely uncharacterized. In this study, we investigated the expression of the cancer/testis antigens ADAM2, CALR3 and SAGE1 in lung and breast cancer, the two most...

  19. 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

  20. Myosin-binding Protein C Compound Heterozygous Variant Effect on the Phenotypic Expression of Hypertrophic Cardiomyopathy

    Science.gov (United States)

    Rafael, Julianny Freitas; Cruz Filho, Fernando Eugênio dos Santos; de Carvalho, Antônio Carlos Campos; Gottlieb, Ilan; Cazelli, José Guilherme; Siciliano, Ana Paula; Dias, Glauber Monteiro

    2017-01-01

    Hypertrophic cardiomyopathy (HCM) is an autosomal dominant genetic disease caused by mutations in genes encoding sarcomere proteins. It is the major cause of sudden cardiac death in young high-level athletes. Studies have demonstrated a poorer prognosis when associated with specific mutations. The association between HCM genotype and phenotype has been the subject of several studies since the discovery of the genetic nature of the disease. This study shows the effect of a MYBPC3 compound variant on the phenotypic HCM expression. A family in which a young man had a clinical diagnosis of HCM underwent clinical and genetic investigations. The coding regions of the MYH7, MYBPC3 and TNNT2 genes were sequenced and analyzed. The proband present a malignant manifestation of the disease, and is the only one to express HCM in his family. The genetic analysis through direct sequencing of the three main genes related to this disease identified a compound heterozygous variant (p.E542Q and p.D610H) in MYBPC3. A family analysis indicated that the p.E542Q and p.D610H alleles have paternal and maternal origin, respectively. No family member carrier of one of the variant alleles manifested clinical signs of HCM. We suggest that the MYBPC3-biallelic heterozygous expression of p.E542Q and p.D610H may cause the severe disease phenotype seen in the proband. PMID:28538763

  1. Myosin-binding Protein C Compound Heterozygous Variant Effect on the Phenotypic Expression of Hypertrophic Cardiomyopathy

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    Julianny Freitas Rafael

    Full Text Available Abstract Hypertrophic cardiomyopathy (HCM is an autosomal dominant genetic disease caused by mutations in genes encoding sarcomere proteins. It is the major cause of sudden cardiac death in young high-level athletes. Studies have demonstrated a poorer prognosis when associated with specific mutations. The association between HCM genotype and phenotype has been the subject of several studies since the discovery of the genetic nature of the disease. This study shows the effect of a MYBPC3 compound variant on the phenotypic HCM expression. A family in which a young man had a clinical diagnosis of HCM underwent clinical and genetic investigations. The coding regions of the MYH7, MYBPC3 and TNNT2 genes were sequenced and analyzed. The proband present a malignant manifestation of the disease, and is the only one to express HCM in his family. The genetic analysis through direct sequencing of the three main genes related to this disease identified a compound heterozygous variant (p.E542Q and p.D610H in MYBPC3. A family analysis indicated that the p.E542Q and p.D610H alleles have paternal and maternal origin, respectively. No family member carrier of one of the variant alleles manifested clinical signs of HCM. We suggest that the MYBPC3-biallelic heterozygous expression of p.E542Q and p.D610H may cause the severe disease phenotype seen in the proband.

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

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    Cecilia González

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

  3. Localization and expression of histone H2A variants during mouse oogenesis and preimplantation embryo development.

    Science.gov (United States)

    Wu, B J; Dong, F L; Ma, X S; Wang, X G; Lin, F; Liu, H L

    2014-08-07

    Epigenetic modifications of the genome, such as histone H2A variants, ensure appropriate gene activation or silencing during oogenesis and preimplantation embryo development. We examined global localization and expression of the histone H2A variants, including H2A.Bbd, H2A.Z and H2A.X, during mouse oogenesis and preimplantation embryo development. Immunocytochemistry with specific antibodies against various histone H2A variants showed their localization and changes during oogenesis and preimplantation development. H2A.Bbd and H2A.Z were almost absent from nuclei of growing oocytes (except 5-day oocyte), whereas H2A.X was deposited in nuclei throughout oogenesis and in preimplantation embryos. In germinal vesicle (GV) oocyte chromatin, H2A.Bbd was detected as a weak signal, whereas no fluorescent signal was detected in GV breakdown (GVBD) or metaphase II (MII) oocytes; H2A.Z showed intense signals in chromatin of GV, GVBD and MII oocytes. H2A. Bbd showed very weak signals in both pronucleus and 2-cell embryo nuclei, but intense signals were detected in nuclei from 4-cell embryo to blastula. The H2A.Z signal was absent from pronucleus to morula chromatin, whereas a fluorescent signal was detected in blastula nuclei. Our results suggest that histone H2A variants are probably involved in reprogramming of genomes during oocyte meiosis or after fertilization.

  4. Selection of Highly Expressed Gene Variants in Escherichia coli Using Translationally Coupled Antibiotic Selection Markers

    DEFF Research Database (Denmark)

    Rennig, Maja; Daley, Daniel O.; Nørholm, Morten H. H.

    2018-01-01

    Strategies to select highly expressed variants of a protein coding sequence are usually based on trial-and-error approaches, which are time-consuming and expensive. We address this problem using translationally coupled antibiotic resistance markers. The system requires that the target gene can...... be fused at the 3'-end with a translational coupling element and an antibiotic resistance gene. Highly expressed target genes can then be selected using a fast and simple whole cell survival assay in the presence of high antibiotic concentrations. Herein we show that the system can be used to select highly...

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

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    Yao-Tseng Chen

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

  6. Genetic variants alter T-bet binding and gene expression in mucosal inflammatory disease.

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    Katrina Soderquest

    2017-02-01

    Full Text Available The polarization of CD4+ T cells into distinct T helper cell lineages is essential for protective immunity against infection, but aberrant T cell polarization can cause autoimmunity. The transcription factor T-bet (TBX21 specifies the Th1 lineage and represses alternative T cell fates. Genome-wide association studies have identified single nucleotide polymorphisms (SNPs that may be causative for autoimmune diseases. The majority of these polymorphisms are located within non-coding distal regulatory elements. It is considered that these genetic variants contribute to disease by altering the binding of regulatory proteins and thus gene expression, but whether these variants alter the binding of lineage-specifying transcription factors has not been determined. Here, we show that SNPs associated with the mucosal inflammatory diseases Crohn's disease, ulcerative colitis (UC and celiac disease, but not rheumatoid arthritis or psoriasis, are enriched at T-bet binding sites. Furthermore, we identify disease-associated variants that alter T-bet binding in vitro and in vivo. ChIP-seq for T-bet in individuals heterozygous for the celiac disease-associated SNPs rs1465321 and rs2058622 and the IBD-associated SNPs rs1551398 and rs1551399, reveals decreased binding to the minor disease-associated alleles. Furthermore, we show that rs1465321 is an expression quantitative trait locus (eQTL for the neighboring gene IL18RAP, with decreased T-bet binding associated with decreased expression of this gene. These results suggest that genetic polymorphisms may predispose individuals to mucosal autoimmune disease through alterations in T-bet binding. Other disease-associated variants may similarly act by modulating the binding of lineage-specifying transcription factors in a tissue-selective and disease-specific manner.

  7. Phenotypic and genetic characterization of a unique variant of serogroup C ET-15 meningococci (with the antigenic formula C:2a:P1.7,1) causing invasive meningococcal disease in Quebec, Canada.

    Science.gov (United States)

    Tsang, Raymond S W; Tsai, Chao Ming; Zhu, Peixuan; Ringuette, Louise; Lorange, Manon; Law, Dennis K S

    2004-04-01

    Serogroup C Neisseria meningitidis belonging to the electrophoretic type (ET) ET-15, a variant of ET-37, is endemic in Canada. Like other serogroup C ET-37 meningococci, the endemic ET-15 strains are usually found to carry the serotype and serosubtype antigens of 2a:P1.5,2. In 2001, a sudden increase in the number of cases of serogroup C meningococcal disease in Quebec, Canada, was caused by an antigenic variant of the ET-15 strain. This antigenic variant carries the unique serosubtype marker of P1.7,1. Strains of C:2a:P1.7,1 meningococci were not isolated in Canada in large numbers prior to 2001, and the characteristics of these meningococcal strains linked to an outbreak in Quebec, Canada, are described in the present study.

  8. Characterization of human FHL2 transcript variants and gene expression regulation in hepatocellular carcinoma.

    Science.gov (United States)

    Ng, Chor-Fung; Zhou, Wayne Jun-Wei; Ng, Patrick Kwok-Shing; Li, Man-Shan; Ng, Yuen-Keng; Lai, Paul Bo-San; Tsui, Stephen Kwok-Wing

    2011-07-15

    The four-and-a-half LIM protein 2 (FHL2) was originally identified to be expressed abundantly in the heart, as well as in a wide range of tissues demonstrated in various studies. The human FHL2 gene expresses different transcripts which are known to differ only in the 5'UTR region. However, little is known about the functional role of the different variants and the mechanism of gene regulation. In the present study, we characterized the different alternative spliced transcripts of FHL2 by in silico analysis and RT-PCR analysis. A novel transcript variant was identified. The FHL2 gene produces transcripts by different 5' exons, which may be responsible for tissue-specific regulation. To study the mechanism of FHL2 gene regulation, the potential promoter region was investigated. We have identified a functional promoter region upstream of the transcription start site. Deletion mutation analysis of 5' flanking region showed that the fragment from -138 to +292 bp have positive regulatory effect. We identified the binding sites of Pax-5/ZF5 in this region and found that Pax-5 and ZF5 expression in HCC samples had a significant positive correlation with FHL2 expression, suggesting a possible role for these transcription factors in the regulation of FHL2 expression. Copyright © 2011 Elsevier B.V. All rights reserved.

  9. Expression, intracellular targeting and purification of HIV Nef variants in tobacco cells

    Directory of Open Access Journals (Sweden)

    Baschieri Selene

    2007-02-01

    Full Text Available Abstract Background Plants may represent excellent alternatives to classical heterologous protein expression systems, especially for the production of biopharmaceuticals and vaccine components. Modern vaccines are becoming increasingly complex, with the incorporation of multiple antigens. Approaches towards developing an HIV vaccine appear to confirm this, with a combination of candidate antigens. Among these, HIV-Nef is considered a promising target for vaccine development because immune responses directed against this viral protein could help to control the initial steps of viral infection and to reduce viral loads and spreading. Two isoforms of Nef protein can be found in cells: a full-length N-terminal myristoylated form (p27, 27 kDa and a truncated form (p25, 25 kDa. Here we report the expression and purification of HIV Nef from transgenic tobacco. Results We designed constructs to direct the expression of p25 and p27 Nef to either the cytosol or the secretory pathway. We tested these constructs by transient expression in tobacco protoplasts. Cytosolic Nef polypeptides are correctly synthesised and are stable. The same is not true for Nef polypeptides targeted to the secretory pathway by virtue of a signal peptide. We therefore generated transgenic plants expressing cytosolic, full length or truncated Nef. Expression levels were variable, but in some lines they averaged 0.7% of total soluble proteins. Hexahistidine-tagged Nef was easily purified from transgenic tissue in a one-step procedure. Conclusion We have shown that transient expression can help to rapidly determine the best cellular compartment for accumulation of a recombinant protein. We have successfully expressed HIV Nef polypeptides in the cytosol of transgenic tobacco plants. The proteins can easily be purified from transgenic tissue.

  10. 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.

  11. Detection of specific antibodies directed against a consistently expressed surface antigen of Mycoplasma gallisepticum using a monoclonal blocking enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Czifra, G; Kleven, S H; Engström, B; Stipkovits, L

    1995-01-01

    Sera from 14 groups of chickens inoculated with different laboratory and field strains of Mycoplasma gallisepticum (MG) were used to compare the diagnostic potential of the hemagglutination-inhibition (HI) test and a recently developed monoclonal blocking enzyme-linked immunosorbent assay (ELISA). HI was performed with strain A5969, commonly used as hemagglutinating antigen, and it could detect 62.7% of the inoculated chickens as positive. Of all sera, 83% proved to be positive when examined with the blocking ELISA. The difference between the sensitivities of the two methods was due to group-specific insensitivity of the HI test. None of the sera from groups inoculated with strains K 1501, K 1503, K 503, or K 703 and only half of the sera from groups inoculated with K 1453 or 236C could inhibit the activity of the A5969 hemagglutinating antigen, indicating antigenic differences between these challenge strains and the diagnostic strain. ELISA detected MG-specific antibodies in every group of sera, although inoculation with variant strains K 503 or K 703 resulted in lower level of antibody production than inoculation with other strains. The monoclonal blocking ELISA can be useful in the serological diagnosis of MG infections, because it is based on a consistently expressed, specific region of MG.

  12. Human leukocyte antigen class II variants and adult-onset asthma: does occupational allergen exposure play a role?

    NARCIS (Netherlands)

    Smit, L.A.M.; Strachan, D.P.; Vermeulen, R.; Bakker, P.I.W. de; Demenais, F.; Dumas, O.; Carsin, A.E.; Cullinan, P.; Curjuric, I.; Ghosh, R.E.; Heederik, D.; Imboden, M.; Jarvis, D.; Lathrop, M.; Moual, N. le; Mehta, A.; Miedinger, D.; Sigsgaard, T.; Siroux, V.; Vernez, D.; Zock, J.P.; Kauffmann, F.; Probst-Hensch, N.; Kogevinas, M.; Bouzigon, E.

    2014-01-01

    Recently, a locus centred on rs9273349 in the HLA-DQ region emerged from genome-wide association studies of adult-onset asthma. We aimed to further investigate the role of human leukocyte antigen (HLA) class II in adult-onset asthma and a possible interaction with occupational exposures. We imputed

  13. Human leukocyte antigen class II variants and adult-onset asthma : does occupational allergen exposure play a role?

    NARCIS (Netherlands)

    Smit, Lidwien A M; Strachan, David P; Vermeulen, Roel; de Bakker, Paul I W; Demenais, Florence; Dumas, Orianne; Carsin, Anne-Elie; Cullinan, Paul; Curjuric, Ivan; Ghosh, Rebecca E; Heederik, Dick; Imboden, Medea; Jarvis, Deborah; Lathrop, Mark; Le Moual, Nicole; Mehta, Amar; Miedinger, David; Sigsgaard, Torben; Siroux, Valérie; Vernez, David; Zock, Jan Paul; Kauffmann, Francine; Probst-Hensch, Nicole; Kogevinas, Manolis; Bouzigon, Emmanuelle

    2014-01-01

    Recently, a locus centred on rs9273349 in the HLA-DQ region emerged from genome-wide association studies of adult-onset asthma. We aimed to further investigate the role of human leukocyte antigen (HLA) class II in adult-onset asthma and a possible interaction with occupational exposures. We imputed

  14. 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.

  15. 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.

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

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    Gupta Namita

    2008-01-01

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

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

    Science.gov (United States)

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

    2013-12-01

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

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

    Science.gov (United States)

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

    2010-08-01

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

  19. 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.

  20. Expression of CD44 standard form and variant isoforms in human bone marrow stromal cells

    Directory of Open Access Journals (Sweden)

    Xiaofeng Wang

    2017-05-01

    Full Text Available Human Bone Marrow Stromal Cells (hBMSCs can migrate from bone marrow to injured tissues, where they may differentiate into different types of new cells for replacement of dysfunctional cells. CD44 plays an important role in stem cell movement. The expression distribution of CD44 standard form (CD44S and CD44 variants (CD44V is closely related to cell movement and tissue migration. The aim of this study was to evaluate the expressions of CD44S and CD44V in hBMSCs. The hBMSCs from four human subjects were cultured in vitro. Phenotypic properties were analyzed by flow cytometry, and adipocyte and osteoblast differentiations were evaluated at passage 4. The expressions of CD44S and CD44V were examined using quantitative real-time polymerase chain reaction (q-PCR. Results showed that hBMSCs were successfully cultured, with positive expressions of markers of mesenchymal cells (CD90, CD73, CD105, and negative expressions of markers of hematopoietic cells (CD34, CD45. The cultured hBMSCs can be induced to differentiate into adipocytes and osteoblasts. Q-PCR results showed that the expression of CD44S was significantly higher than the expressions of different CD44V isoforms in different samples. These results revealed significant differences in the distributions of CD44S and CD44V gene expressions, demonstrating a dominant CD44S expression in hBMCSs.

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

    Science.gov (United States)

    1996-09-01

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

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

    Science.gov (United States)

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

    2015-10-27

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

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2016-12-01

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

  5. 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...

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

    Science.gov (United States)

    Longacre, S; Eisen, H

    1986-05-01

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

  7. Characterization of zinc transporter 8 (ZnT8) antibodies in autoimmune diabetic patients from Argentinian population using monomeric, homodimeric, and heterodimeric ZnT8 antigen variants.

    Science.gov (United States)

    Faccinetti, Natalia I; Guerra, Luciano L; Penas Steinhardt, Alberto; Iacono, Ruben F; Frechtel, Gustavo D; Trifone, Liliana; Poskus, Edgardo; Trabucchi, Aldana; Valdez, Silvina N

    2016-02-01

    In order to gain further knowledge of the structure of zinc transporter 8 (ZnT8) epitopes, we studied the role of the amino acid at position 325 in the antigen and its dimeric conformation for autoantibodies to ZnT8 (ZnT8A) recognition. For this purpose, several ZnT8 C-terminal domain variants were designed: monomer carrying Arg325 or Trp325, homo-dimers ZnT8-Arg-Arg325 and ZnT8-Trp-Trp325, and hetero-dimer ZnT8-Arg-Trp325. Two groups of Argentinian diabetic patients were subjected to analysis using [(35)S]-ZnT8 variants by radioligand binding assay (RBA): i) 100 new-onset, insulin-dependent, type 1 diabetic patients and ii) 282 slowly progressing to insulin requirement, non-obese adult-onset diabetic patients. In addition, 50 type 1 diabetic patients and 100 normal control sera provided by the American Diabetes Association (ADA) were evaluated in order to calculate the sensitivity and specificity of ZnT8A assays for each antigenic variant. Other routine β-cell autoantibodies were also tested by RBA. Of the 100 Argentinian type 1 diabetic patients, 65 were ZnT8A+. Out of them, 8 patients recognized all recombinant forms of ZnT8 and most patients (56) reacted against the heterodimer. Additionally, out of 282 non-obese adult-onset diabetic patients 46 were ZnT8A+, whereas 29 patients recognized only dimers. Besides, exclusive reactivity against ZnT8A was found in 9.0% for type 1 diabetes mellitus and 10.3% for non-obese adult-onset diabetic patients. Significantly higher signal values in RBA were obtained with the heterodimeric variant. An increased detection of humoral autoimmunity was found in both groups when ZnT8A was employed in combination with the other β-cell autoantibodies. The inclusion of homodimeric immunoreactive peptides revealed the existence of quaternary structure-defined epitopes probably resembling the actual state of the autoantigen in vivo. Finally, the differential profiles of ZnT8A exhibited by type 1 and non-obese adult-onset diabetic

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

    Science.gov (United States)

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

    2008-01-01

    Background The number of breast cancer patients diagnosed with ductal carcinoma in situ (DCIS) continues to grow. Laboratory and clinical data indicate that DCIS can progress to invasive disease. Carbohydrate-mediated cell-cell adhesion and tumor-stroma interaction play crucial roles in tumorigenesis and tumor aggressive behavior. Breast carcinogenesis may reflect quantitative as well as qualitative changes in oligosaccharide expression, which may provide a useful tool for early detection of breast cancer. Because tumor-associated carbohydrate antigens (TACA) are implicated in tumor invasion and metastasis, the purpose of this study was to assess the expression of selected TACA by lectin histochemistry on DCIS specimens from the archival breast cancer tissue array bank of the University of Arkansas for Medical Sciences. Methods For detection of TACA expression, specimens were stained with Griffonia simplicifolia lectin-I (GS-I) and Vicia vilosa agglutinin (VVA). We studied associations of lectin reactivity with established prognostic factors, such as tumor size, tumor nuclear grade, and expression of Her-2/neu, p53 mutant and estrogen and progesterone receptors. Results We observed that both lectins showed significant associations with nuclear grade of DCIS. DCIS specimens with nuclear grades II and III showed significantly more intense reactivity than DCIS cases with nuclear grade I to GS-1 (Mean-score chi-square = 17.60, DF = 2; P = 0.0002) and VVA (Mean-score chi-square = 15.72, DF = 2; P = 0.0004). Conclusion The results suggest that the expression of VVA- and GS-I-reactive carbohydrate antigens may contribute to forming higher grade DCIS and increase the recurrence risk. PMID:18479514

  9. 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.

