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Sample records for 51-kilodalton antigen gene

  1. Analysis of 16S rRNA and 51-kilodalton antigen gene and transmission in mice of Ehrlichia risticii in virgulate trematodes from Elimia livescens snails in Ohio.

    PubMed

    Kanter, M; Mott, J; Ohashi, N; Fried, B; Reed, S; Lin, Y C; Rikihisa, Y

    2000-09-01

    Operculate snails (the family Pleuroceridae: Elimia livescens) were collected between June and October 1998 from a river in central Ohio where repeated cases of Potomac horse fever (PHF) have occurred. Of collected snails, consistently 50 to 80% carried a combination of cercariae and sporocysts of digenetic virgulate trematodes. The trematodes obtained from each snail were pooled and tested for Ehrlichia risticii, the agent of PHF, by nested PCR using primers specific to the 16S rRNA gene. Out of a total of 209 trematode pools, 50 pools were found to be positive by PCR. The DNA sequence of the 16S rRNA gene identified in one trematode pool was identical to that of the type strain of E. risticii, and the sequence of the gene identified in another pool differed from that of the type strain by 1 nucleotide. Comparison of the deduced amino acid sequence of the partial 51-kDa antigen gene from various sources revealed that Maryland, Ohio (except Ohio 081), and Kentucky strains are in a cluster distinct from the sequences obtained from sources in California and Oregon. Ohio 081 was shown previously by antigenic composition analysis to be distinct from other groups. However, all sequences examined were not segregated according to their sources: horse blood or infected trematodes. E. risticii was found to be transmittable from trematodes to mice and was subsequently passaged from infected mice to additional mice, as determined by PCR analysis. Our findings suggest the evolution of E. risticii in the natural reservoir in separate geographic regions and persistent infection of trematode populations with E. risticii during summer and early fall. The study also suggests that the mouse can be used to isolate E. risticii from the infected trematode.

  2. 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

  3. Analysis of antigen receptor genes in Hodgkin's disease.

    PubMed Central

    Angel, C A; Pringle, J H; Naylor, J; West, K P; Lauder, I

    1993-01-01

    AIM--To analyse the configuration of the antigen receptor genes in Hodgkin's disease. METHODS--DNA extracted from 45 samples of Hodgkin's disease was analysed using Southern blotting and DNA hybridisation, using probes to the joining region of the immunoglobulin heavy chain gene, the constant region of kappa immunoglobulin light chain gene, and the constant region of the beta chain of the T cell receptor gene. RESULTS--A single case of nodular sclerosing disease showed clonal rearrangement of the immunoglobulin heavy and light chain genes, all other samples having germline immunoglobulin genes. The nature of the clonal population in the diseased tissue is uncertain, because the intensity of the rearranged bands did not correlate with the percentage of Reed-Sternberg cells present. The T cell receptor genes were in germline configuration in all the samples. CONCLUSIONS--Antigen receptor gene rearrangement is a rare finding in unselected cases of Hodgkin's disease. Images PMID:8388407

  4. The TL region gene 37 encodes a Qa-1 antigen

    PubMed Central

    1990-01-01

    Of all the biochemically defined mouse MHC class I molecules, the Qa-1 antigens are the only ones for which a gene has not been identified. Recent evidence has suggested that Qa-1 antigens are functional class I molecules and can function as restriction elements for gamma/delta T cells. We have examined the relationship between Qa-1 and the product of gene 37, a presumed novel class I antigen encoded within the TL region. Immunoprecipitation and polyacrylamide gel electrophoresis analysis of the molecules reactive with anti-Qa-1 and anti-37 sera show that the Qa-1 molecule of Qa-1b (Qa-1.2) mouse strains is identical to the product of gene 37 on the basis of molecular weight, pI, and strain distribution. Immunodepletion, biosynthetic labeling, and tunicamycin treatment confirm that the protein encoded by gene 37 in Qa-1b mice is Qa-1.2. In contrast, the anti-37 serum was unable to recognize the Qa-1 molecule in Qa-1a strains. Given the fact that the only allele to gene 37 thus far identified in a Qa-1a strain (A/J) has a termination codon in the alpha 3 domain, our data lead us to conclude that the Qa-1 molecule expressed in Qa-1a mice is not a true allelic product of the gene 37 encoded antigen of Qa-1b mouse strains. PMID:2258708

  5. Molecular cloning of Taenia taeniaeformis oncosphere antigen genes.

    PubMed

    Cougle, W G; Lightowlers, M W; Bogh, H O; Rickard, M D; Johnson, K S

    1991-03-01

    Infection of mice with the cestode Taenia taeniaeformis exhibits several important features common to other cestode infections, including the ability to vaccinate with crude antigen mixtures. Partial purification of the protective oncosphere antigens has been reported with a cutout from deoxycholate (DOC) acrylamide gels; this cutout was called fraction II (FII), and comprises approximately 10% of total DOC-soluble oncosphere antigen. Western blots of DOC gels probed with anti-FII antisera revealed a series of 3-5 discrete bands within the FII region. Further fractionation of the FII antigens on DOC gels was impractical due to limitations in supply of oncospheres, so a cDNA library was constructed from 150 ng of oncosphere mRNA and screened with alpha-FII antisera. Two distinct clone families were identified, oncA and oncB. Antibodies affinity-purified on either of two representative members, oncA1 and oncB1, recognised all the FII bands. Individual FII bands excised from a DOC gel resolved into an overlapping series of molecules when re-run on SDS-PAGE, indicating that each FII band consisted of several polypeptides of differing molecular weight. Immunoprecipitates resolved on SDS-PAGE revealed that alpha-FII recognised 3 major oncosphere antigens, of 62, 34 and 25 kDa; antisera against oncB precipitated both the 34- and 25-kDa antigens, whereas alpha-oncA antisera precipitated the 62-kDa antigen. We conclude that oncA and oncB encode the major antigens in the FII complex. The 62-kDa antigen encoded by oncA1 was the only common antigen precipitated by anti-FII and two other antisera raised against different protective extracts, suggesting that it may be a protective component in all three. Southern blot results indicate that oncA and oncB are distinct genes present at low copy number in the genome. Evidence is also presented suggesting that some cestode mRNAs, including oncA, may use variant polyadenylation signals.

  6. Evolutionary History of the Cancer Immunity Antigen MAGE Gene Family

    PubMed Central

    Katsura, Yukako; Satta, Yoko

    2011-01-01

    The evolutionary mode of a multi-gene family can change over time, depending on the functional differentiation and local genomic environment of family members. In this study, we demonstrate such a change in the melanoma antigen (MAGE) gene family on the mammalian X chromosome. The MAGE gene family is composed of ten subfamilies that can be categorized into two types. Type I genes are of relatively recent origin, and they encode epitopes for human leukocyte antigen (HLA) in cancer cells. Type II genes are relatively ancient and some of their products are known to be involved in apoptosis or cell proliferation. The evolutionary history of the MAGE gene family can be divided into four phases. In phase I, a single-copy state of an ancestral gene and the evolutionarily conserved mode had lasted until the emergence of eutherian mammals. In phase II, eight subfamily ancestors, with the exception for MAGE-C and MAGE-D subfamilies, were formed via retrotransposition independently. This would coincide with a transposition burst of LINE elements at the eutherian radiation. However, MAGE-C was generated by gene duplication of MAGE-A. Phase III is characterized by extensive gene duplication within each subfamily and in particular the formation of palindromes in the MAGE-A subfamily, which occurred in an ancestor of the Catarrhini. Phase IV is characterized by the decay of a palindrome in most Catarrhini, with the exception of humans. Although the palindrome is truncated by frequent deletions in apes and Old World monkeys, it is retained in humans. Here, we argue that this human-specific retention stems from negative selection acting on MAGE-A genes encoding epitopes of cancer cells, which preserves their ability to bind to highly divergent HLA molecules. These findings are interpreted with consideration of the biological factors shaping recent human MAGE-A genes. PMID:21695252

  7. Regulation of antigen-receptor gene assembly in hagfish.

    PubMed

    Kishishita, Natsuko; Matsuno, Tatsuya; Takahashi, Yoshimasa; Takaba, Hiroyuki; Nishizumi, Hirofumi; Nagawa, Fumikiyo

    2010-02-01

    Variable lymphocyte receptors (VLRs) are antigen receptors in the jawless vertebrates lamprey and hagfish. VLR genes are classified into VLRA and VLRB, and lymphocytes expressing VLRA are T-cell-like, whereas those expressing VLRB are B-cell-like in the sea lamprey. Diverse VLR genes are assembled somatically in lymphocytes; however, how the assembly is regulated is still largely unknown. Here, we analyse VLR gene assembly at the single-cell level in the inshore hagfish (Eptatretus burgeri). Each lymphocyte assembles and transcribes only one type of VLR gene, either VLRA or VLRB. In general, monoallelic assembly of VLR was observed, but diallelic assembly was found in some cases--in many of which, one allele was functional and the other was defective. In fact, all VLR-assembled lymphocytes contained at least one functional VLR gene. Together, these results indicate a feedback inhibition of VLR assembly and selection of VLR-positive lymphocytes.

  8. Regulation of antigen-receptor gene assembly in hagfish

    PubMed Central

    Kishishita, Natsuko; Matsuno, Tatsuya; Takahashi, Yoshimasa; Takaba, Hiroyuki; Nishizumi, Hirofumi; Nagawa, Fumikiyo

    2010-01-01

    Variable lymphocyte receptors (VLRs) are antigen receptors in the jawless vertebrates lamprey and hagfish. VLR genes are classified into VLRA and VLRB, and lymphocytes expressing VLRA are T-cell-like, whereas those expressing VLRB are B-cell-like in the sea lamprey. Diverse VLR genes are assembled somatically in lymphocytes; however, how the assembly is regulated is still largely unknown. Here, we analyse VLR gene assembly at the single-cell level in the inshore hagfish (Eptatretus burgeri). Each lymphocyte assembles and transcribes only one type of VLR gene, either VLRA or VLRB. In general, monoallelic assembly of VLR was observed, but diallelic assembly was found in some cases—in many of which, one allele was functional and the other was defective. In fact, all VLR-assembled lymphocytes contained at least one functional VLR gene. Together, these results indicate a feedback inhibition of VLR assembly and selection of VLR-positive lymphocytes. PMID:20075989

  9. Changes in repeat number, sequence, and reading frame in S-antigen genes of Plasmodium falciparum.

    PubMed Central

    Saint, R B; Coppel, R L; Cowman, A F; Brown, G V; Shi, P T; Barzaga, N; Kemp, D J; Anders, R F

    1987-01-01

    The S antigens from different isolates of Plasmodium falciparum exhibit extensive size, charge, and serological diversity. We show here that the S-antigen genes behave as multiple alleles of a single locus. The size heterogeneity results from different numbers, lengths, and/or sequences of tandem repeat units encoded within the S-antigen genes. Two genes studied here encode antigenically different S antigens but nevertheless have closely related tandem repeat sequences. We show that antigenic differences can arise because repeats are translated in different reading frames. Images PMID:3313007

  10. 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

  11. Recombinant Carcinoembryonic Antigen as a Reporter Gene for Molecular Imaging

    PubMed Central

    Kenanova, Vania; Barat, Bhaswati; Olafsen, Tove; Chatziioannou, Arion; Herschman, Harvey R.; Braun, Jonathan; Wu, Anna M.

    2009-01-01

    Purpose Reporter genes can provide a way of non-invasively assessing gene activity in vivo. However, current reporter gene strategies may be limited by the immunogenicity of foreign reporter proteins, endogenous expression or unwanted biological activity. We have developed a reporter gene based on carcinoembryonic antigen (CEA), a human protein with limited normal tissue expression. Methods To construct a CEA reporter gene for PET, a CEA minigene (N-A3) was fused to the extracellular and transmembrane domains of the human FcγRIIb receptor. The NA3-FcγRIIb recombinant gene, driven by a CMV promoter, was transfected in Jurkat (human T cell leukemia) cells. Expression was analyzed by flow cytometry, immunohistochemistry (IHC), and microPET imaging. Results Flow cytometry identified Jurkat clones stably expressing NA3-FcγRIIb at low, medium, and high levels. High and medium NA3-FcγRIIb expression could also be detected by Western blot. Reporter gene positive and negative Jurkat cells were used to establish xenografts in athymic mice. IHC showed staining of the tumor with high reporter gene expression; medium and low N-A3 expression was not detected. MicroPET imaging, using an anti-CEA 124I-labeled scFv-Fc antibody fragment, demonstrated that only high N-A3 expression could be detected. Specific accumulation of activity was visualized at the N-A3 positive tumor as early as 4h. MicroPET image quantitation showed tumor activity of 1.8(±0.2), 15.2(±1.3) and 4.6(±1.2) %ID/g at 4h, 20h and 48h, respectively. Biodistribution at 48h, demonstrated tumor uptake of 4.8(±0.8) %ID/g. Conclusion The CEA N-A3 minigene has the potential to be used as a reporter gene for imaging cells in vivo. PMID:18719907

  12. Regulation of cancer germline antigen gene expression: implications for cancer immunotherapy.

    PubMed

    Akers, Stacey N; Odunsi, Kunle; Karpf, Adam R

    2010-05-01

    Cancer germline (CG; also known as cancer-testis) antigen genes are normally expressed in germ cells and trophoblast tissues and are aberrantly expressed in a variety of human malignancies. CG antigen genes have high clinical relevance as they encode a class of immunogenic and highly selective tumor antigens. CG antigen-directed immunotherapy is undergoing clinical evaluation for the treatment of a number of solid tumor malignancies and has been demonstrated to be safe, provoke immune responses and be of therapeutic benefit. Achieving an improved understanding of the mechanisms of CG antigen gene regulation will facilitate the continued development of targeted therapeutic approaches against tumors expressing these antigens. Substantial evidence suggests epigenetic mechanisms, particularly DNA methylation, as a primary regulator of CG antigen gene expression in normal and cancer cells as well as in stem cells. The roles of sequence-specific transcription factors and signal transduction pathways in controlling CG antigen gene expression are less clear but are emerging. A combinatorial therapeutic approach involving epigenetic modulatory drugs and CG antigen immunotherapy is suggested based on these data and is being actively pursued. In this article, we review the mechanisms of CG antigen gene regulation and discuss the implications of these mechanisms for the development of cancer immunotherapy approaches targeting CG antigens.

  13. Evidence for horizontal gene transfer of two antigenically distinct O antigens in Bordetella bronchiseptica

    USDA-ARS?s Scientific Manuscript database

    Antigenic variation is one mechanism pathogens use to avoid immune-mediated competition between 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. b...

  14. T-cell intracellular antigens function as tumor suppressor genes.

    PubMed

    Sánchez-Jiménez, C; Ludeña, M D; Izquierdo, J M

    2015-03-05

    Knockdown of T-cell intracellular antigens TIA1 and TIAR in transformed cells triggers cell proliferation and tumor growth. Using a tetracycline-inducible system, we report here that an increased expression of TIA1 or TIAR in 293 cells results in reduced rates of cell proliferation. Ectopic expression of these proteins abolish endogenous TIA1 and TIAR levels via the regulation of splicing of their pre-mRNAs, and partially represses global translation in a phospho-eukaryotic initiation factor 2 alpha-dependent manner. This is accompanied by cell cycle arrest at G1/S and cell death through caspase-dependent apoptosis and autophagy. Genome-wide profiling illustrates a selective upregulation of p53 signaling pathway-related genes. Nude mice injected with doxycycline-inducible cells expressing TIA1 or TIAR retard, or even inhibit, growth of xenotumors. Remarkably, low expressions of TIA1 and TIAR correlate with poor prognosis in patients with lung squamous cell carcinoma. These findings strongly support the concept that TIA proteins act as tumor suppressor genes.

  15. Molecular and genetic analyses of the putative Proteus O antigen gene locus.

    PubMed

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

    2010-08-01

    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.

  16. 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

  17. Proteinase 3, Wegener's autoantigen: from gene to antigen.

    PubMed

    van der Geld, Y M; Limburg, P C; Kallenberg, C G

    2001-02-01

    Proteinase 3 (PR3) is one of four serine protease homologues in the azurophilic granules of neutrophils and granules of monocytes. It is of importance that anti-neutrophil cytoplasmic antibodies (ANCA) in patients with Wegener's granulomatosis (WG) are mainly directed against PR3 only. Furthermore, PR3 is overexpressed in a variety of acute and chronic myeloid leukemia cells. Cytotoxic T lymphocytes specific for a PR3-derived peptide have been shown to specifically lyse leukemia cells that overexpress PR3. This review will focus on PR3 and the characteristics of PR3 that might implicate this particular antigen in the pathogenesis of WG and as target for immunotherapy in myeloid leukemias. We will discuss the genetic localization and gene regulation of PR3, the processing, storage, and expression of the PR3 protein, and the physiological functions of PR3, and compare this with the three other neutrophil-derived serine proteases: human leukocyte elastase, cathepsin G, and azurocidin. Three main differences are described between PR3 and the other serine proteases. This makes PR3 a very intriguing protein with a large array of physiological functions, some of which may play a role in ANCA-associated vasculitidis and myeloid leukemia.

  18. A Human Minor Histocompatibility Antigen Resulting from Differential Expression due to a Gene Deletion

    PubMed Central

    Murata, Makoto; Warren, Edus H.; Riddell, Stanley R.

    2003-01-01

    Minor histocompatibility antigens (minor H antigens) are targets of graft-versus-host disease and graft-versus-leukemia responses after allogeneic human leukocyte antigen identical hematopoietic stem cell transplantation. Only a few human minor H antigens have been molecularly characterized and in all cases, amino acid differences between homologous donor and recipient proteins due to nucleotide polymorphisms in the respective genes were responsible for immunogenicity. Here, we have used cDNA expression cloning to identify a novel human minor H antigen encoded by UGT2B17, an autosomal gene in the multigene UDP-glycosyltransferase 2 family that is selectively expressed in liver, intestine, and antigen-presenting cells. In contrast to previously defined human minor H antigens, UGT2B17 is immunogenic because of differential expression of the protein in donor and recipient cells as a consequence of a homozygous gene deletion in the donor. Deletion of individual members of large gene families is a common form of genetic variation in the population and our results provide the first evidence that differential protein expression as a consequence of gene deletion is a mechanism for generating minor H antigens in humans. PMID:12743171

  19. Novel Aeromonas hydrophila PPD134/91 Genes Involved in O-Antigen and Capsule Biosynthesis

    PubMed Central

    Zhang, Y. L.; Arakawa, E.; Leung, K. Y.

    2002-01-01

    The sequences of the O-antigen and capsule gene clusters of the virulent Aeromonas hydrophila strain PPD134/91 were determined. The O-antigen gene cluster is 17,296 bp long and comprises 17 genes. Seven pathway genes for the synthesis of rhamnose and mannose, six transferase genes, one O unit flippase gene, and one O-antigen chain length determinant gene were identified by amino acid sequence similarity. PCR and Southern blot analysis were performed to survey the distribution of these 17 genes among 11 A. hydrophila strains of different serotypes. A. hydrophila PPD134/91 might belong to serotype O:18, as represented by JCM3980; it contained all the same O-antigen genes as JCM3980 (97 to 100% similarity at the DNA and amino acid levels). The capsule gene cluster of A. hydrophila PPD134/91 is 17,562 bp long and includes 13 genes, which were assembled into three distinct regions similar to those of the group II capsule gene cluster of Escherichia coli and other bacteria. Regions I and III contained four and two capsule transport genes, respectively. Region II had five genes which were highly similar to capsule synthesis pathway genes found in other bacteria. Both the purified O-antigen and capsular polysaccharides increased the ability of the avirulent A. hydrophila strain PPD35/85 to survive in naïve tilapia serum. However, the purified surface polysaccharides had no inhibitory effect on the adhesion of A. hydrophila PPD134/91 to carp epithelial cells. PMID:11953367

  20. Immunophenotypic and antigen receptor gene rearrangement analysis in T cell neoplasia.

    PubMed Central

    Knowles, D. M.

    1989-01-01

    The author reviews the immunophenotypic profiles displayed by the major clinicopathologic categories of T cell neoplasia, the immunophenotypic criteria useful in the immunodiagnosis of T cell neoplasia, and the contributions made by antigen receptor gene rearrangement analysis to the understanding of T cell neoplasia. Neoplasms belonging to distinct clinicopathologic categories of T cell neoplasia often exhibit characteristic immunophenotypic profiles. Approximately 80% of lymphoblastic lymphomas and 20% of acute lymphoblastic leukemias express phenotypes consistent with prethymic and intrathymic stages of T cell differentiation, including intranuclear terminal deoxynucleotidyl transferase. Cutaneous T cell lymphomas of mycosis fungoides type usually express pan-T cell antigens CD2, CD5, and CD3, often lack the pan-T cell antigen CD7, and usually express the mature, peripheral helper subset phenotype, CD4+ CD8-. Cutaneous T cell lymphomas of nonmycosis fungoides type and peripheral T cell lymphomas often lack one or more pan-T cell antigens and, in addition, occasionally express the anomalous CD4+ CD8+ or CD4- CD8- phenotypes. T gamma-lymphoproliferative disease is divisable into two broad categories: those cases that are CD3 antigen positive and exhibit clonal T cell receptor beta chain (TCR-beta) gene rearrangements and those cases that are CD3 antigen negative and exhibit the TCR-beta gene germline configuration. Human T cell lymphotropic virus-I (HTLV-I) associated Japanese, Carribean, and sporadic adult T cell leukemia/lymphomas usually express pan-T cell antigens, the CD4+ CD8- phenotype, and various T cell-associated activation antigens, including the interleukin-2 receptor (CD25). Immunophenotypic criteria useful in the immunodiagnosis of T cell neoplasia include, in increasing order of utility, T cell predominance, T cell subset antigen restriction, anomalous T cell subset antigen expression, and deletion of one or more pan-T cell antigens. Only in

  1. Comparative analysis of prostate-specific membrane antigen (PSMA) versus a prostate-specific membrane antigen-like gene.

    PubMed

    O'Keefe, Denise S; Bacich, Dean J; Heston, Warren D W

    2004-02-01

    Currently prostate-specific membrane antigen (PSMA) is showing promise both as an imaging and therapeutic target for occult prostate cancer metastases. First generation antibodies against PSMA are used for the FDA approved Prostascint trade mark monoclonal antibody scan and second generation antibodies are being developed for therapeutic targeting as well as imaging 1. However, there have been reports describing PSMA expression in non-prostatic tissues including kidney, liver, and brain. As we had previously showed the existence of a human PSMA homolog, we set out to determine if this non-prostatic expression was due to expression of the PSMA or another gene. The PSMA homolog (PSMA-like) cDNA was cloned by screening a liver cDNA library. mRNA expression of the PSMA and PSMA-like genes was determined via Northern blot analysis using two different probes and protein expression confirmed in some tissues via Western blot analysis. Transcriptional regulation of the two genes was examined using reporter constructs driving luciferase expression. The PSMA-like gene possesses 98% identity to the PSMA gene at the nucleotide level and is expressed in kidney and liver under the control of a different promoter to the PSMA gene. The PSMA gene is expressed in several human tissues and is most abundant in the nervous system and the prostate. The non-prostatic expression of PSMA should be taken into consideration when designing clinical strategies targeting PSMA. Copyright 2003 Wiley-Liss, Inc.

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

    USDA-ARS?s Scientific Manuscript database

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

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

    USDA-ARS?s Scientific Manuscript database

    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...

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

    PubMed Central

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

    2011-01-01

    Objectives To confirm genotype diversities of clinical isolates of Bordetella pertussis and to evaluate the risk of pertussis outbreak in Korea. Methods Seven housekeeping genes and 10 antigenic determinant genes from clinical B. pertussis isolates were analyzed by Multilocus sequence typing (MLST). Results More variant pattern was observed in antigenic determinant genes. Especially, PtxS1 gene was the most variant gene; five genotypes were observed from eight global genotypes. In the bacterial type, the number of observed sequence types in the isolates was seven and the most frequent form was type 1 (79.6%). This major sequence type also showed a time-dependent transition pattern. Older isolates (1968 and 1975) showed type 1 and 6 in housekeeping genes and antigenic determinant genes, respectively. However, these were changed to type 2 and 1 in isolates 1999–2008. This transition was mainly attributed to genotype change of PtxS1 and Fim3 gene; the tendency of genotype change was to avoid vaccine-derived genotype. In addition, there was second transition in 2009. In this period, only the sequence type of antigenic determinant genes was changed to type 2. Based Upon Related Sequence Types (BURST) analysis confirmed that there were two clonal complexes (ACCI and ACCII) in the Korean isolates. Moreover, the recently increased sequence type was revealed as AST2 derived from AST 3 in ACCI. Conclusions Genotype changes in Korean distributing strains are still progressing and there was a specific driving force in antigenic determinant genes. Therefore continuous surveillance of genotype change of the distributing strains should be performed to confirm interrelationship of genotype change with vaccine immunity. PMID:24159461

  5. Improved efficiency in amplification of Escherichia coli o-antigen gene clusters using genome-wide sequence comparison

    USDA-ARS?s Scientific Manuscript database

    Background: In many bacteria including E. coli, genes encoding O-antigens are clustered in the chromosome, with a 39-bp JUMPstart sequence and gnd gene located upstream and downstream of the cluster, respectively. For determining the DNA sequence of the E. coli O-antigen gene cluster, one set of P...

  6. Overlapping gene coexpression patterns in human medullary thymic epithelial cells generate self-antigen diversity.

    PubMed

    Pinto, Sheena; Michel, Chloé; Schmidt-Glenewinkel, Hannah; Harder, Nathalie; Rohr, Karl; Wild, Stefan; Brors, Benedikt; Kyewski, Bruno

    2013-09-10

    Promiscuous expression of numerous tissue-restricted self-antigens (TRAs) in medullary thymic epithelial cells (mTECs) is essential to safeguard self-tolerance. A distinct feature of promiscuous gene expression is its mosaic pattern (i.e., at a given time, each self-antigen is expressed only in 1-3% of mTECs). How this mosaic pattern is generated at the single-cell level is currently not understood. Here, we show that subsets of human mTECs expressing a particular TRA coexpress distinct sets of genes. We identified three coexpression groups comprising overlapping and complementary gene sets, which preferentially mapped to certain chromosomes and intrachromosomal gene clusters. Coexpressed gene loci tended to colocalize to the same nuclear subdomain. The TRA subsets aligned along progressive differentiation stages within the mature mTEC subset and, in vitro, interconverted along this sequence. Our data suggest that single mTECs shift through distinct gene pools, thus scanning a sizeable fraction of the overall repertoire of promiscuously expressed self-antigens. These findings have implications for the temporal and spatial (re)presentation of self-antigens in the medulla in the context of tolerance induction.

  7. Structure and gene cluster of the O-antigen of Escherichia coli O133.

    PubMed

    Shashkov, Alexander S; Zhang, Yuanyuan; Sun, Qiangzheng; Guo, Xi; Senchenkova, Sof'ya N; Perepelov, Andrei V; Knirel, Yuriy A

    2016-07-22

    The O-specific polysaccharide (O-antigen) of Escherichia coli O133 was obtained by mild acid hydrolysis of the lipopolysaccharide of E. coli O133. The structure of the hexasaccharide repeating unit of the polysaccharide was elucidated by (1)H and (13)C NMR spectroscopy, including a two-dimensional (1)H-(1)H ROESY experiment: Functions of genes in the O-antigen gene cluster were putatively identified by comparison with sequences in the available databases and, particularly, an encoded predicted multifunctional glycosyltransferase was assigned to three α-l-rhamnosidic linkages.

  8. Antibodies Raised Against Chlamydial Lipopolysaccharide Antigens Reveal Convergence in Germline Gene Usage and Differential Epitope Recognition

    PubMed Central

    Brooks, Cory L; Müller-Loennies, Sven; Borisova, Svetlana N.; Brade, Lore; Kosma, Paul; Hirama, Tomoko; MacKenzie, C. Roger; Brade, Helmut; Evans, Stephen V

    2011-01-01

    In order to explore monoclonal antibody recognition carbohydrate antigens, several structures from two monoclonal antibodies directed against carbohydrate epitopes derived from chlamydial LPS have been solved to high resolution. With the exception of CDR H3, antibodies S54-10 and S73-2 are both derived from the same set of germline gene segments as the previously reported structures S25-2 and S45-18. Despite this similarity, the antibodies differ in specificity and the mechanism by which they recognize their cognate antigen. S54-10 uses an unrelated CDR H3 to recognize its antigen in a fashion analogous to S45-18; however, S73-2 recognizes the same antigen as S45-18 and S54-10 in a wholly unrelated manner. Together, these antibody-antigen structures provide snapshots into how the immune system uses the same set of inherited germline gene segments to generate multiple possible specificities that allow for differential recognition of epitopes, and how unrelated CDR H3 sequences can result in convergent binding of clinically-relevant bacterial antigens. PMID:20000757

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

    PubMed

    Cunha-Neto, E

    1999-02-01

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

  10. Induction of Interferon-Stimulated Genes by Simian Virus 40 T Antigens

    PubMed Central

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

    2010-01-01

    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 (TAgwt) and TAg-mutants in mouse embryo fibroblasts (MEFs). The expression profile of approximately 800 cellular genes was altered by TAgwt and a truncated TAg (TAgN136), 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 TAgwt including many interferon stimulated genes (ISGs) such as ISG56, OAS, Rsad2, Ifi27 and Mx1. Additionally, we also observed activation of STAT1 by TAgwt. 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. PMID:20692676

  11. Characterization of am404, an amber mutation in the simian virus 40 T antigen gene.

    PubMed Central

    Rawlins, D R; Collis, P; Muzyczka, N

    1983-01-01

    We analyzed the biological activity of an amber mutation, am404, at map position 0.27 in the T antigen gene of simian virus 40. Immunoprecipitation of extracts from am404-infected cells demonstrated the presence of an amber protein fragment (am T antigen) of the expected molecular weight (67,000). Differential immunoprecipitation with monoclonal antibody demonstrated that am T antigen was missing the carboxy-terminal antigenic determinants. The amber mutant was shown to be defective for most of the functions associated with wild-type T antigen. The mutant did not replicate autonomously, but this defect could be complemented by a helper virus (D. R. Rawlins and N. Muzyczka, J. Virol. 36:611-616, 1980). The mutant failed to transform nonpermissive rodent cells and did not relieve the host range restriction of adenovirus 2 in monkey cells. However, stimulation of host cell DNA, whose functional region domain has been mapped within that portion of the protein synthesized by the mutant, could be demonstrated in am404-infected cells. A number of unexpected observations were made. First, the am T antigen was produced in unusually large amounts in a simian virus 40-transformed monkey cell line (COS-1), but overproduction was not seen in nontransformed monkey cells regardless of whether or not a helper virus was present. This feature of the mutant was presumably the result of the inability of am T antigen to autoregulate, the level of wild-type T antigen in COS-1 cells, and the unusually short half-life of am T antigen in vivo. Pulse-chase experiments indicated that am T antigen had an intracellular half-life of approximately 10 min. In addition, although the am T antigen retained the major phosphorylation site found in simian virus 40 T antigen, it was not phosphorylated. Thus, phosphorylation of simian virus 40 T antigen is not required for the stimulation of host cell DNA synthesis. Finally, fusion of am404-infected monkey cells with Escherichia coli protoplasts

  12. Trypanosome Surface Antigen Genes: Analysis Using Recombinant DNA.

    DTIC Science & Technology

    1984-06-15

    glycoprotein ( VSG ) genes. umerous syringe passaged and cyclically transmitted, frequently expressed VATs have been isolated, monoclonal antibodies prepared...to their VSGs , and he expressed VSG genes have been cloned. We have shown that many diverse tocks express VSG epitopes related to the early IsTst...epitopes. The VSG ene organization in the genome and sequence organization has been haracterized. We have confirmed sequence homology at the 3’ terminus

  13. Aberrant Cosmc genes result in Tn antigen expression in human colorectal carcinoma cell line HT-29

    PubMed Central

    Yu, Xiaofeng; Du, Zhenzhen; Sun, Xuhong; Shi, Chuanqin; Zhang, Huaixiang; Hu, Tao

    2015-01-01

    The Tn antigen, which arises from mutation in the Cosmc gene is one of the most common tumor associated carbohydrate antigens. Cosmc resides in X24 encoded by a single gene and functions as a specific molecular chaperone for T-synthase. While the Tn antigen cannot be detected in normal cells, Cosmc mutations inactivate T-synthase and consequently result in Tn antigen expression within certain cancers. In addition to this Cosmc mutation-induced expression, the Tn antigen is also expressed in such cell lines as Jurkat T, LSC and LS174T. Whether the Cosmc mutation is present in the colon cancer cell line HT-29 is still unclear. Here, we isolate HT-29-Tn+ cells from HT-29 cells derived from a female colon cancer patient. These HT-29-Tn+ cells show a loss of the Cosmc gene coding sequence (CDS) leading to an absence of T-synthase activity and Tn antigen expression. Additionally, almost no methylation of Cosmc CpG islands was detected in HT-29-Tn+ as well as in HT-29-Tn- and Tn- tumor cells from male patients. In contrast, the methylation frequency of CpG island of Cosmc in normal female cells was ~50%. Only one active allele of Cosmc existed in HT-29-Tn+ and HT-29-Tn- cells as based upon detection of SNP sites. These results indicate that Tn antigens expression and T-synthase inactivity in HT-29-Tn+ cells can be related to the absence of CDS in Cosmc active alleles, while an inactive allele deletion of Cosmc in HT-29 cells has no influence on Cosmc function. PMID:26045765

  14. 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.

  15. No Evidence of Human Leukocyte Antigen Gene Association With Rheumatic Fever Among Children in Samoa.

    PubMed

    Erdem, Guliz; Seifried, Steven E

    2015-03-01

    Human leukocyte antigens (HLAs) have been implicated in rheumatic fever pathogenesis. This pilot whole genome association study compares genotypes of Samoan children with rheumatic fever to unaffected siblings and unrelated healthy controls. No risk-related genotypes were associated with HLA genes. Thirteen Regions of Interest were identified as candidates for further study.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  17. Immunoglobulin heavy chain variable gene usage and (super)-antigen drive in chronic lymphocytic leukemia.

    PubMed

    Bühler, Andreas; Zenz, Thorsten; Stilgenbauer, Stephan

    2010-01-15

    Increasing evidence supports the prognostic relevance of specific immunoglobulin heavy chain variable (IGHV) genes or stereotyped B-cell receptors (BCR) in chronic lymphocytic leukemia (CLL). The clonotypic BCRs differ in their specificity and affinity toward classical antigens and/or superantigens. The BCR-triggered mechanisms are distinct but could explain in part the different clinical behavior among CLL subgroups.

  18. High polymorphism in genes encoding antigen B from human infecting strains of Echinococcus granulosus.

    PubMed

    Kamenetzky, L; Muzulin, P M; Gutierrez, A M; Angel, S O; Zaha, A; Guarnera, E A; Rosenzvit, M C

    2005-12-01

    Echinococcus granulosus antigen B (AgB) is encoded by a gene family and is involved in the evasion of the host immune response. E. granulosus exists as a number of strains (G1-G10) that differ in biological characteristics. We used PCR-SSCP followed by DNA sequencing to evaluate sequence variation and transcription profile of AgB in 5 E. granulosus strains. Twenty-four genomic sequences were isolated and clustered in 3 groups related to 2 of the 5 reported AgB genes. AgB4 genes were present in almost all strains, whereas AgB2 were present as functional genes exclusively in G1/G2 cluster, and as non-functional genes in G5 and the G6/G7 cluster, suggesting inter-strain variation. The AgB transcription patterns, analysed by RT-PCR, showed that AgB2 and AgB4 genes were transcribed in G1, while only the AgB4 gene was transcribed in G7 strain. Cysts from the same strain or cluster shared more genomic and cDNA variants than cysts from different strain or cluster. The level of nucleotide and deduced amino acid sequence variation observed is higher than that reported so far for coding genes of other helminths. Neutrality was rejected for AgB2 genes. These data show the genetic polymorphism of antigen-coding genes among genetically characterized strains of E. granulosus.

  19. Neutralization of reovirus: the gene responsible for the neutralization antigen

    PubMed Central

    1977-01-01

    The S1 genome segment of reovirus is linked to type specificity as determined by neutralization antibody. This gene segment codes for a minor outer capsid polypeptide (sigma1). Therefore, sigma1 is the peptide responsible for induction of neutralization antibody and confers type specificity. This biologic property of reovirus was defined using hybrid recombinants clones between reovirus types 1 and 3 and 2 and 3. PMID:925604

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

    PubMed Central

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

    2012-01-01

    The Vibrio cholerae lipopolysaccharide O1 antigen is a major target of bacteriophages and the human immune system and is of critical importance for vaccine design. We used an O1-specific lytic bacteriophage as a tool to probe the capacity of V. cholerae to alter its O1 antigen and identified a novel mechanism by which this organism can modulate O antigen expression and exhibit intra-strain heterogeneity. We identified two phase variable genes required for O1 antigen biosynthesis, manA and wbeL. manA resides outside of the previously recognized O1 antigen biosynthetic locus, and encodes for a phosphomannose isomerase critical for the initial step in O1 antigen biosynthesis. We determined that manA and wbeL phase variants are attenuated for virulence, providing functional evidence to further support the critical role of the O1 antigen for infectivity. We provide the first report of phase variation modulating O1 antigen expression in V. cholerae, and show that the maintenance of these phase variable loci is an important means by which this facultative pathogen can generate the diverse subpopulations of cells needed for infecting the host intestinal tract and for escaping predation by an O1-specific phage. PMID:23028317

  1. Influence of human leukocyte antigen genes on TCR V gene segment frequencies.

    PubMed

    Genevée, C; Farace, F; Chung, V; Diu, A; Raffoux, C; Charron, D; Hercend, T; Triebel, F

    1994-10-01

    Human leukocyte antigen (HLA)-dependent selection mechanisms exerted during thymic maturation are supposed to be main contributing factors to the genetic predetermination of the TCR repertoire and may have a detectable effect on adult peripheral blood lymphocyte V segment frequencies. Here, we analyzed whether polymorphic or non-polymorphic HLA determinants are associated with selected expression of some V gene segment specificities. We first examined the reactivity of 17 V segment specific mAb on purified CD4+ and CD8+ cell fractions in 10 unrelated people. We found a significant overexpression of only three V segment products (V beta 2, V beta 5.1 and V beta 6.7) in CD4+ and none in CD8+ cell fractions in most individuals. Skewing of certain V beta segments by non-polymorphic HLA determinants (i.e. class II for CD4+ and class I for CD8+ cells) is therefore more limited (3/17) than previously thought. Considering the effects of polymorphic HLA determinants, we compared TCR V segment frequencies in HLA-identical siblings to sibling pairs who differ at one or both HLA haplotypes, using 13 V beta specific mAb. In pairwise comparisons, we found that the HLA complex had no detectable effect on TCR repertoire in five large families with multiple siblings. Together, these observations suggest that HLA-predicted selection mechanisms exerted during thymic maturation might not have a predominant influence shaping the TCR repertoire of normal adults.

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

    PubMed Central

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

    2016-01-01

    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

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

    DTIC Science & Technology

    1988-02-02

    which appear to encode a sIgnal peptide having characteristics in common with those of other secreted proteins. A consensus TATAAT sequence was located ...UNCLASSIFIED 4144MIT? @.MICATION OF TWOS Ph" r~ .Ewa ..4 20. ABSTRACT (cont) was located seven bp upstream of the ATG initiation codon. The codon usage f.’r...TATAAT seqc. e was located at the putative -10 promoter site. A Shine-Dalgarno site similar to that found in genes of other Bacillus sp. was located seven

  4. Induction of Human Blood Group A Antigen Expression on Mouse Cells, Using Lentiviral Gene Transduction

    PubMed Central

    Fan, Xiaohu; Lang, Haili; Zhou, Xianpei; Zhang, Li; Yin, Rong; Maciejko, Jessica; Giannitsos, Vasiliki; Motyka, Bruce; Medin, Jeffrey A.; Platt, Jeffrey L.

    2010-01-01

    Abstract The ABO histo-blood group system is the most important antigen system in transplantation medicine, yet no small animal model of the ABO system exists. To determine the feasibility of developing a murine model, we previously subcloned the human α-1,2-fucosyltransferase (H-transferase, EC 2.4.1.69) cDNA and the human α-1,3-N-acetylgalactosaminyltransferase (A-transferase, EC 2.4.1.40) cDNA into lentiviral vectors to study their ability to induce human histo-blood group A antigen expression on mouse cells. Herein we investigated the optimal conditions for human A and H antigen expression in murine cells. We determined that transduction of a bicistronic lentiviral vector (LvEF1-AH-trs) resulted in the expression of A antigen in a mouse endothelial cell line. We also studied the in vivo utility of this vector to induce human A antigen expression in mouse liver. After intrahepatic injection of LvEF1-AH-trs, A antigen expression was observed on hepatocytes as detected by immunohistochemistry and real-time RT-PCR. In human group A erythrocyte-sensitized mice, A antigen expression in the liver was associated with tissue damage, and deposition of antibody and complement. These results suggest that this gene transfer strategy can be used to simulate the human ABO blood group system in a murine model. This model will facilitate progress in the development of interventions for ABO-incompatible transplantation and transfusion scenarios, which are difficult to develop in clinical or large animal settings. PMID:20163247

  5. Structure and expression of a mouse major histocompatibility antigen gene, H-2Ld.

    PubMed Central

    Evans, G A; Margulies, D H; Camerini-Otero, R D; Ozato, K; Seidman, J G

    1982-01-01

    A genomic clone encoding H-2Ld, a mouse major transplantation antigen, has been identified and the structure of the H-2Ld gene has been partially determined. We isolated 35 genomic clones from a BALB/c (H-2d) genomic library by hybridization to mouse or human probes. One of these clones encodes H-2Ld as determined by two criteria. First, the gene encodes a protein that is identical at the 76 known amino acid positions for H-2Ld. Second, when introduced into L cells by DNA-mediated gene transfer, a new H-2 antigen is expressed that is recognized by anti-H-2Ld monoclonal antibodies. The sequence of the H-2Ld protein predicted by the DNA sequences shows more than 80% homology to known H-2 antigens. H-2L-like sequences are found in mutant H-2Kb molecules, suggesting that gene conversion or reciprocal recombination may play a role in the development of H-2 polymorphism. PMID:6952248

  6. Differential effect of transporter Tap 2 gene introduction into RMA-S cells on viral antigen processing.

    PubMed

    Ossevoort, M A; Sijts, A J; van Veen, K J; Momburg, F; Hämmerling, G J; Seelig, A; Butcher, G W; Howard, J C; Kast, W M; Melief, C J

    1993-12-01

    The protein products of the Tap (Transporter associated with antigen processing) 1 and 2 genes are presumed to deliver peptides across the endoplasmic reticulum (ER) for assembly with major histocompatibility complex (MHC) class I molecules. The antigen processing-defective cell line RMA-S (H-2b) has a premature stop in the Tap 2 gene and probably therefore fails to deliver peptides into the ER, which leads to a low level of cell surface MHC class I molecules. Transfection of a Tap 2 gene restores to RMA-S both MHC class I molecule expression and the ability to present influenza viral antigens. We investigated the ability of RMA-S cells transfected with a Tap 2 gene to process and present alloantigens, Sendai and Rauscher viral antigens to allogeneic and virus-specific cytotoxic T lymphocytes. We found that allogeneic peptides as well as Rauscher and Sendai viral peptides can be processed and presented by RMA-S but at reduced levels. Transfection of a Tap 2 gene of mouse (BALB/c, H-2d) or rat origin into RMA-S increased the presentation of Sendai viral antigens and partially restored the presentation of allogeneic antigens. The already low level of Rauscher viral peptides presented by RMA-S is not elevated by transfection of either Tap 2 gene into RMA-S. This indicates a differential effect of transfection of a Tap 2 gene of rat or allogeneic mouse origin into RMA-S on viral antigen processing.

  7. A physical map linking the five CD1 human thymocyte differentiation antigen genes.

    PubMed Central

    Yu, C Y; Milstein, C

    1989-01-01

    Human CD1 is a family of thymocyte differentiation antigens which consist of heavy chains with mol. wts between 43 and 49 kd binding to beta 2 microglobulin. They are distant relatives of the major histocompatibility complex (MHC) class I and II products. Five human CD1 genes have been described. Three (CD1A, -B and -C) code for the serologically defined CD1a, -b and -c antigens. The protein products of the other two genes, CD1D and CD1E, remain unknown. All CD1 genes are located on chromosome 1 and hence are independent of the MHC locus. In this paper, the tight linkage of the CD1 genes has been established by pulse field gel electrophoresis, cosmid cloning and walking techniques. The 190 kb of DNA linking all five CD1 genes has been spanned by 14 overlapping cosmids. The order of the genes in the CD1 complex is CD1D-CD1A-CD1C-CD1B-CD1E, and, with the exception of CD1B, they are arranged in the same transcriptional orientation. The genes are evenly spaced in the complex except for the distance between CD1D and CD1A, which is two to three times greater than the average. Images PMID:2583117

  8. Contrasting Population Structures of the Genes Encoding Ten Leading Vaccine-Candidate Antigens of the Human Malaria Parasite, Plasmodium falciparum

    PubMed Central

    Barry, Alyssa E.; Schultz, Lee; Buckee, Caroline O.; Reeder, John C.

    2009-01-01

    The extensive diversity of Plasmodium falciparum antigens is a major obstacle to a broadly effective malaria vaccine but population genetics has rarely been used to guide vaccine design. We have completed a meta-population genetic analysis of the genes encoding ten leading P. falciparum vaccine antigens, including the pre-erythrocytic antigens csp, trap, lsa1 and glurp; the merozoite antigens eba175, ama1, msp's 1, 3 and 4, and the gametocyte antigen pfs48/45. A total of 4553 antigen sequences were assembled from published data and we estimated the range and distribution of diversity worldwide using traditional population genetics, Bayesian clustering and network analysis. Although a large number of distinct haplotypes were identified for each antigen, they were organized into a limited number of discrete subgroups. While the non-merozoite antigens showed geographically variable levels of diversity and geographic restriction of specific subgroups, the merozoite antigens had high levels of diversity globally, and a worldwide distribution of each subgroup. This shows that the diversity of the non-merozoite antigens is organized by physical or other location-specific barriers to gene flow and that of merozoite antigens by features intrinsic to all populations, one important possibility being the immune response of the human host. We also show that current malaria vaccine formulations are based upon low prevalence haplotypes from a single subgroup and thus may represent only a small proportion of the global parasite population. This study demonstrates significant contrasts in the population structure of P. falciparum vaccine candidates that are consistent with the merozoite antigens being under stronger balancing selection than non-merozoite antigens and suggesting that unique approaches to vaccine design will be required. The results of this study also provide a realistic framework for the diversity of these antigens to be incorporated into the design of next

  9. [Production of marker-free plants expressing the gene of the hepatitis B virus surface antigen].

    PubMed

    Rukavtsova, E B; Gaiazova, A R; Chebotareva, E N; Bur'ianova, Ia I

    2009-08-01

    The pBM plasmid, carrying the gene of hepatitis B virus surface antigen (HBsAg) and free of any selection markers of antibiotic or herbicide resistance, was constructed for genetic transformation of plants. A method for screening transformed plant seedlings on nonselective media was developed. Enzyme immunoassay was used for selecting transgenic plants with HBsAg gene among the produced regenerants; this method provides for a high sensitivity detection of HBsAg in plant extracts. Tobacco and tomato transgenic lines synthesizing this antigen at a level of 0.01-0.05% of the total soluble protein were obtained. The achieved level of HBsAg synthesis is sufficient for preclinical trials of the produced plants as a new generation safe edible vaccine. The developed method for selecting transformants can be used for producing safe plants free of selection markers.

  10. Analysis of the Borrelia burgdorferi GeHo fla gene and antigenic characterization of its gene product.

    PubMed Central

    Gassmann, G S; Jacobs, E; Deutzmann, R; Göbel, U B

    1991-01-01

    The fla gene of Borrelia burgdorferi GeHo was analyzed and expressed in Escherichia coli. The structural gene encodes a flagellar protein of 336 amino acids. Comparative sequence analysis of the amino acid sequence revealed a high degree of sequence conservation with flagellins from both phylogenetically related and unrelated bacteria. The antigenic properties of the B. burgdorferi Fla protein were studied by synthesizing overlapping octapeptides, which were screened by using a battery of different monoclonal and polyclonal antibodies from various species directed against native and denatured flagellar proteins. No single species-independent immunodominant epitope could be located. However, immunoreactive oligopeptides clustered within the variable middle region (N-180 to I-260). This region could constitute a candidate antigen for more specific and sensitive serodiagnosis of Lyme borreliosis. Images PMID:1704884

  11. Cloning of Plasmodium yoelii Genes Expressing Three Different Sporozoite-Specific Antigens

    DTIC Science & Technology

    1989-01-01

    advances in the development of a recombinant vaccine against Plasmodium falcipatum sporozoites, there is still an urgent need for a reliable rodent model to...carry out complex vaccine protocols difficult to perform in primates or man. The purpose of this research was to clone the P. yoelii genes coding for...sporozoite antigens that have potential as vaccine candidates in a rodent model. Results our positive clones (B10, 885, B143 and 8155) were identified

  12. The induction of antigen-specific CTL by in situ Ad-REIC gene therapy.

    PubMed

    Ariyoshi, Y; Watanabe, M; Eikawa, S; Yamazaki, C; Sadahira, T; Hirata, T; Araki, M; Ebara, S; Nasu, Y; Udono, H; Kumon, H

    2016-05-01

    An adenovirus vector carrying the human Reduced Expression in Immortalized Cell (REIC)/Dkk-3 gene (Ad-REIC) mediates simultaneous induction of cancer-selective apoptosis and augmentation of anticancer immunity. In our preclinical and clinical studies, in situ Ad-REIC gene therapy showed remarkable direct and indirect antitumor effects to realize therapeutic cancer vaccines. We herein aimed to confirm the induction of tumor-associated antigen-specific cytotoxic T lymphocytes (CTLs) by Ad-REIC. Using an ovalbumin (OVA), a tumor-associated antigen, expressing E.G7 tumor-bearing mouse model, we investigated the induction and expansion of OVA-specific CTLs responsible for indirect, systemic effects of Ad-REIC. The intratumoral administration of Ad-REIC mediated clear antitumor effects with the accumulation of OVA-specific CTLs in the tumor tissues and spleen. The CD86-positive dendritic cells (DCs) were upregulated in the tumor draining lymph nodes of Ad-REIC-treated mice. In a dual tumor-bearing mouse model in the left and right back, Ad-REIC injection in one side significantly suppressed the tumor growth on both sides and significant infiltration of OVA-specific CTLs into non-injected tumor was also detected. Consequently, in situ Ad-REIC gene therapy is expected to realize a new-generation cancer vaccine via anticancer immune activation with DC and tumor antigen-specific CTL expansion.

  13. Gene frequencies of human neutrophil antigens in the Tunisian blood donors and Berbers.

    PubMed

    Abid, S; Zili, M; Bouzid, L; Kibech, R; Foudhaili, N; Joudi, K; Ren Regaya, Z; Abdennaji, B; Mrad, R; Boukef, K

    2001-08-01

    Human neutrophil antigens play an important role in provoking immune neutropenia and transfusion-reactions. The aim of this study was to determine granulocyte-specific antigens on the neutrophil Fc gamma receptor IIIb (Fc gamma RIIIb, CD16b), namely, the HNA-1a(NA1) and HNA-1b(NA2) antigens and their gene frequencies in Tunisian blood donors and Berbers. One hundred and ninety-nine unrelated healthy Tunisian blood donors and Berbers were typed for HNA-1a and HNA-1b(NA1 and NA2), using polymerase chain reaction with sequence-specific primers (PCR-SSP). In 24 granulocyte samples, the HNA-1a and HNA-1b phenotypes was additionally determined by the granulocyte immunofluorescence test (GIFT) and correlated with the genotyping results. A subsequent analysis of the genotyping study showed that, the HNA-1a and HNA-1b gene frequencies observed, were 0.342 and 0.658 for Berbers, and 0.311 and 0.668 for blood donors, respectively. In the genotyping study conducted, it was determined that the HNA-1a and HNA-1b gene frequencies observed in Tunisian blood donors and Berbers are similar to those previously reported in other white populations.

  14. Antigen-receptor gene-modified T cells for treatment of glioma.

    PubMed

    Ikeda, Hiroaki; Shiku, Hiroshi

    2012-01-01

    Immunological effector cells and molecules have been shown to access intracranial tumor sites despite the existence of blood brain barrier (BBB) or immunosuppressive mechanisms associated with brain tumors. Recent progress in T-cell biology and tumor immunology made possible to develop strategies of tumor-associated antigen-specific immunotherapeutic approaches such as vaccination with defined antigens and adoptive T-cell therapy with antigen-specific T cells including gene-modified T cells for the treatment of patients with brain tumors. An array of recent reports on the trials of active and passive immunotherapy for patients with brain tumors have documented safety and some preliminary clinical efficacy, although the ultimate judgment for clinical benefits awaits rigorous evaluation in trials of later phases. Nevertheless, treatment with lymphocytes that are engineered to express tumor-specific receptor genes is a promising immunotherapy against glioma, based on the significant efficacy reported in the trials for patients with other types of malignancy. Overcoming the relative difficulty to apply immunotherapeutic approach to intracranial region, current advances in the understanding of human tumor immunology and the gene-therapy methodology will address the development of effective immunotherapy of brain tumors.

  15. Widespread balancing selection and pathogen-driven selection at blood group antigen genes.

    PubMed

    Fumagalli, Matteo; Cagliani, Rachele; Pozzoli, Uberto; Riva, Stefania; Comi, Giacomo P; Menozzi, Giorgia; Bresolin, Nereo; Sironi, Manuela

    2009-02-01

    Historically, allelic variations in blood group antigen (BGA) genes have been regarded as possible susceptibility factors for infectious diseases. Since host-pathogen interactions are major determinants in evolution, BGAs can be thought of as selection targets. In order to verify this hypothesis, we obtained an estimate of pathogen richness for geographic locations corresponding to 52 populations distributed worldwide; after correction for multiple tests and for variables different from selective forces, significant correlations with pathogen richness were obtained for multiple variants at 11 BGA loci out of 26. In line with this finding, we demonstrate that three BGA genes, namely CD55, CD151, and SLC14A1, have been subjected to balancing selection, a process, rare outside MHC genes, which maintains variability at a locus. Moreover, we identified a gene region immediately upstream the transcription start site of FUT2 which has undergone non-neutral evolution independently from the coding region. Finally, in the case of BSG, we describe the presence of a highly divergent haplotype clade and the possible reasons for its maintenance, including frequency-dependent balancing selection, are discussed. These data indicate that BGAs have been playing a central role in the host-pathogen arms race during human evolutionary history and no other gene category shows similar levels of widespread selection, with the only exception of loci involved in antigen recognition.

  16. 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.

  17. A Comprehensive Phylogenetic and Structural Analysis of the Carcinoembryonic Antigen (CEA) Gene Family

    PubMed Central

    Pavlopoulou, Athanasia; Scorilas, Andreas

    2014-01-01

    The carcinoembryonic antigen (CEA) gene family belongs to the immunoglobulin (Ig) superfamily and codes for a vast number of glycoproteins that differ greatly both in amino acid composition and function. The CEA family is divided into two groups, the carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) and the pregnancy-specific glycoproteins. The CEA family members are implicated in pleiotropic (patho)physiological functions including cell–cell adhesion, pregnancy, immunity, neovascularization, regulation of insulin homeostasis, and carcinogenesis. In general, the CEA-encoded proteins are composed of an extracellular region with Ig variable and constant-like domains and a cytoplasmic region containing signaling motifs. Of particular interest, the well-studied human and mouse CEA genes are arranged in clusters in a single chromosome. Taking into account this characteristic, we made an effort to reconstruct the evolutionary history of the CEA gene family. Toward this end, the publicly available genomes were searched extensively for CEA homologs. The domain organization of the retrieved protein sequences was analyzed, and, subsequently, comprehensive phylogenetic analyses of the entire length CEA homologous proteins were performed. A series of evolutionarily conserved amino acid residues, functionally important, were identified. The relative positioning of these residues on the modeled tertiary structure of novel CEA protein domains revealed that they are, also, spatially conserved. Furthermore, the chromosomal arrangement of CEA genes was examined, and it was found that the CEA genes are preserved in terms of position, transcriptional orientation, and number in all species under investigation. PMID:24858421

  18. Widespread balancing selection and pathogen-driven selection at blood group antigen genes

    PubMed Central

    Fumagalli, Matteo; Cagliani, Rachele; Pozzoli, Uberto; Riva, Stefania; Comi, Giacomo P.; Menozzi, Giorgia; Bresolin, Nereo; Sironi, Manuela

    2009-01-01

    Historically, allelic variations in blood group antigen (BGA) genes have been regarded as possible susceptibility factors for infectious diseases. Since host–pathogen interactions are major determinants in evolution, BGAs can be thought of as selection targets. In order to verify this hypothesis, we obtained an estimate of pathogen richness for geographic locations corresponding to 52 populations distributed worldwide; after correction for multiple tests and for variables different from selective forces, significant correlations with pathogen richness were obtained for multiple variants at 11 BGA loci out of 26. In line with this finding, we demonstrate that three BGA genes, namely CD55, CD151, and SLC14A1, have been subjected to balancing selection, a process, rare outside MHC genes, which maintains variability at a locus. Moreover, we identified a gene region immediately upstream the transcription start site of FUT2 which has undergone non-neutral evolution independently from the coding region. Finally, in the case of BSG, we describe the presence of a highly divergent haplotype clade and the possible reasons for its maintenance, including frequency-dependent balancing selection, are discussed. These data indicate that BGAs have been playing a central role in the host–pathogen arms race during human evolutionary history and no other gene category shows similar levels of widespread selection, with the only exception of loci involved in antigen recognition. PMID:18997004

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

    PubMed

    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.

  20. A gene cluster for the synthesis of serotype g-specific polysaccharide antigen in Aggregatibacter actinomycetemcomitans.

