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Sample records for 51-kilodalton antigen gene

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

    PubMed

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

    2013-01-01

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

  2. Genes encoding homologous antigens in taeniid cestode parasites

    PubMed Central

    Gauci, Charles; Lightowlers, Marshall W.

    2013-01-01

    Recombinant vaccine antigens are being evaluated for their ability to protect livestock animals against cysticercosis and related parasitic infections. Practical use of some of these vaccines is expected to reduce parasite transmission, leading to a reduction in the incidence of neurocysticercosis and hydatid disease in humans. We recently showed that an antigen (TSOL16), expressed in Escherichia coli, confers high levels of protection against Taenia solium cysticercosis in pigs, which provides a strategy for control of T. solium parasite transmission. Here, we discuss the characteristics of this antigen that may affect the utility of TSOL16 and related antigens for development as recombinant vaccines. We also report that genes encoding antigens closely related to TSOL16 from T. solium also occur in other related species of parasites. These highly homologous antigens have the potential to be used as vaccines and may provide protection against related species of Taenia that cause infection in other hosts. PMID:23090389

  3. Cloning and expression of genes encoding Haemophilus somnus antigens.

    PubMed Central

    Corbeil, L B; Chikami, G; Yarnall, M; Smith, J; Guiney, D G

    1988-01-01

    A genomic library of Haemophilus somnus 2336, a virulent isolate from a calf with pneumonia (later used to reproduce H. somnus experimental pneumonia), was constructed in the cosmid vector pHC79. The gene bank in Escherichia coli DH1 was screened by filter immunoassay with convalescent-phase serum, which reacted with several outer membrane antigens of H. somnus. On Western blotting (immunoblotting) of immunoreactive colonies, five clones were found to express proteins which comigrated with H. somnus surface antigens. Three clones (DH1 pHS1, pHS3, and pHS4) expressed both a 120-kilodalton (kDa) antigen and a 76-kDa antigen, one clone (DH1 pHS2) expressed only the 76-kDa antigen, and the fifth clone (DH1 pHS5) expressed a 60-kDa antigen. The 120-kDa and 76-kDa antigens were found internally, whereas the 60-kDa protein was detected in the DH1 pHS5 culture supernatant as membrane blebs or insoluble protein. Both the H. somnus 120-kDa antigen and the recombinant 120-kDa antigen had immunoglobulin Fc-binding activity. Restriction endonuclease mapping demonstrated that the genomic DNA inserts of clones expressing the 76-kDa antigen shared a common 28.4-kilobase-pair region, and the three clones also expressing the 120-kDa antigen shared an additional 7.0-kilobase-pair region. The restriction endonuclease map of pHS5, which expressed the 60-kDa antigen, was not similar to the maps of the other four plasmids. Since these three H. somnus antigens reacted with protective convalescent-phase serum, the recombinants which express these proteins should be useful in further studies of protective immunity in bovine H. somnus disease. Images PMID:2843469

  4. Defining specificities, genes, antigens, and antibodies- A matrix approach.

    PubMed

    Wohlgemuth, A

    1978-12-01

    We study the consequences of assigning single letter symbols to operationally defined entities such as genes, antigens, specificities, and antibodies. If this is to be done and if reagents are not specific in recognizing the products of single genes or single antigens, then these entities must be defined by a 'definition matrix' to avoid mislabeling a matrix of data. A method is given whereby for a given matrix of data all possible definition matrices consistent with this data can be obtained. In particular, all the ways of labeling by the complex-complex code of Hirschfeld can be so obtained.

  5. [Immunoglobulin genes encoding antibodies directed to oncodevelopmental carbohydrate antigens].

    PubMed

    Zenita, K; Yago, K; Fujimoto, E; Kannagi, R

    1990-07-01

    We investigated the immunoglobulin genes which encode the variable region of the monoclonal antibodies directed to the onco-developmental carbohydrate antigens such SSEA-1, fucosyl SSEA-1, SSEA-3 and SSEA-4. The VH region of these antibodies was preferentially encoded by the gene members of the X24, VH7183 and Q52 families, the families which are known to be located at the 3'-end region of the murine germ line VH gene. This result is interesting particularly when considering that the members of the 3'-end VH families are known to be preferentially expressed in embryonic B lymphocytes by an intrinsic genetic program. The comparative study of the nucleic acid sequences of mRNAs encoding these antibodies and the sequences of the corresponding germ line VH genes disclosed that the sequences encoding the antibodies contain no mutation from the germ line VH genes, or contain only a few somatic mutations, which are thought to be insignificant for the reactivity of the antibodies to the nominal antigens. These results imply that some of the embryonic B lymphocytes that express the unmutated germ line VH genes of the 3'-end families can be reactive with embryonic carbohydrate antigens, albeit rearranged with appropriate D-JH gene segments, and coupled with proper light chains. The VH region of the syngenic monoclonal anti-idiotypic antibodies directed to these anti-carbohydrate antibodies were also encoded preferentially by the members of the 3'-end VH families. We propose here that a part of the virgin embryonic B lymphocytes, which express the antibody encoded by the gene members of the 3'-end VH families at the cell surface, will be stimulated by the embryonic carbohydrate antigens which are abundantly present in the internal milieu of the embryo. The clonally expanded B lymphocytes, in turn, will facilitate the proliferation of other populations of embryonic B lymphocytes expressing the corresponding anti-idiotypic antibodies, which are also encoded by the gene members

  6. Human erythrocyte antigens. Regulation of expression of a novel erythrocyte surface antigen by the inhibitor Lutheran In(Lu) gene.

    PubMed Central

    Telen, M J; Eisenbarth, G S; Haynes, B F

    1983-01-01

    Our study describes a novel human erythrocyte protein antigen, the expression of which is regulated by the rare Lutheran inhibitor In(Lu) gene. We have produced a monoclonal antibody (A3D8) that bound strongly to erythrocytes from subjects with Lutheran phenotypes Lu(a+b+), Lu(a+b-), and Lu(a-b+) but bound negligibly to erythrocytes from subjects with the dominant form of Lu(a-b-) phenotype, reflecting inheritance of the In(Lu) gene. Importantly, erythrocytes from an individual with the recessive form of Lu(a-b-) phenotype (i.e., absence of the In(Lu) gene and absence of genes encoding for Lutheran antigens) showed reactivity with A3D8 antibody comparable to that seen with Lu(a+) or Lu(b+) erythrocytes. A3D8 antigen activity was also found on all leukocytes and in serum and plasma; this activity also appeared to be regulated by the In(Lu) gene in serum, plasma, and on a subset of leukocytes. Thus, we have identified a human erythrocyte protein whose expression is modified by the In(Lu) gene. This knowledge that such an antigen exists on erythrocytes and in normal plasma should allow further studies into the molecular genetics of the In(Lu) gene and into the functional and structural significance of the A3D8 antigen. PMID:6863545

  7. DNA sequence of a gene encoding a BALB/c mouse Ld transplantation antigen.

    PubMed

    Moore, K W; Sher, B T; Sun, Y H; Eakle, K A; Hood, L

    1982-02-01

    The sequence of a gene, denoted 27.5, encoding a transplantation antigen for the BALB/c mouse has been determined. Gene transfer studies and comparison of the translated sequence with the partial amino acid sequence of the Ld transplantation antigen establish that gene 27.5 encodes an Ld polypeptide. A comparison of the gene 27.5 sequence with several complementary DNA sequences suggests that the BALB/c mouse may contain a number of closely related L-like genes. Gene 27.5 has eight exons that correlate with the structural domains of the transplantation antigen. PMID:7058332

  8. Evidence for Horizontal Gene Transfer of Two Antigenically Distinct O Antigens in Bordetella bronchiseptica▿ †

    PubMed Central

    Buboltz, Anne M.; Nicholson, Tracy L.; Karanikas, Alexia T.; Preston, Andrew; Harvill, Eric T.

    2009-01-01

    Host immunity is a major driving force of antigenic diversity, resulting in pathogens that can evade immunity induced by closely related strains. Here we show that two Bordetella bronchiseptica strains, RB50 and 1289, express two antigenically distinct O-antigen serotypes (O1 and O2, respectively). When 18 additional B. bronchiseptica strains were serotyped, all were found to express either the O1 or O2 serotype. Comparative genomic hybridization and PCR screening showed that the expression of either the O1 or O2 serotype correlated with the strain containing either the classical or alternative O-antigen locus, respectively. Multilocus sequence typing analysis of 49 B. bronchiseptica strains was used to build a phylogenetic tree, which revealed that the two O-antigen loci did not associate with a particular lineage, evidence that these loci are horizontally transferred between B. bronchiseptica strains. From experiments using mice vaccinated with purified lipopolysaccharide from strain RB50 (O1), 1289 (O2), or RB50Δwbm (O antigen deficient), our data indicate that these O antigens do not confer cross-protection in vivo. The lack of cross-immunity between O-antigen serotypes appears to contribute to inefficient antibody-mediated clearance between strains. Together, these data are consistent with the idea that the O-antigen loci of B. bronchiseptica are horizontally transferred between strains and encode antigenically distinct serotypes, resulting in inefficient cross-immunity. PMID:19528223

  9. Autoregulation of simian virus 40 gene A by T antigen.

    PubMed Central

    Reed, S I; Stark, G R; Alwine, J C

    1976-01-01

    During lytic infection by simian virus 40, gene A is transcribed into early RNA, which is translated into A protein (T antigen). Both the rate of synthesis and the intracellular amount of early RNA are higher in cells infected by temperature-sensitive A (tsA) mutants than in cells infected by wild-type virus. These differences are observed at permissive temperature (32 degrees) and are amplified greatly after a shift to restrictive temperature (41 degrees). For example, at 32 degrees cells infected by tsA mutants synthesize early RNA approximately twice as fast as cells infected by wild-type virus. After the shift to 41 degrees, the rate of synthesis in the tsA infection increases to 15 times the rate in the wild-type infection. In contrast, cells infected by tsA mutants do not overproduce late RNA. We suggest that the A protein regulates its own synthesis by negative feedback control of gene A transcription. PMID:184459

  10. Cell-type specific regulation of gene expression by simian virus 40 T antigens

    SciTech Connect

    Cantalupo, Paul G.; Saenz-Robles, Maria Teresa; Rathi, Abhilasha V.; Beerman, Rebecca W.; Patterson, William H.; Whitehead, Robert H.; Pipas, James M.

    2009-03-30

    SV40 transforms cells through the action of two oncoproteins, large T antigen and small t antigen. Small t antigen targets phosphatase PP2A, while large T antigen stimulates cell proliferation and survival by action on multiple proteins, including the tumor suppressors Rb and p53. Large T antigen also binds components of the transcription initiation complex and several transcription factors. We examined global gene expression in SV40-transformed mouse embryo fibroblasts, and in enterocytes obtained from transgenic mice. SV40 transformation alters the expression of approximately 800 cellular genes in both systems. Much of this regulation is observed in both MEFs and enterocytes and is consistent with T antigen action on the Rb-E2F pathway. However, the regulation of many genes is cell-type specific, suggesting that unique signaling pathways are activated in different cell types upon transformation, and that the consequences of SV40 transformation depends on the type of cell targeted.

  11. Wide Distribution of O157-Antigen Biosynthesis Gene Clusters in Escherichia coli

    PubMed Central

    Seto, Kazuko; Ooka, Tadasuke; Ogura, Yoshitoshi; Hayashi, Tetsuya; Osawa, Kayo; Osawa, Ro

    2011-01-01

    Most Escherichia coli O157-serogroup strains are classified as enterohemorrhagic E. coli (EHEC), which is known as an important food-borne pathogen for humans. They usually produce Shiga toxin (Stx) 1 and/or Stx2, and express H7-flagella antigen (or nonmotile). However, O157 strains that do not produce Stxs and express H antigens different from H7 are sometimes isolated from clinical and other sources. Multilocus sequence analysis revealed that these 21 O157:non-H7 strains tested in this study belong to multiple evolutionary lineages different from that of EHEC O157:H7 strains, suggesting a wide distribution of the gene set encoding the O157-antigen biosynthesis in multiple lineages. To gain insight into the gene organization and the sequence similarity of the O157-antigen biosynthesis gene clusters, we conducted genomic comparisons of the chromosomal regions (about 59 kb in each strain) covering the O-antigen gene cluster and its flanking regions between six O157:H7/non-H7 strains. Gene organization of the O157-antigen gene cluster was identical among O157:H7/non-H7 strains, but was divided into two distinct types at the nucleotide sequence level. Interestingly, distribution of the two types did not clearly follow the evolutionary lineages of the strains, suggesting that horizontal gene transfer of both types of O157-antigen gene clusters has occurred independently among E. coli strains. Additionally, detailed sequence comparison revealed that some positions of the repetitive extragenic palindromic (REP) sequences in the regions flanking the O-antigen gene clusters were coincident with possible recombination points. From these results, we conclude that the horizontal transfer of the O157-antigen gene clusters induced the emergence of multiple O157 lineages within E. coli and speculate that REP sequences may involve one of the driving forces for exchange and evolution of O-antigen loci. PMID:21876740

  12. Molecular and Genetic Analyses of the Putative Proteus O Antigen Gene Locus▿ †

    PubMed Central

    Wang, Quan; Torzewska, Agnieszka; Ruan, Xiaojuan; Wang, Xiaoting; Rozalski, Antoni; Shao, Zhujun; Guo, Xi; Zhou, Haijian; Feng, Lu; Wang, Lei

    2010-01-01

    Proteus species are well-characterized opportunistic pathogens primarily associated with urinary tract infections (UTI) of humans. The Proteus O antigen is one of the most variable constituents of the cell surface, and O antigen heterogeneity is used for serological classification of Proteus isolates. Even though most Proteus O antigen structures have been identified, the O antigen locus has not been well characterized. In this study, we identified the putative Proteus O antigen locus and demonstrated this region's high degree of heterogeneity by comparing sequences of 40 Proteus isolates using PCR-restriction fragment length polymorphism (RFLP). This analysis identified five putative Proteus O antigen gene clusters, and the probable functions of these O antigen-related genes were proposed, based on their similarity to genes in the available databases. Finally, Proteus-specific genes from these five serogroups were identified by screening 79 strains belonging to the 68 Proteus O antigen serogroups. To our knowledge, this is the first molecular characterization of the putative Proteus O antigen locus, and we describe a novel molecular classification method for the identification of different Proteus serogroups. PMID:20581173

  13. Structure and gene cluster of the O-antigen of Escherichia coli O68.

    PubMed

    Jiang, Lingyan; Perepelov, Andrei V; Filatov, Andrei V; Liu, Bin; Shashkov, Alexander S; Senchenkova, Sof'ya N; Wang, Lei; Knirel, Yuriy A

    2014-10-01

    The O-polysaccharide (O-antigen) of Escherichia coli O68 was studied by sugar analysis, partial solvolysis with anhydrous trifluoroacetic acid, and 1D and 2D (1)H and (13)C NMR spectroscopies. The following structure of the branched heptasaccharide repeating unit was established: [structure: see text]. The O-antigen gene cluster of E. coli O68 was sequenced. The gene functions were tentatively assigned by comparison with sequences in the available databases and found to be in full agreement with the O-antigen structure.

  14. [Relation between Ia antigens and Ir gene products. A theory on the development of the immune response].

    PubMed

    Seignalet, J

    1983-11-01

    To explain the relations between Ia and Ir, we propose the following hypothesis. It would exist an antigen specific factor released by T lymphocytes. This factor would be constituted by two parts: a variable fragment forming the antigenic receptor of T cells and coded by Ir genes, a constant fragment transmitting a helper or suppressor signal and carrying Ia antigens. Ia antigens would allow a mutual recognition between macrophages, T lymphocytes and B lymphocytes and a recognition by these cells of some mediators released during immune response. Ir genes products would allow the antigen recognition, if they correspond to the antigenic receptor of T lymphocytes.

  15. [Relations between Ia antigens and the products of Ir genes. A theory of the evolution of the immune response].

    PubMed

    Seignalet, J

    1983-01-01

    To explain the relations between Ia and Ir, we propose the following hypothesis. It would exist an antigen specific factor released by T lymphocytes. This factor would be constituted by two parts: a variable fragment forming the antigenic receptor of T cells and coded by Ir genes, a constant fragment transmitting an helper or suppressor signal and carrying Ia antigens. Ia antigens would allow a mutual recognition between macrophages, T lymphocytes and B lymphocytes and a recognition by these cells of some mediators released during immune response. Ir genes products would allow the antigen recognition, if they correspond to the antigenic receptor of T lymphocytes.

  16. Molecular characterization of swine leukocyte antigen (SLA) class II genes in outbred pig populations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The highly polymorphic swine leukocyte antigen (SLA) genes are one of the most important determinants in swine immune, disease and vaccine responses. Thus, understanding how SLA gene polymorphism affects immunity, especially in outbred pig populations with a diverse genetic background, requires accu...

  17. Cellular gene expression induced by parasite antigens and allergens in neonates from parasite-infected mothers.

    PubMed

    Soboslay, Peter T; Orlikowsky, Thorsten; Huang, Xiangsheng; Gille, Christian; Spring, Bärbel; Kocherscheidt, Lars; Agossou, Abram; Banla, Meba; Bonin, Michael; Köhler, Carsten

    2016-05-01

    Prenatal exposure to parasite antigens or allergens will influence the profile and strength of postnatal immune responses, such contact may tolerize and increase susceptibility to future infections or sensitize to environmental allergens. Exposure in utero to parasite antigens will distinctly alter cellular gene expression in newborns. Gene microarrays were applied to study gene expression in umbilical cord blood cell (UCBC) from parasite-exposed (Para-POS) and non-exposed (Para-NEG) neonates. UCBC were activated with antigens of helminth (Onchocerca volvulus), amoeba (Entamoeba histolytica) or allergens of mite (Dermatophagoides farinae). When UCBC from Para-POS and Para-NEG newborns were exposed to helminth antigens or allergens consistent differences occurred in the expression of genes encoding for MHC class I and II alleles, signal transducers of activation and transcription (STATs), cytokines, chemokines, immunoglobulin heavy and light chains, and molecules associated with immune regulation (SOCS, TLR, TGF), inflammation (TNF, CCR) and apoptosis (CASP). Expression of genes associated with innate immune responses were enhanced in Para-NEG, while in Para-POS, the expression of MHC class II and STAT genes was reduced. Within functional gene networks for cellular growth, proliferation and immune responses, Para-NEG neonates presented with significantly higher expression values than Para-POS. In Para-NEG newborns, the gene cluster and pathway analyses suggested that gene expression profiles may predispose for the development of immunological, hematological and dermatological disorders upon postnatal helminth parasite infection or allergen exposure. Thus, prenatal parasite contact will sensitize without generating aberrant inflammatory immune responses, and increased pro-inflammatory but decreased regulatory gene expression profiles will be present in those neonates lacking prenatal parasite antigen encounter. PMID:27062712

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  19. Population structuring of multi-copy, antigen-encoding genes in Plasmodium falciparum

    PubMed Central

    Artzy-Randrup, Yael; Rorick, Mary M; Day, Karen; Chen, Donald; Dobson, Andrew P; Pascual, Mercedes

    2012-01-01

    The coexistence of multiple independently circulating strains in pathogen populations that undergo sexual recombination is a central question of epidemiology with profound implications for control. An agent-based model is developed that extends earlier ‘strain theory’ by addressing the var gene family of Plasmodium falciparum. The model explicitly considers the extensive diversity of multi-copy genes that undergo antigenic variation via sequential, mutually exclusive expression. It tracks the dynamics of all unique var repertoires in a population of hosts, and shows that even under high levels of sexual recombination, strain competition mediated through cross-immunity structures the parasite population into a subset of coexisting dominant repertoires of var genes whose degree of antigenic overlap depends on transmission intensity. Empirical comparison of patterns of genetic variation at antigenic and neutral sites supports this role for immune selection in structuring parasite diversity. DOI: http://dx.doi.org/10.7554/eLife.00093.001 PMID:23251784

  20. Rearrangement and expression of T cell antigen receptor and gamma genes during thymic development

    PubMed Central

    1986-01-01

    Rearrangement and expression of the T cell antigen receptor and the gamma genes during T cell ontogeny is a regulated process; the gamma genes are rearranged and expressed first, followed by the beta and then the alpha genes. Expression of both functional alpha and beta gene RNA first occurs at day 17 of gestation, along with the expression of T3 delta chain RNA. T cell antigen receptor gene rearrangements occur primarily or exclusively in the thymus, although some gamma gene rearrangements occur outside the thymus in fetal liver cells that may be committed T cell progenitors. There is no gross difference in the extent of beta and gamma gene rearrangements in the adult thymocyte subpopulations that were analyzed, despite the fact that some of these populations cannot respond to antigen and never emigrate from the thymus. Quantitative analysis of rearrangements in total adult thymocyte DNA shows that beta gene rearrangements generally occur on both chromosomal homologs, and that rearrangements occur preferentially to the J beta 2 gene segment cluster. PMID:3487610

  1. Structure and gene cluster of the O-antigen of Escherichia coli O140.

    PubMed

    Perepelov, Andrei V; Wang, Quan; Senchenkova, Sof'ya N; Mei, Zhu; Shashkov, Alexander S; Wang, Lei; Knirel, Yuriy A

    2015-06-26

    An acidic O-polysaccharide (O-antigen) was isolated from the lipopolysaccharide of Escherichia coli O140 and studied by sugar analysis along with 1D and 2D (1)H and (13)C NMR spectroscopy. The following structure of the branched hexasaccharide repeating unit was established: [Formula: see text]. The O-antigen gene cluster of E. coli O140 was sequenced. The gene functions were tentatively assigned by a comparison with sequences in the available databases and found to be in full agreement with the E. coli O140 polysaccharide structure.

  2. Structure and gene cluster of the O-antigen of Escherichia coli O43.

    PubMed

    Perepelov, Andrei V; Guo, Xi; Filatov, Andrei V; Liu, Bin; Knirel, Yuriy A

    2015-10-30

    The O-polysaccharide (O-antigen) of Escherichia coli O43 was isolated from the lipopolysaccharide and studied by chemical methods, including sugar analyses, Smith degradation, and solvolysis with anhydrous trifluoroacetic acid, along with (1)H and (13)C NMR spectroscopy. The following structure of the pentasaccharide repeating unit of the O-polysaccharide was established: [Formula: see text] Functions of genes in the O-antigen gene cluster of E. coli O43 were assigned by a comparison with sequences in the available databases and found to be in agreement with the O-polysaccharide structure.

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

    PubMed

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

    2016-08-01

    Antigen uptake is a critical process for activation of the immune system, and therefore the ability to enhance antigen uptake is a primary consideration in the development of an immersion vaccination of fish. In the present work, flounders (Paralichthys olivaceus) were immersed in three hyperosmotic solutions with 40, 50 and 60‰ salinities, then transferred into seawater of normal salinity (i.e. 30‰) containing formalin-inactivated Edwardsiella tarda for 30 min. The antigen uptake in vaccinated flounder was determined using an absolute quantitative PCR (qPCR). The results showed significantly higher antigen uptake in the tissues of flounders immersed in solutions with 50‰ and 60‰ salinity compared to the control group directly immersed in vaccine (DI) (P < 0.05), and the highest amount of antigen was detected in flounders immersed in the 50‰ salinity solution, whereas there was no significant difference in antigen uptake between the 40‰ salinity group and the DI group (P > 0.05). A rapid and significant increase in antigen uptake was detected in the mucosal-associated tissues including the gill, skin and intestine (P < 0.05) compared with the spleen, kidney and liver. Antigen uptake in the gill and skin both peaked at 30 min post immersion, which was significantly higher than the levels of uptake measured in the other tissues (P < 0.05), and then quickly declined. In contrast, antigen uptake in the spleen, kidney and liver gradually increased 3 h post immersion (hpi). The expression profiles of four antigen presentation-related immune genes (MHC Iα, MHC IIα, CD4-1 and CD8α) were investigated after immersion. These four genes showed a significantly stronger response in the immersed flounders exposed to 50‰ salinity compared with the DI group (P < 0.05). In the mucosal-associated tissues, the expression of MHC Iα and CD8α genes peaked at 24 hpi, while the expression of MHC IIα and CD4-1 genes showed up-regulation in the gill and skin

  4. An evolutionarily mobile antigen receptor variable region gene: doubly rearranging NAR-TcR genes in sharks.

    PubMed

    Criscitiello, Michael F; Saltis, Mark; Flajnik, Martin F

    2006-03-28

    Distinctive Ig and T cell receptor (TcR) chains define the two major lineages of vertebrate lymphocyte yet similarly recognize antigen with a single, membrane-distal variable (V) domain. Here we describe the first antigen receptor chain that employs two V domains, which are generated by separate VDJ gene rearrangement events. These molecules have specialized "supportive" TcRdeltaV domains membrane-proximal to domains with most similarity to IgNAR V. The ancestral NAR V gene encoding this domain is hypothesized to have recombined with the TRD locus in a cartilaginous fish ancestor >200 million years ago and encodes the first V domain shown to be used in both Igs and TcRs. Furthermore, these data support the view that gamma/delta TcRs have for long used structural conformations recognizing free antigen.

  5. An evolutionarily mobile antigen receptor variable region gene: Doubly rearranging NAR-TcR genes in sharks

    PubMed Central

    Criscitiello, Michael F.; Saltis, Mark; Flajnik, Martin F.

    2006-01-01

    Distinctive Ig and T cell receptor (TcR) chains define the two major lineages of vertebrate lymphocyte yet similarly recognize antigen with a single, membrane-distal variable (V) domain. Here we describe the first antigen receptor chain that employs two V domains, which are generated by separate VDJ gene rearrangement events. These molecules have specialized “supportive” TcRδV domains membrane-proximal to domains with most similarity to IgNAR V. The ancestral NAR V gene encoding this domain is hypothesized to have recombined with the TRD locus in a cartilaginous fish ancestor >200 million years ago and encodes the first V domain shown to be used in both Igs and TcRs. Furthermore, these data support the view that γ/δ TcRs have for long used structural conformations recognizing free antigen. PMID:16549799

  6. Structure and gene cluster of the O-antigen of Escherichia coli O137.

    PubMed

    Perepelov, Andrei V; Guo, Xi; Senchenkova, Sof'ya N; Li, Yayue; Shashkov, Alexander S; Liu, Bin; Knirel, Yuriy A

    2016-03-01

    The O-polysaccharide (O-antigen) was isolated from the lipopolysaccharide of Escherichia coli O137 and studied by sugar analysis and NMR spectroscopy. The following structure of the branched tetrasaccharide repeating unit was established: Formula: see text] Both structure and gene cluster of the E. coli O137 polysaccharide are related to those of the E. coli K40 polysaccharide (Amor et al., 1999), which lacks the side-chain glucosylation but contains serine that is amide-linked to GlcA. Functions of genes in the O137-antigen gene cluster were assigned by a comparison with those in K40 and sequences in the available databases. Particularly, predicted glycosyltransferases encoded in the gene cluster were assigned to the formation of three glycosidic linkages in the O-polysaccharide repeating unit.

  7. An antigenic protein gene of a phytoplasma associated with sweet potato witches' broom.

    PubMed

    Yu, Y L; Yeh, K W; Lin, C P

    1998-05-01

    A gene encoding the major antigenic protein of phytoplasma associated with sweet potato witches' broom (SPWB) was cloned and analysed by screening the genomic library of SPWB phytoplasma with monoclonal antibodies for SPWB phytoplasma. The entire predicted structural gene encoded an antigenic protein composed of 172 amino acids with a computed molecular mass of 19.15 kDa and a pl value of 9.78. The -10 region of the promoter and the terminator region of the gene were identified and found to be similar to those of prokaryotes. The hydropathy profile of the deduced amino acid sequence consisted of two distinct regions, a strongly hydrophobic N-terminus and a highly hydrophilic C-terminus. This major antigenic protein was also present in phytoplasma associated with peanut witches' broom (PNWB) and the two showed homology based on the results of Western blot analysis, Southern hybridization, Northern hybridization, primer extension analysis and PCR. The homologous genes of the antigenic protein of SPWB phytoplasma and PNWB phytoplasma were not found in other phytoplasmas tested.

  8. MOLECULAR CHARACTERIZATION OF SWINE LEUKOCYTE ANTIGEN (SLA) CLASS I GENES IN OUTBRED PIG POPULATIONS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The highly polymorphic swine leukocyte antigen (SLA) genes are one of the most important determinants in swine immune responses to infectious diseases, vaccines, and in transplantation. Study of SLA influence requires accurate and effective typing methods. We developed a simple and rapid method to t...

  9. Evolutionary origin and human-specific expansion of a cancer/testis antigen gene family.

    PubMed

    Zhang, Qu; Su, Bing

    2014-09-01

    Cancer/testis (CT) antigens are encoded by germline genes and are aberrantly expressed in a number of human cancers. Interestingly, CT antigens are frequently involved in gene families that are highly expressed in germ cells. Here, we presented an evolutionary analysis of the CTAGE (cutaneous T-cell-lymphoma-associated antigen) gene family to delineate its molecular history and functional significance during primate evolution. Comparisons among human, chimpanzee, gorilla, orangutan, macaque, marmoset, and other mammals show a rapid and primate specific expansion of CTAGE family, which starts with an ancestral retroposition in the haplorhini ancestor. Subsequent DNA-based duplications lead to the prosperity of single-exon CTAGE copies in catarrhines, especially in humans. Positive selection was identified on the single-exon copies in comparison with functional constraint on the multiexon copies. Further sequence analysis suggests that the newly derived CTAGE genes may obtain regulatory elements from long terminal repeats. Our result indicates the dynamic evolution of primate genomes, and the recent expansion of this CT antigen family in humans may confer advantageous phenotypic traits during early human evolution.

  10. The Yersinia kristensenii O11 O-antigen gene cluster was acquired by lateral gene transfer and incorporated at a novel chromosomal locus.

    PubMed

    Cunneen, Monica M; Reeves, Peter R

    2007-06-01

    We have sequenced the O-antigen gene clusters for the Escherichia coli O98 and Yersinia kristensenii O11 O antigens. The basic structures of these O antigens are identical, and the sequence data indicate that Y. kristensenii O11 gained its O-antigen gene cluster by lateral gene transfer (LGT). Escherichia coli O98 has a typical O-antigen gene cluster between galF and gnd as is usual in E. coli. However, the O-antigen gene cluster of Y. kristensenii O11 is not located at the traditional Yersinia O-antigen gene cluster locus, between hemH and gsk, but at a novel chromosomal locus between aroA and cmk where it is flanked by remnant galF and gnd genes that indicate the probable source of the gene cluster. Phylogenetic analysis indicated that the source was not E. coli itself but a species in the Escherichia, Salmonella, and Klebsiella group of genera. Although other O-antigen studies imply LGT on the basis of the hypervariability of the loci and GC content, this report also identifies a potential donor and provides evidence for the mechanism involved. Remnant insertion sequence (IS) sequences flank the galF and gnd remnants and suggest that LGT of the gene cluster was IS mediated.

  11. Alternative haplotypes of antigen processing genes in zebrafish diverged early in vertebrate evolution.

    PubMed

    McConnell, Sean C; Hernandez, Kyle M; Wcisel, Dustin J; Kettleborough, Ross N; Stemple, Derek L; Yoder, Jeffrey A; Andrade, Jorge; de Jong, Jill L O

    2016-08-23

    Antigen processing and presentation genes found within the MHC are among the most highly polymorphic genes of vertebrate genomes, providing populations with diverse immune responses to a wide array of pathogens. Here, we describe transcriptome, exome, and whole-genome sequencing of clonal zebrafish, uncovering the most extensive diversity within the antigen processing and presentation genes of any species yet examined. Our CG2 clonal zebrafish assembly provides genomic context within a remarkably divergent haplotype of the core MHC region on chromosome 19 for six expressed genes not found in the zebrafish reference genome: mhc1uga, proteasome-β 9b (psmb9b), psmb8f, and previously unknown genes psmb13b, tap2d, and tap2e We identify ancient lineages for Psmb13 within a proteasome branch previously thought to be monomorphic and provide evidence of substantial lineage diversity within each of three major trifurcations of catalytic-type proteasome subunits in vertebrates: Psmb5/Psmb8/Psmb11, Psmb6/Psmb9/Psmb12, and Psmb7/Psmb10/Psmb13. Strikingly, nearby tap2 and MHC class I genes also retain ancient sequence lineages, indicating that alternative lineages may have been preserved throughout the entire MHC pathway since early diversification of the adaptive immune system ∼500 Mya. Furthermore, polymorphisms within the three MHC pathway steps (antigen cleavage, transport, and presentation) are each predicted to alter peptide specificity. Lastly, comparative analysis shows that antigen processing gene diversity is far more extensive than previously realized (with ancient coelacanth psmb8 lineages, shark psmb13, and tap2t and psmb10 outside the teleost MHC), implying distinct immune functions and conserved roles in shaping MHC pathway evolution throughout vertebrates. PMID:27493218

  12. Network-based gene expression analysis of intracranial aneurysm tissue reveals role of antigen presenting cells.

    PubMed

    Krischek, B; Kasuya, H; Tajima, A; Akagawa, H; Sasaki, T; Yoneyama, T; Ujiie, H; Kubo, O; Bonin, M; Takakura, K; Hori, T; Inoue, I

    2008-07-17

    Little is known about the pathology and pathogenesis of the rupture of intracranial aneurysms. For a better understanding of the molecular processes involved in intracranial aneurysm (IA) formation we performed a gene expression analysis comparing ruptured and unruptured aneurysm tissue to a control artery. Tissue samples of six ruptured and four unruptured aneurysms, and four cerebral arteries serving as controls, were profiled using oligonucleotide microarrays. Gene ontology classification of the differentially expressed genes was analyzed and regulatory functional networks and canonical pathways were identified with a network-based computational pathway analysis tool. Real time reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical staining were performed as confirmation. Analysis of aneurysmal and control tissue revealed 521 differentially expressed genes. The most significantly associated gene ontology term was antigen processing (P=1.64E-16). Further network-based analysis showed the top scoring regulatory functional network to be built around overexpressed major histocompatibility class (MHC) I and II complex related genes and confirmed the canonical pathway "Antigen Presentation" to have the highest upregulation in IA tissue (P=7.3E-10). Real time RT-PCR showed significant overexpression of MHC class II genes. Immunohistochemical staining showed strong positivity for MHC II molecule specific antibody (HLA II), for CD68 (macrophages, monocytes), for CD45RO (T-cells) and HLA I antibody. Our results offer strong evidence for MHC class II gene overexpression in human IA tissue and that antigen presenting cells (macrophages, monocytes) play a key role in IA formation. PMID:18538937

  13. A novel mechanism for control of antigenic variation in the haemagglutinin gene family of mycoplasma synoviae.

    PubMed

    Noormohammadi, A H; Markham, P F; Kanci, A; Whithear, K G; Browning, G F

    2000-02-01

    High-frequency phase and antigenic variation of homologous lipoprotein haemagglutinins has been seen in both the major avian mycoplasma pathogens, Mycoplasma synoviae and Mycoplasma gallisepticum. The expression and, hence, antigenic variation of the pMGA gene family (encoding these lipoproteins in M. gallisepticum) is controlled by variation in the length of a trinucleotide repeat motif 5' to the promoter of each gene. However, such a mechanism was not detected in preliminary observations on M. synoviae. Thus, the basis for control of variation in the vlhA gene family (which encodes the homologous haemagglutinin in M. synoviae) was investigated to enable comparison with its homologue in M. gallisepticum and with other lipoprotein gene families in mycoplasmas. The start point of transcription was identified 119 bp upstream of the initiation codon, but features associated with control of transcription in other mycoplasma lipoprotein genes were not seen. Comparison of three copies of vlhA revealed considerable sequence divergence at the 3' end of the gene, but conservation of the 5' end. Southern blot analysis of M. synoviae genomic DNA revealed that the promoter region and part of the conserved 5' coding sequence occurred as a single copy, whereas the remainder of the coding sequence occurred as multiple copies. A 9.7 kb fragment of the genome was found to contain eight tandemly repeated regions partially homologous to vlhA, all lacking the putative promoter region and the single-copy 5' end of vlhA, but extending over one of four distinct overlapping regions of the 3' coding sequence. Examination of sequential clones of M. synoviae established that unidirectional recombination occurs between the pseudogenes and the expressed vlhA, with duplication of pseudogene sequence and loss of the corresponding region previously seen in the expressed gene. Expression of the 5' end of two variants of the vlhA gene showed that they differed in their reaction with monoclonal

  14. Structure and gene cluster of the o-antigen of Escherichia coli o96.

    PubMed

    Guo, Xi; Senchenkova, Sof'ya N; Shashkov, Alexander S; Perepelov, Andrei V; Liu, Bin; Knirel, Yuriy A

    2016-02-01

    Mild acid degradation of the lipopolysaccharide of Escherichia coli O96 afforded a mixture of two polysaccharides. The following structure of the pentasaccharide repeating unit of the major polymer was established by sugar analysis, Smith degradation, and (1)H and (13)C NMR spectroscopy: [Formula: see text]. The O-antigen gene cluster of E. coli O96 between conserved galF and gnd genes was found to be consistent with this structure, and hence, the major polysaccharide represents the O96-antigen. The O96-antigen structure and gene cluster are similar to those of E. coli O170, and two proteins encoded in the gene clusters of both bacteria were putatively assigned a function of galactofuranosyltransferases. The minor polymer has the same structure as a peptidoglycan-related polysaccharide reported earlier in Providencia alcalifeciens O45 and several other O-serogoups of this species (Ovchinnikova OG, Liu B, Kocharova NA, Shashkov AS, Kondakova AN, Siwinska M, Feng L, Rozalski A, Wang L, Knirel YA. Biochemistry (Moscow) 2012;77:609-15) → 4)-β-D-GlcpNAc-(1 → 4)-β-D-GlcpNAc3(Rlac-lAla)-(1 → where Rlac-lAla indicates (R)-1-[(S)-1-carboxyethylaminocarbonyl]ethyl.

  15. Comparison of Z and R3 antigen expression and of genes encoding other antigenic markers in invasive human and bovine Streptococcus agalactiae strains from Norway.

    PubMed

    Maeland, Johan A; Radtke, Andreas

    2013-12-27

    Streptococcus agalactiae (GBS) may cause a variety of infectious diseases in humans caused by human GBS and mastitis in cattle caused by bovine GBS. Over the last few years molecular testing has provided evidence that human and bovine GBS have evolved along diverse phylogenetic lines. In the present study 173 invasive human GBS strains and 52 invasive bovine strains were tested for altogether 18 strain-variable and surface-localized antigenic markers including all 10 capsular polysaccharides (CPS) and proteins including Cβ, the alpha-like proteins, R3 and the recently described Z1 and Z2 antigens. PCR was used to detect encoding genes and antibody-based methods to detect expression of antigens. Thirteen of the 18 markers were detected in isolates of both strain categories. Seven of the ten CPS antigens were detected in both groups with types III and V predominating in the human GBS strains, types IV and V in the bovine isolates. Z1, Z2 and/or R3 expression and the genes encoding Cβ, Cα, Alp1, Alp2/3 or R4 (Rib) were detected in both groups. Protein antigen-CPS associations well known for human strains were essentially the same in the bovine isolates. The results show that in spite of evolution along different lines, human and bovine GBS share a variety of surface-exposed antigenic markers, substantiating close relationship between the two GBS subpopulations. PMID:24120184

  16. 5'-structural analysis of genes encoding polymorphic antigens of chemically induced tumors

    SciTech Connect

    Srivastava, R.K.; Chen, Y.T.; Old, L.J.

    1987-06-01

    The authors have proposed that the distinct tumor rejection antigens of chemically induced sarcomas in inbred mice belong to a family of M/sub r/ 96,000 glycoproteins (gp96). An identical 14-amino acid sequence was found at the amino terminus of gp96 from two antigenically distinct BALB/c sarcomas. Oligonucleotide probes, end-labeled with (/sup 12/P)-ATP, derived from this sequence permitted isolation of 5' cDNA and genomic fragments coding for gp96. Three short exons interrupted by relatively long introns were identified at the 5' terminus of the gp96 gene. The first exon encodes a signal peptide, which is consistent with gp96 being a cell surface antigen. Southern blot analysis indicated that the gp96 family is encoded by a single gene, and 3-kilobase transcripts were detected in all normal and tumor cells tested. Nucleotide and deduced amino acid sequences from 311 base pairs at the 5' terminus showed no homology with any know protein. The availability of molecular probes for the gp96 system permits analysis of the structural polymorphism of these antigens.

  17. Genetic diversity and population structure of genes encoding vaccine candidate antigens of Plasmodium vivax

    PubMed Central

    2012-01-01

    Background A major concern in malaria vaccine development is genetic polymorphisms typically observed among Plasmodium isolates in different geographical areas across the world. Highly polymorphic regions have been observed in Plasmodium falciparum and Plasmodium vivax antigenic surface proteins such as Circumsporozoite protein (CSP), Duffy-binding protein (DBP), Merozoite surface protein-1 (MSP-1), Apical membrane antigen-1 (AMA-1) and Thrombospondin related anonymous protein (TRAP). Methods Genetic variability was assessed in important polymorphic regions of various vaccine candidate antigens in P. vivax among 106 isolates from the Amazon Region of Loreto, Peru. In addition, genetic diversity determined in Peruvian isolates was compared to population studies from various geographical locations worldwide. Results The structured diversity found in P. vivax populations did not show a geographic pattern and haplotypes from all gene candidates were distributed worldwide. In addition, evidence of balancing selection was found in polymorphic regions of the trap, dbp and ama-1 genes. Conclusions It is important to have a good representation of the haplotypes circulating worldwide when implementing a vaccine, regardless of the geographic region of deployment since selective pressure plays an important role in structuring antigen diversity. PMID:22417572

  18. Molecular Characterization of Cronobacter Lipopolysaccharide O-Antigen Gene Clusters and Development of Serotype-Specific PCR Assays ▿

    PubMed Central

    Jarvis, K. G.; Grim, C. J.; Franco, A. A.; Gopinath, G.; Sathyamoorthy, V.; Hu, L.; Sadowski, J. A.; Lee, C. S.; Tall, B. D.

    2011-01-01

    Cronobacter (formerly Enterobacter sakazakii) is a recently defined genus consisting of six species, C. sakazakii, C. malonaticus, C. dublinensis, C. muytjensii, C. turicensis, and Cronobacter genomospecies 1. In this study, MboII restriction fragment length polymorphism (RFLP) patterns of O-antigen gene clusters, located between galF and gnd, were used to identify serotypes in Cronobacter spp. Seven O-antigen RFLP clusters were generated, including three C. sakazakii clusters, previously identified as serotypes O1, O2, and O3. The O-antigen regions of six strains with unique RFLP patterns, including two C. sakazakii strains, two C. malonaticus strains, one C. turicensis strain, and one C. muytjensii strain, revealed three O-antigen gene clusters shared among Cronobacter species. PCR assays were developed, targeting the wzx O-antigen polymerase gene, and used to screen 231 Cronobacter strains to determine the frequency of these newly identified serotypes. PMID:21531829

  19. Induction of interferon-stimulated genes by Simian virus 40 T antigens

    SciTech Connect

    Rathi, Abhilasha V.; Cantalupo, Paul G.; Sarkar, Saumendra N.; Pipas, James M.

    2010-10-25

    Simian virus 40 (SV40) large T antigen (TAg) is a multifunctional oncoprotein essential for productive viral infection and for cellular transformation. We have used microarray analysis to examine the global changes in cellular gene expression induced by wild-type T antigen (TAg{sup wt}) and TAg-mutants in mouse embryo fibroblasts (MEFs). The expression profile of approximately 800 cellular genes was altered by TAg{sup wt} and a truncated TAg (TAg{sup N136}), including many genes that influence cell cycle, DNA-replication, transcription, chromatin structure and DNA repair. Unexpectedly, we found a significant number of immune response genes upregulated by TAg{sup wt} including many interferon-stimulated genes (ISGs) such as ISG56, OAS, Rsad2, Ifi27 and Mx1. Additionally, we also observed activation of STAT1 by TAg{sup wt}. Our genetic studies using several TAg-mutants reveal an unexplored function of TAg and indicate that the LXCXE motif and p53 binding are required for the upregulation of ISGs.

  20. Identification and characterization of Tu88, an antigenic gene from Theileria uilenbergi.

    PubMed

    Lu, Yizhu; Wang, Yuefeng; Li, Youquan; Gou, Huitian; Luo, Jianxun; Yin, Hong; Liu, Zhijie

    2015-06-01

    Theileria uilenbergi is a pathogen that causes ovine theileriosis. Prevention and control of theileriosis relies on its diagnosis at early stages of occurrence and requires understanding of proteins with antigenic properties from the pathogen. Despite its prevalence in China, only a few molecules with antigenic properties have been characterized from T. uilenbergi. In this study, we identified a cDNA named Tu88 by immunoscreening a T. uilenbergi merozoite cDNA library with T. uilenbergi-positive sera from infected sheep. Recombinant Tu88 (rTu88) expressed in bacteria reacted strongly with the positive sera of T. uilenbergi in western blot analysis indicating its potential as an antigen. Southern blot analysis showed that it is a single copy gene. Protein localization by immunostaining blood smears from an infected sheep demonstrated the presence of native Tu88 in merozoites. These findings suggest that Tu88 is a potential candidate antigen for the development of a sero-diagnostic tool.

  1. Cloning and disruption of the antigenic catalase gene of Aspergillus fumigatus.

    PubMed Central

    Calera, J A; Paris, S; Monod, M; Hamilton, A J; Debeaupuis, J P; Diaquin, M; López-Medrano, R; Leal, F; Latgé, J P

    1997-01-01

    Aspergillus fumigatus possesses two catalases (described as fast and slow on the basis of their electrophoretic mobility). The slow catalase has been recognized as a diagnostic antigen for aspergillosis in immunocompetent patients. The antigenic catalase has been purified. The enzyme is a tetrameric protein composed of 90-kDa subunits. The corresponding cat1 gene was cloned, and sequencing data show that the cat1 gene codes for a 728-amino-acid polypeptide. A recombinant protein expressed in Pichia pastoris is enzymatically active and has biochemical and antigenic properties that are similar to those of the wild-type catalase. Molecular experiments reveal that CAT1 contains a signal peptide and a propeptide of 15 and 12 amino acid residues, respectively. cat1-disrupted mutants that were unable to produce the slow catalase were as sensitive to H2O2 and polymorphonuclear cells as the wild-type strain. In addition, there was no difference in pathogenicity between the cat1 mutant and its parental cat1+ strain in a murine model of aspergillosis. PMID:9353056

  2. Mathematical analysis of antigen selection in somatically mutated immunoglobulin genes associated with autoimmunity.

    PubMed

    MacDonald, C M; Boursier, L; D'Cruz, D P; Dunn-Walters, D K; Spencer, J

    2010-09-01

    Affinity maturation is a process by which low-affinity antibodies are transformed into highly specific antibodies in germinal centres. This process occurs by hypermutation of immunoglobulin heavy chain variable (IgH V) region genes followed by selection for high-affinity variants. It has been proposed that statistical tests can identify affinity maturation and antigen selection by analysing the frequency of replacement and silent mutations in the complementarity determining regions (CDRs) that contact antigen and the framework regions (FRs) that encode structural integrity. In this study three different methods that have been proposed for detecting selection: the binomial test, the multinomial test and the focused binomial test, have been assessed for their reliability and ability to detect selection in human IgH V genes. We observe first that no statistical test is able to identify selection in the CDR antigen-binding sites, second that tests can reliably detect selection in the FR and third that antibodies from nasal biopsies from patients with Wegener's granulomatosis and pathogenic antibodies from systemic lupus erythematosus do not appear to be as stringently selected for structural integrity as other groups of functional sequences.

  3. DNA methylation and nucleosome occupancy regulate the cancer germline antigen gene MAGEA11

    PubMed Central

    James, Smitha R; Cedeno, Carlos D; Sharma, Ashok; Zhang, Wa; Mohler, James L; Odunsi, Kunle; Wilson, Elizabeth M; Karpf, Adam R

    2013-01-01

    MAGEA11 is a cancer germline (CG) antigen and androgen receptor co-activator. Its expression in cancers other than prostate, and its mechanism of activation, has not been reported. In silico analyses reveal that MAGEA11 is frequently expressed in human cancers, is increased during tumor progression, and correlates with poor prognosis and survival. In prostate and epithelial ovarian cancers (EOC), MAGEA11 expression was associated with promoter and global DNA hypomethylation, and with activation of other CG genes. Pharmacological or genetic inhibition of DNA methyltransferases (DNMTs) and/or histone deacetylases (HDACs) activated MAGEA11 in a cell line specific manner. MAGEA11 promoter activity was directly repressed by DNA methylation, and partially depended on Sp1, as pharmacological or genetic targeting of Sp1 reduced MAGEA11 promoter activity and endogenous gene expression. Importantly, DNA methylation regulated nucleosome occupancy specifically at the -1 positioned nucleosome of MAGEA11. Methylation of a single Ets site near the transcriptional start site (TSS) correlated with -1 nucleosome occupancy and, by itself, strongly repressed MAGEA11 promoter activity. Thus, DNA methylation regulates nucleosome occupancy at MAGEA11, and this appears to function cooperatively with sequence-specific transcription factors to regulate gene expression. MAGEA11 regulation is highly instructive for understanding mechanisms regulating CG antigen genes in human cancer. PMID:23839233

  4. Difference in number of loci of swine leukocyte antigen classical class I genes among haplotypes.

    PubMed

    Tanaka-Matsuda, Maiko; Ando, Asako; Rogel-Gaillard, Claire; Chardon, Patrick; Uenishi, Hirohide

    2009-03-01

    The structure of the entire genomic region of swine leukocyte antigen (SLA)-the porcine major histocompatibility complex--was recently elucidated in a particular haplotype named Hp-1.0 (H01). However, it has been suggested that there are differences in the number of loci of SLA genes, particularly classical class I genes, among haplotypes. To clarify the between-haplotype copy number variance in genes of the SLA region, we sequenced the genomic region carrying SLA classical class I genes on two different haplotypes, revealing increments of up to six in the number of classical class I genes in a single haplotype. All of the SLA-1(-like) (SLA-1 and newly designated SLA-12) and SLA-3 genes detected in the haplotypes thus analyzed were transcribed in the individual. The process by which duplication of SLA classical class I genes was likely to have occurred was interpreted from an analysis of repetitive sequences adjacent to the duplicated class I genes.

  5. Expression of complete transplantation antigens by mammalian cells transformed with truncated class I genes.

    PubMed

    Goodenow, R S; Stroynowski, I; McMillan, M; Nicolson, M; Eakle, K; Sher, B T; Davidson, N; Hood, L

    1983-02-01

    Mouse L cells transformed with the cloned class I genes of the major histocompatibility complex of the mouse express transplantation antigens with serological determinants of the donor haplotype. However, transformation with the truncated subclones of a BALB/c H-2Ld gene containing the exons encoding the external domains also leads to the production of cells which express complete cell-surface molecules. Moreover, full-length products of the foreign haplotype, as judged by serological and biochemical criteria, are generated independently of the use of carrier DNA in transformation. However, the frequency of productive transformation is substantially less than that obtained with a complete gene. The most plausible explanation for these phenomena involves homologous recombination between host chromosomal and donor class I sequences. PMID:6823314

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

    PubMed Central

    Jirholt, Pernilla; Turesson, Olof; Wing, Kajsa; Holmdahl, Rikard; Kihlberg, Jan; Stern, Anna; Mårtensson, Inga-Lill; Henningsson, Louise; Gustafsson, Kenth; Gjertsson, Inger

    2016-01-01

    Here, we investigate induction of immunological tolerance by lentiviral based gene therapy in a mouse model of rheumatoid arthritis, collagen II-induced arthritis (CIA). Targeting the expression of the collagen type II (CII) to antigen presenting cells (APCs) induced antigen-specific tolerance, where only 5% of the mice developed arthritis as compared with 95% of the control mice. In the CII-tolerized mice, the proportion of Tregs as well as mRNA expression of SOCS1 (suppressors of cytokine signaling 1) increased at day 3 after CII immunization. Transfer of B cells or non-B cell APC, as well as T cells, from tolerized to naïve mice all mediated a certain degree of tolerance. Thus, sustainable tolerance is established very early during the course of arthritis and is mediated by both B and non-B cells as APCs. This novel approach for inducing tolerance to disease specific antigens can be used for studying tolerance mechanisms, not only in CIA but also in other autoimmune diseases. PMID:27159398

  7. Transcription analysis, physical mapping, and molecular characterization of a nonclassical human leukocyte antigen class I gene.

    PubMed Central

    Chorney, M J; Sawada, I; Gillespie, G A; Srivastava, R; Pan, J; Weissman, S M

    1990-01-01

    The human major histocompatibility complex contains approximately 20 class I genes, pseudogenes, and gene fragments. These include the genes for the three major transplantation antigens, HLA-A, HLA-B, and HLA-C, as well as a number of other genes or pseudogenes of unknown biological significance. Most of the latter have C + G-rich sequences in their 5' ends that are unmethylated in the B-lymphoblastoid cell line 3.1.0. We investigated one of these genes, HLA-H, in more detail. The gene is, overall, strongly homologous in sequence to HLA-A but differs in several potentially significant ways, including changes in conserved promoter sequences, a single-base deletion producing a translation termination codon in exon 4, and a region of sequence divergence downstream of the transcribed portion of the gene. Nevertheless, mouse L cells transfected with the gene accumulated small amounts of apparently full-length polyadenylated RNA. A portion of this RNA begins at the transcription site predicted by analogy to certain class I cDNA clones, while another portion appears to begin shortly upstream. L cells transfected with a hybrid gene containing the first three exons of HLA-H and the last five exons of HLA-B27 accumulated full-length HLA transcripts at the same level as cells transfected with an HLA-B27 gene; both levels are at least 15- to 20-fold higher than that directed by HLA-H alone. In addition, we isolated a cDNA clone for HLA-H that contains a portion of intron 3 attached to a normally spliced sequence comprising exons 4 through 8. These results suggest that low levels of translatable mRNA for the truncated class I heavy chain encoded by HLA-H are produced under physiologic circumstances and that sequences 3' of intron 3 decrease the levels of stable transcripts. Images PMID:2294403

  8. Repression of the Drosophila proliferating-cell nuclear antigen gene promoter by zerknuellt protein

    SciTech Connect

    Yamaguchi, Masamitsu; Hirose, Fumiko; Nishida, Yasuyoshi; Matsukage, Akio )

    1991-10-01

    A 631-bp fragment containing the 5{prime}-flanking region of the Drosophila melanogaster proliferating-cell nuclear antigen (PCNA) gene was placed upstream of the chloramphenicol acetyltransferase (CAT) gene of a CAT vector. A transient expression assay of CAT activity in Drosophila Kc cells transfected with this plasmid and a set of 5{prime}-deletion derivatives revealed that the promoter function resided within a 192-bp region. Cotransfection with a zerknuellt (zen)-expressing plasmid specifically repressed CAT expression. However, cotransfection with expression plasmids for a nonfunctional zen mutation, even skipped, or bicoid showed no significant effect on CAT expression. RNase protection analysis revealed that the repression by zen was at the transcription step. The target sequence of zen was mapped within the 34-bp region of the PCNA gene promoter, even though it lacked zen protein-binding sites. Transgenic flies carrying the PCNA gene regulatory region fused with lacZ were established. These results indicate that zen indirectly represses PCNA gene expression, probably by regulating the expression of some transcription factor(s) that binds to the PCNA gene promoter.

  9. Generation of Antigenic Diversity in Plasmodium falciparum by Structured Rearrangement of Var Genes During Mitosis

    PubMed Central

    Kekre, Mihir; Otto, Thomas D.; Faizullabhoy, Adnan; Rayner, Julian C.; Kwiatkowski, Dominic

    2014-01-01

    The most polymorphic gene family in P. falciparum is the ∼60 var genes distributed across parasite chromosomes, both in the subtelomeres and in internal regions. They encode hypervariable surface proteins known as P. falciparum erythrocyte membrane protein 1 (PfEMP1) that are critical for pathogenesis and immune evasion in Plasmodium falciparum. How var gene sequence diversity is generated is not currently completely understood. To address this, we constructed large clone trees and performed whole genome sequence analysis to study the generation of novel var gene sequences in asexually replicating parasites. While single nucleotide polymorphisms (SNPs) were scattered across the genome, structural variants (deletions, duplications, translocations) were focused in and around var genes, with considerable variation in frequency between strains. Analysis of more than 100 recombination events involving var exon 1 revealed that the average nucleotide sequence identity of two recombining exons was only 63% (range: 52.7–72.4%) yet the crossovers were error-free and occurred in such a way that the resulting sequence was in frame and domain architecture was preserved. Var exon 1, which encodes the immunologically exposed part of the protein, recombined in up to 0.2% of infected erythrocytes in vitro per life cycle. The high rate of var exon 1 recombination indicates that millions of new antigenic structures could potentially be generated each day in a single infected individual. We propose a model whereby var gene sequence polymorphism is mainly generated during the asexual part of the life cycle. PMID:25521112

  10. Plasmodium falciparum complicated malaria: Modulation and connectivity between exportome and variant surface antigen gene families.

    PubMed

    Subudhi, Amit Kumar; Boopathi, P A; Pandey, Isha; Kohli, Ramandeep; Karwa, Rohan; Middha, Sheetal; Acharya, Jyoti; Kochar, Sanjay K; Kochar, Dhanpat K; Das, Ashis

    2015-05-01

    In temperate and sub-tropical regions of Asia and Latin America, complicated malaria manifested as hepatic dysfunction or renal dysfunction is seen in all age groups. There has been a concerted focus on understanding the patho-physiological and molecular basis of complicated malaria in children, much less is known about it in adults. We report here, the analysis of data from a custom, cross strain microarray (Agilent Platform) using material from adult patient samples, showing hepatic dysfunction or renal failure. These are the most common manifestations seen in adults along with cerebral malaria. The data has been analyzed with reference to variant surface antigens, encoded by the var, rifin and stevor gene families. The differential regulation profiles of key genes (comparison between Plasmodium falciparum complicated and uncomplicated isolates) have been observed. The exportome has been analyzed using similar parameters. Gene ontology term based functional enrichment of differentially regulated genes identified, up-regulated genes statistically enriched (P<0.05) to critical biological processes like generation of precursor metabolite and energy, chromosome organization and electron transport chain. Systems network based functional enrichment of overall differentially regulated genes yielded a similar result. We are reporting here, up-regulation of var group B and C genes whose proteins are predicted to interact with CD36 receptor in the host, the up-regulation of domain cassette 13 (DC13) containing var group A, as also the up-regulation of group A rifins and many of the stevors. This is contrary to most other reports from pediatric patients, with cerebral malaria where the up-regulation of mostly var A group genes have been seen. A protein-protein interaction based network has been created and analysis performed. This co-expression and text mining based network has shown overall connectivity between the variant surface antigens (VSA) and the exportome. The up

  11. Epstein-Barr virus nuclear antigen 3C regulated genes in lymphoblastoid cell lines.

    PubMed

    Zhao, Bo; Mar, Jessica C; Maruo, Seiji; Lee, Sungwook; Gewurz, Benjamin E; Johannsen, Eric; Holton, Kristina; Rubio, Renee; Takada, Kenzo; Quackenbush, John; Kieff, Elliott

    2011-01-01

    EBV nuclear antigen 3C (EBNA3C) is an essential transcription factor for EBV transformed lymphoblast cell line (LCL) growth. To identify EBNA3C-regulated genes in LCLs, microarrays were used to measure RNA abundances in each of three different LCLs that conditionally express EBNA3C fused to a 4-OH-Tamoxifen-dependent estrogen receptor hormone binding domain (EBNA3CHT). At least three RNAs were assayed for each EBNA3CHT LCL under nonpermissive conditions, permissive conditions, and nonpermissive conditions with wild-type EBNA3C transcomplementation. Using a two-way ANOVA model of EBNA3C levels, we identified 550 regulated genes that were at least 1.5-fold up- or down-regulated with false discovery rates < 0.01. EBNA3C-regulated genes overlapped significantly with genes regulated by EBNA2 and EBNA3A consistent with coordinated effects on cell gene transcription. Of the 550 EBNA3C-regulated genes, 106 could be placed in protein networks. A seeded Bayesian network analysis of the 80 most significant EBNA3C-regulated genes suggests that RAC1, LYN, and TNF are upstream of other EBNA3C-regulated genes. Gene set enrichment analysis found enrichment for MAP kinase signaling, cytokine-cytokine receptor interactions, JAK-STAT signaling, and cell adhesion molecules, implicating these pathways in EBNA3C effects on LCL growth or survival. EBNA3C significantly up-regulated the CXCL12 ligand and its CXCR4 receptor and increased LCL migration. CXCL12 up-regulation depended on EBNA3C's interaction with the cell transcription factor, RBPJ, which is essential for LCL growth. EBNA3C also up-regulated MYC 1.3-fold and down-regulated CDKN2A exons 2 and 3, shared by p16 and p14, 1.4-fold, with false discovery rates < 5 × 10(-4).

  12. Plasmodium falciparum: genetic polymorphism in apical membrane antigen-1 gene from Indian isolates.

    PubMed

    Rajesh, Vidya; Singamsetti, Vijay Kumar; Vidya, S; Gowrishankar, M; Elamaran, M; Tripathi, Jyotsna; Radhika, N B; Kochar, Dhanpat; Ranjan, Akash; Roy, S K; Das, Ashis

    2008-05-01

    A number of stage-specific antigens have been characterized for vaccine development against Plasmodium falciparum malaria. This study presents a comprehensive analysis of the sequence polymorphism in Plasmodium falciparum apical membrane antigen-1 (PfAMA-1) in population samples from the eastern and western parts of India. This is the first study of its kind for the nearly full length PfAMA-1 gene from these regions in India. Our observations confirmed that sequence diversity of PfAMA-1 confines only to point mutations and shows 4-8% variation as compared to the prototypes. As opposed to the previous studies on PfAMA-1, our study revealed a greater degree of polymorphism in the Domain II region of PfAMA-1 protein, though signature for diversifying selection is seen throughout the gene. Our present investigation also indicates a very high degree of variation in the reported T- and B-cell epitopes of PfAMA-1. Few noteworthy and unique observations made in this study are the substitution of Cysteine residues responsible for the disulfide bond structure of the protein and the presence of premature termination after 595 amino acids in 3 of the 13 isolates under consideration. These crucial findings add new perspectives to the future of AMA-1 research and could have major implications in establishing AMA-1 as a vaccine candidate. PMID:18343371

  13. Molecular genotyping of human Ureaplasma species based on multiple-banded antigen (MBA) gene sequences.

    PubMed

    Kong, F; Ma, Z; James, G; Gordon, S; Gilbert, G L

    2000-09-01

    Ureaplasma urealyticum has been divided into 14 serovars. Recently, subdivision of U. urealyticum into two species has been proposed: U. parvum (previously U. urealyticum parvo biovar), comprising four serovars (1, 3, 6, 14) and U. urealyticum (previously U. urealyticum T-960 biovar), 10 serovars (2, 4, 5, 7-13). The multiple-banded antigen (MBA) genes of these species contain both species and serovar/subtype specific sequences. Based on whole sequences of the 5'-ends of MBA genes of U. parvum serovars and partial sequences of the 5'-ends of MBA genes of U. urealyticum serovars, we previously divided each of these species into three MBA genotypes. To further elucidate the relationships between serovars, we sequenced the whole 5'-ends of MBA genes of all 10 U. urealyticum serovars and partial repetitive regions of these genes from all serovars of U. parvum and U. urealyticum. For the first time, all four serovars of U. parvum were clearly differentiated from each other. In addition, the 10 serovars of U. urealyticum were divided into five MBA genotypes, as follows: MBA genotype A comprises serovars 2, 5, 8; MBA genotype B, serovar 10 only; MBA genotype C, serovars 4, 12, 13; MBA genotype D, serovar 9 only; and MBA genotype E comprises serovars 7 and 11. There were no sequence differences between members within each MBA genotype. Further work is required to identify other genes or other regions of the MBA genes that may be used to differentiate U. urealyticum serovars within MBA genotypes A, C and E. A better understanding of the molecular basis of serotype differentiation will help to improve subtyping methods for use in studies of the pathogenesis and epidemiology of these organisms.

  14. Cloning of the gene coding for a shared human melanoma antigen recognized by autologous T cells infiltrating into tumor.

    PubMed Central

    Kawakami, Y; Eliyahu, S; Delgado, C H; Robbins, P F; Rivoltini, L; Topalian, S L; Miki, T; Rosenberg, S A

    1994-01-01

    By cDNA expression cloning we have isolated a gene encoding a shared human melanoma antigen recognized by HLA-A2 restricted autologous and allogenic tumor-infiltrating lymphocytes (TILs) from patients with metastatic melanoma. By using both transient and stable expression systems, transfection of this gene into non-antigen-expressing HLA-A2+ cell lines resulted in recognition by the antigen-specific TILs. The sequence of this cDNA revealed a previously undescribed putative transmembrane protein whose expression was restricted to melanoma and melanocyte cell lines and human retina but no other fresh or cultured normal tissues tested or other tumor histologies. Thus, we have identified a gene encoding a melanocyte lineage-specific protein (MART-1; melanoma antigen recognized by T cells 1) that is a widely shared melanoma antigen recognized by the T lymphocytes of patients with established malignancy. Identification of this gene opens possibilities for the development of immunotherapies for patients with melanoma. PMID:8170938

  15. Characterization of the Aspergillus nidulans aspnd1 gene demonstrates that the ASPND1 antigen, which it encodes, and several Aspergillus fumigatus immunodominant antigens belong to the same family.

    PubMed Central

    Calera, J A; Ovejero, M C; López-Medrano, R; Segurado, M; Puente, P; Leal, F

    1997-01-01

    For the first time, an immunodominant Aspergillus nidulans antigen (ASPND1) consistently reactive with serum samples from aspergilloma patients has been purified and characterized, and its coding gene (aspnd1) has been cloned and sequenced. ASPND1 is a glycoprotein with four N-glycosidically-bound sugar chains (around 2.1 kDa each) which are not necessary for reactivity with immune human sera. The polypeptide part is synthesized as a 277-amino-acid precursor of 30.6 kDa that after cleavage of a putative signal peptide of 16 amino acids, affords a mature protein of 261 amino acids with a molecular mass of 29 kDa and a pI of 4.24 (as deduced from the sequence). The ASPND1 protein is 53.1% identical to the AspfII allergen from Aspergillus fumigatus and 48% identical to an unpublished Candida albicans antigen. All of the cysteine residues and most of the glycosylation sites are perfectly conserved in the three proteins, suggesting a similar but yet unknown function. Analysis of the primary structure of the ASPND1 coding gene (aspnd1) has allowed the establishment of a clear relationship between several previously reported A. fumigatus and A. nidulans immunodominant antigens. PMID:9119471

  16. Effect of Genetic Diversity in Swine Leukocyte Antigen-DRA Gene on Piglet Diarrhea.

    PubMed

    Huang, Xiaoyu; Yang, Qiaoli; Yuan, Junhu; Liu, Lixia; Sun, Wenyang; Jiang, Yingdi; Zhao, Shengguo; Zhang, Shengwei; Huang, Wangzhou; Gun, Shuangbao

    2016-07-15

    The swine leukocyte antigens (SLAs) are the multigene families related to immune responses. Little is known about the effect of the DRA gene on diarrheal disease. This study reported the genetic diversity of the DRA gene in exons 1, 3 and 4 in 290 Chinese Yantai black pigs. No variation was identified in exon 3. In exon 1, three genotypes and two alleles were identified, generated by two single nucleotide polymorphisms (SNPs). In exon 4, there were eight genotypes and five alleles containing seven SNPs were detected with four SNPs being novel SNPs. The low polymorphism found in swine DRA is consistent with the concept that the DRA gene is highly conserved among all mammalian species. Statistical analyses indicated that the genotypes of exon 1 were not significantly associated with piglet diarrhea (p > 0.05); however, genotypes C₄C₄ (1.80 ± 0.33) and A₄E₄ (1.66 ± 0.25) of exon 4 were significantly susceptible to diarrhea (p < 0.01). These indicate that the particular genotypes of the DRA gene are susceptible to diarrheal disease, which provides valuable information for disease-resistance breeding in swine.

  17. Effect of Genetic Diversity in Swine Leukocyte Antigen-DRA Gene on Piglet Diarrhea.

    PubMed

    Huang, Xiaoyu; Yang, Qiaoli; Yuan, Junhu; Liu, Lixia; Sun, Wenyang; Jiang, Yingdi; Zhao, Shengguo; Zhang, Shengwei; Huang, Wangzhou; Gun, Shuangbao

    2016-01-01

    The swine leukocyte antigens (SLAs) are the multigene families related to immune responses. Little is known about the effect of the DRA gene on diarrheal disease. This study reported the genetic diversity of the DRA gene in exons 1, 3 and 4 in 290 Chinese Yantai black pigs. No variation was identified in exon 3. In exon 1, three genotypes and two alleles were identified, generated by two single nucleotide polymorphisms (SNPs). In exon 4, there were eight genotypes and five alleles containing seven SNPs were detected with four SNPs being novel SNPs. The low polymorphism found in swine DRA is consistent with the concept that the DRA gene is highly conserved among all mammalian species. Statistical analyses indicated that the genotypes of exon 1 were not significantly associated with piglet diarrhea (p > 0.05); however, genotypes C₄C₄ (1.80 ± 0.33) and A₄E₄ (1.66 ± 0.25) of exon 4 were significantly susceptible to diarrhea (p < 0.01). These indicate that the particular genotypes of the DRA gene are susceptible to diarrheal disease, which provides valuable information for disease-resistance breeding in swine. PMID:27429004

  18. Effect of Genetic Diversity in Swine Leukocyte Antigen-DRA Gene on Piglet Diarrhea

    PubMed Central

    Huang, Xiaoyu; Yang, Qiaoli; Yuan, Junhu; Liu, Lixia; Sun, Wenyang; Jiang, Yingdi; Zhao, Shengguo; Zhang, Shengwei; Huang, Wangzhou; Gun, Shuangbao

    2016-01-01

    The swine leukocyte antigens (SLAs) are the multigene families related to immune responses. Little is known about the effect of the DRA gene on diarrheal disease. This study reported the genetic diversity of the DRA gene in exons 1, 3 and 4 in 290 Chinese Yantai black pigs. No variation was identified in exon 3. In exon 1, three genotypes and two alleles were identified, generated by two single nucleotide polymorphisms (SNPs). In exon 4, there were eight genotypes and five alleles containing seven SNPs were detected with four SNPs being novel SNPs. The low polymorphism found in swine DRA is consistent with the concept that the DRA gene is highly conserved among all mammalian species. Statistical analyses indicated that the genotypes of exon 1 were not significantly associated with piglet diarrhea (p > 0.05); however, genotypes C4C4 (1.80 ± 0.33) and A4E4 (1.66 ± 0.25) of exon 4 were significantly susceptible to diarrhea (p < 0.01). These indicate that the particular genotypes of the DRA gene are susceptible to diarrheal disease, which provides valuable information for disease-resistance breeding in swine. PMID:27429004

  19. Molecular Analysis of the O-Antigen Gene Cluster of Escherichia coli O86:B7 and Characterization of the Chain Length Determinant Gene (wzz)

    PubMed Central

    Guo, Hongjie; Yi, Wen; Shao, Jun; Lu, Yuquan; Zhang, Wenpeng; Song, Jing; Wang, Peng George

    2005-01-01

    Escherichia coli O86:B7 has long been used as a model bacterial strain to study the generation of natural blood group antibody in humans, and it has been shown to possess high human blood B activity. The O-antigen structure of O86:B7 was solved recently in our laboratory. Comparison with the published structure of O86:H2 showed that both O86 subtypes shared the same O unit, yet each of the O antigens is polymerized from a different terminal sugar in a different glycosidic linkage. To determine the genetic basis for the O-antigen differences between the two O86 strains, we report the complete sequence of O86:B7 O-antigen gene cluster between galF and hisI, each gene was identified based on homology to other genes in the GenBank databases. Comparison of the two O86 O-antigen gene clusters revealed that the encoding regions between galF and gnd are identical, including wzy genes. However, deletion of the two wzy genes revealed that wzy in O86:B7 is responsible for the polymerization of the O antigen, while the deletion of wzy in O86:H2 has no effect on O-antigen biosynthesis. Therefore, we proposed that there must be another functional wzy gene outside the O86:H2 O-antigen gene cluster. Wzz proteins determine the degree of polymerization of the O antigen. When separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the lipopolysaccharide (LPS) of O86:B7 exhibited a modal distribution of LPS bands with relatively short O units attached to lipid A-core, which differs from the LPS pattern of O86:H2. We proved that the wzz genes are responsible for the different LPS patterns found in the two O86 subtypes, and we also showed that the very short type of LPS is responsible for the serum sensitivity of the O86:B7 strain. PMID:16332778

  20. Gene-modified T-cell therapy using chimeric antigen receptors for pediatric hematologic malignancies.

    PubMed

    Nakazawa, Yozo

    2016-06-01

    Chimeric antigen receptor (CAR) is the generic name for synthetic T cell receptors redirected to tumor-associated antigens. Most CARs consist of an ectodomain (scFv or ligand), a hinge region, a transmembrane domain, and signaling endodomains derived from one or two co-stimulatory molecules (CD28, 4-1BB, etc) and from a CD3-ζ chain. CD19-targeted CAR T cell therapy has achieved major success in the treatment of B cell malignancies. CD19 CAR-T cells elicited complete remission in 70-90% of adult and pediatric patients with relapsed/refractory acute lymphoblastic leukemia (ALL). CD19 CAR T cell therapy from allogeneic donors including third party donors is a potential option for B-cell malignancies. CAR T cell therapies for myeloma, acute myeloid leukemia, and T-cell leukemia are still under development. Our group is currently preparing a phase I study of CD19 CAR T cell therapy in pediatric and young adult patients with ALL using a non-viral gene transfer method, the piggyBac-transposon system. PMID:27384848

  1. Antigen-specific T cell–mediated gene therapy in collagen-induced arthritis

    PubMed Central

    Nakajima, Atsuo; Seroogy, Christine M.; Sandora, Matthew R.; Tarner, Ingo H.; Costa, Gina L.; Taylor-Edwards, Cariel; Bachmann, Michael H.; Contag, Christopher H.; Fathman, C. Garrison

    2001-01-01

    Autoantigen-specific T cells have tissue-specific homing properties, suggesting that these cells may be ideal vehicles for the local delivery of immunoregulatory molecules. We tested this hypothesis by using type II collagen–specific (CII-specific) CD4+ T hybridomas or primary CD4+ T cells after gene transfer, as vehicles to deliver an immunoregulatory protein for the treatment of collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA). CII-specific T cells or hybridomas were transduced using retroviral vectors to constitutively express the IL-12 antagonist, IL-12 p40. Transfer of engineered CD4+ T cells after immunization significantly inhibited the development of CIA, while cells transduced with vector control had no effect. The beneficial effect on CIA of IL-12 p40-transduced T cells required TCR specificity against CII, since transfer of T cells specific for another antigen producing equivalent amounts of IL-12 p40 had no effect. In vivo cell detection using bioluminescent labels and RT-PCR showed that transferred CII-reactive T-cell hybridomas accumulated in inflamed joints in mice with CIA. These results indicate that the local delivery of IL-12 p40 by T cells inhibited CIA by suppressing autoimmune responses at the site of inflammation. Modifying antigen-specific T cells by retroviral transduction for local expression of immunoregulatory proteins thus offers a promising strategy for treating RA. PMID:11375419

  2. Prostate specific antigen gene expression in androgen insensitive prostate carcinoma subculture cell line.

    PubMed

    Tsui, Ke-Hung; Feng, Tsui-Hsia; Chung, Li-Chuan; Chao, Chun-Hsiang; Chang, Phei-Lang; Juang, Horng-Heng

    2008-01-01

    A novel prostate cancer cell line (PC-J) was isolated from an androgen independent non-prostate specific antigen (non-PSA) producing carcinoma cell line. The homologous correlation between PC-J and PC-3 was determined by short tandem repeat analysis. The PSA promoter activity was detected by transient expression assay in the PC-J and LNCaP cells but not in androgen insensitive PC-3 cells. When the PC-J cells were cotransfected with androgen receptor, androgen receptor coactivators and PSA reporter vector cells, the reporter assays indicated that nuclear receptor coactivator 4 (NCOA4) but not androgen receptor activator 24 (ARA24) increased the sensitivity and maximum stimulation of dihydrotestosterone (DHT)-inducing PSA promoter activity. The RT-PCR assays revealed that the expression of several tumor markers, including interleukin-6, prostate stem cell antigen (PSCA), prostate epithelium-specific Ets transcription factor (PDEF) and matriptase, was lower in the PC-J cells than in the PC-3 cells. This cell model elucidated the regulation of PSA expression and enabled comparison of the gene profile at different stages of metastasis in prostatic carcinoma.