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

    Directory of Open Access Journals (Sweden)

    Nagendra Suryanarayana

    2016-01-01

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

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

    Science.gov (United States)

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

    2011-03-31

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

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

    Science.gov (United States)

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

    2013-05-20

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

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Science.gov (United States)

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

    1995-07-01

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

  15. Breaking tolerance in hepatitis B surface antigen (HBsAg) transgenic mice by vaccination with cross-reactive, natural HBsAg variants

    DEFF Research Database (Denmark)

    Schirmbeck, Reinhold; Dikopoulos, Nektarios; Kwissa, Marcin

    2003-01-01

    Processing exogenous hepatitis B surface antigen (HBsAg) of the hepatitis B virus (HBV) generates the K(b)-binding S(208-215) epitope 1; processing endogenous HBsAg generates the K(b)-binding S(190-197) epitope 2. Cross-reactive CD8(+) T cell responses were primed to epitope 1 but not epitope 2......-tg mice by HBsAg(adw2); these specific CD8(+) T cells cross-reacted with epitope 1 processed from the transgene-encoded HBsAg(ayw). The liver of vaccinated HBsAg(ayw) transgenic mice showed severe histopathology and contained functional (IFNgamma-producing), cross-reactive CD8(+) T cells, and vaccinated...... HBs-tg mice showed reduced antigenemia. Hence, vaccination with natural HBsAg variants from different HBV sero/genotypes can prime cross-reactive, specific CD8(+) T cell immunity that breaks tolerance to HBsAg....

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

    Directory of Open Access Journals (Sweden)

    Moshe eIsraeli

    2014-10-01

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

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

    Science.gov (United States)

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

    2000-09-01

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

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

    Science.gov (United States)

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

    2013-12-01

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

  19. 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.

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

    Science.gov (United States)

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

    2016-11-15

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

  1. 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.

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

    Science.gov (United States)

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

    2015-05-01

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

  4. 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.

  5. HE4 Transcription- and Splice Variants-Specific Expression in Endometrial Cancer and Correlation with Patient Survival

    Directory of Open Access Journals (Sweden)

    Shi-Wen Jiang

    2013-11-01

    Full Text Available We investigated the HE4 variant-specific expression patterns in various normal tissues as well as in normal and malignant endometrial tissues. The relationships between mRNA variants and age, body weight, or survival are analyzed. ICAT-labeled normal and endometrial cancer (EC tissues were analyzed with multidimensional liquid chromatography followed by tandem mass spectrometry. Levels of HE4 mRNA variants were measured by real-time PCR. Mean mRNA levels were compared among 16 normal endometrial samples, 14 grade 1 and 14 grade 3 endometrioid EC, 15 papillary serous EC, and 14 normal human tissue samples. The relationship between levels of HE4 variants and EC patient characteristics was analyzed with the use of Pearson correlation test. We found that, although all five HE4 mRNA variants are detectable in normal tissue samples, their expression is highly tissue-specific, with epididymis, trachea, breast and endometrium containing the highest levels. HE4-V0, -V1, and -V3 are the most abundant variants in both normal and malignant tissues. All variants are significantly increased in both endometrioid and papillary serous EC, with higher levels observed in grade 3 endometrioid EC. In the EC group, HE4-V1, -V3, and -V4 levels inversely correlate with EC patient survival, whereas HE4-V0 levels positively correlate with age. HE4 variants exhibit tissue-specific expression, suggesting that each variant may exert distinct functions in normal and malignant cells. HE4 levels appear to correlate with EC patient survival in a variant-specific manner. When using HE4 as a biomarker for EC management, the effects of age should be considered.

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

    DEFF Research Database (Denmark)

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

    2014-01-01

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

  7. A Splice Variant of Bardet-Biedl Syndrome 5 (BBS5 Protein that Is Selectively Expressed in Retina.

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    Susan N Bolch

    Full Text Available Bardet-Biedl syndrome is a complex ciliopathy that usually manifests with some form of retinal degeneration, amongst other ciliary-related deficiencies. One of the genetic causes of this syndrome results from a defect in Bardet-Biedl Syndrome 5 (BBS5 protein. BBS5 is one component of the BBSome, a complex of proteins that regulates the protein composition in cilia. In this study, we identify a smaller molecular mass form of BBS5 as a variant formed by alternative splicing and show that expression of this splice variant is restricted to the retina.Reverse transcription PCR from RNA was used to isolate and identify potential alternative transcripts of Bbs5. A peptide unique to the C-terminus of the BBS5 splice variant was synthesized and used to prepare antibodies that selectively recognized the BBS5 splice variant. These antibodies were used on immunoblots of tissue extracts to determine the extent of expression of the alternative transcript and on tissue slices to determine the localization of expressed protein. Pull-down of fluorescently labeled arrestin1 by immunoprecipitation of the BBS5 splice variant was performed to assess functional interaction between the two proteins.PCR from mouse retinal cDNA using Bbs5-specific primers amplified a unique cDNA that was shown to be a splice variant of BBS5 resulting from the use of cryptic splicing sites in Intron 7. The resulting transcript codes for a truncated form of the BBS5 protein with a unique 24 amino acid C-terminus, and predicted 26.5 kD molecular mass. PCR screening of RNA isolated from various ciliated tissues and immunoblots of protein extracts from these same tissues showed that this splice variant was expressed in retina, but not brain, heart, kidney, or testes. Quantitative PCR showed that the splice variant transcript is 8.9-fold (+/- 1.1-fold less abundant than the full-length transcript. In the retina, the splice variant of BBS5 appears to be most abundant in the connecting cilium

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

    National Research Council Canada - National Science Library

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

    2002-01-01

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

  9. DETERMINATION OF LEVEL EXPRESSION OF mRNA SPLICING VARIANTS FOR DR3 IN BLOOD CELLS IN INFECTIOUS MONONUCLEOSIS

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    V. D. Cvetkova

    2016-01-01

    Full Text Available The DR3 «death receptor» plays an important role in the initiation of apoptosis, proliferation, or inflammation. This receptor is shown to be involved in various diseases, including infectious conditions. Different variants of mRNA DR3 are formed as a result of alternative splicing. These variant transcripts encode membrane and soluble forms of the receptor which have different functions. Features of their expression and contribution of individual DR3 variants to the immune pathogenesis of infectious mononucleosis (IM are poorely understood.The purpose of this work was to develop, validate and test the techniques of DR3 gene expression assays, as well as to evaluate the DR3 mRNA splice variants by means of real-time RT-PCR and RT-PCR in the IM patients.The original version of real-time RT-PCR allowed to determine relative amounts of DR3 mRNA, DR3 membrane variants (LARD1a + LARD8, and ratios of mRNAs encoding membrane and soluble forms of the receptor. The technique proved to be specific and sensitive (a semi-quantitative detection limit = 34-35 cycles when tested in healthy volunteers and patients with acute infectious mononucleosis (AIM. Lower expression levels were shown for two alternative membrane variants of DR3 mRNA (LARD1b and DR3beta thus regarding these isoforms as minor fractions. The relative levels of total DR3 mRNA expression were decreased in patients with AIM, as compared to healthy volunteers, whereas mRNA expression of membrane receptor variants did not differ between IM and controls.To determine a qualitative contribution of either LARD1a and LARD8 variants into the expression of membrane forms of DR3, a two-step «nested» version of RT-PCR has been developed. It was shown that, in majority of control and IM samples, both main LARD1a, and alternative LARD8 membrane forms are contributing to mRNA expression of membrane DR3 variants.The presented methods for evaluation of expression and occurrence of DR3 mRNA variants allow

  10. Characterization of the Ala62Pro polymorphic variant of human cytochrome P450 1A1 using recombinant protein expression

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Seung Heon; Kang, Sukmo [College of Veterinary Medicine, BK21plus Program for Creative Veterinary Science Research, and Research Institute for Veterinary Science, Seoul National University, Seoul (Korea, Republic of); Dong, Mi Sook [School of Life Sciences and Biotechnology, Korea University, Seoul (Korea, Republic of); Park, Jung-Duck [College of Medicine, Chung-Ang University, Seoul (Korea, Republic of); Park, Jinseo; Rhee, Sangkee [College of Agriculture of Life Science, Seoul National University, Seoul (Korea, Republic of); Ryu, Doug-Young, E-mail: dyryu@snu.ac.kr [College of Veterinary Medicine, BK21plus Program for Creative Veterinary Science Research, and Research Institute for Veterinary Science, Seoul National University, Seoul (Korea, Republic of)

    2015-06-15

    Cytochrome P450 (CYP) 1A1 is a heme-containing enzyme involved in detoxification of hydrophobic pollutants. Its Ala62Pro variant has been identified previously. Ala62 is located in α-helix A of CYP1A1. Residues such as Pro and Gly are α-helix breakers. In this study, the Ala62Pro variant was characterized using heterologous expression. E. coli expressing the Ala62Pro variant, and the purified variant protein, had lower CYP (i.e. holoenzyme) contents than their wild-type (WT) equivalents. The CYP variant from E. coli and mammalian cells exhibited lower 7-ethoxyresorufin O-dealkylation (EROD) and benzo[a]pyrene hydroxylation activities than the WT. Enhanced supplementation of a heme precursor during E. coli culture did not increase CYP content in E. coli expressing the variant, but did for the WT. As for Ala62Pro, E. coli expressing an Ala62Gly variant had a lower CYP content than the WT counterpart, but substitution of Ala62 with α-helix-compatible residues such as Ser and Val partially recovered the level of CYP produced. Microsomes from mammalian cells expressing Ala62Pro and Ala62Gly variants exhibited lower EROD activities than those expressing the WT or Ala62Val variant. A region harboring α-helix A has interactions with another region containing heme-interacting residues. Site-directed mutagenesis analyses suggest the importance of interactions between the two regions on holoenzyme expression. Together, these findings suggest that the Ala62Pro substitution leads to changes in protein characteristics and function of CYP1A1 via structural disturbance of the region where the residue is located. - Highlights: • Ala62 is located in α-helix A of the carcinogen-metabolizing enzyme CYP1A1. • Pro acts as an α-helix breaker. • A variant protein of CYP1A1, Ala62Pro, had lower heme content than the wild-type. • The variant of CYP1A1 had lower enzyme activities than the wild-type.

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

    Directory of Open Access Journals (Sweden)

    Vera L. Tunitskaya

    2016-10-01

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

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

    Science.gov (United States)

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

    1994-10-01

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

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

    Science.gov (United States)

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

    2015-06-01

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

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

    Science.gov (United States)

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

    2008-08-01

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

  15. A novel splice variant of folate receptor 4 predominantly expressed in regulatory T cells

    Directory of Open Access Journals (Sweden)

    Tian Yi

    2012-06-01

    Full Text Available Abstract Background Regulatory T cells (Tregs are required for proper maintenance of immunological self-tolerance and immune homeostasis. Folate receptor 4 (FR4 is expressed at high levels in transforming growth factor-beta (TGF-β-induced Tregs and natural Tregs. Moreover, antibody-mediated targeting of FR4 is sufficient to mediate Treg depletion. Results In this study, we describe a novel FR4 transcript variant, FR4D3, in which exon 3 is deleted. The mRNA of FR4D3 encodes a FR4 variant truncated by 189 bp. FR4D3 was found to be predominantly expressed in CD4+CD25+ Treg cells. Overexpression of FR4D3 in CD4+CD25+ Treg cells in vitro stimulated proliferation, which may modulate the ability of these cells to bind and incorporate folic acid. Conclusions Our results suggested that high levels of FR4D3 may be critical to support the substantial proliferative capacity of Treg cells.

  16. Mesenchymal stem cells expressing osteoprotegerin variants inhibit osteolysis in a murine model of multiple myeloma.

    Science.gov (United States)

    Higgs, Jerome T; Lee, Joo Hyoung; Wang, Hong; Ramani, Vishnu C; Chanda, Diptiman; Hardy, Cherlene Y; Sanderson, Ralph D; Ponnazhagan, Selvarangan

    2017-11-28

    The current treatment options for multiple myeloma (MM) osteolytic lesions are mainly combinations of chemotherapy and other small-molecule inhibitors, but toxic side effects still remain a major concern. Studies have shown that osteoclast activity is enhanced in MM patients through increased expression of receptor activator of nuclear factor κB ligand (RANKL), triggering RANK signaling on osteoclast precursors, which results in aggressive bone resorption. Furthermore, osteoprotegerin (OPG), a decoy receptor for RANKL, and the osteogenic potential of mesenchymal stem cells (MSCs) are significantly decreased in myeloma patients with multiple bone lesions. Thus, the use of OPG as a therapeutic molecule would greatly decrease osteolytic damage and reduce morbidity. However, in addition to inhibiting osteoclast activation, OPG binds to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), thereby rendering the tumor cells resistant to TRAIL-induced apoptosis and limiting the use of OPG for therapy. The present study developed a bone-disseminated myeloma disease model in mouse and successfully tested a cell therapy approach using MSCs, genetically engineered to express OPG variants that retain the capacity to bind RANKL, but do not bind TRAIL. Our results of skeletal remodeling following this regenerative stem cell therapy with OPG variants indicated a significant protection against myeloma-induced osteolytic bone damage in areas of major myeloma skeletal dissemination, suggesting the potential of this therapy for treating osteolytic damage in myeloma patients.

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Science.gov (United States)

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

    2004-08-01

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

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

    Directory of Open Access Journals (Sweden)

    L. E. Esteban

    2013-01-01

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

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

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    LSF Boogerd

    2017-05-01

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

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

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    Seri Jeong

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

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

    Science.gov (United States)

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

    2017-12-15

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

  3. A variant of Runx2 that differs from the bone isoform in its splicing is expressed in spermatogenic cells

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    Satoru Kanto

    2016-04-01

    Full Text Available Background. Members of the Runx gene family encode transcription factors that bind to DNA in a sequence-specific manner. Among the three Runx proteins, Runx2 comprises 607 amino acid (aa residues, is expressed in bone, and plays crucial roles in osteoblast differentiation and bone development. We examined whether the Runx2 gene is also expressed in testes. Methods. Murine testes from 1-, 2-, 3-, 4-, and 10-week-old male mice of the C57BL/6J strain and W∕Wv strain were used throughout the study. Northern Blot Analyses were performed using extracts form the murine testes. Sequencing of cDNA clones and 5′-rapid amplification of cDNA ends were performed to determine the full length of the transcripts, which revealed that the testicular Runx2 comprises 106 aa residues coding novel protein. Generating an antiserum using the amino-terminal 15 aa of Runx2 (Met1 to Gly15 as an antigen, immunoblot analyses were performed to detect the predicted polypeptide of 106 aa residues with the initiating Met1. With the affinity-purified anti-Runx2 antibody, immunohistochemical analyses were performed to elucidate the localization of the protein. Furthermore, bioinformatic analyses were performed to predict the function of the protein. Results. A Runx2 transcript was detected in testes and was specifically expressed in germ cells. Determination of the transcript structure indicated that the testicular Runx2 is a splice isoform. The predicted testicular Runx2 polypeptide is composed of only 106 aa residues, lacks a Runt domain, and appears to be a basic protein with a predominantly alpha-helical conformation. Immunoblot analyses with an anti-Runx2 antibody revealed that Met1 in the deduced open reading frame of Runx2 is used as the initiation codon to express an 11 kDa protein. Furthermore, immunohistochemical analyses revealed that the Runx2 polypeptide was located in the nuclei, and was detected in spermatocytes at the stages of late pachytene, diplotene and

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

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    Marcin Cebula

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

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

    Science.gov (United States)

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

    2011-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Lilija Miller

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

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

    Science.gov (United States)

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

    2009-01-01

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

  8. 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.

  9. Myosin-binding Protein C Compound Heterozygous Variant Effect on the Phenotypic Expression of Hypertrophic Cardiomyopathy.

    Science.gov (United States)

    Rafael, Julianny Freitas; Cruz, Fernando Eugênio Dos Santos; Carvalho, Antônio Carlos Campos de; Gottlieb, Ilan; Cazelli, José Guilherme; Siciliano, Ana Paula; Dias, Glauber Monteiro

    2017-04-01

    Hypertrophic cardiomyopathy (HCM) is an autosomal dominant genetic disease caused by mutations in genes encoding sarcomere proteins. It is the major cause of sudden cardiac death in young high-level athletes. Studies have demonstrated a poorer prognosis when associated with specific mutations. The association between HCM genotype and phenotype has been the subject of several studies since the discovery of the genetic nature of the disease. This study shows the effect of a MYBPC3 compound variant on the phenotypic HCM expression. A family in which a young man had a clinical diagnosis of HCM underwent clinical and genetic investigations. The coding regions of the MYH7, MYBPC3 and TNNT2 genes were sequenced and analyzed. The proband present a malignant manifestation of the disease, and is the only one to express HCM in his family. The genetic analysis through direct sequencing of the three main genes related to this disease identified a compound heterozygous variant (p.E542Q and p.D610H) in MYBPC3. A family analysis indicated that the p.E542Q and p.D610H alleles have paternal and maternal origin, respectively. No family member carrier of one of the variant alleles manifested clinical signs of HCM. We suggest that the MYBPC3-biallelic heterozygous expression of p.E542Q and p.D610H may cause the severe disease phenotype seen in the proband. Resumo A cardiomiopatia hipertrófica (CMH) é uma doença autossômica dominante causada por mutações em genes que codificam as proteínas dos sarcômeros. É a principal causa de morte súbita cardíaca em atletas jovens de alto nível. Estudos têm demonstrado um pior prognóstico associado a mutações específicas. A associação entre genótipo e fenótipo em CMH tem sido objeto de diversos estudos desde a descoberta da origem genética dessa doença. Este trabalho apresenta o efeito de uma mutação composta em MYBPC3 na expressão fenotípica da CMH. Uma família na qual um jovem tem o diagnóstico clínico de CMH foi

  10. Splice variants of enigma homolog, differentially expressed during heart development, promote or prevent hypertrophy.