    PubMed

    Tsuzukibashi, Osamu; Saito, Masanori; Kobayashi, Taira; Umezawa, Koji; Nagahama, Fumio; Hiroi, Takachika; Hirasawa, Masatomo; Takada, Kazuko

    2014-04-01

    Aggregatibacter actinomycetemcomitans is an important pathogen related to aggressively progressive periodontal breakdown in adolescents and adults. The species can be divided into six serotypes (a-f) according to their surface carbohydrate antigens. Recently, a new serotype g of A. actinomycetemcomitans was proposed. The aim of the present study was to sequence the gene cluster associated with the biosynthesis of the serotype g-specific polysaccharide antigen and develop serotype-specific primers for PCR assay to identify serotype g strains of A. actinomycetemcomitans. The serotype-specific polysaccharide (SSPS) gene cluster of the NUM-Aa 4039 strain contained 21 genes in 21,842-bp nucleotides. The similarity of the SSPS gene cluster sequence was 96.7 % compared with that of the serotype e strain. Seventeen serotype g genes showed more than 90 % homology both in nucleotide and amino acids to the serotype e strain. Three additional genes with 1,579 bp in NUM-Aa 4039 were inserted into the corresponding ORF13 of the serotype e strain. The serotype g-specific primers were designed from the insertion region of NUM-Aa 4039. Serotypes of the a-f strains were not amplified by serotype-specific g primers; only NUM-Aa 4039 showed an amplicon band. The NUM-Aa 4039 strain was three genes in the SSPS gene cluster different from those of serotype e strain. The specific primers derived from these different regions are useful for identification and distribution of serotype g strain among A. actinomycetemcomitans from clinical samples.

  1. A Gene Encoding Antigenic Peptides of Human Squamous Cell Carcinoma Recognized by Cytotoxic T Lymphocytes

    PubMed Central

    Shichijo, Shigeki; Nakao, Masanobu; Imai, Yasuhisa; Takasu, Hideo; Kawamoto, Mayumi; Niiya, Fumihiko; Yang, Damu; Toh, Yuji; Yamana, Hideaki; Itoh, Kyogo

    1998-01-01

    Except for melanomas, tumor antigens recognized by cytotoxic T lymphocytes (CTLs) are yet unidentified. We have identified a gene encoding antigenic peptides of human squamous cell carcinomas (SCCs) recognized by human histocompatibility leukocyte antigens (HLA)- A2601–restricted CTLs. This gene showed no similarity to known sequences, and encoded two (125- and 43-kilodalton [kD]) proteins. The 125-kD protein with the leucine zipper motif was expressed in the nucleus of the majority of proliferating cells tested, including normal and malignant cells. The 43-kD protein was expressed in the cytosol of most SCCs from various organs and half of lung adenocarcinomas, but was not expressed in other cancers nor in a panel of normal tissues. The three nonapeptides shared by the two proteins were recognized by the KE4 CTLs, and one of the peptides induced in vitro from peripheral blood mononuclear cells (PBMCs) the CTLs restricted to the autologous tumor cells. The 43-kD protein and this nonapeptide (KGSGKMKTE) may be useful for the specific immunotherapy of HLA-A2601+ epithelial cancer patients. PMID:9449708

  2. Characterization of the lymphocyte activation gene 3-encoded protein. A new ligand for human leukocyte antigen class II antigens

    PubMed Central

    1992-01-01

    The lymphocyte activation gene 3 (LAG-3), expressed in human activated T and natural killer (NK) cells, is closely related to CD4 at the gene and protein levels. We report here the initial characterization of the LAG-3-encoded protein. We have generated two monoclonal antibodies after immunization of mice with a 30-amino acid peptide that corresponds to an exposed extra loop region present in the LAG-3 immunoglobulin-like first domain. The reactivity of these reagents is directed against LAG-3 since they recognize both membrane-expressed and soluble recombinant LAG-3 molecules produced in a baculovirus expression system. The two antibodies are likely to react with the same or closely related epitope (termed LAG-3.1) exposed on the LAG-3 first domain extra loop, as assessed in competition experiments on LAG-3- expressing activated lymphocytes. Cellular distribution analysis indicated that the LAG-3.1 epitope is expressed on activated T (both CD4+ and CD8+ subsets) and NK cells, and not on activated B cells or monocytes. In immunoprecipitation experiments performed on activated T and NK cell lysates, a 70-kD protein was detected after SDS-PAGE analysis. 45-kD protein species were also immunoprecipitated. Both the 70- and 45-kD proteins were shown to be N-glycosylated. In Western blot analysis, only the former molecule was recognized by the anti-LAG-3 antibodies, demonstrating that it is LAG-3 encoded. These anti-LAG-3 antibodies were used to investigate whether the LAG-3 protein interacts with the CD4 ligands. By using a high-level expression cellular system based on COS-7 cell transfection with recombinant CDM8 vectors and a quantitative cellular adhesion assay, we demonstrate that rosette formation between LAG-3-transfected COS-7 cells and human leukocyte antigen (HLA) class II-bearing B lymphocytes is specifically dependent on LAG-3/HLA class II interaction. In contrast to CD4, LAG-3 does not bind the human immunodeficiency virus gp120. This initial

  3. IgM antigen receptor complex contains phosphoprotein products of B29 and mb-1 genes.

    PubMed Central

    Campbell, K S; Hager, E J; Friedrich, R J; Cambier, J C

    1991-01-01

    Membrane immunoglobulin M (mIgM) and mIgD are major B-lymphocyte antigen receptors, which function by internalizing antigens for processing and presentation to T cells and by transducing essential signals for proliferation and differentiation. Although ligation of mIgM or mIgD results in rapid activation of a phospholipase C and a tyrosine kinase(s), these receptors have cytoplasmic tails of only three amino acid residues (Lys-Val-Lys), which seem ill suited for direct physical coupling with cytoplasmic signal transduction structures. In this report, we identify the alpha, beta, and gamma components of the mIgM-associated phosphoprotein complex, which may play a role in signal transduction. Proteolytic peptide mapping demonstrated that the IgM-alpha chain differs from Ig-beta and Ig-gamma. The chains were purified, and amino-terminal sequencing revealed identity with two previously cloned B-cell-specific genes. One component, IgM-alpha, is a product of the mb-1 gene, and the two additional components, Ig-beta and Ig-gamma, are products of the B29 gene. Immunoblotting analysis using rabbit antibodies prepared against predicted peptide sequences of each gene product confirmed the identification of these mIgM-associated proteins. The deduced sequence indicates that these receptor subunits lack inherent protein kinase domains but include common tyrosine-containing sequence motifs, which are likely sites of induced tyrosine phosphorylation. Images PMID:2023945

  4. A Killer Immunoglobulin - Like Receptor Gene - Content Haplotype and A Cognate Human Leukocyte Antigen Ligand are Associated with Autism

    PubMed Central

    Torres, Anthony; Westover, Jonna; Benson, Michael; Johnson, Randall; Dykes, Annelise

    2016-01-01

    The killing activity of natural killer cells is largely regulated by the binding of class I human leukocyte antigen cognate ligands to killer cell immunoglobulin - like receptor proteins. The killer cell immunoglobulin - like receptor gene - complex contains genes that activate and others that inhibit the killing state of natural killer cells depending on the binding of specific human leukocyte antigen cognate ligands. It has been suggested in previous publications that activating human leukocyte antigen/killer - cell immunoglobulin - like receptor complexes are increased in people with autism. We present data, which suggests that an activating cB01/tA01 killer cell immunoglobulin - like receptor gene - content haplotype and the cognate ligand human leukocyte antigen - C1k that activates this haplotype is significantly increased in autism. This is an important observation suggesting that the interaction between two proteins encoded on different chromosomes increases natural killer cell killing in autism. PMID:27853655

  5. A Killer Immunoglobulin - Like Receptor Gene - Content Haplotype and A Cognate Human Leukocyte Antigen Ligand are Associated with Autism.

    PubMed

    Torres, Anthony; Westover, Jonna; Benson, Michael; Johnson, Randall; Dykes, Annelise

    2016-04-01

    The killing activity of natural killer cells is largely regulated by the binding of class I human leukocyte antigen cognate ligands to killer cell immunoglobulin - like receptor proteins. The killer cell immunoglobulin - like receptor gene - complex contains genes that activate and others that inhibit the killing state of natural killer cells depending on the binding of specific human leukocyte antigen cognate ligands. It has been suggested in previous publications that activating human leukocyte antigen/killer - cell immunoglobulin - like receptor complexes are increased in people with autism. We present data, which suggests that an activating cB01/tA01 killer cell immunoglobulin - like receptor gene - content haplotype and the cognate ligand human leukocyte antigen - C1k that activates this haplotype is significantly increased in autism. This is an important observation suggesting that the interaction between two proteins encoded on different chromosomes increases natural killer cell killing in autism.

  6. Identification of a Mouse Cytomegalovirus Gene Selectively Targeting CD86 Expression on Antigen-Presenting Cells

    PubMed Central

    Loewendorf, Andrea; Krüger, Corinna; Borst, Eva Maria; Wagner, Markus; Just, Ursula; Messerle, Martin

    2004-01-01

    We and others have shown that infection of dendritic cells with murine cytomegalovirus (MCMV) leads to severe functional impairment of these antigen-presenting cells (D. M. Andrews, C. E. Andoniou, F. Granucci, P. Ricciardi-Castagnoli, and M. A. Degli-Esposti, Nat. Immunol. 2:1077-1084, 2001; S. Mathys, T. Schroeder, J. Ellwart, U. H. Koszinowski, M. Messerle, and U. Just, J. Infect. Dis. 187:988-999, 2003). Phenotypically, reduced surface expression of costimulatory molecules and major histocompatibility complex molecules was detected. In order to identify the molecular basis for the observed effects, we generated MCMV mutants with large deletions of nonessential genes. The study was facilitated by the finding that a monocyte-macrophage cell line displayed similar phenotypic alterations after MCMV infection. By analyzing the expression of cell surface molecules on infected cells, we identified a mutant virus which is no longer able to downmodulate the expression of the costimulatory molecule CD86. Additional mutants with smaller deletions allowed us to pin down the responsible gene to a certain genomic region. RNA analysis led to the identification of the spliced gene m147.5, encoding a protein with 145 amino acids. Experiments with an m147.5 mutant revealed that the protein affects CD86 expression only, suggesting that additional MCMV genes are responsible for downmodulation of the other surface molecules. Identification of viral gene products interfering with functionally important proteins of antigen-presenting cells will provide the basis to dissect the complex interaction of CMV with these important cells and to evaluate the biological importance of these viral genes in vivo. PMID:15542658

  7. Sir2 paralogues cooperate to regulate virulence genes and antigenic variation in Plasmodium falciparum.

    PubMed

    Tonkin, Christopher J; Carret, Céline K; Duraisingh, Manoj T; Voss, Till S; Ralph, Stuart A; Hommel, Mirja; Duffy, Michael F; Silva, Liliana Mancio da; Scherf, Artur; Ivens, Alasdair; Speed, Terence P; Beeson, James G; Cowman, Alan F

    2009-04-14

    Cytoadherance of Plasmodium falciparum-infected erythrocytes in the brain, organs and peripheral microvasculature is linked to morbidity and mortality associated with severe malaria. Parasite-derived P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) molecules displayed on the erythrocyte surface are responsible for cytoadherance and undergo antigenic variation in the course of an infection. Antigenic variation of PfEMP1 is achieved by in situ switching and mutually exclusive transcription of the var gene family, a process that is controlled by epigenetic mechanisms. Here we report characterisation of the P. falciparum silent information regulator's A and B (PfSir2A and PfSir2B) and their involvement in mutual exclusion and silencing of the var gene repertoire. Analysis of P. falciparum parasites lacking either PfSir2A or PfSir2B shows that these NAD(+)-dependent histone deacetylases are required for silencing of different var gene subsets classified by their conserved promoter type. We also demonstrate that in the absence of either of these molecules mutually exclusive expression of var genes breaks down. We show that var gene silencing originates within the promoter and PfSir2 paralogues are involved in cis spreading of silenced chromatin into adjacent regions. Furthermore, parasites lacking PfSir2A but not PfSir2B have considerably longer telomeric repeats, demonstrating a role for this molecule in telomeric end protection. This work highlights the pivotal but distinct role for both PfSir2 paralogues in epigenetic silencing of P. falciparum virulence genes and the control of pathogenicity of malaria infection.

  8. DNA secondary structures are associated with recombination in major Plasmodium falciparum variable surface antigen gene families

    PubMed Central

    Sander, Adam F.; Lavstsen, Thomas; Rask, Thomas S.; Lisby, Michael; Salanti, Ali; Fordyce, Sarah L.; Jespersen, Jakob S.; Carter, Richard; Deitsch, Kirk W.; Theander, Thor G.; Pedersen, Anders Gorm; Arnot, David E.

    2014-01-01

    Many bacterial, viral and parasitic pathogens undergo antigenic variation to counter host immune defense mechanisms. In Plasmodium falciparum, the most lethal of human malaria parasites, switching of var gene expression results in alternating expression of the adhesion proteins of the Plasmodium falciparum-erythrocyte membrane protein 1 class on the infected erythrocyte surface. Recombination clearly generates var diversity, but the nature and control of the genetic exchanges involved remain unclear. By experimental and bioinformatic identification of recombination events and genome-wide recombination hotspots in var genes, we show that during the parasite’s sexual stages, ectopic recombination between isogenous var paralogs occurs near low folding free energy DNA 50-mers and that these sequences are heavily concentrated at the boundaries of regions encoding individual Plasmodium falciparum-erythrocyte membrane protein 1 structural domains. The recombinogenic potential of these 50-mers is not parasite-specific because these sequences also induce recombination when transferred to the yeast Saccharomyces cerevisiae. Genetic cross data suggest that DNA secondary structures (DSS) act as inducers of recombination during DNA replication in P. falciparum sexual stages, and that these DSS-regulated genetic exchanges generate functional and diverse P. falciparum adhesion antigens. DSS-induced recombination may represent a common mechanism for optimizing the evolvability of virulence gene families in pathogens. PMID:24253306

  9. A 55-kilodalton immunodominant antigen of Porphyromonas gingivalis W50 has arisen via horizontal gene transfer.

    PubMed

    Hanley, S A; Aduse-Opoku, J; Curtis, M A

    1999-03-01

    A 55-kDa outer membrane protein of Porphyromonas gingivalis W50 is a significant target of the serum immunoglobulin G antibody response of periodontal disease patients and hence may play an important role in host-bacterium interactions in periodontal disease. The gene encoding the 55-kDa antigen (ragB, for receptor antigen B) was isolated on a 9.5-kb partial Sau3AI fragment of P. gingivalis W50 chromosomal DNA in pUC18 by immunoscreening with a monoclonal antibody to this antigen. The 1.6-kb open reading frame (ORF) encoding RagB was located via subcloning and nested-deletion analysis. Sequence analysis demonstrated the presence of an upstream 3.1-kb ORF (ragA) which is cotranscribed with ragB. A number of genetic characteristics suggest that the ragAB locus was acquired by a horizontal gene transfer event. These include a significantly reduced G+C content relative to that of the P. gingivalis chromosome (42 versus 48%) and the presence of mobility elements flanking this locus in P. gingivalis W50. Furthermore, Southern blotting and PCR analyses showed a restricted distribution of this locus in laboratory and clinical isolates of this bacterium. The association of ragAB+ P. gingivalis with clinical status was examined by PCR analysis of subgingival samples. ragAB+ was not detected in P. gingivalis-positive shallow pockets from periodontal disease patients but was present in 36% of the P. gingivalis-positive samples from deep pockets. These data suggest that the ragAB locus was acquired by certain P. gingivalis strains via horizontal gene transfer and that the acquisition of this locus may facilitate the survival of these strains at sites of periodontal destruction.

  10. 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

  11. Cloning and characterization of the merozoite surface antigen 1 gene of Plasmodium berghei.

    PubMed

    Zhong, H; Fan, J Y; Yang, S; Davidson, E A

    1999-06-01

    Merozoite surface antigen 1 (MSA1) is a promising candidate for vaccine development against malaria parasites. Here, we report the complete nucleotide sequence of the gene encoding the precursor to this major surface antigen of Plasmodium berghei strain ANKA using cDNA library screening and polymerase chain reaction techniques. A single open reading frame of 5,376 basepairs encoding a protein with a calculated molecular mass of 197 kD was defined. The protein contains a putative signal peptide of 19 amino acids, a membrane anchor sequence of 18 residues, and shows two epidermal growth factor-like domains rich in Cys residues at the C-terminus. There are four repeat sequences of oligopeptides in the molecule: tetrapeptide (Ser-Thr-Thr-Thr), tripeptide (Pro-Thr-Pro and Pro-Ala-Ala), and dipeptide (Ser-Gly). Furthermore, three nine-residue stretches of a motif (Ala-Ser-Asn-Pro-Gly-Ala-Ser-Ala-Ser) are located near each other. All of these repeat sequences are unexceptionally located in the variable regions when compared with other MSA1 molecules. The molecule displays 79% overall identity to the analogous antigen of P. yoelii yoelii strain YM, 70% to that of P. chabaudi chabaudi strain AS, and 38% to that of P. falciparum strain Wellcome.

  12. The antigens - Volume VII

    SciTech Connect

    Sela, M. )

    1987-01-01

    This book contains four chapters. They are: Ir Genes: Antigen-Specific Genetic Regulation of the Immune Response; Molecular Genetics of Class II (Ia) Antigens; Antigen-Specific T Cell Clones and T Cell Factors; and Infection and Autoimmunity.

  13. Predominant role for directly transfected dendritic cells in antigen presentation to CD8+ T cells after gene gun immunization.

    PubMed

    Porgador, A; Irvine, K R; Iwasaki, A; Barber, B H; Restifo, N P; Germain, R N

    1998-09-21

    Cutaneous gene (DNA) bombardment results in substantial expression of the encoded antigen in the epidermal layer as well as detectable expression in dendritic cells (DC) in draining lymph nodes (LNs). Under these conditions, two possible modes of DC antigen presentation to naive CD8+ T cells might exist: (a) presentation directly by gene-transfected DC trafficking to local lymph nodes, and (b) cross-presentation by untransfected DC of antigen released from or associated with transfected epidermal cells. The relative contributions of these distinct modes of antigen presentation to priming for cytotoxic T cell (CTL) responses have not been clearly established. Here we show that LN cells directly expressing the DNA-encoded antigen are rare; 24 h after five abdominal skin bombardments, the number of these cells does not exceed 50-100 cells in an individual draining LN. However, over this same time period, the total number of CD11c+ DC increases more than twofold, by an average of 20,000-30,000 DC per major draining node. This augmentation is due to gold bombardment and is independent of the presence of plasmid DNA. Most antigen-bearing cells in the LNs draining the site of DNA delivery appear to be DC and can be depleted by antibodies to an intact surface protein encoded by cotransfected DNA. This finding of predominant antigen presentation by directly transfected cells is also consistent with data from studies on cotransfection with antigen and CD86-encoding DNA, showing that priming of anti-mutant influenza nucleoprotein CTLs with a single immunization is dependent upon coexpression of the DNAs encoding nucleoprotein and B7.2 in the same cells. These observations provide insight into the relative roles of direct gene expression and cross-presentation in CD8+ T cell priming using gene gun immunization, and indicate that augmentation of direct DC gene expression may enhance such priming.

  14. Polymorphism in the gene encoding the Pfs48/45 antigen of Plasmodium falciparum. XI. Asembo Bay Cohort Project.

    PubMed

    Escalante, Ananias A; Grebert, Heather M; Chaiyaroj, Sansanee C; Riggione, Flavia; Biswas, Sukla; Nahlen, Bernard L; Lal, Altaf A

    2002-01-01

    We have investigated the genetic diversity of the gene encoding the transmission-blocking vaccine antigen Pfs48/45 of Plasmodium falciparum parasites from western Kenya and compared it with parasite populations from Thailand, India, and Venezuela. We report 44 complete new sequences. Overall, the antigen is less polymorphic as compared with other pre-erythrocytic and blood stage antigens. Contrary to other P. falciparum antigens, the number of synonymous substitutions per synonymous site exceeds the number of non-synonymous substitutions per non-synonymous site. We have found that the Pfs48/45 gene of Kenyan parasites is more polymorphic than parasites from other geographic origins. Our analysis reveals that positive natural selection is involved in the maintenance of the observed polymorphism. No evidence of intragenic recombination was found. F(st) values reveal high levels of gene flow between India and Thailand, however, there are strong constraints in gene flow among Kenyan, Southeast Asian, and Venezuelan parasites. No alleles could be linked to a specific geographic region. The results of this study suggest that this gametocyte antigen, like other asexual blood stage antigens, is under selection pressure.

  15. Consistent quantitative gene product expression: #2. Antigen intensities on bone marrow cells are invariant between individuals

    PubMed Central

    Voigt, Andrew P.; Eidenschink Brodersen, Lisa; Fritschle, Wayne; Menssen, Andrew J.; Meshinchi, Soheil; Wells, Denise A.

    2016-01-01

    Abstract Five reference populations in bone marrow specimens were identified by flow cytometry using specific combinations of reagents in order define the variation of gene product expression intensities both within and between individuals. Mature lymphocytes, uncommitted progenitor cells, promyelocytes, mature monocytes and mature neutrophils can be reproducibly identified as distinct clusters of events in heterogeneous, maturing bone marrow specimens. Support Vector Machines were used to identify the reference populations in order to reduce subjective bias in manually defining boundaries of these populations since they were not discretely separated from the remainder of the cells. Reference populations were identified in 50 randomly selected bone marrow aspirates obtained over a period spanning 3 years and 6 months from pediatric patients following chemotherapy for acute myeloid leukemia (AML). The quantitative expression of gene products (cell surface antigens) and light scattering characteristics on these stressed specimens were demonstrated to be tightly regulated both within individuals and between individuals. Within an individual most gene products (CD45, CD34, CD14, CD16, CD64, CD33) demonstrated limited variability with a standard deviation of <0.20 log units while CD13 and CD36 exhibited broader variation >0.25 log units. Surprisingly, with the exception of CD33, the variation of the mean intensities of each antigen between individuals was even less than the variation within an individual. These data confirm that the amounts of gene products expressed on normal developing cells are highly regulated but differ in intensities between different lineages and during the maturational pathway of those lineages. The amounts of gene products expressed at specific stages of development of each lineage are a biologic constant with minimal variation within or between individuals. © 2016 The Authors. Cytometry Part A Published by Wiley Periodicals, Inc. on behalf of

  16. Mannose receptor-mediated gene delivery into antigen presenting dendritic cells.

    PubMed

    Diebold, Sandra S; Plank, Christian; Cotten, Matt; Wagner, Ernst; Zenke, Martin

    2002-11-01

    Dendritic cells are professional antigen presenting cells and are unique in their ability to prime naïve T cells. Gene modification of dendritic cells is of particular interest for immunotherapy of diseases where the immune system has failed or is aberrantly regulated, such as in cancer or autoimmune disease, respectively. Dendritic cells abundantly express mannose receptor and mannose receptor-related receptors, and receptor-mediated gene transfer via mannose receptor offers a versatile tool for targeted gene delivery into these cells. Accordingly, mannose polyethylenimine DNA transfer complexes were generated and used for gene delivery into dendritic cells. Mannose receptor belongs to the group of scavenger receptors that allow dendritic cells to take up pathogenic material, which is directed for degradation and MHC class II presentation. Therefore, a limiting step of transgene expression by mannose receptor-mediated gene delivery is endosomal degradation of DNA. Several strategies have been explored to overcome this limitation including the addition of endosomolytic components to DNA transfer complexes like adenovirus particles and influenza peptides. Here, we review the current understanding of mannose receptor-mediated gene delivery into dendritic cells and discuss strategies to identify appropriate endosomolytic agents to improve DNA transfer efficacy.

  17. 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.

  18. 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

  19. Gene Transfer Efficiency in Gonococcal Biofilms: Role of Biofilm Age, Architecture, and Pilin Antigenic Variation

    PubMed Central

    Kouzel, Nadzeya; Oldewurtel, Enno R.

    2015-01-01

    ABSTRACT Extracellular DNA is an important structural component of many bacterial biofilms. It is unknown, however, to which extent external DNA is used to transfer genes by means of transformation. Here, we quantified the acquisition of multidrug resistance and visualized its spread under selective and nonselective conditions in biofilms formed by Neisseria gonorrhoeae. The density and architecture of the biofilms were controlled by microstructuring the substratum for bacterial adhesion. Horizontal transfer of antibiotic resistance genes between cocultured strains, each carrying a single resistance, occurred efficiently in early biofilms. The efficiency of gene transfer was higher in early biofilms than between planktonic cells. It was strongly reduced after 24 h and independent of biofilm density. Pilin antigenic variation caused a high fraction of nonpiliated bacteria but was not responsible for the reduced gene transfer at later stages. When selective pressure was applied to dense biofilms using antibiotics at their MIC, the double-resistant bacteria did not show a significant growth advantage. In loosely connected biofilms, the spreading of double-resistant clones was prominent. We conclude that multidrug resistance readily develops in early gonococcal biofilms through horizontal gene transfer. However, selection and spreading of the multiresistant clones are heavily suppressed in dense biofilms. IMPORTANCE Biofilms are considered ideal reaction chambers for horizontal gene transfer and development of multidrug resistances. The rate at which genes are exchanged within biofilms is unknown. Here, we quantified the acquisition of double-drug resistance by gene transfer between gonococci with single resistances. At early biofilm stages, the transfer efficiency was higher than for planktonic cells but then decreased with biofilm age. The surface topography affected the architecture of the biofilm. While the efficiency of gene transfer was independent of the

  20. Epstein–Barr virus nuclear antigen 3C regulated genes in lymphoblastoid cell lines

    PubMed Central

    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. PMID:21173222

  1. Gene Transfer Efficiency in Gonococcal Biofilms: Role of Biofilm Age, Architecture, and Pilin Antigenic Variation.

    PubMed

    Kouzel, Nadzeya; Oldewurtel, Enno R; Maier, Berenike

    2015-07-01

    Extracellular DNA is an important structural component of many bacterial biofilms. It is unknown, however, to which extent external DNA is used to transfer genes by means of transformation. Here, we quantified the acquisition of multidrug resistance and visualized its spread under selective and nonselective conditions in biofilms formed by Neisseria gonorrhoeae. The density and architecture of the biofilms were controlled by microstructuring the substratum for bacterial adhesion. Horizontal transfer of antibiotic resistance genes between cocultured strains, each carrying a single resistance, occurred efficiently in early biofilms. The efficiency of gene transfer was higher in early biofilms than between planktonic cells. It was strongly reduced after 24 h and independent of biofilm density. Pilin antigenic variation caused a high fraction of nonpiliated bacteria but was not responsible for the reduced gene transfer at later stages. When selective pressure was applied to dense biofilms using antibiotics at their MIC, the double-resistant bacteria did not show a significant growth advantage. In loosely connected biofilms, the spreading of double-resistant clones was prominent. We conclude that multidrug resistance readily develops in early gonococcal biofilms through horizontal gene transfer. However, selection and spreading of the multiresistant clones are heavily suppressed in dense biofilms. Biofilms are considered ideal reaction chambers for horizontal gene transfer and development of multidrug resistances. The rate at which genes are exchanged within biofilms is unknown. Here, we quantified the acquisition of double-drug resistance by gene transfer between gonococci with single resistances. At early biofilm stages, the transfer efficiency was higher than for planktonic cells but then decreased with biofilm age. The surface topography affected the architecture of the biofilm. While the efficiency of gene transfer was independent of the architecture, spreading of

  2. A Survey of ABO, Rhesus (D) Antigen and Haemoglobin Genes Variants in Oyo State, Nigeria.

    PubMed

    Omotosho, Ishiaq

    2015-12-20

    A survey of ABO and Rhesus (Rh D) antigens and variants of haemoglobin genes (HbGen) in Oyo state was carried out. This longitudinal study involved the determination of ABO and Rh(D) antigens in 3241 and HbGen in 2622 male and female adults (aged 26-65years) respectively using standard methods. 94.5% of the subjects were Rh(D) positive while 5.5% were Rh(D) negative respectively based on the detection (Positive) or absence (Negative) of Rh(D) antigen. 22.8% of the subjects had ABO blood group A, 26.4% were group B, 4.1% were group AB while 46.7% were group O. Further analysis revealed that 695 (21.4%) of the group A were Apositive while 44 (1.4%) were Anegative. 800 of these subjects (24.7%) were Bpositive while 56 (1.7%) were group Bnegative. 133 (4.1%) showed group AB out of which 125 (3.8%) were ABpositive and 8 (0.3%) were ABnegative. 1513 (46.7%) were group O out of which 1444 (44.6%) were Opositive while 69 (2.1%) were Onegative. HbGen determination showed that 1933 of the subjects (73.7%) had HbGen AA; 553 (21.1%) were AS; 119 (4.5%) were AC; 11 (0.4%) were SC while 3 subjects representing 0.1% and 0.2% each had HbGen SS and CC respectively. Although the results were similar to earlier ones; however, the need for sustained counselling towards eradication of SS genes and increased research towards identifying artificial blood substitutes was highlighted in this work. The increasing need for blood transfusion especially with the increase in various politically/communally motivated emergency situations underscores this fact.

  3. Structure and gene cluster of the O-antigen of Enterobacter cloacae G3421.

    PubMed

    Perepelov, Andrei V; Filatov, Andrei V; Wang, Min; Shashkov, Alexander S; Wang, Lei; Knirel, Yuriy A

    2016-06-02

    The O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Enterobacter cloacae G3421 and studied by sugar analysis along with 1D and 2D (1)H and (13)C NMR spectroscopy. In addition, partial solvolysis with anhydrous trifluoroacetic acid was applied, which cleaved selectively the α-l-rhamnopyranosidic linkages. The following structure of the branched hexasaccharide repeating unit was established. The O-polysaccharide studied shares the β-l-Rhap-(1→4)-α-l-Rhap-(1→2)-α-l-Rhap trisaccharide fragment with the O-polysaccharide of Shigella boydii type 18. The O-antigen gene cluster of E. cloacae G3421 was sequenced. Functions of genes in the cluster, including those for glycosyltransferases, were tentatively assigned by a comparison with sequences in the available databases and found to be consistent with the O-polysaccharide structure.

  4. Structure elucidation and gene cluster characterization of the O-antigen of Escherichia coli O80.

    PubMed

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

    2016-09-02

    Mild alkaline degradation of the lipopolysaccharide of Escherichia coli O80 afforded a polysaccharide, which was studied by sugar analysis, selective cleavage of glycosidic linkages, and (1)H and (13)C NMR spectroscopy. Solvolysis of the polysaccharide with CF3CO2H cleaved the linkages of α-Fuc and β-linked GlcNAc and GalNAc residues to give two disaccharides. The following structure of the hexasaccharide repeating unit of the O-polysaccharide was established: The polysaccharide repeat also contains a minor O-acetyl group but its position was not determined. The O-antigen gene cluster of E. coli O80 between the conserved galF and gnd genes was analyzed and found to be consistent with the O-polysaccharide structure established.

  5. Molecular Pathways: Breaking the Epithelial Cancer Barrier for Chimeric Antigen Receptor and T-cell Receptor Gene Therapy.

    PubMed

    Hinrichs, Christian S

    2016-04-01

    Adoptive transfer of T cells genetically engineered to express a tumor-targeting chimeric antigen receptor (CAR) or T-cell receptor (TCR) can mediate cancer regression in some patients. CARs are synthetic single-chain proteins that use antibody domains to target cell surface antigens. TCRs are natural heterodimeric proteins that can target intracellular antigens through recognition of peptides bound to human leukocyte antigens. CARs have shown promise in B-cell malignancies and TCRs in melanoma, but neither approach has achieved clear success in an epithelial cancer. Treatment of epithelial cancers may be particularly challenging because of a paucity of target antigens expressed by carcinomas and not by important healthy tissues. In addition, epithelial cancers may be protected by inhibitory ligands and soluble factors in the tumor microenvironment. One strategy to overcome these negative regulators is to modulate expression of T-cell genes to enhance intrinsic T-cell function. Programmable nucleases, which can suppress inhibitory genes, and inducible gene expression systems, which can enhance stimulatory genes, are entering clinical testing. Other work is delineating whether control of genes for immune checkpoint receptors (e.g.,PDCD1, CTLA4) and cytokine and TCR signaling regulators (e.g.,CBLB, CISH, IL12, IL15) can increase the antitumor activity of therapeutic T cells.

  6. Cloning, sequencing, and expression of the gene coding for an antigenic 120-kilodalton protein of Rickettsia conorii.

    PubMed Central

    Schuenke, K W; Walker, D H

    1994-01-01

    Several high-molecular-mass (above 100 kDa) antigens are recognized by sera from humans infected with spotted fever group rickettsiae and may be important stimulators of the host immune response. Molecular cloning techniques were used to make genomic Rickettsia conorii (Malish 7 strain) libraries in expression vector lambda gt11. The 120-kDa R. conorii antigen was identified by monospecific antibodies to the recombinant protein expressed on construct lambda 4-7. The entire gene DNA sequence was obtained by using this construct and two other overlapping constructs. An open reading frame of 3,068 bp with a calculated molecular mass of approximately 112 kDa was identified. Promoters and a ribosome-binding site were identified on the basis of their DNA sequence homology to other rickettsial genes and their relative positions in the sequence. The DNA coding region shares no significant homology with other spotted fever group rickettsial antigen genes (i.e., the R. rickettsii 190-, 135-, and 17-kDa antigen-encoding genes). The PCR technique was used to amplify the gene from eight species of spotted fever group rickettsiae. A 75-kDa portion of the 120-kDa antigen was overexpressed in and purified from Escherichia coli. This polypeptide was recognized by antirickettsial antibodies and may be a useful diagnostic reagent for spotted fever group rickettsioses. Images PMID:8112862

  7. The inhibition of Escherichia coli lac operon gene expression by antigene oligonucleotides-mathematical modeling.

    PubMed

    Cheng, B; Fournier, R L; Relue, P A

    2000-11-20

    Gene transcription is regulated by transcription factors that can bind to specific regions on DNA. Antigene oligonucleotides (oligos) can bind to specific regions on DNA and form a triplex with the double-stranded DNA. The triplex can competitively inhibit the binding of transcription factors and, as a result, transcription can be inhibited. A genetically structured model has been developed to quantitatively describe the inhibition of the Escherichia coli lac operon gene expression by triplex-forming oligos. The model predicts that the effect of triplex-forming oligos on the lac operon gene expression depends on their target sites. Oligonucleotides targeted to the operator are much more effective than those targeted to other regulatory sites on the lac operon. In some cases, the effect of oligo binding is similar to that of a mutation in the lac operon. The model provides insight as to the specific binding site to be targeted to achieve the most effective inhibition of gene expression. The model is also capable of predicting the oligo concentration needed to inhibit gene expression, which is in general agreement with results reported by other investigators.

  8. Detection of circulating tumor cells using GeneScan analysis for antigen receptor gene rearrangements in canine lymphoma patients

    PubMed Central

    HIYOSHI-KANEMOTO, Saaya; GOTO-KOSHINO, Yuko; FUKUSHIMA, Kenjiro; TAKAHASHI, Masashi; KANEMOTO, Hideyuki; UCHIDA, Kazuyuki; FUJINO, Yasuhito; OHNO, Koichi; TSUJIMOTO, Hajime

    2016-01-01

    The presence of circulating tumor cells (CTCs) serves as a prognostic marker and indicator of disease relapse, as well as a means of evaluating treatment efficacy in human and canine lymphoma patients. As an extension of our previous study for the construction of clinically useful GeneScan system, we utilized the GeneScan system for detecting CTCs in canine lymphoma patients. Samples from the primary lesion and peripheral blood mononuclear cells (PBMCs) were obtained from 32 dogs with lymphoma at initial diagnosis. All samples were subjected to polymerase chain reaction (PCR) for antigen receptor gene rearrangements (PARR) followed by GeneScan analysis. Common clonal rearrangements with identical amplified fragments were detected in both the primary lesion and PBMCs in 19 of the 32 dogs (59.4%). However, the detection rate of CTCs varied among the anatomical classification of lymphoma studied. GeneScan analysis following PARR would facilitate studies on determining the clinical significance of CTCs in canine lymphoma patients. PMID:26888583

  9. 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.

  10. A gene cluster at an unusual chromosomal location responsible for the novel O-antigen synthesis in Escherichia coli O62 by the ABC transporter-dependent pathway.

    PubMed

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

    2017-04-10

    The O-antigen is a part of the outer membrane of Gram-negative bacteria and is related to bacterial virulence. It is one of the most variable cell constituents, and its structural diversity is almost entirely due to genetic variation of the O-antigen gene cluster. In this study, the O-antigen structure of Escherichia coli O62 was elucidated by chemical analysis and nuclear magnetic resonance spectroscopy, but showing not consistent with the O-antigen gene cluster between conserved genes galF and gnd reported earlier. The complete genome of E. coli O62 was then sequenced and analyzed, and another O-antigen gene cluster was found and characterized that correlated perfectly with the established O-antigen structure. A deletion and complementation experiment confirmed the functionality of the novel gene cluster and demonstrated that the O62-antigen is synthesized by the ABC transporter-dependent system. To our knowledge, this is the first report that the O-antigen gene cluster is positioned at a novel locus in E. coli. Comparative analysis indicated that E. coli O62 likely originated from E. coli O68 via an IS event resulting in the repression of the O68-antigen synthesis, followed by the acquisition of a novel O-antigen gene cluster from Enterobacter aerogenes.

  11. 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

  12. BGMUT: NCBI dbRBC database of allelic variations of genes encoding antigens of blood group systems.

    PubMed

    Patnaik, Santosh Kumar; Helmberg, Wolfgang; Blumenfeld, Olga O

    2012-01-01

    Analogous to human leukocyte antigens, blood group antigens are surface markers on the erythrocyte cell membrane whose structures differ among individuals and which can be serologically identified. The Blood Group Antigen Gene Mutation Database (BGMUT) is an online repository of allelic variations in genes that determine the antigens of various human blood group systems. The database is manually curated with allelic information collated from scientific literature and from direct submissions from research laboratories. Currently, the database documents sequence variations of a total of 1251 alleles of all 40 gene loci that together are known to affect antigens of 30 human blood group systems. When available, information on the geographic or ethnic prevalence of an allele is also provided. The BGMUT website also has general information on the human blood group systems and the genes responsible for them. BGMUT is a part of the dbRBC resource of the National Center for Biotechnology Information, USA, and is available online at http://www.ncbi.nlm.nih.gov/projects/gv/rbc/xslcgi.fcgi?cmd=bgmut. The database should be of use to members of the transfusion medicine community, those interested in studies of genetic variation and related topics such as human migrations, and students as well as members of the general public.

  13. Metastability of Helicobacter pylori bab adhesin genes and dynamics in Lewis b antigen binding

    PubMed Central

    Bäckström, Anna; Lundberg, Carina; Kersulyte, Dangeruta; Berg, Douglas E.; Borén, Thomas; Arnqvist, Anna

    2004-01-01

    Heterogeneity among Helicobacter pylori strains in gastric epithelial adherence is postulated to contribute to pathogen fitness in the physiologically diverse human population. H. pylori adherence to ABO and Lewis b (Leb) blood group antigens in the human stomach is mediated by the blood group antigen-binding adhesin BabA. Approximately 70% of Swedish and U.S. H. pylori clinical isolates exhibit Leb binding, but here we show that the babA gene is present in each of 10 Leb-nonbinding strains. Fluorescence microscopy identified occasional bacterial cells with a Leb-binding phenotype in populations of Leb-nonbinding strains. Thus, nonbinding seemed to be a metastable phenotype. To model metastable transition into the virulence-associated Leb-binding mode, Leb-binding clones were isolated from nonadherent strains by panning with Leb-magnetic beads and characterized. Strain 17875 has two babA genes, babA1 (silent) and babA2 (expressed). We found that a babA2-cam derivative of strain 17875 regained Leb binding by recombination of the formerly silent babA1 gene into the expressed and partially homologous babB locus. The chimeric BabB/A adhesin binds Leb with an affinity similar to that of wild-type BabA adhesin, but its expression level was lower and was subject to phase variation through slipped-strand mispairing. Equivalent results were obtained with strain NCTC11638. We propose that adhesin metastability and heterogeneity contributes to bacterial fitness and results in some clones having potential for periodic activation and deactivation of virulence appropriate for intensity of the host response to infection. PMID:15557006

  14. Isolation and characterization of a human sperm antigen gene h-Sp-1.

    PubMed

    Kanazawa, Ri-Ichiro; Komori, Shinji; Sakata, Kazuko; Tanaka, Hiroyuki; Sawai, Hideaki; Tsuji, Yoshiyuki; Koyama, Koji

    2003-08-01

    We isolated and characterized a human sperm antigen gene (h-Sp-1) from human testis complementary DNA using antiserum against the human sperm membrane. Northern blot analysis detected two transcripts (2.3 and 1.1 kb) of the h-Sp-1 gene. The 2.3-kb transcript is ubiquitous, whereas the 1.1-kb transcript is specific to the human testis with a high level of expression. Determination of the base sequence of h-Sp-1 showed a size of 2170 bp and 43.4% homology with human synaptophysin. The base sequence indicates a molecule consisting of 259 amino acids, with four hydrophilic and four hydrophobic regions. In order to further characterize the h-Sp-1 molecule, we synthesized the probable region of amino acids with high antigenicity based on the amino acid sequence (amino acid nos. 174-198) and immunized rabbits to prepare an antiserum. In our experimental model of fertilization between human sperm and zona pellucida-free hamster ova, partial inhibition of fertilization was observed. We were able to synthesize a large quantity of recombinant protein by inserting the h-Sp-1 gene into a baculovirus vector and infecting spodoptera frugiperda culture cells (sf9 insect cells). The synthesized protein had a molecular weight of 30 kDa. We then immunized Balb/c mice with this protein to prepare a monoclonal antibody (G3G9), which was used to localize the h-Sp-1 molecule in sperm and tissues (e.g. testis). The h-Sp-1 molecule was present in the cell membrane from the head to tail of human sperm. Staining of the testis and epididymis also showed h-Sp-1 to be present in spermatogonia, spermatocyte, sperm and epididymal duct epithelium. These findings suggest that the h-Sp-1 molecule is expressed in sperm and testes and plays a role in fertilization.

  15. 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

  16. Molecular Variation in the Variable-Length PCR Target and 120-Kilodalton Antigen Genes of Ehrlichia chaffeensis from White-Tailed Deer (Odocoileus virginianus)

    PubMed Central

    Yabsley, Michael J.; Little, Susan E.; Sims, Ethan J.; Dugan, Vivien G.; Stallknecht, David E.; Davidson, William R.

    2003-01-01

    Genes encoding two surface-expressed antigens of Ehrlichia chaffeensis, the variable-length PCR target (VLPT) and the 120-kDa antigen, which contain variable numbers of tandem repeats, were characterized for E. chaffeensis from white-tailed deer (Odocoileus virginianus). Both genes from infected deer contained numbers of repeats similar to those reported in genes from humans and ticks, although a new variant of the 120-kDa antigen gene containing five repeat units and coinfection with multiple VLPT and 120-kDa antigen gene genetic types were detected. Sequence analysis of both genes revealed more nucleotide variation than previously reported for E. chaffeensis from infected humans or ticks. This is the most extensive study of E. chaffeensis VLPT and 120-kDa antigen gene genetic variation to date and is the first to examine genetic variation in E. chaffeensis from a nonhuman vertebrate host. PMID:14605163

  17. In vivo gene transfer targeting in pancreatic adenocarcinoma with cell surface antigens

    PubMed Central

    2012-01-01

    Background Pancreatic ductal adenocarcinoma is a deadly malignancy resistant to current therapies. It is critical to test new strategies, including tumor-targeted delivery of therapeutic agents. This study tested the possibility to target the transfer of a suicide gene in tumor cells using an oncotropic lentiviral vector. Results Three cell surface markers were evaluated to target the transduction of cells by lentiviruses pseudotyped with a modified glycoprotein from Sindbis virus. Only Mucin-4 and the Claudin-18 proteins were found efficient for targeted lentivirus transductions in vitro. In subcutaneous xenografts of human pancreatic cancer cells models, Claudin-18 failed to achieve efficient gene transfer but Mucin-4 was found very potent. Human pancreatic tumor cells were modified to express a fluorescent protein detectable in live animals by bioimaging, to perform a direct non invasive and costless follow up of the tumor growth. Targeted gene transfer of a bicistronic transgene bearing a luciferase gene and the herpes simplex virus thymidine kinase gene into orthotopic grafts was carried out with Mucin-4 oncotropic lentiviruses. By contrast to the broad tropism VSV-G carrying lentivirus, this oncotropic lentivirus was found to transduce specifically tumor cells, sparing normal pancreatic cells in vivo. Transduced cells disappeared after ganciclovir treatment while the orthotopic tumor growth was slowed down. Conclusion This work considered for the first time three aspect of pancreatic adenocarcinoma targeted therapy. First, lentiviral transduction of human pancreatic tumor cells was possible when cells were grafted orthotopically. Second, we used a system targeting the tumor cells with cell surface antigens and sparing the normal cells. Finally, the TK/GCV anticancer system showed promising results in vivo. Importantly, the approach presented here appeared to be a safer, much more specific and an as efficient way to perform gene delivery in pancreatic tumors

  18. Prostate-Specific Antigen Modulates the Expression of Genes Involved in Prostate Tumor Growth1

    PubMed Central

    Bindukumar, B; Schwartz, Stanley A; Nair, Madhavan P N; Aalinkeel, Ravikumar; Kawinski, Elzbieta; Chadha, Kailash C

    2005-01-01

    Abstract Prostate-specific antigen (PSA) is a serine protease that is widely used as a surrogate marker in the early diagnosis and management of prostate cancer. The physiological relevance of tissue PSA levels and their role in prostate tumor growth and metastasis are not known. Free-PSA (f-PSA) was purified to homogeneity from human seminal plasma by column chromatography, eliminating hk2 and all known PSA complexes and retaining its protease activity. Confluent mono-layers of prostate cancer cell lines, PC-3M and LNCaP, were treated with f-PSA in a series of in vitro experiments to determine the changes in expression of various genes that are known to regulate tumor growth and metastasis. Gene array, quantitative polymerase chain reaction (QPCR), and enzyme-linked immunosorbent assay (ELISA) results show significant changes in the expression of various cancer-related genes in PC-3M and LNCaP cells treated with f-PSA. In a gene array analysis of PC-3M cells treated with 10 µM f-PSA, 136 genes were upregulated and 137 genes were downregulated. In LNCaP cells treated with an identical concentration of f-PSA, a total of 793 genes was regulated. QPCR analysis reveals that the genes for urokinase-type plasminogen activator (uPA), VEGF, and Pim-1 oncogene, known to promote tumor growth, were significantly downregulated, whereas IFN-γ, known to be a tumor-suppressor gene, was significantly upregulated in f-PSA-treated PC-3M cells. The effect of f-PSA on VEGF and IFN-γ gene expression and on protein release in PC-3M cells was distinctly dose-dependent. In vivo studies showed a significant reduction (P = .03) in tumor load when f-PSA was administered in the tumor vicinity of PC-3M tumor-bearing BALB/c nude mice. Our data support the hypothesis that f-PSA plays a significant role in prostate tumor growth by regulating various proangiogenic and antiangiogenic growth factors. PMID:15799824

  19. 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

  20. Crosstalk between ABO and Forssman (FORS) blood group systems: FORS1 antigen synthesis by ABO gene-encoded glycosyltransferases

    PubMed Central

    Yamamoto, Miyako; Cid, Emili; Yamamoto, Fumiichiro

    2017-01-01

    A and B alleles at the ABO genetic locus specify A and B glycosyltransferases that catalyze the biosynthesis of A and B oligosaccharide antigens, respectively, of blood group ABO system which is important in transfusion and transplantation medicine. GBGT1 gene encodes Forssman glycolipid synthase (FS), another glycosyltransferase that produces Forssman antigen (FORS1). Humans are considered to be Forssman antigen-negative species without functional FS. However, rare individuals exhibiting Apae phenotype carry a dominant active GBGT1 gene and express Forssman antigen on RBCs. Accordingly, FORS system was recognized as the 31st blood group system. Mouse ABO gene encodes a cis-AB transferase capable of producing both A and B antigens. This murine enzyme contains the same GlyGlyAla tripeptide sequence as FSs at the position important for the determination of sugar specificity. We, therefore, transfected the expression construct into appropriate recipient cells and examined whether mouse cis-AB transferase may also exhibit FS activity. The result was positive, confirming the crosstalk between the ABO and FORS systems. Further experiments have revealed that the introduction of this tripeptide sequence to human A transferase conferred some, although weak, FS activity, suggesting that it is also involved in the recognition/binding of acceptor substrates, in addition to donor nucleotide-sugars. PMID:28134301

  1. 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

  2. Gene for the major antigenic structural protein (p100) of human herpesvirus 6.

    PubMed Central

    Neipel, F; Ellinger, K; Fleckenstein, B

    1992-01-01

    A human herpesvirus 6 (HHV-6) structural protein of 100 kDa (p100) is the polypeptide most frequently and intensively reactive in immunoblotting analyses with human sera on HHV-6-infected cells or partially purified virions. The gene for p100 was identified by screening a bacteriophage lambda library with monospecific rabbit antisera. The gene codes for a polypeptide of 870 amino acids with a calculated molecular size of 97 kDa. Its amino-terminal third is weakly homologous to the immunogenic basic matrix phosphoprotein pp150 of human cytomegalovirus. Five fragments representing more than 93% of HHV-6 p100 were prokaryotically expressed. The antigenic epitopes of p100 were preliminary mapped by immunoblotting with human sera. They are located within the carboxy-terminal part which is neither homologous nor cross-reactive to pp150 of human cytomegalovirus. Availability of the gene for the immunodominant structural protein should provide tools for studies of pathogenesis by HHV-6. Images PMID:1374813

  3. Cloning and sequence analysis of a gene encoding a 67-kilodalton myosin-cross-reactive antigen of Streptococcus pyogenes reveals its similarity with class II major histocompatibility antigens.

    PubMed Central

    Kil, K S; Cunningham, M W; Barnett, L A

    1994-01-01

    The group A streptococcal sequela acute rheumatic fever (ARF) has been associated with immunological cross-reactivity between streptococcal and heart proteins. To identify Streptococcus pyogenes genes that encode a myosin cross-reactive antigen(s) recognized by ARF sera, a genomic library from an emm deletion strain (T28/51/4) was screened with a single ARF serum. A positively identified lambda EMBL3 clone (T.2.18) produced a protein which reacted with myosin-specific antibodies affinity purified from individual ARF sera. The recombinant protein was initially estimated to be 60 kDa in size by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; however, upon sequence analysis it had a molecular mass equivalent to 67 kDa. Sera from patients with streptococcal infections, acute glomerulonephritis, and ARF were reactive with the recombinant 67-kDa protein. However, individual sera from healthy persons were negative or demonstrated low levels of reactivity with the 67-kDa antigen. The gene encoding the 67-kDa myosin-cross-reactive antigen was subcloned, and its nucleotide sequence was determined by using a combined strategy of DNA sequencing of the cloned gene and N-terminal amino acid sequencing of the protein expressed in Escherichia coli. The amino-terminal sequence deduced from the nucleotide sequence of an open reading frame was identical to that determined from the 67-kDa protein expressed in E. coli. The gene encoded 590 amino acids with a calculated molecular weight of 67,381. No cleavable signal peptide was detected with the 67-kDa protein expressed in E. coli. The deduced amino acid sequence of the 67-kDa protein did not exhibit significant similarity to any known streptococcal proteins. However, it was found to be 19% identical and 62% similar over 151 amino acid residues to the beta chain of mouse major histocompatibility complex class II antigen (I-Au). Similar degrees of homology to the beta chains of other murine and human class II haplotypes were

  4. Characterization of an equine herpesvirus type 1 gene encoding a glycoprotein (gp13) with homology to herpes simplex virus glycoprotein C.

    PubMed

    Allen, G P; Coogle, L D

    1988-08-01

    The molecular structure of the equine herpesvirus type 1 (EHV-1) gene encoding glycoprotein 13 (gp13) was analyzed. The gene is contained within a 1.8-kilobase AccI-EcoRI restriction fragment mapping at map coordinates 0.136 to 0.148 in the UL region of the EHV-1 genome and is transcribed from right to left. Determination of the nucleotide sequence of the DNA fragment revealed a complete transcriptional unit composed of typical regulatory promoter elements upstream to a long open reading frame (1,404 base pairs) that encoded a 468-amino-acid primary translation product of 51 kilodaltons. The predicted protein has the characteristic features of a membrane-spanning protein: an N-terminal signal sequence, a hydrophobic membrane anchor region, a charged C-terminal cytoplasmic tail, and an exterior domain with nine potential N-glycosylation sites. The EHV-1 DNA sequences expressed in lambda gt11 as gp13 epitopes were present in the open reading frame. Amino acid sequences composing a major antigenic site, recognized by 35% of a panel of 42 anti-gp13 monoclonal antibodies, were identified in the N-terminal surface domain of the deduced gp13 molecule. Comparison of the EHV-1 gp13 DNA sequence with that encoding glycoproteins of other alphaherpesviruses revealed no detectable homology. However, a search for homology at the amino acid level showed regions of significant sequence similarity between the amino acids of the carboxy half of EHV-1 gp13 and those of the same region of gC-like glycoproteins of herpes simplex virus (gC-1 and gC-2), pseudorabies herpesvirus (gIII), and varicella-zoster virus (gp66). The sequences of the N-terminal portion of gp13, by contrast, were much less conserved. The results of these studies indicate that EHV-1 gp13 is the structural homolog of herpes simplex virus glycoprotein C and further suggest that the epitope-containing N-terminal amino acid sequences of the herpesvirus gC-like glycoproteins have undergone more extensive evolutionary

  5. Prediction of G gene epitopes of viral hemorrhagic septicemia virus and eukaryotic expression of major antigen determinant sequence.

    PubMed

    Sun, T; Yin, W-L; Fang, B-H; Wang, Q; Liang, C-Z; Yue, Z-Q

    2017-08-15

    This study aims to express fish Viral hemorrhagic septicemia virus (VHSV) G main antigen domain by using Bac-to-bac expression system. Using bioinformatics tools, B cell epitope of VHSV G gene was predicted, and G main antigen domain was optimized. GM gene was inserted into pFastBac1 vector, then transferred recombinant plasmid into DH10Bac to get recombinant rBacmid-GM. Obtained shuttle plasmid rBacmid-GM was transfected into sf9 cells. GM expression was examined using by PCR and western-blot. Results indicated that G main antigen domain gene of VHSV was successfully cloned and sequenced which contains 1209 bp. PCR proved that shuttle plasmid rBacmid-GM was constructed correctly. SDS-PAGE electrophoresis analysis detected a band of protein about 45kD in expression product of G gene. Obtained recombinant G protein reacted with VHSV-positive serum that was substantiated by western-blot analysis. In conclusion, the main antigen domain of VHSV G was successfully expressed in the Bac-to-Bac baculovirus system.

  6. Adoptive immunotherapy for hematological malignancies using T cells gene-modified to express tumor antigen-specific receptors.

    PubMed

    Fujiwara, Hiroshi

    2014-12-15

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

  7. 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

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

    PubMed Central

    Adair, Patrick R.; Kim, Yong Chan; Zhang, Ai-Hong; Yoon, Jeongheon; Scott, David W.

    2017-01-01

    Human regulatory CD4+ T cells (Tregs) are potent immunosuppressive lymphocytes responsible for immune tolerance and homeostasis. Since the seminal reports identifying Tregs, vast research has been channeled into understanding their genesis, signature molecular markers, mechanisms of suppression, and role in disease. This research has opened the doors for Tregs as a potential therapeutic for diseases and disorders such as multiple sclerosis, type I diabetes, transplantation, and immune responses to protein therapeutics, like factor VIII. Seminal clinical trials have used polyclonal Tregs, but the frequency of antigen-specific Tregs among polyclonal populations is low, and polyclonal Tregs may risk non-specific immunosuppression. Antigen-specific Treg therapy, which uses genetically modified Tregs expressing receptors specific for target antigens, greatly mitigates this risk. Building on the principles of T-cell receptor cloning, chimeric antigen receptors (CARs), and a novel CAR derivative, called B-cell antibody receptors, our lab has developed different types of antigen-specific Tregs. This review discusses the current research and optimization of gene-modified antigen-specific human Tregs in our lab in several disease models. The preparations and considerations for clinical use of such Tregs also are discussed. PMID:28983300

  9. Anti-colorectal cancer effect of interleukin-2 and interferon-β fusion gene driven by carcinoembryonic antigen promoter

    PubMed Central

    Wang, Yan; Wang, Mengchun; Li, Yan

    2016-01-01

    This study was designed to investigate the antitumor effects of combined interleukin-2/interferon-β-based gene therapy in colorectal cancer. Transfection of the fusion gene expression plasmid induced significant apoptosis of Lovo cells. Additionally, the fusion gene exhibited strong inhibitory activity against tumor growth and apoptosis when being injected into the nude mice implanted with human colon cancer cells. Furthermore, the tail-vein injection showed a more notable effect than direct injection into tumor. These results suggest that the combined interleukin-2/interferon-β-based gene therapy with the carcinoembryonic antigen promoter might be an effective antitumor strategy. PMID:27313471

  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. A common gene pool for the Neisseria FetA antigen.

    PubMed

    Bennett, Julia S; Thompson, Emily A L; Kriz, Paula; Jolley, Keith A; Maiden, Martin C J

    2009-02-01

    Meningococcal FetA is an iron-regulated, immunogenic outer membrane protein and vaccine component. The most diverse region of this protein is a previously defined variable region (VR) that has been shown to be immunodominant. In this analysis, a total of 275 Neisseria lactamica isolates, collected during studies of nasopharyngeal bacterial carriage in infants, were examined for the presence of a fetA gene. The fetA VR nucleotide sequence was determined for 217 of these isolates, with fetA apparently absent from 58 isolates, the majority of which belonged to the ST-624 clonal complex. The VR in N. lactamica was compared to the same region in N. meningitidis, N. gonorrhoeae, and a number of other commensal Neisseria. Identical fetA variable region sequences were identified among commensal and pathogenic Neisseria, suggesting a common gene pool, differing from other antigens in this respect. Carriage of commensal Neisseria species, such as N. lactamica, that express FetA may be involved in the development of natural immunity to meningococcal disease.

  12. A common gene pool for the Neisseria FetA antigen

    PubMed Central

    Bennett, Julia S.; Thompson, Emily A. L.; Kriz, Paula; Jolley, Keith A.; Maiden, Martin C. J.

    2014-01-01

    Meningococcal FetA is an iron-regulated, immunogenic outer membrane protein and vaccine component. The most diverse region of this protein is a previously defined variable region (VR) that has been shown to be immunodominant. In this analysis, a total of 275 Neisseria lactamica isolates, collected during studies of nasopharyngeal bacterial carriage in infants were examined for the presence of a fetA gene. The fetA VR nucleotide sequence was determined for 217 of these isolates, with fetA apparently absent from 58 isolates, the majority of which belonged to the ST-624 clonal complex. The VR in N. lactamica was compared to the same region in Neisseria meningitidis, Neisseria gonorrhoeae and a number of other commensal Neisseria. Identical fetA variable region sequences were identified among commensal and pathogenic Neisseria, suggesting a common gene pool, differing from other antigens in this respect. Carriage of commensal Neisseria species, such as N. lactamica, that express FetA may be involved in the development of natural immunity to meningococcal disease. PMID:18718812

  13. An experimental and theoretical study of the inhibition of Escherichia coli lac operon gene expression by antigene oligonucleotides.