  3. Altered transcription of genes coding for class I histocompatibility antigens in murine tumor cells

    PubMed Central

    1983-01-01

    Three murine tumors induced by Moloney murine leukemia virus (M-MLV) which exhibited loss of some or all H-2 class I antigens at the cell surface were analyzed at the DNA and RNA level with molecular probes specific of H-2 heavy chains and beta 2-microglobulin sequences. No observable difference could be detected at the DNA level between the tumors and the parent animals. However, a decrease in H-2 mRNA was observed, especially in phenotypically H-2 negative tumor, BM5R, where H-2 transcripts were at least 30-fold less abundant. These results show that an H-2-negative character may result from a general alteration in the transcription of H-2 genes, which could reflect some kind of regulatory process. PMID:6311935

  4. Pasteurella haemolytica serotype 2 contains the gene for a noncapsular serotype 1-specific antigen.

    PubMed Central

    Gonzalez, C T; Maheswaran, S K; Murtaugh, M P

    1995-01-01

    An ssa1-homologous genomic fragment cloned from Pasteurella haemolytica serotype 2 (ST2) enabled transformation of Escherichia coli DH5 alpha to a serotype 1 (ST1) phenotype through expression of the ST1-specific antigen (Ssa1). The Ssa1 protein expressed by ssa1-transformed E. coli was susceptible to heat and protease treatment and was distinct from P. haemolytica ST1-specific capsular polysaccharide. Electrophoretic analysis of in vitro-translated proteins, as well as the predicted amino acid sequence, demonstrated that Ssa1 proteins encoded from either ST1- or ST2-derived ssa1 genes were essentially identical. A comparison of the nucleotide sequences of ssa1 genes derived from P. haemolytica ST1 and ST2 revealed greater than 99% homology. Amino acid sequence homology of the predicted products of ST1- and ST2-derived ssa1 genes was greater than 98%. Northern (RNA) blot studies revealed that the presence of an increased level of ssa1 transcript in P. haemolytica ST1 grown as surface-adherent cultures on solid medium was correlated with a serologically detectable Ssa1 protein. Expression of the ssa1 transcript in ST1 was similarly upregulated by a high iron concentration in the growth medium. PMID:7890392

  5. Characterization and allelic variation of the transporters associated with antigen processing (TAP) genes in the domestic dog (Canis lupus familiaris).

    PubMed

    Gojanovich, Gregory S; Ross, Peter; Holmer, Savannah G; Holmes, Jennifer C; Hess, Paul R

    2013-12-01

    The function of the transporters associated with antigen processing (TAP) complex is to shuttle antigenic peptides from the cytosol to the endoplasmic reticulum to load MHC class I molecules for CD8(+) T-cell immunosurveillance. Here we report the promoter and coding regions of the canine TAP1 and TAP2 genes, which encode the homologous subunits forming the TAP heterodimer. By sampling genetically divergent breeds, polymorphisms in both genes were identified, although there were few amino acid differences between alleles. Splice variants were also found. When aligned to TAP genes of other species, functional regions appeared conserved, and upon phylogenetic analysis, canine sequences segregated appropriately with their orthologs. Transfer of the canine TAP2 gene into a murine TAP2-defective cell line rescued surface MHC class I expression, confirming exporter function. This data should prove useful in investigating the association of specific TAP defects or alleles with immunity to intracellular pathogens and cancer in dogs. PMID:23892057

  6. Adoptive Immunotherapy for Hematological Malignancies Using T Cells Gene-Modified to Express Tumor Antigen-Specific Receptors

    PubMed Central

    Fujiwara, Hiroshi

    2014-01-01

    Accumulating clinical evidence suggests that adoptive T-cell immunotherapy could be a promising option for control of cancer; evident examples include the graft-vs-leukemia effect mediated by donor lymphocyte infusion (DLI) and therapeutic infusion of ex vivo-expanded tumor-infiltrating lymphocytes (TIL) for melanoma. Currently, along with advances in synthetic immunology, gene-modified T cells retargeted to defined tumor antigens have been introduced as “cellular drugs”. As the functional properties of the adoptive immune response mediated by T lymphocytes are decisively regulated by their T-cell receptors (TCRs), transfer of genes encoding target antigen-specific receptors should enable polyclonal T cells to be uniformly redirected toward cancer cells. Clinically, anticancer adoptive immunotherapy using genetically engineered T cells has an impressive track record. Notable examples include the dramatic benefit of chimeric antigen receptor (CAR) gene-modified T cells redirected towards CD19 in patients with B-cell malignancy, and the encouraging results obtained with TCR gene-modified T cells redirected towards NY-ESO-1, a cancer-testis antigen, in patients with advanced melanoma and synovial cell sarcoma. This article overviews the current status of this treatment option, and discusses challenging issues that still restrain the full effectiveness of this strategy, especially in the context of hematological malignancy. PMID:25517545

  7. An immobilization antigen gene of the fish-parasitic protozoan Ichthyophthirius multifiliis strain ARS-6

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ichthyophthirius multifiliis (Ich) is a severe fish parasite that causes ‘white spot’ disease in many freshwater fish and leads to high mortality. The antigens on the parasite surface are involved in the antibody-mediated immobilization and hence designated as immobilization antigens (i-antigens). ...

  8. HLA-DR, DQ and T cell antigen receptor constant beta genes in Japanese patients with ulcerative colitis.

    PubMed Central

    Kobayashi, K; Atoh, M; Konoeda, Y; Yagita, A; Inoko, H; Sekiguchi, S

    1990-01-01

    We studied the T cell antigen receptor (TcR) constant beta chain genes on HLA typed Japanese patients with ulcerative colitis (UC). A TcR constant beta EcoRI 6.0-kb fragment was present in all Japanese UC patients (n = 17) but completely absent in the controls (n = 35) (chi2 = 47.6, P less than 0.001). The frequency of HLA-DR2 antigen was significantly higher in UC patients (85% versus 28% in controls, P less than 0.001). Furthermore, HLA-DQw1 antigen was also increased in UC patients (96% versus 60% in controls, P less than 0.001). However, HLA-DR4 antigen was significantly decreased in UC patients (12% versus 37%, P = 0.02). HLA-DR1 antigen was not found in UC patients and was present in only 15% of the controls. These results suggest that TcR beta chain and HLA-DQw1 antigen may be important in the pathogenesis of Japanese UC. Images Fig. 1 PMID:1973647

  9. Recombinative events of the T cell antigen receptor delta gene in peripheral T cell lymphomas.

    PubMed Central

    Kanavaros, P; Farcet, J P; Gaulard, P; Haioun, C; Divine, M; Le Couedic, J P; Lefranc, M P; Reyes, F

    1991-01-01

    Recombinative events of the T cell antigen receptor (TCR) delta-chain gene were studied in 37 cases of peripheral T cell lymphoma (PTCL) and related to their clinical presentation and the expression of the alpha beta or gamma delta heterodimers as determined by immunostaining of frozen tissue samples. There were 22 cases of alpha beta, 5 cases of gamma delta, and 10 cases of silent TCR expressing neither the alpha beta nor gamma delta TCR. 5 different probes were used to examine the delta locus. The 22 cases of alpha beta PTCL displayed biallelic and monoallelic deletions; a monoallelic V delta 1 J delta 1 rearrangement was observed in 1 case and a monoallelic germ line configuration in 7 cases. The 5 cases of gamma delta PTCL displayed biallelic rearrangements: the productive rearrangements could be ascribed to V delta 1J delta 1 joining in 3 cases and VJ delta 1 joining in 2 cases according to the combined pattern of DNA hybridization with the appropriate probes and of cell reactivity with the TCR delta-1, delta TCS-1, and anti-V delta 2 monoclonal antibodies. In the VJ delta 1 joining, the rearranged V segments were located between V delta 1 and V delta 2. Interestingly, in the third group of 10 cases of silent PTCL, 5 cases were found to have a TCR gene configuration identical to that in the TCR alpha beta PTCL, as demonstrated by biallelic delta gene deletion. These 5 cases were CD3 positive. The 5 remaining cases showed a monoallelic delta gene rearrangement with a monoallelic germ line configuration in 4 and a monoallelic deletion in 1. Four of these cases were CD3 negative, which was consistent with an immature genotype the TCR commitent of which could not be ascertained. Finally, TCR gamma delta PTCL consisted of a distinct clinical morphological and molecular entity whereas TCR alpha beta and silent PTCL had a similar presentation. Images PMID:1991851

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

    SciTech Connect

    Min, Hyehyun; Lee, Ji-Yeon; Bok, Jinwoong; Chung, Hyun Joo; Kim, Myoung Hee

    2010-02-19

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

  11. Transfection, expression, and DNA sequence of a gene encoding a BoLA-A11 antigen

    SciTech Connect

    Sawhney, S.M.S.; Hasima, N.N.; Glass, E.J.; Al-Murrani, S.W.K.; Nichani, A.K.; Spooner, R.L.; Williams, J.L.; Russell, G.C.

    1995-02-01

    Cattle major histocompatibility complex [(MHC) (BoLA)] class I molecules are heterodimeric glycoproteins which present endogenous antigenic peptides to CD8{sup +} T lymphocytes, initiating a cellular immune response. The MHC-encoded heavy chains are highly polymorphic and, in cattle, have been characterized mainly by using allo-antisera raised by reciprocal calf/dam immunizations. This has enabled the identification of about 50 serlogical specificities, most of which behave as alleles of a single highly polymorphic class I locus. However, evidence from biochemical and molecular biological studies suggest that more than one BoLA class I locus is expressed. These loci are apparently in linkage disequilibrium, making them difficult to distinguish by conventional methods. In order to investigate the expression and function of individual class I locus products, we are correlating BoLA class I gene sequences with the expressed products by the transfection and characterization of genomic class I clones. Shotgun transfection and expression of BoLA class I molecules has been described previously, but the genes involved were not isolated. In this paper we report the isolation, DNA sequencing, transfection, and expression of a genomic clone encoding a BoLA-A11 determinant from an animal expressing A10 and A11 serological specificities. 23 refs., 3 figs.

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

    PubMed

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

    2012-06-01

    Antigen 5 (Ag5) has been identified as a dominant component of cyst fluid of Echinococcus granulosus and is considered as a member of serine proteases family, which in other helminth, plays an important role in the egg hatch and larva invasion. However, whether Ag5 is expressed and secreted in all life stages is unknown. In this study, according to the sequence in GenBank, we cloned and sequenced the open reading frame (ORF) of Ag5 gene from the protoscolices of E. granulosus isolated from the sheep in Qinhai Province of China, and found several substitutions and a base insert and deletion in a short region near the stop code, leading to a frameshift mutation which is conserved with the homologue of other cestode. The ORF is 1,455 bp in length, encoding 484 amino acids with a secretory signal peptide. Bioinformatics analysis predicted several phosphorylation and myristoylation sites and a N-glycosylation site and a species-specific linear B epitope in the protein. The ORF was cloned into the plasmid pET28a(+) vector and expressed in Escherichia coli . The recombinant protein was purified by affinity chromatography. Anti-rEgAg5 antiserum was prepared in rats and used to analyze the localization of Ag5 in protoscolex and adult worm by immunofluorescence technique. Results demonstrated that the Ag5 is strongly expressed in the tegument of protoscolex and the embryonic membrane of egg and surface of oncosphere; meanwhile, it is also weakly expressed in tegument of the adult. This study showed that Ag5 is expressed in all stages of life cycle, secreted from the surface of the worm and may be anchored in membrane by its myristoylation sites; these characteristics make it a candidate antigen for diagnosis and vaccine for both intermediate and definitive hosts. PMID:22200957

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

    PubMed

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

    2012-06-01

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

  14. T antigen expression and tumorigenesis in transgenic mice containing a mouse major urinary protein/SV40 T antigen hybrid gene.

    PubMed Central

    Held, W A; Mullins, J J; Kuhn, N J; Gallagher, J F; Gu, G D; Gross, K W

    1989-01-01

    A hybrid mouse major urinary protein (MUP)/SV40 T antigen gene was microinjected into fertilized mouse embryos and the resulting transgenic mice analyzed for the regulated expression of the transgene. Available evidence indicates that the MUP gene used for the hybrid gene construct is expressed in both male and female liver and possibly mammary gland. Three different transgenic lines exhibited a consistent pattern of tissue specific expression of the transgene. As a consequence of transgene expression and T antigen synthesis in the liver, both male and female transgenic animals developed liver hyperplasia and tumors. Transgene expression and liver hyperplasia commenced at approximately 2-4 weeks of age, the same time that MUP gene expression is first detected in the liver. The expression of the transgene resulted in an immediate strong suppression of liver MUP mRNA levels but had relatively little effect on other liver specific mRNAs. From 4 to 8 weeks, the liver increased several fold in size, relative to non-transgenic littermates. Definitive tumor nodules were not apparent until 8-10 weeks. The transgene was also consistently found to be expressed in the skin sebaceous glands and the preputial gland, a modified sebaceous gland. The expression of the transgene in the skin sebaceous glands is consistent with the presence of MUP mRNA in the skin and a putative role for MUPs in the transport and excretion of small molecules. Occasional expression of the transgene in other tissues (kidney and mammary connective tissues) was also noted.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:2714250

  15. Diversity of Babesia bovis merozoite surface antigen genes in the Philippines.

    PubMed

    Tattiyapong, Muncharee; Sivakumar, Thillaiampalam; Ybanez, Adrian Patalinghug; Ybanez, Rochelle Haidee Daclan; Perez, Zandro Obligado; Guswanto, Azirwan; Igarashi, Ikuo; Yokoyama, Naoaki

    2014-02-01

    Babesia bovis is the causative agent of fatal babesiosis in cattle. In the present study, we investigated the genetic diversity of B. bovis among Philippine cattle, based on the genes that encode merozoite surface antigens (MSAs). Forty-one B. bovis-positive blood DNA samples from cattle were used to amplify the msa-1, msa-2b, and msa-2c genes. In phylogenetic analyses, the msa-1, msa-2b, and msa-2c gene sequences generated from Philippine B. bovis-positive DNA samples were found in six, three, and four different clades, respectively. All of the msa-1 and most of the msa-2b sequences were found in clades that were formed only by Philippine msa sequences in the respective phylograms. While all the msa-1 sequences from the Philippines showed similarity to those formed by Australian msa-1 sequences, the msa-2b sequences showed similarity to either Australian or Mexican msa-2b sequences. In contrast, msa-2c sequences from the Philippines were distributed across all the clades of the phylogram, although one clade was formed exclusively by Philippine msa-2c sequences. Similarities among the deduced amino acid sequences of MSA-1, MSA-2b, and MSA-2c from the Philippines were 62.2-100, 73.1-100, and 67.3-100%, respectively. The present findings demonstrate that B. bovis populations are genetically diverse in the Philippines. This information will provide a good foundation for the future design and implementation of improved immunological preventive methodologies against bovine babesiosis in the Philippines. The study has also generated a set of data that will be useful for futher understanding of the global genetic diversity of this important parasite.

  16. Association of Polymorphisms in HLA Antigen Presentation-Related Genes with the Outcomes of HCV Infection

    PubMed Central

    Lu, Xiaomei; Xu, Yin; Wang, Jie; Zhang, Yun; Yu, Rongbin; Su, Jing

    2015-01-01

    Antigen-presentation genes play a vital role in the pathogenesis of HCV infection. However, the relationship of variants of these genes with spontaneous outcomes of HCV infection has not been fully investigated. To explore novel loci in the Chinese population, 34 tagging-SNPs in 9 candidate genes were genotyped for their associations with the outcomes of HCV infection. The distributions of different genotypes and haplotypes were compared among 773 HCV-negative controls, 246 subjects with HCV natural clearance, and 218 HCV persistent carriers recruited from hemodialysis patients and intravenous drug users. Our study implicated that TAP2, HLA-DOA, HLA-DOB, and tapasin loci were novel candidate regions for susceptibility to HCV infection and viral clearance in the Chinese population. Logistic regression analyses showed that TAP2 rs1800454 A (OR = 1.48, P = 0.002) and HLA-DOB rs2071469 G (OR = 1.23, P = 0.048) were significantly associated with increased susceptibility to establishment of HCV infection. However, high-risk behavior exposure and age were stronger predictors of HCV infection. Mutation of tapasin rs9277972 T (OR = 1.57, P =0.043) increased the risk of HCV chronicity, and HLA-DOA rs3128935 C (OR = 0.62, P = 0.019) increased the chance of viral resolution. With regards to the effect of rs3128925, interactions were found with high-risk behavior (P = 0.013) and age (P = 0.035). The risk effect of rs3128925 T for persistent HCV infection was higher in injecting drug users (vs. dialysis patients) and in subjects ≥ 40 years old (vs. < 40 years old). PMID:25874709

  17. In silico prediction of tumor antigens derived from functional missense mutations of the cancer gene census

    PubMed Central

    Khalili, Jahan S.; Hanson, Russell W.; Szallasi, Zoltan

    2012-01-01

    Antigen-specific immune responses against peptides derived from missense gene mutations have been identified in multiple cancers. The application of personalized peptide vaccines based on the tumor mutation repertoire of each cancer patient is a near-term clinical reality. These peptides can be identified for pre-validation by leveraging the results of massive gene sequencing efforts in cancer. In this study, we utilized NetMHC 3.2 to predict nanomolar peptide binding affinity to 57 human HLA-A and B alleles. All peptides were derived from 5,685 missense mutations in 312 genes annotated as functionally relevant in the Cancer Genome Project. Of the 26,672,189 potential 8–11 mer peptide-HLA pairs evaluated, 0.4% (127,800) display binding affinities < 50 nM, predicting high affinity interactions. These peptides can be segregated into two groups based on the binding affinity to HLA proteins relative to germline-encoded sequences: peptides for which both the mutant and wild-type forms are high affinity binders, and peptides for which only the mutant form is a high affinity binder. Current evidence directs the attention to mutations that increase HLA binding affinity, as compared with cognate wild-type peptide sequences, as these potentially are more relevant for vaccine development from a clinical perspective. Our analysis generated a database including all predicted HLA binding peptides and the corresponding change in binding affinity as a result of point mutations. Our study constitutes a broad foundation for the development of personalized peptide vaccines that hone-in on functionally relevant targets in multiple cancers in individuals with diverse HLA haplotypes. PMID:23243591

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

    PubMed

    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.

  19. Novel meningococcal 4CMenB vaccine antigens - prevalence and polymorphisms of the encoding genes in Neisseria gonorrhoeae.

    PubMed

    Hadad, Ronza; Jacobsson, Susanne; Pizza, Mariagrazia; Rappuoli, Rino; Fredlund, Hans; Olcén, Per; Unemo, Magnus

    2012-09-01

    The first cross-protective Neisseria meningitidis vaccine (focus on serogroup B), the protein-based 4 component meningococcus serogroup B (4CMenB), includes the New Zealand outer membrane vesicle and three main genome-derived neisserial antigens (GNAs). These GNAs are fHbp (fused to GNA2091), NHBA (fused to GNA1030) and NadA. In this study, the prevalence and polymorphisms of the nucleotide and amino acid sequences of the 4CMenB antigens in a temporally and geographically diverse collection of N. gonorrhoeae isolates (n = 111) were investigated. All the examined GNA genes, except the nadA gene, were present in all gonococcal isolates. However, 25 isolates contained premature stop codons in the fHbp gene and/or the nhba gene, resulting in truncated proteins. Compared with the 4CMenB antigen sequences in reference strain MC58, the gonococcal strains displayed 67.0-95.4% and 60.9-94.9% identity in nucleotide sequence and amino acid sequence, respectively, in the equivalent GNA antigens. The absence of NadA, lack of universal expression of fHbp and NHBA and the uncertainty regarding the surface exposure of fHbp as well as the function of NHBA in N. gonorrhoeae will likely limit the use of the identical 4CMenB antigens in a gonococcal vaccine. However, possible cross-immunity of 4CMenB with gonococci and expression and function of the equivalent gonococcal GNAs, as well as of more appropriate GNAs for a gonococcal vaccine, need to be further examined. PMID:22882265

  20. Polymorphism at the apical membrane antigen 1 gene (AMA1) of the malaria parasite Plasmodium falciparum in a Vietnamese population.

    PubMed

    Quang, Nguyen Duc; Hoa, Phan Thi Phuong; Tuan, Mai Sy; Viet, Nguyen Xuan; Jalloh, Amadu; Matsuoka, Hiroyuki

    2009-06-01

    The patterns of molecular evolution of the most diverse region of the apical membrane antigen 1 (AMA1) gene in Plasmodium falciparum from a Vietnamese subpopulation (Bao Loc) were investigated. Within the Bao Loc population, the sequenced gene region showed relatively high allelic and nucleotide diversity (0.985 and 0.02694, respectively). Further, the level of population recombination was substantial, resulting in a significant decay of linkage disequilibrium along the gene region. The results suggest that AMA1 is a useful genetic marker for studying the relationships between adaptation of parasite populations (to the human host immune system) and malaria epidemiology.

  1. Determination of serotyping antigens, clonal analysis and genetic characterization of the 4CMenB vaccine antigen genes in invasive Neisseria meningitidis from Western Canada, 2009 to 2013.

    PubMed

    Law, Dennis K S; Zhou, Jianwei; Deng, Saul; Hoang, Linda; Tyrrell, Gregory; Horsman, Greg; Wylie, John; Tsang, Raymond S W

    2014-11-01

    This study examined invasive Neisseria meningitidis recovered from invasive meningococcal disease (IMD) cases in Western Canada between 2009 and 2013. A total of 161 isolates from individual IMD cases were analysed for serogroup, serotype, serosubtype, PorA genotype, multi-locus sequence type and nucleotide sequence of their 4CMenB vaccine antigen genes. Sixty-nine isolates were serogroup B (MenB), 47 were serogroup Y (MenY), 22 were serogroup C (MenC), 19 were serogroup W (MenW), three were serogroup E and one was non-encapsulated. MenC, MenY and MenW were mainly clonal, represented primarily by clonal complex (cc) 11, cc23 or cc167, and cc22, respectively. In contrast, MenB were composed of eight different ccs together with 11 isolates not assigned to any known cc. Antigenic analysis and PorA genotyping confirmed the heterogeneity of MenB isolates, while such results supported the clonal nature of most MenC, MenY and MenW isolates. Thirty-four (21.1%) isolates had at least one gene that encoded one matching vaccine protein component of the 4CMenB vaccine (i.e. PorA P1.4; fHbp variant 1.1; NHBA peptide 2; and NadA-1, -2, or -3). An additional 18 isolates had genes that encoded variant 1 or subfamily B factor H binding proteins of this same vaccine.

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

    SciTech Connect

    Li Hongwei; Li Jinzhong; Helm, Gregory A.; Pan Dongfeng . E-mail: Dongfeng_pan@yahoo.com

    2005-09-09

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

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

    PubMed

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

    1995-03-01

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

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

    PubMed Central

    Jackson, Andrew P.; Otto, Thomas D.; Darby, Alistair; Ramaprasad, Abhinay; Xia, Dong; Echaide, Ignacio Eduardo; Farber, Marisa; Gahlot, Sunayna; Gamble, John; Gupta, Dinesh; Gupta, Yask; Jackson, Louise; Malandrin, Laurence; Malas, Tareq B.; Moussa, Ehab; Nair, Mridul; Reid, Adam J.; Sanders, Mandy; Sharma, Jyotsna; Tracey, Alan; Quail, Mike A.; Weir, William; Wastling, Jonathan M.; Hall, Neil; Willadsen, Peter; Lingelbach, Klaus; Shiels, Brian; Tait, Andy; Berriman, Matt; Allred, David R.; Pain, Arnab

    2014-01-01

    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. PMID:24799432

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

    PubMed

    Jackson, Andrew P; Otto, Thomas D; Darby, Alistair; Ramaprasad, Abhinay; Xia, Dong; Echaide, Ignacio Eduardo; Farber, Marisa; Gahlot, Sunayna; Gamble, John; Gupta, Dinesh; Gupta, Yask; Jackson, Louise; Malandrin, Laurence; Malas, Tareq B; Moussa, Ehab; Nair, Mridul; Reid, Adam J; Sanders, Mandy; Sharma, Jyotsna; Tracey, Alan; Quail, Mike A; Weir, William; Wastling, Jonathan M; Hall, Neil; Willadsen, Peter; Lingelbach, Klaus; Shiels, Brian; Tait, Andy; Berriman, Matt; Allred, David R; Pain, Arnab

    2014-06-01

    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.

  6. Mta, the maternally transmitted antigen, is determined jointly by the chromosomal Hmt and the extrachromosomal Mtf genes

    PubMed Central

    1986-01-01

    Mus spretus from four stocks, originating in Spain, Portugal, and Morocco, were tested for the maternally transmitted antigen, Mta. All expressed a variant form not found in other species of mice. Analysis of appropriate crosses with inbred mice showed that the spretus form of Mta is determined by a new allele, c, of the Hmt gene. The Hmtc allele has been isolated in coupling with four different H-2 haplotypes. It is possible to raise CTL specific for the spretus form of Mta. The maternally transmitted factor, Mtf alpha s, of spretus mice determines, in conjunction with the Hmta allele of C57BL/6, an Mta that is indistinguishable from the common form found in C57BL/6 and most other inbred mice. Our experiments show that the specificity of the cell surface antigen Mta is governed jointly by the cytoplasmic gene Mtf and the chromosomal gene Hmt. We propose that Hmt encodes a class I histocompatibility antigen that acts as a restricting element for the Mtf gene product, thus meeting the requirements of T killer cell recognition. PMID:3511170

  7. Serotype O:8 isolates in the Yersinia pseudotuberculosis complex have different O-antigen gene clusters and produce various forms of rough LPS.

    PubMed

    Kenyon, Johanna J; Duda, Katarzyna A; De Felice, Antonia; Cunneen, Monica M; Molinaro, Antonio; Laitinen, Juha; Skurnik, Mikael; Holst, Otto; Reeves, Peter R; De Castro, Cristina

    2016-04-01

    In Yersinia pseudotuberculosis complex, the O-antigen of LPS is used for the serological characterization of strains, and 21 serotypes have been identified to date. The O-antigen biosynthesis gene cluster and corresponding O-antigen structure have been described for 18, leaving O:8, O:13 and O:14 unresolved. In this study, two O:8 isolates were examined. The O-antigen gene cluster sequence of strain 151 was near identical to serotype O:4a, though a frame-shift mutation was found in ddhD, while No. 6 was different to 151 and carried the O:1b gene cluster. Structural analysis revealed that No. 6 produced a deeply truncated LPS, suggesting a mutation within the waaF gene. Both ddhD and waaF were cloned and expressed in 151 and No. 6 strains, respectively, and it appeared that expression of ddhD gene in strain 151 restored the O-antigen on LPS, while waaF in No. 6 resulted in an LPS truncated less severely but still without the O-antigen, suggesting that other mutations occurred in this strain. Thus, both O:8 isolates were found to be spontaneous O-antigen-negative mutants derived from other validated serotypes, and we propose to remove this serotype from the O-serotyping scheme, as the O:8 serological specificity is not based on the O-antigen.

  8. Detection of Cytotoxic T-Lymphocyte Associated Antigen-4 Gene Polymorphism in Type 1 Diabetes Mellitus.

    PubMed

    Arafa, Roshdan M; Desouky, Somaya M; Emam, Sherin M; Abed, Neveen Tawfik; Mohamed, Sahar Y

    2015-01-01

    Type 1 diabetes is one of the most common chronic childhood illnesses. Interplay between genetic susceptibility and environmental factors is thought to provide the fundamental element for the disease. It has been shown that more than 40 genetic loci are associated with T1DM. Important one among these is the CTLA-4. This work aimed to detect Cytotoxic T Lymphocyte-associated antigen 4 (CTLA-4) gene polymorphism in patients with type 1 diabetes mellitus T1DM using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to clarify its role in the susceptibility to T1DM. The study was carried out on forty unrelated Egyptian children with TIDM. Twenty unrelated healthy children were enrolled as a control group. Blood samples were collected from patients and control groups and subjected to CTLA-4 gene polymorphism analysis using polymerase chain reaction followed by restriction fragment length polymorphism (PCR-RFLP). CTLA-4 G allele and GG homozygous genotype were significantly increased in T1DM patients than in control group (P < 0.001, P = 0.002 respectively). There was significant association between the three CTLA-4 genotypes (AA, AG, GG) and diabetic complications (p = 0.002), AG and GG polymorphisms were associated with complications of diabetes with ratio 84.6% and 100% respectively. While no association was found with sex, weight, height, risk factors of diabetes or insulin treatment. It was concluded that there is a strong association between AG polymorphism and T1DM (P = 0.002). PMID:26415372

  9. Genomic polymorphism, recombination, and linkage disequilibrium in human major histocompatibility complex-encoded antigen-processing genes

    SciTech Connect

    van Endert, P.M.; Lopez, M.T.; Patel, S.D.; McDevitt, H.O. ); Monaco, J.J. )

    1992-12-01

    Recently, two subunits of a large cytosolic protease and two putative peptide transporter proteins were found to be encoded by genes within the class II region of the major histocompatibility complex (MHC). These genes have been suggested to be involved in the processing of antigenic proteins for presentation by MHC class I molecules. Because of the high degree of polymorphism in MHC genes, and previous evidence for both functional and polypeptide sequence polymorphism in the proteins encoded by the antigen-processing genes, we tested DNA from 27 consanguineous human cell lines for genomic polymorphism by restriction fragment length polymorphism (RFLP) analysis. These studies demonstrate a strong linkage disequilibrium between TAP1 and LMP2 RFLPs. Moreover, RFLPs, as well as a polymorphic stop codon in the telomeric TAP2 gene, appear to be in linkage disequilibrium with HLA-DR alleles and RFLPs in the HLA-DO gene. A high rate of recombination, however, seems to occur in the center of the complex, between the TAP1 and TAP2 genes.

  10. Structure and gene cluster organization of the O-antigen of Providencia alcalifaciens O45:H25.

    PubMed

    Ovchinnikova, Olga G; Shashkov, Alexander S; Moryl, Magdalena; Liu, Bin; Rozalski, Antoni; Knirel, Yuriy A

    2014-10-29

    O-Polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Providencia alcalifaciens O45:H25 and studied by sugar analysis, Smith degradation, and (1)H and (13)C NMR spectroscopy. The following structure of the pentasaccharide repeat of the O-polysaccharide was established: [structure: see text]. The O-antigen gene cluster of P. alcalifaciens O45 was sequenced and found to be in full agreement with the O-polysaccharide structure established.

  11. A search for novel cancer/testis antigens in lung cancer identifies VCX/Y genes expanding the repertoire of potential immunotherapeutic targets

    PubMed Central

    Taguchi, Ayumu; Taylor, Allen D.; Rodriguez, Jaime; Çeliktaş, Müge; Liu, Hui; Ma, Xiaotu; Zhang, Qing; Wong, Chee-Hong; Chin, Alice; Girard, Luc; Behrens, Carmen; Lam, Wan L.; Lam, Stephen; Minna, John D.; Wistuba, Ignacio I.; Gazdar, Adi F.; Hanash, Samir M.

    2015-01-01

    Cancer/testis (CT) antigens are potential immunotherapeutic targets in cancer. However, the expression of particular antigens is limited to a subset of tumors of a given type. Thus, there is a need to identify antigens with complementary expression patterns for effective therapeutic intervention. In this study, we searched for genes that were distinctly expressed at a higher level in lung tumor tissue and the testes compared to other non-tumor tissues and identified members of the VCX/Y gene family as novel CT antigens. VCX3A, a member of the VCX/Y gene family, was expressed at the protein level in approximately 20% of lung adenocarcinomas and 35% of squamous cell carcinomas, but not expressed in normal lung tissues. Among CT antigens with concordant mRNA and protein expression levels, four CT antigens, XAGE1, VCX, IL13RA2, and SYCE1, were expressed, alone or in combination, in about 80% of lung adenocarcinoma tumors. The CT antigen VCX/Y gene family broadens the spectrum of CT antigens expressed in lung adenocarcinomas for clinical applications. PMID:24970476

  12. IgE-associated IGHV genes from venom and peanut allergic individuals lack mutational evidence of antigen selection.

    PubMed

    Wang, Yan; Jackson, Katherine J L; Davies, Janet; Chen, Zhiliang; Gaeta, Bruno A; Rimmer, Janet; Sewell, William A; Collins, Andrew M

    2014-01-01

    Antigen selection of B cells within the germinal center reaction generally leads to the accumulation of replacement mutations in the complementarity-determining regions (CDRs) of immunoglobulin genes. Studies of mutations in IgE-associated VDJ gene sequences have cast doubt on the role of antigen selection in the evolution of the human IgE response, and it may be that selection for high affinity antibodies is a feature of some but not all allergic diseases. The severity of IgE-mediated anaphylaxis is such that it could result from higher affinity IgE antibodies. We therefore investigated IGHV mutations in IgE-associated sequences derived from ten individuals with a history of anaphylactic reactions to bee or wasp venom or peanut allergens. IgG sequences, which more certainly experience antigen selection, served as a control dataset. A total of 6025 unique IgE and 5396 unique IgG sequences were generated using high throughput 454 pyrosequencing. The proportion of replacement mutations seen in the CDRs of the IgG dataset was significantly higher than that of the IgE dataset, and the IgE sequences showed little evidence of antigen selection. To exclude the possibility that 454 errors had compromised analysis, rigorous filtering of the datasets led to datasets of 90 core IgE sequences and 411 IgG sequences. These sequences were present as both forward and reverse reads, and so were most unlikely to include sequencing errors. The filtered datasets confirmed that antigen selection plays a greater role in the evolution of IgG sequences than of IgE sequences derived from the study participants.

  13. IgE-associated IGHV genes from venom and peanut allergic individuals lack mutational evidence of antigen selection.

    PubMed

    Wang, Yan; Jackson, Katherine J L; Davies, Janet; Chen, Zhiliang; Gaeta, Bruno A; Rimmer, Janet; Sewell, William A; Collins, Andrew M

    2014-01-01

    Antigen selection of B cells within the germinal center reaction generally leads to the accumulation of replacement mutations in the complementarity-determining regions (CDRs) of immunoglobulin genes. Studies of mutations in IgE-associated VDJ gene sequences have cast doubt on the role of antigen selection in the evolution of the human IgE response, and it may be that selection for high affinity antibodies is a feature of some but not all allergic diseases. The severity of IgE-mediated anaphylaxis is such that it could result from higher affinity IgE antibodies. We therefore investigated IGHV mutations in IgE-associated sequences derived from ten individuals with a history of anaphylactic reactions to bee or wasp venom or peanut allergens. IgG sequences, which more certainly experience antigen selection, served as a control dataset. A total of 6025 unique IgE and 5396 unique IgG sequences were generated using high throughput 454 pyrosequencing. The proportion of replacement mutations seen in the CDRs of the IgG dataset was significantly higher than that of the IgE dataset, and the IgE sequences showed little evidence of antigen selection. To exclude the possibility that 454 errors had compromised analysis, rigorous filtering of the datasets led to datasets of 90 core IgE sequences and 411 IgG sequences. These sequences were present as both forward and reverse reads, and so were most unlikely to include sequencing errors. The filtered datasets confirmed that antigen selection plays a greater role in the evolution of IgG sequences than of IgE sequences derived from the study participants. PMID:24586993

  14. Characterization and phylogenetic analysis of the swine leukocyte antigen 3 gene from Korean native pigs.

    PubMed

    Chung, H Y; Choi, Y C; Park, H N

    2015-05-18

    We investigated the phylogenetic relationships between pig breeds, compared the genetic similarity between humans and pigs, and provided basic genetic information on Korean native pigs (KNPs), using genetic variants of the swine leukocyte antigen 3 (SLA-3) gene. Primers were based on sequences from GenBank (accession Nos. AF464010 and AF464009). Polymerase chain reaction analysis amplified approximately 1727 bp of segments, which contained 1086 bp of coding regions and 641 bp of the 3'- and 5'-untranslated regions. Bacterial artificial chromosome clones of miniature pigs were used for sequencing the SLA-3 genomic region, which was 3114 bp in total length, including the coding (1086 bp) and non-coding (2028 bp) regions. Sequence analysis detected 53 single nucleotide polymorphisms (SNPs), based on a minor allele frequency greater than 0.01, which is low compared with other pig breeds, and the results suggest that there is low genetic variability in KNPs. Comparative analysis revealed that humans possess approximately three times more genetic variation than do pigs. Approximately 71% of SNPs in exons 2 and 3 were detected in KNPs, and exon 5 in humans is a highly polymorphic region. Newly identified sequences of SLA-3 using KNPs were submitted to GenBank (accession No. DQ992512-18). Cluster analysis revealed that KNPs were grouped according to three major alleles: SLA-3*0502 (DQ992518), SLA-3*0302 (DQ992513 and DQ992516), and SLA-3*0303 (DQ992512, DQ992514, DQ992515, and DQ992517). Alignments revealed that humans have a relatively close genetic relationship with pigs and chimpanzees. The information provided by this study may be useful in KNP management.

  15. Efficacy of a DNA Vaccine Carrying Eimeria maxima Gam56 Antigen Gene against Coccidiosis in Chickens

    PubMed Central

    Xu, Jinjun; Zhang, Yan

    2013-01-01

    To control coccidiosis without using prophylactic medications, a DNA vaccine targeting the gametophyte antigen Gam56 from Eimeria maxima in chickens was constructed, and the immunogenicity and protective effects were evaluated. The ORF of Gam56 gene was cloned into an eukaryotic expression vector pcDNA3.1(zeo)+. Expression of Gam56 protein in COS-7 cells transfected with recombinant plasmid pcDNA-Gam56 was confirmed by indirect immunofluorescence assay. The DNA vaccine was injected intramuscularly to yellow feathered broilers of 1-week old at 3 dosages (25, 50, and 100 µg/chick). Injection was repeated once 1 week later. One week after the second injection, birds were challenged orally with 5×104 sporulated oocysts of E. maxima, then weighed and killed at day 8 post challenge. Blood samples were collected and examined for specific peripheral blood lymphocyte proliferation activity and serum antibody levels. Compared with control groups, the administration of pcDNA-Gam56 vaccine markedly increased the lymphocyte proliferation activity (P<0.05) at day 7 and 14 after the first immunization. The level of lymphocyte proliferation started to decrease on day 21 after the first immunization. A similar trend was seen in specific antibody levels. Among the 3 pcDNA-Gam56 immunized groups, the median dosage group displayed the highest lymphocyte proliferation and antibody levels (P<0.05). The median dosage group had the greatest relative body weight gain (89.7%), and the greatest oocyst shedding reduction (53.7%). These results indicate that median dosage of DNA vaccine had good immunogenicity and immune protection effects, and may be used in field applications for coccidiosis control. PMID:23710081

  16. Development of PCR Assays Targeting Genes in O-Antigen Gene Clusters for Detection and Identification of Escherichia coli O45 and O55 Serogroups

    PubMed Central

    DebRoy, Chitrita; Fratamico, Pina M.; Roberts, Elisabeth; Davis, Michael A.; Liu, Yanhong

    2005-01-01

    The Escherichia coli O45 O-antigen gene cluster of strain O45:H2 96-3285 was sequenced, and conventional (singleplex), multiplex, and real-time PCR assays were designed to amplify regions in the wzx (O-antigen flippase) and wzy (O-antigen polymerase) genes. In addition, PCR assays targeting the E. coli O55 wzx and wzy genes were designed based on previously published sequences. PCR assays targeting E. coli O45 showed 100% specificity for this serogroup, whereas by PCR assays specific for E. coli O55, 97/102 strains serotyped as E. coli O55 were positive for wzx and 98/102 for wzy. Multiplex PCR assays targeting the E. coli O45 and the E. coli O55 wzx and wzy genes were used to detect the organisms in fecal samples spiked at levels of 106 and 108 CFU/0.2 g feces. Thus, the PCR assays can be used to detect and identify E. coli serogroups O45 and O55. PMID:16085897

  17. Development of Primers to O-Antigen Biosynthesis Genes for Specific Detection of Escherichia coli O157 by PCR

    PubMed Central

    Maurer, John J.; Schmidt, Denise; Petrosko, Patricia; Sanchez, Susan; Bolton, Lance; Lee, Margie D.

    1999-01-01

    The chemical composition of each O-antigen subunit in gram-negative bacteria is a reflection of the unique DNA sequences within each rfb operon. By characterizing DNA sequences contained with each rfb operon, a diagnostic serotype-specific probe to Escherichia coli O serotypes that are commonly associated with bacterial infections can be generated. Recently, from an E. coli O157:H7 cosmid library, O-antigen-positive cosmids were identified with O157-specific antisera. By using the cosmid DNAs as probes, several DNA fragments which were unique to E. coli O157 serotypes were identified by Southern analysis. Several of these DNA fragments were subcloned from O157-antigen-positive cosmids and served as DNA probes in Southern analysis. One DNA fragment within plasmid pDS306 which was specific for E. coli O157 serotypes was identified by Southern analysis. The DNA sequence for this plasmid revealed homology to two rfb genes, the first of which encodes a GDP-mannose dehydratase. These rfb genes were similar to O-antigen biosynthesis genes in Vibrio cholerae and Yersinia enterocolitica serotype O:8. An oligonucleotide primer pair was designed to amplify a 420-bp DNA fragment from E. coli O157 serotypes. The PCR test was specific for E. coli O157 serotypes. PCR detected as few as 10 cells with the O157-specific rfb oligonucleotide primers. Coupled with current enrichment protocols, O157 serotyping by PCR will provide a rapid, specific, and sensitive method for identifying E. coli O157. PMID:10388689

  18. Immunogenicity of recombinant attenuated Salmonella enterica serovar Typhimurium vaccine strains carrying a gene that encodes Eimeria tenella antigen SO7.

    PubMed

    Konjufca, Vjollca; Jenkins, Mark; Wang, Shifeng; Juarez-Rodriguez, Maria Dolores; Curtiss, Roy

    2008-12-01

    Recombinant attenuated Salmonella vaccines against avian coccidiosis were developed to deliver Eimeria species antigens to the lymphoid tissues of chickens via the type 3 secretion system (T3SS) and the type 2 secretion system (T2SS) of Salmonella. For antigen delivery via the T3SS, the Eimeria tenella gene encoding sporozoite antigen SO7 was cloned downstream of the translocation domain of the Salmonella enterica serovar Typhimurium sopE gene in the parental pYA3868 and pYA3870 vectors to generate pYA4156 and pYA4157. Newly constructed T3SS vectors were introduced into host strain chi8879 (Delta phoP233 Delta sptP1033::xylE Delta asdA16), an attenuated derivative of the highly virulent UK-1 strain. The SopE-SO7 fusion protein was secreted by the T3SS of Salmonella. The vector pYA4184 was constructed for delivery of the SO7 antigen via the T2SS. The SO7 protein was toxic to Salmonella when larger amounts were synthesized; thus, the synthesis of this protein was placed under the control of the lacI repressor gene, whose expression in turn was dependent on the amount of available arabinose in the medium. The pYA4184 vector was introduced into host strain chi9242 (Delta phoP233 Delta asdA16 Delta araBAD23 Delta relA198::araC P(BAD) lacI TT [TT is the T4ipIII transcription terminator]). In addition to SO7, for immunization and challenge studies we used the EAMZ250 antigen of Eimeria acervulina, which was previously shown to confer partial protection against E. acervulina challenge when it was delivered via the T3SS. Immunization of chickens with a combination of the SO7 and EAMZ250 antigens delivered via the T3SS induced superior protection against challenge by E. acervulina. In contrast, chickens immunized with SO7 that was delivered via the T2SS of Salmonella were better protected from challenge by E. tenella.

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

    PubMed

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

    2010-06-01

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

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

    PubMed

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

    2010-06-01

    T-cell receptor (TCR) gene transfer is an attractive strategy to generate antigen-specific T-cells for adoptive immunotherapy of cancer and chronic viral infection. However, current TCR gene transfer protocols trigger T-cell differentiation into terminally differentiated effector cells, which likely have reduced ability to mediate disease protection in vivo. We have developed a lentiviral gene transfer strategy to generate TCR-transduced human T-cells without promoting T-cell differentiation. We found that a combination of interleukin-15 (IL15) and IL21 facilitated lentiviral TCR gene transfer into non-proliferating T-cells. The transduced T-cells showed redirection of antigen specificity and produced IL2, IFNgamma and TNFalpha in a peptide-dependent manner. A significantly higher proportion of the IL15/IL21-stimulated T-cells were multi-functional and able to simultaneously produce all three cytokines (P<0.01), compared with TCR-transduced T-cells generated by conventional anti-CD3 plus IL2 stimulation, which primarily secreted only one cytokine. Similarly, IL15/IL21 maintained high levels of CD62L and CD28 expression in transduced T-cells, whereas anti-CD3 plus IL2 accelerated the loss of CD62L/CD28 expression. The data demonstrate that the combination of lentiviral TCR gene transfer together with IL15/IL21 stimulation can efficiently redirect the antigen specificity of resting primary human T-cells and generate multi-functional T-cells. PMID:20164855

  1. Control by H-2 genes of the Th1 response induced against a foreign antigen expressed by attenuated Salmonella typhimurium.

    PubMed Central

    Lo-Man, R; Martineau, P; Dériaud, E; Newton, S M; Jehanno, M; Clément, J M; Fayolle, C; Hofnung, M; Leclerc, C D

    1996-01-01

    Attenuated salmonellae represent an attractive vehicle for the delivery of heterologous protective antigens to the immune system. Here, we have investigated the influence of the genetic background of the host which regulates the growth and elimination of Salmonella cells on the cellular response induced against a foreign antigen delivered by an aroA Salmonella strain. We have tested CD4+ T-cell responses (cell proliferation and cytokine production) in various mouse strains following immunization with Salmonella typhimurium SL3261 expressing a high level of the recombinant Escherichia coli MalE protein. We were able to detect a CD4+ T-cell response against the recombinant MalE protein only in a restricted number of mouse strains, whereas all mice produced good levels of anti-MalE immunoglobulin G antibodies. The Ity gene did not play a major role in these differences in T-cell responses, since both Ity-resistant and -susceptible strains of mice were found to be unresponsive to MalE delivered by recombinant salmonellae. In contrast, when B10 congenic mice were used, a correlation was established between MalE-specific T-cell unresponsiveness and H-2 genes. The discrepancies described in this paper in the ability of various strains of mice to develop an efficient Th1 response against a recombinant antigen displayed by a live Salmonella vaccine underscore the difficulties that can be encountered in the vaccination of human populations by such a strategy. PMID:8890187

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

    PubMed Central

    Amin, Ruhul; Jesmin; Jamil, Hasan; Hossain, M. Anwar

    2009-01-01

    Melanoma antigen family (MAGE) genes are widely expressed in various tumor types but silent in normal cells except germ-line cells lacking human leukocyte antigen (HLA) expression. Over 25 MAGE genes have been identified in different tissues, mostly located in Xq28 of human chromosome and some of them in chromosome 3 and 15, containing either single or multiple-exons. This in silico study predicted the genes on hTERT location and identified a distant relative of MAGE gene located on chromosome 5. The study identified a single exon coding ~850 residues polypeptide sharing ~30% homology with Macfa-MAGE E1 and hMAGE-E1. dbEST search of the predicted transcript matches 5′ and 3′ flanking ESTs. The predicted protein showed sequence homology within the MAGE homology domain 2 (MHD2). UCSC genome annotation of CpG Island around the coding region reveals that this gene could be silent by methylation. Affymetrix all-exon track indicates the gene could be expressed in different tissues particularly in cancer cells as they widely undergo a genome wide demethylation process. PMID:20011463

  3. Cloning and Characterization of PRA1, a Gene Encoding a Novel pH-Regulated Antigen of Candida albicans

    PubMed Central

    Sentandreu, Maria; Elorza, M. Victoria; Sentandreu, Rafael; Fonzi, William A.

    1998-01-01

    Candida albicans is an opportunistic fungal pathogen of humans. The cell wall of the organism defines the interface between the pathogen and host tissues and is likely to play an essential and pivotal role in the host-pathogen interaction. The components of the cell wall critical to this interaction are undefined. Immunoscreening of a lambda expression library with sera raised against mycelial cell walls of C. albicans was used to identify genes encoding cell surface proteins. One of the positive clones represented a candidal gene that was differentially expressed in response to changes in the pH of the culture medium. Maximal expression occurred at neutral pH, with no expression detected below pH 6.0. On the basis of the expression pattern, the corresponding gene was designated PRA1, for pH-regulated antigen. The protein predicted from the nucleotide sequence was 299 amino acids long with motifs characteristic of secreted glycoproteins. The predicted surface localization and N glycosylation of the protein were directly demonstrated by cell fractionation and immunoblot analysis. Deletion of the gene imparted a temperature-dependent defect in hypha formation, indicating a role in morphogenesis. The PRA1 protein was homologous to surface antigens of Aspergillus spp. which react with serum from aspergillosis patients, suggesting that the PRA1 protein may have a role in the host-parasite interaction during candidal infection. PMID:9440517

  4. Splice acceptor site mutation of the transporter associated with antigen processing-1 gene in human bare lymphocyte syndrome

    PubMed Central

    Furukawa, Hiroshi; Murata, Shigeo; Yabe, Toshio; Shimbara, Naoki; Keicho, Naoto; Kashiwase, Kouichi; Watanabe, Kaoru; Ishikawa, Yoshihide; Akaza, Tatsuya; Tadokoro, Kenji; Tohma, Shigeto; Inoue, Tetsufumi; Tokunaga, Katsushi; Yamamoto, Kazuhiko; Tanaka, Keiji; Juji, Takeo

    1999-01-01

    Expression of histocompatibility leukocyte antigen (HLA) class I molecules on the cell surface depends on the heterodimer of the transporter associated with antigen processing 1 and 2 (TAP1 and TAP2), which transport peptides cleaved by proteasome to the class I molecules. Defects in the TAP2 protein have been reported in two families with HLA class I deficiency, the so-called bare lymphocyte syndrome (BLS) type I. We have, to our knowledge, identified for the first time a splice site mutation in the TAP1 gene of another BLS patient. In addition, class I heavy chains (HCs) did not form the normal complex with tapasin in the endoplasmic reticulum (ER) of the cells of our patient. J. Clin. Invest. 103:649–652 (1999) PMID:10074494

  5. Genes that can be mutated to unmask hidden antigenic determinants in the cuticle of the nematode Caenorhabditis elegans.

    PubMed Central

    Politz, S M; Philipp, M; Estevez, M; O'Brien, P J; Chin, K J

    1990-01-01

    Rabbit antisera directed against a mixture of proteins solubilized from the wild-type adult Caenorhabditis elegans cuticle were used to isolate mutants, induced by ethyl methanesulfonate treatment, that exhibit alterations in surface antigenicity by immunofluorescence. Genetic mapping and complementation data for four such mutations define two genes, srf-2(I) and srf-3(IV). The mutant phenotypes observed by immunofluorescence appear to result from unmasking of antigenic determinants that are normally hidden in the wild-type cuticle. In support of this hypothesis, surface radioiodination experiments indicate that components labeled on the wild-type surface are missing or less readily labeled on the surface of srf-2 and srf-3 mutants. Images PMID:1691498

  6. Transfection of neonatal rat Schwann cells with SV-40 large T antigen gene under control of the metallothionein promoter

    PubMed Central

    1987-01-01

    Secondary cultures of Schwann cells were transfected with a plasmid containing the SV-40 T antigen gene expressed under the control of the mouse metallothionein-I promoter. We used the calcium phosphate method for transfection and obtained a transfection efficiency of 0.01%. The colonies were cloned by limited dilution, and these cloned cell lines were carried in medium containing zinc chloride (100 microM). One cloned cell line, which has now been carried for 180 doublings, appears to have a transformed phenotype with a doubling time of 20 h. These cells express SV-40 T antigen while maintaining established Schwann cell properties (positive staining for 217c, Ran-2, A5E3, glial fibrillary acidic protein, presence of 2',3'-cyclic nucleotide phosphohydrolase [CNPase] activity, and the ability to synthesize sulfogalactosylceramide and mRNA for the myelin protein, P0). Removal of zinc chloride from the medium resulted in reduced expression of T antigen and a change in the appearance of the cells to a more bipolar shape, although they still did not exhibit contact inhibition and maintained a doubling time of 20 h. These cells now became Ran-2- negative and showed increases in CNPase activity and in their ability to synthesize sulfogalactosylceramide. The amount of P0 mRNA remained unchanged. Transfected Schwann cells, however, stopped dividing when they contacted either basal lamina or neurites and became bipolar in appearance. The Schwann cells in contact with the neurites then extended processes to wrap around bundles of neurites. Transfection with the SV-40 T antigen gene therefore provides a method for obtaining Schwann cell lines that continue to express properties associated with untransfected cells in culture and may be used to study axon-Schwann cell interaction. PMID:2824529

  7. Analysis of gene selection in reassortant formation between canine rotavirus K9 and human rotaviruses with different antigenic specificities.

    PubMed

    Kobayashi, N; Taniguchi, K; Urasawa, T; Urasawa, S

    1993-01-01

    A number of antigenic mosaic reassortants which have neutralization proteins VP4 and VP7 derived from different parental strains were analysed in order to study gene selection in reassortant formation between animal and human rotaviruses (HRV). These reassortants were isolated from mixed infection of MA-104 cells with canine rotavirus strain K9 (subgroup I and G serotype 3) and HRV strains (with subgroup I or II antigen and G serotype 1-4, 9 or 12 antigen), through repeated selections with anti-VP4 and anti-VP7 neutralizing monoclonal antibodies directed specifically at HRV and K9, respectively. By serological and genomic analyses, all the isolated clones were found to be antigenic mosaic reassortants possessing VP4 of K9 and VP7 of HRV. In the reassortants between strain K9 and one of the six strains of subgroup II HRV, a single or a few genotypes with particular constellations of RNA segments were predominant, with only a few RNA segments including gene 4 (encoding VP4) being derived from K9. In contrast, in the reassortants between strain K9 and any one of the subgroup I HRV, more than nine different genotypes were identified and various RNA segments, except for segments 8 and 10, were derived from K9. These findings indicated that the RNA segments of K9 might be reassorted more readily with those of subgroup I HRV than with those of subgroup II HRV, suggesting the possible existence of functional mechanisms which determine the extent of diversity of genome selection depending on the pairs of parent strains in the reassortant formation. PMID:7506839

  8. A novel translational regulation function for the simian virus 40 large-T antigen gene.

    PubMed Central

    Rajan, P; Swaminathan, S; Zhu, J; Cole, C N; Barber, G; Tevethia, M J; Thimmapaya, B

    1995-01-01

    Cells use the interferon-induced, double-stranded-RNA-dependent protein kinase PKR as a defense against virus infections. Upon activation, PKR phosphorylates and thereby inactivates the protein synthesis initiation factor eIF-2, resulting in the cessation of protein synthesis. Viruses have evolved various strategies to counteract this cellular defense. In this paper, we show that simian virus 40 (SV40) large-T antigen can antagonize the translational inhibitory effect resulting from the activation of PKR in virus-infected cells. Unlike the situation with other virus-host cell interactions, SV40 large-T antigen does not block the activation of PKR, suggesting that SV40 counteracts the cellular antiviral response mediated by PKR at a step downstream of PKR activation. Mutational analysis of large-T antigen indicates that a domain located between amino acids 400 and 600 of large-T antigen is responsible for this function. These results define a novel translational regulatory function for the SV40 large-T antigen. PMID:7815544

  9. Nitrocellulose immunoblotting for identification and molecular gene cloning of Eimeria maxima antigens that stimulate lymphocyte proliferation.

    PubMed Central

    Bumstead, J M; Dunn, P P; Tomley, F M

    1995-01-01

    An immunoblotting technique was used to identify lymphostimulatory antigens within sized polypeptide fractions of Eimeria maxima sporozoites. Six fractions contained polypeptides that specifically stimulated the proliferation of immune lymphocytes in an in vitro assay, and polyclonal antisera were made in rabbits against these fractions. cDNA clones, isolated with antisera against a lymphostimulatory fraction of around 70 kDa, were found to encode four different antigens including a classical hsp70, a molecule homologous to an endoplasmic reticulum chaperonin (BiP/GRP), and a calcium-dependent serine/threonine protein kinase that appears homologous to a recently described molecule from Plasmodium falciparum. The protein kinase cDNA clone was overexpressed in Escherichia coli, and the recombinant antigen was found to induce both antibody and lymphoproliferative responses in chickens when administered subcutaneously. Thus, immunoblotting, in combination with in vitro lymphoproliferation assays, can be used as an initial screen for the identification of lymphostimulatory antigens from a complex pool of polypeptides, and a combination of cDNA cloning, expression, and immunization allows assessment of the lymphostimulatory activity of individual polypeptides. These studies should facilitate further evaluation of antigens that are potential candidates for inclusion in a recombinant vaccine against poultry coccidiosis. PMID:8548529

  10. Low expression of human histocompatibility leukocyte antigen-DR is associated with hypermethylation of human histocompatibility leukocyte antigen-DR alpha gene regions in B cells from patients with systemic lupus erythematosus.

    PubMed Central

    Sano, H; Compton, L J; Shiomi, N; Steinberg, A D; Jackson, R A; Sasaki, T

    1985-01-01

    The relationship between the expression of HLA-DR antigens and the HLA-DR alpha gene methylation was examined in systemic lupus erythematosus (SLE). Using permanent B cell lines, we found reduced DR expression in SLE. The low DR expression was correlated with high anti-DNA antibody titers in patients' sera. The amounts of DR alpha message were lower in SLE cells than in normal controls, suggesting that the low expression of DR antigens is associated with gene functions. The extent of DNA methylation was examined at five CCGG sites in the HLA-DR alpha locus. DNA from both SLE and normal cells showed variable methylation patterns. Since the DR alpha gene is a single-copy gene, such a variability is the result of assaying a mixture of transformed clones containing methylated DR alpha gene, with other clones containing unmethylated DR alpha gene. A distinctive feature of normal cells was a consistent methylation pattern: 12 normal cell lines showed exactly the same pattern. In contrast, 28 SLE cell lines showed a cell-line-specific methylation, and hypermethylation at the DR alpha locus. The hypermethylation is often associated with transcriptionally inactive genes. Thus, our results suggest that (a) B cells with hypermethylated DR genes might express no or few DR antigens; (b) the ratio of cells with differently methylated DR genes is consistent in normal individuals, while, in SLE patients, cells with hypermethylated DR genes predominate, resulting in apparently reduced DR antigen expression; and (c) the aberrant DR expression could be associated directly with immunoregulatory dysfunctions in SLE disease. Images PMID:2997276

  11. Cloning, sequencing, and expression of the apa gene coding for the Mycobacterium tuberculosis 45/47-kilodalton secreted antigen complex.

    PubMed

    Laqueyrerie, A; Militzer, P; Romain, F; Eiglmeier, K; Cole, S; Marchal, G

    1995-10-01

    Effective protection against a virulent challenge with Mycobacterium tuberculosis is induced mainly by previous immunization with living attenuated mycobacteria, and it has been hypothesized that secreted proteins serve as major targets in the specific immune response. To identify and purify molecules present in culture medium filtrate which are dominant antigens during effective vaccination, a two-step selection procedure was used to select antigens able to interact with T lymphocytes and/or antibodies induced by immunization with living bacteria and to counterselect antigens interacting with the immune effectors induced by immunization with dead bacteria. A Mycobacterium bovis BCG 45/47-kDa antigen complex, present in BCG culture filtrate, has been previously identified and isolated (F. Romain, A. Laqueyrerie, P. Militzer, P. Pescher, P. Chavarot, M. Lagranderie, G. Auregan, M. Gheorghiu, and G. Marchal, Infect. Immun. 61:742-750, 1993). Since the cognate antibodies recognize the very same antigens present in M. tuberculosis culture medium filtrates, a project was undertaken to clone, express, and sequence the corresponding gene of M. tuberculosis. An M. tuberculosis shuttle cosmid library was transferred in Mycobacterium smegmatis and screened with a competitive enzyme-linked immunosorbent assay to detect the clones expressing the proteins. A clone containing a 40-kb DNA insert was selected, and by means of subcloning in Escherichia coli, a 2-kb fragment that coded for the molecules was identified. An open reading frame in the 2,061-nucleotide sequence codes for a secreted protein with a consensus signal peptide of 39 amino acids and a predicted molecular mass of 28,779 Da. The gene was referred to as apa because of the high percentages of proline (21.7%) and alanine (19%) in the purified protein. Southern hybridization analysis of digested total genomic DNA from M. tuberculosis (reference strains H37Rv and H37Ra) indicated that the apa gene was present as a

  12. Epigenetic changes within the promoter regions of antigen processing machinery family genes in Kazakh primary esophageal squamous cell carcinoma.

    PubMed

    Sheyhidin, Ilyar; Hasim, Ayshamgul; Zheng, Feng; Ma, Hong

    2014-01-01

    The esophageal squamous cell carcinoma (ESCC) is thought to develop through a multi-stage process. Epigenetic gene silencing constitutes an alternative or complementary mechanism to mutational events in tumorigenesis. Posttranscriptional regulation of human leukocyte antigen class I (HLA-I) and antigen processing machinery (APM) proteins expression may be associated with novel epigenetic modifications in cancer development. In the present study, we determined the expression levels of HLA-I antigen and APM components by immunohistochemistry. Then by a bisulfite-sequencing PCR (BSP) approach, we identified target CpG islands methylated at the gene promoter region of APM family genes in a ESCC cell line (ECa109), and further quantitative analysis of CpG site specific methylation of these genes in cases of Kazakh primary ESCCs with corresponding non-cancerous esophageal tissues using the Sequenom MassARRAY platform. Here we showed that the development of ESCCs was accompanied by partial or total loss of protein expression of HLA-B, TAP2, LMP7, tapasin and ERp57. The results demonstrated that although no statistical significance was found of global target CpG fragment methylation level sof HLA-B, TAP2, tapasin and ERp57 genes between ESCC and corresponding non-cancerous esophageal tissues, there was significant differences in the methylation level of several single sites between the two groups. Of thesse only the global methylation level of LMP7 gene target fragments was statistically higher (0.0517±0.0357) in Kazakh esophageal cancer than in neighboring normal tissues (0.0380±0.0214, p<0.05). Our results suggest that multiple CpG sites, but not methylation of every site leads to down regulation or deletion of gene expression. Only some of them result in genetic transcription, and silencing of HLA-B, ERp57, and LMP7 expression through hypermethylation of the promoters or other mechanisms may contribute to mechanisms of tumor escape from immune surveillance in Kazakh

  13. Francisella tularensis Schu S4 O-antigen and capsule biosynthesis gene mutants induce early cell death in human macrophages.

    PubMed

    Lindemann, Stephen R; Peng, Kaitian; Long, Matthew E; Hunt, Jason R; Apicella, Michael A; Monack, Denise M; Allen, Lee-Ann H; Jones, Bradley D

    2011-02-01

    Francisella tularensis is capable of rampant intracellular growth and causes a potentially fatal disease in humans. Whereas many mutational studies have been performed with avirulent strains of Francisella, relatively little has been done with strains that cause human disease. We generated a near-saturating transposon library in the virulent strain Schu S4, which was subjected to high-throughput screening by transposon site hybridization through primary human macrophages, negatively selecting 202 genes. Of special note were genes in a locus of the Francisella chromosome, FTT1236, FTT1237, and FTT1238. Mutants with mutations in these genes demonstrated significant sensitivity to complement-mediated lysis compared with wild-type Schu S4 and exhibited marked defects in O-antigen and capsular polysaccharide biosynthesis. In the absence of complement, these mutants were phagocytosed more efficiently by macrophages than wild-type Schu S4 and were capable of phagosomal escape but exhibited reduced intracellular growth. Microscopic and quantitative analyses of macrophages infected with mutant bacteria revealed that these macrophages exhibited signs of cell death much earlier than those infected with Schu S4. These data suggest that FTT1236, FTT1237, and FTT1238 are important for polysaccharide biosynthesis and that the Francisella O antigen, capsule, or both are important for avoiding the early induction of macrophage death and the destruction of the replicative niche.

  14. Serological characterization and gene localization of an Escherichia coli-expressed 37-kilodalton Treponema pallidum antigen.

    PubMed Central

    Rodgers, G C; Laird, W J; Coates, S R; Mack, D H; Huston, M; Sninsky, J J

    1986-01-01

    A recombinant plasmid containing a 5.6-kilobase-pair DNA fragment of the Treponema pallidum genome was characterized by endonuclease mapping, and the encoded proteins were expressed in Escherichia coli and analyzed by use of in vitro transcription and translation. One of the proteins, identified as having a molecular weight of 37,000 (37K protein), was selected for further study. Initially, the seroreactivity of the partially purified 37K antigen was demonstrated by immunoblotting. After its purification to near homogeneity, the cloned T. pallidum protein was assessed for diagnostic significance by radioimmunoassay. Although first identified as seroreactive by screening with secondary syphilitic sera (T. E. Fehniger, A. M. Walfield, T. M. Cunningham, J. D. Radolf, J. N. Miller, and M. A. Lovett, Abstr. Annu. Meet. Am. Soc. Microbiol. 1985, B156, p. 44), the antigen was shown to be serologically reactive with antibodies in serum from all stages of syphilis but was not recognized by serum from controls by both immunoblotting and radioimmune assay. Further, a monospecific polyclonal rabbit antiserum generated to the 37K antigen recognized a polypeptide of the same molecular weight from T. pallidum but did not efficiently recognize proteins from five nonpathogenic treponemes tested. Therefore, because of reactivity with and specificity for T. pallidum antibodies, the 37K antigen may be of serodiagnostic value in the detection of syphilis. Images PMID:3522427

  15. Point mutation in essential genes with loss or mutation of the second allele: relevance to the retention of tumor-specific antigens.

    PubMed

    Beck-Engeser, G B; Monach, P A; Mumberg, D; Yang, F; Wanderling, S; Schreiber, K; Espinosa, R; Le Beau, M M; Meredith, S C; Schreiber, H

    2001-08-01

    Antigens that are tumor specific yet retained by tumor cells despite tumor progression offer stable and specific targets for immunologic and possibly other therapeutic interventions. Therefore, we have studied two CD4(+) T cell-recognized tumor-specific antigens that were retained during evolution of two ultraviolet-light-induced murine cancers to more aggressive growth. The antigens are ribosomal proteins altered by somatic tumor-specific point mutations, and the progressor (PRO) variants lack the corresponding normal alleles. In the first tumor, 6132A-PRO, the antigen is encoded by a point-mutated L9 ribosomal protein gene. The tumor lacks the normal L9 allele because of an interstitial deletion from chromosome 5. In the second tumor, 6139B-PRO, both alleles of the L26 gene have point mutations, and each encodes a different tumor-specific CD4(+) T cell-recognized antigen. Thus, for both L9 and L26 genes, we observe "two hit" kinetics commonly observed in genes suppressing tumor growth. Indeed, reintroduction of the lost wild-type L9 allele into the 6132A-PRO variant suppressed the growth of the tumor cells in vivo. Since both L9 and L26 encode proteins essential for ribosomal biogenesis, complete loss of the tumor-specific target antigens in the absence of a normal allele would abrogate tumor growth.

  16. Investigating the impact of hepatitis B virus surface gene polymorphism on antigenicity using ex vivo phenotyping.

    PubMed

    Ijaz, Samreen; Szypulska, Renata; Andrews, Nick; Tedder, Richard S

    2012-11-01

    The hepatitis B virus (HBV) surface antigen (HBsAg) is a complex protein, and understanding accurately the impact of amino acid changes on the antigenicity of the immunodominant a determinant must take this complexity into consideration. Epitope mapping with four mAbs was used to phenotype HBsAg directly from patients' sera to investigate the effect of mutations in their native genetic backbone. The expected mAb reactivity was established initially for samples harbouring 'wild-type' HBsAg sequences across genotypes A-E. The alteration of HBsAg antigenicity, defined by mAb epitope loss, was demonstrated in a number of samples with sequence-inferred amino acid changes. Individual mutations within the mapped epitopes to which the mAbs were directed usually affected their binding. However, the loss of more than one epitope was observed as the number of mutations within a sequence increased. Conversely, not all mutations occurring in the a determinant altered the HBsAg conformation. The genotype backbone, the specific amino acid substitution and amino acid changes occurring outside the major antigenic region appeared to be important in determining expression of the predicted epitope loss. These data clearly demonstrate that sequence-based methods alone may not accurately define HBsAg phenotype. This phenotyping methodology allows for the rapid and accurate identification of antigenically altered viruses and will greatly enhance current HBV surveillance, research and diagnostic activities. The data generated can be used to inform on public health issues relating to prevalence, transmission and impact of HBsAg mutants in HBV-infected populations. PMID:22855781

  17. The promoter of the human cystic fibrosis transmembrane conductance regulator gene directing SV40 T antigen expression induces malignant proliferation of ependymal cells in transgenic mice.

    PubMed

    Perraud, F; Yoshimura, K; Louis, B; Dalemans, W; Ali-Hadji, D; Schultz, H; Claudepierre, M C; Chartier, C; Danel, C; Bellocq, J P

    1992-05-01

    Transgenic mice bearing a human cystic fibrosis transmembrane conductance regulator (CFTR) promoter-SV40 T antigen fusion transgene were generated in order to localize in vivo the potential oncogenesis linked to the tissue-specific activity of the promoter for the CFTR gene. Surprisingly, the only site of tumors resulting from expression of the reporter onc gene was ependymal cells lining the brain ventricles. SV40 T antigen expression in these cells led to a consistent pathology in the first weeks of age: ependymoma and consequent hydrocephaly. Tumor-derived cell lines were established, characterized and shown to originate from SV40 T antigen-induced ependymoma. No pathological alterations were found in other organs, such as lungs and pancreas, in which cystic fibrosis is pathologically manifest in humans. Such transgenic mice and derived cell lines may represent valid models for analysing (1) the role of SV40 T antigen in ependymoma formation and (2) CFTR function in ependymal cells. PMID:1373882

  18. Distribution, gene sequence and expression in vivo of the plasmid encoded fimbrial antigen of Salmonella serotype Enteritidis.

    PubMed Central

    Woodward, M. J.; Allen-Vercoe, E.; Redstone, J. S.

    1996-01-01

    The pefA gene which encoded the serotype associated plasmid (SAP) mediated fimbrial major subunit antigen of Salmonella enterica serotype Typhimurium shared genetic identity with 128 of 706 salmonella isolates as demonstrated by dot (colony) hybridization. Seventy-seven of 113 isolates of Typhimurium and individual isolates of serotypes Bovis-morbificans, Cholerae-suis and Enteritidis phage type 9b hybridized pefA strongly, whereas 48 isolates of Enteritidis hybridized pefA weakly and one Enteritidis isolate of phage type 14b failed to hybridize. Individual isolates of 294 serotypes and 247 individual isolates of serotype Dublin did not hybridize pefA. Southern hybridization of plasmids extracted from Enteritidis demonstrated that the pefA gene probe hybridized strongly an atypical SAP of 80 kb in size harboured by one Enteritidis isolate of phage-type 9b, whereas the typical SAP of 58 kb in size harboured by 48 Enteritidis isolates hybridized weakly. One Enteritidis isolate of phage type 14b which failed to hybridize pefA in dot (colony) hybridization experiments was demonstrated to be plasmid free. A cosmid library of Enteritidis phage type 4 expressed in Escherichia coli K12 was screened by hybridization for the presence of pef sequences. Recombinant clones which were deduced to harbour the entire pef operon elaborated a PEF-like fimbrial structure at the cell surface. The PEF-like fimbrial antigen was purified from one cosmid clone and used in western blot experiments with sera from chickens infected with Enteritidis phage-type 4. Seroconversion to the fimbrial antigen was observed which indicated that the Enteritidis PEF-like fimbrial structure was expressed at some stage during infection. Nucleotide sequence analysis demonstrated that the pefA alleles of Typhimurium and Enteritidis phage-type 4 shared 76% DNA nucleotide and 82% deduced amino acid sequence identity. Images Fig. 1 Fig. 4 Fig. 5 PMID:8760946

  19. Modification of Hemodynamic and Immune Responses to Exposure with a Weak Antigen by the Expression of a Hypomorphic BMPR2 Gene

    PubMed Central

    Park, Sung-Hyun; Chen, Wen-Chi; Hoffman, Carol; Marsh, Leigh M.; West, James; Grunig, Gabriele

    2013-01-01

    Background Hypomorphic mutations in the bone morphogenic protein receptor (BMPR2) confer a much greater risk for developing pulmonary arterial hypertension (PAH). However, not all carriers of a mutation in the BMPR2 gene suffer from PAH. We have previously shown that prolonged T helper 2 (Th2) responses in the lungs to a mild antigen delivered via the airways induce severe pulmonary arterial remodeling, but no pulmonary hypertension. The current studies were designed to test the idea that Th2 responses to a mild antigen together with the expression of a hypomorphic BMPR2 gene would trigger pulmonary hypertension. Methodology/Principal Findings Mice that expressed a hypomorphic BMPR2 transgene (transgene-positive) and transgene-negative mice were either exposed to saline, or primed and exposed to a mild antigen (Ovalbumin) over a prolonged period of time. Only transgene-positive but not transgene-negative mice exposed to antigen developed significantly increased right ventricular systolic pressures, while both groups showed pulmonary artery remodeling with severe muscularization and airway inflammation to a similar degree. Antigen exposure resulted in a smaller increase in the percentage of Interleukin (IL)-13 positive T cells in the lymph nodes, and in a smaller increase in resistin-like-molecule (RELM)α expression and a decreased ratio of expression of IL-33 relative to its receptor (IL-1-receptor-like 1, IL1RL1-ST2) in the right ventricles of transgene-positive mice compared to transgene-negative animals. Furthermore, only antigen-challenged transgene-positive mice showed a significant increase in Interferon (IFN)γ positive T cells over saline-exposed controls. Conclusions/Significance Our study suggests that exposure with a mild Th2 antigen can trigger pulmonary hypertension on the background of the expression of a hypomorphic BMPR2 gene and that conversely, the expression of the hypomorphic BMPR2 gene can alter the immune response to a mild, inhaled antigen

  20. Maternal inhibition of hepatitis B surface antigen gene expression in transgenic mice correlates with de novo methylation.

    PubMed

    Hadchouel, M; Farza, H; Simon, D; Tiollais, P; Pourcel, C

    Differential modifications of the genome during gametogenesis result in a functional difference between the paternal and maternal genomes at the moment of fertilization. A possible cause of this imprinting is the methylation of DNA. The insertion of foreign DNA into transgenic mice allows the tagging of regions that are differentially methylated during gametogenesis. We describe here a transgenic mouse strain in which the expression of the hepatitis B surface antigen gene is irreversibly repressed following its passage through the female germ line. This inhibition is accompanied by the methylation of all the HpaII and HhaI sites within the foreign gene, which we have shown to be integrated into a site on chromosome 13. The irreversibility reported here contrasts with what is found with other transgenic mice sequences which are reversibly methylated after passage through the male or female germ line, though in both cases methylation appears to be important in the imprinting process.

  1. The human application of gene therapy to re-program T-cell specificity using chimeric antigen receptors

    PubMed Central

    Guerrero, Alan D; Moyes, Judy S; Cooper, Laurence JN

    2014-01-01

    The adoptive transfer of T cells is a promising approach to treat cancers. Primary human T cells can be modified using viral and non-viral vectors to promote the specific targeting of cancer cells via the introduction of exogenous T-cell receptors (TCRs) or chimeric antigen receptors (CARs). This gene transfer displays the potential to increase the specificity and potency of the anticancer response while decreasing the systemic adverse effects that arise from conventional treatments that target both cancerous and healthy cells. This review highlights the generation of clinical-grade T cells expressing CARs for immunotherapy, the use of these cells to target B-cell malignancies and, particularly, the first clinical trials deploying the Sleeping Beauty gene transfer system, which engineers T cells to target CD19+ leukemia and non-Hodgkin's lymphoma. PMID:25189715

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

    PubMed

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

    2015-06-04

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

  3. Escherichia coli serogroup O2 and O28ac O-antigen gene cluster sequences and detection of pathogenic E. coli O2 and O28ac by PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The O antigen gene clusters of Escherichia coli serogroups O2 and O28ac were sequenced. Thirteen open reading frames (ORFs) were identified in the O antigen gene cluster of E. coli O2 strain O2:H4 U 9-41, and 7 ORFs were identified in the O antigen gene cluster of E. coli serogroup O28ac strain O2...

  4. The SPANX gene family of cancer/testis-specific antigens: rapid evolution and amplification in African great apes and hominids.

    PubMed

    Kouprina, Natalay; Mullokandov, Michael; Rogozin, Igor B; Collins, N Keith; Solomon, Greg; Otstot, John; Risinger, John I; Koonin, Eugene V; Barrett, J Carl; Larionov, Vladimir

    2004-03-01

    Human sperm protein associated with the nucleus on the X chromosome (SPANX) genes comprise a gene family with five known members (SPANX-A1, -A2, -B, -C, and -D), encoding cancer/testis-specific antigens that are potential targets for cancer immunotherapy. These highly similar paralogous genes cluster on the X chromosome at Xq27. We isolated and sequenced primate genomic clones homologous to human SPANX. Analysis of these clones and search of the human genome sequence revealed an uncharacterized group of genes, SPANX-N, which are present in all primates as well as in mouse and rat. In humans, four SPANX-N genes comprise a series of tandem duplicates at Xq27; a fifth member of this subfamily is located at Xp11. Similarly to SPANX-A/D, human SPANX-N genes are expressed in normal testis and some melanoma cell lines; testis-specific expression of SPANX is also conserved in mouse. Analysis of the taxonomic distribution of the long and short forms of the intron indicates that SPANX-N is the ancestral form, from which the SPANX-A/D subfamily evolved in the common ancestor of the hominoid lineage. Strikingly, the coding sequences of the SPANX genes evolved much faster than the intron and the 5' untranslated region. There is a strong correlation between the rates of evolution of synonymous and nonsynonymous codon positions, both of which are accelerated 2-fold or more compared to the noncoding sequences. Thus, evolution of the SPANX family appears to have involved positive selection that affected not only the protein sequence but also the synonymous sites in the coding sequence.

  5. Chromosomal localization of the gene for human B-cell antigen CD40

    SciTech Connect

    Ramesh, N.; Geha, R. ); Ramesh, V.; Gusella, J.F. )

    1993-05-01

    CD40 is a surface glycoprotein expressed on all human B lymphocytes and plays an important role in B-cell development, growth, and differentiation. Anti-CD40 monoclonal antibodies cause isotype switching in B cells treated with IL-4. CD40 is a member of a family of proteins that include low-affinity nerve growth factor receptor, TNF receptor, and the antigen Fas. The ligand for CD40 had been recently identified and had been assigned to the X chromosome. Using a panel of human-rodent somatic cell hybrids, the authors now show that CD40 maps to human chromosome 20.

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

    SciTech Connect

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

    1988-01-15

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

  7. Identification of the gene encoding the major latency-associated nuclear antigen of the Kaposi's sarcoma-associated herpesvirus.