    Science.gov (United States)

    Yamazaki, Tomoko; Wälchli, Sébastien; Fujita, Toshitsugu; Ryser, Stephan; Hoshijima, Masahiko; Schlegel, Werner; Kuroda, Shun'ichi; Maturana, Andrés D

    2010-06-01

    Proteins with a PDZ (for PSD-95, DLG, ZO-1) and one to three LIM (for Lin11, Isl-1, Mec-3) domains are scaffolding sarcomeric and cytoskeletal elements that form structured muscle fibres and provide for the link to intracellular signalling by selectively associating protein kinases, ion channels, and transcription factors with the mechanical stress-strain sensors. Enigma homolog (ENH) is a PDZ-LIM protein with four splice variants: ENH1 with an N-terminal PDZ domain and three C-terminal LIM domains and ENH2, ENH3, and ENH4 without LIM domains. We addressed the functional role of ENH alternative splicing. We studied the expression of the four ENH isoforms in the heart during development and in a mouse model of heart hypertrophy. All four isoforms are expressed in the heart but the pattern of expression is clearly different between embryonic, neonatal, and adult stages. ENH1 appears as the embryonic isoform, whereas ENH2, ENH3, and ENH4 are predominant in adult heart. Moreover, alternative splicing of ENH was changed following induction of heart hypertrophy, producing an ENH isoform pattern similar to that of neonatal heart. Next, we tested a possible causal role of ENH1 and ENH4 in the development of cardiac hypertrophy. When overexpressed in rat neonatal cardiomyocytes, ENH1 promoted the expression of hypertrophy markers and increased cell volume, whereas, on the contrary, ENH4 overexpression prevented these changes. Antagonistic splice variants of ENH may play a central role in the adaptive changes of the link between mechanical stress-sensing and signalling occurring during embryonic development and/or heart hypertrophy.

  11. Physiological relation between respiration activity and heterologous expression of selected benzoylformate decarboxylase variants in Escherichia coli

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    Pohl Martina

    2010-10-01

    Full Text Available Abstract Background The benzoylformate decarboxylase (BFD from Pseudomonas putida is a biotechnologically interesting biocatalyst. It catalyses the formation of chiral 2-hydroxy ketones, which are important building blocks for stereoselective syntheses. To optimise the enzyme function often the amino acid composition is modified to improve the performance of the enzyme. So far it was assumed that a relatively small modification of the amino acid composition of a protein does not significantly influence the level of expression or media requirements. To determine, which effects these modifications might have on cultivation and product formation, six different BFD-variants with one or two altered amino acids and the wild type BFD were expressed in Escherichia coli SG13009 pKK233-2. The oxygen transfer rate (OTR as parameter for growth and metabolic activity of the different E. coli clones was monitored on-line in LB, TB and modified PanG mineral medium with the Respiratory Activity MOnitoring System (RAMOS. Results Although the E. coli clones were genetically nearly identical, the kinetics of their metabolic activity surprisingly differed in the standard media applied. Three different types of OTR curves could be distinguished. Whereas the first type (clones expressing Leu476Pro-Ser181Thr or Leu476Pro had typical OTR curves, the second type (clones expressing the wild type BFD, Ser181Thr or His281Ala showed an early drop of OTR in LB and TB medium and a drastically reduced maximum OTR in modified PanG mineral medium. The third type (clone expressing Leu476Gln behaved variable. Depending on the cultivation conditions, its OTR curve was similar to the first or the second type. It was shown, that the kinetics of the metabolic activity of the first type depended on the concentration of thiamine, which is a cofactor of BFD, in the medium. It was demonstrated that the cofactor binding strength of the different BFD-variants correlated with the differences

  12. 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.

  13. The chemokine receptor CXCR3 and its splice variant are expressed in human airway epithelial cells.

    Science.gov (United States)

    Kelsen, Steven G; Aksoy, Mark O; Yang, Yi; Shahabuddin, Syed; Litvin, Judith; Safadi, Fayez; Rogers, Thomas J

    2004-09-01

    Activation of the chemokine receptor CXCR3 by its cognate ligands induces several differentiated cellular responses important to the growth and migration of a variety of hematopoietic and structural cells. In the human respiratory tract, human airway epithelial cells (HAEC) release the CXCR3 ligands Mig/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11. Simultaneous expression of CXCR3 by HAEC would have important implications for the processes of airway inflammation and repair. Accordingly, in the present study we sought to determine whether HAEC also express the classic CXCR3 chemokine receptor CXCR3-A and its splice variant CXCR3-B and hence may respond in autocrine fashion to its ligands. We found that cultured HAEC (16-HBE and tracheocytes) constitutively expressed CXCR3 mRNA and protein. CXCR3 mRNA levels assessed by expression array were approximately 35% of beta-actin expression. In contrast, CCR3, CCR4, CCR5, CCR8, and CX3CR1 were <5% beta-actin. Both CXCR3-A and -B were expressed. Furthermore, tracheocytes freshly harvested by bronchoscopy stained positively for CXCR3 by immunofluorescence microscopy, and 68% of cytokeratin-positive tracheocytes (i.e., the epithelial cell population) were positive for CXCR3 by flow cytometry. In 16-HBE cells, CXCR3 receptor density was approximately 78,000 receptors/cell when assessed by competitive displacement of 125I-labeled IP-10/CXCL10. Finally, CXCR3 ligands induced chemotactic responses and actin reorganization in 16-HBE cells. These findings indicate constitutive expression by HAEC of a functional CXC chemokine receptor, CXCR3. Our data suggest the possibility that autocrine activation of CXCR3 expressed by HAEC may contribute to airway inflammation and remodeling in obstructive lung disease by regulating HAEC migration.

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

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    Joseph M Catanzaro

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

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

    Science.gov (United States)

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

    1999-04-01

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

  16. Serodiagnosis of bovine trypanosomosis caused by non-tsetse transmitted Trypanosoma (Duttonella) vivax parasites using the soluble form of a Trypanozoon variant surface glycoprotein antigen.

    Science.gov (United States)

    Uzcanga, Graciela L; Pérez-Rojas, Yenis; Camargo, Rocío; Izquier, Adriana; Noda, José A; Chacín, Ronny; Parra, Nereida; Ron, Lenin; Rodríguez-Hidalgo, Richar; Bubis, José

    2016-03-15

    Previous studies have shown that a 64-kDa antigen (p64) that was purified from the Venezuelan TeAp-N/D1 isolate of Trypanosoma (Trypanozoon) equiperdum corresponds to the soluble form of its predominant variant surface glycoprotein (VSG), and exhibited cross-reactivity with Trypanosoma (Duttonella) vivax. The course of experimental acute infections of bovines with T. vivax were followed by measuring whole anti-p64 antibodies and specific anti-p64 IgG and IgM antibodies in animal sera by indirect enzyme-linked immunosorbent assay (ELISA). The value of p64 to diagnose bovine trypanosomosis was also examined using 350 sera from healthy and T. vivax-infected cows living in a trypanosomosis-endemic and enzootic stable area, and 48 sera obtained during a trypanosomosis outbreak. Serological assays showed that ∼ 70-80% of the infected sera contained anti-p64 antibodies, based on the comparative immunodetection of the T. equiperdum clarified antigenic fraction used as a reference test. In the absence of a gold standard, Bayesian analysis for multiple testing estimated a sensitivity and specificity of 71.6% and 98.8%, respectively, for the indirect ELISA using p64 as antigen. An apparent prevalence of 37.7% for bovine trypanosomosis infection was also estimated with a Bayesian approach when the p64 ELISA test was used. Employing blood from acute infected cows, the indirect ELISA response against p64 was contrasted with the microhematocrit centrifuge method and analyses by polymerase chain reaction (PCR) using specific primers targeting the inter-specific length variation of the internal transcribed spacer 1 region of the 18S ribosomal gene. The efficiency of p64 for the detection of anti-trypanosome antibodies in acute infected bovines was also corroborated serologically by comparing its response to that of the Indonesian Trypanosoma evansi Rode Trypanozoon antigen type (RoTat) 1.2 VSG, which possesses high specificity and sensitivity. As expected, PCR was the best

  17. Differential expression of splicing variants of the human caldesmon gene (CALD1) in glioma neovascularization versus normal brain microvasculature

    NARCIS (Netherlands)

    P.P. Zheng (Pingpin); A.M. Sieuwerts (Anieta); T.M. Luider (Theo); M.M. van der Weiden (Marcel); J.M. Kros (Johan); P.A.E. Sillevis Smitt (Peter)

    2004-01-01

    textabstractCaldesmon is a cytoskeleton-associated protein which has not yet been related to neoplastic angiogenesis. In this study we investigated the expression of the caldesmon gene (CALD1) splicing variants and the protein expression level in glioma microvessels versus normal

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

    DEFF Research Database (Denmark)

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

    1983-01-01

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

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

    Science.gov (United States)

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

    2016-02-01

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

  20. Modeling malaria infection and immunity against variant surface antigens in Príncipe island, West Africa

    DEFF Research Database (Denmark)

    Bandeiras, Cátia; Trovoada, Maria Jesus; Gonçalves, Lígia A

    2014-01-01

    After remarkable success of vector control campaigns worldwide, concerns about loss of immunity against Plasmodium falciparum due to lack of exposure to the parasite are relevant since an increase of severe cases in less immune individuals is expected. We present a mathematical model to investigate...... the impact of reducing exposure to the parasite on the immune repertoire against P. falciparum erythrocyte membrane protein 1 (PfEMP1) variants. The model was parameterized with data from Príncipe Island, West Africa, and applied to simulate two alternative transmission scenarios: one where control measures...... are continued to eventually drive the system to elimination; and another where the effort is interrupted after 6 years of its initiation and the system returns to the initial transmission potential. Population dynamics of parasite prevalence predict that in a few years infection levels return to the pre...

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

    Science.gov (United States)

    Strzadała, L; Opolski, A; Kisielow, P

    1981-01-01

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

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

    Science.gov (United States)

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

    1985-02-01

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

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

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    Uffe B. Skov Sørensen

    2016-11-01

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

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

    Science.gov (United States)

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

    2003-10-01

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

  5. Analysis of NPM1 splice variants reveals differential expression patterns of prognostic value in acute myeloid leukemia

    Science.gov (United States)

    Stasiak, Grazyna; Zaleska, Joanna; Kielbus, Michal; Czapinski, Jakub; Schunn, Matthias; Correa, Stephany C.; Glodkowska-Mrowka, Eliza; Sundaram, Reddy Chakkarappan; Jankowska-Lecka, Olga; Schlenk, Richard F.; Döhner, Hartmut; Döhner, Konstanze; Stepulak, Andrzej; Bullinger, Lars; Giannopoulos, Krzysztof

    2017-01-01

    Mutations of the nucleophosmin-1 (NPM1) gene in cytogenetically normal (CN) acute myeloid leukemia (AML) identify a group of patients with more favorable prognosis. NPM1 encodes three main alternatively spliced isoforms R1(B23.1), R2(B23.2), and R3(B23.3). The expression of splice variants R1, R2 and R3 were higher in AML patients compared to normal cells of healthy volunteers (HVs), although RNA-seq analysis revealed enhanced R2 expression also in less differentiated cells of HVs as well as in AML cells. The variant R2, which lacks exons 11 and 12 coding for the nucleolar localization domain, might behave similar to the mutant form of NPM1 (NPM1mut). In accordance, in CN-AML high R2 expression was associated with favorable impact on outcome. Moreover, functional studies showed nucleolar localization of the eGFP-NPM1 wildtype and cytoplasmic localization of the eGFP-NPM1 mut protein. While the eGFP-NPM1 R2 splice variant localized predominantly in the nucleoplasm, we also could detect cytoplasmic expression for the R2 variant. These results support a unique biological consequence of R2 overexpression and in part explain our clinical observation, where that high R2 variant expression was associated with a better prognosis in CN-AML patients. PMID:29221119

  6. Alternative splicing variations in mouse CAPS2: differential expression and functional properties of splicing variants

    Directory of Open Access Journals (Sweden)

    Furuichi Teiichi

    2007-04-01

    Full Text Available Abstract Background Ca2+-dependent activator protein 2 (CAPS2/CADPS2 is a secretory vesicle-associated protein involved in the release of neurotrophin. We recently reported that an aberrant, alternatively spliced CAPS2 mRNA that lacks exon 3 (CAPS2Δexon3 is detected in some patients with autism. Splicing variations in mouse CAPS2 and their expression and functions remain unclear. Results In this study, we defined 31 exons in the mouse CAPS2 gene and identified six alternative splicing variants, CAPS2a-f. CAPS2a is an isoform lacking exons 22 and 25, which encode part of the Munc13-1-homologous domain (MHD. CAPS2b lacks exon 25. CAPS2c lacks exons 11 and 22. CAPS2d, 2e, and 2f have C-terminal deletions from exon 14, exon 12, and exon 5, respectively. On the other hand, a mouse counterpart of CAPS2Δexon3 was not detected in the mouse tissues tested. CAPS2b was expressed exclusively in the brain, and the other isoforms were highly expressed in the brain, but also in some non-neural tissues. In the brain, all isoforms showed predominant expression patterns in the cerebellum. In the developing cerebellum, CAPS2b showed an up-regulated expression pattern, whereas the other isoforms exhibited transiently peaked expression patterns. CAPS2 proteins were mostly recovered in soluble fractions, but some were present in membrane fractions, except for CAPS2c and 2f, both of which lack the PH domain, suggesting that the PH domain is important for membrane association. In contrast to CAPS2a and 2b, CAPS2c showed slightly decreased BDNF-releasing activity, which is likely due to the C-terminal truncation of the PH domain in CAPS2c. Conclusion This study indicates that, in mouse, there are six splicing variants of CAPS2 (CAPS2a-f, and that these are subdivided into two groups: a long form containing the C-terminal MHD and a short form lacking the C-terminal MHD. These results demonstrate that the splicing variations correlate with their expression patterns and

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Science.gov (United States)

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

    2009-01-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

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

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    Tanzer Fiona L

    2008-06-01

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

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

    Science.gov (United States)

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

    2008-03-01

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

  12. Expression of human placental lactogen and variant growth hormone genes in placentas.

    Science.gov (United States)

    Martinez-Rodriguez, H G; Guerra-Rodriguez, N E; Iturbe-Cantu, M A; Martinez-Torres, A; Barrera-Saldaña, H A

    1997-01-01

    Previous studies comparing the expression levels of human placental lactogen (hPL) genes have shown varying results, due to, perhaps, the fact that in all of them only one placenta was being analyzed. Here, the expression of hPL and growth hormone variant (hGH-V) genes in fifteen term placentas was comparatively analyzed at the RNA level, using reverse transcription coupled to polymerase chain reaction (RT-PCR). The abundance of the combined RNA transcripts derived from these genes varied from one placenta to another. The authors found that hPL-4 transcripts were more abundant than those of hPL-3 in most samples (ratios from 1:1 to 6:1), transcripts from the putative hPL-1 pseudogene were more abundant at the unprocessed stage while those of the hGH-V gene were mostly processed. Again, the authors of this study observed wide variation from placenta to placenta in the abundance of both of these types of transcripts. The same was observed when a group of six placentas from abortuses and nine from pregnancies complicated by preclampsia, diabetes and hypertension was studied. The authors conclude that the disagreeing results reported in the literature which are not in agreement concerning the expression levels of hPL genes could be explained by normal variations of their expression levels among the different placentas analyzed.

  13. Extracellular Matrix Proteins Expression Profiling in Chemoresistant Variants of the A2780 Ovarian Cancer Cell Line

    Directory of Open Access Journals (Sweden)

    Radosław Januchowski

    2014-01-01

    Full Text Available Ovarian cancer is the leading cause of death among gynaecological malignancies. Extracellular matrix (ECM can affect drug resistance by preventing the penetration of the drug into cancer cells and increased resistance to apoptosis. This study demonstrates alterations in the expression levels of ECM components and related genes in cisplatin-, doxorubicin-, topotecan-, and paclitaxel-resistant variants of the A2780 ovarian cancer cell line. Affymetrix Gene Chip Human Genome Array Strips were used for hybridisations. The genes that had altered expression levels in drug-resistant sublines were selected and filtered by scatter plots. The genes that were up- or downregulated more than fivefold were selected and listed. Among the investigated genes, 28 genes were upregulated, 10 genes were downregulated, and two genes were down- or upregulated depending on the cell line. Between upregulated genes 12 were upregulated very significantly—over 20-fold. These genes included COL1A2, COL12A1, COL21A1, LOX, TGFBI, LAMB1, EFEMP1, GPC3, SDC2, MGP, MMP3, and TIMP3. Four genes were very significantly downregulated: COL11A1, LAMA2, GPC6, and LUM. The expression profiles of investigated genes provide a preliminary insight into the relationship between drug resistance and the expression of ECM components. Identifying correlations between investigated genes and drug resistance will require further analysis.

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

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    Warren L Denning

    2011-02-01

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

  15. [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.

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

    Directory of Open Access Journals (Sweden)

    Rancés Blanco

    2013-01-01

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

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

    OpenAIRE

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

    2008-01-01

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

  18. Expression of a Splice Variant of CYP26B1 in Betel Quid-Related Oral Cancer

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    Ping-Ho Chen

    2014-01-01

    Full Text Available Betel quid (BQ is a psychostimulant, an addictive substance, and a group 1 carcinogen that exhibits the potential to induce adverse health effects. Approximately, 600 million users chew a variety of BQ. Areca nut (AN is a necessary ingredient in BQ products. Arecoline is the primary alkaloid in the AN and can be metabolized through the cytochrome P450 (CYP superfamily by inducing reactive oxygen species (ROS production. Full-length CYP26B1 is related to the development of oral pharyngeal cancers. We investigated whether a splice variant of CYP26B1 is associated with the occurrence of ROS related oral and pharyngeal cancer. Cytotoxicity assays were used to measure the effects of arecoline on cell viability in a dose-dependent manner. In vitro and in vivo studies were conducted to evaluate the expression of the CYP26B1 splice variant. The CYP26B1 splice variant exhibited lower expression than did full-length CYP26B1 in the human gingival fibroblast-1 and Ca9-22 cell models. Increased expression of the CYP26B1 splice variant was observed in human oral cancer tissue compared with adjacent normal tissue, and increased expression was observed in patients at a late tumor stage. Our results suggested that the CYP26B1 splice variant is associated with the occurrence of BQ-related oral cancer.