    PubMed

    Cheng, B; Fournier, R L; Relue, P A; Schisler, J

    2001-08-05

    Previously, we have developed a genetically structured mathematical model to describe the inhibition of Escherichia coli lac operon gene expression by antigene oligos. Our model predicted that antigene oligos targeted to the operator region of the lac operon would have a significant inhibitory effect on beta-galactosidase production. In this investigation, the E. coli lac operon gene expression in the presence of antigene oligos was studied experimentally. A 21-mer oligo, which was designed to form a triplex with the operator, was found to be able to specifically inhibit beta-galactosidase production in a dose-dependent manner. In contrast to the 21-mer triplex-forming oligonucleotide (TFO), several control oligos showed no inhibitory effect. The ineffectiveness of the various control oligos, along with the fact that the 21-mer oligo has no homology sequence with lacZYA, and no mRNA is transcribed from the operator, suggests that the 21-mer oligo inhibits target gene expression by an antigene mechanism. To simulate the kinetics of lac operon gene expression in the presence of antigene oligos, a genetically structured kinetic model, which includes transport of oligo into the cell, growth of bacteria cells, and lac operon gene expression, was developed. Predictions of the kinetic model fit the experimental data quite well after adjustment of the value of the oligonucleotide transport rate constant (9.0 x 10(-)(3) min(-)(1)) and oligo binding affinity constant (1.05 x 10(6) M(-)(1)). Our values for these two adjusted parameters are in the range of reported literature values.

  14. Molecular characterization of swine leucocyte antigen class I genes in outbred pig populations.

    PubMed

    Ho, C-S; Lunney, J K; Franzo-Romain, M H; Martens, G W; Lee, Y-J; Lee, J-H; Wysocki, M; Rowland, R R R; Smith, D M

    2009-08-01

    The highly polymorphic swine leucocyte antigen (SLA) genes are one of the most important determinants in swine immune responses to infectious diseases, vaccines, and in transplantation success. Study of SLA influence requires accurate and effective typing methods. We developed a simple and rapid method to type alleles at the three classical SLA class I loci (SLA-1, SLA-3 and SLA-2) using the PCR-sequence-specific primer (PCR-SSP) strategy. This typing system relies on 47 discriminatory PCR primer pairs designed to amplify the SLA class I alleles by groups that have similar sequence motifs. We applied this low-resolution group-specific typing method to characterize the SLA class I alleles present in three outbred pig populations (n = 202). Alleles from 24 class I allele groups corresponding to 56 class I genotypes were detected. We also identified 23 low-resolution SLA class I haplotypes in these pigs and found haplotypes Lr-1.0 (SLA-1*01XX-SLA-3*01XX-SLA-2*01XX) and Lr-4.0 (SLA-1*04XX-SLA-3*04XX-SLA-2*04XX) in all three pig populations with a high prevalence. Over 80% of the pigs examined (n = 162) were found to bear at least one of these haplotypes, resulting in a combined haplotype frequency of nearly 50%. This PCR-SSP-based typing system demonstrates a reliable and unambiguous detection of SLA class I alleles, and can be used to effectively investigate the SLA diversity in outbred pig populations. It will help to identify the role of SLA antigens in disease-resistant pigs and may facilitate the development of effective vaccines.

  15. 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

  16. Role of Escherichia coli K-12 rfa genes and the rfp gene of Shigella dysenteriae 1 in generation of lipopolysaccharide core heterogeneity and attachment of O antigen.

    PubMed Central

    Klena, J D; Ashford, R S; Schnaitman, C A

    1992-01-01

    The rfp gene of Shigella dysenteriae 1 and the rfa genes of Escherichia coli K-12 and Salmonella typhimurium LT2 have been studied to determine their relationship to lipopolysaccharide (LPS) core heterogeneity and their role in the attachment of O antigen to LPS. It has been inferred from the nucleotide sequence that the rfp gene encodes a protein of 41,864 Da which has a structure similar to that of RfaG protein. Expression of this gene in E. coli K-12 results in the loss of one of the three bands seen in gel analysis of the LPS and in the appearance of a new, more slowly migrating band. This is consistent with the hypothesis that Rfp is a sugar transferase which modifies a subset of core molecules so that they become substrates for attachment of S. dysenteriae O antigen. A shift in gel migration of the bands carrying S. dysenteriae O antigen and disappearance of the Rfp-modified band in strains producing O antigen suggest that the core may be trimmed or modified further before attachment of O antigen. Mutation of rfaL results in a loss of the rough LPS band which appears to be modified by Rfp and prevents the appearance of the Rfp-modified band. Thus, RfaL protein is involved in core modification and is more than just a component of the O-antigen ligase. The products of rfaK and rfaQ also appear to be involved in modification of the core prior to attachment of O antigen, and the sites of rfaK modification are different in E. coli K-12 and S. typhimurium. In contrast, mutations in rfaS and rfaZ result in changes in the LPS core but do not affect the attachment of O antigen. We propose that these genes are involved in an alternative pathway for the synthesis of rough LPS species which are similar to lipooligosaccharides of other species and which are not substrates for O-antigen attachment. All of these studies indicate that the apparent heterogeneity of E. coli K-12 LPS observed on gels is not an artifact but instead a reflection of functional differences among

  17. Extensive Variation in the O-Antigen Gene Cluster within One Salmonella enterica Serogroup Reveals an Unexpected Complex History

    PubMed Central

    Wang, Lei; Andrianopoulos, Kanella; Liu, Dan; Popoff, Michel Y.; Reeves, Peter R.

    2002-01-01

    The 46 serogroups of Salmonella enterica have different O-antigens, and each is thought to have a specific form of the O-antigen cluster. Comparison of the 145 serovars of serogroup B revealed much more intraserogroup genetic diversity than expected. The O27 factor, due to an α 1-6 linkage between O units in place of the more common α 1-2 linkage and previously thought to be due to a converting bacteriophage, is now shown to be due to a wzyα(1-6) gene located within the major gene cluster. Surprisingly a remnant of this gene in all O27− serovars shows that the ancestor was O27+. There are six distinct gene cluster forms, five apparently derived by a series of deletions and one by an insertion from an ancestral O27+ form present in 57 serovars. The history of the gene cluster and movement between subspecies I and II can be traced. Two of the derivative forms still have a functional wzyα(1-6) gene, while in three it has been inactivated by deletion or insertion. Two of the forms lacking a functional wzyα(1-6) gene have the wzyα(1-2) gene first described for strain LT2 as rfc, whereas for the third the wzy gene has not been located. PMID:11872718

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

    DTIC Science & Technology

    1986-11-26

    SIE cop AD nCloning and Expression of Genes for Dengue Virus ,4. CJ Type 2 Encoded Antigens for Rapid Diagnosis and Vaccine Development 0ANNUAL...Type 2 Encoded Antigens for Rapid Diagnosis and Vaccine Development 12. PERSONAL AUTHOR(S) Radha K. Padmanabhan, Ph.D. 13a. TYPE OF REPORT 13b. TIME...pVVI and pVVI7 cDNA clones, synthetic peptides homologous to NS5 and NSI regions were synthesized. These peptides are being used at Walter Reed Army

  19. O-antigen structure of Shigella flexneri serotype Yv and effect of the lpt-O gene variation on phosphoethanolamine modification of S. flexneri O-antigens.

    PubMed

    Knirel, Yuriy A; Lan, Ruiting; Senchenkova, Sof'ya N; Wang, Jianping; Shashkov, Alexander S; Wang, Yan; Perepelov, Andrei V; Xiong, Yanwen; Xu, Jianguo; Sun, Qiangzheng

    2013-04-01

    Shigella flexneri is the major human pathogen causing shigellosis. O-antigens of all S. flexneri serotypes (except for serotype 6) share the →2)-α-l-Rhap(III)-(1 → 2)-α-l-Rhap(II)-(1 → 3)-α-l-Rhap(I)-(1 → 3)-β-d-GlcpNAc-(1→ basic O-unit, whereas differences between the serotypes are conferred by phage-encoded glucosylation and/or O-acetylation at various positions. Recently, in serotype X and 4a variants called Xv and 4av, respectively, O-antigen modification with phosphoethanolamine (PEtN) has been identified, which is encoded by a plasmid-borne gene (lpt-O) for a PEtN-transferase and confers the monoclonal antibody IV-1(MASF IV-1) determinant to the bacteria. In this study, we elucidated the O-antigen structure of serotype Yv, another MASF IV-1-positive novel variant of S. flexneri. The serotype Yv O-antigen has the same basic carbohydrate backbone structure as that of the "classical" serotype Y, but differs in the presence of PEtN at position 3 of Rha(III) (major) or both Rha(II) and Rha(III) (minor). This pattern is similar to that of serotype 4av, but different from the pattern of serotype Xv, which is characterized by major PEtN modification on Rha(II). In serotype Yv, mono- and bisphosphorylated O-units generate a block-copolymeric structure, the former being partially O-acetylated at position 6 of GlcNAc and the latter lacking O-acetylation. Functional analysis revealed a correlation between the serotype-specific PEtN modification pattern and the lpt-O variation in different serotypes: lpt-O(RII) in serotype Xv is better tuned for phosphorylation of Rha(II) and lpt-O(RIII) in serotypes Yv and 4av for phosphorylation of Rha(III). These data enhance our knowledge of S. flexneri serotype conversion mechanisms and help to understand the biosynthesis process of the new O-antigen variants.

  20. 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. © 2013.

  1. Cytotoxic T Lymphocyte Antigen-4 Gene Variants in Type 2 Diabetic Patients with or without Neuropathy.

    PubMed

    Kiani, Javad; Khadempar, Saedeh; Hajilooi, Mehrdad; Rezaei, Hamzeh; Keshavarzi, Fatemeh; Solgi, Ghasem

    2016-06-01

    Many studies have shown that cytotoxic T lymphocyte antigen-4 (CTLA-4) gene variants are associated with several autoimmune diseases particularly type 1 diabetes. Due to the lack of consistent data for this association with type 2 diabetes (T2D), this study explored the possible influence of CTLA-4 gene polymorphisms at -1722 (T/C), -318 (C/T), and +49 (G/A) positions for susceptibility to T2D in relation with neuropathy. One hundred and eleven unrelated patients with T2D [49 patients with diabetic peripheral neuropathy (DPN) and 62 patients without PDN] and 100 healthy ethnic- and gender-matched controls were included in this study. The dimorphisms at -1722 (C/T), -318 (C/T) and +49 (A/G) for CTLA-4 gene were determined using ARMS-PCR. The CTLA-4 (+49 G/G) and (+49 A/A) genotypes were found to be positively and negatively associated with T2D, respectively (p=0.03). The -318 C/T and T/T genotypes were more frequent in patients than controls and -318 C/C genotype was shown to be protective for T2D (p=0.003). ACT and GTT Haplotypes were less and more frequent in controls and patients, respectively (p=3.86×10-7 and p=2.29×10-5). Genotypes distribution among T2D patients with and without DPN compared to healthy controls showed significantly lower frequencies for -318 C/C and +49 A/A genotypes and significantly higher frequencies for -318 C/T and T/T genotypes as well. Our findings indicate that CTLA-4 (+49 A/G) and (-318 C/T) genotypes could be considered as genetic risk factors associated with susceptibility or protection for T2D.

  2. Structure and antigenicity analysis of the IgG gene for Nyctereutes procyonoides

    PubMed Central

    Zhao, Cui; Guo, Shuyuan; Pang, Xiaoru; Song, Daozhen; Fu, Shijun

    2015-01-01

    Objective Nyctereutes procyonoides immunoglobulin G (IgG) gene is partially cloned. Material and methods In order to obtain a certain length (966bp) of Nyctereutes procyonoides immunoglobulin G (IgG), two pairs of primers are designed according to the conserved nucleotide sequence of canine (GenBank:AF354265, AF354265, AF354266, AF354267) and mink (GenBank: L07789). Using Bioinformatics technology and Western-blot to analyze antigenicity of Nyctereutes procyonoides IgG-B gene. Results The homology for nucleotide sequence of IgG between Nyctereutes procyonoides and canine (IgG A, IgG B, IgG C, IgG D), mink, Homo sapiens, Oryctolagus cuniculus, Mus musculus, Anas platyrhynchos and gallus were respectively (88.1%, 93.6%, 85.4%, 87.2%), 83.7%, 74.8%, 71.8%, 69.2%, 51.6%, 48.4%. It can be seen that there was high homology of aminoacid sequence between IgG of Nyctereutes procyonoides and IgG (A, B, C, D) of canine. And the serum antibody of Nyctereutes procyonoides had obviously cross-reaction with HRP conjugated rabbit anti-dog IgG, compared with those of canine, oryctolagus cuniculus, mus musculus, mink, gallus. Conclusions We successfully got Nyctereutes procyonoides immuneglobulin G (IgG) gene (Gen- Bank: KM010191). There is the closest ties of consanguinity of IgG exist between Nyctereutes procyonoides and canine among the mammal through the genetic evolution. The detection and treament of canine distemper can be used on Nyctereutes procyonoides. PMID:26648768

  3. 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

  4. [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.

  5. Genomic analysis of isolates from the United Kingdom 2012 pertussis outbreak reveals that vaccine antigen genes are unusually fast evolving.

    PubMed

    Sealey, Katie L; Harris, Simon R; Fry, Norman K; Hurst, Laurence D; Gorringe, Andrew R; Parkhill, Julian; Preston, Andrew

    2015-07-15

    A major outbreak of whooping cough, or pertussis, occurred in 2012 in the United Kingdom (UK), with nearly 10 000 laboratory-confirmed cases and 14 infant deaths attributed to pertussis. A worldwide resurgence of pertussis has been linked to switch to the use of acellular pertussis vaccines and the evolution of Bordetella pertussis away from vaccine-mediated immunity. We have conducted genomic analyses of multiple strains from the UK outbreak. We show that the UK outbreak was polyclonal in nature, caused by multiple distinct but closely related strains. Importantly, we demonstrate that acellular vaccine antigen-encoding genes are evolving at higher rates than other surface protein-encoding genes. This was true even prior to the introduction of pertussis vaccines but has become more pronounced since the introduction of the current acellular vaccines. The fast evolution of vaccine antigen-encoding genes has serious consequences for the ability of current vaccines to continue to control pertussis.

  6. Patterns of human genetic variation inferred from comparative analysis of allelic mutations in blood group antigen genes.

    PubMed

    Patnaik, Santosh Kumar; Blumenfeld, Olga O

    2011-03-01

    Comparative analysis of allelic variation of a gene sheds light on the pattern and process of its diversification at the population level. Gene families for which a large number of allelic forms have been verified by sequencing provide a useful resource for such studies. In this regard, human blood group-encoding genes are unique in that differences of cell surface traits among individuals and populations can be readily detected by serological screening, and correlation between the variant cell surface phenotype and the genotype is, in most cases, unequivocal. Here, we perform a comprehensive analysis of allelic forms, compiled in the Blood Group Antigen Gene Mutation database, of ABO, RHD/CE, GYPA/B/E and FUT1/2 gene families that encode the ABO, RH, MNS, and H/h blood group system antigens, respectively. These genes are excellent illustrative examples showing distinct mutational patterns among the alleles, and leading to speculation on how their origin may have been driven by recurrent but different molecular mechanisms. We illustrate how alignment of alleles of a gene may provide an additional insight into the DNA variation process and its pathways, and how this approach may serve to catalog alleles of a gene, simplifying the task and content of mutation databases.

  7. In Vitro Generation of Human NK cells Expressing Chimeric Antigen Receptor through Differentiation of Gene-Modified Hematopoietic Stem Cells

    PubMed Central

    Lowe, Emily; Truscott, Laurel C.; De Oliveira, Satiro N.

    2016-01-01

    Summary NK cells represent a very promising source for adoptive cellular approaches for cancer immunotherapy, and extensive research has been conducted, including clinical trials. Gene modification of NK cells can direct their specificity and enhance their function, but the efficiency of gene transfer techniques is very limited. Here we describe two protocols designed to generate mature human NK cells from gene-modified hematopoietic stem cells. These protocols use chimeric antigen receptor as the transgene, but could potentially be modified for the expression any particular transgene in human NK cells. PMID:27177671

  8. Analysis of the role of human leukocyte antigen class-I genes to understand the etiopathology of schizophrenia

    PubMed Central

    Singh, Bisu; Banerjee, Sikta; Bera, Nirmal K.; Nayak, Chitta R.; Chaudhuri, Tapas K.

    2008-01-01

    Background: Schizophrenia is the paradigmatic illness of psychiatry. The involvement of immunological and immunopathological mechanisms in the etiopathogenesis of schizophrenia has been a matter of research, with recently increasing effort. Aims: In this study, we investigated the incidence of human leukocyte antigen (HLA) Class I antigens to understand the role of HLA genes in schizophrenia. Materials and Methods: India born schizophrenic patients in and around Siliguri who attended outpatient department (OPD) of Department of Psychiatry, North Bengal Medical College and Hospital were considered for the present study. After the longitudinal follow up, 50 patients were enrolled for the study. The same number of age, sex and ethnically matched healthy subjects were considered as control. Low resolution polymerase chain reaction-sequence specific primer method was applied for typing the HLA antigens. Statistics: The phenotype frequencies were calculated by direct count. χ2 test was done to compare the frequency of each antigen among the patients and control group and it was followed by Fisher's exact test. Relative risk was estimated by using Haldane's method. Results: The result showed that some of the HLA antigens are associated with the schizophrenia and significant increase were observed for HLA A*03 antigen along with the significant decrease for HLA A*25, A*31 and HLA B*51. Conclusions: The study provides the evidence for the possible existence of susceptibility locus for schizophrenia within the HLA region. This preliminary observation may help to understand the etiological basis of this disorder and the study may further strengthen the HLA antigens as the marker for schizophrenia. PMID:19742184

  9. 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.

  10. 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.

  11. The enterobacterial common antigen-like gene cluster of Haemophilus ducreyi contributes to virulence in humans.

    PubMed

    Banks, Keith E; Fortney, Kate R; Baker, Beth; Billings, Steven D; Katz, Barry P; Munson, Robert S; Spinola, Stanley M

    2008-06-01

    Haemophilus ducreyi 35000HP contains a cluster of homologues of genes required for the synthesis of enterobacterial common antigen (ECA), suggesting that H. ducreyi may express a putative ECA-like glycoconjugate. WecA initiates the synthesis of ECA by transferring N-acetylglucosamine to undecaprenyl-P, to form lipid I. A wecA mutant (35000HPwecA) was constructed, and 5 volunteers were inoculated at 3 sites with fixed doses of 35000HP on one arm and at 3 sites with varying doses of 35000HPwecA on the other arm. 35000HPwecA caused pustules to form at 3 sites inoculated with a dose 2.5-fold higher than that of 35000HP. However, at sites inoculated with similar doses of 35000HP and 35000HPwecA, pustules developed at 46.7% (95% confidence interval [CI], 23.3%-70.0%) of 15 parent-strain sites and at 8.3% (95% CI, 0.01%-23.6%) of 12 mutant-strain sites (P = .013). Thus, the expression of wecA contributes to the ability of H. ducreyi to cause pustules in humans.

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

    PubMed

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

    2016-11-01

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

  13. 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

  14. Human leukocyte antigen-DRB1 class II genes in Mexican Amerindian Mazahuas: genes and languages do not correlate.

    PubMed

    Arnaiz-Villena, Antonio; Abd-El-Fatah, Sedeka; Granados-Silvestre, María Angeles; Parga-Lozano, Carlos; Gómez-Prieto, Pablo; Rey, Diego; Areces, Cristina; Peñaranda, Patricia; Menjívar, Martha; Rodríguez-Pérez, José Manuel; Granados, Julio; Vargas-Alarcón, Gilberto

    2011-01-01

    The major histocompatibility complex genes are located on the short arm of the human sixth chromosome; they are highly polymorphic and therefore have been very advantageous in population genetic studies. A Mazahua group established in North Mexico State and also in nearby Michoacan state in the rainy mountain highlands (Mexico) was studied for their human leukocyte antigen (HLA)-DRB1 alleles. The relationship with other Amerindians and worldwide populations was studied by using 14,996 chromosomes from 75 different populations and calculating neighbor-joining dendrograms and correspondence multidimensional values. Five principal HLA allele frequencies were found in our group: DRB1*0802 (the most frequent one in this population), DRB1*0407, DRB1*0403, DRB1*0101, and DRB1*1406. Both genetic distances and correspondence analyses clearly show that our Mazahua group is genetically close to some of the most ancient groups living in Mexico (Mayos, Zapotecans, Tennek) and South American Amerindians. Amerindians remain as a group apart from the rest of the world. The results analyzing the HLA-DR locus suggest that Mazahua language (Otomangue) does not correlate with those of the most closely HLA-correlated ethnic groups. The present data may be useful for future transplantation programs, HLA and disease diagnosis, and pharmacogenetic studies.

  15. Role of the rfe gene in the synthesis of the O8 antigen in Escherichia coli K-12.

    PubMed Central

    Rick, P D; Hubbard, G L; Barr, K

    1994-01-01

    The Escherichia coli O8 antigen is a mannan composed of the trisaccharide repeat unit -->3)-alpha-Man-(1-->2)-alpha-Man-(1-->2)-alpha-Man-(1--> (K. Reske and K. Jann, Eur. J. Biochem. 67:53-56, 1972), and synthesis of the O8 antigen is rfe dependent (G. Schmidt, H. Mayer, and P. H. Mäkelä, J. Bacteriol. 127:755-762, 1976). The rfe gene has recently been identified as encoding a tunicamycin-sensitive UDP-GlcNAc:undecaprenylphosphate GlcNAc-1-phosphate transferase (U. Meier-Dieter, K. Barr, R. Starman, L. Hatch, and P. D. Rick, J. Biol. Chem. 267:746-753, 1992). However, the role of rfe in O8 side chain synthesis is not understood. Thus, the role of the rfe gene in the synthesis of the O8 antigen was investigated in an rfbO8+ (rfb genes encoding O8 antigen) derivative of E. coli K-12 mutant possessing a defective phosphoglucose isomerase (pgi). The in vivo synthesis of O8 side chains was inhibited by the antibiotic tunicamycin. In addition, putative lipid carrier-linked O8 side chains accumulated in vivo when lipopolysaccharide outer core synthesis was precluded by growing cells in the absence of exogenously supplied glucose. The lipid carrier-linked O8 antigen was extracted from cells and treated with mild acid in order to release free O8 side chains. The water-soluble O8 side chains were then purified by affinity chromatography using Sepharose-bound concanavalin A. Characterization of the affinity-purified O8 side chains revealed the occurrence of glucosamine in the reducing terminal position of the polysaccharide chains. The data presented suggest that GlcNAc-pyrophosphorylundecaprenol functions as the acceptor of mannose residues for the in vivo synthesis of O8 side chains in E. coli K-12. Images PMID:7514591

  16. 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.

  17. Efficient nonviral Sleeping Beauty transposon-based TCR gene transfer to peripheral blood lymphocytes confers antigen-specific antitumor reactivity

    PubMed Central

    Peng, PD; Cohen, CJ; Yang, S; Hsu, C; Jones, S; Zhao, Y; Zheng, Z; Rosenberg, SA; Morgan, RA

    2012-01-01

    Genetically engineered lymphocytes hold promise for the treatment of genetic disease, viral infections and cancer. However, current methods for genetic transduction of peripheral blood lymphocytes rely on viral vectors, which are hindered by production and safety-related problems. In this study, we demonstrated an efficient novel nonviral platform for gene transfer to lymphocytes. The Sleeping Beauty transposon-mediated approach allowed for long-term stable expression of transgenes at ~50% efficiency. Utilizing transposon constructs expressing tumor antigen-specific T-cell receptor genes targeting p53 and MART-1, we demonstrated sustained expression and functional reactivity of transposon-engineered lymphocytes on encountering target antigen presented on tumor cells. We found that transposon- and retroviral-modified lymphocytes had comparable transgene expression and phenotypic function. These results demonstrate the promise of nonviral ex vivo genetic modification of autologous lymphocytes for the treatment of cancer and immunologic disease. PMID:19494842

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

    DTIC Science & Technology

    1988-10-31

    and the Administrative Practices Suppliments, as indicated in the Memorandum of Understanding and Agreement, approved by the Institutional Biosafety...00 0 Cloning and Expression of Genes for Dengue Virus (Type-2 Encoded-Antigens for Rapid ODiagnosis and Vaccine DevelopmentN| ANNUAL PROGRESS REPORT...CI DOD DISTRIBUTION STATEMENT Approved for Public Release; distribution unlimited The findings in this report are not to be construed as an official

  19. Identification of Actinobacillus pleuropneumoniae Genes Preferentially Expressed During Infection Using In Vivo-Induced Antigen Technology (IVIAT).

    PubMed

    Zhang, Fei; Zhang, Yangyi; Wen, Xintian; Huang, Xiaobo; Wen, Yiping; Wu, Rui; Yan, Qigui; Huang, Yong; Ma, Xiaoping; Zhao, Qin; Cao, Sanjie

    2015-10-01

    Porcine pleuropneumonia is an infectious disease caused by Actinobacillus pleuropneumoniae. The identification of A. pleuropneumoniae genes, specially expressed in vivo, is a useful tool to reveal the mechanism of infection. IVIAT was used in this work to identify antigens expressed in vivo during A. pleuropneumoniae infection, using sera from individuals with chronic porcine pleuropneumonia. Sequencing of DNA inserts from positive clones showed 11 open reading frames with high homology to A. pleuropneumoniae genes. Based on sequence analysis, proteins encoded by these genes were involved in metabolism, replication, transcription regulation, and signal transduction. Moreover, three function-unknown proteins were also indentified in this work. Expression analysis using quantitative real-time PCR showed that most of the genes tested were up-regulated in vivo relative to their expression levels in vitro. IVI (in vivoinduced) genes that were amplified by PCR in different A. pleuropneumoniae strains showed that these genes could be detected in almost all of the strains. It is demonstrated that the identified IVI antigen may have important roles in the infection of A. pleuropneumoniae.

  20. Variant surface antigens of malaria parasites: functional and evolutionary insights from comparative gene family classification and analysis

    PubMed Central

    2013-01-01

    Background Plasmodium parasites, the causative agents of malaria, express many variant antigens on cell surfaces. Variant surface antigens (VSAs) are typically organized into large subtelomeric gene families that play critical roles in virulence and immune evasion. Many important aspects of VSA function and evolution remain obscure, impeding our understanding of virulence mechanisms and vaccine development. To gain further insights into VSA function and evolution, we comparatively classified and examined known VSA gene families across seven Plasmodium species. Results We identified a set of ultra-conserved orthologs within the largest Plasmodium gene family pir, which should be considered as high-priority targets for experimental functional characterization and vaccine development. Furthermore, we predict a lipid-binding domain in erythrocyte surface-expressed PYST-A proteins, suggesting a role of this second largest rodent parasite gene family in host cholesterol salvage. Additionally, it was found that PfMC-2TM proteins carry a novel and putative functional domain named MC-TYR, which is conserved in other P. falciparum gene families and rodent parasites. Finally, we present new conclusive evidence that the major Plasmodium VSAs PfEMP1, SICAvar, and SURFIN are evolutionarily linked through a modular and structurally conserved intracellular domain. Conclusion Our comparative analysis of Plasmodium VSA gene families revealed important functional and evolutionary insights, which can now serve as starting points for further experimental studies. PMID:23805789

  1. Transcript origin analysis identifies antigen-presenting cells as primary targets of socially regulated gene expression in leukocytes

    PubMed Central

    Cole, Steven W.; Hawkley, Louise C.; Arevalo, Jesusa M. G.; Cacioppo, John T.

    2011-01-01

    To clarify the biological rationale for social regulation of gene expression, this study sought to identify the specific immune cell types that are transcriptionally sensitive to subjective social isolation (loneliness). Using reference distributions for the expression of each human gene in each major leukocyte subtype, we mapped the cellular origin of transcripts found to be differentially expressed in the circulating immune cells from chronically lonely individuals. Loneliness-associated genes derived primarily from plasmacytoid dendritic cells, monocytes, and, to a lesser extent, B lymphocytes. Those dynamics reflected per-cell changes in the expression of inducible genes and related more strongly to the subjective experience of loneliness than to objective social network size. Evolutionarily ancient myeloid antigen-presenting cells appear to have evolved a transcriptional sensitivity to socioenvironmental conditions that may allow them to shift basal gene expression profiles to counter the changing microbial threats associated with hostile vs. affine social conditions. PMID:21300872

  2. Inhibition of antigen-specific T lymphocyte activation by structurally related Ir gene-controlled polymers. II. Competitive inhibition of I-E- restricted, antigen-specific T cell responses

    PubMed Central

    1984-01-01

    Our previous studies have defined a highly specific competitive inhibition between a pair of structurally related antigens (GT and GAT) for antigen presentation by accessory cells. The present report investigates this phenomenon in a second antigenic system, which is controlled by a distinct Ir gene product. Two GL phi-specific, I-Ed- restricted, interleukin 2-producing T cell hybridomas were constructed. The antigenic fine specificity of these two hybrid clones was distinct. One hybrid reacted solely with GL phi while the second cross-reacted with GLleu and GLT. These latter two copolymers, as well as the antigen GL, were found to inhibit the GL phi response of the non-cross-reactive hybrid. The structurally related antigen G phi was not inhibitory for this clone's response. The cross-reactive GL phi hybrid could also be inhibited, but, in this case, G phi and not GL caused the inhibition. Reciprocal inhibitions could be demonstrated between these and other hybrids (e.g., GAT responsive), indicating a very high degree of specificity to the inhibition. The inhibition caused by the various copolymers was reversible by increasing the concentration of GL phi, This effect was localized to the antigen-presenting cell and not the T cell hybridoma. Functionally, this competition did not appear to be for antigen uptake or general antigen processing. These findings generalize the phenomenon of antigen competition to a second antigen system in the context of a second Ia molecule. The possible mechanisms accounting for the complex pattern of specificities in this system are discussed. PMID:6210339

  3. 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.

  4. Molecular cloning and sequence analysis of the Sta58 major antigen gene of Rickettsia tsutsugamushi: sequence homology and antigenic comparison of Sta58 to the 60-kilodalton family of stress proteins.

    PubMed Central

    Stover, C K; Marana, D P; Dasch, G A; Oaks, E V

    1990-01-01

    The scrub typhus 58-kilodalton (kDa) antigen (Sta58) of Rickettsia tsutsugamushi is a major protein antigen often recognized by humans infected with scrub typhus rickettsiae. A 2.9-kilobase HindIII fragment containing a complete sta58 gene was cloned in Escherichia coli and found to express the entire Sta58 antigen and a smaller protein with an apparent molecular mass of 11 kDa (Stp11). DNA sequence analysis of the 2.9-kilobase HindIII fragment revealed two adjacent open reading frames encoding proteins of 11 (Stp11) and 60 (Sta58) kDa. Comparisons of deduced amino acid sequences disclosed a high degree of homology between the R. tsutsugamushi proteins Stp11 and Sta58 and the E. coli proteins GroES and GroEL, respectively, and the family of primordial heat shock proteins designated Hsp10 Hsp60. Although the sequence homology between the Sta58 antigen and the Hsp60 protein family is striking, the Sta58 protein appeared to be antigenically distinct among a sample of other bacterial Hsp60 homologs, including the typhus group of rickettsiae. The antigenic uniqueness of the Sta58 antigen indicates that this protein may be a potentially protective antigen and a useful diagnostic reagent for scrub typhus fever. Images PMID:2108930

  5. Polymorphisms in prostate stem cell antigen gene rs2294008 increase gastric cancer risk in Chinese.

    PubMed

    Zeng, Zhirong; Wu, Xiaoqin; Chen, Fanggen; Yu, Jun; Xue, Ling; Hao, Yuantao; Wang, Yiming; Chen, Minhu; Sung, Joseph J Y; Hu, Pinjin

    2011-05-01

    A recent genome-wide study identified a strong association between polymorphisms in the prostate stem cell antigen (PSCA) gene and the risk of diffuse-type of gastric cancer in Japanese and Korean population. In this case-control study, we aimed to investigate the possible association between PSCA rs2294008 C/T with clinicopathological features and the prognosis of gastric cancer in a Southern Chinese population. Genotypes of 460 gastric cancer patients and 549 controls were determined by PCR-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing. We found that individuals with at least one copy of the rs2294008T allele (CT or TT genotype) had an increased risk for gastric cancer compared with CC genotype (OR = 1.42, 95% CI = 1.10-1.82, P = 0.006). Further stratification analyses indicated that the effect of PSCA rs2294008T carriers was noteworthy in intestinal type (OR = 1.55, 95% CI = 1.18-2.04, P = 0.002), poorly differentiated (OR = 1.59, 95% CI = 1.19-2.13, P = 0.002), noncardia (OR = 1.55, 95% CI = 1.17-2.04, P = 0.002) subtypes of gastric cancer. Cox proportional hazards analyses demonstrated that TT genotype (HR = 2.12, 95% CI = 1.22-3.69, P = 0.008) as well as TNM staging were prognostic factors of gastric cancer patients. In conclusion, The T allele of PSCA rs2294008 is associated with increased risk of gastric cancer, especially intestinal type, poorly differentiated, early onset, and noncardia gastric cancer in Chinese population. TNM staging and TT genotype might be involved in the prognosis of gastric cancer patients.

  6. 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.

  7. Transcriptionally targeted in vivo gene therapy for carcinoembrionic antigen-producing adenocarcinoma.

    PubMed

    Konishi, F; Maeda, H; Yamanishi, Y; Hiyama, K; Ishioka, S; Yamakido, M

    1999-09-01

    Inoperable adenocarcinoma in colon or lung shows resistance to conventional anti-cancer therapy. For these cancers, the feasibility of transcriptionally targeted killing of carcinoembryonic antigen (CEA)-producing adenocarcinoma cells was investigated. Adenovirus vectors carrying a CEA promoter to express E. coli lacZ (AdCEALacZ) or herpes simplex thymidine kinase (AdCEATK) were made and their in vitro and in vivo tumoricidal effects on CEA-producing or non-producing colon and lung cancer cells were evaluated. In vitro infection with AdCEALacZ showed significantly higher CEA promoter-driven lacZ expression in CEA-producing adenocarcinoma cells including VMRC-LCD and LoVo than in CEA-non-producing cells. AdCEATK-infected LoVo showed higher sensitivity to ganciclovir than control vector-infected LoVo or AdCEATK-infected HeLa both in vitro and in subcutaneously implanted tumors of nude mice. Moreover, total tumor elimination in vivo was achieved by either pre-infection of as few as 30% of cells comprising tumors or by direct in vivo injection of AdCEATK to pre-established LoVo tumors. In addition, CEA promoter-driven lacZ expression in LoVo cells was enhanced by the addition of interleukin-6 (IL-6) in vitro. These results provide a rationale for CEA-promoter-driven, adenovirus-mediated gene therapy for CEA-producing adenocarcinomas in colon and lung with reduced toxicity to normal cells.

  8. 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

  9. Structural Analysis and Biosynthesis Gene Cluster of an Antigenic Glycopeptidolipid from Mycobacterium intracellulare▿ †

    PubMed Central

    Fujiwara, Nagatoshi; Nakata, Noboru; Naka, Takashi; Yano, Ikuya; Doe, Matsumi; Chatterjee, Delphi; McNeil, Michael; Brennan, Patrick J.; Kobayashi, Kazuo; Makino, Masahiko; Matsumoto, Sohkichi; Ogura, Hisashi; Maeda, Shinji

    2008-01-01

    Mycobacterium avium-Mycobacterium intracellulare complex (MAC) is the most common isolate of nontuberculous mycobacteria and causes pulmonary and extrapulmonary diseases. MAC species can be grouped into 31 serotypes by the epitopic oligosaccharide structure of the species-specific glycopeptidolipid (GPL) antigen. The GPL consists of a serotype-common fatty acyl peptide core with 3,4-di-O-methyl-rhamnose at the terminal alaninol and a 6-deoxy-talose at the allo-threonine and serotype-specific oligosaccharides extending from the 6-deoxy-talose. Although the complete structures of 15 serotype-specific GPLs have been defined, the serotype 16-specific GPL structure has not yet been elucidated. In this study, the chemical structure of the serotype 16 GPL derived from M. intracellulare was determined by using chromatography, mass spectrometry, and nuclear magnetic resonance analyses. The result indicates that the terminal carbohydrate epitope of the oligosaccharide is a novel N-acyl-dideoxy-hexose. By the combined linkage analysis, the oligosaccharide structure of serotype 16 GPL was determined to be 3-2′-methyl-3′-hydroxy-4′-methoxy-pentanoyl-amido-3,6-dideoxy-β-hexose-(1→3)-4-O-methyl-α-l-rhamnose-(1→3)-α-l-rhamnose-(1→3)-α-l-rhamnose-(1→2)-6-deoxy-α-l-talose. Next, the 22.9-kb serotype 16-specific gene cluster involved in the glycosylation of oligosaccharide was isolated and sequenced. The cluster contained 17 open reading frames (ORFs). Based on the similarity of the deduced amino acid sequences, it was assumed that the ORF functions include encoding three glycosyltransferases, an acyltransferase, an aminotransferase, and a methyltransferase. An M. avium serotype 1 strain was transformed with cosmid clone no. 253 containing gtfB-drrC of M. intracellulare serotype 16, and the transformant produced serotype 16 GPL. Together, the ORFs of this serotype 16-specific gene cluster are responsible for the biosynthesis of serotype 16 GPL. PMID:18326570

  10. Analysis of Dengue Virus Enhancing Epitopes Using Peptide Antigens Derived from the Envelope Glycoprotein Gene Sequence

    DTIC Science & Technology

    1990-11-27

    AD________ AD-A230 976 ARMY PROJECT NO: 89PP9961 TITLE: ANALYSIS OF DENGUE VIRUS ENHANCING EPITOPES USING PEPTIDE ANTIGENS DERIVED FROM THE ENVELOPE...INO. INO r CCESSION NO I1I TITLE (Include Security Classification) Analysis of Dengue Virus Enhancing Epitopes Using Peptide Antigens Derived From the...necessary and identify by block number) Antibody-dependent enhancement (ADE) ot dengue (DEN) virus infection in human mononuclear cells in vitro has been

  11. 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.

  12. 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.

  13. Evolutionary conserved gene co-expression drives generation of self-antigen diversity in medullary thymic epithelial cells.

    PubMed

    Rattay, Kristin; Meyer, Hannah Verena; Herrmann, Carl; Brors, Benedikt; Kyewski, Bruno

    2016-02-01

    Promiscuous expression of a plethora of tissue-restricted antigens (TRAs) in medullary thymic epithelial cells (mTECs) is essential for central tolerance. This promiscuous gene expression (pGE) is characterized by inclusion of a broad range of TRAs and by its mosaic expression patterns, i.e. each antigen is only expressed in 1-3% of mTECs. It is currently unclear to which extent random and/or deterministic mechanisms are involved in the regulation of pGE. In order to address this issue, we deconstructed the transcriptional heterogeneity in mTEC to minor subsets expressing a particular TRA. We identified six delineable co-expression groups in mouse mTECs. These co-expression groups displayed a variable degree of mutual overlap and mapped to different stages of mTEC development. Co-expressed genes showed chromosomal preference and clustered within delimited genomic regions. Moreover, co-expression groups in mice and humans selected by a pair of orthologous genes preferentially co-expressed sets of orthologous genes attesting to the species conservation of pGE between mouse and human. Furthermore, co-expressed genes were enriched for specific transcription factor binding motifs concomitant with up-regulation of the corresponding transcription factors, implicating additional factors in the regulation of pGE besides the Autoimmune Regulator (Aire). Thus promiscuous transcription of self-antigens in mTECs entails a highly coordinated process, which is evolutionary strictly conserved between species. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. The Escherichia coli Serogroup O1 and O2 Lipopolysaccharides Are Encoded by Multiple O-antigen Gene Clusters.

    PubMed

    Delannoy, Sabine; Beutin, Lothar; Mariani-Kurkdjian, Patricia; Fleiss, Aubin; Bonacorsi, Stéphane; Fach, Patrick

    2017-01-01

    Escherichia coli strains belonging to serogroups O1 and O2 are frequently associated with human infections, especially extra-intestinal infections such as bloodstream infections or urinary tract infections. These strains can be associated with a large array of flagellar antigens. Because of their frequency and clinical importance, a reliable detection of E. coli O1 and O2 strains and also the frequently associated K1 capsule is important for diagnosis and source attribution of E. coli infections in humans and animals. By sequencing the O-antigen clusters of various O1 and O2 strains we showed that the serogroups O1 and O2 are encoded by different sets of O-antigen encoding genes and identified potentially new O-groups. We developed qPCR-assays to detect the various O1 and O2 variants and the K1-encoding gene. These qPCR assays proved to be 100% sensitive and 100% specific and could be valuable tools for the investigations of zoonotic and food-borne infection of humans with O1 and O2 extra-intestinal (ExPEC) or Shiga toxin-producing E. coli (STEC) strains.

  15. The Escherichia coli Serogroup O1 and O2 Lipopolysaccharides Are Encoded by Multiple O-antigen Gene Clusters

    PubMed Central

    Delannoy, Sabine; Beutin, Lothar; Mariani-Kurkdjian, Patricia; Fleiss, Aubin; Bonacorsi, Stéphane; Fach, Patrick

    2017-01-01

    Escherichia coli strains belonging to serogroups O1 and O2 are frequently associated with human infections, especially extra-intestinal infections such as bloodstream infections or urinary tract infections. These strains can be associated with a large array of flagellar antigens. Because of their frequency and clinical importance, a reliable detection of E. coli O1 and O2 strains and also the frequently associated K1 capsule is important for diagnosis and source attribution of E. coli infections in humans and animals. By sequencing the O-antigen clusters of various O1 and O2 strains we showed that the serogroups O1 and O2 are encoded by different sets of O-antigen encoding genes and identified potentially new O-groups. We developed qPCR-assays to detect the various O1 and O2 variants and the K1-encoding gene. These qPCR assays proved to be 100% sensitive and 100% specific and could be valuable tools for the investigations of zoonotic and food-borne infection of humans with O1 and O2 extra-intestinal (ExPEC) or Shiga toxin-producing E. coli (STEC) strains. PMID:28224115

  16. T-cell receptor gene therapy targeting melanoma-associated antigen-A4 inhibits human tumor growth in non-obese diabetic/SCID/γcnull mice.

    PubMed

    Shirakura, Yoshitaka; Mizuno, Yukari; Wang, Linan; Imai, Naoko; Amaike, Chisaki; Sato, Eiichi; Ito, Mamoru; Nukaya, Ikuei; Mineno, Junichi; Takesako, Kazutoh; Ikeda, Hiroaki; Shiku, Hiroshi

    2012-01-01

    Adoptive cell therapy with lymphocytes that have been genetically engineered to express tumor-reactive T-cell receptors (TCR) is a promising approach for cancer immunotherapy. We have been exploring the development of TCR gene therapy targeting cancer/testis antigens, including melanoma-associated antigen (MAGE) family antigens, that are ideal targets for adoptive T-cell therapy. The efficacy of TCR gene therapy targeting MAGE family antigens, however, has not yet been evaluated in vivo. Here, we demonstrate the in vivo antitumor activity in immunodeficient non-obese diabetic/SCID/γc(null) (NOG) mice of human lymphocytes genetically engineered to express TCR specific for the MAGE-A4 antigen. Polyclonal T cells derived from human peripheral blood mononuclear cells were transduced with the αβ TCR genes specific for MAGE-A4, then adoptively transferred into NOG mice inoculated with MAGE-A4 expressing human tumor cell lines. The transferred T cells maintained their effector function in vivo, infiltrated into tumors, and inhibited tumor growth in an antigen-specific manner. The combination of adoptive cell therapy with antigen peptide vaccination enhanced antitumor activity, with improved multifunctionality of the transferred cells. These data suggest that TCR gene therapy with MAGE-A4-specific TCR is a promising strategy to treat patients with MAGE-A4-expressing tumors; in addition, the acquisition of multifunctionality in vivo is an important factor to predict the quality of the T-cell response during adoptive therapy with human lymphocytes.

  17. Tandem insertion sequence-like elements define the expression site for variable antigen genes of Borrelia hermsii.

    PubMed Central

    Barbour, A G; Carter, C J; Burman, N; Freitag, C S; Garon, C F; Bergström, S

    1991-01-01

    The spirochete Borrelia hermsii avoids the immune response of its mammalian host through multiphasic antigenic variation. Serotype specificity is determined by variable antigens, Vmp proteins, in the outer membrane. Through nonreciprocal recombination between linear plasmids, a formerly silent vmp gene replaces another vmp gene downstream from a common expression site. To further characterize this activating site, we determined the nucleotide sequence of 6.9 kb of the common upstream expression region of strain HS1 of B. hermsii. Preceding the vmp gene promoter and a poly(dT.dA) run were three imperfectly repeated segments of 2 kb. Each of the 2-kb segments contained 1-kb elements with inverted repeats of approximately 0.2 kb each at their termini. The potential of the 1-kb elements to form stem-and-loop structures was demonstrated by heteroduplex analysis. There was no evidence of the presence of the elements elsewhere in the genome of B. hermsii. One or more of these elements may confer the unidirectionality that characterizes vmp gene switches. Images PMID:1987053

  18. In vitro generation of tumor specific T cells that recognize a shared antigen of AML: molecular characterization of TCR genes.

    PubMed

    Coppage, Myra; Belanger, Todd; Zauderer, Maurice; Sahasrabudhe, Deepak

    2007-02-01

    The identification of immunologically relevant tumor antigens is hampered by the difficulty of generating tumor-specific cytotoxic T cells (CTL). We present data demonstrating in vitro induction of autologous acute myelogenous leukemia (AML)-specific CTL. The specific T cell receptor has been identified and cloned. The CTL demonstrated specific lysis to autologous tumor blasts, but not to autologous BLCL or the NK-sensitive target K562. The clone secreted GM-CSF, TNFa, and IFNg when stimulated with AML blasts from 3 of 11 patients or cell lines tested, but not with K562 or autologous B-LCL. These three AML samples share a single HLA Class I antigen, HLA-A24. The T cell receptor genes identified by molecular methods are Vbeta7.9-J2.3-Cbeta2 and Valpha17-J49-Calpha.

  19. 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

  20. 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

  1. 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.

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

    PubMed Central

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

    1995-01-01

    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

  3. T-cell tolerance toward a transgenic beta-cell antigen and transcription of endogenous pancreatic genes in thymus.

    PubMed Central

    Jolicoeur, C; Hanahan, D; Smith, K M

    1994-01-01

    Transgenic mice expressing T antigen (Tag) in pancreatic beta cells establish systemic tolerance toward this self-protein. The self-tolerance in two families of rat insulin promoter (RIP)-Tag mice, expressing different levels of Tag protein, has been characterized. These mice have impaired antibody responses to Tag, show diminished Tag-specific T-cell proliferation, and evidence an inability to generate Tag-specific cytotoxic T cells. The existence of systemic tolerance toward a beta-cell-specific protein motivated examination of transgene expression in the thymus. Indeed, low levels of Tag mRNA were detected intrathymically. Remarkably, this expression is a valid property of the insulin gene regulatory region, since insulin RNA was also expressed in the thymus of nontransgenic mice. RNA for other pancreatic genes was also detected in the thymus, thus raising the possibility that many tissue-specific genes could be expressed intrathymically during immunological development and induction of self-tolerance. These results raise important questions for future research into the role of the thymus in tolerance induction toward so-called tissue-specific antigens. Images PMID:8022837

  4. A mutated beta-catenin gene encodes a melanoma-specific antigen recognized by tumor infiltrating lymphocytes

    PubMed Central

    1996-01-01

    A number of antigens recognized by tumor-reactive T cells have recently been identified. The antigens identified in mouse model systems appear, with one exception, to represent the products of mutated genes. In contrast, most of the antigens recognized by human tumor-reactive T cells reported to date appear to represent the products of non-mutated genes. Here we report the isolation of a cDNA clone encoding beta- catenin, which was shown to be recognized by the tumor-infiltrating lymphocyte (TIL) 1290, a HLA-A24 restricted melanoma-specific CTL line from patient 888. The cDNA clone, which was isolated from the autologous melanoma cDNA library, differed by a single base pair from the published beta-catenin sequence, resulting in a change from a serine to a phenylalanine residue at position 37. Normal tissues from this patient did not express the altered sequence, nor did 12 allogeneic melanomas, indicating that this represented a unique mutation in this patient's melanoma. A peptide corresponding to the sequence between amino acids 29 and 37 of the mutant gene product was identified as the T cell epitope recognized by TIL 1290. The observation that HLA-A24 binding peptides contain an aromatic or hydrophobic residue at position 9 suggested that the change at position 37 may have generated a peptide (SYLDSGIHF) which was capable of binding to HLA-A24, and a competitive binding assay confirmed this hypothesis. The beta-catenin protein has been shown previously to be involved in cell adhesion mediated through the cadherin family of cell surface adhesion molecules. The high frequency of mutations found in members of cellular adhesion complexes in a variety of cancers suggests that these molecules may play a role in development of the malignant phenotype. PMID:8642260

  5. Prostate stem cell antigen gene TT genotype and development of intestinal metaplasia in Helicobacter pylori infection

    PubMed Central

    Uotani, Takahiro; Sugimoto, Mitsushige; Ichikawa, Hitomi; Tanaka, Shingo; Nagashima, Hiroyuki; Uchida, Tomohisa; Graham, David Y.; Yamaoka, Yoshio

    2016-01-01

    Aim Gastric cancer is etiologically related to interactions between Helicobacter pylori infection, environmental, and host factors. Gastric carcinoma is associated with a cascade of increasing atrophic gastric mucosal damage. Prostate stem cell antigen polymorphisms have been associated with an increased risk of gastric cancer. Here, we examined the interaction between prostate stem cell antigen polymorphisms and H. pylori in the progression of H. pylori gastritis. Methods Prostate stem cell antigen polymorphisms (TT, TC and CC) among H. pylori infected and uninfected Bhutanese were compared with the severity of H. pylori gastritis (neutrophils, monocytes, atrophy scores, H. pylori density, and the presence and extent of intestinal metaplasia) using the updated Sydney system. Results Biopsies from 339 patients were included. The proportion of biopsies with intestinal metaplasia was also significantly (P<0.05) greater among those with the TT genotype than with either the CT or CC genotype. Despite no significant differences in inflammation or H. pylori density scores, the scores for the premalignant condition, intestinal metaplasia in both the gastric corpus and antrum, among H. pylori infected with the TT genotype was significantly (P <0.05) greater than C allele carriers. Conclusions Prostate stem cell antigen TT genotype was associated with more than a 3-fold increase in the prevalence and extent of gastric mucosal intestinal metaplasia compared to C allele carriers among H. pylori infected Bhutanese. PMID:26706772

  6. Identification of the size and antigenic determinants of the human C4 gene by a polymerase chain-reaction-based amplification method.

    PubMed

    Lee, Hsien-Hsiung; Chang, Shwu-Fen; Tseng, Yung-Te; Lee, Yann-Jinn

    2006-10-01

    The human C4 complement components of the C4 locus are encoded by two genes, C4A and C4B, located on chromosome 6p21.3 of the major histocompatibility complex of the human leukocyte antigen class III region. The size difference between the two genes is due to the presence of HERV-K (C4), an endogenous retroviral sequence (6.7 kb long), in intron 9 of the long C4 gene. Whether the C4 is the long (L) or short (S) gene was determined by the Southern blot method, and the antigenic determinants in residues 1,054-1,106 of Rodgers and Chido were generally identified by immunoblot analysis. Herein, we explore a polymerase chain reaction (PCR) amplification method for directly determining the size of C4 loci adjacent to the respective RP1 and RP2 genes and antigenic determinants by DNA sequencing. From the results of this study, we concluded that all of the C4 genes adjacent to the RP1 gene presented the long gene. In addition, 47% of the C4 genes adjacent to the RP2 gene were the short gene and 53% were the long gene. This result was consistent with that of the Southern blot analysis. The PCR method is practical for identifying the C4 genotype and can be used to detect other polymorphisms among variants of C4 genes.

  7. Structure and gene cluster of the O-antigen of Escherichia coli O156 containing a pyruvic acid acetal.

    PubMed

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

    2016-07-22

    The lipopolysaccharide of Escherichia coli O156 was degraded under mild acidic and alkaline conditions and the resulting polysaccharides were studied by sugar analysis and (1)H and (13)C NMR spectroscopy. The following structure of the pentasaccharide repeating unit of the O-polysaccharide was established: where Rpyr indicates R-configurated pyruvic acid acetal. Minor O-acetyl groups also were present and tentatively localized on the Gal residues. The gene cluster for biosynthesis of the O-antigen of E. coli O156 was analyzed and shown to be consistent with the O-polysaccharide structure.

  8. [Effective agrobacterium-mediated transformation of chicory (Cichorium intybus L.) using vector with gene of tuberculosis antigene ESAT6].

    PubMed

    Matveeva, N A; Vasilenko, M Iu; Shakhovskiĭ, A M; Bannikova, M A; Kvasko, O Iu; Kuchuk, N V

    2011-01-01

    The conditions of high efficient chicory transformation with Mycobacterium tuberculosis antigene ESAT6 have been determined. Transformation frequency was up to 86% when the cotyledons were cultivated within 3 days without cefotaxime and then 1 day without kanamycine. DNA PCR-analysis has shown the presence both of selective nptII and target esxA genes in all analysed plants. At the same time RT-PCR has shown the presence of nptII transcripts for eight analysed lines and esxA transcripts for only five analysed lines.

  9. Use of Conversion Adaptors to Clone Antigen Genes in Lambda gt11

    DTIC Science & Technology

    1987-01-01

    80*C for fu- estimated by comparison to molecular ture use. weight standards on ethidium bromide- DNA purification and manipulation. R. stained...restriction fragments suit- lated prior to ligation. High- molecular - ably sized (upper limit 8 kb) for insertion weight insert DNA was selectively precipi...epitopes identified in each library are listed in column 5 (antigen molecular mass in kDa). + Sau96I, Ddel, and Hinfl libraries are corn- fled sera were

  10. Isotypic and allotypic variation of human class II histocompatibility antigen alpha-chain genes.

    PubMed

    Auffray, C; Lillie, J W; Arnot, D; Grossberger, D; Kappes, D; Strominger, J L

    DNA sequences of four human class II histocompatibility antigen alpha chain DNA sequences (derived from cDNA and genomic clones representing DC1 alpha, DC4 alpha, DX alpha and SB alpha) are presented and compared to DR alpha and to mouse I-A alpha and I-E alpha sequences. These data suggest possible mechanisms for the generation of polymorphism and the evolution of the DR, DC and SB families.

  11. Analysis of Dengue Virus Enhancing Epitopes Using Peptide Antigens Derived From the Envelope Glycoprotein Gene Sequence.

    DTIC Science & Technology

    1991-11-29

    AD-A261 707 AD____ ARMY PROJECT ORDER NO: 89PP9961 TITLE: ANALYSIS OF DENGUE VIRUS ENHANCING EPITOPES USING PEPTIDE ANTIGENS DERIVED FROM THE...DATES COVERED 29 Nov 91 Final Report (9/1/89 - 11/30/91) 4. TITLE AND SUBTITLE Ana ysis or Dengue Vnrus nancing 5. FUNDING NUMBERS Epitopes Using...biological events leading to the development of severe disease manifestations of dengue infections ( dengue hemorrhagic fever/ dengue shock syndrome

  12. The gene corresponding to the putative Goodpasture antigen is present in Alport's syndrome.

    PubMed Central

    Savige, J A

    1991-01-01

    Alport's syndrome is a heterogeneous group of inherited abnormalities of basement membranes that may result in progressive renal failure, defective hearing and lens abnormalities. The glomerular basement membrane (GBM) characteristically has areas of reduplication, lamellation and attenuation on electron microscopic examination. In the majority of affected males and some females, there is reduced or variable binding of serum from patients with anti-GBM disease (Goodpasture's syndrome) to these basement membranes. These sera contain antibodies directed against the Goodpasture antigen which has been thought to be located in the non-collagenous domain of the alpha3 chain of type IV collagen and is presumed to be important in cross-linking of the collagen molecules. The reduced staining for the Goodpasture antigen suggests that this structure is either absent or masked in Alport's syndrome. We have tested DNA from six unrelated individuals with Alport's syndrome. All had been transplanted for renal failure. The diagnosis of Alport's syndrome was made on the characteristic electron microscopic appearance of the renal basement membranes (n = 4), the presence of sensori-neural deafness (n = 4), a family history of Alport's syndrome (n = 5) and the presence of circulating inhibitable anti-GBM antibody activity post-transplant (n = 2). Oligonucleotides (20mers) corresponding to the 5' and 3' ends of the known 25 amino acid sequence for the putative Goodpasture antigen were used as primers for amplification of genomic DNA. The products were then blotted and probed with an intermediate 19-mer DNA. All Alport's patients contained a 75-bp fragment corresponding to the published peptide sequence for the non-collagenous domain of the alpha 3 chain of type IV collagen, suggesting that a large deletion of this region, the putative Goodpasture antigen, is unlikely to account for the defect in Alport's syndrome. Images Fig. 2 PMID:1864003

  13. The gene corresponding to the putative Goodpasture antigen is present in Alport's syndrome.

    PubMed

    Savige, J A

    1991-08-01

    Alport's syndrome is a heterogeneous group of inherited abnormalities of basement membranes that may result in progressive renal failure, defective hearing and lens abnormalities. The glomerular basement membrane (GBM) characteristically has areas of reduplication, lamellation and attenuation on electron microscopic examination. In the majority of affected males and some females, there is reduced or variable binding of serum from patients with anti-GBM disease (Goodpasture's syndrome) to these basement membranes. These sera contain antibodies directed against the Goodpasture antigen which has been thought to be located in the non-collagenous domain of the alpha3 chain of type IV collagen and is presumed to be important in cross-linking of the collagen molecules. The reduced staining for the Goodpasture antigen suggests that this structure is either absent or masked in Alport's syndrome. We have tested DNA from six unrelated individuals with Alport's syndrome. All had been transplanted for renal failure. The diagnosis of Alport's syndrome was made on the characteristic electron microscopic appearance of the renal basement membranes (n = 4), the presence of sensori-neural deafness (n = 4), a family history of Alport's syndrome (n = 5) and the presence of circulating inhibitable anti-GBM antibody activity post-transplant (n = 2). Oligonucleotides (20mers) corresponding to the 5' and 3' ends of the known 25 amino acid sequence for the putative Goodpasture antigen were used as primers for amplification of genomic DNA. The products were then blotted and probed with an intermediate 19-mer DNA. All Alport's patients contained a 75-bp fragment corresponding to the published peptide sequence for the non-collagenous domain of the alpha 3 chain of type IV collagen, suggesting that a large deletion of this region, the putative Goodpasture antigen, is unlikely to account for the defect in Alport's syndrome.