    PubMed Central

    Kedes, D H; Lagunoff, M; Renne, R; Ganem, D

    1997-01-01

    Over 85% of patients with Kaposi's sarcoma (KS) are seropositive for antibodies to the latency-associated nuclear antigen (LANA) expressed in B cell lines infected with Kaposi's sarcoma-associated herpesvirus (KSHV). The presence of antibodies to LANA strongly correlates with the risk of developing the disease. However, the identity of the protein(s) comprising LANA and the corresponding gene(s) has remained unclear. To identify potential latent gene candidates for LANA, we probed total RNA extracted from BCBL-1 cells (a B cell line latently infected with KSHV) using lambda clones that span the KSHV genome. One region encoding latent transcripts spanned KSHV open reading frames (orfs) 71 (K13), 72 (v-cyclin), and 73. Among these, however, only orf 73, when expressed in heterologous mammalian cell systems, reacted with KSHV antibody-positive human sera, resulting in a punctate nuclear staining pattern reminiscent of LANA in BCBL-1 cells. Furthermore, extracts from cells expressing the orf 73 protein product specifically blocked the binding of KS patient antibodies to LANA. Finally, seroreactivity with recombinant orf 73 protein exactly paralleled reactivity with classical LANA as expressed in BCBL-1 cells, both in KS patients and in other groups. Together, these data support the identification of KSHV orf 73 as the gene encoding the dominant immunogenic component of LANA. PMID:9366576

  8. A BTP1 prophage gene present in invasive non-typhoidal Salmonella determines composition and length of the O-antigen of the lipopolysaccharide.

    PubMed

    Kintz, Erica; Davies, Mark R; Hammarlöf, Disa L; Canals, Rocío; Hinton, Jay C D; van der Woude, Marjan W

    2015-04-01

    Salmonella Typhimurium isolate D23580 represents a recently identified ST313 lineage of invasive non-typhoidal Salmonellae (iNTS). One of the differences between this lineage and other non-iNTS S. Typhimurium isolates is the presence of prophage BTP1. This prophage encodes a gtrC gene, implicated in O-antigen modification. GtrC(BTP) (1) is essential for maintaining O-antigen length in isolate D23580, since a gtr(BTP) (1) mutant yields a short O-antigen. This phenotype can be complemented by gtrC(BTP) (1) or very closely related gtrC genes. The short O-antigen of the gtr(BTP) (1) mutant was also compensated by deletion of the BTP1 phage tailspike gene in the D23580 chromosome. This tailspike protein has a putative endorhamnosidase domain and thus may mediate O-antigen cleavage. Expression of the gtrC(BTP) (1) gene is, in contrast to expression of many other gtr operons, not subject to phase variation and transcriptional analysis suggests that gtrC is produced under a variety of conditions. Additionally, GtrC(BTP) (1) expression is necessary and sufficient to provide protection against BTP1 phage infection of an otherwise susceptible strain. These data are consistent with a model in which GtrC(BTP) (1) mediates modification of the BTP1 phage O-antigen receptor in lysogenic D23580, and thereby prevents superinfection by itself and other phage that uses the same O-antigen co-receptor. PMID:25586744

  9. Gene Encoding Duffy Antigen/Receptor for Chemokines Is Associated with Asthma and IgE in Three Populations

    PubMed Central

    Vergara, Candelaria; Tsai, Yuhjung J.; Grant, Audrey V.; Rafaels, Nicholas; Gao, Li; Hand, Tracey; Stockton, Maria; Campbell, Monica; Mercado, Dilia; Faruque, Mezbah; Dunston, Georgia; Beaty, Terri H.; Oliveira, Ricardo Riccio; Ponte, Eduardo V.; Cruz, Alvaro A.; Carvalho, Edgar; Araujo, Maria Ilma; Watson, Harold; Schleimer, Robert P.; Caraballo, Luis; Nickel, Renate G.; Mathias, Rasika A.; Barnes, Kathleen C.

    2008-01-01

    Rationale: Asthma prevalence and severity are high among underserved minorities, including those of African descent. The Duffy antigen/receptor for chemokines is the receptor for Plasmodium vivax on erythrocytes and functions as a chemokine-clearing receptor. Unlike European populations, decreased expression of the receptor on erythrocytes is common among populations of African descent, and results from a functional T-46C polymorphism (rs2814778) in the promoter. This variant provides an evolutionary advantage in malaria-endemic regions, because Duffy antigen/receptor for chemokines-negative erythrocytes are more resistant to infection by P. vivax. Objectives: To determine the role of the rs2814778 polymorphism in asthma and atopy as measured by total serum IgE levels among four populations of African descent (African Caribbean, African American, Brazilian, and Colombian) and a European American population. Methods: Family-based association tests were performed in each of the five populations to test for association between the rs2814778 polymorphism and asthma or total IgE concentration. Measurements and Main Results: Asthma was significantly associated with the rs2814778 polymorphism in the African Caribbean, Colombian, and Brazilian families (P < 0.05). High total IgE levels were associated with this variant in African Caribbean and Colombian families (P < 0.05). The variant allele was not polymorphic among European Americans. Conclusions: Susceptibility to asthma and atopy among certain populations of African descent is influenced by a functional polymorphism in the gene encoding Duffy antigen/receptor for chemokines. This genetic variant, which confers resistance to malarial parasitic infection, may also partially explain ethnic differences in morbidity of asthma. PMID:18827265

  10. An African swine fever virus gene with similarity to the T-lymphocyte surface antigen CD2 mediates hemadsorption.

    PubMed

    Borca, M V; Kutish, G F; Afonso, C L; Irusta, P; Carrillo, C; Brun, A; Sussman, M; Rock, D L

    1994-03-01

    An open reading frame, LMW8-DR, in the African swine fever virus (ASFV) genome possesses striking similarity to the lymphocyte membrane antigen CD2. All characterized CD2 domains, including the amino-terminal signal sequence, IgV, hinge, IgC2, stalk, transmembrane, and proline-rich carboxy cytoplasmic domains, are highly conserved in the ASFV gene. Critical residues for the binding of the lymphocyte function-associated antigen (LFA-3) and CD59 and for T-cell activation are also partially conserved. LMW8-DR is actively transcribed in ASFV-infected swine macrophages and Vero cells at late times in the infection cycle and Vero and COS cells transiently expressing the LMW8-DR open reading frame hemadsorbed swine red blood cells. The structural and functional similarities of LMW8-DR to CD2, a protein that is involved in cell-cell adhesion and immune response modulation, suggest a possible role in the pathogenesis of ASFV infection.

  11. Gene frequencies of the HPA-1 to -6 and -15 human platelet antigens in Tunisian blood donors.

    PubMed

    Hadhri, S; Gandouz, R; Chatti, N; Bierling, P; Skouri, H

    2010-09-01

    Gene frequencies of mainly human platelet antigens (HPA) -1 to -6 and -15 were determined in 116 Tunisian blood donors. The distribution of HPA-1, -3 and -5 systems approach those found in other Maghrebian populations. Tunisians have the highest frequency of HPA-1b and -5b alleles. The distribution of HPA-1a allele and HPA-4, -6 and -15 systems is similar to Caucasians. Phylogenetic study using the neighbor-joining method and principal component multivariate analysis demonstrate that Tunisians are more closely related to western than to eastern Mediterraneans. This immunogenetic study highlights the relatedness between Mediterranean populations and will serve as a baseline study for future clinical research involving platelet disorders among Tunisians. PMID:20492600

  12. Fifteen Years of Gene Therapy Based on Chimeric Antigen Receptors: “Are We Nearly There Yet?”

    PubMed Central

    Savoldo, Barbara; Brenner, Malcolm

    2009-01-01

    Abstract “T-body” or chimeric antigen receptor (CAR) technology, which combines the specificity of an antibody with the homing, tissue penetration, and target cell destruction of T cells, was first described in 1993. After many years of unmet promise, significant improvements in gene transfer, including the development of efficient retroviral vectors for transduction of human T cells, and better understanding of immunological pathways and immune cell interactions, are allowing this technology to reach a critical phase of evaluation, in which we will learn whether the approach can truly meet expectations. In this review we summarize the concept of CAR-based immunotherapy, describe the steps accomplished, and outline the future progress we need to make if this approach is truly to improve cancer immunotherapy. PMID:19702437

  13. Role of human leukocyte antigen, killer-cell immunoglobulin-like receptors, and cytokine gene polymorphisms in leptospirosis.

    PubMed

    Fialho, Raquel Nunes; Martins, Luís; Pinheiro, João Paulo; Bettencourt, Bruno Filipe; Couto, Ana Rita; Santos, Margarida Rodrigues; Peixoto, Maria José; Garrett, Francisco; Leal, João; Tomás, Ana Maria; Bruges-Armas, Jácome

    2009-11-01

    Leptospirosis is an emerging zoonotic disease caused by pathogenic species of the genus Leptospira. It has a broad range of clinical presentations in humans. Although progress has been made in the characterization of the host immune system factors that may affect disease progression and outcome, to date few reports have addressed the role of genetic polymorphisms in the susceptibility to leptospirosis. In this work a group of patients with a history of leptospiral infection and a control group were compared for polymorphisms in the human leukocyte antigen (HLA), in killer-cell immunoglobulin-like receptors (KIR), and in cytokine genes. Alleles in the HLA-A and -B loci were associated with susceptibility, as were the class I haplotype A*01-B*08-Cw*07 and the 8.1 ancestral haplotype (A*01-B*08-Cw*07-DRB1*03-DQB1*02). Single nucleotide polymorphisms in the interleukin (IL)-4 and IL-4Ralpha genes also had significantly higher frequencies in the patient group. No association was reported between KIR gene profile and leptospirosis. This work highlights the importance of using genetic polymorphisms to better understand the mechanisms involved in the immune response to leptospirosis.

  14. Somatic hypermutation of immunoglobulin genes: lessons from proliferating cell nuclear antigenK164R mutant mice.

    PubMed

    Langerak, Petra; Krijger, Peter H L; Heideman, Marinus R; van den Berk, Paul C M; Jacobs, Heinz

    2009-03-12

    Proliferating cell nuclear antigen (PCNA) encircles DNA as a ring-shaped homotrimer and, by tethering DNA polymerases to their template, PCNA serves as a critical replication factor. In contrast to high-fidelity DNA polymerases, the activation of low-fidelity translesion synthesis (TLS) DNA polymerases seems to require damage-inducible monoubiquitylation (Ub) of PCNA at lysine residue 164 (PCNA-Ub). TLS polymerases can tolerate DNA damage, i.e. they can replicate across DNA lesions. The lack of proofreading activity, however, renders TLS highly mutagenic. The advantage is that B cells use mutagenic TLS to introduce somatic mutations in immunoglobulin (Ig) genes to generate high-affinity antibodies. Given the critical role of PCNA-Ub in activating TLS and the role of TLS in establishing somatic mutations in immunoglobulin genes, we analysed the mutation spectrum of somatically mutated immunoglobulin genes in B cells from PCNAK164R knock-in mice. A 10-fold reduction in A/T mutations is associated with a compensatory increase in G/C mutations-a phenotype similar to Poleta and mismatch repair-deficient B cells. Mismatch recognition, PCNA-Ub and Poleta probably act within one pathway to establish the majority of mutations at template A/T. Equally relevant, the G/C mutator(s) seems largely independent of PCNAK(164) modification.

  15. Genetic variations in merozoite surface antigen genes of Babesia bovis detected in Vietnamese cattle and water buffaloes.

    PubMed

    Yokoyama, Naoaki; Sivakumar, Thillaiampalam; Tuvshintulga, Bumduuren; Hayashida, Kyoko; Igarashi, Ikuo; Inoue, Noboru; Long, Phung Thang; Lan, Dinh Thi Bich

    2015-03-01

    The genes that encode merozoite surface antigens (MSAs) in Babesia bovis are genetically diverse. In this study, we analyzed the genetic diversity of B. bovis MSA-1, MSA-2b, and MSA-2c genes in Vietnamese cattle and water buffaloes. Blood DNA samples from 258 cattle and 49 water buffaloes reared in the Thua Thien Hue province of Vietnam were screened with a B. bovis-specific diagnostic PCR assay. The B. bovis-positive DNA samples (23 cattle and 16 water buffaloes) were then subjected to PCR assays to amplify the MSA-1, MSA-2b, and MSA-2c genes. Sequencing analyses showed that the Vietnamese MSA-1 and MSA-2b sequences are genetically diverse, whereas MSA-2c is relatively conserved. The nucleotide identity values for these MSA gene sequences were similar in the cattle and water buffaloes. Consistent with the sequencing data, the Vietnamese MSA-1 and MSA-2b sequences were dispersed across several clades in the corresponding phylogenetic trees, whereas the MSA-2c sequences occurred in a single clade. Cattle- and water-buffalo-derived sequences also often clustered together on the phylogenetic trees. The Vietnamese MSA-1, MSA-2b, and MSA-2c sequences were then screened for recombination with automated methods. Of the seven recombination events detected, five and two were associated with the MSA-2b and MSA-2c recombinant sequences, respectively, whereas no MSA-1 recombinants were detected among the sequences analyzed. Recombination between the sequences derived from cattle and water buffaloes was very common, and the resultant recombinant sequences were found in both host animals. These data indicate that the genetic diversity of the MSA sequences does not differ between cattle and water buffaloes in Vietnam. They also suggest that recombination between the B. bovis MSA sequences in both cattle and water buffaloes might contribute to the genetic variation in these genes in Vietnam.

  16. Expression of the gene encoding secretor type galactoside 2 α fucosyltransferase (FUT2) and ABH antigens in patients with oral lesions

    PubMed Central

    Campi, Carlos; Escovich, Livia; Moreno, Alejandra; Racca, Liliana; Racca, Amelia; Cotorruelo, Carlos

    2012-01-01

    Objective: The aim of this work was to evaluate the expression of FUT2 gene in saliva and histo ABH antigens of patients with oral lesions. Study Design: In total 178 subjects were examined, half of whom suffered from oral pre-cancerous and cancerous lesions, while the other half were the healthy control group We analyzed the FUT 2 polymorphism by ASO-PCR (allele specific oligonucleotid – polymerase chain reaction) with specific primers for G428 allele and the wild type allele of FUT2 gene. To reveal A, B and H antigens in tissue sections of the patients (n= 89) we used a modified specific red cell adherence technique. Results: We found a high intensity of oral disease in the non-secretor group (OR = 2.43). A total of 58% of the patients with oral pre-cancerous and cancerous lesions was non secretors (se_/_), in contrast with the healthy population (21.5%). A strongly positive reaction was defined as a sheet of indicator erythrocytes adhered to the epithelial cells. In 31 of the 54 samples analyzed the test showed slightly positive results on atypical areas, and there was a complete antigen deletion in areas affected by neoplasia. Nineteen samples showed a total absence of ABH antigens in both histologically normal and pathological areas. Blood group antigens were expressed at a high level in benign and highly differentiated malignant tumors. In poorly differentiated malignant tumors, they were mostly absent. Conclusion: Considering these results we suggest the use of this method to monitor probable preneoplastic lesions in risk population, especially in those with no secretor status (absence of FUT2 gene). Key words: FUT2 gene, ABH antigens, secretor status, oral lesions. PMID:22157667

  17. Escherichia coli O-antigen gene clusters of serogroups O62, O68, O131, O140, O142, and O163: DNA sequences and similarity between O62 and O68, and PCR-based serogrouping

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The DNA sequences of the O-antigen gene clusters of the Escherichia coli serogroups O62, O131, O140, O142 and O163 were determined. There were 9-14 open reading frames (ORFs) identified, encoding genes required for O-antigen sugar biosynthesis, transfer, and processing. Primers based on the wzx (O...

  18. DNA sequence and analysis of the O-antigen gene clusters of Escherichia coli serogroups O62, O68, O131, O140, O142, and O163 and serogroup-specific PCR assays

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The DNA sequence of the O-antigen gene clusters of Escherichia coli serogroups O62, O68, O131, O140, O142, and O163 was determined. There were 9 to 12 open reading frames (ORFs) identified, encoding genes required for O-antigen sugar biosynthesis, transfer, and processing. Primers based on the wzx...

  19. Lack of cosegregation of the subgroup II antigens on genes 2 and 6 in porcine rotaviruses.

    PubMed Central

    Svensson, L; Padilla-Noriega, L; Taniguchi, K; Greenberg, H B

    1990-01-01

    The rotavirus subgroup I and II specificities associated with gene 2 and 6 products (vp2 and vp6, respectively) were shown not to cosegregate in a number of porcine rotavirus strains. The porcine OSU rotavirus strain and OSU-vp7-like strains were all found to possess a subgroup II-specific region on vp2 and a subgroup I-specific region on vp6. Of interest is the observation that the subgroup II-specific epitope on vp2 appears to be present only in human and porcine rotavirus strains, suggesting a possible human-pig ancestral lineage for gene 2. Images PMID:1688386

  20. Targeted gene delivery to the synovial pannus in antigen-induced arthritis by ultrasound-targeted microbubble destruction in vivo.

    PubMed

    Xiang, Xi; Tang, Yuanjiao; Leng, Qianying; Zhang, Lingyan; Qiu, Li

    2016-02-01

    The purpose of this study was to optimize an ultrasound-targeted microbubble destruction (UTMD) technique to improve the in vivo transfection efficiency of the gene encoding enhanced green fluorescent protein (EGFP) in the synovial pannus in an antigen-induced arthritis rabbit model. A mixture of microbubbles and plasmids was locally injected into the knee joints of an antigen-induced arthritis (AIA) rabbits. The plasmid concentrations and ultrasound conditions were varied in the experiments. We also tested local articular and intravenous injections. The rabbits were divided into five groups: (1) ultrasound+microbubbles+plasmid; (2) ultrasound+plasmid; (3) microbubble+plasmid; (4) plasmid only; (5) untreated controls. EGFP expression was observed by fluorescent microscope and immunohistochemical staining in the synovial pannus of each group. The optimal plasmid dosage and ultrasound parameter were determined based on the results of EGFP expression and the present and absent of tissue damage under light microscopy. The irradiation procedure was performed to observe the duration of the EGFP expression in the synovial pannus and other tissues and organs, as well as the damage to the normal cells. The optimal condition was determined to be a 1-MHz ultrasound pulse applied for 5 min with a power output of 2 W/cm(2) and a 20% duty cycle along with 300 μg of plasmid. Under these conditions, the synovial pannus showed significant EGFP expression without significant damage to the surrounding normal tissue. The EGFP expression induced by the local intra-articular injection was significantly more increased than that induced by the intravenous injection. The EGFP expression in the synovial pannus of the ultrasound+microbubbles+plasmid group was significantly higher than that of the other four groups (P<0.05). The expression peaked on day 5, remained detectable on day 40 and disappeared on day 60. No EGFP expression was detected in the other tissues and organs. The UTMD

  1. Polymorphism in the Gene Coding for the Immunodominant Antigen gp43 from the Pathogenic Fungus Paracoccidioides brasiliensis

    PubMed Central

    Morais, Flavia V.; Barros, Tânia F.; Fukada, Márcio K.; Cisalpino, Patrícia S.; Puccia, Rosana

    2000-01-01

    The gp43 glycoprotein is an immune-dominant antigen in patients with paracoccidioidomycosis (PCM). It is protective against murine PCM and is a putative virulence factor. The gp43 gene of Paracoccidioides brasiliensis B-339 is located in a 1,329-bp DNA fragment that includes two exons, a 78-bp intron, and a leader peptide-coding region of 105 bp. Polymorphism in gp43 has been suggested by the occurrence, in the same isolate or among different fungal samples, of isoforms with distinct isoelectric points. In the present study we aligned and compared with a consensus sequence the gp43 precursor genes of 17 P. brasiliensis isolates after sequencing two PCR products from each fungal sample. The genotypic types detected showed 1 to 4 or 14 to 15 informative substitution sites, preferentially localized between 578 and 1166 bp. Some nucleotide differences within individual isolates (noninformative sites) resulted in a second isoelectric point for the deduced protein. The most polymorphic sequences were also phylogenetically distant from the others and encoded basic gp43 isoforms. The three isolates in this group were from patients with chronic PCM, and their DNA restriction patterns were distinct in Southern blots. The nucleotides encoding the inner core of the murine T-cell-protective epitope of gp43 were conserved, offering hope for the development of a universal vaccine. PMID:11060052

  2. A non-stop S-antigen gene mutation is associated with late onset hereditary retinal degeneration in dogs

    PubMed Central

    Jordan, Julie Ann; Aguirre, Gustavo D.; Acland, Gregory M.

    2013-01-01

    Purpose To identify the causative mutation of canine progressive retinal atrophy (PRA) segregating as an adult onset autosomal recessive disorder in the Basenji breed of dog. Methods Basenji dogs were ascertained for the PRA phenotype by clinical ophthalmoscopic examination. Blood samples from six affected cases and three nonaffected controls were collected, and DNA extraction was used for a genome-wide association study using the canine HD Illumina single nucleotide polymorphism (SNP) array and PLINK. Positional candidate genes identified within the peak association signal region were evaluated. Results The highest -Log10(P) value of 4.65 was obtained for 12 single nucleotide polymorphisms on three chromosomes. Homozygosity and linkage disequilibrium analyses favored one chromosome, CFA25, and screening of the S-antigen (SAG) gene identified a non-stop mutation (c.1216T>C), which would result in the addition of 25 amino acids (p.*405Rext*25). Conclusions Identification of this non-stop SAG mutation in dogs affected with retinal degeneration establishes this canine disease as orthologous to Oguchi disease and SAG-associated retinitis pigmentosa in humans, and offers opportunities for genetic therapeutic intervention. PMID:24019744

  3. Detection of antigens using a protein-DNA chimera developed by enzymatic covalent bonding with phiX gene A*.

    PubMed

    Akter, Farhima; Mie, Masayasu; Grimm, Sebastian; Nygren, Per-Åke; Kobatake, Eiry

    2012-06-01

    The chemical reactions used to make antibody-DNA conjugates in many immunoassays diminish antigen-binding activity and yield heterogeneous products. Here, we address these issues by developing an antibody-based rolling circle amplification (RCA) strategy using a fusion of φX174 gene A* protein and Z(mab25) (A*-Zmab). The φX174 gene A* protein is an enzyme that can covalently link with DNA, while the Z(mab25) protein moiety can bind to specific species of antibodies. The DNA in an A*-Zmab conjugate was attached to the A* protein at a site chosen to not interfere with protein function, as determined by enzyme-linked immunosorbent assay (ELISA) and gel mobility shift analysis. The novel A*-Zmab-DNA conjugate retained its binding capabilities to a specific class of murine immunoglobulin γ1 (IgG1) but not to rabbit IgG. This indicates the generality of the A*-Zmab-based immuno-RCA assay that can be used in-sandwich ELISA format. Moreover, the enzymatic covalent method dramatically increased the yields of A*-Zmab-DNA conjugates up to 80% after a 15 min reaction. Finally, sensitive detection of human interferon-γ (IFN-γ) was achieved by immuno-RCA using our fusion protein in sandwich ELISA format. This new approach of the use of site-specific enzymatic DNA conjugation to proteins should be applicable to fabrication of novel immunoassays for biosensing.

  4. Non-essential genes in the vaccinia virus HindIII K fragment: a gene related to serine protease inhibitors and a gene related to the 37K vaccinia virus major envelope antigen.

    PubMed

    Boursnell, M E; Foulds, I J; Campbell, J I; Binns, M M

    1988-12-01

    The complete nucleotide sequence of a cloned copy of the HindIII K fragment of the WR strain of vaccinia virus has been determined. Eight open reading frames (ORFs) have been identified, on the basis of size and codon usage. The predicted amino acid sequences of the putative genes have been compared to the Protein Identification Resource and to published vaccinia virus sequences. One gene, predicted to encode a 42.2K protein, is highly related to the family of serine protease inhibitors. It shows approximately 25% identity to human antithrombin III and 19% identity to the cowpox virus 38K protein gene which is also related to serine protease inhibitors. The product of another gene shows a similar high level of identity to the 37K vaccinia virus major envelope antigen. The existence of viable deletion mutants and recombinants containing foreign DNA inserted into both these genes indicates that they are non-essential.

  5. vls Antigenic Variation Systems of Lyme Disease Borrelia: Eluding Host Immunity through both Random, Segmental Gene Conversion and Framework Heterogeneity.

    PubMed

    Norris, Steven J

    2014-12-01

    Spirochetes that cause Lyme borreliosis (also called Lyme disease) possess the vls locus, encoding an elaborate antigenic variation system. This locus contains the expression site vlsE as well as a contiguous array of vls silent cassettes, which contain variations of the central cassette region of vlsE. The locus is present on one of the many linear plasmids in the organism, e.g. plasmid lp28-1 in the strain Borrelia burgdorferi B31. Changes in the sequence of vlsE occur continuously during mammalian infection and consist of random, segmental, unidirectional recombination events between the silent cassettes and the cassette region of vlsE. These gene conversion events do not occur during in vitro culture or the tick portion of the infection cycle of B. burgdorferi or the other related Borrelia species that cause Lyme disease. The mechanism of recombination is largely unknown, but requires the RuvAB Holliday junction branch migrase. Other features of the vls locus also appear to be required, including cis locations of vlsE and the silent cassettes and high G+C content and GC skew. The vls system is required for long-term survival of Lyme Borrelia in infected mammals and represents an important mechanism of immune evasion. In addition to sequence variation, immune selection also results in significant heterogeneity in the sequence of the surface lipoprotein VlsE. Despite antigenic variation, VlsE generates a robust antibody response, and both full-length VlsE and the C6 peptide (corresponding to invariant region 6) are widely used in immunodiagnostic tests for Lyme disease.

  6. Identification and mapping of Epstein-Barr virus early antigens and demonstration of a viral gene activator that functions in trans.

    PubMed Central

    Wong, K M; Levine, A J

    1986-01-01

    The BamHI M DNA fragment of the Epstein-Barr virus (EBV) genome was inserted in two orientations into a simian virus 40-based expression vector, and the EBV-specific proteins produced in COS-7 monkey cells were examined. In one orientation, termed BamHI-M rightward reading frame 1 (BMRF1), a set of phosphoproteins ranging in size from 47,000 to 54,000 daltons was synthesized. These proteins reacted with monoclonal and polyclonal antisera, defining them as components of the EBV early antigen diffuse set of proteins (EA-D). The BamHI M DNA fragment in the opposite orientation, termed BamHI-M leftward reading frame 1 (BMLF1), directed the synthesis of a nuclear antigen detected by antibodies in serum from a patient with nasopharyngeal carcinoma. The BMLF1 antigen was not detected by monoclonal or polyclonal antibodies directed against the EA-D complex. A series of deletion mutants were constructed in the BamHI M DNA fragment, and the EA-D complex and BMLF1 antigen were mapped to discrete open reading frames in this DNA fragment. A test for several possible functions of these antigens showed that the BMLF1 antigen had the ability to activate or enhance, in trans, the level of expression of a gene under the control of the adenovirus early region 3 promoter or the simian virus 40 early promoter in the absence of its cis-acting enhancer. These experiments demonstrate a new gene function, encoded by EBV, that may be important in the positive regulation of viral or cellular genes. Images PMID:3018282

  7. The context of gene expression defines the immunodominance hierarchy of cytomegalovirus antigens.

    PubMed

    Dekhtiarenko, Iryna; Jarvis, Michael A; Ruzsics, Zsolt; Čičin-Šain, Luka

    2013-04-01

    Natural immunity to CMV dominates the CD4 and CD8 memory compartments of the CMV-seropositive host. This property has been recently exploited for experimental CMV-based vaccine vector strategies, and it has shown promise in animal models of AIDS and Ebola disease. Although it is generally agreed that CMV-based vaccine vectors may induce highly protective and persistent memory T cells, the influence of the gene expression context on Ag-specific T cell memory responses and immune protection induced by CMV vectors is not known. Using murine CMV (MCMV) recombinants expressing a single CD8 T cell epitope from HSV-1 fused to different MCMV genes, we show that magnitude and kinetics of T cell responses induced by CMV are dependent on the gene expression of CMV Ags. Interestingly, the kinetics of the immune response to the HSV-1 epitope was paralleled by a reciprocal depression of immune responses to endogenous MCMV Ags. Infection with a recombinant MCMV inducing a vigorous initial immune response to the recombinant peptide resulted in a depressed early response to endogenous MCMV Ag. Another recombinant virus, which induced a slowly developing "inflationary" T cell response to the HSV-1 peptide, induced weaker long-term responses to endogenous CMV Ags. Importantly, both mutants were able to protect mice from a challenge with HSV-1, mediating strong sterilizing immunity. Our data suggest that the context of gene expression markedly influences the T cell immunodominance hierarchy of CMV Ags, but the immune protection against HSV-1 does not require inflationary CD8 responses against the recombinant CMV-expressed epitope. PMID:23460738

  8. Application of high-resolution, massively parallel pyrosequencing for estimation of haplotypes and gene expression levels of swine leukocyte antigen (SLA) class I genes.

    PubMed

    Kita, Yuki F; Ando, Asako; Tanaka, Keiko; Suzuki, Shingo; Ozaki, Yuki; Uenishi, Hirohide; Inoko, Hidetoshi; Kulski, Jerzy K; Shiina, Takashi

    2012-03-01

    The swine is an important animal model for allo- and xeno-transplantation donor studies, which necessitates an extensive characterization of the expression and sequence variations within the highly polygenic and polymorphic swine leukocyte antigen (SLA) region. Massively parallel pyrosequencing is potentially an effective new 2ndGen method for simultaneous high-throughput genotyping and detection of SLA class I gene expression levels. In this study, we compared the 2ndGen method using the Roche Genome Sequencer 454 FLX with the conventional method using sub-cloning and Sanger sequencing to genotype SLA class I genes in five pigs of the Clawn breed and four pigs of the Landrace breed. We obtained an average of 10.4 SLA class I sequences per pig by the 2ndGen method, consistent with the inheritance data, and an average of only 6.0 sequences by the conventional method. We also performed a correlation analysis between the sequence read numbers obtained by the 2ndGen method and the relative expression values obtained by quantitative real-time PCR analysis at the allele level. A significant correlation coefficient (r = 0.899, P < 0.01) was observed between the sequence read numbers and the relative quantitative values for the expressed classical SLA class I genes SLA-1, SLA-2, and SLA-3, suggesting that the sequence read numbers closely reflect the gene expression levels in white blood cells. Overall, five novel class I sequences, different haplotype-specific expression patterns and a splice variant for one of the SLA class I genes were identified by the 2ndGen method at greater efficiency and sensitivity than the conventional method.

  9. p53 gene product expression in resected non-small cell carcinoma of the lung, with studies of concurrent cytological preparations and microwave antigen retrieval.

    PubMed Central

    Binks, S; Clelland, C A; Ronan, J; Bell, J

    1997-01-01

    AIM: To document the frequency and extent of p53 gene product expression in paraffin sections of resected non-small cell carcinoma of the lung and in cytological preparations of the same tumours; to determine the effect of microwave antigen retrieval on antigen detection. METHODS: Representative paraffin sections of 50 non-small cell carcinomas were stained with an antibody to p53 gene product (DO-7) both with and without prior microwave antigen retrieval. Cytoblocks and cell smears obtained from 19 cases were similarly stained. RESULTS: Using a histochemical scoring system (0-300) which takes into account staining intensity and extent, 78% (n = 39) of microwave pretreated paraffin sections and 52% (n = 26) of non-pretreated sections scored between 5 and 300; p = 0.001; 56% (n = 28) of microwave pretreated sections and only 2% (n = 1) of non-pretreated sections scored between 100 and 300 (p = 0.0001); 75% of direct smears of tumours and 80% of cytoblocks stained similarly to the paraffin sections of the resected specimens. No smears or cytoblocks stained positively when the sections of the resected specimen were negative. CONCLUSIONS: As up to 78% of non-small cell lung carcinomas overexpress p53 gene product, this may prove to be a valuable diagnostic method in biopsy or cytological material when the morphological diagnosis is uncertain. Microwave antigen retrieval is effective on formalin fixed tissue. Images PMID:9215149

  10. Dual oncogenic and tumor suppressor roles of the promyelocytic leukemia gene in hepatocarcinogenesis associated with hepatitis B virus surface antigen.

    PubMed

    Chung, Yih-Lin; Wu, Mei-Ling

    2016-05-10

    Proteasome-mediated degradation of promyelocytic leukemia tumor suppressor (PML) is upregulated in many viral infections and cancers. We previously showed that PML knockdown promotes early-onset hepatocellular carcinoma (HCC) in hepatitis B virus surface antigen (HBsAg)-transgenic mice. Here we report the effects of PML restoration on late-onset HBsAg-induced HCC. We compared protein expression patterns, genetic mutations and the effects of pharmacologically targeting PML in wild-type, PML-/-, PML+/+HBsAgtg/o and PML-/-HBsAgtg/o mice. PML-/- mice exhibited somatic mutations in DNA repair genes and developed severe steatosis and proliferative disorders, but not HCC. PML-/-HBsAgtg/o mice exhibited early mutations in cancer driver genes and developed hyperplasia, fatty livers and indolent adipose-like HCC. In PML+/+HBsAg-transgenic mice, HBsAg expression declined over time, and HBsAg-associated PML suppression was concomitantly relieved. Nevertheless, these mice accumulated mutations in genes contributing to oxidative stress pathways and developed aggressive late-onset angiogenic trabecular HCC. PML inhibition using non-toxic doses of arsenic trioxide selectively killed long-term HBsAg-affected liver cells in PML+/+HBsAgtg/o mice with falling HBsAg and rising PML levels, but not normal liver cells or early-onset HCC cells in PML-/-HBsAgtg/0 mice. These findings suggest dual roles for PML as a tumor-suppressor lost in early-onset HBsAg-induced hepatocarcinogenesis and as an oncogenic promoter in late-onset HBsAg-related HCC progression. PMID:27058621

  11. Dual oncogenic and tumor suppressor roles of the promyelocytic leukemia gene in hepatocarcinogenesis associated with hepatitis B virus surface antigen

    PubMed Central

    Chung, Yih-Lin; Wu, Mei-Ling

    2016-01-01

    Proteasome-mediated degradation of promyelocytic leukemia tumor suppressor (PML) is upregulated in many viral infections and cancers. We previously showed that PML knockdown promotes early-onset hepatocellular carcinoma (HCC) in hepatitis B virus surface antigen (HBsAg)-transgenic mice. Here we report the effects of PML restoration on late-onset HBsAg-induced HCC. We compared protein expression patterns, genetic mutations and the effects of pharmacologically targeting PML in wild-type, PML−/−, PML+/+HBsAgtg/o and PML−/−HBsAgtg/o mice. PML−/− mice exhibited somatic mutations in DNA repair genes and developed severe steatosis and proliferative disorders, but not HCC. PML−/−HBsAgtg/o mice exhibited early mutations in cancer driver genes and developed hyperplasia, fatty livers and indolent adipose-like HCC. In PML+/+HBsAg-transgenic mice, HBsAg expression declined over time, and HBsAg-associated PML suppression was concomitantly relieved. Nevertheless, these mice accumulated mutations in genes contributing to oxidative stress pathways and developed aggressive late-onset angiogenic trabecular HCC. PML inhibition using non-toxic doses of arsenic trioxide selectively killed long-term HBsAg-affected liver cells in PML+/+HBsAgtg/o mice with falling HBsAg and rising PML levels, but not normal liver cells or early-onset HCC cells in PML−/−HBsAgtg/0 mice. These findings suggest dual roles for PML as a tumor-suppressor lost in early-onset HBsAg-induced hepatocarcinogenesis and as an oncogenic promoter in late-onset HBsAg-related HCC progression. PMID:27058621

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

    SciTech Connect

    Tabuchi, Yoshiaki . E-mail: ytabu@ms.toyama-mpu.ac.jp; Kondo, Takashi; Suzuki, Yoshihisa; Obinata, Masuo

    2005-04-15

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

  13. Characterization of the gene encoding the polymorphic immunodominant molecule, a neutralizing antigen of Theileria parva

    SciTech Connect

    Toye, P.G.; Metzelaar, M.J.; Wijngaard, P.L.J.

    1995-08-01

    Theileria parva, a tick-transmitted protozoan parasite related to Plasmodium spp., causes the disease East Coast fever, an acute and usually fatal lymphoproliferative disorder of cattle in Africa. Previous studies using sera from cattle that have survived infection identified a polymorphic immunodominant molecule (PIM) that is expressed by both the infective sporozoite stage of the parasite and the intracellular schizont. Here we show that mAb specific for the PIM Ag can inhibit sporozoite invasion of lymphocytes in vitro. A cDNA clone encoding the PIM Ag of the T. parva (Muguga) stock was obtained by using these mAb in a novel eukaryotic expression cloning system that allows isolation of cDNA encoding cytoplasmic or surface Ags. To establish the molecular basis of the polymorphism of PIM, the cDNA of the PIM Ag from a buffalo-derived T. parva stock was isolated and its sequence was compared with that of the cattle-derived Muguga PIM. The two cDNAs showed considerable identity in both the 5{prime} and 3{prime} regions, but there was substantial sequence divergence in the central regions. Several types of repeated sequences were identified in the variant regions. In the Muguga form of the molecule, there were five tandem repeats of the tetrapeptide, QPEP, that were shown, by transfection of a deleted version of the PIM gene, not to react with several anti-PIM mAbs. By isolating and sequencing the genomic version of the gene, we identified two small introns in the 3{prime} region of the gene. Finally, we showed that polyclonal rat Abs against recombinant PIM neutralize sporozoite infectivity in vitro, suggesting that the PIM Ag should be evaluated for its capacity to immunize cattle against East Coast Fever.

  14. Vaccine antigen production in transgenic plants: strategies, gene constructs and perspectives.

    PubMed

    Sala, Francesco; Manuela Rigano, M; Barbante, Alessandra; Basso, Barbara; Walmsley, Amanda M; Castiglione, Stefano

    2003-01-30

    Stable integration of a gene into the plant nuclear or chloroplast genome can transform higher plants (e.g. tobacco, potato, tomato, banana) into bioreactors for the production of subunit vaccines for oral or parental administration. This can also be achieved by using recombinant plant viruses as transient expression vectors in infected plants. The use of plant-derived vaccines may overcome some of the major problems encountered with traditional vaccination against infectious diseases, autoimmune diseases and tumours. They also offer a convenient tool against the threat of bio-terrorism. State of the art, experimental strategies, safety and perspectives are discussed in this article.

  15. Comparison of O-Antigen Gene Clusters of All O-Serogroups of Escherichia coli and Proposal for Adopting a New Nomenclature for O-Typing.

    PubMed

    DebRoy, Chitrita; Fratamico, Pina M; Yan, Xianghe; Baranzoni, GianMarco; Liu, Yanhong; Needleman, David S; Tebbs, Robert; O'Connell, Catherine D; Allred, Adam; Swimley, Michelle; Mwangi, Michael; Kapur, Vivek; Raygoza Garay, Juan A; Roberts, Elisabeth L; Katani, Robab

    2016-01-01

    Escherichia coli strains are classified based on O-antigens that are components of the lipopolysaccharide (LPS) in the cell envelope. O-antigens are important virulence factors, targets of both the innate and adaptive immune system, and play a role in host-pathogen interactions. Because they are highly immunogenic and display antigenic specificity unique for each strain, O-antigens are the biomarkers for designating O-types. Immunologically, 185 O-serogroups and 11 OX-groups exist for classification. Conventional serotyping for O-typing entails agglutination reactions between the O-antigen and antisera generated against each O-group. The procedure is labor intensive, not always accurate, and exhibits equivocal results. In this report, we present the sequences of 71 O-antigen gene clusters (O-AGC) and a comparison of all 196 O- and OX-groups. Many of the designated O-types, applied for classification over several decades, exhibited similar nucleotide sequences of the O-AGCs and cross-reacted serologically. Some O-AGCs carried insertion sequences and others had only a few nucleotide differences between them. Thus, based on these findings, it is proposed that several of the E. coli O-groups may be merged. Knowledge of the O-AGC sequences facilitates the development of molecular diagnostic platforms that are rapid, accurate, and reliable that can replace conventional serotyping. Additionally, with the scientific knowledge presented, new frontiers in the discovery of biomarkers, understanding the roles of O-antigens in the innate and adaptive immune system and pathogenesis, the development of glycoconjugate vaccines, and other investigations, can be explored. PMID:26824864

  16. Swine Leukocyte Antigen-DQA Gene Variation and Its Association with Piglet Diarrhea in Large White, Landrace and Duroc.

    PubMed

    Yang, Q L; Kong, J J; Wang, D W; Zhao, S G; Gun, S B

    2013-08-01

    The swine leukocyte antigen class II molecules are possibly associated with the induction of protective immunity. The study described here was to investigate the relationship between polymorphisms in exon 2 of the swine DQA gene and piglet diarrhea. This study was carried out on 425 suckling piglets from three purebred pig strains (Large White, Landrace and Duroc). The genetic diversity of exon 2 in swine DQA was detected by PCR-SSCP and sequencing analysis, eight unique SSCP patterns (AB, BB, BC, CC, CD, BD, BE and DD) representing five specific allele (A to E) sequences were detected. Sequence analysis revealed 21 nucleotide variable sites and resulting in 12 amino acid substitutions in the populations. A moderate level polymorphism and significant deviations from Hardy-Weinberg equilibrium of the genotypes distribution were observed in the populations (p<0.01). The association analysis indicated that there was a statistically significant difference in the score of piglet diarrhea between different genotypes, individuals with genotype CC showed a lower diarrhea score than genotypes AB (0.98±0.09), BB (0.85±0.77) and BC (1.25±0.23) (p<0.05), and significantly low than genotype BE (1.19±0.19) (p<0.01), CC genotype may be a most resistance genotype for piglet diarrhea.

  17. The RAG2 C-terminus and ATM protect genome integrity by controlling antigen receptor gene cleavage.

    PubMed

    Chaumeil, Julie; Micsinai, Mariann; Ntziachristos, Panagiotis; Roth, David B; Aifantis, Iannis; Kluger, Yuval; Deriano, Ludovic; Skok, Jane A

    2013-01-01

    Tight control of antigen-receptor gene rearrangement is required to preserve genome integrity and prevent the occurrence of leukaemia and lymphoma. Nonetheless, mistakes can happen, leading to the generation of aberrant rearrangements, such as Tcra/d-Igh inter-locus translocations that are a hallmark of ataxia telangiectasia-mutated (ATM) deficiency. Current evidence indicates that these translocations arise from the persistence of unrepaired breaks converging at different stages of thymocyte differentiation. Here we show that a defect in feedback control of RAG2 activity gives rise to bi-locus breaks and damage on Tcra/d and Igh in the same T cell at the same developmental stage, which provides a direct mechanism for generating these inter-locus rearrangements. Both the RAG2 C-terminus and ATM prevent bi-locus RAG-mediated cleavage through modulation of three-dimensional conformation (higher-order loops) and nuclear organization of the two loci. This limits the number of potential substrates for translocation and provides an important mechanism for protecting genome stability. PMID:23900513

  18. The Effect of the Nonionic Block Copolymer Pluronic P85 on Gene Expression in Mouse Muscle and Antigen Presenting Cells

    PubMed Central

    Gaymalov, Zagit Z.; Yang, Zhihui; Pisarev, Vladimir M.; Alakhov, Valery Yu.; Kabanov, Alexander V.

    2008-01-01

    DNA vaccines can be greatly improved by polymer agents that simultaneously increase transgene expression and activate immunity. We describe here Pluronic P85 (P85), a triblock copolymer of ethylene oxide (EO) and propylene oxide (PO) EO26-PO40-EO26,. Using a mouse model we demonstrate that co-administration of a bacterial plasmid DNA with P85 in a skeletal muscle greatly increases gene expression in the injection site and distant organs, especially the draining lymph nodes and spleen. The reporter expression colocalizes with the specific markers of myocytes and keratinocytes in the muscle, as well as dendritic cells (DC) and macrophages in the muscle, lymph nodes and spleen. Furthermore, DNA/P85 and P85 alone increase the systemic expansion of CD11c+ (DC), and local expansion of CD11c+, CD14+ (macrophages) and CD49b+ (natural killer) cell populations. DNA/P85 (but not P85) also increases maturation of local DC (CD11c+CD86+, CD11c+CD80+, and CD11c+CD40+). We suggest that DNA/P85 promotes the activation and recruitment of the antigen-presenting cells, which further incorporate, express and carry the transgene to the immune system organs. PMID:19064283

  19. Effect of human leukocyte antigen class II genes on Hashimoto's thyroiditis requiring replacement therapy with levothyroxine in the Japanese population.

    PubMed

    Katahira, Masahito; Hanakita, Mizuki; Ito, Tatsuo; Suzuki, Mari

    2013-05-01

    Contribution of the human leukocyte antigen (HLA) subtype to Hashimoto's thyroiditis (HT) that requires replacement therapy with levothyroxine remains unclear in the Japanese population. The frequencies of HLA DR-DQ haplotypes were compared between patients with HT requiring levothyroxine replacement therapy and the control individuals. We studied 82 patients with HT requiring levothyroxine replacement therapy. The frequencies of DRB1*08:03-DQB1*06:01 and DRB1*09:01-DQB1*03:03 haplotypes were significantly higher in HT patients, whereas those of DRB1*13:02-DQB1*06:04 and DRB1*15:01-DQB1*06:02 haplotypes were significantly lower in these patients than in the controls. Deduced from known linkage disequilibria, DRB1*13:02-DQB1*06:04 and DRB1*15:01-DQB1*06:02 haplotypes share the same DQA1*01:02 allele. Since DQB1*06:02 and DQB1*06:04 molecules differ in the beta chain by 7 residues, these DQB1 genes are very similar. The DQA1*01:02-DQB1*06 (DQB1*06:02 or DQB1*06:04) haplotype might play a pivotal role in the resistance to HT.

  20. [Site-directed mutagensis of the major antigen E2 gene of CSFV, its high level expression in Escherichia coli and the immunonicity of recombinant E2 protein].

    PubMed

    Yu, Xing-Long; Tu, Chang-Chun; Xu, Xing-Ran; Zhang, Mao-Lin; Chen, Yi-Xiang; Liu, Bo-Hua

    2003-07-01

    Classical swine fever virus (CSFV), an enveloped positive-stranded RNA virus in the genus Pestivirus of the Flaviviridae family, is the causative agent of a highly contagious swine disease characterized by symptoms of hemorrhagic fever and immune depression, usually leading to substantial economic losses. The serological methods for detection of CSFV antibody such as ELISA are important means for the diagnosis of CSFV and immune surveillance. It is difficult to obtain CSFV antigen with high quality using traditional method because its titration titer is low in cell culture. CSFV has four structural protein named C, E0, El and E2. The E2 protein contains major antigenic determinants that are conserved between different CSFV strains and involved in neutralization by antibodies. So recombinant E2 protein can be developed as an alternative to the intact viral antigen. So far, CSFV E2 have not been expressed in E. coli with high level. Many factors, such as the secondary structure, the stability of 5' and 3' terminus of gene, the location of SD sequence and the bias of codes, are involved in the expressing level of foreign gene in E. coli . In this study, two sites of the E2 gene sequence were confirmed to be detrimental to its expression efficiency in E. coli through the computer-aided analysis. So they were mutated using recombinant PCR without changing the amino acids sequence of CSFV E2 gene. A plasmid was constructed by inserting the mutated E2 gene into the prokaryotic expression vector pET-28a(+) and named pETE2. The E. coli competent host BL21 (DE3)lysS transformed with pETE2 could express the E2 gene at high level, amounting to 28% of the total protein of the induced recombinant bacteria at the presence of IPTG. Except the hydrophobic transmembrane domain at C terminus, the recombinant E2 protein includes the total aa sequence. So it contains all the potential linear antigen epitopes of E2 protein because hydrophobic aa region can not form epitope. The

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

    PubMed

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

    2016-04-22

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

  2. TLR21's agonists in combination with Aeromonas antigens synergistically up-regulate functional TLR21 and cytokine gene expression in yellowtail leucocytes.

    PubMed

    Reyes-Becerril, Martha; Ascencio-Valle, Felipe; Hirono, Ikuo; Kondo, Hidehiro; Jirapongpairoj, Walissara; Esteban, Maria Angeles; Alamillo, Erika; Angulo, Carlos

    2016-08-01

    The purpose of this study was to characterize the TLR21 gene from yellowtail (Seriola lalandi) and its functional activity using TLR agonist stimulation and Aeromonas antigens. The TLR21 nucleotide sequence from yellowtail was obtained using the whole-genome shotgun sequencing method and bioinformatics tools. Basal TLR21 gene expression was analyzed in several tissues. Subsequently, the gene expression of TLR21 and cytokines IL-1β and TNF-α was evaluated in TLR agonist (CpG-ODN2006, LPS, and Poly I:C) exposing head kidney leucocytes, which were then subjected to Aeromonas antigen stimulation. The yellowtail full-length cDNA sequence of SlTLR21 was 3615 bp (980 aa) showing a high degree of similarity with the counterparts of other fish species and sharing the common structural architecture of the TLR family, including LRR domains, one C-terminal LRR region, and a TIR domain. Gene expression studies revealed the constitutive expression of TLR21 mRNA in all the analyzed tissues; the highest levels were observed in spleen and head kidney where they play an important role in the fish immune system. Transcripts of TLR21 and the downstream IL-1β and TNF-α cytokine genes were most strongly up-regulated after exposure to the TLR agonists following Aeromonas antigen stimulation, suggesting they are involved in immune response. The results indicated that TLR agonists, in combination with Aeromonas antigens in head kidney leucocytes, synergistically enhance TLR21 and cytokines in yellowtail. PMID:26987525

  3. Cytotoxic T lymphocyte antigen 4 (CTLA4) gene polymorphism with bladder cancer risk in North Indian population.

    PubMed

    Jaiswal, Praveen Kumar; Singh, Vibha; Mittal, Rama Devi

    2014-02-01

    Cytotoxic T Lymphocyte antigen 4 (CTLA4) is a potent immunoregulatory molecule that suppresses antitumor response by down-regulating T cell activation. We examined candidate disease-susceptibility single nucleotide polymorphism (SNPs) of CTLA4 at +49A/G, CT60A/G and -318C/T genes in bladder cancer (BC) patients of North Indian population. Histopathologically confirmed 200 patients of BC and 200 unrelated, healthy controls of similar ethnicity were genotyped by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) and amplification refractory mutation specific (PCR-ARMS) methods. In present study SNP CTLA4 +49A/G, variant genotype showed 3.74-fold risks for BC. While looking at G allele carrier level, risk for BC was high (OR = 1.54). The risk for BC was also evident in case G allele (OR = 1.58). CTLA4 CT60A/G gene polymorphism variant genotype showed 1.36-fold risks for BC. While at G allele carrier and with variant G allele it showed significantly reduced risk for BC. CTLA4 +49A/G genotype exhibited 1.57-fold risks with smoking in BC patients in homozygous mutant condition. In silico analysis further supports the results of SNP at CTLA4 +49A/G and CTLA4 CT60A/G. None of the above SNPs of CTLA4 demonstrated association with tumor stage/grade for BC severity and progression. BCG immunotherapy had no impact on CTLA4 gene polymorphism revealing no significant association. Haplotype GAC showed high risk for BC while other haplotype AGT showed reduced risk for BC. Our results indicated that genetic variations in CTLA4 gene (+49A/G, CT60A/G) play role in susceptibility to BC. Haplotype GAC showed high risk for BC. An association study utilizing a larger sample size and different ethnicity warrant further investigation through replication and advance techniques.

  4. Association between Genetic Polymorphism in the Swine Leukocyte Antigen-DRA Gene and Piglet Diarrhea in Three Chinese Pig Breeds.

    PubMed

    Yang, Q L; Zhao, S G; Wang, D W; Feng, Y; Jiang, T T; Huang, X Y; Gun, S B

    2014-09-01

    The swine leukocyte antigen (SLA)-DRA locus is noteworthy among other SLA class II loci for its limited variation and has not been investigated in depth. This study was investigated to detect polymorphisms of four exons of SLA-DRA gene and its association with piglet diarrhea in Landrace, Large White and Duroc pigs. No polymorphisms were detected in exon 3, while 2 SNPs (c.178G>A and c.211T>C), 2 SNPs (c.3093A>C and c.3104C>T) and 5 SNPs (c.4167A>G, c.4184A>G, c.4194A>G, c.4246A>G and c.4293G>A) were detected in exon 1, exon 2 and exon 4 respectively, and 1 SNP (c.4081T>C) in intron 3. Statistical results showed that genotype had significant effect on piglet diarrhea, individuals with genotype BC had a higher diarrhea score when compared with the genotypes AA, AB, AC and CC. Futhermore, genotype AC had a higher diarrhea score than the genotype CC in exon 1 (p<0.05); diarrhea scores of genotype AA and BB were higher than those of genotypes AC and CC in exon 2 (p<0.05); individuals with genotype AA had a higher diarrhea score than individuals with genotype AB and BB in exon 4 (p<0.05). Fourteen common haplotypes were founded by haplotype constructing of all SNPs in the three exons, its association with piglet diarrhea appeared that Hap2, 5, 8, 10, and 14 may be the susceptible haplotypes and Hap9 may be the resistant haplotype to piglet diarrhea. The genetic variations identified of the SLA-DRA gene may potentially be functional mutations related to piglet diarrhea.

  5. Human Leukocyte Antigen (HLA) A*1101-Restricted Epstein-Barr Virus-Specific T-cell Receptor Gene Transfer to Target Nasopharyngeal Carcinoma.

    PubMed

    Zheng, Yong; Parsonage, Greg; Zhuang, Xiaodong; Machado, Lee R; James, Christine H; Salman, Asmaa; Searle, Peter F; Hui, Edwin P; Chan, Anthony T C; Lee, Steven P

    2015-10-01

    Infusing virus-specific T cells is effective treatment for rare Epstein-Barr virus (EBV)-associated posttransplant lymphomas, and more limited success has been reported using this approach to treat a far more common EBV-associated malignancy, nasopharyngeal carcinoma (NPC). However, current approaches using EBV-transformed lymphoblastoid cell lines to reactivate EBV-specific T cells for infusion take 2 to 3 months of in vitro culture and favor outgrowth of T cells targeting viral antigens expressed within EBV(+) lymphomas, but not in NPC. Here, we explore T-cell receptor (TCR) gene transfer to rapidly and reliably generate T cells specific for the NPC-associated viral protein LMP2. We cloned a human leukocyte antigen (HLA) A*1101-restricted TCR, which would be widely applicable because 40% of NPC patients carry this HLA allele. Studying both the wild-type and modified forms, we have optimized expression of the TCR and demonstrated high-avidity antigen-specific function (proliferation, cytotoxicity, and cytokine release) in both CD8(+) and CD4(+) T cells. The engineered T cells also inhibited LMP2(+) epithelial tumor growth in a mouse model. Furthermore, transduced T cells from patients with advanced NPC lysed LMP2-expressing NPC cell lines. Using this approach, within a few days large numbers of high-avidity LMP2-specific T cells can be generated reliably to treat NPC, thus providing an ideal clinical setting to test TCR gene transfer without the risk of autoimmunity through targeting self-antigens.

  6. Identification and Validation of HCC-specific Gene Transcriptional Signature for Tumor Antigen Discovery.

    PubMed

    Petrizzo, Annacarmen; Caruso, Francesca Pia; Tagliamonte, Maria; Tornesello, Maria Lina; Ceccarelli, Michele; Costa, Valerio; Aprile, Marianna; Esposito, Roberta; Ciliberto, Gennaro; Buonaguro, Franco M; Buonaguro, Luigi

    2016-07-08

    A novel two-step bioinformatics strategy was applied for identification of signatures with therapeutic implications in hepatitis-associated HCC. Transcriptional profiles from HBV- and HCV-associated HCC samples were compared with non-tumor liver controls. Resulting HCC modulated genes were subsequently compared with different non-tumor tissue samples. Two related signatures were identified, namely "HCC-associated" and "HCC-specific". Expression data were validated by RNA-Seq analysis carried out on unrelated HCC samples and protein expression was confirmed according to The Human Protein Atlas" (http://proteinatlas.org/), a public repository of immunohistochemistry data. Among all, aldo-keto reductase family 1 member B10, and IGF2 mRNA-binding protein 3 were found strictly HCC-specific with no expression in 18/20 normal tissues. Target peptides for vaccine design were predicted for both proteins associated with the most prevalent HLA-class I and II alleles. The described novel strategy showed to be feasible for identification of HCC-specific proteins as highly potential target for HCC immunotherapy.

  7. Identification and Validation of HCC-specific Gene Transcriptional Signature for Tumor Antigen Discovery

    PubMed Central

    Petrizzo, Annacarmen; Caruso, Francesca Pia; Tagliamonte, Maria; Tornesello, Maria Lina; Ceccarelli, Michele; Costa, Valerio; Aprile, Marianna; Esposito, Roberta; Ciliberto, Gennaro; Buonaguro, Franco M.; Buonaguro, Luigi

    2016-01-01

    A novel two-step bioinformatics strategy was applied for identification of signatures with therapeutic implications in hepatitis-associated HCC. Transcriptional profiles from HBV- and HCV-associated HCC samples were compared with non-tumor liver controls. Resulting HCC modulated genes were subsequently compared with different non-tumor tissue samples. Two related signatures were identified, namely “HCC-associated” and “HCC-specific”. Expression data were validated by RNA-Seq analysis carried out on unrelated HCC samples and protein expression was confirmed according to The Human Protein Atlas" (http://proteinatlas.org/), a public repository of immunohistochemistry data. Among all, aldo-keto reductase family 1 member B10, and IGF2 mRNA-binding protein 3 were found strictly HCC-specific with no expression in 18/20 normal tissues. Target peptides for vaccine design were predicted for both proteins associated with the most prevalent HLA-class I and II alleles. The described novel strategy showed to be feasible for identification of HCC-specific proteins as highly potential target for HCC immunotherapy. PMID:27387388

  8. Construction of human liver cancer vascular endothelium cDNA expression library and screening of the endothelium-associated antigen genes

    PubMed Central

    Zhong, Xing; Ran, Yu-Liang; Lou, Jin-Ning; Hu, Dong; Yu, Long; Zhang, Yu-Shan; Zhou, Zhuan; Yang, Zhi-Hua

    2004-01-01

    AIM: To gain tumor endothelium associated antigen genes from human liver cancer vascular endothelial cells (HLCVECs) cDNA expression library, so as to find some new possible targets for the diagnosis and therapy of liver tumor. METHODS: HLCVECs were isolated and purified from a fresh hepatocellular carcinoma tissue sample, and were cultured and proliferated in vitro. A cDNA expression library was constructed with the mRNA extracted from HLCVECs. Anti-sera were prepared from immunized BALB/c mice through subcutaneous injection with high dose of fixed HLCVECs, and were then tested for their specificity against HLCVECs and angiogenic effects in vitro, such as inhibiting proliferation and inducing apoptosis of tumor endothelial cells, using immunocytochemistry, immunofluorescence, cell cycle analysis and MTT assays, etc. The identified xenogeneic sera from immunized mice were employed to screen the library of HLCVECs by modified serological analyses of recombinant cDNA expression libraries (SEREX). The positive clones were sequenced and analyzed by bio-informatics. RESULTS: The primary cDNA library consisted of 2 × 106 recombinants. Thirty-six positive clones were obtained from 6 × 105 independent clones by immunoscreening. Bio-informatics analysis of cDNA sequences indicated that 36 positive clones represented 18 different genes. Among them, 3 were new genes previously unreported, 2 of which were hypothetical genes. The other 15 were already known ones. Series analysis of gene expression (SAGE) database showed that ERP70, GRP58, GAPDH, SSB, S100A6, BMP-6, DVS27, HSP70 and NAC alpha in these genes were associated with endothelium and angiogenesis, but their effects on HLCVECs were still unclear. GAPDH, S100A6, BMP-6 and hsp70 were identified by SEREX in other tumor cDNA expression libraries. CONCLUSION: By screening of HLCVECs cDNA expression library using sera from immunized mice with HLCVECs, the functional genes associated with tumor endothelium or angiogenesis

  9. Expression of E2 gene of bovine viral diarrhea virus in Pichia pastoris: a candidate antigen for indirect Dot ELISA.

    PubMed

    Zhao, Yuelan; Ma, Tianyi; Ju, Xingyu; Zhang, Yue; Wang, Min; Liu, Teng; Cao, Wenbo; Bao, Yongzhan; Qin, Jianhua

    2015-02-01

    The E2 gene containing the EcoR I and Not I sites of bovine viral diarrhea virus (BVDV) was amplified from the plasmid pMD-18T-E2 of the HB-bd isolated, and inserted into Pichia pastoris (P. pastoris) expression vector pPIC9K, and transfected into Escherichia coli DH5α. The recombinant plasmid pPIC9K-E2 was digested by the SalI restriction enzyme and transformed into the P. pastoris strain GS115 by electroporation. High copy integrative transformants were obtained by G418 screening and induced for expression with methanol. The expressed products in the culture medium were identified by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the Western blotting and the antibody test for immunity. An indirect Dot-ELISA for the detection of antibody against BVDV was established by the recombinant E2 protein as the coating antigen. The reaction conditions of the indirect Dot-ELISA were optimized. The coating concentration of the E2 recombinant protein antigen, the dilution of serum sample, the optimal concentration of HRP labeled antibody, the optimal blocking reagent and blocking time were studied. 100 sera samples from cows in the field were tested for the antibody against BVDV by the Dot-ELISA and the IDEXX HerdChek BVDV antibody ELISA kit simultaneously to compare the specificity, sensitivity and accuracy. The results showed that the expressed products in the culture medium resulted in single band of 44kDa by SDS-PAGE and Western blotting. The results of the immunogenicity assay indicated that the protein E2 expressed in P. pastoris could induce the experimental animals of the rabbit to produce BVDV specific antibodies. The results of the indirect Dot-ELISA showed that the optimal coating concentration of the E2 recombinant protein was 2.0μg/mL, the bovine serum dilution was 1:100, the optimal concentration of HRP-labeled rabbit anti-bovine antibody IgG was 1:500, and the optimal blocking reagent was 3% glutin-TBS and blocking for 45min. The

  10. Immortalization of epithelial-like cells from human liver tissue with SV40 T-antigen gene.

    PubMed

    Miyazaki, M; Mihara, K; Bai, L; Kano, Y; Tsuboi, S; Endo, A; Seshimo, K; Yoshioka, T; Namba, M

    1993-05-01

    The cells derived from the human embryo liver tissue were transfected with a plasmid pSV3neo containing both the large and small T-antigen gene of the early region of simian virus 40 (SV40), and two cell strains, OUMS-21 and -22, were obtained. OUMS-22 cells, to date, have reached over 100 population doublings through a culture crisis and are considered to have become an immortal cell line. However, OUMS-21 cells failed to become an immortal cell line. Both OUMS-21 and -22 cells were SV40 T-antigen-positive, epithelial-like, and immunoreactive against an anti-keratin 18 monoclonal antibody but against neither an anti-vimentin nor an anti-von Willebrandt factor VIII monoclonal antibody. The staining pattern of cytokeratin in these cells was similar to that in the differentiated human hepatoblastoma and hepatocellular carcinoma cell lines but not to that in the human cholangiocellular carcinoma cell lines. OUMS-21 and -22 cells expressed neither alpha-fetoprotein nor albumin mRNAs. These cells showed no tyrosine aminotransferase activity. However, both OUMS-21 and -22 cells were sensitive to cytotoxicity of aflatoxin B1, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole, and benzo[a]pyrene, whereas human embryo lung fibroblasts were insensitive to the cytotoxicity of these carcinogens. These findings suggest that OUMS-21 and -22 cells may arise from undifferentiated liver stem cells or from hepatocytes that lost their ability to express the liver-specific functions prior to immortalization. Both OUMS-21 and -22 cells expressed glutathione S-transferase pi (GST-pi) mRNA. The expression of GST-pi mRNA highly increased in OUMS-22 cells with their immortalization. Karyotypic analysis showed that numerical and structural aberrations of the chromosomes were profound, but neither specific events nor marker chromosomes were found in OUMS-21 and -22 cells. Both OUMS-21 and -22 cells could grow in soft agar, but they were not tumorigenic when transplanted into nude mice. PMID

  11. Deletion of the Meningococcal fetA Gene Used for Antigen Sequence Typing of Invasive and Commensal Isolates from Germany: Frequencies and Mechanisms▿

    PubMed Central

    Claus, Heike; Elias, Johannes; Meinhardt, Christine; Frosch, Matthias; Vogel, Ulrich

    2007-01-01

    Antigen sequence typing (ST) of FetA is part of the molecular typing scheme of Neisseria meningitidis. Among invasive meningococcal isolates from 2,201 patients in Germany, we identified 11 strains lacking the fetA gene because of deletions mediated by repeat arrays flanking the gene, i.e., Correia elements, repeat sequence 13 (RS13), and duplicated RS3. Geographic mapping and multilocus ST of invasive isolates revealed that fetA deletion was a sporadic event without genetic fixation. Among 821 carrier strains, 12 strains lacked fetA, suggesting that fetA is maintained during asymptomatic carriage. Interestingly, most of these isolates belonged to the multilocus ST-35 clonal complex (cc). ST-35 cc strains and the recently published ST-192 strains from Burkina Faso may benefit from loss of fetA, but their infrequent occurrence among invasive isolates currently does not affect fetA antigen ST. PMID:17626167

  12. Escherichia coli O-Antigen Gene Clusters of Serogroups O62, O68, O131, O140, O142, and O163: DNA Sequences and Similarity between O62 and O68, and PCR-Based Serogrouping

    PubMed Central

    Liu, Yanhong; Yan, Xianghe; DebRoy, Chitrita; Fratamico, Pina M.; Needleman, David S.; Li, Robert W.; Wang, Wei; Losada, Liliana; Brinkac, Lauren; Radune, Diana; Toro, Magaly; Hegde, Narasimha; Meng, Jianghong

    2015-01-01

    The DNA sequence of the O-antigen gene clusters of Escherichia coli serogroups O62, O68, O131, O140, O142, and O163 was determined, and primers based on the wzx (O-antigen flippase) and/or wzy (O-antigen polymerase) genes within the O-antigen gene clusters were designed and used in PCR assays to identify each serogroup. Specificity was tested with E. coli reference strains, field isolates belonging to the target serogroups, and non-E. coli bacteria. The PCR assays were highly specific for the respective serogroups; however, the PCR assay targeting the O62 wzx gene reacted positively with strains belonging to E. coli O68, which was determined by serotyping. Analysis of the O-antigen gene cluster sequences of serogroups O62 and O68 reference strains showed that they were 94% identical at the nucleotide level, although O62 contained an insertion sequence (IS) element located between the rmlA and rmlC genes within the O-antigen gene cluster. A PCR assay targeting the rmlA and rmlC genes flanking the IS element was used to differentiate O62 and O68 serogroups. The PCR assays developed in this study can be used for the detection and identification of E. coli O62/O68, O131, O140, O142, and O163 strains isolated from different sources. PMID:25664526

  13. Loss of the WaaL O-Antigen Ligase Prevents Surface Activation of the Flagellar Gene Cascade in Proteus mirabilis▿

    PubMed Central

    Morgenstein, Randy M.; Clemmer, Katy M.; Rather, Philip N.

    2010-01-01

    Proteus mirabilis is a Gram-negative bacterium that undergoes a physical and biochemical change from a vegetative swimmer cell (a typical Gram-negative rod) to an elongated swarmer cell when grown on a solid surface. In this study, we report that a transposon insertion in the waaL gene, encoding O-antigen ligase, blocked swarming motility on solid surfaces but had little effect on swimming motility in soft agar. The waaL mutant was unable to differentiate into a swarmer cell. Differentiation was also prevented by a mutation in wzz, encoding a chain length determinant for O antigen, but not by a mutation in wzyE, encoding an enzyme that polymerizes enterobacterial common antigen, a surface polysaccharide different from the lipid A::core. In wild-type P. mirabilis, increased expression of the flhDC operon occurs after growth on solid surfaces and is required for the high-level expression of flagellin that is characteristic of swarmer cells. However, in both the waaL and the wzz mutants, the flhDC operon was not activated during growth on agar. A loss-of-function mutation in the rcsB response regulator or overexpression of flhDC restored swarming to the waaL mutant, despite the absence of O antigen. Therefore, although O antigen may serve a role in swarming by promoting wettability, the loss of O antigen blocks a regulatory pathway that links surface contact with the upregulation of flhDC expression. PMID:20382766

  14. Cloning and Expression of Major Surface Antigen 1 Gene of Toxoplasma gondii RH Strain Using the Expression Vector pVAX1 in Chinese Hamster Ovary Cells

    PubMed Central

    Abdizadeh, Rahman; Maraghi, Sharif; Ghadiri, Ata A.; Tavalla, Mehdi; Shojaee, Saeedeh

    2015-01-01

    Background: Toxoplasmosis is an opportunistic protozoan infection with a high prevalence in a broad range of hosts infecting up to one-third of the world human population. Toxoplasmosis leads to serious medical problems in immunocompromised individuals and fetuses and also induces abortion and mortality in domestic animals. Therefore, there is a huge demand for the development of an effective vaccine. Surface Antigen 1 (SAG1) is one of the important immunodominant surface antigens of Toxoplasma gondii, which interacts with host cells and primarily involved in adhesion, invasion and stimulation of host immune response. Surface antigen 1 is considered as the leading candidate for development of an effective vaccine against toxoplasmosis. Objectives: The purpose of this study was to clone the major surface antigen1 gene (SAG1) from the genotype 1 of T. gondii, RH strain into the eukaryotic expression vector pVAX1 in order to use for a DNA vaccine. Materials and Methods: Genomic DNA was extracted from tachyzoite of the parasite using the QIAamp DNA mini kit. After designing the specific primers, SAG1 gene was amplified by Polymerase Chain Reaction (PCR). The purified PCR products were then cloned into a pPrime plasmid vector. The aforementioned product was subcloned into the pVAX1 eukaryotic expression vector. The recombinant pVAX1-SAG1 was then transfected into Chinese Hamster Ovary (CHO) cells and expression of SAG1 antigen was evaluated using Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), Immunofluorescence Assay (IFA) and Western Blotting (WB). Results: The cloning and subcloning products (pPrime-SAG1 and pVAX1-SAG1 plasmid vectors) of SAG1 gene were verified and confirmed by enzyme digestion and sequencing. A 30 kDa recombinant protein was expressed in CHO cells as shown by IFA and WB methods. Conclusions: The pVAX1 expression vector and CHO cells are a suitable system for high-level recombinant protein production for SAG1 gene from T. gondii parasites

  15. Molecular cloning of a 46-kilodalton surface antigen (P46) gene from Mycoplasma hyopneumoniae: direct evidence of CGG codon usage for arginine.

    PubMed

    Futo, S; Seto, Y; Mitsuse, S; Mori, Y; Suzuki, T; Kawai, K

    1995-04-01

    The DNA sequence of the gene encoding the early and specific 46-kDa surface antigen (P46) of Mycoplasma hyopneumoniae has been determined. The P46 gene, encoding a putative lipoprotein, contained three TGA codons and a single CGG codon in a 1,257-bp open reading frame. Edman degradation of peptide fragments showed that at least one TGA codon encodes tryptophan and that the CGG codon, which has been reported to be nonsense or unassigned in other mycoplasmas, is used for arginine in M. hyopneumoniae. PMID:7896725

  16. Molecular cloning of a 46-kilodalton surface antigen (P46) gene from Mycoplasma hyopneumoniae: direct evidence of CGG codon usage for arginine.

    PubMed Central

    Futo, S; Seto, Y; Mitsuse, S; Mori, Y; Suzuki, T; Kawai, K

    1995-01-01

    The DNA sequence of the gene encoding the early and specific 46-kDa surface antigen (P46) of Mycoplasma hyopneumoniae has been determined. The P46 gene, encoding a putative lipoprotein, contained three TGA codons and a single CGG codon in a 1,257-bp open reading frame. Edman degradation of peptide fragments showed that at least one TGA codon encodes tryptophan and that the CGG codon, which has been reported to be nonsense or unassigned in other mycoplasmas, is used for arginine in M. hyopneumoniae. PMID:7896725

  17. Mutations in pattern recognition receptor genes modulate seroreactivity to microbial antigens in patients with inflammatory bowel disease

    PubMed Central

    Henckaerts, Liesbet; Pierik, Marie; Joossens, Marie; Ferrante, Marc; Rutgeerts, Paul; Vermeire, Séverine

    2007-01-01

    Background and aims A number of antibodies against microbial epitopes or self‐antigens have been associated with Crohn's disease. The development of antibodies reflects a loss of tolerance to intestinal bacteria that underlies Crohn's disease, resulting in an exaggerated adaptive immune response to these bacteria. It was hypothesised that the development of antimicrobial antibodies is influenced by the presence of genetic variants in pattern recognition receptor genes. The aim of this study was therefore to investigate the influence of mutations in these innate immune receptor genes (nucleotide oligomerisation domain (NOD) 2/caspase recruitment domain (CARD) 15, NOD1/CARD4, TUCAN/CARDINAL/CARD8, Toll‐like receptor (TLR) 4, TLR2, TLR1 and TLR6) on the development of antimicrobial and antiglycan antibodies in inflammatory bowel disease (IBD). Materials and methods A cohort of 1163 unrelated patients with IBD (874 Crohn's disease, 259 ulcerative colitis, 30 indeterminate colitis) and 312 controls were analysed for anti‐Saccharomyces cerevisiae antibodies (gASCA) IgG, anti‐laminaribioside antibodies (ALCA) IgG, anti‐chitobioside antibodies (ACCA) IgA, anti‐mannobioside antibodies (AMCA) IgG and outer membrane porin (Omp) IgA and were genotyped for variants in NOD2/CARD15, TUCAN/CARDINAL/CARD8, NOD1/CARD4, TLR4, TLR1, TLR2 and TLR6. Results When compared with Crohn's disease patients without CARD15 mutations, the presence of at least one CARD15 variant in Crohn's disease patients more frequently led to gASCA positivity (66.1% versus 51.5%, p < 0.0001) and ALCA positivity (43.3% versus 34.9%, p  =  0.018) and higher gASCA titers (85.7 versus 51.8 ELISA units, p < 0.0001), independent of ileal involvement. A gene dosage effect, with increasing gASCA and ALCA positivity for patients carrying none, one and two CARD15 variants, respectively, was seen for both markers. Similarly, Crohn's disease patients carrying NOD1/CARD4 indel had a higher

  18. Simian virus 40 T antigen can transcriptionally activate and mediate viral DNA replication in cells which lack the retinoblastoma susceptibility gene product.

    PubMed Central

    Trifillis, P; Picardi, J; Alwine, J C

    1990-01-01

    Simian virus 40 T antigen is a multifunctional protein which has recently been shown to form a complex with the retinoblastoma susceptibility gene product (Rb protein) (J.A. DeCaprio, J.W. Ludlow, J. Figge, J.-Y. Shaw, C.-M. Huang, W.-H. Lee, E. Marsilio, E. Paucha, and D.M. Livingston, Cell 54:275-283, 1988; P. Whyte, K.J. Buchkovich, J.M. Horowitz, S.H. Friend, M. Raybuck, R.A. Weinberg, and E. Harlow, Nature (London) 334:124-129, 1988). This interaction may facilitate some of the functions of T antigen. The ability of simian virus 40 T antigen to mediate transcriptional activation and viral DNA replication was tested in human osteosarcoma cell lines U-2OS and Saos-2, which are Rb positive and Rb negative, respectively. Both functions of T antigen were efficient in both cell lines. Hence, these functions can occur in the absence of Rb protein. Images PMID:2154611

  19. Antigens and cytokine genes in antitumor vaccines: the importance of the temporal delivery sequence in antitumor signals.

    PubMed

    Herrero, María José; Botella, Rafael; Dasí, Francisco; Algás, Rosa; Sánchez, María; Aliño, Salvador F

    2006-12-01

    Studies against cancer, including clinical trials, have shown that a correct activation of the immune system can lead to tumor rejection whereas incorrect signaling results in no positive effects or even anergy. We have worked assuming that two signals, GM-CSF (granulocyte and macrophage colony-stimulating factor) and tumor antigens are necessary to mediate an antitumor effective response. To study which is the ideal temporal sequence for their administration, we have used a murine model of antimelanoma vaccine employing whole B16 tumor cells or their membrane protein antigens (TMPs) in combination with gm-csf transfer before or after the antigen delivery. Our results show that: (i) When gm-csf tisular transfection is performed before TMP delivery, a tumor growth inhibition is observed, but with a limit effect when administering high antigen doses; in contrast, when signals are inverted, the limited effect is lost and greater antitumor efficacy is obtained. (ii) A similar behavior, but with stronger positive results, is observed employing gm-csf transfection and whole tumor cells as antigens. While negative results are obtained with gm-csf before cells, the best results (total survival of treated mice) are obtained when GM-CSF is administered in transfected cells. We conclude that optimal antitumoral response can be obtained when the antigen signal is given before (or simultaneous with) GM-CSF production, while the inversion of the signals could result in the undesired inhibition or anergy of the immune response.

  20. Stimulation of host centriolar antigen in TC7 cells by simian virus 40: requirement for RNA and protein syntheses and an intact simian virus 40 small-t gene function.

    PubMed

    Shyamala, M; Atcheson, C L; Kasamatsu, H

    1982-08-01

    Simian virus 40 (SV 40) stimulated a host cell antigen in the centriolar region after infection of African green monkey kidney (AGMK) cells. The addition of puromycin and actinomycin D to cells infected with SV40 within 5 h after infection inhibited the stimulation of the host cell antigen, indicating that de novo protein and RNA syntheses that occurred within the first 5 h after infection were essential for the stimulation. Early viable deletion mutants of SV40 with deletions mapping between 0.54 and 0.59 map units on the SV40 genome, dl2000, dl2001, dl2003, dl2004, dl2005, dl2006, and dl2007, did not stimulate the centriolar antigen above the level of uninfected cells. This indicated that an intact, functional small-t protein was essential for the SV40-mediated stimulation of the host cell antigen. Our studies, using cells infected with nondefective adenovirus-SV40 hybrid viruses that lack the small-t gene region of SV40 (Ad2+ND1, Ad2+ND2, Ad2+ND3, Ad2+ND4, and Ad2+ND5), revealed that the lack of small-t gene function of SV40 could be complemented by a gene function of the adenovirus-SV40 hybrid viruses for the centriolar antigen stimulation. Thus, adenovirus 2 has a gene(s) that is analogous to the small-t gene of SV40 for the stimulation of the host cell antigen in AGMK cells.