  19. Linking protective GAB2 variants, increased cortical GAB2 expression and decreased Alzheimer's disease pathology.

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    Fanggeng Zou

    Full Text Available GRB-associated binding protein 2 (GAB2 represents a compelling genome-wide association signal for late-onset Alzheimer's disease (LOAD with reported odds ratios (ORs ranging from 0.75-0.85. We tested eight GAB2 variants in four North American Caucasian case-control series (2,316 LOAD, 2,538 controls for association with LOAD. Meta-analyses revealed ORs ranging from (0.61-1.20 with no significant association (all p>0.32. Four variants were hetergeneous across the populations (all p<0.02 due to a potentially inflated effect size (OR = 0.61-0.66 only observed in the smallest series (702 LOAD, 209 controls. Despite the lack of association in our series, the previously reported protective association for GAB2 remained after meta-analyses of our data with all available previously published series (11,952-22,253 samples; OR = 0.82-0.88; all p<0.04. Using a freely available database of lymphoblastoid cell lines we found that protective GAB2 variants were associated with increased GAB2 expression (p = 9.5×10(-7-9.3×10(-6. We next measured GAB2 mRNA levels in 249 brains and found that decreased neurofibrillary tangle (r = -0.34, p = 0.0006 and senile plaque counts (r = -0.32, p = 0.001 were both good predictors of increased GAB2 mRNA levels albeit that sex (r = -0.28, p = 0.005 may have been a contributing factor. In summary, we hypothesise that GAB2 variants that are protective against LOAD in some populations may act functionally to increase GAB2 mRNA levels (in lymphoblastoid cells and that increased GAB2 mRNA levels are associated with significantly decreased LOAD pathology. These findings support the hypothesis that Gab2 may protect neurons against LOAD but due to significant population heterogeneity, it is still unclear whether this protection is detectable at the genetic level.

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

    Directory of Open Access Journals (Sweden)

    Harjeet Singh

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

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Science.gov (United States)

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

    2013-01-01

    Adoptive transfer of T cells expressing a CD19-specific chimeric antigen receptor (CAR) is being evaluated in multiple clinical trials. Our current approach to adoptive immunotherapy is based on a second generation CAR (designated CD19RCD28) that signals through a CD28 and CD3-ζ endodomain. T cells are electroporated with DNA plasmids from the Sleeping Beauty (SB) transposon/transposase system to express this CAR. Stable integrants of genetically modified T cells can then be retrieved when co-cultured with designer artificial antigen presenting cells (aAPC) in the presence of interleukin (IL)-2 and 21. Here, we reveal how the platform technologies of SB-mediated transposition and CAR-dependent propagation on aAPC were adapted for human application. Indeed, we have initiated clinical trials in patients with high-risk B-lineage malignancies undergoing autologous and allogeneic hematopoietic stem-cell transplantation (HSCT). We describe the process to manufacture clinical grade CD19-specific T cells derived from healthy donors. Three validation runs were completed in compliance with current good manufacturing practice for Phase I/II trials demonstrating that by 28 days of co-culture on γ-irradiated aAPC ∼1010 T cells were produced of which >95% expressed CAR. These genetically modified and propagated T cells met all quality control testing and release criteria in support of infusion. PMID:23741305

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

    Science.gov (United States)

    Ohtani, Haruo

    2013-02-01

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

  4. Genetic variants in human leukocyte antigen-DP influence both hepatitis C virus persistence and hepatitis C virus F protein generation in the Chinese Han population.

    Science.gov (United States)

    Xu, Xiaodong; Yue, Ming; Jiang, Longfeng; Deng, Xiaozhao; Zhang, Yongxiang; Zhang, Yun; Zhu, Danyan; Xiao, Wen; Zhou, Zhenxian; Yao, Wenjuan; Kong, Jing; Yu, Xiaojie; Wei, Juan

    2014-06-03

    Chronic hepatitis C is a serious liver disease that often results in cirrhosis or hepatocellular carcinoma. The aim of this study was to assess the association of human leukocyte antigen-DP (HLA-DP) variants with risk of chronic hepatitis C virus (HCV) or anti-F antibody generation. We selected two single nucleotide polymorphisms (SNPs) in a region including HLA-DPA1 (rs3077) and HLA-DPB1 (rs9277534) and genotyped SNPs in 702 cases and 342 healthy controls from the Chinese population using TaqMan SNP genotyping assay. Moreover, the exon 2 of the HLA-DPA1 and HLA-DPB1 genes were amplified and determined by sequencing-based typing (SBT). The results showed that rs3077 significantly increased the risk of chronic HCV infection in additive models and dominant models (odds ratio (OR) = 1.32 and 1.53). The rs3077 also contributed to decrease the risk of anti-F antibody generation in additive models and dominant models (OR = 0.46 and 0.56). Subsequent analyses revealed the risk haplotypes (DPA1*0103-DPB1*0501 and DPA1*0103-DPB1*0201) and protective haplotypes (DPA1*0202-DPB1*0501 and DPA1*0202-DPB1*0202) to chronic HCV infection. Moreover, we also found that the haplotype of DPA1*0103-DPB1*0201 and DPA1*0202-DPB1*0202 were associated with the anti-F antibody generation. Our findings show that genetic variants in HLA-DP gene are associated with chronic HCV infection and anti-F antibody generation.

  5. Association of human leukocyte antigen variants and allopurinol-induced Stevens-Johnson syndrome and toxic epidermal necrolysis: A meta-analysis.

    Science.gov (United States)

    Li, Xingang; Zhao, Zhigang; Sun, Shu-Sen

    2017-05-01

    The association between human leukocyte antigen (HLA) variants and allopurinol-induced Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) was evaluated through a pooled analysis of published studies. A comprehensive search was performed in multiple databases, including PubMed, MEDLINE, ISI Web of Knowledge, EMBASE, Cochrane Register of Controlled Trials, and Science Direct. Studies investigating the association between HLA alleles with allopurinol-induced SJS or TEN were retrieved, and the data were independently extracted. The overall odds ratios (ORs) with corresponding 95% confidence intervals were calculated to determine the association between the presence of HLA variant in at least one allele and allopurinol-induced SJS or TEN. To test the robustness of the meta-analysis results, a sensitivity analysis was performed by removing each study one at a time and calculating the pooled ORs of the remaining studies. The fixed-effects and random-effects models were used to pool the collected data. A total of 4 studies with 81 allopurinol-induced SJS or TEN cases and matched controls (allopurinol-tolerant patients) or population controls (general population) were identified. SJS and TEN were found to be significantly associated with HLA-A*33:03 and HLA-C*03:02 alleles in both groups of studies with matched controls and population controls. All of the pooled ORs were not significantly affected by the remaining studies and different modeling methods, indicating robust results. A strong association was found between HLA-A*33:03 and HLA-C*03:02 alleles and allopurinol-induced SJS or TEN, especially in an Asian population. Copyright © 2017 by the American Society of Health-System Pharmacists, Inc. All rights reserved.

  6. Gene expression, single nucleotide variant and fusion transcript discovery in archival material from breast tumors.

    Directory of Open Access Journals (Sweden)

    Nadine Norton

    Full Text Available Advantages of RNA-Seq over array based platforms are quantitative gene expression and discovery of expressed single nucleotide variants (eSNVs and fusion transcripts from a single platform, but the sensitivity for each of these characteristics is unknown. We measured gene expression in a set of manually degraded RNAs, nine pairs of matched fresh-frozen, and FFPE RNA isolated from breast tumor with the hybridization based, NanoString nCounter (226 gene panel and with whole transcriptome RNA-Seq using RiboZeroGold ScriptSeq V2 library preparation kits. We performed correlation analyses of gene expression between samples and across platforms. We then specifically assessed whole transcriptome expression of lincRNA and discovery of eSNVs and fusion transcripts in the FFPE RNA-Seq data. For gene expression in the manually degraded samples, we observed Pearson correlations of >0.94 and >0.80 with NanoString and ScriptSeq protocols, respectively. Gene expression data for matched fresh-frozen and FFPE samples yielded mean Pearson correlations of 0.874 and 0.783 for NanoString (226 genes and ScriptSeq whole transcriptome protocols respectively, p<2x10(-16. Specifically for lincRNAs, we observed superb Pearson correlation (0.988 between matched fresh-frozen and FFPE pairs. FFPE samples across NanoString and RNA-Seq platforms gave a mean Pearson correlation of 0.838. In FFPE libraries, we detected 53.4% of high confidence SNVs and 24% of high confidence fusion transcripts. Sensitivity of fusion transcript detection was not overcome by an increase in depth of sequencing up to 3-fold (increase from ~56 to ~159 million reads. Both NanoString and ScriptSeq RNA-Seq technologies yield reliable gene expression data for degraded and FFPE material. The high degree of correlation between NanoString and RNA-Seq platforms suggests discovery based whole transcriptome studies from FFPE material will produce reliable expression data. The RiboZeroGold ScriptSeq protocol

  7. Gene expression, single nucleotide variant and fusion transcript discovery in archival material from breast tumors.

    Science.gov (United States)

    Norton, Nadine; Sun, Zhifu; Asmann, Yan W; Serie, Daniel J; Necela, Brian M; Bhagwate, Aditya; Jen, Jin; Eckloff, Bruce W; Kalari, Krishna R; Thompson, Kevin J; Carr, Jennifer M; Kachergus, Jennifer M; Geiger, Xochiquetzal J; Perez, Edith A; Thompson, E Aubrey

    2013-01-01

    Advantages of RNA-Seq over array based platforms are quantitative gene expression and discovery of expressed single nucleotide variants (eSNVs) and fusion transcripts from a single platform, but the sensitivity for each of these characteristics is unknown. We measured gene expression in a set of manually degraded RNAs, nine pairs of matched fresh-frozen, and FFPE RNA isolated from breast tumor with the hybridization based, NanoString nCounter (226 gene panel) and with whole transcriptome RNA-Seq using RiboZeroGold ScriptSeq V2 library preparation kits. We performed correlation analyses of gene expression between samples and across platforms. We then specifically assessed whole transcriptome expression of lincRNA and discovery of eSNVs and fusion transcripts in the FFPE RNA-Seq data. For gene expression in the manually degraded samples, we observed Pearson correlations of >0.94 and >0.80 with NanoString and ScriptSeq protocols, respectively. Gene expression data for matched fresh-frozen and FFPE samples yielded mean Pearson correlations of 0.874 and 0.783 for NanoString (226 genes) and ScriptSeq whole transcriptome protocols respectively, pNanoString and RNA-Seq platforms gave a mean Pearson correlation of 0.838. In FFPE libraries, we detected 53.4% of high confidence SNVs and 24% of high confidence fusion transcripts. Sensitivity of fusion transcript detection was not overcome by an increase in depth of sequencing up to 3-fold (increase from ~56 to ~159 million reads). Both NanoString and ScriptSeq RNA-Seq technologies yield reliable gene expression data for degraded and FFPE material. The high degree of correlation between NanoString and RNA-Seq platforms suggests discovery based whole transcriptome studies from FFPE material will produce reliable expression data. The RiboZeroGold ScriptSeq protocol performed particularly well for lincRNA expression from FFPE libraries, but detection of eSNV and fusion transcripts was less sensitive.

  8. 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.

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

    Science.gov (United States)

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

    1995-07-01

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

  10. 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.

  11. AntigenMap 3D: an online antigenic cartography resource.

    Science.gov (United States)

    Barnett, J Lamar; Yang, Jialiang; Cai, Zhipeng; Zhang, Tong; Wan, Xiu-Feng

    2012-05-01

    Antigenic cartography is a useful technique to visualize and minimize errors in immunological data by projecting antigens to 2D or 3D cartography. However, a 2D cartography may not be sufficient to capture the antigenic relationship from high-dimensional immunological data. AntigenMap 3D presents an online, interactive, and robust 3D antigenic cartography construction and visualization resource. AntigenMap 3D can be applied to identify antigenic variants and vaccine strain candidates for pathogens with rapid antigenic variations, such as influenza A virus. http://sysbio.cvm.msstate.edu/AntigenMap3D

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

    Science.gov (United States)

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

    1989-07-01

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

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

    Science.gov (United States)

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

    1996-05-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    DEFF Research Database (Denmark)

    Hokland, M; Larsen, B; Heron, I

    1982-01-01

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

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

    NARCIS (Netherlands)

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

    1996-01-01

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

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

    DEFF Research Database (Denmark)

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

    2003-01-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Dina A Moustafa

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

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

    DEFF Research Database (Denmark)

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

    1989-01-01

    After a peripheral nerve lesion (rat facial and sciatic) an induction of major histocompatibility complex (MHC) antigens class I was detected immunohistochemically in skeletal muscle fibers and motor neurons. This MHC expression was transient after a nerve crush, when regeneration occurred...

  1. Survivin 2α: a novel Survivin splice variant expressed in human malignancies

    Directory of Open Access Journals (Sweden)

    Honsey Laura E

    2005-03-01

    Full Text Available Abstract Background Survivin and its alternative splice forms are involved in critical cellular processes, including cell division and programmed cell death. Survivin is expressed in the majority of human cancers, but minimally in differentiated normal tissues. Expression levels correlate with tumor aggressiveness and resistance to therapy. Results In the present study, we identify and characterize a novel survivin isoform that we designate survivin 2α. Structurally, the transcript consists of 2 exons: exon 1 and exon 2, as well as a 3' 197 bp region of intron 2. Acquisition of a new in-frame stop codon within intron 2 results in an open reading frame of 225 nucleotides, predicting a truncated 74 amino acid protein. Survivin 2α is expressed at high levels in several malignant cell lines and primary tumors. Functional assays show that survivin 2α attenuates the anti-apoptotic activity of survivin. Subcellular localization and immunoprecipitation of survivin 2α suggests a physical interaction with survivin. Conclusion We characterized a novel survivin splice variant that we designated survivin 2α. We hypothesize that survivin 2α can alter the anti-apoptotic functions of survivin in malignant cells. Thus survivin 2α may be useful as a therapeutic tool in sensitizing chemoresistant tumor cells to chemotherapy.

  2. Quasispecies variant of pre-S/S gene in HBV-related hepatocellular carcinoma with HBs antigen positive and occult infection.

    Science.gov (United States)

    Hatazawa, Yuri; Yano, Yoshihiko; Okada, Rina; Tanahashi, Toshihito; Hayashi, Hiroki; Hirano, Hirotaka; Minami, Akihiro; Kawano, Yuki; Tanaka, Motofumi; Fukumoto, Takumi; Murakami, Yoshiki; Yoshida, Masaru; Hayashi, Yoshitake

    2018-01-01

    Hepatocellular carcinoma (HCC) can develop in patients who are negative for the hepatitis B surface antigen (HBsAg) in serum but positive for hepatitis B virus (HBV) DNA in the liver, referred to as occult HBV infection (OBI). Previous reports showed that HBV variants in OBI-related HCC are different from those in HBsAg-positive HCC. In the present study, HBV quasispecies based on the pre-S/S gene in OBI-related HCC patients were examined by high throughput sequencing and compared with those in HBsAg-positive HCC. Nineteen tissue samples (9 OBI-related and 10 HBsAg-positive non-cancerous tissues) were collected at the time of surgery at Kobe University Hospital. The quasispecies with more than 1% variation in the pre-S/S region were isolated and analysed by ultra-deep sequencing. There were no significant differences in the major HBV populations, which exhibit more than 20% variation within the entire pre-S/S region, between OBI-related HCC and HBsAg-positive HCC. However, the prevalences of major populations with pre-S2 region mutations and of minor populations with polymerized human serum albumin-binding domain mutations were significantly higher in OBI-related HCC than in HBsAg-positive HCC. Moreover, the major variant populations associated with the B-cell epitope, located within the pre-S1 region, and the a determinant domain, located in the S region, were detected frequently in HBsAg-positive HCC. The minor populations of variants harbouring the W4R, L30S, Q118R/Stop, N123D and S124F/P mutations in the pre-S region and the L21F/S and L42F/S mutations in the S region were detected more frequently in OBI-related HCC than in HBsAg-positive HCC. Ultra-deep sequencing revealed that the B-cell epitope domain in the pre-S1 region and alpha determinant domain in the S region were variable in HBsAg-positive HCC, although the quasispecies associated with the pre-S2 region were highly prevalent in OBI-related HCC. Ref: R000034382/UMIN000030113; Retrospectively

  3. Expression of murine 5-aminolevulinate synthase variants causes protoporphyrin IX accumulation and light-induced mammalian cell death.

    Directory of Open Access Journals (Sweden)

    Erica J Fratz

    Full Text Available 5-Aminolevulinate synthase (ALAS; EC 2.3.1.37 catalyzes the first committed step of heme biosynthesis in animals. The erythroid-specific ALAS isozyme (ALAS2 is negatively regulated by heme at the level of mitochondrial import and, in its mature form, certain mutations of the murine ALAS2 active site loop result in increased production of protoporphyrin IX (PPIX, the precursor for heme. Importantly, generation of PPIX is a crucial component in the widely used photodynamic therapies (PDT of cancer and other dysplasias. ALAS2 variants that cause high levels of PPIX accumulation provide a new means of targeted, and potentially enhanced, photosensitization. In order to assess the prospective utility of ALAS2 variants in PPIX production for PDT, K562 human erythroleukemia cells and HeLa human cervical carcinoma cells were transfected with expression plasmids for ALAS2 variants with greater enzymatic activity than the wild-type enzyme. The levels of accumulated PPIX in ALAS2-expressing cells were analyzed using flow cytometry with fluorescence detection. Further, cells expressing ALAS2 variants were subjected to white light treatments (21-22 kLux for 10 minutes after which cell viability was determined. Transfection of HeLa cells with expression plasmids for murine ALAS2 variants, specifically for those with mutated mitochondrial presequences and a mutation in the active site loop, caused significant cellular accumulation of PPIX, particularly in the membrane. Light treatments revealed that ALAS2 expression results in an increase in cell death in comparison to aminolevulinic acid (ALA treatment producing a similar amount of PPIX. The delivery of stable and highly active ALAS2 variants has the potential to expand and improve upon current PDT regimes.

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

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    Luciana Rossi

    2014-01-01

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

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

    Science.gov (United States)

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

    2009-05-27

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

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

    Directory of Open Access Journals (Sweden)

    Mangia Anita

    2009-05-01

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

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

    NARCIS (Netherlands)

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

    1985-01-01

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

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

    DEFF Research Database (Denmark)

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

    2013-01-01

    NSCLC has shown promising results. However, little is known about the expression of other cancer/testis antigens in NSCLC. In the present study the expression of cancer/testis antigens GAGE, NY-ESO-1 and SP17 was investigated in patients with completely resected, early stage, primary NSCLC....