  14. Expression of the ’Bacillus anthracis’ Protective Antigen Gene by Baculovirus and Vaccinia Virus Recombinants

    DTIC Science & Technology

    1990-02-01

    ANTIIRACIS PROTI-CTIVE ANTIGEN 367 in medium containing 10% fetal bovine serum. Vaccinia nitrocellulose filters . All cell lysate samples subjected...mM glycine. and 20% methanol at 4°C for 12 to 16 h. by CaCl. precipitation. A. cali irni’, nuclear polyhedrosis Nitrocellulose filters were washed in...and monoclonal antibodies used for these pl[XTJSCRilrr(Stratagcnc. La Jolla. Calif.I. pI3LSCRPPA studies. Filters were washed and incubated with

  15. Association of primary central nervous system vasculitis with the presence of specific human leucocyte antigen gene variant.

    PubMed

    Kraemer, Markus; Becker, Jana; Horn, Peter A; Schwitalla, Jan Claudius; Keyvani, Kathy; Metz, Imke; Wegner, Christiane; Brück, Wolfgang; Schlamann, Marc; Heinemann, Falko M; Berlit, Peter

    2017-09-01

    The etiology and genetic susceptibility of primary central nervous system vasculitis (PCNSV) are still unclear. We analyzed the DNA of 25 Caucasian patients with PCNSV for human leucocyte antigen genes HLA-A, HLA-B, HLA-DRB1, and HLA-DQB1, respectively. HLA-frequencies of the 25 patients with PCNSV were compared with HLA-frequencies of matched Caucasian controls. No statistically significant associations were found for HLA-B, HLA-DR1 and HLA-DQB1 variant. In the PCNSV group, only the HLA-A*69 variant was found more often than expected statistically. The results of this study indicate a potential association of HLA marker with PCNSV in Caucasian patients. Further studies are needed to elucidate the role of genes within the human major histocompatibility complex in the pathogenesis of this angiopathy. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Overlapping gene expression profiles indicative of antigen processing and the interferon pathway characterize inflammatory fibrotic skin diseases.

    PubMed

    Limpers, Annelies; van Royen-Kerkhof, Annet; van Roon, Joel A G; Radstake, Timothy R D J; Broen, Jasper C A

    2014-02-01

    Inflammatory fibrotic disorders have been of high interest both for dermatologists and rheumatologists. Although the phenotypic end stage of this group of diseases is ultimately the same, namely fibrosis, patients present with different clinical features and are often treated with distinct therapeutic modalities. This review addresses whether there is evidence for different underlying molecular pathways in the various inflammatory fibrotic diseases such as localized scleroderma, pediatric lichen sclerosus, adult lichen sclerosus, eosinophilic fasciitis and systemic sclerosis. To investigate this, a large number of gene expression microarray studies performed on skin or fibroblasts from patients with these aforementioned diseases were described, (re-)analysed, and compared. As suspected by the heterogeneous phenotype, most diseases showed unique gene expression features. Intriguingly, a clear overlap was observed between adult and pediatric lichen sclerosus and localized scleroderma, in antigen processing and the interferon pathway. Delineating the cause and consequence of these pathways may generate novel tools to better characterize and more effectively treat these patients.

  17. Definition of the gene encoding the minor histocompatibility antigen HA-1 and typing for HA-1 from genomic DNA.

    PubMed

    Tseng, L H; Lin, M T; Martin, P J; Pei, J; Smith, A G; Hansen, J A

    1998-10-01

    Recipient mismatching for the minor histocompatibility antigen HA-1 has been associated with acute graft-versus-host disease after allogeneic marrow transplantation. Two polymorphic nucleotides near an exon-intron junction of the gene encoding this minor histocompatibility antigen have been identified. In this study, we determined the genomic DNA sequence of the intron downstream from this polymorphic exon. Based on this sequence, primers were designed to amplify the genomic HA-1 gene sequence, and analysis of restriction fragment length polymorphisms was used to assign the HA-1 genotypes of 160 unrelated probands and a paired sibling for each proband. Among probands, the HA-1H allele frequency was 0.441, and the HA-1R allele frequency was 0.559. The distribution of HA-1 genotypes showed close fit with Hardy-Weinberg equilibrium. Likewise, the number of sibling pairs with disparity for HA-1 alleles showed close fit with predictions based on Hardy-Weinberg equilibrium. These results provide a simple and well validated method for future studies correlating HA-1 disparity with clinical outcome after allogeneic marrow transplantation.

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

    SciTech Connect

    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. - Highlights: • We established highly efficient gene transduction protocols for murine T cells. • CD8{sup +} CAR-T cells had antigen-specific cytotoxic activity. • CD4{sup +} CAR-T cells secreted multiple cytokines by antigen stimulation. • This finding can contribute to the development of T-cell biology and immunotherapy.

  19. [Construction and expression of the targeting super-antigen EGF-SEA fusion gene].

    PubMed

    Xie, Yang; Peng, Shaoping; Liao, Zhiying; Liu, Jiafeng; Liu, Xuemei; Chen, Weifeng

    2014-05-01

    To construct expression vector for the SEA-EGF fusion gene. Clone the SEA gene and the EGF gene segment with PCR and RT-PCR independently, and connect this two genes by the bridge PCR. Insert the fusion gene EGF-SEA into the expression vector PET-44. Induced the secretion of the fusion protein SEA-EGF by the antileptic. The gene fragment encoding EGF and SEA mature peptide was successfully cloned. The fusion gene EGF-SEA was successfully constructed and was inserted into expression vector. The new recombinant expression vector for fusion gene EGF-SEA is specific for head and neck cancer, laid the foundation for the further study of fusion protein SEA-EGF targeting immune therapy in head and neck tumors.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  1. Identification of the Drosophila core 1 beta1,3-galactosyltransferase gene that synthesizes T antigen in the embryonic central nervous system and hemocytes.

    PubMed

    Yoshida, Hideki; Fuwa, Takashi J; Arima, Mikiko; Hamamoto, Hiroshi; Sasaki, Norihiko; Ichimiya, Tomomi; Osawa, Ken-Ichi; Ueda, Ryu; Nishihara, Shoko

    2008-12-01

    T antigen (Galbeta1-3GalNAcalpha1-Ser/Thr), the well-known tumor-associated antigen, is a core 1 mucin-type O-glycan structure that is synthesized by core 1 beta1,3-galactosyltransferase (C1beta3GalT), which transfers Gal from UDP-Gal to Tn antigen (GalNAcalpha1-Ser/Thr). Three putative C1beta3GalTs have been identified in Drosophila. However, although all three are expressed in embryos, their roles during embryogenesis have not yet been clarified. In this study, we used P-element inserted mutants to show that CG9520, one of the three putative C1beta3GalTs, synthesizes T antigen expressed on the central nervous system (CNS) during embryogenesis. We also found that T antigen was expressed on a subset of the embryonic hemocytes. CG9520 mutant embryos showed the loss of T antigens on the CNS and on a subset of hemocytes. Then, the loss of T antigens was rescued by precise excision of the P-element inserted into the CG9520 gene. Our data demonstrate that T antigens expressed on the CNS and on a subset of hemocytes are synthesized by CG9520 in the Drosophila embryo. In addition, we found that the number of circulating hemocytes was reduced in third instar larvae of CG9520 mutant. We, therefore, named the CG9520 gene Drosophila core 1 beta1,3-galactosyltransferase 1 because it is responsible for the synthesis and function of T antigen in vivo.

  2. 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

    2004-03-02

    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.

  3. Complete nucleotide sequence of a gene encoding a functional human class I histocompatibility antigen (HLA-CW3).

    PubMed Central

    Sodoyer, R; Damotte, M; Delovitch, T L; Trucy, J; Jordan, B R; Strachan, T

    1984-01-01

    The HLA-CW3 gene contained in a cosmid clone identified by transfection expression experiments has been completely sequenced. This provides, for the first time, data on the structure of HLA-C locus products and constitutes, together with that of the gene coding for HLA-A3, the first complete nucleotide sequences of genes coding for serologically defined class I HLA molecules. In contrast to the organisation of the two class I HLA pseudogenes whose sequences have previously been determined, the sequence of the HLA-CW3 gene reveals an additional cytoplasmic encoding domain, making the organisation of this gene very similar to that of known H-2 class I genes and also the HLA-A3 gene. The deduced amino acid sequences of HLA-CW3 and HLA-A3 now allow a systematic comparison of such sequences of HLA class I molecules from the three classical transplantation antigen loci A, B, C. The compared sequences include the previously determined partial amino acid sequences of HLA-B7, HLA-B40, HLA-A2 and HLA-A28. The comparisons confirm the extreme polymorphism of HLA classical class I molecules, and permit a study of the level of diversity and the location of sequence differences. The distribution of differences is not uniform, most of them being located in the first and second extracellular domains, the third extracellular domain is extremely conserved, and the cytoplasmic domain is also a variable region. Although it is difficult to determine locus-specific regions, we have identified several candidate positions which may be C locus-specific. PMID:6609813

  4. Regression of melanoma metastases after immunotherapy is associated with activation of antigen presentation and interferon-mediated rejection genes

    PubMed Central

    Carretero, Rafael; Wang, Ena; Rodriguez, Ana I.; Reinboth, Jennifer; Ascierto, Maria L.; Engle, Alyson M.; Liu, Hui; Camacho, Francisco M.; Marincola, Francesco M.; Garrido, Federico; Cabrera, Teresa

    2012-01-01

    We present the results of a comparative gene expression analysis of 15 metastases (10 regressing and 5 progressing) obtained from 2 melanoma patients with mixed response following different forms of immunotherapy. Whole genome transcriptional analysis clearly indicate that regression of melanoma metastases is due to an acute immune rejection mediated by the upregulation of genes involved in antigen presentation and interferon mediated response (STAT-1/IRF-1) in all the regressing metastases from both patients. In contrast, progressing metastases showed low transcription levels of genes involved in these pathways. Histological analysis showed T cells and HLA-DR positive infiltrating cells in the regressing but not in the progressing metastases. Quantitative expression analysis of HLA-A, B and C genes on microdisected tumoral regions indicate higher HLA expression in regressing than in progressing metastases. The molecular signature obtained in melanoma rejection appeared to be similar to that observed in other forms of immune-mediated tissue-specific rejection such as allograft, pathogen clearance, graft versus host or autoimmune disease, supporting the immunological constant of rejection. We favor the idea that the major factor determining the success or failure of immunotherapy is the nature of HLA Class I alterations in tumor cells and not the type of immunotherapy used. If the molecular alteration is reversible by the immunotherapy, the HLA expression will be upregulated and the lesion will be recognized and rejected. In contrast, if the defect is structural the MHC Class I expression will remain unchanged and the lesion will progress. PMID:21964766

  5. Construction and application of a Korean reference panel for imputing classical alleles and amino acids of human leukocyte antigen genes.

    PubMed

    Kim, Kwangwoo; Bang, So-Young; Lee, Hye-Soon; Bae, Sang-Cheol

    2014-01-01

    Genetic variations of human leukocyte antigen (HLA) genes within the major histocompatibility complex (MHC) locus are strongly associated with disease susceptibility and prognosis for many diseases, including many autoimmune diseases. In this study, we developed a Korean HLA reference panel for imputing classical alleles and amino acid residues of several HLA genes. An HLA reference panel has potential for use in identifying and fine-mapping disease associations with the MHC locus in East Asian populations, including Koreans. A total of 413 unrelated Korean subjects were analyzed for single nucleotide polymorphisms (SNPs) at the MHC locus and six HLA genes, including HLA-A, -B, -C, -DRB1, -DPB1, and -DQB1. The HLA reference panel was constructed by phasing the 5,858 MHC SNPs, 233 classical HLA alleles, and 1,387 amino acid residue markers from 1,025 amino acid positions as binary variables. The imputation accuracy of the HLA reference panel was assessed by measuring concordance rates between imputed and genotyped alleles of the HLA genes from a subset of the study subjects and East Asian HapMap individuals. Average concordance rates were 95.6% and 91.1% at 2-digit and 4-digit allele resolutions, respectively. The imputation accuracy was minimally affected by SNP density of a test dataset for imputation. In conclusion, the Korean HLA reference panel we developed was highly suitable for imputing HLA alleles and amino acids from MHC SNPs in East Asians, including Koreans.

  6. Gene encoding Duffy antigen/receptor for chemokines is associated with asthma and IgE in three populations.

    PubMed

    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

    2008-11-15

    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. 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. 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. 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. 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.

  7. Mutational analysis of simian virus 40 T antigen: isolation and characterization of mutants with deletions in the T-antigen gene.

    PubMed Central

    Pipas, J M; Peden, K W; Nathans, D

    1983-01-01

    A series of mutants of simian virus 40 has been constructed with deletions in the coding sequence for large T antigen. Nucleotide sequence analysis indicates that 4 mutants have in-phase and 11 have out-of-phase deletions. Mutant DNAs were assayed for the following activities: the ability to form plaques, the ability to produce T antigen as scored by indirect immunofluorescence, viral DNA replication, and morphological transformation of rat cells. Two viable mutants were found, and these had deletions confined to the carboxyl terminus of T antigen. Only those mutants coding for polypeptides greater than 40% of the length of wildtype T antigen produced detectable nuclear fluorescence. The two viable mutants with deletions in the carboxyl terminus of the protein retained the ability both to replicate their DNA, although at a reduced level, and to transform nonpermissive cells. Mutants with sequence changes that result in the loss of more than 117 amino acids from the carboxyl terminus were not viable and were also defective in the DNA replication and transformation functions of T antigen, although several produced detectable nuclear fluorescence. These functions were also sensitive to the removal of amino acids near the amino terminus and in the middle of the protein. Images PMID:6300656

  8. Frequency distribution of HLA-antigens, genes and haplotypes in the migrant population of Magadan as a function of length of residence in the region.

    PubMed

    Pereverzeva, V V; Solovenchuk, L L

    1996-09-01

    Peculiarities of the frequency distribution of antigens, genes, and haplotypes at subloci A, B, and Cw of the HLA system in 1429 Slavic inhabitants of Magadan are presented in dependence on length of residence in extreme conditions. No significant differences were revealed with respect to frequency of genes and antigens in inhabitants with different lengths of residence in northeastern Russian conditions. Analysis of gamete associations shows that the revealed positive or negative associativity in some cases is characteristic for Caucasoids on the whole, but an associativity specific to inhabitants of Magadan was also established. Its character depends on the length of residence in extreme conditions.

  9. Enhanced-affinity murine T-cell receptors for tumor/self-antigens can be safe in gene therapy despite surpassing the threshold for thymic selection

    PubMed Central

    Schmitt, Thomas M.; Aggen, David H.; Stromnes, Ingunn M.; Dossett, Michelle L.; Richman, Sarah A.; Kranz, David M.

    2013-01-01

    Many of the most promising tumor antigens for T-cell-based cancer immunotherapies are unmodified self-antigens. Unfortunately, the avidity of T cells specific for these antigens is limited by central tolerance during T-cell development in the thymus, resulting in decreased anti-tumor efficacy of these T cells. One approach to overcoming this obstacle is to mutate T-cell receptor (TCR) genes from naturally occurring T cells to enhance the affinity for the target antigen. These enhanced-affinity TCRs can then be developed for use in TCR gene therapy. Although TCRs with significantly enhanced affinity have been generated using this approach, it is not clear whether these TCRs, which bypass the affinity limits imposed by negative selection, remain unresponsive to the low levels of self-antigen generally expressed by some normal tissues. Here we show that 2 variants of a high-affinity WT1-specific TCR with enhanced affinity for WT1 are safe and do not mediate autoimmune tissue infiltration or damage when transduced into peripheral CD8 T cells and transferred in vivo. However, if expressed in developing T cells and subjected to thymic selection, the same enhanced-affinity TCRs signal tolerance mechanisms in the thymus, resulting in T cells with attenuated antigen sensitivity in the periphery. PMID:23673862

  10. Characterization and expression of multiple alternatively spliced transcripts of the Goodpasture antigen gene region. Goodpasture antibodies recognize recombinant proteins representing the autoantigen and one of its alternative forms.

    PubMed

    Penadés, J R; Bernal, D; Revert, F; Johansson, C; Fresquet, V J; Cervera, J; Wieslander, J; Quinones, S; Saus, J

    1995-05-01

    Collagen IV, the major component of basement membranes, is composed of six distinct alpha chains (alpha 1-alpha 6). Atypically among the collagen IV genes, the exons encoding the carboxyl-terminal region of the human alpha 3(IV) chain undergo alternative splicing. This region has been designated as the Goodpasture antigen because of its reactivity in the kidney and lung with the pathogenic autoantibodies causing Goodpasture syndrome. The data presented in this report demonstrate that, in human kidney, the gene region encompassing the Goodpasture antigen generates at least six alternatively spliced transcripts predicting five distinct proteins that differ in their carboxyl-terminus and retain, except in one case, the exon that harbors the characteristic amino-terminus of the antigen. Goodpasture antibodies specifically recognize recombinant proteins representing the antigen and the alternative form that retains the amino-half of the antigen, suggesting that this moiety could be involved in the in vivo binding of the pathogenic antibodies. Furthermore, the sera of control individuals contain autoantibodies against the antigen that can be differentiated from those causing the syndrome based on their specific reactivities, suggesting that the binding of the pathogenic autoantibodies to a specific determinant likely trigger a distinct and unique cascade of events causing the disease.

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

    PubMed

    Virnik, Konstantin; Ni, Yisheng; Berkower, Ira

    2013-04-19

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

  12. Active compounds from Saussurea lappa Clarks that suppress hepatitis B virus surface antigen gene expression in human hepatoma cells.

    PubMed

    Chen, H C; Chou, C K; Lee, S D; Wang, J C; Yeh, S F

    1995-05-01

    We have examined the antiviral activity of the crude extract prepared from the root of Saussurea lappa Clarks, a Chinese medicinal herb which is widely used for many illnesses including cancer. Two active components, costunolide and dehydrocostus lactone, were identified which show strong suppressive effect on the expression of the hepatitis B surface antigen (HBsAg) in human hepatoma Hep3B cells, but have little effect on the viability of the cells. Both costunolide and dehydrocostus lactone suppress the HBsAg production by Hep3B cells in a dose-dependent manner with IC50s of 1.0 and 2.0 microM, respectively. Northern blotting analysis shows that the suppression of HBsAg gene expression by both costunolide and dehydrocostus lactone were mainly at the mRNA level. Furthermore, the suppressive effect of costunolide and dehydrocostus lactone on HBsAg and hepatitis B e antigen (HBeAg), a marker for hepatitis B viral genome replication in human liver cells, was also observed in another human hepatoma cell line HepA2 which was derived from HepG2 cells by transfecting a tandemly repeat hepatitis B virus (HBV) DNA. Similarly, the mRNA of HBsAg in HepA2 cells was also suppressed by these two compounds. Our findings suggest that costunolide and dehydrocostus lactone may have potential to develop as specific anti-HBV drugs in the future.

  13. Adoptive T-cell therapy for hematological malignancies using T cells gene-modified to express tumor antigen-specific receptors.

    PubMed

    Fujiwara, Hiroshi

    2014-02-01

    The functional properties of the adoptive immune response mediated by effector T lymphocytes are decisively regulated by their T-cell receptors (TCRs). Transfer of genes encoding target antigen-specific receptors enables polyclonal T cells to redirect toward cancer cells and virally infected cells expressing those defined antigens. Using this technology, a large population of redirected T cells displaying uniform therapeutic properties has been produced, powerfully advancing their clinical application as "cellular drugs" for adoptive immunotherapy against cancer. Clinically, anticancer adoptive immunotherapy using these genetically engineered T cells has an impressive and proven track record. Notable examples include the dramatic benefit of chimeric antigen receptor gene-modified T cells redirected towards B-cell lineage antigen CD19 in patients with chronic lymphocytic leukemia, and the impressive outcomes in the use of TCR gene-modified T cells redirected towards NY-ESO-1, a representative cancer-testis antigen, in patients with advanced melanoma and synovial cell sarcoma. In this review, we briefly overview the current status of this treatment option in the context of hematological malignancy, and discuss a number of challenges that still pose an obstacle to the full effectiveness of this strategy.

  14. Selective gene silencing in activated leukocytes by targeting siRNAs to the integrin lymphocyte function-associated antigen-1

    PubMed Central

    Peer, Dan; Zhu, Pengcheng; Carman, Christopher V.; Lieberman, Judy; Shimaoka, Motomu

    2007-01-01

    Silencing gene expression by RNAi is a powerful method for exploring gene function and validating drug targets and potentially for therapy. Lymphocytes and other primary blood cells are resistant to lipid-based transfection in vitro and are difficult to target in vivo. We show here that antibody-protamine fusion proteins targeting the human integrin lymphocyte function-associated antigen-1 (LFA-1) efficiently deliver siRNAs and specifically induce silencing in primary lymphocytes, monocytes, and dendritic cells. Moreover, a fusion protein constructed from an antibody that preferentially recognizes activation-dependent conformational changes in LFA-1 selectively targets activated leukocytes and can be used to suppress gene expression and cell proliferation only in activated lymphocytes. The siRNA-fusion protein complexes do not cause lymphocyte activation or induce IFN responses. K562 cells expressing latent WT or constitutively activated LFA-1 engrafted in the lungs of SCID mice are selectively targeted by intravenously injected fusion protein–siRNA complexes, demonstrating the potential in vivo applicability of LFA-1-directed siRNA delivery. PMID:17360483

  15. Killer-cell immunoglobulin-like receptor and human leukocyte antigen-C genes in common variable immunodeficiency.

    PubMed

    Kartal, Ozgur; Musabak, Ugur; Yesillik, Sait; Sagkan, Rahsan I; Pekel, Aysel; Demirel, Fevzi; Baysan, Abdullah; Selçuk, Ali; Güleç, Mustafa; Şener, Osman

    2016-11-01

    We aimed herein to investigate the killer-cell immunoglobulin-like receptor (KIR) genes and human leukocyte antigen (HLA)-C alleles in patients with common variable immunodeficiency (CVID), and to reveal their differences from those in healthy population. In all, 18 patients who have been diagnosed with CVID and 15 living donors of kidney transplant recipients were enrolled in the study. Polymerase chain reaction-sequence-specific primer (PCR-SSP) typing method was used in molecular genetic analysis. The frequencies of the genes in the study groups were statistically compared with each other using chi-square or Fisher exact tests, whichever were appropriate. Although there was no significant difference between both study groups with respect to distribution of KIR and HLA-C2 group genes, HLA-Cw7 allele frequency in patients with CVID was significantly lower than that in healthy population (P = 0.008). This present study results support that HLA-Cw7 allele, an inhibitor of KIR ligand, may play a role in the pathogenesis of CVID.

  16. 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. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Construction and expression of a recombinant DNA gene encoding a polyomavirus middle-size tumor antigen with the carboxyl terminus of the vesicular stomatitis virus glycoprotein G

    SciTech Connect

    Templeton, D.; Voronova, A.; Eckhart, W.

    1984-02-01

    The authors constructed a molecular clone encoding the N-terminal 379 amino acids of the polyomavirus middle-size tumor antigen, followed by the C-terminal 60 amino acids of the vesicular stomatitis virus glycoprotein G. This hybrid gene contained the coding region for the C-terminal hydrophobic membrane-spanning domain of the G protein in place of the C-terminal hydrophobic domain of the middle-size tumor antigen. The hybrid gene was expressed in COS-1 cells under the control of the simian virus 40 late promoter. The hybrid protein was located in cell membranes and was associated with a tyrosine-specific protein kinase activity, as was the middle-size tumor antigen. Plasmids encoding the hybrid protein failed to transform mouse NIH 3T3 or rat F2408 cells.

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

    PubMed

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

    2017-02-01

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

  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. Immunoglobulin gene repertoire in ocular adnexal lymphomas: hints on the nature of the antigenic stimulation.

    PubMed

    Dagklis, A; Ponzoni, M; Govi, S; Cangi, M G; Pasini, E; Charlotte, F; Vino, A; Doglioni, C; Davì, F; Lossos, I S; Ntountas, I; Papadaki, T; Dolcetti, R; Ferreri, A J M; Stamatopoulos, K; Ghia, P

    2012-04-01

    Evidence from certain geographical areas links lymphomas of the ocular adnexa marginal zone B-cell lymphomas (OAMZL) with Chlamydophila psittaci (Cp) infection, suggesting that lymphoma development is dependent upon chronic stimulation by persistent infections. Notwithstanding that, the actual immunopathogenetical mechanisms have not yet been elucidated. As in other B-cell lymphomas, insight into this issue, especially with regard to potential selecting ligands, could be provided by analysis of the immunoglobulin (IG) receptors of the malignant clones. To this end, we studied the molecular features of IGs in 44 patients with OAMZL (40% Cp-positive), identifying features suggestive of a pathogenic mechanism of autoreactivity. Herein, we show that lymphoma cells express a distinctive IG repertoire, with electropositive antigen (Ag)-binding sites, reminiscent of autoantibodies (auto-Abs) recognizing DNA. Additionally, five (11%) cases of OAMZL expressed IGs homologous with autoreactive Abs or IGs of patients with chronic lymphocytic leukemia, a disease known for the expression of autoreactive IGs by neoplastic cells. In contrast, no similarity with known anti-Chlamydophila Abs was found. Taken together, these results strongly indicate that OAMZL may originate from B cells selected for their capability to bind Ags and, in particular, auto-Ags. In OAMZL associated with Cp infection, the pathogen likely acts indirectly on the malignant B cells, promoting the development of an inflammatory milieu, where auto-Ags could be exposed and presented, driving proliferation and expansion of self-reactive B cells.

  1. Antigen genes for molecular epidemiology of leishmaniasis: polymorphism of cysteine proteinase B and surface metalloprotease glycoprotein 63 in the Leishmania donovani complex.

    PubMed

    Quispe Tintaya, Kelly Wilber; Ying, Xu; Dedet, Jean-Pierre; Rijal, Suman; De Bolle, Xavier; Dujardin, Jean-Claude

    2004-03-15

    Efficient monitoring of endemic and resurgent visceral leishmaniasis (VL) requires discriminatory molecular tools that allow direct characterization of etiological agents (i.e., the Leishmania donovani complex) in host tissues. This characterization is possible through restriction fragment-length polymorphism (RFLP) analysis of polymerase chain reaction (PCR)-amplified sequences (PCR-RFLP). We present 2 new PCR-RFLP assays that target the gene locus of cysteine proteinase B (cpb), an important Leishmania antigen. The assays were applied to the characterization of 15 reference strains of the L. donovani complex, and their discriminatory power was compared with that of PCR-RFLP analysis of the gp63 gene, another Leishmania antigen, and with that of multilocus enzyme electrophoresis (MLEE), which is the reference standard for parasite typing. Restriction patterns of the cpb locus were polymorphic, but less so than gp63 patterns. When data for both loci were combined, differences between PCR-RFLP and MLEE results were encountered. Antigen gene analysis was more discriminatory and supported a different classification of parasites, one that fitted with their geographic origin. PCR-RFLP analysis of cpb also allowed direct genotyping of parasites in bone marrow aspirate and venous blood samples obtained from patients with VL. Antigen genes constitute valid targets for PCR-based Leishmania typing without the need for isolation of parasites.

  2. 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

    USDA-ARS?s Scientific Manuscript database

    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...

  3. 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

    USDA-ARS?s Scientific Manuscript database

    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...

  4. Physical mapping of the human T-cell antigen receptor (TCR) {beta}-chain gene complex

    SciTech Connect

    Yashim, Y.; So, A.K.

    1994-09-01

    The genetic variation of the TCR loci and their contribution to autoimmune diseases is poorly defined, in direct contrast to the clear examples of disease association with the Class I and II alleles of the major histocompatibility complex. We have therefore started to determine the gene organization and polymorphism of the TCR {beta} locus. Yeast artificial chromosomes (YACs) were used to construct a physical map of the germline human TCR {beta}-chain gene complex. Variable gene (V{beta}) sequences for the 25 known V{beta} subfamilies were amplified by PCR and were used as probes to screen a YAC library. Five positive YACs were identified. YACs designated B3, E11 and H11 of sizes 820, 400 and 600 kbp, respectively, were analyzed for their V{beta} content by pulse-field gel electrophoresis (PFGE). YAC B3 was found to contain all 25 V{beta} subfamilies, E11 for 14 and H11 for 7. B3 was also positive for the constant region genes. Restriction enzyme mapping of B3 located V{beta} and C{beta} gene regions to four Sfi I fragments of 280, 110, 90 and 125 kbp, and was in accordance with published data. The data thus showed that YAC B3 encoded a complete and unrearranged TCR {beta}-gene locus. The map was further resolved by locating restriction sites for Sal I and Bssll II on B3. Fluorescent in situ hybridization to human metaphase chromosomes localized B3 to chromosome 7q35. However, two additional signals were obtained: one attributable to V{beta} orphon cluster on chromosome 9q21; the second to the long arm of chromosome 2. PCR amplification of a chromosome 2 somatic cell hybrid using primers for all 25 V{beta} gene families revealed the signal was not attributable to a second orphon cluster. It is suggested that B3 is a chimeric YAC with an intact TCR {beta} locus flanked by chromosome 2 sequences. The determination of the TCR genomic organization will help extend studies of the role T-cells play in autoimmune diseases.

  5. Dynamics of antigen presentation to transgene product-specific CD4+ T cells and of Treg induction upon hepatic AAV gene transfer

    PubMed Central

    Perrin, George Q; Zolotukhin, Irene; Sherman, Alexandra; Biswas, Moanaro; de Jong, Ype P; Terhorst, Cox; Davidoff, Andrew M; Herzog, Roland W

    2016-01-01

    The tolerogenic hepatic microenvironment impedes clearance of viral infections but is an advantage in viral vector gene transfer, which often results in immune tolerance induction to transgene products. Although the underlying tolerance mechanism has been extensively studied, our understanding of antigen presentation to transgene product-specific CD4+ T cells remains limited. To address this, we administered hepatotropic adeno-associated virus (AAV8) vector expressing cytoplasmic ovalbumin (OVA) into wt mice followed by adoptive transfer of transgenic OVA-specific T cells. We find that that the liver-draining lymph nodes (celiac and portal) are the major sites of MHC II presentation of the virally encoded antigen, as judged by in vivo proliferation of DO11.10 CD4+ T cells (requiring professional antigen-presenting cells, e.g., macrophages) and CD4+CD25+FoxP3+ Treg induction. Antigen presentation in the liver itself contributes to activation of CD4+ T cells egressing from the liver. Hepatic-induced Treg rapidly disseminate through the systemic circulation. By contrast, a secreted OVA transgene product is presented in multiple organs, and OVA-specific Treg emerge in both the thymus and periphery. In summary, liver draining lymph nodes play an integral role in hepatic antigen presentation and peripheral Treg induction, which results in systemic regulation of the response to viral gene products. PMID:27933310

  6. 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

  7. 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

  8. 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

  9. Membrane Ia expression and antigen-presenting accessory cell function of L cells transfected with class II major histocompatibility complex genes

    PubMed Central

    1984-01-01

    To study the relationship between the structure and function of Ia antigens, as well as the physiologic requirements for antigen presentation to major histocompatibility complex-restricted T cells, class II A alpha and A beta genes from the k and d haplotypes were transfected into Ltk- fibroblasts using the calcium phosphate coprecipitation technique. Individually transfected genes were actively transcribed in the L cells without covalent linkage to, or cotransformation with, viral enhancer sequences. However, cell surface expression of detectable I-A required the presence of transfected A alpha dA beta d or A alpha kA beta k pairs in a single cell. The level of I-A expression under these conditions was 1/5-1/10 that of Ia+ B lymphoma cells, or B lymphoma cells expressing transfected class II genes. These I-A-expressing transfectants were tested for accessory cell function and shown to present polypeptide and complex protein antigens to T cell clones and hybridomas in the context of the transfected gene products. One T cell clone, restricted to I-Ak plus GAT (L-glutamic acid60-L-alanine30-L-tyrosine10), had a profound cytotoxic effect on I-Ak- but not I-Ad-expressing transfectants in the presence of specific antigen. Assays of unprimed T cells showed that both Ia+ and Ia- L cells could serve as accessory cells for concanavalin A-induced proliferative responses. These data indicate that L cells can transcribe, translate, and express transfected class II genes and that such I-A-bearing L cells possess the necessary metabolic mechanisms for presenting these antigens to T lymphocytes in the context of their I-A molecules. PMID:6436430

  10. Significance of clonal rearrangements of lymphocyte antigen receptor genes on the prognosis of chronic enteropathy in 22 Shiba dogs.

    PubMed

    Ohmi, Aki; Ohno, Koichi; Uchida, Kazuyuki; Goto-Koshino, Yuko; Tomiyasu, Hirotaka; Kanemoto, Hideyuki; Fukushima, Kenjiro; Tsujimoto, Hajime

    2017-09-29

    Shiba dogs are predisposed to chronic enteropathy (CE) and have poorer prognosis than other dog breeds. The objective of this study was to investigate the significance of polymerase chain reaction for antigen receptor rearrangement (PARR) results on clinical findings and prognosis of Shiba dogs with CE. We retrospectively collected data on 22 Shiba dogs diagnosed as having CE. Fifty-nine percent of the dogs had clonality-positive results on PARR analysis. Furthermore, on histopathology, epitheliotropic behavior of small lymphocytes of the intestinal mucosa was observed significantly more frequently in dogs with clonal rearrangement of antigen receptor genes (P=0.027). The median overall survival time of clonality-positive dogs was 48 days (range, 4-239 days), compared to 271 days (range, 45-1,316+ days) in clonality-negative dogs. The median overall survival time of epitheliotropism-positive dogs was 76 days (range, 30-349 days) compared to 239 days (range, 4-1,316+ days) for epitheliotropism-negative dogs. Statistical analysis revealed that the clonality-positive result was associated with significantly shorter survival time (P=0.036). In contrast, presence or absence of epitheliotropism had no statistically significant effect on survival time (P=0.223). These cases might appropriately be diagnosed as small T-cell intestinal lymphoma; there are some common clinical and pathogenic features with human enteropathy-associated T-cell lymphoma type 2. The pathogenesis and poor prognosis for Shiba dogs with CE seem to be associated with this type of lymphoma, although further investigation is warranted.

  11. Characterization of the genes specifying two metacyclic variable antigen types in Trypanosoma brucei rhodesiense.

    PubMed Central

    Lenardo, M J; Rice-Ficht, A C; Kelly, G; Esser, K M; Donelson, J E

    1984-01-01

    Bloodstream trypanosomes evade the immune system of their mammalian host by sequentially expressing a large number of different variable surface glycoproteins (VSGs). In contrast, metacyclic trypanosomes, the final developmental stage in the tsetse fly, express a much more restricted set of VSGs. These metacyclic VSGs are the first to be exposed to the immune system of the mammalian host after infection and may offer the potential for the eventual development of a vaccine. We have identified cDNAs for two VSGs in cDNA libraries prepared from amplified metacyclic populations of Trypanosoma brucei rhodesiense and show that they correspond to two different metacyclic serotypes. Determination of the cDNA sequences shows that metacyclic VSG mRNAs are similar to VSG mRNAs expressed during the bloodstream stage. Southern blots demonstrate that the metacyclic VSG genes are located near chromosomal telomeres. No evidence of gene rearrangement associated with expression of these VSGs was found. Images PMID:6593722

  12. Epigenetics in Apicomplexa: control of gene expression during cell cycle progression, differentiation and antigenic variation.

    PubMed

    Hakimi, Mohamed-Ali; Deitsch, Kirk W

    2007-08-01

    Apicomplexan parasites are important disease causing organisms that infect both animals and humans, causing extensive health and economic damage to human populations, particularly those in the developing world. The ability to perform genetic crosses, to engineer transgenic parasites lines, and the wealth of information made available through recent genome sequencing projects have made the laboratory study of these parasites important not only for understanding the diseases that they cause, but also for gaining insights into basic biological processes. The control of gene expression and cellular differentiation are particularly interesting in these organisms, as the apparent lack of large families of recognizable transcription factors typically found in other eukaryotic organisms suggests that they may be unusually reliant on epigenetic mechanisms. Here we review recent advances in the study of epigenetic gene regulation in the apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii.

  13. 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.

  14. Antigen-presenting genes and genomic copy number variations in the Tasmanian devil MHC

    PubMed Central

    2012-01-01

    Background The Tasmanian devil (Sarcophilus harrisii) is currently under threat of extinction due to an unusual fatal contagious cancer called Devil Facial Tumour Disease (DFTD). DFTD is caused by a clonal tumour cell line that is transmitted between unrelated individuals as an allograft without triggering immune rejection due to low levels of Major Histocompatibility Complex (MHC) diversity in Tasmanian devils. Results Here we report the characterization of the genomic regions encompassing MHC Class I and Class II genes in the Tasmanian devil. Four genomic regions approximately 960 kb in length were assembled and annotated using BAC contigs and physically mapped to devil Chromosome 4q. 34 genes and pseudogenes were identified, including five Class I and four Class II loci. Interestingly, when two haplotypes from two individuals were compared, three genomic copy number variants with sizes ranging from 1.6 to 17 kb were observed within the classical Class I gene region. One deletion is particularly important as it turns a Class Ia gene into a pseudogene in one of the haplotypes. This deletion explains the previously observed variation in the Class I allelic number between individuals. The frequency of this deletion is highest in the northwestern devil population and lowest in southeastern areas. Conclusions The third sequenced marsupial MHC provides insights into the evolution of this dynamic genomic region among the diverse marsupial species. The two sequenced devil MHC haplotypes revealed three copy number variations that are likely to significantly affect immune response and suggest that future work should focus on the role of copy number variations in disease susceptibility in this species. PMID:22404855

  15. Antigen-presenting genes and genomic copy number variations in the Tasmanian devil MHC.

    PubMed

    Cheng, Yuanyuan; Stuart, Andrew; Morris, Katrina; Taylor, Robyn; Siddle, Hannah; Deakin, Janine; Jones, Menna; Amemiya, Chris T; Belov, Katherine

    2012-03-12

    The Tasmanian devil (Sarcophilus harrisii) is currently under threat of extinction due to an unusual fatal contagious cancer called Devil Facial Tumour Disease (DFTD). DFTD is caused by a clonal tumour cell line that is transmitted between unrelated individuals as an allograft without triggering immune rejection due to low levels of Major Histocompatibility Complex (MHC) diversity in Tasmanian devils. Here we report the characterization of the genomic regions encompassing MHC Class I and Class II genes in the Tasmanian devil. Four genomic regions approximately 960 kb in length were assembled and annotated using BAC contigs and physically mapped to devil Chromosome 4q. 34 genes and pseudogenes were identified, including five Class I and four Class II loci. Interestingly, when two haplotypes from two individuals were compared, three genomic copy number variants with sizes ranging from 1.6 to 17 kb were observed within the classical Class I gene region. One deletion is particularly important as it turns a Class Ia gene into a pseudogene in one of the haplotypes. This deletion explains the previously observed variation in the Class I allelic number between individuals. The frequency of this deletion is highest in the northwestern devil population and lowest in southeastern areas. The third sequenced marsupial MHC provides insights into the evolution of this dynamic genomic region among the diverse marsupial species. The two sequenced devil MHC haplotypes revealed three copy number variations that are likely to significantly affect immune response and suggest that future work should focus on the role of copy number variations in disease susceptibility in this species.

  16. Identification of immediate early gene products of bovine herpes virus 1 (BHV-1) as dominant antigens recognized by CD8 T cells in immune cattle.

    PubMed

    Hart, Jane; MacHugh, Niall D; Sheldrake, Tara; Nielsen, Morten; Morrison, W Ivan

    2017-07-01

    In common with other herpes viruses, bovine herpes virus 1 (BHV-1) induces strong virus-specific CD8 T-cell responses. However, there is a paucity of information on the antigenic specificity of the responding T-cells. The development of a system to generate virus-specific CD8 T-cell lines from BHV-1-immune cattle, employing Theileria-transformed cell lines for antigen presentation, has enabled us to address this issue. Use of this system allowed the study to screen for CD8 T-cell antigens that are efficiently presented on the surface of virus-infected cells. Screening of a panel of 16 candidate viral gene products with CD8 T-cell lines from 3 BHV-1-immune cattle of defined MHC genotypes identified 4 antigens, including 3 immediate early (IE) gene products (ICP4, ICP22 and Circ) and a tegument protein (UL49). Identification of the MHC restriction specificities revealed that the antigens were presented by two or three class I MHC alleles in each animal. Six CD8 T-cell epitopes were identified in the three IE proteins by screening of synthetic peptides. Use of an algorithm (NetMHCpan) that predicts the peptide-binding characteristics of restricting MHC alleles confirmed and, in some cases refined, the identity of the epitopes. Analyses of the epitope specificity of the CD8 T-cell lines showed that a large component of the response is directed against these IE epitopes. The results indicate that these IE gene products are dominant targets of the CD8 T-cell response in BHV-I-immune cattle and hence are prime-candidate antigens for the generation of a subunit vaccine.

  17. 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

  18. Human-leukocyte antigen class II genes in early-onset obsessive-compulsive disorder.

    PubMed

    Rodriguez, Natalia; Morer, Astrid; González-Navarro, E Azucena; Gassó, Patricia; Boloc, Daniel; Serra-Pagès, Carles; Lafuente, Amalia; Lazaro, Luisa; Mas, Sergi

    2017-05-31

    The exact aetiology of obsessive-compulsive disorder (OCD) is unknown, although there is evidence to suggest a gene-environment interaction model. Several lines of evidence support a possible role of the immune system in this model. The present study explores the allele variability in HLA genes of class II (HLA-DRB1, HLA-DQB1) in a sample of 144 early-onset OCD compared with reference samples of general population in the same geographical area. None of the 39 alleles identified (allele frequency >1%) showed significant differences between OCD and reference populations. Pooling the different alleles that comprised HLA-DR4 (including DRB1*04:01, DRB1*04:04 and DRB1*04:05 alleles) we observed a significantly higher frequency (X(2)1 = 5.53, P = 0.018; OR = 1.64, 95% CI 1.08-2.48) of these alleles in the early-onset OCD sample (10.8%) than in the reference population (6.8%). Taking into account the role of HLA class II genes in the central nervous system, the results presented here support a role of the immune system in the pathophysiological model of OCD.

  19. Microarray evidence of glutaminyl cyclase gene expression in melanoma: implications for tumor antigen specific immunotherapy

    PubMed Central

    Gillis, John Stuart

    2006-01-01

    Background In recent years encouraging progress has been made in developing vaccine treatments for cancer, particularly with melanoma. However, the overall rate of clinically significant results has remained low. The present research used microarray datasets from previous investigations to examine gene expression patterns in cancer cell lines with the goal of better understanding the tumor microenvironment. Methods Principal Components Analyses with Promax rotational transformations were carried out with 90 cancer cell lines from 3 microarray datasets, which had been made available on the internet as supplementary information from prior publications. Results In each of the analyses a well defined melanoma component was identified that contained a gene coding for the enzyme, glutaminyl cyclase, which was as highly expressed as genes from a variety of well established biomarkers for melanoma, such as MAGE-3 and MART-1, which have frequently been used in clinical trials of melanoma vaccines. Conclusion Since glutaminyl cyclase converts glutamine and glutamic acid into a pyroglutamic form, it may interfere with the tumor destructive process of vaccines using peptides having glutamine or glutamic acid at their N-terminals. Finding ways of inhibiting the activity of glutaminyl cyclase in the tumor microenvironment may help to increase the effectiveness of some melanoma vaccines. PMID:16820060

  20. Somatic hypermutation of the new antigen receptor gene (NAR) in the nurse shark does not generate the repertoire: Possible role in antigen-driven reactions in the absence of germinal centers

    PubMed Central

    Diaz, Marilyn; Greenberg, Andrew S.; Flajnik, Martin F.

    1998-01-01

    The new antigen receptor (NAR) gene in the nurse shark diversifies extensively by somatic hypermutation. It is not known, however, whether NAR somatic hypermutation generates the primary repertoire (like in the sheep) or rather is used in antigen-driven immune responses. To address this issue, the sequences of NAR transmembrane (Tm) and secretory (Sec) forms, presumed to represent the primary and secondary repertoires, respectively, were examined from the peripheral blood lymphocytes of three adult nurse sharks. More than 40% of the Sec clones but fewer than 11% of Tm clones contained five mutations or more. Furthermore, more than 75% of the Tm clones had few or no mutations. Mutations in the Sec clones occurred mostly in the complementarity-determining regions (CDR) with a significant bias toward replacement substitutions in CDR1; in Tm clones there was no significant bias toward replacements and only a low level of targeting to the CDRs. Unlike the Tm clones where the replacement mutational pattern was similar to that seen for synonymous changes, Sec replacements displayed a distinct pattern of mutations. The types of mutations in NAR were similar to those found in mouse Ig genes rather than to the unusual pattern reported for shark and Xenopus Ig. Finally, an oligoclonal family of Sec clones revealed a striking trend toward acquisition of glutamic/aspartic acid, suggesting some degree of selection. These data strongly suggest that hypermutation of NAR does not generate the repertoire, but instead is involved in antigen-driven immune responses. PMID:9826702

  1. Structural comparison of O-antigen gene clusters of Legionella pneumophila and its application of a serogroup-specific multiplex PCR assay.

    PubMed

    Cao, Boyang; Tian, Zhenyang; Wang, Suwei; Zhu, Zhiyan; Sun, Yamin; Feng, Lu; Wang, Lei

    2015-12-01

    The Legionella pneumophila serogroups O1, O4, O6, O7, O10 and O13 are pathogenic strains associated with pneumonia. The surface O-antigen gene clusters of L. pneumophila serogroups O4, O6, O7, O10 and O13 were sequenced and analyzed, with the function annotated on the basis of homology to that of the genes of L. pneumophila serogroup O1 (L. pneumophila subsp. pneumophila str. Philadelphia 1). The gene locus of the six L. pneumophila serogroups contains genes of yvfE, neuABCD, pseA-like for nucleotide sugar biosynthesis, wecA for sugar transfer, and wzm as well as wzt for O-antigen processing. The detection of O-antigen genes allows the fine differentiation at species and serogroup level without the neccessity of nucleotide sequencing. The O-antigen-processing genes wzm and wzt, which were found to be distinctive for different for different serogroups, have been used as the target genes for the detection and identification of L. pneumophila strains of different O serogroups. In this report, a multiplex PCR assay based on wzm or wzt that diferentiates all the six serogroups by amplicon size was developed with the newly designed specific primer pairs for O1 and O7, and the specific primer pairs for O4, O6, O10, and O13 reported previously. The array was validated by analysis of 34 strains including 15 L. pneumophila O-standard reference strains, eight reference strains of other Legionella non-pneumophila species, six other bacterial species, and five L. pneumophila environmental isolates. The detection sensitivity was one ng genomic DNA. The accurate and sensitive assay is suitable for the identification and detection of strains of these serogroups in environmental and clinical samples.

  2. 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.

  3. 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

  4. The simian virus 40 minimal origin and the 72-base-pair repeat are required simultaneously for efficient induction of late gene expression with large tumor antigen.

    PubMed

    Hartzell, S W; Byrne, B J; Subramanian, K N

    1984-10-01

    We have studied the temporal regulation of simian virus 40 (SV40) late gene expression by construction and transient expression analysis of plasmids containing the transposon Tn9 chloramphenicol acetyltransferase gene placed downstream from the late control region. The SV40 origin region in the early (but not the late) orientation promotes chloramphenicol acetyltransferase gene expression efficiently in monkey cells lacking large tumor (T) antigen. In monkey cells producing T antigen, the promoter activity of the late control region is induced by approximately 1,000-fold above the basal level. By deletion and point mutagenesis, we define two domains of the late control region required for efficient induction with T antigen. Domain I is the minimal replication origin containing T-antigen binding site II. Domain II consists of the 72-base-pair (bp) repeat and a 19-bp downstream sequence up to nucleotide 270. Domains I and II should act synergistically because the absence of either one or the other decreases induction efficiency by 2 orders of magnitude. Though a complete copy of domain II is optimal, the origin-proximal 22-bp portion of this domain is sufficient. The 21-bp repeat, located between domains I and II, is dispensable for this induction, as are sequences located downstream from nucleotide 270 in the late orientation.

  5. Increased expression of TLR-2, COX-2, and SOD-2 genes in the peripheral blood leukocytes of opisthorchiasis patients induced by Opisthorchis viverrini antigen.

    PubMed

    Yongvanit, Puangrat; Thanan, Raynoo; Pinlaor, Somchai; Sithithaworn, Paiboon; Loilome, Watcharin; Namwat, Nisana; Techasen, Anchalee; Dechakhamphu, Somkid

    2012-05-01

    Re-infection with liver fluke, Opisthorchis viverrini, increases proinflammatory molecules involved in inflammation-mediated disease and carcinogenesis in an animal model. To clarify whether these genes respond to parasite antigen in peripheral blood leukocytes (PBL) of opisthorchiasis patients, we examined the transcriptional level of oxidant-generating (toll-like receptor 2 (TLR-2), nuclear factor-kappa B (NF-KB), and cyclooxygenase 2 (COX-2)), anti-oxidant-generating (manganese superoxide dismutase 2 (SOD-2) and catalase (CAT)), proinflammatory cytokine (interleukin (IL)-1β), and anti-inflammatory cytokine (IL-10), in PBL exposed to parasite antigen in O. viverrini-infected patients compared with healthy individuals in an in vitro experiment. After O. viverrini antigen-treated PBL, quantitative RT-PCR analysis revealed that increased expression of cytokines and oxidant-generating genes in PBL was similar between O. viverrini-infected and healthy groups. Interestingly, compared with healthy subjects, increase of TLR-2, COX-2, and SOD-2 and decreased CAT mRNA expression levels were observed in O. viverrini-infected group. The results indicate that O. viverrini antigen induces upregulation of TLR-2, COX-2, and SOD-2 and downregulation of CAT genes in opisthorchiasis patients, suggesting that imbalance of oxidant/anti-oxidant transcripts during re-infection may be involved in the inflammatory-driven carcinogenesis. These molecules may be used as the chemopreventive target for intervention of opisthorchiasis patients in an endemic area.

  6. Comparative antigen-induced gene expression profiles unveil novel aspects of susceptibility/resistance to adjuvant arthritis in rats.

    PubMed

    Yu, Hua; Lu, Changwan; Tan, Ming T; Moudgil, Kamal D

    2013-12-01

    Lewis (LEW) and Wistar Kyoto (WKY) rats of the same major histocompatibility complex (MHC) haplotype (RT.1(l)) display differential susceptibility to adjuvant-induced arthritis (AIA). LEW are susceptible while WKY are resistant to AIA. To gain insights into the mechanistic basis of these disparate outcomes, we compared the gene expression profiles of the draining lymph node cells (LNC) of these two rat strains early (day 7) following a potentially arthritogenic challenge. LNC were tested both ex vivo and after restimulation with the disease-related antigen, mycobacterial heat-shock protein 65. Biotin-labeled fragment cRNA was generated from RNA of LNC and then hybridized with an oligonucleotide-based DNA microarray chip. The differentially expressed genes (DEG) were compared by limiting the false discovery rate to <5% and fold change ≥2.0, and their association with quantitative trait loci (QTL) was analyzed. This analysis revealed overall a more active immune response in WKY than LEW rats. Important differences were observed in the association of DEG with QTL in LEW vs. WKY rats. Both the number of upregulated DEG associated with rat arthritis-QTL and their level of expression were relatively higher in LEW when compared to WKY rat; however, the number of downregulated DEG-associated with rat arthritis-QTL as well as AIA-QTL were found to be higher in WKY than in LEW rats. In conclusion, distinct gene expression profiles define arthritis-susceptible versus resistant phenotype of MHC-compatible inbred rats. These results would advance our understanding of the pathogenesis of autoimmune arthritis and might also offer potential novel targets for therapeutic purposes.

  7. 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.

  8. Allele and haplotype frequencies at human leukocyte antigen class I and II genes in Venezuela's population.

    PubMed

    Del Pilar Fortes, María; Gill, Gisselle; Paredes, María Elena; Gamez, Ligia Elena; Palacios, Marina; Blanca, Isaac; Tassinari, Paolo

    2012-01-01

    Population studies represent an integral part and link in understanding the complex chain of host-pathogen interactions, disease pathogenesis, and MHC gene polymorphisms. Genes of Mongoloid, Caucasoid, and Negroid populations have created a distinctive HLA genetic profile in the Venezuelan population. Our objective was to determine the predominant HLA class I and II alleles and haplotype frequencies in the hybrid population of Venezuela. The study population consisted of 486 healthy unrelated native Venezuelans and 180 families. We examined the frequency of HLA A-B-C, HLA-DQ and HLA-DR genes by polymerase chain reaction and subsequent hybridization with sequence-specific oligonucleotide probes. Phenotypic, allelic and haplotype frequencies were estimated by direct counting and using the maximum-likelihood method. The predominant HLA class I alleles were A*02, A*24, A*68, B*35, B*44, B*51, B*07, B*15 and Cw*07. Regarding HLA class II, the most frequent alleles were DQB1*03 and DRB1*04, DRB1*15, DRB1*13, DRB1*07. The prevailing haplotype was HLA-A*02B*35 DQB1*03 DRB1*04. Some of these alleles and haplotype frequencies were predominantly present in Amerindians (A*02, A*24, B*35, Cw*07, DRB1*04, A*24 B*35). Previous reports have shown high incidence of A*02, B*44, B*51, DRB1*15, DRB1*13, DRB1*07 alleles in several European populations and A*68, B*07, B*15 alleles in African Americans, which could have contributed to the ethnic admixture of the Venezuelan population. We conclude that our results provide strong evidence that Venezuela's population represents an admixture of the primitive Mongoloid Aborigines, Caucasoid Europeans and Western African Negroid migrants.

  9. 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.

  10. Structure and gene cluster of the O antigen of Escherichia coli L-19, a candidate for a new O-serogroup.

    PubMed

    Zdorovenko, Evelina L; Varbanets, Lyudmila D; Liu, Bin; Valueva, Olga A; Wang, Quan; Shashkov, Alexander S; Garkavaya, Ekaterina G; Brovarskaya, Oksana S; Wang, Lei; Knirel, Yuriy A

    2014-09-01

    Escherichia coli L-19 isolated from a healthy individual did not agglutinate with any of 21 polyvalent antisera that cover 174 E. coli O-serogroups. The strain was studied in respect to the O-antigen (O-specific polysaccharide, OPS) structure and genetics. The LPS was isolated by phenol-water extraction of bacterial cells and cleaved by mild acid hydrolysis to yield the OPS. The OPS was studied by sugar and methylation analyses, along with 1D and 2D (1)H and (13)C NMR spectroscopy. The established structure of the linear tetrasaccharide repeating unit was found to be unique among known bacterial polysaccharide structures. A peculiar component of the L-19 OPS was an amide of glucuronic acid with 2-amino-1,3-propanediol (2-amino-2-deoxyglycerol) (GroN). The O-antigen gene cluster of L-19 between the conserved genes galF and gnd was sequenced, and gene functions were tentatively assigned by a comparison with sequences in the available databases and found to be in agreement with the OPS structure. Except for putative genes for synthesis and transfer of GroN, the sequences in the L-19 O-antigen gene cluster were little related to those of reference strains of the 174 known E. coli O-serogroups. The data obtained suggest that L-19 can be considered as a candidate for a new E. coli O-serogroup. © 2014 The Authors.

  11. 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.

  12. C-fos and IL-2 gene expression in rat brain cells and splenic lymphocytes after nonantigenic and antigenic stimuli.

    PubMed

    Korneva, E A; Barabanova, S V; Golovko, O I; Nosov, M A; Novikova, N S; Kazakova, T B

    2000-01-01

    Immunostimulatory or immunosuppressive stress models were used: (1) rotation stress (RS) and (2) immobilization (restraint) stress (IS). Intravenous injection of tetanus toxoid (anatoxin) (TT) was chosen as the antigenic stimulus (500 micrograms/kg weight), and intravenous injection of saline solution was used as the control. Splenic lymphocytes (CBA mice) or different brain structures (Wistar and Sprague-Dawley rats) were analyzed. The c-fos and interleukin-2 (IL-2) mRNA expression was measured using a digoxigenin (Dig)-labeled cDNA probe by spot or in situ hybridization. Rotation stress stimulated IL-2 mRNA synthesis in lymphocytes in the presence of ConA and rIL-2 by 40%. IL-2 mRNA synthesis in lymphoid cells obtained from animals after IS and after IS in combination with the administration in vitro of the cytotoxic drug CsA to the splenic lymphocytes was inhibited (30% and 99%), accordingly, as compared with control rats. Induction of c-fos mRNA synthesis in rat brain cells was noted 30 minutes after RS in the hypothalamus (lateralis hypothalamic area, LHA), thalamus, corpus collosum, and sensorimotor zone of the brain cortex. IL-2 mRNA synthesis was shown two hours after RS in the same structures. The increased number of c-fos mRNA-positive cells two hours after TT injection was shown in the posterior hypothalamus area (PHA), LHA, dorsomedial nucleus (DMH), ventromedial nucleus (VMH), and anterior hypothalamus area (AHA) as compared to the effect of i.v. saline injection. Moreover, IL-2 mRNA-positive cell induction was noted in the PHA, DMH, and VMH. Six hours after TT injection, c-fos mRNA expression was decreased in the PHA, LHA, and AHA. Activation of c-fos and IL-2 mRNA was detected in the paraventricularis nucleus 6 hours after TT i.v. injection. Thus, inhibition or stimulation of IL-2 gene expression in lymphoid cells depends on the nature of the stressors. RS or antigenic stimuli induce c-fos and IL-2 gene expression in definite structures of the brain

  13. The Cryptococcus neoformans Gene DHA1 Encodes an Antigen That Elicits a Delayed-Type Hypersensitivity Reaction in Immune Mice

    PubMed Central

    Mandel, M. Alejandra; Grace, Greg G.; Orsborn, Kris I.; Schafer, Fredda; Murphy, Juneann W.; Orbach, Marc J.; Galgiani, John N.