  1. Insertion of Vaccinia Virus C7L Host Range Gene into NYVAC-B Genome Potentiates Immune Responses against HIV-1 Antigens

    PubMed Central

    Nájera, José Luis; Gómez, Carmen Elena; García-Arriaza, Juan; Sorzano, Carlos Oscar; Esteban, Mariano

    2010-01-01

    Background The highly attenuated vaccinia virus strain NYVAC expressing HIV-1 components has been evaluated as a vaccine candidate in preclinical and clinical trials with encouraging results. We have previously described that the presence of C7L in the NYVAC genome prevents the induction of apoptosis and renders the vector capable of replication in human and murine cell lines while maintaining an attenuated phenotype in mice. Methodology/Principal Findings In an effort to improve the immunogenicity of NYVAC, we have developed a novel poxvirus vector by inserting the VACV host-range C7L gene into the genome of NYVAC-B, a recombinant virus that expresses four HIV-1 antigens from clade B (Env, Gag, Pol and Nef) (referred as NYVAC-B-C7L). In the present study, we have compared the in vitro and in vivo behavior of NYVAC-B and NYVAC-B-C7L. In cultured cells, NYVAC-B-C7L expresses higher levels of heterologous antigen than NYVAC-B as determined by Western blot and fluorescent-activated cell sorting to score Gag expressing cells. In a DNA prime/poxvirus boost approach with BALB/c mice, both recombinants elicited robust, broad and multifunctional antigen-specific T-cell responses to the HIV-1 immunogens expressed from the vectors. However, the use of NYVAC-B-C7L as booster significantly enhanced the magnitude of the T cell responses, and induced a more balanced cellular immune response to the HIV-1 antigens in comparison to that elicited in animals boosted with NYVAC-B. Conclusions/Significance These findings demonstrate the possibility to enhance the immunogenicity of the highly attenuated NYVAC vector by the insertion of the host-range gene C7L and suggest the use of this modified vector as an improved vaccine candidate against HIV/AIDS. PMID:20613977

  2. Cytotoxic T lymphocyte antigen 4 decreases humoral and cellular immunity by adenovirus to enhance target GFP gene transfer in C57BL/6 mice.

    PubMed

    Bai, Dou; Zhu, Wei; Zhang, Yu; Long, Ling; Zhu, Naishuo

    2015-01-01

    Adenoviruses (Ad) are once potential and promising vectors for gene delivery, but the immunogenicity attenuates its transfer efficiency. Cytotoxic T lymphocyte antigen 4 (CTLA-4) can inhibit T cell immunity. Thus, we aimed to study the effect of CTLA-4 in the process of Ad-mediated gene transfer. The C57BL/6 mice were injected by Ad vectors at twice, and CTLA-4 was administrated after the first Ad injection. Then, the CD3(+)CD4(+) T cells and circulating levels of IL-2, IL-4, and anti-Ad IgG were decreased by CTLA-4, while Ad generated immune responses. The green fluorescence protein (GFP) expressions of tissues were enhanced by CTLA-4 till injection of Ad at twice. Our results indicate that CTLA-4 can inhibit humoral and cellular immunity by adenovirus generation to enhance GFP delivery, and provide a potential way to assist in Ad-mediated gene transfer.

  3. Cloning of the complete gene for carcinoembryonic antigen: analysis of its promoter indicates a region conveying cell type-specific expression.

    PubMed Central

    Schrewe, H; Thompson, J; Bona, M; Hefta, L J; Maruya, A; Hassauer, M; Shively, J E; von Kleist, S; Zimmermann, W

    1990-01-01

    Carcinoembryonic antigen (CEA) is a widely used tumor marker, especially in the surveillance of colonic cancer patients. Although CEA is also present in some normal tissues, it is apparently expressed at higher levels in tumorous tissues than in corresponding normal tissues. As a first step toward analyzing the regulation of expression of CEA at the transcriptional level, we have isolated and characterized a cosmid clone (cosCEA1), which contains the entire coding region of the CEA gene. A close correlation exists between the exon and deduced immunoglobulin-like domain borders. We have determined a cluster of transcriptional starts for CEA and the closely related nonspecific cross-reacting antigen (NCA) gene and have sequenced their putative promoters. Regions of sequence homology are found as far as approximately 500 nucleotides upstream from the translational starts of these genes, but farther upstream they diverge completely. In both cases we were unable to find classic TATA or CAAT boxes at their expected positions. To characterize the CEA and NCA promoters, we carried out transient transfection assays with promoter-indicator gene constructs in the CEA-producing adenocarcinoma cell line SW403, as well as in nonproducing HeLa cells. A CEA gene promoter construct, containing approximately 400 nucleotides upstream from the translational start, showed nine times higher activity in the SW403 than in the HeLa cell line. This indicates that cis-acting sequences which convey cell type-specific expression of the CEA gene are contained within this region. Images PMID:2342461

  4. Gene complementation in the T-lymphocyte proliferative response to poly (Glu55Lys36Phe9)n. A demonstration that both immune response gene products must be expressed in the same antigen-presenting cell

    PubMed Central

    1979-01-01

    The immune response (Ir) to the random copolymer GLphi depends upon the function of two Ir genes, Ir-GLphi-beta[beta] and Ir-GLphi- alpha[alpha], mapped to the I-A and I-E/C subregions of the major histocompatibility complex, respectively. In this paper, the site(s) of expression of the products of these two Ir genes was examined by evaluating T-lymphocyte proliferative responses of bone marrow radiation chimeras. Chimeras were created in [alpha+beta- X alpha- beta+]F1 responder mice by lethal irradiation and reconstitution with a mixture of bone marrow cells from both parental strains. These chimeras failed to respond to GLphi, although they were capable or responding to the much weaker antigens, (T,G)-A--L, TEPC-15, pigeon cytochrome c, and (H,G)-A--L. This failure to respond to GLphi was shown not to be the result of a cryptic mixed lymphocyte reaction, as similar chimeras created in (alpha+beta+ X alpha-beta+)F1 mice responded well to GLphi, although they possessed almost the same potential histoincompatibility. Furthermore, the lack of response to GLphi could not be attributed to a general failure of the two parental cell types in the chimeras to collaboratc with each other, as each chimeric parental cell type could respond to dinitrophenyl conjugated ovalbumin presented on nonimmune spleen cells from the other parent. Thus, the failure of low responder parental into F1 high responder chimeras to generate an immune response to GLphi suggests that immune competence for this antigen requires at least one cell type in the immune system to express gene products of both the Ir-glphi-alpha and -beta genes, i.e. one cell must be of high responder genotype. The the antigen-presenting cell is one such cell type was shown by experiments in which GLphi-primed T lymphocytes from responder F1 mice were stimulated with antigen bound to nonimmune spleen cells. Only spleen cells from responder F1 and recombinant mice could present GLphi. Neither of the two complementing

  5. Immune recognition of protein antigens

    SciTech Connect

    Laver, W.G.; Air, G.M.

    1985-01-01

    This book contains 33 papers. Some of the titles are: Antigenic Structure of Influenze Virus Hemagglutinin; Germ-line and Somatic Diversity in the Antibody Response to the Influenza Virus A/PR/8/34 Hemagglutinin; Recognition of Cloned Influenza A Virus Gene Products by Cytotoxic T Lymphocytes; Antigenic Structure of the Influenza Virus N2 Neuraminidase; and The Molecular and Genetic Basis of Antigenic Variation in Gonococcal Pillin.

  6. Identification of the O antigen polymerase (rfc) gene in Escherichia coli O4 by insertional mutagenesis using a nonpolar chloramphenicol resistance cassette.

    PubMed Central

    Lukomski, S; Hull, R A; Hull, S I

    1996-01-01

    Computer analysis of the O4 polysaccharide gene cluster of Escherichia coli revealed the presence of two open reading frames (ORFs) encoding strongly hydrophobic polypeptides. O antigen polymerase, which is encoded by the rfc gene, is a potential membrane protein and therefore should be hydrophobic. To identify the rfc gene, these two ORFs were subjected to insertional mutagenesis. A chloramphenicol resistance cassette was designed which, when properly inserted, does not cause a polar effect in downstream genes. Each of two ORFs, cloned into a plasmid vector, was inactivated with this cassette. Two types of mutants bearing chromosomal insertions of the cassettes in each ORF were constructed by homologous recombination. These mutants were characterized by PCR, Southern blotting, and transverse-alternating-field electrophoresis. Only one class of mutants exhibited the expected O polymerase-deficient phenotype; they produced O4-specific, semirough lipopolysaccharide. Therefore, this ORF was identified as the rfc gene. The chromosomal rfc mutation was complemented in trans by the rfc gene expressed from a plasmid vector. PMID:8550424

  7. Recognition of HLA-A2 and -B7 antigens by cloned cytotoxic T lymphocytes after gene transfer into human and monkey, but not mouse, cells.

    PubMed Central

    Barbosa, J A; Mentzer, S J; Minowada, G; Strominger, J L; Burakoff, S J; Biro, P A

    1984-01-01

    The genes that code for the human major histocompatibility class I antigens, HLA-A2 and HLA-B7, were introduced into human, monkey, and mouse cell lines by cotransfection with suitable biochemical markers and the fluorescence-activated cell sorter was used to identify and/or select stable cell populations expressing high surface levels of these antigens. Levels of expression obtained were similar to those observed for endogenous HLA antigens on various human cell lines and were 25-80% of those observed on the human B-lymphoblastoid cell line JY. Serologically defined HLA-A2 and HLA-B7 polymorphic determinants remained intact on all transfected recipient cells analyzed. Cloned human allospecific cytotoxic T lymphocytes (CTL) specific for HLA-A2 or HLA-B7 were capable of lysing appropriate HLA-transfected human cells with comparable efficiency to JY cell lysis. Two of 10 CTL clones lysed appropriate monkey cell transfectants with approximately equal to 20% the efficiency of human cell transfectants. No specific lysis of any HLA-transfected mouse cell lines, including a B cell lymphoma, was observed despite comparable levels of surface antigen expression or after induction of higher levels by mouse gamma-interferon. Furthermore, L cells expressing human beta 2-microglobulin in addition to HLA-A2 or -B7 were not lysed by these CTL. Thus, an additional species-specific component may be involved in lysis by allogeneic CTL--possibly related to the function(s) of other surface proteins on target cells. PMID:6390442

  8. Differentiation antigens in lymphohemopoietic tissues

    SciTech Connect

    Miyasaka, M.; Trnka, Z.

    1988-01-01

    This book contains 15 chapters. Some of the chapter titles are: In Situ Characterization of Human Lymphoid Cells Using Monoclonal Antibodies; Structural and Functional Aspects of HLA Clas II Genes; Cell-Surface Differentiation Antigens Expressed on Thymocytes and T Cells of the Mouse; and Differentiation Antigens on Lymphoid Cells of the Guinea Pig.

  9. The inducible caspase-9 suicide gene system as a “safety switch” to limit on-target, off-tumor toxicities of chimeric antigen receptor T cells

    PubMed Central

    Gargett, Tessa; Brown, Michael P.

    2014-01-01

    Immune modulation has become a central element in many cancer treatments, and T cells genetically engineered to express chimeric antigen receptors (CAR) may provide a new approach to cancer immunotherapy. Autologous CAR T cells that have been re-directed toward tumor-associated antigens (TAA) have shown promising results in phase 1 clinical trials, with some patients undergoing complete tumor regression. However, this T-cell therapy must carefully balance effective T-cell activation, to ensure antitumor activity, with the potential for uncontrolled activation that may produce immunopathology. An inducible Caspase 9 (iCasp9) “safety switch” offers a solution that allows for the removal of inappropriately activated CAR T cells. The induction of iCasp9 depends on the administration of the small molecule dimerizer drug AP1903 and dimerization results in rapid induction of apoptosis in transduced cells, preferentially killing activated cells expressing high levels of transgene. The iCasp9 gene has been incorporated into vectors for use in preclinical studies and demonstrates effective and reliable suicide gene activity in phase 1 clinical trials. A third-generation CAR incorporating iCasp9 re-directs T cells toward the GD2 TAA. GD2 is over-expressed in melanoma and other malignancies of neural crest origin and the safety and activity of these GD2-iCAR T cells will be investigated in CARPETS and other actively recruiting phase 1 trials. PMID:25389405

  10. Transcription analysis of class II human leukocyte antigen genes from normal and immunodeficient B lymphocytes, using polymerase chain reaction.

    PubMed Central

    Bull, M; van Hoef, A; Gorski, J

    1990-01-01

    The RNA transcript levels of all human leukocyte antigen class II loci were determined from class II congenital immunodeficient B cells by polymerase chain reaction amplification of cDNA. No mRNA was observed under conditions in which 0.01% normal levels could be visualized. Pre-mRNA could be amplified from normal B cells but not from immunodeficient B cells, indicating a transcription defect. Images PMID:2113177

  11. Clonally diverse rfb gene clusters are involved in expression of a family of related D-galactan O antigens in Klebsiella species.

    PubMed Central

    Kelly, R F; Whitfield, C

    1996-01-01

    Klebsiella species express a family of structurally related lipopolysaccharide O antigens which share a common backbone known as D-galactan I. Serotype specificity results from modification of D-galactan I by addition of domains of altered structure or by substitution with O-acetyl and/or alpha-D-Galp side groups with various linkages and stoichiometries. In the prototype, Klebsiella serotype O1, the his-linked rfb gene cluster is required for synthesis of D-galactan I, but genes conferring serotype specificity are unlinked. The D-galactan I part of the O polysaccharide is O acetylated in Klebsiella serotype O8. By cloning the rfb region from Klebsiella serotype O8 and analyzing the O polysaccharide synthesized in Escherichia coli K-12 hosts, we show that, like rfbO1, the rfbO8 region directs formation of unmodified D-galactan I. The rfbAB genes encode an ATP-binding cassette transporter required for export of polymeric D-galactan I across the plasma membrane prior to completion of the lipopolysaccharide molecule by ligation of the O polysaccharide to lipid A-core. Complementation experiments show that the rfbAB gene products in serotypes O1 and O8 are functionally equivalent and interchangeable. Hybridization experiments and physical mapping of the rfb regions in related Klebsiella serotypes suggest the existence of shared rfb genes with a common organization. However, despite the functional equivalence of these rfb gene clusters, at least three distinct clonal groups were detected in different Klebsiella species and subspecies, on the basis of Southern hybridization experiments carried out under high-stringency conditions. The clonal groups cannot be predicted by features of the O-antigen structure. To examine the relationships in more detail, the complete nucleotide sequence of the serotype O8 rfb cluster was determined and compared with that of the serotype O1 prototype. The nucleotide sequences for the six rfb genes showed variations in moles percent G

  12. Gene and antigen markers of Shiga-toxin producing E. coli from Michigan and Indiana river water: Occurrence and relation to recreational water quality criteria

    USGS Publications Warehouse

    Duris, J.W.; Haack, S.K.; Fogarty, L.R.

    2009-01-01

    The relation of bacterial pathogen occurrence to fecal indicator bacteria (FIB) concentrations used for recreational water quality criteria (RWQC) is poorly understood. This study determined the occurrence of Shiga-toxin producing Escherichia coli (STEC) markers and their relation to FIB concentrations in Michigan and Indiana river water. Using 67 fecal coliform (FC) bacteria cultures from 41 river sites in multiple watersheds, we evaluated the occurrence of five STEC markers: the Escherichia coli (EC) O157 antigen and gene, and the STEC virulence genes eaeA, stx1, and stx2. Simple isolations from selected FC cultures yielded viable EC O157. By both antigen and gene assays, EC O157 was detected in a greater proportion of samples exceeding rather than meeting FC RWQC (P < 0.05), but was unrelated to EC and enterococci RWQC. The occurrence of all other STEC markers was unrelated to any FIB RWQC. The eaeA, stx2, and stx1 genes were found in 93.3, 13.3, and in 46.7% of samples meeting FC RWQC and in 91.7, 0.0, and 37.5% of samples meeting the EC RWQC. Although not statistically significant, the percentage of samples positive for each STEC marker except stx1 was lower in samples that met, as opposed to exceeded, FIB RWQC. Viable STEC were common members of the FC communities in river water throughout southern Michigan and northern Indiana, regardless of FIB RWQC. Our study indicates that further information on the occurrence of pathogens in recreational waters, and research on alternative indicators of their occurrence, may help inform water-resource management and public health decision-making. Copyright ?? 2009 by the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America. All rights reserved.

  13. DNA hypomethylation-mediated activation of Cancer/Testis Antigen 45 (CT45) genes is associated with disease progression and reduced survival in epithelial ovarian cancer.

    PubMed

    Zhang, Wa; Barger, Carter J; Link, Petra A; Mhawech-Fauceglia, Paulette; Miller, Austin; Akers, Stacey N; Odunsi, Kunle; Karpf, Adam R

    2015-01-01

    Epithelial ovarian cancer (EOC) is a highly lethal malignancy due to a lack of early detection approaches coupled with poor outcomes for patients with clinically advanced disease. Cancer-testis (CT) or cancer-germline genes encode antigens known to generate spontaneous anti-tumor immunity in cancer patients. CT45 genes are a recently discovered 6-member family of X-linked CT genes with oncogenic function. Here, we determined CT45 expression in EOC and fully defined its epigenetic regulation by DNA methylation. CT45 was silent and hypermethylated in normal control tissues, but a large subset of EOC samples showed increased CT45 expression in conjunction with promoter DNA hypomethylation. In contrast, copy number status did not correlate with CT45 expression in the TCGA database for EOC. CT45 promoter methylation inversely correlated with both CT45 mRNA and protein expression, the latter determined using IHC staining of an EOC TMA. CT45 expression was increased and CT45 promoter methylation was decreased in late-stage and high-grade EOC, and both measures were associated with poor survival. CT45 hypomethylation was directly associated with LINE-1 hypomethylation, and CT45 was frequently co-expressed with other CT antigen genes in EOC. Decitabine treatment induced CT45 mRNA and protein expression in EOC cells, and promoter transgene analyses indicated that DNA methylation directly represses CT45 promoter activity. These data verify CT45 expression and promoter hypomethylation as possible prognostic biomarkers, and suggest CT45 as an immunological or therapeutic target in EOC. Treatment with decitabine or other epigenetic modulators could provide a means for more effective immunological targeting of CT45.

  14. DNA hypomethylation-mediated activation of Cancer/Testis Antigen 45 (CT45) genes is associated with disease progression and reduced survival in epithelial ovarian cancer.

    PubMed

    Zhang, Wa; Barger, Carter J; Link, Petra A; Mhawech-Fauceglia, Paulette; Miller, Austin; Akers, Stacey N; Odunsi, Kunle; Karpf, Adam R

    2015-01-01

    Epithelial ovarian cancer (EOC) is a highly lethal malignancy due to a lack of early detection approaches coupled with poor outcomes for patients with clinically advanced disease. Cancer-testis (CT) or cancer-germline genes encode antigens known to generate spontaneous anti-tumor immunity in cancer patients. CT45 genes are a recently discovered 6-member family of X-linked CT genes with oncogenic function. Here, we determined CT45 expression in EOC and fully defined its epigenetic regulation by DNA methylation. CT45 was silent and hypermethylated in normal control tissues, but a large subset of EOC samples showed increased CT45 expression in conjunction with promoter DNA hypomethylation. In contrast, copy number status did not correlate with CT45 expression in the TCGA database for EOC. CT45 promoter methylation inversely correlated with both CT45 mRNA and protein expression, the latter determined using IHC staining of an EOC TMA. CT45 expression was increased and CT45 promoter methylation was decreased in late-stage and high-grade EOC, and both measures were associated with poor survival. CT45 hypomethylation was directly associated with LINE-1 hypomethylation, and CT45 was frequently co-expressed with other CT antigen genes in EOC. Decitabine treatment induced CT45 mRNA and protein expression in EOC cells, and promoter transgene analyses indicated that DNA methylation directly represses CT45 promoter activity. These data verify CT45 expression and promoter hypomethylation as possible prognostic biomarkers, and suggest CT45 as an immunological or therapeutic target in EOC. Treatment with decitabine or other epigenetic modulators could provide a means for more effective immunological targeting of CT45. PMID:26098711

  15. Analysis of processing and polyadenylation signals of the hepatitis B virus surface antigen gene by using simian virus 40-hepatitis B virus chimeric plasmids

    SciTech Connect

    Simonsen, C.C.; Levinson, A.D.

    1983-12-01

    The authors examined the transcription of the hepatitis B virus surface antigen (HBsAg) gene in COs cells transfected with simian virus 40-based recombinant plasmids. When positioned behind the simian virus 40 late promoter, three transcripts were identified which hybridized to the HBsAg gene: a 2,000-nucleotide transcript colinear with a gene, a 1,100-nucleotide transcript representing a spliced molecule in which a major portion of the sequences encoding HBsAg were deleted, and an 800-nucleotide transcript derived primarily from sequences 3' to the HBsAg gene. The splice acceptor site utilized by the 1,100-nucleotide transcript is located immediately upstream of an open reading frame of unknown function contained within the 3' nontranslated region of the HBsAg gene. The HBsAg-specific mRNA species terminate 12 to 19 base pairs 3' of the sequence UAUAAA, similar to the concensus hexanucleotide which is thought to promote polyadenylation (AAUAAA). They constructed a series of plasmids with progressive deletions from the region surrounding where these transcripts terminate. Analysis of mRNA produced by cells transfected with these plasmids indicated that the signal hexanucleotide is in itself unable to promote the efficient processing of mRNA in the absence of downstream hepatitis B virus sequences. Processing proceeds properly, however, from plasmids containing an additional 30 nucleotides 3' of this signal.

  16. Tissue-specific expression of cell-surface Qa-2 antigen from a transfected Q7b gene of C57BL/10 mice

    PubMed Central

    1987-01-01

    We screened a cDNA library prepared from a BALB.B10 CTL clone that expresses Qa-2 antigen, and isolated four clones derived from Q7b, a Qa region gene of C57BL/10. One of these Q7b cDNAs and the Q7b chromosomal gene were subcloned into expression vectors and transfected into L cells and R1.1 thymoma cells. We found that the chromosomal Q7b gene expresses Qa-2 on the surface of R1.1 cells, but not on L cells while the Q7b cDNA expresses protein on the surface of both cell types. The levels of Qa-2 expression do not correlate with the total levels of Q7b mRNA in these transfectants. Our results suggest that the tissue- specific expression of Qa-2 may be controlled, in part, by mechanisms of alternate RNA splicing. By using hybrid gene constructs, we have mapped the tissue-specific element to the 3' part of the gene, downstream of a site near the middle of exon 4. The hybrid polypeptides differ significantly in their transmembrane and cytoplasmic regions. These portions of the protein also may play a role in the tissue- specific expression of Qa-2. PMID:3502706

  17. Structural organization of the human S-antigen gene. cDNA, amino acid, intron, exon, promoter, in vitro transcription, retina, and pineal gland.

    PubMed

    Yamaki, K; Tsuda, M; Kikuchi, T; Chen, K H; Huang, K P; Shinohara, T

    1990-12-01

    S-Antigen (S-Ag) is a major soluble photoreceptor protein involved in the visual transduction cascade. Several S-Ag cDNAs and a gene coding for human S-Ag were isolated from cDNA and gene libraries. The gene sequences of the coding, noncoding, and 5'-flanking regions of the gene were determined. The S-Ag gene was approximately 50 kbp (kilobase pairs) in length and contained 16 exons and 15 introns. The length of most exons was less than 100 base pairs (bp) and the smallest one was only 10 bp. In contrast, the length of most introns was larger than 2 kbp, and the gene comprised 97% intron and 3% exon. The splice sites for donor and acceptor were in good agreement with the GT/AG rule. The S-Ag protein of 403 amino acid residues was translated from a mRNA of 1.9 kbp, and the mRNA was transcribed from a gene of 50 kbp. The 5'-flanking region of the gene, approximately 1.1 kbp long, had no known regulatory elements for transcription such as TATA, GC, and CCAAT boxes. Interestingly, the 5'-flanking region had promoter activity in an in vitro transcription assay using a nuclear extract of rat brain. A major transcription start site was found at 387 bp upstream from the translation start site ATG. Our results indicate that the sequence of S-Ag promoter differs from other known promoters and may, perhaps, be specific for photoreceptor rod cells and pinealocytes.

  18. Expression profile of peripheral tissue antigen genes in medullary thymic epithelial cells (mTECs) is dependent on mRNA levels of autoimmune regulator (Aire).

    PubMed

    Oliveira, Ernna H; Macedo, Claudia; Donate, Paula B; Almeida, Renata S; Pezzi, Nicole; Nguyen, Catherine; Rossi, Marcos A; Sakamoto-Hojo, Elza T; Donadi, Eduardo A; Passos, Geraldo A

    2013-01-01

    In the thymus of non-obese diabetic (NOD) mice, the expression of the autoimmune regulator (Aire) gene varies with age, and its down-regulation in young mice precedes the later emergence of type 1 diabetes mellitus (T1D). In addition, the insulin (Ins2) peripheral tissue antigen (PTA) gene, which is Aire-dependent, is also deregulated in these mice. Based in these findings, we hypothesized that the imbalance in PTA gene expression in the thymus can be associated with slight variations in Aire transcript levels. To test this, we used siRNA to knockdown Aire by in vivo electro-transfection of the thymus of BALB/c mice. The efficiency of the electro-transfection was monitored by assessing the presence of irrelevant Cy3-labeled siRNA in the thymic stroma. Importantly, Aire-siRNA reached medullary thymic epithelial cells (mTECs) down-regulating Aire. As expected, the in vivo Aire knockdown was partial and transient; the maximum 59% inhibition occurred in 48 h. The Aire knockdown was sufficient to down-regulate PTA genes; however, surprisingly, several others, including Ins2, were up-regulated. The modulation of these genes after in vivo Aire knockdown was comparable to that observed in NOD mice before the emergence of T1D. The in vitro transfections of 3.10 mTEC cells with Aire siRNA resulted in samples featuring partial (69%) and complete (100%) Aire knockdown. In these Aire siRNA-transfected 3.10 mTECs, the expression of PTA genes, including Ins2, was down-regulated. This suggests that the expression profile of PTA genes in mTECs is affected by fine changes in the transcription level of Aire.

  19. The role of B. pertussis vaccine antigen gene variants in pertussis resurgence and possible consequences for vaccine development.

    PubMed

    Preston, Andrew

    2016-05-01

    Whooping cough, or pertussis, caused by Bordetella pertussis is considered resurgent in a number of countries world-wide, despite continued high level vaccine coverage. Among a number of causes for this that have been proposed, is the emergence of B. pertussis strains expressing variants of the antigens contained in acellular pertussis vaccines; i.e. the evolution of B. pertussis toward vaccine escape. This commentary highlights the contradictory nature of evidence for this but also discusses the importance of understanding the role of B. pertussis adaptation to vaccine-mediated immune selection pressures for vaccine-mediated pertussis control strategies.

  20. The role of B. pertussis vaccine antigen gene variants in pertussis resurgence and possible consequences for vaccine development.

    PubMed

    Preston, Andrew

    2016-05-01

    Whooping cough, or pertussis, caused by Bordetella pertussis is considered resurgent in a number of countries world-wide, despite continued high level vaccine coverage. Among a number of causes for this that have been proposed, is the emergence of B. pertussis strains expressing variants of the antigens contained in acellular pertussis vaccines; i.e. the evolution of B. pertussis toward vaccine escape. This commentary highlights the contradictory nature of evidence for this but also discusses the importance of understanding the role of B. pertussis adaptation to vaccine-mediated immune selection pressures for vaccine-mediated pertussis control strategies. PMID:26889694

  1. Antigen-Independent Appearance of Recombination Activating Gene (Rag)-Positive Bone Marrow B Cells in the Spleens of Immunized Mice

    PubMed Central

    Gärtner, Frank; Alt, Frederick W.; Monroe, Robert J.; Seidl, Katherine J.

    2000-01-01

    Splenic B lineage cells expressing recombination activation genes (RAG+) in mice immunized with 4-hydroxy-3-nitrophenyl-acetyl coupled to chicken γ-globulin (NP-CGG) and the adjuvant aluminum-hydroxide (alum) have been proposed to be mature B cells that reexpress RAG after an antigen encounter in the germinal center (GC), a notion supported by findings of RAG expression in peripheral B lymphocyte populations activated in vitro. However, recent studies indicate that these cells might be immature B cells that have not yet extinguished RAG expression. Here, we employ RAG2–green fluorescent protein (GFP) fusion gene knock-in mice to show that RAG+ B lineage cells do appear in the spleen after the administration of alum alone, and that their appearance is independent of T cell interactions via the CD40 pathway. Moreover, splenic RAG+ B lineage cells were detectable in immunized RAG2-deficient mice adoptively transferred with bone marrow (BM) cells, but not with spleen cells from RAG+ mice. Although splenic RAG+ B cells express surface markers associated with GC B cells, we also find the same basic markers on progenitor/precursor BM B cells. Finally, we did not detect RAG gene expression after the in vitro stimulation of splenic RAG− mature B cells with mitogens (lipopolysaccharide and anti-CD40) and cytokines (interleukin [IL]-4 and IL-7). Together, our studies indicate that RAG+ B lineage cells from BM accumulate in the spleen after immunization, and that this accumulation is not the result of an antigen-specific response. PMID:11120771

  2. A serine proteinase inhibitor locus at 18q21.3 contains a tandem duplication of the human squamous cell carcinoma antigen gene.

    PubMed Central

    Schneider, S S; Schick, C; Fish, K E; Miller, E; Pena, J C; Treter, S D; Hui, S M; Silverman, G A

    1995-01-01

    The squamous cell carcinoma antigen (SCCA) is a member of the ovalbumin family of serine proteinase inhibitors (serpins). A neutral form of the protein is found in normal and some malignant squamous cells, whereas an acidic form is detected exclusively in tumor cells and in the circulation of patients with squamous cell tumors. In this report, we describe the cloning of the SCCA gene from normal genomic DNA. Surprisingly, two genes were found. They were tandemly arrayed and flanked by two other closely related serpins, plasminogen activator inhibitor type 2 (PAI2) and maspin at 18q21.3. The genomic structure of the two genes, SCCA1 and SCCA2, was highly conserved. The predicted amino acid sequences were 92% identical and suggested that the neutral form of the protein was encoded by SCCA1 and the acidic form was encoded by SCCA2. Further characterization of the region should determine whether the differential expression of the SCCA genes plays a causal role in development of more aggressive squamous cell carcinomas. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:7724531

  3. High throughput quantitative reverse transcription PCR assays revealing over-expression of cancer testis antigen genes in multiple myeloma stem cell-like side population cells.

    PubMed

    Wen, Jianguo; Li, Hangwen; Tao, Wenjing; Savoldo, Barbara; Foglesong, Jessica A; King, Lauren C; Zu, Youli; Chang, Chung-Che

    2014-09-01

    Multiple myeloma (MM) stem cells, proposed to be responsible for the tumourigenesis, drug resistance and recurrence of this disease, are enriched in the cancer stem cell-like side population (SP). Cancer testis antigens (CTA) are attractive targets for immunotherapy because they are widely expressed in cancers but only in limited types of normal tissues. We designed a high throughput assay, which allowed simultaneous relative quantifying expression of 90 CTA genes associated with MM. In the three MM cell lines tested, six CTA genes were over-expressed in two and LUZP4 and ODF1 were universally up-regulated in all three cell lines. Subsequent study of primary bone marrow (BM) from eight MM patients and four healthy donors revealed that 19 CTA genes were up-regulated in SP of MM compared with mature plasma cells. In contrast, only two CTA genes showed a moderate increase in SP cells of healthy BM. Furthermore, knockdown using small interfering RNA (siRNA) revealed that LUZP4 expression is required for colony-forming ability and drug resistance in MM cells. Our findings indicate that multiple CTA have unique expression profiles in MM SP, suggesting that CTA may serve as targets for immunotherapy that it specific for MM stem cells and which may lead to the long-term cure of MM.

  4. The Severity of Chorioamnionitis in Pregnant Sheep Is Associated with In Vivo Variation of the Surface-Exposed Multiple-Banded Antigen/Gene of Ureaplasma parvum1

    PubMed Central

    Knox, Christine L.; Dando, Samantha J.; Nitsos, Ilias; Kallapur, Suhas G.; Jobe, Alan H.; Payton, Diane; Moss, Timothy J.M.; Newnham, John P.

    2010-01-01

    Ureaplasma species are the bacteria most frequently isolated from human amniotic fluid in asymptomatic pregnancies and placental infections. Ureaplasma parvum serovars 3 and 6 are the most prevalent serovars isolated from men and women. We hypothesized that the effects on the fetus and chorioamnion of chronic ureaplasma infection in amniotic fluid are dependent on the serovar, dose, and variation of the ureaplasma multiple-banded antigen (MBA) and mba gene. We injected high- or low-dose U. parvum serovar 3, serovar 6, or vehicle intra-amniotically into pregnant ewes at 55 days of gestation (term = 150 days) and examined the chorioamnion, amniotic fluid, and fetal lung tissue of animals delivered by cesarean section at 125 days of gestation. Variation of the multiple banded antigen/mba generated by serovar 3 and serovar 6 ureaplasmas in vivo were compared by PCR assay and Western blot. Ureaplasma inoculums demonstrated only one (serovar 3) or two (serovar 6) MBA variants in vitro, but numerous antigenic variants were generated in vivo: serovar 6 passage 1 amniotic fluid cultures contained more MBA size variants than serovar 3 (P = 0.005), and ureaplasma titers were inversely related to the number of variants (P = 0.025). The severity of chorioamnionitis varied between animals. Low numbers of mba size variants (five or fewer) within amniotic fluid were associated with severe inflammation, whereas the chorioamnion from animals with nine or more mba variants showed little or no inflammation. These differences in chorioamnion inflammation may explain why not all women with in utero Ureaplasma spp. experience adverse pregnancy outcomes. PMID:20519696

  5. The severity of chorioamnionitis in pregnant sheep is associated with in vivo variation of the surface-exposed multiple-banded antigen/gene of Ureaplasma parvum.

    PubMed

    Knox, Christine L; Dando, Samantha J; Nitsos, Ilias; Kallapur, Suhas G; Jobe, Alan H; Payton, Diane; Moss, Timothy J M; Newnham, John P

    2010-09-01

    Ureaplasma species are the bacteria most frequently isolated from human amniotic fluid in asymptomatic pregnancies and placental infections. Ureaplasma parvum serovars 3 and 6 are the most prevalent serovars isolated from men and women. We hypothesized that the effects on the fetus and chorioamnion of chronic ureaplasma infection in amniotic fluid are dependent on the serovar, dose, and variation of the ureaplasma multiple-banded antigen (MBA) and mba gene. We injected high- or low-dose U. parvum serovar 3, serovar 6, or vehicle intra-amniotically into pregnant ewes at 55 days of gestation (term = 150 days) and examined the chorioamnion, amniotic fluid, and fetal lung tissue of animals delivered by cesarean section at 125 days of gestation. Variation of the multiple banded antigen/mba generated by serovar 3 and serovar 6 ureaplasmas in vivo were compared by PCR assay and Western blot. Ureaplasma inoculums demonstrated only one (serovar 3) or two (serovar 6) MBA variants in vitro, but numerous antigenic variants were generated in vivo: serovar 6 passage 1 amniotic fluid cultures contained more MBA size variants than serovar 3 (P = 0.005), and ureaplasma titers were inversely related to the number of variants (P = 0.025). The severity of chorioamnionitis varied between animals. Low numbers of mba size variants (five or fewer) within amniotic fluid were associated with severe inflammation, whereas the chorioamnion from animals with nine or more mba variants showed little or no inflammation. These differences in chorioamnion inflammation may explain why not all women with in utero Ureaplasma spp. experience adverse pregnancy outcomes.

  6. Differential, Positional-Dependent Transcriptional Response of Antigenic Variation (var) Genes to Biological Stress in Plasmodium falciparum

    PubMed Central

    Shalev, Oshrit; Sinay, Rosa; Cowman, Alan

    2009-01-01

    1% of the genes of the human malaria causing agent Plasmodium falciparum belong to the heterogeneous var gene family which encodes P. falciparum erythrocyte membrane protein 1 (PFEMP1). This protein mediates part of the pathogenesis of the disease by causing adherence of infected erythrocytes (IE) to the host endothelium. At any given time, only one copy of the family is expressed on the IE surface. The cues which regulate the allelic exclusion of these genes are not known. We show the existence of a differential expression pattern of these genes upon exposure to biological stress in relation to their positional placement on the chromosome – expression of centrally located var genes is induced while sub-telomeric copies of the family are repressed - this phenomenon orchestrated by the histone deacetylase pfsir2. Moreover, stress was found to cause a switch in the pattern of the expressed var genes thus acting as a regulatory cue. By using pharmacological compounds which putatively affect pfsir2 activity, distinct changes of var gene expression patterns were achieved which may have therapeutic ramifications. As disease severity is partly associated with expression of particular var gene subtypes, manipulation of the IE environment may serve as a mechanism to direct transcription towards less virulent genes. PMID:19730749

  7. A trans-acting major histocompatibility complex-linked gene whose alleles determine gain and loss changes in the antigenic structure of a classical class I molecule

    PubMed Central

    1989-01-01

    The RT1.A locus of the rat MHC encodes the H chain of the single classical class I molecule of this species. One of the alleles of this polymorphic locus, RT1.Aa, is present in several laboratory inbred, congenic, and MHC recombinant rat strains. Studies of the RT1.Aa class I molecule from a number of these strains as a target for CTL show that its antigenicity, both as an alloantigen and a restricting element, is subject to gain and loss alterations by the action of a gene mapping in the MHC to the right of RT1.A. This locus is apparently present in two allelic forms (one possibly a null allele) corresponding to the presence or absence of a dominant transacting modifier, and has been named class I modification, or cim. The antigenic change brought about by cim is scarcely detectable serologically but highly immunogenic for CTL. Biochemical investigations show that cim affects the post- translational modification of RT1.Aa. PMID:2475574

  8. Affinity maturation of an anti-V antigen IgG expressed in situ through adenovirus gene delivery confers enhanced protection against Yersinia pestis challenge.

    PubMed

    Van Blarcom, T J; Sofer-Podesta, C; Ang, J; Boyer, J L; Crystal, R G; Georgiou, G

    2010-07-01

    Genetic transfer of neutralizing antibodies (Abs) has been shown to confer strong and persistent protection against bacterial and viral infectious agents. Although it is well established that for many exogenous neutralizing Abs increased antigen affinity correlates with protection, the effect of antigen affinity on Abs produced in situ after adenoviral gene transfer has not been examined. The mouse IgG2b monoclonal Ab, 2C12.4, recognizes the Yersinia pestis type III secretion apparatus protein, LcrV (V antigen), and confers protection in mice when administered as an IgG intraperitoneally or after genetic immunization with engineered, replication-defective serotype 5 human adenovirus (Ad). The 2C12.4 Ab was expressed as a single-chain variable fragment (scFv) in Escherichia coli and was shown to display an equilibrium dissociation constant (K(D))=3.5 nM by surface plasmon resonance analysis. The 2C12.4 scFv was subjected to random mutagenesis, and variants with increased affinity were isolated by flow cytometry using the anchored periplasmic expression bacterial display system. After a single round of mutagenesis, variants displaying up to 35-fold lower K(D) values (H8, K(D)=100 pM) were isolated. The variable domains of the H8 scFv were used to replace those of the parental 2C12.4 IgG encoded in the Ad vector, AdalphaV, giving rise to AdalphaV.H8. The two adenoviral vectors resulted in similar titers of anti-V antigen Abs 3 days after immunization, with 10(9), 10(10) or 10(11) particle units (pu). After intranasal challenge with 363 LD(50) (lethal dose, 50%) of Y. pestis CO92, 54% of the mice immunized with 10(10) pu of AdalphaV.H8 survived through the 14 day end point compared with only 15% survivors for the group immunized with AdalphaV expressing the lower-affinity 2C12.4 (P<0.04; AdalphaV versus AdalphaV.H8). These results indicate that affinity maturation of a neutralizing Ab delivered by genetic transfer may confer increased protection not only for Y. pestis

  9. Characterization Based on the 56-Kda Type-Specific Antigen Gene of Orientia tsutsugamushi Genotypes Isolated from Leptotrombidium Mites and the Rodent Host Post-Infection

    PubMed Central

    Takhampunya, Ratree; Tippayachai, Bousaraporn; Promsathaporn, Sommai; Leepitakrat, Surachai; Monkanna, Taweesak; Schuster, Anthony L.; Melendrez, Melanie C.; Paris, Daniel H.; Richards, Allen L.; Richardson, Jason H.

    2014-01-01

    Characterization of the 56-kDa type-specific antigen (TSA) genes of Orientia tsutsugamushi (OT) from three naturally infected, laboratory-reared mite colonies comprising three species (Leptotrombidium deliense [Ld], Leptotrombidium imphalum [Li], and Leptotrombidium chiangraiensis [Lc]) has revealed the presence of single and coexisting OT genotypes found in individual chiggers. The Karp genotype was found in all of the chiggers examined, whereas Gilliam and UT302 genotypes were only observed in combination with the Karp genotype. From analysis of these OT genotypes after transmission from chiggers to mice it was determined that with the Lc and Li mites, the OT genotype composition in the rodent spleens post-infection had not changed and therefore resembled that observed in the feeding chiggers. However, only the Karp genotype was found in rodents after feeding by Ld chiggers carrying Karp and Gilliam genotypes. The current findings reveal a complex association among the host, pathogen, and vector. PMID:24297814

  10. High-resolution structure of HLA-A*0201 in complex with a tumour-specific antigenic peptide encoded by the MAGE-A4 gene.

    PubMed

    Hillig, R C; Coulie, P G; Stroobant, V; Saenger, W; Ziegler, A; Hülsmeyer, M

    2001-07-27

    The heterotrimeric complex of the human major histocompatibity complex (MHC) molecule HLA-A*0201, beta2-microglobulin and the decameric peptide GVYDGREHTV derived from the melanoma antigen (MAGE-A4 protein has been determined by X-ray crystallography at 1.4 A resolution. MAGE-A4 belongs to a family of genes that are specifically expressed in a variety of tumours. MAGE-A4-derived peptides are presented by MHC molecules at the cell surface to cytotoxic T-lymphocytes. As the HLA-A*0201:MAGE-A4 complex occurs only on tumour cells, it is considered to be an appropriate target for immunotherapy. The structure presented here reveals potential epitopes specific to the complex and indicates which peptide residues could be recognised by T-cell receptors. In addition, as the structure could be refined anisotropically, it was possible to describe the movements of the bound peptide in more detail.

  11. Murine retroviruses control class I major histocompatibility antigen gene expression via a trans effect at the transcriptional level.

    PubMed

    Wilson, L D; Flyer, D C; Faller, D V

    1987-07-01

    Moloney murine leukemia virus (M-MuLV) and Moloney murine sarcoma virus (M-MSV) exert a regulatory effect on the class I genes of the murine major histocompatibility complex (MHC). We have previously shown that M-MuLV infection of mouse fibroblasts results in a substantial increase in cell surface expression of H-2K, H-2D, and H-2L proteins, whereas M-MSV, upon coinfection of the same cells, is apparently able to override the MuLV-induced increase in H-2 expression. As a result of this modulation, immune recognition of the infected cells is profoundly altered. Our efforts have been directed toward elucidating the molecular basis for this phenomenon. We report here that stimulation of interferon production as a result of infection with MuLV does not occur and, therefore, is not the cause of MuLV-induced enhancement of MHC expression. Control of H-2 class I and beta 2-microglobulin gene expression by M-MuLV, and probably by M-MSV, takes place at the transcriptional level as indicated by nuclear runoff studies and analysis of steady-state mRNA levels. Our demonstration that M-MuLV controls expression of widely separated endogenous cellular genes (those coding for H-2D, H-2K, H-2L, and beta 2-microglobulin), transfected class I MHC genes, and unintegrated chimeric genes consisting of fragments of class I MHC genes linked to sequences encoding a procaryotic enzyme, chloramphenicol acetyltransferase, suggests that M-MuLV exerts its effect in trans and not by proviral integration in the vicinity of the H-2 gene complex. Finally, we show that the sequences of at least one MHC gene, which are responsive to trans regulation by M-MuLV, lie within 1.2 kilobases upstream of the initiation codon for that gene.

  12. The African buffalo parasite Theileria. sp. (buffalo) can infect and immortalize cattle leukocytes and encodes divergent orthologues of Theileria parva antigen genes.

    PubMed

    Bishop, R P; Hemmink, J D; Morrison, W I; Weir, W; Toye, P G; Sitt, T; Spooner, P R; Musoke, A J; Skilton, R A; Odongo, D O

    2015-12-01

    African Cape buffalo (Syncerus caffer) is the wildlife reservoir of multiple species within the apicomplexan protozoan genus Theileria, including Theileria parva which causes East coast fever in cattle. A parasite, which has not yet been formally named, known as Theileria sp. (buffalo) has been recognized as a potentially distinct species based on rDNA sequence, since 1993. We demonstrate using reverse line blot (RLB) and sequencing of 18S rDNA genes, that in an area where buffalo and cattle co-graze and there is a heavy tick challenge, T. sp. (buffalo) can frequently be isolated in culture from cattle leukocytes. We also show that T. sp. (buffalo), which is genetically very closely related to T. parva, according to 18s rDNA sequence, has a conserved orthologue of the polymorphic immunodominant molecule (PIM) that forms the basis of the diagnostic ELISA used for T. parva serological detection. Closely related orthologues of several CD8 T cell target antigen genes are also shared with T. parva. By contrast, orthologues of the T. parva p104 and the p67 sporozoite surface antigens could not be amplified by PCR from T. sp. (buffalo), using conserved primers designed from the corresponding T. parva sequences. Collectively the data re-emphasise doubts regarding the value of rDNA sequence data alone for defining apicomplexan species in the absence of additional data. 'Deep 454 pyrosequencing' of DNA from two Theileria sporozoite stabilates prepared from Rhipicephalus appendiculatus ticks fed on buffalo failed to detect T. sp. (buffalo). This strongly suggests that R. appendiculatus may not be a vector for T. sp. (buffalo). Collectively, the data provides further evidence that T. sp. (buffalo). is a distinct species from T. parva. PMID:26543804

  13. The African buffalo parasite Theileria. sp. (buffalo) can infect and immortalize cattle leukocytes and encodes divergent orthologues of Theileria parva antigen genes

    PubMed Central

    Bishop, R.P.; Hemmink, J.D.; Morrison, W.I.; Weir, W.; Toye, P.G.; Sitt, T.; Spooner, P.R.; Musoke, A.J.; Skilton, R.A.; Odongo, D.O.

    2015-01-01

    African Cape buffalo (Syncerus caffer) is the wildlife reservoir of multiple species within the apicomplexan protozoan genus Theileria, including Theileria parva which causes East coast fever in cattle. A parasite, which has not yet been formally named, known as Theileria sp. (buffalo) has been recognized as a potentially distinct species based on rDNA sequence, since 1993. We demonstrate using reverse line blot (RLB) and sequencing of 18S rDNA genes, that in an area where buffalo and cattle co-graze and there is a heavy tick challenge, T. sp. (buffalo) can frequently be isolated in culture from cattle leukocytes. We also show that T. sp. (buffalo), which is genetically very closely related to T. parva, according to 18s rDNA sequence, has a conserved orthologue of the polymorphic immunodominant molecule (PIM) that forms the basis of the diagnostic ELISA used for T. parva serological detection. Closely related orthologues of several CD8 T cell target antigen genes are also shared with T. parva. By contrast, orthologues of the T. parva p104 and the p67 sporozoite surface antigens could not be amplified by PCR from T. sp. (buffalo), using conserved primers designed from the corresponding T. parva sequences. Collectively the data re-emphasise doubts regarding the value of rDNA sequence data alone for defining apicomplexan species in the absence of additional data. ‘Deep 454 pyrosequencing’ of DNA from two Theileria sporozoite stabilates prepared from Rhipicephalus appendiculatus ticks fed on buffalo failed to detect T. sp. (buffalo). This strongly suggests that R. appendiculatus may not be a vector for T. sp. (buffalo). Collectively, the data provides further evidence that T. sp. (buffalo). is a distinct species from T. parva. PMID:26543804

  14. Swine leukocyte antigen class II genes (SLA-DRA, SLA-DRB1, SLA-DQA, SLA-DQB1) polymorphism and genotyping in Guizhou minipigs.

    PubMed

    Liu, Z Z; Xia, J H; Xin, L L; Wang, Z G; Qian, L; Wu, S G; Yang, S L; Li, K

    2015-11-30

    The swine leukocyte antigen (SLA) complex harbors highly polymorphic gene clusters encoding glycoproteins that are involved in responses to vaccines, infectious disease, and production performance. Pigs with well-defined SLA class II genes are useful for the study of disease, immunology, and vaccines. In this study, we analyzed four SLA class II genes (SLA-DRA, SLA-DRB1, SLA-DQA, SLA-DQB1) in 22 founder Guizhou minipigs using a sequence-based typing method. Twelve alleles were detected, compared with the SLA class II allele sequences in the GenBank, and one of twelve alleles was found to be novel in Guizhou minipigs. There are four SLA II haplotypes, and one of them has been previously reported in Meishan pigs. Furthermore, based on sequence information of these alleles, we developed a simple SLA typing method implemented to SLA-typing for unknown offspring of Guizhou minipigs, relying on designed twelve sequence specific primers that could discriminate between each other. According to the combination of sequence-based typing and PCR-SSP, we were able to rapidly check SLA typing of Guizhou breeding stock and identified four SLA haplotypes in the herd. Therefore, SLA-defined Guizhou minipigs will be useful as animal models for xenotransplantation and immunological research.

  15. Redefining the Epstein-Barr virus-encoded nuclear antigen EBNA-1 gene promoter and transcription initiation site in group I Burkitt lymphoma cell lines.

    PubMed Central

    Schaefer, B C; Strominger, J L; Speck, S H

    1995-01-01

    The Epstein-Barr virus-encoded nuclear antigen EBNA-1 gene promoter for the restricted Epstein-Barr virus (EBV) latency program operating in group I Burkitt lymphoma (BL) cell lines was previously identified incorrectly. Here we present evidence from RACE (rapid amplification of cDNA ends) cloning, reverse transcription-PCR, and S1 nuclease analyses, which demonstrates that the EBNA-1 gene promoter in group I BL cell lines is located in the viral BamHI Q fragment, immediately upstream of two low-affinity EBNA-1 binding sites. Transcripts initiated from this promoter, referred to as Qp, have the previously reported Q/U/K exon splicing pattern. Qp is active in group I BL cell lines but not in group III BL cell lines or in EBV immortalized B-lymphoblastoid cell lines. In addition, transient transfection of Qp-driven reporter constructs into both an EBV-negative BL cell line and a group I BL cell line gave rise to correctly initiated transcripts. Inspection of Qp revealed that it is a TATA-less promoter whose architecture is similar to the promoters of housekeeping genes, suggesting that Qp may be a default promoter which ensures EBNA-1 expression in cells that cannot run the full viral latency program. Elucidation of the genetic mechanism responsible for the EBNA-1-restricted program of EBV latency is an essential step in understanding control of viral latency in EBV-associated tumors. Images Fig. 1 Fig. 3 Fig. 4 PMID:7479841

  16. Feline immunodeficiency virus reverse transcriptase: expression, functional characterization, and reconstitution of the 66- and 51-kilodalton subunits.

    PubMed Central

    Amacker, M; Hottiger, M; Hübscher, U

    1995-01-01

    The two subunits of the feline immunodeficiency virus (FIV) reverse transcriptase (RT) were cloned and functionally expressed in Escherichia coli. The recombinant proteins are enzymatically active as homodimers (p66 and p51) as well as a heterodimer p66/p51. The biochemical properties of the FIV RT are very similar to those of the counterpart of the human immunodeficiency virus type 1 in being an RNA-dependent and DNA-dependent DNA polymerase. When a double-stranded DNA containing a small gap of 26 nucleotides was tested, we found a new activity of the FIV RT p66/p51 heterodimer--the cat viral enzyme could perform strand displacement DNA synthesis of approximately 300 bases. The FIV RT homodimer p66 alone could carry out limited strand displacement DNA synthesis, but this activity was stimulated by the p51 subunit at a molar ratio of one molecule of p66 to five molecules of p51. On the other hand, the homodimeric p51 itself was unable to fill a small gap of 26 nucleotides in a double-stranded DNA substrate and was not active by itself in strand displacement DNA synthesis. These data are in agreement with an earlier finding of strand displacement DNA synthesis by human immunodeficiency virus type 1 RT (M. Hottiger, V.N. Podust, R.L. Thimmig, C.S. McHenry, and U. Hübscher. J. Biol. Chem. 269:986-991, 1994). Our data therefore suggest a general and important function of lentiviral p51 subunits in strand displacement DNA synthesis which appears to be required in later stages of the lentiviral replication cycle, when DNA-dependent DNA synthesis occurs on double-stranded DNA. PMID:7545246

  17. Androgen receptor and prostate-specific antigen gene polymorphisms and breast cancer in African-American women.

    PubMed

    Wang, Wei; John, Esther M; Ingles, Sue Ann

    2005-12-01

    Several previous studies have found the CAG repeat polymorphism in exon 1 of the androgen receptor (AR) gene to be associated with breast cancer risk among some groups of Caucasian and Asian women. In a population-based case-control study of 488 African-American women (239 cases and 249 controls), we examined this polymorphism along with a polymorphism (-158 G/A) in an androgen-regulated gene (PSA) whose expression has been correlated with breast cancer prognosis. Overall, we did not observe any significant association between the CAG repeat polymorphism and breast cancer risk. However, among women with a first-degree family history of breast cancer, longer CAG repeats were associated with a significantly increased risk. Women carrying at least one longer allele [(CAG)n > or = 22] had a 3-fold increased risk compared to those with two shorter alleles (odds ratio, 3.18; 95% confidence interval, 1.08-9.36). There was no significant association between the PSA gene polymorphism and breast cancer risk, nor was there significant gene-gene interaction. In summary, our results further support that shorter CAG repeats (stronger AR transactivation activity) may reduce the risk of breast cancer, at least among some groups of women. Our data, however, are unable to provide evidence that PSA is the pathway through which the protective effect of androgens operates.

  18. Influence of molecular weight upon mannosylated bio-synthetic hybrids for targeted antigen presenting cell gene delivery.

    PubMed

    Jones, Charles H; Gollakota, Akhila; Chen, Mingfu; Chung, Tai-Chun; Ravikrishnan, Anitha; Zhang, Guojian; Pfeifer, Blaine A

    2015-07-01

    Given the rise of antibiotic resistant microbes, genetic vaccination is a promising prophylactic strategy that enables rapid design and manufacture. Facilitating this process is the choice of vector, which is often situationally-specific and limited in engineering capacity. Furthermore, these shortcomings are usually tied to an incomplete understanding of the structure-function relationships driving vector-mediated gene delivery. Building upon our initial report of a hybrid bacterial-biomaterial gene delivery vector, a comprehensive structure-function assessment was completed using a class of mannosylated poly(beta-amino esters). Through a top-down screening methodology, an ideal polymer was selected on the basis of gene delivery efficacy and then used for the synthesis of a stratified molecular weight polymer library. By eliminating contributions of polymer chemical background, we were able to complete an in-depth assessment of gene delivery as a function of (1) polymer molecular weight, (2) relative mannose content, (3) polymer-membrane biophysical properties, (4) APC uptake specificity, and (5) serum inhibition. In summary, the flexibility and potential of the hybrid design featured in this work highlights the ability to systematically probe vector-associated properties for the development of translational gene delivery candidates.

  19. Exon/intron structure of the human alpha 3(IV) gene encompassing the Goodpasture antigen (alpha 3(IV)NC1). Identification of a potentially antigenic region at the triple helix/NC1 domain junction.

    PubMed

    Quinones, S; Bernal, D; García-Sogo, M; Elena, S F; Saus, J

    1992-10-01

    The Goodpasture antigen has been identified as the non-collagenous (NC1) domain of alpha 3(IV), a novel collagen IV chain (Saus, J., Wieslander, J., Langeveld, J., Quinones, S., and Hudson, B.G. (1988) J. Biol. Chem. 263, 13374-13380). In the present study, the exon/intron structure and sequence for 285 amino acids of human alpha 3(IV), comprising 53 amino acids of the triple-helical domain and the complete NC1 domain (232 amino acids), were determined. Based on the comparison of the amino acid sequences of the alpha 1(IV), alpha 2(IV), alpha 3(IV), and alpha 5(IV) NC1 domains, a phylogenetic tree was constructed which indicates that alpha 2(IV) was the first chain to evolve, followed by alpha 3(IV), and then by alpha 1(IV) and alpha 5(IV). The exon/intron structure of these domains is consistent with this evolution model. In addition, it appears that alpha 3(IV) changed most after diverging from the parental gene. Analysis of its primary structure reveals that, at the junction between the triple-helical and NC1 domains, there exists a previously unrecognized, highly hydrophilic region (GLKGKRGDSGSPATWTTR) which is unique to the human alpha 3(IV) chain, containing a cell adhesion motif (RGD) as an integral part of a sequence (KRGDSGSP) conforming to a number of protein kinase recognition sites. Based on primary structure data, we outline new aspects to be explored concerning the molecular basis of collagen IV function and Goodpasture syndrome.

  20. Construction of expression systems for flaA and flaB genes of Helicobacter pylori and determination of immunoreactivity and antigenicity of recombinant proteins

    PubMed Central

    Yan, Jie; Liang, Shao-Hui; Mao, Ya-Fei; Li, Li-Wei; Li, Shu-Ping

    2003-01-01

    positive for rFlaA and rFlaB antibodies, respectively. One hundred percent and 98.98% of the 98 tested isolates of H pylori were detectable for rFlaA and rFlaB epitopes, respectively. CONCLUSION: Two prokaryotic expression systems with high efficiency of H pylori flaA and flaB genes were successfully established. The expressed rFlaA and rFlaB showed satisfactory immunoreactivity and antigenicity. High frequencies of FlaA and FlaB expression in different H pylori clinical strains and the general existence of specific antibodies against FlaA and FlaB in H pylori infected patients strongly indicate that FlaA and FlaB are excellent antigen candidates for developing H pylori vaccine. PMID:14562386

  1. Use of antibody gene library for the isolation of specific single chain antibodies by ampicillin-antigen conjugates.

    PubMed

    Neumann-Schaal, Meina; Messerschmidt, Katrin; Grenz, Nicole; Heilmann, Katja

    2013-03-01

    Isolation of recombinant antibodies from antibody libraries is commonly performed by different molecular display formats including phage display and ribosome display or different cell-surface display formats. We describe a new method which allows the selection of Escherichia coli cells producing the required single chain antibody by cultivation in presence of ampicillin conjugated to the antigen of interest. The method utilizes the neutralization of the conjugate by the produced single chain antibody which is secreted to the periplasm. Therefore, a new expression system based on the pET26b vector was designed and a library was constructed. The method was successfully established first for the selection of E. coli BL21 Star (DE3) cells expressing a model single chain antibody (anti-fluorescein) by a simple selection assay on LB-agar plates. Using this selection assay, we could identify a new single chain antibody binding biotin by growing E. coli BL21 Star (DE3) containing the library in presence of a biotin-ampicillin conjugate. In contrast to methods as molecular or cell surface display our selection system applies the soluble single chain antibody molecule and thereby avoids undesired effects, e.g. by the phage particle or the yeast fusion protein. By selecting directly in an expression strain, production and characterization of the selected single chain antibody is possible without any further cloning or transformation steps.

  2. [The antibodies to neurohormonal anti-genes as a criterion of early diagnostic and neurointoxication in workers of chemical enterprises].

    PubMed

    Bodienkova, G M; Alekseev, R Iu

    2014-11-01

    The examination was applied to enterprise workers laboring in conditions of vinyl chloride (79 patients), caustic soda (24 patients) and 10 patients with professional chronic mercury intoxication. The differences are established concerning manifestation of autoimmune reactions of personnel working in conditions of chronic effecting of vinyl chloride distinct of parameters characterizing autoimmune reactions of personnel working under impact of another neuro-toxicants (vapors of metallic mercury). The increasing of auto-antibodies to MAG was detected in healthy personnel and increasing of concentrations of auto-antibodies to protein S-100 and DNA was detected in personnel with initial manifestations of neuro-intoxication. These occurrences testify availability, of different mechanisms underlying formation of neurological disorders. The study data confirms involvement of auto-antibodies to neuronal antigens into derangement of neural activity in personnel working in conditions of effect of vinyl chloride and vapors of metallic mercury. Hence, the new possibilities are opened in studying pathogenesis of occupational neuro-intoxications. The detection of auto-antibodies to proteins of neural tissue can be recommended as a criterion of early identification of damage of neural system in personnel working in conditions of chemical industry.

  3. Molecular and antigenic characterization of reassortant H3N2 viruses from turkeys with a unique constellation of pandemic H1N1 internal genes.

    PubMed

    Berhane, Yohannes; Kehler, Helen; Handel, Katherine; Hisanaga, Tamiko; Xu, Wanhong; Ojkic, Davor; Pasick, John

    2012-01-01

    Triple reassortant (TR) H3N2 influenza viruses cause varying degrees of loss in egg production in breeder turkeys. In this study we characterized TR H3N2 viruses isolated from three breeder turkey farms diagnosed with a drop in egg production. The eight gene segments of the virus isolated from the first case submission (FAV-003) were all of TR H3N2 lineage. However, viruses from the two subsequent case submissions (FAV-009 and FAV-010) were unique reassortants with PB2, PA, nucleoprotein (NP) and matrix (M) gene segments from 2009 pandemic H1N1 and the remaining gene segments from TR H3N2. Phylogenetic analysis of the HA and NA genes placed the 3 virus isolates in 2 separate clades within cluster IV of TR H3N2 viruses. Birds from the latter two affected farms had been vaccinated with a H3N4 oil emulsion vaccine prior to the outbreak. The HAl subunit of the H3N4 vaccine strain had only a predicted amino acid identity of 79% with the isolate from FAV-003 and 80% for the isolates from FAV-009 and FAV-0010. By comparison, the predicted amino acid sequence identity between a prototype TR H3N2 cluster IV virus A/Sw/ON/33853/2005 and the three turkey isolates from this study was 95% while the identity between FAV-003 and FAV-009/10 isolates was 91%. When the previously identified antigenic sites A, B, C, D and E of HA1 were examined, isolates from FAV-003 and FAV-009/10 had a total of 19 and 16 amino acid substitutions respectively when compared with the H3N4 vaccine strain. These changes corresponded with the failure of the sera collected from turkeys that received this vaccine to neutralize any of the above three isolates in vitro.

  4. Human Leukocyte Antigen and Cytokine Receptor Gene Polymorphisms Associated With Heterogeneous Immune Responses to Mumps Viral Vaccine

    PubMed Central

    Ovsyannikova, Inna G.; Jacobson, Robert M.; Dhiman, Neelam; Vierkant, Robert A.; Pankratz, V. Shane; Poland, Gregory A.

    2009-01-01

    OBJECTIVES Mumps outbreaks continue to occur throughout the world, including in highly vaccinated populations. Vaccination against mumps has been successful; however, humoral and cellular immune responses to mumps vaccines vary significantly from person to person. We set out to assess whether HLA and cytokine gene polymorphisms are associated with variations in the immune response to mumps viral vaccine. METHODS To identify genetic factors that might contribute to variations in mumps vaccine–induced immune responses, we performed HLA genotyping in a group of 346 healthy schoolchildren (12–18 years of age) who previously received 2 doses of live mumps vaccine. Single-nucleotide polymorphisms (minor allele frequency of >5%) in cytokine and cytokine receptor genes were genotyped for a subset of 118 children. RESULTS Median values for mumps-specific antibody titers and lymphoproliferative stimulation indices were 729 IU/mL and 4.8, respectively. Girls demonstrated significantly higher mumps antibody titers than boys, indicating gender-linked genetic differences in humoral immune response. Significant associations were found between the HLA-DQB1*0303 alleles and lower mumps-specific antibody titers. An interesting finding was the association of several HLA class II alleles with mumps-specific lymphoproliferation. Alleles of the DRB1 (*0101, *0301, *0801, *1001, *1201, and *1302), DQA1 (*0101, *0105, *0401, and *0501), and DQB1 (*0201, *0402, and *0501) loci were associated with significant variations in lymphoproliferative immune responses to mumps vaccine. Additional associations were observed with single-nucleotide polymorphisms in the interleukin-10RA, interleukin-12RB1, and interleukin-12RB2 cytokine receptor genes. Minor alleles for 4 single-nucleotide polymorphisms within interleukin-10RA and interleukin-12RB genes were associated with variations in humoral and cellular immune responses to mumps vaccination. CONCLUSIONS These data suggest the important role of

  5. Clonality Analysis of Immunoglobulin Gene Rearrangement by Next-Generation Sequencing in Endemic Burkitt Lymphoma Suggests Antigen Drive Activation of BCR as Opposed to Sporadic Burkitt Lymphoma

    PubMed Central

    Amato, Teresa; Abate, Francesco; Piccaluga, Pierpaolo; Iacono, Michele; Fallerini, Chiara; Renieri, Alessandra; De Falco, Giulia; Ambrosio, Maria Raffaella; Mourmouras, Vaselious; Ogwang, Martin; Calbi, Valeria; Rabadan, Roul; Hummel, Michael; Pileri, Stefano; Bellan, Cristiana

    2016-01-01

    Objectives: Recent studies using next-generation sequencing (NGS) analysis disclosed the importance of the intrinsic activation of the B-cell receptor (BCR) pathway in the pathogenesis of sporadic Burkitt lymphoma (sBL) due to mutations of TCF3/ID3 genes. Since no definitive data are available on the genetic landscape of endemic Burkitt (eBL), we first assessed the mutation frequency of TCF3/ID3 in eBL compared with sBL and subsequently the somatic hypermutation status of the BCR to answer whether an extrinsic activation of BCR signaling could also be demonstrated in Burkitt lymphoma. Methods: We assessed the mutations of TCF3/ID3 by RNAseq and the BCR status by NGS analysis of the immunoglobulin genes (IGs). Results: We detected mutations of TCF3/ID3 in about 30% of the eBL cases. This rate is significantly lower than that detected in sBL (64%). The NGS analysis of IGs revealed intraclonal diversity, suggesting an active targeted somatic hypermutation process in eBL compared with sBL. Conclusions: These findings support the view that the antigenic pressure plays a key role in the pathogenetic pathways of eBL, which may be partially distinct from those driving sBL development. PMID:26712879

  6. MOLECULAR CHARACTERIZATION AND SEQUENCE PHYLOGENETIC ANALYSIS OF SURFACE ANTIGEN 3 (SAG3) GENE OF LOCAL INDIAN ISOLATES (CHENNAI AND IZATNAGAR) OF Toxoplasma gondii

    PubMed Central

    SUDAN, Vikrant; TEWARI, Anup Kumar; SINGH, Harkirat

    2015-01-01

    Context and objective: The molecular characterization of local isolates of Toxoplasma gondii is considered significant so as to assess the homologous variations between the different loci of various strains of parasites. Design and setting: The present communication deals with the molecular cloning and sequence analysis of the 1158 bp entire open reading frame (ORF) of surface antigen 3 (SAG3) of two Indian T. gondii isolates (Chennai and Izatnagar) being maintained as cryostock at the IVRI. Method: The surface antigen 3 (SAG3) of two local Indian isolates were cloned and sequenced before being compared with the available published sequences. Results: The sequence comparison analysis revealed 99.9% homology with the standard published RH strain sequence of T. gondii. The strains were also compared with other established published sequences and found to be most related to the P-Br strain and CEP strain (both 99.3%), and least with PRU strain (98.4%). However, the two Indian isolates had 100% homology between them. Conclusion: Finally, it was concluded that the Indian isolates were closer to the RH strain than to the P-Br strain (Brazilian strain), the CEP strain and the PRU strains (USA), with respect to nucleotide homology. The two Indian isolates used in the present study are known to vary between themselves, as far as homologies related to other genes are concerned, but they were found to be 100% homologous as far as SAG3 locus is concerned. This could be attributed to the fact that this SAG3 might be a conserved locus and thereby, further detailed studies are thereby warranted to exploit the use of this particular molecule in diagnostics and immunoprophylactics. The findings are important from the point of view of molecular phylogeny. PMID:26200959

  7. pH-sensitive polymer-liposome-based antigen delivery systems potentiated with interferon-γ gene lipoplex for efficient cancer immunotherapy.

    PubMed

    Yuba, Eiji; Kanda, Yuhei; Yoshizaki, Yuta; Teranishi, Ryoma; Harada, Atsushi; Sugiura, Kikuya; Izawa, Takeshi; Yamate, Jyoji; Sakaguchi, Naoki; Koiwai, Kazunori; Kono, Kenji

    2015-10-01

    Potentiation of pH-sensitive liposome-based antigen carriers with IFN-γ gene lipoplexes was attempted to achieve efficient induction of tumor-specific immunity. 3-Methylglutarylated poly(glycidol) (MGluPG)-modified liposomes and cationic liposomes were used, respectively, for the delivery of antigenic protein ovalbumin (OVA) and IFN-γ-encoding plasmid DNA (pDNA). The MGluPG-modified liposomes and the cationic liposome-pDNA complexes (lipoplexes) formed hybrid complexes via electrostatic interactions after their mixing in aqueous solutions. The hybrid complexes co-delivered OVA and IFN-γ-encoding pDNA into DC2.4 cells, a murine dendritic cell line, as was the case of MGluPG-modified liposomes for OVA or the lipoplexes for pDNA. Both the lipoplexes and the hybrid complexes transfected DC2.4 cells and induced IFN-γ protein production, but transfection activities of the hybrid complexes were lower than those of the parent lipoplexes. Subcutaneous administration of hybrid complexes to mice bearing E.G7-OVA tumor reduced tumor volumes, which might result from the induction of OVA-specific cytotoxic T lymphocytes (CTLs). However, the hybrid complex-induced antitumor effect was the same level of the MGluPG-modified liposome-mediated antitumor immunity. In contrast, an extremely strong antitumor immune response was derived when these liposomes and lipoplexes without complexation were injected subcutaneously at the same site of tumor-bearing mice. Immunohistochemical analysis of tumor sections revealed that immunization through the liposome-lipoplex combination promoted the infiltration of CTLs to tumors at an early stage of treatment compared with liposomes, resulting in strong therapeutic effects.

  8. O Acetylation of the Enterobacterial Common Antigen Polysaccharide Is Catalyzed by the Product of the yiaH Gene of Escherichia coli K-12▿

    PubMed Central

    Kajimura, Junko ; Rahman, Arifur; Hsu, James; Evans, Matthew R.; Gardner, Kevin H.; Rick, Paul D.

    2006-01-01

    The carbohydrate component of the enterobacterial common antigen (ECA) of Escherichia coli K-12 occurs primarily as a water-soluble cyclic polysaccharide located in the periplasm (ECACYC) and as a phosphoglyceride-linked linear polysaccharide located on the cell surface (ECAPG). The polysaccharides of both forms are comprised of the amino sugars N-acetyl-d-glucosamine (GlcNAc), N-acetyl-d-mannosaminuronic acid (ManNAcA), and 4-acetamido-4,6-dideoxy-d-galactose (Fuc4NAc). These amino sugars are linked to one another to form trisaccharide repeat units with the structure →3-α-d-Fuc4NAc-(1→4)-β-d-ManNAcA-(1→4)-α-d-GlcNAc-(1→. The hydroxyl group in the 6 position of the GlcNAc residues of both ECACYC and ECAPG are nonstoichiometrically esterified with acetyl groups. Random transposon insertion mutagenesis of E. coli K-12 resulted in the generation of a mutant defective in the incorporation of O-acetyl groups into both ECACYC and ECAPG. This defect was found to be due to an insertion of the transposon into the yiaH locus, a putative gene of unknown function located at 80.26 min on the E. coli chromosomal map. Bioinformatic analyses of the predicted yiaH gene product indicate that it is an integral inner membrane protein that is a member of an acyltransferase family of enzymes found in a wide variety of organisms. The results of biochemical and genetic experiments presented here strongly support the conclusion that yiaH encodes the O-acetyltransferase responsible for the incorporation of O-acetyl groups into both ECACYC and ECAPG. Accordingly, we propose that this gene be designated wecH. PMID:16936038

  9. Genetic diversity of the Plasmodium falciparum apical membrane antigen I gene in parasite population from the China-Myanmar border area

    PubMed Central

    Zhu, Xiaotong; Zhao, Zhenjun; Feng, Yonghui; Li, Peipei; Liu, Fei; Liu, Jun; Yang, Zhaoqing; Yan, Guiyun; Fan, Qi; Cao, Yaming; Cui, Liwang

    2016-01-01

    To investigate the genetic diversity of the Plasmodium falciparum apical membrane antigen 1 (PfAMA1) gene in Southeast Asia, we determined PfAMA1 sequences from 135 field isolates collected from the China-Myanmar border area and compared them with 956 publically available PfAMA1 sequences from seven global P. falciparum populations. This analysis revealed high genetic diversity of PfAMA1 in global P. falciparum populations with a total of 229 haplotypes identified. The genetic diversity of PfAMA1 gene from the China-Myanmar border is not evenly distributed in the different domains of this gene. Sequence diversity in PfAMA1 from the China-Myanmar border is lower than that observed in Thai, African and Oceanian populations, but higher than that in the South American population. This appeared to correlate well with the levels of endemicity of different malaria-endemic regions, where hyperendemic regions favor genetic cross of the parasite isolates and generation of higher genetic diversity. Neutrality tests show significant departure from neutrality in the entire ectodomain and Domain I of PfAMA1 in the China-Myanmar border parasite population. We found evidence supporting a substantial continent-wise genetic structure among P. falciparum populations, with the highest genetic differentiation detected between the China-Myanmar border and the South American populations. Whereas no alleles were unique to a specific region, there were considerable geographical differences in major alleles and their frequencies, highlighting further necessity to include more PfAMA1 alleles in vaccine designs. PMID:26825252

  10. Genetic diversity of the Plasmodium falciparum apical membrane antigen I gene in parasite population from the China-Myanmar border area.

    PubMed

    Zhu, Xiaotong; Zhao, Zhenjun; Feng, Yonghui; Li, Peipei; Liu, Fei; Liu, Jun; Yang, Zhaoqing; Yan, Guiyun; Fan, Qi; Cao, Yaming; Cui, Liwang

    2016-04-01

    To investigate the genetic diversity of the Plasmodium falciparum apical membrane antigen 1 (PfAMA1) gene in Southeast Asia, we determined PfAMA1 sequences from 135 field isolates collected from the China-Myanmar border area and compared them with 956 publically available PfAMA1 sequences from seven global P. falciparum populations. This analysis revealed high genetic diversity of PfAMA1 in global P. falciparum populations with a total of 229 haplotypes identified. The genetic diversity of PfAMA1 gene from the China-Myanmar border is not evenly distributed in the different domains of this gene. Sequence diversity in PfAMA1 from the China-Myanmar border is lower than that observed in Thai, African and Oceanian populations, but higher than that in the South American population. This appeared to correlate well with the levels of endemicity of different malaria-endemic regions, where hyperendemic regions favor genetic cross of the parasite isolates and generation of higher genetic diversity. Neutrality tests show significant departure from neutrality in the entire ectodomain and Domain I of PfAMA1 in the China-Myanmar border parasite population. We found evidence supporting a substantial continent-wise genetic structure among P. falciparum populations, with the highest genetic differentiation detected between the China-Myanmar border and the South American populations. Whereas no alleles were unique to a specific region, there were considerable geographical differences in major alleles and their frequencies, highlighting further necessity to include more PfAMA1 alleles in vaccine designs.

  11. Intestinal alpha beta T cells differentiate and rearrange antigen receptor genes in situ in the human infant.

    PubMed

    Williams, Amanda M; Bland, Paul W; Phillips, Anne C; Turner, Susan; Brooklyn, Trevor; Shaya, Gabriel; Spicer, Richard D; Probert, Christopher S J

    2004-12-15

    Intestinal Ag exposure during neonatal life influences appropriate adult immune responses. To define the mechanisms shaping the T cell repertoire during this period, we examined T cell differentiation and receptor diversity in the intestine of human infants. Developmental phenotypes of intraepithelial and lamina propria intestinal T cells from infants aged 1 day to 2 years were assessed ex vivo by flow cytometry and in situ by triple-fluorescent immunohistochemistry. Gene recombination-specific enzymes were assessed by PCR. TCR beta-chain V region gene diversity was determined by sequencing. Several different early lineage T cell populations were present neonatally: CD3(+)4(-)8(-) T cells were present at birth and numbers decreased during the neonatal period; CD3(+)4(+)8(+) T cells were present in low numbers throughout infancy; and CD3(+)4(+)8(-) or CD3(+)4(-)8(+) T cells increased with age. Very early lineage T cells, CD3(-)2(-)7(+) and CD3(-)2(+)7(+), were present neonatally, but were essentially absent at 1 year. Most lamina propria T cells differentiated rapidly after birth, but maturation of intraepithelial T cells took place over 1 year. Intestinal samples from infants less than 6 mo old contained transcripts of T early alpha and TdT, and 15 of 19 infant samples contained mRNA for RAG-1, some coexpressing RAG-2. TCR beta-chain repertoires were polyclonal in infants. Immature T cells, pre-T cells, and genes involved in T cell recombination were found in the intestine during infancy. T cell differentiation occurs within the neonatal human intestine, and the TCR repertoire of these developing immature T cells is likely to be influenced by luminal Ags. Thus, mucosal T cell responsiveness to environmental Ag is shaped in situ during early life.