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

    DEFF Research Database (Denmark)

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

    1993-01-01

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

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

    Science.gov (United States)

    1996-05-01

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

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

    Science.gov (United States)

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

    2018-01-30

    Although autoimmune disorders are a significant source of morbidity and mortality in older individuals, the mechanisms governing age-associated increases in susceptibility remain incompletely understood. Central T cell tolerance is mediated through presentation of self-antigens by cells constituting the thymic microenvironment, including epithelial cells, dendritic cells, and B cells. Medullary thymic epithelial cells (mTECs) and B cells express distinct cohorts of self-antigens, including tissue-restricted self-antigens (TRAs), such that developing T cells are tolerized to antigens from peripheral tissues. We find that expression of the TRA transcriptional regulator Aire, as well as Aire-dependent genes, declines with age in thymic B cells in mice and humans and that cell-intrinsic and cell-extrinsic mechanisms contribute to the diminished capacity of peripheral B cells to express Aire within the thymus. Our findings indicate that aging may diminish the ability of thymic B cells to tolerize T cells, revealing a potential mechanistic link between aging and autoimmunity. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

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

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    Sergio Cepeda

    2018-01-01

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

  13. Consensus HIV-1 FSU-A integrase gene variants electroporated into mice induce polyfunctional antigen-specific CD4+ and CD8+ T cells.

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    Olga Krotova

    Full Text Available Our objective is to create gene immunogens targeted against drug-resistant HIV-1, focusing on HIV-1 enzymes as critical components in viral replication and drug resistance. Consensus-based gene vaccines are specifically fit for variable pathogens such as HIV-1 and have many advantages over viral genes and their expression-optimized variants. With this in mind, we designed the consensus integrase (IN of the HIV-1 clade A strain predominant in the territory of the former Soviet Union and its inactivated derivative with and without mutations conferring resistance to elvitegravir. Humanized IN gene was synthesized; and inactivated derivatives (with 64D in the active site mutated to V with and without elvitegravir-resistance mutations were generated by site-mutagenesis. Activity tests of IN variants expressed in E coli showed the consensus IN to be active, while both D64V-variants were devoid of specific activities. IN genes cloned in the DNA-immunization vector pVax1 (pVaxIN plasmids were highly expressed in human and murine cell lines (>0.7 ng/cell. Injection of BALB/c mice with pVaxIN plasmids followed by electroporation generated potent IFN-γ and IL-2 responses registered in PBMC by day 15 and in splenocytes by day 23 after immunization. Multiparametric FACS demonstrated that CD8+ and CD4+ T cells of gene-immunized mice stimulated with IN-derived peptides secreted IFN-γ, IL-2, and TNF-α. The multi-cytokine responses of CD8+ and CD4+ T-cells correlated with the loss of in vivo activity of the luciferase reporter gene co-delivered with pVaxIN plasmids. This indicated the capacity of IN-specific CD4+ and CD8+ T-cells to clear IN/reporter co-expressing cells from the injection sites. Thus, the synthetic HIV-1 clade A integrase genes acted as potent immunogens generating polyfunctional Th1-type CD4+ and CD8+ T cells. Generation of such response is highly desirable for an effective HIV-1 vaccine as it offers a possibility to attack virus

  14. Fertility Defects in Mice Expressing the L68Q Variant of Human Cystatin C

    Science.gov (United States)

    Whelly, Sandra; Serobian, Gaiane; Borchardt, Clinton; Powell, Jonathan; Johnson, Seethal; Hakansson, Katarina; Lindstrom, Veronica; Abrahamson, Magnus; Grubb, Anders; Cornwall, Gail A.

    2014-01-01

    Hereditary cystatin C amyloid angiopathy is an autosomal dominant disorder in which a variant form of cystatin C (L68Q) readily forms amyloid deposits in cerebral arteries in affected individuals resulting in early death. L68Q protein deposits in human cystatin C amyloid angiopathy patients have also been found in tissues outside of the brain including the testis, suggesting possible effects on fertility. Heterozygous transgenic mice (L68Q) that express the human L68Q variant of cystatin C under the control of the mouse cystatin C promoter were unable to generate offspring, suggesting the presence of L68Q cystatin C amyloid affected sperm function. In vitro studies showed that epididymal spermatozoa from L68Q mice were unable to fertilize oocytes and exhibited poor sperm motility. Furthermore, spermatozoa from L68Q mice exhibited reduced cell viability compared with wild type (WT) spermatozoa and often were detected in large agglutinated clumps. Examination of the epididymal fluid and spermatozoa from L68Q mice showed increased levels and distinct forms of cystatin C amyloid that were not present in WT mice. The addition of epididymal fluid from L68Q mice to WT spermatozoa resulted in a recapitulation of the L68Q phenotype in that WT spermatozoa showed reduced cell viability and motility compared with WT spermatozoa incubated in epididymal fluid from WT mice. L68Q epididymal fluid that was depleted of cystatin C amyloids, however, did not impair the motility of WT spermatozoa. Taken together these studies suggest that amyloids in the epididymal fluid can be cytotoxic to the maturing spermatozoa resulting in male infertility. PMID:24500719

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

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    Priya Saikumar Lakshmi

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

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

    Science.gov (United States)

    Yang, Xiangdong; Lloyd, Bethany; Daniell, Henry

    2013-01-01

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

  17. Elevated Oestrogen Receptor Splice Variant ERαΔ5 Expression in Tumour-adjacent Hormone-responsive Tissue

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    Pierre L. Martin-Hirsch

    2010-11-01

    Full Text Available Susceptibility to prostate or endometrial cancer is linked with obesity, a state of oestrogen excess. Oestrogen receptor (ER splice variants may be responsible for the tissue-level of ER activity. Such micro-environmental regulation may modulate cancer initiation and/or progression mechanisms. Real-time reverse transcriptase (RT polymerase chain reaction (PCR was used to quantitatively assess the levels of four ER splice variants (ERαΔ3, ERαΔ5, ERβ2 and ERβ5, plus the full-length parent isoforms ERα and ERβ1, in high-risk [tumour-adjacent prostate (n = 10 or endometrial cancer (n = 9] vs. low-risk [benign prostate (n = 12 or endometrium (n = 9], as well as a comparison of UK (n = 12 vs. Indian (n = 15 benign prostate. All three tissue groups expressed the ER splice variants at similar levels, apart from ERαΔ5. This splice variant was markedly raised in all of the tumour-adjacent prostate samples compared to benign tissues. Immunofluorescence analysis for ERβ2 in prostate tissue demonstrated that such splice variants are present in comparable, if not greater, amounts as the parent full-length isoform. This small pilot study demonstrates the ubiquitous nature of ER splice variants in these tissue sites and suggests that ERαΔ5 may be involved in progression of prostate adenocarcinoma.

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

    DEFF Research Database (Denmark)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2005-01-01

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

  20. 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.

  1. Vaccine Containing the Three Allelic Variants of the Plasmodium vivax Circumsporozoite Antigen Induces Protection in Mice after Challenge with a Transgenic Rodent Malaria Parasite

    Directory of Open Access Journals (Sweden)

    Alba Marina Gimenez

    2017-10-01

    Full Text Available Plasmodium vivax is the most common species that cause malaria outside of the African continent. The development of an efficacious vaccine would contribute greatly to control malaria. Recently, using bacterial and adenoviral recombinant proteins based on the P. vivax circumsporozoite protein (CSP, we demonstrated the possibility of eliciting strong antibody-mediated immune responses to each of the three allelic forms of P. vivax CSP (PvCSP. In the present study, recombinant proteins representing the PvCSP alleles (VK210, VK247, and P. vivax-like, as well as a hybrid polypeptide, named PvCSP-All epitopes, were generated. This hybrid containing the conserved C-terminal of the PvCSP and the three variant repeat domains in tandem were successfully produced in the yeast Pichia pastoris. After purification and biochemical characterization, they were used for the experimental immunization of C57BL/6 mice in a vaccine formulation containing the adjuvant Poly(I:C. Immunization with a recombinant protein expressing all three different allelic forms in fusion elicited high IgG antibody titers reacting with all three different allelic variants of PvCSP. The antibodies targeted both the C-terminal and repeat domains of PvCSP and recognized the native protein on the surface of P. vivax sporozoites. More importantly, mice that received the vaccine formulation were protected after challenge with chimeric Plasmodium berghei sporozoites expressing CSP repeats of P. vivax sporozoites (Pb/PvVK210. Our results suggest that it is possible to elicit protective immunity against one of the most common PvCSP alleles using soluble recombinant proteins expressed by P. pastoris. These recombinant proteins are promising candidates for clinical trials aiming to develop a multiallele vaccine against P. vivax malaria.

  2. Targeting a Single Alternative Polyadenylation Site Coordinately Blocks Expression of Androgen Receptor mRNA Splice Variants in Prostate Cancer.

    Science.gov (United States)

    Van Etten, Jamie L; Nyquist, Michael; Li, Yingming; Yang, Rendong; Ho, Yeung; Johnson, Rachel; Ondigi, Olivia; Voytas, Daniel F; Henzler, Christine; Dehm, Scott M

    2017-10-01

    Prostate cancer is the second leading cause of male cancer deaths due to disease progression to castration-resistant prostate cancer (CRPC). Androgen receptor (AR) splice variants including AR-V7 function as constitutively active transcription factors in CRPC cells, thereby promoting resistance to AR-targeted therapies. To date, there are no AR variant-specific treatments for CRPC. Here we report that the splicing of AR variants AR-V7 as well as AR-V1 and AR-V9 is regulated coordinately by a single polyadenylation signal in AR intron 3. Blocking this signal with morpholino technology or silencing of the polyadenylation factor CPSF1 caused a splice switch that inhibited expression of AR variants and blocked androgen-independent growth of CRPC cells. Our findings support the development of new therapies targeting the polyadenylation signal in AR intron 3 as a strategy to prevent expression of a broad array of AR variants in CRPC. Cancer Res; 77(19); 5228-35. ©2017 AACR. ©2017 American Association for Cancer Research.

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

    Science.gov (United States)

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

    2005-08-01

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

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

    Science.gov (United States)

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

    1989-01-01

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

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

    Science.gov (United States)

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

    2016-12-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-09-10

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

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

    DEFF Research Database (Denmark)

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

    2003-01-01

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

  8. Plasmodium falciparum-infected erythrocyte knob density is linked to the PfEMP1 variant expressed

    DEFF Research Database (Denmark)

    Subramani, Ramesh; Quadt, Katharina; Jeppesen, Anine Engholm

    2015-01-01

    by atomic force microscopy (AFM) and scanning electron microscopy (SEM) and compared to PfEMP1 and knob-associated histidine-rich protein (KAHRP) expression. Selection for uniform expression of IT4VAR04 produced little change in knob density, compared to unselected IEs. In contrast, selection for IT4VAR32b...... regardless of the PfEMP1 expressed. Our study documents for the first time that knob density is related to the PfEMP1 variant expressed. This may reflect topological requirements to ensure optimal adhesive properties of the IEs. IMPORTANCE: Infections with Plasmodium falciparum malaria parasites are still...

  9. A Genetic Variant in the Distal Enhancer Region of the Human Renin Gene Affects Renin Expression.

    Science.gov (United States)

    Makino, Yasukazu; Konoshita, Tadashi; Omori, Atsuhito; Maegawa, Nobuhiro; Nakaya, Takahiro; Ichikawa, Mai; Yamamoto, Katsushi; Wakahara, Shigeyuki; Ishizuka, Tamotsu; Onoe, Tamehito; Nakamura, Hiroyuki

    2015-01-01

    The high heritability of plasma renin activity was confirmed in recent investigations. A variation located near the strong enhancer of the human renin gene (REN), C-5312T, has been shown to have different transcription activity levels depending on its allele: the 5312T allele shows transcription levels that are 45% greater than those of the 5312C allele. The purpose of this study was to confirm the hypothesis that variations in the enhancer region of the REN gene are involved in regulating renal expression of renin. Sixty-four subjects with biopsy-proven renal diseases were included in this study (male/female: 35/29, age 41.9 ± 20.9 years, SBP/DBP 123.1 ± 23.7/73.4 ± 14.8 mmHg, s-Cr 0.93 ± 0.63 mg/dl). A genetic variant of REN, C-5312T, was assayed by PCR-RFLP and the TaqMan method. Total RNAs from a small part of the renal cortex were reverse-transcribed and amplified for REN and GAPDH with a real-time PCR system. Logarithmically transformed expression values of the relative ratio of REN to GAPDH (10-3) were as follows (mean ± SE): CC (26 cases), 0.016 ± 0.005; CT (33 cases), 0.047 ± 0.021 (p = 0.41 vs. CC); TT (5 cases), 0.198 ± 0.194 (p = 0.011 vs. CC, p renin gene have an effect on the expression levels of renin in renal tissue; this observation is in good accordance with the results of the transcriptional assay.

  10. Assessing pathogenicity of MLH1 variants by co-expression of human MLH1 and PMS2 genes in yeast

    Directory of Open Access Journals (Sweden)

    Hudler Petra

    2009-10-01

    Full Text Available Abstract Background Loss of DNA mismatch repair (MMR in humans, mainly due to mutations in the hMLH1 gene, is linked to hereditary nonpolyposis colorectal cancer (HNPCC. Because not all MLH1 alterations result in loss of MMR function, accurate characterization of variants and their classification in terms of their effect on MMR function is essential for reliable genetic testing and effective treatment. To date, in vivo assays for functional characterization of MLH1 mutations performed in various model systems have used episomal expression of the modified MMR genes. We describe here a novel approach to determine accurately the functional significance of hMLH1 mutations in vivo, based on co-expression of human MLH1 and PMS2 in yeast cells. Methods Yeast MLH1 and PMS1 genes, whose protein products form the MutLα complex, were replaced by human orthologs directly on yeast chromosomes by homologous recombination, and the resulting MMR activity was tested. Results The yeast strain co-expressing hMLH1 and hPMS2 exhibited the same mutation rate as the wild-type. Eight cancer-related MLH1 variants were introduced, using the same approach, into the prepared yeast model, and their effect on MMR function was determined. Five variants (A92P, S93G, I219V, K618R and K618T were classified as non-pathogenic, whereas variants T117M, Y646C and R659Q were characterized as pathogenic. Conclusion Results of our in vivo yeast-based approach correlate well with clinical data in five out of seven hMLH1 variants and the described model was thus shown to be useful for functional characterization of MLH1 variants in cancer patients found throughout the entire coding region of the gene.

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

    Science.gov (United States)

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

    1989-01-01

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

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

    Science.gov (United States)

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

    2018-03-01

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

  13. Fractionation of T cell subsets on Ig anti-Ig columns: isolation of helper T cells from nonresponder mice, demonstration of antigen-specific T suppressor cells, and selection of CD-3 negative variants of Jurkat T cells

    DEFF Research Database (Denmark)

    Rubin, B; Geisler, C; Kuhlmann, J

    1989-01-01

    In the present experiments we have explored the possibilities of a modified immunoadsorbent technique to select for (1) mutagenized T cell receptor (Tcr) negative variants of Jurkat T lymphoma cells and (2) purified CD-4+ or CD-8+ T lymphocytes. The basic principle was to make large numbers...... of immunoglobulin (Ig) negative T cells Ig+ by T cell subset-specific monoclonal antibodies (mAb), and to select such cells on Ig anti-Ig columns. Our results demonstrated that Thy-1+, Fc receptor positive, antigen-specific T cells regulate the immune response in mice nonresponders to pork insulin......." The most important finding is the demonstration of antigen-specific Thy-1+, CD-8+, and Fc receptor+ T suppressor cell that apparently react with antigen in a non-major histocompatibility complex-restricted manner....

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

    DEFF Research Database (Denmark)

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

    2007-01-01

    BACKGROUND: Cancer/testis antigens (CTAs) are expressed in several cancers and during normal adult male germ cell differentiation. Little is known about their role in fetal development of human germ cells. METHODS: We examined expression of the CTAs MAGE-A1, GAGE and NY-ESO-1 in fetal gonads...... by single and double immunohistochemical staining. RESULTS: We found that GAGE was expressed in the primordial germ cells of the gonadal primordium, whereas MAGE-A1 and NY-ESO-1 were first detected in germ cells of both testis and ovary after sexual differentiation was initiated. The number of positive germ...... cells and the staining intensity of all three CTAs peaked during the second trimester and gradually decreased towards birth in both male and female germ cells. In oocytes, MAGE-A1 expression terminated around birth, whereas NY-ESO-1 expression persisted through the neonatal stage and GAGE expression...

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

    Directory of Open Access Journals (Sweden)

    B. Bindukumar

    2005-03-01

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

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

    DEFF Research Database (Denmark)

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

    1993-01-01

    Numerous studies have examined kinetics of T-cell functional responses following non-specific and antigen-specific stimulation in vitro. However, while reports of phenotypic T-cell changes after non-specific stimulation are abundant, only little information on phenotypic effects of antigen......-specific stimulation is available. In the present study we have examined phenotypic T-cell changes after in vitro stimulation by the antigens purified derivative of tuberculin (PPD) and tetanus toxoid (TT). We show that the well-established differences in kinetics of mitogen- and antigen-induced T-cell proliferation...... in vitro is paralleled by differential kinetics in the expression of the T-cell adhesion and activation antigens leukocyte function-associated antigen 1 (LFA-1; CD11a/CD18), interleukin-2 receptor (IL-2R; CD25), and intercellular adhesion molecule 1 (ICAM-1; CD54). Furthermore, the changes in expression...

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

    Directory of Open Access Journals (Sweden)

    Sang Mee Hwang

    2013-01-01

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

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

    Science.gov (United States)

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

    2018-01-08

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

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

    Directory of Open Access Journals (Sweden)

    Maha Imam Ahmed

    2004-01-01

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

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

    OpenAIRE

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

    1997-01-01

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

  1. Salt-Sensitive Hypertension and Cardiac Hypertrophy in Transgenic Mice Expressing a Corin Variant Identified in African Americans

    Science.gov (United States)

    Wang, Wei; Cui, Yujie; Shen, Jianzhong; Jiang, Jingjing; Chen, Shenghan; Peng, Jianhao; Wu, Qingyu

    2012-01-01

    African Americans represent a high risk population for salt-sensitive hypertension and heart disease but the underlying mechanism remains unclear. Corin is a cardiac protease that regulates blood pressure by activating natriuretic peptides. A corin gene variant (T555I/Q568P) was identified in African Americans with hypertension and cardiac hypertrophy. In this study, we test the hypothesis that the corin variant contributes to the hypertensive and cardiac hypertrophic phenotype in vivo. Transgenic mice were generated to express wild-type or T555I/Q568P variant corin in the heart under the control of α-myosin heavy chain promoter. The mice were crossed into a corin knockout background to create KO/TgWT and KO/TgV mice that expressed WT or variant corin, respectively, in the heart. Functional studies showed that KO/TgV mice had significantly higher levels of pro-atrial natriuretic peptide in the heart compared with that in control KO/TgWT mice, indicating that the corin variant was defective in processing natriuretic peptides in vivo. By radiotelemetry, corin KO/TgV mice were found to have hypertension that was sensitive to dietary salt loading. The mice also developed cardiac hypertrophy at 12–14 months of age when fed a normal salt diet or at a younger age when fed a high salt diet. The phenotype of salt-sensitive hypertension and cardiac hypertrophy in KO/TgV mice closely resembles the pathological findings in African Americans who carry the corin variant. The results indicate that corin defects may represent an important mechanism in salt-sensitive hypertension and cardiac hypertrophy in African Americans. PMID:22987923

  2. Salt-sensitive hypertension and cardiac hypertrophy in transgenic mice expressing a corin variant identified in blacks.