    2000-01-01

    When mice are vaccinated with a culture filtrate from Cryptococcus neoformans (CneF), they mount a protective cell-mediated immune response as detected by dermal delayed-type hypersensitivity (DTH) to CneF. We have identified a gene (DHA1) whose product accounts at least in part for the DTH reactivity. Using an acapsular mutant (Cap-67) of C. neoformans strain B3501, we prepared a culture filtrate (CneF-Cap67) similar to that used for preparing the commonly used skin test antigen made with C. neoformans 184A (CneF-184A). CneF-Cap67 elicited DTH in mice immunized with CneF-184A. Deglycosylation of CneF-Cap67 did not diminish its DTH activity. Furthermore, size separation by either chromatography or differential centrifugation identified the major DTH activity of CneF-Cap67 to be present in fractions that contained proteins of approximately 19 to 20 kDa. Using N-terminal and internal amino acid sequences derived from the 20-kDa band, oligonucleotide primers were designed, two of which produced a 776-bp amplimer by reverse transcription-PCR (RT-PCR) using RNA from Cap-67 to prepare cDNA for the template. The amplimer was used as a probe to isolate clones containing the full-length DHA1 gene from a phage genomic library prepared from strain B3501. The full-length cDNA was obtained by 5′ rapid amplification of cDNA ends and RT-PCR. Analysis of DHA1 revealed a similarity between the deduced open reading frame and that of a developmentally regulated gene from Lentinus edodes (shiitake mushroom) associated with fruiting-body formation. Also, the gene product contained several amino acid sequences identical to those determined biochemically from the purified 20-kDa peptide encoded by DHA1. Recombinant DHA1 protein expressed in Escherichia coli was shown to elicit DTH reactions similar to those elicited by CneF-Cap67 in mice immunized against C. neoformans. Thus, DHA1 is the first gene to be cloned from C. neoformans whose product has been shown to possess immunologic

  14. Spermine and Spermidine Alter Gene Expression and Antigenic Profile of Borrelia burgdorferi

    PubMed Central

    Lin, Ying-Han; Romo, Jesus A.; Smith, Trever C.; Reyes, Ann N.; Karna, S. L. Rajasekhar; Miller, Christine L.; Van Laar, Tricia A.; Yendapally, Raghunandan; Chambers, James P.

    2017-01-01

    ABSTRACT Borrelia burgdorferi, the agent of Lyme disease, responds to numerous host-derived signals to alter adaptive capabilities during its enzootic cycle in an arthropod vector and mammalian host. Molecular mechanisms that enable B. burgdorferi to detect, channel, and respond to these signals have become an intense area of study for developing strategies to limit transmission/infection. Bioinformatic analysis of the borrelial genome revealed the presence of polyamine transport components (PotA, PotB, PotC, and PotD), while homologs for polyamine biosynthesis were conspicuously absent. Although potABCD is cotranscribed, the level of PotA was elevated under in vitro growth conditions mimicking unfed ticks compared to the level in fed ticks, while the levels of PotD were similar under the aforementioned conditions in B. burgdorferi. Among several polyamines and polyamine precursors, supplementation of spermine or spermidine in the borrelial growth medium induced synthesis of major regulators of gene expression in B. burgdorferi, such as RpoS and BosR, with a concomitant increase in proteins that contribute to colonization and survival of B. burgdorferi in the mammalian host. Short transcripts of rpoS were elevated in response to spermidine, which was correlated with increased protein levels of RpoS. Transcriptional analysis of rpoZ and B. burgdorferi rel (relBbu; bb0198) in the presence of spermidine revealed the interplay of multiple regulatory factors in B. burgdorferi gene expression. The effect of spermidine on the levels of select borrelial proteins was also influenced by serum factors. These studies suggest that multiple host-derived signals/nutrients and their transport systems contribute to B. burgdorferi adaptation during the vector and vertebrate host phases of infection. PMID:28052993

  15. Spermine and Spermidine Alter Gene Expression and Antigenic Profile of Borrelia burgdorferi.

    PubMed

    Lin, Ying-Han; Romo, Jesus A; Smith, Trever C; Reyes, Ann N; Karna, S L Rajasekhar; Miller, Christine L; Van Laar, Tricia A; Yendapally, Raghunandan; Chambers, James P; Seshu, J

    2017-03-01

    Borrelia burgdorferi, the agent of Lyme disease, responds to numerous host-derived signals to alter adaptive capabilities during its enzootic cycle in an arthropod vector and mammalian host. Molecular mechanisms that enable B. burgdorferi to detect, channel, and respond to these signals have become an intense area of study for developing strategies to limit transmission/infection. Bioinformatic analysis of the borrelial genome revealed the presence of polyamine transport components (PotA, PotB, PotC, and PotD), while homologs for polyamine biosynthesis were conspicuously absent. Although potABCD is cotranscribed, the level of PotA was elevated under in vitro growth conditions mimicking unfed ticks compared to the level in fed ticks, while the levels of PotD were similar under the aforementioned conditions in B. burgdorferi Among several polyamines and polyamine precursors, supplementation of spermine or spermidine in the borrelial growth medium induced synthesis of major regulators of gene expression in B. burgdorferi, such as RpoS and BosR, with a concomitant increase in proteins that contribute to colonization and survival of B. burgdorferi in the mammalian host. Short transcripts of rpoS were elevated in response to spermidine, which was correlated with increased protein levels of RpoS. Transcriptional analysis of rpoZ and B. burgdorferirel (relBbu ; bb0198) in the presence of spermidine revealed the interplay of multiple regulatory factors in B. burgdorferi gene expression. The effect of spermidine on the levels of select borrelial proteins was also influenced by serum factors. These studies suggest that multiple host-derived signals/nutrients and their transport systems contribute to B. burgdorferi adaptation during the vector and vertebrate host phases of infection. Copyright © 2017 American Society for Microbiology.

  16. Effect of deletion of genes involved in lipopolysaccharide core and O-antigen synthesis on virulence and immunogenicity of Salmonella enterica serovar typhimurium.

    PubMed

    Kong, Qingke; Yang, Jiseon; Liu, Qing; Alamuri, Praveen; Roland, Kenneth L; Curtiss, Roy

    2011-10-01

    Lipopolysaccharide (LPS) is a major virulence factor of Salmonella enterica serovar Typhimurium and is composed of lipid A, core oligosaccharide (C-OS), and O-antigen polysaccharide (O-PS). While the functions of the gene products involved in synthesis of core and O-antigen have been elucidated, the effect of removing O-antigen and core sugars on the virulence and immunogenicity of Salmonella enterica serovar Typhimurium has not been systematically studied. We introduced nonpolar, defined deletion mutations in waaG (rfaG), waaI (rfaI), rfaH, waaJ (rfaJ), wbaP (rfbP), waaL (rfaL), or wzy (rfc) into wild-type S. Typhimurium. The LPS structure was confirmed, and a number of in vitro and in vivo properties of each mutant were analyzed. All mutants were significantly attenuated compared to the wild-type parent when administered orally to BALB/c mice and were less invasive in host tissues. Strains with ΔwaaG and ΔwaaI mutations, in particular, were deficient in colonization of Peyer's patches and liver. This deficiency could be partially overcome in the ΔwaaI mutant when it was administered intranasally. In the context of an attenuated vaccine strain delivering the pneumococcal antigen PspA, all of the mutations tested resulted in reduced immune responses against PspA and Salmonella antigens. Our results indicate that nonreversible truncation of the outer core is not a viable option for developing a live oral Salmonella vaccine, while a wzy mutant that retains one O-antigen unit is adequate for stimulating the optimal protective immunity to homologous or heterologous antigens by oral, intranasal, or intraperitoneal routes of administration.

  17. Effect of Deletion of Genes Involved in Lipopolysaccharide Core and O-Antigen Synthesis on Virulence and Immunogenicity of Salmonella enterica Serovar Typhimurium▿

    PubMed Central

    Kong, Qingke; Yang, Jiseon; Liu, Qing; Alamuri, Praveen; Roland, Kenneth L.; Curtiss, Roy

    2011-01-01

    Lipopolysaccharide (LPS) is a major virulence factor of Salmonella enterica serovar Typhimurium and is composed of lipid A, core oligosaccharide (C-OS), and O-antigen polysaccharide (O-PS). While the functions of the gene products involved in synthesis of core and O-antigen have been elucidated, the effect of removing O-antigen and core sugars on the virulence and immunogenicity of Salmonella enterica serovar Typhimurium has not been systematically studied. We introduced nonpolar, defined deletion mutations in waaG (rfaG), waaI (rfaI), rfaH, waaJ (rfaJ), wbaP (rfbP), waaL (rfaL), or wzy (rfc) into wild-type S. Typhimurium. The LPS structure was confirmed, and a number of in vitro and in vivo properties of each mutant were analyzed. All mutants were significantly attenuated compared to the wild-type parent when administered orally to BALB/c mice and were less invasive in host tissues. Strains with ΔwaaG and ΔwaaI mutations, in particular, were deficient in colonization of Peyer's patches and liver. This deficiency could be partially overcome in the ΔwaaI mutant when it was administered intranasally. In the context of an attenuated vaccine strain delivering the pneumococcal antigen PspA, all of the mutations tested resulted in reduced immune responses against PspA and Salmonella antigens. Our results indicate that nonreversible truncation of the outer core is not a viable option for developing a live oral Salmonella vaccine, while a wzy mutant that retains one O-antigen unit is adequate for stimulating the optimal protective immunity to homologous or heterologous antigens by oral, intranasal, or intraperitoneal routes of administration. PMID:21768282

  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. Structure of O-antigen and functional characterization of O-antigen gene cluster of Salmonella enterica O47 containing ribitol phosphate and 2-acetimidoylamino-2,6-dideoxy-L-galactose.

    PubMed

    Perepelov, A V; Liu, B; Senchenkova, S N; Shashkov, A S; Feng, L; Wang, L; Knirel, Y A

    2009-04-01

    An O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Salmonella enterica O47 and studied by sugar analysis along with one- and two-dimensional 1H- and 13C-NMR spectroscopy. The following structure of the linear ribitol phosphate-containing repeating unit of the O-polysaccharide was established: -->2)-D-Ribitol-5-P-(O-->6)-alpha-D-Galp-(1-->3)-alpha-L-FucpNAm-(1-->3)-beta-D-GlcpNAc-(1-->, where FucNAm stands for 2-acetimidoylamino-2,6-dideoxy-L-galactose. About 10% of Gal is O-acetylated at position 4 and another minor O-acetyl group is present at an undetermined position. Functions of the S. enterica O47 antigen biosynthetic genes were tentatively assigned by comparison with gene databases and found to be in agreement with the O-polysaccharide structure. A comparison of the O-antigen gene clusters of S. enterica O47 and E. coli O145 suggested their close evolutionary relationship.

  20. Structuraland antigenic analysis of a new Rhoptry Pseudokinase Gene (ROP54) in Toxoplasma gondii.

    PubMed

    Zhou, Jian; Lu, Gang; Wang, Lin; Zhou, Aihua H; Han, Yali L; Guo, Jingjing J; Song, Pengxia X; Zhou, Huaiyu Y; Cong, Hua; Hou, Ming; He, Shenyi Y

    2017-09-26

    Toxoplasma gondii is defined as an obligate intracellular apicomplexan parasite and influences approximatelyone-third of the human all over the world. ROP54 protein is expressed in the rhoptry of Toxoplasma gondii. In the present study, we used SMART software to analyzethe secondary structure of ROP54. The 3D model of ROP54 protein was constructed and analyzed using SWISS-MODEL server and VMD software. The structure results fully showed that ROP54 proteinis an importantmember from the ROP family. Moreover, DNAMAN software and Epitope Database online service were used to analyze liner-B cell epitopes and Th-cell epitopes of the protein. The bioinformatics prediction of ROP54 protein could provide positive information on treatment and vaccine for toxoplasmosis. Furthermore, ROP54 gene was obtained from PCR, and a recombinant eukaryotic expression vector (pEGFP-ROP54) was constructed in the following study. After identification of enzyme digestion, the constructed plasmid was transfected into HEK 293-T cells. The RT-PCR result suggested that the recombinant plasmid could transcribe successfully in HEK 293-T cell.

  1. 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

  2. Cloning, partial sequence, expression, and antigenic analysis of the filamentous hemagglutinin gene of Bordetella pertussis.

    PubMed Central

    Delisse-Gathoye, A M; Locht, C; Jacob, F; Raaschou-Nielsen, M; Heron, I; Ruelle, J L; de Wilde, M; Cabezon, T

    1990-01-01

    The gene coding for the filamentous hemagglutinin (FHA), one of the main factors involved in mediating adherence of Bordetella pertussis to ciliated host cells, was cloned in Escherichia coli, and the 3,500-base-pair nucleotide sequence encoding the amino-terminal region was determined. Molecular cloning, together with the characterization of recombinant FHA-related proteins produced in E. coli, revealed that the primary translation product is a protein of about 370 kilodaltons (kDa). The mature 220-kDa FHA polypeptide secreted by B. pertussis is most probably generated by proteolytic processing that eliminates a carboxy-terminal portion of about 150 kDa. The 1,087 amino-terminal residues of the predicted FHA sequence showed a number of remarkable features. Extensive homology to the Serratia marcescens and Proteus mirabilis hemolysin proteins was found between amino acids 91 and 205 of the FHA sequence, suggesting involvement of this FHA domain in host cell binding or secretion of FHA from B. pertussis. In addition, two regions containing repetitive amino acid sequences were identified. One region, extending from residues 382 to 664, was formed by six repeats, and a second, extending from residues 701 to 912, contained three repeats. The reactivities of several recombinant FHA-derived proteins with a panel of monoclonal antibodies identified at least four epitopes composing an immunoreactive domain present in the carboxy-terminal moiety of the mature FHA. Images PMID:1696934

  3. Sequence Variation Analysis of Epstein-Barr Virus Nuclear Antigen 1 Gene in the Virus Associated Lymphomas of Northern China.

    PubMed

    Sun, Lingling; Zhao, Zhenzhen; Liu, Song; Liu, Xia; Sun, Zhifu; Luo, Bing

    2015-01-01

    Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is the only viral protein expressed in all EBV-positive tumors as it is essential for the maintenance, replication and transcription of the virus genome. According to the polymorphism of residue 487 in EBNA1 gene, EBV isolates can be classified into five subtypes: P-ala, P-thr, V-val, V-leu and V-pro. Whether these EBNA1 subtypes contribute to different tissue tropism of EBV and are consequently associated with certain malignancies remain to be determined. To elucidate the relationship, one hundred and ten EBV-positive lymphoma tissues of different types from Northern China, a non-NPC endemic area, were tested for the five subtypes by nested-PCR and DNA sequencing. In addition, EBV type 1 and type 2 classification was typed by using standard PCR assays across type-specific regions of the EBNA3C genes. Four EBNA1 subtypes were identified: V-val (68.2%, 75/110), P-thrV (15.5%, 17/110), V-leuV (3.6%, 4/110) and P-ala (10.9%, 12/110). The distribution of the EBNA1 subtypes in the four lymphoma groups was not significantly different (p = 0.075), neither was that of the EBV type 1/type 2 (p = 0.089). Compared with the previous data of gastric carcinoma (GC), nasopharyngeal carcinoma (NPC) and throat washing (TW) from healthy donors, the distribution of EBNA1 subtypes in lymphoma differed significantly (p = 0.016), with a little higher frequency of P-ala subtype. The EBV type distribution between lymphoma and the other three groups was significantly different (p = 0.000, p = 0.000, p = 0.001, respectively). The proportion of type 1 and type 2 mixed infections was higher in lymphoma than that in GC, NPC and TW. In lymphomas, the distribution of EBNA1 subtypes in the three EBV types was not significantly different (p = 0.546). These data suggested that the variation patterns of EBNA1 gene may be geographic-associated rather than tumor-specific and the role of EBNA1 gene variations in tumorigenesis needs more extensive and

  4. Sequence Variation Analysis of Epstein-Barr Virus Nuclear Antigen 1 Gene in the Virus Associated Lymphomas of Northern China

    PubMed Central

    Sun, Lingling; Zhao, Zhenzhen; Liu, Song; Liu, Xia; Sun, Zhifu; Luo, Bing

    2015-01-01

    Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is the only viral protein expressed in all EBV-positive tumors as it is essential for the maintenance, replication and transcription of the virus genome. According to the polymorphism of residue 487 in EBNA1 gene, EBV isolates can be classified into five subtypes: P-ala, P-thr, V-val, V-leu and V-pro. Whether these EBNA1 subtypes contribute to different tissue tropism of EBV and are consequently associated with certain malignancies remain to be determined. To elucidate the relationship, one hundred and ten EBV-positive lymphoma tissues of different types from Northern China, a non-NPC endemic area, were tested for the five subtypes by nested-PCR and DNA sequencing. In addition, EBV type 1 and type 2 classification was typed by using standard PCR assays across type-specific regions of the EBNA3C genes. Four EBNA1 subtypes were identified: V-val (68.2%, 75/110), P-thrV (15.5%, 17/110), V-leuV (3.6%, 4/110) and P-ala (10.9%, 12/110). The distribution of the EBNA1 subtypes in the four lymphoma groups was not significantly different (p = 0.075), neither was that of the EBV type 1/type 2 (p = 0.089). Compared with the previous data of gastric carcinoma (GC), nasopharyngeal carcinoma (NPC) and throat washing (TW) from healthy donors, the distribution of EBNA1 subtypes in lymphoma differed significantly (p = 0.016), with a little higher frequency of P-ala subtype. The EBV type distribution between lymphoma and the other three groups was significantly different (p = 0.000, p = 0.000, p = 0.001, respectively). The proportion of type 1 and type 2 mixed infections was higher in lymphoma than that in GC, NPC and TW. In lymphomas, the distribution of EBNA1 subtypes in the three EBV types was not significantly different (p = 0.546). These data suggested that the variation patterns of EBNA1 gene may be geographic-associated rather than tumor-specific and the role of EBNA1 gene variations in tumorigenesis needs more extensive and

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

    PubMed

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

    1997-11-25

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

  6. 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.

  7. 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.

  8. 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

  9. Immunogenicity of recombinant attenuated Salmonella enterica serovar Typhimurium vaccine strains carrying a gene that encodes Eimeria tenella antigen S07

    USDA-ARS?s Scientific Manuscript database

    In an attempt to develop an efficacious vaccine against avian coccidiosis, research was conducted using Type III Secretion System (TTSS) of Salmonella to deliver Eimeria antigens into the cytoplasm of host cells. Once delivered, recombinant protein may enter the MHC I antigen processing pathway for...

  10. [Study on the antigenicity and HA1 gene characteristics of influenza A viruses during 2004-2008 year in China].

    PubMed

    Zhang, Ye; Zhao, Xiang; Huang, Wei-juan; Guo, Jun-feng; Wei, He-jiang; Cheng, Yan-hui; Li, Xin-wan; Li, Xi-yan; Guo, Yuan-ji; Shu, Yue-long

    2009-12-01

    To under stand influenza A viruses epidemic, antigenicity and genetic characteristics variation between the vaccine and Circulation strains during 2004-2008 year in China. The influenza A viruses (H1N1, H3N2) isolated from 2004-2008 year were under took antigenic and sequence analysis. Influenza A virus antigenicity and genetic characteristics were analyzed thought amino acid variation compassion of HA1 protein of influenza A virus isolates. The antigenicity of influenza H1N1 subtype viruses isolated from 2004 to 2007 is very similar with vaccine strain A/New Caledonia/20/1999 (HIN1)-like virus. The influenza H1N1 viruses circulated in 2008 year had similar antigenic characteristics with A/Brisben/59/2007 (H1N1) which is component of influenza vaccines for northern hemisphere 2008-2009 year. The influenza H3N2 subtype viruses of 2004-2005 year had antigenic variation comparatively with vaccine strain A/Fujian/411/12002 (H3N2), The antigenicity of 2006-2007 H3N2 viruses and 2008 year's is A/Wiscansin/67/2006(H3N2) and A/ Brisben/10/2006(H3N2) respectively. There is change of influenza A viruses (H1N1, H3N2) antigenic and genetic characteristics during 2004-2008 in China.

  11. Immune response genes controlling responsiveness to major transplantation antigens. Specific major histocompatibility complex-linked defect for antibody responses to class I alloantigens

    SciTech Connect

    Butcher, G.W.; Corvalan, J.R.; Licence, D.R.; Howard, J.C.

    1982-01-01

    We have identified two major histocompatibility complex (MHC)-linked Ir genes that control the antibody response made by rats against class I major alloantigens. We have named these genes Ir-RT1Aa and Ir-RT1Ac. These Ir genes determine responsiveness of the immunized animal in a typical codominant fashion. There is no evidence so far for trans-complementation between low-responder haplotypes. Detailed studies of Ir-RT1Aa indicate that it controls the antibody response to at least two distinct alloantigenic determinants on RT1Aa molecules. These class I molecules thus behave like hapten-carrier conjugates when the response against the carrier is under Ir gene control. Analysis of the origin of alloantibody-forming cells in tetraparental radiation chimeras indicates that Ir-RT1Aa must control the provision of effective help to B cells. In many respects therefore, the properties of Ir-RT1Aa are broadly similar to those described for Ir genes controlling antibody responses to conventional antigens. The existence of apparently conventional Ir genes controlling the antibody response to major alloantigens strongly suggest that the processing of these transmembrane molecules by host antigen-presenting cells is a prerequisite for immune induction, and that it is the MHC of the responder rather than that of the allograft to which T helper cells are restricted in alloimmune responses in vivo.

  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. Two distinct allotypic determinants on the antigen-specific suppressor and enhancing T cell factors that are encoded by genes linked to the immunoglobulin heavy chain locus

    PubMed Central

    1982-01-01

    The alloantiserum was raised in BALB/c (H-2d, Igh-1a) mice hyperimmunized with spleen cells of Igh allotype congenic mice, CB-20 (H-2d, Igh-1b). It was found to define the new allotypic determinants (distinct from B cell Igh constant region determinants: Igh allotype) expressed only on a small population of T cells belonging to the Thy-1 dull-stained Lyt-2- or Lyt-2+ population of Igh-1b mice. Genes coding for the determinants were shown to be accommodated somewhere in the right side of the Igh variable region gene (Igh-V) cluster, as the antibody activity was completely absorbed with BAB-14 thymocytes. It was also demonstrated that the products detected by the antiserum represent the allotypic determinants (probably constant region determinants) on the antigen-binding moiety of the antigen-specific augmenting (TaF) and suppressor (TsF) T cell factors. Moreover, determinants on TsF were found to be distinct from those on TaF. Therefore, it can be suggested that the two genes coding for the T cell allodeterminants (distinct from those of the B cell Igh) are located in the right side of the B cell Igh-V on the 12th chromosome, and that both encode the antigen-recognition units of the functionally distinct T cell factors. PMID:6172538

  15. Co-transfection of dendritic cells with AFP and IL-2 genes enhances the induction of tumor antigen-specific antitumor immunity.

    PubMed

    Yang, Jing-Yue; Li, Xiao; Gao, Li; Teng, Zeng-Hui; Liu, Wen-Chao

    2012-10-01

    Dendritic cells (DCs) are highly efficient, specialized antigen-presenting cells and DCs transfected with tumor-related antigens are regarded as promising vaccines in cancer immunotherapy. The aim of the present study was to investigate whether DCs co-transfected with the α-fetoprotein (AFP) and human interleukin-2 (IL-2) genes were able to induce stronger therapeutic antitumor immunity in transfected DCs. In this study, DCs from hepatocellular carcinoma (HCC) patients were co-transfected with the IL-2 gene and/or the AFP gene. The reverse transcription-PCR (RT-PCR) data revealed that the DCs transfected with the adenovirus AdAFP/IL-2 expressed AFP and IL-2. The DCs co-transfected with IL-2 and AFP (AFP/IL-2-DCs) enhanced the cytotoxicities of cytotoxic T lymphocytes (CTLs) and increased the production of IL-2 and interferon-γ significantly compared with their AFP-DC, green fluorescent protein (GFP)-DC, DC or phosphate-buffered saline (PBS) counterparts. In vivo data suggested that immunization with AFP-DCs enhances antigen-specific antitumor efficacy more potently than immunization with IL-2-DCs or AFP-DCs. These findings provide a potential strategy to improve the efficacy of DC-based tumor vaccines.

  16. 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

  17. STAT3 promotes CD1d-mediated lipid antigen presentation by regulating a critical gene in glycosphingolipid biosynthesis.

    PubMed

    Iyer, Abhirami K; Liu, Jianyun; Gallo, Richard M; Kaplan, Mark H; Brutkiewicz, Randy R

    2015-11-01

    Cytokines that regulate the immune response signal through the Janus kinase / signal transducer and activation of transcription (JAK/STAT) pathway, but whether this pathway can regulate CD1d-mediated lipid antigen presentation to natural killer T (NKT) cells is unknown. Here, we found that STAT3 promotes antigen presentation by CD1d. Antigen-presenting cells (APCs) in which STAT3 expression was inhibited exhibited markedly reduced endogenous lipid antigen presentation to NKT cells without an impact on exogenous lipid antigen presentation by CD1d. Consistent with this observation, in APCs where STAT3 was knocked down, dramatically decreased levels of UDP glucose ceramide glucosyltransferase (UGCG), an enzyme involved in the first step of glycosphingolipid biosynthesis, were observed. Impaired lipid antigen presentation was reversed by ectopic expression of UGCG in STAT3-silenced CD1d(+) APCs. Hence, by controlling a fundamental step in CD1d-mediated lipid antigen presentation, STAT3 signalling promotes innate immune responses driven by CD1d.

  18. Expression of the Yersinia enterocolitica O:3 LPS O-antigen and outer core gene clusters is RfaH-dependent.

    PubMed

    Leskinen, Katarzyna; Varjosalo, Markku; Li, Zhilin; Li, Chun-Mei; Skurnik, Mikael

    2015-06-01

    The antiterminator RfaH is required for the expression of LPS, capsule, haemolysin, exotoxin, haemin uptake receptor and F pilus. As these structures are critical for bacterial virulence, loss of RfaH usually leads to attenuation. Here, we inactivated the rfaH gene of Yersinia enterocolitica O:3 to study its role in this enteropathogen. RNA sequencing of the WT and ΔrfaH strain transcriptomes revealed that RfaH acted as a highly specific regulator that enhanced the transcription of the operons involved in biosynthesis of LPS O-antigen and outer core (OC), but did not affect the expression of enterobacterial common antigen. Interestingly, the transcriptome of the ΔrfaH strain was very similar to that of an O-antigen-negative mutant. This indicated that some of the changes seen in the ΔrfaH strain, such as the genes involved in outer membrane homeostasis or in the stress-response-associated Cpx pathway, were actually due to indirect responses via the loss of O-antigen. The decreased amount of LPS on the ΔrfaH strain cell surface resulted in an attenuated stress response, and lower resistance to compounds such as SDS and polymyxin B. However, the ΔrfaH strain was significantly more resistant to complement-mediated killing by normal human serum. Taken together, our results revealed a novel role of RfaH acting as a highly specific regulator of O-antigen and OC of LPS in Y. enterocolitica O:3. It may be speculated that RfaH might have an in vivo role in controlling tissue-specific expression of bacterial surface oligo/polysaccharides.

  19. Expression of the developmental I antigen by a cloned human cDNA encoding a member of a beta-1,6-N-acetylglucosaminyltransferase gene family.

    PubMed

    Bierhuizen, M F; Mattei, M G; Fukuda, M

    1993-03-01

    The blood group i/I antigens were the first identified alloantigens that display a dramatic change during human development. The i and I antigens are determined by linear and branched poly-N-acetyllactosaminoglycans, respectively. In human erythrocytes during embryonic development, the fetal (i) antigen is replaced by the adult (I) antigen as a result of the appearance of a beta-1,6-N-acetylglucosaminyltransferase, the I-branching enzyme. Here, we report the cDNA cloning and expression of this branching enzyme that converts linear into branched poly-N-acetyllactosaminoglycans, thus introducing the I antigen in transfected cells. The cDNA sequence predicts a protein with type II membrane topology as has been found for all other mammalian glycosyltransferases cloned to date. The Chinese hamster ovary cells that stably express the isolated cDNA acquire I-branched structures as evidenced by the structural analysis of glycopeptides from these cells. Comparison of the amino acid sequence with those of other glycosyltransferases revealed that this I-branching enzyme and another beta-1,6-N-acetylglucosaminyltransferase that forms a branch in O-glycans are strongly homologous in the center of their putative catalytic domains. Moreover, the genes encoding these two beta-1,6-N-acetylglucosaminyltransferases were found to be located at the same locus on chromosome 9, band q21. These results indicate that the I-branching enzyme represents a member of a beta-1,6-N-acetylglucosaminyltransferase gene family of which expression is controlled by developmental programs.

  20. Use of human antigen presenting cell gene array profiling to examine the effect of human T-cell leukemia virus type 1 Tax on primary human dendritic cells.

    PubMed

    Ahuja, Jaya; Kampani, Karan; Datta, Suman; Wigdahl, Brian; Flaig, Katherine E; Jain, Pooja

    2006-02-01

    Human T-cell leukemia virus type 1 (HTLV-1) is etiologically linked to adult T-cell leukemia and a progressive demyelinating disorder termed HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). One of the most striking features of the immune response in HAM/TSP centers on the expansion of HTLV-1-specific CD8(+) cytotoxic T lymphocyte (CTL) compartment in the peripheral blood and cerebrospinal fluid. More than 90% of the HTLV-1-specific CTLs are directed against the viral Tax (11-19) peptide implying that Tax is available for immune recognition by antigen presenting cells, such as dendritic cells (DCs). DCs obtained from HAM/TSP patients have been shown to be infected with HTLV-1 and exhibit rapid maturation. Therefore, we hypothesized that presentation of Tax peptides by activated DCs to naIve CD8(+) T cells may play an important role in the induction of a Tax-specific CTL response and neurologic dysfunction. In this study, a pathway-specific antigen presenting cell gene array was used to study transcriptional changes induced by exposure of monocyte-derived DCs to extracellular HTLV-1 Tax protein. Approximately 100 genes were differentially expressed including genes encoding toll-like receptors, cell surface receptors, proteins involved in antigen uptake and presentation and adhesion molecules. The differential regulation of chemokines and cytokines characteristic of functional DC activation was also observed by the gene array analyses. Furthermore, the expression pattern of signal transduction genes was also significantly altered. These results have suggested that Tax-mediated DC gene regulation might play a critical role in cellular activation and the mechanisms resulting in HTLV-1-induced disease.

  1. Spontaneous Activation of Antigen-presenting Cells by Genes Encoding Truncated Homo-Oligomerizing Derivatives of CD40.

    PubMed

    Levin, Noam; Pato, Aviad; Cafri, Gal; Eisenberg, Galit; Peretz, Tamar; Margalit, Alon; Lotem, Michal; Gross, Gideon

    The interaction between the CD40 receptor on antigen-presenting cells (APCs) and its trimeric ligand on CD4 T cells is essential for the initiation and progression of the adaptive immune response. Here we undertook to endow CD40 with the capacity to trigger spontaneous APC activation through ligand-independent oligomerization. To this end we exploited the GCN4 yeast transcriptional activator, which contains a leucine zipper DNA-binding motif that induces homophilic interactions. We incorporated GCN4 variants forming homodimers, trimers, or tetramers at the intracellular domain of human and mouse CD40 and replaced the extracellular portion with peptide-β2m or other peptide tags. In parallel we examined similarly truncated CD40 monomers lacking a GCN4 motif. The oligomeric products appeared to arrange in high-molecular-weight aggregates and were considerably superior to the monomer in their ability to trigger nuclear factor kB signaling, substantiating the anticipated constitutively active (ca) phenotype. Cumulative results in human and mouse APC lines transfected with caCD40 mRNA revealed spontaneous upregulation of CD80, IL-1β, TNFα, IL-6, and IL-12, which could be further enhanced by caTLR4 mRNA. In mouse bone-marrow-derived dendritic cells caCD40 upregulated CD80, CD86, MHC-II, and IL-12 and in human monocyte-derived dendritic cells it elevated surface CD80, CD83 CD86, CCR7, and HLA-DR. Oligomeric products carrying the peptide-β2m extracellular portion could support MHC-I presentation of the linked peptide up to 4 days post-mRNA transfection. These findings demonstrate that the expression of a single caCD40 derivative in APCs can exert multiple immunostimulatory effects, offering a new powerful tool in the design of gene-based cancer vaccines.

  2. Variations of Epstein-Barr virus nuclear antigen 1 gene in gastric carcinomas and nasopharyngeal carcinomas from Northern China.

    PubMed

    Wang, Yun; Liu, Xia; Xing, Xiaoming; Cui, Ying; Zhao, Chengquan; Luo, Bing

    2010-02-01

    The Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1), the only viral protein consistently expressed in all EBV-associated tumors, is classified into five distinct subtypes: P-ala, P-thr, V-leu, V-val and V-pro based on the signature changes at amino acid residue 487. By now, whether the EBNA1 subtypes preferentially associate with particular malignancies or represent geographical polymorphism remains controversial. In China, most studies of the EBNA1 variations focused on nasopharyngeal carcinoma (NPC) in endemic area, among which some suggested the V-val subtype is preferentially associated with NPC. To characterize the variations of EBNA1 in NPC non-endemic area in China and to explore the association of EBNA1 variations with EBV-associated gastric carcinoma (EBVaGC) and NPC, the C-terminal sequences of EBNA1 were analyzed for 41 EBVaGC, 41 NPC biopsies and 55 throat washing (TW) samples from healthy donors in Northern China. Three major patterns of the EBNA1 variations, V-val, P-thrV and V-leuV, were observed, and V-val was the most common subtype in all the three groups, followed by P-thrV and V-leuV. The distribution of the EBNA1 subtypes among EBVaGC, NPC and healthy donors was not significantly different (P>0.05). In addition, preferential linkages between EBNA1 subtypes and EBNA3C variants were found to exist. There was no evidence that particular EBNA1 subtypes are preferentially associated with EBVaGC or NPC in Northern China, suggesting that EBNA1 gene variations are geographically restricted rather than tumor-specific polymorphisms. (c) 2009 Elsevier B.V. All rights reserved.

  3. Isolation of a phylogenetically conserved and testis-specific gene using a monoclonal antibody against the serological H-Y antigen.

    PubMed

    Su, H; Kozak, C A; Veerhuis, R; Lau, Y F; Wiberg, U

    1992-04-01

    Several cDNA clones of a gene termed male-enhanced antigen-2 (Mea-2), have been isolated from a mouse testicular expression cDNA library using a monoclonal histocompatability Y (H-Ys) antibody which detects specific protein(s) present in the mouse testis but not the ovary. The Mea-2 gene is phylogenetically conserved among various mammalian species examined, and is expressed at high levels in adult mouse testis. The expression pattern of Mea-2 is very similar to that of another gene, the male-enhanced antigen-1 (Mea-1), previously isolated using a polyclonal H-Ys antibody. Northern blotting and RT-PCR analyses demonstrated that Mea-2 is also expressed in other adult and fetal mouse organs at low levels. The testis-enhanced expression of this gene is associated with germ cell development at mid- to late-meiotic stages of spermatogenesis. Analysis of an intersubspecies mouse backcross has assigned this gene to chromosome 5, between the loci Gus and Hnf-1.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    Bruske, Ellen Inga; Dimonte, Sandra; Enderes, Corinna; Tschan, Serena; Flötenmeyer, Matthias; Koch, Iris; Berger, Jürgen; Kremsner, Peter; Frank, Matthias

    2016-01-01

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

  6. 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.

  7. Selective killing of lung cancer cells using carcinoembryonic antigen promoter and double suicide genes, thymidine kinase and cytosine deaminase (pCEA-TK/CD).

    PubMed

    Qiu, Yuan; Peng, Gui-Lin; Liu, Qi-Cai; Li, Fu-Li; Zou, Xu-Sen; He, Jian-Xing

    2012-03-01

    The application of gene therapy in cancer treatment is limited by non-specific targeting. In the present study, we constructed a recombinant plasmid, containing a carcinoembryonic antigen (CEA) promoter and double suicide genes thymidine kinase (TK) and cytosine deaminase (CD), henceforth referred to as pCEA-TK/CD. Our results showed that the CEA promoter can specifically drive target gene expression in CEA-positive lung cancer cells. In the presence of prodrugs 5-flucytosine and ganciclovir, pCEA-TK/CD transfection decreased inhibitory concentration 50 and increased apoptosis and cyclomorphosis. Our result suggests that gene therapy using pCEA-TK/CD may be a promising new approach for treating lung cancer.

  8. A short polypeptide from the herpes simplex virus type 2 ICP10 gene can induce antigen aggregation and autophagosomal degradation for enhanced immune presentation.

    PubMed

    Fu, Xinping; Tao, Lihua; Zhang, Xiaoliu

    2010-12-01

    It has been reported that certain polypeptides derived from aggregation-prone cellular proteins can turn soluble green fluorescent protein (GFP) into aggregates. Here we report our finding that a short peptide derived from a viral gene, ICP10 of herpes simplex virus (HSV)-2, also possesses such a property. A sequence as short as 13 amino acids from the middle region of the gene can convert GFP into an aggregation-prone toxic protein once it is fused to the C terminus. Moreover, this short peptide can direct a surrogate tumor antigen into the autophagosome/lysosome degradation pathway, drastically increasing both MHC class I and class II antigen presentation. The simultaneous induction of both arms of the T cell immune response to the tumor antigen effectively protects the immunized animals from tumor challenge. Designated VIPA (i.e., viral inducer of protein aggregation), this unique viral sequence may represent an attractive candidate as a molecular adjuvant for cancer immunotherapy and for other immunologically preventable diseases.

  9. The PANE1 gene encodes a novel human minor histocompatibility antigen that is selectively expressed in B-lymphoid cells and B-CLL

    PubMed Central

    Brickner, Anthony G.; Evans, Anne M.; Mito, Jeffrey K.; Xuereb, Suzanne M.; Feng, Xin; Nishida, Tetsuya; Fairfull, Liane; Ferrell, Robert E.; Foon, Kenneth A.; Hunt, Donald F.; Shabanowitz, Jeffrey; Engelhard, Victor H.; Riddell, Stanley R.; Warren, Edus H.

    2006-01-01

    Minor histocompatibility antigens (mHAg's) are peptides encoded by polymorphic genes that are presented by major histocompatibility complex (MHC) molecules and recognized by T cells in recipients of allogeneic hematopoietic cell transplants. Here we report that an alternative transcript of the proliferation-associated nuclear element 1 (PANE1) gene encodes a novel human leukocyte antigen (HLA)-A*0301-restricted mHAg that is selectively expressed in B-lymphoid cells. The antigenic peptide is entirely encoded within a unique exon not present in other PANE1 transcripts. Sequencing of PANE1 alleles in mHAg-positive and mHAg-negative cells demonstrates that differential T-cell recognition is due to a single nucleotide polymorphism within the variant exon that replaces an arginine codon with a translation termination codon. The PANE1 transcript that encodes the mHAg is expressed at high levels in resting CD19+ B cells and B-lineage chronic lymphocytic leukemia (B-CLL) cells, and at significantly lower levels in activated B cells. Activation of B-CLL cells through CD40 ligand (CD40L) stimulation decreases expression of the mHAg-encoding PANE1 transcript and reciprocally increases expression of PANE1 transcripts lacking the mHAg-encoding exon. These studies suggest distinct roles for different PANE1 isoforms in resting compared with activated CD19+ cells, and identify PANE1 as a potential therapeutic target in B-CLL. PMID:16391015

  10. Human Leukocyte Antigen (HLA) A*1101-restricted Epstein-Barr Virus-specific T-cell Receptor Gene Transfer to Target Nasopharyngeal Carcinoma

    PubMed Central

    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.

    2015-01-01

    Infusing virus-specific T cells is effective treatment for rare Epstein-Barr virus (EBV)-associated post-transplant 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 favour 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 HLA A*1101-restricted TCR, which would be widely applicable since 40% of NPC patients carry this HLA allele. Studying both the wild-type and modified forms we have optimised expression of the TCR and demonstrated high avidity antigen-specific function (proliferation, cytotoxicity, cytokine release) in both CD8+ and CD4+ T cells. The engineered T cells also inhibited LMP2+ epithelial tumour 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. PMID:25711537

  11. 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.

  12. Identification of Brucella abortus genes in elk (Cervus elaphus) using in vivo-induced antigen technology (IVIAT) reveals novel markers of infection.

    PubMed

    Lowry, J E; Goodridge, L; Vernati, G; Fluegel, A M; Edwards, W H; Andrews, G P

    2010-05-19

    Elk in the Greater Yellowstone Area are a major reservoir for brucellosis, which represents an obstacle to eradication of the disease in domestic livestock. Furthermore, immune responses to Brucella abortus infection in the wild host are not well-understood. In this regard, in vivo-induced antigen technology (IVIAT) was employed to identify novel B. abortus antigens expressed during infection in elk. Sera collected from sero-positive Wyoming elk were pooled and absorbed against in vitro-grown cultures of B. abortus. Approximately 35,000 E. coli clones, expressing B. abortus DNA, were then screened by colony immunoblot, yielding ten genes with immuno-reactive products, to include seven proteins secreted beyond the inner membrane. Three products, an outer membrane protein (D15), malate dehydrogenase (Mdh), and an ion transporter (AfuA), were examined by Western blot against individual elk serum samples. Sero-reactivity was significantly more frequent for both Mdh and D15 in naturally infected animals, compared to vaccinated and uninfected elk, indicating that antibody to these two antigens is a predictor of natural infection. Cross-reactivity of all three proteins was next examined with serum samples from confirmed brucellosis-positive cattle. While variable patterns of reactivity were seen with the antigens, the sample group was equivalently reactive to AfuA and Mdh, compared to elk, suggesting that these antigens are commonly expressed during infection in both hosts. We conclude that the application of IVIAT to B. abortus may not only facilitate the identification of serologic markers for brucellosis in elk, but may provide further insight into biological processes of the pathogen in different hosts. Copyright 2009 Elsevier B.V. All rights reserved.

  13. Gene Therapy-Induced Antigen-Specific Tregs Inhibit Neuro-inflammation and Reverse Disease in a Mouse Model of Multiple Sclerosis.

    PubMed

    Keeler, Geoffrey D; Kumar, Sandeep; Palaschak, Brett; Silverberg, Emily L; Markusic, David M; Jones, Noah T; Hoffman, Brad E

    2017-09-19

    The devastating neurodegenerative disease multiple sclerosis (MS) could substantially benefit from an adeno-associated virus (AAV) immunotherapy designed to restore a robust and durable antigen-specific tolerance. However, developing a sufficiently potent and lasting immune-regulatory therapy that can intervene in ongoing disease is a major challenge and has thus been elusive. We addressed this problem by developing a highly effective and robust tolerance-inducing in vivo gene therapy. Using a pre-clinical animal model, we designed a liver-targeting gene transfer vector that expresses full-length myelin oligodendrocyte glycoprotein (MOG) in hepatocytes. We show that by harnessing the tolerogenic nature of the liver, this powerful gene immunotherapy restores immune tolerance by inducing functional MOG-specific regulatory T cells (Tregs) in vivo, independent of major histocompatibility complex (MHC) restrictions. We demonstrate that mice treated prophylactically are protected from developing disease and neurological deficits. More importantly, we demonstrate that when given to mice with preexisting disease, ranging from mild neurological deficits to severe paralysis, the gene immunotherapy abrogated CNS inflammation and significantly reversed clinical symptoms of disease. This specialized approach for inducing antigen-specific immune tolerance has significant therapeutic potential for treating MS and other autoimmune disorders. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  14. AntigenDB: an immunoinformatics database of pathogen antigens.

    PubMed

    Ansari, Hifzur Rahman; Flower, Darren R; Raghava, G P S

    2010-01-01

    The continuing threat of infectious disease and future pandemics, coupled to the continuous increase of drug-resistant pathogens, makes the discovery of new and better vaccines imperative. For effective vaccine development, antigen discovery and validation is a prerequisite. The compilation of information concerning pathogens, virulence factors and antigenic epitopes has resulted in many useful databases. However, most such immunological databases focus almost exclusively on antigens where epitopes are known and ignore those for which epitope information was unavailable. We have compiled more than 500 antigens into the AntigenDB database, making use of the literature and other immunological resources. These antigens come from 44 important pathogenic species. In AntigenDB, a database entry contains information regarding the sequence, structure, origin, etc. of an antigen with additional information such as B and T-cell epitopes, MHC binding, function, gene-expression and post translational modifications, where available. AntigenDB also provides links to major internal and external databases. We shall update AntigenDB on a rolling basis, regularly adding antigens from other organisms and extra data analysis tools. AntigenDB is available freely at http://www.imtech.res.in/raghava/antigendb and its mirror site http://www.bic.uams.edu/raghava/antigendb.

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

    DTIC Science & Technology

    1990-12-12

    Della-Porta and Westaway, 1977; Kitano et al., 1974; Heinz et al., 1981). In order to develop a subunit vaccine against dengue virus, it is important to...Antigens for Rapid Diagnosis and Vaccine Development DPC TAB 0 A .. asin]o ANNUAL PROGRESS REPORT ’Q;-10.v&,,,d 0 by By SAv.ailability Caote# Radha Krishnan...Type 2 Encoded Antigens for Rapid Diagnosis and Vaccine Development 12. PERSONAL AUTHOR(S) Radha K. Padmanabhan 13a. TYPE OF REPORT 13b. TIME COVERED

  16. Sensitive and high resolution in situ hybridization to human chromosomes using biotin labelled probes: assignment of the human thymocyte CD1 antigen genes to chromosome 1.

    PubMed Central

    Albertson, D G; Fishpool, R; Sherrington, P; Nacheva, E; Milstein, C

    1988-01-01

    A method for in situ hybridization originally developed for mapping genes in the nematode, Caenorhabditis elegans has been adapted for high resolution cytological mapping of genes in the human. The probe DNAs are labelled by incorporation of biotin dUTP and the site of hybridization detected by immunofluorescence. For the accurate assignment of the hybridization signal to chromosome bands, visualized by staining with Hoechst 33258, a heterologous ribosomal DNA probe is also included in the hybridization reaction. These rDNA signals are used as fiducial markers when aligning the two fluorescent images. We demonstrate the method by assignment of the human thymocyte CD1 antigen genes to human chromosome 1q22-23. Images PMID:3053166

  17. Gene gun-mediated DNA vaccination enhances antigen-specific immunotherapy at a late preclinical stage of type 1 diabetes in nonobese diabetic mice

    PubMed Central

    Goudy, Kevin S.; Wang, Bo; Tisch, Roland

    2008-01-01

    Type 1 diabetes (T1D) is characterized by the T cell mediated destruction of the insulin producing β cells. Antigen-specific immunotherapies are used to selectively tolerize β cell-specific pathogenic T cells either directly, or indirectly through the induction of immunoregulatory T cells. A key concern of antigen-specific immunotherapy is exacerbating autoimmunity. We compared the T cell reactivity and efficacy induced by plasmid DNA (pDNA) encoding glutamic acid decarboxylase 65 (GAD65) administered via intramuscular versus gene gun vaccination in NOD mice at a late preclinical stage of T1D. Whereas intramuscular injection of pGAD65 promoted a predominant type 1 CD4+ T cell response and failed to suppress ongoing β cell autoimmunity, gene gun vaccination preferentially induced IL-4 secreting CD4+ T cells and significantly delayed the onset of diabetes. These findings demonstrate that gene gun delivery of autoantigen-encoding pDNA preferentially elicits immunoregulatory T cells and offers a safe, effective mode of pDNA vaccination for the treatment of T1D and other autoimmune diseases. PMID:18675592

  18. Stability of multiple antigen receptor gene rearrangements and immunophenotype in Hodgkin's disease-derived cell line L428 and variant subline L428KSA.

    PubMed

    Athan, E S; Paietta, E; Papenhausen, P R; Augenlicht, L; Wiernik, P H; Gallagher, R E

    1989-07-01

    The Hodgkin's disease (HD) derived cell line L428 and a phorbol ester-selected subline L428KSA, which have been independently passaged in tissue culture for several years, were studied for possible antigen receptor gene and immunophenotypic differences. Multiple but identical alterations of these genes were found, including: the deletion of one and rearrangement of the other immunoglobulin (Ig) heavy chain allele; the rearrangement of one kappa and one lambda light chain allele; and the rearrangement of one T cell receptor (TCR) beta allele. Restriction mapping of the Ig heavy chain locus indicated that rearrangement of the retained allele produced a JH-C gamma 4 fusion product by an isotype switch mechanism. The 14q+ chromosome [t(14q32;?)] present in both cell cultures derived either from translocation 5' (telomeric) to the rearranged JH allele or 3' (centromeric) to the deleted Ig heavy chain allele and did not involve detectable rearrangement of the c-myc, bcl 1, or bcl 2 oncogenes. No differences in the immunophenotype were found between the L428 and L428KSA cells: both expressed leukocyte activation antigens and some determinants associated with myelomonocytic cells but no lymphoid markers. It is postulated that these phenotypic characteristics derived from secondary genetic events/differentiative reprogramming which produced extinction of primary lymphoid characters, including terminal deoxynucleotidyl transferase (TdT) essential to generation of the Ig and TCR gene rearrangements, and expression of an incomplete set of myelomonocytic markers.

  19. A rns-like regulatory gene for colonization factor antigen I (CFA/I) that controls expression of CFA/I pilin.

    PubMed Central

    Caron, J; Scott, J R

    1990-01-01

    Colonization factor antigens (CFA) are needed for adherence of human enterotoxigenic Escherichia coli (ETEC) strains to their hosts. The CFA/II antigens, CS1 and CS2, which are found in some ETEC strains, require the plasmid-encoded gene rns for expression (J. Caron, L. M. Coffield, and J. R. Scott, Proc. Natl. Acad. Sci. USA 86:963-967, 1989). Other ETEC strains express CFA/I, whose synthesis and assembly require genes on two unlinked regions (regions 1 and 2) of a plasmid (G. A. Willshaw, H. R. Smith, and B. Rowe, FEMS Microbiol. Lett. 16:101-106, 1983). We report that CFA/I region 2 DNA can substitute for rns to cause expression of CS1 and CS2. The cfaR gene in region 2 is defined by a mutation abolishing both expression of CFA/I and complementation of a rns mutant for expression of CS1 or CS2. In a strain containing only region 1, complementation for expression of CFA/I by a plasmid containing rns+ is inefficient but is adequate to cause hemagglutination by the CFA/I adhesin. Images PMID:1969396

  20. A binding site for the transcription factor Grainyhead/Nuclear transcription factor-1 contributes to regulation of the Drosophila proliferating cell nuclear antigen gene promoter.

    PubMed

    Hayashi, Y; Yamagishi, M; Nishimoto, Y; Taguchi, O; Matsukage, A; Yamaguchi, M

    1999-12-03

    The Drosophila proliferating cell nuclear antigen promoter contains multiple transcriptional regulatory elements, including upstream regulatory element (URE), DNA replication-related element, E2F recognition sites, and three common regulatory factor for DNA replication and DNA replication-related element-binding factor genes recognition sites. In nuclear extracts of Drosophila embryos, we detected a protein factor, the URE-binding factor (UREF), that recognizes the nucleotide sequence 5'-AAACCAGTTGGCA located within URE. Analyses in Drosophila Kc cells and transgenic flies revealed that the UREF-binding site plays an important role in promoter activity both in cultured cells and in living flies. A yeast one-hybrid screen using URE as a bait allowed isolation of a cDNA encoding a transcription factor, Grainyhead/nuclear transcription factor-1 (GRH/NTF-1). The nucleotide sequence required for binding to GRH was indistinguishable from that for UREF detected in embryo nuclear extracts. Furthermore, a specific antibody to GRH reacted with UREF in embryo nuclear extracts. From these results we conclude that GRH is identical to UREF. Although GRH has been thought to be involved in regulation of differentiation-related genes, this study demonstrates, for the first time, involvement of a GRH-binding site in regulation of the DNA replication-related proliferating cell nuclear antigen gene.

  1. Immune Responses Induced by Gene Gun or Intramuscular Injection of DNA Vaccines That Express Immunogenic Regions of the Serine Repeat Antigen from Plasmodium falciparum

    PubMed Central

    Belperron, Alexia A.; Feltquate, David; Fox, Barbara A.; Horii, Toshihiro; Bzik, David J.

    1999-01-01

    The liver- and blood-stage-expressed serine repeat antigen (SERA) of Plasmodium falciparum is a candidate protein for a human malaria vaccine. We compared the immune responses induced in mice immunized with SERA-expressing plasmid DNA vaccines delivered by intramuscular (i.m.) injection or delivered intradermally by Gene Gun immunization. Mice were immunized with a pcdna3 plasmid encoding the entire 47-kDa domain of SERA (amino acids 17 to 382) or the N-terminal domain (amino acids 17 to 110) of SERA. Minimal antibody responses were detected following DNA vaccination with the N-terminal domain of SERA, suggesting that the N-terminal domain alone is not highly immunogenic by this route of vaccine delivery. Immunization of mice by Gene Gun delivery of the 47-kDa domain of SERA elicited a significantly higher serum antibody titer to the antigen than immunization of mice by i.m. injection with the same plasmid did. The predominant isotype subclass of the antibodies elicited to the SERA protein following i.m. and Gene Gun immunizations with SERA plasmid DNA was immunoglobulin G1. Coimmunization of mice with SERA plasmid DNA and a plasmid expressing the hepatitis B surface antigen (pCMV-s) by the i.m. route resulted in higher anti-SERA titers than those generated in mice immunized with the SERA DNA plasmid alone. Vaccination with DNA may provide a viable alternative or may be used in conjunction with protein-based subunit vaccines to maximize the efficacy of a human malaria vaccine that includes immunogenic regions of the SERA protein. PMID:10496891

  2. Detection of clonal antigen receptor gene rearrangement in dogs with lymphoma by real-time polymerase chain reaction and melting curve analysis.

    PubMed

    Langner, Kathrin F A; Joetzke, Alexa E; Nerschbach, Verena; Eberle, Nina; Schuberth, Hans-Joachim; Koy, Mirja; Nolte, Ingo; Betz, Daniela

    2014-01-03

    Molecular techniques that detect canine lymphoma cells by their clonal antigen receptor gene rearrangement play an increasing role for diagnosis as well as for monitoring minimal residual disease during and after cytostatic therapy. However, the methods currently available are time-consuming and/or cost-intensive thus impeding the use in clinical routine. The aim of the present study was to develop and evaluate a real-time polymerase chain reaction (PCR) with subsequent melting curve analysis (MCA) for the detection of clonally rearranged antigen receptor genes in dogs with B and T cell lymphoma on non formalin-fixed and paraffin-embedded lymph node samples. In lymph node aspirates from 30 dogs with multicentric B cell lymphoma, real-time PCR with MCA detected clonal rearrangement in 100% and conventional PCR with polyacrylamide gel electrophoresis (PAGE) in 93% of samples. Both methods correctly identified clonality in 80% of lymph node aspirates of 10 dogs with T cell lymphoma. None of the two PCR systems detected clonal rearrangement in samples from 9 dogs with lymph node hyperplasia. Using a dilutional series with regular lymphoid desoxyribonucleic acid (DNA), detection limits of lymphoma DNA were as low as 0.8% and 6.25% for B and T cell clonal rearrangement with real-time PCR and MCA and at 3.13% and 12.5% with the conventional system. Median absolute detection limits of lymphoma DNA were shown to be at 0.1 ng and 1 ng for the B and T cell immunophenotype with the real-time PCR system and at 10 ng each with conventional PCR and PAGE. Real-time PCR with MCA is a convenient and reliable method with a good analytical sensitivity. Thus, the method may assist the detection of clonal antigen receptor gene rearrangement in canine lymphoma patients in a clinical setting also in the presence of small amounts of neoplastic cells.

  3. Screening of early antigen genes of adult-stage Trichinella spiralis using pig serum from different stages of early infection

    USDA-ARS?s Scientific Manuscript database

    The goal of this work was to identify novel, early antigens present in Trichinella spiralis. To this end, a cDNA library generated from 3-day old adult worms (Ad3) was immunologically screened using serum from a pig infected with 20,000 muscle larvae. The serum was obtained from multiple, time cours...

  4. Isolation of a complementary DNA clone for thyroid microsomal antigen. Homology with the gene for thyroid peroxidase.

    PubMed Central

    Seto, P; Hirayu, H; Magnusson, R P; Gestautas, J; Portmann, L; DeGroot, L J; Rapoport, B

    1987-01-01

    The thyroid microsomal antigen (MSA) in autoimmune thyroid disease is a protein of approximately 107 kD. We screened a human thyroid cDNA library constructed in the expression vector lambda gt11 with anti-107-kD monoclonal antibodies. Of five clones obtained, the recombinant beta-galactosidase fusion protein from one clone (PM-5) was confirmed to react with the monoclonal antiserum. The complementary DNA (cDNA) insert from PM-5 (0.8 kb) was used as a probe on Northern blot analysis to estimate the size of the mRNA coding for the MSA. The 2.9-kb messenger RNA (mRNA) species observed was the same size as that coding for human thyroid peroxidase (TPO). The probe did not bind to human liver mRNA, indicating the thyroid-specific nature of the PM-5-related mRNA. The nucleotide sequence of PM-5 (842 bp) was determined and consisted of a single open reading frame. Comparison of the nucleotide sequence of PM-5 with that presently available for pig TPO indicates 84% homology. In conclusion, a cDNA clone representing part of the microsomal antigen has been isolated. Sequence homology with porcine TPO, as well as identity in the size of the mRNA species for both the microsomal antigen and TPO, indicate that the microsomal antigen is, at least in part, TPO. Images PMID:3654979

  5. Evidence for Contemporary Switching of the O-Antigen Gene Cluster between Shiga Toxin-Producing Escherichia coli Strains Colonizing Cattle

    PubMed Central

    Geue, Lutz; Menge, Christian; Eichhorn, Inga; Semmler, Torsten; Wieler, Lothar H.; Pickard, Derek; Berens, Christian; Barth, Stefanie A.