  12. Association of Single Nucleotide Polymorphisms in the ST3GAL4 Gene with VWF Antigen and Factor VIII Activity.

    PubMed

    Song, Jaewoo; Xue, Cheng; Preisser, John S; Cramer, Drake W; Houck, Katie L; Liu, Guo; Folsom, Aaron R; Couper, David; Yu, Fuli; Dong, Jing-Fei

    2016-01-01

    VWF is extensively glycosylated with biantennary core fucosylated glycans. Most N-linked and O-linked glycans on VWF are sialylated. FVIII is also glycosylated, with a glycan structure similar to that of VWF. ST3GAL sialyltransferases catalyze the transfer of sialic acids in the α2,3 linkage to termini of N- and O-glycans. This sialic acid modification is critical for VWF synthesis and activity. We analyzed genetic and phenotypic data from the Atherosclerosis Risk in Communities (ARIC) study for the association of single nucleotide polymorphisms (SNPs) in the ST3GAL4 gene with plasma VWF levels and FVIII activity in 12,117 subjects. We also analyzed ST3GAL4 SNPs found in 2,535 subjects of 26 ethnicities from the 1000 Genomes (1000G) project for ethnic diversity, SNP imputation, and ST3GAL4 haplotypes. We identified 14 and 1,714 ST3GAL4 variants in the ARIC GWAS and 1000G databases respectively, with 46% being ethnically diverse in their allele frequencies. Among the 14 ST3GAL4 SNPs found in ARIC GWAS, the intronic rs2186717, rs7928391, and rs11220465 were associated with VWF levels and with FVIII activity after adjustment for age, BMI, hypertension, diabetes, ever-smoking status, and ABO. This study illustrates the power of next-generation sequencing in the discovery of new genetic variants and a significant ethnic diversity in the ST3GAL4 gene. We discuss potential mechanisms through which these intronic SNPs regulate ST3GAL4 biosynthesis and the activity that affects VWF and FVIII. PMID:27584569

  13. Complement gene variants in relation to autoantibodies to beta cell specific antigens and type 1 diabetes in the TEDDY Study

    PubMed Central

    Törn, Carina; Liu, Xiang; Hagopian, William; Lernmark, Åke; Simell, Olli; Rewers, Marian; Ziegler, Anette-G; Schatz, Desmond; Akolkar, Beena; Onengut-Gumuscu, Suna; Chen, Wei-Min; Toppari, Jorma; Mykkänen, Juha; Ilonen, Jorma; Rich, Stephen S.; She, Jin-Xiong; Sharma, Ashok; Steck, Andrea; Krischer, Jeffrey; Abbondondolo, Michael; Adams, Janey; Adamsson, Annika; Agardh, Daniel; Anderson, Stephen W.; Aronsson, Carin Andrén; Ask, Maria; Austin-Gonzalez, Sarah; Ayres, Stephen; Baethke, Sandra; Bautista, Kimberly; Baxter, Judith; Becker, Dorothy; Bedoy, Ruth; Bennet, Rasmus; Johnson, Suzanne Bennett; Beyerlein, Andreas; Bonifacio, Ezio; Bourcier, Kasia; Bremer, Jenny; Briese, Thomas; Brown, Rasheedah; Burkhardt, Brant; Butterworth, Martha; Carlsson, Ulla-Marie; Cilio, Corrado; Clasen, Joanna; Crouch, Claire Cowen; Cuthbertson, David; Daftary, Ashi; Dalmagro-Elias, MaryEllen; Dunson, Kayleen; Eberhard, Christopher; Larsson, Helena Elding; Ericsson-Hallström, Emelie; Felipe-Morales, Daniel; Fiske, Steven; Foghis, Gabriella; Foterek, Kristina; Fransiscus, Margaret; Fransson, Lina; Frohnert, Brigitte I.; Garcia, Dena; Gard, Thomas; Gardiner, Melissa; Garmeson, Jennifer; Gerardsson, Joanna; Gesualdo, Patricia; Gowda, Veena; Haller, Michael; Hansen, Monica; Hansson, Gertie; Harmby, Cecilia; Hervey, Rachel; Heyman, Kathleen; Hoffman, Michelle; Hopkins, Diane; Hummel, Michael; Hummel, Sandra; Hyberg, Susanne; Hyöty, Heikki; Johansen, Fredrik; Johnson, Corbin; Jokipuu, Sanna; Jonasdottir, Berglind; Kallio, Tiina; Karban, Rachel; Kersting, Mathilde; Killian, Michael; Klein, Beth; Knip, Mikael; Knopff, Annette; Koivu, Annika; Koletzko, Sibylle; Koreasalo, Mirva; Kurppa, Kalle; Kähönen, Miia; Lee, Hye-Seung; Forss, Sigrid Lenrick; Liu, Edwin; Liu, Shu; Lundgren, Markus; Lynch, Kristian; Lyons, Rachel; Lönnrot, Maria; Malloy, Jamie; Markan, Maria; McCarthy, Cristina; McIndoe, Richard; McLeod, Wendy; Melin, Jessica; Mestan, Zeliha; Meulemans, Steven; Meyer, Arlene; Mulenga, Denise; Multasuo, Katja; Månsson-Martinez, Maria; Mäntimäki, Elina; Niinien, Tiina; Norris, Jill; Nyblom, Mia; Peplow, Claudia; Laras, Francisco Perez; Rahmati, Kobra; Rajala, Petra; Ramelius, Anita; Rautanen, Jenna; Riikonen, Anne; Robinson, Richard; Romo, Minna; Rosenquist, Anna; Roth, Roswith; Salami, Falastin; Samper-Imaz, Adela; Scott, Elisabeth; Shaffer, Chris; Sibthorpe, Sara; Silvis, Katherine; Simell, Satu; Simell, Ville; Sjöberg, Maija; Sjöberg, Birgitta; Skidmore, Jennifer; Smith, Laura; Smith, Susan; Stabbert, Joshua; Steed, Leigh; Stenius, Aino; Stock, Joanna; Strauss, Elisabeth; Sulman, Noah; Swartling, Ulrica; Särmä, Maria; Tamura, Roy; Tarr, Alexander; Amboh, Evelyn Tekum; Thomas, Jamie; Triplett, Eric; Trulsson, Erika; Uland, Morgan; Uusitalo, Ulla; Vainionpää, Sini; Wallin, Anne; Varionen, Eeva; Warncke, Katharina; Waugh, Kathleen; Vehik, Kendra; Veijola, Riitta; Vijayakandipan, Ponni; Williams, Joshua; Willis, John; Wimar, Åsa; Winkler, Christiane; Virtanen, Suvi M.; Wood, Keith; Wright, Hali; Vähä-Mäkilä, Mari; Yang, Jimin; Yates, Chrystal; Åberg, Sofie; Åkerlund, Mari

    2016-01-01

    A total of 15 SNPs within complement genes and present on the ImmunoChip were analyzed in The Environmental Determinants of Diabetes in the Young (TEDDY) study. A total of 5474 subjects were followed from three months of age until islet autoimmunity (IA: n = 413) and the subsequent onset of type 1 diabetes (n = 115) for a median of 73 months (IQR 54–91). Three SNPs within ITGAM were nominally associated (p < 0.05) with IA: rs1143678 [Hazard ratio; HR 0.80; 95% CI 0.66–0.98; p = 0.032], rs1143683 [HR 0.80; 95% CI 0.65–0.98; p = 0.030] and rs4597342 [HR 1.16; 95% CI 1.01–1.32; p = 0.041]. When type 1 diabetes was the outcome, in DR3/4 subjects, there was nominal significance for two SNPs: rs17615 in CD21 [HR 1.52; 95% CI 1.05–2.20; p = 0.025] and rs4844573 in C4BPA [HR 0.63; 95% CI 0.43–0.92; p = 0.017]. Among DR4/4 subjects, rs2230199 in C3 was significantly associated [HR 3.20; 95% CI 1.75–5.85; p = 0.0002, uncorrected] a significance that withstood Bonferroni correction since it was less than 0.000833 (0.05/60) in the HLA-specific analyses. SNPs within the complement genes may contribute to IA, the first step to type 1 diabetes, with at least one SNP in C3 significantly associated with clinically diagnosed type 1 diabetes. PMID:27306948

  14. Complement gene variants in relation to autoantibodies to beta cell specific antigens and type 1 diabetes in the TEDDY Study.

    PubMed

    Törn, Carina; Liu, Xiang; Hagopian, William; Lernmark, Åke; Simell, Olli; Rewers, Marian; Ziegler, Anette-G; Schatz, Desmond; Akolkar, Beena; Onengut-Gumuscu, Suna; Chen, Wei-Min; Toppari, Jorma; Mykkänen, Juha; Ilonen, Jorma; Rich, Stephen S; She, Jin-Xiong; Sharma, Ashok; Steck, Andrea; Krischer, Jeffrey

    2016-01-01

    A total of 15 SNPs within complement genes and present on the ImmunoChip were analyzed in The Environmental Determinants of Diabetes in the Young (TEDDY) study. A total of 5474 subjects were followed from three months of age until islet autoimmunity (IA: n = 413) and the subsequent onset of type 1 diabetes (n = 115) for a median of 73 months (IQR 54-91). Three SNPs within ITGAM were nominally associated (p < 0.05) with IA: rs1143678 [Hazard ratio; HR 0.80; 95% CI 0.66-0.98; p = 0.032], rs1143683 [HR 0.80; 95% CI 0.65-0.98; p = 0.030] and rs4597342 [HR 1.16; 95% CI 1.01-1.32; p = 0.041]. When type 1 diabetes was the outcome, in DR3/4 subjects, there was nominal significance for two SNPs: rs17615 in CD21 [HR 1.52; 95% CI 1.05-2.20; p = 0.025] and rs4844573 in C4BPA [HR 0.63; 95% CI 0.43-0.92; p = 0.017]. Among DR4/4 subjects, rs2230199 in C3 was significantly associated [HR 3.20; 95% CI 1.75-5.85; p = 0.0002, uncorrected] a significance that withstood Bonferroni correction since it was less than 0.000833 (0.05/60) in the HLA-specific analyses. SNPs within the complement genes may contribute to IA, the first step to type 1 diabetes, with at least one SNP in C3 significantly associated with clinically diagnosed type 1 diabetes. PMID:27306948

  15. Association of Single Nucleotide Polymorphisms in the ST3GAL4 Gene with VWF Antigen and Factor VIII Activity

    PubMed Central

    Song, Jaewoo; Xue, Cheng; Preisser, John S.; Cramer, Drake W.; Houck, Katie L.; Liu, Guo; Folsom, Aaron R.; Couper, David; Yu, Fuli; Dong, Jing-fei

    2016-01-01

    VWF is extensively glycosylated with biantennary core fucosylated glycans. Most N-linked and O-linked glycans on VWF are sialylated. FVIII is also glycosylated, with a glycan structure similar to that of VWF. ST3GAL sialyltransferases catalyze the transfer of sialic acids in the α2,3 linkage to termini of N- and O-glycans. This sialic acid modification is critical for VWF synthesis and activity. We analyzed genetic and phenotypic data from the Atherosclerosis Risk in Communities (ARIC) study for the association of single nucleotide polymorphisms (SNPs) in the ST3GAL4 gene with plasma VWF levels and FVIII activity in 12,117 subjects. We also analyzed ST3GAL4 SNPs found in 2,535 subjects of 26 ethnicities from the 1000 Genomes (1000G) project for ethnic diversity, SNP imputation, and ST3GAL4 haplotypes. We identified 14 and 1,714 ST3GAL4 variants in the ARIC GWAS and 1000G databases respectively, with 46% being ethnically diverse in their allele frequencies. Among the 14 ST3GAL4 SNPs found in ARIC GWAS, the intronic rs2186717, rs7928391, and rs11220465 were associated with VWF levels and with FVIII activity after adjustment for age, BMI, hypertension, diabetes, ever-smoking status, and ABO. This study illustrates the power of next-generation sequencing in the discovery of new genetic variants and a significant ethnic diversity in the ST3GAL4 gene. We discuss potential mechanisms through which these intronic SNPs regulate ST3GAL4 biosynthesis and the activity that affects VWF and FVIII. PMID:27584569

  16. Prostate-associated gene 4 (PAGE4), an intrinsically disordered cancer/testis antigen, is a novel therapeutic target for prostate cancer

    PubMed Central

    Kulkarni, Prakash; Dunker, A Keith; Weninger, Keith; Orban, John

    2016-01-01

    Prostate-associated gene 4 (PAGE4) is a remarkably prostate-specific Cancer/Testis Antigen that is highly upregulated in the human fetal prostate and its diseased states but not in the adult normal gland. PAGE4 is an intrinsically disordered protein (IDP) that functions as a stress-response protein to suppress reactive oxygen species as well as prevent DNA damage. In addition, PAGE4 is also a transcriptional regulator that potentiates transactivation by the oncogene c-Jun. c-Jun forms the AP-1 complex by heterodimerizing with members of the Fos family and plays an important role in the development and pathology of the prostate gland, underscoring the importance of the PAGE4/c-Jun interaction. HIPK1, also a component of the stress-response pathway, phosphorylates PAGE4 at T51 which is critical for its transcriptional activity. Phosphorylation induces conformational and dynamic switching in the PAGE4 ensemble leading to a new cellular function. Finally, bioinformatics evidence suggests that the PAGE4 mRNA could be alternatively spliced resulting in four potential isoforms of the polypeptide alluding to the possibility of a range of conformational ensembles with latent functions. Considered together, the data suggest that PAGE4 may represent the first molecular link between stress and prostate cancer (PCa). Thus, pharmacologically targeting PAGE4 may be a novel opportunity for treating and managing patients with PCa, especially patients with low-risk disease. PMID:27270343

  17. Breed differences in development of anti-insulin antibodies in diabetic dogs and investigation of the role of dog leukocyte antigen (DLA) genes.

    PubMed

    Holder, Angela L; Kennedy, Lorna J; Ollier, William E R; Catchpole, Brian

    2015-10-15

    Administration of insulin for treatment of diabetes mellitus in dogs can stimulate an immune response, with a proportion of animals developing anti-insulin antibodies (AIA). For an IgG antibody response to occur, this would require B cell presentation of insulin peptides by major histocompatibility complex (MHC) class II molecules, encoded by dog leukocyte antigen (DLA) genes, in order to receive T-cell help for class switching. DLA genes are highly polymorphic in the dog population and vary from breed to breed. The aim of the present study was to evaluate AIA reactivity in diabetic dogs of different breeds and to investigate whether DLA genes influence AIA status. Indirect ELISA was used to determine serological reactivity to insulin in diabetic dogs, treated with either a porcine or bovine insulin preparation. DLA haplotypes for diabetic dogs were determined by sequence-based typing of DLA-DRB1, -DQA1 and -DQB1 loci. Significantly greater insulin reactivity was seen in treated diabetic dogs (n=942) compared with non-diabetic dogs (n=100). Relatively few newly diagnosed diabetic dogs (3/109) were found to be AIA positive, although this provides evidence that insulin autoantibodies might be involved in the pathogenesis of the disease in some cases. Of the diabetic dogs treated with a bovine insulin preparation, 52.3% (182/348) were AIA positive, compared with 12.6% (75/594) of dogs treated with a porcine insulin preparation, suggesting that bovine insulin is more immunogenic. Breeds such as dachshund, Cairn terrier, miniature schnauzer and Tibetan terrier were more likely to develop AIA, whereas cocker spaniels were less likely to develop AIA, compared with crossbreed dogs. In diabetic dogs, DLA haplotype DRB1*0015--DQA1*006--DQB1*023 was associated with being AIA positive, whereas the haplotype DLA-DRB1*006--DQA1*005--DQB1*007 showed an association with being AIA negative. These research findings suggest that DLA genes influence AIA responses in treated diabetic

  18. Molecular cloning and characterization of a proliferating cell nuclear antigen gene by chemically induced male sterility in wheat (Triticum aestivum L.).

    PubMed

    Yu, Y A; Zhang, G S; Zhang, J; Ju, L; Zhu, Q D; Song, Y L; Wang, J W; Niu, N; Ma, S C

    2015-10-05

    Although a number of studies have shown that chemical hybridizing agents (CHAs) affect anther growth and regulate cell-cycle progression, little is known about the molecular and cellular mechanisms involved. Proliferating cell nuclear antigen (PCNA) is an essential factor in DNA replication, and in many other processes in eukaryotic cells. In this study, the open reading frame of TaPCNA, the PCNA in wheat (Triticum aestivum L.), was cloned by reverse transcription polymerase chain reaction (RT-PCR). Sequence analysis revealed that this gene was 792-bp long and encoded a protein with 234 amino acids. Alignment of the TaPCNA-predicted sequence revealed a high degree of identity with PCNAs from other plant species. A subcellular localization assay indicated that TaPCNA was localized in the nucleus. The TaPCNA was cloned into the prokaryotic expression plasmid pET32a, and the recombinant plasmid was transformed into BL21 (DE3). TaPCNA expression was induced by 0.5 mM isopropyl-beta-D-thiogalactopyranoside and verified using sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blot assays, which indicated that the fusion protein was successfully expressed. The gene involved in the G1-to-S transition, Histone H4, was downregulated by 1376- CIMS, which is a chemically induced male sterility line. However, a semi-quantitative RT-PCR revealed that TaPCNA expression was upregulated in 1376-CIMS. Our results suggest that CHAs (SQ-1) induce DNA damage in wheat anthers. DNA damage results in either the delay or arrest of cell-cycle progression, which affects anther development. This study will help to elucidate the mechanisms of SQ-1-induced male sterility.

  19. TCRs Used in Cancer Gene Therapy Cross-React with MART-1/Melan-A Tumor Antigens via Distinct Mechanisms

    SciTech Connect

    Borbulevych, Oleg Y.; Santhanagopolan, Sujatha M.; Hossain, Moushumi; Baker, Brian M.

    2013-09-18

    T cells engineered to express TCRs specific for tumor Ags can drive cancer regression. The first TCRs used in cancer gene therapy, DMF4 and DMF5, recognize two structurally distinct peptide epitopes of the melanoma-associated MART-1/Melan-A protein, both presented by the class I MHC protein HLA-A*0201. To help understand the mechanisms of TCR cross-reactivity and provide a foundation for the further development of immunotherapy, we determined the crystallographic structures of DMF4 and DMF5 in complex with both of the MART-1/Melan-A epitopes. The two TCRs use different mechanisms to accommodate the two ligands. Although DMF4 binds the two with a different orientation, altering its position over the peptide/MHC, DMF5 binds them both identically. The simpler mode of cross-reactivity by DMF5 is associated with higher affinity toward both ligands, consistent with the superior functional avidity of DMF5. More generally, the observation of two diverging mechanisms of cross-reactivity with the same Ags and the finding that TCR-binding orientation can be determined by peptide alone extend our understanding of the mechanisms underlying TCR cross-reactivity.

  20. Transcription of the Epstein-Barr virus nuclear antigen 1 (EBNA1) gene occurs before induction of the BCR2 (Cp) EBNA gene promoter during the initial stages of infection in B cells.

    PubMed

    Schlager, S; Speck, S H; Woisetschläger, M

    1996-06-01

    The purpose of this study was to gain insights into the regulation of Epstein-Barr virus (EBV) gene transcription during the establishment of viral latency in B cells. During the early stages of EBV infection in B lymphocytes, transcription of six viral nuclear antigens (EBNAs) is initiated from an early promoter (Wp). This is followed by a switch of promoter usage to an upstream promoter, Cp, whose activity is autoregulated by both EBNA1 and EBNA2. Previously it was demonstrated that infection of primary B cells with EBNA2-negative (EBNA2-) EBNA4-mutant (EBNA4mut) virus resulted only in the expression of mutant EBNA4 protein and failure to express the other EBNA gene products (C. Rooney H. G. Howe, S. H. Speck, and G. Miller, J. Virol. 63:1531-1539, 1989). We extended this research to demonstrate that Wp-to-Cp switching did not occur upon infection of primary B cells with an EBNA2- EBNA4mut virus (M. Woisetschlaeger, X. W. Jin, C. N. Yandara, L. A. Furmanski, J. L. Strominger, and S. H. Speck, Proc. Natl. Acad. Sci. USA 88:3942-3946, 1991). Further characterization of this phenomenon led to the identification of an EBNA2-dependent enhancer upstream of Cp. On the basis of these data, a model was proposed in which initial transcription from Wp gives rise to the expression of EBNA2 and EBNA4, and then transcription is upregulated from Cp via the EBNA2- dependent enhancer (Woisetschlaeger et al., as noted above). Implicit in this model is that transcription of the EBNA1 and EBNA3a to -3c genes is dependent on the switch from Wp to Cp, since primary cells infected with EBNA2- EBNA4mut virus fail to switch and also fail to express these viral antigens. Here we critically evaluate this model and demonstrate, in contrast to the predictions of the model, that transcription of both the EBNA1 and EBNA2 genes precedes activation of Cp. Furthermore, the level of EBNA1 gene transcription was strongly reduced in primary B cells infected with EBNA2- EBNA4mut virus compared with

  1. Expression and gene transcript of Fc receptors for IgG, HLA class II antigens and Langerhans cells in human cervico-vaginal epithelium.

    PubMed Central

    Hussain, L A; Kelly, C G; Fellowes, R; Hecht, E M; Wilson, J; Chapman, M; Lehner, T

    1992-01-01

    The mechanism of transmission of HIV from the male to the female genital tract or in the reverse order is not clear. CD4 glycoprotein is the receptor for HIV and Langerhans cells and the related dendritic cells could play a role in the initial transmission of HIV. Fc receptors (FcR) for IgG might be involved in antibody-mediated binding of HIV. We carried out an immunohistological study of normal human cervical and vaginal epithelia for the presence of CD4 glycoprotein, Langerhans cells and FcR to IgG. CD4+ glycoprotein was not found in the vaginal or cervical epithelium, with the exception of a few endocervical epithelial cells. A small number of CD4+ mononuclear cells were found in the endocervical epithelium of a third of the specimens but a large number of CD4+ cells was found in the submucosa of most of the cervical and vaginal specimens. Langerhans cells expressing CD4, HLA class II, Fc gamma R2 and Fc gamma R3 were detected in most vaginal, ectocervical and transformation zone epithelia and in 9/14 endocervical tissues. Fc gamma R3 was detected in about two-thirds of the columnar endocervical epithelium and the transformation zone. A smaller number of specimens expressed Fc gamma R2 in these epithelia, but Fc gamma R1 was not detected. We then demonstrated mRNA for Fc gamma R3 in the columnar endocervical epithelial cells and transformation zone by in situ hybridization, using a CD16-RNA probe. Fc gamma R3 and Fc gamma R2 gene transcripts were also found in fetal cervical tissue by applying the polymerase chain reaction to amplify portions of the Fc gamma R3 and Fc gamma R2 coding sequences in cDNA prepared from fetal RNA. HLA-DR was found in the endocervical cells, transformation zone and in Langerhans cells of all specimens. The presence of Langerhans cells, Fc gamma receptors and HLA class II antigen offers three potential mechanisms for cervico-vaginal HIV transmission: (i) direct HIV infection of Langerhans cells, (ii) binding of HIV antibody complexes

  2. Comparison of O-Antigen gene clusters of all O-Serogroups of Escherichia coli and proposal for adopting a new nomenclature for O-Typing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Escherichia coli strains are classified based on O-antigens that are components of the lipopolysaccharide (LPS) in the cell envelope. O-antigens are important virulence factors, targets of both the innate and adaptive immune system, and play a role in host-pathogen interaction. Because they are hi...

  3. Cooperation between the polyomavirus middle-T-antigen gene and the human c-myc oncogene in a rat thyroid epithelial differentiated cell line: model of in vitro progression.

    PubMed Central

    Berlingieri, M T; Portella, G; Grieco, M; Santoro, M; Fusco, A

    1988-01-01

    Two rat thyroid epithelial differentiated cell lines, PC Cl 3 and PC myc, were infected with the polyoma murine leukemia virus (PyMLV) carrying the Middle-T-antigen gene of polyomavirus. After infection, both cell lines acquired the typical markers of neoplastic transformation; however, the PC myc cells showed a greater malignant phenotype. Furthermore, the thyroid differentiated functions were completely suppressed in PC myc cells transformed by PyMLV, whereas they were, at least partially, retained in PC Cl 3 cells transformed by PyMLV, and in particular, thyroglobulin synthesis and secretion were not affected at all. Since no differences in the expression of the middle-T-antigen gene were observed in the two PyMLV-transformed cell lines, the different properties shown by these two infected cell lines must be ascribed to the expression of the c-myc oncogene. Images PMID:2838744

  4. Antigenically Modified Human Pluripotent Stem Cells Generate Antigen-Presenting Dendritic Cells

    PubMed Central

    Zeng, Jieming; Wu, Chunxiao; Wang, Shu

    2015-01-01

    Human pluripotent stem cells (hPSCs) provide a promising platform to produce dendritic cell (DC) vaccine. To streamline the production process, we investigated a unique antigen-loading strategy that suits this novel platform. Specifically, we stably modified hPSCs using tumour antigen genes in the form of a full-length tumour antigen gene or an artificial tumour antigen epitope-coding minigene. Such antigenically modified hPSCs were able to differentiate into tumour antigen-presenting DCs. Without conventional antigen-loading, DCs derived from the minigene-modified hPSCs were ready to prime a tumour antigen-specific T cell response and further expand these specific T cells in restimulation processes. These expanded tumour antigen-specific T cells were potent effectors with central memory or effector memory phenotype. Thus, we demonstrated that immunocompetent tumour antigen-loaded DCs can be directly generated from antigenically modified hPSCs. Using such strategy, we can completely eliminate the conventional antigen-loading step and significantly simplify the production of DC vaccine from hPSCs. PMID:26471005

  5. Reverse mapping of the gene encoding the human fos-related antigen-1 (fra-1) within chromosome band 11q13

    SciTech Connect

    Sinke, R.J. ); Tanigami, A.; Nakamura, Y.; Kessel, A.G. van )

    1993-10-01

    For the identification of sequence-tagged sites within cosmid clones derived from the q13 region of human chromosome 11, the authors subcloned a number of single copy BamHI fragments in a pT7T3 19U vector. Surprisingly, within one of these subclones, they encountered a stretch of DNA that matches perfectly with 5' end of the known cDNA sequence for the human fos-related antigen. This match comes to an abrupt end at nucleotide 328, which coincides with a perfect splice donor site. At position 231 an ATG codon (translation initiation) was found. From these data they conclude that position 328 marks the boundary of the first exon of the fra-1 gene. Homology of this same region with the rat fra-1 cDNA sequence is [+-]70%. This human fra-1 genomic fragment is contained within cosmid clone cCL11-254 (D11S460), which was previously mapped to region V of a long-range contig map of 11q13. The localization of fra-1 on chromosome 11 was cross-checked and confirmed by hybridizing the fra-1 containing fragment to a panel of somatic cell hybrids, including one line that contains chromosome 11 as its only human constituent. In addition to a cross-hybridizing rodent fragment, a single human-specific band was revealed in all the chromosome 11-containing hybrids. This subchromosomal localization of fra-1 to 11q13 adds yet another proto-oncogene to this restricted genomic region, which appears to be involved in a number of neoplastic disorders, e.g., multiple endocrine neoplasia type 1, breast cancer, squamous cell tumors of head and neck, esophageal carcinoma, hepatocellular carcinoma, melanoma, bladder carcinoma, extragonadal germ cell tumors, and some hematopoietic malignancies. 7 refs., 1 fig.

  6. Gene-centric approach identifies new and known loci for FVIII activity and VWF antigen levels in European Americans and African Americans.

    PubMed

    Tang, Weihong; Cushman, Mary; Green, David; Rich, Stephen S; Lange, Leslie A; Yang, Qiong; Tracy, Russell P; Tofler, Geoffrey H; Basu, Saonli; Wilson, James G; Keating, Brendan J; Weng, Lu-Chen; Taylor, Herman A; Jacobs, David R; Delaney, Joseph A; Palmer, Cameron D; Young, Taylor; Pankow, James S; O'Donnell, Christopher J; Smith, Nicholas L; Reiner, Alexander P; Folsom, Aaron R

    2015-06-01

    Coagulation factor VIII and von Willebrand factor (VWF) are key proteins in procoagulant activation. Higher FVIII coagulant activity (FVIII :C) and VWF antigen (VWF :Ag) are risk factors for cardiovascular disease and venous thromboembolism. Beyond associations with ABO blood group, genetic determinants of FVIII and VWF are not well understood, especially in non European-American populations. We performed a genetic association study of FVIII :C and VWF:Ag that assessed 50,000 gene-centric single nucleotide polymorphisms (SNPs) in 18,556 European Americans (EAs) and 5,047 African Americans (AAs) from five population-based cohorts. Previously unreported associations for FVIII :C were identified in both AAs and EAs with KNG1 (most significantly associated SNP rs710446, Ile581Thr, Ile581Thr, P = 5.10 × 10(-7) in EAs and P = 3.88 × 10(-3) in AAs) and VWF rs7962217 (Gly2705Arg,P = 6.30 × 10(-9) in EAs and P = 2.98 × 10(-2) in AAs. Significant associations for FVIII :C were also observed with F8/TMLHE region SNP rs12557310 in EAs (P = 8.02 × 10(-10) ), with VWF rs1800380 in AAs (P = 5.62 × 10(-11) ), and with MAT1A rs2236568 in AAs (P51.69 × 10(-6) ). We replicated previously reported associations of FVIII :C and VWF :Ag with the ABO blood group, VWF rs1063856(Thr789Ala), rs216321 (Ala852Gln), and VWF rs2229446 (Arg2185Gln). Findings from this study expand our understanding of genetic influences for FVIII :C and VWF :Ag in both EAs and AAs.

  7. Complementation of Brucella abortus RB51 with a functional wboA gene results in O-antigen synthesis and enhanced vaccine efficacy but no change in rough phenotype and attenuation.

    PubMed

    Vemulapalli, R; He, Y; Buccolo, L S; Boyle, S M; Sriranganathan, N; Schurig, G G

    2000-07-01

    Brucella abortus RB51 is a stable rough, attenuated mutant vaccine strain derived from the virulent strain 2308. Recently, we demonstrated that the wboA gene in RB51 is disrupted by an IS711 element (R. Vemulapalli, J. R. McQuiston, G. G. Schurig, N. Srirauganathan, S. M. Halling, and S. M. Boyle, Clin. Diagn. Lab. Immunol. 6:760-764, 1999). Disruption of the wboA gene in smooth, virulent B. abortus, Brucella melitensis, and Brucella suis results in rough, attenuated mutants which fail to produce the O polysaccharide (O antigen). In this study, we explored whether the wboA gene disruption is responsible for the rough phenotype of RB51. We complemented RB51 with a functional wboA gene, and the resulting strain was designated RB51WboA. Colony and Western blot analyses indicated that RB51WboA expressed the O antigen; immunoelectron microscopy revealed that the O antigen was present in the cytoplasm. Crystal violet staining, acryflavin agglutination, and polymyxin B sensitivity studies indicated that RB51WboA had rough phenotypic characteristics similar to those of RB51. Bacterial clearance studies of BALB/c mice indicated no increase in the survival ability of RB51WboA in vivo compared to that of RB51. Vaccination of mice with live RB51WboA induced antibodies to the O antigen which were predominantly of the immunoglobulin G2a (IgG2a) and IgG3 isotypes. After in vitro stimulation of splenocytes with killed bacterial cells, quantitation of gamma interferon in the culture supernatants indicated that RB51WboA immunization induced higher levels of gamma interferon than immunization with RB51. Mice vaccinated with RB51WboA were better protected against a challenge infection with the virulent strain 2308 than those vaccinated with RB51. These studies indicate that in addition to the disruption of the wboA gene there is at least one other mutation in RB51 responsible for its rough phenotype. These studies also suggest that the expressed O antigen in RB51WboA is responsible

  8. Vγ9 and Vδ2 T cell antigen receptor genes and butyrophilin 3 (BTN3) emerged with placental mammals and are concomitantly preserved in selected species like alpaca (Vicugna pacos).

    PubMed

    Karunakaran, Mohindar M; Göbel, Thomas W; Starick, Lisa; Walter, Lutz; Herrmann, Thomas

    2014-04-01

    Human Vγ9Vδ2 T cells recognize phosphorylated products of isoprenoid metabolism (phosphoantigens) PAg with TCR comprising Vγ9JP γ-chains and Vδ2 δ-chains dependent on butyrophilin 3 (BTN3) expressed by antigen-presenting cells. They are massively activated in many infections and show anti-tumor activity and so far, they have been considered to exist only in higher primates. We performed a comprehensive analysis of databases and identified the three genes in species of both placental magnorders, but not in rodents. The common occurrence or loss of in silico translatable Vγ9, Vδ2, and BTN3 genes suggested their co-evolution based on a functional relationship. In the peripheral lymphocytes of alpaca (Vicugna pacos), characteristic Vγ9JP rearrangements and in-frame Vδ2 rearrangements were found and could be co-expressed in a TCR-negative mouse T cell hybridoma where they rescued CD3 expression and function. Finally, database sequence analysis of the extracellular domain of alpaca BTN3 revealed complete conservation of proposed PAg binding residues of human BTN3A1. In summary, we show emergence and preservation of Vγ9 and Vδ2 TCR genes with the gene of the putative antigen-presenting molecule BTN3 in placental mammals and lay the ground for analysis of alpaca as candidate for a first non-primate species to possess Vγ9Vδ2 T cells.

  9. Distinct Transcript Isoforms of the Atypical Chemokine Receptor 1 (ACKR1)/Duffy Antigen Receptor for Chemokines (DARC) Gene Are Expressed in Lymphoblasts and Altered Isoform Levels Are Associated with Genetic Ancestry and the Duffy-Null Allele.

    PubMed

    Davis, Melissa B; Walens, Andrea; Hire, Rupali; Mumin, Kauthar; Brown, Andrea M; Ford, DeJuana; Howerth, Elizabeth W; Monteil, Michele

    2015-01-01

    The Atypical ChemoKine Receptor 1 (ACKR1) gene, better known as Duffy Antigen Receptor for Chemokines (DARC or Duffy), is responsible for the Duffy Blood Group and plays a major role in regulating the circulating homeostatic levels of pro-inflammatory chemokines. Previous studies have shown that one common variant, the Duffy Null (Fy-) allele that is specific to African Ancestry groups, completely removes expression of the gene on erythrocytes; however, these individuals retain endothelial expression. Additional alleles are associated with a myriad of clinical outcomes related to immune responses and inflammation. In addition to allele variants, there are two distinct transcript isoforms of DARC which are expressed from separate promoters, and very little is known about the distinct transcriptional regulation or the distinct functionality of these protein isoforms. Our objective was to determine if the African specific Fy- allele alters the expression pattern of DARC isoforms and therefore could potentially result in a unique signature of the gene products, commonly referred to as antigens. Our work is the first to establish that there is expression of DARC on lymphoblasts. Our data indicates that people of African ancestry have distinct relative levels of DARC isoforms expressed in these cells. We conclude that the expression of both isoforms in combination with alternate alleles yields multiple Duffy antigens in ancestry groups, depending upon the haplotypes across the gene. Importantly, we hypothesize that DARC isoform expression patterns will translate into ancestry-specific inflammatory responses that are correlated with the axis of pro-inflammatory chemokine levels and distinct isoform-specific interactions with these chemokines. Ultimately, this work will increase knowledge of biological mechanisms underlying disparate clinical outcomes of inflammatory-related diseases among ethnic and geographic ancestry groups. PMID:26473357

  10. Antigen S1, encoded by the MIC1 gene, is characterized as an epitope of human CD59, enabling measurement of mutagen-induced intragenic deletions in the AL cell system

    NASA Technical Reports Server (NTRS)

    Wilson, A. B.; Seilly, D.; Willers, C.; Vannais, D. B.; McGraw, M.; Waldren, C. A.; Hei, T. K.; Davies, A.; Chatterjee, A. (Principal Investigator)

    1999-01-01

    S1 cell membrane antigen is encoded by the MIC1 gene on human chromosome 11. This antigen has been widely used as a marker for studies in gene mapping or in analysis of mutagen-induced gene deletions/mutations, which utilized the human-hamster hybrid cell-line, AL-J1, carrying human chromosome 11. Evidence is presented here which identifies S1 as an epitope of CD59, a cell membrane complement inhibiting protein. E7.1 monoclonal antibody, specific for the S1 determinant, was found to react strongly with membrane CD59 in Western blotting, and to bind to purified, urinary form of CD59 in ELISAs. Cell membrane expression of S1 on various cell lines always correlated with that of CD59 when examined by immunofluorescent staining. In addition, E7.1 antibody inhibited the complement regulatory function of CD59. Identification of S1 protein as CD59 has increased the scope of the AL cell system by enabling analysis of intragenic mutations, and multiplex PCR analysis of mutated cells is described, showing variable loss of CD59 exons.

  11. Characterization of the rcsA and rcsB genes from Salmonella typhi: rcsB through tviA is involved in regulation of Vi antigen synthesis.

    PubMed Central

    Virlogeux, I; Waxin, H; Ecobichon, C; Lee, J O; Popoff, M Y

    1996-01-01

    Synthesis of Vi antigen, a capsular polysaccharide expressed by Salmonella typhi, is controlled by the viaA and viaB chromosomal loci. It was previously shown that Vi antigen expression was regulated by a system similar to the rcs regulatory system involved in colanic acid synthesis in Escherichia coli. We have cloned the rcsA, rcsB, and rcsC genes from S. typhi. The predicted amino sequences of the RcsA and RcsB proteins showed a high degree of similarity to their E. coli homologs. The nucleotide sequence of the rcsC gene was partially determined and was shown to be homologous to that of its E. coli counterpart. Complementation experiments indicated that rcsB and rcsC were encompassed within the viaA locus. The RcsA protein was not involved in Vi antigen synthesis. In contrast, the RcsB protein acted as a positive regulator of Vi polysaccharide expression. By mRNA and gene fusion analyses, we studied the role of RcsB and TviA, a via-B-encoded regulatory protein characterized previously, in regulating Vi antigen synthesis. The transcriptional start point of tviA mRNA was not influenced by RcsB or TviA. In the absence of RcsB or TviA protein, transcription of tviA gave rise to only a monocistronic tviA-specific mRNA. The presence of RcsB and TriA not only increased the amount of monocistronic tviA-specific mRNA but also resulted in countranscription of tviA and tviB, which is located immediately downstream of tviA on the viaB locus. In addition, TviA protein did not appear to be subject to degradation by the Lon protease. These results strongly suggest that TviA might act in concert with RcsB at the tviA promoter to activate transcription of the genes involved in Vi polymer synthesis in S. typhi in a Lon-independent manner. PMID:8626298

  12. Preparation and diagnostic utility of a hemagglutination inhibition test antigen derived from the baculovirus-expressed hemagglutinin-neuraminidase protein gene of Newcastle disease virus.

    PubMed

    Choi, Kang-Seuk; Kye, Soo-Jeong; Jeon, Woo-Jin; Park, Mi-Ja; Kim, Saeromi; Seul, Hee-Jung; Kwon, Jun-Hun

    2013-01-01

    A recombinant hemagglutinin-neuraminidase (rHN) protein from Newcastle disease virus (NDV) with hemagglutination (HA) activity was expressed in Spodoptera frugiperda cells using a baculovirus expression system. The rHN protein extracted from infected cells was used as an antigen in a hemagglutination inhibition (HI) test for the detection and titration of NDV-specific antibodies present in chicken sera. The rHN antigen produced high HA titers of 2(13) per 25 μL, which were similar to those of the NDV antigen produced using chicken eggs, and it remained stable without significant loss of the HA activity for at least 12 weeks at 4°C. The rHN-based HI assay specifically detected NDV antibodies, but not the sera of other avian pathogens, with a specificity and sensitivity of 100% and 98.0%, respectively, in known positive and negative chicken sera (n = 430). Compared with an NDV-based HI assay, the rHN-based HI assay had a relative sensitivity and specificity of 96.1% and 95.5%, respectively, when applied to field chicken sera. The HI titers of the rHN-based HI assay were highly correlated with those in an NDV-based HI assay (r = 0.927). Overall, these results indicate that rHN protein provides a useful alternative to NDV antigen in HI assays.

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

    PubMed Central

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

    1989-01-01

    The precore/core gene of hepatitis B virus directs the synthesis of two polypeptides, the 21-kilodalton subunit (p21c) forming the viral nucleocapsid (serologically defined as core antigen [HBcAg]) and a secreted processed protein (p17e, serologically defined as HBe antigen [HBeAg]). Although most of their primary amino acid sequences are identical, HBcAg and HBeAg display different antigenic properties that are widely used in hepatitis B virus diagnosis. To locate and to characterize the corresponding determinants, segments of the core gene were expressed in Escherichia coli and probed with a panel of polyclonal or monoclonal antibodies in radioimmunoassays or enzyme-linked immunosorbent assays, Western blots, and competition assays. Three distinct major determinants were characterized. The single conformational determinant responsible for HBc antigenicity in the assembled core (HBc) and a linear HBe-related determinant (HBe1) were both mapped to an overlapping hydrophilic sequence around amino acid 80; a second HBe determinant (HBe2) was assigned to a location in the vicinity of amino acid 138 but found to require for its antigenicity the intramolecular participation of the extended sequence between amino acids 10 and 140. It is postulated that HBcAg and HBeAg share common basic three-dimensional structure exposing the common linear determinant HBe1 but that they differ in the presentation of two conformational determinants that are either introduced (HBc) or masked (HBe2) in the assembled core. The simultaneous presentation of HBe1 and HBc, two distinctly different antigenic determinants with overlapping amino acid sequences, is interpreted to indicate the presence of slightly differently folded, stable conformational states of p21c in the hepatitis B virus nucleocapsid. Images PMID:2463383

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

    PubMed Central

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

    2013-01-01

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

  15. A Prospective Study of Comparing Multi-Gene Biomarker Chip and Serum Carcinoembryonic Antigen in the Postoperative Surveillance for Patients with Stage I-III Colorectal Cancer

    PubMed Central

    Chang, Yu-Tang; Huang, Ming-Yii; Yeh, Yung-Sung; Huang, Ching-Wen; Tsai, Hsiang-Lin; Cheng, Tian-Lu; Wang, Jaw-Yuan

    2016-01-01

    Background Circulating biomarkers can predict clinical outcomes in colorectal cancer patients. The aim of the study was to evaluate the feasibility of our multigene biomarker chip for detecting circulating tumor cells for postoperative surveillance of stage I–III colorectal cancer patients. Materials and Methods In total, 298 stage I–III colorectal cancer patients were analyzed after curative resection between June 2010 and October 2014. During each follow-up, a postoperative surveillance strategy, including ESMO Guidelines Working Group recommendations and the biochip, was used. Results After a 28.4-month median follow-up, 48 (16.1%) patients had postoperative relapse. Univariate analysis revealed that the postoperative relapse risk factors were rectal tumor, perineural invasion, elevated preoperative and postoperative serum carcinoembryonic antigen levels, and positive biochip results (all P < 0.05). Multivariate analyses revealed that postoperative relapse correlated significantly with elevated postoperative serum carcinoembryonic antigen levels (odds ratio = 4.136, P = 0.008) and positive biochip results (odds ratio = 66.878, P < 0.001). However, the sensitivity (P = 0.003), specificity (P = 0.003), positive (P = 0.002) and negative (P = 0.006) predictive values, and accuracy (P < 0.001) of the biochip for predicting postoperative relapse were significantly higher than those of elevated postoperative serum carcinoembryonic antigen levels. Moreover, the median lead time between positive biochip result and postoperative relapse detection was significantly earlier than that between elevated postoperative serum carcinoembryonic antigen level and postoperative relapse detection (10.7 vs. 2.8 months, P < 0.001). Furthermore, positive biochip results correlated strongly with lower disease-free survival and overall survival of colorectal cancer patients (both P < 0.001). Conclusion Compared with conventional serum carcinoembryonic antigen detection, our multigene

  16. Enhancement of the antigen-specific cytotoxic T lymphocyte-inducing ability in the PMDC11 leukemic plasmacytoid dendritic cell line via lentiviral vector-mediated transduction of the caTLR4 gene.

    PubMed

    Iwabuchi, Minami; Narita, Miwako; Uchiyama, Takayoshi; Iwaya, Shunpei; Oiwa, Eri; Nishizawa, Yoshinori; Hashimoto, Shigeo; Bonehill, Aude; Kasahara, Noriyuki; Takizawa, Jun; Takahashi, Masuhiro

    2015-08-01

    The aim of the present study was to enhance the efficiency of leukemia immunotherapy by increasing the antigen-specific cytotoxic T lymphocyte-inducing ability of leukemia cells. The leukemic plasmacytoid dendritic cell line PMDC05 containing the HLA-A02/24 antigen, which was previously established in our laboratory (Laboratory of Hematology and Oncology, Graduate School of Health Sciences, Niigata University, Niigata, Japan), was used in the present study. It exhibited higher expression levels of CD80 following transduction with lentiviruses encoding the CD80 gene. This CD80-expressing PMDC05 was named PMDC11. In order to establish a more potent antigen-presenting cell for cellular immunotherapy of tumors or severe infections, PMDC11 cells were transduced with a constitutively active (ca) toll-like receptor 4 (TLR4) gene using the Tet-On system (caTLR4-PMDC11). CD8(+) T cells from healthy donors with HLA-A02 were co-cultured with mutant WT1 peptide-pulsed PMDC11, lipopolysaccharide (LPS)-stimulated PMDC11 or caTLR4-PMDC11 cells. Interleukin (IL)-2 (50 IU/ml) and IL-7 (10 ng/ml) were added on day three of culture. Priming with mutant WT1 peptide-pulsed PMDC11, LPS-stimulated PMDC11 or caTLR4-PMDC11 cells was conducted once per week and two thirds of the IL-2/IL-7 containing medium was replenished every 3-4 days. Immediately prior to the priming with these various PMDC11 cells, the cultured cells were analyzed for the secretion of interferon (IFN)-γ in addition to the percentage and number of CD8(+)/WT1 tetramer(+) T cells using flow cytometry. caTLR4-PMDC11 cells were observed to possess greater antigen-presenting abilities compared with those of PMDC11 or LPS-stimulated PMDC11 cells in a mixed leukocyte culture. CD8 T cells positive for the WT1 tetramer were generated following 3-4 weeks of culture and CD8(+)/WT1 tetramer+ T cells were markedly increased in caTLR4-PMDC11-primed CD8(+) T cell culture compared with PMDC11 or LPS-stimulated PMDC11-primed CD8(+) T

  17. Enhancement of the antigen-specific cytotoxic T lymphocyte-inducing ability in the PMDC11 leukemic plasmacytoid dendritic cell line via lentiviral vector-mediated transduction of the caTLR4 gene

    PubMed Central

    IWABUCHI, MINAMI; NARITA, MIWAKO; UCHIYAMA, TAKAYOSHI; IWAYA, SHUNPEI; OIWA, ERI; NISHIZAWA, YOSHINORI; HASHIMOTO, SHIGEO; BONEHILL, AUDE; KASAHARA, NORIYUKI; TAKIZAWA, JUN; TAKAHASHI, MASUHIRO

    2015-01-01

    The aim of the present study was to enhance the efficiency of leukemia immunotherapy by increasing the antigen-specific cytotoxic T lymphocyte-inducing ability of leukemia cells. The leukemic plasmacytoid dendritic cell line PMDC05 containing the HLA-A02/24 antigen, which was previously established in our laboratory (Laboratory of Hematology and Oncology, Graduate School of Health Sciences, Niigata University, Niigata, Japan), was used in the present study. It exhibited higher expression levels of CD80 following transduction with lentiviruses encoding the CD80 gene. This CD80-expressing PMDC05 was named PMDC11. In order to establish a more potent antigen-presenting cell for cellular immunotherapy of tumors or severe infections, PMDC11 cells were transduced with a constitutively active (ca) toll-like receptor 4 (TLR4) gene using the Tet-On system (caTLR4-PMDC11). CD8+ T cells from healthy donors with HLA-A02 were co-cultured with mutant WT1 peptide-pulsed PMDC11, lipopolysaccharide (LPS)-stimulated PMDC11 or caTLR4-PMDC11 cells. Interleukin (IL)-2 (50 IU/ml) and IL-7 (10 ng/ml) were added on day three of culture. Priming with mutant WT1 peptide-pulsed PMDC11, LPS-stimulated PMDC11 or caTLR4-PMDC11 cells was conducted once per week and two thirds of the IL-2/IL-7 containing medium was replenished every 3–4 days. Immediately prior to the priming with these various PMDC11 cells, the cultured cells were analyzed for the secretion of interferon (IFN)-γ in addition to the percentage and number of CD8+/WT1 tetramer+ T cells using flow cytometry. caTLR4-PMDC11 cells were observed to possess greater antigen-presenting abilities compared with those of PMDC11 or LPS-stimulated PMDC11 cells in a mixed leukocyte culture. CD8 T cells positive for the WT1 tetramer were generated following 3–4 weeks of culture and CD8+/WT1 tetramer+ T cells were markedly increased in caTLR4-PMDC11-primed CD8+ T cell culture compared with PMDC11 or LPS-stimulated PMDC11-primed CD8+ T cell

  18. Antigen presenting cells - diversity, differentiation, and regulation

    SciTech Connect

    Schook, L.B. ); Tew, J.G. )

    1988-01-01

    This book contains 35 papers. Some of the titles are: DNA-mediated gene transfer as a tool for analyzing Ia structure-function relationships and antigen presentation; Regulation of immune-associated genes during macrophage differentiation; Presentation of arsonate-tyrosine to cloned T-cells by L-Cells transfected with class II genes; and The duration of class II MHC glycoprotein expression by mononuclear phagocytes is regulated by the Bcg gene.

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

    SciTech Connect

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

    1989-02-01

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

  20. Sequence Variations in the Flagellar Antigen Genes fliCH25 and fliCH28 of Escherichia coli and Their Use in Identification and Characterization of Enterohemorrhagic E. coli (EHEC) O145:H25 and O145:H28.

    PubMed

    Beutin, Lothar; Delannoy, Sabine; Fach, Patrick

    2015-01-01

    Enterohemorrhagic E. coli (EHEC) serogroup O145 is regarded as one of the major EHEC serogroups involved in severe infections in humans. EHEC O145 encompasses motile and non-motile strains of serotypes O145:H25 and O145:H28. Sequencing the fliC-genes associated with the flagellar antigens H25 and H28 revealed the genetic diversity of the fliCH25 and fliCH28 gene sequences in E. coli. Based on allele discrimination of these fliC-genes real-time PCR tests were designed for identification of EHEC O145:H25 and O145:H28. The fliCH25 genes present in O145:H25 were found to be very similar to those present in E. coli serogroups O2, O100, O165, O172 and O177 pointing to their common evolution but were different from fliCH25 genes of a multiple number of other E. coli serotypes. In a similar way, EHEC O145:H28 harbor a characteristic fliCH28 allele which, apart from EHEC O145:H28, was only found in enteropathogenic (EPEC) O28:H28 strains that shared some common traits with EHEC O145:H28. The real time PCR-assays targeting these fliCH25[O145] and fliCH28[O145] alleles allow better characterization of EHEC O145:H25 and EHEC O145:H28. Evaluation of these PCR assays in spiked ready-to eat salad samples resulted in specific detection of both types of EHEC O145 strains even when low spiking levels of 1-10 cfu/g were used. Furthermore these PCR assays allowed identification of non-motile E. coli strains which are serologically not typable for their H-antigens. The combined use of O-antigen genotyping (O145wzy) and detection of the respective fliCH25[O145] and fliCH28[O145] allele types contributes to improve identification and molecular serotyping of E. coli O145 isolates.

  1. Sequence Variations in the Flagellar Antigen Genes fliCH25 and fliCH28 of Escherichia coli and Their Use in Identification and Characterization of Enterohemorrhagic E. coli (EHEC) O145:H25 and O145:H28

    PubMed Central

    Beutin, Lothar; Delannoy, Sabine; Fach, Patrick

    2015-01-01

    Enterohemorrhagic E. coli (EHEC) serogroup O145 is regarded as one of the major EHEC serogroups involved in severe infections in humans. EHEC O145 encompasses motile and non-motile strains of serotypes O145:H25 and O145:H28. Sequencing the fliC-genes associated with the flagellar antigens H25 and H28 revealed the genetic diversity of the fliCH25 and fliCH28 gene sequences in E. coli. Based on allele discrimination of these fliC-genes real-time PCR tests were designed for identification of EHEC O145:H25 and O145:H28. The fliCH25 genes present in O145:H25 were found to be very similar to those present in E. coli serogroups O2, O100, O165, O172 and O177 pointing to their common evolution but were different from fliCH25 genes of a multiple number of other E. coli serotypes. In a similar way, EHEC O145:H28 harbor a characteristic fliCH28 allele which, apart from EHEC O145:H28, was only found in enteropathogenic (EPEC) O28:H28 strains that shared some common traits with EHEC O145:H28. The real time PCR-assays targeting these fliCH25[O145] and fliCH28[O145] alleles allow better characterization of EHEC O145:H25 and EHEC O145:H28. Evaluation of these PCR assays in spiked ready-to eat salad samples resulted in specific detection of both types of EHEC O145 strains even when low spiking levels of 1–10 cfu/g were used. Furthermore these PCR assays allowed identification of non-motile E. coli strains which are serologically not typable for their H-antigens. The combined use of O-antigen genotyping (O145wzy) and detection of the respective fliCH25[O145] and fliCH28[O145] allele types contributes to improve identification and molecular serotyping of E. coli O145 isolates. PMID:26000885

  2. Evaluation of Selected Borrelia burgdorferi lp54 Plasmid-Encoded Gene Products Expressed during Mammalian Infection as Antigens To Improve Serodiagnostic Testing for Early Lyme Disease.

    PubMed

    Weiner, Zachary P; Crew, Rebecca M; Brandt, Kevin S; Ullmann, Amy J; Schriefer, Martin E; Molins, Claudia R; Gilmore, Robert D

    2015-11-01

    Laboratory testing for the diagnosis of Lyme disease is performed primarily by serologic assays and is accurate for detection beyond the acute stage of the infection. Serodiagnostic assays to detect the early stages of infection, however, are limited in their sensitivity, and improvement is warranted. We analyzed a series of Borrelia burgdorferi proteins known to be induced within feeding ticks and/or during mammalian infection for their utility as serodiagnostic markers against a comprehensive panel of Lyme disease patient serum samples. The antigens were assayed for IgM and IgG reactivity in line immunoblots and separately by enzyme-linked immunosorbent assay (ELISA), with a focus on reactivity against early Lyme disease with erythema migrans (EM), early disseminated Lyme neuroborreliosis, and early Lyme carditis patient serum samples. By IgM immunoblotting, we found that recombinant proteins BBA65, BBA70, and BBA73 reacted with early Lyme EM samples at levels comparable to those of the OspC antigen used in the current IgM blotting criteria. Additionally, these proteins reacted with serum samples from patients with early neuroborreliosis and early carditis, suggesting value in detecting early stages of this disease progression. We also found serological reactivity against recombinant proteins BBA69 and BBA73 with early-Lyme-disease samples using IgG immunoblotting and ELISA. Significantly, some samples that had been scored negative by the Centers for Disease Control and Prevention-recommended 2-tiered testing algorithm demonstrated positive reactivity to one or more of the antigens by IgM/IgG immunoblot and ELISA. These results suggest that incorporating additional in vivo-expressed antigens into the current IgM/IgG immunoblotting tier in a recombinant protein platform assay may improve the performance of early-Lyme-disease serologic testing.

  3. Relationship between major histocompatibility antigens and disease

    PubMed Central

    Oldstone, Michael B. A.

    1975-01-01

    Histocompatibility antigens, virus infections, and disease are discussed relative to avenues of research in humans with arenavirus infections. The data implicating a relationship between histocompatibility complexes in man and animals and diseases of the central nervous system are reviewed. Histocompatibility antigens may share common antigenic determinants with viruses, act as receptor sites for attachment of viruses, and be altered by viruses. In addition, genes regulating immune responses to a variety of natural and synthetic antigens are linked, in many species, to the major histocompatibility complex. Since injury associated with virus infections may be largely due to the activity of the immune system, study of immune response genes may provide insight into understanding resistance to disease. Further, histoincompatibility reactions can activate latent viruses with resultant disease. PMID:60183

  4. Association of Nucleotide Polymorphisms within the O-Antigen Gene Cluster of Escherichia coli O26, O45, O103, O111, O121, and O145 with Serogroups and Genetic Subtypes

    PubMed Central

    Norman, Keri N.; Strockbine, Nancy A.

    2012-01-01

    Shiga toxin-producing Escherichia coli (STEC) strains are important food-borne pathogens capable of causing hemolytic-uremic syndrome. STEC O157:H7 strains cause the majority of severe disease in the United States; however, there is a growing concern for the amount and severity of illness attributable to non-O157 STEC. Recently, the Food Safety and Inspection Service (FSIS) published the intent to regulate the presence of STEC belonging to serogroups O26, O45, O103, O111, O121, and O145 in nonintact beef products. To ensure the effective control of these bacteria, sensitive and specific tests for their detection will be needed. In this study, we identified single nucleotide polymorphisms (SNPs) in the O-antigen gene cluster that could be used to detect STEC strains of the above-described serogroups. Using comparative DNA sequence analysis, we identified 22 potentially informative SNPs among 164 STEC and non-STEC strains of the above-described serogroups and designed matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF) assays to test the STEC allele frequencies in an independent panel of bacterial strains. We found at least one SNP that was specific to each serogroup and also differentiated between STEC and non-STEC strains. Differences in the DNA sequence of the O-antigen gene cluster corresponded well with differences in the virulence gene profiles and provided evidence of different lineages for STEC and non-STEC strains. The SNPs discovered in this study can be used to develop tests that will not only accurately identify O26, O45, O103, O111, O121, and O145 strains but also predict whether strains detected in the above-described serogroups contain Shiga toxin-encoding genes. PMID:22798363

  5. Age-related deregulation of Aire and peripheral tissue antigen genes in the thymic stroma of non-obese diabetic (NOD) mice is associated with autoimmune type 1 diabetes mellitus (DM-1).

    PubMed

    Fornari, Thaís A; Donate, Paula B; Macedo, Claudia; Marques, Márcia M C; Magalhães, Danielle A; Passos, Geraldo A S

    2010-09-01

    Gene expression of peripheral tissue antigens (PTAs) in stromal medullary thymic epithelial cells (mTECs) is a key process to the negative selection of autoreactive thymocytes. This phenomenon was termed "promiscuous gene expression" (PGE), which is partially controlled by the Aire gene. Nevertheless, reasons for the correlation of Aire and PTAs with the emergence of autoimmune diseases are largely unknown, though it may be a result of a chronological effect. Although the effect of Aire mutations in pathogenic autoimmunity is well know, it could not be a unique cause for autoimmunity. Independently of mutations, temporal deregulation of Aire expression may imbalance Aire-dependent PTAs and/or wide PGE. This deregulation may be an early warning sign for autoimmune diseases as it guarantees autoantigen representation in the thymus. To assess this hypothesis, we studied the expression levels of Aire, Aire-dependent (Ins2) and Aire-independent (Gad67 and Col2a1) PTAs using real-time-PCR of the thymic stromal cells of NOD mice during the development of autoimmune type 1 diabetes mellitus (DM-1). Wide PGE was studied by microarrays in which the PTA genes were identified through parallel CD80(+) mTEC 3.10 cell line expression profiling. The results show that Aire gene was down-regulated in young pre-autoimmune (pre-diabetic) NOD mice. PGE and specific PTA genes were down-regulated in adult autoimmune diabetic animals. These findings represent evidence indicating that chronological deregulation of genes important to negative selection may be associated with the development of an autoimmune disease (DM-1) in mice.

  6. Breed differences in the frequency of bovine lymphocyte antigens.

    PubMed

    Stear, M J; Brown, S C; Dimmock, C K; Dufty, J H; Hetzel, D J; Mackie, J T; Nicholas, F W; Tierney, T J; Wetherall, J D

    1987-01-01

    Lymphocytes from 1,564 cattle of 18 breeds and cross-bred groups in Australia were tested for major histocompatibility system class 1 antigens. Gene frequencies were calculated for the Angus, Belmont Red, Brahman, Hereford and Holstein-Friesian breeds. There were substantial differences among these breeds in antigen and gene frequency. There were striking differences among all 18 breeds in the presence or absence of certain antigens. Two antigens, CA13 and CA36, were strongly associated in Hereford cattle but occurred independently of each other in the other breeds. PMID:3273412

  7. Genetic Diversity of the fliC Genes Encoding the Flagellar Antigen H19 of Escherichia coli and Application to the Specific Identification of Enterohemorrhagic E. coli O121:H19

    PubMed Central

    Beutin, Lothar; Delannoy, Sabine

    2015-01-01

    Enterohemorrhagic Escherichia coli (EHEC) O121:H19 belong to a specific clonal type distinct from other classical EHEC and major enteropathogenic E. coli groups and is regarded as one of the major EHEC serogroups involved in severe infections in humans. Sequencing of the fliC genes associated with the flagellar antigen H19 (fliCH19) revealed the genetic diversity of the fliCH19 gene sequences in E. coli. A cluster analysis of 12 fliCH19 sequences, 4 from O121 and 8 from non-O121 E. coli strains, revealed five different genotypes. All O121:H19 strains fell into one cluster, whereas a second cluster was formed by five non-O121:H19 strains. Cluster 1 and cluster 2 strains differ by 27 single nucleotide exchanges in their fliCH19 genes (98.5% homology). Based on allele discrimination of the fliCH19 genes, a real-time PCR test was designed for specific identification of EHEC O121:H19. The O121 fliCH19 PCR tested negative in 73 E. coli H19 strains that belonged to serogroups other than O121, including 28 different O groups, O-nontypeable H19, and O-rough:H19 strains. The O121 fliCH19 PCR reacted with all 16 tested O121:H19 strains and 1 O-rough:H19 strain which was positive for the O121 wzx gene. A cross-reaction was observed only with E. coli H32 strains which share sequence similarities in the target region of the O121 fliCH19 PCR. The combined use of O-antigen genotyping (O121 wzx) and the detection of O121 fliCH19 allele type contributes to improving the identification and molecular serotyping of EHEC O121:H19 motile and nonmotile strains and variants of these strains lacking stx genes. PMID:25862232

  8. Meningococcal vaccine antigen diversity in global databases.

    PubMed

    Brehony, Carina; Hill, Dorothea M; Lucidarme, Jay; Borrow, Ray; Maiden, Martin C

    2015-01-01

    The lack of an anti-capsular vaccine against serogroup B meningococcal disease has necessitated the exploration of alternative vaccine candidates, mostly proteins exhibiting varying degrees of antigenic variation. Analysis of variants of antigen-encoding genes is facilitated by publicly accessible online sequence repositories, such as the Neisseria PubMLST database and the associated Meningitis Research Foundation Meningococcus Genome Library (MRF-MGL). We investigated six proposed meningococcal vaccine formulations by deducing the prevalence of their components in the isolates represented in these repositories. Despite high diversity, a limited number of antigenic variants of each of the vaccine antigens were prevalent, with strong associations of particular variant combinations with given serogroups and genotypes. In the MRF-MGL and globally, the highest levels of identical sequences were observed with multicomponent/multivariant vaccines. Our analyses further demonstrated that certain combinations of antigen variants were prevalent over periods of decades in widely differing locations, indicating that vaccine formulations containing a judicious choice of antigen variants have potential for long-term protection across geographic regions. The data further indicated that formulations with multiple variants would be especially relevant at times of low disease incidence, as relative diversity was higher. Continued surveillance is required to monitor the changing prevalence of these vaccine antigens.

  9. Meningococcal vaccine antigen diversity in global databases.

    PubMed

    Brehony, Carina; Hill, Dorothea M; Lucidarme, Jay; Borrow, Ray; Maiden, Martin C

    2015-01-01

    The lack of an anti-capsular vaccine against serogroup B meningococcal disease has necessitated the exploration of alternative vaccine candidates, mostly proteins exhibiting varying degrees of antigenic variation. Analysis of variants of antigen-encoding genes is facilitated by publicly accessible online sequence repositories, such as the Neisseria PubMLST database and the associated Meningitis Research Foundation Meningococcus Genome Library (MRF-MGL). We investigated six proposed meningococcal vaccine formulations by deducing the prevalence of their components in the isolates represented in these repositories. Despite high diversity, a limited number of antigenic variants of each of the vaccine antigens were prevalent, with strong associations of particular variant combinations with given serogroups and genotypes. In the MRF-MGL and globally, the highest levels of identical sequences were observed with multicomponent/multivariant vaccines. Our analyses further demonstrated that certain combinations of antigen variants were prevalent over periods of decades in widely differing locations, indicating that vaccine formulations containing a judicious choice of antigen variants have potential for long-term protection across geographic regions. The data further indicated that formulations with multiple variants would be especially relevant at times of low disease incidence, as relative diversity was higher. Continued surveillance is required to monitor the changing prevalence of these vaccine antigens. PMID:26676305

  10. Two amino acid substitutions in apolipoprotein B are in complete allelic association with the antigen group (x/y) polymorphism: Evidence for little recombination in the 3 prime end of the human gene

    SciTech Connect

    Dunning, A.M.; Renges, H.H.; Xu, Chunfang; Peacock, R.; Humphries, S.E.; Talmud, P.; Laxer, G. ); Brasseur, R. ); Tikkanen, M.J. ); Buetler, R. ); Saha, N. ); Hamsten, A. ); Rosseneu, M. )

    1992-01-01

    The authors report the identification of an A-to-G base change, in exon 29 of the apolipoprotein B (apo B) gene, that results in the substitution of serine for asparagine at residue 4,311 of mature apo B100. In a recent publication, Huang et al. have reported a C-to-T base change in exon 26 that causes the substitution of leucine for proline at residue 2712 of apo B. The authors have found complete linkage disequilibrium between the alleles at both these sites and an immunochemical polymorphism of LDL designated antigen group (x/y) (Ag(x/y)) in a sample of 118 Finnish individuals. This implies that either one of these substitutions - or both of them combined - could be the molecular basis of the Ag(x/y) antigenic determinants, with the allele encoding serine{sub 4311} plus leucine{sub 2,712} representing the Ag(x) epitope, and that encoding asparagine{sub 4,311} plus proline{sub 2,712} the Ag(y) epitope. In a sample of 90 healthy Swedish individuals the Leu{sub 2,712}/Ser{sub 4,311} allele is associated both with reduced serum levels of LDL cholesterol and apo B and with raised levels of HDL. They have also genotyped 523 individuals from European, Asian, Chinese, and Afro-Caribbean populations and have found complete association between the sites encoding residues 2,712 and 4,311 in all of these samples, although there are large allele frequency differences between these populations. Taken together, these data suggest that, since the divergence of the major ethnic groups, there has been little or no recombination in the 3' end of the human apo B gene.

  11. Sequencing of the gene encoding the major pilin of pilus colonization factor antigen III (CFA/III) of human enterotoxigenic Escherichia coli and evidence that CFA/III is related to type IV pili.

    PubMed Central

    Taniguchi, T; Fujino, Y; Yamamoto, K; Miwatani, T; Honda, T

    1995-01-01

    The plasmid-encoded structural gene cofA necessary for the production of the major pilin subunit of pilus colonization factor antigen III (CFA/III) of human enterotoxigenic Escherichia coli was identified, and the nucleotide sequence of the gene was determined. cofA consists of 714 nucleotides encoding a 238-amino-acid protein (molecular weight of 25,309). CofA seems to be a precursor of CFA/III pilin, because the first 23 residues of the N-terminal amino acid sequence of the purified CFA/III pili coincided with the deduced amino acid sequence for residues 32 to 54 of CofA. Western blot (immunoblot) analysis of CofA also indicated its processing to form mature pilin in the presence of the downstream region of cofA. These results suggest that the major pilin of CFA/III pili is produced as a precursor form which is posttranslationally modified to the mature pilin and forms morphological pili after cleavage of the Gly-30-Met-31 junction, probably by a protease encoded by an as-yet-unknown gene located downstream of cofA. Interestingly, the N-terminal 30-amino-acid sequence of mature CFA/III shows the highest identity (76.7%) to TcpA pilin of Vibrio cholerae, which is a type IV class B pilin. PMID:7822050

  12. Antigenic variation in African trypanosomes

    PubMed Central

    Horn, David

    2014-01-01

    Studies on Variant Surface Glycoproteins (VSGs) and antigenic variation in the African trypanosome, Trypanosoma brucei, have yielded a remarkable range of novel and important insights. The features first identified in T. brucei extend from unique to conserved-among-trypanosomatids to conserved-among-eukaryotes. Consequently, much of what we now know about trypanosomatid biology and much of the technology available has its origin in studies related to VSGs. T. brucei is now probably the most advanced early branched eukaryote in terms of experimental tractability and can be approached as a pathogen, as a model for studies on fundamental processes, as a model for studies on eukaryotic evolution or often all of the above. In terms of antigenic variation itself, substantial progress has been made in understanding the expression and switching of the VSG coat, while outstanding questions continue to stimulate innovative new approaches. There are large numbers of VSG genes in the genome but only one is expressed at a time, always immediately adjacent to a telomere. DNA repair processes allow a new VSG to be copied into the single transcribed locus. A coordinated transcriptional switch can also allow a new VSG gene to be activated without any detectable change in the DNA sequence, thereby maintaining singular expression, also known as allelic exclusion. I review the story behind VSGs; the genes, their expression and switching, their central role in T. brucei virulence, the discoveries that emerged along the way and the persistent questions relating to allelic exclusion in particular. PMID:24859277

  13. Transcutaneous antigen delivery system

    PubMed Central

    Lee, Mi-Young; Shin, Meong-Cheol; Yang, Victor C.

    2013-01-01

    Transcutaneous immunization refers to the topical application of antigens onto the epidermis. Transcutaneous immunization targeting the Langerhans cells of the skin has received much attention due to its safe, needle-free, and noninvasive antigen delivery. The skin has important immunological functions with unique roles for antigen-presenting cells such as epidermal Langerhans cells and dermal dendritic cells. In recent years, novel vaccine delivery strategies have continually been developed; however, transcutaneous immunization has not yet been fully exploited due to the penetration barrier represented by the stratum corneum, which inhibits the transport of antigens and adjuvants. Herein we review recent achievements in transcutaneous immunization, focusing on the various strategies for the enhancement of antigen delivery and vaccination efficacy. [BMB Reports 2013; 46(1): 17-24] PMID:23351379

  14. Evaluation of immune responses in sheep induced by DNA immunization with genes encoding GRA1, GRA4, GRA6 and GRA7 antigens of Toxoplasma gondii.

    PubMed

    Hiszczyńska-Sawicka, Elżbieta; Olędzka, Gabriela; Holec-Gąsior, Lucyna; Li, Hong; Xu, Janet Boyu; Sedcole, Richard; Kur, Józef; Bickerstaffe, Roy; Stankiewicz, Mirosław

    2011-05-11

    The dense granule proteins of Toxoplasma gondii are investigated as possible vaccine candidates against the parasite. The aim of this research was to evaluate the immune responses of sheep injected twice, intramuscularly, with DNA plasmids encoding T. gondii dense granule antigens GRA1, GRA4, GRA6 and GRA7 formulated into liposomes. Control sheep were injected with an empty vector or received no injections. The injection of sheep with DNA plasmids encoding for GRA1, GRA4, GRA6 or GRA7 elicited an immune response after the first and the second injections as indicated by the moderate to high antibody responses. The injection of pGRA7 induced a significant level of anti-GRA7 IgG2 antibody and IFN-γ responses indicating a Th1-like immune response whereas injection with pGRA1, pGRA4 and pGRA6 stimulated a IgG1 type antibody response with a limited, if any, IFN-γ response. The results demonstrate that the intramuscular injection of sheep with a DNA liposome formulated plasmid coding for GRA proteins is an effective system that induces a significant immune response against T. gondii.

  15. Hybrid genes between HLA-A2 and HLA-A3 constructed by in vivo recombination allow mapping of HLA-A2 and HLA-A3 polymorphic antigenic determinants.

    PubMed

    Sire, J; Chimini, G; Boretto, J; Toubert, A; Kahn-Perles, B; Layet, C; Sodoyer, R; Lemonnier, F; Jordan, B

    1988-04-01

    HLA-A2 and -A3 genes have been modified in their third exon (second domain) by using in vivo recombination. In this method Escherichia coli are transfected with a plasmid which contains two highly homologous sequences (e.g., the third exons of HLA-A2 and -A3) and has been linearized by cleavage between these two sequences. Circularization takes place in the bacteria by homologous recombination leading to hybrid A2-A3 sequences. The analysis by DNA sequencing of a number of such recombinants shows that they indeed occur by homologous recombination (no insertions or deletions) and that the probability of crossing over decreases as the distance from the free end of DNA in the homologous region increases. No double recombinants were observed. These hybrid exons were reinserted into either HLA-A2 or HLA-A3 genes, thus generating a panel of functional hybrid genes containing one or several HLA-A2 specific substitutions in an HLA-A3 background or vice versa. These genes were expressed by transfection into murine P815-high transfection efficiency recipient cells. Serologic analysis leads to the conclusion that expression of polymorphic antigenic determinants specific for HLA-A2 (detected with M58, A2A28M1, and CR11.351 mAb) is linked to the presence of threonine residue (amino acid (AA) 142) and/or histidine residue (AA 145) and valine residue (AA 152). The expression of specific HLA-A3 polymorphic determinants (recognized by GAP-A3 mAb) is correlated with the existence of a asparagine residue (AA 127) and a aspartic residue (AA 161). But aspartic residue 161 contributes with glutamic acid residue 152 in the formation of the A3 epitope recognized by the anti-A3 mAb X1.23.2. PMID:2450922

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

    PubMed

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

    2008-01-01

    Somatostatin (SS) and growth hormone-releasing hormone (GHRH) are synthesized and secreted by the hypothalamus, which can control the synthesis and secretion of the growth hormone (GH) from the hypophysis as well as regulate the GH concentrations in animals and humans. In this article, we describe the regulation of animal growth using plasmid DNA encoding both the GHRH gene and the SS gene fused with the hepatitis B surface antigen (HBsAg) gene. We constructed a series of expression plasmids to express the GHRH and HBsAg-SS fusion genes individually as well as collectively. The fusion gene and GHRH were successfully expressed in Chinese hamster ovary (CHO) cells, as proven by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblotting tests. Poly D, L-lactide-co-glycolic acid (PLGA) plasmid-encapsulating microspheres were prepared and injected intramuscularly into the leg skeletal muscles of rabbits. Weight gain/day and the levels of insulinlike growth factor-I (IGF-I), SS, and hepatitis B surface antibody (HBsAb) were monitored. During days 30 postinjection, increase in weight gain/day and IGF- I concentration and decrease in SS were observed in treatment groups. From days 15 to 30 postinjection, the weight gain/day significantly increased (P < 0.05) by 129.13%, 106.8%, and 72.82% relative to the control group in the co-expression GHRH and fusion gene (named P-G-HS), fusion gene (named P-HS), and GHRH (named P-G) groups, respectively. And most importantly, the P-G-HS group showed significant weight gain/day (P < 0.05) relative to the P-G and P-HS groups. A significant increase in the IGF-I concentration and decrease in the SS level relative to the control group were also observed. The results indicated that the combination of plasmid-mediated GHRH supplementation and positive immunization against SS led to more robust weight gain/day in rabbits.

  17. DMA and DMB are the only genes in the class II region of the human MHC needed for class II-associated antigen processing

    SciTech Connect

    Ceman, S.; Rudersdorf, R.A.; Petersen, J.M.

    1995-03-15

    Previous studies have shown that homozygous mutations between the LMP2 and DNA loci in the human MHC cause class II molecules to be abnormally conformed and unstable in the presence of SDS at low temperature, and impede class II-associated Ag processing and presentation. These abnormalities result from impaired ability to form intracellular class II/peptide complexes that predominate in normal cells. We show in this work that this defect results from deficient expression of either the DMA or the DMB gene. Human B-LCL.174 (DR3) cells, which have a deletion of all known expressible genes in the class II region, express transgene-encoded HLA-DR3, but have the abnormalities. Transfer of cosmid HA14, which contains the DMA and DMB genes, into .174 (DR3) cells restored normal DR3 conformation, stability in 0.4% SDS at 0{degrees}, and ability to process and present tetanus toxoid, but only when both DMA and DMB mRNAs were present. The requirement for both genetic expressions in engendering normal phenotypes was confirmed by transferring the cloned genes into .174 (DR3) cells separately or together. Because normal phenotypes were fully restored in transferent cells expressing DMA plus DMB, other genes in the {approximately} 1-mb homozygous class II region deletion in .174 (DR3) cells either do not participate in or are dispensable for apparently normal production of intracellular class II/peptide complexes. The properties of DM-deficient EBV-transformed B lymphoblastoid cell lines (LCLs) suggest ways of identifying humans in whom DM deficiency contributes to congenital immunodeficiency and malignancy. 67 refs., 5 figs., 1 tab.

  18. The 5' flanking region of the gene for the Epstein-Barr virus-encoded nuclear antigen 2 contains a cell type specific cis-acting regulatory element that activates transcription in transfected B-cells.

    PubMed Central

    Ricksten, A; Olsson, A; Andersson, T; Rymo, L

    1988-01-01

    We have recently identified the promoter that positions the initiation (cap) site for RNA encoding the Epstein-Barr virus (EBV) determined nuclear antigen 2 (EBNA2) in transfected COS-1 cells. The cells were transfected with recombinant vectors that contained the BamHI WYH region of the EBV genome. In order to delineate regulatory DNA sequences required for the expression of EBNA2 the 5' flanking region of the gene was linked to reporter genes in expression vectors and transfected into EBV genome-negative lymphoid DG75 cells. We demonstrate that several cis-acting elements contribute to a transcriptional enhancer activity found in the region between nucleotides-553 and -86 relative to the cap site. The enhancer was active in lymphoid DG75 cells but not in HeLa cells and stimulated transcription also from the heterologous thymidine kinase (TK) and beta-globin promoters. Nuclear extracts of lymphoid cells contained protein factors that bound to the enhancer. The in vitro introduction of a mutation in the enhancer sequence that substantially reduced the transcription stimulatory activity concurrently blocked the binding of one of the factors. Images PMID:2843816

  19. Investigation of the genes involved in antigenic switching at the vlsE locus in Borrelia burgdorferi: an essential role for the RuvAB branch migrase.