    Science.gov (United States)

    Wang, Wei; Cui, Yujie; Shen, Jianzhong; Jiang, Jingjing; Chen, Shenghan; Peng, Jianhao; Wu, Qingyu

    2012-11-01

    Blacks represent a high-risk population for salt-sensitive hypertension and heart disease, but the underlying mechanism remains unclear. Corin is a cardiac protease that regulates blood pressure by activating natriuretic peptides. A corin gene variant (T555I/Q568P) was identified in blacks with hypertension and cardiac hypertrophy. In this study, we tested the hypothesis that the corin variant contributes to the hypertensive and cardiac hypertrophic phenotype in vivo. Transgenic mice were generated to express wild-type (WT) or T555I/Q568P variant corin in the heart under the control of α-myosin heavy chain promoter. The mice were crossed into a corin knockout (KO) background to create KO/TgWT and KO/TgV mice that expressed WT or variant corin, respectively, in the heart. Functional studies showed that KO/TgV mice had significantly higher levels of proatrial natriuretic peptide in the heart compared with that in control KO/TgWT mice, indicating that the corin variant was defective in processing natriuretic peptides in vivo. By radiotelemetry, corin KO/TgV mice were found to have hypertension that was sensitive to dietary salt loading. The mice also developed cardiac hypertrophy at 12 to 14 months of age when fed a normal salt diet or at a younger age when fed a high-salt diet. The phenotype of salt-sensitive hypertension and cardiac hypertrophy in KO/TgV mice closely resembles the pathological findings in blacks who carry the corin variant. The results indicate that corin defects may represent an important mechanism in salt-sensitive hypertension and cardiac hypertrophy in blacks.

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

    OpenAIRE

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

    2009-01-01

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

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

    Science.gov (United States)

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

    2000-03-02

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

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

    Science.gov (United States)

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

    2000-01-01

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

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

    Science.gov (United States)

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

    1999-01-01

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

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

    DEFF Research Database (Denmark)

    Sellebjerg, F; Krakauer, M; Khademi, M

    2012-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Aneel Paulus

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1982-12-01

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

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

    DEFF Research Database (Denmark)

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

    2010-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Franziska Hempel

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

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

    Science.gov (United States)

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

    2011-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Danuta Kowalczyk

    2006-09-01

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

  14. 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.

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

    Directory of Open Access Journals (Sweden)

    Dan eLi

    2015-07-01

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

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

    Science.gov (United States)

    2013-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-01-15

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

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

    Science.gov (United States)

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

    2006-05-01

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

  19. Changes in global gene expression profiles induced by HPV 16 E6 oncoprotein variants in cervical carcinoma C33-A cells

    Energy Technology Data Exchange (ETDEWEB)

    Zacapala-Gómez, Ana Elvira, E-mail: zak_ana@yahoo.com.mx [Laboratorio de Biomedicina Molecular, Unidad Académica de Ciencias Químico Biológicas, Universidad Autónoma de Guerrero, Chilpancingo, Gro., México (Mexico); Del Moral-Hernández, Oscar, E-mail: odelmoralh@gmail.com [Laboratorio de Biomedicina Molecular, Unidad Académica de Ciencias Químico Biológicas, Universidad Autónoma de Guerrero, Chilpancingo, Gro., México (Mexico); Villegas-Sepúlveda, Nicolás, E-mail: nvillega@cinvestav.mx [Departamento de Biomedicina Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), México, D.F., México (Mexico); Hidalgo-Miranda, Alfredo, E-mail: ahidalgo@inmegen.gob.mx [Laboratorio de Genómica del Cáncer, Instituto Nacional de Medicina Genómica (INMEGEN), México, D.F., México (Mexico); Romero-Córdoba, Sandra Lorena, E-mail: sromero_cordoba@hotmail.com [Laboratorio de Genómica del Cáncer, Instituto Nacional de Medicina Genómica (INMEGEN), México, D.F., México (Mexico); and others

    2016-01-15

    We analyzed the effects of the expression of HPV 16 E6 oncoprotein variants (AA-a, AA-c, E-A176/G350, E-C188/G350, E-G350), and the E-Prototype in global gene expression profiles in an in vitro model. E6 gene was cloned into an expression vector fused to GFP and was transfected in C33-A cells. Affymetrix GeneChip Human Transcriptome Array 2.0 platform was used to analyze the expression of over 245,000 coding transcripts. We found that HPV16 E6 variants altered the expression of 387 different genes in comparison with E-Prototype. The altered genes are involved in cellular processes related to the development of cervical carcinoma, such as adhesion, angiogenesis, apoptosis, differentiation, cell cycle, proliferation, transcription and protein translation. Our results show that polymorphic changes in HPV16 E6 natural variants are sufficient to alter the overall gene expression profile in C33-A cells, explaining in part the observed differences in oncogenic potential of HPV16 variants. - Highlights: • Amino acid changes in HPV16 E6 variants modulate the transciption of specific genes. • This is the first comparison of global gene expression profile of HPV 16 E6 variants. • Each HPV 16 E6 variant appears to have its own molecular signature.

  20. Expression defect size among unclassified MLH1 variants determines pathogenicity in Lynch syndrome diagnosis

    DEFF Research Database (Denmark)

    Hinrichsen, Inga; Brieger, Angela; Trojan, Jörg

    2013-01-01

    Lynch syndrome is caused by a germline mutation in a mismatch repair gene, most commonly the MLH1 gene. However, one third of the identified alterations are missense variants with unclear clinical significance. The functionality of these variants can be tested in the laboratory, but the results c...... cannot be used for clinical diagnosis. We therefore aimed to establish a laboratory test that can be applied clinically.......Lynch syndrome is caused by a germline mutation in a mismatch repair gene, most commonly the MLH1 gene. However, one third of the identified alterations are missense variants with unclear clinical significance. The functionality of these variants can be tested in the laboratory, but the results...

  1. Use of Cells Expressing γ Subunit Variants to Identify Diverse Mechanisms of AMPK Activation

    National Research Council Canada - National Science Library

    Hawley, Simon A; Ross, Fiona A; Chevtzoff, Cyrille; Green, Kevin A; Evans, Ashleigh; Evans, A. Mark; Fogarty, Sarah; Towler, Mhairi C; Brown, Laura J; Ogunbayo, Oluseye A; Hardie, D. Grahame

    2010-01-01

    ... (R531G) gamma2 variants. Mitochondrial poisons such as oligomycin and dinitrophenol only activated AMPK in WT cells, as did AICAR, 2-deoxyglucose, hydrogen peroxide, metformin, phenformin, galegine, troglitazone...

  2. Loss of histone variant macroH2A2 expression associates with progression of anal neoplasm.

    Science.gov (United States)

    Hu, Wan-Hsiang; Miyai, Katsumi; Sporn, Judith C; Luo, Linda; Wang, Jean Y J; Cosman, Bard; Ramamoorthy, Sonia

    2016-07-01

    The macroH2A histone variants are epigenetic marks for inactivated chromatin. In this study, we examined the expression of macroH2A2 in anal neoplasm from anal intraepithelial neoplasia (AIN) to anal squamous cell carcinoma (SCC). AIN and anal SCC samples were analysed for macroH2A2 expression, HIV and human papilloma virus (HPV). The association of macroH2A2 expression with clinical grade, disease recurrence, overall survival and viral involvement was determined. macroH2A2 was expressed in normal squamous tissue and lower grade AIN (I and II). Expression was lost in 38% of high-grade AIN (III) and 71% of anal SCC (p=0.002). Patients with AIN with macroH2A2-negative lesions showed earlier recurrence than those with macroH2A2-positive neoplasm (p=0.017). With anal SCC, macroH2A2 loss was more prevalent in the HPV-negative tumours. Loss of histone variant macroH2A2 expression is associated with the progression of anal neoplasm and can be used as a prognostic biomarker for high-grade AIN and SCC. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

  3. Local binary pattern variants-based adaptive texture features analysis for posed and nonposed facial expression recognition

    Science.gov (United States)

    Sultana, Maryam; Bhatti, Naeem; Javed, Sajid; Jung, Soon Ki

    2017-09-01

    Facial expression recognition (FER) is an important task for various computer vision applications. The task becomes challenging when it requires the detection and encoding of macro- and micropatterns of facial expressions. We present a two-stage texture feature extraction framework based on the local binary pattern (LBP) variants and evaluate its significance in recognizing posed and nonposed facial expressions. We focus on the parametric limitations of the LBP variants and investigate their effects for optimal FER. The size of the local neighborhood is an important parameter of the LBP technique for its extraction in images. To make the LBP adaptive, we exploit the granulometric information of the facial images to find the local neighborhood size for the extraction of center-symmetric LBP (CS-LBP) features. Our two-stage texture representations consist of an LBP variant and the adaptive CS-LBP features. Among the presented two-stage texture feature extractions, the binarized statistical image features and adaptive CS-LBP features were found showing high FER rates. Evaluation of the adaptive texture features shows competitive and higher performance than the nonadaptive features and other state-of-the-art approaches, respectively.

  4. Expression of multiple forms of 3'-end variant CCK2 receptor mRNAs in human pancreatic adenocarcinomas

    Directory of Open Access Journals (Sweden)

    Monstein Hans-Jürg

    2011-04-01

    Full Text Available Abstract Background Two main types of receptors for gastrin and cholecystokinin (CCK have been cloned and identified. CCK1 (CCK-A receptors are expressed in the pancreas, the gallbladder, and parts of the brain, while CCK2 (CCK-B/gastrin receptors (CCK2R are expressed in gastric glands and in most of the brain. A splice variant of the CCK2R designated CCKRi4sv (CCK-C, which is constitutively expressed in human pancreatic cancer cells, has also been described. The purpose of the present investigation was to study CCK2R, CCK2i4svR, and gastrin mRNA expression in human pancreatic adenocarcinoma on the assumption that co-expression of CCK2R and gastrin or constitutive CCK2i4svR mRNA expression plays a pivotal role in the progression of pancreatic cancer. Findings PCR amplification using CCK2R specific primer-pairs, followed by ethidium-bromide stained agarose gel electrophoresis revealed the expression of wild-type CCK2R mRNA in 12 of 17 biopsy specimens. A CCK2R intron 4 specific nested PCR assay revealed that CCK2i4svR mRNA was expressed in only one of the biopsy specimen. The authenticity of PCR amplicons was confirmed by cloning of selected amplicons and DNA sequence analysis. Moreover, we found that hitherto undescribed multiple forms of 3'-end variant CCK2R mRNAs with various deletions in the retained intron 4 and exon 5, tentatively generating truncated proteins, were expressed in the pancreatic adenocarcinomas. Conclusion Cloning and DNA sequencing of selected amplicons revealed that CCK2R and multiple CCK2i4svR-like mRNAs are expressed in human pancreatic adenocarcinoma. The originally described CCK2i4svR mRNA was only expressed in one of 17 tumours and appears to be rarely expressed in pancreatic adenocarcinoma. We report that CCK2R- and gastrin mRNA co-expression may play a role in a portion, but not in all of these tumours, and that aberrant splicing takes places in these tissues generating multiple forms of 3'-end variant CCK2R mRNAs.

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

    Science.gov (United States)

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

    2017-12-14

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

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

    NARCIS (Netherlands)

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

    1997-01-01

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

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

    DEFF Research Database (Denmark)

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

    1995-01-01

    The prognostic value of oncogenic antigen 519 (OA-519) expression and tumour proliferative activity was evaluated in a retrospective series of 118 patients with low-risk breast cancer. Low risk was defined as negative axillary nodes, tumour diameter

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

    DEFF Research Database (Denmark)

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

    2016-01-01

    New strategies are needed to develop better tools to control TB, including identification of novel antigens for vaccination. Such Mtb antigens must be expressed during Mtb infection in the major target organ, the lung, and must be capable of eliciting human immune responses. Using genome......-wide transcriptomics of Mtb infected lungs we developed data sets and methods to identify IVE-TB (in-vivo expressed Mtb) antigens expressed in the lung. Quantitative expression analysis of 2,068 Mtb genes from the predicted first operons identified the most upregulated IVE-TB genes during in-vivo pulmonary infection....... By further analysing high-level conservation among whole-genome sequenced Mtb-complex strains (n = 219) and algorithms predicting HLA-class-Ia and II presented epitopes, we selected the most promising IVE-TB candidate antigens. Several of these were recognized by T-cells from in-vitro Mtb-PPD and ESAT6/CFP10...

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

    Science.gov (United States)

    Ozawa, Keiya

    2014-03-01

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

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

    Science.gov (United States)

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

    2008-12-09

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

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

    Science.gov (United States)

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

    1995-10-15

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

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

    Science.gov (United States)

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

    2009-05-01

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

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

    Directory of Open Access Journals (Sweden)

    Daria Jacob

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

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

    Science.gov (United States)

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

    2003-12-01

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

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

    Science.gov (United States)

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

    2010-10-01

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

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

    Directory of Open Access Journals (Sweden)

    Zhao Yingdong

    2008-09-01

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

  17. Identification, expression and functional characterization of M4L, a muscarinic acetylcholine M4 receptor splice variant.

    Directory of Open Access Journals (Sweden)

    Douglas A Schober

    Full Text Available Rodent genomic alignment sequences support a 2-exon model for muscarinic M4 receptor. Using this model a novel N-terminal extension was discovered in the human muscarinic acetylcholine M4 receptor. An open reading frame was discovered in the human, mouse and rat with a common ATG (methionine start codon that extended the N-terminus of the muscarinic acetylcholine M4 receptor subtype by 155 amino acids resulting in a longer variant. Transcriptional evidence for this splice variant was confirmed by RNA-Seq and RT-PCR experiments performed from human donor brain prefrontal cortices. We detected a human upstream exon indicating the translation of the mature longer M4 receptor transcript. The predicted size for the longer two-exon M4 receptor splice variant with the additional 155 amino acid N-terminal extension, designated M4L is 69.7 kDa compared to the 53 kDa canonical single exon M4 receptor (M4S. Western blot analysis from a mammalian overexpression system, and saturation radioligand binding with [3H]-NMS (N-methyl-scopolamine demonstrated the expression of this new splice variant. Comparative pharmacological characterization between the M4L and M4S receptors revealed that both the orthosteric and allosteric binding sites for both receptors were very similar despite the addition of an N-terminal extension.

  18. Nucleolin is required for DNA methylation state and the expression of rRNA gene variants in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Frédéric Pontvianne

    2010-11-01

    Full Text Available In eukaryotes, 45S rRNA genes are arranged in tandem arrays in copy numbers ranging from several hundred to several thousand in plants. Although it is clear that not all copies are transcribed under normal growth conditions, the molecular basis controlling the expression of specific sets of rRNA genes remains unclear. Here, we report four major rRNA gene variants in Arabidopsis thaliana. Interestingly, while transcription of one of these rRNA variants is induced, the others are either repressed or remain unaltered in A. thaliana plants with a disrupted nucleolin-like protein gene (Atnuc-L1. Remarkably, the most highly represented rRNA gene variant, which is inactive in WT plants, is reactivated in Atnuc-L1 mutants. We show that accumulated pre-rRNAs originate from RNA Pol I transcription and are processed accurately. Moreover, we show that disruption of the AtNUC-L1 gene induces loss of symmetrical DNA methylation without affecting histone epigenetic marks at rRNA genes. Collectively, these data reveal a novel mechanism for rRNA gene transcriptional regulation in which the nucleolin protein plays a major role in controlling active and repressed rRNA gene variants in Arabidopsis.

  19. The Survivin −31 Snp in Human Colorectal Cancer Correlates with Survivin Splice Variant Expression and Improved Overall Survival

    Directory of Open Access Journals (Sweden)

    Anna G. Antonacopoulou

    2010-01-01

    Full Text Available Background: Survivin is involved in the regulation of cell division and survival, two key processes in cancer. The majority of studies on survivin in colorectal cancer (CRC have focused on protein expression and less is known about the expression of survivin splicing variants or survivin gene polymorphisms in CRC. In the present study, the mRNA levels of the five known isoforms of survivin as well as survivin protein were assessed in matched normal and neoplastic colorectal tissue. Moreover, the 9386C/T and −31G/C polymorphisms were investigated.

  20. 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...

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

    DEFF Research Database (Denmark)

    Hokland, M; Heron, I; Berg, K

    1982-01-01

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

  2. The role of the brain-derived neurotrophic factor (BDNF) val66met variant in the phenotypic expression of obsessive-compulsive disorder (OCD)

    NARCIS (Netherlands)

    Katerberg, H.; Lochner, C.; Cath, D.C.; de Jonge, P.; Bochdanovits, Z.; Moolman-Smook, J.C.; Hemmings, S.M.J.; Carey, P.D.; Stein, D.J.; Sondervan, D.; den Boer, J.A.; van Balkom, A.J.L.M.; Polman, A.; Heutink, P.

    2009-01-01

    Evidence suggests that the Va166Met variant of the brain-derived neurotrophic factor (BDNF) gene may play a role in the etiology of Obsessive-Compulsive Disorder (OCD). In this study, the role of the BDNF Va166Met variant in the etiology and the phenotypic expression of OCD is investigated.

  3. Re-evaluation of the PBAN receptor (PBANR molecule: characterization of PBANR variants expressed in the pheromone glands of moths

    Directory of Open Access Journals (Sweden)

    Jae Min Lee

    2012-01-01

    Full Text Available Sex pheromone production in most moths is initiated following pheromone biosynthesis activating neuropeptide receptor (PBANR activation. PBANR was initially cloned from pheromone glands (PGs of Helicoverpa zea and Bombyx mori. The B. mori PBANR is characterized by a relatively long C-terminus that is essential for ligand-induced internalization, whereas the H. zea PBANR has a shorter C-terminus that lacks features present in the B. mori PBANR critical for internalization. Multiple PBANRs have been reported to be concurrently expressed in the larval CNS of Heliothis virescens. In the current study, we sought to examine the prevalence of multiple PBANRs in the PGs of three moths and to ascertain their potential functional relevance. Multiple PBANR variants (As, A, B, and C were cloned from the PGs of all species examined with PBANR-C the most highly expressed. Alternative splicing of the C-terminal coding sequence of the PBAN gene gives rise to the variants, which are distinguishable only by the length and composition of their respective C-terminal tails. Transient expression of fluorescent PBANR chimeras in insect cells revealed that PBANR-B and PBANR-C localized exclusively to the cell surface while PBANR-As and PBANR-A exhibited varying degrees of cytosolic localization. Similarly, only the PBANR-B and PBANR-C variants underwent ligand-induced internalization. Taken together, our results suggest that PBANR-C is the principal receptor molecule involved in PBAN signaling regardless of moth species. The high GC content of the C-terminal coding sequence in the B and C variants, which makes amplification using conventional polymerases difficult, likely accounts for previous preferential amplification of PBANR-A like receptors from other species.