    2017-01-01

    Shiga toxin-producing Escherichia coli (STEC) comprise a group of zoonotic enteric pathogens with ruminants, especially cattle, as the main reservoir. O-antigens are instrumental for host colonization and bacterial niche adaptation. They are highly immunogenic and, therefore, targeted by the adaptive immune system. The O-antigen is one of the most diverse bacterial cell constituents and variation not only exists between different bacterial species, but also between individual isolates/strains within a single species. We recently identified STEC persistently infecting cattle and belonging to the different serotypes O156:H25 (n = 21) and O182:H25 (n = 15) that were of the MLST sequence types ST300 or ST688. These STs differ by a single nucleotide in purA only. Fitness-, virulence-associated genome regions, and CRISPR/CAS (clustered regularly interspaced short palindromic repeats/CRISPR associated sequence) arrays of these STEC O156:H25 and O182:H25 isolates were highly similar, and identical genomic integration sites for the stx converting bacteriophages and the core LEE, identical Shiga toxin converting bacteriophage genes for stx1a, identical complete LEE loci, and identical sets of chemotaxis and flagellar genes were identified. In contrast to this genomic similarity, the nucleotide sequences of the O-antigen gene cluster (O-AGC) regions between galF and gnd and very few flanking genes differed fundamentally and were specific for the respective serotype. Sporadic aEPEC O156:H8 isolates (n = 5) were isolated in temporal and spatial proximity. While the O-AGC and the corresponding 5′ and 3′ flanking regions of these aEPEC isolates were identical to the respective region in the STEC O156:H25 isolates, the core genome, the virulence associated genome regions and the CRISPR/CAS elements differed profoundly. Our cumulative epidemiological and molecular data suggests a recent switch of the O-AGC between isolates with O156:H8 strains having served as DNA donors. Such

  6. The Modality of Enterobacterial Common Antigen Polysaccharide Chain Lengths Is Regulated by o349 of the wec Gene Cluster of Escherichia coli K-12

    PubMed Central

    Barr, Kathleen; Klena, John; Rick, Paul D.

    1999-01-01

    The assembly of the phosphoglyceride-linked form of enterobacterial common antigen (ECAPG) occurs by a mechanism that involves modulation of polysaccharide chain length. However, the genetic determinant of this modulation has not been identified. Site-directed mutagenesis of o349 of the Escherichia coli K-12 wec gene cluster revealed that this locus encodes a Wzz protein that specifically modulates the chain length of ECAPG polysaccharides, and we have designated this locus wzzECA. The WzzECA-mediated modulation of ECAPG polysaccharide chains is the first demonstrated example of Wzz regulation involving a polysaccharide that is not linked to the core-lipid A structure of lipopolysaccharide. PMID:10515954

  7. A capripoxvirus detection PCR and antibody ELISA based on the major antigen P32, the homolog of the vaccinia virus H3L gene.

    PubMed

    Heine, H G; Stevens, M P; Foord, A J; Boyle, D B

    1999-07-30

    Sheeppoxvirus (SPV), goatpoxvirus (GPV) and lumpy skin disease virus (LSDV) of cattle belong to the Capripoxvirus genus of the Poxviridae family and can cause significant economic losses in countries where they are endemic. Capripox diagnosis by classical virological methods dependent on live capripox virus is not suitable in countries such as Australia where the virus is exotic and live virus is not available. To develop diagnostic tests based on recombinant material, we cloned and sequenced a 3.7 kb viral DNA fragment of SPV that contained open reading frames homologous to the vaccinia virus J6R, H1L, H2R, H3L and H4L genes. A capripoxvirus specific PCR assay was developed that differentiated between SPV and LSDV on the basis of unique restriction sites in the corresponding PCR fragments. The vaccinia virus H3L homolog was identified as the capripoxvirus P32 antigen. The P32 proteins of SPV and LSDV were expressed in Escherichia coli as a fusion protein with a poly-histidine tag and affinity purified on metal binding resin. The full-length P32 protein contained a transmembrane region close to the carboxy terminus and was membrane associated but could be solubilised in detergent and used as trapping antigen in an antibody detection ELISA. The ELISA was specific for capripoxvirus as only sera from sheep infected with capripoxvirus but not orf or vaccinia virus reacted with the capripoxvirus P32 antigen.

  8. Establishment of renal proximal tubule cell lines by targeted oncogenesis in transgenic mice using the L-pyruvate kinase-SV40 (T) antigen hybrid gene.

    PubMed

    Cartier, N; Lacave, R; Vallet, V; Hagege, J; Hellio, R; Robine, S; Pringault, E; Cluzeaud, F; Briand, P; Kahn, A

    1993-03-01

    Targeted oncogenesis allowed us to obtain two cell lines which have been derived from the proximal tubule of kidney from transgenic mice harbouring the simian virus (SV40) large T and small t antigens placed under the control of the 5' regulatory sequence from the rat L-type pyruvate kinase (L-PK) gene. The cell lines (PKSV-PCT and PKSV-PR cells) were derived from early (PCT) and late (Pars Recta, PR) microdissected proximal tubules grown in D-glucose-enriched medium. In such conditions of culture, both cell lines exhibited L-PK transcripts, a stable expression of SV40-encoded nuclear large T antigen, a prolonged life span but failed to induce tumors when injected sub-cutaneously into athymic (nu-nu) mice. Confluent cells, grown on plastic support or porous filters, were organized as monolayers of polarized cuboid cells with well developed apical microvilli and formed domes. Both cell lines exhibited morphological features of proximal tubule cells with villin located in the apical brush-border and substantial amounts of hydrolase activity. By immunofluorescence studies using specific antibodies, aminopeptidase N appeared restricted to the apical microvillar domain, whereas the H2 histocompatibility antigen was distributed in the cytoplasm and lateral membranes. These results demonstrate that the proximal morphological phenotype has been fully preserved in these cultured cells derived from tissue-specific targeted oncogenesis in transgenic mice.

  9. 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.

  10. Utilizing the O-antigen lipopolysaccharide biosynthesis pathway in Escherichia coli to interrogate the substrate specificities of exogenous glycosyltransferase genes in a combinatorial approach.

    PubMed

    Johansen, Eric B; Szoka, Francis C; Zaleski, Anthony; Apicella, Michael A; Gibson, Bradford W

    2010-06-01

    In previous work, our laboratory generated novel chimeric lipopolysaccharides (LPS) in Escherichia coli transformed with a plasmid containing exogenous lipooligosaccharide synthesis genes (lsg) from Haemophilus influenzae. Analysis of these novel oligosaccharide-LPS chimeras allowed characterization of the carbohydrate structures generated by several putative glycosyltransferase genes within the lsg locus. Here, we adapted this strategy to construct a modular approach to study the synthetic properties of individual glycosyltransferases expressed alone and in combinations. To this end, a set of expression vectors containing one to four putative glycosyltransferase genes from the lsg locus, lsgC-F, were transformed into E. coli K12 (XL-1) which is defective in LPS O-antigen biosynthesis. This strategy relied on the inclusion of the H. influenzae gene product lsgG in every plasmid construct, which partially rescues the E. coli LPS biosynthesis defect by priming uridine diphosphate-undecaprenyl in the WecA-dependent O-antigen synthetic pathway with N-acetyl-glucosamine (GlcNAc). This GlcNAc-undecaprenyl then served as an acceptor substrate for further carbohydrate extension by transformed glycosyltransferases. The resultant LPS-linked chimeric glycans were isolated from their E. coli constructs and characterized by mass spectrometry, methylation analysis and enzyme-linked immunosorbent assays. These structural data allowed the specificity of various glycosyltransferases to be unambiguously assigned to individual genes. LsgF was found to transfer a galactose (Gal) to terminal GlcNAc. LsgE was found to transfer GlcNAc to Gal-GlcNAc, and both LsgF and LsgD were found to transfer Gal to GlcNAc-Gal-GlcNAc but with differing linkage specificities. This method can be generalized and readily adapted to study the substrate specificity of other putative or uncharacterized glycosyltransferases.

  11. Prostate cancer antigen 3 gene expression in peripheral blood and urine sediments from prostate cancer and benign prostatic hyperplasia patients versus healthy individuals.

    PubMed

    Moradi Sardareh, Hemen; Goodarzi, Mohammad Taghi; Yadegar-Azari, Reza; Poorolajal, Jalal; Mousavi-Bahar, Seyed Habibollah; Saidijam, Massoud

    2014-11-30

    To determine the expression of prostate cancer antigen 3 (PCA3) gene in peripheral blood and urine sediments from patients with prostate cancer (PCa) and benign prostatic hyperplasia (BPH) and normal subjects. A total number of 48 patients [24 with biopsy proven prostate cancer (PCa) and 24 with benign prostate hyperplasia (BPH)] were studied. Twenty-four healthy individuals were also recruited as control group. After blood and urine sampling, total RNA was extracted and cDNA was synthesized. Expression of PCA3 gene was assessed by quantitative reverse transcription polymerase chain reaction. Comparison of PCA3 gene expression between control and BPH groups indicated no statistically significant differences in both urine and blood samples. Patients with PCa demonstrated an increased PCA3 gene expression rate compared to control and BPH groups (10.64 and 7.17 folds, respectively). The rate of fold increased PCA3 gene expression in urine was 20.90, 20.90, and 20.35 in patients with PCa, BPH and normal subjects, respectively. Evaluation of PCA3 gene expression can be considered as a reliable marker for detection of PCa. Increased level of this marker in urine sediments is more sensitive than blood for distinguishing between cancerous and non-cancerous groups. 

  12. Molecular analysis of region 1 of the Escherichia coli K5 antigen gene cluster: a region encoding proteins involved in cell surface expression of capsular polysaccharide.

    PubMed Central

    Pazzani, C; Rosenow, C; Boulnois, G J; Bronner, D; Jann, K; Roberts, I S

    1993-01-01

    The nucleotide sequence of region 1 of the K5 antigen gene cluster of Escherichia coli was determined. This region is postulated to encode functions which, at least in part, participate in translocation of polysaccharide across the periplasmic space and onto the cell surface. Analysis of the nucleotide sequence revealed five genes that encode proteins with predicted molecular masses of 75.7, 60.5, 44, 43, and 27 kDa. The 27-kDa protein was 70.7% homologous to the CMP-2-keto-3-deoxyoctulosonic acid synthetase enzyme encoded by the E. coli kdsB gene, indicating the presence of a structural gene for a similar enzyme within the region 1 operon. The 43-kDa protein was homologous to both the Ctrb and BexC proteins encoded by the Neisseria meningitidis and Haemophilus influenzae capsule gene clusters, respectively, indicating common stages in the expression of capsules in these gram-negative bacteria. However, no homology was detected between the 75.7, 60.5-, and 44-kDa proteins and any of the proteins so far described for the H. influenzae and N. meningitidis capsule gene clusters. Images PMID:8397187

  13. Expression of VCA (viral capsid antigen) and EBNA1 (Epstein-Barr-virus-encoded nuclear antigen 1) genes of Epstein-Barr virus in Pichia pastoris and application of the products in a screening test for patients with nasopharyngeal carcinoma.

    PubMed

    Hu, Bo; Hong, Guoqiang; Li, Zhaoxia; Xu, Jue; Zhu, Zhenyu; Li, Lin

    2007-05-01

    EBV (Epstein-Barr virus) serological tests have been used for many years as accessory diagnostic predictors of NPC (nasopharyngeal carcinoma). To date, IF (indirect immunofluorescence) assays still serve as the 'gold standard' for EBV serodiagnosis. However, IF assays are time-consuming, unsuitable for automatic handling and difficult to standardize. This makes their application in mass screening of populations inconvenient. Some of the technical difficulties associated with IF have been overcome by the development of specific ELISAs, but, at present, high sensitivity and specificity cannot be achieved simultaneously by using recombinant protein-based ELISAs, as the diagnostic value of different fragments of EBV in NPC is different. In an attempt to determine a suitable recombinant EBV protein for diagnostic purposes, fragments of EBV VCA (viral capsid antigen) and EBNA1 (Epstein-Barr-virus-encoded nuclear antigen 1) genes were expressed in the methylotrophic yeast Pichia pastoris, and a novel ELISA was established using P. pastoris-expressed VCA-BALF4 [aa (amino acids) 287-623; the BALF4 gene encodes the EBV glycoprotein gp125], EBNA1 (aa 390-641) and VCA-BFRF3 (the gene BFRF3 encodes a viral structural capsid protein or tegument protein VCA p18) proteins. Serum samples were collected from patients with NPC and healthy controls and were tested using this ELISA. The sensitivity of VCA-BFRF3, VCA-BALF4 and EBNA1 tests in the NPC sera were 65.0 (195/300), 76.3 (229/300) and 81.4% (244/300) respectively, whereas the specificity of normal individuals were 92 (460/500), 96 (480/500) and 95.8% (479/500). The optimum combination is VCA-BALF4 plus EBNA1, which identified 90.3% (271/300) of the NPC patients and had a specificity of 92.8% (464/500) for normal individuals. The results obtained from the evaluation of three antibodies to EBV as markers for detecting NPC suggests that a combination of EBNA1 (aa 390-641) and VCA-BALF4 (aa 287-623) assays would give better results

  14. Expression and plasmid transfer of genes coding for the fimbrial antigen F107 in porcine Escherichia coli strains.

    PubMed

    Wittig, W; Prager, R; Stamm, M; Streckel, W; Tschäpe, H

    1994-08-01

    Expression of the fimbrial antigen F107 of porcine E. coli strains on agar plates was achieved by microaerobic cultivation. For part of the strains of some types, addition of alizarin yellow and eosin to the agar medium proved to be necessary. Some of these strains reacted distinctly positive only when the small colonies growing between the larger ones on alizarin-yellow agar were tested. The fimbrial antigen of the Swiss strain 107/86 was provisionally designated the F107ab variant and that of the Hungarian strain 2134P and the Czech strain 8813, the F107ac variant. The F107 genetic determinants were found to be often linked with those encoding haemolysin production and are frequently carried by plasmids.

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

    DTIC Science & Technology

    1991-08-26

    necessary and identify by block number) FIELD GROUP SUB-GROUP cDNA cloning; sequence analysis; E . Coli expression; 06 13 epitope mapping; ELISA titers...antigens in E . coli 6 4.3 Mapping of the neutralization epitope of DEN-2" E protein 7 4.4 Detection of stable secondary structure at the 3-terminus of...based high ievel expression systems and characterize functions of flavivirus proteins. Three notable expression systems are: 1) the E . coli expression

  16. Molecular cloning and sequence analysis of the gene coding for the 57kDa soluble antigen of the salmonid fish pathogen Renibacterium salmoninarum

    USGS Publications Warehouse

    Chien, Maw-Sheng; Gilbert , Teresa L.; Huang, Chienjin; Landolt, Marsha L.; O'Hara, Patrick J.; Winton, James R.

    1992-01-01

    The complete sequence coding for the 57-kDa major soluble antigen of the salmonid fish pathogen, Renibacterium salmoninarum, was determined. The gene contained an opening reading frame of 1671 nucleotides coding for a protein of 557 amino acids with a calculated Mr value of 57190. The first 26 amino acids constituted a signal peptide. The deduced sequence for amino acid residues 27–61 was in agreement with the 35 N-terminal amino acid residues determined by microsequencing, suggesting the protein in synthesized as a 557-amino acid precursor and processed to produce a mature protein of Mr 54505. Two regions of the protein contained imperfect direct repeats. The first region contained two copies of an 81-residue repeat, the second contained five copies of an unrelated 25-residue repeat. Also, a perfect inverted repeat (including three in-frame UAA stop codons) was observed at the carboxyl-terminus of the gene.

  17. Expression of cloned herpesvirus genes. I. Detection of nuclear antigens from herpes simplex virus type 2 inverted repeat regions in transfected mouse cells.

    PubMed Central

    Middleton, M H; Reyes, G R; Ciufo, D M; Buchan, A; Macnab, J C; Hayward, G S

    1982-01-01

    Three different recombinant plasmids containing the entire 15-kilobase L and S inverted repeat sequence of herpes simplex virus type 2 DNA have been introduced into cultured Ltk- or BSC cells by both the calcium and DEAE-dextran transfection procedures. In each case, after 24 h approximately 1% of the cells gave strongly positive nuclear staining when assayed by immunofluorescence with hyperimmune antisera made against early and immediate-early infected-cell polypeptides. The nuclear fluorescence pattern and intensity mimicked that observed within 2 to 3 h after infection of Ltk- cells with either herpes simplex virus type 1 or type 2 wild-type virus. Herpes simplex virus type 1 (KOStsB2)-infected Ltk- cells under nonpermissive conditions did not express these antigens in the nucleus. Therefore, we conclude that either one or both of the 185,000- and 110,000-molecular-weight immediate early proteins, or some other as yet unknown gene product encoded entirely within the inverted repeats, can be transiently expressed in large amounts in transfected cells in the absence of other viral genes or accompanying virion components. Permanent mouse cell lines derived from transfection with these plasmids by using the thymidine kinase coselection procedure did not express sufficient nuclear antigen to be detectable by immunofluorescence. Images PMID:6292452

  18. Vaccine Properties of a Novel Marker Gene-Free Recombinant Modified Vaccinia Ankara Expressing Immunodominant CMV Antigens pp65 and IE1

    PubMed Central

    Wang, Zhongde; La Rosa, Corinna; Li, Zhongqi; Ly, Heang; Krishnan, Aparna; Martinez, Joy; Britt, William J.; Diamond, Don

    2007-01-01

    CMV tegument protein pp65 and CMV immediate early gene product IE1 are both considered immunodominant targets of cell-mediated immunity (CMI) and potentially capable of controlling CMV infection. To better assess their role in host defense, we have constructed a novel MVA transfer vector named pZWIIA and generated a recombinant MVA (rMVA) expressing both full-length pp65 and exon4 of IE1 (pp65-IE1-MVA) at high levels, followed by the genetic removal of the bacterial marker gene used to distinguish recombinant forms. Immunogenicity evaluation indicates that pp65-IE1-MVA not only can induce robust primary CMI to both antigens in HLA A2.1 Tg mice, but also can stimulate vigorous expansion of memory T lymphocyte responses to pp65 and IE1 in PBMC of CMV-positive donors. These properties make the MVA-based vaccine ideal for the dual role of priming and boosting CMV-specific T cell immunity as a means to control CMV disease in recipients of hematopoietic cell or solid organ transplantation (HCT or SOT). pZWIIA alone or in combination with other MVA transfer vectors can be used to generate MVA based multiple-antigen vaccine which have application in vaccine development for a wide spectrum of infectious diseases and cancer. PMID:17049414

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

    PubMed Central

    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

  20. 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.

  1. 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.

  2. 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

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

    PubMed Central

    Pascal, Laura E; True, Lawrence D; Campbell, David S; Deutsch, Eric W; Risk, Michael; Coleman, Ilsa M; Eichner, Lillian J; Nelson, Peter S; Liu, Alvin Y

    2008-01-01

    Background: Expression levels of mRNA and protein by cell types exhibit a range of correlations for different genes. In this study, we compared levels of mRNA abundance for several cluster designation (CD) genes determined by gene arrays using magnetic sorted and laser-capture microdissected human prostate cells with levels of expression of the respective CD proteins determined by immunohistochemical staining in the major cell types of the prostate – basal epithelial, luminal epithelial, stromal fibromuscular, and endothelial – and for prostate precursor/stem cells and prostate carcinoma cells. Immunohistochemical stains of prostate tissues from more than 50 patients were scored for informative CD antigen expression and compared with cell-type specific transcriptomes. Results: Concordance between gene and protein expression findings based on 'present' vs. 'absent' calls ranged from 46 to 68%. Correlation of expression levels was poor to moderate (Pearson correlations ranged from 0 to 0.63). Divergence between the two data types was most frequently seen for genes whose array signals exceeded background (> 50) but lacked immunoreactivity by immunostaining. This could be due to multiple factors, e.g. low levels of protein expression, technological sensitivities, sample processing, probe set definition or anatomical origin of tissue and actual biological differences between transcript and protein abundance. Conclusion: Agreement between these two very different methodologies has great implications for their respective use in both molecular studies and clinical trials employing molecular biomarkers. PMID:18501003

  4. Prevalence of genes encoding virulence factors among Escherichia coli with K1 antigen and non-K1 E. coli strains.

    PubMed

    Kaczmarek, Agnieszka; Budzynska, Anna; Gospodarek, Eugenia

    2012-10-01

    Multiplex PCR was used to detect genes encoding selected virulence determinants associated with strains of Escherichia coli with K1 antigen (K1(+)) and non-K1 E. coli (K1(-)). The prevalence of the fimA, fimH, sfa/foc, ibeA, iutA and hlyF genes was studied for 134 (67 K1(+) and 67 K1(-)) E. coli strains isolated from pregnant women and neonates. The fimA gene was present in 83.6 % of E. coli K1(+) and in 86.6 % of E. coli K1(-) strains. The fimH gene was present in all tested E. coli K1(+) strains and in 97.0 % of non-K1 strains. E. coli K1(+) strains were significantly more likely to possess the following genes than E. coli K1(-) strains: sfa/foc (37.3 vs 16.4 %, P = 0.006), ibeA (35.8 vs 4.5 %, P<0.001), iutA (82.1 vs 35.8 %, P<0.001) and hlyF (28.4 vs 6.0 %, P<0.001). In conclusion, E. coli K1(+) seems to be more virulent than E. coli K1(-) strains in developing severe infections, thereby increasing possible sepsis or neonatal bacterial meningitis.

  5. 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

  6. In Vitro Pre-Clinical Validation of Suicide Gene Modified Anti-CD33 Redirected Chimeric Antigen Receptor T-Cells for Acute Myeloid Leukemia

    PubMed Central

    Minagawa, Kentaro; Jamil, Muhammad O.; AL-Obaidi, Mustafa; Pereboeva, Larisa; Salzman, Donna; Erba, Harry P.; Lamb, Lawrence S.; Bhatia, Ravi; Mineishi, Shin

    2016-01-01

    Background Approximately fifty percent of patients with acute myeloid leukemia can be cured with current therapeutic strategies which include, standard dose chemotherapy for patients at standard risk of relapse as assessed by cytogenetic and molecular analysis, or high-dose chemotherapy with allogeneic hematopoietic stem cell transplant for high-risk patients. Despite allogeneic hematopoietic stem cell transplant about 25% of patients still succumb to disease relapse, therefore, novel strategies are needed to improve the outcome of patients with acute myeloid leukemia. Methods and findings We developed an immunotherapeutic strategy targeting the CD33 myeloid antigen, expressed in ~ 85–90% of patients with acute myeloid leukemia, using chimeric antigen receptor redirected T-cells. Considering that administration of CAR T-cells has been associated with cytokine release syndrome and other potential off-tumor effects in patients, safety measures were here investigated and reported. We genetically modified human activated T-cells from healthy donors or patients with acute myeloid leukemia with retroviral supernatant encoding the inducible Caspase9 suicide gene, a ΔCD19 selectable marker, and a humanized third generation chimeric antigen receptor recognizing human CD33. ΔCD19 selected inducible Caspase9-CAR.CD33 T-cells had a 75±3.8% (average ± standard error of the mean) chimeric antigen receptor expression, were able to specifically lyse CD33+ targets in vitro, including freshly isolated leukemic blasts from patients, produce significant amount of tumor-necrosis-factor-alpha and interferon-gamma, express the CD107a degranulation marker, and proliferate upon antigen specific stimulation. Challenging ΔCD19 selected inducible Caspase9-CAR.CD33 T-cells with programmed-death-ligand-1 enriched leukemia blasts resulted in significant killing like observed for the programmed-death-ligand-1 negative leukemic blasts fraction. Since the administration of 10 nanomolar of a

  7. Polymorphisms within the human leucocyte antigen-E gene and their associations with susceptibility to rheumatoid arthritis as well as clinical outcome of anti-tumour necrosis factor therapy.

    PubMed

    Iwaszko, M; Świerkot, J; Kolossa, K; Jeka, S; Wiland, P; Bogunia-Kubik, K

    2015-12-01

    Involvement of the non-classical human leucocyte antigen-E (HLA-E) in both innate and acquired immune response suggests its possible role in development of autoimmune pathologies. This study was undertaken to investigate relationships between the HLA-E gene single nucleotide polymorphisms (SNPs) and a risk of rheumatoid arthritis (RA), as well as to evaluate a potential of these polymorphisms to modulate clinical outcome of anti-tumour necrosis factor (TNF) treatment in female patients. A total of 223 female patients with RA receiving anti-TNF biological therapy and 134 female healthy subjects were enrolled into the study. Genotypings for two SNPs within the HLA-E gene (rs1264457 HLA-E*01:01/01:03; rs1059510 HLA-E*01:03:01/01:03:02) were performed using a polymerase chain reaction (PCR) amplification employing LightSNiP assays. Clinical response was evaluated according to the European League Against Rheumatism (EULAR) criteria at 12 and 24 weeks after initiation of the therapy. The frequency of the HLA-E*01:01/01:01 genotype was decreased significantly in RA patients in comparison to controls (P = 0.031). The presence of the HLA-E*01:01/01:01 genotype in patients correlated with better EULAR response after 12 weeks of anti-TNF treatment, while 01:03 allele carriers were generally unresponsive to the treatment (P = 0.014). The HLA-E*01:03/01:03 genotype was also over-represented among non-responding patients in comparison to HLA-E*01:01/01:01 homozygotes (P = 0.021). With respect to the HLA-E rs1059510 variation, a better response after 12 weeks was observed more frequently in patients carrying the HLA-E*01:03:01/01:03:01 genotype than other genotypes (P = 0.009). The results derived from this study imply that HLA-E polymorphisms may influence RA susceptibility and affect clinical outcome of anti-TNF therapy in female RA patients.

  8. Polymorphisms within the human leucocyte antigen-E gene and their associations with susceptibility to rheumatoid arthritis as well as clinical outcome of anti-tumour necrosis factor therapy

    PubMed Central

    Iwaszko, M; Świerkot, J; Kolossa, K; Jeka, S; Wiland, P; Bogunia-Kubik, K

    2015-01-01

    Involvement of the non-classical human leucocyte antigen-E (HLA-E) in both innate and acquired immune response suggests its possible role in development of autoimmune pathologies. This study was undertaken to investigate relationships between the HLA-E gene single nucleotide polymorphisms (SNPs) and a risk of rheumatoid arthritis (RA), as well as to evaluate a potential of these polymorphisms to modulate clinical outcome of anti-tumour necrosis factor (TNF) treatment in female patients. A total of 223 female patients with RA receiving anti-TNF biological therapy and 134 female healthy subjects were enrolled into the study. Genotypings for two SNPs within the HLA-E gene (rs1264457 HLA-E*01:01/01:03; rs1059510 HLA-E*01:03:01/01:03:02) were performed using a polymerase chain reaction (PCR) amplification employing LightSNiP assays. Clinical response was evaluated according to the European League Against Rheumatism (EULAR) criteria at 12 and 24 weeks after initiation of the therapy. The frequency of the HLA-E*01:01/01:01 genotype was decreased significantly in RA patients in comparison to controls (P = 0·031). The presence of the HLA-E*01:01/01:01 genotype in patients correlated with better EULAR response after 12 weeks of anti-TNF treatment, while 01:03 allele carriers were generally unresponsive to the treatment (P = 0·014). The HLA-E*01:03/01:03 genotype was also over-represented among non-responding patients in comparison to HLA-E*01:01/01:01 homozygotes (P = 0·021). With respect to the HLA-E rs1059510 variation, a better response after 12 weeks was observed more frequently in patients carrying the HLA-E*01:03:01/01:03:01 genotype than other genotypes (P = 0·009). The results derived from this study imply that HLA-E polymorphisms may influence RA susceptibility and affect clinical outcome of anti-TNF therapy in female RA patients. PMID:26307125

  9. Effects of humanization and gene shuffling on immunogenicity and antigen binding of anti-TAG-72 single-chain Fvs.

    PubMed

    Pavlinkova, G; Colcher, D; Booth, B J; Goel, A; Wittel, U A; Batra, S K

    2001-12-01

    One major constraint in the clinical application of murine monoclonal antibodies (MAbs) is the development of a human antimurine antibody response. The immunogenicity of MAbs can be minimized by replacing nonhuman regions with corresponding human sequences. The studies reported in our article were undertaken to analyze the immunoreactivity and the immunogenicity of the CC49 single-chain antibody fragments (scFvs): (i) an scFv construct comprised of mouse CC49 VL and VH (m/m scFv), (ii) a light chain shuffled scFv with human VL (Hum4 VL) and mouse CC49 VH (h/m scFv), and (iii) a humanized scFv assembled from Hum4 VL and CC49 VH complementary determining regions (CDRs) grafted onto a VH framework of MAb 21/28' CL (h/CDR scFv). The CC49 scFvs competed for an antigen binding site with CC49 IgG in a similar fashion in a competition radioimmunoassay and were able to inhibit the binding of CC49 IgG to the antigen completely. The immunogenicity of CC49 scFvs was tested using sera with antiidiotypic antibodies to MAb CC49 obtained from patients treated by CC49 IgG in clinical trials. All tested sera exhibited the highest reactivity to the m/m scFv. However, the sera demonstrated differential reactivities to h/CDR scFv and h/m scFv. Replacement of the mouse chain in h/m scFv and h/CDR scFv decreased or completely averted serum reactivity. Our studies compared for the first time the antigen binding and immunogenicity of different scFv constructs containing the mouse, CDR grafted or human variable chains. These results indicate that the humanized CC49 scFv is potentially an important agent for imaging and therapeutic applications with TAG-72-positive tumors.

  10. 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. Copyright © 2012 Elsevier GmbH. All rights reserved.

  11. Influence of Cationic Lipid Composition on Gene Silencing Properties of Lipid Nanoparticle Formulations of siRNA in Antigen-Presenting Cells

    PubMed Central

    Basha, Genc; Novobrantseva, Tatiana I; Rosin, Nicole; Tam, Yuen Yi C; Hafez, Ismail M; Wong, Matthew K; Sugo, Tsukasa; Ruda, Vera M; Qin, June; Klebanov, Boris; Ciufolini, Marco; Akinc, Akin; Tam, Ying K; Hope, Michael J; Cullis, Pieter R

    2011-01-01

    Lipid nanoparticles (LNPs) are currently the most effective in vivo delivery systems for silencing target genes in hepatocytes employing small interfering RNA. Antigen-presenting cells (APCs) are also potential targets for LNP siRNA. We examined the uptake, intracellular trafficking, and gene silencing potency in primary bone marrow macrophages (bmMΦ) and dendritic cells of siRNA formulated in LNPs containing four different ionizable cationic lipids namely DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA. LNPs containing DLinKC2-DMA were the most potent formulations as determined by their ability to inhibit the production of GAPDH target protein. Also, LNPs containing DLinKC2-DMA were the most potent intracellular delivery agents as indicated by confocal studies of endosomal versus cytoplamic siRNA location using fluorescently labeled siRNA. DLinK-DMA and DLinKC2-DMA formulations exhibited improved gene silencing potencies relative to DLinDMA but were less toxic. In vivo results showed that LNP siRNA systems containing DLinKC2-DMA are effective agents for silencing GAPDH in APCs in the spleen and peritoneal cavity following systemic administration. Gene silencing in APCs was RNAi mediated and the use of larger LNPs resulted in substantially reduced hepatocyte silencing, while similar efficacy was maintained in APCs. These results are discussed with regard to the potential of LNP siRNA formulations to treat immunologically mediated diseases. PMID:21971424

  12. Intergenic Region 3 of Modified Vaccinia Ankara is a Functional Site for Insert Gene Expression and Allows for Potent Antigen-Specific Immune Responses

    PubMed Central

    Manuel, Edwin R.; Wang, Zhongde; Li, Zhongqi; La Rosa, Corinna; Zhou, Wendi; Diamond, Don J.

    2010-01-01

    Integration of exogenous DNA into modified vaccinia Ankara (MVA) is often accomplished using mapped deletion sites in the viral genome. Since MVA has a large capacity (≥30kb) for foreign gene inserts and a limited number of unique integration sites, development of additional integration sites is needed to take full advantage of the extraordinary capacity for foreign gene insertion. In this report, we evaluate an alternative insertion site known as intergenic region 3 (IGR3). Recombinant MVA carrying the cytomegalovirus pp65 gene in IGR3 (rMVA-pp65-IGR3) demonstrated expression and genetic stability of the insert gene upon passage. Immunization of transgenic HLA-A2 mice with rMVA-pp65-IGR3 induced robust antigen-specific immune responses. Moreover, rMVA-pp65-IGR3-infected human EBV-transformed B cell lines were able to stimulate high levels of pp65-specific memory T cell responses in human PBMCs. These data support the usage of IGR3 for the development of highly immunogenic rMVA vaccines for clinical or veterinary use. PMID:20471051

  13. Major deletions in the gene encoding the p53 tumor antigen cause lack of p53 expression in HL-60 cells.

    PubMed Central

    Wolf, D; Rotter, V

    1985-01-01

    The tumor antigen p53 is overproduced in transformed cells of various species, including man. HL-60 is an exceptional human tumor cell line that does not express this protein. Hybridization of polyadenylylated mRNA of these cells with a human p53 cDNA probe (p53-H14), which we cloned, had indicated a total absence of the mature-size (3.0 kilobases) or any aberrant p53 mRNA species. Analysis of the genomic HL-60 DNA indicated that the p53 gene in these cells was significantly altered. Most of the gene was deleted, and the residual p53 sequences of these cells, which show weak homology, mapped to the corresponding 5' region of the p53 gene. In agreement with previously documented results, we found that HL-60 cells have an amplified c-myc gene. We suggest that the deficiency of the p53 protein in HL-60 cells could have been overcome by using an alternative metabolic pathway. The c-myc product is a candidate for such an alternative protein. Images PMID:2858093

  14. Drosophila proliferating cell nuclear antigen (cyclin) gene: structure, expression during development, and specific binding of homeodomain proteins to its 5'-flanking region.

    PubMed Central

    Yamaguchi, M; Nishida, Y; Moriuchi, T; Hirose, F; Hui, C C; Suzuki, Y; Matsukage, A

    1990-01-01

    The genomic and cDNA clones for a Drosophila melanogaster proliferating cell nuclear antigen (PCNA) (cyclin) were isolated and sequenced. The coding sequence for a 260-amino-acid residue polypeptide was interrupted by a single short intron of 60 base pairs (bp), and about 70% of the deduced amino acid sequence of the Drosophila PCNA was identical to the rat and human PCNA polypeptides, with conserved unique repeats of leucine in the C-terminal region. Genomic Southern blot hybridization analysis indicates the presence of a single gene for PCNA per genome. The PCNA mRNA was detected at a high level in adult ovaries, unfertilized eggs, and early embryos and at low levels in the other developmental stages. The major transcription initiation site (cap site) was localized at 89 bp upstream from the ATG codon. Neither a TATA box nor a CAAT box was found within the 600-bp region upstream of the cap site. Clusters of 10 bp of sequence similar to the binding sites for Drosophila proteins containing homeodomains were found in the region from -127 to -413. DNase I footprint analysis revealed that the Drosophila homeodomain proteins coded by even-skipped and zerknüllt genes can specifically bind to these sites. These results suggest that the expression of the PCNA gene is under the control of genes coding for homeodomain proteins. Images PMID:1968224

  15. 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

  16. 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 Central

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

    1982-01-01

    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. Images PMID:6180184

  17. Use of in vivo-induced antigen technology (IVIAT) to identify genes uniquely expressed during human infection with Vibrio cholerae

    PubMed Central

    Hang, Long; John, Manohar; Asaduzzaman, Muhammad; Bridges, Emily Anna; Vanderspurt, Cecily; Kirn, Thomas J.; Taylor, Ronald K.; Hillman, Jeffrey D.; Progulske-Fox, Ann; Handfield, Martin; Ryan, Edward T.; Calderwood, Stephen B.

    2003-01-01

    In vivo-induced antigen technology is a method to identify proteins expressed by pathogenic bacteria during human infection. Sera from 10 patients convalescing from cholera infection in Bangladesh were pooled, adsorbed against in vitro-grown El Tor Vibrio cholerae O1, and used to probe a genomic expression library in Escherichia coli constructed from El Tor V. cholerae O1 strain N16961. We identified 38 positive clones in the screen, encoding pili (PilA and TcpA), cell membrane proteins (PilQ, MshO, MshP, and CapK), methyl-accepting chemotaxis proteins, chemotaxis and motility proteins (CheA and CheR), a quorum-sensing protein (LuxP), and four hypothetical proteins. Analysis of immune responses to purified PilA and TcpA in individual patients demonstrated that the majority seroconverted to these proteins, confirming results with pooled sera. These results suggest that PilA and its outer membrane secretin, PilQ, are expressed during human infection and may be involved in colonization of the gastrointestinal tract. These results also demonstrate substantial immune responses to TcpA in patients infected with El Tor V. cholerae O1. In vivo-induced antigen technology provides a simple method for identifying microbial proteins expressed during human infection, but not during in vitro growth. PMID:12826608

  18. Use of in vivo-induced antigen technology (IVIAT) to identify genes uniquely expressed during human infection with Vibrio cholerae.

    PubMed

    Hang, Long; John, Manohar; Asaduzzaman, Muhammad; Bridges, Emily Anna; Vanderspurt, Cecily; Kirn, Thomas J; Taylor, Ronald K; Hillman, Jeffrey D; Progulske-Fox, Ann; Handfield, Martin; Ryan, Edward T; Calderwood, Stephen B

    2003-07-08

    In vivo-induced antigen technology is a method to identify proteins expressed by pathogenic bacteria during human infection. Sera from 10 patients convalescing from cholera infection in Bangladesh were pooled, adsorbed against in vitro-grown El Tor Vibrio cholerae O1, and used to probe a genomic expression library in Escherichia coli constructed from El Tor V. cholerae O1 strain N16961. We identified 38 positive clones in the screen, encoding pili (PilA and TcpA), cell membrane proteins (PilQ, MshO, MshP, and CapK), methyl-accepting chemotaxis proteins, chemotaxis and motility proteins (CheA and CheR), a quorum-sensing protein (LuxP), and four hypothetical proteins. Analysis of immune responses to purified PilA and TcpA in individual patients demonstrated that the majority seroconverted to these proteins, confirming results with pooled sera. These results suggest that PilA and its outer membrane secretin, PilQ, are expressed during human infection and may be involved in colonization of the gastrointestinal tract. These results also demonstrate substantial immune responses to TcpA in patients infected with El Tor V. cholerae O1. In vivo-induced antigen technology provides a simple method for identifying microbial proteins expressed during human infection, but not during in vitro growth.

  19. 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

  20. Detection of clonal antigen receptor gene rearrangement in dogs with lymphoma by real-time polymerase chain reaction and melting curve analysis

    PubMed Central

    2014-01-01

    Background Molecular techniques that detect canine lymphoma cells by their clonal antigen receptor gene rearrangement play an increasing role for diagnosis as well as for monitoring minimal residual disease during and after cytostatic therapy. However, the methods currently available are time-consuming and/or cost-intensive thus impeding the use in clinical routine. The aim of the present study was to develop and evaluate a real-time polymerase chain reaction (PCR) with subsequent melting curve analysis (MCA) for the detection of clonally rearranged antigen receptor genes in dogs with B and T cell lymphoma on non formalin-fixed and paraffin-embedded lymph node samples. Results In lymph node aspirates from 30 dogs with multicentric B cell lymphoma, real-time PCR with MCA detected clonal rearrangement in 100% and conventional PCR with polyacrylamide gel electrophoresis (PAGE) in 93% of samples. Both methods correctly identified clonality in 80% of lymph node aspirates of 10 dogs with T cell lymphoma. None of the two PCR systems detected clonal rearrangement in samples from 9 dogs with lymph node hyperplasia. Using a dilutional series with regular lymphoid desoxyribonucleic acid (DNA), detection limits of lymphoma DNA were as low as 0.8% and 6.25% for B and T cell clonal rearrangement with real-time PCR and MCA and at 3.13% and 12.5% with the conventional system. Median absolute detection limits of lymphoma DNA were shown to be at 0.1 ng and 1 ng for the B and T cell immunophenotype with the real-time PCR system and at 10 ng each with conventional PCR and PAGE. Conclusions Real-time PCR with MCA is a convenient and reliable method with a good analytical sensitivity. Thus, the method may assist the detection of clonal antigen receptor gene rearrangement in canine lymphoma patients in a clinical setting also in the presence of small amounts of neoplastic cells. PMID:24383544

  1. Gene cloning of an Actinobacillus actinomycetemcomitans Y4 antigen which reacts with peripheral blood sera in patients with advanced destructive periodontitis.

    PubMed

    Arakawa, S; Hata, S; Ishikawa, I; Tsuchida, N

    1990-01-01

    Actinobacillus actinomycetemcomitans has been implicated in the aetiology of juvenile periodontitis and advanced destructive periodontitis. Levels of IgG antibody against A. actinomycetemcomitans in peripheral blood sera of patients with advanced destructive periodontitis are high, as are those against Bacteroides gingivalis. To clone the genes of antigens reactive with sera of such patients, a library of the A. actinomycetemcomitans strain Y4 DNA in lambda L47 was constructed and then screened, using an immunochemical detection method, with serum from a patient with the advanced disease. Six clones from among nearly 1000 reacted with the serum and also with that of another patient. They were designated 3, 4, 6, 7, 8 and 9. Restriction enzyme and Southern blot analyses indicated that clones 8 and 9 were identical and that all the clones were overlapping because they shared in common the 4 and 5 kbp HincII DNA fragments of A. actinomycetemcomitans. The cloned DNA fragment hybridized to the DNA of two other strains of A. actinomycetemcomitans but not to those of six periodontopathic bacteria examined. These findings suggest that a DNA sequence encoding an A. actinomycetemcomitans strain Y4 antigen strongly reactive with sera of patients with advanced destructive periodontitis was cloned. This sequence is present specifically in A. actinomycetemcomitans but not in other bacteria isolated from patients with periodontal diseases. Thus, the cloned DNA could serve as a probe for the diagnosis of periodontitis.

  2. HLA class I gene expression on human primary tumours and autologous metastases: demonstration of selective losses of HLA antigens on colorectal, gastric and laryngeal carcinomas.

    PubMed Central

    López-Nevot, M. A.; Esteban, F.; Ferrón, A.; Gutiérrez, J.; Oliva, M. R.; Romero, C.; Huelin, C.; Ruiz-Cabello, F.; Garrido, F.

    1989-01-01

    The expression of HLA class I antigens was studied in 99 primary tumour (colorectal, gastric and laryngeal carcinomas) and 57 autologous metastases using immunohistological techniques and monoclonal antibodies against class I monomorphic determinants, HLA-B isotypic determinants and HLA polymorphic determinants. Fourteen per cent of colorectal, 9.6% of gastric and 20% of laryngeal carcinomas completely lacked class I molecules. Selective losses of HLA-B antigens were also detected in 8.8, 3.4 and 5.8% of these tumours respectively. Taking into account complete and selective loss of HLA-B the average alteration in the class I molecules expression totalled 21%. The comparison of class I expression between primary tumours and autologous metastases showed differences in 24% of the patients. These differences consisted mainly in a decrease of class I expression by metastases. Nevertheless, four types of divergence were detected in laryngeal carcinomas, namely: +/-, +/+, -/+, -/-. In addition, a clear correlation between degree of differentiation and class I expression was observed in laryngeal tumours. Finally, when class I gene RFLPs were compared with DNA from 15 tumours and autologous normal mucosa or peripheral lymphocytes, no differences were detected between these samples. Images Figure 1 Figure 2 PMID:2649129

  3. Mechanisms of co-modified liver-targeting liposomes as gene delivery carriers based on cellular uptake and antigens inhibition effect.

    PubMed

    Zhang, Yuan; Rong Qi, Xian; Gao, Yan; Wei, Lai; Maitani, Yoshie; Nagai, Tsuneji

    2007-02-12

    In order to deliver antisense oligonucleotides (asODN) into hepatocytes orientedly in the treatment of hepatitis B virus (HBV) infection, the liver-targeting cationic liposomes was developed as a gene carrier, which was co-modified with the ligand of the asialoglycoprotein receptor (ASGPR), beta-sitosterol-beta-d-glucoside (sito-G) and the nonionic surfactant, Brij 35. Flow cytometry (FCM) analysis and enzyme-linked immunosorbent assay (ELISA) showed that the asODN-encapsulating cationic liposomes exhibited high transfection efficiency and strong antigens inhibition effect in primary rat hepatocytes and HepG2.2.15 cells, respectively. With the help of several inhibitors acting on different steps during the targeting lipofection, the cellular uptake mechanisms of the co-modified liver-targeting cationic liposomes were investigated through antigens inhibition effect assay and confocal laser scanning microscopy (CLSM) analysis. The cellular uptake with high transfection efficiency seemed to involve both endocytosis and membrane fusion. The ligand sito-G was confirmed to be able to enhance ASGPR-mediated endocytosis, the nonionic surfactant Brij 35 seemed to be able to facilitate membrane fusion, and the co-modification resulted in the most efficient transfection but no enhanced cytotoxicity. These results suggested that the co-modified liver-targeting cationic liposomes would be a specific and effective carrier to transfer asODN into hepatocytes infected with HBV orientedly.

  4. Selecting soluble/foldable protein domains through single-gene or genomic ORF filtering: structure of the head domain of Burkholderia pseudomallei antigen BPSL2063.

    PubMed

    Gourlay, Louise J; Peano, Clelia; Deantonio, Cecilia; Perletti, Lucia; Pietrelli, Alessandro; Villa, Riccardo; Matterazzo, Elena; Lassaux, Patricia; Santoro, Claudio; Puccio, Simone; Sblattero, Daniele; Bolognesi, Martino

    2015-11-01

    The 1.8 Å resolution crystal structure of a conserved domain of the potential Burkholderia pseudomallei antigen and trimeric autotransporter BPSL2063 is presented as a structural vaccinology target for melioidosis vaccine development. Since BPSL2063 (1090 amino acids) hosts only one conserved domain, and the expression/purification of the full-length protein proved to be problematic, a domain-filtering library was generated using β-lactamase as a reporter gene to select further BPSL2063 domains. As a result, two domains (D1 and D2) were identified and produced in soluble form in Escherichia coli. Furthermore, as a general tool, a genomic open reading frame-filtering library from the B. pseudomallei genome was also constructed to facilitate the selection of domain boundaries from the entire ORFeome. Such an approach allowed the selection of three potential protein antigens that were also produced in soluble form. The results imply the further development of ORF-filtering methods as a tool in protein-based research to improve the selection and production of soluble proteins or domains for downstream applications such as X-ray crystallography.

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

    PubMed Central

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

    2013-01-01

    Genetic transfer of neutralizing antibodies has been shown to confer strong and persistent protection against bacterial and viral infectious agents. While it is well established that for many exogenous neutralizing antibodies increased antigen affinity correlates with protection, the effect of antigen affinity on antibodies produced in situ following adenoviral gene transfer has not been examined. The mouse IgG2b monoclonal antibody 2C12.4 recognizes the Yersinia pestis Type III secretion apparatus protein LcrV (V antigen) and confers protection in mice when administered as an IgG intraperitoneally or, following genetic immunization with engineered, replication-defective serotype 5 human adenovirus (Ad) 1. 2C12.4 was expressed as a scFv fragment in E. coli and was shown to display a KD=3.5 nM by surface plasmon resonance (SPR) analysis. The 2C12.4 scFv was subjected to random mutagenesis and variants with increased affinity were isolated by flow cytometry using the Anchored Periplasmic Expression (APEx) bacterial display system. After a single round of mutagenesis, variants displaying up to 35-fold lower KD values (H8, KD=100 pM) were isolated. The variable domains of the H8 scFv were used to replace those of the parental 2C12.4 IgG encoded in the Ad vector, AdαV giving rise to AdαV.H8. The two adenoviral vectors resulted in similar titers of anti-V antigen antibodies 3 days post-immunization with 109, 1010 or 1011 particle units. Following intranasal challenge with 363 LD50Y. pestis CO92, 54% of the mice immunized with 1010 pu of AdαV.H8 survived at the 14 day end point compared to only 15% survivors for the group immunized with AdαV expressing the lower affinity 2C12.4 (P<0.04, AdαV versus AdαV.H8). These results indicate that affinity maturation of a neutralizing antibody delivered by genetic transfer may confer increased protection not only for Y. pestis challenge but possibly for other pathogens. PMID:20393511

  6. 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

  7. Phylogenetic relationships of Mongolian Babesia bovis isolates based on the merozoite surface antigen (MSA)-1, MSA-2b, and MSA-2c genes.

    PubMed

    Altangerel, Khukhuu; Sivakumar, Thillaiampalam; Battsetseg, Badgar; Battur, Banzragch; Ueno, Akio; Igarashi, Ikuo; Yokoyama, Naoaki

    2012-03-23

    We conducted a molecular epidemiological study on Babesia bovis in Mongolia. Three hundred blood samples collected from cattle grazed in seven different districts were initially screened using a previously established diagnostic polymerase chain reaction (PCR) assay for the detection of B. bovis-specific DNA. Positive samples were then used to amplify and sequence the hyper-variable regions of three B. bovis genes encoding the merozoite surface antigen (MSA)-1, MSA-2b, and MSA-2c. The diagnostic PCR assay detected B. bovis among cattle populations of all districts surveyed (4.4-26.0%). Sequences of each of the three genes were highly homologous among the Mongolian isolates, and found in a single phylogenetic cluster. In particular, a separate branch was formed only by the Mongolian isolates in the MSA-2b gene-based phylogenetic tree. Our findings indicate that effective preventative and control strategies are essential to control B. bovis infection in Mongolian cattle populations, and suggest that a careful approach must be adopted when using immunization techniques.

  8. Tat engagement of p38 MAP kinase and IRF7 pathways leads to activation of interferon-stimulated genes in antigen-presenting cells.

    PubMed

    Kim, Nayoung; Kukkonen, Sami; Martinez-Viedma, Maria Del Pilar; Gupta, Sumeet; Aldovini, Anna

    2013-05-16

    As a result of its interaction with transcription factors, HIV type 1 (HIV-1) Tat can modulate the expression of both HIV and cellular genes. In antigen-presenting cells Tat induces the expression of a subset of interferon (IFN)-stimulated genes (ISGs) in the absence of IFNs. We investigated the genome-wide Tat association with promoters in immature dendritic cells and in monocyte-derived macrophages. Among others, Tat associated with the MAP2K6, MAP2K3, and IRF7 promoters that are functionally part of IL-1 and p38 mitogen-activated protein kinase (MAPK) signaling pathways. The association correlated with their increased gene expression, increased activation of p38 MAPK and of phosphorylated signal transducer and activator of transcription 1 (STAT1), and consequent induction of ISGs. Probing these pathways with RNA interference, pharmacological p38 MAPK inhibition, and in cell lines lacking STAT1s or the type I IFN receptor chain confirmed the role of MAPKKs and IRF7 in Tat-mediated modulation of ISGs and excluded the involvement of IFNs in this modulation. Tat interaction with the 2 MAPKK and IRF7 promoters in HIV-1-infected cells and the resulting persistent activation of ISGs, which include inflammatory cytokines and chemokines, can contribute to the increased immune activation that characterizes HIV infection.

  9. 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.

  10. The predisposition to type 1 diabetes linked to the human leukocyte antigen complex includes at least one non-class II gene.

    PubMed Central

    Lie, B A; Todd, J A; Pociot, F; Nerup, J; Akselsen, H E; Joner, G; Dahl-Jørgensen, K; Rønningen, K S; Thorsby, E; Undlien, D E

    1999-01-01

    The human leukocyte antigen (HLA) complex, encompassing 3.5 Mb of DNA from the centromeric HLA-DPB2 locus to the telomeric HLA-F locus on chromosome 6p21, encodes a major part of the genetic predisposition to develop type 1 diabetes, designated "IDDM1." A primary role for allelic variation of the class II HLA-DRB1, HLA-DQA1, and HLA-DQB1 loci has been established. However, studies of animals and humans have indicated that other, unmapped, major histocompatibility complex (MHC)-linked genes are participating in IDDM1. The strong linkage disequilibrium between genes in this complex makes mapping a difficult task. In the present paper, we report on the approach we have devised to circumvent the confounding effects of disequilibrium between class II alleles and alleles at other MHC loci. We have scanned 12 Mb of the MHC and flanking chromosome regions with microsatellite polymorphisms and analyzed the transmission of these marker alleles to diabetic probands from parents who were homozygous for the alleles of the HLA-DRB1, HLA-DQA1, and HLA-DQB1 genes. Our analysis, using three independent family sets, suggests the presence of an additional type I diabetes gene (or genes). This approach is useful for the analysis of other loci linked to common diseases, to verify if a candidate polymorphism can explain all of the association of a region or if the association is due to two or more loci in linkage disequilibrium with each other. PMID:10053014

  11. DNA fragmentation, caspase 3 and prostate-specific antigen genes expression induced by arsenic, cadmium, and chromium on nontumorigenic human prostate cells.

    PubMed

    El-Atta, Hend M Abo; El-Bakary, Amal A; Attia, Afaf M; Lotfy, Ahmed; Khater, Shery S; Elsamanoudy, Ayman Z; Abdalla, Hussein Abdelaziz

    2014-12-01

    Prostate cancer is one of the most common cancers and the second cause of cancer-related deaths among men. Metals are recognized as chemical carcinogens where chronic exposures to such metals are implicated in the development of cancer, including prostate cancer. This in vitro study demonstrates the relative death sensitivity of prostatic (RWPE-1) cells to arsenic (As), cadmium (Cd), and chromium (Cr) as environmental pollutants through its apoptotic effects and the effect of these chemicals on prostate-specific antigen (PSA) gene expression as a marker for their carcinogecity. RWPE-1 cells were divided into three groups that were treated with As, Cd, and Cr in three replicates, at three different concentrations for each metal for 48 h. A control group consisted of untreated RWPE1 cells was used. Apoptosis was assessed using comet assay and caspase 3 gene expression; meanwhile, PSA gene expression was evaluated by semiqualitative real-time PCR (RT-PCR). One of the novel findings of this study is that arsenic and cadmium at low concentrations decreased apoptosis of RWPE-1 cells in a concentration-dependent manner while chromium induced significant concentration-dependent increase in apoptosis. Yet, at the highest concentrations, apoptosis was relatively more induced by all chemicals. Arsenic was the most chemical inhibiting apoptosis in RWPE-1 cells at low concentration. While at the moderate and highest concentrations, cadmium was the most inhibiting chemical of RWPE-1 cells' apoptosis. No distinct differences between treated and untreated cells for PSA gene expression were observed. It can be concluded that As and Cd, at low concentrations, can reduce apoptosis of prostatic cells in a concentration-dependent manner while chromium induced it; however, all metal salts used in this study did not induce PSA gene expression.

  12. Human anti-V3 HIV-1 monoclonal antibodies encoded by the VH5-51/VL lambda genes define a conserved antigenic structure.

    PubMed

    Gorny, Miroslaw K; Sampson, Jared; Li, Huiguang; Jiang, Xunqing; Totrov, Maxim; Wang, Xiao-Hong; Williams, Constance; O'Neal, Timothy; Volsky, Barbara; Li, Liuzhe; Cardozo, Timothy; Nyambi, Phillipe; Zolla-Pazner, Susan; Kong, Xiang-Peng

    2011-01-01

    Preferential usage of immunoglobulin (Ig) genes that encode antibodies (Abs) against various pathogens is rarely observed and the nature of their dominance is unclear in the context of stochastic recombination of Ig genes. The hypothesis that restricted usage of Ig genes predetermines the antibody specificity was tested in this study of 18 human anti-V3 monoclonal Abs (mAbs) generated from unrelated individuals infected with various subtypes of HIV-1, all of which preferentially used pairing of the VH5-51 and VL lambda genes. Crystallographic analysis of five VH5-51/VL lambda-encoded Fabs complexed with various V3 peptides revealed a common three dimensional (3D) shape of the antigen-binding sites primarily determined by the four complementarity determining regions (CDR) for the heavy (H) and light (L) chains: specifically, the H1, H2, L1 and L2 domains. The CDR H3 domain did not contribute to the shape of the binding pocket, as it had different lengths, sequences and conformations for each mAb. The same shape of the binding site was further confirmed by the identical backbone conformation exhibited by V3 peptides in complex with Fabs which fully adapted to the binding pocket and the same key contact residues, mainly germline-encoded in the heavy and light chains of five Fabs. Finally, the VH5-51 anti-V3 mAbs recognized an epitope with an identical 3D structure which is mimicked by a single mimotope recognized by the majority of VH5-51-derived mAbs but not by other V3 mAbs. These data suggest that the identification of preferentially used Ig genes by neutralizing mAbs may define conserved epitopes in the diverse virus envelopes. This will be useful information for designing vaccine immunogen inducing cross-neutralizing Abs.

  13. Genetic control of T cell responsiveness to the Friend murine leukemia virus envelope antigen. Identification of class II loci of the H-2 as immune response genes

    PubMed Central

    1988-01-01

    T cells primed specifically for the envelope glycoprotein of Friend murine leukemia helper virus (F-MuLV) were prepared by immunizing mice with a recombinant vaccinia virus that expressed the entire env gene of F-MuLV. Significant proliferative responses of F-MuLV envelope- specific, H-2a/b T cells were observed when the T cells were stimulated with antigen-pulsed peritoneal exudate cells (PEC) having the b allele at the K, A beta, A alpha, and E beta loci of the H-2. On the other hand, PEC having only the kappa allele at these loci did not induce the envelope-specific T cell proliferation, even when the PEC had the b allele at the E alpha, S, or D loci. F-MuLV envelope-specific proliferation of H-2a/b T cells under the stimulation of antigen- pulsed, H-2a/b PEC was specifically blocked with anti-I-Ab and anti-I- Ek mAbs but not with anti-Kb, anti-Kk, or anti-I-Ak mAbs. Moreover, (B10.MBR x A/WySn)F1 mice that have the b allele only at the K locus but not in I-A subregion were nonresponders to the envelope glycoprotein, and the bm12 mutation at the A beta locus completely abolished the T cell responsiveness to this antigen. These results indicate that proliferative T cells recognize a limited number of epitopes on F-MuLV envelope protein in the context of I-Ab, hybrid I- Ak/b, and/or hybrid I-Ek/b class II MHC molecules but fail to recognize the same envelope protein in the context of I-Ak or I-Ek molecules. This influence of the H-2I region on T cell recognition of the envelope glycoprotein appeared to control in vivo induction of protective immunity against Friend virus complex after immunization with the vaccinia-F-MuLV env vaccine. Thus, these results provide, for the first time, direct evidence for Ir gene-controlled responder/nonresponder phenotypes influencing the immune response to a pathogenic virus of mice. PMID:3141552

  14. CCL3 and CCL20-recruited dendritic cells modified by melanoma antigen gene-1 induce anti-tumor immunity against gastric cancer ex vivo and in vivo

    PubMed Central

    2010-01-01

    Background To investigate whether dendritic cell (DC) precursors, recruited by injection of chemokine ligand 3 (CCL3) and CCL20, induce anti-tumor immunity against gastric cancer induced by a DC vaccine expressing melanoma antigen gene-1 (MAGE-1) ex vivo and in vivo. Methods B6 mice were injected with CCL3 and CCL20 via the tail vein. Freshly isolated F4/80-B220-CD11c+ cells cultured with cytokines were analyzed by phenotype analysis and mixed lymphocyte reaction (MLR). For adenoviral (Ad)-mediated gene transduction, cultured F4/80-B220-CD11c+ cells were incubated with Ad-MAGE-1. Vaccination of stimulated DC induced T lymphocytes. The killing effect of these T cells against gastric carcinoma cells was assayed by MTT. INF-γ production was determined with an INF-γ ELISA kit. In the solid tumor and metastases model, DC-based vaccines were used for immunization after challenge with MFC cells. Tumor size, survival of mice, and number of pulmonary metastatic foci were used to assess the therapeutic effect of DC vaccines. Results F4/80-B220-CD11c+ cell numbers increased after CCL3 and CCL20 injection. Freshly isolated F4/80-B220-CD11c+ cells cultured with cytokines were phenotyically identical to typical DC and gained the capacity to stimulate allogeneic T cells. These DCs were transduced with Ad-MAGE-1, which were prepared for DC vaccines expressing tumor antigen. T lymphocytes stimulated by DCs transduced with Ad-MAGE-1 exhibited specific killing effects on gastric carcinoma cells and produced high levels of INF-γ ex vivo. In vivo, tumor sizes of the experimental group were much smaller than both the positive control group and the negative control groups (P < 0.05). Kaplan-Meier survival curves showed that survival of the experimental group mice was significantly longer than the control groups (P < 0.05). In addition, MAGE-1-transduced DCs were also a therapeutic benefit on an established metastatic tumor, resulting in a tremendous decrease in the number of pulmonary

  15. Mutation of a Salmonella serogroup-C1-specific gene abrogates O7-antigen biosynthesis and triggers NaCl-dependent motility deficiency.