    PubMed

    Dresser, Ashley R; Hardy, Pierre-Olivier; Chaconas, George

    2009-12-01

    Persistent infection by pathogenic organisms requires effective strategies for the defense of these organisms against the host immune response. A common strategy employed by many pathogens to escape immune recognition and clearance is to continually vary surface epitopes through recombinational shuffling of genetic information. Borrelia burgdorferi, a causative agent of Lyme borreliosis, encodes a surface-bound lipoprotein, VlsE. This protein is encoded by the vlsE locus carried at the right end of the linear plasmid lp28-1. Adjacent to the expression locus are 15 silent cassettes carrying information that is moved into the vlsE locus through segmental gene conversion events. The protein players and molecular mechanism of recombinational switching at vlsE have not been characterized. In this study, we analyzed the effect of the independent disruption of 17 genes that encode factors involved in DNA recombination, repair or replication on recombinational switching at the vlsE locus during murine infection. In Neisseria gonorrhoeae, 10 such genes have been implicated in recombinational switching at the pilE locus. Eight of these genes, including recA, are either absent from B. burgdorferi, or do not show an obvious requirement for switching at vlsE. The only genes that are required in both organisms are ruvA and ruvB, which encode subunits of a Holliday junction branch migrase. Disruption of these genes results in a dramatic decrease in vlsE recombination with a phenotype similar to that observed for lp28-1 or vls-minus spirochetes: productive infection at week 1 with clearance by day 21. In SCID mice, the persistence defect observed with ruvA and ruvB mutants was fully rescued as previously observed for vlsE-deficient B. burgdorferi. We report the requirement of the RuvAB branch migrase in recombinational switching at vlsE, the first essential factor to be identified in this process. These findings are supported by the independent work of Lin et al. in the

  20. Genetic Analysis and Detection of fliCH1 and fliCH12 Genes Coding for Serologically Closely Related Flagellar Antigens in Human and Animal Pathogenic Escherichia coli

    PubMed Central

    Beutin, Lothar; Delannoy, Sabine; Fach, Patrick

    2016-01-01

    The E. coli flagellar types H1 and H12 show a high serological cross-reactivity and molecular serotyping appears an advantageous method to establish a clear discrimination between these flagellar types. Analysis of fliCH1 and fliCH12 gene sequences showed that they were 97.5% identical at the nucleotide level. Because of this high degree of homology we developed a two-step real-time PCR detection procedure for reliable discrimination of H1 and H12 flagellar types in E. coli. In the first step, a real-time PCR assay for common detection of both fliCH1 and fliCH12 genes is used, followed in a second step by real-time PCR assays for specific detection of fliCH1 and fliCH12, respectively. The real-time PCR for common detection of fliCH1 and fliCH12 demonstrated 100% sensitivity and specificity as it reacted with all tested E. coli H1 and H12 strains and not with any of the reference strains encoding all the other 51 flagellar antigens. The fliCH1 and fliCH12 gene specific assays detected all E. coli H1 and all E. coli H12 strains, respectively (100% sensitivity). However, both assays showed cross-reactions with some flagellar type reference strains different from H1 and H12. The real-time PCR assays developed in this study can be used in combination for the detection and identification of E. coli H1 and H12 strains isolated from different sources. PMID:26913025

  1. Targeted disruption of a ring-infected erythrocyte surface antigen (RESA)-like export protein gene in Plasmodium falciparum confers stable chondroitin 4-sulfate cytoadherence capacity.

    PubMed

    Goel, Suchi; Muthusamy, Arivalagan; Miao, Jun; Cui, Liwang; Salanti, Ali; Winzeler, Elizabeth A; Gowda, D Channe

    2014-12-01

    The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family proteins mediate the adherence of infected erythrocytes to microvascular endothelia of various organs, including the placenta, thereby contributing to cerebral, placental, and other severe malaria pathogenesis. Several parasite proteins, including KAHRP and PfEMP3, play important roles in the cytoadherence by mediating the clustering of PfEMP1 in rigid knoblike structures on the infected erythrocyte surface. The lack of a subtelomeric region of chromosome 2 that contains kahrp and pfemp3 causes reduced cytoadherence. In this study, microarray transcriptome analysis showed that the absence of a gene cluster, comprising kahrp, pfemp3, and four other genes, results in the loss of parasitized erythrocytes adhering to chondroitin 4-sulfate (C4S). The role of one of these genes, PF3D7_0201600/PFB0080c, which encodes PHISTb (Plasmodium helical interspersed subtelomeric b) domain-containing RESA-like protein 1 expressed on the infected erythrocyte surface, was investigated. Disruption of PFB0080c resulted in increased var2csa transcription and VAR2CSA surface expression, leading to higher C4S-binding capacity of infected erythrocytes. Further, PFB0080c-knock-out parasites stably maintained the C4S adherence through many generations of growth. Although the majority of PFB0080c-knock-out parasites bound to C4S even after culturing for 6 months, a minor population bound to both C4S and CD36. These results strongly suggest that the loss of PFB0080c markedly compromises the var gene switching process, leading to a marked reduction in the switching rate and additional PfEMP1 expression by a minor population of parasites. PFB0080c interacts with VAR2CSA and modulates knob-associated Hsp40 expression. Thus, PFB0080c may regulate VAR2CSA expression through these processes. Overall, we conclude that PFB0080c regulates PfEMP1 expression and the parasite's cytoadherence.

  2. H-Y antigen in transsexuality, and how to explain testis differentiation in H-Y antigen-negative males and ovary differentiation in H-Y antigen-positive females.

    PubMed

    Engel, W; Pfäfflin, F; Wiedeking, C

    1980-01-01

    H-Y antigen was determined in eight transsexual patients. Two of the four male-to-female transsexual patients typed as H-Y antigen-negative, while the other two typed as expected from their phenotypic and gonadal sex, namely H-Y antigen-positive. Of the four female-to-male transsexual patients, three typed as H-Y antigen-positive and one was H-Y antigen-negative, as expected. The presence of normal testes in H-Y antigen-negative males is assumed to result from a mutation of nucleotide sequences of the H-Y structural gene for antigenic determinants. Thus, an H-Y is produced with normal receptor-binding activity which can sustain the testis determination of the bipotent gonadal anlage. In the case of H-Y antigen-positive females with normal ovaries a deletion of the autosomally located H-Y structural gene is assumed. This deletion should affect sequences for repressor-binding (as was suggested for H-Y antigen-positive XX-males) and for receptor-binding activity of the H-Y antigen molecule. The resulting H-Y antigen is unable to bind to the gonadal receptor of the bipotent gonadal anlage. Thus an ovary is determined. The relevance of H-Y antigen for the aetiology of transsexualism is discussed.

  3. In vivo imaging and quantitation of adoptively transferred human antigen-specific T cells transduced to express a human norepinephrine transporter gene.

    PubMed

    Doubrovin, Mikhail M; Doubrovina, Ekaterina S; Zanzonico, Pat; Sadelain, Michel; Larson, Steven M; O'Reilly, Richard J

    2007-12-15

    Sequential imaging of genetically marked effector cells after adoptive transfer in vivo has greatly enhanced analyses of their biodistribution, growth, and activity both in animal models and in clinical trials of cellular immunotherapy. However, the immunogenicity of cells expressing xenogeneic reporter constructs limits their survival and clinical utility. To address this limitation, we have evaluated a human norepinephrine transporter (hNET) permitting imaging of transduced cells in vivo with a previously approved clinical grade radiolabeled probe, metaiodobenzylguanidine (MIBG). The hNET gene cDNA was cloned from the SK-N-SH cell line and inserted into a bicistronic retroviral vector also encoding green fluorescent protein. Following transfection, human EBV-specific T lymphocytes seemed fully functional in vitro and also selectively accumulated [(123)I]MIBG. In nonobese diabetic/severe combined immunodeficient mice bearing human EBV lymphoma xenografts, as few as 10(4) transduced T cells injected into the tumors could be imaged by single-photon emission computed tomography (SPECT) or positron emission tomography (PET) after i.v. infusion of [(123)I]MIBG or [(124)I]MIBG, respectively. When hNET(+) EBV-specific T cells were infused i.v., their migration and specific accumulation in EBV(+) tumors expressing their restricting HLA allele could be imaged by SPECT or PET over 28 days. Image intensity was closely correlated with the number of T cells accumulated in targeted tumors. The use of two reporter probes (MIBG and 2'-deoxy-2'-fluoro-beta-d-arabinofuranosyl-5-iodouracil) permitted independent contemporaneous tracking of two distinct EBV-specific T-cell subpopulations expressing different reporter genes (hNET-CD4(+) T cells and HSV-TK-CD8(+) T cells) in the same animal using three-dimensional nuclear modalities (SPECT and PET). The hNET-based system described may thus have significant potential as a nonimmunogenic reporter for extended repeated quantitative in

  4. Idiopathic focal segmental glomerulosclerosis and HLA antigens.

    PubMed

    Gerbase-DeLima, M; Pereira-Santos, A; Sesso, R; Temin, J; Aragão, E S; Ajzen, H

    1998-03-01

    The objective of the present study was to investigate a possible association between HLA class II antigens and idiopathic focal segmental glomerulosclerosis (FSGS). HLA-A, -B, -DR and -DQ antigens were determined in 19 Brazilian patients (16 white subjects and three subjects of Japanese origin) with biopsy-proven FSGS. Comparison of the HLA antigen frequencies between white patients and white local controls showed a significant increase in HLA-DR4 frequency among FSGS patients (37.7 vs 17.2%, P < 0.05). In addition, the three patients of Japanese extraction, not included in the statistical analysis, also presented HLA-DR4. In conclusion, our data confirm the association of FSGS with HLA-DR4 previously reported by others, thus providing further evidence for a role of genes of the HLA complex in the susceptibility to this disease. PMID:9698788

  5. Sequence analysis of the Bs-Ag1 gene of Baylisascaris schroederi from the giant panda and an evaluation of the efficacy of a recombinant Baylisascaris schroederi Bs-Ag1 antigen in mice.

    PubMed

    He, Guangzhi; Chen, Sijie; Wang, Tao; Yan, Yubo; Zhang, Zhihe; Li, Desheng; Yu, Hua; Xie, Yue; Wang, Chengdong; Gu, Xiaobin; Wang, Shuxian; Peng, Xuerong; Yang, Guangyou

    2012-07-01

    The Baylisascaris schroederi infection rate among wild giant pandas may reach over 50% or even 100%, making it one of the leading causes of death from primary or secondary infection in wild populations. Until now, little was known about how protective immunity to B. schroederi infection could be achieved. The present study was conducted to evaluate the immunogenicity and protective efficacy of recombinant Bs-Ag1 from B. schroederi, by cloning the full-length Bs-Ag1 gene of B. schroederi and expressing it in a heterologous host, Escherichia coli BL21. In mice vaccinated with rBs-Ag1 coupled with Freund's complete adjuvant (FCA), there was a significant reduction (69.2%) in the recovery of challenged B. schroederi L3 compared with either nonvaccinated controls or mice vaccinated with FCA alone. Our study supports the use of Bs-Ag1 as a potential candidate for vaccination against B. schroederi infection and provides basic data for further vaccination trials with mixtures of antigens (with Bs-Ag2 and Bs-Ag3) to B. schroederi. PMID:22339267

  6. Discriminating antigen and non-antigen using proteome dissimilarity: bacterial antigens

    PubMed Central

    Ramakrishnan, Kamna; Flower, Darren R

    2010-01-01

    It has been postulated that immunogenicity results from the overall dissimilarity of pathogenic proteins versus the host proteome. We have sought to use this concept to discriminate between antigens and non-antigens of bacterial origin. Sets of 100 known antigenic and nonantigenic peptide sequences from bacteria were compared to human and mouse proteomes. Both antigenic and non-antigenic sequences lacked human or mouse homologues. Observed distributions were compared using the non-parametric Mann-Whitney test. The statistical null hypothesis was accepted, indicating that antigen and non-antigens did not differ significantly. Likewise, we were unable to determine a threshold able to separate meaningfully antigen from non-antigen. Thus, antigens cannot be predicted from pathogen genomes based solely on their dissimilarity to the human genome. PMID:20975907

  7. Presentation of hepatocellular antigens

    PubMed Central

    Grakoui, Arash; Crispe, Ian Nicholas

    2016-01-01

    The liver is an organ in which antigen-specific T-cell responses manifest a bias toward immune tolerance. This is clearly seen in the rejection of allogeneic liver transplants, and multiple other phenomena suggest that this effect is more general. These include tolerance toward antigens introduced via the portal vein, immune failure to several hepatotropic viruses, the lack of natural liver-stage immunity to malaria parasites, and the frequent metastasis of cancers to the liver. Here we review the mechanisms by which T cells engage with hepatocellular antigens, the context in which such encounters occur, and the mechanisms that act to suppress a full T-cell response. While many mechanisms play a role, we will argue that two important processes are the constraints on the cross-presentation of hepatocellular antigens, and the induction of negative feedback inhibition driven by interferons. The constant exposure of the liver to microbial products from the intestine may drive innate immunity, rendering the local environment unfavorable for specific T-cell responses through this mechanism. Nevertheless, tolerance toward hepatocellular antigens is not monolithic and under specific circumstances allows both effective immunity and immunopathology. PMID:26924525

  8. Probing the human antibody repertoire to exogenous antigens: Characterization of the H and L chain V region gene segments from anti-hepatitis B virus antibodies

    SciTech Connect

    Andris, J.S.; Capra, J.D. ); Ehrlich, P.H.; Oestberg, L. )

    1992-12-15

    Structural studies of human antibody V regions have been largely limited to those involving the fetal repertoire, autoantibodies, and malignant cell rearrangements, leaving the normal' repertoire relatively unexplored. In this study the authors describe the nucleotide sequences of the H and L chain V regions of four antibodies specific for the surface Ag of the hepatitis B virus. Monoclonal cell lines were derived from healthy individuals who received standard immunizations with the serum-derived or recombinant hepatitis B virus vaccines by fusion of PBL to a heterohybridoma cell line, SPAZ-4. They utilized the polymerase chain reaction to aimplify the H and L chain V regions for cloning and sequencing. The four antibodies express the following V region combinations: V[sub H]III/V[lambda]V, V[sub H]III/V[kappa]II, V[sub H]IV/V[kappa]I, V[sub H]V/V[lambda]III. When compared to germline genes with the closest sequence homology, all of the V regions appear to have undergone somatic mutation, ranging from 3.4 to 11.3% for the H chain, and 5.1 to 9.2% for the L chain. Analysis of the mutations shows them to be typical for an Ag-driven immune response. 50 refs., 3 figs., 2 tabs.

  9. Deep sequencing and Circos analyses of antibody libraries reveal antigen-driven selection of Ig VH genes during HIV-1 infection.

    PubMed

    Xiao, Madelyne; Prabakaran, Ponraj; Chen, Weizao; Kessing, Bailey; Dimitrov, Dimiter S

    2013-12-01

    The vast diversity of antibody repertoires is largely attributed to heavy chain (V(H)) recombination of variable (V), diversity (D) and joining (J) gene segments. We used 454 sequencing information of the variable domains of the antibody heavy chain repertoires from neonates, normal adults and an HIV-1-infected individual, to analyze, with Circos software, the VDJ pairing patterns at birth, adulthood and a time-dependent response to HIV-1 infection. Our comparative analyses of the Ig VDJ repertoires from these libraries indicated that, from birth to adulthood, VDJ recombination patterns remain the same with some slight changes, whereas some V(H) families are selected and preferentially expressed after long-term infection with HIV-1. We also demonstrated that the immune system responds to HIV-1 chronic infection by selectively expanding certain HV families in an attempt to combat infection. Our findings may have implications for understanding immune responses in pathology as well as for development of new therapeutics and vaccines.

  10. Fungal Antigens Expressed during Invasive Aspergillosis

    PubMed Central

    Denikus, Nicole; Orfaniotou, Foteini; Wulf, Gerald; Lehmann, Paul F.; Monod, Michel; Reichard, Utz

    2005-01-01

    Rabbits that had been infected intravenously with conidiospores of Aspergillus fumigatus were used as sources of antibody for screening a λ phage cDNA expression library. The cDNA was derived from A. fumigatus mRNA that had been extracted from newly formed, germling hyphae. Thirty-six antigens were identified using antisera from six rabbits. Though many of these antigens were expected to be intracellular proteins because their genes did not encode a signal sequence, the antisera showed consistently a stronger immunoblot reaction with a cell fraction enriched for the fungal cell wall than with a fraction of predominantly intracellular components. Antibodies to eight antigens, including the glycosylhydrolase Asp f 16, were produced by more than one rabbit. In current vaccine studies, Asp f 16 is the only single antigen which has been reported to be capable of inducing protection against invasive aspergillosis in mice (S. Bozza et al., Microb. Infect. 4:1281-1290, 2002). Enolase and Aspergillus HSP90 were detected also; their homologues in Candida albicans have been tested as vaccines and have been reported to provide a partially protective response against invasive candidiasis in mice. The Aspergillus antigens reported here may have value both in diagnostic tests for different forms of aspergillosis and as vaccine candidates for protection against invasive disease. PMID:16040983

  11. Five novel cell surface antigens of CNS neoplasms.

    PubMed

    Jennings, M T; Jennings, V D; Asadourian, L L; Rosenblum, M; Albino, A P; Cairncross, J G; Old, L J

    1989-01-01

    Optimal monoclonal antibody-mediated immunotherapy requires the identification of tumor-restricted cell surface antigens. We have identified and partially characterized 5 new monoclonal antibodies generated against malignant astrocytoma, medulloblastoma, neuroblastoma and melanoma which were used to define 5 neuroectodermal tumor antigenic systems. CNT/1 identifies a 57-kDa, heat-stable, trypsin-sensitive neuroblastoma surface antigen, which is expressed intracellularly in many malignant gliomas, medulloblastomas, ependymomas, breast and ovarian carcinomas. CNT/2 reacts with a 130-kDa, heat-labile, trypsin- and neuraminidase-resistant antigen restricted to low-grade astrocytomas and malignant gliomas. CNT/11 reacts with a 70-kDa, heat-labile, trypsin-sensitive antigen coded for by a gene on chromosome 12, and is restricted to astrocytomas, neuroblastomas and sarcomas. CNT/8 identifies a heat-labile, trypsin-sensitive antigen whose gene has been localized to chromosome 15 and is expressed by neuroectodermal and mesodermally derived tumors and few epithelial cancers. The B2.6 antigen is identified only in terms of serologic reactivity with a subset of cultured astrocytomas and melanomas. Neuroectodermal tumor-associated antigens may be categorized as lineage-consistent, lineage-independent and putatively tumor-restricted in their expression. These restricted antibodies may be potentially useful reagents to consider for monoclonal antibody-mediated immunotherapy of CNS neoplasms.

  12. Genetic regulation of variable Vi antigen expression in a strain of Citrobacter freundii.

    PubMed

    Snellings, N J; Johnson, E M; Kopecko, D J; Collins, H H; Baron, L S

    1981-02-01

    Certain strains of the genus Citrobacter exhibit a variable expression of the Vi surface antigen that appears to involve a special mechanism for regulation of gene expression. Two nonlinked chromosomal loci, viaA and viaB, are known to determine nonvariable Vi antigen expression in strains of Salmonella. To confirm the presence of analogous loci in Citrobacter and to ascertain whether either of them is involved in variable Vi antigen expression in this organism, donor strains were constructed from Citrobacter freundii WR7004 and used to transfer their Vi antigen-determining genes to ViaA- and ViaB- Salmonella typhi recipient strains. Vi antigen expression in C. freundii was found to be controlled by loci analogous to the Salmonella via genes. S. typhi recipients of the C. freundii viaA+ genes were restored to the full, continuous expression of the Vi antigen normally seen in S. typhi. Thus, the C. freundii viaA genes appeared to play no role in the variable expression of the Vi antigen. In contrast, S. typhi recipients of the C. freundii viaB+ genes exhibited the rapid, reversible alternation between full Vi antigen expression and markedly reduced Vi antigen expression that was seen to occur in the C. freundii parent. The C. freundii viaB locus was thus identified as the one whose genes are regulated so as to produce variable Vi antigen expression. Genes determining another C. freundii surface antigen, the synthesis of which is not affected by the mechanism regulating Vi expression, were coinherited with the C. freundii viaB+ genes. An invertible, insertion sequence element located within the C. freundii viaB locus is proposed to account for the regulation of variable Vi antigen expression.

  13. Pathways of Antigen Processing

    PubMed Central

    Blum, Janice S.; Wearsch, Pamela A.; Cresswell, Peter

    2014-01-01

    T cell recognition of antigen presenting cells depends on their expression of a spectrum of peptides bound to Major Histocompatibility Complex class I (MHC-I) and class II (MHC-II) molecules. Conversion of antigens from pathogens or transformed cells into MHC-I and MHC-II-bound peptides is critical for mounting protective T cell responses, and similar processing of self proteins is necessary to establish and maintain tolerance. Cells use a variety of mechanisms to acquire protein antigens, from translation in the cytosol to variations on the theme of endocytosis, and to degrade them once acquired. In this review we highlight the aspects of MHC-I and MHC-II biosynthesis and assembly that have evolved to intersect these pathways and sample the peptides that are produced. PMID:23298205

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

    SciTech Connect

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

    1984-08-01

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

  15. Antigen detection systems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Infectious agents or their constituent parts (antigens or nucleic acids) can be detected in fresh, frozen, or fixed tissues or other specimens, using a variety of direct or indirect assays. The assays can be modified to yield the greatest sensitivity and specificity but in most cases a particular m...

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

    PubMed

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

    2016-10-01

    We examined whether aqueous extract of Lepidium meyenii (red Maca) could inhibit growth, potentiate apoptotic activity of two anticancer drugs Taxol and 2-methoxyestradiol (2ME) or change mRNA expression for the androgen target genes, androgen receptor (Ar) and prostate-specific antigen (Psa) in the human prostate cancer cell line LNCaP. Red Maca aqueous extract at 0, 10, 20, 40 or 80 μg/ml was added to LNCaP cells, and viability was evaluated by the MTS assay at 24 or 48 hr after treatment. Furthermore, LNCaP cells were treated with 80 μg/ml of red Maca plus Taxol or 2ME 5 μM and viability was assessed 48 hr later. Finally, LNCaP cells were treated with red Maca 0, 20, 40 or 80 μg/ml, and 12 hr later, mRNA level for Ar or Psa was assessed by real-time PCR. Treatment with red Maca did not affect viability of LNCaP cells. Apoptotic activity induced by Taxol and 2ME in LNCaP cells was not altered with red Maca treatment. Relative expression of the mRNA for Ar and Psa increased with red Maca 20 and 40 μg/ml, but not at 80 μg/ml. We conclude that red Maca aqueous extract does not have toxic effects, but stimulates androgen signalling in LNCaP cells. PMID:27681649

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

    PubMed

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

    2016-10-01

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

  18. Effect of yeast-derived products and distillers dried grains with solubles (DDGS) on antibody-mediated immune response and gene expression of pattern recognition receptors and cytokines in broiler chickens immunized with T-cell dependent antigens.

    PubMed

    Alizadeh, M; Rodriguez-Lecompte, J C; Echeverry, H; Crow, G H; Slominski, B A

    2016-04-01

    This study evaluated the effect of yeast-derived products on innate and antibody mediated immune response in broiler chickens following immunization with sheep red blood cells (SRBC) and bovine serum albumin (BSA). One-day-old male broiler chickens (Ross-308) were randomly assigned to 6 dietary treatments of 9 replicate cages of 5 birds each per treatment. Dietary treatments consisted of a Control diet without antibiotic, and diets containing 11 mg/kg of virginiamycin, 0.25% of yeast cell wall (YCW), 0.2% of a commercial product Maxi-Gen Plus containing processed yeast and nucleotides, 0.05% of nucleotides, or a diet containing 10% of DDGS. On days 21 and 28 post-hatching, 5 birds per treatment were immunized intramuscularly with both SRBC and BSA. One week after each immunization, blood samples were collected. Serum samples were analyzed by hemagglutination test for antibody response to SRBC, and by ELISA for serum IgM and IgG response to BSA. On d 35, 5 birds per treatment were euthanized and the tissue samples from the cecal tonsils were collected to assess the gene expression of toll-like receptors TLR2b, TLR4, and TLR21, monocyte mannose receptor (MMR), and cytokines IL-10, IL-13, IL-4, IL-12p35, and IFN-γ. The results for gene expression analysis demonstrated that the diet supplemented with YCW increased the expression of TLR2b and T-helper type 2 cytokines IL-10, IL-4, and IL-13 relative to the Control; and the expression of TLR4 and IL-13 was upregulated in the nucleotide-containing diet. However, the diets containing antibiotics or Maxi-Gen Plus downregulated the expression of IFN-γ compared to the control. The primary antibody response to SRBC was not affected by diets. However, the diet containing YCW increased the secondary antibody response to SRBC compared to the antibiotic treatment. Neither primary nor secondary IgG and IgM response against BSA were affected by diets. In conclusion, supplementation of the diet with YCW stimulated Th2 cell

  19. Cloning of a developmentally regulated tegument antigen of Schistosoma mansoni.

    PubMed

    Stein, L D; David, J R

    1986-09-01

    We have cloned a gene encoding a 22.6 kDa antigen from a Schistosoma mansoni cDNA library. Northern blots indicate that transcription of this antigen occurs in adults and sporocysts but not in cercariae, eggs or in newly-transformed schistosomula. Immunoprecipitation and Western blotting with specific antisera indicate that the antigen is not detectable in the newly transformed schistosomulum but appears within 24 h of schistosomulum transformation. Indirect immunofluorescence of adult worms shows this protein to be located in the tegument.

  20. Expression of Plasmodium falciparum surface antigens in Escherichia coli.

    PubMed Central

    Ardeshir, F; Flint, J E; Reese, R T

    1985-01-01

    The asexual blood stages of the human malarial parasite Plasmodium falciparum produce many antigens, only some of which are important for protective immunity. Most of the putative protective antigens are believed to be expressed in schizonts and merozoites, the late stages of the asexual cycle. With the aim of cloning and characterizing genes for important parasite antigens, we used late-stage P. falciparum mRNA to construct a library of cDNA sequences inserted in the Escherichia coli expression vector pUC8. Nine thousand clones from the expression library were immunologically screened in situ with serum from Aotus monkeys immune to P. falciparum, and 95 clones expressing parasite antigens were identified. Mice were immunized with lysates from 49 of the bacterial clones that reacted with Aotus sera, and the mouse sera were tested for their reactivity with parasite antigens by indirect immunofluorescence, immunoprecipitation, and immunoblotting assays. Several different P. falciparum antigens were identified by these assays. Indirect immunofluorescence studies of extracellular merozoites showed that three of these antigens appear to be located on the merozoite surface. Thus, we have identified cDNA clones to three different P. falciparum antigens that may be important in protective immunity. Images PMID:3887406

  1. Cancer vaccine--Antigenics.

    PubMed

    2002-01-01

    Antigenics is developing a therapeutic cancer vaccine based on heat-shock proteins (HSPs). The vaccine [HSPPC-96, Oncophage] is in a pivotal phase III clinical trial for renal cancer at 80 clinical sites worldwide. The trial is enrolling at least 500 patients who are randomised to receive surgical removal of the primary tumour followed by out-patient treatment with Oncophage((R)) or surgery only. This study was initiated on the basis of results from a pilot phase I/II study and preliminary results from a phase II study in patients with renal cell cancer. In October 2001, Oncophage was designated as a fast-track product by the Food and Drug Administration in the US for the treatment of renal cell carcinoma. Oncophage is in phase I/II trials in Italy for colorectal cancer (30 patients) and melanoma. The trials in Italy are being conducted at the Istituto dei Tumouri, Milan (in association with Sigma-Tau). Preliminary data from the phase II trial for melanoma was presented at the AACR-NCI-EORTC International Conference in Florida, USA, in October 2001. Oncophage is also in a phase I/II (42 patients) and a phase II trial (84 patients) in the US for renal cell cancer, a phase II trial in the US for non-Hodgkin's lymphoma (35 patients), a phase II trial in the US for sarcoma (20-35 patients), a phase I/II trial in the US for melanoma (36 patients), and phase I/II trials in Germany for gastric (30 patients) and pancreatic cancers. A pilot phase I trial in patients with pancreatic cancer began in the US in 1997 with 5 patients enrolled. In November 2000, Antigenics announced that this trial had been expanded to a phase I/II study which would now include survival as an endpoint and would enroll 5 additional patients. The US trials are being performed at Memorial Sloan-Kettering Cancer Center and the M.D. Anderson Cancer Center. The trials in Germany are being carried out at Johannes Gutenberg-University Hospital, Mainz. Oncophage is an autologous vaccine consisting of

  2. Antigens and allergic asthma

    SciTech Connect

    Reed, C.E.; Swanson, M.C.

    1987-06-01

    There are few reliable epidemiologic data on the overall frequency and importance of allergy. We describe a practical method for quantifying the concentration of both amorphous and morphologically defined antigens in the air. A high volume air sampler is used to collect airborne particles and has a facility to separate samples into different particle sizes. Samples are tested for allergenic activity by radioallergosorbent test inhibition assay. Preliminary findings from studies of community wide, amorphous and common household allergens are reported.

  3. Beyond model antigens: high-dimensional methods for the analysis of antigen-specific T cells

    PubMed Central

    Newell, Evan W.; Davis, Mark M.

    2014-01-01

    Adaptive immune responses often begin with the formation of a molecular complex between a T cell receptor (TCR) and a peptide antigen bound to a major histocompatibility complex (MHC) molecule. These complexes are highly variable, however, due to the polymorphism of MHC genes, the random, inexact recombination of TCR gene segments and the vast array of possible self and pathogen peptide antigens. As a result, it has been very difficult to comprehensively study the TCR repertoire or identify and track more than a few antigen-specific T cells in mice or humans. For mouse studies, this had led to a reliance on model antigens and TCR transgenes. The study of limited human clinical samples, in contrast, requires techniques that can simultaneously survey phenotype, function and reactivity to many T cell epitopes. Thanks to recent advances in single-cell and cytometry methodologies, as well as high-throughput sequencing of the TCR repertoire, we now have or will soon have the tools needed to comprehensively analyze T-cell responses during health and disease. PMID:24441473

  4. Antigenic Relationships among Human Pathogenic Orientia tsutsugamushi Isolates from Thailand

    PubMed Central

    Nawtaisong, Pruksa; Tanganuchitcharnchai, Ampai; Smith, Derek J.; Day, Nicholas P. J.; Paris, Daniel H.

    2016-01-01

    Background Scrub typhus is a common cause of undiagnosed febrile illness in certain tropical regions, but can be easily treated with antibiotics. The causative agent, Orientia tsutsugamushi, is antigenically variable which complicates diagnosis and efforts towards vaccine development. Methodology/Principal Findings This study aimed to dissect the antigenic and genetic relatedness of O. tsutsugamushi strains and investigate sero-diagnostic reactivities by titrating individual patient sera against their O. tsutsugamushi isolates (whole-cell antigen preparation), in homologous and heterologous serum-isolate pairs from the same endemic region in NE Thailand. The indirect immunofluorescence assay was used to titrate Orientia tsutsugamushi isolates and human sera, and a mathematical technique, antigenic cartography, was applied to these data to visualise the antigenic differences and cross-reactivity between strains and sera. No functional or antigen-specific analyses were performed. The antigenic variation found in clinical isolates was much less pronounced than the genetic differences found in the 56kDa type-specific antigen genes. The Karp-like sera were more broadly reactive than the Gilliam-like sera. Conclusions/Significance Antigenic cartography worked well with scrub typhus indirect immunofluorescence titres. The data from humoral responses suggest that a Karp-like strain would provide broader antibody cross-reactivity than a Gilliam-like strain. Although previous exposure to O. tsutsugamushi could not be ruled out, scrub typhus patient serum antibody responses were characterised by strong homologous, but weak heterologous antibody titres, with little evidence for cross-reactivity by Gilliam-like sera, but a broader response from some Karp-like sera. This work highlights the importance of antigenic variation in O. tsutsugamushi diagnosis and determination of new serotypes. PMID:27248711

  5. Soy milk digestion extract inhibits progression of prostate cancer cell growth via regulation of prostate cancer‑specific antigen and cell cycle-regulatory genes in human LNCaP cancer cells.

    PubMed

    Kang, Nam-Hee; Shin, Hee-Chang; Oh, Seunghyun; Lee, Kyun-Hee; Lee, Yoon-Bok; Choi, Kyung-Chul

    2016-08-01

    Soy milk, which is produced from whole soybeans, contains a variety of biologically active components. Isoflavones are a class of soy-derived phytoestrogens with beneficial effects, among which genistein (GEN) has been previously indicated to reduce the risk of prostate cancer. The present study evaluated the effects of soy milk digestion extract (SMD) on the progression of prostate cancer via the estrogen receptor (ER)β in human LNCaP prostate cancer cells. To evaluate the effects of SMD (daizein, 1.988 mg/100g, glycitein, 23.537 mg/100 g and GEN, 0.685 mg/100g) on cell proliferation, LNCaP cells were cultured in media containing vehicle (0.1% dimethyl sulfoxide), 17β‑estradiol (E2; 2.7x10‑7 mg/ml), GEN (2.7x10-2 mg/ml) of SMD (total aglycon concentration, 0.79 mg/ml), after which the cell viability was examined using an MTT assay. The cell viability was significantly elevated by E2 (by 45±0.18%), while it was markedly reduced by GEN (73.2±0.03%) or SMD (74.8±0.09%). Semi‑quantitative reverse transcription polymerase chain reaction analysis was performed to assess the mRNA expression levels of target genes, including ERβ, prostate cancer‑specific antigen (PSA) and cell cycle regulators p21, Cyclin D1 and cyclin-dependent kinase (CDK)4. The expression of ERβ was almost completely diminished by E2, whereas it was significantly elevated by SMD. In addition, the expression levels of PSA were considerably reduced by SMD. The expression of p21 was significantly elevated by SMD, while it was markedly reduced by E2. Of note, the expression levels of Cyclin D1 and CDK4 were considerably elevated by E2, while being significantly reduced by GEN and SMD. All of these results indicated that SMD may inhibit the proliferation of human prostate cancer cells via regulating the expression of ERβ, PSA, p21, Cyclin D1 and CDK4 in an ER-dependent manner.

  6. DNA Double-Strand Breaks and Telomeres Play Important Roles in Trypanosoma brucei Antigenic Variation

    PubMed Central

    2015-01-01

    Human-infecting microbial pathogens all face a serious problem of elimination by the host immune response. Antigenic variation is an effective immune evasion mechanism where the pathogen regularly switches its major surface antigen. In many cases, the major surface antigen is encoded by genes from the same gene family, and its expression is strictly monoallelic. Among pathogens that undergo antigenic variation, Trypanosoma brucei (a kinetoplastid), which causes human African trypanosomiasis, Plasmodium falciparum (an apicomplexan), which causes malaria, Pneumocystis jirovecii (a fungus), which causes pneumonia, and Borrelia burgdorferi (a bacterium), which causes Lyme disease, also express their major surface antigens from loci next to the telomere. Except for Plasmodium, DNA recombination-mediated gene conversion is a major pathway for surface antigen switching in these pathogens. In the last decade, more sophisticated molecular and genetic tools have been developed in T. brucei, and our knowledge of functions of DNA recombination in antigenic variation has been greatly advanced. VSG is the major surface antigen in T. brucei. In subtelomeric VSG expression sites (ESs), VSG genes invariably are flanked by a long stretch of upstream 70-bp repeats. Recent studies have shown that DNA double-strand breaks (DSBs), particularly those in 70-bp repeats in the active ES, are a natural potent trigger for antigenic variation in T. brucei. In addition, telomere proteins can influence VSG switching by reducing the DSB amount at subtelomeric regions. These findings will be summarized and their implications will be discussed in this review. PMID:25576484

  7. Genetics of O-Antigen Biosynthesis in Pseudomonas aeruginosa

    PubMed Central

    Rocchetta, H. L.; Burrows, L. L.; Lam, J. S.

    1999-01-01

    Pathogenic bacteria produce an elaborate assortment of extracellular and cell-associated bacterial products that enable colonization and establishment of infection within a host. Lipopolysaccharide (LPS) molecules are cell surface factors that are typically known for their protective role against serum-mediated lysis and their endotoxic properties. The most heterogeneous portion of LPS is the O antigen or O polysaccharide, and it is this region which confers serum resistance to the organism. Pseudomonas aeruginosa is capable of concomitantly synthesizing two types of LPS referred to as A band and B band. The A-band LPS contains a conserved O polysaccharide region composed of d-rhamnose (homopolymer), while the B-band O-antigen (heteropolymer) structure varies among the 20 O serotypes of P. aeruginosa. The genes coding for the enzymes that direct the synthesis of these two O antigens are organized into two separate clusters situated at different chromosomal locations. In this review, we summarize the organization of these two gene clusters to discuss how A-band and B-band O antigens are synthesized and assembled by dedicated enzymes. Examples of unique proteins required for both A-band and B-band O-antigen synthesis and for the synthesis of both LPS and alginate are discussed. The recent identification of additional genes within the P. aeruginosa genome that are homologous to those in the A-band and B-band gene clusters are intriguing since some are able to influence O-antigen synthesis. These studies demonstrate that P. aeruginosa represents a unique model system, allowing studies of heteropolymeric and homopolymeric O-antigen synthesis, as well as permitting an examination of the interrelationship of the synthesis of LPS molecules and other virulence determinants. PMID:10477307

  8. T Cells as Antigen Carriers for Anti-tumor Vaccination.

    PubMed

    Traversari, Catia; Russo, Vincenzo

    2016-01-01

    The exploitation of the physiologic processing and presenting machinery of dendritic cells (DCs) by in vivo loading of tumor-associated antigens may improve the immunogenic potential and clinical efficacy of DC-based cancer vaccines. The approach developed by our group was based on the clinical observation that some patients treated with the infusion of donor lymphocytes transduced to express the HSV-TK suicide gene for relapse of hematologic malignancies, after allogeneic hematopoietic stem cell transplantation, developed a T cell-mediated immune response specifically directed against the HSV-TK gene product.We demonstrated that lymphocytes genetically modified to express HSV-TK as well as self/tumor antigens, acting as antigen carriers, efficiently target DCs in vivo in tumor-bearing mice. The infusion of TRP-2-transduced lymphocytes induced the establishment of protective immunity and long-term memory in tumor-bearing mice by cross-presentation of the antigen mediated by the CD11c(+)CD8a(+) DCs subset. A similar approach was applied in a clinical setting. Ten patients affected by MAGE-3(+) metastatic melanoma were treated with autologous lymphocytes retrovirally transduced to express the MAGE-3 tumor antigen. In three patients, the treatment led to the increase of MAGE-3 specific CD8+ and CD4+ effectors and the development of long-term memory, which ultimately correlated with a favorable clinical outcome. Transduced lymphocytes represent an efficient way for in vivo loading of tumor-associated antigens of DCs.

  9. Microbial antigenic variation mediated by homologous DNA recombination

    PubMed Central

    Vink, Cornelis; Rudenko, Gloria; Seifert, H. Steven

    2012-01-01

    Pathogenic microorganisms employ numerous molecular strategies in order to delay or circumvent recognition by the immune system of their host. One of the most widely used strategies of immune evasion is antigenic variation, in which immunogenic molecules expressed on the surface of a microorganism are continuously modified. As a consequence, the host is forced to constantly adapt its humoral immune response against this pathogen. An antigenic change thus provides the microorganism with an opportunity to persist and/or replicate within the host (population) for an extended period of time or to effectively infect a previously infected host. In most cases, antigenic variation is caused by genetic processes that lead to modification of the amino acid sequence of a particular antigen or to alterations in the expression of biosynthesis genes that induce changes in expression of a variant antigen. Here, we will review antigenic variation systems that rely on homologous DNA recombination and which are found in a wide range of cellular, human pathogens, including bacteria (such as Neisseria spp., Borrelia spp., Treponema pallidum and Mycoplasma spp.), fungi (like Pneumocystis carinii) and parasites (such as the African trypanosome Trypanosoma brucei). Specifically, the various DNA recombination-based antigenic variation systems will be discussed with a focus on the employed mechanisms of recombination, the DNA substrates, and the enzymatic machinery involved. PMID:22212019

  10. Polyethyleneimine is a potent systemic adjuvant for glycoprotein antigens.

    PubMed

    Sheppard, Neil C; Brinckmann, Sarah A; Gartlan, Kate H; Puthia, Manoj; Svanborg, Catharina; Krashias, George; Eisenbarth, Stephanie C; Flavell, Richard A; Sattentau, Quentin J; Wegmann, Frank

    2014-10-01

    Polyethyleneimine (PEI) is an organic polycation used extensively as a gene and DNA vaccine delivery reagent. Although the DNA targeting activity of PEI is well documented, its immune activating activity is not. We recently reported that PEI has robust mucosal adjuvanticity when administered intranasally with glycoprotein antigens. Here, we show that PEI has strong immune activating activity after systemic delivery. PEI administered subcutaneously with viral glycoprotein (HIV-1 gp140) enhanced antigen-specific serum IgG production in the context of mixed Th1/Th2-type immunity. PEI elicited higher titers of both antigen binding and neutralizing antibodies than alum in mice and rabbits and induced an increased proportion of antibodies reactive with native antigen. In an intraperitoneal model, PEI recruited neutrophils followed by monocytes to the site of administration and enhanced antigen uptake by antigen-presenting cells. The Th bias was modulated by PEI activation of the Nlrp3 inflammasome; however its global adjuvanticity was unchanged in Nlrp3-deficient mice. When coformulated with CpG oligodeoxynucleotides, PEI adjuvant potency was synergistically increased and biased toward a Th1-type immune profile. Taken together, these data support the use of PEI as a versatile systemic adjuvant platform with particular utility for induction of secondary structure-reactive antibodies against glycoprotein antigens. PMID:24844701

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

    PubMed Central

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

    2016-01-01

    Mass spectrometry-based phenotypic H-antigen typing (MS-H) combined with whole-genome-sequencing-based genetic identification of H antigens, O antigens, and toxins (WGS-HOT) was used to type 60 clinical Escherichia coli isolates, 43 of which were previously identified as nonmotile, H type undetermined, or O rough by serotyping or having shown discordant MS-H and serotyping results. Whole-genome sequencing confirmed that MS-H was able to provide more accurate data regarding H antigen expression than serotyping. Further, enhanced and more confident O antigen identification resulted from gene cluster based typing in combination with conventional typing based on the gene pair comprising wzx and wzy and that comprising wzm and wzt. The O antigen was identified in 94.6% of the isolates when the two genetic O typing approaches (gene pair and gene cluster) were used in conjunction, in comparison to 78.6% when the gene pair database was used alone. In addition, 98.2% of the isolates showed the existence of genes for various toxins and/or virulence factors, among which verotoxins (Shiga toxin 1 and/or Shiga toxin 2) were 100% concordant with conventional PCR based testing results. With more applications of mass spectrometry and whole-genome sequencing in clinical microbiology laboratories, this combined phenotypic and genetic typing platform (MS-H plus WGS-HOT) should be ideal for pathogenic E. coli typing. PMID:27307455

  12. Antigenic variation in ciliates: antigen structure, function, expression.

    PubMed

    Simon, Martin C; Schmidt, Helmut J

    2007-01-01

    In the past decades, the major focus of antigen variation research has been on parasitic protists. However, antigenic variation occurs also in free-living protists. The antigenic systems of the ciliates Paramecium and Tetrahymena have been studied for more than 100 yr. In spite of different life strategies and distant phylogenetic relationships of free-living ciliates and parasitic protists, their antigenic systems have features in common, such as the presence of repeated protein motifs and multigene families. The function of variable surface antigens in free-living ciliates is still unknown. Up to now no detailed monitoring of antigen expression in free-living ciliates in natural habitats has been performed. Unlike stochastic switching in parasites, antigen expression in ciliates can be directed, e.g. by temperature, which holds great advantages for research on the expression mechanism. Regulated expression of surface antigens occurs in an exclusive way and the responsible mechanism is complex, involving both transcriptional and post-transcriptional features. The involvement of homology-dependent effects has been proposed several times but has not been proved yet.

  13. Small, clonally variant antigens expressed on the surface of the Plasmodium falciparum-infected erythrocyte are encoded by the rif gene family and are the target of human immune responses.

    PubMed

    Fernandez, V; Hommel, M; Chen, Q; Hagblom, P; Wahlgren, M

    1999-11-15

    Disease severity in Plasmodium falciparum infections is a direct consequence of the parasite's efficient evasion of the defense mechanisms of the human host. To date, one parasite-derived molecule, the antigenically variant adhesin P. falciparum erythrocyte membrane protein 1 (PfEMP1), is known to be transported to the infected erythrocyte (pRBC) surface, where it mediates binding to different host receptors. Here we report that multiple additional proteins are expressed by the parasite at the pRBC surface, including a large cluster of clonally variant antigens of 30-45 kD. We have found these antigens to be identical to the rifins, predicted polypeptides encoded by the rif multigene family. These parasite products, formerly called rosettins after their identification in rosetting parasites, are prominently expressed by fresh isolates of P. falciparum. Rifins are immunogenic in natural infections and strain-specifically recognized by human immune sera in immunoprecipitation of surface-labeled pRBC extracts. Furthermore, human immune sera agglutinate pRBCs digested with trypsin at conditions such that radioiodinated PfEMP1 polypeptides are not detected but rifins are detected, suggesting the presence of epitopes in rifins targeted by agglutinating antibodies. When analyzed by two-dimensional electrophoresis, the rifins resolved into several isoforms in the pI range of 5.5-6.5, indicating molecular microheterogeneity, an additional potential novel source of antigenic diversity in P. falciparum. Prominent polypeptides of 20, 22, 76-80, 140, and 170 kD were also detected on the surfaces of pRBCs bearing in vitro-propagated or field-isolated parasites. In this report, we describe the rifins, the second family of clonally variant antigens known to be displayed by P. falciparum on the surface of the infected erythrocyte.

  14. Novel antigen delivery systems

    PubMed Central

    Trovato, Maria; Berardinis, Piergiuseppe De

    2015-01-01

    Vaccines represent the most relevant contribution of immunology to human health. However, despite the remarkable success achieved in the past years, many vaccines are still missing in order to fight important human pathologies and to prevent emerging and re-emerging diseases. For these pathogens the known strategies for making vaccines have been unsuccessful and thus, new avenues should be investigated to overcome the failure of clinical trials and other important issues including safety concerns related to live vaccines or viral vectors, the weak immunogenicity of subunit vaccines and side effects associated with the use of adjuvants. A major hurdle of developing successful and effective vaccines is to design antigen delivery systems in such a way that optimizes antigen presentation and induces broad protective immune responses. Recent advances in vector delivery technologies, immunology, vaccinology and system biology, have led to a deeper understanding of the molecular and cellular mechanisms by which vaccines should stimulate both arms of the adaptive immune responses, offering new strategies of vaccinations. This review is an update of current strategies with respect to live attenuated and inactivated vaccines, DNA vaccines, viral vectors, lipid-based carrier systems such as liposomes and virosomes as well as polymeric nanoparticle vaccines and virus-like particles. In addition, this article will describe our work on a versatile and immunogenic delivery system which we have studied in the past decade and which is derived from a non-pathogenic prokaryotic organism: the “E2 scaffold” of the pyruvate dehydrogenase complex from Geobacillus stearothermophilus. PMID:26279977

  15. Stool Test: H. Pylori Antigen

    MedlinePlus

    ... Things to Know About Zika & Pregnancy Stool Test: H. Pylori Antigen KidsHealth > For Parents > Stool Test: H. Pylori Antigen Print A A A Text Size ... en español Muestra de materia fecal: antígeno de H. pylori What It Is Helicobacter pylori ( H. pylori ) ...

  16. Radioimmunoassays of hidden viral antigens

    SciTech Connect

    Neurath, A.R.; Strick, N.; Baker, L.; Krugman, S.

    1982-07-01

    Antigens corresponding to infectious agents may be present in biological specimens only in a cryptic form bound to antibodies and, thus, may elude detection. We describe a solid-phase technique for separation of antigens from antibodies. Immune complexes are precipitated from serum by polyethylene glycol, dissociated with NaSCN, and adsorbed onto nitrocellulose or polystyrene supports. Antigens remain topographically separated from antibodies after removal of NaSCN and can be detected with radiolabeled antibodies. Genomes from viruses immobilized on nitrocellulose can be identified by nucleic acid hybridization. Nanogram quantities of sequestered hepatitis B surface and core antigens and picogram amounts of hepatitis B virus DNA were detected. Antibody-bound adenovirus, herpesvirus, and measles virus antigens were discerned by the procedure.

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

    PubMed

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

    2014-09-01

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

  18. A surface antigen influenza vaccine. 2. Pyrogenicity and antigenicity.

    PubMed Central

    Brady, M. I.; Furminger, I. G.

    1976-01-01

    Conventional influenza vaccine containing whole virus particles purified on a zonal centrifuge is pyrogenic and can cause systemic and local adverse side effects. An improved vaccine was therefore prepared which contained only the surface antigens of the virus adsorbed to aluminium hydroxide. The antigenicity of this vaccine was compared with conventional vaccine in chickens. Both vaccines induced similar titres of serum haemagglutination-inhibition and neuraminidase inhibition antibody. The dose response curves, however, were different. The surface antigens at vaccine strength without aluminium hydroxide were of negligible pyrogenicity in rabbits. PMID:1068196

  19. [Antigenic response against PPD and antigen 60 in tubercular patients: single antigen versus the combined test].

    PubMed

    Máttar, S; Broquetas, J M; Gea, J; Aran, X; el-Banna, N; Sauleda, J; Torres, J M

    1992-05-01

    We analyze serum samples from 70 patients with pulmonary tuberculosis and 50 healthy individuals. The antigenic activity (IgG) against protein purified antigen (PPD) and antigen 60 (A60) from M. tuberculosis. Thirteen patients were also HIV infected, and three patients had AIDS defined by the presence of disseminated tuberculosis. The test using antigen alone showed a 77% sensitivity and 74% specificity when PPD is used. When A60 was used, both values improved (81% sensitivity, 94% specificity). The use of a combined test (PPD and A60) improves the sensitivity (89%) but reduces the specificity (82%). The HIV infected patients showed similar responses to those of other patients. The combined use of different antigens might be useful for diagnosing tuberculosis. PMID:1390996

  20. Antigen processing and presentation: Evolution from a bird's eye view☆

    PubMed Central

    Kaufman, Jim

    2013-01-01

    Most detailed knowledge of the MHC outside of mammals has come from studies of chickens, originally due to the economic importance of the poultry industry. We have used our discoveries about the chicken MHC to develop a framework for understanding the evolution of the MHC, based on the importance of genomic organisation for gene co-evolution. In humans, MHC class I molecules are polymorphic and determine the specificity of peptide presentation, while the molecules involved in antigen processing are functionally monomorphic. The genes for tapasin, transporters associated with antigen presentation (TAPs) and inducible proteasome components (LMPs) are located in and beyond the class II region, far away from the class I genes in the class I region. In contrast, chickens express only one class I locus at high levels, which can result in strong MHC associations with resistance to particular infectious pathogens. The chicken TAP and tapasin genes are located very close to the class I genes, and have high levels of allelic polymorphism and moderate sequence diversity, co-evolving their specificities to work optimally with the dominantly expressed class I molecule. The salient features of the chicken MHC are found in many if not most non-mammalian species examined, and are likely to represent the ancestral organisation of the MHC. Comparison with the MHC organisation of humans and typical mammals suggests that a large inversion brought the class III region into the middle of the MHC, separating the antigen processing genes from the class I gene, breaking the co-evolutionary relationships and allowing a multigene family of well-expressed class I genes. Such co-evolution in the primordial MHC was likely responsible for the appearance of the antigen presentation pathways and receptor–ligand interactions at the birth of the adaptive immune system. Of course, much further work is required to understand this evolutionary framework in more detail. PMID:23182425

  1. Serospecific antigens of Legionella pneumophila.

    PubMed Central

    Otten, S; Iyer, S; Johnson, W; Montgomery, R

    1986-01-01

    Serospecific antigens isolated by EDTA extraction from four serogroups of Legionella pneumophila were analyzed for their chemical composition, molecular heterogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunological properties. The antigens were shown to be lipopolysaccharides and to differ from the lipopolysaccharides of other gram-negative bacteria. The serospecific antigens contained rhamnose, mannose, glucosamine, and two unidentified sugars together with 2-keto-3-deoxyoctonate, phosphate, and fatty acids. The fatty acid composition was predominantly branched-chain acids with smaller amounts of 3-hydroxymyristic acid. The antigens contain periodate-sensitive groups; mannosyl residues were completely cleaved by periodate oxidation. Hydrolysis of the total lipopolysaccharide by acetic acid resulted in the separation of a lipid A-like material that cross-reacted with the antiserum to lipid A from Salmonella minnesota but did not comigrate with it on sodium dodecyl sulfate gels. None of the four antigens contained heptose. All of the antigen preparations showed endotoxicity when tested by the Limulus amebocyte lysate assay. The results of this study indicate that the serogroup-specific antigens of L. pneumophila are lipopolysaccharides containing an unusual lipid A and core structure and different from those of other gram-negative bacteria. Images PMID:3017918

  2. Analysis of antigen presentation by metabolically inactive accessory cells and their isolated membranes.

    PubMed Central

    Falo, L D; Sullivan, K; Benacerraf, B; Mescher, M F; Rock, K L

    1985-01-01

    Several amino acid copolymers are potent immunogens under the control of major histocompatibility complex (MHC)-encoded Ir genes. We have further characterized their accessory-cell-dependent, MHC-restricted presentation to T lymphocytes. We initially characterized their processing requirements by investigating the ability of paraformaldehyde-fixed antigen-presenting cells (APC) to present these copolymers. Fixed APC can present poly(Glu56Lys35Phe9) and poly(Glu60Ala30Tyr10) provided that they have been incubated with antigen prior to fixation. The inability of these same fixed preparations to present soluble antigen indicates a fixation-sensitive antigen-processing step. In contrast, the antigens poly(Glu55Lys35Leu10) and poly(Glu55Lys35Tyr10) can be presented by APC fixed before antigen exposure. This differential requirement for antigen processing was exploited to analyze the events of antigen presentation in two related systems. First, the ability of isolated APC membranes to process and present antigen was assessed. APC membranes can present the antigens poly(GluLysLeu) and poly(GluLysTyr) in a specific and MHC-restricted manner. However, the isolated membranes fail to present either poly(GluLysPhe) or poly(GluAlaTyr), suggesting that such preparations can present but not process antigen. Second, the distinct properties of the various copolymers were used with fixed APC to test the effects of antigen processing on the phenomenon of antigen competition. APC that had processed poly(GluLysPhe) or poly(GluAlaTyr) were subsequently fixed and used to present antigen in the presence or absence of various antagonists. Under these conditions, poly(GluLysLeu) and poly(Glu50Tyr50) could effect specific inhibition, clearly indicating that antigen competition occurs distal to and does not require antigen processing. In contrast, native antigen with an absolute processing requirement is not capable of competing with preprocessed antigen on fixed APC. Taken together, these

  3. Antigenic and molecular characterization of rabies virus in Argentina.

    PubMed

    Cisterna, Daniel; Bonaventura, Romina; Caillou, Susana; Pozo, Oscar; Andreau, Maria Lidia; Fontana, Liliana Dalla; Echegoyen, Cristina; de Mattos, Carlos; de Mattos, Cecilia; Russo, Susana; Novaro, Laura; Elberger, Diana; Freire, María Cecilia

    2005-05-01

    The nucleoprotein genes of 54 human, domestic and wild animals rabies isolates obtained in Argentina between 1995 and 2002 were characterized using monoclonal antibodies and partial gene sequence analysis. The antigenic and genetic diversities of rabies virus in samples from bat and bat-related cases were studied, leading to the identification of five distinct genetic variants. Rabies viruses isolated from vampire bat related cases were very similar to each other, showing 98.9% overall similarity. Specific antigenic variants (AgV) were detected associated with different insectivorous bats species, in samples from Tadarida brasiliensis and Eumops patagonicus bats. In contrast, isolates from Myotis sp. and Histiotus sp. bats could not be matched to any antigenic type. Additionally, bat rabies cases were also detected in southern provinces previously considered rabies-free. Finally, two independent antigenic and genetic variants co-circulating in northern Argentina were found in isolates obtained from dogs and dog-related cases, suggesting two independent cycles of virus transmission. This is the first national coordinated study of antigenic as well as molecular epidemiology of rabies in Argentina. The information presented here will improve our knowledge about rabies epidemiology and therefore, will assist preventing fatal human cases. PMID:15763144

  4. Antigen Retrieval Immunohistochemistry

    PubMed Central

    Shi, Shan-Rong; Shi, Yan; Taylor, Clive R.

    2011-01-01

    As a review for the 20th anniversary of publishing the antigen retrieval (AR) technique in this journal, the authors intend briefly to summarize developments in AR-immunohistochemistry (IHC)–based research and diagnostics, with particular emphasis on current challenges and future research directions. Over the past 20 years, the efforts of many different investigators have coalesced in extending the AR approach to all areas of anatomic pathology diagnosis and research and further have led to AR-based protein extraction techniques and tissue-based proteomics. As a result, formalin-fixed paraffin-embedded (FFPE) archival tissue collections are now seen as a literal treasure of materials for clinical and translational research to an extent unimaginable just two decades ago. Further research in AR-IHC is likely to focus on tissue proteomics, developing a more efficient protocol for protein extraction from FFPE tissue based on the AR principle, and combining the proteomics approach with AR-IHC to establish a practical, sophisticated platform for identifying and using biomarkers in personalized medicine. PMID:21339172

  5. [Duffy blood group antigens: structure, serological properties and function].

    PubMed

    Łukasik, Ewa; Waśniowska, Kazimiera

    2016-01-01

    Duffy (Fy) blood group antigens are located on seven-transmembrane glycoprotein expressed on erythrocytes and endothelial cells, which acts as atypical chemokine receptor (ACKR1) and malarial receptor. The biological role of the Duffy glycoprotein has not been explained yet. It is suggested that Duffy protein modulate the intensity of the inflammatory response. The Duffy blood group system consists of two major antigens, Fy(a) and Fy(b), encoded by two codominant alleles designated FY*A and FY*B which differ by a single nucleotide polymorphism (SNP) at position 125G>A of the FY gene that results in Gly42Asp amino acid change in the Fy(a) and Fy(b) antigens, respectively. The presence of antigen Fy(a) and/or Fy(b) on the erythrocytes determine three Duffy-positive phenotypes: Fy(a+b-), Fy(a-b+) and Fy(a+b+), identified in Caucasian population. The Duffy-negative phenotype Fy(a-b-), frequent in Africans, but very rare in Caucasians, is defined by the homozygous state of FY*B-33 alleles. The FY*B-33 allele is associated with a SNP -33T>C in the promoter region of the FY gene, which suppresses erythroid expression of this gene without affecting its expression in other tissues. The FY*X allele, found in Caucasians, is correlated with weak expression of Fy(b) antigen. Fy(x) antigen differs from the native Fy(b) by the Arg89Cys and Ala100Thr amino acid substitutions due to SNPs: 265C>T and 298G>A in FY*B allele. The frequency of the FY alleles shows marked geographic disparities, the FY*B-33 allele is predominant in Africans, the FY*B in Caucasians, while the FY*A allele is dominant in Asians and it is the most prevalent allele globally. PMID:26943312

  6. Active immunization with recombinant V antigen from Yersinia pestis protects mice against plague.

    PubMed Central

    Leary, S E; Williamson, E D; Griffin, K F; Russell, P; Eley, S M; Titball, R W

    1995-01-01

    The gene encoding V antigen from Yersinia pestis was cloned into the plasmid expression vector pGEX-5X-2. When electroporated into Escherichia coli JM109, the recombinant expressed V antigen as a stable fusion protein with glutathione S-transferase. The glutathione S-transferase-V fusion protein was isolated from recombinant E. coli and cleaved with factor Xa to yield purified V antigen as a stable product. Recombinant V antigen was inoculated intraperitoneally into mice and shown to induce a protective immune response against a subcutaneous challenge with 3.74 x 10(6) CFU of virulent Y. pestis. Protection correlated with the induction of a high titer of serum antibodies and a T-cell response specific for recombinant V antigen. These results indicate that V antigen should be a major component of an improved vaccine for plague. PMID:7622205

  7. Natural selection promotes antigenic evolvability.

    PubMed

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

    2013-01-01

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

  8. Vaccines and viral antigenic diversity.

    PubMed

    Mumford, J A

    2007-04-01

    Antigenic diversity among ribonucleic acid (RNA) viruses occurs as a result of rapid mutation during replication and recombination/reassortment between genetic material of related strains during co-infections. Variants which have a selective advantage in terms of ability to spread or to avoid host immunity become established within populations. Examples of antigenically diverse viruses include influenza, foot and mouth disease (FMD) and bluetongue (BT). Effective vaccination against such viruses requires surveillance programmes to monitor circulating serotypes and their evolution to ensure that vaccine strains match field viruses. A formal vaccine strain selection scheme for equine influenza has been established under the auspices of the World Organisation for Animal Health (OIE) based on an international surveillance programme. A regulatory framework has been put in place to allow rapid updating of vaccine strains withoutthe need to provide full registration data for licensing the updated vaccine. While there is extensive surveillance of FMD worldwide and antigenic and genetic characterisation of isolates, there is no formal vaccine strain selection system. A coordinated international effort has been initiated to agree harmonised approaches to virus characterisation which is aimed at providing the basis for an internationally agreed vaccine matching system for FMD supported by the OIE. The emergence and spread of BT in Europe have resulted in an intensification of vaccine evaluation in terms of safety and efficacy, particularly cross-protection within and between serotypes. The most important requirement for producing vaccines against viruses displaying antigenic diversity is a method of measuring antigenic distances between strains and developing an understanding of how these distances relate to cross-protection. Antigenic cartography, a new computational method of quantifying antigenic distances between strains has been applied to human and equine influenza to

  9. The Structure and Function of the Rh antigen Complex

    PubMed Central

    Westhoff, Connie M.

    2007-01-01

    The Rh system is one of the most important and complex blood group systems because of the large number of antigens and the serious complications for the fetus of a woman sensitized by transfusion or pregnancy. Major advances in our understanding of the Rh system have occurred with the cloning of the genes and with functional evidence that the Rh blood group proteins belong to an ancient family of membrane proteins involved in ammonia transport. The arrangement and configuration of the genes at the RH locus promotes genetic exchange, generating new antigens. Importantly, RH genetic testing can now be applied to clinical transfusion medicine and prenatal practice. This includes testing for RHD zygosity, confirmation or resolution of D antigen status, and detection of altered RHD and RHCE genes in individuals at risk for producing antibodies to high incidence Rh antigens, particularly sickle cell disease patients. The Rh proteins form a core complex that is critical to the structure of the erythrocyte membrane, and may play a physiologically role in the sequestration of blood ammonia. The Rh family of proteins now includes non-erythroid Rh homologs present in many other tissues, and comparative genomics reveals Rh homologs in all domains of life. PMID:17198846

  10. Comprehensive red blood cell and platelet antigen prediction from whole genome sequencing: proof of principle

    PubMed Central

    Westhoff, Connie M.; Uy, Jon Michael; Aguad, Maria; Smeland‐Wagman, Robin; Kaufman, Richard M.; Rehm, Heidi L.; Green, Robert C.; Silberstein, Leslie E.

    2015-01-01

    BACKGROUND There are 346 serologically defined red blood cell (RBC) antigens and 33 serologically defined platelet (PLT) antigens, most of which have known genetic changes in 45 RBC or six PLT genes that correlate with antigen expression. Polymorphic sites associated with antigen expression in the primary literature and reference databases are annotated according to nucleotide positions in cDNA. This makes antigen prediction from next‐generation sequencing data challenging, since it uses genomic coordinates. STUDY DESIGN AND METHODS The conventional cDNA reference sequences for all known RBC and PLT genes that correlate with antigen expression were aligned to the human reference genome. The alignments allowed conversion of conventional cDNA nucleotide positions to the corresponding genomic coordinates. RBC and PLT antigen prediction was then performed using the human reference genome and whole genome sequencing (WGS) data with serologic confirmation. RESULTS Some major differences and alignment issues were found when attempting to convert the conventional cDNA to human reference genome sequences for the following genes: ABO, A4GALT, RHD, RHCE, FUT3, ACKR1 (previously DARC), ACHE, FUT2, CR1, GCNT2, and RHAG. However, it was possible to create usable alignments, which facilitated the prediction of all RBC and PLT antigens with a known molecular basis from WGS data. Traditional serologic typing for 18 RBC antigens were in agreement with the WGS‐based antigen predictions, providing proof of principle for this approach. CONCLUSION Detailed mapping of conventional cDNA annotated RBC and PLT alleles can enable accurate prediction of RBC and PLT antigens from whole genomic sequencing data. PMID:26634332

  11. Molecular cloning of a gene encoding a Chlamydia psittaci 57-kDa protein that shares antigenic determinants with ca. 60-kDa proteins present in many gram-negative bacteria.

    PubMed

    Menozzi, F D; Menozzi-Dejaiffe, C; Nano, F E

    1989-03-01

    In order to develop reagents to study the immune response of guinea pigs to infection by Chlamydia psittaci guinea pig inclusion conjunctivitis strain (GPIC), we constructed a plasmid clone bank with C. psittaci DNA. One of the recombinant clones isolated produced large amounts of a 57-kilodalton (kDa) protein that was immunoreactive with sera from GPIC infected guinea pigs. While investigating this recombinant protein, we discovered that all the Gram-negative bacteria analyzed so far have immunoreactive proteins of similar size. This protein seems to be a 'common antigen' already described in various Gram-negative bacteria.

  12. Identification of the class I genes of the mouse major histocompatibility complex by DNA-mediated gene transfer.

    PubMed

    Goodenow, R S; McMillan, M; Nicolson, M; Sher, B T; Eakle, K; Davidson, N; Hood, L

    1982-11-18

    DNA-mediated gene transfer was used to identify cloned class I genes from the major histocompatibility complex of the BALB/c mouse. Three genes encoding the transplantation antigens H-2 Kd, Dd and Ld were identified as well as genes encoding the Qa-2,3 and two TL differentiation antigens. As many as 10 putative novel class I genes were detected by the association of their gene products with beta 2-microglobulin. Alloantiserum prepared to one of the novel antigens was used to demonstrate the expression of the previously undetected antigen on spleen cells of various inbred, congeneic, and recombinant congeneic strains of mice. PMID:6815535

  13. Comparative Analysis of Gingival Tissue Antigen Presentation Pathways in Aging and Periodontitis

    PubMed Central

    Gonzalez, O.A.; Novak, M.J.; Kirakodu, S.; Orraca, L.; Chen, K.C.; Strom-berg, A.; Gonzalez-Martinez, J.; Ebersole, J. L.

    2014-01-01

    Aim Gingival tissues of periodontitis lesions contribute to local elevations in mediators, including both specific T cell and antibody immune responses to oral bacterial antigens. Thus, antigen processing and presentation activities must exist in these tissues to link antigen-presenting cells with adaptive immunity. We hypothesized that alterations in the transcriptome of antigen processing and presentation genes occur in aging gingival tissues and that periodontitis enhances these differences reflecting tissues less capable of immune resistance to oral pathogens. Materials and Methods Rhesus monkeys (n=34) from 3–23 years of age were examined. A buccal gingival sample from healthy or periodontitis sites were obtained, total RNA isolated, and microarray analysis was used to describe the transcriptome. Results The results demonstrated increased transcription of genes related to the MHC class II and negative regulation of NK cells with aging in healthy gingival tissues. In contrast, both adult and aging periodontitis tissues showed decreased transcription of genes for MHC class II antigens, coincident with up-regulation of MHC class I-associated genes. Conclusion These transcriptional changes suggest a response of healthy aging tissues through the class II pathway (i.e., endocytosed antigens) and altered responses in periodontitis that could reflect host-associated self-antigens or targeting cytosolic intra-cellular microbial pathogens. PMID:24304139

  14. Common antigenic structures of HL-A antigens

    PubMed Central

    Nakamuro, K.; Tanigaki, N.; Kreiter, V. P.; Pressman, D.

    1974-01-01

    Spent culture media of all the human cell lines tested have been found to contain the antigenic activity present on the 11,000-Dalton HL-A common portion fragment of the HL-A antigen molecule that appears to be a characteristic, invariant portion of HL-A antigen molecules. From the culture medium of one of these lines, RPMI 1788, a lymphoid cell line, the substance carrying HL-A common activity was isolated, which was shown to be identical to the HL-A common portion fragment with respect to molecular size, electrophoretic mobility, isoelectric focusing patterns, and certain antigenic characteristics. By an isolation procedure involving differential ultrafiltration, gel filtration, and column electrophoresis, 8 litres of the culture medium yielded 1.5–2.0 A280 units of the substance representing 15–20 per cent of the HL-A common antigenic activity originally present. A single protein band with a Rf of 0.47 was obtained by disc electrophoresis. The molecular size was shown to be about 11,000 Daltons by gel filtration and by sodium dodecyl sulphate—acrylamide gel electrophoresis. Upon isoelectric focusing two bands were obtained which corresponded exactly to those obtained with HL-A common portion fragment prepared from papain-solubilized HL-A antigen preparations by acid dissociation. The isoelectric point of the major band was 5.0. The reactions of this substance with rabbit antisera against human lymphoid cell membrane and against the substance were essentially identical to the reactions of HL-A common portion fragment with these same antisera. ImagesFIG. 3Fig. 4Fig. 5 PMID:4476726

  15. Organ-Specific Membrane Antigens

    PubMed Central

    Sell, K. W.; Mori, W.; Rack, J. H.; Gurner, B. W.; Coombs, R. R. A.

    1969-01-01

    A satisfactory system for testing the reaction of rabbit antisera with membrane antigens of human tissue cells is described. This method allows the differentiation between IgG and IgM antibodies and provides an extremely sensitive method for the detection of antigens on all cells including non-viable fixed cells. Anti-organ serum before selective absorption showed very little organ specificity in their reactions, but may be made specific by extensive absorption although often the resulting specific titre was very low. Organ-specific membrane antigens were also identified and shown to be represented on tumour cells, although in some cases such as the colon the reactions were weaker with tumour cells than with normal parenchymal cells of an organ. On the other hand, in one case of carcinoma of the kidney the organ-specific antigens were detectably stronger on tumour cells than on normal kidney cells. Preliminary studies on human ascitic tumour cells from 4 different cancer patients show that species-specific membrane antigens can be demonstrated. Unfortunately none of the cases were derived from organs whose origin could be identified with the antisera which had been prepared for this series of experiments. ImagesFigs. 2-3 PMID:5806432

  16. Adenoviral-vector-mediated gene transfer to dendritic cells.

    PubMed

    Song, W; Crystal, R G

    2001-01-01

    Dendritic cells (DC) are the most potent antigen presenting cells capable of initiating T-cell-dependent immune responses (1-5). This biologic potential can be harnessed to elicit effective antigen-specific immune responses by transferring the relevant antigens to the DC. Once the DC have been mobilized and purified, the relevant antigens can be transferred to the DC as intact proteins, or as peptides representing specific epitopes, or with gene transfer using sequences of DNA or RNA coding for the pertinent antigen(s) (6-15). Theoretically, genetically modifying DC with genes coding for specific antigens has potential advantages over pulsing the DC with peptides repeating the antigen or antigen fragment. First, the genetically modified DC may present previously unknown epitopes in association with different MHC molecules. Second, gene transfer to DC ensures that the gene product is endogenously processed, leading to the generation of MHC class I-restricted cytotoxic T lymphocytes (CTL), the effector arm of cell-mediated immune responses. Finally, in addition to genes coding for the antigen(s), genetic modification of the DC can induce genes coding for mediators relevant to generation of the immune response to the antigen(s), further boosting host responses to the antigens presented by the modified DC. Different gene transfer approaches have been explored to genetically modify DC, including retroviral vectors (16-18), recombinant vaccinia virus vectors (19), and recombinant adenovirus (Ad) vectors (19-23). The focus of this chapter is on using recombinant Ad vectors to transfer genes to murine DC. We have used a similar strategy to transfer genes to human DC (24). As an example of the power of this technology, we will describe the use of Ad-vector-modified DC to suppress the growth of tumor cells modified to express a specific antigen.

  17. The human E48 antigen, highly homologous to the murine Ly-6 antigen ThB, is a GPI-anchored molecule apparently involved in keratinocyte cell-cell adhesion

    PubMed Central

    1995-01-01

    The E48 antigen, a putative human homologue of the 20-kD protein present in desmosomal preparations of bovine muzzle, and formerly called desmoglein III (dg4), is a promising target antigen for antibody- based therapy of squamous cell carcinoma in man. To anticipate the effect of high antibody dose treatment, and to evaluate the possible biological involvement of the antigen in carcinogenesis, we set out to molecularly characterize the antigen. A cDNA clone encoding the E48 antigen was isolated by expression cloning in COS cells. Sequence analysis revealed that the clone contained an open reading frame of 128 amino acids, encoding a core protein of 13,286 kD. Database searching showed that the E48 antigen has a high level of sequence similarity with the mouse ThB antigen, a member of the Ly-6 antigen family. Phosphatidylinositol-specific (PI-specific) phospholipase-C treatment indicated that the E48 antigen is glycosylphosphatidylinositol-anchored (GPI-anchored) to the plasma membrane. The gene encoding the E48 antigen is a single copy gene, located on human chromosome 8 in the 8q24-qter region. The expression of the gene is confined to keratinocytes and squamous tumor cells. The putative mouse homologue, the ThB antigen, originally identified as an antigen on cells of the lymphocyte lineage, was shown to be highly expressed in squamous mouse epithelia. Moreover, the ThB expression level is in keratinocytes, in contrast to that in lymphocytes, not mouse strain related. Transfection of mouse SV40-polyoma transformed mouse NIH/3T3 cells with the E48 cDNA confirmed that the antigen is likely to be involved in cell-cell adhesion. PMID:7790363

  18. A T cell-independent antitumor response in mice with bone marrow cells retrovirally transduced with an antibody/Fc-gamma chain chimeric receptor gene recognizing a human ovarian cancer antigen.

    PubMed

    Wang, G; Chopra, R K; Royal, R E; Yang, J C; Rosenberg, S A; Hwu, P

    1998-02-01

    In order to treat common cancers with immunotherapy, chimeric receptors have been developed that combine the tumor specificity of antibodies with T-cell effector functions. Previously, we demonstrated that T cells transduced with a chimeric receptor gene against human ovarian cancer were able to recognize ovarian cancer cells in vitro and in vivo. We now report that recipients of bone marrow cells transduced with these genes exhibited significant antitumor activity in vivo. Moreover, in vivo depletion of T cells in reconstituted mice did not affect antitumor activity, suggesting that other immune cells expressing the chimeric receptor gene may play an important role in tumor rejection.

  19. A multiplex method for the detection of serum antibodies against in silico-predicted tumor antigens.

    PubMed

    Reuschenbach, Miriam; Dörre, Jonathan; Waterboer, Tim; Kopitz, Jürgen; Schneider, Martin; Hoogerbrugge, Nicoline; Jäger, Elke; Kloor, Matthias; von Knebel Doeberitz, Magnus

    2014-12-01

    Humoral immune responses against tumor antigens are studied as indirect markers of antigen exposure and in cancer vaccine studies. An increasing number of tumor antigens potentially translated from mutant genes is identified by advances in genomic sequencing. They represent an interesting source for yet unknown immunogenic epitopes. We here describe a multiplex method using the Luminex technology allowing for the detection of antibodies against multiple in silico-predicted linear neo-antigens in large sets of sera. The approach included 32 synthetic biotinylated peptides comprising a predicted set of frameshift mutation-induced neo-antigens. The antigens were fused to a FLAG epitope to ensure monitoring antigen binding to avidin-linked microspheres in the absence of monoclonal antibodies. Analytical specificity of measured serum antibody reactivity was proven by the detection of immune responses in immunized rabbits and a colorectal cancer patient vaccinated with peptides included in the assay. The measured antibody responses were comparable to peptide ELISA, and inter-assay reproducibility of the multiplex approach was excellent (R (2) > 0.98) for 20 sera tested against all antigens. Our methodic approach represents a valuable platform to monitor antibody responses against predicted antigens. It may be used in individualized cancer vaccine studies, thereby extending the relevance beyond the model system in the presented approach.

  20. Identification of Enterococcus faecalis antigens specifically expressed in vivo

    PubMed Central

    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

  1. Surface antigens of smooth brucellae.

    PubMed

    Diaz, R; Jones, L M; Leong, D; Wilson, J B

    1968-10-01

    Surface antigens of smooth brucellae were extracted by ether-water, phenol-water, trichloroacetic acid, and saline and examined by immunoelectrophoresis and gel diffusion with antisera from infected and immunized rabbits. Ether-water extracts of Brucella melitensis contained a lipopolysaccharide protein component, which was specific for the surface of smooth brucellae and was correlated with the M agglutinogen of Wilson and Miles, a polysaccharide protein component devoid of lipid which was not restricted to the surface of smooth brucellae and was not correlated with the smooth agglutinogen (component 1), and several protein components which were associated with internal antigens of rough and smooth brucellae. Immunoelectrophoretic analysis of ether-water extracts of B. abortus revealed only two components, a lipopolysaccharide protein component, which was correlated with the A agglutinogen, and component 1. Component 1 from B. melitensis and B. abortus showed identity in gel diffusion tests, whereas component M from B. melitensis and component A from B. abortus showed partial identity with unabsorbed antisera and no cross-reactions with monospecific sera. Attempts to prepare monospecific sera directly by immunization of rabbits with cell walls or ether-water extracts were unsuccessful. Absorption of antisera with heavy fraction of ether-water extracts did not always result in monospecific sera. It was concluded (as has been described before) that the A and M antigens are present on a single antigenic complex, in different proportions depending upon the species and biotype, and that this component is a lipopolysaccharide protein complex of high molecular weight that diffuses poorly through agar gel. Components 1, A, and M were also demonstrated in trichloroacetic acid and phenol-water extracts. With all extracts, B. melitensis antigen showed greater diffusibility in agar than B. abortus antigens. After mild acid hydrolysis, B. abortus ether-water extract was able

  2. [Detection and typing for swine leukocyte antigen].