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

    Science.gov (United States)

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

    2016-07-01

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

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

    Science.gov (United States)

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

    1994-04-01

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

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

    Science.gov (United States)

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

    2013-10-08

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

  7. Novel kinin B₁ receptor splice variant and 5'UTR regulatory elements are responsible for cell specific B₁ receptor expression.

    Directory of Open Access Journals (Sweden)

    Faang Y Cheah

    Full Text Available The kinin B₁ receptor (B₁R is rapidly upregulated after tissue trauma or inflammation and is involved in cancer and inflammatory diseases such as asthma. However, the role of the: promoter; a postulated alternative promoter; and spliced variants in airway epithelial and other lung cells are poorly understood. We identified, in various lung cell lines and leucocytes, a novel, naturally occurring splice variant (SV of human B₁R gene with a shorter 5'untranslated region. This novel SV is ≈35% less stable than the wild-type (WT transcript in lung adenocarcinoma cells (H2126, but does not influence translation efficiency. Cell-specific differences in splice variant expression were observed post des[Arg10]-kallidin stimulation with delayed upregulation of SV compared to WT suggesting potentially different regulatory responses to inflammation. Although an alternative promoter was not identified in our cell-lines, several cell-specific regulatory elements within the postulated alternative promoter region (negative response element (NRE -1020 to -766 bp in H2126; positive response element (PRE -766 to -410 bp in 16HBE; -410 to +1 region acts as a PRE in H2126 and NRE in 16HBE cells were found. These findings reveal complex regulation of B₁R receptor expression in pulmonary cells which may allow future therapeutic manipulation in chronic pulmonary inflammation and cancer.

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

    Science.gov (United States)

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

    2014-03-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2011-06-01

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

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

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    Qiang Zou

    2010-01-01

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

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

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    Flávia Márcia Oliveira

    2013-12-01

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

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

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    Fereshteh Mohammadizadeh

    2012-01-01

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

  14. Embryonic stem cells and mice expressing different GFP variants for multiple non-invasive reporter usage within a single animal

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    Macmaster Suzanne

    2002-06-01

    Full Text Available Abstract Background Non-invasive autofluorescent reporters have revolutionized lineage labeling in an array of different organisms. In recent years green fluorescent protein (GFP from the bioluminescent jellyfish Aequoria Victoria has gained popularity in mouse transgenic and gene targeting regimes 1. It offers several advantages over conventional gene-based reporters, such as lacZ and alkaline phosphatase, in that its visualization does not require a chromogenic substrate and can be realized in vivo. We have previously demonstrated the utility and developmental neutrality of enhanced green fluorescent protein (EGFP in embryonic stem (ES cells and mice 2. Results In this study we have used embryonic stem (ES cell-mediated transgenesis to test the enhanced cyan fluorescent protein (ECFP and enhanced yellow fluorescent protein (EYFP, two mutant and spectrally distinct color variants of wild type (wt GFP. We have also tested DsRed1, the novel red fluorescent protein reporter recently cloned from the Discostoma coral by virtue of its homology to GFP. To this end, we have established lines of ES cells together with viable and fertile mice having widespread expression of either the ECFP or EYFP GFP-variant reporters. However, we were unable to generate equivalent DsRed1 lines, suggesting that DsRed1 is not developmentally neutral or that transgene expression cannot be sustained constitutively. Balanced (diploid diploid and polarized (tetraploid diploid chimeras comprising combinations of the ECFP and EYFP ES cells and/or embryos, demonstrate that populations of cells expressing each individual reporter can be distinguished within a single animal. Conclusions GFP variant reporters are unique in allowing non-invasive multi-spectral visualization in live samples. The ECFP and EYFP-expressing transgenic ES cells and mice that we have generated provide sources of cells and tissues for combinatorial, double-tagged recombination experiments, chimeras or

  15. Behavioral and Neural Manifestations of Reward Memory in Carriers of Low-Expressing versus High-Expressing Genetic Variants of the Dopamine D2 Receptor

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    Anni Richter

    2017-05-01

    Full Text Available Dopamine is critically important in the neural manifestation of motivated behavior, and alterations in the human dopaminergic system have been implicated in the etiology of motivation-related psychiatric disorders, most prominently addiction. Patients with chronic addiction exhibit reduced dopamine D2 receptor (DRD2 availability in the striatum, and the DRD2 TaqIA (rs1800497 and C957T (rs6277 genetic polymorphisms have previously been linked to individual differences in striatal dopamine metabolism and clinical risk for alcohol and nicotine dependence. Here, we investigated the hypothesis that the variants of these polymorphisms would show increased reward-related memory formation, which has previously been shown to jointly engage the mesolimbic dopaminergic system and the hippocampus, as a potential intermediate phenotype for addiction memory. To this end, we performed functional magnetic resonance imaging (fMRI in 62 young, healthy individuals genotyped for DRD2 TaqIA and C957T variants. Participants performed an incentive delay task, followed by a recognition memory task 24 h later. We observed effects of both genotypes on the overall recognition performance with carriers of low-expressing variants, namely TaqIA A1 carriers and C957T C homozygotes, showing better performance than the other genotype groups. In addition to the better memory performance, C957T C homozygotes also exhibited a response bias for cues predicting monetary reward. At the neural level, the C957T polymorphism was associated with a genotype-related modulation of right hippocampal and striatal fMRI responses predictive of subsequent recognition confidence for reward-predicting items. Our results indicate that genetic variations associated with DRD2 expression affect explicit memory, specifically for rewarded stimuli. We suggest that the relatively better memory for rewarded stimuli in carriers of low-expressing DRD2 variants may reflect an intermediate phenotype of

  16. Molecular Cloning and Expression of Sequence Variants of Manganese Superoxide Dismutase Genes from Wheat

    Science.gov (United States)

    Reactive oxygen species (ROS) are very harmful to living organisms due to the potential oxidation of membrane lipids, DNA, proteins, and carbohydrates. Transformed E.coli strain QC 871, superoxide dismutase (SOD) double-mutant, with three sequence variant MnSOD1, MnSOD2, and MnSOD3 manganese supero...

  17. Large-Scale Identification of Common Trait and Disease Variants Affecting Gene Expression

    DEFF Research Database (Denmark)

    Hauberg, Mads Engel; Zhang, Wen; Giambartolomei, Claudia

    2017-01-01

    Genome-wide association studies (GWASs) have identified a multitude of genetic loci involved with traits and diseases. However, it is often unclear which genes are affected in such loci and whether the associated genetic variants lead to increased or decreased gene function. To mitigate this, we ...

  18. Molecular characterization of "inconsistent" variants of Salmonella Typhimurium isolated in Italy

    DEFF Research Database (Denmark)

    Barco, Lisa; Longo, Alessandra; Lettini, Antonia Anna

    2014-01-01

    Salmonella 4,[5],12:i:- is a variant of Salmonella Typhimurium, which lacks the expression of phase-2 flagellar antigen, generally associated with the deletion of the fljB gene. Additional mechanisms involving the fljAB operon ( fljA, fljB, and hin genes) lead to the lack of expression of phase-2...

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

    NARCIS (Netherlands)

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

    2005-01-01

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

  20. Diagnostic implications of IDH1-R132H and OLIG2 expression patterns in rare and challenging glioblastoma variants.

    Science.gov (United States)

    Joseph, Nancy M; Phillips, Joanna; Dahiya, Sonika; M Felicella, Michelle; Tihan, Tarik; Brat, Daniel J; Perry, Arie

    2013-03-01

    Recent work has demonstrated that nearly all diffuse gliomas display nuclear immunoreactivity for the bHLH transcription factor OLIG2, and the R132H mutant isocitrate dehydrogenase 1 (IDH1) protein is expressed in the majority of diffuse gliomas other than primary glioblastoma. However, these antibodies have not been widely applied to rarer glioblastoma variants, which can be diagnostically challenging when the astrocytic features are subtle. We therefore surveyed the expression patterns of OLIG2 and IDH1 in 167 non-conventional glioblastomas, including 45 small cell glioblastomas, 45 gliosarcomas, 34 glioblastomas with primitive neuroectodermal tumor-like foci (PNET-like foci), 23 with an oligodendroglial component, 11 granular cell glioblastomas, and 9 giant cell glioblastomas. OLIG2 was strongly expressed in all glioblastomas with oligodendroglial component, 98% of small cell glioblastomas, and all granular cell glioblastomas, the latter being particularly helpful in ruling out macrophage-rich lesions. In 74% of glioblastomas with PNET-like foci, OLIG2 expression was retained in the PNET-like foci, providing a useful distinction from central nervous system PNETs. The glial component of gliosarcomas was OLIG2 positive in 93% of cases, but only 14% retained focal expression in the sarcomatous component; as such this marker would not reliably distinguish these from pure sarcoma in most cases. OLIG2 was expressed in 67% of giant cell glioblastomas. IDH1 was expressed in 55% of glioblastomas with oligodendroglial component, 15% of glioblastomas with PNET-like foci, 7% of gliosarcomas, and none of the small cell, granular cell, or giant cell glioblastomas. This provides further support for the notion that most glioblastomas with oligodendroglial component are secondary, while small cell glioblastomas, granular cell glioblastomas, and giant cell glioblastomas are primary variants. Therefore, in one of the most challenging differential diagnoses, IDH1 positivity could

  1. Expression of Caspase-1 Gene Transcript Variant mRNA in Peripheral Blood Mononuclear Cells of Patients with Primary Gout in Different TCM Syndromes

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    Wan-Tai Dang

    2015-01-01

    Full Text Available A large number of studies have shown that cysteinyl aspartate specific protease-1 (CASP1 played an important role in the inflammatory response of primary gout, but the decreased expression of different CASP1 transcript variant could inhibit the activation of IL-1β. Our study mainly analyzed the expression level and function of CASP1 gene transcript variant mRNA in peripheral blood mononuclear cells of patients with gout in different TCM syndromes. The expression of CASP1 gene transcript variant and IL-1β mRNA in PBMCs were detected in patients with PG [acute phase (AP: 44 cases; nonacute phase (NAP: 52 cases] and healthy controls (HC: 30 cases by reverse transcription-polymerase chain reaction and/or real-time quantitative polymerase chain reaction. The expressions of plasma IL-1β in patients with PG and HC were detected by enzyme-linked immunosorbent assay. Dysregulated expression of the CASP1 gene and its transcript variant, plasma proinflammatory cytokines in all patients with primary gout in different TCM syndromes, correlation analysis showed that there was negative correlation between the expression of CASP1-gamma gene transcript variant mRNA and IL-1β protein in APPG group. The study suggested that CASP1 gene and its transcript variant may play a critical role in the inflammatory response of patients with PG in different phases and TCM syndromes.

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

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    T. Bujas

    2011-03-01

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

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

    Science.gov (United States)

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

    2011-03-21

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

  4. Identification of a novel splice variant form of the influenza A virus M2 ion channel with an antigenically distinct ectodomain.

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    Helen M Wise

    Full Text Available Segment 7 of influenza A virus produces up to four mRNAs. Unspliced transcripts encode M1, spliced mRNA2 encodes the M2 ion channel, while protein products from spliced mRNAs 3 and 4 have not previously been identified. The M2 protein plays important roles in virus entry and assembly, and is a target for antiviral drugs and vaccination. Surprisingly, M2 is not essential for virus replication in a laboratory setting, although its loss attenuates the virus. To better understand how IAV might replicate without M2, we studied the reversion mechanism of an M2-null virus. Serial passage of a virus lacking the mRNA2 splice donor site identified a single nucleotide pseudoreverting mutation, which restored growth in cell culture and virulence in mice by upregulating mRNA4 synthesis rather than by reinstating mRNA2 production. We show that mRNA4 encodes a novel M2-related protein (designated M42 with an antigenically distinct ectodomain that can functionally replace M2 despite showing clear differences in intracellular localisation, being largely retained in the Golgi compartment. We also show that the expression of two distinct ion channel proteins is not unique to laboratory-adapted viruses but, most notably, was also a feature of the 1983 North American outbreak of H5N2 highly pathogenic avian influenza virus. In identifying a 14th influenza A polypeptide, our data reinforce the unexpectedly high coding capacity of the viral genome and have implications for virus evolution, as well as for understanding the role of M2 in the virus life cycle.

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

    Science.gov (United States)

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

    2009-12-01

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

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

    Science.gov (United States)

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

    2000-04-01

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

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

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    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.

  8. Biological, immunological and functional properties of two novel multi-variant chimeric recombinant proteins of CSP antigens for vaccine development against Plasmodium vivax infection.

    Science.gov (United States)

    Shabani, Samaneh H; Zakeri, Sedigheh; Salmanian, Ali H; Amani, Jafar; Mehrizi, Akram A; Snounou, Georges; Nosten, François; Andolina, Chiara; Mourtazavi, Yousef; Djadid, Navid D

    2017-10-01

    The circumsporozoite protein (CSP) of the malaria parasite Plasmodium vivax is a major pre-erythrocyte vaccine candidate. The protein has a central repeat region that belongs to one of repeat families (VK210, VK247, and the P. vivax-like). In the present study, computer modelling was employed to select chimeric proteins, comprising the conserved regions and different arrangements of the repeat elements (VK210 and VK247), whose structure is similar to that of the native counterparts. DNA encoding the selected chimeras (named CS127 and CS712) were synthetically constructed based on E. coli codons, then cloned and expressed. Mouse monoclonal antibodies (mAbs; anti-Pv-210-CDC and -Pv-247-CDC), recognized the chimeric antigens in ELISA, indicating correct conformation and accessibility of the B-cell epitopes. ELISA using IgG from plasma samples collected from 221 Iranian patients with acute P. vivax showed that only 49.32% of the samples reacted to both CS127 and CS712 proteins. The dominant subclass for the two chimeras was IgG1 (48% of the positive responders, OD492=0.777±0.420 for CS127; 48.41% of the positive responders, OD492=0.862±0.423 for CS712, with no statistically significant difference P>0.05; Wilcoxon signed ranks test). Binding assays showed that both chimeric proteins bound to immobilized heparan sulphate and HepG2 hepatocyte cells in a concentration-dependent manner, saturable at 80μg/mL. Additionally, anti-CS127 and -CS712 antibodies raised in mice recognized the native protein on the surface of P. vivax sporozoite with high intensity, confirming the presence of common epitopes between the recombinant forms and the native proteins. In summary, despite structural differences at the molecular level, the expression levels of both chimeras were satisfactory, and their conformational structure retained biological function, thus supporting their potential for use in the development of vivax-based vaccine. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Stefan L Karlsson

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

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

    Directory of Open Access Journals (Sweden)

    Myriam Arévalo-Herrera

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

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

    National Research Council Canada - National Science Library

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

    .... In this study we analyzed the expression of PSA, PSAP and androgen receptor (AR) by immunohistochemistry in 26 cases of male breast carcinomas and correlated these with the expression of other prognostic markers...

  12. 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.

  13. Reduced Penetrance and Variable Expression of SCN5A Mutations and the Importance of Co-inherited Genetic Variants: Case Report and Review of the Literature

    Directory of Open Access Journals (Sweden)

    T. Robyns, MD.

    2014-05-01

    Full Text Available Mutations in the SCN5A gene are responsible for multiple phenotypical presentations including Brugada syndrome, long QT syndrome, progressive familial heart block, sick sinus syndrome, dilated cardiomyopathy, lone atrial fibrillation and multiple overlap syndromes. These different phenotypic expressions of a mutation in a single gene can be explained by variable expression and reduced penetrance. One of the possible explanations of these phenomena is the co-inheritance of genetic variants. We describe a family where the individuals exhibit a compound heterozygosity in the SCN5A gene including a mutation (R1632H and a new variant (M858L. Individuals with both the mutation and new variant present with a more severe phenotype including spontaneous atrial tachyarrhythmia at young age. We give an overview of the different phenotypes of "SCN5A disease" and discuss the importance of co-inherited genetic variants in the expression of SCN5A disease.

  14. Two variants of the major serine protease inhibitor from the sea anemone Stichodactyla helianthus, expressed in Pichia pastoris.

    Science.gov (United States)

    García-Fernández, Rossana; Ziegelmüller, Patrick; González, Lidice; Mansur, Manuel; Machado, Yoan; Redecke, Lars; Hahn, Ulrich; Betzel, Christian; Chávez, María de Los Ángeles

    2016-07-01

    The major protease inhibitor from the sea anemone Stichodactyla helianthus (ShPI-1) is a non-specific inhibitor that binds trypsin and other trypsin-like enzymes, as well as chymotrypsin, and human neutrophil elastase. We performed site-directed mutagenesis of ShPI-1 to produce two variants (rShPI-1/K13L and rShPI/Y15S) that were expressed in Pichia pastoris, purified, and characterized. After a single purification step, 65 mg and 15 mg of protein per liter of culture supernatant were obtained for rShPI-1/K13L and rShPI/Y15S, respectively. Functional studies demonstrated a 100-fold decreased trypsin inhibitory activity as result of the K13L substitution at the reactive (P1) site. This protein variant has a novel tight-binding inhibitor activity of pancreatic elastase and increased activity toward neutrophil elastase in comparison to rShPI-1A. In contrast, the substitution Y15S at P2' site did not affect the Ki value against trypsin, but did reduce activity 10-fold against chymotrypsin and neutrophil elastase. Our results provide two new ShPI-1 variants with modified inhibitory activities, one of them with increased biomedical potential. This study also offers new insight into the functional impact of the P1 and P2' sites on ShPI-1 specificity. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    1995-05-01

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

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

    Science.gov (United States)

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

    1997-12-01

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

  17. Expression of Tetrahymena snRNA gene variants including a U1 gene with mutations in the 5' splice site recognition sequence

    DEFF Research Database (Denmark)

    Eugen-Olsen, J; Hagemeister, J J; Hellung-Larsen, P

    1997-01-01

    The expression of U1, U2 and U5 snRNA gene variants has been studied under different physiological states of Tetrahymena. Variants of all three snRNA genes are expressed. Among the snRNAs detected is U1-3, a variant with 66 mutations compared to the normal U1 snRNA. Three of these mutations affec...... the 5' splice site recognition sequence. The U1-3 snRNA is present in a few hundred copies per cell. The expression of Tetrahymena snRNA genes is independent of the physiological state of the cell.......The expression of U1, U2 and U5 snRNA gene variants has been studied under different physiological states of Tetrahymena. Variants of all three snRNA genes are expressed. Among the snRNAs detected is U1-3, a variant with 66 mutations compared to the normal U1 snRNA. Three of these mutations affect...

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

    DEFF Research Database (Denmark)

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

    2014-01-01

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

  19. 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.