    PubMed

    Zhou, Xiujuan; Liu, Bin; Shi, Chunlei; Shi, Xianming

    2014-01-01

    Several molecular detection marker genes specific for a number of individual Salmonella serogroups have been recently identified in our lab by comparative genomics for the genotyping of diverse serogroups. To further understand the correlation between serotype and genotype, the function of a Salmonella serogroup-C1-specific gene (SC_2092) was analyzed in this study. It was indicated from the topological prediction using the deduced amino acid sequence of SC_2092 that this putative protein was highly similar to the confirmed Wzx flippases. Furthermore, SDS-PAGE revealed that lipopolysaccharide (LPS) biosynthesis, specifically O-antigen synthesis, was incomplete in an SC_2092 in-frame deletion mutant, and no agglutination reaction with the O7 antibody was exhibited in this mutant. Therefore, it was revealed that this Salmonella serogroup-C1-specific gene SC_2092 encoded a putative flippase, which was required for O7-polysaccharide biosynthesis, and was designated here as wzxC1. Subsequently, the effects of the deletion of wzxC1 on bacterial motility and sodium chloride (NaCl) tolerance were evaluated. The wzxC1 mutant lacked swarming motility on solid surfaces and was impaired in swimming motility in soft agar. Moreover, microscopic examination and RT-qPCR exhibited that an increased auto-aggregation and a strong defect in flagella expression, respectively, were responsible for the reduced motility in this mutant. In addition, the wzxC1 mutant was more sensitive than the wild-type strain to NaCl, and auto-aggregation of mutant cells was observed immediately up on the addition of 1% NaCl to the medium. Interestingly, the motility deficiency of the mutant strain, as well as the cell agglomeration and the decrease in flagellar expression, were relieved in a NaCl-free medium. This is the first study to experimentally demonstrate a connection between a Salmonella serogroup specific gene identified by comparative genomics with the synthesis of a specific O-antigen

  16. The Widespread Multidrug-Resistant Serotype O12 Pseudomonas aeruginosa Clone Emerged through Concomitant Horizontal Transfer of Serotype Antigen and Antibiotic Resistance Gene Clusters.

    PubMed

    Thrane, Sandra Wingaard; Taylor, Véronique L; Freschi, Luca; Kukavica-Ibrulj, Irena; Boyle, Brian; Laroche, Jérôme; Pirnay, Jean-Paul; Lévesque, Roger C; Lam, Joseph S; Jelsbak, Lars

    2015-09-22

    The O-specific antigen (OSA) in Pseudomonas aeruginosa lipopolysaccharide is highly varied by sugar identity, side chains, and bond between O-repeats. These differences classified P. aeruginosa into 20 distinct serotypes. In the past few decades, O12 has emerged as the predominant serotype in clinical settings and outbreaks. These serotype O12 isolates exhibit high levels of resistance to various classes of antibiotics. Here, we explore how the P. aeruginosa OSA biosynthesis gene clusters evolve in the population by investigating the association between the phylogenetic relationships among 83 P. aeruginosa strains and their serotypes. While most serotypes were closely linked to the core genome phylogeny, we observed horizontal exchange of OSA biosynthesis genes among phylogenetically distinct P. aeruginosa strains. Specifically, we identified a "serotype island" ranging from 62 kb to 185 kb containing the P. aeruginosa O12 OSA gene cluster, an antibiotic resistance determinant (gyrA(C248T)), and other genes that have been transferred between P. aeruginosa strains with distinct core genome architectures. We showed that these genes were likely acquired from an O12 serotype strain that is closely related to P. aeruginosa PA7. Acquisition and recombination of the "serotype island" resulted in displacement of the native OSA gene cluster and expression of the O12 serotype in the recipients. Serotype switching by recombination has apparently occurred multiple times involving bacteria of various genomic backgrounds. In conclusion, serotype switching in combination with acquisition of an antibiotic resistance determinant most likely contributed to the dissemination of the O12 serotype in clinical settings. Infection rates in hospital settings by multidrug-resistant (MDR) Pseudomonas aeruginosa clones have increased during the past decades, and serotype O12 is predominant among these epidemic strains. It is not known why the MDR phenotype is associated with serotype

  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. Molecular cloning, expression, and chromosomal localization of the gene encoding a human myeloid membrane antigen (gp150).

    PubMed Central

    Look, A T; Peiper, S C; Rebentisch, M B; Ashmun, R A; Roussel, M F; Lemons, R S; Le Beau, M M; Rubin, C M; Sherr, C J

    1986-01-01

    DNA from a tertiary mouse cell transformant containing amplified human sequences encoding a human myeloid membrane glycoprotein, gp150, was used to construct a bacteriophage lambda library. A single recombinant phage containing 12 kilobases (kb) of human DNA was isolated, and molecular subclones were then used to isolate the complete gp150 gene from a human placental genomic DNA library. The intact gp150 gene, assembled from three recombinant phages, proved to be biologically active when transfected into NIH 3T3 cells. Molecular probes from the gp150 locus annealed with a 4.0-kb polyadenylated RNA transcript derived from human myeloid cell lines and from tertiary mouse cell transformants. The gp150 gene was assigned to human chromosome 15, and was subchromosomally localized to bands q25-26 by in situ hybridization. The chromosomal location of the gp150 gene coincides cytogenetically with the region assigned to the c-fes proto-oncogene, another human gene specifically expressed by myeloid cells. Images PMID:2428842

  20. ANTIGENIC MODULATION

    PubMed Central

    Old, Lloyd J.; Stockert, Elisabeth; Boyse, Edward A.; Kim, Jae Ho

    1968-01-01

    Antigenic modulation (the loss of TL antigens from TL+ cells exposed to TL antibody in the absence of lytic complement) has been demonstrated in vitro. An ascites leukemia, phenotype TL.1,2,3, which modulates rapidly and completely when incubated with TL antiserum in vitro, was selected for further study of the phenomenon. Over a wide range of TL antibody concentrations modulation at 37°C was detectable within 10 min and was complete within approximately 1 hr. The cells were initially sensitized to C' by their contact with antibody, thereafter losing this sensitivity to C' lysis together with their sensitivity to TL antibody and C' in the cytotoxic test. The capacity of the cells to undergo modulation was abolished by actinomycin D and by iodoacetamide, and by reducing the temperature of incubation to 0°C. Thus modulation apparently is an active cellular process. Antigens TL. 1,2, and 3 are all modulated by anti-TL.1,3 serum and by anti-TL.3 serum. This modulation affects all three TL components together, even when antibody to one or two of them is lacking. aAnti-TL.2 serum does not induce modulation and in fact impairs modulation by the other TL antibodies. The influence of the TL phenotype of cells upon the demonstrable content of H-2 (D region) isoantigen, first shown in cells modulated in vivo, has been observed with cells modulated in vitro. Cells undergoing modulation show a progressive increase in H-2 (D region) antigen over a period of 4 hr, with no change in H-2 antigens of the K region. Restoration of the TL+ phenotype of modulated cells after removal of antibody is less rapid than TL+ → TL- modulation and may require several cell divisions. PMID:5636556

  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. 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.

  3. Evidence that Mls-2 antigens which delete V beta 3+ T cells are controlled by multiple genes.

    PubMed

    Pullen, A M; Marrack, P; Kappler, J W

    1989-05-01

    V beta 3+ T cells are eliminated in Mls-2a mice carrying some, but not all, H-2 types. Analysis of AKXD and BXD recombinant inbred strains showed that Mls-2a (formerly Mlsc) was not the product of a single gene and suggested that at least two non-H-2 genes control V beta 3 levels. Studies of the progeny of a B10.BR x (C3H/HeJ x B10.BR)F1 backcross confirmed the existence of two V beta 3+ T cell deleting genes: one unlinked and one linked to Ly-7, which we propose be called Mls-2 and Mls-3, respectively. Mls-2a induces partial deletion of V beta 3+ T cells with a bias toward deleting CD4+ cells. It stimulates V beta 3+ hybrids and may be linked to Mtv-13 on chromosome 4. A third non-H-2 gene is implicated in enhancing the presentation of Mls-2a. Mls-3a causes elimination of all V beta 3+ T cells in H-2k and H-2d mice but poorly stimulates V beta 3+ hybrids.

  4. 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

  5. Association of cytotoxic T-lymphocyte antigen 4 +49A/G gene polymorphism with acute rejection risk in renal transplantation.

    PubMed

    Yang, Chun-Hua; Chen, Xue-Xia; Chen, Li; Zheng, Dong-Hua; Liu, Qiong-Shan; Xie, Wen-Feng

    2017-03-23

    The conclusions on the association between cytotoxic T-lymphocyte antigen 4 (CTLA4) +49A/G gene polymorphism and acute rejection risk in renal transplantation are still debated. This meta-analysis was performed to update the association between CTLA4 +49A/G and acute rejection risk in renal transplantation. The association investigations were identified from PubMed and Cochrane Library, and eligible studies were included and synthesized using meta-analysis method. Fourteen reports were included into this meta-analysis for the association of CTLA4 A/G gene polymorphism and acute rejection risk in renal transplantation, consisting of 962 acute rejection patients and 2084 non-acute rejection controls. The association between CTLA4 G allele/GG genotype and acute rejection risk in renal transplantation was found in this meta-analysis (G allele: OR=1.21, 95% CI: 1.03-1.44, P=.02; GG genotype: OR=1.37, 95% CI: 1.10-1.69, P=.004). However, the AA genotype was not associated with acute rejection risk in renal transplantation. In conclusion, CTLA4 G allele/GG genotype is associated with the acute rejection risk in renal transplantation.

  6. Gene variations of Epstein-Barr virus nuclear antigen 3A in nasopharyngeal carcinomas, gastric carcinomas and healthy carriers in northern China.

    PubMed

    Wang, Xiaofeng; Wu, Guocai; Wang, Yun; Sun, Zhifu; Luo, Bing

    2013-10-01

    The Epstein-Barr virus (EBV) nuclear antigen protein 3A (EBNA-3A), a protein of 944 amino acids, is one of five EBNAs (EBNA-1, -2, -LP, -3A and -3C) essential for conversion of primary B lymphocytes to lymphoblastoid cell lines. To characterize the variations of the EBNA-3A gene and explore the association between EBNA-3A gene variations and EBV-associated diseases, we sequenced the key regions of EBNA-3A in the isolates of 30 EBV-associated gastric carcinomas (EBVaGCs), 44 nasopharyngeal carcinomas (NPCs) and 48 samples from healthy donors in northern China. We found that EBNA-3A shares a common evolutionary origin with isolates from southern China and Japan but has the character of a geographical variant. Based on a phylogenetic tree, all of the samples can be subdivided into three patterns, named 3A-8, 3A-5 and B95-8-like. The distribution of EBNA-3A subtypes among EBVaGC, NPC and healthy donors is not significantly different. The subtype 3A-8 is predominant not only in northern China but also in southern China; it is a geographically associated polymorphism in China.

  7. Transcription of a subset of human class II major histocompatibility complex genes is regulated by a nucleoprotein complex that contains c-fos or an antigenically related protein.

    PubMed Central

    Ono, S J; Bazil, V; Levi, B Z; Ozato, K; Strominger, J L

    1991-01-01

    Transcriptional regulation of the human major histocompatibility complex class II genes requires at least two upstream elements, the X and Y boxes, located in the -50- to -150-base-pair region of all class II promoters. The DRA and DPB promoters contain phorbol ester-responsive elements overlapping the 3' side of their X boxes. Mutation of this sequence down-regulates the efficiency of the DRA promoter, suggesting that a positive regulator(s) binds to this site. In this report, anti-sense c-fos RNA and an anti-c-fos antibody were used to show that the product of the protooncogene c-fos or an antigenically related protein is a component of a complex that binds to the X box and is required for maximal transcription from the DRA and DPB promoters. As c-fos (or its related proteins) cannot bind alone to DNA, these results suggest that it may dimerize with other members of the JUN/AP-1 family, such as hXBP1, to participate in the activation of a subset of class II major histocompatibility complex genes. Images PMID:1709740

  8. Differential regulation of Salmonella typhimurium genes involved in O-antigen capsule production and their role in persistence within tomato fruit.

    PubMed

    Marvasi, Massimiliano; Cox, Clayton E; Xu, Yimin; Noel, Jason T; Giovannoni, James J; Teplitski, Max

    2013-07-01

    Enteric pathogens, including non-typhoidal Salmonella spp. and enterovirulent Escherichia coli, are capable of persisting and multiplying within plants. Yet, little is still known about the mechanisms of these interactions. This study identified the Salmonella yihT gene (involved in synthesis of the O-antigen capsule) as contributing to persistence in immature tomato fruit. Deletion of yihT reduced competitive fitness of S. enterica sv. Typhimurium in green (but not ripe, regardless of color) tomato fruit by approximately 3 logs. The yihT recombinase-based in vivo expression technology (RIVET) reporter was strongly activated in unripe tomato fruit, and fitness of the mutant inversely correlated with the level of the yihT gene expression. Expression of yihT in mature tomato fruit was low, and yihT did not affect competitive fitness within mature fruit. To better understand the molecular basis of the phenotype, behaviors of the yihT RIVET reporter and the yihT mutant were tested in tomato fruit defective in ethylene signaling. These experiments suggest a role for functional ethylene-mediated signaling in the persistence of Salmonella spp. within tomato fruit. Furthermore, jasmonic acid and its precursors strongly reduced expression of yihT.

  9. Highly specific antibody to Rous sarcoma virus src gene product recognizes nuclear and nucleolar antigens in human cells.

    PubMed Central

    David-Pfeuty, T; Nouvian-Dooghe, Y

    1995-01-01

    An antiserum to the Rous sarcoma virus-transforming protein pp60v-src, raised in rabbits immunized with the bacterially produced protein alpha p60 serum (M. D. Resh and R. L. Erikson, J. Cell Biol. 100:409-417, 1985) previously reported to detect very specifically a novel population of pp60v-src and pp60c-src molecules associated with juxtareticular nuclear membranes in normal and Rous sarcoma virus-infected cells of avian and mammalian origin, was used here to investigate by immunofluorescence microscopy localization patterns of Src molecules in human cell lines, either normal or derived from spontaneous tumors. We found that the alpha p60 serum reveals nuclear and nucleolar concentrations of antigens in all the human cell lines tested and in two rat and mouse hepatoma cell lines derived from adult tumorous tissues but not in any established rat and mouse cell lines either untransformed or transformed by the src and ras oncogenes. Both the nuclear and nucleolar stainings can be totally extinguished by preincubation of the serum with highly purified chicken c-Src. We show also that the partitioning of the alpha p60-reactive proteins among the whole nucleus and the nucleolus depends mostly on two different parameters: the position in the cell cycle and the degree of cell confluency. Our observations raise the attractive possibility that, in differentiated cells, pp60c-src and related proteins might be involved not only in mediating the transduction of mitogenic signals at the plasma membrane level but also in controlling progression through the cell cycle and entry in mitosis by interacting with cell division cycle regulatory components at the nuclear level. PMID:7853507

  10. Serological analysis of human tumor antigens: molecular definition and implications.

    PubMed

    Türeci, O; Sahin, U; Pfreundschuh, M

    1997-08-01

    Specific vaccines for the immunotherapy of human neoplasms require specific human tumor antigens. While efforts to identify such antigens by the analysis of the T-cell repertoire have yielded few antigens, the application of SEREX, the serological identification of antigens by recombinant expression cloning, has brought a cornucopia of new antigens. Several specific antigens have been identified in each tumor tested, suggesting that many human tumors elicit multiple immune responses in the autologous host. The frequency of human tumor antigens, which can be readily defined at the molecular level, facilitates the identification of T-cell-dependent antigens and provides a basis for peptide and gene-therapeutic vaccine strategies.

  11. A pilot study of the association of manganese superoxide dismutase and glutathione peroxidase 1 single gene polymorphisms with prostate cancer and serum prostate specific antigen levels

    PubMed Central

    Atilgan, Dogan; Gencten, Yusuf; Benli, Ismail; Ozyurt, Huseyin; Uluocak, Nihat; Erdemir, Fikret

    2015-01-01

    Introduction The aim of the study was to evaluate the potential association of single gene polymorphisms of the antioxidant enzymes manganese superoxide dismutase (MnSOD) and glutathione peroxidase (GPX1) with prostate cancer (PCa). Material and methods Manganese superoxide dismutase and glutathione peroxidase 1 genotypes and allele frequencies in 49 prostate cancer cases (PCa group) and 98 control subjects were determined. Analysis of genotypes in control group individuals were performed in two subgroups according to serum prostate-specific antigen levels: the control group (n = 49), with prostate specific antigen (PSA) level < 4 ng/ml; and the nonPCa-high PSA control group (n = 49), with serum PSA > 4 ng/ml. Determination of MnSOD Ala-9Val and GPX1 Pro198Leu polymorphisms was performed using real-time polymerase chain reaction amplification. Results No association was found between GPX1 polymorphisms and PCa in all groups (p > 0.05). In the PCa group, the frequency of homozygote Val allele carriers was significantly higher in comparison to nonPCa-high PSA control cases. Therefore, Val/Val genotype was found significantly suspicious for PCa risk (OR = 2.48; 95% CI: 1.37–4.48; p = 0.002). Furthermore, an overall protective effect of the Ala allele of the MnSOD polymorphism on PCa risk was detected. These findings in this small Turkish population suggested that individual risk of PCa may be modulated by MnSOD polymorphism especially in patients with high PSA, but GPX1 polymorphism seemed to have no effect on PCa risk. Conclusions The presence of genetic variants of antioxidant enzymes could have a potential influence on genesis of prostatic malignancy. PMID:26528342

  12. Cloning of the Gene Encoding a 22-Kilodalton Cell Surface Antigen of Mycobacterium bovis BCG and Analysis of Its Potential for DNA Vaccination against Tuberculosis

    PubMed Central

    Lefèvre, Philippe; Denis, Olivier; De Wit, Lucas; Tanghe, Audrey; Vandenbussche, Paul; Content, Jean; Huygen, Kris

    2000-01-01

    Using spleen cells from mice vaccinated with live Mycobacterium bovis BCG, we previously generated three monoclonal antibodies reactive against a 22-kDa protein present in mycobacterial culture filtrate (CF) (K. Huygen et al., Infect. Immun. 61:2687–2693, 1993). These monoclonal antibodies were used to screen an M. bovis BCG genomic library made in phage λgt11. The gene encoding a 233-amino-acid (aa) protein, including a putative 26-aa signal sequence, was isolated, and sequence analysis indicated that the protein was 98% identical with the M. tuberculosis Lppx protein and that it contained a sequence 94% identical with the M. leprae 38-mer polypeptide 13B3 recognized by T cells from killed M. leprae-immunized subjects. Flow cytometry and cell fractionation demonstrated that the 22-kDa CF protein is also highly expressed in the bacterial cell wall and membrane compartment but not in the cytosol. C57BL/6, C3H, and BALB/c mice were vaccinated with plasmid DNA encoding the 22-kDa protein and analyzed for immune response and protection against intravenous M. tuberculosis challenge. Whereas DNA vaccination induced elevated antibody responses in C57BL/6 and particularly in C3H mice, Th1-type cytokine response, as measured by interleukin-2 and gamma interferon secretion, was only modest, and no protection against intravenous M. tuberculosis challenge was observed in any of the three mouse strains tested. Therefore, the 22-kDa antigen seems to have little potential for a DNA vaccine against tuberculosis, but it may be a good candidate for a mycobacterial antigen detection test. PMID:10678905

  13. 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.

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

    DTIC Science & Technology

    1989-12-11

    of template, 4 unit of Taq polymerase, 125 mM dNTP, I g.M each of primer. The reactions were perFormed for 25 cycles . The amplified DNA were...25 cycles of denaturation, annealing and DNA synthesis was digested with KpnI and then purified by electrophoresis on an agarose gel. For cloning...Lication (Send deletion mnutant) MMH294 MC k. Lysogen carrying wuild type repressor gene * N5 151 Lysogen carrying temperature sensitive mutation in

  15. The upstream sequence minus 537 to minus 278 is necessary for transcription of the human nucleolar antigen p120 gene

    SciTech Connect

    Haidar, M.A.; Henning, D.; Busch, H. . Coll. of Medicine)

    1990-06-01

    This paper discusses how two {ital cis}-acting elements in the p120 genes play important roles in transcription; the region from {minus}537 to {minus}278 is necessary for initiation of transcription, and the region from {minus}1426 to {minus}1223 is necessary for efficient transcription. The distal element(s) which lies upstream of {minus}278 is required for initiation of transcription.

  16. Measuring T cell receptor and T cell gene expression diversity in antigen-responsive human CD4+ T cells.

    PubMed

    Eugster, Anne; Lindner, Annett; Heninger, Anne-Kristin; Wilhelm, Carmen; Dietz, Sevina; Catani, Mara; Ziegler, Anette-G; Bonifacio, Ezio

    2013-12-31

    T cells have diversity in TCR, epitope recognition, and cytokine production, and can be used for immune monitoring. Furthermore, clonal expansion of TCR families in disease may provide opportunities for TCR-directed therapies. We developed methodology for sequencing expressed genes of TCR alpha and beta chains from single cells and applied this to vaccine (tetanus-toxoid)-responsive CD4(+) T cells. TCR alpha and beta chains were both successfully sequenced in 1309 (43%) of 3038 CD4(+) T cells yielding 677 different receptors. TRAV and TRBV gene usage differed between tetanus-toxoid-responsive and non-responsive cells (p=0.004 and 0.0002), and there was extensive TCR diversity in tetanus-toxoid-responsive cells within individuals. Identical TCRs could be recovered in different samples from the same subject: TCRs identified after booster vaccination were frequent in pre-booster memory T cells (31% of pre-booster TCR), and also identified in pre-booster vaccination naïve cells (6.5%). No TCR was shared between subjects, but tetanus toxoid-responsive cells sharing one of their TCR chains were observed within and between subjects. Coupling single-cell gene expression profiling to TCR sequencing revealed examples of distinct cytokine profiles in cells bearing identical TCR. Novel molecular methodology demonstrates extensive diversity of Ag-responsive CD4(+) T cells within and between individuals. Copyright © 2013 Elsevier B.V. All rights reserved.

  17. Structure of the CD59-encoding gene: further evidence of a relationship to murine lymphocyte antigen Ly-6 protein.

    PubMed

    Petranka, J G; Fleenor, D E; Sykes, K; Kaufman, R E; Rosse, W F

    1992-09-01

    The gene for CD59 [membrane inhibitor of reactive lysis (MIRL), protectin], a phosphatidylinositol-linked surface glycoprotein that regulates the formation of the polymeric C9 complex of complement and that is deficient on the abnormal hematopoietic cells of patients with paroxysmal nocturnal hemoglobinuria, consists of four exons spanning 20 kilobases. The untranslated first exon is preceded by a G+C-rich promoter region that lacks a consensus TATA or CAAT motif. The second exon encodes the hydrophobic leader sequence of the protein, and the third exon encodes the amino-terminal portion of the mature protein. The fourth exon encodes the remainder of the mature protein, including the hydrophobic sequence necessary for glycosyl-phosphatidylinositol anchor attachment. The structure of the CD59 gene is very similar to that encoding Ly-6, a murine glycoprotein with which CD59 has some structural similarity. The striking similarity in gene structure is further evidence that the two proteins belong to a superfamily of proteins that may also include the urokinase plasminogen-activator receptor and a squid glycoprotein of unknown function.

  18. Structure of the CD59-encoding gene: further evidence of a relationship to murine lymphocyte antigen Ly-6 protein.

    PubMed Central

    Petranka, J G; Fleenor, D E; Sykes, K; Kaufman, R E; Rosse, W F

    1992-01-01

    The gene for CD59 [membrane inhibitor of reactive lysis (MIRL), protectin], a phosphatidylinositol-linked surface glycoprotein that regulates the formation of the polymeric C9 complex of complement and that is deficient on the abnormal hematopoietic cells of patients with paroxysmal nocturnal hemoglobinuria, consists of four exons spanning 20 kilobases. The untranslated first exon is preceded by a G+C-rich promoter region that lacks a consensus TATA or CAAT motif. The second exon encodes the hydrophobic leader sequence of the protein, and the third exon encodes the amino-terminal portion of the mature protein. The fourth exon encodes the remainder of the mature protein, including the hydrophobic sequence necessary for glycosyl-phosphatidylinositol anchor attachment. The structure of the CD59 gene is very similar to that encoding Ly-6, a murine glycoprotein with which CD59 has some structural similarity. The striking similarity in gene structure is further evidence that the two proteins belong to a superfamily of proteins that may also include the urokinase plasminogen-activator receptor and a squid glycoprotein of unknown function. Images PMID:1381503

  19. The DNA sequence of the H-2kb gene: evidence for gene conversion as a mechanism for the generation of polymorphism in histocompatibilty antigens.

    PubMed Central

    Weiss, E; Golden, L; Zakut, R; Mellor, A; Fahrner, K; Kvist, S; Flavell, R A

    1983-01-01

    We have determined the DNA sequence of the H-2Kb gene of the C57B1/10 mouse. Comparison of this sequence with that of the allelic H-2Kd shows surprisingly that the exons have accumulated more mutations than their introns. Moreover, many of these changes in the exons are clustered in short regions or hot spots. Additional comparison of these sequences with the H-2Ld and H-2Db sequences shows that, in several cases, the altered sequence generated at the hot spot is identical to the corresponding region of a non-allelic H-2 gene. The clustered changes are responsible for 60% of the amino acid differences between the H-2Kb and H-2Kd genes and suggest that micro-gene conversion events occurring within the exons and involving only tens of nucleotides are an important mechanism for the generation of polymorphic differences between natural H-2 alleles. Images Fig. 3. PMID:11894963

  20. MicroRNA-762 is upregulated in human corneal epithelial cells in response to tear fluid and Pseudomonas aeruginosa antigens and negatively regulates the expression of host defense genes encoding RNase7 and ST2.

    PubMed

    Mun, James; Tam, Connie; Chan, Gary; Kim, Jong Hun; Evans, David; Fleiszig, Suzanne

    2013-01-01

    Mucosal surfaces regulate defenses against infection and excessive inflammation. We previously showed that human tears upregulated epithelial expression of genes encoding RNase7 and ST2, which inhibited Pseudomonas aeruginosa invasion of human corneal epithelial cells. Here, microRNA microarrays were used to show that a combination of tear fluid exposure (16 h) then P. aeruginosa antigens (3 h) upregulated miR-762 and miR-1207, and down-regulated miR-92 and let-7b (all > 2-fold) in human corneal epithelial cells compared to P. aeruginosa antigens alone. RT-PCR confirmed miR-762 upregulation ∼ 3-fold in tear-antigen exposed cells. Without tears or antigens, an antagomir reduced miR-762 expression relative to scrambled controls by ∼50%, increased expression of genes encoding RNase7 (∼80 %), ST2 (∼58%) and Rab5a (∼75%), without affecting P. aeruginosa internalization. However, P. aeruginosa invasion was increased > 3-fold by a miR-762 mimic which reduced RNase7 and ST2 gene expression. Tear fluid alone also induced miR-762 expression ∼ 4-fold, which was reduced by the miR-762 antagomir. Combination of tear fluid and miR-762 antagomir increased RNase7 and ST2 gene expression. These data show that mucosal fluids, such as tears, can modulate epithelial microRNA expression to regulate innate defense genes, and that miR-762 negatively regulates RNase7, ST2 and Rab5a genes. Since RNase7 and ST2 inhibit P. aeruginosa internalization, and are upregulated by tear fluid, other tear-induced mechanisms must counteract inhibitory effects of miR-762 to regulate resistance to bacteria. These data also suggest a complex relationship between tear induction of miR-762, its modulation of innate defense genes, and P. aeruginosa internalization.

  1. Deletion of the Vaccinia Virus N2L Gene Encoding an Inhibitor of IRF3 Improves the Immunogenicity of Modified Vaccinia Virus Ankara Expressing HIV-1 Antigens

    PubMed Central

    García-Arriaza, Juan; Gómez, Carmen E.; Sorzano, Carlos Óscar S.

    2014-01-01

    ABSTRACT A modified vaccinia virus Ankara poxvirus vector expressing the HIV-1 Env, Gag, Pol, and Nef antigens from clade B (MVA-B) is currently being tested in clinical trials. To improve its immunogenicity, we have generated and characterized the immune profile of MVA-B containing a deletion of the vaccinia viral gene N2L, which codes for an inhibitor of IRF3 (MVA-B ΔN2L). Deletion of N2L had no effect on virus growth kinetics or on the expression of HIV-1 antigens; hence, the N2 protein is not essential for MVA replication. The innate immune responses triggered by MVA-B ΔN2L revealed an increase in beta interferon, proinflammatory cytokines, and chemokines. Mouse prime-boost protocols showed that MVA-B ΔN2L improves the magnitude and polyfunctionality of HIV-1-specific CD4+ and CD8+ T cell adaptive and memory immune responses, with most of the HIV-1 responses mediated by CD8+ T cells. In the memory phase, HIV-1-specific CD8+ T cells with an effector phenotype were predominant and in a higher percentage with MVA-B ΔN2L than with MVA-B. In both immunization groups, CD4+ and CD8+ T cell responses were directed mainly against Env. Furthermore, MVA-B ΔN2L in the memory phase enhanced levels of antibody against Env. For the vector immune responses, MVA-B ΔN2L induced a greater magnitude and polyfunctionality of VACV-specific CD8+ T memory cells than MVA-B, with an effector phenotype. These results revealed the immunomodulatory role of N2L, whose deletion enhanced the innate immunity and improved the magnitude and quality of HIV-1-specific T cell adaptive and memory immune responses. These findings are relevant for the optimization of poxvirus vectors as vaccines. IMPORTANCE On the basis of the limited efficacy of the RV144 phase III clinical trial, new optimized poxvirus vectors as vaccines against HIV/AIDS are needed. Here we have generated and characterized a new HIV/AIDS vaccine candidate on the basis of the poxvirus MVA vector expressing HIV-1 Env, Gag, Pol

  2. Correlation between preferentially expressed antigen of melanoma and tumour necrosis factor-related apoptosis-inducing ligand gene expression in different types of leukaemia patients.

    PubMed

    Zhang, Wenhui; Chi, Kaikai; Zhang, Yin; Ma, Baogen; Shi, Jie; Chen, Yuqing; Lei, Pingchong; Li, Yulong; Sun, Kai

    2013-01-01

    Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) down-regulation by preferentially expressed antigen of melanoma (PRAME) is a general phenomenon in different types of solid tumours, but research on the correlation between PRAME and TRAIL gene expression in leukaemia patients is rare. PRAME and TRAIL expression was detected in bone marrow samples from 80 newly diagnosed acute leukaemia (AL) patients and 40 chronic myeloid leukaemia (CML) patients using TaqMan-based real-time quantitative PCR methods, and a linear correlation analysis was performed on their levels of expression. A total of 15 normal bone marrow samples from individuals with non-malignant haematological diseases served as normal controls. PRAME expression was higher in both AL and CML patients compared to controls (both p < 0.001). CML patients in both blast crisis (BC) and the accelerated phase (AP) had significantly higher PRAME levels than CML patients in the chronic phase (CP) (p = 0.006 and 0.0461, respectively). TRAIL expression was higher in both the acute myeloid leukaemia (AML) group and the acute lymphoblastic leukaemia (ALL) group than in the controls (p = 0.039 and 0.047, respectively). In contrast, CML patients had lower TRAIL levels than controls (p = 0.043), and TRAIL expression in CML patients in the advanced phases (BC and AP) was significantly lower than in CML-CP patients (p = 0.006). In CML patients, there was a significant inverse correlation (Spearman's R = -0.6669, p < 0.0001) between PRAME and TRAIL gene expression, while a greater significant inverse correlation was found in patients in the advanced phases (BC and AP) (R = -0.6764). In addition, no correlation was observed in AML and ALL patients. The simultaneous detection of PRAME and TRAIL gene expression may be helpful to monitor condition changes in leukaemia patients and evaluate therapeutic effects in clinical practice, particularly in CML patients. © 2013 S. Karger AG, Basel.

  3. 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.

  4. Cloning and Expression of the dnaK Gene of Campylobacter jejuni and Antigenicity of Heat Shock Protein 70

    PubMed Central

    Thies, Frank L.; Karch, Helge; Hartung, Hans-Peter; Giegerich, Gerhard

    1999-01-01

    Campylobacter jejuni is a leading cause of infectious diarrhea throughout the world. In addition, there is growing evidence that Guillain-Barré syndrome, an inflammatory demyelinating disease of the peripheral nervous system, is frequently preceded by C. jejuni infection. In the present study, the hrcA-grpE-dnaK gene cluster of C. jejuni was cloned and sequenced. The dnaK gene consists of an open reading frame of 1,869 bp and encodes a protein with a high degree of homology to other bacterial 70-kDa heat shock proteins (HSPs). The overall percentages of identity to the HSP70 proteins of Helicobacter pylori, Borrelia burgdorferi, Chlamydia trachomatis, and Bacillus subtilis were calculated to be 78.1, 60.5, 57.2, and 53.8%, respectively. Regions similar to the Escherichia coli ς70 promoter consensus sequence and to a cis-acting regulatory element (CIRCE) are located upstream of the hrcA gene. Following heat shock, a rapid increase of dnaK mRNA was detectable, which reached its maximum after 20 to 30 min. A 6-His-tagged recombinant DnaK protein (rCjDnaK-His) was generated in E. coli, after cloning of the dnaK coding region into pET-22b(+), and purified by affinity and gel filtration chromatography. Antibody responses to rCjDnaK-His were significantly elevated, compared to those of healthy individuals, in about one-third of the serum specimens obtained from C. jejuni enteritis patients. PMID:10024560

  5. Immunogenic Profiling in Mice of a HIV/AIDS Vaccine Candidate (MVA-B) Expressing Four HIV-1 Antigens and Potentiation by Specific Gene Deletions

    PubMed Central

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

    2010-01-01

    Background The immune parameters of HIV/AIDS vaccine candidates that might be relevant in protection against HIV-1 infection are still undefined. The highly attenuated poxvirus strain MVA is one of the most promising vectors to be use as HIV-1 vaccine. We have previously described a recombinant MVA expressing HIV-1 Env, Gag, Pol and Nef antigens from clade B (referred as MVA-B), that induced HIV-1-specific immune responses in different animal models and gene signatures in human dendritic cells (DCs) with immunoregulatory function. Methodology/Principal Findings In an effort to characterize in more detail the immunogenic profile of MVA-B and to improve its immunogenicity we have generated a new vector lacking two genes (A41L and B16R), known to counteract host immune responses by blocking the action of CC-chemokines and of interleukin 1β, respectively (referred as MVA-B ΔA41L/ΔB16R). A DNA prime/MVA boost immunization protocol was used to compare the adaptive and memory HIV-1 specific immune responses induced in mice by the parental MVA-B and by the double deletion mutant MVA-B ΔA41L/ΔB16R. Flow cytometry analysis revealed that both vectors triggered HIV-1-specific CD4+ and CD8+ T cells, with the CD8+ T-cell compartment responsible for >91.9% of the total HIV-1 responses in both immunization groups. However, MVA-B ΔA41L/ΔB16R enhanced the magnitude and polyfunctionality of the HIV-1-specific CD4+ and CD8+ T-cell immune responses. HIV-1-specific CD4+ T-cell responses were polyfunctional and preferentially Env-specific in both immunization groups. Significantly, while MVA-B induced preferentially Env-specific CD8+ T-cell responses, MVA-B ΔA41L/ΔB16R induced more GPN-specific CD8+ T-cell responses, with an enhanced polyfunctional pattern. Both vectors were capable of producing similar levels of antibodies against Env. Conclusions/Significance These findings revealed that MVA-B and MVA-B ΔA41L/ΔB16R induced in mice robust, polyfunctional and durable T

  6. DNA binding and antigene activity of a daunomycin-conjugated triplex-forming oligonucleotide targeting the P2 promoter of the human c-myc gene

    PubMed Central

    Carbone, Giuseppina M.; McGuffie, Eileen; Napoli, Sara; Flanagan, Courtney E.; Dembech, Chiara; Negri, Umberto; Arcamone, Federico; Capobianco, Massimo L.; Catapano, Carlo V.

    2004-01-01

    Triplex-forming oligonucleotides (TFO) that bind DNA in a sequence-specific manner might be used as selective repressors of gene expression and gene-targeted therapeutics. However, many factors, including instability of triple helical complexes in cells, limit the efficacy of this approach. In the present study, we tested whether covalent linkage of a TFO to daunomycin, which is a potent DNA-intercalating agent and anticancer drug, could increase stability of the triple helix and activity of the oligonucleotide in cells. The 11mer daunomycin-conjugated GT (dauno-GT11) TFO targeted a sequence upstream of the P2 promoter, a site known to be critical for transcription of the c-myc gene. Band-shift assays showed that the dauno-GT11 formed triplex DNA with enhanced stability compared to the unmodified TFO. Band shift and footprinting experiments demonstrated that binding of dauno-GT11 was highly sequence-specific with exclusive binding to the 11 bp target site in the c-myc promoter. The daunomycin-conjugated TFO inhibited transcription in vitro and reduced c-myc promoter activity in prostate and breast cancer cells. The daunomycin-conjugated TFO was taken up by cells with a distinctive intracellular distribution compared to free daunomycin. However, cationic lipid-mediated delivery was required for enhanced cellular uptake, nuclear localization and biological activity of the TFO in cells. Dauno-GT11 reduced transcription of the endogenous c-myc gene in cells, but did not affect expression of non-target genes, such as ets-1 and ets-2, which contained very similar target sequences in their promoters. Daunomycin-conjugated control oligonucleotides unable to form triplex DNA with the target sequence did not have any effect in these assays, indicating that daunomycin was not directly responsible for the activity of daunomycin-conjugated TFO. Thus, attachment of daunomycin resulted in increased triplex stability and biological activity of the 11mer GT-rich TFO without

  7. Analysis of HLA-DR glycoproteins by DNA-mediated gene transfer. Definition of DR2 beta gene products and antigen presentation to T cell clones from leprosy patients

    PubMed Central

    1988-01-01

    We have used DNA-mediated gene transfer to express HLA class II molecules in mouse L cells for serological, biochemical, and functional analysis. cDNA clones encoding the DR2 beta a and DR2 beta b products of the DR2Dw2 haplotype were subcloned into a mouse Moloney leukemia virus-based expression vector (pJ4) and transfected separately into mouse L cells together with a HLA-DR alpha/pJ4 construct. These transfectants have allowed differential analysis of the two DR2 beta products in a manner normally prohibited by the concomitant expression seen in B cells. Two-dimensional SDS-PAGE analysis of the transfectants defines the more acidic beta chain as the product of the DR2 beta a sequence, and the more basic chain as the product of the DR2 beta b sequence. The LDR2a transfectants present antigen efficiently to M.leprae-specific T cell clones and are capable of presenting synthetic peptide, 65-kD recombinant mycobacterial antigen and M.leprae. Of the DR2Dw2-restricted T cell clones we have tested, all use the DR2 beta a chain as their restriction element. Inhibition studies with mAbs demonstrate the dependence of presentation by the transfectant on class II and CD4, while mAbs against LFA-1, which substantially inhibit presentation by B-lymphoblastoid cell lines, do not inhibit transfectant presentation. PMID:3128633

  8. 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

  9. 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

    2016-06-16

    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.

  10. Is there an inter-relationship between prostate specific antigen, kallikrein-2 and androgen receptor gene polymorphisms with risk of prostate cancer in north Indian population?

    PubMed

    Mittal, Rama D; Mishra, Dhruva K; Thangaraj, K; Singh, Rajender; Mandhani, Anil

    2007-04-01

    Androgen receptor (AR) and kallikrein (KLK-2) regulates the PSA (prostate specific antigen) transcription and activation, respectively. We investigated the individual and combined risk of KLK-2, PSA and AR gene polymorphism in histologically confirmed CaP patients and healthy controls from north India. DNA was extracted from peripheral blood leucocytes pellet of 277 subjects. AR repeats analysis was done by PCR-Genscan method. PSA and KLK-2 were genotyped by PCR-RFLP method. Kruskal-Wallis test and logistic regression was applied for mean comparison and risk determination. A significant association for CaP risk was observed with short AR-CAG repeats (OR=3.36, p<0.001) and CC genotype of KLK-2 (OR=2.78, p=0.031), however, no association was found with PSA and AR-GGN repeat polymorphism. PSA/GG genotype was significantly associated with higher Gleason score (> or =7) of tumor (OR=6.23, p<0.01). No association was observed with other confounding variables such as PSA and age with any of these polymorphisms. Thus, we hypothesize that these polymorphisms may influence the etiology of CaP and may have the probability to become appropriate marker either independently or in combination. The combined information on serum PSA level, PSA (G/A), KLK-2 (C/T) genotypes and AR (CAG; GGN repeat) may assist in the deterrence of unnecessary biopsies.

  11. Evolution of the capsid protein genes of foot-and-mouth disease virus: antigenic variation without accumulation of amino acid substitutions over six decades.

    PubMed Central

    Martínez, M A; Dopazo, J; Hernández, J; Mateu, M G; Sobrino, F; Domingo, E; Knowles, N J

    1992-01-01

    The genetic diversification of foot-and-mouth disease virus (FMDV) of serotype C over a 6-decade period was studied by comparing nucleotide sequences of the capsid protein-coding regions of viruses isolated in Europe, South America, and The Philippines. Phylogenetic trees were derived for VP1 and P1 (VP1, VP2, VP3, and VP4) RNAs by using the least-squares method. Confidence intervals of the derived phylogeny (significance levels of nodes and standard deviations of branch lengths) were placed by application of the bootstrap resampling method. These procedures defined six highly significant major evolutionary lineages and a complex network of sublines for the isolates from South America. In contrast, European isolates are considerably more homogeneous, probably because of the vaccine origin of several of them. The phylogenetic analysis suggests that FMDV CGC Ger/26 (one of the earliest FMDV isolates available) belonged to an evolutionary line which is now apparently extinct. Attempts to date the origin (ancestor) of the FMDVs analyzed met with considerable uncertainty, mainly owing to the stasis noted in European viruses. Remarkably, the evolution of the capsid genes of FMDV was essentially associated with linear accumulation of silent mutations but continuous accumulation of amino acid substitutions was not observed. Thus, the antigenic variation attained by FMDV type C over 6 decades was due to fluctuations among limited combinations of amino acid residues without net accumulation of amino acid replacements over time. PMID:1316467

  12. Detection of monoclonality in intestinal lymphoma with polymerase chain reaction for antigen receptor gene rearrangement analysis to differentiate from enteritis in dogs.

    PubMed

    Ohmura, S; Leipig, M; Schöpper, I; Hergt, F; Weber, K; Rütgen, B C; Tsujimoto, H; Hermanns, W; Hirschberger, J

    2017-03-01

    The diagnosis of canine intestinal lymphoma by morphological examination is challenging, especially when endoscopic tissue specimens are used. The utility of detection of antigen receptor gene rearrangement by polymerase chain reaction (PARR) in canine lymphoma has been well established, but its usefulness to distinguish enteritis and intestinal lymphoma remains unclear. In this retrospective study we assessed clonality of 29 primary canine intestinal lymphoma, 14 enteritis and 15 healthy control cases by PARR analysis, using formalin-fixed, paraffin-embedded full-thickness tissue specimens. We could detect monoclonal rearrangements in 22 of 29 canine intestinal lymphomas [76%; 95% confidence interval (CI) 56-90%] and polyclonal rearrangements in all of the enteritis and healthy control cases (100%; CI 88-100%). We revealed a predominance of T-cell phenotype compared to B-cell phenotype (85%; CI 65-96% and 15%; CI 4-35%, respectively). We showed that PARR analysis contributes to differentiation of canine intestinal lymphoma from enteritis and to phenotyping of lymphomas.

  13. CAGE, a novel cancer/testis antigen gene, promotes cell motility by activation ERK and p38 MAPK and downregulating ROS.

    PubMed

    Shim, Hyeeun; Shim, Eunsook; Lee, Hansoo; Hahn, Janghee; Kang, Dongmin; Lee, Yun-Sil; Jeoung, Dooil

    2006-06-30

    We previously identified a novel cancer/testis antigen gene CAGE by screening cDNA expression libraries of human testis and gastric cancer cell lines with sera of gastric cancer patients. CAGE is expressed in many cancers and cancer cell lines, but not in normal tissues apart from the testis. In the present study, we investigated its role in the motility of cells of two human cancer cell lines: HeLa and the human hepatic cancer cell line, SNU387. Induction of CAGE by tetracycline or transient transfection enhanced the migration and invasiveness of HeLa cells, but not the adhesiveness of either cell line. Overexpression of CAGE led to activation of ERK and p38 MAPK but not Akt, and inhibition of ERK by PD98059 or p38 MAPK by SB203580 counteracted the CAGE-promoted increase in motility in both cell lines. Overexpression of CAGE also resulted in a reduction of ROS and an increase of ROS scavenging, associated with induction of catalase activity. Inhibition of ERK and p38 MAPK increased ROS levels in cells transfected with CAGE, suggesting that ROS reduce the motility of both cell lines. Inhibition of ERK and p38 MAPK reduced the induction of catalase activity resulting from overexpression of CAGE, and inhibition of catalase reduced CAGE-promoted motility. We conclude that CAGE enhances the motility of cancer cells by activating ERK and p38 MAPK, inducing catalase activity, and reducing ROS levels.

  14. siRNA associated with immunonanoparticles directed against cd99 antigen improves gene expression inhibition in vivo in Ewing's sarcoma.

    PubMed

    Ramon, A L; Bertrand, J R; de Martimprey, H; Bernard, G; Ponchel, G; Malvy, C; Vauthier, C

    2013-07-01

    Ewing's sarcoma is a rare, mostly pediatric bone cancer that presents a chromosome abnormality called EWS/Fli-1, responsible for the development of the tumor. In vivo, tumor growth can be inhibited specifically by delivering small interfering RNA (siRNA) associated with nanoparticles. The aim of the work was to design targeted nanoparticles against the cell membrane glycoprotein cd99, which is overexpressed in Ewing's sarcoma cells to improve siRNA delivery to tumor cells. Biotinylated poly(isobutylcyanoacrylate) nanoparticles were conceived as a platform to design targeted nanoparticles with biotinylated ligands and using the biotin-streptavidin coupling method. The targeted nanoparticles were validated in vivo for the targeted delivery of siRNA after systemic administration to mice bearing a tumor model of the Ewing's sarcoma. The expression of the gene responsible of Ewing's sarcoma was inhibited at 78% ± 6% by associating the siRNA with the cd99-targeted nanoparticles compared with an inhibition of only 41% ± 9% achieved with the nontargeted nanoparticles.

  15. 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.

  16. TCRs used in cancer gene therapy cross-react with MART-1/Melan-A tumor antigens via distinct mechanisms.

    PubMed

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

    2011-09-01

    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.

  17. Improvement of neurological deficits in 6-hydroxydopamine-lesioned rats after transplantation with allogeneic simian virus 40 large tumor antigen gene-induced immortalized dopamine cells

    PubMed Central

    Clarkson, Edward D.; Rosa, Francisco G. La; Edwards-Prasad, Judith; Weiland, David A.; Witta, Samir E.; Freed, Curt R.; Prasad, Kedar N.

    1998-01-01

    The replacement of dopamine (DA) by DA neuron transplants in the treatment of advanced Parkinson disease (PD) is a rational approach. Because of limitations associated with fetal tissue transplants, a clone (1RB3AN27) of simian virus 40 large tumor antigen (LTa) gene-induced immortalized DA neurons were used in this study. These allogeneic immortalized dopamine neurons, when grafted into striata of normal rats, did not divide, did not form tumors, did not produce LTa, did not extend neurites to host neurons, and were not rejected, for as long as 13 months after transplantation. Grafted cells when recultured in vitro resumed cell proliferation and LTa production, suggesting the presence of a LTa gene-inhibiting factor in the brain. The grafting of undifferentiated and differentiated 1RB3AN27 cells or differentiated murine neuroblastoma (NBP2) cells into striata of 6-hydroxydopamine-lesioned rats (an animal model of PD) caused a time-dependent improvement in neurological deficits (reduction in the methamphetamine-induced turning rate). At 3 months after transplantation, 100% of the animals receiving differentiated 1RB3AN27 cells, 63% of the animals receiving undifferentiated 1RB3AN27 cells, 56% of the animals receiving differentiated NBP2 cells, and 0% of the sham-transplanted animals showed improvements in neurological deficits. At 6 months after transplantation, there was a progressive increase in spontaneous recovery in sham-transplanted animals. These results suggest that immortalized DA neurons should be further studied for their potential use in transplant therapy in advanced PD patients. PMID:9448320

  18. 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

  19. Nucleotide sequence and expression of the gene encoding the major 25-kilodalton outer membrane protein of Brucella ovis: Evidence for antigenic shift, compared with other Brucella species, due to a deletion in the gene.

    PubMed Central

    Cloeckaert, A; Verger, J M; Grayon, M; Zygmunt, M S; Grépinet, O

    1996-01-01

    The nucleotide sequences encoding the major 25-kDa outer membrane protein (OMP) (omp25 genes) of Brucella ovis 63/290, Brucella melitensis 16M, Brucella suis 1330, Brucella canis RM6/66, and Brucella neotomae 5K33 (all reference strains) were determined and compared with that of Brucella abortus 544 (P. de Wergifosse, P. Lintermans, J. N. Limet, and A. Cloeckaert, J. Bacteriol. 177:1911-1914, 1995). The major difference found was between the omp25 gene of B. ovis and those of the other Brucella species; the B. ovis gene had a 36-bp deletion located at the 3' end of the gene. The corresponding regions of other Brucella species contain two 8-bp direct repeats and two 4-bp inverted repeats, which could have been involved in the genesis of the deletion. The mechanism responsible for the genesis of the deletion appears to be related to the "slipped mispairing" mechanism described in the literature. Expression of the 25-kDa outer membrane protein (Omp25) in Brucella spp. or expression from the cloned omp25 gene in Escherichia coli cells was studied with a panel of anti-Omp25 monoclonal antibodies (MAbs). As shown by enzyme-linked immunosorbent assay (ELISA) and immunoelectron microscopy, Omp25 was exported to the outer membrane in E. coli expressing either the truncated omp25 gene of B. ovis or the entire omp25 genes of the other Brucella species. Size and antigenic shifts due to the 36-bp deletion were demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting and by the differences in binding patterns in ELISA of the anti-Omp25 MAbs at the cell surface of E. coli cells harboring the appropriate gene and of cells of B. ovis and other Brucella species. In particular, MAbs directed against discontinuous epitopes of the entire Omp25 showed the absence of, or a significant reduction in, antibody reactivity with the B. ovis truncated Omp25. The results indicated that, as defined by the MAbs, exported Omp25 probably presents similar

  20. Analysis of the VSG gene silent archive in Trypanosoma brucei reveals that mosaic gene expression is prominent in antigenic variation and is favored by archive substructure.

    PubMed

    Marcello, Lucio; Barry, J David

    2007-09-01

    Trypanosoma brucei evades host acquired immunity through differential activation of its large archive of silent variant surface glycoprotein (VSG) genes, most of which are pseudogenes in subtelomeric arrays. We have analyzed 940 VSGs, representing one half to two thirds of the arrays. Sequence types A and B of the VSG N-terminal domains were confirmed, while type C was found to be a constituent of type A. Two new C-terminal domain types were found. Nearly all combinations of domain types occurred, with some bias to particular combinations. One-third of encoded N-terminal domains, but only 13% of C-terminal domains, are intact, indicating a particular need for silent VSGs to gain a functional C-terminal domain to be expressed. About 60% of VSGs are unique, the rest occurring in subfamilies of two to four close homologs (>50%-52% peptide identity). We found a subset of VSG-related genes, differing from VSGs in genomic environment and expression patterns, and predict they have distinct function. Almost all (92%) full-length array VSGs have the partially conserved flanks associated with the duplication mechanism that activates silent genes, and these sequences have also contributed to archive evolution, mediating most of the conversions of segments, containing >/=1 VSG, within and between arrays. During infection, intact array genes became activated by duplication after two weeks, and mosaic VSGs assembled from pseudogenes became expressed by week three and predominated by week four. The small subfamily structure of the archive appears to be fundamental in providing the interacting donors for mosaic formation.

  1. Antigenic variation in Giardia lamblia.

    PubMed

    Prucca, Cesar G; Lujan, Hugo D

    2009-12-01

    Giardia lamblia undergoes antigenic variation, both in vitro and within the intestines of infected individuals. Variant-specific surface proteins (VSPs) cover the entire surface of the trophozoites and are the main antigens recognized by the host. Only 1 of about 200 VSP genes encoded by the Giardia genome is expressed on the surface of individual Giardia cells at any time; however, VSP antigen switching occurs spontaneously. In the recent year, significant advances in the knowledge of the antigen switching process have been achieved, which strongly suggests that antigenic variation in Giardia is regulated at the post-transcriptional level by a mechanism similar to RNA interference (RNAi). Several enzymes of the RNAi pathway are directly involved in VSP mRNA silencing and/or translational repression. Although several questions remain regarding how individual VSP antigens are selected for expression on the parasite surface, it is clear that an epigenetic mechanism is involved. In this review, we summarize the characteristics of this fascinating mechanism, analyse conflicting information regarding the structure of VSPs as it relates to the host's immune response, and highlight the major issues that need to be resolved to fully understand antigenic variation in this important pathogen.

  2. [Characteristic of nuclear antigen 1 gene and latent membrane protein 1 gene of Epstein-Barr virus in primary EBV infection in children in Beijing area in 2005-2010].

    PubMed

    Ai, Jun-Hong; Xie, Zheng-De; Liu, Chun-Yan; Gao, Li-Wei; Yan, Jing

    2012-10-01

    To analyze the characteristic of nuclear antigen 1 gene and latent membrane protein 1 gene of Epstein-Barr virus in primary EBV infection in children in Beijing area in 2005-2012. Polymerase chain reaction (PCR) was used to amplify the EBNA-3C, EBNA1 and LMP1 genes. The amplified products were sequenced directly and the sequences were analyzed by BioEdit 7. 0. 9 and MEGA 4. 0. 2. Type A EBV was detected in 98% samples. Nucleotide sequence analysis of the carboxy-terminal region of EBNA1 showed that Vvvl was deteted in 98% samples. DNA sequence analysis of LMP1 C-terminus indicated that China 1 was 90% in this study. There were no significant differences in the frequency of Vvv1 and China 1 between the IM and HLH samples (P = 1.00). Linkage analysis of EBV types, EBNA1 and LMP1 variants indicated that 90% of EBV type A was associated with EBNA1-Vvv1 variant and LMP1-China 1 variant in 40 cases. Full length of LMP1 gene was successfully amplified in 35 cases. Four Chinese groups (CG1-4) were identified. The percentage of CG1-CG4 were 85%, 6%, 6% and 3%, respectively. EBV type A is predominant in primary EBV infection in children in Beijing Area. EBNA1-Vvv1 and LMP1-China 1 variants were predominant genotypes in this area. There is a high linkage between EBNA1-Vvv1 variant and LMP1-China 1 variant. Four Chinese groups (CG1-4) were identified according to the full length of LMP1 gene and CG1 was the most prevalent.

  3. In vivo induced antigen technology (IVIAT).

    PubMed

    Rollins, Sean M; Peppercorn, Amanda; Hang, Long; Hillman, Jeffrey D; Calderwood, Stephen B; Handfield, Martin; Ryan, Edward T

    2005-01-01

    In vivo induced antigen technology (IVIAT) is a technique that identifies pathogen antigens that are immunogenic and expressed in vivo during human infection. IVIAT is complementary to other techniques that identify genes and their products expressed in vivo. Genes and gene pathways identified by IVIAT may play a role in virulence or pathogenesis during human infection, and may be appropriate for inclusion in therapeutic, vaccine or diagnostic applications.

  4. Assessment of long-term outcomes associated with urinary prostate cancer antigen 3 and TMPRSS2:ERG gene fusion at repeat biopsy.

    PubMed

    Merdan, Selin; Tomlins, Scott A; Barnett, Christine L; Morgan, Todd M; Montie, James E; Wei, John T; Denton, Brian T

    2015-11-15

    In men with clinically localized prostate cancer who have undergone at least 1 previous negative biopsy and have elevated serum prostate-specific antigen (PSA) levels, long-term health outcomes associated with the assessment of urinary prostate cancer antigen 3 (PCA3) and the transmembrane protease, serine 2 (TMPRSS2):v-ets erythroblastosis virus E26 oncogene homolog (avian) (ERG) gene fusion (T2:ERG) have not been investigated previously in relation to the decision to recommend a repeat biopsy. The authors performed a decision analysis using a decision tree for men with elevated PSA levels. The probability of cancer was estimated using the Prostate Cancer Prevention Trial Risk Calculator (version 2.0). The use of PSA alone was compared with the use of PCA3 and T2:ERG scores, with each evaluated independently, in combination with PSA to trigger a repeat biopsy. When PCA3 and T2:ERG score evaluations were used, predefined thresholds were established to determine whether the patient should undergo a repeat biopsy. Biopsy outcomes were defined as either positive (with a Gleason score of <7, 7, or >7) or negative. Probabilities and estimates of 10-year overall survival and 15-year cancer-specific survival were derived from previous studies and a literature review. Outcomes were defined as age-dependent and Gleason score-dependent 10-year overall and 15-year cancer-specific survival rates and the percentage of biopsies avoided. Incorporating the PCA3 score (biopsy threshold, 25; generated based on the urine PCA3 level normalized to the amount of PSA messenger RNA) or the T2:ERG score (biopsy threshold, 10; based on the urine T2:ERG level normalized to the amount of PSA messenger RNA) into the decision to recommend repeat biopsy would have avoided 55.4% or 64.7% of repeat biopsies for the base-case patient, respectively, and changes in the 10-year survival rate were only 0.93% or 1.41%, respectively. Multi-way sensitivity analyses suggested that these results were robust

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

    USDA-ARS?s Scientific Manuscript database

    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...