    PubMed

    Li, Hua; Luo, Huai-Rong; Zhang, Ya-Ping; Qiu, Xiang-Pin; Ye, Chun

    2004-03-01

    Traditionally the cluster of swine leukocyte antigen (SLA) was typed by serological, cytological and biochemical methods. Many special molecular typing methods have been developed with the progress of molecular biological technology, such as PCR-RFLP, PCR-SSCP , MS and DNA sequencing. Here we discussed the advantages and disadvantages of each method based on the polymorphic and conservative (from the functional aspect, such as supertype and supermotif) characteristics of SLA, and illustrated the development of typing for SLA in the future. In addition, we pointed out the editorial mistakes about the serological haplotype of SLA in reference book and emphasized that the accurate polymorphism of SLA-DQB gene must be based on the cloning sequencing. PMID:15639990

  3. Haemophilus parainfluenzae expresses diverse lipopolysaccharide O-antigens using ABC transporter and Wzy polymerase-dependent mechanisms

    PubMed Central

    Young, Rosanna E.B.; Twelkmeyer, Brigitte; Vitiazeva, Varvara; Power, Peter M.; Schweda, Elke K.H.; Hood, Derek W.

    2013-01-01

    Lipopolysaccharide O-antigens are the basis of serotyping schemes for Gram negative bacteria and help to determine the nature of host–bacterial interactions. Haemophilus parainfluenzae is a normal commensal of humans but is also an occasional pathogen. The prevalence, diversity and biosynthesis of O-antigens were investigated in this species for the first time. 18/18 commensal H. parainfluenzae isolates contain a O-antigen biosynthesis gene cluster flanked by glnA and pepB, the same position as the hmg locus for tetrasaccharide biosynthesis in Haemophilus influenzae. The O-antigen loci show diverse restriction digest patterns but fall into two main groups: (1) those encoding enzymes for the synthesis and transfer of FucNAc4N in addition to the Wzy-dependent mechanism of O-antigen synthesis and transport and (2) those encoding galactofuranose synthesis/transfer enzymes and an ABC transporter. The other glycosyltransferase genes differ between isolates. Three H. parainfluenzae isolates fell outside these groups and are predicted to synthesise O-antigens containing ribitol phosphate or deoxytalose. Isolates using the ABC transporter system encode a putative O-antigen ligase, required for the synthesis of O-antigen-containing LPS glycoforms, at a separate genomic location. The presence of an O-antigen contributes significantly to H. parainfluenzae resistance to the killing effect of human serum in vitro. The discovery of O-antigens in H. parainfluenzae is striking, as its close relative H. influenzae lacks this cell surface component. PMID:24035104

  4. Multiplex PCR-based detection and identification of the most common Salmonella second-phase flagellar antigens.

    PubMed

    Echeita, M Aurora; Herrera, Silvia; Garaizar, Javier; Usera, Miguel A

    2002-03-01

    Most Salmonella serotypes alternatively express phase 1 or phase 2 flagellar antigens encoded by fliC and fljB genes respectively. Flagellar phase reversal to identify both flagellar antigens is not necessary at the genetic level. Variable internal regions of the fljB genes encoding H:1,w, H:e,n,x and H:e,n,z15 antigens have been sequenced and the specific sites for each antigen determined in selected Salmonella serotypes. These results, together with flagellar H1 complex variable internal sequences previously published, have been used to design a multiplex-PCR to identify H:1,2, H:1,5, H:1,6, H:1,7, H:1,w, H:e,n,x and H:e,n,z15 second-phase antigens. These antigens are part of the most common Salmonella serotypes possessing second-phase flagellar antigens. This multiplex-PCR includes 10 primers. A total of 140 Salmonella strains associated with 49 different serotypes were tested. Each strain generated one second-phase-specific antigen fragment, ranging between 50 and 400 bps. Twenty-five strains associated with 17 serotypes, with no second-phase antigen or with an antigen different from those tested in this work, did not generate any fragments. The method is quick, specific and reproducible and is independent of the phase expressed by the bacteria when tested.

  5. Identification of Novel Pre-Erythrocytic Malaria Antigen Candidates for Combination Vaccines with Circumsporozoite Protein

    PubMed Central

    Sahu, Tejram; Malkov, Vlad; Morrison, Robert; Pei, Ying; Juompan, Laure; Milman, Neta; Zarling, Stasya; Anderson, Charles; Wong-Madden, Sharon; Wendler, Jason; Ishizuka, Andrew; MacMillen, Zachary W.; Garcia, Valentino; Kappe, Stefan H. I.; Krzych, Urszula; Duffy, Patrick E.

    2016-01-01

    Malaria vaccine development has been hampered by the limited availability of antigens identified through conventional discovery approaches, and improvements are needed to enhance the efficacy of the leading vaccine candidate RTS,S that targets the circumsporozoite protein (CSP) of the infective sporozoite. Here we report a transcriptome-based approach to identify novel pre-erythrocytic vaccine antigens that could potentially be used in combination with CSP. We hypothesized that stage-specific upregulated genes would enrich for protective vaccine targets, and used tiling microarray to identify P. falciparum genes transcribed at higher levels during liver stage versus sporozoite or blood stages of development. We prepared DNA vaccines for 21 genes using the predicted orthologues in P. yoelii and P. berghei and tested their efficacy using different delivery methods against pre-erythrocytic malaria in rodent models. In our primary screen using P. yoelii in BALB/c mice, we found that 16 antigens significantly reduced liver stage parasite burden. In our confirmatory screen using P. berghei in C57Bl/6 mice, we confirmed 6 antigens that were protective in both models. Two antigens, when combined with CSP, provided significantly greater protection than CSP alone in both models. Based on the observations reported here, transcriptional patterns of Plasmodium genes can be useful in identifying novel pre-erythrocytic antigens that induce protective immunity alone or in combination with CSP. PMID:27434123

  6. Identification of Novel Pre-Erythrocytic Malaria Antigen Candidates for Combination Vaccines with Circumsporozoite Protein.

    PubMed

    Speake, Cate; Pichugin, Alexander; Sahu, Tejram; Malkov, Vlad; Morrison, Robert; Pei, Ying; Juompan, Laure; Milman, Neta; Zarling, Stasya; Anderson, Charles; Wong-Madden, Sharon; Wendler, Jason; Ishizuka, Andrew; MacMillen, Zachary W; Garcia, Valentino; Kappe, Stefan H I; Krzych, Urszula; Duffy, Patrick E

    2016-01-01

    Malaria vaccine development has been hampered by the limited availability of antigens identified through conventional discovery approaches, and improvements are needed to enhance the efficacy of the leading vaccine candidate RTS,S that targets the circumsporozoite protein (CSP) of the infective sporozoite. Here we report a transcriptome-based approach to identify novel pre-erythrocytic vaccine antigens that could potentially be used in combination with CSP. We hypothesized that stage-specific upregulated genes would enrich for protective vaccine targets, and used tiling microarray to identify P. falciparum genes transcribed at higher levels during liver stage versus sporozoite or blood stages of development. We prepared DNA vaccines for 21 genes using the predicted orthologues in P. yoelii and P. berghei and tested their efficacy using different delivery methods against pre-erythrocytic malaria in rodent models. In our primary screen using P. yoelii in BALB/c mice, we found that 16 antigens significantly reduced liver stage parasite burden. In our confirmatory screen using P. berghei in C57Bl/6 mice, we confirmed 6 antigens that were protective in both models. Two antigens, when combined with CSP, provided significantly greater protection than CSP alone in both models. Based on the observations reported here, transcriptional patterns of Plasmodium genes can be useful in identifying novel pre-erythrocytic antigens that induce protective immunity alone or in combination with CSP. PMID:27434123

  7. Concepts and applications for influenza antigenic cartography

    PubMed Central

    Cai, Zhipeng; Zhang, Tong; Wan, Xiu-Feng

    2011-01-01

    Influenza antigenic cartography projects influenza antigens into a two or three dimensional map based on immunological datasets, such as hemagglutination inhibition and microneutralization assays. A robust antigenic cartography can facilitate influenza vaccine strain selection since the antigenic map can simplify data interpretation through intuitive antigenic map. However, antigenic cartography construction is not trivial due to the challenging features embedded in the immunological data, such as data incompleteness, high noises, and low reactors. To overcome these challenges, we developed a computational method, temporal Matrix Completion-Multidimensional Scaling (MC-MDS), by adapting the low rank MC concept from the movie recommendation system in Netflix and the MDS method from geographic cartography construction. The application on H3N2 and 2009 pandemic H1N1 influenza A viruses demonstrates that temporal MC-MDS is effective and efficient in constructing influenza antigenic cartography. The web sever is available at http://sysbio.cvm.msstate.edu/AntigenMap. PMID:21761589

  8. Multiplex PCR for distinguishing the most common phase-1 flagellar antigens of Salmonella spp.

    PubMed

    Herrera-León, Silvia; McQuiston, John R; Usera, Miguel A; Fields, Patricia I; Garaizar, Javier; Echeita, M Aurora

    2004-06-01

    Most Salmonella serotypes alternatively express either phase-1 or phase-2 flagellar antigens, encoded by the fliC and fljB genes, respectively. Flagellar phase reversal for the identification of both flagellar antigens is not necessary at the genetic level. Variable internal regions of the fliC genes encoding the H:i, H:r, H:l,v, H:e,h, H:z(10), H:b, and H:d antigens have been sequenced; and the specific sites for each antigen in selected Salmonella serotypes have been determined. These results, together with flagellar G-complex variable internal sequences obtained by the Foodborne and Diarrheal Diseases Branch at the Centers for Disease Control and Prevention in Atlanta, GA, have been used to design a multiplex PCR to identify the G-complex antigens as well as the H:i, H:r, H:l,v, H:e,h, Hz(10), H:b, and H:d first-phase antigens. These antigens are part of the most common Salmonella serotypes possessing first-phase flagellar antigens. Salmonella enterica serotype Enteritidis is identified by adding a specific primer pair published previously. This multiplex PCR includes 13 primers. A total of 161 Salmonella strains associated with 72 different serotypes were tested. Each strain generated one first-phase-specific antigen fragment ranging from 100 to 500 bp; Salmonella serotype Enteritidis, however, generated two amplicons of 500 bp that corresponded to the G complex and a 333-bp serotype-specific amplicon, respectively. Twenty-three strains representing 19 serotypes with flagellar genes different from those targeted in this work did not generate any fragments. The method is quick, specific, and reproducible and is independent of the phase expressed by the bacteria when they are tested.

  9. Enhanced production of recombinant Mycobacterium tuberculosis antigens in Escherichia coli by replacement of low-usage codons.

    PubMed

    Lakey, D L; Voladri, R K; Edwards, K M; Hager, C; Samten, B; Wallis, R S; Barnes, P F; Kernodle, D S

    2000-01-01

    A major obstacle to development of subunit vaccines and diagnostic reagents for tuberculosis is the inability to produce large quantities of these proteins. To test the hypothesis that poor expression of some mycobacterial genes in Escherichia coli is due, in part, to the presence of low-usage E. coli codons, we used site-directed mutagenesis to convert low-usage codons to high-usage codons for the same amino acid in the Mycobacterium tuberculosis genes for antigens 85A and 85B and superoxide dismutase. Replacement of five codons in the wild-type gene for antigen 85B increased recombinant protein production in E. coli 54-fold. The recombinant antigen elicited proliferation and gamma interferon production by lymphocytes from healthy tuberculin reactors and was recognized by monoclonal antibodies to native antigen 85, indicating that the recombinant antigen contained T-cell and B-cell epitopes. Northern blotting demonstrated only a 1.7- to 2.5-fold increase in antigen 85B mRNA, suggesting that the enhanced protein production was due primarily to enhanced efficiency of translation. Codon replacement in the genes encoding antigen 85A and superoxide dismutase yielded four- to sixfold increases in recombinant protein production, suggesting that this strategy may be generally applicable to overexpression of mycobacterial genes in E. coli. PMID:10603393

  10. Plasmodium knowlesi Sporozoite Antigen: Expression by Infectious Recombinant Vaccinia Virus

    NASA Astrophysics Data System (ADS)

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

    1984-04-01

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

  11. Posttranscriptional control is a strong factor enabling exclusive expression of surface antigens in Paramecium tetraurelia.

    PubMed

    Simon, Martin C; Marker, Simone; Schmidt, Helmut J

    2006-01-01

    Variable antigens are large proteins located on the outer membrane of parasitic but also of free-living protists. Multigene families encoding surface antigens demonstrate an exclusive expression of proteins. The resulting presence of just one protein species on the cell surface is required for surface antigen function; therefore, the molecular mechanism of exclusive expression is of main interest. Regulation of gene expression and mechanisms establishing switching of antigens are hardly understood in any organism. Here we report on the reaction of Paramecium to the artificial knock down of surface antigen 51A expression by bacteria-mediated RNAi. This technique involves the feeding of dsRNA-producing bacteria. We analyzed different fragments of the target gene for dsRNA template regarding their specific knock down efficiency and found great differences. Treatment of Paramecia with RNAi against the 51A antigen demonstrated that although a massive amount of mRNA was present, the protein was not detected on the cell surface. Moreover, a minor abundance of 51D transcripts resulted in an exclusive presence of 51D proteins on the cell surface. This posttranscriptional regulation was confirmed by the transcript ratio (51A/51D) determined by real-time (RT) PCR of single cells. Because we were able to document unexclusive transcription also in wild-type cells our results indicate that this posttranscriptional regulation is a main factor of enabling exclusive gene expression. The comparison of serotype shifts, caused by efficient and inefficient knock down, indicates an involvement of full-length transcripts in regulation of gene expression. Thus, our study gives new insights into the mechanism of exclusive expression on the molecular level: (i) exclusive transcription does not occur, (ii) posttranscriptional regulation is a powerful factor enabling exclusive antigen expression, and (iii) surface antigen mRNA is shown to be involved in this mechanism in a regulating way.

  12. Impaired Tumor Antigen Processing by Immunoproteasome-expressing CD40-Activated B cells and Dendritic Cells

    PubMed Central

    Anderson, Karen S.; Zeng, Wanyong; Sasada, Tetsuro; Su, Mei; Choi, Jaewon; Drakoulakos, Donna; Kang, Yoon-Joong; Brusic, Vladimir; Wu, Catherine; Reinherz, Ellis L.

    2012-01-01

    Professional APCs, such as dendritic cells, are routinely used in vitro for the generation of cytotoxic T lymphocytes specific for tumor antigens. In addition to dendritic cells, CD40-activated B cells and variant K562 leukemic cells can be readily transfected with nucleic acids for in vitro and in vivo antigen presentation. However, the expression of immunoproteasome components in dendritic cells may preclude display of tumor antigens such as Mart1/MelanA. Here, we use three target epitopes, two derived from tumor antigens [Mart126–34 (M26) and Cyp1B1239–247 (Cyp239)] and one derived from the Influenza A viral antigen [FluM158–66 (FluM58)], to demonstrate that CD40-activated B cells, like dendritic cells, have a limited capability to process certain tumor antigens. In contrast, the K562 HLA-A*0201 transfectant efficiently processes and presents M26 and Cyp239 as well as the influenza FluM58 epitopes to T cells. These results demonstrate that the choice of target APC for gene transfer of tumor antigens may be limited by the relative efficacy of proteasome components to process certain tumor epitopes. Importantly, K562 can be exploited as an artificial APC, efficient in processing both M26 and Cyp239 epitopes and presumably, by extension, other relevant tumor antigens. PMID:21400024

  13. Identification of novel Mycobacterium tuberculosis CD4 T-cell antigens via high throughput proteome screening

    PubMed Central

    Nayak, Kaustuv; Jing, Lichen; Russell, Ronnie M.; Davies, D. Huw; Hermanson, Gary; Molina, Douglas M.; Liang, Xiaowu; Sherman, David R.; Kwok, William W.; Yang, Junbao; Kenneth, John; Ahamed, Syed F.; Chandele, Anmol; Kaja, Murali-Krishna; Koelle, David M.

    2015-01-01

    Elicitation of CD4 IFN-gamma T cell responses to Mycobacterium tuberculosis (MTB) is a rational vaccine strategy to prevent clinical tuberculosis. Diagnosis of MTB infection is based on T-cell immune memory to MTB antigens. The MTB proteome contains over four thousand open reading frames (ORFs). We conducted a pilot antigen identification study using 164 MTB proteins and MTB-specific T-cells expanded in vitro from 12 persons with latent MTB infection. Enrichment of MTB-reactive T-cells from PBMC used cell sorting or an alternate system compatible with limited resources. MTB proteins were used as single antigens or combinatorial matrices in proliferation and cytokine secretion readouts. Overall, our study found that 44 MTB proteins were antigenic, including 27 not previously characterized as CD4 T-cell antigens. Antigen truncation, peptide, NTM homology, and HLA class II tetramer studies confirmed malate synthase G (encoded by gene Rv1837) as a CD4 T-cell antigen. This simple, scalable system has potential utility for the identification of candidate MTB vaccine and biomarker antigens. PMID:25857935

  14. Genetic Variation at the O-Antigen Biosynthetic Locus in Pseudomonas aeruginosa

    PubMed Central

    Raymond, Christopher K.; Sims, Elizabeth H.; Kas, Arnold; Spencer, David H.; Kutyavin, Tanya V.; Ivey, Richard G.; Zhou, Yang; Kaul, Rajinder; Clendenning, James B.; Olson, Maynard V.

    2002-01-01

    The outer carbohydrate layer, or O antigen, of Pseudomonas aeruginosa varies markedly in different isolates of these bacteria, and at least 20 distinct O-antigen serotypes have been described. Previous studies have indicated that the major enzymes responsible for O-antigen synthesis are encoded in a cluster of genes that occupy a common genetic locus. We used targeted yeast recombinational cloning to isolate this locus from the 20 internationally recognized serotype strains. DNA sequencing of these isolated segments revealed that at least 11 highly divergent gene clusters occupy this region. Homology searches of the encoded protein products indicated that these gene clusters are likely to direct O-antigen biosynthesis. The O15 serotype strains lack functional gene clusters in the region analyzed, suggesting that O-antigen biosynthesis genes for this serotype are harbored in a different portion of the genome. The overall pattern underscores the plasticity of the P. aeruginosa genome, in which a specific site in a well-conserved genomic region can be occupied by any of numerous islands of functionally related DNA with diverse sequences. PMID:12057956

  15. Antigen Export Reduces Antigen Presentation and Limits T Cell Control of M. tuberculosis.

    PubMed

    Srivastava, Smita; Grace, Patricia S; Ernst, Joel D

    2016-01-13

    Persistence of Mycobacterium tuberculosis results from bacterial strategies that manipulate host adaptive immune responses. Infected dendritic cells (DCs) transport M. tuberculosis to local lymph nodes but activate CD4 T cells poorly, suggesting bacterial manipulation of antigen presentation. However, M. tuberculosis antigens are also exported from infected DCs and taken up and presented by uninfected DCs, possibly overcoming this blockade of antigen presentation by infected cells. Here we show that the first stage of this antigen transfer, antigen export, benefits M. tuberculosis by diverting bacterial proteins from the antigen presentation pathway. Kinesin-2 is required for antigen export and depletion of this microtubule-based motor increases activation of antigen-specific CD4 T cells by infected cells and improves control of intracellular infection. Thus, although antigen transfer enables presentation by bystander cells, it does not compensate for reduced antigen presentation by infected cells and represents a bacterial strategy for CD4 T cell evasion.

  16. Antigen Export Reduces Antigen Presentation and Limits T Cell Control of M. tuberculosis.

    PubMed

    Srivastava, Smita; Grace, Patricia S; Ernst, Joel D

    2016-01-13

    Persistence of Mycobacterium tuberculosis results from bacterial strategies that manipulate host adaptive immune responses. Infected dendritic cells (DCs) transport M. tuberculosis to local lymph nodes but activate CD4 T cells poorly, suggesting bacterial manipulation of antigen presentation. However, M. tuberculosis antigens are also exported from infected DCs and taken up and presented by uninfected DCs, possibly overcoming this blockade of antigen presentation by infected cells. Here we show that the first stage of this antigen transfer, antigen export, benefits M. tuberculosis by diverting bacterial proteins from the antigen presentation pathway. Kinesin-2 is required for antigen export and depletion of this microtubule-based motor increases activation of antigen-specific CD4 T cells by infected cells and improves control of intracellular infection. Thus, although antigen transfer enables presentation by bystander cells, it does not compensate for reduced antigen presentation by infected cells and represents a bacterial strategy for CD4 T cell evasion. PMID:26764596

  17. Antigenic Variation in Plasmodium falciparum.

    PubMed

    Petter, Michaela; Duffy, Michael F

    2015-01-01

    Plasmodium falciparum is the protozoan parasite that causes most malaria-associated morbidity and mortality in humans with over 500,000 deaths annually. The disease symptoms are associated with repeated cycles of invasion and asexual multiplication inside red blood cells of the parasite. Partial, non-sterile immunity to P. falciparum malaria develops only after repeated infections and continuous exposure. The successful evasion of the human immune system relies on the large repertoire of antigenically diverse parasite proteins displayed on the red blood cell surface and on the merozoite membrane where they are exposed to the human immune system. Expression switching of these polymorphic proteins between asexual parasite generations provides an efficient mechanism to adapt to the changing environment in the host and to maintain chronic infection. This chapter discusses antigenic diversity and variation in the malaria parasite and our current understanding of the molecular mechanisms that direct the expression of these proteins. PMID:26537377

  18. HLA antigens and insulin dependent diabetes mellitus. A family study.

    PubMed

    Savi, M; Neri, T M; Zavaroni, I; Coscelli, I

    1977-12-01

    Sixteen insulin dependent diabetic patients (age at onset less than 35 years) and their families were tissue typed for HLA antigens. Glucose tolerance of relatives was also tested. Among diabetic patients two HLA antigens were found with increased frequency: B8 (31 percent, control 15 percent) and Bw35 (38 percent, control 23 percent). Among normal relatives B8 and Bw35 had the same frequency as the control group. Bw15 frequency was not increased in either group. In relatives, no correlation between HLA antigens (B8 or Bw35) and abnormal glucose tolerance, obesity and over-weight at birth was found. Present data confirm previous reports of high B8 frequency in early onset diabetic patients, but fail to demonstrate a raised frequency of abnormal glucose tolerance among relatives bearing B8 (or, in our cases, Bw35). B8 may be considered a genetic indicator for susceptibility to juvenile diabetes. On the basis of present results in families, however non genetic factors clearly also play a determinant role. Furthermore, that diabetogenesis arises from a link between Ir-genes and HLA-B8 antigen should only be considered a suggestive hypothesis. PMID:413751

  19. [HLA antigens in juvenile rheumatoid arthritis].

    PubMed

    Rumba, I V; Sochnev, A M; Kukaĭne, E M; Burshteĭn, A M; Benevolenskaia, L I

    1990-01-01

    Antigens of I class HLA system (locus A and B) were investigated in 67 patients of Latvian nationality suffering from juvenile rheumatoid arthritis (JRA). Associations of HLA antigens with juvenile rheumatoid arthritis partially coincided with the ones revealed earlier. Typing established an increased incidence of antigen B27 (p less than 0.01) and gaplotype A2, B40 (p less than 0.01). Antigen B15 possessed a protective action with respect to JRA. Interlocus combinations demonstrated a closer association with the disease than a single antigen. The authors also revealed markers of various clinico-anatomical variants of JRA.

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

    PubMed

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

    2015-01-01

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

  1. Immunization with viral antigens: infectious haematopoietic necrosis.

    PubMed

    Winton, J R

    1997-01-01

    Infectious haematopoietic necrosis (IHN) is one of the most important viral diseases of salmonids, especially among juvenile fish where losses can be high. For over 20 years, researchers have tested a variety of preparations for control of IHN. Early vaccines consisted of killed virus and were effective when delivered by injection, but too costly to be practical on a large scale. Attenuated vaccines were developed by serial passage in cell culture and by monoclonal antibody selection. These offered excellent protection and were cost-effective, but residual virulence and uncertainty about their effects on other aquatic species made them poor candidates for licensing. Subunit vaccines using part of the IHNV glycoprotein gene cloned into E. coli or into an attenuated strain of A. salmonicida have been tested, appeared safe and were inexpensive. These vaccines were reported to provide some protection when delivered by immersion. Information on the location of antigenic sites on the glycoprotein led to trials using synthetic peptides, but these did not seem to be economically viable. Recently, plasmid vectors encoding the glycoprotein gene under control of a cytomegalovirus promoter were developed for genetic immunization. The constructs were highly protective when delivered by injection, but a more practical delivery system is needed. Thus, while several vaccine strategies have been tried in order to stimulate specific immunity against IHN, more research is needed to develop a commercially viable product for control of this important disease. PMID:9270850

  2. Immunization with viral antigens: infectious haematopoietic necrosis.

    PubMed

    Winton, J R

    1997-01-01

    Infectious haematopoietic necrosis (IHN) is one of the most important viral diseases of salmonids, especially among juvenile fish where losses can be high. For over 20 years, researchers have tested a variety of preparations for control of IHN. Early vaccines consisted of killed virus and were effective when delivered by injection, but too costly to be practical on a large scale. Attenuated vaccines were developed by serial passage in cell culture and by monoclonal antibody selection. These offered excellent protection and were cost-effective, but residual virulence and uncertainty about their effects on other aquatic species made them poor candidates for licensing. Subunit vaccines using part of the IHNV glycoprotein gene cloned into E. coli or into an attenuated strain of A. salmonicida have been tested, appeared safe and were inexpensive. These vaccines were reported to provide some protection when delivered by immersion. Information on the location of antigenic sites on the glycoprotein led to trials using synthetic peptides, but these did not seem to be economically viable. Recently, plasmid vectors encoding the glycoprotein gene under control of a cytomegalovirus promoter were developed for genetic immunization. The constructs were highly protective when delivered by injection, but a more practical delivery system is needed. Thus, while several vaccine strategies have been tried in order to stimulate specific immunity against IHN, more research is needed to develop a commercially viable product for control of this important disease.

  3. Meningococcal protein antigens and vaccines.

    PubMed

    Feavers, Ian M; Pizza, Mariagrazia

    2009-06-24

    The development of a comprehensive vaccine against meningococcal disease has been challenging. Recent developments in molecular genetics have provided both explanations for these challenges and possible solutions. Since genome sequence data became available there has been a marked increase in number of protein antigens that have been suggested as prospective vaccine components. This review catalogues the proposed vaccine candidates and examines the evidence for their inclusion in potential protein vaccine formulations.

  4. Adoptive transfer of cytotoxic T lymphocytes targeting two different antigens limits antigen loss and tumor escape.

    PubMed

    Kaluza, Karen M; Kottke, Timothy; Diaz, Rosa Maria; Rommelfanger, Diana; Thompson, Jill; Vile, Richard

    2012-10-01

    An antitumor T-cell response can lead to tumor control without clearing all tumor cells. As long as residual tumor cells remain, there is a constant risk of escape from that T-cell response. We previously showed that adoptive transfer of anti-ova OT-I T cells into B16ova-bearing mice led to tumor regression followed by escape of tumors that had lost the ova gene, rendering the OT-I T cells ineffective. In this study, we hypothesized that simultaneous transfer of cytotoxic T lymphocytes targeted against two independent antigens would reduce selection for single-antigen-loss cells, thereby limiting tumor escape. Using OT-I and Pmel T cells to treat B16ova tumors, we found that early cotransfer could prevent tumor emergence in most mice, whereas neither T-cell specificity alone was able to do so. When combined with total body irradiation for the treatment of larger 7-day tumors, cotransfer was also better at limiting tumor recurrence, and the tumors that did escape combination therapy continued to express both target antigens. As adoptively transferred T cells also persisted in vivo, even in mice with recurrent tumors, we hypothesized that restimulation of these antitumor T cells would prolong survival of mice with recurrent tumors. Consistent with this hypothesis, administration of a low-dose regimen of cyclophosphamide following tumor escape slowed tumor growth in mice that had previously received T-cell therapy, but not in control-treated mice, an effect that was associated with increased activation of T cells in vitro by low- but not high-dose cyclophosphamide.

  5. Common antigens between hydatid cyst and cancers

    PubMed Central

    Daneshpour, Shima; Bahadoran, Mehran; Hejazi, Seyed Hossein; Eskandarian, Abas Ali; Mahmoudzadeh, Mehdi; Darani, Hossein Yousofi

    2016-01-01

    Background: Different research groups reported a negative correlation between cancers and parasitical infections. As an example, the prevalence of a hydatid cyst among patients with cancer was significantly lower than its prevalence among normal population. Tn antigens exist both in cancer and hydatid cyst. This common antigen may be involved in the effect of parasite on cancer growth. So in this work, common antigens between hydatid cyst and cancers have been investigated. Materials and Methods: Different hydatid cyst antigens including hydatid fluid, laminated and germinal layer antigens, and excretory secretory antigens of protoscolices were run in SDS PAGE and transferred to NCP paper. In western immunoblotting, those antigens were probed with sera of patients with different cancer and also sera of non-cancer patients. Also, cross reaction among excretory secretory products of cancer cells and antisera raised against different hydatid cyst antigen was investigated. Results: In western immunoblotting, antisera raised against laminated and germinal layers of hydatid cyst reacted with excretory secretory products of cancer cells. Also, a reaction was detected between hydatid cyst antigens and sera of patients with some cancers. Conclusion: Results of this work emphasize existence of common antigens between hydatid cyst and cancers. More investigation about these common antigens is recommended. PMID:26962511

  6. Stable solid-phase Rh antigen.

    PubMed

    Yared, M A; Moise, K J; Rodkey, L S

    1997-12-01

    Numerous investigators have attempted to isolate the Rh antigens in a stable, immunologically reactive form since the discovery of the Rh system over 56 years ago. We report here a successful and reproducible approach to solubilizing and adsorbing the human Rh antigen(s) to a solid-phase matrix in an antigenically active form. Similar results were obtained with rabbit A/D/F red blood cell antigens. The antigen preparation was made by dissolution of the red blood cell membrane lipid followed by fragmentation of the residual cytoskeleton in an EDTA solution at low ionic strength. The antigenic activity of the soluble preparations was labile in standard buffers but was stable in zwitterionic buffers for extended periods of time. Further studies showed that the antigenic activity of these preparations was enhanced, as was their affinity for plastic surfaces, in the presence of acidic zwitterionic buffers. Adherence to plastic surfaces at low pH maintained antigenic reactivity and specificity for antibody was retained. The data show that this approach yields a stable form of antigenically active human Rh D antigen that could be used in a red blood cell-free assay for quantitative analysis of Rh D antibody and for Rh D antibody immunoadsorption and purification.

  7. Molecular cloning, gene organization and expression of the human UDP-GalNAc:Neu5Acalpha2-3Galbeta-R beta1,4-N-acetylgalactosaminyltransferase responsible for the biosynthesis of the blood group Sda/Cad antigen: evidence for an unusual extended cytoplasmic domain.

    PubMed Central

    Montiel, Maria-Dolores; Krzewinski-Recchi, Marie-Ange; Delannoy, Philippe; Harduin-Lepers, Anne

    2003-01-01

    The nucleotide sequence of the short and long transcripts of beta1,4- N -acetylgalactosaminyltransferase have been submitted to the DDBJ, EMBL, GenBank(R) and GSDB Nucleotide Sequence Databases under accession nos AJ517770 and AJ517771 respectively. The human Sd(a) antigen is formed through the addition of an N -acetylgalactosamine residue via a beta1,4-linkage to a sub-terminal galactose residue substituted with an alpha2,3-linked sialic acid residue. We have taken advantage of the previously cloned mouse cDNA sequence of the UDP-GalNAc:Neu5Acalpha2-3Galbeta-R beta1,4- N -acetylgalactosaminyltransferase (Sd(a) beta1,4GalNAc transferase) to screen the human EST and genomic databases and to identify the corresponding human gene. The sequence spans over 35 kb of genomic DNA on chromosome 17 and comprises at least 12 exons. As judged by reverse transcription PCR, the human gene is expressed widely since it is detected in various amounts in almost all cell types studied. Northern blot analysis indicated that five Sd(a) beta1,4GalNAc transferase transcripts of 8.8, 6.1, 4.7, 3.8 and 1.65 kb were highly expressed in colon and to a lesser extent in kidney, stomach, ileum and rectum. The complete coding nucleotide sequence was amplified from Caco-2 cells. Interestingly, the alternative use of two first exons, named E1(S) and E1(L), leads to the production of two transcripts. These nucleotide sequences give rise potentially to two proteins of 506 and 566 amino acid residues, identical in their sequence with the exception of their cytoplasmic tail. The short form is highly similar (74% identity) to the mouse enzyme whereas the long form shows an unusual long cytoplasmic tail of 66 amino acid residues that is as yet not described for any other mammalian glycosyltransferase. Upon transient transfection in Cos-7 cells of the common catalytic domain, a soluble form of the protein was obtained, which catalysed the transfer of GalNAc residues to alpha2,3-sialylated acceptor

  8. Natural micropolymorphism in human leukocyte antigens provides a basis for genetic control of antigen recognition

    SciTech Connect

    Archbold, Julia K.; Macdonald, Whitney A.; Gras, Stephanie; Ely, Lauren K.; Miles, John J.; Bell, Melissa J.; Brennan, Rebekah M.; Beddoe, Travis; Wilce, Matthew C.J.; Clements, Craig S.; Purcell, Anthony W.; McCluskey, James; Burrows, Scott R.; Rossjohn, Jamie

    2009-07-10

    Human leukocyte antigen (HLA) gene polymorphism plays a critical role in protective immunity, disease susceptibility, autoimmunity, and drug hypersensitivity, yet the basis of how HLA polymorphism influences T cell receptor (TCR) recognition is unclear. We examined how a natural micropolymorphism in HLA-B44, an important and large HLA allelic family, affected antigen recognition. T cell-mediated immunity to an Epstein-Barr virus determinant (EENLLDFVRF) is enhanced when HLA-B*4405 was the presenting allotype compared with HLA-B*4402 or HLA-B*4403, each of which differ by just one amino acid. The micropolymorphism in these HLA-B44 allotypes altered the mode of binding and dynamics of the bound viral epitope. The structure of the TCR-HLA-B*4405EENLLDFVRF complex revealed that peptide flexibility was a critical parameter in enabling preferential engagement with HLA-B*4405 in comparison to HLA-B*4402/03. Accordingly, major histocompatibility complex (MHC) polymorphism can alter the dynamics of the peptide-MHC landscape, resulting in fine-tuning of T cell responses between closely related allotypes.

  9. Increased frequency of HLA B17 antigen in girls with Turner syndrome and their fathers.

    PubMed

    Dacou-Voutetakis, C; Georgopoulos, N; Pappa, H; Vlachos, K; Tarassi, K; Chryssovergi, D; Papasteriades, C

    1993-12-01

    HLA-A, -B and -DR antigen distribution was studied in 49 girls with Turner Syndrome (TS), in 43 of their parents, as well as in 433 controls. No increased frequency of DR3, DR4 was found in our group. However, an increased frequency of HLA B17 antigen was disclosed (18.3% in TS versus 6.4% in the controls, p < 0.001 and pc < 0.01). Furthermore, the HLA B17 antigen was of paternal origin in 77.7% of the cases. The interpretation of the present findings is quite difficult. Most likely, the findings are related to the chromosomal abnormality rather than to autoimmunity. It is quite possible that genes within the region of class I genes create unfavorable circumstances leading to the loss of the sex chromosome or, alternatively, genes in this region confer protection and prevent miscarriage of the affected fetus.

  10. Roles of Predicted Glycosyltransferases in the Biosynthesis of the Rhizobium etli CE3 O Antigen

    PubMed Central

    Ojeda, Kristylea J.; Simonds, Laurie

    2013-01-01

    The Rhizobium etli CE3 O antigen is a fixed-length heteropolymer. The genetic regions required for its synthesis have been identified, and the nucleotide sequences are known. The structure of the O antigen has been determined, but the roles of specific genes in synthesizing this structure are relatively unclear. Within the known O-antigen genetic clusters of this strain, nine open reading frames (ORFs) were found to contain a conserved glycosyltransferase domain. Each ORF was mutated, and the resulting mutant lipopolysaccharide (LPS) was analyzed. Tricine SDS-PAGE revealed stepwise truncations of the O antigen that were consistent with differences in mutant LPS sugar compositions and reactivity with O-antigen-specific monoclonal antibodies. Based on these results and current theories of O-antigen synthesis, specific roles were deduced for each of the nine glycosyltransferases, and a model for biosynthesis of the R. etli CE3 O antigen was proposed. In this model, O-antigen biosynthesis is initiated with the addition of N-acetyl-quinovosamine-phosphate (QuiNAc-P) to bactoprenol-phosphate by glycosyltransferase WreU. Glycosyltransferases WreG, WreE, WreS, and WreT would each act once to attach mannose, fucose, a second fucose, and 3-O-methyl-6-deoxytalose (3OMe6dTal), respectively. WreH would then catalyze the addition of methyl glucuronate (MeGlcA) to complete the first instance of the O-antigen repeat unit. Four subsequent repeats of this unit composed of fucose, 3OMe6dTal, and MeGlcA would be assembled by a cycle of reactions catalyzed by two additional glycosyltransferases, WreM and WreL, along with WreH. Finally, the O antigen would be capped by attachment of di- or tri-O-methylated fucose as catalyzed by glycosyltransferase WreB. PMID:23435981

  11. Mechanism of induction of class I major histocompatibility antigen expression by murine leukemia virus.

    PubMed

    Faller, D V; Wilson, L D; Flyer, D C

    1988-03-01

    Alterations in expression of major histocompatibility complex (MHC) antigens on tumor cells clearly correlate with the tumorgenicity and metastatic potential of those cells. These changes in the biological behavior of the tumor cells are presumably secondary to resulting changes in their susceptibility to immune recognition and destruction. Murine leukemia viruses (MuLV) exert regulatory effects on class I genes of the MHC locus. MuLV infection results in substantial increases in cell surface expression of all three class I MHC antigens. These viral effects on MHC antigen expression profoundly influence immune-mediated interaction with the infected cells, as assessed by cytotoxic T lymphocyte recognition and killing. Control of class I MHC and beta-2 microglobulin genes by MuLV takes place via a trans-acting molecular mechanism. MuLV controls expression of widely separated endogenous cellular MHC genes, transfected xenogeneic class I MHC genes, and unintegrated chimeric genes consisting of fragments of class I MHC genes linked to a bacterial reporter gene. These findings indicate that MuLV exerts its effects on MHC expression via a trans mechanism. The MuLV-responsive sequences on the MHC genes appear to lie within 1.2 kilobases upstream of the initiation codon for those genes.

  12. Cell Wall Anchoring of the Campylobacter Antigens to Lactococcus lactis.

    PubMed

    Kobierecka, Patrycja A; Olech, Barbara; Książek, Monika; Derlatka, Katarzyna; Adamska, Iwona; Majewski, Paweł M; Jagusztyn-Krynicka, Elżbieta K; Wyszyńska, Agnieszka K

    2016-01-01

    Campylobacter jejuni is the most frequent cause of human food-borne gastroenteritis and chicken meat is the main source of infection. Recent studies showed that broiler chicken immunization against Campylobacter should be the most efficient way to lower the number of human infections by this pathogen. Induction of the mucosal immune system after oral antigen administration should provide protective immunity to chickens. In this work we tested the usefulness of Lactococcus lactis, the most extensively studied lactic acid bacterium, as a delivery vector for Campylobacter antigens. First we constructed hybrid protein - CjaA antigen presenting CjaD peptide epitopes on its surface. We showed that specific rabbit anti-rCjaAD serum reacted strongly with both CjaA and CjaD produced by a wild type C. jejuni strain. Next, rCjaAD and CjaA were fused to the C-terminus of the L. lactis YndF containing the LPTXG motif. The genes expressing these proteins were transcribed under control of the L. lactis Usp45 promoter and their products contain the Usp45 signal sequences. This strategy ensures a cell surface location of both analyzed proteins, which was confirmed by immunofluorescence assay. In order to evaluate the impact of antigen location on vaccine prototype efficacy, a L. lactis strain producing cytoplasm-located rCjaAD was also generated. Animal experiments showed a decrease of Campylobacter cecal load in vaccinated birds as compared with the control group and showed that the L. lactis harboring the surface-exposed rCjaAD antigen afforded greater protection than the L. lactis producing cytoplasm-located rCjaAD. To the best of our knowledge, this is the first attempt to employ Lactic Acid Bacteria (LAB) strains as a mucosal delivery vehicle for chicken immunization. Although the observed reduction of chicken colonization by Campylobacter resulting from vaccination was rather moderate, the experiments showed that LAB strains can be considered as an alternative vector to

  13. Cell Wall Anchoring of the Campylobacter Antigens to Lactococcus lactis

    PubMed Central

    Kobierecka, Patrycja A.; Olech, Barbara; Książek, Monika; Derlatka, Katarzyna; Adamska, Iwona; Majewski, Paweł M.; Jagusztyn-Krynicka, Elżbieta K.; Wyszyńska, Agnieszka K.

    2016-01-01

    Campylobacter jejuni is the most frequent cause of human food-borne gastroenteritis and chicken meat is the main source of infection. Recent studies showed that broiler chicken immunization against Campylobacter should be the most efficient way to lower the number of human infections by this pathogen. Induction of the mucosal immune system after oral antigen administration should provide protective immunity to chickens. In this work we tested the usefulness of Lactococcus lactis, the most extensively studied lactic acid bacterium, as a delivery vector for Campylobacter antigens. First we constructed hybrid protein – CjaA antigen presenting CjaD peptide epitopes on its surface. We showed that specific rabbit anti-rCjaAD serum reacted strongly with both CjaA and CjaD produced by a wild type C. jejuni strain. Next, rCjaAD and CjaA were fused to the C-terminus of the L. lactis YndF containing the LPTXG motif. The genes expressing these proteins were transcribed under control of the L. lactis Usp45 promoter and their products contain the Usp45 signal sequences. This strategy ensures a cell surface location of both analyzed proteins, which was confirmed by immunofluorescence assay. In order to evaluate the impact of antigen location on vaccine prototype efficacy, a L. lactis strain producing cytoplasm-located rCjaAD was also generated. Animal experiments showed a decrease of Campylobacter cecal load in vaccinated birds as compared with the control group and showed that the L. lactis harboring the surface-exposed rCjaAD antigen afforded greater protection than the L. lactis producing cytoplasm-located rCjaAD. To the best of our knowledge, this is the first attempt to employ Lactic Acid Bacteria (LAB) strains as a mucosal delivery vehicle for chicken immunization. Although the observed reduction of chicken colonization by Campylobacter resulting from vaccination was rather moderate, the experiments showed that LAB strains can be considered as an alternative vector to

  14. Differential roles of homologous recombination pathways in Neisseria gonorrhoeae pilin antigenic variation, DNA transformation and DNA repair.

    PubMed

    Mehr, I J; Seifert, H S

    1998-11-01

    Neisseria gonorrhoeae (Gc) pili undergo antigenic variation when the amino acid sequence of the pilin protein is changed, aiding in immune avoidance and altering pilus expression. Pilin antigenic variation occurs by RecA-dependent unidirectional transfer of DNA sequences from a silent pilin locus to the expressed pilin gene through high-frequency recombination events that occur at limited regions of homology. We show that the Gc recQ and recO genes are essential for pilin antigenic and phase variation and DNA repair but are not involved in natural DNA transformation. This suggests that a RecF-like pathway of recombination exists in Gc. In addition, mutations in the Gc recB, recC or recD genes revealed that a Gc RecBCD pathway also exists and is involved in DNA transformation and DNA repair but not in pilin antigenic variation. PMID:10094619

  15. Killed poliovirus antigen titration in humans.

    PubMed

    Salk, J; Cohen, H; Fillastre, C; Stoeckel, P; Rey, J L; Schlumberger, M; Nicolas, A; van Steenis, G; van Wezel, A L; Triau, R; Saliou, P; Barry, L F; Moreau, J P; Mérieux, C

    1978-01-01

    To establish the antigen content of a killed poliovirus vaccine sufficiently potent to induce immunity with one or two doses and to establish a reference standard vaccine which has been tested under field conditions, a titration was carried out in infants to determine the amount of each of the three antigenic types of poliovirus vaccine required to induce seroconversion with a single dose. It has been observed that over a critical range of antigen concentration there is an essentially linear relationship between antibody response and quantity of antigen administered. More than 90 percent of the groups studied had detectable antibody after receiving single injections of 80, 8 and 64 D-antigen units of Types I, II and III, respectively. Four-fold less antigen for each of the three types was less effective. The implications of these findings for an efficient immunization procedure are discussed.

  16. Acquired loss of red cell Kell antigens.

    PubMed

    Vengelen-Tyler, V; Gonzalez, B; Garratty, G; Kruppe, C; Johnson, C L; Mueller, K A; Marsh, W L

    1987-02-01

    A 19-year-old patient with a long history of idiopathic thrombocytopenic purpura developed a potent antibody against a high-incidence antigen in the Kell blood group system. The direct antiglobulin test on his red cells was negative. His cells exhibited profound depression of Kell blood group antigens, but antigens of other blood groups were normal. Transfusion of incompatible blood was well tolerated and differential agglutination tests, using selected Rh antisera, showed in vivo survival of the transfused red cells for more than 8 weeks. However, the transfused red cells also showed acquired loss of Kell antigens. Five months after the initial findings, Kell-related antibody disappeared and Kell antigens reappeared on his red cells. The patient's serum stored from the initial investigation now reacted with his freshly collected red cells. These data suggest that an environmental agent in the patient's plasma was responsible for the temporary loss of Kell antigens from red cells in his circulation.

  17. Identification of the antigen content of electroimmunoprecipitates.

    PubMed

    Beyer, N Helena; Heegaard, Niels H H

    2013-01-01

    Polyclonal antibodies including purified antibody fractions and animal or human antisera may react with unknown antigens or antigens other than their main specificity in reactions that are best visualized by gel electroimmunoprecipitation methods, e.g., when analyzing complex antigen mixtures. The great advantage of gel immunoprecipitation approaches is that each immunoprecipitate contains antigen in a pure form and that the precipitate is separated by position, shape, and size from other precipitates in the complex patterns of crossed immunoelectrophoresis. The identification of the antigen content of such immunoprecipitates is important but challenging because of the very stable, high affinity complex formation leading to precipitation in the gels. Here, we present detailed step-by-step recipes for identifying the antigen content of electroimmunoprecipitates.

  18. The ABCs of artificial antigen presentation.

    PubMed

    Kim, Jiyun V; Latouche, Jean-Baptiste; Rivière, Isabelle; Sadelain, Michel

    2004-04-01

    Artificial antigen presentation aims to accelerate the establishment of therapeutic cellular immunity. Artificial antigen-presenting cells (AAPCs) and their cell-free substitutes are designed to stimulate the expansion and acquisition of optimal therapeutic features of T cells before therapeutic infusion, without the need for autologous antigen-presenting cells. Compelling recent advances include fibroblast AAPCs that process antigens, magnetic beads that are antigen specific, novel T-cell costimulatory combinations, the augmentation of therapeutic potency of adoptively transferred T lymphocytes by interleukin-15, and the safe use of dendritic cell-derived exosomes pulsed with tumor antigen. Whereas the safety and potency of the various systems warrant further preclinical and clinical studies, these emerging technologies are poised to have a major impact on adoptive T-cell therapy and the investigation of T cell-mediated immunity. PMID:15060556

  19. Mini-review: Strategies for Variation and Evolution of Bacterial Antigens

    PubMed Central

    Foley, Janet

    2015-01-01

    Across the eubacteria, antigenic variation has emerged as a strategy to evade host immunity. However, phenotypic variation in some of these antigens also allows the bacteria to exploit variable host niches as well. The specific mechanisms are not shared-derived characters although there is considerable convergent evolution and numerous commonalities reflecting considerations of natural selection and biochemical restraints. Unlike in viruses, mechanisms of antigenic variation in most bacteria involve larger DNA movement such as gene conversion or DNA rearrangement, although some antigens vary due to point mutations or modified transcriptional regulation. The convergent evolution that promotes antigenic variation integrates various evolutionary forces: these include mutations underlying variant production; drift which could remove alleles especially early in infection or during life history phases in arthropod vectors (when the bacterial population size goes through a bottleneck); selection not only for any particular variant but also for the mechanism for the production of variants (i.e., selection for mutability); and overcoming negative selection against variant production. This review highlights the complexities of drivers of antigenic variation, in particular extending evaluation beyond the commonly cited theory of immune evasion. A deeper understanding of the diversity of purpose and mechanisms of antigenic variation in bacteria will contribute to greater insight into bacterial pathogenesis, ecology and coevolution with hosts. PMID:26288700

  20. The symbiotic role of O-antigen of Burkholderia symbiont in association with host Riptortus pedestris.

    PubMed

    Kim, Jiyeun Kate; Park, Ha Young; Lee, Bok Luel

    2016-07-01

    Riptortus pedestris harboring Burkholderia symbiont is a useful symbiosis model to study the molecular interactions between insects and bacteria. We recently reported that the lipopolysaccharide O-antigen is absent in the Burkholderia symbionts isolated from Riptortus guts. Here, we investigated the symbiotic role of O-antigen comprehensively in the Riptortus-Burkholderia model. Firstly, Burkholderia mutant strains deficient of O-antigen biosynthesis genes were generated and confirmed for their different patterns of the lipopolysaccharide by electrophoretic analysis. The O-antigen-deficient mutant strains initially exhibited a reduction of infectivity, having significantly lower level of symbiont population at the second-instar stage. However, both the wild-type and O-antigen mutant symbionts exhibited a similar level of symbiont population from the third-instar stage, indicating that the O-antigen deficiency did not affect the bacterial persistence in the host midgut. Taken together, we showed that the lipopolysaccharide O-antigen of gut symbiont plays an exclusive role in the initial symbiotic association.

  1. Tandem repeat protein as potential diagnostic antigen for Trypanosoma evansi infection.

    PubMed

    Thuy, Nguyen Thu; Goto, Yasuyuki; Lun, Zhao-Rong; Kawazu, Shin-Ichiro; Inoue, Noboru

    2012-02-01

    Trypanosoma evansi infection (surra) causes significant losses in livestock production in tropical and sub-tropical areas. The current ELISA recommended by OIE for diagnosis of the disease is based on trypanosome lysate antigen. However, antigenic variation and unstable nature of cell lysate antigen make it difficult to standardize the assay. Thus, there are needs to develop recombinant antigen-based ELISA that improve stability, sensitivity, and specificity of the test. Since tandem repeat (TR) proteins of trypanosomatid parasites generally possess high antigenicity, they have been considered to be the promising antigens for trypanosomosis and leishmaniosis. In this study, IgG responses against 14 recombinant TR proteins of trypanosomes were examined by ELISA. Serum samples were obtained from three water buffaloes experimentally infected with T. evansi. Since Trypanosoma congolense GM6 (TcoGM6) elicited highest IgG responses to all water buffaloes, we further bioinformatically and molecular biologically identified Trypanosoma brucei brucei GM6 (TbbGM6) and T. evansi GM6 (TeGM6) TR genes, respectively. As expected, predicted amino acid sequences of TbbGM6 and TeGM6 were identical while the nucleic acid sequence homology between TbbGM6 and TcoGM6 was 63.8%. All buffaloes became clearly positive in recombinant TbbGM6 (rTbbGM6)-based ELISA at 48 days post-infection, suggesting that rTbbGM6 is usable as a serodiagnostic antigen for chronic T. evansi infection.

  2. Genetic Analysis for the Lack of Expression of the O157 Antigen in an O Rough:H7 Escherichia coli Strain ▿

    PubMed Central

    Rump, Lydia V.; Feng, Peter C. H.; Fischer, Markus; Monday, Steven R.

    2010-01-01

    The O-antigen (rfb) operon and related genes of MA6, an O rough:H7 Shiga-toxigenic Escherichia coli strain, were examined to determine the cause of the lack of O157 expression. A 1,310-bp insertion, homologous to IS629, was observed within its gne gene. trans complementation with a functional gne gene from O157:H7 restored O157 antigen expression in MA6. PMID:19948859

  3. Stimulation of mouse lymphocytes by a mitogen derived from Mycoplasma arthritidis. VII. Responsiveness is associated with expression of a product(s) of the V beta 8 gene family present on the T cell receptor alpha/beta for antigen.

    PubMed

    Cole, B C; Kartchner, D R; Wells, D J

    1989-06-15

    Mycoplasma arthritidis produces a potent soluble T lymphocyte mitogen (MAM) which is dependent upon accessory cells bearing the alpha-chain of the I-E molecule (E alpha). Lymphocytes from the RIIIS mouse strain which possess E alpha yet whose T cells fail to recognize the MAM-E alpha complex were shown not to express V beta 8.1, 8.2 or 8.3 gene products present on the TCR-alpha/beta by virtue of their lack of reactivity with the F23.1 mAb. Because lymphocytes from the congenic B10.RIII mouse strain reacted positively with F23.1, we examined the progeny from (RIIIS x B10.RIII)F1 x RIIIS backcross mice for cosegregation of lymphocytes expressing F23.1-reactive sites and ability to proliferate in response to MAM. Whereas lymphocytes of all mice responded to Con A, only lymphocytes from progeny expressing F23.1-reactive cells responded to MAM. Similar studies were conducted on progeny from the F23.1- SWR mouse which was backcrossed to (SWR x B10.RIII)F1 mice. Of the E alpha-bearing progeny, there was a direct correlation between lymphocyte expression of F23.1 determinants and ability to respond to MAM. These results established that MAM reactivity was dependent upon a product(s) of the V beta locus of the TCR-alpha/beta. Nodal and thymic lymphocytes cultured for 3 days in vitro with MAM exhibited clonal expansion of F23.1 and F23.2-reactive cells as compared with cultures treated with Con A. We also demonstrated that F23.1 and F23.2 mAb inhibited the ability of lymphocytes to proliferate in response to MAM but had little effect on responses to Con A. The combined data suggest that the MAM-E alpha complex can utilize a V beta 8 gene product(s) on the TCR-alpha/beta.

  4. Somatic cell genetic analysis of human cell surface antigens 5.1H11 and F35/9 (gp45).

    PubMed

    Rettig, W J; Triche, T J; Bander, N H

    1990-01-01

    Serologic analysis of rodent-human somatic cell hybrids has permitted the assignment of loci coding for cell surface differentiation antigens 5.1H11 (gene symbol MSK39) and F35/9 (MSK40) to human chromosomes 11q13-qter and 22, respectively. Both antigens are expressed in hybrids constructed with antigen-positive human cells and certain hybrids constructed with antigen-negative human cells, indicating that the coding genes are not irreversibly silenced in human nonexpressor cells. Antigens 5.1H11 and F35/9, and at least six additional cell surface antigens encoded by chromosomes 11 and 22, are expressed on human Ewing sarcoma and peripheral neuroepithelioma cells, providing selectable markers for isolating and characterizing the specific t(11;22)(q24;q12) marker chromosomes of these tumors in interspecies hybrids.

  5. Minor histocompatibility antigens: past, present, and future.

    PubMed

    Spierings, Eric

    2014-10-01

    Minor histocompatibility (H) antigens are key molecules driving allo-immune responses in both graft-versus-host-disease (GvHD) and in graft-versus-leukemia (GvL) reactivity in human leukocyte antigen (HLA)-matched hematopoietic stem-cell transplantation (HSCT). Dissection of the dual function of minor H antigens became evident through their different modes of tissue and cell expression, i.e. hematopoietic system-restricted or broad. Broadly expressed minor H antigens can cause both GvHD and GvL effects, while hematopoietic system-restricted minor H antigens are more prone to induce GvL responses. This phenomenon renders the latter group of minor H antigens as curative tools for HSCT-based immunotherapy of hematological malignancies and disorders, in which minor H antigen-specific responses are enhanced in order to eradicate the malignant cells. This article describes the immunogenetics of minor H antigens and methods that have been developed to identify them. Moreover, it summarizes the clinical relevance of minor H antigens in transplantation, with special regards to allogeneic HSCT and solid-organ transplantation.

  6. Tumor-associated antigens in gynecologic cancer.

    PubMed

    DiSaia, P J

    1975-12-01

    If the study of tumor immunology is to have a profound impact on clinical medicine, certain hypotheses must be proven to be valid. First and foremost, it must be demonstrated that malignant tissue possesses antigenic substances (probably protein moieties) that are unique to that particular malignant process. In addition, these antigenic substances must be very similar in histologically similar tumors. Second, the host defense mechanisms must be capable of reacting to these tumor-associated antigens. The reaction is, of course, necessary in order to develop both diagnostic and therapeutic routes of application. The reaction of the immunologic system to these tumor-associated antigens could be monitored as an early serodiagnostic tool for subclinical cancer, and the cytotoxic reaction holds great promise as an immunotherapeutic tool. The essence of tumor immunologic research can thus be stated in the form of the following questions: 1. Do histologically similar cancers from identical primary sites share common tumor-associated antigens? 2. Does the immunologic system react to these antigens? 3. Can this reaction be assayed on one hand for serodiagnosis and augmented on the other for immunotherapy? Specific antigens have been found in animal tumors and have been divided into two classes: the viral induced tumors, which share common antigens when caused by the same viral agent, and carcinogen-induced tumors, which appear to have unique antigenic determinants for each tumor. In recent years a great many human tumors have been found to have tumor-associated antigens; these include colonic carcinoma, neuroblastoma, melanoma, soft tissue and osteogenic sarcoma, bladder carcinoma and Burkitt's lymphoma. This report includes evidence for the existence of such antigens in adenocarcinoma of the ovary and squamous cell carcinoma of the cervix. The laboratory evidence that has been presented would suggest that there are both a cell-mediated response and humoral response to the

  7. Integrating influenza antigenic dynamics with molecular evolution

    PubMed Central

    Bedford, Trevor; Suchard, Marc A; Lemey, Philippe; Dudas, Gytis; Gregory, Victoria; Hay, Alan J; McCauley, John W; Russell, Colin A; Smith, Derek J; Rambaut, Andrew

    2014-01-01

    Influenza viruses undergo continual antigenic evolution allowing mutant viruses to evade host immunity acquired to previous virus strains. Antigenic phenotype is often assessed through pairwise measurement of cross-reactivity between influenza strains using the hemagglutination inhibition (HI) assay. Here, we extend previous approaches to antigenic cartography, and simultaneously characterize antigenic and genetic evolution by modeling the diffusion of antigenic phenotype over a shared virus phylogeny. Using HI data from influenza lineages A/H3N2, A/H1N1, B/Victoria and B/Yamagata, we determine patterns of antigenic drift across viral lineages, showing that A/H3N2 evolves faster and in a more punctuated fashion than other influenza lineages. We also show that year-to-year antigenic drift appears to drive incidence patterns within each influenza lineage. This work makes possible substantial future advances in investigating the dynamics of influenza and other antigenically-variable pathogens by providing a model that intimately combines molecular and antigenic evolution. DOI: http://dx.doi.org/10.7554/eLife.01914.001 PMID:24497547

  8. Research progress on surface antigen 1 (SAG1) of Toxoplasma gondii

    PubMed Central

    2014-01-01

    Toxoplasma gondii is an obligate intracellular parasitic protozoan that has a wide host range and causes a zoonotic parasitosis called toxoplasmosis. This infection causes significant morbidity, costs for care and loss of productivity and suffering. The most effective measures to minimize this parasite’s harm to patients are prompt diagnosis and treatment and preventing infection. A parasite surface antigen, SAG1, is considered an important antigen for the development of effective diagnostic tests or subunit vaccines. This review covers several aspects of this antigen, including its gene structure, contribution to host invasion, mechanisms of the immune responses and its applications for diagnosis and vaccine development. This significant progress on this antigen provides foundations for further development of more effective and precise approaches to diagnose toxoplasmosis in the clinic, and also have important implications for exploring novel measures to control toxoplasmosis in the near future. PMID:24726014

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

    SciTech Connect

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

    2010-03-15

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

  10. Lamprey VLRB response to influenza virus supports universal rules of immunogenicity and antigenicity.

    PubMed

    Altman, Meghan O; Bennink, Jack R; Yewdell, Jonathan W; Herrin, Brantley R

    2015-01-01

    Immunoglobulins (Igs) are a crown jewel of jawed vertebrate evolution. Through recombination and mutation of small numbers of genes, Igs can specifically recognize a vast variety of natural and man-made organic molecules. Jawless vertebrates evolved a parallel system of humoral immunity, which recognizes antigens not with Ig, but with a structurally unrelated receptor called the variable lymphocyte receptor B (VLRB). We exploited the convergent evolution of Ig and VLRB antibodies (Abs) to investigate if intrinsic chemical features of foreign proteins determine their antigenicity and immunogenicity. Surprisingly, we find lamprey VLRB and mouse Ig responses to influenza A virus are extremely similar. Each focuses ~80% of the response on hemagglutinin (HA), mainly through recognition of the major antigenic sites in the HA globular head domain. Our findings predict basic conservation of Ab responses to protein antigens, strongly supporting the use of animal models for understanding human Ab responses to viruses and protein immunogens. PMID:26252514

  11. Lamprey VLRB response to influenza virus supports universal rules of immunogenicity and antigenicity.

    PubMed

    Altman, Meghan O; Bennink, Jack R; Yewdell, Jonathan W; Herrin, Brantley R

    2015-08-07

    Immunoglobulins (Igs) are a crown jewel of jawed vertebrate evolution. Through recombination and mutation of small numbers of genes, Igs can specifically recognize a vast variety of natural and man-made organic molecules. Jawless vertebrates evolved a parallel system of humoral immunity, which recognizes antigens not with Ig, but with a structurally unrelated receptor called the variable lymphocyte receptor B (VLRB). We exploited the convergent evolution of Ig and VLRB antibodies (Abs) to investigate if intrinsic chemical features of foreign proteins determine their antigenicity and immunogenicity. Surprisingly, we find lamprey VLRB and mouse Ig responses to influenza A virus are extremely similar. Each focuses ~80% of the response on hemagglutinin (HA), mainly through recognition of the major antigenic sites in the HA globular head domain. Our findings predict basic conservation of Ab responses to protein antigens, strongly supporting the use of animal models for understanding human Ab responses to viruses and protein immunogens.

  12. Selective distribution of Pseudomonas aeruginosa O-antigen among strains producing group I pilin.

    PubMed

    Allison, Tara M; Castric, Peter

    2016-02-01

    Strains of Pseudomonas aeruginosa that produce type IVa pili categorized as group I have the potential to covalently attach an O-antigen repeating unit to the pilin C-terminal residue. PCR, employing primers targeting a conserved region of a group-I-specific gene, was used to provide evidence that 110 of 206 clinical isolates studied had the capability of producing this type of pilus. The potential of P. aeruginosa to produce a particular O-antigen type is determined by the presence of a specific biosynthetic gene cluster. The distribution of these gene clusters among the isolates studied was determined using a second PCR procedure. The results of these studies showed that the O-antigen repeating unit types associated with group I pilin producers were significantly different from those found in the non-group I pilin strains. In addition, the predicted ability to express O-antigen repeating units composed of four sugars, and the ability of the glycan to express a negative charge were associated with group I pilin producing strains. The results presented suggest that these properties specifically enhance group I pilus function and that the commonality of pilus and O-antigen types may be useful as targets in disease intervention.

  13. A computational framework for influenza antigenic cartography.

    PubMed

    Cai, Zhipeng; Zhang, Tong; Wan, Xiu-Feng

    2010-10-07

    Influenza viruses have been responsible for large losses of lives around the world and continue to present a great public health challenge. Antigenic characterization based on hemagglutination inhibition (HI) assay is one of the routine procedures for influenza vaccine strain selection. However, HI assay is only a crude experiment reflecting the antigenic correlations among testing antigens (viruses) and reference antisera (antibodies). Moreover, antigenic characterization is usually based on more than one HI dataset. The combination of multiple datasets results in an incomplete HI matrix with many unobserved entries. This paper proposes a new computational framework for constructing an influenza antigenic cartography from this incomplete matrix, which we refer to as Matrix Completion-Multidimensional Scaling (MC-MDS). In this approach, we first reconstruct the HI matrices with viruses and antibodies using low-rank matrix completion, and then generate the two-dimensional antigenic cartography using multidimensional scaling. Moreover, for influenza HI tables with herd immunity effect (such as those from Human influenza viruses), we propose a temporal model to reduce the inherent temporal bias of HI tables caused by herd immunity. By applying our method in HI datasets containing H3N2 influenza A viruses isolated from 1968 to 2003, we identified eleven clusters of antigenic variants, representing all major antigenic drift events in these 36 years. Our results showed that both the completed HI matrix and the antigenic cartography obtained via MC-MDS are useful in identifying influenza antigenic variants and thus can be used to facilitate influenza vaccine strain selection. The webserver is available at http://sysbio.cvm.msstate.edu/AntigenMap.

  14. BINDING OF ANTIGEN BY IMMUNOCYTES

    PubMed Central

    Bystryn, Jean-Claude; Siskind, Gregory W.; Uhr, Jonathan W.

    1973-01-01

    The binding of antigen to cells with antibody on their surface has been studied in a model system consisting of murine myeloma cells (MOPC 315) and DNP conjugates. Specific binding occurred between the DNP groups of DNP conjugates and cell surface immunoglobulin. Using this model, the binding affinities of multivalent and univalent DNP conjugates were measured directly by equilibrium-binding techniques and indirectly by displacement of bound conjugate with univalent hapten. With both approaches the multivalent conjugate was shown to bind to cells with an avidity 100–300 fold greater than the univalent hapten. Nonspecific binding of unrelated protein and repeated washing of cells was found to markedly dedecrease the specific binding of univalent conjugates, presumably because the relatively weak bonds dissociate readily. PMID:4734402

  15. Recent improvements in antigene technology.

    PubMed

    Buchini, Sabrina; Leumann, Christian J

    2003-12-01

    DNA triple-helix-based approaches to control and modulate cellular functions on the level of genomic DNA (antigene technology) suffered in the past from a stepmother-like treatment in comparison to the flourishing field of oligonucleotide-based control of translation (antisense technology). This was mostly due to lack of affinity of triplex-forming oligonucleotides to their DNA target, to sequence restriction constraints imposed by the triple helical recognition motifs and by open questions to the accessibility of the target DNA. Recent developments in the area have brought about new bases that specifically recognize pyrimidine-purine inversion sites as well as sugar modifications, for example, the 2'-aminoethoxy-oligonucleotides or oligonucleotides based on the locked nucleic acid sugar unit, which greatly enhance triplex stability and alleviate in part the sequence restriction constraints. With this, sequence-specific genomic DNA manipulation is starting to become a useful tool in biotechnology.

  16. [Clonality lymphoid study through rearrangement analysis of antigen receptor].

    PubMed

    Villamizar-Rivera, Nicolás; Olaya, Natalia

    2015-01-01

    As a rule, malignant lymphoid proliferations are clonal. While most of the time the biological potential can be established through routine pathologic examination and auxiliary techniques, some cases are difficult to classify. Moreover, there are situations in which there are dominant clones whose analysis are important, such as occur in autoimmune diseases and immunodeficiency. This paper presents in an understandable way the main techniques for the study of clonality in lymphoid lesions, i.e. the analysis of rearrangements of antigen receptor genes by multiplex polymerase chain reaction (PCR) based tests.

  17. Direct Introduction of Genes into Rats and Expression of the Genes

    NASA Astrophysics Data System (ADS)

    Benvenisty, Nissim; Reshef, Lea

    1986-12-01

    A method of introducing actively expressed genes into intact mammals is described. DNA precipitated with calcium phosphate has been injected intraperitoneally into newborn rats. The injected genes have been taken up and expressed by the animal tissues. To examine the generality of the method we have injected newborn rats with the chloramphenicol acetyltransferase prokaryotic gene fused with various viral and cellular gene promoters and the gene for hepatitis B surface antigen, and we observed appearance of chloramphenicol acetyltransferase activity and hepatitis B surface antigen in liver and spleen. In addition, administration of genes coding for hormones (insulin or growth hormone) resulted in their expression.

  18. Pecking order among tumor-specific antigens.

    PubMed

    Urban, J L; Van Waes, C; Schreiber, H

    1984-02-01

    The ultraviolet light-induced fibrosarcoma 1591 is regularly rejected upon transplantation into young syngeneic mice; in rare instances, however, this tumor grows progressively and the tumors that develop are then heritably stable variant progressor tumors (1591-PRO tumors). In this study, we have induced transplantation resistance to 1591-PRO tumors and determined which antigens were recognized by mice that rejected these progressor tumors. We found that cytolytic T cells of such mice recognized a 1591-specific antigen that was present not only on all the independently derived 1591-PRO tumors but also on the parental regressor tumor (1591-RE). However, the cytolytic immune response of mice that rejected 1591-RE lysed 1591-RE tumor cells but not 1591-PRO tumor cells. Thus, the 1591-RE tumor seemed to express two antigens that were specific for tumors of the 1591 lineage, one that was lost and a second that was retained by 1591-PRO tumor cells. Mice challenged with 1591-R# tumor cells mounted a response to only one of the tumor-specific antigens which was therefore "immunodominant" over the other "immunorecessive" antigen. This immunorecessive antigen became the target of the immune response once the immunodominant antigen was lost. This "pecking order" interfered with the simultaneous recognition of two tumor-specific antigens and this mechanism may favor immune escape.

  19. Lipase Processing of Complex Lipid Antigens.

    PubMed

    Sander, Peter; Becker, Katja; Molin, Michael Dal

    2016-09-22

    Mycobacterium tuberculosis synthesizes a wide variety of complex lipids that can serve as antigens in immune recognition of the bacterium. In this issue of Cell Chemical Biology, Gilleron et al. (2016) identify key enzymes essential for lipid antigen processing, which is required for CD1b-restricted T cell activation. PMID:27662250

  20. Vaccinia Virus A35R Inhibits MHC Class II Antigen Presentation

    PubMed Central

    Rehm, Kristina E.; Connor, Ramsey F.; Jones, Gwendolyn J.B.; Yimbu, Kenneth; Roper, Rachel L.

    2009-01-01

    The Vaccinia virus gene A35R (Copenhagen designation) is highly conserved in mammalian-tropic poxviruses and is an important virulence factor, but its function was unknown. We show herein that A35 does not affect viral infectivity, apoptosis induction, or replication; however, we found that A35 significantly inhibited MHC class II-restricted antigen presentation, immune priming of T lymphocytes, and subsequent chemokine and cytokine synthesis. A35 localized to endosomes and reduced the amount of a model antigenic peptide displayed in the cleft of class II MHC. In addition, A35 decreased VV specific T cell responses in vivo. Thus, this is the first report identifying a function for the A35 protein in virulence as well as the first report identifying a VV gene that inhibits peptide antigen presentation. PMID:19954808

  1. Designing chimeric antigen receptors to effectively and safely target tumors.

    PubMed

    Jensen, Michael C; Riddell, Stanley R

    2015-04-01

    The adoptive transfer of T cells engineered to express artificial chimeric antigen receptors CARs) that target a tumor cell surface molecule has emerged as an exciting new approach for cancer immunotherapy. Clinical trials in patients with advanced B cell malignancies treated with CD19-specific CAR-modified T cells (CAR-T) have shown impressive antitumor efficacy, leading to optimism that this approach will be useful for treating common solid tumors. Because CAR-T cells recognize tumor cells independent of their expression of human leukocyte antigen (HLA) molecules, tumors that escape conventional T cells by downregulating HLA and/or mutating components of the antigen processing machinery can be eliminated. The ability to introduce or delete additional genes in T cells has the potential to provide therapeutic cell products with novel attributes that overcome impediments to immune mediated tumor elimination in immunosuppressive tumor microenvironments. This review will discuss recent concepts in the development of effective and safe synthetic CARs for adoptive T cell therapy (ACT).

  2. Accurate identification of paraprotein antigen targets by epitope reconstruction

    PubMed Central

    Sompuram, Seshi R.; Bastas, Gerassimos; Vani, Kodela

    2008-01-01

    We describe the first successful clinical application of a new discovery technology, epitope-mediated antigen prediction (E-MAP), to the investigation of multiple myeloma. Until now, there has been no reliable, systematic method to identify the cognate antigens of paraproteins. E-MAP is a variation of previous efforts to reconstruct the epitopes of paraproteins, with the significant difference that it provides enough epitope sequence data so as to enable successful protein database searches. We first reconstruct the paraprotein's epitope by analyzing the peptides that strongly bind. Then, we compile the data and interrogate the nonredundant protein database, searching for a close match. As a clinical proof-of-concept, we apply this technology to uncovering the protein targets of para-proteins in multiple myeloma (MM). E-MAP analysis of 2 MM paraproteins identified human cytomegalovirus (HCMV) as a target in both. E-MAP sequence analysis determined that one para-protein binds to the AD-2S1 epitope of HCMV glycoprotein B. The other binds to the amino terminus of the HCMV UL-48 gene product. We confirmed these predictions using immunoassays and immunoblot analyses. E-MAP represents a new investigative tool for analyzing the role of chronic antigenic stimulation in B-lymphoproliferative disorders. PMID:17878398

  3. Lymphoid-specific antigen: distribution and behaviour

    PubMed Central

    Potworowski, E. F.; Nairn, R. C.

    1968-01-01

    The distribution of a lymphoid-specific antigen has been studied by immunofluorescence in the thymus and other lymphoid organs of the rat and in the thymuses of other vertebrate species. It was demonstrable in all rat lymphocytes except those of the bone marrow. The thymic lymphocytes of all warm-blooded vertebrates also reacted with the lymphoid-specific serum. Plasma cells in antigenically stimulated lymph nodes did not seem to possess the antigen but similar cells appearing in lymph nodes of rats which had been irradiated and injected with marrow cells of the same strain showed a strong reaction with the antiserum. The antigen is depleted in human and murine leukaemic lymphocytes. X-irradiation did not appear to affect the antigen significantly in cells surviving the treatment. ImagesFIG. 1-6 PMID:4871349

  4. Expression of SV40 T antigen under control of rabbit uteroglobin promoter in transgenic mice.

    PubMed

    DeMayo, F J; Finegold, M J; Hansen, T N; Stanley, L A; Smith, B; Bullock, D W

    1991-08-01

    The rabbit uteroglobin gene is expressed in the lungs and reproductive tracts of male and female rabbits. To examine whether the promoter region of the uteroglobin gene could be used to target a heterologous gene to the lungs of transgenic mice, a fusion gene consisting of 3.3 kb of the 5'-flanking region of the rabbit uteroglobin gene and the large T antigen gene of the SV40 virus was constructed and microinjected into the pronuclei of one-cell mouse embryos. Eleven founder transgenic mice (5 female and 6 male) were generated. Seven of these mice developed bronchioalveolar neoplasms. Four of the founder males also developed primitive undifferentiated urogenital tract tumors. One founder female and one female offspring of a founder male developed glandular paraovarian tumors. Northern analysis revealed that the predominant site of expression of the transgene was the lung. Immunohistochemical staining showed T antigen predominantly in epithelial cells lining the bronchioles, the submucosal glands of the trachea, and the neoplasms. There appeared to be a high level of mosaicism for the transgene in the founder mice, with poor transmission of the transgene to subsequent generations. This suggests that, under the control of the uteroglobin promoter, the T antigen gene may be lethal to the fetus.

  5. Calcium-dependent antigen binding as a novel modality for antibody recycling by endosomal antigen dissociation.

    PubMed

    Hironiwa, N; Ishii, S; Kadono, S; Iwayanagi, Y; Mimoto, F; Habu, K; Igawa, T; Hattori, K

    2016-01-01

    The pH-dependent antigen binding antibody, termed a recycling antibody, has recently been reported as an attractive type of second-generation engineered therapeutic antibody. A recycling antibody can dissociate antigen in the acidic endosome, and thus bind to its antigen multiple times. As a consequence, a recycling antibody can neutralize large amounts of antigen in plasma. Because this approach relies on histidine residues to achieve pH-dependent antigen binding, which could limit the epitopes that can be targeted and affect the rate of antigen dissociation in the endosome, we explored an alternative approach for generating recycling antibodies. Since calcium ion concentration is known to be lower in endosome than in plasma, we hypothesized that an antibody with antigen-binding properties that are calcium-dependent could be used as recycling antibody. Here, we report a novel anti-interleukin-6 receptor (IL-6R) antibody, identified from a phage library that binds to IL-6R only in the presence of a calcium ion. Thermal dynamics and a crystal structure study revealed that the calcium ion binds to the heavy chain CDR3 region (HCDR3), which changes and possibly stabilizes the structure of HCDR3 to make it bind to antigen calcium dependently (PDB 5AZE). In vitro and in vivo studies confirmed that this calcium-dependent antigen-binding antibody can dissociate its antigen in the endosome and accelerate antigen clearance from plasma, making it a novel approach for generating recycling antibody.