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

    OpenAIRE

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

    2013-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Elizabeth N. De Gaspari

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

  2. A case of cervical cancer expressed three mRNA variant of Hyaluronan-mediated motility receptor

    Science.gov (United States)

    Villegas-Ruíz, Vanessa; Salcedo, Mauricio; Zentella-Dehesa, Alejandro; de Oca, Edén V Montes; Román-Basaure, Edgar; Mantilla-Morales, Alejandra; Dávila-Borja, Víctor M; Juárez-Méndez, Sergio

    2014-01-01

    Cervical cancer is the second malignancy in Mexico, little is known about the prognostic factors associated with this disease. Several cellular components are important in their transformation and progression. Alternative mRNA splice is an important mechanism for generating protein diversity, nevertheless, in cancer unknown mRNA diversity is expressed. Hyaluronan-mediated motility receptor (HMMR, RHAMM, CD168) is a family member of proteins, hyaluronan acid dependent, and has been associated with different malignant processes such as: angiogenesis, cell invasiveness, proliferation, metastasis and poor outcome in some tumors. In the present study we identified expression of HMMR in cervical cancer by means of RT-PCR and sequencing. Our results indicate co-expression of two HMMR variants in all samples, and one case expressed three alternative HMMR splice transcripts. These results showed the heterogeneity of mRNA transcripts of HMMR that could express in cancer and the expression of HMMR could be marker of malignancy in CC. PMID:24966934

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

    Science.gov (United States)

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

    2017-08-01

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

  4. Mechanisms Mediating Antigenic Variation in Trypanosoma brucei

    Directory of Open Access Journals (Sweden)

    Rudenko Gloria

    1999-01-01

    Full Text Available Antigenic variation in Trypanosoma brucei is a highly sophisticated survival strategy involving switching between the transcription of one of an estimated thousand variant surface glycoprotein (VSG genes. Switching involves either transcriptional control, resulting in switching between different VSG expression sites; or DNA rearrangement events slotting previously inactive VSG genes into an active VSG expression site. In recent years, considerable progress has been made in techniques allowing us to genetically modify infective bloodstream form trypanosomes. This is allowing us to reengineer VSG expression sites, and look at the effect on the mechanisms subsequently used for antigenic variation. We can now begin a dissection of a highly complicated survival strategy mediated by many different mechanisms operating simultaneously.

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

    National Research Council Canada - National Science Library

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

    2016-01-01

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

  6. MDS shows a higher expression of hTERT and alternative splice variants in unactivated T-cells.

    Science.gov (United States)

    Dong, Wen; Wu, Lei; Sun, Houfang; Ren, Xiubao; Epling-Burnette, Pearlie K; Yang, Lili

    2016-11-01

    Telomere instability and telomerase reactivation are believed to play an important role in the development of myelodysplastic syndromes (MDS). Abnormal enzymatic activity of human telomerase reverse transcriptase (hTERT), and its alternative splice variants have been reported to account for deregulated telomerase function in many cancers. In this study, we aim to compare the differences in expression of hTERT and hTERT splice variants, as well as telomere length and telomerase activity in unstimulated T-cells between MDS subgroups and healthy controls. Telomere length in MDS cases was significantly shorter than controls (n = 20, pMDS using World Health Organization classification (WHO subgroups versus control: RARS, p= 0.009; RCMD, p=0.0002; RAEB1/2, p=0.004, respectively) and the International Prognostic Scoring System (IPSS subgroups: Low+Int-1, pMDS patients (n=20) had significantly higher telomerase activity (p=0.002), higher total hTERT mRNA levels (p=0.001) and hTERT α+β- splice variant expression (pMDS (r=0.58, p=0.007). This data is in sharp contrast to data published previously by our group showing a reduction in telomerase and hTERT mRNA in MDS T-cells after activation. In conclusion, this study provides additional insight into hTERT transcript patterns and activity in peripheral T-cells of MDS patients. Additional studies are necessary to better understand the role of this pathway in MDS development and progression.

  7. TLR9 polymorphisms in African populations: no association with severe malaria, but evidence of cis-variants acting on gene expression

    Directory of Open Access Journals (Sweden)

    Pinder Margaret

    2009-03-01

    Full Text Available Abstract Background During malaria infection the Toll-like receptor 9 (TLR9 is activated through induction with plasmodium DNA or another malaria motif not yet identified. Although TLR9 activation by malaria parasites is well reported, the implication to the susceptibility to severe malaria is not clear. The aim of this study was to assess the contribution of genetic variation at TLR9 to severe malaria. Methods This study explores the contribution of TLR9 genetic variants to severe malaria using two approaches. First, an association study of four common single nucleotide polymorphisms was performed on both family- and population-based studies from Malawian and Gambian populations (n>6000 individual. Subsequently, it was assessed whether TLR9 expression is affected by cis-acting variants and if these variants could be mapped. For this work, an allele specific expression (ASE assay on a panel of HapMap cell lines was carried out. Results No convincing association was found with polymorphisms in TLR9 for malaria severity, in either Gambian or Malawian populations, using both case-control and family based study designs. Using an allele specific expression assay it was observed that TLR9 expression is affected by cis-acting variants, these results were replicated in a second experiment using biological replicates. Conclusion By using the largest cohorts analysed to date, as well as a standardized phenotype definition and study design, no association of TLR9 genetic variants with severe malaria was found. This analysis considered all common variants in the region, but it is remains possible that there are rare variants with association signals. This report also shows that TLR9 expression is potentially modulated through cis-regulatory variants, which may lead to differential inflammatory responses to infection between individuals.

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

    Directory of Open Access Journals (Sweden)

    Bappaditya Dey

    Full Text Available BACKGROUND: In spite of a consistent protection against tuberculosis (TB in children, Mycobacterium bovis Bacille Calmette-Guerin (BCG fails to provide adequate protection against the disease in adults as well as against reactivation of latent infections or exogenous reinfections. It has been speculated that failure to generate adequate memory T cell response, elicitation of inadequate immune response against latency-associated antigens and inability to impart long-term immunity against M. tuberculosis infections are some of the key factors responsible for the limited efficiency of BCG in controlling TB. METHODS/PRINCIPAL FINDINGS: In this study, we evaluated the ability of a DNA vaccine expressing α-crystallin--a key latency antigen of M. tuberculosis to boost the BCG induced immunity. 'BCG prime-DNA boost' regimen (B/D confers robust protection in guinea pigs along with a reduced pathology in comparison to BCG vaccination (1.37 log(10 and 1.96 log(10 fewer bacilli in lungs and spleen, respectively; p<0.01. In addition, B/D regimen also confers enhanced protection in mice. Further, we show that B/D immunization in mice results in a heightened frequency of PPD and antigen specific multi-functional CD4 T cells (3(+ simultaneously producing interferon (IFNγ, tumor necrosis factor (TNFα and interleukin (IL2. CONCLUSIONS/SIGNIFICANCE: These results clearly indicate the superiority of α-crystallin based B/D regimen over BCG. Our study, also demonstrates that protection against TB is predictable by an increased frequency of 3(+ Th1 cells with superior effector functions. We anticipate that this study would significantly contribute towards the development of superior booster vaccines for BCG vaccinated individuals. In addition, this regimen can also be expected to reduce the risk of developing active TB due to reactivation of latent infection.

  9. Molecular Cloning, Carbohydrate Specificity and the Crystal Structure of Two Sclerotium rolfsii Lectin Variants.

    Science.gov (United States)

    Peppa, Vassiliki I; Venkat, Hemalatha; Kantsadi, Anastassia L; Inamdar, Shashikala R; Bhat, Ganapati G; Eligar, Sachin; Shivanand, Anupama; Chachadi, Vishwanath B; Satisha, Gonchigar J; Swamy, Bale M; Skamnaki, Vassiliki T; Zographos, Spyridon E; Leonidas, Demetres D

    2015-06-12

    SRL is a cell wall associated developmental-stage specific lectin secreted by Sclerotium rolfsii, a soil-born pathogenic fungus. SRL displays specificity for TF antigen (Galβ1→3GalNAc-α-Ser//Thr) expressed in all cancer types and has tumour suppressing effects in vivo. Considering the immense potential of SRL in cancer research, we have generated two variant gene constructs of SRL and expressed in E. coli to refine the sugar specificity and solubility by altering the surface charge. SSR1 and SSR2 are two different recombinant variants of SRL, both of which recognize TF antigen but only SSR1 binds to Tn antigen (GalNAcα-Ser/Thr). The glycan array analysis of the variants demonstrated that SSR1 recognizes TF antigen and their derivative with high affinity similar to SRL but showed highest affinity towards the sialylated Tn antigen, unlike SRL. The carbohydrate binding property of SSR2 remains unaltered compared to SRL. The crystal structures of the two variants were determined in free form and in complex with N-acetylglucosamine at 1.7 Å and 1.6 Å resolution, respectively. Structural analysis highlighted the structural basis of the fine carbohydrate specificity of the two SRL variants and results are in agreement with glycan array analysis.

  10. Molecular Cloning, Carbohydrate Specificity and the Crystal Structure of Two Sclerotium rolfsii Lectin Variants

    Directory of Open Access Journals (Sweden)

    Vassiliki I. Peppa

    2015-06-01

    Full Text Available SRL is a cell wall associated developmental-stage specific lectin secreted by Sclerotium rolfsii, a soil-born pathogenic fungus. SRL displays specificity for TF antigen (Galβ1→3GalNAc-α-Ser//Thr expressed in all cancer types and has tumour suppressing effects in vivo. Considering the immense potential of SRL in cancer research, we have generated two variant gene constructs of SRL and expressed in E. coli to refine the sugar specificity and solubility by altering the surface charge. SSR1 and SSR2 are two different recombinant variants of SRL, both of which recognize TF antigen but only SSR1 binds to Tn antigen (GalNAcα-Ser/Thr. The glycan array analysis of the variants demonstrated that SSR1 recognizes TF antigen and their derivative with high affinity similar to SRL but showed highest affinity towards the sialylated Tn antigen, unlike SRL. The carbohydrate binding property of SSR2 remains unaltered compared to SRL. The crystal structures of the two variants were determined in free form and in complex with N-acetylglucosamine at 1.7 Å and 1.6 Å resolution, respectively. Structural analysis highlighted the structural basis of the fine carbohydrate specificity of the two SRL variants and results are in agreement with glycan array analysis.

  11. MC1R variants affect the expression of melanocortin and melanogenic genes and the association between melanocortin genes and coloration.

    Science.gov (United States)

    San-Jose, Luis M; Ducrest, Anne-Lyse; Ducret, Valérie; Simon, Céline; Richter, Hannes; Wakamatsu, Kazumasa; Roulin, Alexandre

    2017-01-01

    The melanocortin-1 receptor (MC1R) gene influences coloration by altering the expression of genes acting downstream in the melanin synthesis. MC1R belongs to the melanocortin system, a genetic network coding for the ligands that regulate MC1R and other melanocortin receptors controlling different physiological and behavioural traits. The impact of MC1R variants on these regulatory melanocortin genes was never considered, even though MC1R mutations could alter the influence of these genes on coloration (e.g. by decreasing MC1R response to melanocortin ligands). Using barn owl growing feathers, we investigated the differences between MC1R genotypes in the (co)expression of six melanocortin and nine melanogenic-related genes and in the association between melanocortin gene expression and phenotype (feather pheomelanin content). Compared to the MC1R rufous allele, responsible for reddish coloration, the white allele was not only associated with an expected lower expression of melanogenic-related genes (TYR, TYRP1, OCA2, SLC45A2, KIT, DCT) but also with a lower MC1R expression and a higher expression of ASIP, the MC1R antagonist. More importantly, the expression of PCSK2, responsible for the maturation of the MC1R agonist, α-melanocyte-stimulating hormone, was positively related to pheomelanin content in MC1R white homozygotes but not in individuals carrying the MC1R rufous allele. These findings indicate that MC1R mutations not only alter the expression of melanogenic-related genes but also the association between coloration and the expression of melanocortin genes upstream of MC1R. This suggests that MC1R mutations can modulate the regulation of coloration by the pleiotropic melanocortin genes, potentially decoupling the often-observed associations between coloration and other phenotypes. © 2016 John Wiley & Sons Ltd.

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

    African Journals Online (AJOL)

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

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

    NARCIS (Netherlands)

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

    1999-01-01

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

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

    NARCIS (Netherlands)

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

    1989-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Athanasios Niarchos

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

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

    Science.gov (United States)

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

    2004-06-01

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

  17. Expression of Immunoglobulin Receptors with Distinctive Features Indicating Antigen Selection by Marginal Zone B Cells from Human Spleen

    Science.gov (United States)

    Colombo, Monica; Cutrona, Giovanna; Reverberi, Daniele; Bruno, Silvia; Ghiotto, Fabio; Tenca, Claudya; Stamatopoulos, Kostas; Hadzidimitriou, Anastasia; Ceccarelli, Jenny; Salvi, Sandra; Boccardo, Simona; Calevo, Maria Grazia; De Santanna, Amleto; Truini, Mauro; Fais, Franco; Ferrarini, Manlio

    2013-01-01

    Marginal zone (MZ) B cells, identified as surface (s)IgMhighsIgDlowCD23low/−CD21+CD38− B cells, were purified from human spleens, and the features of their V(D)J gene rearrangements were investigated and compared with those of germinal center (GC), follicular mantle (FM) and switched memory (SM) B cells. Most MZ B cells were CD27+ and exhibited somatic hypermutations (SHM), although to a lower extent than SM B cells. Moreover, among MZ B-cell rearrangements, recurrent sequences were observed, some of which displayed intraclonal diversification. The same diversifying sequences were detected in very low numbers in GC and FM B cells and only when a highly sensitive, gene-specific polymerase chain reaction was used. This result indicates that MZ B cells could expand and diversify in situ and also suggested the presence of a number of activation-induced cytidine deaminase (AID)-expressing B cells in the MZ. The notion of antigen-driven expansion/selection in situ is further supported by the VH CDR3 features of MZ B cells with highly conserved amino acids at specific positions and by the finding of shared (“stereotyped”) sequences in two different spleens. Collectively, the data are consistent with the notion that MZ B cells are a special subset selected by in situ antigenic stimuli. PMID:23877718

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

    Directory of Open Access Journals (Sweden)

    Aziz Alami Chentoufi

    2011-01-01

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

  19. Resistance to Multiple Tuber Diseases Expressed in Somaclonal Variants of the Potato Cultivar Russet Burbank

    Directory of Open Access Journals (Sweden)

    Tamilarasan Thangavel

    2014-01-01

    Full Text Available Multiple disease resistance is an aim of many plant breeding programs. Previously, novel somatic cell selection was used to generate potato variants of “Russet Burbank” with resistance to common scab caused by infection with an actinomycete pathogen. Coexpression of resistance to powdery scab caused by a protozoan pathogen was subsequently shown. This study sought to define whether this resistance was effective against additional potato tuber diseases, black scurf, and tuber soft rot induced by fungal and bacterial pathogens. Pot trials and in vitro assays with multiple pathogenic strains identified significant resistance to both tuber diseases across the potato variants examined; the best clone A380 showed 51% and 65% reductions in disease severity to tuber soft rot and black scurf, respectively, when compared with the parent line. The resistance appeared to be tuber specific as no enhanced resistance was recorded in stolons or stem material when challenged Rhizoctonia solani that induces stolon pruning and stem canker. The work presented here suggests that morphological characteristics associated with tuber resistance may be the predominant change that has resulted from the somaclonal cell selection process, potentially underpinning the demonstrated broad spectrum of resistance to tuber invading pathogens.

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

    Science.gov (United States)

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

    2011-08-01

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

  1. A 17.6 kbp region located upstream of the rabbit WAP gene directs high level expression of a functional human protein variant in transgenic mouse milk

    NARCIS (Netherlands)

    Bischoff, Rainer; Degryse, E.; Perraud, F.; Dalemans, W.; Ali-Hadji, D.; Thepot, D.; Devinoy, E.; Houdebine, L.M.; Pavirani, A.

    1992-01-01

    We have investigated whether DNA regions present in the rabbit whey acidic protein (WAP) promoter/5' flanking sequence could potentially confer, in vivo, high level expression of reporter genes. Transgenic mice were generated expressing a variant of human alpha 1-antitrypsin, which has inhibitory

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

    NARCIS (Netherlands)

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

    2011-01-01

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

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

    National Research Council Canada - National Science Library

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

    2009-01-01

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

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

    National Research Council Canada - National Science Library

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

    2010-01-01

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

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

    Science.gov (United States)

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

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

    Directory of Open Access Journals (Sweden)

    Hanady A Amoudy

    2016-01-01

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

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

    Science.gov (United States)

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

    2005-01-01

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

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

    Science.gov (United States)

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

    2017-02-01

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

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

    DEFF Research Database (Denmark)

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

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Smith Daniel C.

    2003-01-01

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

  11. 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.

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

    Directory of Open Access Journals (Sweden)

    Anu Sharma

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

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

    Science.gov (United States)

    Marshall, Joanna M; Gunn, John S

    2015-10-01

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

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

    Science.gov (United States)

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

    1995-10-24

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

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

    Directory of Open Access Journals (Sweden)

    Qiu Yan

    2008-11-01

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

  16. Unexpected dependence of RyR1 splice variant expression in human lower limb muscles on fiber-type composition.

    Science.gov (United States)

    Willemse, Hermia; Theodoratos, Angelo; Smith, Paul N; Dulhunty, Angela F

    2016-02-01

    The skeletal muscle ryanodine receptor Ca(2+) release channel (RyR1), essential for excitation-contraction (EC) coupling, demonstrates a known developmentally regulated alternative splicing in the ASI region. We now find unexpectedly that the expression of the splice variants is closely related to fiber type in adult human lower limb muscles. We examined the distribution of myosin heavy chain isoforms and ASI splice variants in gluteus minimus, gluteus medius and vastus medialis from patients aged 45 to 85 years. There was a strong positive correlation between ASI(+)RyR1 and the percentage of type 2 fibers in the muscles (r = 0.725), and a correspondingly strong negative correlation between the percentages of ASI(+)RyR1 and percentage of type 1 fibers. When the type 2 fiber data were separated into type 2X and type 2A, the correlation with ASI(+)RyR1 was stronger in type 2X fibers (r = 0.781) than in type 2A fibers (r = 0.461). There was no significant correlation between age and either fiber-type composition or ASI(+)RyR1/ASI(-)RyR1 ratio. The results suggest that the reduced expression of ASI(-)RyR1 during development may reflect a reduction in type 1 fibers during development. Preferential expression of ASI(-) RyR1, having a higher gain of in Ca(2+) release during EC coupling than ASI(+)RyR1, may compensate for the reduced terminal cisternae volume, fewer junctional contacts and reduced charge movement in type 1 fibers.

  17. 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.

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

    Directory of Open Access Journals (Sweden)

    Rikke Christensen

    2017-08-01

    Full Text Available The cell surface protein Stem Cell Antigen-1 (Sca-1 marks stem or progenitor cells in several murine tissues and is normally upregulated during cancer development. Although the specific function of S