  6. A novel baculovirus vector shows efficient gene delivery of modified porcine reproductive and respiratory syndrome virus antigens and elicits specific immune response.

    PubMed

    Karuppannan, Anbu K; Qiang, Jia; Chang, C C; Kwang, Jimmy

    2013-11-04

    Porcine reproductive and respiratory syndrome (PRRS) is an economically devastating epizootic of porcine species. Current vaccines are inadequate to control the disease burden and outbreaks in the field. We report a novel baculovirus vaccine vector with White spot syndrome virus immediate early 1 shuttle promoter, with strong activity in both insect cells and mammalian cells, for immunization against PRRSV. The insect cell cultured baculovirus vector produces PRRSV envelope glycoproteins ORF2a, ORF3, ORF4 and ORF5, which are similar to the antigens in the infectious PRRS virion, and these antigens are stably incorporated on the surface of the baculovirus. Further, the baculovirus vector efficiently transduces these antigens in cells of porcine origin, thereby simulating a live infection. The baculovirus vectored PRRSV antigens, upon inoculation in mice, elicits robust neutralizing antibodies against the infective PRRS virus. Further, the experiments indicate that hitherto under emphasized ORF2a and ORF4 are important target antigens for neutralizing PRRSV infectivity.

  7. 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

    SciTech Connect

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

    1988-05-01

    Two rat thyroid epithelial differentiated cell lines, PC CI 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 CI 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.

  8. 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

  9. [Features of the distribution of BoLA-A antigens and alleles of the BoLA-DRB3 gene in Black Pied cattle in relation to association with leukemia].

    PubMed

    Ernst, L K; Sulimova, G E; Orlova, A R; Udina, I G; Pavlenko, S P

    1997-01-01

    The character of distribution of BoLA class-I antigens was studied in Black Pied cattle populations differing in status in relation to leukemia. Associative relationships of distinct antigens with resistance and susceptibility to leukemia were revealed. Using the statusmetria method, an integral estimate of predisposition to leukemia (Z) was calculated taking into consideration the contribution of each antigen in the immunogenetic status of the animal. The interval of Z values was determined, which allowed animals to be divided into groups according to resistance or susceptibility to leukemia. Alleles of the BoLA-DRB3 gene were typed in subsamples of animals with leukemia and healthy animals by the PCR-RFLP method. Twenty alleles of the gene were detected, and their frequencies were determined in both subsamples. Alleles mediating resistance of animals to leukemia (BoLA-DRB3.2*11, *23, and *28) were distributed in the group of healthy animals with frequencies of 0.079, 0.132, and 0.053, respectively; they were completely absent in animals with leukemia. The data on the estimate of animal status in relation to leukemia, which were obtained by the method of statusmetria taking in consideration the real contribution of the each class-I antigen in the detection of the disease risk (value Z), and data of allele typing by the PCR-RFLP method were shown to be in good agreement. The possibility of using BoLA class-I antigen typing in herds to determine the number of animals with leukemia was demonstrated.

  10. First systematic experience of preimplantation genetic diagnosis for single-gene disorders, and/or preimplantation human leukocyte antigen typing, combined with 24-chromosome aneuploidy testing.

    PubMed

    Rechitsky, Svetlana; Pakhalchuk, Tatiana; San Ramos, Geraldine; Goodman, Adam; Zlatopolsky, Zev; Kuliev, Anver

    2015-02-01

    To study the feasibility, accuracy, and reproductive outcome of 24-chromosome aneuploidy testing (24-AT), combined with preimplantation genetic diagnosis (PGD) for single-gene disorders (SGDs) or human leukocyte antigen (HLA) typing in the same biopsy sample. Retrospective study. Preimplantation genetic diagnosis center. A total of 238 PGD patients, average age 36.8 years, for whom 317 combined PGD cycles were performed, involving 105 different conditions, with or without HLA typing. Whole-genome amplification product, obtained in 24-AT, was used for PGD and/or HLA typing in the same blastomere or blastocyst biopsy samples. Proportion of the embryos suitable for transfer detected in these blastomere or blastocyst samples, and the resulting pregnancy and spontaneous abortion rates. Embryos suitable for transfer were detected in 42% blastocyst and 25.1% blastomere samples, with a total of 280 unaffected, HLA-matched euploid embryos detected for transfer in 212 cycles (1.3 embryos per transfer), resulting in 145 (68.4%) unaffected pregnancies and birth of 149 healthy, HLA-matched children. This outcome is significantly different from that of our 2,064 PGD cycle series without concomitant 24-AT, including improved pregnancy (68.4% vs. 45.4%) and 3-fold spontaneous abortion reduction (5.5% vs. 15%) rates. The introduced combined approach is a potential universal PGD test, which in addition to achieving extremely high diagnostic accuracy, significantly improves reproductive outcomes of PGD for SGDs and HLA typing in patients of advanced reproductive age. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  11. B4GALNT2 gene expression controls the biosynthesis of Sda and sialyl Lewis X antigens in healthy and cancer human gastrointestinal tract.

    PubMed

    Groux-Degroote, Sophie; Wavelet, Cindy; Krzewinski-Recchi, Marie-Ange; Portier, Lucie; Mortuaire, Marlène; Mihalache, Adriana; Trinchera, Marco; Delannoy, Philippe; Malagolini, Nadia; Chiricolo, Mariella; Dall'Olio, Fabio; Harduin-Lepers, Anne

    2014-08-01

    The histo blood group carbohydrate Sd(a) antigen and its cognate biosynthetic enzyme B4GALNT2 show the highest level of expression in normal colon. Their dramatic down regulation previously observed in colon cancer tissues could play a role in the concomitant elevation of the selectin ligand sLe(x), involved in metastasis. However, down regulation of sLe(x) expression by B4GALNT2 has been so far demonstrated in vitro, but not in tissues. The human B4GALNT2 gene specifies at least two transcripts, diverging in the first exon, never studied in normal and cancer tissues. The long form contains a 253 nt exon 1L; the short form contains a 38 nt exon 1S. Using qPCR, we showed that cell lines and normal or cancerous colon, expressed almost exclusively the short form, while the long form was mainly expressed by the embryonic colon fibroblast cell line CCD112CoN. Immunochemistry approaches using colon cancer cells permanently expressing either B4GALNT2 cDNAs as controls, led to the observation of several protein isoforms in human normal and cancerous colon, and cell lines. We showed that tissues expressing B4GALNT2 protein isoforms were able to induce Sd(a) and to inhibit sLe(x) expression; both of which are expressed mainly on PNGase F-insensitive carbohydrate chains. Concomitant expression of B4GALNT2 and siRNA-mediated inhibition of FUT6, the major fucosyltransferase involved in sLe(x) synthesis in colon, resulted in a cumulative inhibition of sLe(x). In normal colon samples a significant relationship between sLe(x) expression and the ratio between FUT6/B4GALNT2 activities exists, demonstrating for the first time a role for B4GALNT2 in sLe(x) inhibition in vivo. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Identification and Migration of Primordial Germ Cells in Atlantic Salmon, Salmo salar: Characterization of Vasa, Dead End, and Lymphocyte Antigen 75 Genes

    PubMed Central

    Nagasawa, Kazue; Fernandes, Jorge MO; Yoshizaki, Goro; Miwa, Misako; Babiak, Igor

    2013-01-01

    No information exists on the identification of primordial germ cells (PGCs) in the super-order Protacanthopterygii, which includes the Salmonidae family and Atlantic salmon (Salmo salar L.), one of the most commercially important aquatic animals worldwide. In order to identify salmon PGCs, we cloned the full-length cDNA of vasa, dead end (dnd), and lymphocyte antigen 75 (ly75/CD205) genes as germ cell marker candidates, and analyzed their expression patterns in both adult and embryonic stages of Atlantic salmon. Semi-quantitative RT-PCR results showed that salmon vasa and dnd were specifically expressed in testis and ovary, and vasa, dnd, and ly75 mRNA were maternally deposited in the egg. vasa mRNA was consistently detected throughout embryogenesis while dnd and ly75 mRNA were gradually degraded during cleavages. In situ analysis revealed the localization of vasa and dnd mRNA and Ly75 protein in PGCs of hatched larvae. Whole-mount in situ hybridization detected vasa mRNA during embryogenesis, showing a distribution pattern somewhat different to that of zebrafish; specifically, at mid-blastula stage, vasa-expressing cells were randomly distributed at the central part of blastodisc, and then they migrated to the presumptive region of embryonic shield. Therefore, the typical vasa localization pattern of four clusters during blastulation, as found in zebrafish, was not present in Atlantic salmon. In addition, salmon PGCs could be specifically labeled with a green fluorescence protein (GFP) using gfp-rt-vasa 3′-UTR RNA microinjection for further applications. These findings may assist in understanding PGC development not only in Atlantic salmon but also in other salmonids. PMID:23239145

  13. Antitumor activity of chimeric immunoreceptor gene-modified Tc1 and Th1 cells against autologous carcinoembryonic antigen-expressing colon cancer cells.

    PubMed

    Sasaki, Takeshi; Ikeda, Hiroaki; Sato, Masayoshi; Ohkuri, Takayuki; Abe, Hiroyuki; Kuroki, Masahide; Onodera, Masafumi; Miyamoto, Masaki; Kondo, Satoshi; Nishimura, Takashi

    2006-09-01

    To generate tumor-specific and interferon (IFN)-gamma-producing Tc1 and Th1 cells applicable for many cancer patients, we previously developed a protocol for generating carcinoembryonic antigen (CEA)-specific Tc1 and Th1 cells from healthy human T cells by transduction with a lentivirus containing a chimeric immunoglobulin T-cell receptor (cIgTCR) gene composed of single-chain variable fragments from an anti-CEA-specific monoclonal antibody fused to an intracellular signaling domain of CD28 and CD3zeta. These cells, designated Tc1-T and Th1-T bodies, respectively, showed strong antitumor activity against CEA-expressing tumor cells in RAG2-/- mice when both of them were transferred. However, it remains unclear whether it is possible to generate Tc1-T and Th1-T bodies from cancer patients with defective T-cell function because of significant immunosuppression. Here, we prepared Tc1-T and Th1-T bodies from T cells of a colon cancer patient, and asked whether these T bodies can exert effective T-cell function against autologous tumor cells. These T bodies showed high cytotoxicity and produced IFN-gamma in response to CEA-expressing autologous tumor cells, even in the presence of soluble CEA. It was also demonstrated that Th1-T bodies supported the survival of Tc1-T bodies through cell-to-cell interactions. Furthermore, our protocol utilized retrovirus for cIgTCR transduction to achieve better induction efficiency compared to lentivirus-mediated transduction. Taken together, our findings here indicate that retrovirally transduced Tc1-T and Th1-T bodies will become a promising strategy for adoptive immunotherapy of human cancer.

  14. Carriage of a Tumor Necrosis Factor Polymorphism Amplifies the Cytotoxic T-Lymphocyte Antigen 4 Attributed Risk of Primary Biliary Cirrhosis: Evidence for a Gene–Gene Interaction

    PubMed Central

    Juran, Brian D.; Atkinson, Elizabeth J.; Larson, Joseph J.; Schlicht, Erik M.; Liu, Xiangdong; Heathcote, E. Jenny; Hirschfield, Gideon M.; Siminovitch, Katherine A.; Lazaridis, Konstantinos N.

    2010-01-01

    Common genetic variants significantly influence complex diseases such as primary biliary cirrhosis (PBC). We recently reported an association between PBC and a single nucleotide polymorphism (rs231725) of the immunoreceptor gene cytotoxic T-lymphocyte antigen 4 (CTLA4). We hypothesized that PBC risk attributed to this polymorphism might be increased by propensity to an overly robust inflammatory response. Thus, we examined its potential interaction with the commonly studied −308AG promoter polymorphism (rs1800629) of the tumor necrosis factor (TNF) gene for which the variant TNF2A allele causes increased TNF production. The polymorphisms were genotyped in 866 PBC patients and 761 controls from independent US and Canadian registries; the effects of individual single nucleotide polymorphisms (SNPs) and their interaction on PBC risk was assessed by logistic regression. The reported association of PBC with the CTLA4 “A/A” genotype was replicated in the Canadian cohort and significant for PBC risk in the combined data (odds ratio [OR], 1.68; P = 0.0005). TNF2A allele frequency was elevated in PBC patients, but only reached borderline significance using the combined data (OR, 1.21; P = 0.042). Analysis showed that TNF2A carriage was significantly increased in CTLA4 “A/A” PBC patients compared with CTLA4 “A/A” controls (39.7% versus 16.5%, P = 0.0004); no apparent increase of TNF2A carriage was noted in CTLA4 “A/G” or “G/G” individuals. Finally, interaction under a logistic model was highly significant, as TNF2A carriage in combination with the CTLA4 “A/A” genotype was present in 6.5% of PBC patients, compared with 1.7% of controls (OR, 3.98; P < 0.0001). Conclusion TNF2A amplifies the CTLA4 rs231725 “A/A” genotype risk for PBC. Although the mechanisms remain unclear, the premise that deficiency in T-cell regulation resulting in an increased risk of PBC is amplified by overexpression of an important proinflammatory cytokine provides a basis

  15. Analysis of T cell antigen receptor (TCR) expression by human peripheral blood CD4-8- alpha/beta T cells demonstrates preferential use of several V beta genes and an invariant TCR alpha chain

    PubMed Central

    1993-01-01

    CD4-CD8- (double negative [DN]) alpha/beta T cells are a largely uncharacterized subpopulation of unknown function. To investigate whether these cells are selected to recognize particular antigens or antigen-presenting molecules, DN alpha/beta T cells were purified from the peripheral blood of five normal donors and their T cell receptor (TCR) alpha and beta chains were examined. Random cloning of TCR alpha chains by single-sided polymerase chain reaction (PCR) amplification identified an invariant rearrangement between V alpha 24 and J alpha Q, with no N region diversity, which was expressed preferentially by DN alpha/beta T cells from all donors. Random cloning also identified a precise V alpha 7.2-J alpha (IGRJa14) rearrangement, with two variable amino acids encoded in the V-J junction, which was enriched in the DN alpha/beta T cell preparations from some, but not all, donors. Analysis of TCR beta chains by quantitative PCR amplification demonstrated that the expression of four V beta gene families, V beta 2, 8, 11, and 13, was markedly increased in these DN alpha/beta T cell preparations. The expression of particular TCRs by DN alpha/beta T cells from multiple donors indicates that these cells, or at least a subpopulation of cells with this phenotype, recognize a limited spectrum of antigens and suggests that they may use nonpolymorphic antigen-presenting molecules. PMID:8391057

  16. Construction of a fusion plasmid containing the PSCA gene and cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) and its anti-tumor effect in an animal model of prostate cancer

    PubMed Central

    Mai, T.J.; Ma, R.; Li, Z.; Bi, S.C.

    2016-01-01

    Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is a negative regulator of T cell activation, which competes with CD28 for B7.1/B7.2 binding, and which has a greater affinity. Fusion of specific antigens to extracellular domain of CTLA4 represents a promising approach to increase the immunogenicity of DNA vaccines. In this study, we evaluated this interesting approach for CTLA4 enhancement on prostate stem cell antigen (PSCA)-specific immune responses and its anti-tumor effects in a prostate cancer mouse model. Consequently, we constructed a DNA vaccine containing the PSCA and the CTLA-4 gene. Vaccination with the CTLA4-fused DNA not only induced a much higher level of anti-PSCA antibody, but also increased PSCA-specific T cell response in mice. To evaluate the anti-tumor efficacy of the plasmids, murine models with PSCA-expressing tumors were generated. After injection of the tumor-bearing mouse model, the plasmid carrying the CTLA4 and PSCA fusion gene showed stronger inhibition of tumor growth than the plasmid expressing PSCA alone. These observations emphasize the potential of the CTLA4-fused DNA vaccine, which could represent a promising approach for tumor immunotherapy. PMID:27783810

  17. Antigen Recognition By Variable Lymphocyte Receptors

    SciTech Connect

    Han, B.W.; Herrin, B.R.; Cooper, M.D.; Wilson, I.A.

    2009-05-18

    Variable lymphocyte receptors (VLRs) rather than antibodies play the primary role in recognition of antigens in the adaptive immune system of jawless vertebrates. Combinatorial assembly of leucine-rich repeat (LRR) gene segments achieves the required repertoire for antigen recognition. We have determined a crystal structure for a VLR-antigen complex, VLR RBC36 in complex with the H-antigen trisaccharide from human blood type O erythrocytes, at 1.67 angstrom resolution. RBC36 binds the H-trisaccharide on the concave surface of the LRR modules of the solenoid structure where three key hydrophilic residues, multiple van der Waals interactions, and the highly variable insert of the carboxyl-terminal LRR module determine antigen recognition and specificity. The concave surface assembled from the most highly variable regions of the LRRs, along with diversity in the sequence and length of the highly variable insert, can account for the recognition of diverse antigens by VLRs.

  18. HLA antigens in patients with adrenocortical hyperfunction.

    PubMed

    Lada, G; Gyódi, E; Gláz, E

    1977-01-01

    The HLA antigen frequencies in 100 Caucasian patients with adrenocortical hyperfunction were compared with those found in 352 healthy unrelated subjects. Fourteen antigens on the HLA--A locus, seventeen antigens on the HLA--B locus and three antigens on the HLA--C locus were determined using the standard NIH microlymphocytotoxicity test. The frequency of HLA--A1 antigen in the patient group was 49% as compared with 28% in the controls (pcorr less than 0.01). An increased frequency of HLA--B8 and HLA-BW35 antigens was also found, but the difference was not significant. Increased A1--B8 and A1--BW35 haplotype frequencies were observed. The relationship between the HLA system and various endogenous and exogenous factors eliciting hypercorticism, together with complementary family studies indicate that the HLA system may be a useful genetic marker of the disease susceptibility gene.

  19. 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.

  20. 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 Central

    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

  1. Genes encoding two Theileria parva antigens recognized by CD8+ T-cells exhibit sequence diversity in South Sudanese cattle populations but the majority of alleles are similar to the Muguga component of the live vaccine cocktail

    PubMed Central

    Pelle, Roger; Mwacharo, Joram M.; Njahira, Moses N.; Marcellino, Wani L.; Kiara, Henry; Malak, Agol K.; EL Hussein, Abdel Rahim M.; Bishop, Richard; Skilton, Robert A.

    2017-01-01

    East Coast fever (ECF), caused by Theileria parva infection, is a frequently fatal disease of cattle in eastern, central and southern Africa, and an emerging disease in South Sudan. Immunization using the infection and treatment method (ITM) is increasingly being used for control in countries affected by ECF, but not yet in South Sudan. It has been reported that CD8+ T-cell lymphocytes specific for parasitized cells play a central role in the immunity induced by ITM and a number of T. parva antigens recognized by parasite-specific CD8+ T-cells have been identified. In this study we determined the sequence diversity among two of these antigens, Tp1 and Tp2, which are under evaluation as candidates for inclusion in a sub-unit vaccine. T. parva samples (n = 81) obtained from cattle in four geographical regions of South Sudan were studied for sequence polymorphism in partial sequences of the Tp1 and Tp2 genes. Eight positions (1.97%) in Tp1 and 78 positions (15.48%) in Tp2 were shown to be polymorphic, giving rise to four and 14 antigen variants in Tp1 and Tp2, respectively. The overall nucleotide diversity in the Tp1 and Tp2 genes was π = 1.65% and π = 4.76%, respectively. The parasites were sampled from regions approximately 300 km apart, but there was limited evidence for genetic differentiation between populations. Analyses of the sequences revealed limited numbers of amino acid polymorphisms both overall and in residues within the mapped CD8+ T-cell epitopes. Although novel epitopes were identified in the samples from South Sudan, a large number of the samples harboured several epitopes in both antigens that were similar to those in the T. parva Muguga reference stock, which is a key component in the widely used live vaccine cocktail. PMID:28231338

  2. Time-dependent transition of the immunoglobulin G subclass and immunoglobulin E response in cancer patients vaccinated with cholesteryl pullulan-melanoma antigen gene-A4 nanogel

    PubMed Central

    Kyogoku, Noriaki; Ikeda, Hiroaki; Tsuchikawa, Takahiro; Abiko, Takehiro; Fujiwara, Aki; Maki, Takehiro; Yamamura, Yoshiyuki; Ichinokawa, Masaomi; Tanaka, Kimitaka; Imai, Naoko; Miyahara, Yoshihiro; Kageyama, Shinichi; Shiku, Hiroshi; Hirano, Satoshi

    2016-01-01

    A phase I+II clinical trial of vaccination with MAGE-A4 protein complexed with cholesteryl pullulan melanoma antigen gene-A4 nanogel (CHP-MAGE-A4) is currently underway in patients with MAGE-A4-expressing cancer. In the present study, the primary phase I endpoint was to test the safety of the administration of 300 µg CHP-MAGE-A4 with and without OK-432. Another aim of the study was to clarify the details of the specific humoral immune response to vaccination. The 9 patients enrolled for phase I were vaccinated 6 times, once every 2 weeks: 3 patients with 100 µg and 3 patients with 300 µg CHP-MAGE-A4, and 3 patients with 300 µg CHP-MAGE-A4 plus 0.5 clinical units of OK-432. Toxicities were assessed using Common Terminology Criteria for Adverse Events v3.0. Clinical response was evaluated by modified Response Evaluation Criteria in Solid Tumours. Immunological monitoring of anti-MAGE-A4-specific antibodies was performed by ELISA of pre- and post-vaccination patient sera. The 6 vaccinations produced no severe adverse events. Stable disease was assessed in 4/9 patients. Anti-MAGE-A4 total immunoglobulin (Ig)G titers increased in 7/9 patients. Efficacious anti-MAGE-A4 IgG1, 2 and 3 antibody responses were observed in 7/9 patients. Among them, positive conversions to T helper 2 (Th2)-type antibody responses (IgG4 and IgE) were observed after frequent vaccination in 4/7 patients. The Th2 conversion was possibly associated with undesirable clinical observations, including progressive disease and the appearance of a new relapse lesion. The present study suggested that frequent vaccinations activated a Th2-dominant status in the cancer patients. The identification of a time-dependent IgG subclass and IgE antibody production during vaccination protocols may be a useful surrogate marker indicating a potentially undesirable change of the immunological environment for an effective antitumor immune response in cancer patients. PMID:28105158

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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

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

    2016-01-01

    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. 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. 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). Compared with conventional serum carcinoembryonic antigen detection, our multigene chip aided more accurate and earlier prediction of

  8. 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

  9. Frequency analysis of functional immunoglobulin C- and V-gene expression by mitogen-reactive B cells in germfree mice fed chemically defined ultra-filtered "antigen-free" diet.

    PubMed

    Hooijkaas, H; van der Linde-Preesman, A A; Bitter, W M; Benner, R; Pleasants, J R; Wostmann, B S

    1985-04-01

    The frequencies of lipopolysaccharide (LPS)-reactive B cells and their antibody specificity repertoire have been determined in the spleen and bone marrow (BM) of conventional (CV) and "antigen-free" C3H/HeCr mice of various ages. The antigen-free mice were germfree (GF)-raised and were fed an ultrafiltered solution of chemically defined (CD) low m.w. nutrients, and were thus devoid of exogenous antigenic stimulation. Spleen and BM cells were grown in a limiting dilution culture system that allows the growth and development of every newly formed LPS-reactive B cell into a clone of IgM-secreting cells which are capable of switching to other immunoglobulin (Ig) heavy chain isotypes (C-gene expression). The secretion of IgM and IgG1 was determined in the protein A plaque assay, whereas specific IgM antibody-secreting cells (V-gene expression) were detected in plaque assays specific for various heterologous erythrocytes and sheep red blood cells (SRBC) coupled with a number of different haptens. The absolute frequency of LPS-reactive B cells and their capacity to switch to IgG1-secretion was not significantly different in 8- to 12-wk-old and 52-wk-old GF-CD mice and their age-matched CV controls. Moreover, no differences were observed in the frequencies of antigen-specific B cells within the pool of LPS reactive B cells. These frequencies ranged from 1 in 20 to 1 in 50 for NIP4-SRBC and NNP2-SRBC, from 1 in 100 to 1 in 150 for NIP0.4-SRBC, from 1 in 50 to 1 in 100 for TNP30-SRBC, and from 1 in 1000 to 1 in 2000 for SRBC and horse red blood cells. Within the limitations of having determined the switching capacity of IgM to IgG1 only and having assessed only a minor fraction of the total B cell antibody-specificity repertoire, the data indicate that young and old GF-CD mice, although devoid of exogenous antigenic and/or mitogenic stimulation, generate B cells with a similar switching capacity and a similar IgM antibody specificity repertoire as CV mice.

  10. 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

  11. Induction of MTG8-specific cytotoxic T-cell lines: MTG8 is probably a tumour antigen that is recognized by cytotoxic T cells in AML1-MTG8-fused gene-positive acute myelogenous leukaemia.

    PubMed

    Maeda, M; Otsuka, T; Kimura, N; Kozu, T; Fukuyama, T; Uchida, N; Sugio, Y; Itoh, Y; Iino, T; Inaba, S; Niho, Y

    2000-11-01

    Several reports have demonstrated the persistent detection of AML1-MTG8 fusion products, representing minimal residual disease (MRD), in patients with t(8;21) acute myelogenous leukaemia (AML) who are in long-term remission. It is probable that immune-mediated mechanisms that are able to suppress the expansion of MRD may result in the continuance of remission. It was previously shown that some t(8;21) AML patients had high anti-MTG8 antibody titres. MTG8 expression in normal adult tissues is limited to the brain or heart in which human leucocyte antigen (HLA) class I cell-surface antigens are either not or are only faintly detectable. We hypothesized that the overexpression of the MTG8 gene in t(8;21) AML cells could act as a possible tumour antigen, which might be able to induce the immune-mediated suppression of the expansion of MRD. We were able to induce HLA-A0201-restricted cytotoxic T-lymphocyte (CTL) lines against an MTG8 peptide (MTG8b amino acids 182-191) using monocyte-derived dendritic cells from a healthy donor. T-cell receptor (TCR)Valpha17, TCRVbeta14 and 15, and TCRJbeta2.1 and 2.3 are predominantly used in these CTL lines. Our data, which suggest that the MTG8 protein could be one of the tumour antigens recognized by CTLs, may be helpful in further investigations of TCR analysis in t(8;21) AML patients with HLA-A0201 who are in long-term remission.

  12. NE1: a new neutrophil specific antigen.

    PubMed

    Claas, F H; Langerak, J; Sabbe, L J; van Rood, J J

    1979-02-01

    The sera of three children with chronic benign neutropenia, due to anti-neutrophil antibodies, were studied with respect to their antibody specificity. This was done by screening the sera against a panel of leukocyte donors in the EDTA micro-agglutination test and in the indirect fluorescence test. Two of the sera contained antibodies against the known neutrophil-specific antigen NA2. The third serum was directed against a new neutrophil-specific antigen. Genetic analysis showed no correlation between this antigen and the already known neutrophil-specific antigens: 9A, NA1, NA2, NB1, and NC1. In the Dutch population the frequency of the new antigen, tentatively called NE1, is 23%, which gives a gene frequency of 0.12.

  13. The role of the prostate cancer gene 3 urine test in addition to serum prostate-specific antigen level in prostate cancer screening among breast cancer, early-onset gene mutation carriers.

    PubMed

    Cremers, Ruben G; Eeles, Rosalind A; Bancroft, Elizabeth K; Ringelberg-Borsboom, Janneke; Vasen, Hans F; Van Asperen, Christi J; Schalken, Jack A; Verhaegh, Gerald W; Kiemeney, Lambertus A

    2015-05-01

    To evaluate the additive value of the prostate cancer gene 3 (PCA3) urine test to serum prostate-specific antigen (PSA) in prostate cancer (PC) screening among breast cancer, early-onset gene (BRCA) mutation carriers. This study was performed among the Dutch participants of IMPACT, a large international study on the effectiveness of PSA screening among BRCA mutation carriers. Urinary PCA3 was measured in 191 BRCA1 mutation carriers, 75 BRCA2 mutation carriers, and 308 noncarriers. The physicians and participants were blinded for the results. Serum PSA level ≥ 3.0 ng/ml was used to indicate prostate biopsies. PCA3 was evaluated (1) as an independent indicator for prostate b iopsies and (2) as an indicator for prostate biopsies among men with an elevated PSA level. PC detected up to the 2-year screening was used as gold standard as end-of-study biopsies were not performed. Overall, 23 PCs were diagnosed, 20 of which were in men who had an elevated PSA level in the initial screening round. (1) PCA3, successfully determined in 552 participants, was elevated in 188 (cutoff ≥ 25; 34%) or 134 (cutoff ≥ 35; 24%) participants, including 2 of the 3 PCs missed by PSA. PCA3 would have added 157 (≥ 25; 28%) or 109 (≥ 35; 20%) biopsy sessions to screening with PSA only. (2) Elevated PCA3 as a requirement for biopsies in addition to PSA would have saved 37 (cutoff ≥ 25) or 43 (cutoff ≥ 35) of the 68 biopsy sessions, and 7 or 11 PCs would have been missed, respectively, including multiple high-risk PCs. So far, PCA3 performed best among BRCA2 mutation carriers, but the numbers are still small. Because PCA3 was not used to indicate prostate biopsies, its true diagnostic value cannot be calculated. The results do not provide evidence for PCA3 as a useful additional indicator of prostate biopsies in BRCA mutation carriers, as many participants had an elevated PCA3 in the absence of PC. This must be interpreted with caution because PCA3 was not used to indicate biopsies

  14. 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. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  15. 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 Central

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

    2015-01-01

    Laboratory testing for the diagnosis of Lyme disease is performed primarily by serologic assays and is accurate for detection beyond the acute stage of the infection. Serodiagnostic assays to detect the early stages of infection, however, are limited in their sensitivity, and improvement is warranted. We analyzed a series of Borrelia burgdorferi proteins known to be induced within feeding ticks and/or during mammalian infection for their utility as serodiagnostic markers against a comprehensive panel of Lyme disease patient serum samples. The antigens were assayed for IgM and IgG reactivity in line immunoblots and separately by enzyme-linked immunosorbent assay (ELISA), with a focus on reactivity against early Lyme disease with erythema migrans (EM), early disseminated Lyme neuroborreliosis, and early Lyme carditis patient serum samples. By IgM immunoblotting, we found that recombinant proteins BBA65, BBA70, and BBA73 reacted with early Lyme EM samples at levels comparable to those of the OspC antigen used in the current IgM blotting criteria. Additionally, these proteins reacted with serum samples from patients with early neuroborreliosis and early carditis, suggesting value in detecting early stages of this disease progression. We also found serological reactivity against recombinant proteins BBA69 and BBA73 with early-Lyme-disease samples using IgG immunoblotting and ELISA. Significantly, some samples that had been scored negative by the Centers for Disease Control and Prevention-recommended 2-tiered testing algorithm demonstrated positive reactivity to one or more of the antigens by IgM/IgG immunoblot and ELISA. These results suggest that incorporating additional in vivo-expressed antigens into the current IgM/IgG immunoblotting tier in a recombinant protein platform assay may improve the performance of early-Lyme-disease serologic testing. PMID:26376927

  16. 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.

  17. 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

  18. Identification of human leukocyte antigen-A24-restricted epitope peptides derived from gene products upregulated in lung and esophageal cancers as novel targets for immunotherapy.

    PubMed

    Suda, Takako; Tsunoda, Takuya; Daigo, Yataro; Nakamura, Yusuke; Tahara, Hideaki

    2007-11-01

    For the development of cancer vaccine therapies, we have searched for possible epitope peptides that can elicit cytotoxic T lymphocytes (CTL) to the TTK protein kinase (TTK), lymphocyte antigen 6 complex locus K (LY6K) and insulin-like growth factor (IGF)-II mRNA binding protein 3 (IMP-3), which were previously identified to be transactivated in the majority of lung and esophageal cancers. We screened 31, 17 and 17 candidate human leukocyte antigen (HLA)-A*2402-binding peptides to parts of TTK, LY6K and IMP-3, respectively. As a result, we successfully established strong CTL clones stimulated by TTK-567 (SYRNEIAYL), LY6K-177 (RYCNLEGPPI) and IMP-3-508 (KTVNELQNL) that have specific cytotoxic activities against the HLA-A24-positive target cells pulsed with the candidate peptides. Subsequent analysis of the CTL clones also revealed their cytotoxic activities against lung and esophageal tumor cells that endogenously express TTK, LY6K or IMP-3. A cold target inhibition assay further confirmed that the CTL cell clones specifically recognized the MHC class I–peptide complex. Our results strongly imply that TTK, LY6K and IMP-3 are novel tumor-associated antigens recognized by CTL, and TTK-567 (SYRNEIAYL), LY6K-177 (RYCNLEGPPI) and IMP-3-508 (KTVNELQNL) are HLA-A24-restricted epitope peptides that can induce potent and specific immune responses against lung and esophageal cancer cells expressing TTK, LY6K and IMP-3.

  19. Antigenic variation in malaria: in situ switching, relaxed and mutually exclusive transcription of var genes during intra-erythrocytic development in Plasmodium falciparum.

    PubMed Central

    Scherf, A; Hernandez-Rivas, R; Buffet, P; Bottius, E; Benatar, C; Pouvelle, B; Gysin, J; Lanzer, M

    1998-01-01

    Members of the Plasmodium falciparum var gene family encode clonally variant adhesins, which play an important role in the pathogenicity of tropical malaria. Here we employ a selective panning protocol to generate isogenic P.falciparum populations with defined adhesive phenotypes for CD36, ICAM-1 and CSA, expressing single and distinct var gene variants. This technique has established the framework for examining var gene expression, its regulation and switching. It was found that var gene switching occurs in situ. Ubiquitous transcription of all var gene variants appears to occur in early ring stages. However, var gene expression is tightly regulated in trophozoites and is exerted through a silencing mechanism. Transcriptional control is mutually exclusive in parasites that express defined adhesive phenotypes. In situ var gene switching is apparently mediated at the level of transcriptional initiation, as demonstrated by nuclear run-on analyses. Our results suggest that an epigenetic mechanism(s) is involved in var gene regulation. PMID:9736619

  20. A major allogenic leukocyte antigen in the agnathan hagfish.

    PubMed

    Takaba, Hiroyuki; Imai, Takeshi; Miki, Shoji; Morishita, Yasuyuki; Miyashita, Akihiro; Ishikawa, Naoko; Nishizumi, Hirofumi; Sakano, Hitoshi

    2013-01-01

    All vertebrates, from jawless fish to mammals, possess adaptive immune systems that can detect and inactivate non-self-antigens through a vast repertoire of antigen receptors. Unlike jawed vertebrates, the hagfish utilizes variable lymphocyte receptors (VLRs) that are unrelated to immunoglobulin molecules but are diversified by copy-choice gene conversion mechanism. Here, we report that hagfish VLRs react with allogenic leukocyte antigens but not with self-antigens. We found that a highly polymorphic membrane protein, NICIR3, is recognized by VLRs as an allogenic leukocyte antigen (ALA). In a serological cross-reactivity test, a close correlation was observed between the amino acid differences in the protein sequences and the VLR cross-reactivities. This leukocyte antigen was predominantly expressed in phagocytic leukocytes, where it was associated with phagocytosed protein antigens. These findings suggest that a polymorphic leukocyte antigen, NICIR3/ALA, plays a pivotal role in jawless vertebrate adaptive immunity.

  1. A major allogenic leukocyte antigen in the agnathan hagfish

    PubMed Central

    Takaba, Hiroyuki; Imai, Takeshi; Miki, Shoji; Morishita, Yasuyuki; Miyashita, Akihiro; Ishikawa, Naoko; Nishizumi, Hirofumi; Sakano, Hitoshi

    2013-01-01

    All vertebrates, from jawless fish to mammals, possess adaptive immune systems that can detect and inactivate non-self-antigens through a vast repertoire of antigen receptors. Unlike jawed vertebrates, the hagfish utilizes variable lymphocyte receptors (VLRs) that are unrelated to immunoglobulin molecules but are diversified by copy-choice gene conversion mechanism. Here, we report that hagfish VLRs react with allogenic leukocyte antigens but not with self-antigens. We found that a highly polymorphic membrane protein, NICIR3, is recognized by VLRs as an allogenic leukocyte antigen (ALA). In a serological cross-reactivity test, a close correlation was observed between the amino acid differences in the protein sequences and the VLR cross-reactivities. This leukocyte antigen was predominantly expressed in phagocytic leukocytes, where it was associated with phagocytosed protein antigens. These findings suggest that a polymorphic leukocyte antigen, NICIR3/ALA, plays a pivotal role in jawless vertebrate adaptive immunity. PMID:23612706

  2. Expression of SV40 T antigen polypeptides in cells biochemically transformed by plasmids containing the herpes simplex virus thymidine kinase gene and the genome of an SV40tsA mutant.

    PubMed

    Kit, S; Otsuka, H; Qavi, H; Trkula, D; Dubbs, D R

    1981-10-15

    To study the expression of SV40 tsA genomes that had been non-selectively introduced into mouse cells, SV40 tsA207 DNA was cleaved with BamH I and ligated to BamH I-cleaved plasmid pAGO DNA, which contains a functional HSV-1 thymidine kinase (TK) gene in the form of 2 kbp Pvu II fragment inserted at the Pvu II site of pBR322. Recombinant plasmids (11-12 kbp) were isolated and amplified in E. coli K12 strain RRI. Restriction nuclease analyses demonstrated that recombinant plasmids pSB15 and pSB10 contained intact SV40 genomes with the polarity of transcription oriented in the same direction (clockwise) or the opposite direction (counterclockwise), respectively, in relation to that of the HSV-1 TK gene. Cla I-cleaved pSB10 and pSB15 DNAs were used to transform LM(TK-) cells to TK+. Serological and disc PAGE analyses showed that clonal lines transformed by these plasmids all expressed the selected marker, HSV-1 TK. Molecular hybridization experiments showed that transformed clonal lines TF pSB10 C7 and TF pSB15 C10 had integrated intact SV40 genomes at one integration site, TF pSB10 C3 had integrated an SV40 genome with a small deletion near the BamH I site, but TF pSB15 Cl had integrated a plasmid from which most of the SV40 nucleotide sequences had been deleted. IF assays with hamster anti-SV40 tumor sera showed that TF pSB10 C7 and TF pSB15 C10 strongly expressed SV40 T antigens in over 90% of the cells, TF pSB10 C3 expressed SV40 T antigens in a minority of the cells, and TF pSB15 C1 did not express SV40 T antigens at all. [35S]-methionine labelling and immunoprecipitation experiments showed that, at 36.5 degrees C: (1) TF pSB10 C7 and TF pSB15 C10 expressed 92K and 20K mol. wt. species of SV40 T antigens and 50-55K cellular protein; (2) expression of all three was reduced in TF pSB10 C3 cells; and (3) TF pSB15 C1 expressed none of the SV40 T antigens, nor did parental LM(TK-) or TF 8-2 transformed cells (which contained the HSV-1 TK gene but not SV40 DNA). At 40

  3. Global Population Structure of the Genes Encoding the Malaria Vaccine Candidate, Plasmodium vivax Apical Membrane Antigen 1 (PvAMA1)

    PubMed Central

    Arnott, Alicia; Mueller, Ivo; Ramsland, Paul A.; Siba, Peter M.; Reeder, John C.; Barry, Alyssa E.

    2013-01-01

    Background The Plasmodium vivax Apical Membrane Antigen 1 (PvAMA1) is a promising malaria vaccine candidate, however it remains unclear which regions are naturally targeted by host immunity and whether its high genetic diversity will preclude coverage by a monovalent vaccine. To assess its feasibility as a vaccine candidate, we investigated the global population structure of PvAMA1. Methodology and Principal Findings New sequences from Papua New Guinea (PNG, n = 102) were analysed together with published sequences from Thailand (n = 158), India (n = 8), Sri Lanka (n = 23), Venezuela (n = 74) and a collection of isolates from disparate geographic locations (n = 8). A total of 92 single nucleotide polymorphisms (SNPs) were identified including 22 synonymous SNPs and 70 non-synonymous (NS) SNPs. Polymorphisms and signatures of balancing (positive Tajima's D and low FST values) selection were predominantly clustered in domain I, suggesting it is a dominant target of protective immune responses. To estimate global antigenic diversity, haplotypes comprised of (i) non-singleton (n = 40) and (ii) common (≥10% minor allele frequency, n = 23) polymorphic amino acid sites were then analysed revealing a total of 219 and 210 distinct haplotypes, respectively. Although highly diverse, the 210 haplotypes comprised of only common polymorphisms were grouped into eleven clusters, however substantial geographic differentiation was observed, and this may have implications for the efficacy of PvAMA1 vaccines in different malaria-endemic areas. The PNG haplotypes form a distinct group of clusters not found in any other geographic region. Vaccine haplotypes were rare and geographically restricted, suggesting potentially poor efficacy of candidate PvAMA1 vaccines. Conclusions It may be possible to cover the existing global PvAMA1 diversity by selection of diverse alleles based on these analyses however it will be important to first define the

  4. Presence of interchain disulfide bonds between two gene products that compose the secreted form of an antigen-specific suppressor factor

    PubMed Central

    1981-01-01

    The secreted form of the suppressor T cell factor specific for keyhole limpet hemocyanin derived from the hybridoma 34S-704 was found to consist of the two distinct polypeptide chains, i.e., the antigen- binding and the I-J-encoded chains. They were linked in covalent association with disulfide bonds. The two chains were cleaved by the reduction with dithiothreitol and were easy to reconstitute the active form of TsF. The association of the two distinct chains was suggested to be essential for the expression of the TsF activity. PMID:6166720

  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. A Luciferase Gene Driven by an Alphaherpesviral Promoter Also Responds to Immediate Early Antigens of the Betaherpesvirus HCMV, Allowing Comparative Analyses of Different Human Herpesviruses in One Reporter Cell Line

    PubMed Central

    Villinger, Clarissa; Schubert, Axel; Walther, Paul; Sinzger, Christian; Lieber, Diana

    2017-01-01

    Widely used methods for quantification of human cytomegalovirus (HCMV) infection in cell culture such as immunoblotting or plaque reduction assays are generally restricted to low throughput and require time-consuming evaluation. Up to now, only few HCMV reporter cell lines have been generated to overcome these restrictions and they are afflicted with other limitations because permanently expandable cell lines are normally not fully permissive to HCMV. In this work, a previously existing epithelial cell line hosting a luciferase gene under control of a Varicella-zoster virus promoter was adopted to investigate HCMV infection. The cells were susceptible to different HCMV strains at infection efficiencies that corresponded to their respective degree of epithelial cell tropism. Expression of early and late viral antigens, formation of nuclear inclusions, release of infectious virus progeny, and focal growth indicated productive viral replication. However, viral release and spread occurred at lower levels than in primary cell lines which appears to be due to a malfunction of virion morphogenesis during the nuclear stage. Expression of the luciferase reporter gene was specifically induced in HCMV infected cultures as a function of the virus dose and dependent on viral immediate early gene expression. The level of reporter activity accurately reflected infection efficiencies as determined by viral antigen immunostaining, and hence could discriminate the cell tropism of the tested virus strains. As proof-of-principle, we demonstrate that this cell line is applicable to evaluate drug resistance of clinical HCMV isolates and the neutralization capacity of human sera, and that it allows comparative and simultaneous analysis of HCMV and human herpes simplex virus type 1. In summary, the permanent epithelial reporter cell line allows robust, rapid and objective quantitation of HCMV infection and it will be particularly useful in higher throughput analyses as well as in

  7. Interferon-gamma down-regulates expression of the 230-kDa bullous pemphigoid antigen gene (BPAG1) in epidermal keratinocytes via novel chimeric sequences of ISRE and GAS.

    PubMed

    Kaneko, Takahide; Tamai, Katsuto; Matsuzaki, Yasushi; Yamazaki, Takehiko; Nakano, Hajime; Kon, Atsushi; Hashimoto, Isao; Hanada, Katsumi; Kaneda, Yasuhumi; Uitto, Jouni

    2006-04-01

    The 230-kDa bullous pemphigoid antigen (BPAG1) is an integral component of hemidesmosomes. We have previously reported that interferon-gamma (IFNgamma) inhibits the transcription of the BPAG1 gene (1). Here we investigated the target sequences of IFNgamma-signal transduction pathway in the BPAG1 promoter in epidermal keratinocytes. Transient transfections with 5'-deletion constructs of BPAG1 promoter-luciferase reporter gene plasmids in cultured normal human epidermal keratinocytes (NHEK) allowed us to narrow the DNA region containing IFNgamma inhibitory element (IGIE) to between -1 and -89, upstream from the transcription initiation site (+1). Homology search in this region identified a chimeric sequence, consisting of IFN-stimulated responsive element (ISRE) with a partial 7-bp sequence of IFNgamma activation site (GAS), as identified in the guanylate-binding protein (GBP) gene, inserted at its center. Functional analysis of IGIE, inserted in front of the heterologous thymidine kinase promoter, indicated that IGIE acts as a down-regulatory element of the promoter through IFNgamma-dependent signal pathway. Transient transfection studies with BPAG1 promoter-reporter gene constructs containing mutated IGIE (with TT to GG transversions in the region of 5'ISRE, GAS, and 3'ISRE) demonstrated that disruption of the ISRE sequences, but not GAS, markedly suppressed the BPAG1 basal promoter activity and resulted in attenuated IFNgamma response in keratinocytes. Our findings provide novel insight into the mechanism of IFNgamma regulation in keratinocyte differentiation and proliferation.

  8. Gene Electrotransfer of Plasmid-Encoding IL-12 Recruits the M1 Macrophages and Antigen-Presenting Cells Inducing the Eradication of Aggressive B16F10 Murine Melanoma

    PubMed Central

    Lampreht Tratar, Ursa; Loiacono, Luisa; Kamensek, Urska; Fazio, Vito Michele

    2017-01-01

    Cancer immunotherapy is currently one of the leading approaches in cancer treatment. Gene electrotransfer of plasmids encoding interleukin 12 (IL-12) into the cells leads to the production of IL-12, which drives immune cell polarization to an antitumoral response. One of the cell types that shows great promise in targeting tumor cells under the influence of IL-12 cytokine milieu is that of macrophages. Therefore, the aim of this study was to evaluate gene electrotransfer of antibiotic resistance-free plasmid DNA-encoding murine IL-12 (mIL-12) in mice bearing aggressive B16F10 murine melanoma. IL-12 electrotransfer resulted in the complete long-term eradication of the tumors. Serum mIL-12 and murine interferon γ (mIFNγ) were increased after IL-12 gene electrotransfer. Further on, hematoxylin and eosin (HE) staining showed increased infiltration of immune cells that lasted from day 4 until day 14. Immunohistochemistry (IHC) staining of F4/80, MHCII, and CD11c showed higher positive staining in the IL-12 gene electrotransfer group than in the control groups. Immune cell infiltration into the tumors and the high density of MHCII- and CD11c-positive cells suggest an antitumor polarization of macrophages and the presence of antigen-presenting cells that contributes to the important antitumor effectiveness of IL-12. PMID:28596641

  9. Two classes of transformation-deficient, immortalization-positive simian virus 40 mutants constructed by making three-base insertions in the T antigen gene.

    PubMed Central

    Sugano, S; Yamaguchi, N

    1984-01-01

    We constructed two mutants of simian virus 40 (SV40) by introducing a three-base duplication at AvaII cutting sites within the large T antigen coding region, and we examined these mutants for their abilities to replicate in monkey GC7 cells, to transform rat cell line 3Y1 cells, and to transform and immortalize primary cells from newborn rats. Neither of the mutants could replicate in GC7 cells. One mutant with the duplication at 0.335 SV40 map units (m.u.) (inA942) could transform 3Y1 cells, but the other mutant with the duplication at 0.636 m.u. (inA941) could not. The two mutants could not transform primary rat cells but retained immortalization activity. The results suggest that transformation of primary cells by SV40 requires at least two distinct activities of the large T antigen, one of which can be replaced by a cellular function(s) expressed in immortalized 3Y1 cells. Images PMID:6092718

  10. 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

  11. 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

    Beutin, Lothar; Delannoy, Sabine; Fach, Patrick

    2015-06-15

    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. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  12. TL antigen as a transplantation antigen recognized by TL-restricted cytotoxic T cells

    PubMed Central

    1994-01-01

    In contrast to broadly expressed classical class I antigens of the major histocompatibility complex, structurally closely related TL antigens are expressed in a highly restricted fashion. Unlike classical class I antigens, TL antigens are not known to be targets of cytotoxic T cells or to mediate graft rejection. Whereas classical class I antigens function as antigen-presenting molecules to T cell receptors (TCR), the role of TL is yet to be defined. To elucidate the function of TL, we have derived transgenic mice expressing TL in most tissues including skin by introducing a TL gene, T3b of C57BL/6 mouse origin, driven by the H-2Kb promoter. By grafting the skin of transgenic mice, we demonstrate that TL can serve as a transplantation antigen and mediate a TCR-alpha/beta+ CD8+ cytotoxic T cell response. This T cell recognition of TL does not require antigen presentation by H-2 molecules. Furthermore, we show that C57BL/6 F1 mice develop CD8+ T cells that are cytotoxic for C57BL/6 TL+ leukemia cells, providing further support for the concept that aberrantly expressed nonmutated proteins such as TL can be recognized as tumor antigens. PMID:8113675

  13. Identification of MEF2B, EBF1, and IL6R as Direct Gene Targets of Epstein-Barr Virus (EBV) Nuclear Antigen 1 Critical for EBV-Infected B-Lymphocyte Survival

    PubMed Central

    Tempera, Italo; De Leo, Alessandra; Kossenkov, Andrew V.; Cesaroni, Matteo; Song, Hui; Dawany, Noor; Showe, Louise; Lu, Fang; Wikramasinghe, Priyankara

    2015-01-01

    ABSTRACT Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is the EBV-encoded nuclear antigen and sequence-specific DNA binding protein required for viral origin binding and episome maintenance during latency. EBNA1 can also bind to numerous sites in the cellular genome and can provide a host cell survival function, but it is not yet known how EBNA1 sequence-specific binding is responsible for host cell survival. Here, we integrate EBNA1 chromatin immunoprecipitation sequencing (ChIP-Seq) with transcriptome sequencing (RNA-Seq) after EBNA1 depletion to identify cellular genes directly regulated by EBNA1 that are also essential for B-cell survival. We first compared EBNA1 ChIP-Seq patterns in four different EBV-positive cell types, including Burkitt lymphoma (BL) cells, nasopharyngeal carcinoma (NPC) cells, and lymphoblastoid cell lines (LCLs). EBNA1 binds to ∼1,000 sites that are mostly invariant among cell types and share a consensus recognition motif. We found that a large subset of EBNA1 binding sites are located proximal to transcription start sites and correlate genome-wide with transcription activity. EBNA1 bound to genes of high significance for B-cell growth and function, including MEF2B, IL6R, and EBF1. EBNA1 depletion from latently infected LCLs results in the loss of cell proliferation and the loss of gene expression for some EBNA1-bound genes, including MEF2B, EBF1, and IL6R. Depletion of MEF2B, EBF1, or IL6R partially phenocopies EBNA1 depletion by decreasing the cell growth and viability of cells latently infected with EBV. These findings suggest that EBNA1 binds to a large cohort of cellular genes important for cell viability and implicates EBNA1 as a critical regulator of transcription of host cell genes important for enhanced survival of latently infected cells. IMPORTANCE Epstein-Barr virus (EBV) latent infection is responsible for a variety of lymphoid and epithelial cell malignancies. EBNA1 is the EBV-encoded nuclear antigen that is

  14. Detection of aberrant transcription of major histocompatibility complex class II antigen presentation genes in chronic lymphocytic leukaemia identifies HLA-DOA mRNA as a prognostic factor for survival.

    PubMed

    Souwer, Yuri; Chamuleau, Martine E D; van de Loosdrecht, Arjan A; Tolosa, Eva; Jorritsma, Tineke; Muris, Jettie J F; Dinnissen-van Poppel, Marion J; Snel, Sander N; van de Corput, Lisette; Ossenkoppele, Gert J; Meijer, Chris J L M; Neefjes, Jacques J; Marieke van Ham, S

    2009-05-01

    In human B cells, effective major histocompatibility complex (MHC) class II-antigen presentation depends not only on MHC class II, but also on the invariant chain (CD74 or Ii), HLA-DM (DM) and HLA-DO (DO), the chaperones regulating the antigen loading process of MHC class II molecules. We analysed immediate ex vivo expression of HLA-DR (DR), CD74, DM and DO in B cell chronic lymphocytic leukaemia (B-CLL). Real-time reverse transcription polymerase chain reaction demonstrated a highly significant upregulation of DRA, CD74, DMB, DOA and DOB mRNA in purified malignant cells compared to B cells from healthy donors. The increased mRNA levels were not translated into enhanced protein levels but could reflect aberrant transcriptional regulation. Indeed, upregulation of DRA, DMB, DOA and DOB mRNA correlated with enhanced expression of class II transactivator (CIITA). In-depth analysis of the various CIITA transcripts demonstrated a significant increased activity of the interferon-gamma-inducible promoter CIITA-PIV in B-CLL. Comparison of the aberrant mRNA levels with clinical outcome identified DOA mRNA as a prognostic indicator for survival. Multivariate analysis revealed that the prognostic value of DOA mRNA was independent of the mutational status of the IGHV genes. Thus, aberrant transcription of DOA forms a novel and additional prognostic indicator for survival in B-CLL.

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

    PubMed

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

    2012-05-01

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

  16. Age-related changes in the gene expression profile of antigen-specific mouse CD8+ T cells can be partially reversed by blockade of the BTLA/CD160 pathways during vaccination

    PubMed Central

    Dawany, Noor; Parzych, Elizabeth M; Showe, Louise C; Ertl, Hildegund CJ

    2016-01-01

    We analyzed gene expression profiles of young and aged mouse CD8+ T cells specific for the nucleoprotein (NP) of influenza A/PR8/34 virus. CD8+ T cells were stimulated either by the NP antigen expressed in its native form or fused into the herpes virus (HSV)-1 glycoprotein D (gD) protein, which blocks signaling through the immunoinhibitory B and T lymphocyte attenuator (BTLA) and CD160 pathways. We show that NP-specific CD8+ T cells from aged mice exhibit numerous differences in gene expression compared to NP-specific CD8+ T cells from young mice, including a significant reduction of expression in genes involved in T cell receptor (TcR) and CD28 signaling. We also show that these changes can be reversed in a sub-population (∼50%) of the aged mice by a BTLA/CD160 checkpoint blockade. These results suggest that BTLA/CD160 checkpoint blockade has potential value as a vaccine additive to induce better CD8+ T cell responses in the aged. PMID:27922818

  17. 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

  18. O-antigen modifications providing antigenic diversity of Shigella flexneri and underlying genetic mechanisms.

    PubMed

    Knirel, Y A; Sun, Qiangzheng; Senchenkova, S N; Perepelov, A V; Shashkov, A S; Xu, Jianguo

    2015-07-01

    O-Antigens (O-specific polysaccharides) of Shigella flexneri, a primary cause of shigellosis, are distinguished by a wide diversity of chemical modifications following the oligosaccharide O-unit assembly. The present review is devoted to structural, serological, and genetic aspects of these modifications, including O-acetylation and phosphorylation with phosphoethanolamine that have been identified recently. The modifications confer the host with specific immunodeterminants (O-factors or O-antigen epitopes), which accounts for the antigenic diversity of S. flexneri considered as a virulence factor of the pathogen. Totally, 30 O-antigen variants have been recognized in these bacteria, the corresponding O-factors characterized using specific antibodies, and a significant extension of the serotyping scheme of S. flexneri on this basis is suggested. Multiple genes responsible for the O-antigen modifications and the resultant serotype conversions of S. flexneri have been identified. The genetic mechanisms of the O-antigen diversification by acquisition of mobile genetic elements, including prophages and plasmids, followed occasionally by gene mobilization and inactivation have been revealed. These findings further our understanding of the genetics and antigenicity of S. flexneri and assist control of shigellosis.

  19. [Over-expression of CCL21 up-regulates the antigen presentation-related genes of CK8/18 positive thymic epithelial cells in patients with myasthenia gravis].

    PubMed

    Zhang, Yun; Zhao, Juntao; Zhu, Zhengkun; Yin, Tingting; Xuan, Xiaoyan; Liu, Pingping; Li, Qianru; DU, Ying

    2015-07-01

    To identify the distribution of chemokine (C-C motif) ligand 21 (CCL21) in the thymus of patients with myasthenia gravis (MG) and explore the effects of up-regulation of CCL21 on the expressions of antigen presentation-related genes in cytokeratin 8/18 (CK8/18) positive thymic epithelial cells (TECs) after transfected with CCL21 genes. The expressions and distributions of CK8/18 and CCL21 in the thymus tissue of MG patients were detected by immunohistochemistry. The mRNA levels of CCL21, CCL19 and their receptor chemokine (C-C motif) receptor 7 (CCR7) in the thymus tissue of MG patients were determined by real-time quantitative PCR (qRT-PCR). Primary cultured CK8/18⁺ TECs were transfected with pCMV-CCL21, and the relative mRNA expressions of function-associated genes (CD80, ICAM-1, CD86, HLA-DR, HLA-A) in CK8/18⁺ TECs before and after the transfection were investigated by qRT-PCR. Immunohistochemical results showed that the number of CK8/18 positive cells in the hyperplastic thymus tissues of MG patients was significantly more than that in the normal controls, and the protein expression of CCL21 was also much higher in the hyperplastic thymus tissues. The qRT-PCR showed that the expressions of CCL21 and CCR7 mRNA increased significantly in hyperplastic thymus tissues of MG patients compared with those in normal controls, while there was no difference in the expression of CCL19. Furthermore, CK8/18 positive cells were found mainly located in cortico-medullary junction and medulla area. The relative mRNA expression levels of HLA-A, HLA-DR, ICAM and CD80 rose significantly in CK8/18⁺ TECs after tran