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

    PubMed Central

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

    2015-01-01

    Human leukocyte antigen-E (HLA-E) is one of the most extensively studied non-classical MHC class I molecules that is almost non-polymorphic. Only two alleles (HLA-E*0101 and HLA-E*0103) are found in worldwide populations, and suggested to be functional differences between these variants. The HLA-E molecule can contribute to the escape of cancer cells from host immune surveillance. However, it is still unknown whether HLA-E gene polymorphisms might play a role in cancer immune escape. To explore the association between HLA-E alleles and the susceptibility to serous ovarian cancer (SOC), 85 primary SOC patients and 100 healthy women were enrolled. Here, we indicated that high frequency of HLA-E*0103 allele existed in SOC patients by the allele-specific quantitative real-time PCR method. The levels of HLA-E protein expression in SOC patients with the HLA-E*0103 allele were higher than those with the HLA-E*0101 allele using immunohistochemistry analysis. The cell surface expression and functional differences between the two alleles were verified by K562 cells transfected with HLA-E*0101 or HLA-E*0103 allelic heavy chains. The HLA-E*0103 allele made the transfer of the HLA-E molecule to the cell surface easier, and HLA-E/peptides complex more stable. These differences ultimately influenced the function of natural killer cells, showing that the cells transfected with HLA-E*0103 allele inhibited natural killer cells to lysis. This study reveals a novel mechanism regarding the susceptibility to SOC, which is correlated with the HLA-E*0103 allele. PMID:25711417

  7. Tiny T antigen: an autonomous polyomavirus T antigen amino-terminal domain.

    PubMed Central

    Riley, M I; Yoo, W; Mda, N Y; Folk, W R

    1997-01-01

    Three mRNAs from the murine polyomavirus early region encode the three well-characterized tumor antigens. We report the existence of a fourth alternatively spliced mRNA which encodes a fourth tumor antigen, tiny T antigen, which comprises the amino-terminal domain common to all of the T antigens but is extended by six unique amino acid residues. The amount of tiny T antigen in infected cells is small because of its short half-life. Tiny T antigen stimulates the ATPase activity of Hsc70, most likely because of its DnaJ-like motif. The common amino-terminal domain may interface with chaperone complexes to assist the T antigens in carrying out their diverse functions of replication, transcription, and transformation in the appropriate cellular compartments. PMID:9223500

  8. Universal artificial antigen presenting cells to selectively propagate T cells expressing chimeric antigen receptor independent of specificity.

    PubMed

    Rushworth, David; Jena, Bipulendu; Olivares, Simon; Maiti, Sourindra; Briggs, Neima; Somanchi, Srinivas; Dai, Jianliang; Lee, Dean; Cooper, Laurence J N

    2014-05-01

    T cells genetically modified to stably express immunoreceptors are being assessed for therapeutic potential in clinical trials. T cells expressing a chimeric antigen receptor (CAR) are endowed with a new specificity to target tumor-associated antigen (TAA) independent of major histocompatibility complex. Our approach to nonviral gene transfer in T cells uses ex vivo numeric expansion of CAR T cells on irradiated artificial antigen presenting cells (aAPC) bearing the targeted TAA. The requirement for aAPC to express a desired TAA limits the human application of CARs with multiple specificities when selective expansion through coculture with feeder cells is sought. As an alternative to expressing individual TAAs on aAPC, we expressed 1 ligand that could activate CAR T cells for sustained proliferation independent of specificity. We expressed a CAR ligand (designated CARL) that binds the conserved IgG4 extracellular domain of CAR and demonstrated that CARL aAPC propagate CAR T cells of multiple specificities. CARL avoids technical issues and costs associated with deploying clinical-grade aAPC for each TAA targeted by a given CAR. Using CARL enables 1 aAPC to numerically expand all CAR T cells containing the IgG4 domain, and simplifies expansion, testing, and clinical translation of CAR T cells of any specificity. PMID:24714354

  9. Universal Artificial Antigen Presenting Cells to Selectively Propagate T Cells Expressing Chimeric Antigen Receptor Independent of Specificity

    PubMed Central

    Rushworth, David; Jena, Bipulendu; Olivares, Simon; Maiti, Sourindra; Briggs, Neima; Somanchi, Srinivas; Dai, Jianliang; Lee, Dean; Cooper, Laurence J. N.

    2014-01-01

    T cells genetically modified to stably express immunoreceptors are being assessed for therapeutic potential in clinical trials. T cells expressing a chimeric antigen receptor (CAR) are endowed with a new specificity to target tumor-associated antigen (TAA) independent of major histocompatibility complex. Our approach to non-viral gene transfer in T cells uses ex vivo numeric expansion of CAR+ T cells on irradiated artificial antigen presenting cells (aAPC) bearing the targeted TAA. The requirement for aAPC to express a desired TAA limits the human application of CARs with multiple specificities when selective expansion through co-culture with feeder cells is sought. As an alternative to expressing individual TAAs on aAPC, we expressed one ligand that could activate CAR+ T cells for sustained proliferation independent of specificity. We expressed a CAR ligand (designated CARL) that binds the conserved IgG4 extracellular domain of CAR and demonstrated CARL+ aAPC propagate CAR+ T cells of multiple specificities. CARL avoids technical issues and costs associated with deploying clinical-grade aAPC for each TAA targeted by a given CAR. Employing CARL enables one aAPC to numerically expand all CAR+ T cells containing the IgG4 domain, and simplifies expansion, testing, and clinical translation of CAR+ T cells of any specificity. PMID:24714354

  10. Novel mouse model for carcinoembryonic antigen-based therapy.

    PubMed

    Chan, Carlos H F; Stanners, Clifford P

    2004-06-01

    Many novel cancer therapies, including immunotherapy and gene therapy, are specifically targeted to tumor-associated molecules, among which carcinoembryonic antigen (CEA) represents a popular example. Discrepancies between preclinical experimental data in animal models and clinical outcome in terms of therapeutic response and toxicity, however, often arise. Preclinical testing can be compromised by the lack of CEA and other closely related human CEA family members in rodents, which lack analogous genes for most human CEA family members. Here, we report the construction of a transgenic mouse with a 187-kb human bacterial artificial chromosome (CEABAC) that contains part of the human CEA family gene cluster including complete human CEA (CEACAM5), CEACAM3, CEACAM6, and CEACAM7 genes. The spatiotemporal expression pattern of these genes in the CEABAC mice was found to be remarkably similar to that of humans. This novel mouse will ensure better assessment than previously utilized models for the preclinical testing of CEA-targeted therapies and perhaps allow the testing of CEACAM6, which is overexpressed in many solid tumors and leukemias, as a therapeutic target. Moreover, expression of CEA family genes in gastrointestinal, breast, hematopoietic, urogenital, and respiratory systems could facilitate other clinical applications, such as the development of therapeutic agents against Neisseria gonorrhoeae infections, which use CEA family members as major receptors. PMID:15194045

  11. Modification of a salmonid alphavirus replicon vector for enhanced expression of heterologous antigens.

    PubMed

    Guo, Tz-Chun; Johansson, Daniel X; Liljeström, Peter; Evensen, Øystein; Haugland, Øyvind

    2015-03-01

    A salmonid alphavirus (SAV) replicon has been developed to express heterologous antigens but protein production was low to modest compared with terrestrial alphavirus replicons. In this study, we have compared several modifications to a SAV replicon construct and analysed their influence on foreign gene expression. We found that an insertion of a translational enhancer consisting of the N-terminal 102 nt of the capsid gene, together with a nucleotide sequence encoding the foot-and-mouth disease virus (FMDV) 2A peptide, caused a significant increase in EGFP reporter gene expression. The importance of fusing a hammerhead (HH) ribozyme sequence at the 5' end of the viral genome was also demonstrated. In contrast, a hepatitis D virus ribozyme (HDV-RZ) sequence placed at the 3' end did not augment expression of inserted genes. Taken together, we have developed a platform for optimized antigen production, which can be applied for immunization of salmonid fish in the future. PMID:25395591

  12. Posttranslational modification at the N terminus of the human adenovirus type 12 E1A 235R tumor antigen.

    PubMed Central

    Lucher, L A; Brackmann, K H; Symington, J S; Green, M

    1986-01-01

    The adenovirus E1A transforming region, which encodes immortalization, partial cell transformation, and gene activation functions, expresses two early mRNAs, 13S and 12S. Multiple-T antigen species with different electrophoretic mobilities are formed from each mRNA, presumably by unknown posttranslational modifications. The adenovirus type 12 (Ad12) 13S and 12S mRNAs encode E1A T antigens of 266 and 235 amino acid residues (266R and 235R), respectively. To study possible posttranslational processing at the N and C termini and to distinguish between the Ad12 266R and 235R T antigens, we prepared antibodies targeted to synthetic peptides encoded at the common C (peptide 204) and N (peptide 202) termini of the 266R and 235R T antigens and at the unique internal domain of the 266R T antigen (peptide 206). The specificity of each anti-peptide antibody was confirmed by immunoprecipitation of the 266R and 235R T antigens produced in Escherichia coli. Immunoprecipitation analysis of the E1A T antigens synthesized in Ad12-infected KB cells revealed the following. Antibody to the common C terminus recognized three T antigens with apparent Mrs of 43,000, 42,000, and 39,000 (43K, 42K, and 39K). All three forms were phosphorylated and were present in both the nucleus and the cytoplasm. The 43K and 42K T antigens were rapidly synthesized during a 10-min pulse with [35S]methionine in Ad12-infected cells. The 43K T antigen had a half-life of 20 min, the 42K T antigen had a longer half-life of about 40 min, and the 39K T antigen became the predominant E1A T antigen. Antibodies to the unique region immunoprecipitated the 43K T antigen but not the 42K and 39K T antigens. Antibody to the N terminus immunoprecipitated the 43K and 42K T antigens but not the 39K T antigen, suggesting that the 39K T antigen possessed a modified N terminus. Partial N-terminal amino acid sequence analysis showed that the 43K and 42K T antigens contain methionine at residues 1 and 5, as predicted from the

  13. Bacteriophage-mediated Glucosylation Can Modify Lipopolysaccharide O-Antigens Synthesized by an ATP-binding Cassette (ABC) Transporter-dependent Assembly Mechanism*

    PubMed Central

    Mann, Evan; Ovchinnikova, Olga G.; King, Jerry D.; Whitfield, Chris

    2015-01-01

    Lysogenic bacteriophages may encode enzymes that modify the structures of lipopolysaccharide O-antigen glycans, altering the structure of the bacteriophage receptor and resulting in serotype conversion. This can enhance virulence and has implications for antigenic diversity and vaccine development. Side chain glucosylation is a common modification strategy found in a number of bacterial species. To date, glucosylation has only been observed in O-antigens synthesized by Wzy-dependent pathways, one of the two most prevalent O-antigen synthesis systems. Here we exploited a heterologous system to study the glucosylation potential of a model O-antigen produced in an ATP-binding cassette (ABC) transporter-dependent system. Although O-antigen production is cryptic in Escherichia coli K-12, because of a mutation in the synthesis genes, it possesses a prophage glucosylation cluster, which modifies the GlcNAc residue in an α-l-Rha-(1→3)-d-GlcNAc motif found in the original O16 antigen. Raoultella terrigena ATCC 33257 produces an O-antigen possessing the same disaccharide motif, but its assembly uses an ABC transporter-dependent system. E. coli harboring the R. terrigena O-antigen biosynthesis genes produced an O-antigen displaying reduced reactivity toward antisera raised against the native R. terrigena repeat structure, indicative of an altered chemical structure. Structural determination using NMR revealed the addition of glucose side chains to the repeat units. O-antigen modification was dependent on a functional ABC transporter, consistent with modification in the periplasm, and was eliminated by deletion of the glucosylation genes from the E. coli chromosome, restoring native level antisera sensitivity and structure. There are therefore no intrinsic mechanistic barriers for bacteriophage-mediated O-antigen glucosylation in ABC transporter-dependent pathways. PMID:26330553

  14. Bacteriophage-mediated Glucosylation Can Modify Lipopolysaccharide O-Antigens Synthesized by an ATP-binding Cassette (ABC) Transporter-dependent Assembly Mechanism.

    PubMed

    Mann, Evan; Ovchinnikova, Olga G; King, Jerry D; Whitfield, Chris

    2015-10-16

    Lysogenic bacteriophages may encode enzymes that modify the structures of lipopolysaccharide O-antigen glycans, altering the structure of the bacteriophage receptor and resulting in serotype conversion. This can enhance virulence and has implications for antigenic diversity and vaccine development. Side chain glucosylation is a common modification strategy found in a number of bacterial species. To date, glucosylation has only been observed in O-antigens synthesized by Wzy-dependent pathways, one of the two most prevalent O-antigen synthesis systems. Here we exploited a heterologous system to study the glucosylation potential of a model O-antigen produced in an ATP-binding cassette (ABC) transporter-dependent system. Although O-antigen production is cryptic in Escherichia coli K-12, because of a mutation in the synthesis genes, it possesses a prophage glucosylation cluster, which modifies the GlcNAc residue in an α-l-Rha-(1→3)-d-GlcNAc motif found in the original O16 antigen. Raoultella terrigena ATCC 33257 produces an O-antigen possessing the same disaccharide motif, but its assembly uses an ABC transporter-dependent system. E. coli harboring the R. terrigena O-antigen biosynthesis genes produced an O-antigen displaying reduced reactivity toward antisera raised against the native R. terrigena repeat structure, indicative of an altered chemical structure. Structural determination using NMR revealed the addition of glucose side chains to the repeat units. O-antigen modification was dependent on a functional ABC transporter, consistent with modification in the periplasm, and was eliminated by deletion of the glucosylation genes from the E. coli chromosome, restoring native level antisera sensitivity and structure. There are therefore no intrinsic mechanistic barriers for bacteriophage-mediated O-antigen glucosylation in ABC transporter-dependent pathways. PMID:26330553

  15. Bacteriophage-mediated Glucosylation Can Modify Lipopolysaccharide O-Antigens Synthesized by an ATP-binding Cassette (ABC) Transporter-dependent Assembly Mechanism.

    PubMed

    Mann, Evan; Ovchinnikova, Olga G; King, Jerry D; Whitfield, Chris

    2015-10-16

    Lysogenic bacteriophages may encode enzymes that modify the structures of lipopolysaccharide O-antigen glycans, altering the structure of the bacteriophage receptor and resulting in serotype conversion. This can enhance virulence and has implications for antigenic diversity and vaccine development. Side chain glucosylation is a common modification strategy found in a number of bacterial species. To date, glucosylation has only been observed in O-antigens synthesized by Wzy-dependent pathways, one of the two most prevalent O-antigen synthesis systems. Here we exploited a heterologous system to study the glucosylation potential of a model O-antigen produced in an ATP-binding cassette (ABC) transporter-dependent system. Although O-antigen production is cryptic in Escherichia coli K-12, because of a mutation in the synthesis genes, it possesses a prophage glucosylation cluster, which modifies the GlcNAc residue in an α-l-Rha-(1→3)-d-GlcNAc motif found in the original O16 antigen. Raoultella terrigena ATCC 33257 produces an O-antigen possessing the same disaccharide motif, but its assembly uses an ABC transporter-dependent system. E. coli harboring the R. terrigena O-antigen biosynthesis genes produced an O-antigen displaying reduced reactivity toward antisera raised against the native R. terrigena repeat structure, indicative of an altered chemical structure. Structural determination using NMR revealed the addition of glucose side chains to the repeat units. O-antigen modification was dependent on a functional ABC transporter, consistent with modification in the periplasm, and was eliminated by deletion of the glucosylation genes from the E. coli chromosome, restoring native level antisera sensitivity and structure. There are therefore no intrinsic mechanistic barriers for bacteriophage-mediated O-antigen glucosylation in ABC transporter-dependent pathways.

  16. Shared themes of antigenic variation and virulence in bacterial, protozoal, and fungal infections.

    PubMed Central

    Deitsch, K W; Moxon, E R; Wellems, T E

    1997-01-01

    Pathogenic microbes have evolved highly sophisticated mechanisms for colonizing host tissues and evading or deflecting assault by the immune response. The ability of these microbes to avoid clearance prolongs infection, thereby promoting their long-term survival within individual hosts and, through transmission, between hosts. Many pathogens are capable of extensive antigenic changes in the face of the multiple constitutive and dynamic components of host immune defenses. As a result, highly diverse populations that have widely different virulence properties can arise from a single infecting organism (clone). In this review, we consider the molecular and genetic features of antigenic variation and corresponding host-parasite interactions of different pathogenic bacterial, fungal, and protozoan microorganisms. The host and microbial molecules involved in these interactions often determine the adhesive, invasive, and antigenic properties of the infecting organisms and can dramatically affect the virulence and pathobiology of individual infections. Pathogens capable of such antigenic variation exhibit mechanisms of rapid mutability in confined chromosomal regions containing specialized genes designated contingency genes. The mechanisms of hypermutability of contingency genes are common to a variety of bacterial and eukaryotic pathogens and include promoter alterations, reading-frame shifts, gene conversion events, genomic rearrangements, and point mutations. PMID:9293182

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

    PubMed Central

    Klaenhammer, Todd R.

    2016-01-01

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

  18. Protective immunity against a lethal respiratory Yersinia pestis challenge induced by V antigen or the F1 capsular antigen incorporated into adenovirus capsid.

    PubMed

    Boyer, Julie L; Sofer-Podesta, Carolina; Ang, John; Hackett, Neil R; Chiuchiolo, Maria J; Senina, Svetlana; Perlin, David; Crystal, Ronald G

    2010-07-01

    The aerosol form of the bacterium Yersinia pestis causes pneumonic plague, a rapidly fatal disease that is a biothreat if deliberately released. At present, no plague vaccines are available for use in the United States, but subunit vaccines based on the Y. pestis V antigen and F1 capsular protein show promise when administered with adjuvants. In the context that adenovirus (Ad) gene transfer vectors have a strong adjuvant potential related to the ability to directly infect dendritic cells, we hypothesized that modification of the Ad5 capsid to display either the Y. pestis V antigen or the F1 capsular antigen on the virion surface would elicit high V antigen- or F1-specific antibody titers, permit boosting with the same Ad serotype, and provide better protection against a lethal Y. pestis challenge than immunization with equivalent amounts of V or F1 recombinant protein plus conventional adjuvant. We constructed AdYFP-pIX/V and AdLacZ-pIX/F1, E1(-), E3(-) serotype 5 Ad gene transfer vectors containing a fusion of the sequence for either the Y. pestis V antigen or the F1 capsular antigen to the carboxy-terminal sequence of pIX, a capsid protein that can accommodate the entire V antigen (37 kDa) or F1 protein (15 kDa) without disturbing Ad function. Immunization with AdYFP-pIX/V followed by a single repeat administration of the same vector at the same dose resulted in significantly better protection of immunized animals compared with immunization with a molar equivalent amount of purified recombinant V antigen plus Alhydrogel adjuvant. Similarly, immunization with AdLacZ-pIX/F1 in a prime-boost regimen resulted in significantly enhanced protection of immunized animals compared with immunization with a molar-equivalent amount of purified recombinant F1 protein plus adjuvant. These observations demonstrate that Ad vaccine vectors containing pathogen-specific antigens fused to the pIX capsid protein have strong adjuvant properties and stimulate more robust protective

  19. Molecular Characterization of Antigen-Peptide Pulsed Dendritic Cells: Immature Dendritic Cells Develop a Distinct Molecular Profile when Pulsed with Antigen Peptide

    PubMed Central

    Jiang, Yufei; Catalano, Jennifer; Puri, Raj K.; Khleif, Samir N.

    2014-01-01

    As dendritic cells (DCs) are the most potent professional antigen-presenting cells, they are being tested as cancer vaccines for immunotherapy of established cancers. Although numerous studies have characterized DCs by their phenotype and function, few have identified potential molecular markers of antigen presentation prior to vaccination of host. In this study we generated pre-immature DC (piDC), immature DC (iDC), and mature DC (mDC) from human peripheral blood monocytes (PBMC) obtained from HLA-A2 healthy donors, and pulsed them with human papillomavirus E7 peptide (p11-20), a class I HLA-A2 binding antigen. We then characterized DCs for cell surface phenotype and gene expression profile by microarray technology. We identified a set of 59 genes that distinguished three differentiation stages of DCs (piDC, iDC and mDC). When piDC, iDC and mDC were pulsed with E7 peptide for 2 hrs, the surface phenotype did not change, however, iDCs rather than mDCs showed transcriptional response by up-regulation of a set of genes. A total of 52 genes were modulated in iDC upon antigen pulsing. Elongation of pulse time for iDCs to 10 and 24 hrs did not significantly bring further changes in gene expression. The E7 peptide up-modulated immune response (KPNA7, IGSF6, NCR3, TREM2, TUBAL3, IL8, NFKBIA), pro-apoptosis (BTG1, SEMA6A, IGFBP3 and SRGN), anti-apoptosis (NFKBIA), DNA repair (MRPS11, RAD21, TXNRD1), and cell adhesion and cell migration genes (EPHA1, PGF, IL8 and CYR61) in iDCs. We confirmed our results by Q-PCR analysis. The E7 peptide but not control peptide (PADRE) induced up-regulation of NFKB1A gene only in HLA-A2 positive iDCs and not in HLA-A2 negative iDCs. These results suggest that E7 up-regulation of genes is specific and HLA restricted and that these genes may represent markers of antigen presentation and help rapidly assess the quality of dendritic cells prior to administration to the host. PMID:24475103

  20. Characterization of thymus-derived lymphocytes expressing Ti alpha-beta CD3 gamma delta epsilon zeta-zeta, Ti alpha-beta CD3 gamma delta epsilon eta-eta or Ti alpha-beta CD3 gamma delta epsilon zeta-zeta/zeta- eta antigen receptor isoforms: analysis by gene transfection

    PubMed Central

    1990-01-01

    To characterize the function of the CD3 eta subunit of the T cell receptor (TCR), we have used cDNAs encoding CD3 zeta, CD3 eta, or both to reconstitute a variant of a cytochrome c-specific, I-Ek-restricted murine T cell hybridoma, termed MA5.8, which lacks CD3 zeta and CD3 eta proteins. We provide direct evidence that assembly and surface expression of TCRs can be mediated by either of these subunits separately or together. However, the level of TCR expression on zeta transfectants is up to one order of magnitude greater than that on eta transfectants, implying that CD3 eta is weakly associated with the pentameric Ti alpha-beta CD3 gamma delta epsilon complex and/or inefficient at salvaging the incomplete TCR from lysosomal degradation. As a component of the TCR, the CD3 eta subunit preferentially forms a heterodimer with CD3 zeta, but is also able to form a CD3 eta-eta homodimer. Crosslinking of Ti alpha-beta CD3 gamma delta epsilon zeta- zeta, Ti alpha-beta CD3 gamma delta epsilon eta-eta, or Ti alpha-beta CD3 gamma delta epsilon zeta-zeta/zeta-eta TCR isotypes with anti-CD3 epsilon monoclonal antibody or a cytochrome c peptide epitope on I-Ek antigen-presenting cells mediates signal transduction resulting in reversible cell-cycle arrest of transfected clones. Given the potential for diversity of signals generated by these functional TCR isotypes and the expression of the CD3 eta gene product in the thymus, CD3 eta is likely to play a role in selection and/or activation of thymocytes during development. PMID:2145389

  1. Antigenic variation: Molecular and genetic mechanisms of relapsing disease

    SciTech Connect

    Cruse, J.M.; Lewis, R.E.

    1987-01-01

    This book contains 10 chapters. They are: Contemporary Concepts of Antigenic Variation; Antigenic Variation in the Influenza Viruses; Mechanisms of Escape of Visna Lentiviruses from Immunological Control; A Review of Antigenic Variation by the Equine Infectious Anemia Virus; Biologic and Molecular Variations in AIDS Retrovirus Isolates; Rabies Virus Infection: Genetic Mutations and the Impact on Viral Pathogenicity and Immunity; Immunobiology of Relapsing Fever; Antigenic Variation in African Trypanosomes; Antigenic Variation and Antigenic Diversity in Malaria; and Mechanisms of Immune Evasion in Schistosomiasis.

  2. The use of marsupial x eutherian somatic cell hybrids to study marsupial cell surface antigens.

    PubMed

    Sykes, P J; Hope, R M

    1978-12-01

    Buck and Bodmer (1976) have developed a technique for identifying an antigen on the surface of human x mouse somatic cell hybrids, specified by a gene on a particular human chromosome. We have successfully adapted this technique to a study of marsupial cell surface antigens. Somatic cell hybrids between Macropus rufus (Marsupialia) lymphocytes and the mouse cell lines PG19 and 1R were injected intraperitoneally into mice of the same inbred strain from which the above cell lines were derived (C57B16J and C3H, respectively). The only identified M. rufus chromosome present in the hybrid cells was the X chromosome. The antisera, after adsorption with PG19 or 1R, were tested using indirect immunofluorescence, against the hybrid cells, and also against sub-clones (derived from hybrids) which had apparently lost the M. rufus X chromosome, or at least its long arm. The results of these tests showed that the absorbed antisera contained reactivity against an M. rufus cell surface antigen (or antigens). The reactions of one of the antisera were most simply interpreted by supposing that it was detecting an M. rufus X-lined antigen(s).

  3. Ku-related antigens are associated with transcriptionally active loci in Chironomus polytene chromosomes.

    PubMed

    Gorab, E; Botella, L M; Quinn, J P; Amabis, J M; Díez, J L

    1996-09-01

    Antigens of Chironomus reactive with human sera containing anti-Ku antibodies and also with specific antibodies to each Ku subunit were characterized by immunoblot analysis. Three main antigen species were identified in nuclear-enriched extracts from salivary gland cells of Chironomus thummi, ranging in Mr from 55000 to 67000. The nuclear localization of Ku-related antigens in the dipteran Chironomus was studied by immunofluorescent labeling in polytene chromosomes of the salivary glands. Balbiani rings, loci highly active in transcription, were found to be strongly labeled by anti-Ku antibodies. Sugar-induced changes in the activity of the Balbiani ring genes were accompanied by the redistribution of Ku-related antigens as visualized by their absence in regressed Balbiani ring loci, and continued presence only in those that were transcriptionally active. A drastic change in the distribution of Ku-related antigens was also observed when C. thummi larvae underwent heat treatment as the immunofluorescent staining was restricted to previously described heat shock puffs. Anti-Ku sera reacted in addition with several chromosomal bands in which the presence of RNA polymerase II was also immunologically detected. The results show that Chironomus antigens reactive with anti-Ku antibodies are related to transcription in polytene chromosomes.

  4. Characterization of antigen receptor response elements within the interleukin-2 enhancer.

    PubMed Central

    Durand, D B; Shaw, J P; Bush, M R; Replogle, R E; Belagaje, R; Crabtree, G R

    1988-01-01

    T-cell activation and induction of interleukin-2 (IL-2) expression in human T lymphocytes require both interaction of foreign antigen with the T-cell antigen receptor and protein kinase C (PKC) stimulation. Agents such as phorbol 12-myristate 13-acetate (PMA) that stimulate PKC augment the effects of antigen but are not sufficient for IL-2 activation. By analysis of deletion mutants, we identified three DNA sequences extending from -73 to -89, -217 to -255, and -263 to -279, designated IL-2 sites A, D, and E, respectively, that are required for maximal induction of IL-2 expression. One of these regions, site E, interacted with a protein (NF-IL-2E) present only in the nuclei of cells which have been stimulated. The other two sequences interacted with a protein (NF-IL-2A) that is constitutively expressed in T cells. When multiple tandem copies of either the E site or the A site were placed upstream of the gamma-fibrinogen promoter, they activated expression via this promoter in response to signals initiated at the antigen receptor but not following PMA stimulation. For this reason, we denoted them antigen receptor response elements. The uncoupling of antigen receptor and PKC requirements in these studies indicates that these signal pathways are, at least in part, distinct and integrated at the level of the gene. Images PMID:3260003

  5. Tumor antigen analysis in neuroblastoma by serological interrogation of bioinformatic data.

    PubMed

    Kohler, M Eric; Johnson, Bryon D; Palen, Katie; Chen, Qing-Rong; Khan, Javed; Orentas, Rimas J

    2010-11-01

    The identification of tumor antigens remains a major objective in tumor immunology, especially in pediatric malignancies where solid tumors often do not express a single dominant antigen. Methods such as the Serological Screening of Recombinant cDNA Expression Libraries (SEREX) have been used in the discovery of tumor-expressed proteins by virtue of their ability to induce an antibody response. To focus and accelerate this approach, we first identified candidate antigens by gene expression profiling data from clinical neuroblastoma specimens and then used an animal model to generate an antibody response to an engineered cell-based vaccine. Candidate tumor antigens were expressed as recombinant proteins in a mammalian system and screened for antibody recognition using serum from mice vaccinated with a neuroblastoma cell-based vaccine engineered to express CD80 and CD86, with or without Treg depletion. Through this procedure, the never in mitosis A (NIMA)-related kinase NEK2 was identified as a tumor-associated antigen. Direct testing of serum from patients newly diagnosed with neuroblastoma showed specific serological responses in two of 20 patients. Although NEK2 was not universally recognized, it may serve as a tumor antigen for some patients.

  6. A Mutant Library Approach to Identify Improved Meningococcal Factor H Binding Protein Vaccine Antigens

    PubMed Central

    Konar, Monica; Rossi, Raffaella; Walter, Helen; Pajon, Rolando; Beernink, Peter T.

    2015-01-01

    Factor H binding protein (FHbp) is a virulence factor used by meningococci to evade the host complement system. FHbp elicits bactericidal antibodies in humans and is part of two recently licensed vaccines. Using human complement Factor H (FH) transgenic mice, we previously showed that binding of FH decreased the protective antibody responses to FHbp vaccination. Therefore, in the present study we devised a library-based method to identify mutant FHbp antigens with very low binding of FH. Using an FHbp sequence variant in one of the two licensed vaccines, we displayed an error-prone PCR mutant FHbp library on the surface of Escherichia coli. We used fluorescence-activated cell sorting to isolate FHbp mutants with very low binding of human FH and preserved binding of control anti-FHbp monoclonal antibodies. We sequenced the gene encoding FHbp from selected clones and introduced the mutations into a soluble FHbp construct. Using this approach, we identified several new mutant FHbp vaccine antigens that had very low binding of FH as measured by ELISA and surface plasmon resonance. The new mutant FHbp antigens elicited protective antibody responses in human FH transgenic mice that were up to 20-fold higher than those elicited by the wild-type FHbp antigen. This approach offers the potential to discover mutant antigens that might not be predictable even with protein structural information and potentially can be applied to other microbial vaccine antigens that bind host proteins. PMID:26057742

  7. Erythrocyte Membrane Antigen Frequencies in Patients with Type II Congenital Smell Loss

    PubMed Central

    Stateman, William A.; Henkin, Robert I.; Knöppel, Alexandra; Flegel, Willy A.

    2014-01-01

    OBJECTIVE The objective of this study was to determine whether there are genetic factors associated with Type II congenital smell loss. STUDY DESIGN The expression frequencies of 16 erythrocyte antigens among patients with Type II congenital smell loss were determined and compared to those of a large control group. METHODS Blood samples were obtained from 99 patients with Type II congenital smell loss. Presence of the erythrocyte surface antigens A, B, M, N, S, s, Fya, Fyb, D, C, c, E, e, K, Jka, and Jkb was analyzed by blood group serology. Comparisons of expression frequencies of these antigens were made between the patients and a large control group. RESULTS Patients tested for the Duffy b antigen (Fyb haplotype) exhibited a statistically significant 11% decrease in expression frequency compared to the controls. There were no significant differences between patients and controls in the expression frequencies for all other erythrocyte antigens (A, B, M, N, S, s, Fya, D, C, c, E, e, K, Jka, or Jkb). CONCLUSIONS These findings describe the presence of a previously unrevealed genetic tendency among patients with Type II congenital smell loss related to erythrocyte surface antigen expression. The deviation in expression rate of Duffy b suggests a target gene and chromosome region in which future research into this form of congenital smell loss may reveal a more specific genetic basis for Type II congenital smell loss. PMID:25456515

  8. Cyclosporine inhibits macrophage-mediated antigen presentation

    SciTech Connect

    Ziegler, H.K.; Palay, D.; Wentworth, P.; Cluff, C.

    1986-03-01

    The influence of cyclosporine on antigen-specific, macrophage-dependent T cell activation was analyzed in vitro. Murine T cell activation by antigens derived from Listeria monocytogenes was monitored by the production of interleukin-2. Pretreatment (2 hrs., 37/sup 0/C) of macrophages with cyclosporine resulted in a population of macrophages with a markedly diminished capacity to support the activation of T lymphocytes. When cyclosporine-pretreated macrophages were added to cultures of antigen and untreated T cells, the dose of cyclosporine which produced 50% inhibition was 1.5 ..mu..g/ml. Appropriate control experiments indicated that cyclosporine was indeed inhibiting at the macrophage level. The addition of interleukin-1 or indomethacin to the cultures did not alter the inhibitory effect of cyclosporine. Under conditions which produced >90% inhibition of antigen presentation, macrophage surface Ia expression was not altered, and the uptake and catabolism of radiolabelled antigen was normal. Thus, cyclosporine inhibits antigen presentation by a mechanism which appears unrelated to changes in Il-1 elaboration, prostaglandin production, Ia expression, or antigen uptake and catabolism.

  9. Human immunodeficiency virus type 1 Nef does not alter T-cell sensitivity to antigen-specific stimulation.

    PubMed Central

    Page, K A; van Schooten, W C; Feinberg, M B

    1997-01-01

    We have developed an in vitro model to study the influence that human immunodeficiency virus type 1 (HIV-1) may have on the ability of T cells to respond to antigenic challenge. We have examined consequences of HIV-1 gene expression on T-cell activation in antigen-dependent T cells that have stably integrated copies of replication-defective proviral HIV-1. Virus production by HIV-infected, antigen-dependent T cells was induced in response to antigenic stimulation and then decreased as infected cells returned to a state of quiescence. Contrary to the predictions of models proposing that Nef alters signal transduction pathways in T lymphocytes and thereby alters cellular activation, Nef expression in antigen-dependent T-cell clones did not influence their proliferative responses to low or intermediate concentrations of antigen and did not affect other measures of T-cell activation, such as induction of interleukin 2 receptor alpha-chain expression and cytokine production. In addition, we found no evidence for alteration of T-cell responsiveness to antigen by the gag, pol, vif, tat, or rev gene of HIV-1. PMID:9094653

  10. Late-phase expression of a murine cytomegalovirus immediate-early antigen recognized by cytolytic T lymphocytes.

    PubMed Central

    Reddehase, M J; Fibi, M R; Keil, G M; Koszinowski, U H

    1986-01-01

    The cloned murine cytolytic T-lymphocyte line IE1-IL and several sublines detect a murine cytomegalovirus immediate-early (IE) membrane determinant in conjunction with Ld class I major histocompatibility glycoprotein. The lines retained cytolytic activity, strict antigen specificity, and self-restriction even when adapted to long-term, antigen-independent growth in the presence of interleukin-2 only (M. J. Reddehase, H.-J. Bühring, and U. H. Koszinowski, J. Virol. 57:408-412). These attributes allowed us to use IE1-IL as a stable, monospecific probe for tracing the expression of the IE membrane antigen throughout the viral replication cycle. Presentation of the antigen at the cell membrane proved to be most effective when expression of IE genes in infected mouse embryo fibroblasts was selectively enhanced by consecutive cycloheximide-actinomycin D treatment, whereas without enhancement high numbers of IE1-IL cytolytic T lymphocytes were required to demonstrate the antigen in the IE phase. In the early phase of infection when IE genes were no longer transcribed, cytolysis was not observed, although IE proteins were detectable in the nuclei of the infected cells. Without application of inhibitors IE membrane antigen expression was most prominent during the late phase of infection. Reinitiation of transcription from the genomic region encoding the major IE protein (pp89) and de novo synthesis of pp89 correlated with this reexpression of the IE membrane antigen. Images PMID:2431160

  11. VSTM-v1, a potential myeloid differentiation antigen that is downregulated in bone marrow cells from myeloid leukemia patients.

    PubMed

    Xie, Min; Li, Ting; Li, Ning; Li, Jinlan; Yao, Qiumei; Han, Wenling; Ruan, Guorui

    2015-01-01

    Leukocyte differentiation antigens often represent important markers for the diagnosis, classification, prognosis, and therapeutic targeting of myeloid leukemia. Herein, we report a potential leukocyte differentiation antigen gene VSTM1 (V-set and transmembrane domain-containing 1) that was downregulated in bone marrow cells from leukemia patients and exhibited a higher degree of promoter methylation. The expression level of its predominant encoded product, VSTM1-v1, was positively correlated with myeloid cell maturation state. Restoration of VSTM1-v1 expression inhibited myeloid leukemia cells' growth. Therefore, VSTM1-v1 might represent an important myeloid leukocyte differentiation antigen and provide a potential target for the diagnosis and treatment of leukemia.

  12. The significance of erythrocyte antigen site density

    PubMed Central

    Hoyer, Leon W.; Trabold, Norma C.

    1971-01-01

    The importance of antigen site density has been studied by means of a model passive hemolysis system using red cells coupled with sulfanilic acid groups. Relative site numbers were estimated from the covalent linkage of sulfanilic acid-35S to red cell membrane protein, and the effective antigen site number was determined with 125I-labeled rabbit IgG anti-sulfanilic acid (anti-S). Immune hemolysis was demonstrated for red cells which had greater than a threshold number of antigen sites, the value of which was different for normal human cells (80,000 sites/cell), cells from a patient with paroxysmal nocturnal hemoglobinuria (PNH) (40,000 sites/cell), and sheep red blood cells (RBC) (15,000 sites/cell). Cells with antigen site densities below these values did not hemolyze when tested with 1 mg/ml purified rabbit IgM anti-S. 2-8 times greater antigen site densities were required to obtain hemolysis with IgG anti-S. Above the threshold value, hemolysis titers were proportional to the antigen site number until maximal values were obtained. The greater hemolytic efficiency of IgM antibody was demonstrated in this system, and it was established that the magnitude of the difference was related to the test cell antigen site density. These data, taken with previously reported hemagglutination studies, have been used to develop a general classification of immune hemolysis and hemagglutination based on antigen site density and antibody class. It is suggested that the heterogeneity of blood group systems is caused by differences in the site separation of erythrocyte membrane antigens. PMID:5105661

  13. Limited Antigenic Diversity in Contemporary H7 Avian-Origin Influenza A Viruses from North America.

    PubMed

    Xu, Yifei; Bailey, Elizabeth; Spackman, Erica; Li, Tao; Wang, Hui; Long, Li-Ping; Baroch, John A; Cunningham, Fred L; Lin, Xiaoxu; Jarman, Richard G; DeLiberto, Thomas J; Wan, Xiu-Feng

    2016-01-01

    Subtype H7 avian-origin influenza A viruses (AIVs) have caused at least 500 confirmed human infections since 2003 and culling of >75 million birds in recent years. Here we antigenically and genetically characterized 93 AIV isolates from North America (85 from migratory waterfowl [1976-2010], 7 from domestic poultry [1971-2012], and 1 from a seal [1980]). The hemagglutinin gene of these H7 viruses are separated from those from Eurasia. Gradual accumulation of nucleotide and amino acid substitutions was observed in the hemagglutinin of H7 AIVs from waterfowl and domestic poultry. Genotype characterization suggested that H7 AIVs in wild birds form diverse and transient internal gene constellations. Serologic analyses showed that the 93 isolates cross-reacted with each other to different extents. Antigenic cartography showed that the average antigenic distance among them was 1.14 units (standard deviation [SD], 0.57 unit) and that antigenic diversity among the H7 isolates we tested was limited. Our results suggest that the continuous genetic evolution has not led to significant antigenic diversity for H7 AIVs from North America. These findings add to our understanding of the natural history of IAVs and will inform public health decision-making regarding the threat these viruses pose to humans and poultry. PMID:26858078

  14. Limited Antigenic Diversity in Contemporary H7 Avian-Origin Influenza A Viruses from North America

    PubMed Central

    Xu, Yifei; Bailey, Elizabeth; Spackman, Erica; Li, Tao; Wang, Hui; Long, Li-Ping; Baroch, John A.; Cunningham, Fred L.; Lin, Xiaoxu; Jarman, Richard G.; DeLiberto, Thomas J.; Wan, Xiu-Feng

    2016-01-01

    Subtype H7 avian–origin influenza A viruses (AIVs) have caused at least 500 confirmed human infections since 2003 and culling of >75 million birds in recent years. Here we antigenically and genetically characterized 93 AIV isolates from North America (85 from migratory waterfowl [1976–2010], 7 from domestic poultry [1971–2012], and 1 from a seal [1980]). The hemagglutinin gene of these H7 viruses are separated from those from Eurasia. Gradual accumulation of nucleotide and amino acid substitutions was observed in the hemagglutinin of H7 AIVs from waterfowl and domestic poultry. Genotype characterization suggested that H7 AIVs in wild birds form diverse and transient internal gene constellations. Serologic analyses showed that the 93 isolates cross-reacted with each other to different extents. Antigenic cartography showed that the average antigenic distance among them was 1.14 units (standard deviation [SD], 0.57 unit) and that antigenic diversity among the H7 isolates we tested was limited. Our results suggest that the continuous genetic evolution has not led to significant antigenic diversity for H7 AIVs from North America. These findings add to our understanding of the natural history of IAVs and will inform public health decision-making regarding the threat these viruses pose to humans and poultry. PMID:26858078

  15. The immunodominant Eimeria acervulina sporozoite antigen previously described as p160/p240 is a 19-kilodalton antigen present in several Eimeria species.

    PubMed

    Laurent, F; Bourdieu, C; Kazanji, M; Yvoré, P; Péry, P

    1994-01-01

    A lambda Zap II cDNA expression library, constructed from Eimeria acervulina (PAPa46 strain) sporulated oocyst stage, was screened with sera raised to E. acervulina or Eimeria tenella oocysts in order to isolate clones coding for antigens common to the two species. Most of the clones isolated were derived from the same gene. Antisera raised to a recombinant glutathione-S-transferase fusion protein 1P reacted with an antigen of 19 kDa in immunoblot of E. acervulina sporulated and unsporulated oocysts. Immunofluorescence of E. acervulina sporozoites indicated that the antigen is located in the cytoplasm. The anti-1P antisera reacted on immunoblots of E. tenella with a 19-kDa antigen and by immunofluorescence on E. tenella, Eimeria maxima and Eimeria falciformis sporozoites, indicating that the antigen is conserved in Eimeria species. DNA sequencing indicated that the sequence was almost identical to that of clone cSZ1 previously described by Jenkins et al. using E. acervulina strain #12. The 1P insert hybridized to a 1150-nt mRNA from E. acervulina PAPa46 strain and strain #12, a size consistent with the observed molecular weight of the protein.

  16. Bombyx mori nucleopolyhedrovirus displaying neospora caninum antigens as a vaccine candidate against N. caninum infection in mice.

    PubMed

    Kato, Tatsuya; Otsuki, Takahiro; Yoshimoto, Mai; Itagaki, Kohei; Kohsaka, Tetsuya; Matsumoto, Yumino; Ike, Kazunori; Park, Enoch Y

    2015-02-01

    Baculovirus display systems have been utilized for cell-specific gene transfer, regenerative medicine, and as vaccine vectors. In particular, baculovirus particles displaying surface antigens have been used as vaccines against some parasites and viruses. In this study, Bombyx mori nucleopolyhedrovirus (BmNPV) particles displaying Neospora caninum antigens (NcSAG1, NcSRS2, and NcMIC3) purified from the hemolymph or fat body of silkworm larvae were prepared to vaccinate mice against N. caninum. Each antigen was expressed on the surface of BmNPV particles through glycoprotein 64 transmembrane and cytoplasmic domains. Antigen-specific antibody production was induced in mice by immunization with each recombinant BmNPV particle. NcMIC3-displaying BmNPV particles purified from the fat body induced a lower antibody titer than particles purified from the hemolymph. Antigen-specific IgG2a was predominantly produced in mice by immunization with NcSAG1-displaying BmNPV particles compared to IgG1, and induction of IFN-γ was dominant, indicating that antigen-displaying BmNPV particles can elicit a Th1 immune response in mice. Semi-quantitative PCR analysis revealed that immunization with each antigen-displaying BmNPV particle partially protected mice from cerebral N. caninum infection. These results suggest that antigen-displaying BmNPV particles can provide an alternative method of controlling neosporosis in cattle and represent a new generation of N. caninum vaccines.

  17. NY-ESO-1 antigen-reactive T cell receptors exhibit diverse therapeutic capability.

    PubMed

    Sommermeyer, Daniel; Conrad, Heinke; Krönig, Holger; Gelfort, Haike; Bernhard, Helga; Uckert, Wolfgang

    2013-03-15

    The cancer-testis antigen NY-ESO-1 has been used as a target for different immunotherapies like vaccinations and adoptive transfer of antigen-specific cytotoxic T cells, as it is expressed in various tumor types and has limited expression in normal cells. The in vitro generation of T cells with defined antigen specificity by T cell receptor (TCR) gene transfer is an established method to create cells for immunotherapy. However, an extensive characterization of TCR which are candidates for treatment of patients is crucial for successful therapies. The TCR has to be efficiently expressed, their affinity to the desired antigen should be high enough to recognize low amounts of endogenously processed peptides on tumor cells, and the TCR should not be cross-reactive to other antigens. We characterized three NY-ESO-1 antigen-reactive cytotoxic T lymphocyte clones which were generated by different approaches of T cell priming (autologous, allogeneic), and transferred their TCR into donor T cells for more extensive evaluations. Although one TCR most efficiently bound MHC-multimers loaded with NY-ESO-1 peptide, T cells expressing this transgenic TCR were not able to recognize endogenously processed antigen. A second TCR recognized HLA-A2 independent of the bound peptide beside its much stronger recognition of NY-ESO-1 bound to HLA-A2. A third TCR displayed an intermediate but peptide-specific performance in all functional assays and, therefore, is the most promising candidate TCR for further clinical development. Our data indicate that multiple parameters of TCR gene-modified T cells have to be evaluated to identify an optimal TCR candidate for adoptive therapy.

  18. Antigen retrieval techniques: current perspectives.

    PubMed

    Shi, S R; Cote, R J; Taylor, C R

    2001-08-01

    Development of the antigen retrieval (AR) technique, a simple method of boiling archival paraffin-embedded tissue sections in water to enhance the signal of immunohistochemistry (IHC), was the fruit of pioneering efforts guided by the philosophy of rendering IHC applicable to routine formalin-fixed, paraffin-embedded tissues for wide application of IHC in research and clinical pathology. On the basis of thousands of articles and many reviews, a book has recently been published that summarizes basic principles for practice and further development of the AR technique. Major topics with respect to several critical issues, such as the definition, application, technical principles, and further studies of the AR technique, are highlighted in this article. In particular, a further application of the heat-induced retrieval approach for sufficient extraction of nucleic acids in addition to proteins, and standardization of routine IHC based on the AR technique in terms of a test battery approach, are also addressed. Furthermore, understanding the mechanism of the AR technique may shed light on facilitating the development of molecular morphology.

  19. Hepatitis C Virus Antigenic Convergence

    PubMed Central

    Campo, David S.; Dimitrova, Zoya; Yokosawa, Jonny; Hoang, Duc; Perez, Nestor O.; Ramachandran, Sumathi; Khudyakov, Yury

    2012-01-01

    Vaccine development against hepatitis C virus (HCV) is hindered by poor understanding of factors defining cross-immunoreactivity among heterogeneous epitopes. Using synthetic peptides and mouse immunization as a model, we conducted a quantitative analysis of cross-immunoreactivity among variants of the HCV hypervariable region 1 (HVR1). Analysis of 26,883 immunological reactions among pairs of peptides showed that the distribution of cross-immunoreactivity among HVR1 variants was skewed, with antibodies against a few variants reacting with all tested peptides. The HVR1 cross-immunoreactivity was accurately modeled based on amino acid sequence alone. The tested peptides were mapped in the HVR1 sequence space, which was visualized as a network of 11,319 sequences. The HVR1 variants with a greater network centrality showed a broader cross-immunoreactivity. The entire sequence space is explored by each HCV genotype and subtype. These findings indicate that HVR1 antigenic diversity is extensively convergent and effectively limited, suggesting significant implications for vaccine development. PMID:22355779

  20. Flaviviruses and their antigenic structure.

    PubMed

    Heinz, F X; Stiasny, Karin

    2012-12-01

    Flaviviruses comprise important arthropod-transmitted human pathogens, including yellow fever (YF), dengue (Den), Japanese encephalitis (JE), West Nile (WN) and tick-borne encephalitis (TBE) viruses that have the potential of expanding their endemic areas due to global climatic, ecological and socio-economic changes. While effective vaccines against YF, JE and TBE are in widespread use, the development of a dengue vaccine has been hampered for a long time because of concerns of immunopathological consequences of vaccination. Phase III clinical trials with a recombinant chimeric live vaccine are now ongoing and will show whether the enormous problem of dengue can be resolved or at least reduced by vaccination in the future. Unprecedented details of the flavivirus particle structure have become available through the combined use of X-ray crystallography and cryo-electron microscopy that led to novel and surprising insights into the antigenic structure of these viruses. Recent studies provided evidence for an important role of virus maturation as well as particle dynamics in virus neutralization by antibodies and thus added previously unknown layers of complexity to our understanding of flavivirus immune protection. This information is invaluable for interpreting current investigations on the functional activities of polyclonal antibody responses to flavivirus infections and vaccinations and may open new avenues for studies on flavivirus cell biology and vaccine design.

  1. Human immune response to Mycobacterium tuberculosis antigens.

    PubMed Central

    Havlir, D V; Wallis, R S; Boom, W H; Daniel, T M; Chervenak, K; Ellner, J J

    1991-01-01

    Little is known about the immunodominant or protective antigens of Mycobacterium tuberculosis in humans. Cell-mediated immunity is necessary for protection, and healthy tuberculin-positive individuals are relatively resistant to exogenous reinfection. We compared the targets of the cell-mediated immune response in healthy tuberculin-positive individuals to those of tuberculosis patients and tuberculin-negative persons. By using T-cell Western blotting (immunoblotting) of nitrocellulose-bound M. tuberculosis culture filtrate, peaks of T-cell blastogenic activity were identified in the healthy tuberculin reactors at 30, 37, 44, 57, 64, 71 and 88 kDa. Three of these fractions (30, 64, and 71 kDa) coincided with previously characterized proteins: antigen 6/alpha antigen, HSP60, and HSP70, respectively. The blastogenic responses to purified M. tuberculosis antigen 6/alpha antigen and BCG HSP60 were assessed. When cultured with purified antigen 6/alpha antigen, lymphocytes of healthy tuberculin reactors demonstrated greater [3H]thymidine incorporation than either healthy tuberculin-negative controls or tuberculous patients (8,113 +/- 1,939 delta cpm versus 645 +/- 425 delta cpm and 1,019 +/- 710 delta cpm, respectively; P less than 0.01). Healthy reactors also responded to HSP60, although to a lesser degree than antigen 6/alpha antigen (4,276 +/- 1,095 delta cpm; P less than 0.05). Partially purified HSP70 bound to nitrocellulose paper elicited a significant lymphocyte blastogenic response in two of six of the tuberculous patients but in none of the eight healthy tuberculin reactors. Lymphocytes of none of five tuberculin-negative controls responded to recombinant antigens at 14 or 19 kDa or to HSP70. Antibody reactivity generally was inversely correlated with blastogenic response: tuberculous sera had high titer antibody to M. tuberculosis culture filtrate in a range from 35 to 180 kDa. This is the first systematic evaluation of the human response to a panel of native

  2. Antigen stimulation in the development of chronic lymphocytic leukemia.

    PubMed

    Karp, Marta; Giannopoulos, Krzysztof

    2013-12-05

    Chronic lymphocytic leukemia (CLL) is the most common leukemia in the western world. The mechanism the mechanism of the disease development still remains unrevealed. In recent years new unique molecular and clinical features of CLL have emerged leading to a unified hypothesis of CLL origin. Major progress in understanding CLL biology was made after identification of mutational status of immunoglobulin variable heavy chain (IGHV) genes, which also improved prediction of patients' clinical outcome. Preferential usage of IGHV genes has led to recognition of CLL-specific B cell receptors (BCRs), called stereotyped BCRs. Taken together, these data point to antigen stimulation of CLL progenitor cells. Studies on CLL antibody reactivity have shown affinity to molecular motifs on apoptotic cells and bacterial cell structures, supporting the current hypothesis of the CLL pathomechanism. In this paper we have summarized information available to date regarding current theory of cellular origin and pathology of CLL.

  3. Characterization of the O-antigen Polymerase (Wzy) of Francisella tularensis*

    PubMed Central

    Kim, Tae-Hyun; Sebastian, Shite; Pinkham, Jessica T.; Ross, Robin A.; Blalock, LeeAnn T.; Kasper, Dennis L.

    2010-01-01

    The O-antigen polymerase of Gram-negative bacteria has been difficult to characterize. Herein we report the biochemical and functional characterization of the protein product (Wzy) of the gene annotated as the putative O-antigen polymerase, which is located in the O-antigen biosynthetic locus of Francisella tularensis. In silico analysis (homology searching, hydropathy plotting, and codon usage assessment) strongly suggested that Wzy is an O-antigen polymerase whose function is to catalyze the addition of newly synthesized O-antigen repeating units to a glycolipid consisting of lipid A, inner core polysaccharide, and one repeating unit of the O-polysaccharide (O-PS). To characterize the function of the Wzy protein, a non-polar deletion mutant of wzy was generated by allelic replacement, and the banding pattern of O-PS was observed by immunoblot analysis of whole-cell lysates obtained by SDS-PAGE and stained with an O-PS-specific monoclonal antibody. These immunoblot analyses showed that O-PS of the wzy mutant expresses only one repeating unit of O-antigen. Further biochemical characterization of the subcellular fractions of the wzy mutant demonstrated that (as is characteristic of O-antigen polymerase mutants) the low molecular weight O-antigen accumulates in the periplasm of the mutant. Site-directed mutagenesis based on protein homology and topology, which was carried out to locate a catalytic residue of the protein, showed that modification of specific residues (Gly176, Asp177, Gly323, and Tyr324) leads to a loss of O-PS polymerization. Topology models indicate that these amino acids most likely lie in close proximity on the bacterial surface. PMID:20605777

  4. Antigen-specific tolerogenic and immunomodulatory strategies for the treatment of autoimmune arthritis

    PubMed Central

    Satpute, Shailesh R.; Durai, Malarvizhi; Moudgil, Kamal D.

    2008-01-01

    Objectives To review various antigen-specific tolerogenic and immunomodulatory approaches for arthritis in animal models and patients in regard to their efficacy, mechanisms of action and limitations. Methods We reviewed the published literature in Medline (PubMed) on the induction of antigen-specific tolerance and its effect on autoimmune arthritis, as well as the recent work on B cell-mediated tolerance from our laboratory. The prominent key words used in different combinations included arthritis, autoimmunity, immunotherapy, innate immunity, tolerance, treatment, and rheumatoid arthritis (RA). Although this search spanned the years 1975 to 2007, the majority of the short-listed articles belonged to the period 1990 to 2007. The relevant primary as well as cross-referenced articles were then collected from links within PubMed and reviewed. Results Antigen-specific tolerance has been successful in the prevention and/or treatment of arthritis in animal models. The administration of soluble native antigen or an altered peptide ligand intravenously, orally, or nasally, and the delivery of the DNA encoding a particular antigen by gene therapy have been the mainstay of immunomodulation. Recently, the methods for in vitro-expansion of CD4+CD25+ regulatory T cells have been optimized. Furthermore, interleukin-17 has emerged as a promising new therapeutic target in arthritis. However, in RA patients, non-antigen-specific therapeutic approaches have been much more successful than antigen-specific tolerogenic regimens. Conclusion An antigen-specific treatment against autoimmune arthritis is still elusive. However, insights into newly emerging mechanisms of disease pathogenesis provide hope for the development of effective and safe immunotherapeutic strategies in the near future. PMID:18177689

  5. Melanocyte antigen triggers autoimmunity in human psoriasis.

    PubMed

    Arakawa, Akiko; Siewert, Katherina; Stöhr, Julia; Besgen, Petra; Kim, Song-Min; Rühl, Geraldine; Nickel, Jens; Vollmer, Sigrid; Thomas, Peter; Krebs, Stefan; Pinkert, Stefan; Spannagl, Michael; Held, Kathrin; Kammerbauer, Claudia; Besch, Robert; Dornmair, Klaus; Prinz, Jörg C

    2015-12-14

    Psoriasis vulgaris is a common T cell-mediated inflammatory skin disease with a suspected autoimmune pathogenesis. The human leukocyte antigen (HLA) class I allele, HLA-C*06:02, is the main psoriasis risk gene. Epidermal CD8(+) T cells are essential for psoriasis development. Functional implications of HLA-C*06:02 and mechanisms of lesional T cell activation in psoriasis, however, remained elusive. Here we identify melanocytes as skin-specific target cells of an HLA-C*06:02-restricted psoriatic T cell response. We found that a Vα3S1/Vβ13S1 T cell receptor (TCR), which we had reconstituted from an epidermal CD8(+) T cell clone of an HLA-C*06:02-positive psoriasis patient specifically recognizes HLA-C*06:02-positive melanocytes. Through peptide library screening, we identified ADAMTS-like protein 5 (ADAMTSL5) as an HLA-C*06:02-presented melanocytic autoantigen of the Vα3S1/Vβ13S1 TCR. Consistent with the Vα3S1/Vβ13S1-TCR reactivity, we observed numerous CD8(+) T cells in psoriasis lesions attacking melanocytes, the only epidermal cells expressing ADAMTSL5. Furthermore, ADAMTSL5 stimulation induced the psoriasis signature cytokine, IL-17A, in CD8(+) T cells from psoriasis patients only, supporting a role as psoriatic autoantigen. This unbiased analysis of a TCR obtained directly from tissue-infiltrating CD8(+) T cells reveals that in psoriasis HLA-C*06:02 directs an autoimmune response against melanocytes through autoantigen presentation. We propose that HLA-C*06:02 may predispose to psoriasis via this newly identified autoimmune pathway.

  6. Melanocyte antigen triggers autoimmunity in human psoriasis.

    PubMed

    Arakawa, Akiko; Siewert, Katherina; Stöhr, Julia; Besgen, Petra; Kim, Song-Min; Rühl, Geraldine; Nickel, Jens; Vollmer, Sigrid; Thomas, Peter; Krebs, Stefan; Pinkert, Stefan; Spannagl, Michael; Held, Kathrin; Kammerbauer, Claudia; Besch, Robert; Dornmair, Klaus; Prinz, Jörg C

    2015-12-14

    Psoriasis vulgaris is a common T cell-mediated inflammatory skin disease with a suspected autoimmune pathogenesis. The human leukocyte antigen (HLA) class I allele, HLA-C*06:02, is the main psoriasis risk gene. Epidermal CD8(+) T cells are essential for psoriasis development. Functional implications of HLA-C*06:02 and mechanisms of lesional T cell activation in psoriasis, however, remained elusive. Here we identify melanocytes as skin-specific target cells of an HLA-C*06:02-restricted psoriatic T cell response. We found that a Vα3S1/Vβ13S1 T cell receptor (TCR), which we had reconstituted from an epidermal CD8(+) T cell clone of an HLA-C*06:02-positive psoriasis patient specifically recognizes HLA-C*06:02-positive melanocytes. Through peptide library screening, we identified ADAMTS-like protein 5 (ADAMTSL5) as an HLA-C*06:02-presented melanocytic autoantigen of the Vα3S1/Vβ13S1 TCR. Consistent with the Vα3S1/Vβ13S1-TCR reactivity, we observed numerous CD8(+) T cells in psoriasis lesions attacking melanocytes, the only epidermal cells expressing ADAMTSL5. Furthermore, ADAMTSL5 stimulation induced the psoriasis signature cytokine, IL-17A, in CD8(+) T cells from psoriasis patients only, supporting a role as psoriatic autoantigen. This unbiased analysis of a TCR obtained directly from tissue-infiltrating CD8(+) T cells reveals that in psoriasis HLA-C*06:02 directs an autoimmune response against melanocytes through autoantigen presentation. We propose that HLA-C*06:02 may predispose to psoriasis via this newly identified autoimmune pathway. PMID:26621454

  7. The use of high-throughput DNA sequencing in the investigation of antigenic variation: application to Neisseria species.

    PubMed

    Davies, John K; Harrison, Paul F; Lin, Ya-Hsun; Bartley, Stephanie; Khoo, Chen Ai; Seemann, Torsten; Ryan, Catherine S; Kahler, Charlene M; Hill, Stuart A

    2014-01-01

    Antigenic variation occurs in a broad range of species. This process resembles gene conversion in that variant DNA is unidirectionally transferred from partial gene copies (or silent loci) into an expression locus. Previous studies of antigenic variation have involved the amplification and sequencing of individual genes from hundreds of colonies. Using the pilE gene from Neisseria gonorrhoeae we have demonstrated that it is possible to use PCR amplification, followed by high-throughput DNA sequencing and a novel assembly process, to detect individual antigenic variation events. The ability to detect these events was much greater than has previously been possible. In N. gonorrhoeae most silent loci contain multiple partial gene copies. Here we show that there is a bias towards using the copy at the 3' end of the silent loci (copy 1) as the donor sequence. The pilE gene of N. gonorrhoeae and some strains of Neisseria meningitidis encode class I pilin, but strains of N. meningitidis from clonal complexes 8 and 11 encode a class II pilin. We have confirmed that the class II pili of meningococcal strain FAM18 (clonal complex 11) are non-variable, and this is also true for the class II pili of strain NMB from clonal complex 8. In addition when a gene encoding class I pilin was moved into the meningococcal strain NMB background there was no evidence of antigenic variation. Finally we investigated several members of the opa gene family of N. gonorrhoeae, where it has been suggested that limited variation occurs. Variation was detected in the opaK gene that is located close to pilE, but not at the opaJ gene located elsewhere on the genome. The approach described here promises to dramatically improve studies of the extent and nature of antigenic variation systems in a variety of species.

  8. Multiplex PCR for Distinguishing the Most Common Phase-1 Flagellar Antigens of Salmonella spp.

    PubMed Central

    Herrera-León, Silvia; McQuiston, John R.; Usera, Miguel A.; Fields, Patricia I.; Garaizar, Javier; Echeita, M. Aurora

    2004-01-01

    Most Salmonella serotypes alternatively express either phase-1 or phase-2 flagellar antigens, encoded by the fliC and fljB genes, respectively. Flagellar phase reversal for the identification of both flagellar antigens is not necessary at the genetic level. Variable internal regions of the fliC genes encoding the H:i, H:r, H:l,v, H:e,h, H:z10, H:b, and H:d antigens have been sequenced; and the specific sites for each antigen in selected Salmonella serotypes have been determined. These results, together with flagellar G-complex variable internal sequences obtained by the Foodborne and Diarrheal Diseases Branch at the Centers for Disease Control and Prevention in Atlanta, Ga., have been used to design a multiplex PCR to identify the G-complex antigens as well as the H:i, H:r, H:l,v, H:e,h, Hz10, H:b, and H:d first-phase antigens. These antigens are part of the most common Salmonella serotypes possessing first-phase flagellar antigens. Salmonella enterica serotype Enteritidis is identified by adding a specific primer pair published previously (P. G. Agron, R. L. Walker, H. Kinde, S. J. Sawyer, D. C. Hayes, J. Wollard, and G. L. Andersen, Appl. Environ. Microbiol. 67:4984-4991, 2001). This multiplex PCR includes 13 primers. A total of 161 Salmonella strains associated with 72 different serotypes were tested. Each strain generated one first-phase-specific antigen fragment ranging from 100 to 500 bp; Salmonella serotype Enteritidis, however, generated two amplicons of 500 bp that corresponded to the G complex and a 333-bp serotype-specific amplicon, respectively. Twenty-three strains representing 19 serotypes with flagellar genes different from those targeted in this work did not generate any fragments. The method is quick, specific, and reproducible and is independent of the phase expressed by the bacteria when they are tested. PMID:15184437

  9. Serological detection of cutaneous T-cell lymphoma-associated antigens

    PubMed Central

    Eichmüller, Stefan; Usener, Dirk; Dummer, Reinhard; Stein, Angelika; Thiel, Daniela; Schadendorf, Dirk

    2001-01-01

    Cutaneous T-cell lymphomas (CTCL) are a group of skin neoplasms that originate from T lymphocytes and are difficult to treat in advanced stages. The present study is aimed at the identification of tumor-specific antigens from a human testis cDNA library using human sera known as the SEREX (serological identification of recombinantly expressed genes) approach. A cDNA library from normal testicle tissue was prepared and approximately 2 million recombinants were screened with sera from Sézary Syndrome and Mycosis fungoides patients. A total of 28 positive clones belonging to 15 different genes/ORFs were identified, including five hitherto unknown sequences. Whereas control sera did not react with most clones, 11–71% sera from CTCL patients were reactive against the identified clones. Expression analysis on 28 normal control and 17 CTCL tissues by reverse transcription–PCR (RT-PCR) and Northern blotting revealed seven ubiquitously distributed antigens, six differentially expressed antigens (several normal tissues were positive), and two tumor-specific antigens that were expressed only in testis and tumor tissues: (i) A SCP-1-like sequence, which has already been detected in various tumors, has been found in one CTCL tumor and four sera of CTCL patients reacted with various SCP-1-like clones and (ii) a new sequence named cTAGE-1 (CTCL-associated antigen 1) was detected in 35% of CTCL tumor tissues and sera of 6/18 patients reacted with this clone. The present study unravels CTCL-associated antigens independent of the T-cell receptor. The SCP-1-like gene and cTAGE-1 were shown to be immunogenic and immunologically tumor-specific and may therefore be candidates for immunotherapy targeting CTCL. PMID:11149944

  10. Novel methods for expression of foreign antigens in live vector vaccines

    PubMed Central

    Wang, Jin Yuan; Harley, Regina H.; Galen, James E.

    2013-01-01

    Bacterial live vector vaccines represent a vaccine development strategy that offers exceptional flexibility. In this approach, genes encoding protective antigens of unrelated bacterial, viral or parasitic pathogens are expressed in an attenuated bacterial vaccine strain that delivers these foreign antigens to the immune system, thereby eliciting relevant immune responses. Rather than expressing these antigens using low copy expression plasmids, here we pursue expression of foreign proteins from the live vector chromosome. Our strategy is designed to compensate for the inherent disadvantage of loss of gene dosage (vs. plasmid-based expression) by integrating antigen-encoding gene cassettes into multiple chromosomal sites already inactivated in an attenuated Salmonella enterica serovar Typhi vaccine candidate. We tested expression of a cassette encoding the green fluorescent protein (GFPuv) integrated separately into native guaBA, htrA or clyA chromosomal loci. Using single integrations, we show that expression levels of GFPuv are significantly affected by the site of integration, regardless of the inclusion of additional strong promoters within the incoming cassette. Using cassettes integrated into both guaBA and htrA, we observe cumulative synthesis levels from two integration sites superior to single integrations. Most importantly, we observe that GFPuv expression increases in a growth phase-dependent manner, suggesting that foreign antigen synthesis may be “tuned” to the physiology of the live vaccine. We expect this novel platform expression technology to prove invaluable in the development of a wide variety of multivalent live vector vaccines, capable of expressing multiple antigens from both chromosomal and plasmid-based expression systems within a single strain. PMID:23406777

  11. Targeted Surface Expression of an Exogenous Antigen in Stably Transfected Babesia bovis

    PubMed Central

    Laughery, Jacob M.; Knowles, Donald P.; Schneider, David A.; Bastos, Reginaldo G.; McElwain, Terry F.; Suarez, Carlos E.

    2014-01-01

    Babesia bovis is a tick-borne intraerythocytic protozoan responsible for acute disease in cattle which can be controlled by vaccination with attenuated B. bovis strains. Emerging B. bovis transfection technologies may increase the usefulness of these live vaccines. One use of transfected B. bovis parasites may be as a vaccine delivery platform. Previous transfection methods for B. bovis were limited by single expression sites and intracellular expression of transfected antigens. This study describes a novel transfection system in which two exogenous genes are expressed: one for selection and the other for a selected antigen designed to be delivered to the surface of the parasites. The strategy for duplicating the number of transfected genes was based on the use of the putative bidirectional promoter of the B. bovis 1.4 Kb ef-1α intergenic region. The ability of this region to regulate two independent expression sites was demonstrated using a luciferase assay on transiently transfected B. bovis parasites and then incorporated into a stable transfection plasmid to control independent expression of the selectable marker GFP-BSD and another gene of interest. A chimeric gene was synthetized using sequences from the protective B-cell epitopes of Rhipicephalus microplus tick antigen Bm86 along with sequences from the surface exposed B. bovis major surface antigen-1. This chimeric gene was then cloned into the additional expression site of the transfection plasmid. Transfection of the B. bovis Mo7 strain with this plasmid resulted in stable insertion into the ef-1α locus and simultaneous expression of both exogenous genes. Expression of the Bm86 epitopes on the surface of transfected merozoites was demonstrated using immunofluorescence analyses. The ability to independently express multiple genes by the inclusion of a bidirectional promoter and the achievement of surface expression of foreign epitopes advances the potential of transfected B. bovis as a future vaccine

  12. Lymphocyte activation by streptococcal antigens in psoriasis.

    PubMed

    Gross, W L; Packhäuser, U; Hahn, G; Westphal, E; Christophers, E; Schlaak, M

    1977-11-01

    Cell-mediated immune responses in 28 hospitalized patients with psoriasis and in 36 healthy controls were studied using the two-step leukocyte migration agarose test. Specific cell-mediated immunity to A-streptococcal cell wall and cell membrane antigens occurred significantly more often in patients with psoriasis than in the control group. A statistically significant correlation between psoriasis-associated antigens of the HLA-B locus and cellular immune reactivity to A-streptococcal antigens or clinical course was not found. When patients with guttate psoriasis were compared separately with the control group, leukocyte migration inhibition induced by cell-free supernatants of A-streptococcal antigen-exposed mononuclear cell cultures was found to be more frequent than in other forms of psoriasis.

  13. THE SUBCELLULAR DISTRIBUTION OF ANTIGEN IN MACROPHAGES

    PubMed Central

    Kölsch, E.; Mitchison, N. A.

    1968-01-01

    The intracellular fate of phagocytosed antigens in cells from peritoneal exudate in CBA mice has been studied by using 126I and 131I labeled antigens. After uptake of labeled antigen, cells were homogenized and the subcellular fractions were analyzed by isopycnic centrifugation in a sucrose gradient. The uptake of heat-denatured BSA (c BSA) by these cells in vivo is 3.5 µg/mg c BSA injected/108 cells. The uptake by cells in animals which were exposed 2 days earlier to 900 r whole body irradiation is slightly lower but does not differ significantly. 90% of the phagocytosed material is degraded within 2–3 hr, the residual 10% is retained at least over an 8 hr periods. Using a pulse and chase technique, with 125I and 131I c BSA in vitro and in vivo it was shown that newly phagocytosed antigen is found mainly in a lysosomal turnover compartment of a density 1.19 g cm–3. Antigen which has been in the cells for longer was found in a denser fraction (1.26 g cm–3). In a comparison of nhrmal and X-irradiated cells it can be shown that after irradiation with 900 r less c BSA is found in this storage compartment. Binding of the antigen to the subcellular fractions, and its behavior towards several detergents has been studied. Subcellular fractions do not have the increased immunogenic capacity of antigen enclosed in living macrophages. Two synthetic polypeptide antigens, poly(D-Tyr, D-Glu, D-Ala) and poly-(L-Tyr, L-Glu) have a different subcellular distribution from c BSA, BSA, or bovine gamma globulin. Apart from also being found in the 1.26 storage compartment the polypeptide antigens are mainly located in a 1.15 compartment and only to a small extent in the 1.19 compartment. The half-life of these antigens in the cells is much longer than the half-life of the protein antigens studied. The finding of several subcellular compartments is discussed in connection with the functions possibly performed by macrophages. PMID:5682940

  14. Mapping Epitopes on a Protein Antigen by the Proteolysis of Antigen-Antibody Complexes

    NASA Astrophysics Data System (ADS)

    Jemmerson, Ronald; Paterson, Yvonne

    1986-05-01

    A monoclonal antibody bound to a protein antigen decreases the rate of proteolytic cleavage of the antigen, having the greatest effect on those regions involved in antibody contact. Thus, an epitope can be identified by the ability of the antibody to protect one region of the antigen more than others from proteolysis. By means of this approach, two distinct epitopes, both conformationally well-ordered, were characterized on horse cytochrome c.

  15. [Hepatitis B virus core antigen as a carrier for virus-like partical vaccine: a review].

    PubMed

    Yang, Xing-Yu; Bo, Hong; Shu, Yue-Long

    2012-05-01

    Hepatitis B virus core antigen (HBcAg) is a major viral nucleocapsid protein of HBV. It is a 21-22kD protein consisting of 183-185 amino acids. Because of its easy purification, strong immunogenicity, high expression level, and self-assembles into the virus-like particles (VLP), HBcAg could be an efficient and safe VLP carrier for developing vaccines for various pathogens. Up to now, HBcAg VLP carrier has been an important system to develop novel vaccines and many antigen epitope genes from viruses, bacteria and parasites were expressed successfully using the system.

  16. Vertebrate Cells Express Protozoan Antigen after Hybridization

    NASA Astrophysics Data System (ADS)

    Crane, Mark St. J.; Dvorak, James A.

    1980-04-01

    Epimastigotes, the invertebrate host stage of Trypanosoma cruzi, the protozoan parasite causing Chagas' disease in man, were fused with vertebrate cells by using polyethylene glycol. Hybrid cells were selected on the basis of T. cruzi DNA complementation of biochemical deficiencies in the vertebrate cells. Some clones of the hybrid cells expressed T. cruzi-specific antigen. It might be possible to use selected antigens obtained from the hybrids as vaccines for immunodiagnosis or for elucidation of the pathogenesis of Chagas' disease.

  17. Tales of Antigen Evasion from CAR Therapy.

    PubMed

    Sadelain, Michel

    2016-06-01

    Both T cells bearing chimeric antigen receptors and tumor-specific antibodies can successfully target some malignancies, but antigen escape can lead to relapse. Two articles in this issue of Cancer Immunology Research explore what effective countermeasures may prevent it. Cancer Immunol Res; 4(6); 473-473. ©2016 AACRSee articles by Zah et al., p. 498, and Rufener et al., p. 509. PMID:27252092

  18. Antigens of Streptococcus sanguis: purification and characterization of the b antigen.

    PubMed Central

    Appelbaum, B; Rosan, B

    1978-01-01

    The antigen defining Streptococcus sanguis serotype 2 has been designated the b antigen. This antigen can be detected in extracts, obtained from whole cells by autoclaving (Rantz and Randall extraction), as a single precipitin band using a reference antiserum (M-5). However, the extract can also be shown to contain a teichoic acid using anti-polyglycerol phosphate serum. This teichoic acid does not contain the antigenic determinant for group H specificity. Studies of the b antigen have been hampered because of the difficulty in separating the b antigen from the teichoic acid using ion-exchange and molecular sieve chromatography. However, a relatively pure preparation has been obtained by affinity chromatography using anti-polyglycerol phosphate serum coupled to Sepharose. The isolated b antigen is a typical streptococcal cell wall polysaccharide composed of glucose, rhamnose, and N-acetylglucosamine in a molar ratio of 2.5:1.0:0.1. The antigen appears to have a single antigenic determinant closely related to isomaltose (glucose alpha-1,6-glucoside) based upon hapten inhibition studies. Images PMID:711341

  19. Heat shock protein derivatives for delivery of antigens to antigen presenting cells.

    PubMed

    Nishikawa, Makiya; Takemoto, Seiji; Takakura, Yoshinobu

    2008-04-16

    Delivery of antigens to antigen presenting cells (APCs) is a key issue for developing effective cancer vaccines. Controlling the tissue distribution of antigens can increase antigen-specific immune responses, including the induction of cytotoxic T lymphocytes (CTL). Heat shock protein 70 (Hsp70) forms complexes with a variety of tumor-related antigens via its polypeptide-binding domain. Because Hsp70 is taken up by APCs through recognition by Hsp receptors, such as CD91 and LOX-1, its application to antigen delivery systems has been examined both in experimental and clinical settings. A tissue distribution study revealed that Hsp70 is mainly taken up by the liver, especially by hepatocytes, after intravenous injection in mice. A significant amount of Hsp70 was also delivered to regional lymph nodes when it was injected subcutaneously, supporting the hypothesis that Hsp70 is a natural targeting system for APCs. Model antigens were complexed with or conjugated to Hsp70, resulting in greater antigen-specific immune responses. Cytoplasmic delivery of Hsp70-antigen further increased the efficacy of the Hsp70-based vaccines. These findings indicate that effective cancer therapy can be achieved by developing Hsp70-based anticancer vaccines when their tissue and intracellular distribution is properly controlled. PMID:17980980

  20. Microscale purification of antigen-specific antibodies

    PubMed Central

    Brown, Eric P.; Normandin, Erica; Osei-Owusu, Nana Yaw; Mahan, Alison E.; Chan, Ying N.; Lai, Jennifer I.; Vaccari, Monica; Rao, Mangala; Franchini, Genoveffa; Alter, Galit; Ackerman, Margaret E.

    2015-01-01

    Glycosylation of the Fc domain is an important driver of antibody effector function. While assessment of antibody glycoform compositions observed across total plasma IgG has identified differences associated with a variety of clinical conditions, in many cases it is the glycosylation state of only antibodies against a specific antigen or set of antigens that may be of interest, for example, in defining the potential effector function of antibodies produced during disease or after vaccination. Historically, glycoprofiling such antigen-specific antibodies in clinical samples has been challenging due to their low prevalence, the high sample requirement for most methods of glycan determination, and the lack of high-throughput purification methods. New methods of glycoprofiling with lower sample requirements and higher throughput have motivated the development of microscale and automatable methods for purification of antigen-specific antibodies from polyclonal sources such as clinical serum samples. In this work, we present a robot-compatible 96-well plate-based method for purification of antigen-specific antibodies, suitable for such population level glycosylation screening. We demonstrate the utility of this method across multiple antibody sources, using both purified plasma IgG and plasma, and across multiple different antigen types, with enrichment factors greater than 1000-fold observed. Using an on-column IdeS protease treatment, we further describe staged release of Fc and Fab domains, allowing for glycoprofiling of each domain. PMID:26078040

  1. Microscale purification of antigen-specific antibodies.

    PubMed

    Brown, Eric P; Normandin, Erica; Osei-Owusu, Nana Yaw; Mahan, Alison E; Chan, Ying N; Lai, Jennifer I; Vaccari, Monica; Rao, Mangala; Franchini, Genoveffa; Alter, Galit; Ackerman, Margaret E

    2015-10-01

    Glycosylation of the Fc domain is an important driver of antibody